Natural deep eutectic solvent combined with static magnetic field assisted freezing method and application thereof

Abstract

The application discloses a natural deep eutectic solvent combined with a static magnetic field assisted freezing method and application thereof. The natural deep eutectic solvent is prepared by taking trehalose as a hydrogen bond donor and proline as a hydrogen bond acceptor, and the water content is 20% (w / w). The natural deep eutectic solvent is added to aquatic products, and then the aquatic products are frozen at 35 DEG C under the condition of a 60 mT static magnetic field. Through the synergistic effect of the hydrogen bond network of the natural deep eutectic solvent and the static magnetic field, the freezing denaturation of protein in the aquatic products can be significantly inhibited, and the myofibrillar protein structure can be maintained. Meanwhile, the water retention capacity of the aquatic products is effectively improved, the thawing loss is reduced, and the water holding capacity is improved. The aquatic products prepared by the method have excellent freeze-thaw stability. Therefore, the method has a good application prospect in the field of aquatic product freezing and preservation.

Description

Technical Field

[0001] This invention relates to the field of aquatic product processing technology, specifically to a method for freezing with natural deep eutectic solvents combined with static magnetic field-assisted freezing and its application. Background Technology

[0002] South American white shrimp ( Litopenaeus vannamei Litopenaeus vannamei, also known as Pacific white shrimp, is highly favored by consumers for its delicious meat and rich nutrition, and is one of the highest-yielding shrimp species in global aquaculture. Shrimp paste, an important product of the deep processing of Litopenaeus vannamei, also possesses characteristics of high protein, high moisture, and high endogenous enzyme activity, making it prone to spoilage during processing and storage. Freezing is the most commonly used preservation technology in aquatic product processing, effectively extending shelf life. However, in actual production, transportation, and sales processes, repeated freeze-thaw cycles caused by temperature fluctuations are unavoidable. This leads to the recrystallization and continuous growth of ice crystals within the system, damaging the integrity of muscle tissue, exacerbating myofibril protein denaturation, and ultimately causing a deterioration in the quality of the shrimp paste. Therefore, effectively inhibiting ice crystal growth and mitigating protein denaturation during freezing and freeze-thaw processes is crucial to ensuring the quality of frozen aquatic products.

[0003] Currently, technologies for regulating ice crystal growth during freezing are mainly divided into two categories: physical-assisted freezing technology and antifreeze additives. Static magnetic field-assisted freezing technology has attracted attention in the field of aquatic product freezing and preservation because it can regulate ice crystal nucleation and growth behavior, promote the formation of small, uniform ice crystals, and reduce mechanical damage to muscle tissue caused by ice crystals. However, the protective effect of physical field technology alone on proteins still has room for improvement.

[0004] Natural deep eutectic solvents (NADES) are a class of green solvents formed by intermolecular hydrogen bond interactions between hydrogen bond donors and acceptors. They possess advantages such as being non-toxic, biodegradable, and easy to prepare. Some NADES systems have shown potential as novel cryoprotectants due to their ability to form stable hydrogen bonds with water molecules, reduce the proportion of freezeable water, and inhibit ice crystal growth. However, current research has not yet clarified which NADES systems can improve the frozen meat quality of Litopenaeus vannamei, nor has it clarified whether the combined application of NADES and static magnetic field-assisted freezing technology can produce a synergistic antifreeze effect.

[0005] Therefore, developing a method for freezing Litopenaeus vannamei using NADES combined with a static magnetic field-assisted freezing of aquatic product proteins that can synergistically inhibit protein denaturation during freezing and enhance freeze-thaw water retention capacity is of significant application value. Summary of the Invention

[0006] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides a method for freezing with natural deep eutectic solvent combined with static magnetic field assisted freezing and its application.

[0007] The first object of the present invention is to provide a composition.

[0008] A second object of the present invention is to provide the use of the composition in the preparation of natural deep eutectic solvents.

[0009] The third objective of this invention is to provide a method for preparing a natural deep eutectic solvent.

[0010] A fourth objective of this invention is to provide the application of the natural deep eutectic solvent prepared by the aforementioned method as an antifreeze agent or cryoprotectant for aquatic products.

[0011] The fifth objective of this invention is to provide a method for freezing with a natural deep eutectic solvent combined with a static magnetic field.

[0012] A sixth object of the present invention is to provide the application of the method in inhibiting the denaturation of aquatic products during freezing.

[0013] A seventh objective of this invention is to provide the application of the method in enhancing the water retention capacity of aquatic products.

[0014] An eighth object of the present invention is to provide aquatic products prepared using the method described herein.

[0015] To achieve the above objectives, the present invention is implemented through the following solution: This invention claims protection for the following: A composition comprising trehalose, proline and water, wherein the water content of the composition is 20% (w / w) and the molar ratio of trehalose to proline is 3:8.

[0016] Application of the composition in the preparation of natural deep eutectic solvents.

[0017] A method for preparing a natural deep eutectic solvent, wherein the natural deep eutectic solvent is prepared using the aforementioned composition, the specific method being as follows: trehalose and proline are mixed and ground for 3-7 min, the mixture is transferred to a container, mixed with water, and stirred in a water bath at 80-100℃ and 800-1200 rpm for 1.5-2.5 h until a uniform and transparent liquid is formed.

[0018] Preferably, the grinding time is 5 min, the stirring temperature is 90℃, the stirring speed is 1000 rpm, and the stirring time is 2 h.

