Freezing liquid for freezing aquatic products and preparation and application thereof

Abstract

The application discloses a freezing solution for aquatic product freezing process, which comprises the following raw materials in percentage by mass: antifreeze protein 0.3-0.4%, glycine trimethylamine inner salt 8-9%, beta-D-furan fructosyl-(2→1)-alpha-D-glucopyranose 8-9%, 4-O-alpha-D-glucopyranosyl-D-glucose 10-12%, methyl 4-O-beta-D-galactosyl-beta-D-glucopyranoside 2-4%, methyl alpha-D-glucopyranoside 3-5%, kaempferol-7-O-beta-D-glucoside 4-6%, sodium D-erythorbate 0.03-0.04%, sodium glutamate 0.06-0.07%, and water. In the application, the antifreeze protein can be adsorbed on the ice surface, inhibit the recrystallization of ice, and significantly reduce the freezing point of the aqueous solution.

Description

Technical Field

[0001] This invention relates to a freezing process and technology in the food cryogenic processing industry, and more particularly to a novel freezing liquid for freezing aquatic products. Background Technology

[0002] Freezing technology for aquatic products can preserve aquatic products such as fish, shrimp, and shellfish for a long time at low temperatures, effectively improving people's quality of life.

[0003] Current methods of freezing aquatic products primarily use low-temperature air as the cooling medium. This traditional air-cooling method (referred to as air cooling or air chilling) is slow, time-consuming, and damages the cells of aquatic products, resulting in poor color, taste, and texture, as well as nutrient loss. Furthermore, air cooling can cause surface moisture evaporation, leading to dry skin and reduced overall mass, affecting the quality and value of frozen aquatic products – a phenomenon known in the industry as "dry loss." Liquid nitrogen freezing is a new technology in the freezing industry, significantly improving freezing speed compared to traditional air cooling. Liquid nitrogen-frozen aquatic products are of higher quality, but the continuous consumption of liquid nitrogen during the freezing process makes them very expensive. Recently, another new freezing method has emerged, using liquid as the cooling medium, called liquid-phase freezing. This technology can achieve the same effect as liquid nitrogen freezing at a very low cost. Because liquids have much higher thermal conductivity, specific heat capacity, and density than gases, liquid-phase freezing can significantly increase freezing speed, reduce damage to aquatic cell and tissue, and improve the quality of frozen aquatic products compared to traditional air cooling. Aquatic products processed using liquid-phase freezing technology can maintain their color, flavor, and texture when fresh, with almost no drying out, giving them a significant competitive advantage. The liquid freezing medium used in liquid-phase freezing technology is known in the industry as a cryogenic fluid. The cryogenic fluid needs to remain unfrozen at low temperatures and possess good fluidity; therefore, the lower the freezing point and viscosity of the cryogenic fluid at low temperatures, the better for the liquid freezing process. Because conventional aqueous solutions have difficulty lowering their freezing points to very low temperatures, most cryogenic fluids currently used in liquid-phase freezing processes contain organic solvents. This makes these cryogenic fluids flammable, volatile, and even harmful to human health and polluting the environment, thus posing safety hazards. Developing and preparing novel cryogenic fluids that are free of organic solvents, do not freeze at low temperatures, are non-volatile, non-flammable, non-toxic, and environmentally friendly for use in liquid-phase freezing processes has become a new trend in the industry. Summary of the Invention

[0004] To address the problems and defects of conventional freezing fluids, this invention provides a novel freezing fluid for aquatic product freezing processes.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A novel freezing liquid for aquatic product freezing process comprises, by mass percentage: 0.1%-1% antifreeze protein, 5%-10% glycine trimethylamine inner salt (CAS Registry No.: 107-43-7), 2%-10% kaempferol-7-O-β-D glucoside (CAS Registry No. 16290-07-6), and the remainder being water;

[0007] Preferred options include:

[0008] The composition consists of 0.3%-0.4% antifreeze protein, 8%-9% glycine trimethylamine inner salt, 4%-6% kaempferol-7-O-β-D glucoside, and the remainder is water.

[0009] Substances with the following final mass concentrations may be added to water, based on a percentage by mass:

[0010] β-D-fructofuranosyl-(2→1)-α-D-glucopyranose (CAS Registry No.: 57-50-1) 5%-10%, 4-O-α-D-glucopyranosyl-D-glucopyranose (CAS Registry No.: 69-79-4) 5%-15%, methyl 4-O-BETA-D-galactosyl-BETA-D-glucopyranoside (CAS Registry No.: 7216-69-5) 1%-5%, methyl α-D-glucopyranoside (CAS Registry No.: 97-30-3) 1%-10%, sodium D-isoascorbate (CAS Registry No.: 7378-23-6) 0.01%-0.1%, monosodium glutamate (CAS Registry No.: 142-47-2) 0.01%-0.1%.

[0011] Preferred components: 8%-9% β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, 10%-12% 4-O-α-D-glucopyranosyl-D-glucopyranoside, 2%-4% methyl 4-O-BETA-D-galactosyl-BETA-D-glucopyranoside, 3%-5% methyl α-D-glucopyranoside, 0.03%-0.04% sodium D-isoascorbate, and 0.06%-0.07% monosodium glutamate.

