Method for improving stability of frozen-thawed minced fillet protein by using natural deep eutectic solvent

Abstract

The invention discloses a method for improving the protein stability of freeze-thawed minced fillet by using a natural deep eutectic solvent, and belongs to the field of food processing. NADES is prepared from citric acid and trehalose according to the molar ratio of 2: 1 and added into mirror carp minced carp (w / w = 4%), after freezing and thawing treatment, the protein myofibril fragmentation index, the TCA-dissolved peptide content and the amino nitrogen content of the minced carp minced carp are remarkably reduced, meanwhile, the nitrogen solubility index and the solubility of water-soluble protein and salt-soluble protein are improved, and the content of soluble protein is increased. Protein denaturation, degradation and aggregation are effectively inhibited. According to the invention, environment-friendly natural degradable materials are adopted, so that a new thought is provided for development of a novel green antifreeze agent while the protein stability of the frozen and thawed minced fillet is effectively improved, and a new insight is provided for freezing and fresh-keeping of the minced fillet and more types of muscle foods.

Description

Technical Field

[0001] The invention relates to a method for improving the stability of freeze-thawed surimi protein by utilizing a natural deep eutectic solvent, and belongs to the field of food processing. Background Art

[0002] Frozen surimi is popular among consumers as a raw material for the production of surimi products due to its high protein, low fat, and low cholesterol properties. However, due to imperfect cold chain technology, surimi is subject to repeated freezing and thawing during storage, transportation, and sales, causing ice crystal growth and recrystallization. The mechanical effects of ice crystals and the processing of surimi can disrupt the intact muscle tissue structure, leading to the leakage of endogenous proteases, protein degradation, and quality deterioration. Therefore, the development of ice crystal control technology is key to improving the quality of frozen surimi. Antifreeze, as a raw material for the production of frozen surimi, is economical, efficient, and convenient. However, the high sweetness of commercial antifreeze, the chronic disease risks of polyphosphates, and the high production costs of antifreeze proteins, antifreeze peptides, and polyphenols make it difficult to apply on a large scale.

[0003] In nature, certain cold-tolerant organisms produce metabolites (sugars, amino acids, organic acids, and choline derivatives). These metabolites can form hydrogen-bonded antifreeze systems in specific molar ratios, known as natural deep eutectic solvents (NADES). NADES are nontoxic, biodegradable mixtures with advantages such as low production cost, simple synthesis, and biodegradability. They also possess antioxidant, antibacterial, low or no toxicity, and high viscosity, making them a promising alternative to traditional solvents. Furthermore, NADES are highly pure and waste-free, making them suitable for direct addition to surimi as a new green solvent, a key advantage over traditional green solvents. Studies have shown that NADES can effectively maintain the structural integrity of frozen mirror carp surimi and inhibit its quality deterioration. However, studies have not yet reported on their use as antifreeze agents to maintain the integrity of surimi proteins. Therefore, the use of natural deep eutectic solvents to improve the freeze-thaw stability of surimi proteins holds great research potential. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the stability of freeze-thawed surimi protein by using NADES. This method can effectively improve the stability of freeze-thawed surimi protein and improve the quality of freeze-thawed surimi. In addition, the prepared raw materials are green, biodegradable, and environmentally friendly.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] Step 1: Preparation of natural deep eutectic solvent:

[0007] NADES consisting of citric acid and trehalose in varying molar ratios were prepared using a heat-stirring method. The mixture was heated to 60°C using a magnetic stirrer kit (IKA, Staufen, Germany) equipped with a C-MAG HS10 hotplate and stirred at a constant speed of 100 rpm until a homogeneous, transparent liquid was formed. The mixture was cooled to room temperature and stored in a storage bottle for further analysis. The optimal performance of the natural deep eutectic solvent was achieved with a molar ratio of citric acid to trehalose of 2:1.

[0008] Step 2: Preparation of freeze-thawed mirror carp minced fish:

[0009] Fresh mirror carp (1000±50g) were purchased from Harbin Haoyouduo Supermarket and anesthetized in 0.4% benzocaine solution for 20 minutes. The cleaned fish dorsal muscle was put into a meat grinder at low temperature (<10℃) and minced into mince. The minced fish was washed twice with 3 times the volume of cold water and once with 0.5% (w / v) NaCl solution. The sample was then centrifuged and minced fish with a moisture content of 80% was prepared. The minced fish was divided into 3 groups according to mass. The sample was frozen at -18℃ for 7 days and then thawed at 4℃ for 12 hours. The first freeze-thaw cycle was completed when the center temperature was 0-4℃. The above freeze-thaw process was repeated several times.

