Natural ice crystal control agent derived from cacao husk

Abstract

To provide an ice crystal control agent that can be applied to foods that have not been solved so far by utilizing the function of suppressing quality deterioration caused by ice crystals enlarged during freezing in the food field, further in frozen processed foods, and an ice crystal control agent containing excellent safety and productivity, and including supercooling promotion activity (anti-ice nucleation activity), ice recrystallization inhibition activity (RI activity), and thermal hysteresis activity.SOLUTION: An ice crystal control agent containing cacao husk hot water extract.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a natural ice crystal regulator having supercooling promoting activity and ice recrystallization inhibiting activity. More specifically, the present invention relates to a natural ice crystal regulator contained in an extract extracted from cacao husk, and having a molecular weight of 10,000 or more. [Background technology]

[0002] Generally, water freezes (forms ice nuclei) at temperatures below its melting point (0°C), but pure water that does not contain any foreign matter does not freeze (form ice nuclei) until it reaches -39°C. This phenomenon in which water does not freeze (form ice nuclei) even when cooled to a temperature below its melting point is called the supercooling phenomenon. If the formation of ice nuclei is suppressed, the supercooled state is expanded (accelerated state) and the unfrozen state is maintained. This supercooling phenomenon is released when foreign matter in the water becomes a nucleus and forms ice nuclei (heterogeneous nucleation).

[0003] Once a foreign object becomes an ice nucleus and ice nucleation occurs, the latent heat generated at that time raises the temperature of the water to around 0°C, and the tiny ice crystals formed by the foreign object grow, causing the entire water to turn into ice and become frozen. Natural substances that can inhibit the growth of ice crystals, lower the ice crystal growth temperature (thermal hysteresis activity), and further inhibit ice recrystallization during freezing include antifreeze proteins (Non-Patent Documents 1 and 2; Patent Documents 1, 2, and 3) and antifreeze polysaccharides (Non-Patent Documents 3 and Patent Document 4) when active ingredients dissolved in the aqueous solution bind to the ice crystals when the ice nuclei form and turn into tiny ice crystals, inhibiting the growth of ice crystals and lowering the ice crystal growth temperature (thermal hysteresis activity).

[0004] Antifreeze proteins with this ice recrystallization inhibitory activity have been extensively studied, including antifreeze proteins derived from fish and plants, and antifreeze peptides derived from insects. In the food industry, there is an antifreeze protein derived from radish sprouts (Patent Document 5), and frozen foods have been manufactured and sold by commercializing antifreeze proteins using non-genetic recombination technology. However, antifreeze proteins are generally derived from psychrophilic organisms, and genes are expressed and do not accumulate within the organism unless exposed to low temperatures. For this reason, there have been few reports of active ingredients with similar activity from non-psychrophilic organisms.

[0005] Antifreeze proteins, which are representative natural substances with ice recrystallization inhibitory activity, have two functions: the ability to completely inhibit the growth of ice crystals by binding to ice crystals themselves, thereby lowering the temperature at which ice crystals grow at 0°C (thermal hysteresis activity), and ice crystal inhibition activity, which inhibits ice recrystallization during long-term frozen storage (Non-Patent Document 4). This latter function is important for inhibiting quality deterioration during long-term storage of frozen foods.

[0006] Substances known to promote supercooling include, for example, low molecular weight compounds such as eugenpol, a spice component (Non-Patent Document 4), hinokitiol-iron, a component of Taiwanese cypress (Non-Patent Document 5), kaempferol-7-glucoside present in the cells of the coniferous tree katsura (Non-Patent Document 6), and high molecular weight compounds such as proteins derived from Acinetobacter calcoaceticus (Non-Patent Document 7) and polysaccharides derived from Bacillus thuringiensis (Non-Patent Document 8). However, these anti-ice nucleation active substances have a high function of reducing the rise in the supercooling point caused by the ice nucleation activity contained in water, but are less active than bacteria in reducing the rise in the supercooling point caused by foreign matter such as silver iodide that exhibits ice nucleation activity. In addition, these compounds are difficult to use in the food and chemical fields due to safety, productivity, and cost issues.

