JP7793166B2 - antifreeze - Google Patents

Description

The present invention relates to an antifreeze agent and an antifreeze method using the antifreeze agent.

In cold, snowy regions, many winter-specific traffic accidents occur. Therefore, it is necessary to ensure road traffic safety even under harsh winter conditions. Measures to prevent road ice from melting and freezing include installing road snow melting facilities such as road heating and mechanical snow removal, but these require a large amount of money, manpower, and time and are reaching their limits. Studded tires were widely used to improve vehicle driving performance in winter, but their use was banned by law in 1990 to prevent dust pollution.

Subsequently, tires without spikes (studless tires) began to be used, but they were less effective at preventing slippage on ice-covered road surfaces and created very slippery roads, making it necessary to take measures to prevent winter road conditions. As a result, the use of inexpensive and easy-to-use antifreeze has increased in recent years. The most commonly used antifreezes are chloride-based, such as sodium chloride (NaCl), and account for 99% of the antifreeze sprayed by the Hokkaido Regional Development Bureau.

While their use is increasing, concerns have arisen over the impact that chloride-based deicers can have on road structures due to salt damage. Due to this impact, the use and development of non-chloride deicers is progressing. The most commonly used non-chloride deicer is calcium magnesium acetate (CMA). While CMA causes much less metal corrosion than chloride-based deicers, its high cost remains a problem.

It has been discovered that organisms that live at low temperatures produce antifreeze proteins (AFPs), which have effects such as inhibiting ice recrystallization and controlling ice crystal shape, and are used as a means of protecting cells from freezing. AFPs have been found in, for example, fish, insects, plants, fungi, and microorganisms.

Furthermore, Non-Patent Document 1 reports that collagen hydrolysates with sizes between 600 and 2700 Da, obtained by hydrolyzing bovine collagen with Alcalase, can inhibit ice recrystallization and have the same ice recrystallization inhibitory effect as natural antifreeze proteins. It has also been reported that collagen hydrolysates derived from chicken, pork, salmon, etc. have the same ice recrystallization inhibitory activity as antifreeze proteins.

Several supercooling-promoting substances (anti-ice nucleation substances) that accelerate the supercooling phenomenon have been reported. By using supercooling-promoting substances, it is possible to create water that does not freeze even at sub-zero temperatures. Anti-ice nucleation substances can also prevent damage to plant or animal cells caused by freezing. Even if cells do freeze, the temperature at which ice nuclei form is lowered, making it possible to reduce the size of ice crystals at the moment of freezing. This has potential applications in fields such as food and biomaterials (organ preservation).

As a result of extensive research into applications in the food industry, the inventors have proposed supercooling-promoting substances that utilize peptides contained in bean paste lees extract (Patent Document 1), sake extract (Patent Document 2), compounds extracted from coffee beans that have an aromatic hydrocarbon structure and a carboxyl group (Patent Document 3), and melanoidin (Patent Document 4).

Patent No. 5322602 Patent No. 5608435 Patent No. 6423998 Japanese Patent Application Laid-Open No. 2019-6883

J. Agric. Food Chem. 2009, 57, 12, 5501-5509

The present invention aims to provide an antifreeze agent that can suppress salt damage, is environmentally safe, places little burden on the environment, and has excellent antifreeze performance, as well as an antifreeze method using the antifreeze agent.

As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that a composition obtained by combining a small amount of sodium chloride with a mixture of collagen peptides, which are ice control substances with ice recrystallization inhibitory activity, and a supercooling-promoting substance, has excellent anti-freeze performance while having a favorable effect on metal corrosion and vegetation.

The present invention was completed based on these findings and further research, and provides the following antifreeze agent and antifreeze method using said antifreeze agent.