[0012] like:

[0013] Antifreeze protein 0.1%-1%, glycine trimethylamine inner salt (CAS Registry No.: 107-43-7) 5%-10%, β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside (CAS Registry No.: 57-50-1) 5%-10%, 4-O-α-D-glucopyranosyl-D-glucopyranoside (CAS Registry No.: 69-79-4) 5%-15%, methyl 4-O-BETA-D-galactosyl-BETA-D-glucopyranoside (CA S Registry No.: 7216-69-5) 1%-5%, Methyl α-D-glucopyranoside (CAS Registry No. 97-30-3) 1%-10%, Kaempferol-7-O-β-D-glucopyranoside (CAS Registry No. 16290-07-6) 2%-10%, Sodium D-isoascorbate (CAS Registry No. 7378-23-6) 0.01%-0.1%, Monosodium glutamate (CAS Registry No.: 142-47-2) 0.01%-0.1%, the remainder being water;

[0014] Preferably, the cryosol comprises, by mass percentage: 0.3%-0.4% antifreeze protein, 8%-9% glycine trimethylamine inner salt, 8%-9% β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, 10%-12% 4-O-α-D-glucopyranosyl-D-glucopyranoside, 2%-4% methyl 4-O-BETA-D-galactosyl-BETA-D-glucopyranoside, 3%-5% methyl α-D-glucopyranoside, 4%-6% kaempferol-7-O-β-D-glucopyranoside, 0.03%-0.04% sodium D-isoascorbate, 0.06%-0.07% monosodium glutamate, and the remainder being water;

[0015] Preferably, the special antifreeze protein is a biopolymer antifreeze compound that is creatively designed and artificially synthesized. It has very strong ice recrystallization inhibition activity and thermal hysteresis activity, which can inhibit ice recrystallization and significantly reduce the freezing point of the aqueous solution.

[0016] Preferably, the special antifreeze protein is a biopolymer compound formed by creatively designing a combination of multiple amino acids in a specific sequence, the amino acid sequence being: Lys Asp Ser Ser Gly Glu His Arg ArgAsp Lys Asp Ser Ser Gly Glu His Arg Arg Asp Lys Asp Ser Ser Gly Glu His ArgArg Asp Lys Asp Ser Ser Gly Glu His Arg

[0017] The aforementioned antifreeze protein is prepared by expression, isolation, and purification in transgenic bacteria. The preparation method is as follows:

[0018] a. Transfection. The plasmid containing the above amino acid sequence was labeled with 7 histidine residues. The labeled plasmid was then added to thawed competent cells and incubated on ice for 20 minutes. The competent cells were then heated in 40°C water for 30 seconds and immediately returned to the ice bath for 3 minutes. Next, kanamycin-containing medium was added to the competent cells and the cells were shaken at 37°C for 2 hours. Afterward, the bacterial suspension was plated onto kanamycin-containing medium and incubated at 37°C for 12 hours. Subsequently, a small number of colonies were picked from the medium and placed in kanamycin-containing medium, incubated overnight at 37°C.

[0019] b. Expression and purification. The bacterial suspension was placed in a medium containing kanamycin and incubated at 37°C according to standard procedures until OD500 was achieved. 600 The concentration was adjusted to 0.6. Isopropyl β-D-thiogalactoside was added and the final concentration was adjusted to 1 mM. Culture was continued for 4-5 hours. The bacterial suspension was centrifuged at 4°C for 30 minutes. The supernatant was discarded, and the precipitate was collected. A lysis buffer [a solution of 60 mM NaH₂PO₄ and 350 mM NaCl, pH = 8] was added to the precipitate, along with phenylmethylsulfonyl fluoride as a protease inhibitor. Lysozyme was added and the final concentration was adjusted to 1 mg / mL. The bacteria were then sonicated in an ice bath. The bacterial lysate was sheared at high speed to remove DNA fragments. After centrifugation at 4°C for 1 hour, the collected supernatant was mixed thoroughly with purification resin in a shaker. The novel antifreeze protein compound was then purified using chromatographic purification equipment.

[0020] The present invention also provides a method for preparing the special antifreeze protein.

[0021] Preferably, the refrigerant does not contain toxic or harmful substances, and the ingredients used are safe and environmentally friendly.

[0022] Preferably, the refrigerant is free of organic solvents, non-volatile, non-flammable, and has higher safety and stability.

[0023] The present invention also provides a method for preparing the cryogenic fluid.

[0024] Antifreeze proteins can adsorb onto the ice surface, inhibiting ice recrystallization and significantly lowering the freezing point of the aqueous solution. This invention's novel freezing fluid contains no toxic substances or organic solvents, solving problems in the aquatic product freezing industry such as easy freezing, volatility, flammability, and toxic pollution at low temperatures.