[0007] The present inventors have conducted extensive research with a view to application in the food industry, and have proposed supercooling-promoting substances that utilize low molecular weight peptides contained in bean paste cake extract (Patent Document 6), an extract of sake extract (Patent Document 7), a compound having an aromatic hydrocarbon structure and a carboxyl group extracted from coffee beans (Patent Document 8), melanoidin formed by the reaction of glucose and glycine (Patent Document 9), a tripeptide of the amino acid tyrosine (Patent Document 10), and a polymer of the nucleic acid adenine (Patent Document 11), etc.

[0008] Cocoa husk is the outer skin of cocoa beans, and when the cocoa beans are roasted and crushed, they are separated into cocoa nibs and cocoa husk, with the cocoa nibs being used as ingredients for chocolate and other products, and the cocoa husk being generally discarded. Cocoa beans contain cocoa polyphenols, which are mainly composed of epicatechin, catechin and procyanidins (compounds in which catechin and epicatechin are combined), and these are called flavans (flavan-3-ols). When ingested, these cocoa polyphenols are expected to have three effects: lowering blood pressure, preventing arteriosclerosis and preventing aging.

[0009] One of the natural products extracted from cacao husk is water-soluble polysaccharides extracted with hot water (Patent Documents 12 and 13). Extracts of cacao husk extracted with hot water under a high temperature of 100 to 130°C and a weak acidity of pH 6.5 or less show various functions, and have a characteristic function different from conventional stabilizers, which is to disperse and stabilize proteins in an acidic pH range above the isoelectric point of the protein and give the prepared food an appropriate viscosity. By utilizing this function, it has become possible to produce acidic protein foods that have not been produced before. Furthermore, the produced acidic protein foods can be given the function of maintaining a stable state even when heated at high temperatures such as retort sterilization. When the water-soluble dietary fiber derived from cacao husk in the present invention is added to foods as a shelf life enhancer, a small amount of the added amount shows a sufficient shelf life enhancing effect and good preservability (Patent Documents 12 and 13).

[0010] Cocoa husk components contain many polyphenol-related compounds derived from cocoa beans. Among them, it has been reported that flavals (flavan-3-ols) exhibit supercooling promotion activity, which acts to delay the ice nucleation temperature formed when water freezes (Patent Documents 14 and 15). Compounds with the structure of catechin-type tannins are involved in this activity (molecular weight 834 or less). Furthermore, it has been revealed that theobromine (molecular weight 180), a type of alkaloid with a structure similar to that of purine bases, is a purine base such as adenine and caffeine, and that these purine bases have supercooling promotion activity (Patent Document 11). In addition to these activities, it has not been known until now that hot water extracts from cocoa husk have ice recrystallization inhibition activity or sublimation inhibition activity. Furthermore, there have been no reports of components with a molecular weight of 10,000 or more that have supercooling promotion activity. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent 4228068 [Patent Document 2] Patent 4235083 [Patent Document 3] Patent 4257970 [Patent Document 4] Patent 5881118 [Patent Document 5] Patent 5681494 [Patent Document 6] Patent 5322602 [Patent Document 7] Patent 5608435 [Patent Document 8] Patent 6423998 [Patent Document 9] Patent 6963225 [Patent Document 10] Patent 6704143 [Patent Document 11] Patent 6826730 [Patent Document 12] Patent 3772833 [Patent Document 13] Patent 3804562 [Patent Document 14] Patent 6035371 [Patent Document 15] Patent 6220427 [Non-patent literature]

[0012] [Non-Patent Document 1] PL Davies & CL Hew, FASEB J., 4, 2460-2468, 1990. [Non-Patent Document 2] CHC Cheng, Curr. Opin. Genet. Dev., 8, 715-720, 1998 [Non-Patent Document 3] H. Kawahara et al, Biocontrol Sci., 21, 153-159, 2016. [Non-Patent Document 4] H. Kawahara et al, J. Antibact. Antifung. Agents, 24,95-100, 2006 [Non-Patent Document 5] H. Kawahara et al, Biosci. Biotech. Biochem., 64, 2651-2656, 2000 [Non-Patent Document 6] Kasuga J. et al, Cryobiology, 60, 240-243, 2010 [Non-Patent Document 7] H. Kawahara et al, Biocontrol Sci., 1, 11-17, 1996 [Non-Patent Document 8] Y. Yamashita et al, Biosci. Biotech. Biochem., 66, 948-954, 2002 Summary of the Invention [Problem to be solved by the invention]