[0010] Preferably, the three groups in step 2 are: negative control: surimi without antifreeze; positive control: commercial antifreeze group (SS group); experimental group: NADES with a molar ratio of citric acid to trehalose of 2:1.

[0011] Preferably, the freeze-thaw process in step 2 is repeated 0, 1, 3, and 5 times respectively.

[0012] The present invention adds prepared NADES (citric acid:trehalose = 2:1) to mirror carp surimi (w / w = 4%) and compares the results with surimi without antifreeze and surimi with commercial antifreeze. NADES inhibits the free flow of water molecules through its strong hydrogen bond network, thereby hindering the further growth of ice crystals, effectively maintaining the integrity of myofibrillar tissue and significantly improving the freeze-thaw stability of surimi protein. The NADES raw material used in this method is environmentally friendly and has a simple preparation process. This method provides new ideas for the development of new green antifreeze agents and offers new insights into the frozen preservation of surimi and other types of muscle foods.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) Highly environmentally friendly:

[0015] The present invention uses citric acid and trehalose as raw materials of the natural deep eutectic solvent, both of which are derived from nature, and the formed natural deep eutectic solvent is non-toxic, biodegradable, and extremely environmentally friendly.

[0016] (2) It has the effect of improving the stability of freeze-thawed surimi protein:

[0017] This invention utilizes citric acid and trehalose to form a natural deep eutectic solvent, which is then applied to frozen mirror carp surimi. NADES significantly reduces myofibril fragmentation, TCA-soluble peptide content, and amino nitrogen content after the same number of freeze-thaw cycles; it also improves the nitrogen solubility index and the solubility of water-soluble and salt-soluble proteins. It exhibits excellent resistance to denaturation, degradation, and aggregation, significantly enhancing the stability of freeze-thawed surimi protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Effect of NADES on the myofibril fragmentation index of freeze-thawed surimi of Cyprinus carpio in Example 3;

[0019] Figure 2 Effect of NADES on freeze-thawed mirror carp surimi TCA-solubilized peptides in Example 3;

[0020] Figure 3 Effect of NADES on nitrogen solubility index of freeze-thawed mirror carp minced fish in Example 3;

[0021] Figure 4 Effect of NADES on the content of different soluble proteins in freeze-thawed mirror carp surimi in Example 3;

[0022] Figure 5 Effects of NADES on total nitrogen content, water-soluble nitrogen content, non-protein nitrogen content, and protein degradation index of freeze-thawed surimi in Example 3;

[0023] Figure 6 Effect of NADES on amino nitrogen content in freeze-thawed mirror carp surimi in Example 3;

[0024] Figure 7 Effect of NADES on the degradation of myofibrillar protein in freeze-thawed surimi of Cyprinus carpio;

[0025] Figure 8 Effect of NADES on the degradation of sarcoplasmic protein in freeze-thawed mirror carp minced fish in Example 3;

[0026] Figure 9 Effect of NADES on the degradation of desmin and troponin T in frozen-thawed mirror carp surimi in Example 3. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings, but the protection scope of the present invention is not limited thereto;

[0028] Example 1:

[0029] Step 1: Preparation of natural deep eutectic solvent:

[0030] NADES consisting of citric acid and trehalose in varying molar ratios were prepared using a heat-stirring method. The mixture was heated to 60°C using a magnetic stirrer kit (IKA, Staufen, Germany) equipped with a C-MAG HS10 hotplate and stirred at a constant speed of 100 rpm until a homogeneous, transparent liquid was formed. The mixture was cooled to room temperature and stored in a storage bottle for further analysis. The optimal performance of the natural deep eutectic solvent was achieved with a molar ratio of citric acid to trehalose of 2:1.

