Application of annexin A1 in improving quality of slaughtered meat

Abstract

The invention discloses an application of annexin A1 in improvement of post-slaughter meat quality, and the annexin A1 can effectively improve meat tenderness, enhance water retention, improve moisture distribution, reduce free fatty acid content, enhance cell membrane integrity, inhibit protein oxidation, improve rheological properties and improve fluorescence intensity. The overall quality of meat is improved; the invention deeply studies the influence of annexin A1 on rheological properties, water retention, tenderness and related chemical properties of myofibrillar protein, thereby developing the new application of annexin A1, providing a new idea and method for the improvement of meat processing technology, and having broad market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of biomacromolecule application, in particular to the application of annexin A1 in improving the quality of post-slaughter meat. Background Art

[0002] Annexin A1 is a protein with a molecular weight of about 37 kDa, belonging to the annexin superfamily. Its structure contains multiple repeated annexin core domains, which have the ability to bind to calcium ions and phospholipids, allowing Annexin A1 to play important biological functions inside and outside cells. Annexin A1 plays a key role in a variety of physiological and pathological processes, including participating in cell apoptosis, regulating cell signal transduction, mediating cell adhesion and migration, participating in inflammatory response, and affecting adipogenesis.

[0003] At present, the application of Annexin A1 is mainly concentrated in the following fields: Medical research: Annexin A1 has attracted extensive attention in the research of various diseases such as cancer, cardiovascular disease and diabetic nephropathy. Its role in cell proliferation, migration and apoptosis makes it a potential disease marker and therapeutic target. Inflammatory response: Annexin A1 is considered to be a pro-resolving mediator of inflammation, which can participate in the occurrence, development and resolution of inflammation by regulating cell behavior. Cell signal transduction: Annexin A1 plays an important role in the signal transduction pathway, and can interact with a variety of signal molecules to regulate cell proliferation and differentiation. It can be seen that Annexin A1 is a biological macromolecule with important scientific research and application value in medicine and physiology.

[0004] Water retention, tenderness and other qualities are the problems that restrict the development of my country's yellow-feathered broiler processing industry. Not only do they make the weight and quality losses much higher than those in developed countries, but they also limit consumers' acceptance and willingness to buy fresh yellow-feathered broilers. Therefore, exploring effective ways to improve water retention and other qualities is a challenge and major demand facing the yellow-feathered broiler industry. Annexin A1 is a member of the calcium-dependent phospholipid binding protein Annexins superfamily. It is widely present in eukaryotic cells, participates in a variety of membrane-related physiological activities, and is beneficial to the maintenance of cell membrane integrity and stability. However, the role of Annexin A1 in the formation and regulation of fresh meat quality remains blank.

[0005] In this context, further application development of annexin A1 has attracted the attention of researchers. However, although there are literatures that have explored the role of annexin A1 in cell biology, its specific mechanism in improving the quality of post-slaughter meat has not been systematically studied. Therefore, it is necessary to improve the research on annexin A1 in improving the quality of post-slaughter meat. Summary of the invention

[0006] The technical problem to be solved by the present invention is to develop new uses of the biological macromolecule annexin A1.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] Application of Annexin A1 in improving tenderness and texture of post-slaughter meat.

[0009] Application of annexin A1 in improving water retention of post-slaughter meat.

[0010] Further preferably, the improving water retention capacity includes inhibiting the oxidation of myofibrillar protein in post-slaughter meat, increasing the total thiol content and improving the hydrophilicity of the protein.

[0011] The application of annexin A1 in improving the moisture distribution of meat after slaughter includes increasing the proportion of immobile water and reducing the proportion of free water in meat, and enhancing the binding between water and protein.

[0012] Application of annexin A1 in reducing the content of free unsaturated fatty acids in post-slaughter meat.

[0013] Application of annexin A1 in promoting the hydrolysis of myofibrillar proteins in post-slaughter meat, increasing the activity of calpains and enhancing the integrity of cell membranes.

[0014] Application of annexin A1 in improving the dissociation degree of actomyosin in post-slaughter meat, reducing the cross-linking between myofibrillar proteins and enhancing the integrity of cell membrane structure.

[0015] Application of annexin A1 in inducing the disordered transformation of myofibrillar protein structure in post-slaughter meat, enhancing the sulfhydryl content, inhibiting protein oxidation and improving fluorescence intensity.

[0016] Application of annexin A1 in improving the rheological properties of myofibrillar proteins in post-mortem meat and enhancing the formation and strength of gel networks.

[0017] More preferably, the above-mentioned meat is chicken.

[0018] The beneficial effects of adopting the above technical scheme are: based on the technical scheme of the present invention, membrane-annexin A1 can effectively improve the tenderness of meat, enhance water retention, improve water distribution, reduce free fatty acid content, enhance cell membrane integrity, inhibit protein oxidation, improve rheological properties, increase fluorescence intensity, and improve the overall quality of meat; the present invention conducts in-depth research on the influence of membrane-annexin A1 on the rheological properties, water retention, tenderness and related chemical properties of myofibrillar protein, thereby developing new uses of membrane-annexin A1, providing new ideas and methods for the improvement of meat processing technology, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the effect of annexin A1 of the present invention on the water loss of chicken after slaughter;

[0020] Figure 2 Schematic diagram of the effect of the annexin A1 of the present invention on the water distribution and hydrogen proton distribution in post-slaughter chicken, wherein A: water distribution diagram; B: hydrogen proton distribution diagram; a: control group; b: 50 μg / ml annexin A1 treatment group; c: 100 μg / ml annexin A1 treatment group; d: 150 μg / ml annexin A1 treatment group;

[0021] Figure 3 Schematic diagram showing the effect of annexin A1 of the present invention on the myofibril fragmentation index in post-slaughter chicken;

[0022] Figure 4 The SDS-PAGE diagram of myofibrillar protein and the quantitative analysis diagram of actin of the present invention, wherein A: SDS-PAGE diagram; B: quantitative analysis diagram; 1: control group; 2.: 50 μg / ml annexin A1 treatment group; 3.: 100 μg / ml annexin A1 treatment group; 4: 150 μg / ml annexin A1 treatment group; M.: standard molecular weight (unit is kDa);