[0013] The problem that the present invention aims to solve is to provide an ice crystal control agent extract that contains a polymeric supercooling promotion activator (supercooling promotion activity) and a highly active ice recrystallization inhibition activator (ice recrystallization inhibition activity), which can be applied in various fields including the food industry and is suitable in terms of safety, productivity, and cost. [Means for solving the problem]

[0014] The present inventors have conducted extensive research to solve the above problems, and have unexpectedly found that hot water extract of cacao husk has a strong ice recrystallization inhibitory activity as well as a supercooling promoting activity for molecules having a molecular weight of 10k or more, and have thus completed the present invention. That is, the present invention is as follows. [1] Two types of ice crystal-controlling activity were found in the extract obtained by hot water extraction under normal pressure. [2] The ice crystal control activity of the extract obtained by hot water extraction is a polymer-type supercooling ice recrystallization inhibition activity. [3] The two components with ice crystal control activity contained in the hot water extract of cacao husk are proteins, mainly with molecular weights of 10k or more and 100k or less. Effect of the Invention

[0015] The ice recrystallization inhibitor of the present invention is a natural extract obtained by extracting cacao husk with hot water, and is excellent in terms of productivity and safety. In the case of an extract having this activity, ice crystals in frozen foods can be controlled during freezing and storage, and frozen foods can be stored with better quality. In particular, in frozen sweets with a high moisture content such as ice cream and ice candies, suppressing ice recrystallization is an important function for maintaining quality. Furthermore, in the field of processed food, for example, by making the ice crystals smaller during freezing, it is possible to prevent deterioration of frozen foods during long-term storage and improve their quality. Furthermore, by preventing ice crystals from becoming gigantic (recrystallization) during frozen storage, physical damage caused by ice in foods can be prevented, and dripping during thawing can be reduced. In the agricultural field, for example, frost damage to agricultural crops during late frosts and early frosts in cold regions can also be reduced. In the medical field, it becomes possible to stably store cells for medical or experimental use. Furthermore, the component having a polymer-type supercooling promotion activity makes it possible to store tissues and the like without freezing. In the livestock industry, it will be possible to stably store livestock sperm and fertilized eggs, and in the fisheries industry, it will be possible to stably store fish sperm and fertilized eggs in frozen or unfrozen form. Furthermore, in the environmental field, it will be possible to reduce ice adhesion to roads, vehicles, signs, concrete, aircraft, etc. In addition to these, various other applications related to freezing will be possible. [Brief description of the drawings]

[0016] [Figure 1] Gel filtration chromatography of HE (3k or less) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 1. Cacao husk Cocoa beans have been used as a raw material for chocolate since ancient times, but the outer skin of the cocoa beans, i.e. cocoa husk, is partially used as compost or livestock feed. However, most of it is discarded, and the current situation is that the percentage of it being effectively utilized is low. From the perspective of SDGs, there have been many reports of businesses that recycle and upcycle unused resources produced during food processing, and companies that manufacture chocolate and companies involved in importing cocoa beans are attempting to commercialize various products using this cocoa husk. For example, there are beers and gins that use the aroma of cocoa husk, and tumblers and ties that use the cocoa husk itself as part of the raw material. Since cocoa husk contains polyphenols with strong antioxidant properties, attempts are being made to develop products that use cocoa husk, and cocoa husk tea is consumed as a tea with a rich chocolate flavor. As one of the functional ingredients contained in cacao husk, theobromine and the cacao polyphenols flavour and procyanidin have been reported to promote blood flow and relieve chills and swelling in the hands and feet. Furthermore, theobromine is the source of bitterness and aroma, and is thought to increase serotonin, a brain hormone related to healing, resulting in a sense of relaxation. It has a milder effect than caffeine, so it is less stimulating, and is expected to be effective. In particular, it works to lower LDL cholesterol, and is expected to have a preventive effect against arteriosclerosis.