Item 1. An antifreeze agent containing an ice control substance having ice recrystallization inhibitory activity and a supercooling promoting substance.
Item 2. The antifreeze agent according to Item 1, further comprising sodium chloride.
Item 3. The antifreeze agent according to Item 1 or 2, wherein the ice control substance is an antifreeze polysaccharide and/or an antifreeze protein or peptide.
Item 4. The antifreeze agent according to any one of Items 1 to 3, wherein the ice-controlling substance is a gelatin decomposition product.
Item 5. The antifreeze agent according to any one of Items 1 to 4, wherein the supercooling-accelerating substance is at least one selected from the group consisting of melanoidin, bean paste residue extract, coffee grounds extract, miso extract, banana peel extract, sake extract, purine bases or polymers having purine bases, and tyrosine peptides.
Item 6. The antifreeze agent according to any one of Items 1 to 5, which is used to prevent freezing of roads, parking lots, runways, or parking lots.
Item 7. A method for preventing freezing on a target surface, comprising a step of spraying the antifreeze agent according to any one of Items 1 to 6 onto the target surface.
Item 8. The anti-freezing method according to Item 7, wherein the object is a road, a parking lot, a runway, or an airplane apron.

The antifreeze agent of the present invention can suppress salt damage such as corrosion of steel materials on bridges and highways, and also reduces the adverse effects on vegetation, making it safe for the environment, placing less strain on the environment, and providing excellent antifreeze performance. Furthermore, the antifreeze agent of the present invention is highly economical because it uses inexpensive materials.

This graph shows the results (clotting onset time) of a coagulation test using antifreeze polysaccharide III (100, 10 μg/ml), melanoidin (1000, 10 μg/ml), coffee grounds extract (50, 10 μg/ml), or miso extract (50, 10 μg/ml) alone. The concentrations shown in the graph are final concentrations. This graph shows the results (clotting onset time) of a coagulation test using antifreeze polysaccharide III (100, 10 μg/ml), melanoidin (1000, 10 μg/ml), coffee grounds extract (50, 10 μg/ml), or miso extract (50, 10 μg/ml) mixed with sodium chloride. The concentrations shown in the graph are final concentrations. This graph shows the results (amount of corrosion) of a metal corrosion test using antifreeze polysaccharide III (10 μg/ml), melanoidin (10 μg/ml), coffee grounds extract (10 μg/ml), or miso extract (10 μg/ml) alone, mixed with sodium chloride, or mixed with CMA. The concentrations shown in the figure are final concentrations.

The following describes in detail the embodiments of the present invention.

In this specification, the term "comprise" encompasses the meanings of "essentially consist of" and "consist of only."

The antifreeze agent of the present invention is characterized by containing an ice control substance that has ice recrystallization inhibitory activity and a supercooling-promoting substance.

Antifreeze proteins and peptides are proteins and peptides that have the function of lowering the freezing point (temperature at which ice freezes) and the function of adsorbing to the surface of ice crystals to inhibit the recrystallization growth of ice and control crystallization. The ice control substance used in this invention has ice recrystallization inhibitory activity and is a substance that inhibits "ice recrystallization," which occurs when ice is made up of countless single ice crystals (ice nuclei) that repeatedly grow and fuse to form large chunks over time. Antifreeze proteins and peptides have been found in fish (see JP 2004-83546 A), insects, plants (see JP 2001-245659 A, WO 92/22581 A, EP 843010 A, Plant Physiology, 119, 1361-1369 (1999), Biochem. J., 340, 285-291 (1999)), fungi, microorganisms (see JP 2004-161761 A), and the like, and these antifreeze proteins and peptides are known to have ice recrystallization inhibitory activity. Antifreeze proteins and peptides are also known as thermal hysteresis proteins, antifreeze proteins, ice crystal-binding proteins, recrystallization-inhibiting proteins, antifreeze proteins (AFPs), ice-binding proteins (IBPs), ice structuring proteins (ISPs), and the like. The ice-controlling substances used in the present invention also include the above-mentioned antifreeze proteins and peptides.

Furthermore, the ice-controlling substances used in the present invention include peptides that have the function of inhibiting ice recrystallization growth and controlling crystallization, glycoproteins and glycopeptides (e.g., cyclic antifreeze glycopeptides) that have the function of inhibiting ice recrystallization growth and controlling crystallization, substances extracted and purified from natural resources such as fish, insects, plants, fungi, and microorganisms, substances produced using bacterial culture and genetic engineering technology, and substances produced by chemical synthesis.