[0025] The present invention also provides an application of using the aforementioned freezing liquid to freeze aquatic products, characterized by the following steps: cooling the freezing liquid to -40°C to -70°C using a refrigeration device, maintaining the temperature of the freezing liquid between -40°C and -70°C using the refrigeration device, and placing frozen aquatic products with a total mass not exceeding 70% of the mass of the freezing liquid in the freezing liquid for freezing for 10-100 minutes.

[0026] Preferably, the freezing liquid is cooled to -45°C to -65°C, and the temperature of the freezing liquid is maintained between -45°C and -65°C by a refrigeration device. Aquatic products with a total mass not exceeding 50% of the mass of the freezing liquid are placed in the freezing liquid and frozen for 15-40 minutes. After that, the frozen aquatic products are washed with water at 0-4°C and an ice coating with a thickness of 0.05-1mm is formed on the outer surface of the aquatic products.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. Among the raw materials disclosed in this invention, the special antifreeze protein is a high-molecular-weight antifreeze compound that is creatively designed and artificially synthesized. It has strong ice recrystallization inhibition activity and thermal hysteresis activity, which can inhibit ice growth and significantly lower the freezing point of the aqueous solution. The addition and proportioning of the special antifreeze protein enable the freezing liquid to remain unfrozen at -65°C without crystallization, exhibiting good fluidity and high transparency, making it an ideal medium for the low-temperature cryopreservation of aquatic products.

[0029] 2. The special antifreeze protein disclosed in this invention has a special protective effect on aquatic products such as fish, shrimp, and shellfish, enabling the structural cells of aquatic products such as fish, shrimp, and shellfish to maintain the same state as when they were fresh at low temperatures, thereby improving the quality of aquatic products after freezing.

[0030] 3. The cryogenic liquid disclosed in this invention can significantly improve the heat transfer speed and efficiency, enabling the cells in aquatic products to pass through the "maximum ice crystal formation zone" in the shortest time. At the same time, it utilizes crystallization thermodynamics and the interaction between radio frequency electromagnetic waves and water molecules to regulate the ice crystal nucleation process, resulting in very small ice crystals formed in the cells of aquatic product tissues. This reduces damage to cells and tissues, further ensuring that the quality of aquatic products remains the same after freezing as when they are fresh, thus locking in the original freshness of aquatic products to the greatest extent.

[0031] 4. The refrigerant disclosed in this invention does not contain organic solvents, all ingredients are safe and non-toxic, and it has the characteristics and advantages of being non-volatile, non-flammable, highly safe and stable, and environmentally friendly and pollution-free. Detailed Implementation

[0032] The cryogenic fluid disclosed in this invention can be used for the safe, efficient, and high-quality cryopreservation of aquatic products. To clearly illustrate the technical features of this invention, specific embodiments are described below for further explanation.

[0033] Example 1

[0034] The special antifreeze protein was synthesized according to the preparation method disclosed in this invention. The amino acid sequence is shown in SEQ ID No. 1:

[0035] The sequence number SEQ ID No. 1 is:

[0036] Sequence characteristics:

[0037] ●Length: 38

[0038] ●Type: Amino acid sequence

[0039] ●Chain type: Single chain

[0040] ●Topology: Linear

[0041] (b) Molecular type: protein

[0042] (c) Assumption: No

[0043] (d) Antonym: No

[0044] (e) Original source: Artificial synthesis

[0045] Lys Asp Ser Ser Gly Glu His Arg Arg Asp Lys Asp Ser Ser Gly Glu HisArg Arg Asp Lys Asp Ser Ser Gly Glu His Arg Arg Asp Lys Asp Ser Ser Gly GluHis Arg

[0046] Procedure a. Transfection. The bases corresponding to the amino acids in SEQ ID No. 1 were transferred into the PET-24a plasmid, which was custom-made and provided by GenScript Biotech Corporation, USA (the plasmid can also be obtained by referring to the following literature: Du, F., Liu, YQ., Xu, YS. et al. Regulating the T7 RNA polymerase expression in E. coli BL21(DE3) to provide more host options for recombinant protein production. Microb Cell Fact 20, 189 (2021).). The C-terminus of the plasmid was labeled with 7 tandem histidine residues. BL21(DE3) competent cells provided by New England Biolabs Inc. were removed from -80°C and transfected according to the BL21(DE3) competent cell product manual. First, the BL21(DE3) competent cells were thawed on ice. After thawing, plasmids containing the gene corresponding to the amino acid sequence of the aforementioned specific antifreeze protein were added to competent cells using a pipette and placed in an ice bath at -5°C for 30 minutes. Then, the competent cells were heated in 40°C water for 30 seconds and immediately returned to the ice bath for 3 minutes. Luria-Bertani medium (liquid) containing 50 μg / mL kanamycin (provided by New England Biolabs, Inc.) was added to the competent cells, and the cells were incubated for 2 hours at 37°C and 200 rpm on a shaker. The bacterial suspension was then spread onto Luria-Bertani medium (solid) containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12 hours. Subsequently, a small number of colonies (6 single colonies) were picked from the medium and placed in 100 mL of Luria-Bertani medium (liquid) containing 50 μg / mL kanamycin, and incubated overnight at 37°C.

