Natural deep-eutectic solvent for maintaining quality of frozen penaeus vannamei boone and application of natural deep-eutectic solvent

Abstract

The invention relates to the technical field of aquatic product preservation, and discloses a natural eutectic solvent for maintaining the quality of frozen penaeus vannamei boone and application, the natural eutectic solvent is composed of choline chloride, sorbitol and water, the molar ratio of choline chloride to sorbitol is (1-5): (1-5), and the mass of water is 15-25% of the total mass of choline chloride and sorbitol. The invention also provides an application of the solvent, and the application comprises the following steps: soaking the penaeus vannamei meat in the solvent, and then freezing and storing the penaeus vannamei meat. A supramolecular network system is constructed through hydrogen-bond interaction between choline chloride and sorbitol, growth of ice crystals in the freezing storage process is inhibited, a protein structure is stabilized, and loss of unfrozen juice is reduced, so that the quality of the penaeus vannamei, such as texture, color and flavor, is maintained. The natural deep-eutectic solvent is natural and safe in component, low in heat, simple to prepare and convenient to apply, the application limitation of a traditional anti-freezing agent is solved, and the natural deep-eutectic solvent has a good industrial prospect.

Description

Technical Field

[0001] This invention relates to the field of aquatic product preservation technology, specifically to a natural eutectic solvent for maintaining the quality of frozen whiteleg shrimp and its application. Background Technology

[0002] Whiteleg shrimp are widely popular among consumers due to their high nutritional value and unique flavor. However, due to their high water content, they are prone to quality deterioration during storage and transportation caused by microbial contamination, protein denaturation, and lipid oxidation. Freezing is currently the main method for extending the shelf life of whiteleg shrimp, but prolonged freezing can lead to a series of problems. During freezing, water forms ice crystals. As storage time increases, the growth and recrystallization of these ice crystals cause irreversible mechanical damage to the shrimp's cells, resulting in significant juice loss and protein denaturation after thawing. This leads to a decline in texture and tenderness, among other quality degradation phenomena.

[0003] To address the aforementioned issues, adding antifreeze agents before freezing has become a common and effective technique in aquatic product processing. However, existing antifreeze agents have significant limitations. For example, while phosphate antifreeze agents effectively improve the water retention of products, excessive intake poses a potential health risk and does not meet current food safety requirements. Another commonly used type of antifreeze agent is sugars such as sucrose. Although they possess good antifreeze effects, their high sweetness and high calorie content limit their widespread application in the pursuit of healthy, low-sugar foods. Therefore, developing a novel antifreeze agent that can effectively maintain the quality of frozen aquatic products while also possessing natural, safe, and low-calorie properties has become an urgent technical problem to be solved in this field.

[0004] In recent years, natural eutectic solvents, as supramolecular network systems formed by hydrogen bond donors and acceptors through hydrogen bonding, have shown great potential in the field of food cryopreservation due to their green, biodegradable, and easy-to-prepare properties. However, currently, the application of natural eutectic solvents as antifreeze agents is mainly concentrated in livestock and poultry products, and research on their application in frozen aquatic products such as Litopenaeus vannamei is still insufficient, lacking an industrial solution with a simple preparation process, convenient application, and significant effects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a natural eutectic solvent for maintaining the quality of frozen whiteleg shrimp and its application. This solves the problem that existing antifreeze agents either pose safety risks (such as phosphates) or are not in line with healthy consumption trends due to their high calorie and sweetness (such as sucrose), resulting in a lack of a seafood quality maintenance solution that combines efficient freezing protection with natural, safe, and low-calorie characteristics.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei and its application.

[0007] In a first aspect, the present invention provides a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei, employing the following technical solution: A natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei shrimp, comprising choline chloride, sorbitol and water; wherein the molar ratio of choline chloride to sorbitol is (1-5):(1-5), and the mass of water is 15-25% of the total mass of choline chloride and sorbitol.

[0008] By employing the above technical solution, this invention utilizes natural, edible components to construct a eutectic solvent with unique physicochemical properties. This natural eutectic solvent can serve as a novel cryoprotectant, effectively reducing quality deterioration of Litopenaeus vannamei during frozen storage, such as moisture loss, tissue structure damage, protein denaturation, lipid oxidation, and decline in color and flavor. Its innovation and mechanism are mainly reflected in the following aspects: Excellent freeze-thaw protection: The natural eutectic solvent, through hydrogen bond donor and acceptor interactions between choline chloride, sorbitol, and water, forms a unique intermolecular network structure, lowering the freezing point of the mixture and allowing it to remain liquid at low temperatures. When Pacific white shrimp are immersed in this solvent, solvent molecules can penetrate into the shrimp tissue, replacing some free water molecules and lowering the ice crystal formation temperature and growth rate. During freezing, sorbitol, as a polyol, effectively reduces ice crystal size and minimizes mechanical damage to cell structures; simultaneously, its excellent water-holding capacity helps maintain the water content of the shrimp tissue, reducing dripping water loss during thawing. The addition of choline chloride further enhances the interaction between the solvent and biomolecules (such as proteins), stabilizing protein structure and inhibiting freeze-induced protein denaturation.

[0009] Inhibiting Protein Denaturation: During frozen storage, ice crystal formation and pH changes are the main causes of protein denaturation. The natural eutectic solvent of this invention reduces the degree of protein denaturation by decreasing ice crystal growth, stabilizing the tissue environment, and reducing the contact between proteins and ice crystals. Furthermore, the small molecules in the solvent, especially sorbitol, act as molecular chaperones, maintaining protein folding by forming hydrogen bonds or constructing a hydration layer, thus inhibiting protein aggregation and inactivation. This is crucial for maintaining the tenderness, elasticity, and water retention of shrimp meat.

