Preparation of sunflower seed antifreeze protein peptide and application thereof

Abstract

The application discloses a kind of sunflower seed antifreeze protein peptide preparation and its application, belong to biological base fresh-keeping material and soft material science and technology field.The present application is prepared with defatted sunflower seed meal as raw material, and after heat treatment and alkaline protease enzymolysis, short peptide-rich sunflower seed meal protein solution is prepared;And 3 segments with antifreeze activity peptide segment GTAQAAKEKAGETKA, ASEGGQTRKEQLGE and GTAQAAKEKAGETKASGGQTRKEQLG are screened from it.The peptide segment screened by the present application has excellent antifreeze effect, and has broad-spectrum and long-acting fresh-keeping antifreeze application potential in complex high-fat high-protein meat matrix.

Description

Technical Field

[0001] This invention relates to the preparation and application of sunflower seed antifreeze protein peptides, belonging to the field of bio-based preservation materials and soft matter science and technology. Background Technology

[0002] Low-temperature freezing is the mainstream storage method for extending the shelf life and preserving the quality of food, and it is widely used in the processing and storage of various foods such as meat products, aquatic products, flour products, and pre-prepared foods. However, repeated freeze-thaw cycles often occur during the low-temperature freezing, long-term freezing, and cold chain transportation of food. This causes a phase change between free water and bound water in the food system, generating a large number of irregular ice crystals and resulting in ice crystal recrystallization. The continuous aggregation and growth of small ice crystals form large, sharp-edged ice crystals, which cause irreversible mechanical damage to the microstructure of food, leading to cell rupture, loosening of the tissue structure, and a series of quality deterioration problems. These problems manifest as damage to the spatial structure of food proteins, denaturation and inactivation of functional proteins, precipitation of fat oxidation, increased juice loss, soft and loose texture, and flavor degradation, significantly reducing the edible quality and commercial value of frozen foods.

[0003] To address the quality deterioration caused by ice crystal growth and recrystallization in frozen foods, current methods primarily rely on the exogenous addition of food antifreeze agents. These agents regulate the moisture content of the system and inhibit ice crystal proliferation to mitigate freezing damage. However, existing traditional antifreeze additives, such as small-molecule alcohols, synthetic polymers, and sugars, have limitations including poor taste, poor biocompatibility, or a single antifreeze mechanism. These limitations make it difficult to effectively inhibit ice crystal growth in complex, high-salt, and high-fat food matrices for extended periods.

[0004] In recent years, natural biomolecules and polypeptides have become a research hotspot in low-temperature food preservation due to their excellent biocompatibility and moisture regulation capabilities. Sunflower seed meal is a core byproduct of sunflower seed oil pressing and extraction processes. It has a huge annual output, is widely available, inexpensive to obtain, and is rich in high-quality plant protein, making it a natural plant protein resource with significant development value. Currently, the industry's resource utilization methods for sunflower seed meal are relatively limited. Existing research mostly focuses on primary applications such as simple extraction of crude protein, food fortification, feed additives, and organic fertilizer preparation. The utilization rate of deep processing is extremely low, resulting in the waste of a large amount of high-quality sunflower seed protein resources and failing to achieve high-value resource utilization.

[0005] Therefore, developing a method that uses sunflower seed meal, an agricultural byproduct, as raw material, and through specific directional enzymatic hydrolysis, fine separation, and activity screening, to discover a series of specific protein peptide sequences with high-target ice-controlling and protective functions, and applying them to the low-temperature protection system of complex food matrices, can not only overcome the industry pain points of poor matrix compatibility and low antifreeze efficiency of existing antifreeze materials, but also has important industrial significance for improving the high-value utilization level of agricultural byproducts. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for preparing and applying sunflower seed antifreeze protein peptides. Using defatted sunflower seed meal as raw material, this invention prepares a sunflower seed meal protein solution rich in short peptides through heat treatment and alkaline protease hydrolysis. Three peptides with antifreeze activity, GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, and GTAQAAKEKAGETKASGGQTRKEQLG, are then screened from this solution. The peptides obtained by this invention exhibit excellent antifreeze effects and have broad-spectrum and long-lasting preservation and antifreeze application potential in complex high-fat, high-protein meat matrices.

[0007] The first objective of this invention is to improve an engineered Escherichia coli strain that expresses the following protein peptides: GTAQAAKEKAGETKA (amino acid sequence as shown in SEQ ID NO.1), ASEGGQTRKEQLGE (amino acid sequence as shown in SEQ ID NO.2), or GTAQAAKEKAGETKASGGQTRKEQLG (amino acid sequence as shown in SEQ ID NO.3).