[0018] 2. Ice crystal control agent The hot water extract of cacao husk has two kinds of ice crystal control activities excluding sublimation inhibition activity. The ice crystal control activity contained in the hot water extract of cacao husk of the present invention includes, for example, supercooling promotion activity, which inhibits the formation of ice nuclei, thereby maintaining the supercooled state of water and delaying the formation of ice, and ice recrystallization inhibition activity, which forms ice nuclei and inhibits the growth of fine ice crystals after they are formed. The ice crystal control agent of the present invention can be applied in various fields including the food field, and is excellent in terms of safety because cacao husk cooked with hot water is eaten as cacao husk tea. In addition, the ice crystal control agent of the present invention is excellent in terms of productivity because it can be easily prepared by hot water extraction of cacao husk, which is a by-product of chocolate production and is a partially unused resource. In the food material field, for example, it can improve the freezing resistance of agricultural products in cold regions, and further, in frozen processed foods, it can reduce tissue destruction caused by the enlargement of ice crystals during frozen storage, suppress the amount of dripping after thawing, and maintain the texture even after thawing. In particular, in the case of ice cream and ice confectionery, it is possible to control the amount of ice crystals adhering to the surface and maintain the quality of a smoother state. In the medical field, it becomes possible to stably store organs and tissues for transplantation or experiments, etc., without freezing, and further to improve the viability of cells and store them frozen. In the livestock field, it is possible to stably store livestock cells, organs, sperm, and fertilized eggs. Furthermore, in the environmental (chemical) field, it will be possible to reduce ice adhesion to road antifreeze, road signs, heat exchangers, aircraft wings, etc. In addition to this, various other applications will be possible.

[0019] The ice crystal regulator of the present invention is used in a suitable manner depending on the application. For example, in the food field, an extract containing the ice crystal regulator of the present invention can be added at a suitable concentration to the raw materials of frozen foods and applied. In some cases, an aqueous solution diluted to a suitable concentration for food ingredients or processed foods can be sprayed and applied. In the medical and livestock fields, an aqueous solution containing the ice crystal regulator of the present invention can be prepared and applied. In the environmental (chemical) field, in preventing freezing of roads, etc., an aqueous solution containing the ice crystal regulator of the present invention can be prepared and directly sprayed for application. Furthermore, in applications such as signs and heat exchangers, an aqueous solution containing the ice crystal regulator of the present invention can be prepared and sprayed in combination with another formulation having adhesive ability for application. EXAMPLES

[0020] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Among the ice crystal control effects, the supercooling promotion activity (anti-ice nucleation activity) and ice recrystallization inhibition activity (RI activity) were evaluated by the following methods.

[0021] Test Example 1. Evaluation of supercooling promotion activity (anti-ice nucleation activity) The samples to be evaluated were diluted with 50 mM phosphate buffer (pH 7.0) so that the protein concentration of the samples was 1.0 mg / mL. 500 μL of each of the diluted solutions was prepared. Meanwhile, 500 μL of an aqueous solution in which silver iodide was dissolved in 50 mM phosphate buffer (pH 7.0) was prepared so that the silver iodide concentration was 10 mg / mL. Each sample for evaluation was prepared by mixing the above two aqueous solutions. A blank sample was also prepared by mixing 500 μL of 50 mM phosphate buffer (pH 7.0) and 500 μL of the above silver iodide solution.

[0022] The supercooling promotion activity was measured by the Vali droplet freezing method. Aluminum foil was placed on the copper plate of a cold plate cooling device (TCP-12, Sanwa Seisakusho), and after a thin layer of silicone grease was applied, 10 μL of each evaluation sample and blank sample was dropped onto the surface at 30 locations. The temperature was then lowered at a rate of 1.0°C per minute, and the temperature at which 50% of the 30 droplets were frozen was taken as T50. The temperature at which the droplets of each sample were frozen was taken as SampleT50, and the temperature at which the blank sample was frozen was taken as BlankT50. The supercooling promotion activity value (anti-ice nucleation activity value) ΔT50 (°C) was calculated by the following formula. ΔT50(℃)= BlankT50-SampleT50

[0023] Test Example 2. Evaluation of ice recrystallization inhibitory activity (RI activity) The samples were diluted with deionized water to a protein concentration of 1.0 mg / ml. In addition, 60% (w / v) sucrose aqueous solution prepared with deionized water was prepared, and 100 μL of each solution was mixed to prepare a final sucrose concentration of 30% (w / v). A blank sample was prepared by mixing 100 μL of deionized water and 100 μL of 60% (w / v) sucrose aqueous solution.

