Antarctic krill-derived antifreeze peptide for maintaining quality of squid and preparation and screening method thereof

Abstract

The application discloses a source of antifreeze peptide of euphausia superba for quality keeping of squid and a preparation and screening method thereof, and is prepared from the euphausia superba as raw material, and different proteases are used for enzymolysis under certain material-liquid ratio conditions, and the enzymolysis liquid with the optimal antifreezing effect is screened through a freeze-thaw cycle food model. The euphausia superba enzymolysis liquid obtained through neutral protease enzymolysis can significantly improve the water holding capacity of food, and reduce thawing loss rate and cooking loss rate. After the euphausia superba enzymolysis liquid is prepared under the optimal hydrolysis condition, the molecular weight distribution is determined, then the enzymolysis product is separated and purified through ultrafiltration and dextran gel chromatography, the active component is identified through liquid chromatography-tandem mass spectrometry, finally, the potential active peptide is screened and mechanism analysis is carried out, and the antifreeze peptide is successfully identified. The prepared antifreeze peptide is natural, safe and effective in source, is suitable for quality keeping of frozen aquatic products and other frozen foods, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food freezing and preservation technology, specifically relating to a method for preparing food-grade antifreeze peptides using Antarctic krill as raw material, the antifreeze peptides obtained by this method, and their application in food freezing and preservation. Background Technology

[0002] Freezing is one of the most commonly used technologies for food preservation and cold chain transportation. However, during freezing and thawing, the internal moisture of food forms large ice crystals and recrystallizes, leading to cell structure damage, decreased water-holding capacity, and textural deterioration, severely affecting the quality of frozen foods. Currently, common antifreeze methods in the food industry mainly include adding sugars, phosphates, or synthetic cryoprotectants, but these methods have problems such as large dosage requirements, significant impact on taste, or insufficient safety and naturalness. Therefore, developing natural, safe, and effective antifreeze agents for food has become a research hotspot.

[0003] Antifreeze peptides are a class of functional peptides that can interact with the surface of ice crystals, inhibiting ice crystal growth and recrystallization. They have advantages such as low dosage, significant effects, and high safety. Antarctic krill is rich in high-quality protein resources, and its protein hydrolysates may contain functional peptides with antifreeze activity. However, there is still limited research on the systematic screening, preparation, and application of food-grade antifreeze peptides derived from Antarctic krill, especially lacking technical solutions based on food models to verify their antifreeze effects. Therefore, it is necessary to develop a method for preparing food-grade antifreeze peptides using Antarctic krill as a raw material and to verify their application effects in food freezing and preservation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing food-grade antifreeze peptides using Antarctic krill as raw material, and further to provide the antifreeze peptides obtained by this method and their application in food freezing and preservation, so as to solve the problems of limited sources, insufficient naturalness, and unstable freezing and preservation effects of existing food antifreeze agents.

[0005] This invention is achieved through the following technical solution:

[0006] An Antarctic krill antifreeze peptide, the amino acid sequence of which is SEQ ID NO.1.

[0007] A method for preparing Antarctic krill antifreeze peptides, specifically including the following steps:

[0008] (1) Enzymatic hydrolysis: Antarctic krill was used as raw material, and neutral protease, alkaline protease, trypsin and papain were added respectively under the same process conditions;

[0009] (2) Evaluation of antifreeze effect: Different enzymatic hydrolysates were used in food model systems. After multiple freeze-thaw cycles, the water holding capacity and changes in tissue structure of the food were measured, and the enzymatic hydrolysate with the best antifreeze effect was screened.

[0010] (3) Separation and purification: The enzymatic hydrolysis products are separated and purified by ultrafiltration, Sephadex G-15 gel chromatography and other techniques;

[0011] (4) Antifreeze peptide analysis and simulation: Antifreeze peptides are screened by combining mass spectrometry and bioinformatics tools, and the action sites and mechanisms of target peptides are revealed by molecular docking.

[0012] In a preferred embodiment, the preparation method specifically includes the following steps:

[0013] (1) Enzymatic hydrolysis

[0014] Antarctic krill is mixed with water, and under certain material-to-liquid ratio conditions, protease is added for enzymatic hydrolysis. The protease includes one or more of neutral protease, alkaline protease, trypsin, and papain. The enzymatic hydrolysis is carried out under suitable temperature and pH conditions.

