Shellfish cell-level fresh-keeping freezing method based on magnetic control ice crystal form

Abstract

The invention relates to the technical field of aquatic product freezing and fresh-keeping, and discloses a shellfish cell-level fresh-keeping freezing method based on a magnetic control ice crystal form. The method specifically comprises the following steps: cleaning fresh and live shellfish, firstly treating the shellfish with an antifreeze agent, and then performing directional segmented freezing under the assistance of a static magnetic field at the freezing temperature of-18 DEG C to-23 DEG C and the magnetic field intensity range of 2mT to 20mT. According to the method, the orientation and movement of water molecule clusters can be effectively interfered under the assistance of a static magnetic field, and a large number of uniform nucleation of water molecules is promoted, so that a large number of intracellular ice crystals with small sizes are formed, the damage of the ice crystals to shellfish cell structures is reduced, the integrity of the cells is kept, and the water loss and texture damage of the cells are reduced; the shellfish cell-level fresh-keeping freezing and the freezing quality improvement are realized.

Description

Technical Field

[0001] This invention relates to the field of aquatic product freezing and preservation technology, specifically to a cell-level preservation freezing method for shellfish based on magnetically controlled ice crystal morphology. Background Technology

[0002] The shellfish industry is the largest sector in my country's marine aquaculture, accounting for over 80% of Asia's total production and over 65% of the world's total. Shellfish (such as scallops, oysters, and mussels) have tender flesh, rich in water and protein, but their cell membranes are thin and their tissue structure is fragile. During traditional freezing processes, they easily form large needle-like ice crystals. These ice crystals pierce the cell walls and membranes, causing significant loss of juices during thawing, resulting in the loss of nutrients and flavor compounds, a soft and mushy texture, and severe quality deterioration. Current technologies primarily use cryogenic media such as liquid nitrogen and liquid CO2 for rapid freezing to improve freezing quality, greatly increasing the cooling rate to reduce ice crystal size. However, these methods involve high equipment investment and operating costs, and for shellfish with inherently low thermal conductivity, the freezing rate in the central part is still limited, making it difficult to completely avoid ice crystal damage.

[0003] In recent years, static magnetic field technology has been explored for its use in assisted freezing, showing great application potential due to its residue-free, low-energy, and high-safety characteristics. Studies have shown that magnetic fields with appropriate parameters can affect the hydrogen bond network of water molecules, altering the state of supercooled water and thus influencing the nucleation rate and growth morphology of ice crystals. However, current technologies are mostly still in the laboratory stage, with unstable magnetic field parameters and a lack of synergistic optimization with food freezing processes. Particularly for high-moisture, easily damaged shellfish, a complete, efficient, and industrially applicable magnetically controlled cell-level preservation freezing technology system has not yet been established.

[0004] Therefore, developing a freezing method that can precisely control the morphology of ice crystals, maximize the preservation of the integrity of shellfish cell structures, and is cost-effective is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention proposes a cell-level preservation freezing method for shellfish based on magnetically controlled ice crystal morphology. The aim is to regulate the nucleation and growth process of shellfish ice crystals through a static magnetic field, so as to minimize the damage of ice crystals to cells and provide a new solution for shellfish freezing and preservation technology.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for cell-level preservation freezing of shellfish based on magnetically controlled ice crystal morphology includes the following steps:

[0008] S1: Shellfish pretreatment: Clean the fresh shellfish and soak them in a certain concentration of antifreeze at 4℃±1℃ (the temperature of the refrigeration equipment is set at 4℃ and the temperature fluctuation of the equipment is controlled within 1℃). Then drain the water and place them in a sealed plastic container.

[0009] S2: Static magnetic field-assisted freezing: Place the shellfish processed in step S1 under a static magnetic field for assisted freezing. The temperature of magnetic field-assisted freezing is not higher than -18℃ and the magnetic field strength is not higher than 20mT. Freezing is terminated when the temperature at the center of the shellfish reaches -18℃.