Examples of antifreeze proteins and peptides derived from fish include AFPI, which has a molecular weight of approximately 3,000-5,000 and is composed of an Ala-rich alpha helix; AFPII, which has a molecular weight of approximately 14,000-24,000 and is composed of a C-type lectin-like structural motif; AFPIII, which has a molecular weight of approximately 7,000 and is composed of a globular structure containing multiple beta structures; AFPIV, which has a molecular weight of approximately 12,000 and is composed of a bundle of alpha helices; and AFGP, which has a molecular weight of approximately 3,000-24,000 and is composed of a repeating triplet of -Ala-Thr-Ala-, in which the side chain of the Thr residue is glycosylated.

Examples of plant-derived antifreeze proteins and peptides include those derived from grasses (oat, four-row barley, annual bluegrass, longgrass, common brome, winter rye, winter wheat, triticale, and ryegrass), lilies (daylily), Brassicaceae (garlic mustard, winter pepper, rapeseed, Brussels sprouts, and cabbage), carrots, Dicentra, snowdrops, Asteraceae (Asteraceae, dandelion), conton wood, periwinkle, Plantaginaceae (Plantago major, Plantago major), white oak, Solanaceae (Dulcamara, potato), chickweed, violets, and forsythia. Other examples include radish sprout extract (see JP 2007-153834 A), radish sprout extract, and winter vegetable sprout extract (see JP 2007-169246 A).

Other specific examples of antifreeze proteins and peptides include antifreeze proteins secreted by basidiomycetes (mushrooms such as shiitake mushroom, enoki mushroom, buna-shimeji mushroom, king oyster mushroom, and nameko mushroom) (see Japanese Patent Application Laid-Open Nos. 2004-24237 and 2004-275008), peptides derived from insect larvae (see Japanese Patent Application Laid-Open No. 2002-507889), and antifreeze proteins derived from lichens (see Japanese Patent Application Laid-Open No. 2002-508303).

Another example of an antifreeze protein or peptide is a gelatin (collagen) hydrolyzate. Gelatin hydrolyzate can be produced by hydrolyzing gelatin (collagen) with an enzyme (protease) or the like, and contains collagen peptides. As long as it can be used as an antifreeze protein or peptide, there are no particular limitations on the type of animal from which the gelatin is derived, the type of enzyme (protease) used for hydrolysis, or the production method. The molecular weight of the collagen peptide contained in the gelatin hydrolyzate is not particularly limited as long as it can be used as an antifreeze protein or peptide, and is, for example, 5,000 Da or less, preferably 500 to 3,000 Da, and more preferably 1,000 to 2,500 Da.

The ice control substances used in the present invention can be any of the various antifreeze proteins and peptides listed above, and are not particularly limited and can be selected and used as appropriate. The following antifreeze polysaccharides can also be used as ice control substances in the present invention.

Antifreeze polysaccharides are polysaccharides that inhibit the growth of ice crystals and are components found in the polysaccharides that make up the cell walls of fungi such as enokitake mushrooms (see Patent Publication No. 5881118). In this specification, the term "polysaccharide" generally refers to a linear or branched chain polymer of 10 or more monosaccharides linked by glycosidic bonds.

The antifreeze polysaccharide is not particularly limited, and examples include polysaccharides containing at least one selected from the group consisting of galactose, mannose, xylose, glucose, and rhamnose. Preferably, the polysaccharide contains two or more of these monosaccharides, more preferably, a polysaccharide containing xylose and mannose, and even more preferably, xylomannan.

Xylomannan is a general term for heteropolysaccharides in which a mannan main chain composed of α-1,3-mannose is linked via 1,4-bonds to one xylose molecule as a side chain. However, xylomannans are not limited to those composed only of mannose and xylose, and can also have other sugars in addition to xylose as side chains.

The ratio of mannose to xylose that make up xylomannan is not particularly limited, but is, for example, 1.5 to 2.5 moles, preferably 1.7 to 2.3 moles, more preferably 1.9 to 2.1 moles, and even more preferably about 2 moles, of mannose per mole of xylose.

The molecular weight of the antifreeze polysaccharide is not particularly limited, and is, for example, 100,000 to 1,000,000, as measured by gel filtration chromatography. The lower limit of the average molecular weight is preferably 150,000, more preferably 200,000, even more preferably 240,000, and particularly preferably 280,000. The upper limit of the average molecular weight is preferably 500,000, more preferably 400,000, even more preferably 370,000, and particularly preferably 340,000.