[0024] The ice recrystallization inhibitory activity was measured by the sucrose sandwich method. 1 μL of the above mixture was sandwiched between two cover glasses and fixed to the sample stage of a temperature-controllable microscope system (temperature control device KL-600P, manufactured by Linkam Co., Ltd., phase contrast microscope BX50, manufactured by Olympus Co., Ltd.). The sample was rapidly frozen to -40°C at a rate of 100°C per minute using liquid nitrogen to form minute ice crystals. The temperature was then raised to -6°C, and a photograph of the ice crystals at 0 hours was taken. The sample was maintained at -6°C for 30 minutes, and a photograph of the ice crystals that had recrystallized 30 minutes later was taken. Three photographs were taken with one sample, and deionized water was used as a blank.

[0025] The images taken after 30 minutes were digitized (apparent area per ice crystal in a given area is called pixel) using the free image analysis software Image J. Using the trimming function, the same position of three images for each evaluation sample was cut out to a size of 500 x 500 pixels. The ice crystals in the cut-out images were filled in black, and the area (pixels) was calculated. The area was divided by the number of ice crystals to calculate the average area per ice crystal. The ice recrystallization inhibition activity (RI activity) value was calculated using the following formula. Apparent area (pixel) per ice crystal of sample / Apparent area (pixel) per ice crystal of blank (deionized water)

[0026] Test Example 3. Evaluation of thermal hysteresis activity The samples were diluted with deionized water to a protein concentration of 1.0 mg / ml. In addition, 60% (w / v) sucrose aqueous solution prepared with deionized water was prepared, and 100 μL of each solution was mixed to prepare a final sucrose concentration of 30% (w / v). A blank sample was prepared by mixing 100 μL of deionized water and 100 μL of 60% (w / v) sucrose aqueous solution.

[0027] Thermal hysteresis activity was measured using the same apparatus used for the measurement of the sucrose sandwich method. 1 μL of the above mixture was sandwiched between two cover glasses and fixed on the sample stage. Using liquid nitrogen, the mixture was rapidly frozen to -40°C at a rate of 100°C per minute to form minute ice crystals. The temperature was then raised to -6°C to make the ice crystals visible as single crystals. The temperature was then slowly raised to around 0°C, and the temperature was controlled and adjusted until the diameter of the ice single crystals in the field of view of the microscope was around 10 μm. The sample stage temperature was kept constant when the ice single crystals formed. The time at which the temperature was held was set to 0 hours, and the temperature was then lowered at a rate of 1°C per minute to measure the time (seconds) until the ice single crystals began to grow. The measured time (seconds) was divided by 60 to obtain the thermal hysteresis activity (°C). In addition, the morphology of the ice single crystals as they grew was also photographed.

[0028] Example 1 50g of cacao husk was placed in a household pot (inner diameter 28cm), and 950mL of tap water was added to make 5% (w / v), and mixed by stirring. The pot was covered and heated on a cooking heater (IH) for 5 minutes over high heat. After confirming that it was boiling, the heat was changed to low heat and heated for 30 minutes (approximately 99℃). After treatment, the mixture was left at room temperature until it reached room temperature. The obtained hot water extract was strained through gauze and separated into a solid residue and a filtrate. The filtrate was further filtered under suction through No. 2 and No. 5C filter papers. The filtrate after the final filtration was used as the extract.

[0029] The protein, polyphenol, and sugar contents of the extract prepared in Example 1 were measured. These are shown in Table 1 below. Furthermore, the activity of each of the extracts obtained was measured. These are shown in Table 2 below.

[0030] [Table 1]

[0031] Test Example 4. Test of supercooling promotion activity (anti-ice nucleation activity) and ice recrystallization inhibition activity (RI activity) The protein content and polyphenol content of the extract obtained in Example 1 were measured. The protein content was measured by the BCA protein assay method (manufactured by Thermo Fisher). The polyphenol content was measured by the Folin-Ciocalteu method, and gallic acid was used as the standard substance. The sugar content was measured by the phenol-sulfuric acid method, and glucose was used as the standard substance.

[0032] [Table 2]

[0033] As shown in Table 1, it was found that the extract had the highest protein content. Furthermore, it has been revealed that the low molecular weight polyphenols contained in the cacao husk extract have supercooling promotion activity, and it was found that the amount of polyphenols extracted was greater than that when the coffee grounds extract was prepared. Therefore, both activities were measured after preparing 200μg / ml of polyphenols as the standard. As a result, as shown in Table 2, the supercooling promotion activity was 2.3℃, and when the concentration was increased, it became constant at 2.8℃. In addition, the ice recrystallization inhibition activity (RI activity) was 0.46, which was higher than the activity of various extracts from unused resources (coffee grounds, miso, soybean pulp, etc.) that have been reported so far. From the above results, it was found that cacao husk exhibits two ice crystal control activities, and next, it was examined what molecular weight components were responsible for both activities.