[0010] S3: Freezing preservation. Shellfish frozen with the assistance of a static magnetic field are frozen and preserved at -18℃ to -23℃.

[0011] Preferably, the antifreeze agent is a mixed aqueous solution of trehalose and skim milk, with concentrations of 3%–5% (mass percentage) for trehalose and 1%–2% (mass percentage) for skim milk, and the soaking time is 10–15 minutes.

[0012] Preferably, the static magnetic field-assisted freezing has a freezing temperature of -20℃±1℃ (the freezing equipment is set to -20℃, and the equipment's temperature fluctuation is controlled within 1℃).

[0013] Preferably, the static magnetic field-assisted freezing is carried out in a segmented freezing manner. When the core temperature of the shellfish is above -1℃, the magnetic field strength is 2mT to 10mT; when the core temperature of the shellfish is between -1℃ and -5℃, the magnetic field strength is adjusted to 12mT to 15mT; and when the core temperature of the shellfish is below -5℃, the magnetic field strength is 2mT to 10mT.

[0014] Compared with related technologies, the present invention has the following beneficial effects:

[0015] This invention employs segmented magnetic field-assisted freezing. At different freezing stages, the magnetic field strength is dynamically adjusted based on ice crystal formation. When the shellfish's core temperature is above -1℃, the magnetic field strength is 2mT to 10mT; when the core temperature is between -1℃ and -5℃, the magnetic field strength is adjusted to 12mT to 15mT; and when the core temperature is below -5℃, the magnetic field strength is 2mT to 10mT. This method significantly shortens the freezing time of shellfish, reduces ice crystal size, and effectively reduces water loss. The scallop hardness increases by 19.75%, the total sulfhydryl content of myofibrillar protein increases by 8.47%, and the α-helix content in the secondary structure increases by 10.42%, effectively improving the quality of the shellfish. Attached Figure Description

[0016] Figure 1 Freezing curves, centrifugation loss rate, freezeable water content, and hardness of scallops under different freezing conditions;

[0017] Figure 2 The results of ice crystal growth simulation of scallop muscle tissue structure, ice crystal morphology between muscle tissues, and molecular kinetics under different freezing conditions;

[0018] Figure 3 To investigate the effects of different freezing conditions on the physicochemical properties of scallop myofibril proteins. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0020] Example 1:

[0021] (1) Clean the fresh shellfish thoroughly, soak them in a mixture of 3% trehalose and 1% skim milk at 4℃±1℃ for 12 minutes, then drain the water and store them in a sealed plastic container.

[0022] (2) Place the processed shellfish under a static magnetic field for assisted freezing. The magnetic field assisted freezing temperature is -20℃±1℃. Freezing is stopped when the core temperature of the shellfish reaches -18℃.

[0023] (3) During the magnetic field-assisted freezing process, when the core temperature of the shellfish is above -1℃, the magnetic field strength is 2mT. When the core temperature of the shellfish is in the range of -1℃ to -5℃, the magnetic field strength is adjusted to 12mT. When the core temperature of the shellfish is below -5℃, the magnetic field strength is 2mT.

[0024] (4) Freezing: The shellfish frozen with the assistance of static magnetic field are frozen at -18℃.

[0025] Example 2:

[0026] (1) Clean the fresh shellfish thoroughly, soak them in a mixture of 3% trehalose and 2% skim milk at 4℃±1℃ for 12 minutes, then drain the water and store them in a sealed plastic container.

[0027] (2) Place the processed shellfish under a static magnetic field for assisted freezing. The magnetic field assisted freezing temperature is -18℃±1℃. Freezing is stopped when the core temperature of the shellfish reaches -18℃.

[0028] (3) During the magnetic field-assisted freezing process, when the core temperature of the shellfish is above -1℃, the magnetic field strength is 4mT; when the core temperature of the shellfish is in the range of -1℃ to -5℃, the magnetic field strength is adjusted to 14mT; and when the core temperature of the shellfish is below -5℃, the magnetic field strength is 4mT.