[0008] A second objective of this invention is to provide a protein peptide, wherein the protein peptide is GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, or GTAQAAKEKAGETKASGGQTRKEQLG, and its amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3, respectively.

[0009] A third objective of this invention is to provide a method for preparing a sunflower seed meal protein solution, characterized in that the sunflower seed meal protein solution contains the aforementioned protein peptides, and characterized by comprising the following steps: (1) Mix defatted sunflower seed meal powder with water at a ratio of 1~5 g: 10~25 mL, stir, centrifuge and collect the supernatant; heat the supernatant and centrifuge again to collect the supernatant and freeze dry to obtain sunflower seed meal protein powder; (2) Prepare a sunflower seed meal protein solution with a mass fraction of 5-10% by the sunflower seed meal protein powder obtained in step (1), and add alkaline protease of 2% by mass of sunflower seed meal protein powder to it for enzymatic hydrolysis, enzyme inactivation, cooling, centrifugation and ultrafiltration to obtain sunflower seed meal protein solution. In step (2), the enzyme activity of alkaline protease is 2000~80000 U / g.

[0010] In one embodiment, the stirring conditions in step (1) are 25~50℃ and 300~600rpm for 1~2 hours.

[0011] In one embodiment, the centrifugation conditions in step (1) are 6000~8000 rpm for 15~20 min.

[0012] In one embodiment, the heating conditions in step (1) are 90~100℃ for 10~15 min.

[0013] In one embodiment, the freeze-drying conditions in step (1) are freeze-drying at -40~-60℃ for 24~48 h.

[0014] In one embodiment, the enzymatic hydrolysis in step (2) is performed at pH 7.5-9 and 45-55°C for 0.5-3 h.

[0015] In one embodiment, the enzyme inactivation conditions in step (2) are 90-100°C for 10-15 min.

[0016] In one embodiment, the centrifugation conditions in step (2) are 5000~8000 rpm for 10~20 min.

[0017] In one embodiment, the ultrafiltration in step (2) is for ultrafiltration collection of components with a molecular weight of less than 3000 Da.

[0018] A fourth object of the present invention is to provide an expression vector that encodes the protein peptides described above.

[0019] A fifth object of the present invention is to provide a product, characterized in that the product contains the protein peptides described above.

[0020] In one embodiment, the product may be food, medicine, or health product.

[0021] In one embodiment, the pharmaceutical product further comprises pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field; the excipients include one or more of the following: binders: cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents: pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers: starch and sucrose; humectants: glycerol; disintegrants: sodium carboxymethyl starch, croscarmellose, and dry starch; absorption enhancers: quaternary ammonium compounds; surfactants: polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants: hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials: acrylic resins, hydroxypropyl methylcellulose, povidone, and cellulose acetate; additionally, other excipients may be added to the composition: flavoring agents and sweeteners.

[0022] In one embodiment, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form; The oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, and oral suspensions; The injectable dosage forms include, but are not limited to, injectable solutions and injectable powders for injection; The inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0023] In one embodiment, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods. In one embodiment, the health product also contains acceptable excipients.

[0024] The sixth objective of this invention is to provide a method for improving the antifreeze properties of meat products, wherein the method involves treating the meat products with the aforementioned protein peptides; The treatment may be at least one of soaking, marinating, spraying, injection, tumbling, coating, and vacuum impregnation.

[0025] In one embodiment, the improvement of the antifreeze properties of meat products is to reduce the loss rate of thawed juices from meat products, reduce the degree of myofibrillar protein oxidation in meat products, and stabilize the texture of meat products.

[0026] A seventh object of the present invention is to provide the use of the protein peptides described above, or any of the antifreeze drugs, antifreeze reagents, antifreeze additives, or any of the methods described above, in the preparation of antifreeze products.

[0027] Beneficial effects (1) This invention utilizes specific alkaline protease-targeted enzymatic degreasing of sunflower seed meal, combined with fine purification and screening using gel chromatography and reversed-phase liquid chromatography, to successfully prepare and identify characteristic antifreeze protein peptides GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, and GTAQAAKEKAGETKASGGQTRKEQLG, which possess amphiphilic α-helices and disordered flexible regions. This method not only achieves high-value utilization of sunflower seed meal but also overcomes the limitation of traditional plant antifreeze peptides having only a single function, resulting in a novel bio-based preservation material with both physical antifreeze and biochemical protection mechanisms.