[0019] The application of the natural deep eutectic solvent prepared by the above preparation method as an antifreeze agent or cryoprotectant for aquatic products.

[0020] A method for freezing aquatic products using a natural deep eutectic solvent and a static magnetic field assisted by means of adding a natural deep eutectic solvent prepared by the aforementioned method to aquatic products at an amount of 6% of the product's mass, and freezing the product under static magnetic field conditions of -35 to -20°C and a magnetic field strength of 20 to 80 mT.

[0021] Preferably, the magnetic field strength is 60 mT and the temperature is -35℃.

[0022] The method is applied to suppressing the denaturation of aquatic products during freezing.

[0023] The method is applied to improve the water retention capacity of aquatic products.

[0024] The aquatic products prepared by the method described above are also within the scope of protection of this invention.

[0025] Preferably, the aquatic product is Litopenaeus vannamei.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a combination of natural deep eutectic solvent (NADES) and static magnetic field technology for freezing Pacific white shrimp. A 20% trehalose-proline NADES system, added at a 6% concentration, effectively delays the deterioration of shrimp paste quality under repeated freeze-thaw cycles. The combination of NADES and static magnetic field technology further produces a synergistic effect, reducing moisture loss from the shrimp paste, inhibiting protein oxidation, delaying protein aggregation and conformational degradation, and effectively mitigating the quality decline caused by freeze-thaw cycles. The method of freezing aquatic products using a natural deep eutectic solvent combined with static magnetic field assistance provided by this invention can significantly inhibit freeze-drying denaturation and improve the water retention of aquatic products, showing promising application prospects in the field of aquatic product antifreeze. Attached Figure Description

[0027] Figure 1 Macroscopic morphology and polarized light microscopy images of NADES formed by different hydrogen bond acceptors and trehalose.

[0028] Figure 2 The pH values ​​at which different hydrogen bond acceptors form NADES with trehalose.

[0029] Figure 3 The changes in the epimorphic and micromorphic morphology of NADES formed by different hydrogen bond acceptors and trehalose during storage.

[0030] Figure 4 The viscosity changes of NADES formed by different hydrogen bond acceptors and trehalose.

[0031] Figure 5 FTIR changes for NADES formation by different hydrogen bond acceptors and trehalose.

[0032] Figure 6DSC changes for NADES formation by different hydrogen bond acceptors and trehalose.

[0033] Figure 7 The effect of NADES addition on thawing loss of frozen-thawed shrimp paste.

[0034] Figure 8 The effect of NADES addition on the water-holding capacity of frozen-thawed shrimp paste.

[0035] Figure 9 The effect of NADES addition on the sensory evaluation of frozen-thawed shrimp paste gel.

[0036] Figure 10 The effects of thawing loss on frozen-thawed shrimp paste after treatment with control, single component, physical mixture, and NADES were investigated.

[0037] Figure 11 The effects of NADES treatment on the water-holding capacity of frozen-thawed shrimp paste, as a control, a single component, a physical mixture, and the water-holding capacity of the shrimp paste.

[0038] Figure 12 The effects of NADES treatment on the sensory evaluation of frozen-thawed shrimp paste, including control, single-component, physical mixture, and NADES treatment.

[0039] Figure 13 The effect of NADES combined with static magnetic field-assisted freezing on myofibrillar protein solubility.

[0040] Figure 14 The effect of NADES combined with static magnetic field-assisted freezing on the circular dichroism spectrum of myofibrillar proteins.

[0041] Figure 15 The effect of NADES combined with static magnetic field-assisted freezing on the relative content of secondary structure of myofibrillar protein in frozen and thawed shrimp surimi.

[0042] Figure 16 The effect of NADES combined with static magnetic field-assisted freezing on the fluorescence intensity of myofibrillar proteins.

[0043] Figure 17 The effect of NADES combined with static magnetic field-assisted freezing on thawing loss.

[0044] Figure 18 The effect of NADES combined with static magnetic field-assisted freezing on water holding capacity. Detailed Implementation

[0045] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] The Litopenaeus vannamei shrimp were purchased from the aquatic product wholesale market in Xiaoshan District, Zhanjiang City, with a size of 50 shrimp / kg. They were transported to the laboratory within 30 minutes using oxygenated seawater.

[0048] Example 1: Screening of natural deep eutectic solvent systems I. Experimental Methods 1. Screening of different hydrogen bond acceptors for NADES Using trehalose as a hydrogen bond donor, a systematic screening of composite NADES systems involving sugars (glucose, fructose), choline derivatives (choline chloride, betaine), polyols (glycerol, xylitol, sorbitol, mannitol), and amino acids (glycine, tryptophan, serine, alanine, lysine, proline) was conducted. The optimal NADES was selected through hydrogen bond network characterization and ice crystal inhibition efficacy evaluation. The hydrogen bond network characterization and ice crystal inhibition efficacy of the composite NADES systems were systematically evaluated using macroscopic and microscopic observation, viscosity analysis, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The optimal trehalose-based composite NADES system with suitable viscosity, high stability, and excellent cryoprotection effect was selected.

[0049] The main reagents used are shown in Table 1.