[0034] Example 2 In order to determine the molecular weight of the compound and the component in the extract that is involved in the supercooling promotion activity (anti-ice nucleation activity) and ice recrystallization inhibition activity (RI activity), the extract of Example 1 was first fractionated by molecular weight using a dialysis tube. For the fractionation molecular weight of 100k or more, a dialysis tube (Spectrum 100kDa, 31mm) was used, and for the fraction of molecular weight 100k or less, a VIVASPIN6 (Sartorius 100kDa) was used. Furthermore, for the fraction of molecular weight 10k to 100k and the fraction of molecular weight 10k or less, the fraction of 100k or less was fractionated with VIVASPIN6 (Sartorius 10kDa), and for the fraction of molecular weight 10k or less and 3k or less, the fraction was fractionated with VIVASPIN20 (Sartorius 10kDa) and VIVASPIN20 (Sartorius 3kDa). The protein, polyphenol and sugar contents of each separated fraction sample were measured and compared.

[0035] The protein, polyphenol and sugar amounts of each fraction separated by molecular weight of the extract prepared in Example 2 were measured (see Table 3), and then the polyphenol content of each fraction separated by molecular weight was adjusted to 200 μg / ml. The results of measuring both activities are shown in Table 4 below.

[0036] [Table 3]

[0037] [Table 4]

[0038] As shown in Table 3, the fraction with a molecular weight of 100k or more contained no protein or polyphenols, but contained a small amount of sugar (0.5mg / ml). Most of the protein and polyphenols were present at a molecular weight of 100k or less, and the concentrations of the three components were the same in the fraction with a molecular weight of 10k-100k and the fraction with a molecular weight of 10k and 3k or less. As a result, when the volume of each fraction was converted, the proportions of proteins with both activities in the extract, with molecular weights of 10k or more, 3k-10k, and 3k or less, were 15.7%, 49.0%, and 35.3%, respectively. Furthermore, the proportions of polyphenols with molecular weights of 10k or more and 10k or less were 16.7% and 83.3%, respectively.

[0039] As shown in Table 4, in terms of supercooling promotion activity (anti-ice nucleation activity), the extract without fractionation in Example 1 had an activity of 2.3°C, while the fraction with a molecular weight of 10k to 100k and the fraction with a molecular weight of 10k or less in Example 2 had an activity of 2.1°C and 1.9°C, respectively, showing almost the same level of activity. The low molecular weight polyphenols contained in cacao husk reported so far have a molecular weight of 10k or less, so there have been no reports of high molecular weight supercooling activators such as the fraction with a molecular weight of 10k to 100k. In addition, while the ice recrystallization inhibition activity (RI activity) (Table 2) of the extract of cacao husk (Example 1) was 0.46, as shown in Table 4, the RI activity of the fraction with a molecular weight of 10k or less was high (0.45), and the RI activity of the fraction with a molecular weight of 3k or less was even higher (0.40), proving that it is a low molecular weight ice recrystallization inhibition activator. This result indicates that a low molecular weight compound is the active ingredient, and based on previous reports, it was determined that peptides or sugars are involved in the activity. From the activity of the fraction with a molecular weight of 10k or more and 100k or less, it was determined that the components with supercooling-promoting activity (anti-ice nucleation activity) in the ice crystal control agent of the present invention also contain polymer-type active agents. However, it was necessary to confirm which of the components shown in Table 3 was this polymer active ingredient, and we investigated whether the removal action of protein, which is most likely to be the active ingredient, would affect supercooling-promoting activity (anti-ice nucleation activity) and ice recrystallization inhibition activity (RI activity).

[0040] Test Example 5. Measurement of the amount of components and their activities in the extract subjected to proteolysis and dialysis 300 μl of Alcalase (Merck 126741, 2.995 U / ml) was added to 5.0 ml of the extract prepared in Example 1 in an amount equivalent to 1 / 50 of the protein amount in the extract, and the mixture was treated at 60° C. for 2 hours. Molecular weight fraction of 10 k or less was fractionated using a molecular weight cutoff VIVASPIN6 (Sartorius 10 kDa). The protein, polyphenol and sugar amounts of the separated fraction samples were measured and compared.