[0028] (2) The peptides screened in this invention have excellent antifreeze effects. Specifically, the specific thermal hysteresis activity (THA) of the peptides GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, and GTAQAAKEKAGETKASGGQTRKEQLG screened in this invention can reach 0.52℃, 0.55℃, and 0.49℃, respectively, all significantly higher than that of commercially available fish skin collagen peptides (0.45℃). In the microscopic ice crystal recrystallization inhibition (IRI) evaluation, the peptides of this invention can effectively compress the relative ice crystal area ratio to between 33.9% and 41.6% (100% in the negative pure water blank group), which has better microscopic ice inhibition and steric hindrance efficiency than commercially available fish skin collagen peptides (45.4%).

[0029] (3) The antifreeze peptides screened in this invention exhibit macroscopic protective properties with great commercial value in actual freeze-thaw cycles of fresh meat, specifically: In the pork tenderloin model: after three freeze-thaw cycles, the thawing juice loss rate of the experimental group treated with the peptide GTAQAAKEKAGETKASGGQTRKEQLG was only 13.2%, which was 9.2 percentage points lower than the unprotected negative control group (22.4%) and 3.0 percentage points lower than the commercially available control group (16.2%). The total sulfhydryl content of its myofibrillar protein remained at 55.2 nmol / mg, a significant increase of 69.8% compared to the control group (32.5 nmol / mg) and 31.1% compared to the commercially available control group (42.1 nmol / mg), maintaining the natural elasticity of the meat and preventing textural deterioration.

[0030] In the chicken breast model, the peptide group GTAQAAKEKAGETKASGGQTRKEQLG also suppressed the thawing juice loss rate to 10.4% (18.2% in the control group and 13.5% in the commercial comparison group), and protected the total thiol content of myofibrillar protein at a high level of 50.8 nmol / mg (only 37.4 nmol / mg in the commercial comparison group), which fully demonstrates the broad-spectrum and long-lasting preservation and antifreeze application potential of the present invention in complex high-fat and high-protein meat matrices. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0032] In the examples, solutions without a specified solvent use water; operations without a specified temperature refer to room temperature, which is 15-35°C; and percentages without a specified meaning refer to mass percentage. Raw material source: The defatted sunflower seed meal was purchased from Wenji Food Co., Ltd. in Zhaoyuan City, and its protein content was 43%. The alkaline protease was purchased from Novozymes, with an enzyme activity of 2.4 AU-A / g, and the product name was Alcalase® 2.4 L FG. Commercially available fish skin collagen was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a molecular weight of 2000-3000.

[0033] The measurement methods involved in the examples are as follows: Extraction of myofibrillar protein (MP): Accurately weigh 2.0 g of meat sample, mince it, and add 4 volumes of low-salt wash buffer (0.1 M NaCl, 2 mM MgCl2, 1 mM EGTA, 10 mM phosphate buffer, pH 7.0). Homogenize using a high-speed homogenizer under ice bath conditions for 2 min. Then centrifuge at 4℃, 4000×g for 10 min and discard the supernatant (mainly water-soluble sarcoplasmic proteins). Repeat the low-salt washing step twice with the precipitate. Resuspend the washed precipitate in 4 volumes of high-salt extraction buffer (0.6 M NaCl, 10 mM phosphate buffer, pH 7.0), homogenize, and incubate at 4℃ for 2 h. Centrifuge again under the same conditions, and collect the supernatant as the purified myofibrillar protein (MP) solution. Measure and adjust the MP solution concentration to 2 mg / mL using a BCA protein quantification kit.

[0034] Example 1: Preparation of sunflower seed meal protein solution and screening of peptides 1. Preparation of sunflower seed meal protein solution (1) After mixing defatted sunflower seed meal powder with ultrapure water at a ratio of 1 g: 15 mL, the pH was adjusted to 8.0 with 1.0 M NaOH. After stirring at 600 rpm for 2 h at 25℃, the supernatant was collected by centrifugation at 8000 rpm for 15 min. The supernatant was heated at 100℃ for 15 min to denature and precipitate a large amount of conventional miscellaneous proteins, while retaining highly hydrophilic and heat-resistant late embryonic rich proteins (LEA protein components). The supernatant was collected by centrifugation at 8000 rpm for 15 min again, and then freeze-dried (freeze-dried at -50℃ for 48 h) to obtain sunflower seed meal protein powder. (2) Prepare a sunflower seed meal protein solution with a mass fraction of 5% by the sunflower seed meal protein powder obtained in step (1). Add 2% alkaline protease (Alcalase® 2.4 LFG, solution) of sunflower seed meal protein to the sunflower seed meal protein solution. After enzymatic hydrolysis at pH 8.0 and 50 ℃ for 2 h, treat at 100 ℃ for 10 min to inactivate the enzyme, cool, centrifuge to collect the supernatant, and use an ultrafiltration membrane to retain the components with a molecular weight of less than 3000 Da in the sunflower seed meal protease hydrolysate to obtain a sunflower seed meal protein solution rich in short peptides.