[0023] Figure 5 It is the oxidation change of myofibrillar protein after the annexin A1 treatment of the present invention, wherein A: total thiol content diagram; B: protein solubility diagram;

[0024] Figure 6 Schematic diagram of the effect of annexin A1 of the present invention on the structure of myofibrillar protein, wherein A: circular dichroism spectrum; B: relative percentage of secondary structure; C: Fourier transform infrared spectrum; D: endogenous fluorescence chromatogram;

[0025] Figure 7 Schematic diagram of the effect of annexin A1 of the present invention on the rheological properties of myofibrillar protein, wherein A: change in viscosity; B: change in storage modulus. DETAILED DESCRIPTION

[0026] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained by purchasing from the market. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0027] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or combinations thereof.

[0028] It should also be understood that the term “and / or” used in the specification and appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0029] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0030] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0031] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The research team of this invention will explore the effect of annexin A1 on the quality of post-slaughter meat, develop the application of annexin A1 in improving the quality of post-slaughter meat, and through a series of experiments analyze the changes in myofibrillar proteins and free fatty acids treated with annexin A1, explore the possible ways in which annexin A1 affects the quality of post-slaughter meat, and provide ideas for the development of new meat quality regulation technologies.

[0033] Example 1 Materials and Reagents

[0034] Main ingredient: chicken breast.

[0035] Main reagents: magnesium chloride, EGTA, potassium chloride, and EDTA were purchased from Tianjin Komiou Chemical Reagent Co., Ltd.; urea and potassium hydroxide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; petroleum ether, ether, chloroform, isopropanol, acetic acid, n-hexane, and DTNB were purchased from Shanghai McLean Biochemical Technology Co., Ltd.; 14% boron trifluoride-methanol solution and 2,2-dimethoxypropane were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Coomassie Brilliant Blue Kit was purchased from Shanghai Sangon Bioengineering Co., Ltd.; membrane annexin A1 was purchased from MCE; sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, methanol and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0036] MFI buffer: 0.1 M potassium chloride, 8.8 mM potassium dihydrogen phosphate, 11.2 mM potassium dihydrogen phosphate, 1 mM magnesium chloride, 1 mM EGTA, pH 7.0.

[0037] Myofibrillar protein isolation buffer A: 0.1 M NaCl, 10 mM KH2PO4, 10 mM KH2PO4, 1 mM EGTA, 2 mM MgCl2, pH 7.0.

[0038] Myofibrillar protein isolation buffer B: 0.1 M NaCl, pH 6.0.

[0039] PB buffer: 0.6 M potassium chloride, 10 mM potassium phosphate dibasic, pH 6.0.

[0040] Thiol buffer: 0.2 M Tris-HCl, 3 mM EDTA, 8 M urea, pH 8.0.

[0041] DTNB solution: 0.2 M Tris-HCl, 10 mM DTNB, pH 8.0.

[0042] Example 2 Instruments and Equipment

[0043] Table 1 Experimental instruments and equipment

[0044]

[0045] Example 3 Experimental methods

[0046] 3.1 Experimental material processing

[0047] The left and right breasts of the same chicken were used as the control group and the treatment group, respectively. The treatment group was injected with different concentrations (50μg / mL, 100μg / mL, 150μg / mL) of annexin A1 protein solution at a meat-liquid ratio of 1:0.5 (w / v), with an injection interval of 1.5cm, and the control group was injected with PBS buffer at the same ratio. Both groups of samples were incubated at 4℃, and samples were taken after 24 hours for determination and analysis of various indicators.

[0048] 3.2 Determination of water loss

[0049] Specifically, it includes drip loss determination, centrifugal loss determination and cooking loss determination.

[0050] (1) The specific steps for determining drip loss are as follows:

[0051] The drip loss of chicken was determined according to the method of Liu et al. (2022). First, the chicken breast sample was cut into 40mm×20mm×20mm long strips, and the excess water on the surface was wiped off and weighed, recorded as m1. Then it was hung in a plastic bag, placed in a 4℃ environment for 24 hours, and weighed again, recorded as m2. The drip loss rate was calculated according to the following formula:

[0052]

[0053] (2) The specific steps of centrifugal loss determination are as follows:

[0054] The centrifugal loss of chicken was determined by referring to the method of Xu et al. (2023) and making appropriate adjustments thereto. Wrap about 10 g of chopped chicken breast sample (m1) with filter paper and place it in a 100 mL centrifuge tube. Then centrifuge at 2500 rpm for 20 min in a low temperature environment of 4°C. After that, remove the filter paper, wipe off the excess water, weigh the minced meat again (m2), and calculate the centrifugal loss rate according to the following formula:

[0055]

[0056] (3) The specific steps for determining the cooking loss are as follows:

[0057] According to the method of Liu et al. (2023), the cooking loss of chicken was determined. The chicken breast was cut into 80mm×40mm×40mm pieces and weighed as m1. Subsequently, a digital thermometer was inserted into the geometric center of the meat piece as an indicator that the meat had reached the target temperature. The meat piece was then placed at the bottom of the cooking bag, and 4 marbles were added to each bag to prevent the bag from floating in the water bath. The sample was then placed in an 85°C water bath and removed when the center temperature of the meat reached 75°C. After the temperature of the meat dropped to room temperature, excess moisture on the surface was wiped off and weighed again as m2. The cooking loss rate was calculated according to the following formula:

[0058]

[0059] 3.3 Shear force measurement

[0060] Cut the steamed chicken breast into 75mm×10mm×10mm long strips along the muscle fiber direction, then place them in the tenderness tester and cut perpendicularly to the fiber direction of the chicken breast. The maximum stress value recorded in this process is the shear force value (N). Each sample is tested at least six times, and the average value is taken as the final result.