[0015] (2) Preliminary screening of antifreeze effect

[0016] Squid meat was soaked in the enzymatic hydrolysates obtained from different proteases. The water-holding capacity and tissue structure of the squid samples were evaluated after four freeze-thaw cycles, and the enzymatic hydrolysate with the best antifreeze effect was screened.

[0017] (3) Separation and purification

[0018] The enzymatic hydrolysate with the best antifreeze effect was separated by ultrafiltration and gel filtration chromatography, and its antifreeze properties were tested to obtain a low molecular weight peptide component with high antifreeze activity.

[0019] (4) Detection of antifreeze peptides and bioinformatics analysis of peptide fragments

[0020] The low molecular weight peptide components were identified by LC-MS / MS mass spectrometry; online tools were used to predict the potential biological activities of the peptides, and antifreeze peptides with significant antifreeze activity were screened.

[0021] (5) Study on antifreeze peptides and ice crystal molecular simulation

[0022] The screened antifreeze peptides were docked with ice crystals, and the total interaction energy was calculated. Finally, the docking results were visualized and analyzed using PyMOL 2.3.0.

[0023] In a preferred embodiment, in step (1), Antarctic krill is mixed with water, and the ratio of Antarctic krill to water is 1:10.

[0024] In a preferred embodiment, the amount of enzyme added during enzymatic hydrolysis in step (1) is 2000 U / g protein, the hydrolysis temperature is 40–45℃, and the hydrolysis time is 4h.

[0025] As a preferred embodiment, the food model in step (2) is squid. The squid is frozen at -20℃ for 7 days and thawed at 4℃ for 4 hours. The freeze-thaw cycle is repeated 4 times. The squid's moisture content, thawing loss rate, cooking loss rate, and longitudinal and cross-sectional microstructure of the muscle tissue are detected.

[0026] In a preferred embodiment, the separation and purification method in step (3) is ultrafiltration separation, to obtain low molecular weight peptide components with a molecular weight of 1–3 kDa.

[0027] In a preferred embodiment, the thermal hysteresis activity of the low molecular weight peptide component is 0.38°C.

[0028] The analysis of antifreeze peptides involved identifying peptide fragments of active components using liquid chromatography-tandem mass spectrometry. By employing bioinformatics analysis and molecular docking, potential active peptides were screened and their mechanisms elucidated. One antifreeze peptide, namely the Antarctic krill antifreeze peptide, was successfully identified.

[0029] Application of an Antarctic krill antifreeze peptide in food freezing and preservation.

[0030] The present invention has the following advantages:

[0031] Compared with existing technologies, this invention uses Antarctic krill as raw material, which is natural and safe, suitable for food applications. Through comparative screening of various proteases, it was determined that the neutral protease hydrolysis product has the best antifreeze effect, and the technical route is clear and reliable. The obtained antifreeze peptides have small molecular weight and well-defined structure, which can effectively inhibit ice crystal growth and recrystallization during food freeze-thaw cycles, significantly improve the water-holding capacity of food, maintain the integrity of muscle tissue structure and slow down textural deterioration. They can be used for the freezing and preservation of frozen aquatic products and other foods, and have good application prospects and industrialization value. Attached Figure Description

[0032] Figure 1 The effect of different enzymatic hydrolysates on the water-holding capacity of frozen and thawed squid is shown in the figure. (A) represents the moisture content; (B) represents the thawing loss rate; and (C) represents the cooking loss rate.

[0033] Figure 2 shows the effects of different enzymatic hydrolysates on the tissue structure of frozen and thawed squid (400X), where 2A represents the longitudinal section and 2B represents the cross section.

[0034] Figure 3 This refers to the antifreeze properties of different components after ultrafiltration treatment.

[0035] Figure 4Elution curves (A) and antifreeze properties (B) of the components separated by Sephadex G-15 chromatography.

[0036] Figure 5 The spatial structure and docking results of the antifreeze peptide and ice crystals. Detailed Implementation

[0037] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0038] Raw material pretreatment:

[0039] The Antarctic krill was purchased from a factory in Shandong and transported to its destination via cold chain. Upon arrival, it was immediately placed in a -80 degree freezer and thawed before use.