[0035] 2. Screening and identification of peptides in sunflower seed meal protein solution The sunflower seed meal protein solution rich in short peptides prepared above was subjected to separation, purification, and peptidomics analysis. The specific steps are as follows: (1) Crude extraction and ultrafiltration: The sunflower seed meal protein solution prepared above was centrifuged at 4℃ and 10000×g for 20 min, and the supernatant was obtained by filtration. The supernatant was transferred to an ultrafiltration centrifuge tube and centrifuged at 5000×g for 15 min. Ultrafiltration separation was performed using an ultrafiltration membrane with a molecular weight cutoff (MWCO) of 5000 Da, and the filtrate with a molecular weight <5000 Da was collected. Subsequently, the collected filtrate was dialyzed in 100 mL of ultrapure water for 24 h using a dialysis bag (MWCO 500 Da) to remove salt ions and very small molecular impurities from the system.

[0036] (2) Preliminary purification by gel chromatography: The hydrolyzed protein solution obtained by dialysis was separated by a HiLoad 16 / 600 Superdex 30 pg gel filtration column. 50 mM phosphate buffer with pH 7.0-7.5 was used as the mobile phase, the flow rate was set to 1 mL / min, and the elution time was 90 min. The UV absorption peak was monitored in real time at a wavelength of 220 nm, and the target eluted fractions with molecular weights in the range of 1000-3000 Da were collected. After collection, the preliminarily purified peptide powder was obtained by freeze drying.

[0037] (3) Fine purification by reversed-phase high-performance liquid chromatography (RP-HPLC): The pre-purified peptides were finely separated using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm particle size). Mobile phase A was ultrapure water (containing 0.1% TFA), and mobile phase B was acetonitrile (containing 0.1% TFA). The gradient elution program was set as follows: the initial condition was 5% B phase, which was held for 5 minutes for column equilibration; within 30-40 minutes, the linear gradient of B phase was increased to 50%; during column washing, B phase was increased to 90% within 2 minutes and held for 5 minutes; finally, it was rapidly reduced to 5% B phase and reequilibrated for 10-15 minutes. The lyophilized sample obtained by gel filtration was dissolved in a small amount of mobile phase A and loaded onto the column, and the above elution gradient was run. Each single fraction was collected manually or automatically based on the 220 nm UV absorption peak, and only the central part of each chromatographic peak was collected each time to ensure peptide purity. The collected fractions were freeze-dried to obtain high-purity white powder peptide fragments.

[0038] (4) LC-MS / MS Sequence Identification: The amino acid sequences of each purified peptide were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The mass spectrometry data were compared with those obtained by UniProt Sunflower (… Helianthus annuus Protein databases were searched and compared to identify target peptides with strong amphiphilicity and potential antifreeze activity. Target components were typically eluted with an organic phase concentration between 20% and 40%, and their b-ion and y-ion fragments were resolved by tandem mass spectrometry (MS / MS) to ultimately confirm their amino acid sequences.

[0039] Finally, the peptides that may have antifreeze properties were screened and identified as GTAQAAKEKAGETKA (SEQ ID NO.1), ASEGGQTRKEQLGE (SEQ ID NO.2), and GTAQAAKEKAGETKASGGQTRKEQLG (SEQ ID NO.3). The information of each peptide is shown in Table 1.

[0040] Table 1

[0041] Example 2: Performance determination of peptides The peptides GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, and GTAQAAKEKAGETKASGGQTRKEQLG were sent to a biotechnology company for synthesis and performance testing.