[0050] Table 1: Reagents and sources used in screening natural deep eutectic solvent systems

[0051] (1) Preparation of NADES Accurately weigh trehalose and 14 hydrogen bond acceptors (D-glucose, fructose, choline chloride, betaine, glycerol, xylitol, sorbitol, mannitol, glycine, tryptophan, serine, alanine, lysine, and proline) according to the molar ratio in Table 2, mix them, and grind them thoroughly in a mortar for 5 min. Transfer the mixture to sample vials and add 10%, 20%, 30%, 40%, and 50% (w / w) pure water in gradients, respectively. After sealing, place the vials in a constant temperature magnetic stirring water bath (ISJ-4A, Changzhou Aohua Instrument Co., Ltd., China) at 90℃ and 1000 rpm for 2 h until a uniform and transparent liquid is formed. The synthesized substances were named as follows: trehalose-glucose (Tre-Glu), trehalose-fructose (Tre-Fru), trehalose-choline chloride (Tre-ChCl), trehalose-betaine (Tre-Bet), trehalose-glycerol (Tre-Glyc), trehalose-xylitol (Tre-Xyl), trehalose-sorbitol (Tre-Sor), trehalose-mannitol (Tre-Mann), trehalose-glycine (Tre-Gly), trehalose-tryptophan (Tre-Try), trehalose-serine (Tre-Ser), trehalose-alanine (Tre-Ala), trehalose-lysine (Tre-Lys), and trehalose-proline (Tre-Pro).

[0052] Table 2: Monomer components and their molar ratios for preparing NADES

[0053] (2) Macroscopic photography and observation by polarized light microscope (POM) Macroscopic images of NADES were taken using the built-in digital camera of a smartphone (Honor Magic6) and collected in JPG format, with a size of 4096×3072 pixels. The smartphone's camera settings were as follows: Shooting mode (Professional Shooting), ISO (200), shutter speed (1 / 320 s), aperture (1.9), focal length (54 mm), magnification (2×). The images were captured in a camera box (40×40×40 cm). 3 The process was carried out by placing a sample vial containing NADES at a 45° angle on a black acrylic plate, with the camera positioned approximately 10 ± 1 cm apart for taking pictures.

[0054] The presence of crystals in the NADES system was observed at 50x magnification using a polarized light microscope (CX43MP, Suzhou Omit Optoelectronics Technology Co., Ltd., China).

[0055] (3) pH measurement The pH of NADES was measured at room temperature using a pH meter (PHS-25, Shanghai Yitian Scientific Instruments Co., Ltd., China). The pH meter was calibrated with a standard buffer solution before each measurement to ensure accuracy.

[0056] (4) Observation of NADES stability NADES samples were sealed and stored at room temperature. Their morphology was observed and POM (Polymer Oxidation Membrane) was performed on days 0, 3, 6, 9, and 12 to assess storage stability.

[0057] (5) Viscosity determination The apparent viscosity (η) of NADES at 25°C was determined using a rotational viscometer (DV2T LV, Borelfeld, USA). 45 mL of NADES was placed in a 50 mL centrifuge tube, and the rotor was vertically immersed in the sample until the liquid level reached the groove mark on the rotor rod.

[0058] (8) Determination of Fourier Transform Infrared Spectroscopy (FT-IR) The attenuated total reflectance (ATR) method was used to test 10 μL liquid samples using a Fourier transform infrared spectroscopy (Nicolet iN 10 MX, Thermo Fisher Scientific, USA), while the potassium bromide pellet method was used to test solid samples. The analytes were measured at wavelengths between 4000 and 400 cm⁻¹. -1 The FTIR spectrum within the range was set with 16 scan stacks and a spectral resolution of 4 cm⁻¹. -1 To improve the signal-to-noise ratio, infrared spectra of all samples were acquired at room temperature. The signal from pure KBr pellets was used as the background spectrum, and the background spectrum was subtracted using OPUS 8.0 software to obtain the FTIR spectra of NADES. The wavelengths were located between 3700 and 3000 cm⁻¹. -1 The hydroxyl stretching vibration frequency within the range is used to evaluate the hydrogen bond strength in the NADES system.

[0059] (9) Determination of low-temperature thermodynamic behavior The low-temperature thermodynamic behavior of NADES was studied using differential scanning calorimetry (DSC3, Mettler Toledo, Switzerland). Approximately 10.0 mg of NADES was encapsulated in a standard aluminum crucible, and the heat flux change of NADES between 25 and -80 °C was measured. Under a nitrogen atmosphere, the initial equilibrium temperature was 25 °C, and the temperature was lowered to -80 °C at a cooling rate of 10 °C / min, held at that temperature for 10 min, and then raised to 25 °C at a heating rate of 10 °C / min. The low-temperature thermodynamic behavior was analyzed by plotting the heat flux curve per unit mass of NADES, and the glass transition temperature was determined. T g ).

[0060] 2. Screening of NADES addition amount (1) Preparation of shrimp paste samples Shrimp samples were euthanized using crushed ice, decapitated, and the heads and tails removed. The shrimp meat was ground into a homogeneous paste using a blender at 4°C, and then rinsed three times with ice water containing 0.15% sodium chloride. The shrimp paste and ice water were mixed in a 1:5 ratio, gently stirred, and washed for 15 minutes. It was then placed in a filter cloth and centrifuged at 7000 × g for 20 minutes to dehydrate. The moisture content of the centrifuged shrimp paste was adjusted to 78–80% using cold distilled water. Finally, it was air-shredded and salted with 1.5% sodium chloride.