[0040] Experimental methods:

[0041] A method for preparing Antarctic krill antifreeze peptides, specifically including the following steps:

[0042] (1) Screening of the antifreeze properties of different protease hydrolysis products

[0043] Antarctic krill were mixed at a feed-to-liquid ratio of 1:10. Under the same processing conditions, neutral protease, alkaline protease, trypsin, and papain were added separately, with each enzyme added at a concentration of 2000 U / g. Enzymatic hydrolysis was carried out for 4 hours under suitable temperature and pH conditions. After hydrolysis, the enzymes were inactivated by heating and centrifugation, and the supernatant was collected as samples of different hydrolysates.

[0044] Different enzymatic hydrolysates were used to soak squid at a material-to-liquid ratio of 1:2, and the soaking time was 4 hours at 4°C. The soaked squid were then subjected to a freeze-thaw cycle: frozen at -20°C for 7 days, thawed at 4°C for 4 hours, repeated 4 times for a total cycle of 22 days. The samples after the freeze-thaw cycle were analyzed for moisture content, thawing loss rate, cooking loss rate, and the microstructure of longitudinal and cross-sectional sections of the muscle tissue. The enzymatic hydrolysate with the best antifreeze effect was selected.

[0045] (2) Separation and purification

[0046] The enzymatic hydrolysate was fractionated using 50 mL ultrafiltration centrifuge tubes with molecular weights of 10 kDa, 3 kDa, and 1 kDa. The permeate was used as the initial feed for the next stage of ultrafiltration, resulting in four peptide fractions with different molecular weight ranges: M1 (molecular weight < 1 kDa), M2 (molecular weight 1-3 kDa), M3 (molecular weight 3-10 kDa), and M4 (molecular weight > 10 kDa). The fractions were centrifuged at 5000 × g for 15 min at low temperature, then lyophilized and their freeze resistance was determined.

[0047] Thermal hysteresis activity is used as an indicator to measure antifreeze properties. Thermal hysteresis activity refers to the ability of a substance to lower the freezing point of a solution without affecting its melting point; the difference between the freezing point and the melting point is the magnitude of the thermal hysteresis activity. The greater the thermal hysteresis activity, the stronger the substance's ability to inhibit ice crystal growth. Therefore, it is often used to determine the strength of the antifreeze activity of protein-based antifreeze agents such as antifreeze peptides and proteins. The specific procedure involves dissolving the lyophilized hydrolysate in deionized water to prepare a 20 mg / mL solution. The control group is a 20 mg / mL bovine serum albumin solution. 5 μL of the solution is sealed in a crucible, and the sealed crucible is placed in a DSC device to measure the thermal hysteresis activity of the hydrolysate. The initial temperature is set to 20 °C, and the temperature is lowered to -20 °C at a rate of 5 °C / min and held for 2 min; then the temperature is raised to 20 °C at the same rate and held for 2 min, yielding the melting curve of the hydrolysate. Then, the temperature is lowered again to -20 °C at the same rate, held for 2 min, and then increased at a rate of 1 °C / min until the solution is in a partially molten state. This temperature is called the holding temperature (T). h Hold at this temperature for 2 minutes, then increase the temperature to 20 °C at a rate of 1 °C / min. Repeat this process continuously, selecting different temperatures. h The THA value is recorded when the maximum value is reached.

[0048] Calculate the thermal hysteresis activity according to formula (1):

[0049] THA = T ℎ − T0 (1)

[0050] In the formula, THA refers to thermal hysteresis activity, in °C; T h T0 refers to the retention temperature, in °C; T0 refers to the initial crystallization temperature, in °C.

[0051] The group with the highest frost resistance was further separated and purified. A Sephadex G-15 gel filtration chromatography column (1.6 cm × 60 cm) was used for further separation and purification of the ultrafiltration fraction with the highest frost resistance. A certain mass of the lyophilized sample of the fraction with the highest frost resistance was weighed, added to 5.0 mL of ultrapure water to prepare a sample solution with a concentration of 20 mg / mL. After filtration through a disposable syringe filter (0.45 µm, aqueous system), the solution was added to the Sephadex G-15 chromatography column. The sample solution was eluted with Wahaha purified water at a flow rate of 1 mL / min, and the eluent was collected in 5 mL tubes. An elution curve was plotted with the absorbance of the eluent at 220 nm as the ordinate and the collection sequence of the eluent as the abscissa. Based on the elution curve, two major fractions (F1 and F2) were identified, and each fraction was collected, lyophilized, and stored at -20 °C. The frost resistance of fractions F1 and F2 was tested.