[0042] (1) Determination of ice crystal recrystallization inhibition (IRI) activity Peptide samples (GTAQAAKEKAGETKA, ASEGGQTRKEQLG, GTAQAAKEKAGETKASGGQTRKEQLG, and commercially available fish skin collagen) were prepared into a 10 mg / mL peptide solution with water. This solution was then mixed with an equal volume of 30% (w / w) sucrose solution. 10 μL of this mixture was dropped from a height of 2 meters onto a glass slide pre-cooled to -80 °C, forming a thin, instantaneously frozen ice film. The slide was then rapidly transferred to a polarizing microscope with a heating and cooling stage and annealed at -6 °C for 30 min. The average area of ​​the 10 largest ice crystals in the field of view was calculated using ImageJ software; a smaller area indicates stronger IRI activity. A pure water group was used as a control during the measurement process. (The lower the relative proportion of ice crystal area, the better the IRI effect), the calculation formula is as follows:

[0043] The results are shown in Table 2. The results show that the peptides GTAQAAKEKAGETKA, ASEGGQTRKEQLGE and GTAQAAKEKAGETKASGGQTRKEQLG have good ice crystal recrystallization inhibition activity. Among them, the peptide ASEGGQTRKEQLGE showed the best effect, with a relative ice crystal area ratio of only 33.9%.

[0044] Table 2 IRI Activity Assay

[0045] (2) Thermal hysteresis activity (THA) determination Peptides GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, GTAQAAKEKAGETKASGGQTRKEQLG, and commercially available fish skin collagen were each prepared into peptide solutions with a concentration of 10 mg / mL. 10 μL of each test solution was accurately pipetted into a DSC (Differential Scanning Calorimeter) dedicated aluminum liquid crucible and compacted and sealed to prevent moisture evaporation. At the same time, an equal volume of ultrapure water was set up as a control group. Throughout the test, high-purity nitrogen was used for purging protection at a rate of 50 mL / min. The initial temperature was set at 5 ℃ and held at this temperature for 5 min. Subsequently, the temperature was lowered to -30 ℃ at a constant cooling rate of 2 ℃ / min and held at this temperature for 5 min to ensure complete crystallization and freezing of the water in the sample system. After crystallization, the temperature was raised back to 5 ℃ at the same heating rate (2 ℃ / min). The heat flow curve was analyzed using the instrument's accompanying data analysis software. The exothermic peak initiation temperature during the cooling process was extracted as the crystallization temperature (Tc), and the endothermic peak peak value during the heating process was extracted as the melting temperature (Tm). Finally, the natural supercooling background value of the pure water blank group was subtracted. The specific thermal hysteresis activity (THA) of the antifreeze peptide was calculated using the formula THA (°C) = (Tm(sample) - Tc(sample)) - (Tm(blank) - Tc(blank)). In the formula, Tm (sample) and Tc (sample) are the melting temperature and crystallization temperature of the polypeptide sample group, respectively, and Tm (blank) and Tc (blank) are the melting temperature and crystallization temperature of the pure water blank group, respectively. The units are all ℃.

[0046] The results are shown in Table 3. The results indicate that, compared with pure water, all peptide treatment groups can effectively delay the freezing point of the system (lower the crystallization temperature Tc) and produce significant specific thermal hysteresis activity (THA). Among them, commercially available fish skin collagen peptides showed a certain basic antifreeze activity (THA of 0.45 °C).

[0047] In comparison, the three characteristic peptides synthesized in this invention all exhibited superior antifreeze potential compared to commercially available products. The shorter peptides ASEGGQTRKEQLGE and GTAQAAKEKAGETKA showed significantly better thermal hysteresis activity (THA values ​​of 0.55°C and 0.52°C, respectively), thanks to their high conformational flexibility in solution, enabling them to anchor themselves highly efficiently on the surface of tiny ice crystals. While the longer peptide GTAQAAKEKAGETKASGGQTRKEQLG showed slightly lower thermal hysteresis activity (0.49°C) than the two shorter peptides, its ice-suppressing efficacy still significantly surpassed that of high-end commercially available control products. This fully demonstrates the core technological advantages of the amphiphilic α-helical polypeptide sequences screened in this invention in targeted ice control and cryogenic protection.

[0048] Table 3

[0049] Example 3: Application of antifreeze protein peptides in pork tenderloin 1. Sample processing Fresh, commercially available lean pork tenderloin was used as the experimental material. The surface fascia and visible fat tissue were removed, and the tenderloin was cut into uniform pieces approximately 1 cm × 1 cm × 0.5 cm in size and weighing approximately 4 g each. A randomized controlled trial was conducted, with 5 experimental groups and 6 replicates in each group. The specific grouping method is as follows: Control group of pork tenderloin: The meat pieces were soaked in 20 mL of sterile saline.