[0047] Procedure b. Expression and purification. The bacterial suspension obtained above (5% inoculum volume) was placed in Luria-Bertani medium (liquid) containing 50 μg / mL kanamycin and incubated at 37°C as usual until OD500. 600Reach 0.6. Add isopropyl-β-D-thiogalactoside and adjust the final concentration to 1 mM, then continue culturing for 4 hours. Centrifuge the bacterial suspension at 5000 g for 30 minutes at 4°C. Discard the supernatant and collect the precipitate. Add 5-10 ml (8 ml in this case) of lysis buffer [a solution of 60 mM NaH2PO4 and 350 mM NaCl, pH = 8] to the precipitate, and add phenylmethylsulfonyl fluoride to a final concentration of 1 mg / mL as a protease inhibitor. Add lysozyme and adjust the final concentration to 1 mg / mL, then sonicate the bacteria in an ice bath at -5°C. Repeatedly aspirate the bacterial lysate 10-20 times (16 times in this case) using a fine syringe needle to cut the sticky DNA fragments. After centrifugation at 10000g for 1 hour at 4°C, the collected supernatant was mixed evenly with His-tag purification resin (i.e. His-tag protein purification resin (nickel column)) in a shaker, and the novel antifreeze protein compound was separated and purified using chromatographic purification equipment.

[0048] Example 2

[0049] 1000g of the cryosol was prepared according to the preparation method disclosed in this invention.

[0050] The composition and mass percentage of the prepared cryogenic solution were as follows: 0.36% of the special antifreeze protein prepared in Example 1, 8.5% of glycine trimethylamine inner salt, 8.5% of β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, 11.5% of 4-O-α-D-glucopyranosyl-D-glucopyranoside, 3% of methyl 4-D-glucopyranoside, 4% of methyl α-D-glucopyranoside, 5% of kaempferol-7-O-β-D-glucopyranoside, 0.035% of sodium D-isoascorbate, 0.065% of monosodium glutamate, and the remainder being water.

[0051] Step a. First, pour water into a mixing vessel and heat it to 35-40℃ (38℃ in this case). Then add all other components except for the antifreeze protein and water, stir thoroughly for 10-15 minutes (12 minutes in this case), and let it stand at room temperature for 5-10 minutes (8 minutes in this case).

[0052] Step b. Add the artificially synthesized antifreeze protein into a mixing vessel, stir thoroughly for 10-20 minutes (15 minutes in this case), and let it stand at 0-4°C (4°C in this case) for 6-12 hours (8 hours in this case) to prepare the cryosol.

[0053] The cryosol crystallized when cooled to -68°C using a low-temperature freezing point viscosity multi-function instrument (Anton Paar, USA, model SVM3001 Cold Properties), and completely froze when cooled to -72°C.

[0054] Example 3

[0055] Frozen and thawed sea bass were compared with live sea bass. The experimental samples were live sea bass weighing 500-700g each and measuring 30-40cm in length. A thermocouple probe was inserted 10cm into the sea bass's mouth along its length, and the internal temperature was measured using a Thermo Fisher Scientific Temp 10 series single-channel thermocouple temperature measuring instrument. The cryogenic solution prepared in Example 2 was cooled to -65°C using a refrigeration device. Live sea bass (40% of the cryogenic solution's mass) were then placed in the cryogenic solution and frozen. During the freezing process, the cryogenic solution was maintained at -65°C. According to the thermocouple temperature measuring instrument, the time required for the sea bass's internal temperature to reach -18°C was approximately 20 minutes. Afterward, the sea bass were removed, the thermocouples were disconnected, and the sea bass were washed with water at 0-4°C, forming an ice layer approximately 0.2mm thick on the surface. The frozen sea bass was then stored in a -18℃ freezer (Haier BC / BD-300GH00W0). One month later, the sea bass were removed from the freezer and thawed at room temperature for about 6 hours to obtain samples of the thawed frozen sea bass. One live fresh sea bass and one thawed frozen sea bass were taken, and the following procedures were performed on each fish for comparison.

[0056] 1. Sensory evaluation was conducted on the sea bass samples, assessing five aspects: odor, color, texture, juiciness, and tenderness. The sensory scoring criteria for each indicator are shown in Table 1. The results are shown in Table 2.

[0057] 2. Take the fish meat from the back of the fish, remove the skin, and trim it into three rectangular pieces of 3cm × 2cm × 1cm. Use a colorimeter (Konica Minolta CR-400) to measure the brightness, redness, and yellowness of the fish pieces at room temperature. Each sample was measured three times, for a total of nine measurements. After removing outliers, the average value was taken. The results are shown in Table 3.

[0058] 3. The texture analysis method was employed, and the texture was measured using a secondary compression method. A TMS-PRO texture analyzer from FTC (Factory Direct) was used, with a flat-bottomed cylindrical probe of 5 mm diameter, a trigger point load of 6.0 g, a target value of 5.00 mm, and a test speed of 1.50 mm / s. Each sample was measured six times, and the average value was taken after removing outliers. The results are shown in Table 4.