[0061] The shrimp paste was divided into 6 groups. Negative control (CC): no cryoprotectant. Positive control (SS): 4% sucrose + 4% sorbitol. The remaining 4 groups were treated with different mass fractions of NADES (2%, 4%, 6%, and 8%). The samples were frozen at -18℃ for 7 days, then thawed at 4℃ for 12 hours. The first freeze-thaw cycle (FT) was completed when the core temperature reached 0–4℃. This freeze-thaw process was repeated 7 times, denoted as FT7.

[0062] (2) Determination of thawing loss Weigh the sample before thawing and record the weight as ( ). W 0), then place the frozen sample in a 4℃ freezer to thaw for 12 hours until the core temperature of the shrimp paste reaches 4℃. After wiping the surface moisture of the shrimp paste with paper, weigh it and record it as ( W 1). Thawing losses are calculated according to Formula 1:

[0063] In the formula, W 0 refers to the weight of the shrimp paste before thawing (g); W 1 refers to the weight of the thawed shrimp paste (g).

[0064] (3) Measurement of water holding capacity Weigh out 5 g ( W 2) After thawing, wrap the shrimp paste in three layers of filter paper and place it in a 50 mL centrifuge tube. Incubate at 4°C with centrifugation at 5000× g Centrifuge for 15 minutes. After centrifugation, remove the sample, remove the filter paper, and weigh it. W 3. Water-holding capacity is calculated according to formula 2:

[0065] In the formula, W 2 refers to the weight of the shrimp paste before centrifugation (g); W 3. Weight of shrimp paste after centrifugation (g).

[0066] (4) Sensory evaluation measurement After thawing frozen shrimp paste, it was stuffed into plastic sausage casings with a diameter of 32 mm and a length of 15 cm, and the ends were tied tightly with string. The shrimp sausages underwent two main heating stages: gelling at 40°C for 60 min, and then gelling at 90°C for 30 min. The heat-treated shrimp sausages were then placed in a 4°C refrigerator overnight.

[0067] The sensory evaluation protocol employed a rigorously trained panel of 10 evaluators, consisting of 5 men and 5 women, aged 18–25 years. Cooked shrimp gut samples, after freeze-thaw cycles, were evaluated using a standardized 10-point scale, according to Table 3. After being stuffed with shrimp paste and cooked, the samples were consumed in partitioned compartments under controlled lighting conditions (1000 lux simulated fluorescent bulbs). Random coding and taste-cleaning procedures (mineral water) were implemented between evaluations to ensure impartial scoring and methodological repeatability consistent with international sensory analysis standards for aquatic products.

[0068] Table 3: Sensory Evaluation Table

[0069] II. Experimental Results 1. Screening of different hydrogen bond acceptors for NADES (1) Analysis of the epimorphic and micromorphic morphology of NADESs NADES were prepared by mixing trehalose with 15 different hydrogen bond acceptors at different water contents (10%, 20%, 30%, 40%, 50%, w / w) for screening. The experimental results are as follows: Figure 1 As shown, the screening results are as follows: Under water content conditions of 20%–50% (w / w), the Tre-Xyl, Tre-Glu, Tre-Sor, Tre-Fru, Tre-Bet, and Tre-Glyc systems can form homogeneous and transparent liquids, exhibiting isotropic dark-field behavior under polarized light microscopy with no crystallization, indicating successful NADES preparation. Within a water content range of 10%–50% (w / w), the Tre-Lys, Tre-ChCl, and Tre-Pro systems with full water content gradients can all be successfully prepared, exhibiting homogeneity and transparency with no crystal precipitation under polarized light microscopy. However, all water content gradient systems of Tre-Man, Tre-Gly, Tre-Trp, Tre-Ser, and Tre-Ala were not successfully prepared; the systems appeared turbid, and different crystal morphologies could be observed under polarized light microscopy.

[0070] In summary, the effective NADES preparation systems are: Tre-Xyl, Tre-Glu, Tre-Sor, Tre-Fru, Tre-Bet, and Tre-Glyc systems with a water content of 20%–50%, and Tre-Lys, Tre-ChCl, and Tre-Pro systems with a water content of 10%–50%. pH values ​​of these effective systems will be determined subsequently.

[0071] (2) pH analysis of NADESs The pH of aquatic products is approximately between 6.50 and 8.00. To evaluate the potential application of trehalose-based NADES in frozen shrimp paste, the pH value at which NADES was successfully prepared was systematically analyzed. Experimental results are as follows: Figure 2 As shown, the systems ultimately selected that meet the application pH range (6.50–8.00) for shrimp paste include 40% Tre-Glu, 50% Tre-Glu, 50% Tre-ChCl, 50% Tre-Glyc, 20%–50% Tre-Bet, 10%–50% Tre-Pro, and 20%–50% Tre-Xyl. Therefore, the storage stability and properties of these systems will be analyzed and characterized next.