[0050] Commercially available fish skin collagen group: The meat pieces were soaked in 20 mL of sterile saline containing commercially available fish skin collagen at a final concentration of 20,000 mg / L; GTAQAAKEKAGETKA group: Meat pieces were soaked in 20 mL of sterile saline containing a final concentration of 20,000 mg / L GTAQAAKEKAGETKA.

[0051] ASEGGQTRKEQLGE group: Meat pieces were soaked in 20 mL of sterile saline containing a final concentration of 20,000 mg / L ASEGGQTRKEQLGE.

[0052] Group GTAQAAKEKAGETKASGGQTRKEQLG: Meat chunks were soaked in 20 mL of sterile saline containing a final concentration of 20,000 mg / L GTAQAAKEKAGETKASGGQTRKEQLG.

[0053] Each group of meat pieces was completely immersed in the corresponding treatment solution and left to soak at a constant temperature of 4 ℃ for 2 h. After soaking, the meat pieces were removed and the residual solution on the surface was drained. Subsequently, all meat pieces were subjected to three rigorous freeze-thaw cycles (FTCs). The procedure for each freeze-thaw cycle was as follows: the meat pieces were frozen at -20 ℃ for 12 h, and then transferred to 4 ℃ to thaw for 4 h. This process was repeated three times.

[0054] 2. Performance testing of each group of samples After the above samples underwent freeze-thaw cycles, the thawing juice loss rate, myofibril protein oxidation degree, and textural properties of each group of meat pieces were tested. The specific testing methods are as follows: (1) Determination of thawing juice loss rate Accurately weigh the initial weight of each group of meat pieces before the freeze-thaw cycle, and record it as W1. After all three freeze-thaw cycles are completed, use filter paper to absorb the free moisture on the surface of the meat pieces, and accurately weigh the final weight of the meat pieces, recording it as W2. Calculate the thawing juice loss rate of the meat pieces according to the following formula:

[0055] The results are shown in Table 4. The results indicate that the freezing damage experienced by the small-sized meat pieces (high specific surface area) underwent three consecutive rigorous freeze-thaw cycles in this experiment was significantly amplified. In the pork tenderloin control group, without any added antifreeze, the large number of coarse ice crystals repeatedly generated inside the muscle fibers severely ruptured the cell membranes, leading to the collapse of the water-holding capacity of the myofibril protein network and a thawing juice loss rate as high as 22.4%. The commercially available fish skin collagen group showed a certain basic antifreeze and water-retention capacity, controlling its loss rate to 16.2%.

[0056] In comparison, the peptides GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, and GTAQAAKEKAGETKASGGQTRKEQLG exhibited superior preservation and protection effects. Among them, the GTAQAAKEKAGETKASGGQTRKEQLG peptide resulted in the lowest rate of juice loss during thawing of meat (13.2%). This confirms that GTAQAAKEKAGETKASGGQTRKEQLG, with its unique amphiphilic conformation and block structure, can more effectively anchor ice crystals and inhibit recrystallization at the microscopic level, effectively reducing the mechanical puncture damage to muscle cell membranes caused by ice crystals. Without using traditional chemical water-retaining agents such as phosphates, its effectiveness in preventing water loss surpasses the protective level of currently available fish skin collagen.

[0057] Table 4

[0058] Note: Data in the table are expressed as "mean ± standard deviation (Mean ± SD, n=6)". Different lowercase letters (a, b, c, d) after the data in the same column indicate that there is a significant difference between the groups (P < 0.05).

[0059] (2) Determination of the degree of oxidation of myofibril proteins (determination of thiol content) Myofibrillar protein (MP) was extracted from meat pieces after freeze-thaw treatment in each group. The total sulfhydryl content of the protein was determined by the DTNB colorimetric method. Specifically, 0.5 mL of 2 mg / mL MP solution was added to 4.5 mL of Tris-HCl buffer (0.2 M, pH 8.0, urea is used to expand the protein macromolecule conformation to fully expose the internal sulfhydryl groups). After mixing thoroughly, 0.5 mL of 0.1 M DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) reagent was added, and the mixture was reacted at 25℃ in the dark for 25 min. The absorbance of the mixture without protein solution was measured at a wavelength of 412 nm. The total sulfhydryl content was determined by the molar extinction coefficient (13600 M). -1 cm -1The results were calculated and expressed as nmol / mg protein. A higher thiol content in the sample indicates lower oxidative damage to myofibrillar proteins and better freeze-thaw resistance.