[0059] 4. The moisture content was determined according to the direct drying method in GB5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food". Each sample was measured three times, for a total of nine measurements of the three samples. After removing outliers, the average value was taken. The results are shown in Table 5.

[0060] 5. The myofibril protein concentration of the sample was determined using the following method. Take 1.5g of fish meat and add 10mL of pre-cooled (0℃-4℃) KCl solution (0.1mol / L, pH 7.0). Homogenize (10000r / min) for 2min in an ice-water bath, pausing for 10s every 10s to prevent overheating. Then centrifuge the dispersion at 12000r / min for 15min at 4℃, remove the supernatant, add 8 volumes of KCl solution (0.6mol / L, pH 7.0) to the resulting precipitate, homogenize again, and incubate at 4℃ for 1h. Centrifuge at 15000r / min for 20min at 4℃. Take the supernatant and determine the myofibril protein concentration using a microplate reader according to the method provided by the protein concentration assay kit provided by Thermo Fisher Scientific. The results are shown in Table 5.

[0061] 6. The total thiol content of myofibrillar protein in the samples was measured using the total thiol assay kit and microplate reader provided by Beijing Solarbio Science & Technology Co., Ltd.; referencing the company's ultra-micro Ca... 2+ -The method provided by the ATPase test kit, used in conjunction with an ELISA reader, measures actomyosin Ca. 2+ - ATPase activity. The results are shown in Table 5.

[0062] Example 4

[0063] The cryogenic solution was prepared according to the steps in Example 2, except that the composition and mass percentage of the prepared cryogenic solution were as follows: 0.36% of the special antifreeze protein prepared in Example 1, 8.5% of glycine trimethylamine inner salt, 5% of kaempferol-7-O-β-D glucoside, and the remainder was water.

[0064] The cryosol crystallized when cooled to -48°C in a low-temperature freezing point viscosity multi-functional instrument, and completely froze when cooled to -52°C.

[0065] Sea bass were frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass were compared with live sea bass. The difference from Example 3 is that, since the lowest temperature at which the freezing liquid does not crystallize is approximately -48°C, the freezing liquid was cooled to -45°C using a refrigeration device before the live sea bass (sea bass weight was 40% of the freezing liquid weight) was placed in the freezing liquid. During the freezing process, the freezing liquid temperature was maintained at -45°C. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 35 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5.

[0066] Example 5

[0067] The cryogenic solution was prepared according to the steps in Example 2, except that the composition and mass percentage of the prepared cryogenic solution were as follows: 0.1% of the special antifreeze protein prepared in Example 1, 5% of glycine trimethylamine inner salt, 5% of β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, 5% of 4-O-α-D-glucopyranosyl-D-glucopyranoside, 1% of methyl 4-O-BETA-D-galactosyl-BETA-D-glucopyranoside, 1% of methyl α-D-glucopyranoside, 2% of kaempferol-7-O-β-D-glucopyranoside, 0.01% of sodium D-isoascorbate, 0.01% of monosodium glutamate, and the remainder was water.

[0068] The cryosol crystallized when cooled to -54°C in a low-temperature freezing point viscosity multi-functional instrument, and completely froze when cooled to -58°C.

[0069] The sea bass was frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass was compared with live sea bass. The difference from Example 3 is that, because the lowest temperature at which the freezing liquid does not crystallize is approximately -54°C, the freezing liquid was cooled to -50°C using a refrigeration device before the live sea bass was placed in the freezing liquid. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 28 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5.

[0070] Comparative Example 1

[0071] The cryogenic solution was prepared according to the steps in Example 2, except that it did not contain the special antifreeze protein prepared in Example 1. The other components and mass percentages were the same as in Example 2, with the remainder being water. Crystallization occurred when the cryogenic solution was cooled to -35°C, and it was completely frozen when cooled to -40°C. This comparative example illustrates the significant effect of the antifreeze protein component disclosed in this invention on the freezing point of the cryogenic solution.

[0072] Comparative Example 2

[0073] The cryogenic solution was prepared according to the steps in Example 2, except that the special antifreeze protein prepared in Example 1 was replaced with antifreeze protein EF1 provided by Nichirei Corporation (Japan). All other components and mass percentages were the same as in Example 2. Crystallization occurred when the cryogenic solution was cooled to -38°C, and it was completely frozen when cooled to -42°C. This comparative example illustrates the uniqueness of the antifreeze protein component disclosed in this invention and its significant effect on the freezing point of the cryogenic solution.

[0074] Comparative Example 3

[0075] The antifreeze protein was prepared according to the steps in Example 1, except that the amino acid sequence was changed as follows:

[0076] Ser Asp Gly Ser Gly Glu Arg Arg Arg Asp Lys Asp Ser Ser Gly Glu HisArg Arg Asp Lys Asp Ser Ser Gly Glu His Arg Arg Asp Lys Asp Ser Ser Gly GluHis Arg

[0077] The cryosol was prepared according to the steps in Example 2, except that the antifreeze protein prepared in Example 1 was replaced with the protein in this comparative example, while the other components and mass percentages were the same as in Example 2. Crystallization occurred when the cryosol was cooled to -36°C, and it was completely frozen when cooled to -40°C. This comparative example illustrates the uniqueness of the amino acid sequence in the antifreeze protein component disclosed in this invention.