[0072] (3) Stability analysis of NADESs The NADES system, conforming to the pH range (6.66–8.10) for shrimp paste application, was subjected to a 12-day storage stability observation at 25°C to evaluate its physical stability as an antifreeze agent from both macroscopic and microscopic perspectives. The experimental results are as follows: Figure 3 As shown, after 12 days of storage, the 20%–50% Tre-Pro, 30%–50% Tre-Bet, and 50% Tre-ChCl systems were stable, with no crystallization observed throughout the storage period. The 40%–50% Tre-Glu, 10% Tre-Pro, and 20% Tre-Bet systems were unstable, showing crystallization after 12 days of storage; the 40%–50% Tre-Glu, 10% Tre-Pro, and 20% Tre-Bet systems were also unstable, all showing crystallization after 12 days of storage. Ultimately, the systems exhibiting good storage stability were 50% Tre-ChCl, 50% Tre-Glyc, 30%–50% Tre-Bet, 20%–50% Tre-Pro, and 20%–50% Tre-Xyl. The properties of these systems will be characterized subsequently.

[0073] (4) Viscosity analysis of NADESs Experimental results are as follows Figure 4As shown, under the same water content, the Tre-Pro system exhibits the highest viscosity and the best structural stability, while the Tre-ChCl system has the lowest viscosity and the weakest structural binding ability. With increasing water content, the viscosity of all NADES systems shows a decreasing trend. Based on the comprehensive viscosity performance characterization results, the 10% Tre-Pro, 20% Tre-Pro, 20% Tre-Bet, and 20% Tre-Xyl systems have a denser hydrogen bond network structure and stronger water molecule binding ability, effectively inhibiting the migration of free water in the system. These systems are suitable for food freezing systems and represent the preferred screening groups for antifreeze agents.

[0074] (5) Fourier transform infrared spectroscopy analysis of NADESs The above characterization revealed that four systems—10% Tre-Pro, 20% Tre-Pro, 20% Tre-Bet, and 20% Tre-Xyl—have stronger water molecule binding capabilities and stronger hydrogen bond networks. However, due to the high viscosity of 10% Tre-Pro, high-viscosity NADES is limited in practical applications. Therefore, the individual NADES types 20% Tre-Pro, 20% Tre-Bet, and 20% Tre-Xyl will be characterized next. To avoid interference from the -OH groups in H2O, D2O was used instead of H2O to more clearly observe the hydrogen bond vibration signals.

[0075] like Figure 5 As shown, the OD vibration peak is located at 2500 cm⁻¹. -1 Nearby. Based on NADESs and its monomeric components at 4000–400 cm⁻¹ -1 Infrared spectroscopy within the specified wavelength range revealed that no new characteristic peaks were generated in the NADESs system compared to the monomeric components, indicating that no chemical reaction occurred during its synthesis; only changes in peak intensity and position were observed. The hydrogen bond strengths of the various preferred systems differed, with the order being: 20% Tre-Pro > 20% Tre-Xyl > 20% Tre-Bet. The 20% Tre-Pro system exhibited the strongest hydrogen bond strength and is more suitable for applications in antifreeze technology.

[0076] (6) DSC analysis of NADESs like Figure 6 The results show the thermal behavior of different NADES in the temperature range of 25℃ to -80℃. All three NADES exhibit significant glass transition behavior. T g Between -36.38 and -50.41°C, 20% of Tre-Pro... T g The highest is Tre-Xyl, followed by Tre-Bet at 20%. Tg Lowest, Highest T g The easier it is for a substance to enter the glassy state at normal freezing temperatures, the stronger the movement of molecules and water is inhibited, thus more effectively suppressing ice crystal growth and recrystallization, resulting in better freeze protection. Therefore, 20% Tre-Pro exhibits the superior theoretical freeze protection performance and glassy stability among the three systems.

[0077] Among all systems, the 20% Tre-Pro system stands out: its infrared spectrum exhibits the largest redshift of the OH peak, confirming the strongest intermolecular hydrogen bond interactions; simultaneously, it possesses low water activity, suitable viscosity, and a short LF-NMR transverse relaxation time, indicating the formation of the densest and most water-binding three-dimensional hydrogen bond network within it. This system also exhibits the highest glass transition temperature (…). T g It exhibits the best antifreeze properties and glassy stability. In summary, 20% Tre-Pro demonstrates outstanding overall performance and has the potential to be used as a cryoprotectant for shrimp paste.

[0078] 2. Screening of NADES addition amount (1) Effect of NADES addition on thawing loss of frozen and thawed shrimp paste The loss from thawing shrimp paste is as follows: Figure 7 As shown in the diagram, there was no significant difference in thawing loss among the groups during the first freeze-thaw cycle. From the third freeze-thaw cycle onwards, significant differences in thawing loss appeared between the different treatments. After seven freeze-thaw cycles, the thawing loss of all groups increased significantly. The CC group consistently maintained the highest thawing loss throughout the entire freeze-thaw cycle. After seven freeze-thaw cycles, with increasing NADES dosage, the thawing loss of shrimp paste showed a trend of first decreasing and then increasing, with the 6% NADES group exhibiting the lowest thawing loss. Therefore, 6% NADES was the most effective in delaying the thawing loss of shrimp paste during freeze-thaw cycles.

[0079] (2) Effect of NADES addition on water-holding capacity of frozen-thawed shrimp paste like Figure 8 As shown, the water-holding capacity of all treatment groups decreased significantly with increasing freeze-thaw cycles. The CC group consistently maintained the lowest water-holding capacity. Compared to the SS group, the CC group and the 2% NADES addition group had significantly lower water-holding capacity than the SS group, while the 6% NADES addition group had significantly higher water-holding capacity than the SS group. With increasing NADES dosage, water-holding capacity initially increased and then decreased, with the 6% NADES group exhibiting the highest water-holding capacity. Therefore, 6% NADES is the most effective in maintaining the water-holding capacity of freeze-thawed shrimp paste.