[0029] (4) Freezing: The shellfish frozen with the assistance of static magnetic field are frozen at -20℃.

[0030] Example 3:

[0031] (1) Clean the fresh shellfish thoroughly, soak them in a mixture of 4% trehalose and 1% skim milk at 4℃±1℃ for 12 minutes, then drain the water and store them in a sealed plastic container.

[0032] (2) Place the processed shellfish under a static magnetic field for assisted freezing. The magnetic field assisted freezing temperature is -22℃±1℃. Freezing is stopped when the core temperature of the shellfish reaches -18℃.

[0033] (3) During the magnetic field-assisted freezing process, when the core temperature of the shellfish is above -1℃, the magnetic field strength is 6mT; when the core temperature of the shellfish is in the range of -1℃ to -5℃, the magnetic field strength is adjusted to 15mT; and when the core temperature of the shellfish is below -5℃, the magnetic field strength is 6mT.

[0034] (4) Freezing: The shellfish frozen with the assistance of static magnetic field are frozen at -21℃.

[0035] Example 4:

[0036] (1) Clean the fresh shellfish thoroughly, soak them in a mixture of 5% trehalose and 1% skim milk at 4℃±1℃ for 12 minutes, then drain the water and store them in a sealed plastic container.

[0037] (2) Place the processed shellfish under a static magnetic field for assisted freezing. The magnetic field assisted freezing temperature is -23℃±1℃. Freezing is stopped when the core temperature of the shellfish reaches -18℃.

[0038] (3) During the magnetic field-assisted freezing process, when the core temperature of the shellfish is above -1℃, the magnetic field strength is 10mT; when the core temperature of the shellfish is in the range of -1℃ to -5℃, the magnetic field strength is adjusted to 12mT; and when the core temperature of the shellfish is below -5℃, the magnetic field strength is 10mT.

[0039] (4) Freezing: The shellfish frozen with the assistance of static magnetic field are frozen at -23℃.

[0040] Example 5:

[0041] (1) Clean the fresh shellfish thoroughly, soak them in a mixture of 5% trehalose and 2% skim milk at 4℃±1℃ for 12 minutes, then drain the water and store them in a sealed plastic container.

[0042] (2) Place the processed shellfish under a static magnetic field for assisted freezing. The magnetic field assisted freezing temperature is -20℃±1℃. Freezing is stopped when the core temperature of the shellfish reaches -18℃.

[0043] (3) During the magnetic field-assisted freezing process, when the core temperature of the shellfish is above -1℃, the magnetic field strength is 5mT; when the core temperature of the shellfish is in the range of -1℃ to -5℃, the magnetic field strength is adjusted to 12mT; and when the core temperature of the shellfish is below -5℃, the magnetic field strength is 10mT.

[0044] (4) Freezing: The shellfish frozen with the assistance of static magnetic field are frozen at -23℃.

[0045] Comparative Example 1:

[0046] (1) Clean the fresh shellfish thoroughly, soak them in a mixture of 3% trehalose and 1% skim milk at 4℃±1℃ for 12 minutes, then drain the water and store them in a sealed plastic container.

[0047] (2) Place the processed shellfish in a low temperature of -20℃±1℃ and freeze directly until the core temperature of the shellfish reaches -18℃.

[0048] (3) Freezing: Freeze the frozen shellfish at -18°C.

[0049] Comparative Example 2:

[0050] Clean the fresh shellfish thoroughly, soak them in a mixture of 3% trehalose and 1% skim milk at 4℃±1℃ for 12 minutes, then drain the water and store them in a sealed plastic container without freezing.