[0010] Antioxidant effect: Freezing accelerates lipid oxidation, leading to off-flavors and discoloration in shrimp meat. Sorbitol, as a natural antioxidant precursor or synergist, can effectively capture free radicals and slow down the chain reaction of lipid oxidation. Simultaneously, the dense environment created by this eutectic solvent can isolate oxygen to a certain extent, further inhibiting the oxidation process and thus delaying the quality deterioration of Litopenaeus vannamei during frozen storage.

[0011] Improving Sensory Quality: Through the mechanism described above, this natural eutectic solvent can enhance the sensory quality of frozen whiteleg shrimp after thawing, including appearance, color, texture (tenderness, elasticity), and flavor. The shrimp meat retains better integrity, less juice loss, and a color and texture closer to fresh shrimp.

[0012] Preferably, the molar ratio of choline chloride to sorbitol is (1.5-2.5):(4.5-5.0).

[0013] By adopting the above technical solution, the natural eutectic solvent at the preferred molar ratio exhibits better low-temperature stability, permeability, and protective effect on shrimp meat quality. At this specific ratio, the hydrogen bond network between components reaches optimal balance, maximizing its performance as a cryoprotectant. It can penetrate into shrimp meat tissue more efficiently, providing more stable protection, while avoiding unnecessary component excess.

[0014] Preferably, the mass of the water is 18-22% of the total mass of the choline chloride and the sorbitol.

[0015] By employing the above technical solution, at an optimal water content of 18-22%, this natural eutectic solvent maximizes its cryoprotective effect while maintaining its eutectic properties, fluidity, and permeability to biological tissues. Too little water affects the solvent's solubility and fluidity, while too much water weakens its eutectic properties and cryoprotective capacity, and may even promote ice crystal formation. This optimal range achieves the best synergy between solvent performance and protective effect.

[0016] Preferably, the natural eutectic solvent is a homogeneous and transparent liquid at room temperature.

[0017] By adopting the above technical solution, this characteristic shows that the prepared solvent has good stability, uniformity and clarity, with no precipitation or phase separation, providing a stable medium for subsequent soaking treatment, ensuring the consistency and reliability of the treatment effect, and also demonstrating its excellent solubility and intermolecular interaction.

[0018] Preferably, the preparation steps of the natural eutectic solvent are as follows: choline chloride and sorbitol are mixed, water is added, and the mixture is stirred at 50-70°C until a homogeneous and transparent liquid is formed, thereby obtaining the natural eutectic solvent.

[0019] By employing the above-described technical solution, the preparation method is simple and efficient. Mild heating and stirring promote thorough mixing of the components and the formation of a stable eutectic system. A temperature range of 50-70℃ ensures rapid dissolution of the components and effective formation of hydrogen bond networks, while avoiding degradation of heat-sensitive components due to excessively high temperatures. Stirring ensures the homogeneity of the system, ultimately resulting in a uniform and transparent liquid, verifying the success of the preparation process and the stability of the solvent.

[0020] Secondly, this invention provides an application of a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei, employing the following technical solution: The application of a natural eutectic solvent for maintaining the quality of frozen whiteleg shrimp includes the following steps: soaking whiteleg shrimp meat in the natural eutectic solvent; and freezing and storing the soaked whiteleg shrimp meat.

[0021] By adopting the above technical solution, this invention provides a simple, effective, and economical method for maintaining the frozen storage quality of Litopenaeus vannamei using a natural eutectic solvent. The core of this application lies in the soaking treatment, which allows the active ingredients in the natural eutectic solvent to penetrate into the shrimp tissue, thereby playing a role in cryoprotection, anti-oxidation, and protein structure stabilization during subsequent frozen storage. This avoids the risks of directly adding chemical preservatives and meets consumers' demand for natural and healthy food. Its innovation and mechanism are as follows: Highly efficient soaking and penetration mechanism: When Litopenaeus vannamei shrimp meat is soaked in a natural eutectic solvent, choline chloride and sorbitol molecules in the solvent, utilizing their small molecular weight and hydrophilicity, effectively penetrate into the intermuscular spaces and cells of the shrimp meat through a concentration gradient. This penetration is crucial, allowing the active ingredients to directly contact water molecules, proteins, and lipids within the shrimp meat, thus fulfilling their cryoprotection and quality maintenance functions. The solvent replaces some of the free water, lowering the freezing point and reducing mechanical damage during ice crystal formation.

[0022] Synergistic Protective Effect: After soaking, sorbitol and choline chloride in the natural eutectic solvent exert a synergistic effect during the frozen storage of Litopenaeus vannamei. Sorbitol, as a polyol, can effectively bind water molecules, reduce water activity, and inhibit the growth and recrystallization of ice crystals, thereby mitigating physical damage to cell membranes and protein structures. Simultaneously, sorbitol and choline chloride act as osmotic regulators, stabilizing the intracellular and extracellular environments and reducing shrinkage and damage caused by cell dehydration. Furthermore, this solvent forms a protective film on the shrimp surface, further slowing down the freeze-drying and oxidation processes, effectively preserving the texture and color of the shrimp.

[0023] The comprehensive advantages of maintaining frozen storage quality: Through the above-mentioned soaking and subsequent frozen storage, the quality of Litopenaeus vannamei shrimp after long-term frozen storage is improved. This application not only effectively reduces protein denaturation, maintains the elasticity and tenderness of the shrimp meat, and reduces drip loss after freezing and thawing, but also delays lipid oxidation through antioxidant effects, thereby effectively maintaining the natural flavor and color of the shrimp meat. This natural, gentle, and efficient treatment method provides a sustainable solution for maintaining the quality of Litopenaeus vannamei shrimp and other aquatic products.