[0080] (3) Effect of NADES addition on sensory evaluation of frozen and thawed shrimp paste like Figure 9 As shown, increasing the number of freeze-thaw cycles continuously reduces the sensory quality of the shrimp paste. The blank CC group had the lowest overall scores in color, texture, mouthfeel, and flavor, indicating the most severe deterioration in sensory quality. The overall sensory quality of the shrimp paste in all NADES-treated groups was superior to the blank CC group, and generally superior to or close to the conventional antifreeze agent SS group. The 6% NADES-added group exhibited the best mouthfeel and the highest overall sensory acceptability, effectively maintaining the edible quality of the shrimp paste during freeze-thaw cycles, resulting in the best overall sensory performance.

[0081] Among all addition levels, 6% NADES showed the best treatment effect, with the lowest thawing loss, highest water retention, and highest sensory score. Low addition levels (2%) and 4% had limited protective effects, while high addition levels (8%) caused excessive protein cross-linking, damaging the tissue structure and leading to quality deterioration. In summary, 6% NADES can effectively delay the deterioration of shrimp paste quality, providing better protection than traditional commercial antifreeze agents, and has good application prospects in the field of aquatic product antifreeze.

[0082] Example 2: Effect of natural deep eutectic solvent on the quality of frozen shrimp paste I. Experimental Methods 1. Preparation of NADES The NADES system with 20% Tre-Pro selected in Example 1 was prepared using the following method: Weigh trehalose and proline according to a molar ratio of 3:8, mix them in a mortar and grind them thoroughly for 5 min. Transfer the mixture to a sample bottle, add 20% (w / w) pure water, seal it, and place it in a constant temperature magnetic stirring water bath at 90 ℃ and 1000 rpm for 2 h until a uniform and transparent liquid is formed.

[0083] 2. Preparation of shrimp paste samples The shrimp paste was divided into 6 groups. CC group: no cryoprotectant; SS group: commercial cryoprotectant (4% sucrose + 4% sorbitol); NADES group: 6% NADES; TP group: 6% trehalose and proline physical mixture (trehalose:proline molar ratio 3:8); Tre group: 6% trehalose; Pro group: 6% proline. The samples were frozen at -18 ℃ for 7 days, then thawed at 4 ℃ for 12 h. The first freeze-thaw cycle was completed when the core temperature was between 0 and 4 ℃. This freeze-thaw process was repeated 7 times, denoted as FT7.

[0084] 3. Determination of thawing loss Same as Example 1.

[0085] 4. Measurement of water-holding capacity Same as Example 1.

[0086] 5. Sensory evaluation measurement Same as Example 1.

[0087] II. Experimental Results 1. The effect of different antifreeze agents on the thawing loss of frozen and thawed shrimp paste like Figure 10 As shown, the thawing loss of each group increased continuously with the number of freeze-thaw cycles. Throughout the entire freeze-thaw cycle, the thawing loss of the CC group was significantly higher than that of the other treatment groups. After 7 freeze-thaw cycles, the thawing loss of the TP group was significantly lower than that of the Tre and Pro groups, while the thawing loss of NADES was the lowest. Therefore, NADES treatment can effectively reduce the thawing loss of frozen-thawed shrimp paste.

[0088] 2. Effects of different antifreeze agents on the water-holding capacity of frozen and thawed shrimp paste like Figure 11 As shown, the water-holding capacity of all groups decreased with increasing freeze-thaw cycles. After 7 freeze-thaw cycles, the water-holding capacity of each antifreeze group was significantly higher than that of the CC group. Compared with the SS group, the CC group had lower water-holding capacity, while NADES, Tre, Pro, and TP all had higher water-holding capacity. Among them, the NADES group had the highest water-holding capacity, indicating that NADES can minimize the mechanical damage of ice crystals to myofibril structures and effectively inhibit the unfolding of myofibril protein structures, thus inhibiting the decline in water-holding capacity. In summary, NADES has the best effect on inhibiting the decrease in water-holding capacity.

[0089] 9. Effects of different cryoprotectants on sensory evaluation of frozen and thawed shrimp paste like Figure 12 As shown, with the continuous increase in the number of freeze-thaw cycles, the sensory scores of all treatment groups showed a gradual downward trend, indicating that the problems of muscle protein denaturation, juice loss, and flavor deterioration caused by repeated freeze-thaw cycles continued to worsen. Regarding color, the CC group scored the lowest, while the NADES group scored the highest. This may be because the ice crystals generated by repeated freeze-thaw cycles damage the muscle fiber structure, deteriorate the gel network structure, leading to water loss, reduced surface brightness of the shrimp paste gel, and severe oxidation of proteins and fats, resulting in a yellowish and dull color. In terms of texture and structure, the NADES group scored the highest. Regarding taste and aroma, the shrimp paste gel with added NADES had a better taste. These results indicate that the shrimp paste gel with added NADES had the highest acceptability score and the best overall sensory score.

[0090] Example 3: NADES combined with static magnetic field inhibits protein denaturation in aquatic products I. Experimental Methods 1. Processing of shrimp paste samples The preparation of the shrimp paste sample was the same as in Example 1.

[0091] The shrimp paste was divided into 6 processing groups.