[0052] (3) Detection of antifreeze peptides and bioinformatics analysis of peptide fragments

[0053] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection conditions: Injection volume 5 mL; Column: Acclaim PepMap RPLCC18 (150 mm × 150 μm, 1.9 μm); Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% acetonitrile; Flow rate: 600 nL / min; Analysis time for each component: 66 min. Gradient elution: 0–2 min 4%–8% B phase, 2–45 min 8%–28% B phase, 45–55 min 28%–40% B phase, 55–56 min 40%–95% B phase, 56–66 min 95% B phase. Mass spectrometry conditions: Level 1 mass spectrometry, resolution: 70000, automatic gain control target: 3e6, maximum IT: 100 ms, scan range: 100–1500 m / z; Level 2 mass spectrometry, resolution: 17500, automatic gain control target: 1e5, maximum IT: 50 ms, top N: 20, normalized fragmentation energy: 28.

[0054] Physicochemical properties of peptide sequences were analyzed using ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php). CryoProtect (http: / / codes.bio / cryoprotect / ) was used to assess whether the peptide sequences belonged to the antifreeze peptide family. Subsequently, peptide sequences with lengths of 10-14 amino acids were screened, and their origin was identified using UniProt (https: / / www.uniprot.org / ).

[0055] (4) Study on antifreeze peptides and ice crystal molecular simulation

[0056] Antifreeze peptide structure prediction was performed using AlphaFold Server (https: / / alphafoldserver.com / ). Crystal structure models of ice crystals were obtained in PDB format using GenIce 1.0.10 software (https: / / pypi.org / project / GenIce / 1.0.10 / ) and used as acceptors for molecular docking. Docking was performed using AutoDock Tools-1.5.6, and visualization analysis was performed using PyMOL.

[0057] Experimental data analysis:

[0058] Data analysis was performed using Excel and SPASS, and graphs were generated using Origin 2018 (Origin Lab Corp, Hampton, USA).

[0059] Analysis of experimental results:

[0060] 1. Changes in the water-holding capacity of squid

[0061] Analysis of the results of measuring the moisture content, thawing loss rate, and cooking loss rate of squid samples under different freeze-thaw cycles shows that different enzymatic hydrolysates have a significant impact on the squid's moisture retention capacity during freeze-thaw cycles. For example... Figure 1 As shown, after four freeze-thaw cycles, the moisture content of the squid sample in the blank control group decreased significantly from approximately 77.63% to 63.65%, indicating that repeated freeze-thaw cycles resulted in a large loss of moisture. In contrast, the Antarctic krill hydrolysate treatment group, after being hydrolyzed by neutral protease, still maintained a moisture content of approximately 80.01% after the fourth freeze-thaw cycle, which was significantly higher than that of the blank group and other hydrolysate treatment groups.

[0062] Further analysis of the thawing loss rate data reveals that the thawing loss rate of all treatment groups increased with the number of freeze-thaw cycles. However, the thawing loss rate of the neutral protease hydrolysate treatment group remained consistently lower than that of the other treatment groups. For example, after the fourth freeze-thaw cycle, the thawing loss rate of the neutral protease hydrolysate treatment group was approximately 14.21%, while the thawing loss rates of the alkaline protease, trypsin, and papain treatment groups increased to approximately 25.93%–27.06%, respectively, showing a significant difference.

[0063] The cooking loss rate results were consistent with the trends in moisture content and thawing loss rate. The neutral protease hydrolysate treatment group showed a smaller increase in cooking loss rate after multiple freeze-thaw cycles, indicating that it effectively maintained bound water and structural water during heating. Considering the moisture content, thawing loss rate, and cooking loss rate, it can be seen that the Antarctic krill hydrolysate hydrolyzed with neutral protease significantly slows down water migration during freeze-thaw cycles, reduces water loss during thawing and cooking, and thus more effectively maintains the integrity of the squid's tissue structure and quality stability.