[0024] Preferably, the solid-liquid ratio of the shrimp meat of the whiteleg shrimp to the natural eutectic solvent is 1:(1.8-2.2) (w / v).

[0025] By adopting the above technical solution, this solid-liquid ratio ensures that the shrimp meat is fully soaked, allowing the eutectic solvent to efficiently penetrate into the shrimp meat while avoiding unnecessary solvent waste. Within the preferred range, the contact area between the solvent and the shrimp meat is maximized, and the penetration rate and depth are optimized, thereby ensuring the uniform distribution and maximum effectiveness of the cryoprotectant, which makes a good contribution to maintaining the quality of the shrimp meat.

[0026] Preferably, the soaking process parameters are: temperature 3-5℃, time 25-35 minutes.

[0027] By adopting the above technical solution, the preferred soaking temperature and time parameters were obtained through experimental optimization. A temperature range of 3-5℃ ensures both the stability and penetration rate of the eutectic solvent, while preventing microbial proliferation or autolysis of the shrimp at higher temperatures. A soaking time of 25-35 minutes ensures that the eutectic solvent fully penetrates the shrimp tissue to achieve an effective protective concentration, while avoiding excessive dehydration or solvent accumulation caused by prolonged soaking, thus achieving the optimal balance between processing efficiency and quality protection.

[0028] Preferably, the temperature for the frozen storage is between -20°C and -16°C.

[0029] By adopting the above technical solution, the preferred frozen storage temperature range is the result of combining industry standards and scientific research. Within this temperature range, microbial growth is effectively inhibited, enzyme activity is reduced, and the rate of lipid oxidation is slowed down. Compared with higher or lower temperatures, -20℃ to -16℃ achieves the best balance between energy consumption and quality preservation. Temperatures below this range lead to unnecessary energy consumption, while temperatures above this range accelerate quality deterioration; therefore, this temperature is crucial for maintaining the long-term frozen storage quality of Litopenaeus vannamei.

[0030] Preferably, during the soaking process, the mixture is stirred once every 5-15 minutes.

[0031] By employing the above-mentioned technical solution, regular stirring during the soaking process is crucial for ensuring the uniform distribution and efficient penetration of the eutectic solvent. Stirring breaks up the concentration gradient boundary layer formed on the shrimp surface, promoting contact and material exchange between the solvent and the shrimp tissue, ensuring that all shrimp are evenly exposed to the eutectic solvent. A stirring frequency of 5-15 minutes ensures uniform penetration while avoiding unnecessary physical damage to the shrimp tissue caused by frequent stirring, thereby optimizing the overall treatment effect and further improving the frozen storage quality of Litopenaeus vannamei.

[0032] This invention provides a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei and its application. It offers the following beneficial effects: 1. This invention forms a stable supramolecular network system by combining a specific hydrogen bond donor (sorbitol) and a hydrogen bond acceptor (choline chloride) at a certain molar ratio and water content. This system restricts the movement of water molecules through hydrogen bonding, effectively lowering the freezing point and inhibiting the formation and growth of large ice crystals during freezing. Therefore, this solvent can reduce the mechanical damage of ice crystals to shrimp tissue cells, effectively inhibit protein denaturation during freezing, and reduce the juice loss rate after thawing, thereby better preserving the original tenderness, elastic texture, and nutritional value of Litopenaeus vannamei.

[0033] 2. The components used in this invention are natural and safe, meeting the modern food industry's demands for clean labeling and health benefits. The hydrogen bond donor selected in this invention, sorbitol, is a sugar alcohol widely found in natural plants. Its calorific value is lower than that of traditional sugar-based antifreeze agents (such as sucrose), avoiding the problems of high calories and high sweetness in the product. The hydrogen bond acceptor, choline chloride, is a biocompatible and low-toxicity compound. Both are safe and edible substances. Therefore, the natural eutectic solvent of this invention overcomes the health risks associated with phosphate-based antifreeze agents and also solves the application limitations of sugar-based antifreeze agents such as sucrose, making the treated Litopenaeus vannamei shrimp products more competitive in the market and more easily accepted by health-conscious consumers.

[0034] 3. The natural eutectic solvent of this invention has a simple preparation process, convenient application, and good prospects for industrial application. The solvent is prepared simply by mixing the components and stirring under mild conditions, requiring no complex equipment or harsh reaction conditions. It has low production costs and is easy to scale up. Its application involves a simple soaking treatment, which can be seamlessly integrated into existing aquatic product processing lines. The operation is simple and easy to control, thus providing aquatic product processing enterprises with an economical, efficient, and easily promoted frozen storage and preservation technology solution. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the thawing loss rate of frozen shrimp. Figure 2 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the water holding capacity of frozen shrimp. Figure 3 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the pH of frozen shrimp. Figure 4 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the total volatile basic nitrogen of frozen shrimp. Figure 5 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the thiobarbituric acid value of frozen shrimp. Figure 6 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the brightness value of frozen shrimp. Figure 7 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the red-green color value of frozen shrimp. Figure 8 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the elasticity of frozen shrimp. Figure 9 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the hardness of frozen shrimp. Figure 10 This is a schematic diagram illustrating the effect of the natural eutectic solvent of this invention on the chewiness of frozen shrimp. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0038] Preparation Examples 1-5: Preparation Example 1: Preparation of the natural eutectic solvent ChS11 Choline chloride and sorbitol were mixed in a molar ratio of 1:1. Distilled water of 20% (w / w) of the total mass was added to the mixture. The mixture was then placed in a water bath at 60°C and magnetically stirred until a homogeneous, clear, and transparent liquid was formed. Heating was stopped and the mixture was cooled to room temperature to obtain the natural eutectic solvent ChS11, which was then sealed for later use.