Antifreeze polysaccharides that can be used include those chemically synthesized according to known methods, and extracts obtained by extracting antifreeze polysaccharides from fungi according to known methods.

Among fungi, basidiomycetes are preferred. Examples of basidiomycetes include those belonging to the Agaricales order. Examples of basidiomycetes belonging to the Agaricales order include the family Polytrichum japonicum (such as Goat's mushroom), Tricholomataceae (Kishimeji, Purashimeji, Oshiitake mushroom, Kakuminoshimeji, Shakashimeji, Harushimashimeji, Hatakeshimeji, Bunashimeji, Honshimeji, Owakuitake mushroom, Sugihiratake, Hariganeichibatake, Kitsunetake, Armillaria mellea, Mukitake mushroom, Matsutake mushroom, Shiromatsutake false, Shiitake mushroom, Perilla edodes, etc.). Examples include those belonging to the following families: Amanita plicata (Amanita plicata, Amanita nigricans, etc.), Agaricaceae (Agaricaceae, Agaricus blazei, etc.), Coprinus family (Coprinus capricornus, etc.), Pleurotus family (Nameko, etc.), Cardinalaceae (Siberian ginger, etc.), Boletaceae (Boletus edulis, etc.), Russula family (Amanita plicata, etc.), Polyporaceae (Grafic mushroom, etc.), Pleurotus eryngii (Pleurotus velutipes, etc.). Of these, those belonging to the Tricholomataceae, Pleurotus velutipes, Pleurotus velutipes, etc. are preferred, those belonging to the Tricholomataceae family are more preferred, and even more preferred is Flammulina velutipes.

Related and improved varieties of the above basidiomycetes can also be used as appropriate.

When using basidiomycetes as the organisms containing antifreeze polysaccharides, it is preferable to culture them at low temperatures. Antifreeze polysaccharides can be obtained more efficiently by using basidiomycetes that have been cultured at relatively low temperatures (cold-acclimated) as the extraction source. The culture temperature is preferably 25°C or lower, and more preferably 20°C or lower. On the other hand, a temperature of 0°C or higher is preferable, as there is a risk of the liquid medium freezing below the freezing point.

The culture period is not particularly limited, but is preferably 3 days or longer, more preferably 1 week or longer, even more preferably 2 weeks or longer, and particularly preferably 1 month or longer. There is also no particular upper limit to the culture period, and it can be continued until the basidiomycete fungus reaches a confluent state or until the concentration of the ice crystal growth inhibitor in the medium no longer increases, for example, 6 months or shorter, preferably 5 months or shorter, more preferably 4 months or shorter, and even more preferably 3 months or shorter.

Antifreeze polysaccharides can be extracted from fungi according to known methods. For example, it is known that antifreeze polysaccharides are hardly extractable with hot water, but can be extracted by heat treatment in an alkaline aqueous solution (see Patent Publication No. 5881118). Based on this knowledge, for example, residues from fungal hot water extractions, fungal hot alkaline extracts, etc. can be used as antifreeze polysaccharides.

Fungal hot water extraction residue can be obtained, for example, by recovering the residue after subjecting fungi to hot water extraction treatment.

Hot water extraction can be carried out by immersing the fungus in hot water and stirring as necessary. The water temperature is, for example, 80°C or higher, preferably 90°C or higher, more preferably 95°C or higher, and even more preferably 99°C or higher. The treatment time is not particularly limited and is, for example, about 0.5 to 8 hours. After hot water extraction, the supernatant can be removed by centrifugation, filtration, or other methods to obtain the fungal hot water extraction residue.

Fungal hot alkaline extracts can be obtained, for example, by subjecting fungi to heat extraction in an alkaline aqueous solution.

The fungus can be subjected to the above-mentioned hot water extraction treatment before the heat extraction treatment in an alkaline aqueous solution. This allows for the removal of hot water-soluble components other than antifreeze polysaccharides.

The alkaline substance used to prepare the alkaline aqueous solution is not particularly limited, but examples include sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium polyphosphate, trisodium citrate, sodium bicarbonate, sodium acetate, sodium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, and calcined calcium, and can be used alone or in combination of two or more.