[0060] The results are shown in Table 5. The results indicate that after three freeze-thaw cycles, the pork tenderloin control group, lacking protection, suffered mechanical damage from ice crystals accompanied by intense oxidative stress, leading to disruption of the myofibril spatial structure and severe oxidative cross-linking of thiol groups (disulfide bond formation), resulting in a sharp drop in total thiol content to 32.5 nmol / mg. The commercially available fish skin collagen group showed some antioxidant protection. The GTAQAAKEKAGETKASGGQTRKEQLG group, benefiting from its excellent ice crystal physical barrier effect, largely maintained the natural conformation of myofibril protein, significantly maintaining the total thiol content at 55.2 nmol / mg, demonstrating superior microscopic antioxidant stress protection performance.

[0061] Table 5

[0062] Note: Data in the table are expressed as "mean ± standard deviation (Mean ± SD, n=6)". Different lowercase letters (a, b, c, d) after the data in the same column indicate that there is a significant difference between the groups (P < 0.05).

[0063] (3) Determination of textural properties The texture analyzer was used to perform secondary compression tests on the TPA texture profiles of meat blocks and fresh pork tenderloin after freeze-thaw cycles. A P / 36R dedicated probe was used, and the sample compression ratio was set to 40%. After the test, the two core texture indicators of meat block hardness and elasticity were collected and recorded to evaluate the changes in meat quality after freeze-thaw treatment.

[0064] The results are shown in Table 6. The results indicate that the control group experienced severe juice loss during repeated freeze-thaw cycles. Muscle fibers shrank and clustered together due to water loss, resulting in severe protein cross-linking and denaturation, leading to an increase in hardness to 58.6 N and a significant drop in elasticity to 0.62. Commercially available fish skin collagen alleviated meat aging to some extent. Meanwhile, the peptide GTAQAAKEKAGETKASGGQTRKEQLG successfully induced a liquid-liquid phase separation protective film at the cell interface, effectively anchoring ice crystals, locking in free water, and inhibiting protein denaturation. Its macroscopic texture indicators were closest to those of fresh raw meat, significantly avoiding the "lignification" and "dryness" common in frozen meat.

[0065] Table 6

[0066] Note: Data in the table are expressed as "mean ± standard deviation (Mean ± SD, n=6)". Different lowercase letters (a, b, c, d) after the data in the same column indicate that there is a significant difference between the groups (P < 0.05).

[0067] Example 4: Application of antifreeze protein peptides in chicken breast 1. Sample processing The specific implementation method is the same as step 1 in Example 3, except that the raw material pork tenderloin is replaced with fresh commercially available white-feathered chicken breast. The raw material pretreatment steps and the treatment methods of each experimental group are consistent with step 1 in Example 3.

[0068] 2. Performance testing of each group of samples After the above samples underwent freeze-thaw cycles, the thawing juice loss rate, myofibril protein oxidation degree, and textural properties of each group of meat pieces were tested. The specific test results are as follows: (1) Determination of thawing juice loss rate The thawing juice loss rate of each treated meat piece was determined using the same method as the thawing juice loss rate determination in Example 3.

[0069] The results are shown in Table 7. The results indicate that chicken breast muscle fibers are relatively fragile. After three rigorous freeze-thaw cycles, the saline-treated chicken breast control group suffered severe mechanical damage from ice crystals, with a thawing juice loss rate as high as 18.2%. All peptide treatment groups reduced water loss to varying degrees. The short peptides screened in this invention exhibited superior water-retention properties compared to fish skin collagen, with the GTAQAAKEKAGETKASGGQTRKEQLG group significantly reducing the loss rate to 10.4%.

[0070] Table 7

[0071] Note: Data in the table are expressed as "mean ± standard deviation (Mean ± SD, n=6)". Different lowercase letters (a, b, c, d) after the data in the same column indicate that there is a significant difference between the groups (P < 0.05).

[0072] (2) Determination of the degree of oxidation of myofibril proteins (determination of thiol content) Myofibrillar protein (MP) was extracted from meat pieces after freeze-thaw treatment in each group, and the total thiol content of the protein was determined by DTNB colorimetric method. The determination method was the same as that for the determination of myofibrillar protein oxidation degree in Example 3.