[0039] Preparation Example 2: Preparation of natural eutectic solvent ChS23 Choline chloride and sorbitol were mixed in a molar ratio of 2:3. Distilled water of 20% (w / w) of the total mass was added to the mixture. The mixture was then placed in a 60°C water bath and magnetically stirred until a homogeneous, clear, and transparent liquid was formed. Heating was stopped and the mixture was cooled to room temperature to obtain the natural eutectic solvent ChS23, which was then sealed for later use.

[0040] Preparation Example 3: Preparation of the natural eutectic solvent ChS32 Choline chloride and sorbitol were mixed in a molar ratio of 3:2. Distilled water of 20% (w / w) of the total mass was added to the mixture. The mixture was then placed in a 60°C water bath and magnetically stirred until a homogeneous, clear, and transparent liquid was formed. Heating was stopped and the mixture was cooled to room temperature to obtain the natural eutectic solvent ChS32, which was then sealed for later use.

[0041] Preparation Example 4: Preparation of the natural eutectic solvent ChS25 Choline chloride and sorbitol were mixed in a molar ratio of 2:5. Distilled water of 20% (w / w) of the total mass was added to the mixture. The mixture was then placed in a 60°C water bath and magnetically stirred until a homogeneous, clear, and transparent liquid was formed. Heating was stopped and the mixture was cooled to room temperature to obtain the natural eutectic solvent ChS25, which was then sealed for later use.

[0042] Preparation Example 5: Preparation of natural eutectic solvent ChS52 Choline chloride and sorbitol were mixed in a molar ratio of 5:2. Distilled water of 20% (w / w) of the total mass was added to the mixture. The mixture was then placed in a water bath at 60°C and magnetically stirred until a homogeneous, clear, and transparent liquid was formed. Heating was stopped and the mixture was cooled to room temperature to obtain the natural eutectic solvent ChS52, which was then sealed for later use.

[0043] Examples 1-5: Example 1: This embodiment describes the application of the natural eutectic solvent ChS11 in maintaining the quality of frozen Litopenaeus vannamei, including the following steps: Fresh whiteleg shrimp were cleaned at 0-4℃, and after removing the head, tail and shell, whole shrimp samples of uniform size were selected. The pre-treated fresh whiteleg shrimp meat was completely immersed in the ChS11 solution prepared in Preparation Example 1, with a solid-liquid ratio of 1:2 (w / v), and soaked at 4°C for 30 minutes, stirring once every 10 minutes during the soaking period. After soaking, remove the shrimp and drain them. Then vacuum pack them and freeze them at -18°C for 60 days.

[0044] Example 2: This embodiment describes the application of the natural eutectic solvent ChS23 in maintaining the quality of frozen Litopenaeus vannamei shrimp, including the following steps: Fresh whiteleg shrimp were cleaned at 0-4℃, and after removing the head, tail and shell, whole shrimp samples of uniform size were selected. The pre-treated fresh whiteleg shrimp meat was completely immersed in the ChS23 solution prepared in Example 2, with a solid-liquid ratio of 1:2 (w / v), and soaked at 4°C for 30 minutes, stirring once every 10 minutes during the soaking period. After soaking, remove the shrimp and drain them. Then vacuum pack them and freeze them at -18°C for 60 days.

[0045] Example 3: This embodiment describes the application of the natural eutectic solvent ChS32 in maintaining the quality of frozen Litopenaeus vannamei, including the following steps: Fresh whiteleg shrimp were cleaned at 0-4℃, and after removing the head, tail and shell, whole shrimp samples of uniform size were selected. The pre-treated fresh whiteleg shrimp meat was completely immersed in the ChS32 solution prepared in Preparation Example 3, with a solid-liquid ratio of 1:2 (w / v), and soaked at 4°C for 30 minutes, stirring once every 10 minutes during the soaking period. After soaking, remove the shrimp and drain them. Then vacuum pack them and freeze them at -18°C for 60 days.

[0046] Example 4: This embodiment describes the application of the natural eutectic solvent ChS25 in maintaining the quality of frozen Litopenaeus vannamei shrimp, including the following steps: Fresh whiteleg shrimp were cleaned at 0-4℃, and after removing the head, tail and shell, whole shrimp samples of uniform size were selected. The pre-treated fresh whiteleg shrimp meat was completely immersed in the ChS25 solution prepared in Example 4, with a solid-liquid ratio of 1:2 (w / v), and soaked at 4°C for 30 minutes, stirring once every 10 minutes during the soaking period. After soaking, remove the shrimp and drain them. Then vacuum pack them and freeze them at -18°C for 60 days.

[0047] Example 5: This embodiment describes the application of the natural eutectic solvent ChS52 in maintaining the quality of frozen Litopenaeus vannamei shrimp, including the following steps: Fresh whiteleg shrimp were cleaned at 0-4℃, and after removing the head, tail and shell, whole shrimp samples of uniform size were selected. The pre-treated fresh whiteleg shrimp meat was completely immersed in the ChS52 solution prepared in Example 5, with a solid-liquid ratio of 1:2 (w / v), and soaked at 4°C for 30 minutes, stirring once every 10 minutes during the soaking period. After soaking, remove the shrimp and drain them. Then vacuum pack them and freeze them at -18°C for 60 days.