[0061] 3.4 Texture

[0062] Cut the steamed chicken into uniform pieces (20mm×20mm×15mm) along the direction of the muscle fibers, and keep the surface of the pieces smooth and flat. Then place them steadily on the horizontal platform of the tester, and select the TA-35 flat-bottomed cylindrical probe as the test tool. Under room temperature, measure the key parameters of chicken such as hardness, elasticity, chewiness and stickiness. Each group of samples is measured at least 6 times and the average is taken.

[0063] 3.5 Low-field NMR determination of water distribution

[0064] Chicken breast (2 g) was carefully cut along the direction of the muscle fibers. It was then placed in a 60 mm cylindrical tube and the NMR probe was inserted. The Carr-Purcell-Meiboom-Gillsequences (CPMG) pulse sequence was used to measure the transverse relaxation time (T2). The specific parameters were set as follows: the sampling wait time (TR) was 2000 ms, the sampling frequency (SW) was set to 100 kHz, the number of repeated scans (NS) was 8, and the number of echoes (NECH) was set to 3500. Finally, we used the MultiExpInv analysis software to invert the measured attenuation curve to obtain detailed moisture distribution information.

[0065] 3.6 Determination of free fatty acids

[0066] 3.6.1 Extraction of total lipids from muscle

[0067] The processed chicken samples were freeze-dried and then crushed, 4 g of meat powder was accurately weighed, and a petroleum ether-ether mixed solution (3:2, v / v) was added at a ratio of 1:10 (w / v), mixed well and allowed to stand overnight. The next day, the upper liquid was aspirated into a clean centrifuge tube, and centrifuged at 4°C, 3000 rpm for 5 min. The supernatant was then transferred to a clean centrifuge tube, dried with nitrogen, and stored in a -40°C refrigerator for use.

[0068] 3.6.2 Separation and determination of lipids

[0069] Chloroform was added to the total lipid sample at a ratio of 1:20 (w / v). After fully dissolved, 1 mL of the total lipid solution was pipetted into an aminopropyl silica gel column pre-activated with 2 mL of chloroform. After the lipid solution flowed through, the aminopropyl silica gel column was eluted with a chloroform-isopropanol mixed solution (2:1, v / v), an acetic acid-ether mixed solution (1:49, w / w), and a methanol solution in sequence to obtain neutral lipids, free fatty acids, and phospholipids. Finally, the free fatty acid solution was blown dry with nitrogen, sealed, and stored in a -40°C refrigerator for later use.

[0070] 3.6.3 Composition and content of free fatty acids

[0071] Add 1 mL of tridecanonic acid internal standard solution (5 mg / mL) to the above free fatty acid sample, and then perform methyl esterification. After the reaction is completed, add 1 mL of saturated sodium chloride solution and n-hexane solution, and shake vigorously to mix thoroughly. Then let it stand in a low temperature environment of 4°C. After the mixed solution is naturally separated, carefully pipette the upper layer of liquid into a clean centrifuge tube, blow dry with nitrogen, and dissolve it again in 200 μL of n-hexane to prepare for gas chromatography analysis.

[0072] 3.7 Myofibrillar fragmentation index (MFI)

[0073] Weigh about 10g of chicken breast, add MFI buffer at a ratio of 1:5 (w / v), homogenize at high speed for 90s, then centrifuge at 5000rpm at 4℃, and collect the precipitate after 15min. Repeat this process twice. Then resuspend the precipitate obtained for the third time with 25mL of pre-cooled MFI buffer, filter, add 25mL of buffer to rinse the filter and collect the filtrate. After determining the protein concentration of the filtrate, adjust the concentration to 0.2mg / mL, and finally use an enzyme marker to measure the absorbance of the filtrate at 540nm. The obtained value × 200 is the MFI value.

[0074] 3.8 Myofibrillar protein extraction and SDS-PAGE analysis

[0075] 3.8.1 Myofibrillar protein extraction

[0076] Myofibrillar protein was extracted according to the method of Cheng et al. (2023) and some adjustments were made. Weigh about 10 g of chicken breast meat, add MP separation buffer A at a ratio of 1:5 (w / v), stir evenly with a glass rod, homogenize at high speed for 90 s (10000 r / min, 30 s / time, 3 times), centrifuge at 4°C, 5000 rpm for 15 min, discard the supernatant and take the precipitate, and repeat this step 3 times. Then add pre-cooled MP separation buffer B to the obtained precipitate at a ratio of 1:5 (v / v), homogenize again at high speed for 60 s (10000 r / min, 30 s / time, 2 times), centrifuge at 4°C, 5000 rpm for 15 min, take the precipitate, repeat this step 2 times, pass through 80 mesh medical filter cloth (4 layers) to remove insoluble connective tissue, and finally centrifuge at 4°C, 5000 rpm for 15 min to obtain purified MP precipitate. In the subsequent determination step, the MP precipitate can be dissolved in an appropriate amount of PB buffer to prepare a protein solution. Store in sealed container at 4℃ and use up within 48 hours.

[0077] 3.8.2 SDS-PAGE electrophoresis detection of myofibrillar proteins

[0078] Using bovine serum albumin as the standard, the concentration of the MP solution extracted above was determined by the biuret method. After adjusting the protein concentration to 1 mg / mL, 100 μL of MP solution was mixed with 25 μL of protein loading buffer (5×), and heated at 95°C for 5 min. Subsequently, 5 μL of the supernatant after centrifugation was aspirated into the SDS-PAGE protein gel well, and the concentrated gel was run at a constant voltage of 80 V and then at a constant voltage of 120 V for 50 min.