The concentration of the alkaline aqueous solution can be adjusted as appropriate. From the perspective of more efficient extraction of antifreeze polysaccharides, the lower limit is, for example, 0.1 w/v%, preferably 1.0 w/v%, more preferably 2.0 w/v%, even more preferably 5.0 w/v%, even more preferably 10.0 w/v%, even more preferably 15.0 w/v%, and particularly preferably 20.0 w/v%. From the perspective of cost and safety, the upper limit is, for example, 50 w/v%, preferably 30 w/v%, and more preferably 25 w/v%.

The temperature for the heat extraction treatment is preferably 70°C or higher, more preferably 80°C or higher, even more preferably 90°C or higher, and most preferably approximately 100°C. For example, heat extraction can be performed by adding an alkaline aqueous solution and then heating it to a predetermined temperature while extracting, or by adding an alkaline aqueous solution that has been heated to a predetermined temperature in advance and then maintaining the temperature while extracting.

The time for the heat extraction treatment can be adjusted as appropriate depending on the temperature, concentration of the alkaline substance, etc. The time for the heat extraction treatment is, for example, 0.5 to 8 hours, preferably 1 to 5 hours, and more preferably 2 to 3 hours.

Extraction may be performed once, or, to obtain a larger amount of antifreeze polysaccharide, the residue obtained after a single extraction may be subjected to a similar extraction process once or multiple times.

The extract obtained as described above can be used as is, or after removing alkaline substances by well-known methods such as neutralization or dialysis. It can also be further purified as needed. For example, impurities can be removed by a suitable combination of decantation, filtration, and centrifugation. It can also be purified by a suitable combination of methods such as salting out or precipitation with organic solvents, affinity chromatography, ion exchange column chromatography, gel filtration, and binding to ice using a slow-cooling device, as well as concentration by dialysis or ultrafiltration.

The ice control substance may be used alone or in combination of two or more types.

Supercooling-promoting substances suppress ice nucleation at the freezing point in solutions to which they are added, and promote supercooling by lowering the lower limit of the temperature at which the supercooling phenomenon occurs. This lowers the temperature at which ice crystals (initial ice crystal particles) begin to form, making it more difficult for ice crystal particles to form (making it more difficult for antifreeze solutions to freeze). Using a combination of ice control substances and supercooling-promoting substances provides excellent anti-freeze effects.

The supercooling-promoting substance is not particularly limited as long as it promotes the supercooling phenomenon (a substance with anti-ice nucleation activity). Examples of such substances include melanoidin (see JP 2019-6883 A), bean paste residue extract (see JP 5322602 A), coffee grounds extract (see JP 6423998 A), miso extract, banana peel extract (see JP 2019-6876 A), sake extract (see JP 5608435 A), purine bases or polymers containing purine bases (see WO 2016/167284 A), and tyrosine peptides (see WO 2016/178426 A).

Melanoidins include Maillard reaction products obtained by the reaction of reducing sugars with amino acids. Reducing sugars include reducing aldoses and ketoses, preferably glucose, galactose, mannose, xylose, etc. Amino acids include natural amino acids, preferably glycine, serine, threonine, etc. Melanoidins are also found in foods such as miso and soy sauce.

The bean paste residue used in the bean paste residue extract is generated during the process of making raw bean paste, and is made from beans such as soybeans. There are no particular restrictions on the extraction method for the bean paste residue extract, but extraction with hot water is preferred. Furthermore, since most of the anti-ice nucleation activity is present in fractions with a molecular weight of 3500 or less, it is preferable to use an extract of bean paste residue that contains a fraction with a molecular weight of 3500 or less.

The coffee grounds extract preferably contains a compound having at least an aromatic hydrocarbon structure and a carboxyl group in its molecule. The coffee beans used as coffee grounds are preferably roasted, as this enhances the supercooling-promoting effect. Furthermore, one example of a method for producing the coffee grounds extract is to subject a water extract extracted from the coffee grounds with water to an extraction treatment using an organic solvent. Extraction using this method allows the above compounds to be extracted.

There are no particular limitations on the banana peel extract, so long as it is an extract obtained by extracting banana peel with a solvent or a purified product thereof. Examples of methods for producing banana peel extract include solid-liquid extraction of banana peel with alcohol, concentrating the resulting solution, mixing it with water, and then liquid-liquid extraction of the resulting mixture with alkyl acetate. Banana peel extracts are preferably those that give a negative ferric chloride color reaction.