[0048] Comparative Examples 1-2: Comparative Example 1: Compared with Example 4, the only difference is that the soaking solution is replaced with an equal volume of distilled water, otherwise the same.

[0049] Comparative Example 2: Compared with Example 4, the difference is that the soaking solution is replaced with an aqueous sorbitol solution with a sorbitol concentration equivalent to that in the ChS25 solution of Example 4 (i.e., a 10% (w / v) aqueous sorbitol solution), otherwise the same.

[0050] Test Examples 1-7: Test Example 1: The effect of natural eutectic solvents on the thawing loss rate of frozen Litopenaeus vannamei. Experimental steps: Sample preparation: Following the methods of Examples 1-5 and Comparative Examples 1-2, the shrimp meat of Litopenaeus vannamei was processed and frozen.

[0051] Sampling and thawing: Frozen shrimp samples were randomly taken from each group's freezer on days 0, 10, 20, 30, 40, 50, and 60 of the frozen storage period. Three parallel samples were taken from each sampling point for each treatment group. The frozen samples were thawed in a 4°C freezer for 12 hours or at room temperature (25°C) for 2 hours until the core temperature of the sample reached 4°C.

[0052] Weighing and Calculation: After thawing, gently blot the surface moisture of the sample with filter paper and weigh the thawed shrimp. Record the initial weight of the shrimp meat before freezing. ).

[0053] The formula for calculating the thawing loss rate is as follows: %.

[0054] Data analysis: Statistical analysis was performed on the obtained thawing loss rate data to assess the differences between different treatment groups.

[0055] Experimental data: Table 1: Changes in thawing loss rate with frozen storage time (unit: %) The results of this experiment (such as...) Figure 1 As shown in Table 1, during the 60-day frozen storage period, the thawing loss rate of Litopenaeus vannamei shrimp meat in all treatment groups showed an increasing trend. This phenomenon is due to the physical damage to muscle tissue caused by the growth and recrystallization of ice crystals during frozen storage, as well as the resulting protein denaturation and water redistribution. The thawing loss rate of the distilled water treatment group was higher than that of the other treatment groups, reaching 12.50% at the end of frozen storage, indicating that it had the weakest inhibitory effect on water loss. In contrast, the various natural eutectic solvent (NADES) treatment groups and the sorbitol treatment group of this invention could reduce the thawing loss rate to varying degrees. Among them, the shrimp meat treated with a natural eutectic solvent (ChS25) of choline chloride and sorbitol in a molar ratio of 2:5 had the lowest thawing loss rate, only 9.21% after 60 days of frozen storage, which was better than the distilled water group and all other treatment groups (p<0.05, where p<0.05 indicates that the observed difference was less than 5% due to random factors). The results indicate that the ChS25 system has the most significant inhibitory effect on moisture loss during shrimp thawing.

[0056] The mechanism behind this superior effect lies in the better molar ratio of choline chloride to sorbitol in the ChS25 system, which forms a dense hydrogen bond network between them. This hydrogen bond network effectively binds free water molecules within the shrimp tissue, promoting their conversion to bound water, thereby reducing the formation and growth of ice crystals during frozen storage, and particularly inhibiting the mechanical damage to muscle fiber structures caused by large ice crystals. Simultaneously, the improved surface tension characteristics further enhance its ability to restrict water molecule migration, ultimately reducing the loss of thawing juices due to cell structure damage and water distribution imbalance, thus maintaining the quality of frozen shrimp.

[0057] Test Example 2: The effect of natural eutectic solvents on the water holding capacity of frozen Litopenaeus vannamei shrimp Experimental steps: The shrimp meat samples of Litopenaeus vannamei that had been frozen and stored (prepared according to Examples 1-5 and Comparative Examples 1-2) were removed from a -18°C freezer and thawed at room temperature (25±1°C) for 2 hours. Approximately 2g of the thawed shrimp meat sample was taken and its mass was accurately weighed. Then, place it in a 50 mL centrifuge tube and centrifuge at 5000 rpm for 15 minutes. After centrifugation, discard the supernatant, blot the surface of the precipitate with filter paper, and accurately weigh the precipitate again. ).

[0058] The formula for calculating water holding capacity (%) is as follows: %.

[0059] Experimental data: Table 2: Effects of natural eutectic solvents on water holding capacity of Litopenaeus vannamei during frozen storage The results of this experiment (such as...) Figure 2 As shown in Table 2, the water holding capacity of Litopenaeus vannamei shrimp meat decreased in all treatment groups during the entire 60-day frozen storage period. This phenomenon is mainly related to the damage to muscle tissue and functional changes in proteins during frozen storage, including myofibril degradation and contraction induced by endogenous proteases, leading to increased intermuscular gaps and water loss, as well as the destruction of muscle structural integrity caused by protein and fat decomposition under the action of microorganisms and enzymes. In the initial stage, the water holding capacity of fresh shrimp muscle was 95.56%. After 60 days of frozen storage, the water holding capacity of the distilled water treatment group decreased most significantly, dropping to 88.00%, indicating that it had the worst effect on maintaining the water holding capacity of shrimp meat. In contrast, the sorbitol treatment group and the various natural eutectic solvent (NADES) treatment groups all showed varying degrees of water holding capacity maintenance effect. Among them, the shrimp treated with a natural eutectic solvent (ChS25) of choline chloride and sorbitol in a molar ratio of 2:5 showed the slowest decrease in water holding capacity, which remained at 91.79% at the end of frozen storage, which was better than all other treatment groups (p<0.05, where p<0.05 means that the observed difference was less than 5% due to random factors).