[0079] 3.9 Determination of myofibrillar protein solubility

[0080] The concentration of the extracted myofibrillar protein was adjusted to 2.5 mg / mL using PB buffer, and then placed in a 4°C environment for 2 hours. The protein solution was then centrifuged at 6000 rpm at 4°C, and the concentration of the supernatant was measured after 15 minutes. The MP protein solubility was calculated according to the following formula:

[0081] Protein solubility = supernatant protein concentration / protein concentration before centrifugation × 100%;

[0082] 3.10 Determination of total thiol content of myofibrillar protein

[0083] Referring to the method of Yongsawatdigul et al. (2003), the 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) method was used to determine the total thiol content in the sample. Use PB buffer to adjust the concentration of MP solution to 4 mg / mL, add 4.5 mL of thiol buffer to 0.5 mL of MP solution and mix thoroughly (use PB buffer instead of MP solution for blank control). After that, take out 4 mL of the mixed solution into a clean centrifuge tube, add 0.5 mL of DTNB solution and mix again, then place the mixed solution in a 40°C water bath and incubate for 25 minutes. Use an enzyme marker to measure the absorbance of the mixed solution at 412 nm, and use the absorbance coefficient of 13600M-1cm-1 to calculate the total number of thiols. Calculate the total thiol content according to the following formula:

[0084]

[0085] Where: A—absorbance value at 412nm;

[0086] d—myofibrillar protein dilution multiple;

[0087] c—concentration of the myofibrillar protein solution to be tested;

[0088] ξ—molecular absorption coefficient (13600M-1cm-1)

[0089] 3.11 Determination of myofibrillar protein structure

[0090] 3.11.1 Circular dichroism (CD)

[0091] The MP concentration was adjusted to 0.2 mg / mL using PB buffer and then detected using a circular dichroism spectrometer. During the measurement, a quartz cell with an optical path length of 1 mm was selected. The scanning wavelength was set to 190-250 nm, the scanning speed was 2 nm / s, and the scanning time was 1 s. Subsequently, the circular dichroism data were analyzed using SELCON3 software and expressed by the average residue ellipticity (θ, deg·cm2·dmol-1).

[0092] 3.11.2 Intrinsic fluorescence spectroscopy

[0093] After adjusting the MP concentration to 0.4 mg / mL, the fluorescence spectrum was measured at room temperature using a fluorescence spectrophotometer. The excitation wavelength was set to 280 nm, the emission wavelength was set to 290-400 nm, the slit was set to 5 nm, and the scanning speed was set to 600 nm / min. PB buffer was used as the blank solution for the MP sample.

[0094] 3.11.3 Fourier transform infrared spectroscopy (FTIR)

[0095] After the MP precipitate was freeze-dried in vacuum, FTIR was used to collect its spectral data in the range of 4000-400 cm-1 (32 scans / sample or background) with a resolution of 4 cm-1. Before each acquisition, the ATR crystal was wiped with ethanol and blown dry to prevent the residual MP in the measurement gap from affecting the results.

[0096] 3.12 Rheological properties determination

[0097] Adjust the concentration of MP solution to 8 mg / mL, and use a rheometer to analyze the rheological properties of MP solution. Pipette an appropriate amount of solution and place it on a 40 mm flat sample stage, and set the plate spacing to 1.0 mm. Measure the apparent viscosity of the sample in the shear rate range of 0.1 to 100 s-1; perform a frequency scan on the MP solution sample in the angular frequency range of 0.1 to 100 rad / sω, set the strain to 0.5%, and analyze the storage modulus (G') of the sample.

[0098] Example 4 Results and Discussion

[0099] 4.1 Effect of Annexin A1 on tenderness and texture

[0100] Texture is widely used in the evaluation of the quality of various foods. Its main indicators include hardness, elasticity, chewiness and stickiness, which are usually highly correlated with meat quality (Choe et al., 2016). Shear force is often used to evaluate the tenderness of meat. The results of texture (TPA) and shear force are shown in Table 2. After treatment with annexin A1, the hardness, elasticity, chewiness, stickiness and shear force of post-slaughter chicken meat changed. Compared with the control group, treatment with different concentrations of annexin A1 significantly reduced the hardness of chicken meat (P<0.05), which decreased by 40.29%, 37.29% and 40.20%, respectively, accompanied by lower chewiness and stickiness. When the concentration of annexin A1 was 50 μg / mL, the chewiness and stickiness of chicken meat were the lowest (P<0.05). Changes in hardness are usually related to water loss. Guo et al. (2024) treated beef with a pulsed electric field, measured its texture and cooking loss, and found that beef samples with high water loss also had high hardness. Annexin A1 treatment reduced hardness and reduced water loss in chicken. In addition, the shear force of chicken was the smallest when the concentration of Annexin A1 was 100 μg / mL, which was 45.32% lower than that of the control group. The increase in shear force is due to the tighter muscle fiber structure caused by water loss, and Annexin A1 improves the tenderness of chicken by reducing water loss.

[0101] Table 2 Effects of annexin A1 on tenderness and texture of post-slaughter chicken

[0102]

[0103] Note: Values ​​are expressed as mean ± standard deviation; values ​​with different lowercase letters in the same row indicate significant differences (P<0.05).

[0104] 4.2 Effect of annexin A1 on water retention

[0105] Water retention is one of the important indicators for the meat industry and consumers to measure meat quality, which directly affects the taste, edible value and processing characteristics of meat. A decrease in water retention may lead to increased economic losses in the meat industry, and it will also damage the sensory quality of meat and affect consumers' purchasing desire (Barbera, 2019; Zhang et al., 2019). Therefore, in meat processing, quality control and product development, water retention is a quality that needs to be focused on and optimized. This study analyzed the water retention of post-slaughter chicken by measuring drip loss, centrifugal loss and cooking loss. The measurement results are as follows: Figure 1 As shown in the data, there was a significant difference in water loss between the control group and the annexin A1 treatment group (P<0.05), and different concentrations of annexin A1 had different effects on water loss. When the concentration of annexin A1 was 50 μg / mL, the cooking loss was the lowest (P<0.05), which was 33.15% lower than that of the control group. The dripping loss was the lowest when the concentration of annexin A1 was 100 μg / mL, which was 43.05% lower than that of the control group. The centrifugal loss showed a trend of first decreasing and then increasing. When the concentration of annexin A1 was 50 μg / mL, the centrifugal loss of meat was the lowest, which was 26.32% lower than that of the control group. In summary, the addition of annexin A1 significantly improved the water retention of chicken. In addition, the loss of muscle water after slaughter will increase the hardness and chewiness of meat (Nishimura, 2010), so the improvement of the water retention of chicken by annexin A1 helps to improve tenderness.