Preferably, the sake extract is prepared by adsorbing the components contained in sake onto a synthetic adsorbent (for example, a styrene-divinylbenzene synthetic adsorbent), and containing the adsorbate adsorbed onto the synthetic adsorbent as an extracted component from sake. It is also preferable to extract the adsorbate with ethyl acetate, and contain the extract extracted with ethyl acetate as an extracted component from sake. It is also preferable to fractionate the adsorbate or extract into a fraction with a molecular weight of 3000 or less by ultrafiltration, and contain this fraction as an extracted component from sake.

Purine bases are not particularly limited as long as they are compounds having a purine skeleton, and examples include adenine, purine, guanine, hypoxanthine, xanthine, theobromine, caffeine, urea, isoguanine, and theophylline. Among these, adenine, adenine derivatives, caffeine, caffeine derivatives, urea, and urea derivatives are preferred. A polymer having purine bases refers to a polymer having purine bases as repeating units, with the purine bases present as side chains in the polymer. An example of such a polymer is a polynucleotide, preferably deoxyribonucleic acid (DNA).

Tyrosine peptides are typically tyrosine dimers to hexamers, preferably dimers to pentamers, more preferably dimers to tetramers, and particularly preferably dimers or trimers.

The supercooling-promoting substance may be used alone or in combination of two or more.

The amount of supercooling-promoting substance contained is not particularly limited as long as anti-freezing performance is obtained, but the mass ratio is, for example, 0.001 to 10, preferably 0.01 to 1, parts by weight of ice-controlling substance to 1 part by weight.

The antifreeze agent of the present invention preferably contains sodium chloride, which has traditionally been used as an antifreeze agent, in addition to an ice-controlling substance and a supercooling-accelerating substance. By using sodium chloride in combination with an ice-controlling substance and a supercooling-accelerating substance, it is possible to reduce the amount of sodium chloride used, thereby reducing salt damage and the adverse effects of sodium chloride on vegetation.

The sodium chloride content should be as low as possible to prevent salt damage and ensure environmental safety, and can be set appropriately depending on the purpose.

The antifreeze agent of the present invention may be in a solid form such as a powder, or may be in a liquid form. In the case of a liquid form, examples of the solvent include water.

The antifreeze agent of the present invention may contain other components in addition to those listed above, as long as it exhibits an antifreeze effect.

The antifreeze agent of the present invention is used, for example, to prevent freezing of roads (including bridges and highways), parking lots, runways, and parking lots.

The method for preventing freezing of a target surface of the present invention is characterized by including a step of spraying an antifreeze agent onto the target surface. The antifreeze agent is sprayed, for example, in liquid form using a liquid spraying device. The amount of the antifreeze agent of the present invention to be sprayed is not particularly limited as long as the antifreeze effect is obtained, and can be set appropriately.

The surfaces to which the antifreezing agent of the present invention may be sprayed are not particularly limited, and examples include roads (including bridges and highways), parking lots, runways, and parking lots. The antifreezing agent of the present invention may be sprayed on snow-covered or frozen surfaces, or may be sprayed on the surface in advance to prevent freezing.

Compared to conventional deicers containing only sodium chloride, the deicer of the present invention is safer and less burdensome to the environment because it can inhibit salt damage such as corrosion of steel materials on bridges and highways and reduce adverse effects on vegetation, such as the withering of street trees, shrubs, and flower beds. Furthermore, by using a combination of two components, an ice control substance and a supercooling accelerator, or even a combination of three components including sodium chloride, excellent deicing performance is achieved. Furthermore, the ice control substance and supercooling accelerator used in the deicer of the present invention are less expensive than conventional sodium chloride and CMA, making them highly economical.

The following examples are provided to further explain the present invention. However, the present invention is not limited to these examples.

Test Method
(1) Drugs Used: "Antifreeze polysaccharide III" is an antifreeze polysaccharide derived from Enoki mushrooms discovered by the present inventors. "Collagen peptide-based supercooling-promoting substance" is a collagen peptide obtained by enzymatically decomposing gelatin into low molecular weight collagen peptides, mixed with the supercooling-promoting substances melanoidin, coffee grounds extract, and miso extract. Hereinafter, the names of various collagen peptide-based supercooling-promoting substances will be expressed as the names of the supercooling-promoting substances mixed together (melanoidin, coffee grounds extract, miso extract). In all cases, the final concentration ratio of collagen peptide to supercooling-promoting substance is 10:1, meaning that 1 ml of solution contains 10 mg of collagen peptide and 1 mg of supercooling-promoting substance. When this is diluted for use, the concentration will be expressed below as the collagen peptide concentration.