[0031] Step 2: Preparation of freeze-thawed mirror carp minced fish:

[0032] Fresh mirror carp (1000±50g) were purchased from Harbin Haoyouduo Supermarket and anesthetized in 0.4% benzocaine solution for 20 minutes. The cleaned fish dorsal muscle was put into a meat grinder at low temperature (<10℃) and minced into mince. The minced fish was washed twice with 3 times the volume of cold water and once with 0.5% (w / v) NaCl solution. The sample was then centrifuged and a minced fish with a moisture content of 80% was prepared. No antifreeze was added to the minced fish. The sample was frozen at -18℃ for 7 days and then thawed at 4℃ for 12 hours. The first freeze-thaw cycle was completed when the center temperature was 0-4℃. The above freeze-thaw process was repeated 0, 1, 3, and 5 times, respectively.

[0033] Example 2:

[0034] Step 1: Preparation of natural deep eutectic solvent:

[0035] NADES consisting of citric acid and trehalose in varying molar ratios were prepared using a heat-stirring method. The mixture was heated to 60°C using a magnetic stirrer kit (IKA, Staufen, Germany) equipped with a C-MAG HS10 hotplate and stirred at a constant speed of 100 rpm until a homogeneous, transparent liquid was formed. The mixture was cooled to room temperature and stored in a storage bottle for further analysis. The optimal performance of the natural deep eutectic solvent was achieved with a molar ratio of citric acid to trehalose of 2:1.

[0036] Step 2: Preparation of freeze-thawed mirror carp minced fish:

[0037] Fresh mirror carp (1000±50g) were purchased from Harbin Haoyouduo Supermarket and anesthetized in 0.4% benzocaine solution for 20 minutes. The cleaned fish dorsal muscle was put into a meat grinder at low temperature (<10℃) and minced into mince. The minced fish was washed twice with 3 times the volume of cold water and once with 0.5% (w / v) NaCl solution. The sample was then centrifuged and a minced fish with a moisture content of 80% was prepared. Commercial antifreeze was added to the minced fish, and the sample was frozen at -18℃ for 7 days and then thawed at 4℃ for 12 hours. The first freeze-thaw cycle was completed when the center temperature was 0-4℃. The above freeze-thaw process was repeated 0, 1, 3, and 5 times, respectively.

[0038] Example 3:

[0039] Step 1: Preparation of natural deep eutectic solvent:

[0040] NADES consisting of citric acid and trehalose in varying molar ratios were prepared using a heat-stirring method. The mixture was heated to 60°C using a magnetic stirrer kit (IKA, Staufen, Germany) equipped with a C-MAG HS10 hotplate and stirred at a constant speed of 100 rpm until a homogeneous, transparent liquid was formed. The mixture was cooled to room temperature and stored in a storage bottle for further analysis. The optimal performance of the natural deep eutectic solvent was achieved with a molar ratio of citric acid to trehalose of 2:1.

[0041] Step 2: Preparation of freeze-thawed mirror carp minced fish:

[0042] Fresh mirror carp (1000±50g) were purchased from Harbin Haoyouduo Supermarket and anesthetized in 0.4% benzocaine solution for 20 minutes. The cleaned fish dorsal muscle was placed in a meat grinder at low temperature (<10°C) and minced into mince. The minced fish was washed twice with 3 volumes of cold water and once with 0.5% (w / v) NaCl solution. The sample was then centrifuged and a minced fish with a moisture content of 80% was prepared. NADES, consisting of citric acid and trehalose in a molar ratio of 2:1, was added to the minced fish. The sample was frozen at -18°C for 7 days and then thawed at 4°C for 12 hours. The first freeze-thaw cycle was completed when the core temperature was 0-4°C. The above freeze-thaw process was repeated 0, 1, 3, and 5 times, respectively.

[0043] Example 3: Indicator test of the effect of natural deep eutectic solvent on freeze-thawed carp surimi protein degradation:

[0044] (1) Determination of myofibril fragmentation index:

[0045] Take 4g of surimi and put it into a homogenizer. Add 40mL of pre-cooled MFI buffer (0.1mol / L KCl, 11.2mmol / LK2HPO4, 8.8mmol / LKH2PO4, 1mmol / L EGTA, 1mmol / L MgCl2), homogenize at high speed, centrifuge the homogenate at 10000g, 15min, 4℃, discard the supernatant, repeat the centrifugation once, and fully resuspend the precipitate with 10mL MFI buffer. Filter the suspension through 4 layers of gauze to remove connective tissue, and then wash repeatedly with 10mL MFI buffer. The protein concentration of the filtered suspension is measured by the biuret method, and then the suspension concentration is adjusted to 0.5mg / mL. The absorbance is measured at 540nm, and the MFI value is obtained by multiplying the absorbance reading by 200.