[0106] The ischemic and hypoxic environment after slaughter induces an increase in calcium ion concentration, a decrease in phospholipid creatine and ATP, and thus activates PLA2 to hydrolyze cell membrane phospholipids, which ultimately leads to increased cell membrane permeability, decreased fluidity, and the outflow of substances that maintain osmotic pressure in the cell (such as K+, protein, sulfuric acid, etc.), which causes hypertonicity outside the cell and a large outflow of intracellular water to form juice loss. In addition, the destruction of the cell membrane makes the cell more sensitive to external oxidants, and the free fatty acids produced by membrane lipid hydrolysis, especially unsaturated fatty acids, are more susceptible to attack by various oxidative factors (Shen et al., 2021), and PLA2 can promote membrane lipid oxidation (Tatiyaborworntham and Richards, 2015), thereby further damaging the cell membrane and causing juice loss. Annexin A1, as a PLA2 inhibitory protein, inhibits the activity of PLA2, reduces cell membrane lipid hydrolysis, and improves the water holding capacity of chicken by maintaining the integrity of the muscle cell membrane structure after slaughter.

[0107] 4.3 Effect of annexin A1 on water distribution

[0108] Traditional methods for studying water retention, such as centrifugation, drip loss, and cooking loss, cannot reflect the spatial distribution information of water in meat, and will destroy the structure and properties of meat during detection (Oswell et al., 2021). Low-field nuclear magnetic resonance technology (LF-NMR) can provide information about the interaction between water protons and exchangeable protons in proteins, thereby providing the chemical and physical state of water in muscle. Among them, LF-NMR transverse relaxation (T2) can quantify the water distribution and fluidity changes in meat without destroying the muscle structure, and has been widely used in the study of water retention of meat products (Liu et al., 2022).

[0109] In this study, the effect of annexin A1 on the moisture distribution of chicken meat after slaughter was as follows: Figure 2 As shown in Figure 1, there are three T2 intervals in the post-slaughter muscle, namely, T 2b (0~10ms), T 21 (10~100ms), T 22 (100~1000ms), which is consistent with the research results of Guo et al. (2023). 2b Water that is tightly bound to other macromolecules through molecular forces is called bound water and is not affected by post-mortem muscle maturation (Zhao et al., 2023). 21 The water between the thick and thin filaments of myofibrils is called immobile water and is the main water in skeletal muscle. 22 The free water in the extracellular space is called free water, which is the main factor in the formation of drip loss (Wolkers et al., 2019). 2b 、T 21 、T 22 ) corresponds to the exponential area ratio (P 2b , P 21 , P 22 ) represents the relative content of water in three different states (Zhao et al., 2023). As shown in Table 3, chicken meat treated with different concentrations of annexin A1 had a 21 The values ​​decreased, P 21 The proportion increased significantly, P 22 The ratio was significantly reduced (P<0.05), indicating that annexin A1 causes a faster relaxation rate in meat, which can increase the proportion of immobile water in chicken and reduce the proportion of free water, suggesting that the binding between water molecules and proteins is tighter, which helps to reduce the drip loss of chicken. In addition, NMR can show the distribution of hydrogen protons in meat, such as Figure 2-B, the red areas in the image correspond to higher hydrogen protons, indicating that these areas have higher moisture content. The red areas in the Annexin A1-treated group were significantly higher than those in the control group, indicating that Annexin A1 helps to increase the moisture content in chicken.

[0110] T 21 It is closely related to the contraction of muscle fibers (Li et al., 2018). Yang et al. (2022) found that when myofibrils were treated under conditions similar to PSE, the distance between thick filaments and thin filaments was reduced to the maximum. At this time, T 21 The value is the smallest, the water retention of myofibrils is the lowest, and the centrifugal loss is the largest. Therefore, the contraction of myofibrils will lead to water loss and reduce the proportion of water that is not easily mobile in the muscle. When cytoskeletal proteins are degraded, the connection between myofibrils and the entire muscle fiber can be weakened or eliminated, thereby weakening the contraction of the myofibril network structure, allowing more water to be retained in the myofibrils and improving water retention (Li et al., 2018). Annexin A1 helps reduce the contraction of myofibrils, thereby improving the water retention of chicken.

[0111] Table 3 Effects of annexin A1 on post-mortem chicken T2

[0112]

[0113] Note: The values ​​are expressed as mean ± standard deviation; different lowercase letters in the same column indicate significant differences (P<0.05)

[0114] 4.4 Effect of Annexin A1 on the composition and content of free fatty acids

[0115] Free fatty acids were separated and extracted from chicken using aminopropyl silica gel column, and the composition and content of free fatty acids were analyzed by gas chromatography after the total free fatty acid mass was determined. The test results are shown in Table 4. There are 17 kinds of free fatty acids in chicken after slaughter, among which cis-10-pentadecenoic acid, palmitoleic acid, oleic acid, linoleic acid, γ-linolenic acid and Homo-γ-linolenic acid are unsaturated fatty acids. Compared with the control group, the total free fatty acid content decreased by 53.32% and 50.44% when the concentration of annexin A1 was 50μg / mL and 100μg / mL, respectively (P<0.05). In the control group, total unsaturated fatty acids accounted for about 10.40% of the total free fatty acids. After treatment with different concentrations of annexin A1, the proportion of total unsaturated fatty acids in the total free fatty acids decreased by 25%, 39.62% and 46.83%, respectively (P<0.01). The contents of butyric acid, myristic acid, palmitoleic acid, oleic acid, linoleic acid, γ-linolenic acid, heneicosanoic acid and Homo-γ-linolenic acid were lowest when the concentration of annexin A1 was 100 μg / mL (P<0.01). Most of these fatty acids were unsaturated fatty acids, which was consistent with the result that the content of total unsaturated fatty acids was lowest when the concentration of annexin A1 was 100 μg / mL.