(2) Coagulation test The coagulation test was performed by placing 10 g of the test agent (9 g of tap water + 1 g of agent) in a test tube, storing it frozen (-20°C), and measuring the change in water temperature over approximately 6 to 8 hours. The test was performed using the agent alone or a mixture of the agent and sodium chloride in a mass ratio of 5:5.

(3) Antifreeze test: The antifreeze agent must be sprayed in advance to prevent the water on the road surface from freezing and to keep it in a moist, sherbet state. In order to clarify the antifreeze properties, the test was conducted by visually observing the changes in the freezing state at regular intervals.

20 g of test agent was placed in each dish (18 g tap water + 2 g agent). The dishes containing the test agent were stored frozen (-20°C) and visually observed at regular intervals (30, 60, 120, 180, 300 minutes) (3 dishes per test agent). Visual observations were performed using a five-point numerical evaluation scale as shown in Table 1 below. The test used the above agent alone or a mixture of sodium chloride and the above agent in a 5:5 mass ratio.

(4) Metal corrosion test The most commonly used chloride-based antifreeze agents have the problem of corroding metals. This problem leads to a decrease in the durability of structures and is extremely dangerous. With the background of wanting to reduce metal corrosion caused by the spraying of antifreeze agents, we attempted to clarify the amount of corrosion.

Iron metal test pieces (50 mm x 50 mm x 1 mm) were immersed in the test agent (291 g of tap water + 9 g of agent) and dried daily for seven days (two metal test pieces per test agent). On the eighth day, the metal pieces were immersed in a 20% aqueous ammonium hydrogen citrate solution for 30 minutes to precipitate corrosion, and the amount of corrosion on the metal pieces was measured. Tests were conducted using the above agent alone, mixtures of sodium chloride and the above agent in mass ratios of 5:5, 3:7, and 1:9, and mixtures of CMA and the above agent in mass ratios of 3:7 and 1:9.

(5) Vegetation Impact Test There are several methods for spraying antifreeze, but all of them are sprayed in solid or liquid form. As a result, antifreeze has secondary effects on the surrounding area when it is dispersed by traffic and wind. One of these is that it can kill plants. In real road environments, there are two types of environments: those where plants grow, such as tree and flower beds and private gardens, and those where treatment is used to prevent plants from growing. We attempted to clarify the impact on plant growth.

500 ml of soil, 2 g of fertilizer, and the test agent were placed in a plastic bag and the bag was shaken well to mix evenly. The soil prepared in this way was placed in a Neubauer pot, and 20 komatsuna seeds were sown. The plants were grown for 21 days in a sunny location maintained at around 20°C. After 21 days, the above-ground parts of the komatsuna were weighed, and the fresh weight index was calculated using the following formula. The target fresh weight index is 80; if it is below this, it is possible that a growth inhibitor was present.
Fresh weight index = (Weight of the aboveground part of komatsuna treated with the test agent (g)) ÷ (Weight of the aboveground part of komatsuna grown without the test agent (g)) × 100

The test used 1 g, 2 g, 4 g, and 8 g of the above drug alone at 10 μg/ml, or a mixture of 1 g of the above drug (10 μg/ml) and 1 g of sodium chloride.

(6) Testing for environmental impact In order to clarify the environmental impact of the above chemicals, the above chemicals were analyzed using the measurement methods in Table 2 for the items related to the Water Pollution Control Law.

result
(1) Coagulation Test: Figure 1 shows the results of the coagulation test using the agent alone, and Figure 2 shows the results of the test using a 5:5 mixture of agent and sodium chloride by mass. Figures 1 and 2 show the coagulation initiation time. A longer coagulation initiation time indicates a longer supercooling time, indicating a more difficult freezing. Figure 1 shows that each agent exhibited a superior effect on coagulation initiation time compared to sodium chloride, and that the agent was effective even at low concentrations, not proportional to concentration. This suggests that the agent exhibits properties different from the molar freezing point depression that is proportional to concentration, and is different from the commonly known case of NaCl, where the coagulation initiation time increases proportionally with concentration. Figure 2 also shows that even when mixed with sodium chloride, 10 μg/ml was the most effective.