[0092] (1) AF: Place the processed shrimp paste in a regular freezer at -35℃ and freeze until the center temperature of the shrimp paste reaches -35℃; (2) IF group: The shrimp paste was frozen at -35°C and 0 mT using MF equipment. The shrimp paste was placed on a freezer rack and frozen until the geometric center temperature of the shrimp paste reached -35°C. (3) MF: The shrimp paste was frozen at -35°C and 60 mT magnetic field strength using an MF device. The shrimp paste was placed on a freezer rack and frozen until the geometric center temperature of the shrimp paste reached -35°C. (4) AF-NADES: Add 6% NADES to the shrimp paste, freeze the shrimp paste in a regular refrigerator at -35°C until the center temperature of the shrimp paste reaches -35°C, and then take it out. (5) IF-NADES: Add 6% NADES to the shrimp paste and freeze it using an MF device at -35°C and 0 mT magnetic field strength. The shrimp paste is placed on a freezer rack and frozen until the geometric center temperature of the shrimp paste reaches -35°C. (6) MF-NADES: Add 6% NADES to the shrimp paste. MF: Freeze the shrimp paste at -35°C and 60 mT magnetic field strength using an MF device. The shrimp paste is placed on a freezer rack and frozen until the geometric center temperature of the shrimp paste reaches -35°C.

[0093] All samples that were not frozen were designated as FT0. After freezing, the samples were placed in a -18°C freezer for 24 hours, and then thawed at 4°C for 12 hours. The first FT cycle was completed when the core temperature was between 0 and 4°C. This freeze-thaw process was repeated 7 times and designated as FT7.

[0094] 2. Extraction of shrimp myofibrillar protein Place approximately 50 g of shrimp paste into a blender, then add 4 times the volume of pre-cooled phosphate-buffered saline (PBS, 20 mM, 0.1 M NaCl, pH=7.0) to the homogenizer and homogenize for 30 s. Centrifuge the mixture (4℃, 8000 × 10⁻⁶) g Centrifuge for 15 min). After centrifugation, mix the precipitate with four volumes of pre-cooled PBS (20 mM, 0.1 M NaCl, pH=7.0) and centrifuge again (4℃, 8000 × 10⁻⁶). g (15 min). Then, wash the precipitate with 4 volumes of pre-cooled NaCl solution (0.1 M) and centrifuge (4℃, 8000 × 10⁻⁶). gThe extraction was repeated twice (15 min each time). Finally, the precipitate was washed with NaCl solution (0.1 M) and filtered through four layers of gauze. The filtrate was centrifuged under the same conditions, and the resulting paste was myofibrillar protein (MP). During MP extraction, the temperature was maintained at 3–5 °C. Protein concentration was determined using biuret reagent, with bovine serum albumin (BSA) as the standard.

[0095] 3. Determination of solubility Centrifuge the MP solution (1.0 mg / mL) in a centrifuge (4°C, 5000 × 10⁻⁶). g (20 min). The ratio of protein concentration in the supernatant to that before centrifugation is the solubility.

[0096] 4. Determination of circular dichroism spectrum The secondary structure of the protein was determined using circular dichroism spectroscopy. The concentration of the protein solution was adjusted to 0.1 mg / mL with pure water. 200 μL of the protein solution was placed in a 1 mm quartz cuvette. The measurement parameters were as follows: scan range 190–260 nm, bandwidth 1.0 nm, step size 1 nm, with pure water as the baseline. Secondary structure proportion analysis was performed using the instrument's built-in CDNN software, which deconvolves the CD spectrum into... α -spiral, β - Fold (Antiparallel + Parallel) β - Four secondary structures: corners, irregular curls, etc.

[0097] 5. Determination of fluorescence spectra The fluorescence spectrum of 0.1 mg / mL MP solution was measured using a fluorescence spectrophotometer with the following parameters: excitation wavelength 280 nm, scanning wavelength range 290–460 nm, sampling interval 1 nm, slit width 5 nm, scanning speed Fast, and response time Auto.

[0098] II. Experimental Results 1. Effects of NADES combined with static magnetic field-assisted freezing on myofibrillar protein solubility like Figure 13 As shown, the solubility of shrimp surimi myofibrillar protein in all treatment groups decreased with increasing freeze-thaw cycles. After 7 freeze-thaw cycles, there were significant differences in protein solubility among the groups. The AF group had the lowest solubility, the IF group's solubility was between that of the AF and MF groups, and the MF group's solubility was significantly higher than the aforementioned two groups. Compared with single freezing treatment, NADES combined with freezing treatment can effectively alleviate the decrease in shrimp surimi myofibrillar protein solubility. Among all treatment groups, the MF-NADES group had the highest shrimp surimi myofibrillar protein solubility, demonstrating the best effect in inhibiting the decrease in shrimp surimi protein solubility during freeze-thaw cycles.

[0099] 2. Effects of NADES combined with static magnetic field-assisted freezing on the circular dichroism of myofibrillar proteins like Figure 14 and 15 As shown, with increasing freeze-thaw cycles, the proportion of ordered secondary structure in shrimp surimi myofibrillar proteins decreased while the proportion of disordered secondary structure increased, leading to decreased protein secondary structure stability. After 7 freeze-thaw cycles, significant differences in protein secondary structure stability were observed among the groups. The AF group exhibited the highest degree of protein secondary structure disorder, the IF group showed better secondary structure stability than the AF group, and the MF group effectively inhibited the disordered transition of protein secondary structure, demonstrating significantly better structural stability than the AF and IF groups. Combined NADES treatment with each freezing method significantly improved the secondary structure stability of shrimp surimi myofibrillar proteins compared to the corresponding single freezing treatment groups. Among these, the MF-NADES group maximally inhibited the transition from ordered to disordered protein secondary structure, exhibiting the highest content of ordered structure and the lowest content of disordered structure, thus demonstrating the best effect in maintaining the stability of myofibrillar protein secondary structure during freeze-thaw cycles.