[0116] Annexin A1 significantly reduced the content of free fatty acids in post-slaughter chicken, especially the content of free unsaturated fatty acids. Free fatty acids in post-slaughter muscle are mainly hydrolyzed by phospholipids in the muscle through phospholipases (Huang et al., 2014). Therefore, the reduction in the content of free fatty acids suggests that the addition of annexin A1 inhibits the hydrolysis of phospholipids in post-slaughter muscle. PLA2 is a key phospholipase that promotes the hydrolysis of intramuscular phospholipids and is closely related to the production of unsaturated fatty acids (Wang Daoying, 2014). Annexin A1 is an important inhibitory molecule of PLA2 (Vecchi et al., 2021). Recombinant protein annexin A1 can effectively inhibit PLA2 activity. Therefore, annexin A1 inhibits the hydrolysis of intramuscular phospholipids after slaughter by inhibiting PLA2 activity, reducing the content of free fatty acids in chicken, especially free unsaturated fatty acids. In addition, intramuscular phospholipids are mainly present on the cell membrane, including phosphatidylcholine, phosphatidylinositol, phosphatidylserine, etc., which play an important role in the structure and function of the cell membrane. PLA2 destroys the integrity of cell membrane lipids by releasing arachidonic acid (AA) from the sn-2 position of membrane phospholipids and increases lipid peroxidation (Oh et al., 2023). Annexin A1 maintains the integrity of cell membrane lipids by inhibiting PLA2 activity, thereby improving meat quality.

[0117] Table 4 Effects of annexin A1 on the composition and content of free fatty acids in post-mortem chicken

[0118]

[0119]

[0120] Note: Unit of free fatty acid content (mg / 100g meat); Values ​​are expressed as mean ± standard deviation; Different lowercase letters in the same row indicate significant differences (P<0.05), and different uppercase letters in the same row indicate extremely significant differences (P<0.01)

[0121] 4.5 Effect of annexin A1 on the chemical properties of myofibrillar proteins

[0122] 4.5.1 Changes in the hydrolysis of myofibrillar proteins

[0123] Water retention and tenderness are both important quality characteristics of meat consumption, which are closely related to consumer satisfaction and willingness to repurchase, and there is also a certain relationship between water retention and tenderness (Font-I-Furnols and Guerrero, 2014). MFI can reflect the degree of myofibrillar protein hydrolysis, is often used to evaluate the tenderness of meat, and is positively correlated with the tenderness of meat (Yan et al., 2022). Figure 3 As shown in the figure, there was a significant difference between the annexin A1 treatment group and the control group (P<0.05). Annexin A1 significantly increased the MFI, and the MFI value was the largest at a concentration of 100 μg / mL, which was 23.65% higher than that of the control group. The increase in the MFI value indicates that annexin A1 promotes the hydrolysis of myofibrillar protein and tenderization of meat. Calpains are the main endogenous enzymes responsible for the degradation of myofibrillar protein in postmortem muscle of livestock and poultry (Huang et al., 2019). Annexin A1 plays a role in improving meat quality by acting on calpains.

[0124] The post-mortem oxidative stress process will aggravate protein oxidation, lipid oxidation, DNA and cell damage in muscle (Srinivas et al., 2019). Calpains are very sensitive to oxidation due to the sulfhydryl groups on the cysteine ​​residues in their active sites. They are easily oxidized and lose their activity during the post-mortem muscle oxidative stress process, thereby inhibiting the tenderization of meat (Yin et al., 2014). The reason why the tenderness of meat is significantly improved after treatment with annexin A1 is that when the cell membrane is damaged by oxidation, annexin A1 quickly binds to the damaged membrane site to participate in membrane repair, maintain the integrity and stability of the cell membrane, thereby reducing oxidative damage to the cells, improving the antioxidant capacity of post-mortem muscle cells, inhibiting the oxidation of calpains in the muscle, and then increasing the activity of calpains, accelerating the degradation of skeleton proteins such as desmin, and improving the tenderness of meat. In addition, the degradation of cytoskeletal proteins can remove the connections between myofibrillar proteins, reducing or eliminating the connection between the lateral contraction of myofibrils and the contraction of the entire muscle fiber (Xing et al., 2016), thereby retaining more water in the myofibrils and further improving the water retention of meat.

[0125] 4.5.2 SDS-PAGE analysis of myofibrillar proteins

[0126] Myosin and actin are the two most abundant proteins in skeletal muscle myofibrils, and 85% of the water is retained between myosin and actin filaments. After animal slaughter, the ATP content decreases, forming an actomyosin complex, which leads to muscle stiffness, and the thick and thin filaments are close to each other, leaving less space for water, thus affecting the water retention of meat (Li et al., 2023). The dissociation of actomyosin is closely related to the water retention and tenderness of meat. Therefore, we used SDS-PAGE gel electrophoresis to detect the dissociation of actomyosin. The molecular weight of myosin in MP is about 510 kDa, which is difficult to characterize with an electrophoresis diagram, while the molecular weight of actin is about 43 kDa, so the dissociation of actomyosin is often characterized by actin content (Wang et al., 2016). The SDS-PAGE results are shown in the figure. Figure 4 -A, all protein bands in the control group and the treatment group were intact, indicating that MP in the chicken was not destroyed after slaughter. Then, the free actin was quantitatively analyzed, and the results were as follows Figure 4-B shows that the optical density of free actin was significantly increased after treatment with annexin A1 (P<0.05), and the optical density of actin was the highest when the concentration of annexin A1 was 100μg / mL. This indicates that annexin A1 can significantly increase the degree of actomyosin dissociation in MP and reduce the cross-linking between myofibrillar proteins, thereby improving the water retention and tenderness of chicken after slaughter. Annexin A1 maintains the integrity of cell membrane structure and function, enhances the ability of its own antioxidant defense system, increases the activity of calpains, accelerates the hydrolysis of structural proteins, thereby weakening the connection between actomyosin and producing more free actin. The destruction of the connection between myofibrils reduces the connection between the transverse contraction of myofibrils caused by rigidity and the contraction of the entire muscle fiber, thereby improving the water retention of chicken.