[0046] (2) Analysis of myofibril fragmentation index results:

[0047] Effects of NADES on the myofibril fragmentation index of freeze-thawed carp minced carp Figure 1 As shown, means with the same freeze-thaw cycle are assigned different capital letters (AC) indicating significant differences (P < 0.05). Mean values ​​with the same NADES concentration are assigned different lowercase letters (ad) indicating significant differences (P < 0.05). Within the same freeze-thaw cycle, the treatment groups are as follows: control, commercial cryoprotectant, and NADES (from left to right). The same applies below. The myofibril fragmentation index (MFII) of all samples increased significantly with increasing freeze-thaw cycles (P < 0.05). This is primarily due to the continued growth of ice crystals, which disrupts the internal structure of muscle tissue and accelerates the destruction of myofibril integrity. At the same freeze-thaw cycle, the MFII of the SS and NADES-treated groups was lower than that of the uncryoprotected control group, and the MFII of the NADES-treated group was significantly lower than that of the SS-treated group (P < 0.05). The presence of carbohydrates in SS binds water molecules, delaying their formation into ice crystals. NADES, with its strong hydrogen-bonded network, inhibits the free flow of water molecules, thereby hindering the further growth of ice crystals. Both of these factors lead to a decrease in the degree of MP decomposition and better maintenance of the integrity of myofibril tissue. The lower myofibril fragmentation index indicates that NADES has better antifreeze performance than SS.

[0048] (3) Determination of TCA-soluble peptides:

[0049] 3 g of surimi was added to 27 mL of 5% TCA solution and homogenized, followed by refrigerated centrifugation (centrifugal force: 10,000 g, time: 10 min, temperature: 4° C.). The TCA-soluble peptide content in the supernatant was determined by the Folin-phenol method.

[0050] (4) TCA-dissolved peptide analysis:

[0051] Changes in TCA-soluble peptide content in frozen-thawed carp surimi after NADES treatment Figure 2 As shown, TCA stands for trichloroacetic acid. Mean values ​​for the same freeze-thaw cycles are assigned different capital letters (AC) indicating significant differences (P < 0.05), while mean values ​​for the same NADES concentration are assigned different lowercase letters (ad) indicating significant differences (P < 0.05). After five freeze-thaw cycles, the TCA-soluble peptide content of the three groups increased by 63.46%, 58.33%, and 42.00%, respectively, compared to the fresh samples (P < 0.05). During freeze-thaw cycles, protein macromolecules are degraded into low-molecular-weight peptides by endogenous enzymes. Furthermore, although low-temperature freezing inhibits microbial growth, some microorganisms are activated after thawing. The release of cellular contents, such as vitamins and minerals, caused by ice crystal growth, provides a favorable growth environment for microorganisms, particularly psychrophiles. Therefore, exogenous microbial enzymes can also play a role in degrading muscle proteins into peptides. After the same freeze-thaw treatment, the TCA-soluble peptide content of the samples with antifreeze significantly decreased. At freeze-thaw cycles of 3 and 5, the TCA-soluble peptide content of the NADES samples decreased significantly by 1.52% and 6.58%, respectively, compared to the SS samples (P < 0.05), both significantly lower than the control samples. This indicates that NADES has a significant advantage in resisting protein degradation. This is because NADES effectively inhibits the growth and recrystallization of ice crystals by trapping water molecules, thus effectively reducing the mechanical damage of ice crystals.

[0052] (5) Determination of nitrogen solubility index:

[0053] After mixing 5 g of surimi with 10 mL of distilled water, adjust the pH of the solution to 7 with 1 mol / L NaOH or HCl solution, and then centrifuge at 8000 rpm for 20 minutes at 4°C. The protein content of the supernatant after centrifugation was determined by the biuret method. The nitrogen solubility index is expressed as the percentage of supernatant protein concentration to total protein concentration.