[0041] Example 3 In Example 3, the protein, polyphenol and sugar amounts were measured for the fraction with a molecular weight of 10k or more prepared by decomposing the protein (Table 5). Then, the protein was decomposed and fractionated under the same conditions as in Example 1, using the same dilution ratio to obtain a polyphenol content of 200 μg / ml. The results of measuring the activities of the extract of Example 3 were compared with those of Example 1, as shown in Table 5 below.

[0042] [Table 5]

[0043] As shown in Table 6, the protein components of the extract of Example 1 were decomposed by the Alcalase treatment, and the low molecular weight peptides were removed by ultrafiltration with a molecular weight cutoff of 10k. As a result, the protein amount in Example 3 was about one tenth of that in Example 1. Polyphenols (such as catechins) that were thought to have been adsorbed to this protein (polyphenols generally have the property of being adsorbed to proteins) were also removed by the same ultrafiltration treatment. Furthermore, sugar components present as glycoproteins were also removed. As a result, the amount of polyphenols and sugars were both less than one tenth of that in the extract of Example 1.

[0044] [Table 6]

[0045] As shown in Table 6, when the protein fraction with a molecular weight of 10k or more was decomposed, the supercooling activity (anti-ice nucleation activity) was 0.20°C, making the fraction inactive. Furthermore, the fraction also had no ice recrystallization inhibitory activity (RI activity). From these results, it was determined that proteins are involved in the active components with a molecular weight of 10k or more. Similar results were also confirmed for active components with a molecular weight of 10k or less.

[0046] Example 4 Fractions with molecular weights of 3k or less were fractionated using VIVASPIN20 (3kDa, manufactured by Sartorius). The obtained fractions were fractionated by gel filtration chromatography using a Sepadex LH-20 (Cytiba) column with a diameter of 1.5 cm and a length of 100 cm, which has a molecular weight exclusion limit of 5000. The fraction volume per fraction was 3 ml. Since this was peptide separation, the absorbance was measured at a wavelength of 230 nm (solid line), which is an index of peptide bonds, and at 280 nm (dashed line), which is an index of aromatic compounds.

[0047] As shown in Figure 1, the gel filtration fractionation allowed separation into four peaks, peaks (1) to (4), and each peak was collected to measure the protein and polyphenol content. The results are shown in Table 7 below. Next, each of the four separated peak solutions was concentrated using an evaporator, and then the supercooling activity, ice recrystallization inhibition activity, and thermal hysteresis were measured as ice crystal control activity, with the same peptide content. As shown in Table 8, peak (3) showed a thermal hysteresis value and high ice recrystallization inhibition activity, just like antifreeze proteins. Peak (3) showed the highest ice crystal control ability, and the shape of the ice crystals during ice crystal growth was hexagonal, indicating that peak (3) contains antifreeze peptides. [Table 7] [Table 8] [Industrial Applicability]

[0048] INDUSTRIAL APPLICABILITY The present invention provides an ice crystal regulator which can be applied in various fields including the food field, particularly the frozen food field, and which is excellent in safety and productivity.

Claims

1. An ice crystal control agent obtained from cacao husk extract.

2. The ice crystal control agent according to claim 1, which is an agent having any one of supercooling promotion activity, ice recrystallization inhibition activity, and thermal hysteresis activity.

3. The ice crystal control agent according to claim 1, which is an agent having two or more of a supercooling promotion activity, an ice recrystallization inhibition activity, or a thermal hysteresis activity.

4. The ice crystal control agent according to any one of claims 1 to 3, which is prepared by hot water extraction under normal pressure.

5. The ice crystal control agent according to claim 4, wherein the active substance is a component having a molecular weight of 10,000 or more and 100,000 or less in the hot water extract.

6. The antifreeze agent according to claim 4, wherein the active substance is a component having a molecular weight of 10,000 or less in an extract extracted with hot water.

7. The antifreeze agent according to claim 4, wherein the active substance is a component having a molecular weight of 3,000 or less in an extract extracted with hot water.

8. 8. An ice crystal control agent, wherein the active substance having supercooling promoting activity and ice recrystallization inhibiting activity among the active substances according to any one of claims 4 to 7 is a protein or a peptide.

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