[0060] The mechanism behind this superior effect lies in the stable hydrogen bond network formed between choline chloride and sorbitol in the ChS25 system. This network structure can interact strongly with water molecules through hydrogen bonds, effectively fixing water molecules and reducing free water content, thereby inhibiting cell damage and water loss caused by ice crystal growth during freezing. Furthermore, the viscosity characteristics of the ChS25 system further hinder the diffusion and evaporation of water molecules, working together to maintain the structural integrity of shrimp muscle tissue, thus effectively delaying the decline in water retention.

[0061] Test Example 3: Effect of natural eutectic solvents on pH value of frozen Litopenaeus vannamei. Experimental steps: Shrimp meat samples of Litopenaeus vannamei that had been frozen and stored (prepared according to Examples 1-5 and Comparative Examples 1-2) were removed from a -18°C freezer and thawed at room temperature (25±1°C) for 2 hours. Approximately 5g of the thawed shrimp meat sample was taken, added to 45mL of distilled water, and homogenized using a homogenizer at 10000 rpm for 1 minute to prepare a shrimp meat homogenate. The pH value of the shrimp meat homogenate was measured using an FG2 portable pH meter. The pH meter was calibrated with a standard buffer solution before each measurement. Three replicates were set up for each treatment group.

[0062] Experimental data: Table 3: Effects of natural eutectic solvents on pH value of Litopenaeus vannamei during frozen storage The results of this experiment (such as...) Figure 3 As shown in Table 3, the pH value of Litopenaeus vannamei shrimp meat during frozen storage exhibited a trend of first decreasing and then increasing. In the early stage of frozen storage (0-20 days), the pH value of all groups decreased significantly. This was mainly attributed to the production of lactic acid from the anaerobic respiration of microorganisms and the generation of acidic substances from lipid and carbohydrate degradation. During this stage, the pH value of the ChS25 treatment group decreased to 6.72, while that of the distilled water group decreased to 6.62. The ChS25 group showed a smaller decrease in pH value compared to other treatment groups. This is because the ChS25 system can better inhibit the metabolic activity of microorganisms, thereby reducing their lactic acid production.

[0063] After 20 days of frozen storage, the pH value continued to rise, mainly due to protein oxidative denaturation and the production of alkaline compounds, such as ammonia, by microbial degradation. By day 60 of frozen storage, the pH value of the distilled water group reached 7.25, higher than all other treatment groups (p<0.05, where p<0.05 indicates that the observed difference was less than 5% likely due to random factors). The ChS25 treatment group showed the slowest pH increase, reaching only 7.07 at the end of frozen storage. The sorbitol group had a pH value of 7.19, higher than the ChS25 group, indicating that sorbitol alone was less effective at inhibiting pH increases than the ChS25 system.

[0064] These results indicate that the ChS25 system most effectively maintains the chemical stability of shrimp throughout the entire frozen storage period. Its mechanism of action includes: the sorbitol component effectively inhibits the metabolic activity of spoilage microorganisms by reducing water activity, thereby reducing the production of alkaline substances; simultaneously, the specific hydrogen bond network system of 2:5 choline chloride and sorbitol NADES can better inhibit the freeze-drying deformation and lipid oxidation of muscle proteins, reducing the accumulation of protein degradation products and lipid oxidation products, thus ensuring the relative stability of the pH value of shrimp during frozen storage and maintaining its quality.

[0065] Test Example 4: Effect of natural eutectic solvents on total volatile basic nitrogen in frozen Litopenaeus vannamei. Experimental steps: The determination of nitrogen was performed using a fully automated Kjeldahl nitrogen analyzer based on the semi-micro Kjeldahl method. After thawing frozen samples of Litopenaeus vannamei (prepared according to Examples 1-5 and Comparative Examples 1-2), 10 g of the homogenized sample was placed in a distillation reaction tube, and 5 mL of magnesium oxide solution (10 g / L) was added. Steam distillation was immediately initiated, and the distillate was collected in a receiving flask containing 20 mL of boric acid solution (20 g / L) and a mixed indicator (methyl red and bromocresol green). Distillation was stopped when the collected liquid volume reached 150 mL. The distillate was titrated with 0.01 mol / L hydrochloric acid standard solution until the solution changed from blue-green to gray-red. A reagent blank test was performed simultaneously.

[0066] Total volatile basic nitrogen ( The formula for calculating the content is as follows: % in, The volume of hydrochloric acid standard solution consumed by the sample is expressed in mL. The volume of hydrochloric acid standard solution consumed in the blank test, in mL; This represents the molar concentration of the hydrochloric acid standard solution, expressed in mol / L. The sample mass is expressed in grams.

[0067] Experimental data: Table 4: Effects of natural eutectic solvents on total volatile basic nitrogen in Litopenaeus vannamei during frozen storage The results of this experiment (such as...) Figure 4 (As shown in Table 4) indicates that during the entire frozen storage period, the shrimp meat in each treatment group... The content increased over time, reflecting the continuous degradation of proteins into basic nitrogenous compounds under the action of endogenous enzymes and microorganisms. At the end of cryopreservation (day 60), the distilled water treatment group... The value reached 10.62 mg / 100g, which was higher than all other treatment groups, indicating that its quality deterioration was the highest.