[0064] Figure 2 shows the effects of different enzymatic hydrolysates on the tissue structure of frozen-thawed squid (400X), 2A longitudinal section; 2B cross section. As can be seen from the figure, significant differences in the microstructure of squid muscle tissue were observed after four freeze-thaw cycles. Histological results indicated that, compared with other enzymatic hydrolysates and the control group, the Antarctic krill hydrolysate treatment group treated with neutral protease hydrolysate exhibited superior tissue integrity in both longitudinal and cross sections. Its muscle fibers were more regularly arranged and showed better continuity, with significantly reduced muscle bundle breakage and collapse, and significantly smaller interfiber gaps and freeze-thaw induced vacuolar structures, resulting in a denser overall tissue. In contrast, other treatment groups still showed varying degrees of muscle fiber loosening and structural damage. These results indicate that the hydrolysate obtained from Antarctic krill hydrolysate using neutral protease can more effectively inhibit ice crystal growth and its damage to muscle tissue under multiple freeze-thaw conditions, thus exhibiting a more prominent antifreeze protection effect. Therefore, Antarctic krill hydrolysate hydrolysate obtained from neutral protease hydrolysate was selected for separation and purification.

[0065] 2. Evaluation of the antifreeze properties of the separated and purified components:

[0066] In the process of peptide separation and purification, ultrafiltration can effectively remove insoluble substrates, large protein molecules, and peptides, thereby obtaining bioactive peptides with smaller molecular weights. For example... Figure 3As shown, Antarctic krill enzymatic hydrolysate can be separated into four components using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa, 3 kDa, and 1 kDa. Thermal hysteresis activity (THA) refers to the ability of a substance to lower the freezing point of a solution without affecting its melting point; the difference between the freezing point and the melting point is the magnitude of the thermal hysteresis activity. Substances with thermal hysteresis activity can effectively inhibit ice crystal growth; the greater the thermal hysteresis activity, the more significant the effect. Specifically, THA values ​​of 0.2–0.6 °C indicate moderately active antifreeze peptides, while those of 0.6–6 °C indicate extremely active antifreeze peptides. Figure 3 As shown, the hysteresis activity of peptides with a molecular weight (MW) of 1-3 kDa is the highest, at 0.35℃, meeting the requirements for moderately active antifreeze peptides. Similar to the results of this experiment, numerous studies have found a certain correlation between the molecular weight of peptides and their antifreeze properties, and low molecular weight (<3 kDa) peptides often have higher antifreeze properties. This may be because low molecular weight peptides have shorter chains, less steric hindrance, and are more likely to enter and bind to ice crystals, inhibiting the growth and recrystallization of ice crystals during freeze-thaw cycles.

[0067] The fractions with a molecular weight of 1-3 kDa were further finely separated using a Sephadex G-15 gel chromatography column. Figure 4 As shown, the elution curves of the 1-3 kDa fractions at a detection wavelength of 220 nm exhibited two main peaks. The fractions corresponding to the two peaks were collected, and their thermal hysteresis activity was measured. At a concentration of 2 mg / mL, fraction 2 showed the highest thermal hysteresis activity at 0.38 °C, which was significantly different from the other fraction (P < 0.05). Since ultrafiltration removes long peptides with high molecular weights, it can enrich active small-molecule antifreeze peptides. Furthermore, removing interference from contaminants may help the antifreeze peptides interact better with ice crystals, enhancing their antifreeze properties. Therefore, fraction 2 was ultimately selected for subsequent peptidomics identification and analysis.

[0068] 3. Peptide composition identification and analysis

[0069] The antifreeze activity of peptides is closely related to their sequence. Initially, the ToxinPred database was used to screen for non-toxic peptide sequences with a hydrophilicity score greater than 0. Subsequently, non-antifreeze peptide (AFP) sequences were eliminated using the CryoProtect database. Previous studies have shown that antifreeze peptides prepared by enzymatic hydrolysis typically have a molecular weight of less than 2000 Daltons and a sequence length of less than 20 amino acids. The top 20 sequences were finally obtained, as shown in Table 1. The secondary structure of antifreeze peptides has a significant impact on their cryoprotective activity. Therefore, the peptide sequence structure was constructed using AlphaFold Server.