[0054] (6) Analysis of nitrogen solubility index results:

[0055] Changes in nitrogen solubility index of freeze-thawed mirror carp surimi Figure 3Figures are shown in Figure 1. Mean values ​​for the same freeze-thaw cycle are assigned different capital letters (AC) indicating significant differences (P < 0.05). Mean values ​​for the same NADES concentration are assigned different lowercase letters (ad) indicating significant differences (P < 0.05). The nitrogen solubility index (NSI) of all samples decreased significantly with increasing freeze-thaw cycles (P < 0.05). The trend in the figure indicates a gradual decrease in protein solubility. During freeze-thaw treatment, some proteins bind to water to form ice crystals, which then precipitate. This leads to the formation of non-covalent bonds such as hydrophobic interactions and hydrogen bonds, resulting in the formation of large, insoluble aggregates, which in turn decreases their solubility. Furthermore, the decrease in NSI may be due to changes in the regular spatial structure of protein molecules caused by hydrogen, ionic, and hydrophobic bonds during freeze-thaw cycles. At the same freeze-thaw cycle, the NSI of samples treated with antifreeze was higher than that of samples without antifreeze, and the NADES sample had the highest NSI, indicating that NADES is a better choice than SS for increasing protein solubility. After adding NADES to the frozen-thawed surimi, the sugar molecules in it bind to the surface of ice crystals through interaction with water or ice to prevent the appearance of large ice crystals, effectively reduce the degree of protein denaturation, and reduce the formation of large molecules or insoluble aggregates to improve protein solubility.

[0056] (7) Determination of the content of different soluble proteins:

[0057] Take 3g of surimi, add 30mL of buffer solution A (15.6mmol / LNaHPO4, 3.5mmol / L KH2PO4, pH 7.5), homogenize (10000rpm / min, 1min), and then centrifuge at 10000r / min, 15min, 4℃. Repeat the extraction twice, and collect the supernatant twice, which is the water-soluble protein. After the second centrifugation, the precipitate obtained is mixed with buffer solution B (0.45mol / L KCl, 15.6mmol / L KH2PO4, 3.5mmol / L KH2PO4, pH 7.5), homogenize (10000rpm / min, 1min), and then centrifuge at 10000r / min, 15min, 4℃. Repeat the extraction twice, and collect the supernatant twice, which is the salt-soluble protein. The protein content of all water-soluble and salt-soluble proteins was determined using the biuret method.

[0058] (8) Analysis of different soluble protein content results:

[0059] Changes in the content of different soluble proteins in frozen and thawed carp surimi Figure 4As shown, the mean values ​​for the same freeze-thaw cycle are assigned different capital letters (AC) indicating significant differences (P < 0.05), while the mean values ​​for the same NADES concentration are assigned different lowercase letters (ad) indicating significant differences (P < 0.05). The water-soluble and salt-soluble protein contents of all samples decreased significantly with increasing freeze-thaw cycles (P < 0.05). Low temperatures during freeze-thaw cycles strongly inhibit microbial growth and reproduction, limiting the degradation of water-soluble and salt-soluble proteins by exogenous microbial enzymes. For the same freeze-thaw cycle, the water-soluble and salt-soluble protein contents of samples treated with antifreeze were higher than those of the control group without antifreeze, and the water-soluble and salt-soluble protein contents of samples in the SS group were lower than those in the NADES group. Small sugars such as sucrose and sorbitol in SS significantly inhibit the freeze-denaturation of water-soluble and salt-soluble proteins. NADES, through its strong hydrogen bond network, intercepts water molecules, restricting their migration to ice nuclei and thus preventing the formation of giant ice crystals. In addition, NADES also improves the solubility of water-soluble and salt-soluble proteins by destroying the formation of intermolecular hydrogen bonds, ionic bonds, and disulfide bonds.

[0060] (9) Determination of total nitrogen, water-soluble nitrogen, and non-protein nitrogen content:

[0061] Determination of total nitrogen content: There are three main steps: digestion, distillation, and titration. Digestion: Accurately weigh 2g of fresh meat and thawed meat samples, 15g of anhydrous potassium sulfate, 0.5g of copper sulfate pentahydrate, and 20mL of concentrated sulfuric acid in a Kjeldahl flask, mix thoroughly to soak the sample, and slowly heat until all the contents are carbonized. Switch to high temperature and heat until the liquid turns blue-green and transparent, and then heat for another 1.5 hours. The entire digestion process should not be less than 2 hours. After digestion, when the digestate cools to 40°C, add 50mL of water, mix thoroughly, and cool. Distillation: Add 50mL of boric acid solution and 4 drops of methyl red and methylene blue as a mixed indicator to the receiving flask. After the distillation apparatus is installed, start distillation and boil the digestate in the Kjeldahl flask for 30 minutes. Stop distillation when the distillate reaches 150mL. Titration: Titrate the distillate with a hydrochloric acid standard solution until the solution turns dark red, which is the titration endpoint. Record the volume of hydrochloric acid consumed. Perform a blank test at the same time. The formula for calculating total nitrogen content is as follows:

[0062]

[0063] Where: X-total nitrogen content in the sample, unit: g / 100g

[0064] 0.014-1mol / L hydrochloric acid standard solution 1mL is equivalent to the mass of nitrogen

[0065] V1-The volume of hydrochloric acid standard solution required to measure the sample, unit: mL

[0066] V0-The volume of hydrochloric acid standard solution required for blank determination, unit: mL

[0067] M-mass of the sample, unit: g

[0068] C-Concentration of hydrochloric acid standard solution, unit: mol / L

[0069] Determination of water-soluble nitrogen content: Take a certain amount of surimi and add deionized water at a ratio of 1:10. Homogenize and centrifuge (10,000 rpm, 10 minutes, 4°C). Collect the supernatant. Add 10 times the volume of deionized water to the precipitate and repeat the centrifugation. Combine the two supernatants, filter through qualitative filter paper, and determine the water-soluble nitrogen content of 10 mL of the filtrate by Kjeldahl method.

[0070] Determination of non-protein nitrogen content: Take 20 mL of the above water-soluble nitrogen extract, add an equal volume of 15% TCA solution by mass and mix evenly. After standing at room temperature for 60 minutes, centrifuge (10000r / min, 10min, 4°C) to collect the supernatant, filter with qualitative filter paper, take 10 mL of the filtrate, and determine the non-protein nitrogen content by Kjeldahl method.

[0071] (10) Analysis of total nitrogen, water-soluble nitrogen and non-protein nitrogen content:

[0072] Effects of NADES on total nitrogen content, water-soluble nitrogen content, non-protein nitrogen content and protein degradation index of freeze-thawed surimi Figure 5As shown. Mean values ​​for the same freeze-thaw cycle are assigned different capital letters (AB) indicating significant differences (P < 0.05). Mean values ​​for the same NADES concentration are assigned different lowercase letters (ad) indicating significant differences (P < 0.05). With increasing freeze-thaw cycles, the total nitrogen content and water-soluble nitrogen content of all samples decreased, while the non-protein nitrogen content and protein degradation index showed the opposite trend, indicating a gradual increase in protein degradation. During freeze-thaw cycles, MP in mirror carp surimi, particularly myosin heavy chain and actin, are degraded by endogenous proteases. This degradation, in addition to the production of numerous short peptides and amino acids, also produces nitrogen-containing volatile substances, leading to an increase in non-protein nitrogen content and a decrease in total nitrogen content. Furthermore, ice crystals formed during freeze-thaw cycles grow large enough to puncture muscle cells, causing a large amount of fluid to leak out, including nitrogen-containing substances such as water-soluble proteins. Consequently, the water-soluble nitrogen content decreases dramatically. Under the same freeze-thaw cycle, the NADES group had the lowest non-protein nitrogen content and protein degradation index, while the total nitrogen content and water-soluble nitrogen content were the highest compared to the control group, followed by the SS group, indicating that NADES has superior resistance to protein freeze-degradation. SS can stabilize proteins by competing with surface-bound water for protein through hydrogen and ionic bonds. NADES enhances the viscosity of the system through its hydrogen bond network, thereby reducing water migration and redistribution, hindering the migration and diffusion of liquid water to the surface of solid ice crystals, reducing the formation of large ice crystals, and preventing the elution of tissue proteases attached to lysosomes from the cell.