[0073] The results are shown in Table 8. The results indicate that after three freeze-thaw cycles, the chicken breast control group, lacking protection, suffered from mechanical damage from ice crystals accompanied by intense oxidative stress, leading to disruption of the myofibril structure and severe oxidative cross-linking of thiol groups, resulting in a sharp drop in total thiol content to 28.6 nmol / mg. The commercially available fish skin collagen group showed some antioxidant protection (37.4 nmol / mg). The peptide GTAQAAKEKAGETKASGGQTRKEQLG, benefiting from its excellent ice crystal physical barrier effect, largely maintained the natural conformation of chicken breast myofibrils, significantly maintaining the total thiol content at 50.8 nmol / mg, demonstrating superior microscopic antioxidant stress protection performance.

[0074] Table 8

[0075] Note: Data in the table are expressed as "mean ± standard deviation (Mean ± SD, n=6)". Different lowercase letters (a, b, c, d) after the data in the same column indicate that there is a significant difference between the groups (P < 0.05).

[0076] (3) Determination of textural properties The texture of each treated meat piece and fresh chicken breast was measured using the same method as the texture property determination in Example 3.

[0077] The results are shown in Table 9. The results indicate that the chicken breast control group experienced severe juice loss during repeated freeze-thaw cycles. The muscle fibers shrank and clustered due to water loss, leading to an increase in hardness to 45.3 N and a decrease in elasticity to 0.58. Commercially available fish skin collagen alleviated the aging of the chicken meat to some extent. Meanwhile, the peptide GTAQAAKEKAGETKASGGQTRKEQLG successfully induced a phase separation protective membrane at the cell interface, effectively anchoring ice crystals and locking in free water. Its macroscopic texture indicators (hardness 29.5 N, elasticity 0.79) were closest to those of fresh chicken meat, significantly avoiding the "dry" and "wood-like" feel commonly found in thawed frozen chicken breast.

[0078] Table 9

[0079] Note: Data in the table are expressed as "mean ± standard deviation (Mean ± SD, n=6)". Different lowercase letters (a, b, c, d) after the data in the same column indicate that there is a significant difference between the groups (P < 0.05).

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An engineered strain of *Escherichia coli*, characterized in that, The engineered Escherichia coli expressed the following protein peptides: GTAQAAKEKAGETKA (as shown in SEQ ID NO.1), ASEGGQTRKEQLGE (as shown in SEQ ID NO.2), or GTAQAAKEKAGETKASGGQTRKEQLG (as shown in SEQ ID NO.3).

2. A protein peptide, characterized in that, The protein peptide is GTAQAAKEKAGETKA, ASEGGQTRKEQLGE, or GTAQAAKEKAGETKASGGQTRKEQLG, and its amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3, respectively.

3. A method for preparing sunflower seed meal protein solution, characterized in that, The sunflower seed meal protein solution contains the protein peptides described in claim 2, characterized by comprising the following steps: (1) Mix defatted sunflower seed meal powder with water at a ratio of 1~5 g: 10~25 mL, stir, centrifuge and collect the supernatant; heat the supernatant and centrifuge again to collect the supernatant and freeze dry to obtain sunflower seed meal protein powder; (2) Prepare a sunflower seed meal protein solution with a mass fraction of 5-10% by the sunflower seed meal protein powder obtained in step (1), and add alkaline protease of 2% by mass of sunflower seed meal protein powder to it for enzymatic hydrolysis, enzyme inactivation, cooling, centrifugation and ultrafiltration to obtain sunflower seed meal protein solution. In step (2), the enzyme activity of alkaline protease is 2000~80000 U / g.

4. The method according to claim 3, characterized in that, The enzymatic hydrolysis described in step (2) is carried out at pH 7.5~9 and 45~55℃ for 0.5~3 h.

5. An expression carrier, characterized in that, The expression vector encodes the protein peptide of claim 2.

6. A product characterized in that, The product contains the protein peptide as described in claim 2.

7. The product according to claim 6, characterized in that, The products mentioned may be food, medicine, or health products.

8. A method for improving the antifreeze properties of meat products, characterized in that, The method involves processing meat products using the protein peptides of claim 2; The treatment may be at least one of soaking, marinating, spraying, injection, tumbling, coating, and vacuum impregnation.

9. The method according to claim 8, characterized in that, The improvement of the antifreeze properties of meat products includes reducing the loss of juices during thawing, reducing the degree of myofibril protein oxidation, and stabilizing the texture of meat products.

10. The use of the protein peptide according to claim 2 in the preparation of antifreeze products.

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