[0070] Table 1 Physicochemical Properties Analysis of Antifreeze Peptides

[0071] Serial Number sequence length Molecular weight / Da Source protein Score hydrophilic 1 VYLEGTL 7 793.40 Fructose diphosphate aldolase 46.02 0.84 2 LVDDHFLFMS 10 1222.55 Arginine kinase 46.02 0.83 3 GIKVDKGVVPLM 12 1254.74 Fructose diphosphate aldolase 54.46 0.76 4 LVDDHFL 7 857.42 Arginine kinase 49.48 0.63 5 LGGLGERVLG 10 969.56 Sodium-potassium ATPase α subunit 83.91 0.60 6 FVDWIMTN 8 1040.46 trypsin 58.6 0.60 7 FVDWIMTNTA 10 1212.55 trypsin 50.87 0.59 8 VDDHFLFMS 10 122.569 Arginine kinase 45.95 0.50 9 VHVDLPGWA 9 992.51 Arginine kinase 67.55 0.49 10 QLVDDHFLF 9 1132.56 Arginine kinase 47.13 0.41 11 MPPAVPG 7 667.34 Fructose diphosphate aldolase 54.47 0.39 12 MIDGVNT 7 748.34 Arginine kinase 51.05 0.36 13 SVHVDLPG 8 822.42 Arginine kinase 64.37 0.34 14 VHVDLPGW 9 1008.50 Arginine kinase 60.6 0.33 15 LGYSEVELVQ 10 1135.58 Arginine kinase 53.13 0.30 16 LDQVSEAL 11 1231.56 Tropomyosin 42.3 0.29 17 VDWIMTN 8 1040.46 trypsin 44.99 0.29 18 TMPPAVPG 8 768.38 Fructose diphosphate aldolase 54.42 0.25 19 SVHVDLPGW 9 1008.50 Arginine kinase 44.18 0.20 20 QQLVDDHFLF 10 1260.61 Arginine kinase 75.08 0.02

[0072] 4. Molecular docking study on the inhibition of ice crystal growth by antifreeze peptides

[0073] By predicting the binding mode of antifreeze peptide (AFP) to ice crystal surfaces, molecular docking revealed the mechanism by which AFP interacts with ice crystal surfaces through specific amino acid residues.

[0074] SEQ ID NO.1: VHVDLPGWA.

[0075] Table 2 shows the total binding energy (Etotal) and the number of hydrogen bonds between peptides and ice crystals. A higher absolute value of the total energy indicates a more stable binding. The total binding energies of AFP1, AFP2, and AFP3 with ice crystals are -206, -197, and -103 kcal / mol, respectively, indicating different degrees of interaction. The docking results of AFP1, AFP2, and AFP3 with the ice crystal surface are shown below. Figure 5 As shown in A−5C, the yellow dashed lines represent hydrogen bonds. Studies have shown that AFPs typically bind to the ice crystal surface through their hydrophobic regions or hydrogen bond networks, preventing water molecules from further adhering to and arranging on the ice crystal surface, thereby inhibiting ice crystal growth. AFP1 forms four hydrogen bonds with the ice crystal, involving the residues Ala9, Trp8, His2, and Val1. AFP2 forms one hydrogen bond with the ice crystal, involving the residue Ile1. Similarly, AFP3 forms two hydrogen bonds with the ice crystal, involving the residues His3 and Ser1.

[0076] Table 2. Total binding energy (Etotal) and number of hydrogen bonds between peptides and the ice crystal system.

[0077] sequence Binding energy / kcal / mol Number of hydrogen bonds docking site AFP1 VHVDLPGWA -206 4 Ala9, Trp8, His2, Val1 AFP2 MIDGVNT -197 1 Ile1 AFP3 SVHVDLPG -103 2 His3, Ser1

[0078] This invention utilizes Antarctic krill as raw material and, through comparative screening using different proteases, successfully prepared and obtained a class of food-grade antifreeze peptides with significant antifreeze activity. Experimental results show that, after four freeze-thaw cycles, the Antarctic krill hydrolysate hydrolyzed with neutral protease exhibited the best antifreeze protection effect in a food model system. Compared with other hydrolysates and a blank control, this hydrolysate significantly increased the moisture content of frozen-thawed squid, reduced thawing and cooking losses, and effectively maintained the integrity of the muscle tissue's microstructure, resulting in a more compact and continuous arrangement of muscle fibers, significantly mitigating the damage to tissue structure caused by ice crystal growth and recrystallization during freeze-thaw cycles. Further separation and purification results showed that the low-molecular-weight peptide components with a molecular weight of 1–3 kDa possessed high thermal hysteresis activity, and molecular docking verified their mechanism of action in stably binding to the ice crystal surface and inhibiting ice crystal growth. In summary, the Antarctic krill antifreeze peptides prepared in this invention are naturally derived, safe, and effective, with stable antifreeze effects, suitable for maintaining the quality of frozen aquatic products and other frozen foods, and have good application prospects and industrialization value.