[0051] The performance indicators are as follows:

[0052] 1. Effects of magnetic field-assisted freezing on freezing time, thawing loss rate, centrifugation loss rate, and hardness of scallops (1) Determination of freezing time

[0053] The probe of a multichannel temperature recorder was inserted into the geometric center of the scallop adductor sample to monitor internal temperature changes during freezing. Temperature was recorded every 10 seconds, and freezing was considered complete when the internal temperature reached -18°C. A freezing curve was plotted with time as the independent variable and center temperature as the dependent variable.

[0054] (2) Determination of centrifugal loss rate

[0055] Weigh the thawed sample (G0) and place it in a cylindrical centrifuge tube lined with filter paper. Centrifuge at 4°C and 5000 rpm for 10 minutes. Then remove the filter paper and weigh the sample (G1). Centrifugation loss rate (%) = (G0 - G1) / G0 × 100.

[0056] (3) Determination of freezeable water content

[0057] The determination was performed using a differential scanning calorimeter. 10 mg ± 1 mg of sample was accurately weighed into a crucible using an analytical balance and then covered with a lid for pressure. An empty crucible served as a control, and nitrogen was used as the carrier gas for both purging and protection. The sample was first cooled from 20 °C to -40 °C at a rate of 5 °C / min and held constant for 5 min. Then, it was heated from -40 °C to 10 °C at a rate of 5 °C / min. Freezeable water content = ΔH / (ΔH0 × W) A )×100, where: ΔH is the enthalpy change of water in the sample, J / g; ΔH0 is the latent heat of melting of a unit mass of pure water into ice (334 J / g); W A The total water content of the sample, %.

[0058] (4) Hardness test

[0059] Cut the fresh and thawed samples into 1×1×1cm pieces. 3 The samples were cubed, and their hardness and elasticity were measured using a texture analyzer. Two rounds of compression tests were conducted on the samples using a P / 10 flat-bottomed cylindrical probe. The test conditions were: compression deformation rate of 40% and test rate of 1.00 mm / s.

[0060] (5) Experimental Results

[0061] The freeze curves of each embodiment and comparative example are as follows: Figure 1 As shown in Figure A, compared to Comparative Example 1, the total freezing time of Examples 1, 2, 3, 4, and 5 was reduced by approximately 3.88%, 9.67%, 15.30%, 9.87%, and 8.86%, respectively. This indicates that applying a magnetic field during the freezing process of scallops can effectively improve freezing efficiency. The results for centrifugation loss rates are shown below. Figure 1 As shown in B, compared with Comparative Example 1, the centrifugation loss rate of each embodiment showed varying degrees of reduction. Under magnetic field-assisted freezing conditions, the freezing speed of scallops accelerated, promoting the rapid formation of extracellular ice crystals, inhibiting osmotic imbalance, reducing intracellular water diffusion, and thus maintaining the water-holding capacity of macromolecules.

[0062] The results of the freezeable water content are as follows Figure 1As shown in C, compared with Comparative Example 1, the proportion of freezeable water (including free water and loose bound water) in Examples 1, 2, 3, 4, and 5 decreased by 3.43%, 1.62%, 8.05%, 6.80%, and 5.83%, respectively. This reduction is due to the breakdown of large water clusters into smaller clusters by the magnetic field. These smaller clusters bind more tightly to proteins, limiting the conversion of bound water to free water, thereby reducing the freezeable water content of the scallops.

[0063] The results of hardness are as follows Figure 1 As shown in D, the hardness of all frozen scallop samples was lower than that of the fresh samples (Comparative Example 2). However, the hardness of the samples corresponding to Examples 2-5 was significantly higher than that of Comparative Example 1 (P<0.05), indicating that magnetic field-assisted freezing reduces the damage of ice crystals to muscle cells, thereby maintaining the texture characteristics of the scallops.

[0064] 2. Effects of magnetic field-assisted freezing on scallop muscle tissue structure and ice crystal formation

[0065] (1) Observation of scallop muscle tissue structure

[0066] Frozen samples were cut into 10×10×5 mm cubes perpendicular to the muscle fibers and fixed in 4% paraformaldehyde solution (v / v) for 48 h. They were then embedded in paraffin, sectioned, dewaxed, and stained with hematoxylin-eosin solution. The samples were observed using an optical microscope.