[0068] In contrast, all NADES-treated and sorbitol-treated groups effectively inhibited... The accumulation of [something]. Among them, the sample treated with a natural eutectic solvent (ChS25) of choline chloride and sorbitol at a molar ratio of 2:5 [was effective]. The value was the lowest, only 7.80 mg / 100g, for The generated suppression effect was the most significant (p<0.05, where p<0.05 indicates that the probability of the observed difference being caused by random factors is less than 5%).

[0069] The mechanism of this inhibitory effect lies in the fact that the ChS25 system effectively reduces the release of cathepsins, thereby delaying the initial degradation of proteins. Simultaneously, the high proportion of sorbitol in the system acts as a strong hydrogen bond donor, forming a dense hydrogen bond network with water molecules, effectively binding water within the system and reducing water activity. This reduction in water activity directly inhibits the proliferation of putrefactive microorganisms and their enzymatic reaction rates, reducing the generation of protein degradation products (such as ammonia and amines) at the source. Therefore, ChS25 treatment effectively controls microbial growth and enzyme activity, thus delaying protein degradation and effectively controlling... The formation of this substance plays an important role in maintaining the quality of frozen shrimp.

[0070] Test Example 5: Effect of natural eutectic solvents on TBARS in frozen Litopenaeus vannamei Experimental steps: Thiobarbituric acid (TBA) values ​​were determined using steam distillation. After thawing frozen samples of Litopenaeus vannamei (prepared according to Examples 1-5 and Comparative Examples 1-2), 10 g of homogenized sample was taken, and 50 mL of 7.5% trichloroacetic acid (TCA) solution was added. The mixture was shaken for 30 minutes and then filtered. 5 mL of the filtrate was placed in a test tube, and 5 mL of 0.02 mol / L TBA solution was added and mixed well. The test tube was heated in a 95°C water bath for 40 minutes, and then cooled to room temperature with cold water. The absorbance was measured at 532 nm using a spectrophotometer. A reagent blank test was performed simultaneously.

[0071] TBARS values ​​are expressed as the number of milligrams of malondialdehyde (MDA) contained in each kilogram of sample (mg MDA / kg).

[0072] Experimental data: Table 5: Effects of natural eutectic solvents on TBARS values ​​of Litopenaeus vannamei during frozen storage The results of this experiment (such as...) Figure 5 As shown in Table 5, the TBARS values ​​of all treatment groups showed an increasing trend during the 60-day frozen storage period, reflecting the continuous lipid oxidation in the shrimp samples. At the end of the frozen storage period, the TBARS value of the distilled water treatment group reached the highest, at 0.44 mg MDA / kg. The TBARS values ​​of all NADES treatment groups and sorbitol treatment groups were lower than those of the distilled water group, indicating that these treatments could effectively inhibit lipid oxidation.

[0073] Among the samples treated with a natural eutectic solvent of choline chloride and sorbitol at a molar ratio of 2:5 (ChS25), the TBARS value was 0.35 mg MDA / kg, which was lower than that of the distilled water group (p<0.05, where p<0.05 indicates that the observed difference was less than 5% due to random factors), indicating that the ChS25 system had the best effect on inhibiting lipid oxidation. Meanwhile, its TBARS value was slightly lower than that of the sorbitol-treated group, the ChS11 group, and the ChS23 group, but the difference did not reach a statistically significant level.

[0074] The mechanism of this inhibitory effect lies in the multiple synergistic effects of the ChS25 system. First, the sorbitol component acts as a free radical scavenger, effectively eliminating free radicals that induce lipid oxidation. Second, the metal chelating ability of choline chloride can inhibit metal ion-catalyzed oxidation reactions. More importantly, the hydrogen bond network formed by the ChS25 component can create a physical barrier around lipid molecules, reducing the contact between oxygen and lipids, thereby lowering the rate of oxidation. Therefore, the optimal ChS25 ratio achieves the best balance between physical barrier protection, free radical scavenging, and metal ion chelation, thus exhibiting the best lipid oxidation inhibition effect.

[0075] Test Example 6: The Effect of Natural Eutectic Solvents on the Color of Frozen Litopenaeus vannamei Experimental steps: After thawing the frozen shrimp meat samples (prepared according to Examples 1-5 and Comparative Examples 1-2), the surface moisture was blotted dry with filter paper. The surface color of the second dorsal muscle of the shrimp meat was measured using a precision colorimeter. Before measurement, the instrument was calibrated using a standard white plate. The L* value (lightness), a* value (red-green hue), and b* value (yellow-blue hue) of the samples were recorded. Three parallel samples were set up for each treatment group, and three different parts of each sample were randomly selected for measurement. The results were averaged.

[0076] Experimental data: Table 6: Effects of natural eutectic solvents on L* value of Litopenaeus vannamei during frozen storage Table 7: Effects of natural eutectic solvents on the a* value of Litopenaeus vannamei during frozen storage The results of this experiment (such as...) Figure 6 , Figure 7As shown in Tables 6 and 7, the color of shrimp changed during frozen storage. The L* values ​​of all groups of shrimp showed an increasing trend (p<0.05, where p<0.05 indicates that the observed difference was less than 5% due to random factors). This is related to the destruction of muscle tissue structure caused by ice crystal formation during frozen storage, and the increase in surface free water after thawing, thus enhancing light reflection. At 60 days of frozen storage, the L* value of the distilled water group increased from 43.58 to 51.30, showing the largest increase. The L* value of the ChS25 treatment group increased the slowest, with a final value of 47.89, lower than the distilled water group and other treatment groups. This indicates that the ChS25 system can effectively inhibit ice crystal growth, reduce mechanical damage to muscle tissue, and better maintain the original tissue structure and optical properties of shrimp by binding free water through a hydrogen bond network.