[0127] 4.5.3 Oxidation and solubility of myofibrillar proteins

[0128] Protein oxidation is an irreversible oxidative modification that inevitably occurs in post-slaughter muscle. Protein oxidation may lead to reduced water retention and deterioration of tenderness in meat (Pan et al., 2022). Thiol groups have been widely used to evaluate the oxidation of muscle proteins (Liu et al., 2022). Thiol groups with high antioxidant activity are easily oxidized to -SOH, -SOOH, and -SS-, resulting in a decrease in the thiol content (Pan et al., 2022). In this study, the total thiol content of myofibrillar proteins was significantly increased after treatment with different concentrations of annexin A1 ( Figure 5 -A). When the concentration of annexin A1 was 100 μg / mL, the total thiol content was the highest, which was 26.92% higher than that of the control group (P<0.05). This shows that the addition of annexin A1 inhibits the oxidation of protein in post-slaughter chicken. Protein oxidation destroys the order and integrity of muscle cells, inhibits the ability of myofibrils to bind water, and causes the non-flowing water to be converted into free water to a certain extent, increasing the drip loss of post-slaughter muscle, thereby reducing the water retention capacity of meat. At the same time, protein oxidation destroys the structure of muscle protein, leading to protein aggregation and reduced activity of related enzymes, which has an adverse effect on protein solubility (Liu et al., 2022; Yulong and Per, 2018). Most of the water in the muscle exists in myofibrils. The higher the solubility of myofibrillar protein, the more water it binds to it, and the higher the water retention of the meat (Wang et al., 2022). Figure 5 -B shows that the solubility of myofibrillar protein was significantly increased after being treated with different concentrations of annexin A1 (P<0.05). This shows that annexin A1 enhances the ability of myofibrillar protein to bind to water. Therefore, annexin A1 improves the water retention of chicken by inhibiting the oxidation of myofibrillar protein and enhancing its hydrophilicity.

[0129] 4.6 Effect of annexin A1 on myofibrillar protein structure

[0130] Circular dichroism can reflect the conformation of the peptide bonds in the main chain of proteins and characterize the secondary structure of proteins. The α-helix has two negative peaks at 208nm and 222nm, a positive peak at 192nm, the β-fold has a negative peak at 217-220nm, a positive peak at 200-205nm, and the β-turn is attributed to the random coil at 195nm and is only defined as a negative peak near 200nm (Hou et al., 2019). Figure 6 -A shows the CD spectra of myofibrillar proteins under different treatments. Figure 6 -B shows the content of the corresponding secondary structure. After treatment with annexin A1, the α-helix in myofibrillar protein did not change significantly (P>0.05), and the β-turn angle decreased significantly when the concentration of annexin A1 was 50 and 100 μg / mL (P<0.05). In addition, the random curling was significantly increased after treatment with different concentrations of annexin A1 (P<0.05), increasing by 12.82%, 17.95% and 10.26%, respectively. The increased random curling may be transformed from the β-turn angle. Random curling is a key condition for myofibrillar protein gel to improve the gel network structure, which can promote the improvement of the water retention of protein gel, thereby improving the water retention of meat (Wang et al., 2023).

[0131] In addition, FTIR is a powerful means to study protein backbone vibration bands, which are closely related to the secondary structure of proteins. Generally, proteins have several different absorption bands in the infrared spectrum. The amide A band (3100-3500 cm-1) reflects the symmetric stretching vibration of alcohols and amides or OH and NH bonds in proteins, which is usually related to the interaction between proteins and water molecules. The amide I band (1600-1700 cm-1) is often used for protein secondary structure analysis through the C=O stretching vibration of peptide bonds. In addition, the amide II band (1500-1600 cm-1) and amide III band (1200-1300 cm-1) are usually associated with CN and C-OH (Abd Rashid et al., 2020). Figure 6-C shows the FTIR spectra of myofibrillar protein under different treatments. After treatment with 100 and 150 μg / mL of annexin A1, the peak intensities of amide A band and amide I band were significantly enhanced. The enhanced peak intensity of amide A band indicates that the stretching vibration of OH bond is enhanced and there is more water in the protein (Roobab et al., 2024). The enhanced peak intensity of amide I band indicates that the irregular curling in myofibrillar protein is increased (Li et al., 2019). At the same time, it can be seen from Table 5 that compared with the control group, the peak position of amide A band shifts to a lower wave number after annexin A1 treatment, and the NH stretching vibration of amide A band forms an association with hydrogen bond (Kang Huaibin et al., 2018). In addition, after annexin A1 treatment, the absorption peaks of amide I band, amide II band and amide III band all shift to the right, and the shift increases with the increase of concentration, indicating that the structure of myofibrillar protein changes to a random curling structure (Yan Wei, 2009). This is consistent with the CD results, indicating that annexin A1 induces the structure of myofibrillar protein to change to disorder.

[0132] Intrinsic fluorescence spectroscopy uses the oxidation sensitivity of aromatic amino acids to reflect the tertiary structure of proteins through the inherent changes in the fluorescence of tryptophan residues (Zhang and Ertbjerg, 2019). The conformation of proteins changes during oxidation, and moderate oxidation promotes the unfolding of protein structure, exposing tryptophan residues and enhancing fluorescence intensity. However, excessive oxidation of myofibrillar proteins will cause them to aggregate, thereby burying tryptophan residues and resulting in reduced fluorescence intensity. The lower the fluorescence intensity, the higher the degree of protein oxidation (Li et al., 2022; Pan et al., 2024). Figure 6 -D shows the fluorescence spectrum of myofibrillar protein excited at 283nm after treatment with annexin A1. Compared with the control group, the fluorescence intensity after treatment with different concentrations of annexin A1 was enhanced to varying degrees. When the concentration of annexin A1 was 100μg / mL, the fluorescence intensity was the highest, followed by concentrations of 150μg / mL, 50μg / mL and the control group. This shows that annexin A1 can inhibit excessive oxidation of myofibrillar protein. Wang et al. measured the fluorescence intensity of myofibrillar protein in Atlantic salmon, and the results showed that the greater the fluorescence intensity, the less protein oxidation (Wang et al., 2024). Therefore, the tryptophan fluorescence intensity of myofibrillar protein further proves that annexin A1 can inhibit protein oxidation, thereby improving the water retention and tenderness of chicken.