[0067] (2) Observation of ice crystal morphology in scallop muscle tissue

[0068] The frozen sample was cut into 2 mm thick sections perpendicular to the muscle fiber arrangement, and then the sections were freeze-dried. The dried sample was sputter-coated with gold for 60 seconds using an ion sputtering apparatus, and the ice crystal morphology of the sample was observed using a tungsten filament scanning electron microscope.

[0069] (3) Molecular dynamics simulation to investigate the effect of magnetic field on ice crystal growth during scallop freezing process

[0070] Molecular dynamics simulations were performed using GROMACS 2024 software. A static magnetic field was simulated by adding Lorentz forces to the usual GROMACS calculations. The simulation system included ice, water, and the most abundant fragment of scallop myofibril protein. Water molecules were modeled using the TIP4P / Ice model, and the ice crystal structure consisted of…

[0071] GenIce software was used for model construction. The myofibrillar protein fragment model was predicted and constructed using the PEP-FOLD3 program, and a topology file was generated based on the amber99sb force field. The myofibrillar protein fragments were placed in an ice-water box near an ice layer, and Na+ was added... +The system charge was neutralized. A 5000-step energy minimization was then performed. The system was equilibrated at 253.15 K using a V-rescale thermostat and a Berendsen pressure coupler. A 200 ns simulation was then performed at 235.15 K with a time step of 2 fs. In Example 3, a static magnetic field of 6 mT was applied along the z-axis, while in Comparative Example 1, no magnetic field was applied.

[0072] (4) Experimental Results

[0073] Images of scallop muscle tissue structure, such as Figure 2 As shown in Figure A, the tissue structure of Comparative Example 2 was intact, with smaller gaps between muscle fibers. In contrast, the intercellular gaps between fiber bundles in the sample of Comparative Example 1 were significantly increased, indicating that the muscle tissue suffered more severe damage under air freezing conditions. Examples 1-5, especially Example 3, showed smaller tissue gaps and less muscle tissue damage, demonstrating that magnetic field-assisted freezing effectively reduced damage to scallop muscle cells.

[0074] The morphology of ice crystals in scallop muscle tissue, such as Figure 2 As shown in Figure B, after vacuum freeze-drying, the microstructure of the sample exhibits numerous pores formed by ice crystal sublimation. These residual pores can serve as indirect indicators of the original size and spatial distribution of the ice crystals. In Comparative Example 1, a large number of irregularly shaped and large pores were observed, indicating that large and unevenly distributed ice crystals were generated during freezing, causing severe structural damage to muscle fibers and consequently compromising tissue integrity and moisture retention during thawing. In contrast, the pores in Examples 1-5 are significantly smaller and more uniformly distributed. In particular, in Example 3, the pore size is relatively consistent across the entire tissue cross-section, indicating that the magnetic field effectively modulates the size of the ice crystals during freezing, thereby reducing damage to the muscle tissue.

[0075] Mass spectrometry analysis results showed that the amino acid sequence of the fragment with the highest abundance of scallop myofibril protein was:

[0076] LADELRGEQDHSSQVEK. This fragment was placed in an ice-water mixture for 200 ns molecular dynamics simulations, the process of which is as follows: Figure 2 As shown in C. Analysis of the number of hydrogen bonds in the system ( Figure 2 D) As can be seen, the number of hydrogen bonds in Example 3 was significantly higher than that in Comparative Example 1 in the later stages of the simulation. Since the number of hydrogen bonds in ice crystal structures is generally greater than that in liquid water structures, combined with the freezing time results shown in Table 1, it can be inferred that the magnetic field accelerated the formation rate of ice crystals during the freezing process. Meanwhile, from the results of the change in radius of gyration ( Figure 2 E) It can be seen that Example 3 exhibits a lower radius of gyration value in the later stage of the simulation compared to Comparative Example 1, indicating that the myofibrillar protein fragment structure is more compact and stable under magnetic field conditions.