[0079] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An Antarctic krill antifreeze peptide, characterized in that its amino acid sequence is SEQ ID NO.

1.

2. The method for preparing Antarctic krill antifreeze peptides as described in claim 1, characterized in that the preparation method specifically includes the following steps: (1) Enzymatic hydrolysis: Antarctic krill was used as raw material, and neutral protease, alkaline protease, trypsin and papain were added respectively under the same process conditions; (2) Evaluation of antifreeze effect: Different enzymatic hydrolysates were used in food model systems. After multiple freeze-thaw cycles, the water holding capacity and changes in tissue structure of the food were measured, and the enzymatic hydrolysate with the best antifreeze effect was screened. (3) Separation and purification: The enzymatic hydrolysis products are separated and purified by ultrafiltration, Sephadex G-15 gel chromatography and other techniques; (4) Antifreeze peptide analysis and simulation: Antifreeze peptides are screened by combining mass spectrometry and bioinformatics tools, and the action sites and mechanisms of target peptides are revealed by molecular docking.

3. The method for preparing Antarctic krill antifreeze peptides according to claim 2, characterized in that the preparation method specifically includes the following steps: (1) Enzymatic hydrolysis Antarctic krill is mixed with water, and under certain material-to-liquid ratio conditions, protease is added for enzymatic hydrolysis. The protease includes one or more of neutral protease, alkaline protease, trypsin and papain. The enzyme is enzymatically hydrolyzed under suitable temperature and pH conditions. (2) Preliminary screening of antifreeze effect Squid meat was soaked in the enzymatic hydrolysates obtained from different proteases. The water-holding capacity and tissue structure of the squid samples were evaluated after four freeze-thaw cycles, and the enzymatic hydrolysate with the best antifreeze effect was screened. (3) Separation and purification The enzymatic hydrolysate with the best antifreeze effect was separated by ultrafiltration and gel filtration chromatography, and its antifreeze properties were tested to obtain a low molecular weight peptide component with high antifreeze activity. (4) Detection of antifreeze peptides and bioinformatics analysis of peptide fragments The low molecular weight peptide components were identified by LC-MS / MS mass spectrometry; online tools were used to predict the potential biological activities of the peptides, and antifreeze peptides with significant antifreeze activity were screened. (5) Study on antifreeze peptides and ice crystal molecular simulation The screened antifreeze peptides were docked with ice crystals, and the total interaction energy was calculated. Finally, the docking results were visualized and analyzed using PyMOL 2.3.

0.

4. The method for preparing Antarctic krill antifreeze peptides according to claim 2, characterized in that, in step (1), Antarctic krill is mixed with water, and the ratio of Antarctic krill to water is 1:

10.

5. The method for preparing Antarctic krill antifreeze peptides according to claim 2, characterized in that, in step (1), the amount of enzyme added during enzymatic hydrolysis is 2000 U / g protein, the enzymatic hydrolysis temperature is 40–45℃, and the enzymatic hydrolysis time is 4h.

6. The method for preparing Antarctic krill antifreeze peptides according to claim 2, characterized in that the food model in step (2) is squid, the squid is frozen at -20℃ for 7 days, thawed at 4℃ for 4 hours, and the freeze-thaw cycle is repeated 4 times, and the squid moisture content, thawing loss rate, cooking loss rate and the microstructure of longitudinal and cross sections of muscle tissue are detected.

7. The method for preparing Antarctic krill antifreeze peptides according to claim 2, characterized in that the separation and purification method in step (3) is ultrafiltration separation to obtain low molecular weight peptide components with a molecular weight of 1–3 kDa.

8. The method for preparing Antarctic krill antifreeze peptides according to claim 7, characterized in that the thermal hysteresis activity of the low molecular weight peptide component is 0.38℃.

9. The screening method for Antarctic krill antifreeze peptides as described in claim 1, characterized in that the antifreeze peptide analysis is performed by liquid chromatography-tandem mass spectrometry to identify peptide fragments of active components, and by using bioinformatics analysis and molecular docking to screen potential active peptides and analyze their mechanisms, and a single antifreeze peptide is successfully identified, which is the Antarctic krill antifreeze peptide.

10. The application of the Antarctic krill antifreeze peptide as described in claim 1 in food freezing and preservation.

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