[0073] (11) Determination of amino nitrogen content:

[0074] Weigh 5g of surimi and add water to 100g of slurry. Mix thoroughly and place 20mL in a beaker. Add 60mL of water and titrate with standard sodium hydroxide solution [c(NaOH)=0.05mol / L] to a pH of 8.2. Record the volume of sodium hydroxide solution consumed. Add 10mL of formaldehyde solution, mix thoroughly, and continue titrating with sodium hydroxide solution to a pH of 9.2. Read the volume of standard sodium hydroxide solution consumed. Simultaneously, perform a blank test with 80mL of water. The amino nitrogen content is calculated as follows:

[0075]

[0076] Where: X-the content of amino acid nitrogen in the sample, unit: g / 100g

[0077] V1-The volume of sodium hydroxide solution consumed after adding formaldehyde to the test sample, unit: mL

[0078] V2-the volume of sodium hydroxide solution consumed after adding formaldehyde to the blank, unit: mL

[0079] c-Concentration of sodium hydroxide standard solution, unit: mol / L

[0080] 0.014 The mass of nitrogen equivalent to 1 mL of sodium hydroxide standard titration solution [c(NaOH) = 1.000 mol / L]

[0081] m-mass of the sample, unit: g

[0082] (12) Analysis of amino nitrogen content:

[0083] Changes in amino nitrogen content in freeze-thawed mirror carp surimi Figure 6 As shown, the mean values ​​for the same freeze-thaw cycle are assigned different capital letters (AB) to indicate significant differences (P < 0.05). The mean values ​​for the same NADES concentration are assigned different lowercase letters (ad) to indicate significant differences (P < 0.05). The amino nitrogen content of all samples increased with increasing freeze-thaw cycles, indicating that the protein in the surimi was severely degraded. Amino nitrogen is produced by protein degradation by exopeptidases, which can be produced by microorganisms or endogenous proteases in fish, particularly cathepsin B and cathepsin H. Furthermore, although microbial activity is inhibited by low temperatures, it partially recovers during thawing. Therefore, microbial proliferation can also lead to continued protein degradation in mirror carp surimi, the production of free amino acids, and a corresponding increase in amino nitrogen content. Compared to the control group, the amino nitrogen content in both groups treated with antifreeze treatment decreased significantly (P < 0.05), with the decrease being greater in the NADES group, indicating that protein degradation was effectively inhibited. NADES prevents the formation of large and irregular ice crystals by restricting the free flow of water molecules, thereby reducing the degree of mechanical damage to muscle cells and, accordingly, reducing the leakage of tissue proteases, thereby reducing the degree of protein degradation.

[0084] (13) SDS-PAGE determination:

[0085] The concentration of the separating gel and stacking gel was 12%, and the concentration of the stacking gel was 5%. The preparation method is shown in Table 1. The required solutions are: SDS-PAGE lysing solution: 0.25 mmol / L Tris-HCl, 10% SDS, 0.5% bromophenol blue, and 50% glycerol, pH 6.8; staining solution: 50% methanol, 6.8% glacial acetic acid, and 1 mg / mL Coomassie Brilliant Blue; destaining solution: 5% methanol, 7.5% glacial acetic acid. Adjust the concentration of MP solution to 3 mg / mL and mix it evenly with the SDS-PAGE lysing solution. After boiling in a water bath for 3 minutes, cool to room temperature and set aside. Add 12 μL of protein marker to the first well of the gel, followed by equal volumes of sample. Apply 80V when the sample enters the stacking gel and 120V when it enters the separating gel. After staining, remove the film and stain in staining solution for 15 minutes. Finally, destain with destaining solution until transparent before photographing.

[0086] Table 1 SDS-PAGE 12% separation gel and 5% stacking gel recipe

[0087]

[0088] (14) SDS-PAGE analysis:

[0089] like Figure 7 As shown in the figure, M represents a standard reference; F0 and F5 represent samples subjected to 0 and 5 freeze-thaw cycles, respectively. Compared to fresh samples, the MHC and actin bands of MP extracted from freeze-thawed surimi are narrower and lighter. The former is likely due to freeze-denaturation, which converts sulfhydryl groups into disulfide bonds, leading to covalent cross-linking. The latter is due to the formation of hydrogen bonds or hydrophobic bonds between molecules, resulting in insoluble aggregates. Simultaneously, the colors of tropomyosin and troponin become lighter, while the color of myosin light chain (16-25 kDa) becomes darker, indicating protein degradation during the freeze-thaw cycle. In contrast, compared to the control sample, the four major bands of the SS and NADES samples are darker, and the color of the myosin light chain is lighter, indicating a cryoprotective effect of the cryoprotectant. The color shift trend of the NADES sample is more pronounced than that of the SS sample, indicating that NADES has better resistance to denaturation, degradation, and aggregation.