[0077] 3. Effects of magnetic field-assisted freezing on the oxidation and denaturation of scallop myofibrillar proteins

[0078] (1) Determination of thiol content

[0079] Mix 1 ml of myofibrillar protein solution with 9 ml of phosphate buffer (0.6 M KCl, 10 mM EDTA, and 8 M urea, pH 8.0). Mix 4.5 ml of the above mixture with 0.5 ml of Tris-HCl (0.2 mol / L, 0.1% DTNB, pH 8.0) and react in a 40°C water bath in the dark for 25 min. Take the supernatant and measure the absorbance at 412 nm to calculate the total thiol content.

[0080] (2) Secondary structure

[0081] The myofibrillar protein solution (0.01 mg / mL) was transferred to a quartz cuvette with a light path of 1 mm, and the spectrum was measured using a circular dichroism spectrometer in the wavelength range of 190-300 nm at a scanning speed of 100 nm / min.

[0082] (3) Three-level structure

[0083] The tertiary structure of myofibrillar protein samples (0.02 mg / mL) was analyzed using a fluorescence spectrophotometer. The instrument parameters were set as follows: emission wavelength range of 300-500 nm, excitation wavelength of 295 nm, slit width of 2.5 nm, and scan speed of 1200 nm / min.

[0084] (4) Experimental Results

[0085] The sulfhydryl content of scallop myofibril protein is as follows Figure 3 As shown in Figure A, compared to Comparative Example 1, Examples 3-5 significantly increased the sulfhydryl group content in scallop myofibrillar protein (P<0.05). The secondary and tertiary structures of myofibrillar protein are shown below. Figure 3 As shown in B and 3C, Example 3 exhibited the highest α-helical structure content and the highest fluorescence intensity. Simulated freezing of shellfish myofibril proteins also confirmed that magnetic field treatment can regulate the nucleation and growth of ice crystals, reduce the size and distribution inhomogeneity of ice crystals, thereby reducing the damage of ice crystals to protein structures and effectively protecting the stability of thiol content.

[0086] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for cell-level preservation and freezing of shellfish based on magnetically controlled ice crystal morphology, characterized in that: The following steps are involved: S1: Shellfish pretreatment: Clean the fresh shellfish, soak them in a certain concentration of antifreeze at 4℃±1℃, then drain the water and place them in a sealed plastic container. S2: Static magnetic field-assisted freezing: Place the shellfish processed in step S1 under a static magnetic field for assisted freezing. The temperature of magnetic field-assisted freezing is not higher than -18℃ and the magnetic field strength is not higher than 20mT. Freezing is terminated when the temperature at the center of the shellfish reaches -18℃. S3: Freezing preservation. Shellfish frozen with the assistance of a static magnetic field are frozen and preserved at -18℃ to -23℃.

2. The shellfish cell-level preservation freezing method based on magnetically controlled ice crystal morphology according to claim 1, characterized in that, The antifreeze protectant is a mixed aqueous solution of trehalose and skim milk, with concentrations of 3%–5% trehalose and 1%–2% skim milk, and a soaking time of 10–15 minutes.

3. The shellfish cell-level preservation freezing method based on magnetically controlled ice crystal morphology according to claim 1, characterized in that, The static magnetic field-assisted freezing method has a freezing temperature of -20℃±1℃.

4. The shellfish cell-level preservation freezing method based on magnetically controlled ice crystal morphology according to claim 1, characterized in that, The static magnetic field-assisted freezing method is carried out in stages. When the core temperature of the shellfish is above -1℃, the magnetic field strength is 2mT to 10mT; when the core temperature of the shellfish is between -1℃ and -5℃, the magnetic field strength is adjusted to 12mT to 15mT; and when the core temperature of the shellfish is below -5℃, the magnetic field strength is 2mT to 10mT.

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