[0078] Comparative Example 4

[0079] The antifreeze protein was prepared according to the steps in Example 1, except that the amino acid sequence was changed as follows:

[0080] Glu Asp Gly Arg Gly Glu Arg Lys Arg Asp Lys Asp Gly Ser Gly Glu HisArg Arg Asp Lys Asp Ser Ser Gly Glu His Arg Arg Asp Lys Asp Ser Ser Gly GluHis Arg

[0081] The cryosol was prepared according to the steps in Example 2, except that the antifreeze protein prepared in Example 1 was replaced with the protein in this comparative example, while the other components and mass percentages were the same as in Example 2. Crystallization occurred when the cryosol was cooled to -35°C, and it was completely frozen when cooled to -40°C. This comparative example illustrates the uniqueness of the amino acid sequence in the antifreeze protein component disclosed in this invention.

[0082] Comparative Example 5

[0083] The cryogenic solution was prepared according to the steps in Example 2, except that it did not contain glycine trimethylamine inner salt. The other components and mass percentages were the same as in Example 2, with the remainder being water. Crystallization occurred when the cryogenic solution was cooled to -32°C, and it was completely frozen when cooled to -35°C. This comparative example illustrates the significant effect of the glycine trimethylamine inner salt component disclosed in this invention on the freezing point of the cryogenic solution.

[0084] Comparative Example 6

[0085] The cryogenic solution was prepared according to the steps in Example 2, except that it did not contain kaempferol-7-O-β-D glucoside. The other components and mass percentages were the same as in Example 2, with the balance being water. Crystallization occurred when the cryogenic solution was cooled to -34°C, and it was completely frozen when cooled to -38°C. This comparative example illustrates the significant effect of the kaempferol-7-O-β-D glucoside component disclosed in this invention on the freezing point of the cryogenic solution.

[0086] Comparative Example 7

[0087] Using the freezing liquid from Comparative Example 1, sea bass were frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass were compared with live sea bass. The difference from Example 3 is that, because the lowest temperature at which the freezing liquid does not crystallize is approximately -35°C, the freezing liquid was cooled to -30°C using a refrigeration device before the live sea bass were placed in the freezing liquid. During the freezing process, the freezing liquid was maintained at -30°C. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 50 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5. This comparative example illustrates the significant effect of the antifreeze protein component disclosed in this invention on the freezing point of the freezing liquid, and the resulting significant impact on the quality of the frozen sea bass.

[0088] Comparative Example 8

[0089] Using the freezing liquid from Comparative Example 2, sea bass were frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass were compared with live sea bass. The difference from Example 3 is that, because the lowest temperature at which the freezing liquid does not crystallize is approximately -38°C, the freezing liquid was cooled to -34°C using a refrigeration device before the live sea bass were placed in the freezing liquid. During the freezing process, the freezing liquid was maintained at -34°C. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 46 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5. This comparative example illustrates the uniqueness of the antifreeze protein component disclosed in this invention and its significant impact on the quality of frozen sea bass.

[0090] Comparative Example 9

[0091] Using the freezing liquid from Comparative Example 3, sea bass were frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass were compared with live sea bass. The difference from Example 3 is that, because the lowest temperature at which the freezing liquid does not crystallize is approximately -36°C, the freezing liquid was cooled to -32°C using a refrigeration device before the live sea bass were placed in the freezing liquid. During the freezing process, the freezing liquid was maintained at -32°C. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 48 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5.

[0092] Comparative Example 10

[0093] Using the freezing liquid from Comparative Example 4, sea bass were frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass were compared with live sea bass. The difference from Example 3 is that, because the lowest temperature at which the freezing liquid does not crystallize is approximately -35°C, the freezing liquid was cooled to -30°C using a refrigeration device before the live sea bass were placed in the freezing liquid. During the freezing process, the freezing liquid was maintained at -30°C. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 50 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5.

[0094] Comparative Example 11

[0095] Using the freezing liquid from Comparative Example 5, sea bass were frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass were compared with live sea bass. The difference from Example 3 is that, because the lowest temperature at which the freezing liquid does not crystallize is approximately -32°C, the freezing liquid was cooled to -29°C using a refrigeration device before the live sea bass were placed in the freezing liquid. During the freezing process, the freezing liquid was maintained at -29°C. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 52 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5.

[0096] Comparative Example 12

[0097] Using the freezing liquid from Comparative Example 6, sea bass were frozen according to the steps in Example 3 (process and conditions were the same as in Example 3), and the thawed sea bass were compared with live sea bass. The difference from Example 3 is that, because the lowest temperature at which the freezing liquid does not crystallize is approximately -34°C, the freezing liquid was cooled to -30°C using a refrigeration device before the live sea bass were placed in the freezing liquid. During the freezing process, the freezing liquid was maintained at -30°C. According to a thermocouple temperature measuring instrument, the time required for the internal temperature of the sea bass to reach -18°C was approximately 50 minutes. The remaining operations were the same as in Example 3. The comparison results are shown in Tables 2-5.