(2) Antifreeze Test The results of the coagulation test using the agents alone are shown in Table 3. Table 3 shows values based on the evaluation method shown in Table 1. The results show that each agent remained in a sherbet-like state for a longer period of time than tap water. Furthermore, because tap water freezes rapidly, it causes a rapid decrease in the coefficient of sliding friction, increasing the risk of road hazards. However, each agent gradually hardens the snow and ice on the sherbet-like road surface, preventing a rapid decrease in the coefficient of sliding friction. Furthermore, NaCl is said to have a weak immediate effect, and it is expected that combining NaCl with each of these agents will complement this effect. Furthermore, the results showed that mixing with sodium chloride resulted in less freezing. Note that Table 3 shows the final concentrations.

Table 4 shows the results of coagulation tests using collagen peptides, coffee grounds extract, and miso extract alone or in combination. Each test was performed by mixing them with 10% NaCl at a 1:1 mass ratio, and Table 4 shows the final concentrations. These results demonstrate that combining collagen peptides with coffee grounds extract or miso extract, which are supercooling-promoting substances, provides better antifreeze effects than using either alone.

(3) Metal corrosion test The results of the metal corrosion test are shown in Figure 3. Figure 3 shows that each agent caused less metal corrosion than sodium chloride, and that mixing with CMA had a significant metal corrosion prevention effect, and that this effect was maintained even when the amount of CMA used was reduced.

(4) Vegetation Impact Test The results of the vegetation impact test using each pesticide alone are shown in Table 5. The live weight index is a value calculated with the value of 100 for plants grown without any additives. Table 5 shows that each pesticide does not affect vegetation, but serves as a nutrient. Sodium chloride also had a low live weight index and adversely affected vegetation, with visual observation revealing abnormal growth such as leaf color abnormalities, withering, and leaf curling. Furthermore, mixing with sodium chloride resulted in greater inhibition of growth than each pesticide used alone. Note that Table 5 shows the final concentrations.

(5) Environmental Impact Tests The results of the environmental impact tests are shown in Table 6. As can be seen from Table 6, although only a limited number of items showed values that were not below the lower limit of quantification, for BOD, NaCl was extremely low, while antifreeze polysaccharides and collagen peptides were slightly higher, and CMA was orders of magnitude higher. Also, for COD, NaCl was the lowest, while like BOD, antifreeze polysaccharides and collagen peptides were slightly higher, and CMA was orders of magnitude higher. Furthermore, looking at the nitrogen content, NaCl and CMA were below the lower limit of quantification, but antifreeze polysaccharides contained a small amount, and collagen peptide materials showed values by double digits, indicating that they were higher than the other materials.

When we combine this with the results of the vegetation test, it is thought that the fact that the antifreeze material is acting as fertilizer for vegetation is also related to the nitrogen content in this analysis. The Ministry of the Environment has set the permissible limits for pH between 5.8 and 8.6 (excluding marine areas), BOD and COD at 160 mg/L (daily average 120 mg/L), and nitrogen content at 120 mg/L (daily average 60 mg/L). CMA exceeds the standard values for BOD and COD, raising concerns about its impact on water pollution.

Claims (5)

An antifreeze agent containing an ice control substance having ice recrystallization suppression activity and a supercooling promoting substance ,
the ice-controlling substance is an enzymatic decomposition product of gelatin,
The antifreeze agent, wherein the supercooling-promoting substance is at least one selected from the group consisting of melanoidin, bean paste residue extract, coffee grounds extract, miso extract, banana peel extract, sake extract, purine bases or polymers having purine bases, and tyrosine peptides . The antifreeze agent according to claim 1, further containing sodium chloride. The antifreeze agent according to claim 1 or 2 , which is used to prevent freezing of roads, parking lots, runways, or parking lots. A method for preventing freezing of a target surface, comprising the step of spraying the antifreeze agent according to any one of claims 1 to 3 onto the target surface. The anti-icing method according to claim 4 , wherein the object is a road, a parking lot, a runway, or an airplane apron.