[0077] Meanwhile, the a* values ​​of shrimp in all groups showed an upward trend, indicating that the shrimp color changed from greenish to reddish. This change is mainly related to the Maillard reaction and the oxidative degradation of pigments such as astaxanthin that occurred during frozen storage. At 60 days of frozen storage, the a* value of the distilled water group increased from -2.22 to 0.50, showing the most drastic change. In contrast, the a* value of the ChS25 treatment group showed the most moderate upward trend, with the endpoint value still negative (-0.16), which was better than the other groups. The mechanism of action lies in the fact that the ChS25 system can maintain the relative stability of the system's pH, while its low water activity and high viscosity jointly inhibit the progress of chemical and enzymatic reactions, thus effectively delaying color deterioration.

[0078] Furthermore, the changes in b* values ​​of shrimp in each treatment group were not significant during the study period, indicating that NADES treatment had a relatively small impact on the yellow-blue tint of shrimp. In summary, ChS25 treatment most effectively delayed the deterioration of L* and a* values ​​in frozen shrimp, demonstrating the best effect on maintaining the sensory quality of the product.

[0079] Test Example 7: Effect of natural eutectic solvents on the textural properties of frozen Litopenaeus vannamei. Experimental steps: Texture profile analysis (TPA) was performed using a texture analyzer. Shrimp meat samples of Litopenaeus vannamei (prepared according to Examples 1-5 and Comparative Examples 1-2), after being frozen and stored, were thawed at room temperature (25±1℃) for 2 hours, and surface moisture was blotted dry with filter paper. The shrimp meat samples were placed on the stage of the texture analyzer, and two compression tests were performed using a P / 5 probe. The test parameters were set as follows: pre-test velocity 1.0 mm / s, test velocity 0.5 mm / s, post-test velocity 1.0 mm / s, compression degree 50%, and trigger force 5 g. Hardness (g), elasticity (mm), and chewiness (mJ) were obtained from the TPA curves.

[0080] Experimental data: Table 8: Effect of natural eutectic solvents on the elasticity of Litopenaeus vannamei during frozen storage (mm) Table 9: Effect of natural eutectic solvents on the firmness of Litopenaeus vannamei during frozen storage (g) Table 10: Effects of natural eutectic solvents on the chewiness of Litopenaeus vannamei during frozen storage (mJ) The results of this experiment (such as...) Figure 8 , Figure 9 , Figure 10 As shown in Tables 8, 9, and 10, the textural properties of shrimp in all treatment groups decreased with the extension of the 60-day frozen storage time. The elasticity, firmness, and chewiness of the shrimp decreased, mainly due to the oxidative denaturation of proteins and the irreversible mechanical damage to muscle tissue caused by ice crystal growth.

[0081] Among all treatment groups, the sample treated with a natural eutectic solvent of choline chloride and sorbitol at a molar ratio of 2:5 (ChS25) showed the best performance in maintaining textural properties. After 60 days of frozen storage, the ChS25 group exhibited better elasticity (1.43 mm), hardness (367.60 g), and chewiness (8.60 mJ) than the distilled water group (p < 0.05, where p < 0.05 indicates that the observed difference was less than 5% due to random factors), and was also superior to all other treatment groups.

[0082] The mechanism of this protective effect lies in the unique hydrogen bond network of the ChS25 system. This network stabilizes the three-dimensional conformation of shrimp meat proteins, inhibiting their denaturation and aggregation during frozen storage. Simultaneously, it forms strong hydrogen bonds with water molecules, effectively limiting their migration and thus inhibiting ice crystal growth and recrystallization. This results in smaller and more uniformly distributed ice crystals, reducing physical damage to the myofibril structure, protecting the integrity of the myofibrils, and delaying the deterioration of elasticity, firmness, and chewiness caused by tissue structure damage. Therefore, ChS25 treatment can effectively maintain the textural quality of frozen shrimp.

Claims

1. A natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei, characterized in that, The natural eutectic solvent is composed of choline chloride, sorbitol and water; wherein the molar ratio of choline chloride to sorbitol is (1-5):(1-5), and the mass of water is 15-25% of the total mass of choline chloride and sorbitol.

2. The natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 1, characterized in that, The preferred molar ratio of choline chloride to sorbitol is (1.5-2.5):(4.5-5.0).

3. The natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 1, characterized in that, The water is preferably 18-22% of the total mass of the choline chloride and sorbitol.

4. The natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 1, characterized in that, The natural eutectic solvent is a homogeneous and transparent liquid at room temperature.

5. The natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 4, characterized in that, The natural eutectic solvent is prepared through the following steps: The natural eutectic solvent is prepared by mixing choline chloride and sorbitol, adding water, and stirring at 50-70°C until a homogeneous and transparent liquid is formed.

6. The application of the natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to any one of claims 1 to 5, characterized in that, The application includes the following steps: The shrimp meat of Litopenaeus vannamei is soaked in the aforementioned natural eutectic solvent; The soaked whiteleg shrimp meat is then frozen and stored.

7. The application of a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 6, characterized in that, The solid-liquid ratio of the shrimp meat of the whiteleg shrimp to the natural eutectic solvent is 1:(1.8-2.2) (w / v).

8. The application of a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 6, characterized in that, The soaking process parameters are as follows: Temperature 3-5℃, time 25-35 minutes.

9. The application of a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 6, characterized in that, The temperature for the frozen storage is -20°C to -16°C.

10. The application of a natural eutectic solvent for maintaining the quality of frozen Litopenaeus vannamei according to claim 6, characterized in that, During the soaking process, stir once every 5-15 minutes.

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