[0100] 3. Effects of NADES combined with static magnetic field-assisted freezing on the fluorescence intensity of myofibrillar proteins like Figure 16 As shown, with increasing freeze-thaw cycles, the fluorescence intensity of shrimp myofibrillar proteins in all treatment groups decreased, indicating a gradual impairment of the protein's tertiary structural integrity. After seven freeze-thaw cycles, significant differences in fluorescence intensity were observed among the groups. The AF group exhibited the lowest fluorescence intensity, the IF group had a higher fluorescence intensity than the AF group, and the MF group had a significantly higher fluorescence intensity than both the AF and IF groups. Combining each freezing method with NADES treatment resulted in significantly higher fluorescence intensity of shrimp myofibrillar proteins compared to their respective single-treatment groups, effectively mitigating tertiary structural damage. The MF-NADES group showed the highest fluorescence intensity, demonstrating the best effect in inhibiting the unfolding of the myofibrillar protein's tertiary structure and maintaining protein conformational stability during freeze-thaw cycles.

[0101] Example 4: NADES combined with static magnetic field to improve the water retention capacity of frozen aquatic products I. Experimental Methods 1. Processing of shrimp paste samples The preparation of the shrimp paste sample was the same as in Example 1.

[0102] The shrimp paste was grouped in the same way as in Example 3.

[0103] 2. Determination of thawing loss Same as Example 1.

[0104] 3. Measurement of water holding capacity Same as Example 1.

[0105] II. Experimental Results 1. The effect of NADES combined with static magnetic field-assisted freezing on thawing loss like Figure 17 As shown, the thawing loss of shrimp paste in all groups increased with the number of freeze-thaw cycles. Under the same freeze-thaw conditions, the thawing loss of shrimp paste in the IF and MF groups was significantly lower than that in the AF group. After adding NADES to the AF system, the thawing loss of the AF-NADES group was significantly lower than that of the AF group. Compared with the corresponding groups without added NADES, the thawing losses of the AF-NADES, IF-NADES, and MF-NADES groups were all significantly reduced. Among them, the combined treatment of the IF-NADES and MF-NADES groups showed better results in reducing thawing loss, and the MF-NADES group had the lowest thawing loss and the best overall effect in inhibiting the loss of thawed juice.

[0106] 2. The effect of NADES combined with static magnetic field-assisted freezing on water holding capacity like Figure 18 As shown, the water-holding capacity of shrimp surimi in all treatment groups continuously decreased with increasing freeze-thaw cycles. Whether at the initial freeze-thaw stage or after 7 freeze-thaw cycles, the water-holding capacity of each single-freezing group (AF, IF, MF) was significantly lower than that of the corresponding NADES combined treatment group. Combined treatment with different freezing methods using NADES effectively delayed the decline in shrimp surimi water-holding capacity. Among the single-freezing treatment groups, the AF group had the lowest water-holding capacity, the IF group had a moderate capacity, and the MF group had the highest capacity. Compared to the corresponding single-freezing groups, the AF-NADES, IF-NADES, and MF-NADES combined treatment groups all effectively maintained the water-holding capacity of the shrimp surimi. Among them, the MF-NADES group showed the best effect in maintaining the water-holding capacity of the shrimp surimi during the freeze-thaw process.

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composition, characterized in that, The composition comprises trehalose, proline and water, and the water content of the composition is 20% (w / w), and the molar ratio of trehalose to proline is 3:

8.

2. The use of the composition of claim 1 in the preparation of natural deep eutectic solvents.

3. A method for preparing a natural deep eutectic solvent, characterized in that, The natural deep eutectic solvent is prepared using the composition described in claim 1. The specific method is as follows: trehalose and proline are mixed and ground for 3-7 min, the mixture is transferred to a container, mixed with water, and stirred in a water bath at 80-100°C and 800-1200 rpm for 1.5-2.5 h until a uniform and transparent liquid is formed.

4. The application of the natural deep eutectic solvent prepared by the preparation method described in claim 3 as an antifreeze agent or cryoprotectant for aquatic products.

5. A method for freezing with a natural deep eutectic solvent combined with a static magnetic field, characterized in that, The natural deep eutectic solvent prepared by the method described in claim 3 is added to the aquatic product at an amount of 6% of the product's mass, and the product is then frozen under a static magnetic field condition of -35 to -20°C and a magnetic field strength of 20 to 80 mT.

6. The method according to claim 5, characterized in that, The magnetic field strength is 60 mT, and the temperature is -35℃.

7. The application of the method of claim 5 or 6 in inhibiting the freezing denaturation of aquatic products.

8. The application of the method described in claim 5 or 6 in improving the water retention capacity of aquatic products.

9. Aquatic products prepared using the method described in claim 5 or 6.

10. The aquatic product according to claim 9, characterized in that, The aquatic product in question is Litopenaeus vannamei.

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