[0098] Table 1. Sensory Scoring Criteria for Sea Bass

[0099]

[0100] In the odor assessment, the stronger the fishy smell, the lower the score; in the color assessment, the higher the transparency, the lower the score; in the texture assessment, the worse the elasticity, the lower the score; in the juiciness assessment, the less juice, the lower the score; and in the tenderness assessment, the more tender and smooth, the higher the score.

[0101] Table 2 Comparison of sensory evaluation results of frozen sea bass samples

[0102]

[0103] As shown in Table 2, the frozen sea bass from Examples 3, 4, and 5, prepared using the cryosol disclosed in this invention, remained very close to live sea bass after thawing at -18°C for 30 days. They exhibited almost no fishy odor and were tender and juicy. In contrast, the cryosol used in Comparative Examples 7-12 did not utilize the components and parameters within the scope of this invention's claims, nor did it employ the amino acid sequence disclosed in this invention to synthesize antifreeze proteins. Consequently, the frozen sea bass used in these examples differed significantly in appearance and quality from live sea bass, exhibiting a stronger fishy odor and losing most of its tenderness. Sensory evaluation revealed that the frozen sea bass in Examples 3-5 were slightly inferior to live sea bass in various indicators but significantly superior to the frozen sea bass in Comparative Examples 7-12.

[0104] Table 3 Comparison of color difference results for frozen sea bass samples

[0105]

[0106]

[0107] As can be seen from the results in Table 3, based on the color difference index comparison, the sea bass frozen in Examples 3, 4, and 5, after being thawed in a -18°C freezer for 30 days using the freezing liquid prepared by the technology disclosed in this invention, were very close to the condition of live sea bass. However, the freezing liquid used in Comparative Examples 7-12 did not employ the components and parameters within the scope of protection of this invention's claims, nor did it use the amino acid sequence disclosed in this invention to synthesize antifreeze proteins; consequently, the condition and quality of the frozen sea bass were significantly different from that of live sea bass.

[0108] The color of fish meat is one of the important indicators for evaluating its quality. By measuring the brightness, redness, and yellowness values ​​of fish pieces, the color is quantified, avoiding the interference of human factors in sensory evaluation and obtaining clearer and more reliable data. In Examples 3-5, the color difference indicators of the frozen sea bass were slightly worse than those of the live sea bass, but significantly better than those of the frozen sea bass in Comparative Examples 7-12.

[0109] Table 4 Comparison of textural properties of frozen sea bass samples

[0110]

[0111] As can be seen from the results in Table 4, based on the comparison of textural properties, the sea bass frozen in Examples 3, 4, and 5, after being thawed in a -18°C freezer for 30 days using the cryogenic solution prepared by the technology disclosed in this invention, were very close to the condition of live sea bass. However, the cryogenic solutions used in Comparative Examples 7-12 did not employ the components and parameters within the scope of protection of this invention's claims, nor did they use the amino acid sequences disclosed in this invention to synthesize antifreeze proteins; consequently, the state and quality of the frozen sea bass were significantly different from those of live sea bass.

[0112] Hardness refers to the softness or hardness felt by the human body, the force required to deform food to a certain extent, or the internal binding force required to maintain the shape of food. Elasticity is the height or volume ratio by which a deformed sample returns to its pre-deformation state after the deformation force is removed. Cohesion indicates the degree of cohesion of fish meat or its ability to maintain its integrity. Chewability, as a comprehensive textural evaluation parameter, is a concentrated reflection of the hardness, cohesion, and elasticity of fish meat. In Examples 3-5, the frozen sea bass exhibited slightly worse performance in all the above indicators than the live sea bass, but significantly better performance than the frozen sea bass in Comparative Examples 7-12.

[0113] Table 5 Comparison of moisture content, total sulfhydryl content of myofibrillar protein, and Ca2+-ATPase activity of actomyosin in frozen sea bass samples

[0114]

[0115] As can be seen from the results in Table 5: based on moisture content, total sulfhydryl content of myofibrillar protein, and actomyosin Ca... 2+-Comparison of ATPase activity: Using the cryosol prepared by the technology disclosed in this invention, the sea bass frozen in Examples 3, 4, and 5, after being thawed in a -18°C freezer for 30 days, were very close to the condition of live sea bass. However, the cryosols used in Comparative Examples 7-12 did not use the components and parameters within the scope of protection of this invention's claims, nor did they use the amino acid sequences disclosed in this invention to synthesize antifreeze proteins; consequently, the state and quality of the frozen sea bass were significantly different from those of live sea bass.