[0133] Table 5 Changes in FTIR wavenumber of myofibrillar protein after treatment with annexin A1 Unit: cm -1

[0134]

[0135]

[0136] 4.7 Effect of annexin A1 on the rheological properties of myofibrillar protein gels

[0137] The most important function of myofibrillar protein is to form a gel network structure. The gel has a certain water retention capacity and is closely related to the quality of meat. During the processing and storage of meat, myofibrillar protein is easily affected by the external environment, changing its rheological properties, thereby affecting its ability to form a gel (Lund et al., 2007). Rheological properties are divided into dynamic rheology and static rheology. Viscosity is the main indicator of static rheology. In food, it is often used to reflect the movement speed and movement resistance of food macromolecular slurry or solution under external force. Viscosity represents the resistance of the fluid to flow. The stronger the hydrophilic force, the greater the fluid flow resistance and the higher the viscosity (Xu et al., 2020). The effect curve of annexin A1 on the apparent viscosity of myofibrillar protein solution is shown in Figure 2. Figure 7 -A shows that with the increase of shear rate, the apparent viscosity shows a downward trend, showing a typical shear thinning phenomenon, which is a pseudoplastic fluid (Yuan et al., 2020). Because the linear α-helical tail in the myosin molecule is gradually oriented under the action of shear, the shear force gradually decreases (Liu et al., 2008). Compared with the control group, the apparent viscosity of the myofibrillar protein solution after treatment with different concentrations of annexin A1 increased significantly, indicating that annexin A1 enhances the flow resistance of the myofibrillar protein solution and increases the hydrophilicity. The apparent viscosity can only partially describe the rheological properties of myofibrillar protein. The dynamic rheological properties of myofibrillar protein can reflect the ability of protein to form thermally induced gels (Sun Lechang et al., 2022). Among them, the change of the storage modulus (G′), the main indicator of dynamic rheology, reflects the change of internal energy during the formation of protein gel. The larger the G′, the stronger the ability of myofibrillar protein to form gels (Wang et al., 2023). As Figure 7 -B shows that different concentrations of annexin A1 can increase the storage modulus of myofibrillar protein solution, indicating that annexin A1 is conducive to the formation of myofibrillar protein gel network and can improve gel strength, thereby improving water retention. In summary, annexin A1 can improve the rheological properties of myofibrillar protein, thereby promoting gel formation and enhancing gel degree, and ultimately improving the water retention and tenderness of chicken.

[0138] 4.4 Summary

[0139] The present invention uses recombinantly expressed annexin A1 to process fresh chicken breast, and explores the effect of annexin A1 on meat quality and its mechanism of action.

[0140] (1) Annexin A1 significantly reduces the cooking loss, centrifugal loss, drip loss, hardness, chewiness, stickiness and shear force of chicken, and improves water retention and tenderness.

[0141] (2) During the post-slaughter muscle maturation process, annexin A1 reduces the water freedom, making the connection between water molecules and proteins closer, and significantly increases the proportion of immobile water in myofibrils, thereby improving the water retention of meat.

[0142] (3) During the transformation of muscle into meat, the content of total free fatty acids and unsaturated fatty acids decreased significantly, indicating that annexin A1 can inhibit the hydrolysis of intramuscular lipids and improve the water retention of meat by maintaining the integrity of cell membrane lipids.

[0143] (4) Annexin A1 significantly affects the chemical properties and structure of myofibrillar proteins. Annexin A1 promotes the hydrolysis of myofibrillar proteins and the dissociation of actomyosin, inhibits the oxidation of myofibrillar proteins, and increases their solubility. In addition, Annexin A1 causes the structure of myofibrillar proteins to become disordered, reduces protein cross-linking and aggregation, and the loose structure increases the gap between molecules, which is conducive to the binding of more water molecules.

[0144] (5) Gel formation is an important function of myofibrillar protein, and its rheological properties reflect its gel-forming ability. Annexin A1 can improve the static and dynamic rheological properties of myofibrillar protein, increase the apparent viscosity and storage modulus of myofibrillar protein, promote gel formation, and help improve the water retention and tenderness of meat quality.

[0145] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that numerous changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents.

[0146] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0147] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. Application of annexin A1 in improving tenderness and texture of post-slaughter meat.

2. Application of annexin A1 in improving the water retention of post-slaughter meat.

3. The use according to claim 2, characterized in that: The method for improving water retention includes inhibiting the oxidation of myofibrillar protein in post-slaughter meat, increasing the total sulfhydryl content and improving the hydrophilicity of protein.

4. Application of annexin A1 in improving moisture distribution of meat after slaughter, including increasing the proportion of immobile water and reducing the proportion of free water in meat, and enhancing the binding between water and protein.

5. Application of annexin A1 in reducing the content of free unsaturated fatty acids in post-slaughter meat.

6. Application of annexin A1 in promoting the hydrolysis of myofibrillar proteins in post-slaughter meat, increasing the activity of calpains and enhancing the integrity of cell membranes.

7. Application of annexin A1 in improving the dissociation degree of actomyosin in post-slaughter meat, reducing the cross-linking between myofibrillar proteins and enhancing the integrity of cell membrane structure.

8. Application of annexin A1 in inducing the disordered transformation of myofibrillar protein structure in post-slaughter meat, enhancing the sulfhydryl content, inhibiting protein oxidation and improving fluorescence intensity.

9. Application of annexin A1 in improving the rheological properties of myofibrillar proteins in post-slaughter meat and enhancing the formation and strength of gel networks.

10. The use according to any one of claims 1 to 9, characterized in that: The meat is chicken.

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