[0090] like Figure 8As shown, M represents a standard reference; F0 and F5 represent samples that underwent 0 and 5 freeze-thaw cycles, respectively. Compared with the unfreeze-treated group, the freeze-treated group showed limited enhancement of the electrophoretic band between 20.1 kDa and 29 kDa. The enhancement in the NADES group was weaker than that in the SS group, indicating that cryoprotectants effectively inhibited myoplasmic protein degradation, with NADES being more potent. NADES, by binding water molecules, inhibits ice crystal growth and recrystallization, minimizing mechanical damage to muscle cells by ice crystals and preventing the leakage of endogenous proteases such as cathepsin B and cathepsin L. Therefore, NADES is highly effective in inhibiting myoplasmic protein degradation.

[0091] (15) Immunoblotting assay:

[0092] Desmin and troponin T were immunoblotted using gel preparation, electrophoresis, membrane transfer, blocking, antibody incubation, exposure and development, data processing and statistical analysis. Glyceraldehyde phosphate dehydrogenase was used as an internal control.

[0093] (16) Analysis of immunoblotting results:

[0094] like Figure 9 As shown, means with the same freeze-thaw cycle times are assigned different capital letters (AC) indicating significant differences (P < 0.05), and means with the same NADES concentration are assigned different lowercase letters (ab) indicating significant differences (P < 0.05). Compared with fresh meat samples, immunoreactive bands became less intact after five freeze-thaw cycles. The unfreeze-treated group showed the lowest integrity, followed by the SS group. The NADES group showed relatively intact bands. Immunoblot images of desmin and troponin T were consistent with the grayscale analysis results. The reduction in the integrity of desmin and troponin T is closely related to the increase in degradation products caused by endogenous proteases. In particular, freeze-thaw cycles may induce muscle oxidation and promote myofibril fragmentation by altering the activity of endogenous proteases. The integrity of samples treated with cryoprotectants was better maintained, especially in samples containing NADES. This is likely because NADES inhibits ice crystal expansion, minimizing the impact on the internal structure of the muscle system and the resulting damage to myofibril integrity. In addition, small ice crystals cannot lead to the release of a large amount of endogenous proteases, so the degradation effect of endogenous proteases is limited.

[0095] In summary, the present invention has designed a method for improving the freeze-thaw stability of surimi protein using a natural deep eutectic solvent. All raw materials are naturally derived, making this an environmentally friendly method. Citric acid and trehalose are used to form NADES, which are then added to frozen mirror carp surimi to significantly improve the freeze-thaw stability of surimi protein.

[0096] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for improving the stability of freeze-thawed surimi protein using a natural deep eutectic solvent, characterized in that: The following steps are involved: (1) Preparation of natural deep eutectic solvents: NADES composed of citric acid and trehalose in different molar ratios were prepared by a heating and stirring method. (2) Preparation of freeze-thawed mirror carp surimi: After anesthetizing the mirror carp, the dorsal muscle of the fish was minced into mince, washed with cold water and NaCl, and then centrifuged; the minced fish was divided into three groups according to weight and subjected to freeze-thaw cycles.

2. The preparation method according to claim 1, characterized in that The optimal molar ratio of citric acid to trehalose in step (1) is 2:

1.

3. The preparation method according to claim 1, characterized in that In step (2), the mirror carp was anesthetized with 0.4% benzocaine solution for 20 minutes.

4. The preparation method according to claim 1, characterized in that In step (2), the volume of cold water is 3 times that of the surimi, and the fish is washed twice.

5. The preparation method according to claim 1, characterized in that The mass concentration of the NaCl solution in step (2) is 0.5% (w / v), and washing is performed once.

6. The preparation method according to claim 1, characterized in that The three groups in step (2) are: negative control: fish paste without antifreeze; positive control: commercial antifreeze group; experimental group: NADES with a molar ratio of citric acid to trehalose of 2:

1.

7. The preparation method according to claim 1, characterized in that The freeze-thaw cycle in step (2) is as follows: the sample is frozen at -18°C for 7 days and then thawed at 4°C for 12 hours. The first freeze-thaw cycle is completed when the core temperature is 0-4°C. The above freeze-thaw process is repeated 0, 1, 3, and 5 times, respectively.

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