[0116] Moisture content is an important indicator of the water-holding capacity of sea bass meat. Protein is the main component of muscle tissue, playing a crucial structural support role and contributing significantly to various physiological functions. Protein degradation and denaturation have a significant impact on the flavor and texture of fish meat. Myofibril protein accounts for 60-70% of total muscle protein and is the main structural protein of fish muscle tissue, closely related to the processing characteristics and water-holding properties of meat products. Therefore, studying the changes in myofibril protein mass concentration during freezing is crucial. Total thiol groups, including active and latent thiol groups, are among the most reactive functional groups in fish meat proteins. They are temperature-sensitive and easily oxidized or exchanged with disulfide bonds, leading to a decrease in their content. The stability and denaturation of protein structures are largely related to thiol groups and disulfide bonds; therefore, changes in total thiol content can reflect the degree of change in fish meat during freezing. 2+ -ATPase activity is an important indicator for assessing protein quality, related to the gelation properties of proteins, and is also an important indicator for evaluating the quality of sea bass. The frozen sea bass in Examples 3-5 showed slightly lower performance in all the above indicators compared to live sea bass, but significantly better performance than the frozen sea bass in Comparative Examples 7-12.

[0117] Although specific embodiments have been described in detail above, those skilled in the art can make appropriate changes to the adaptive description of different embodiments without departing from the principles and spirit of the present invention, but all such changes should fall within the protection scope of the present invention.

Claims

1. A freezing solution for freezing aquatic products, characterized by, By weight percentage, it includes: 0.1%-1% antifreeze protein, 5%-10% glycine trimethylamine inner salt, 2%-10% kaempferol-7-O-β-D glucoside, and the remainder is water; The amino acid sequence of the antifreeze protein is as follows: Lys Asp Ser Ser Gly Glu His Arg Arg Asp Lys Asp Ser Ser Gly Glu His ArgArg Asp Lys Asp Ser Ser Gly Glu His Arg Arg Asp Lys Asp Ser Ser Gly Glu HisArg.

2. The refrigerant according to claim 1, characterized in that, The cryogenic fluid comprises, by mass percentage: Antifreeze protein 0.3%-0.4%, glycine trimethylamine inner salt 8%-9%, kaempferol-7-O-β-D glucoside 4%-6%, the remainder is water.

3. The cryogenic fluid according to claim 1 or 2, characterized in that, Substances with the following final mass concentrations may be added to water, based on a percentage by mass: β-D-fructofuranosyl-(2→1)-α-D-glucopyranose 5%-10%, 4-O-α-D-glucopyranosyl-D-glucopyranose 5%-15%, methyl 4-O-BETA-D-galactosyl-BETA-D-glucopyranoside 1%-5%, methyl α-D-glucopyranoside 1%-10%, sodium D-isoascorbate 0.01%-0.1%, monosodium glutamate 0.01%-0.1%.

4. The cryogenic fluid according to claim 3, characterized in that, Substances with the following final mass concentrations may be added to water, based on a percentage by mass: β-D-fructofuranosyl-(2→1)-α-D-glucopyranose 8%-9%, 4-O-α-D-glucopyranosyl-D-glucopyranose 10%-12%, methyl 4-O-BETA-D-galactosyl-BETA-D-glucopyranoside 2%-4%, methyl α-D-glucopyranoside 3%-5%, sodium D-isoascorbate 0.03%-0.04%, monosodium glutamate 0.06%-0.07%.

5. A method for preparing the cryogenic fluid according to claim 1, 2, 3, or 4, characterized in that, Includes the following steps: a. First, pour water into a mixing vessel and heat it to 35-40℃. Then add all other components except for the antifreeze protein and water. Stir thoroughly for 10-15 minutes and let it stand at room temperature for 5-10 minutes. b. Add the antifreeze protein to a mixing vessel, stir thoroughly for 10-20 minutes, and let it stand at 0-4°C for 6-12 hours to prepare the freezing solution.

6. An application of the refrigerant according to claim 1, 2, 3, or 4, characterized in that, The process includes the following steps: cooling the freezing liquid described in claim 1, 2, 3, or 4 to -40°C to -70°C; placing aquatic products to be frozen, with a total mass not exceeding 70% of the mass of the freezing liquid, into the freezing liquid and freezing for 10-100 minutes; and maintaining the temperature of the freezing liquid between -40°C and -70°C during the freezing process. Then the frozen seafood is washed with water.

7. The application of the refrigerant according to claim 6, characterized in that, include: The freezing liquid described in claim 1, 2, 3, or 4 is cooled to -45°C to -65°C, and the freezing liquid is maintained at a temperature between -45°C and -65°C. Aquatic products with a total mass not exceeding 50% of the mass of the freezing liquid are placed in the freezing liquid and frozen for 15-40 minutes to freeze the aquatic products. During the freezing process of the aquatic products, the temperature of the freezing liquid is maintained between -45°C and -65°C. The frozen aquatic products are then washed with water at 0-4℃, forming an ice coating 0.05-1mm thick on the outer surface of the aquatic products.

8. The application of the refrigerant according to claim 6 or 7, characterized in that, The aquatic products mentioned include one or more of the following: freshwater fish, saltwater fish, shellfish, scallops, clams, and snails.

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