Evaluation method for establishing formation of conditioned fish and garlic clove meat structure based on Teethhonin protein content change

Abstract

The invention belongs to the technical field of preparation of conditioned fish products, and particularly relates to an evaluation method for establishing conditioned fish and garlic clove meat structure formation based on Teethhonin protein content change. Proteomics analysis finds that the content of Teethonin protein on a myofibril Z disc is significantly related to the garlic clove meat structure, the expression level of the Teethonin protein has a certain positive correlation with the score of the garlic clove meat structure, and the Teethonin protein can be used as a biomarker for indicating the formation of the garlic clove meat structure and is used for establishing a conditioning fish and garlic clove meat structure formation evaluation method. Furthermore, based on the correlation between the biomarker and other evaluation indexes, the invention also establishes a conditioning fish and garlic clove meat structure formation evaluation method integrating multiple indexes. The invention provides an effective means and approach for optimizing the conditioned fish product with a typical garlic clove meat structure, and has positive significance for developing the conditioned fish product with excellent quality.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of conditioned fish products, and in particular to an evaluation method for establishing formation of garlic clove structure of conditioned fish based on changes in Telethonin protein content. Background Art

[0002] Freshwater fish farming has low cost and high yield. Fish meat is nutritious and rich in protein and fat, with protein accounting for about 15%-20% and fat accounting for about 1%-10%. In addition, fish meat also contains a large amount of minerals and other nutrients, which helps to improve human immunity and has beauty and skin care effects. With the accelerated pace of life, the semi-finished or finished dishes of conditioned fish meat made from fresh fish as raw materials, through slaughtering, cleaning, cutting, conditioning and pickling, and then packaging and quick freezing or frozen storage, can be eaten after thawing or simply cooked, which saves consumers from the complicated daily labor of killing fish and conditioning, and gradually becomes a hot spot for consumption by Chinese residents. However, after freezing and refrigeration, the structural deterioration of conditioned fish products affects the market acceptability of conditioned grass carp products and consumers' willingness to buy and eat. Therefore, providing a structure-maintaining technology is conducive to broadening the market demand for conditioned fish products and improving consumers' willingness to buy.

[0003] The organizational structure, texture characteristics, and flavor quality of fish meat are important evaluation indicators for measuring the quality of pre-prepared conditioned fish products. After being pickled (salted), conditioned, cooked, and processed, some fresh fish form an organizational structure similar to "garlic cloves" in appearance and exhibit a good taste. It is called "garlic clove meat" in the market. Its slices are crystal clear, firm in texture, and fresh and tender in taste. After being frozen and then cooked, the garlic clove structure can still be obtained. The formation of garlic clove meat is conducive to improving consumer acceptability, and is of great significance to broadening the market demand for conditioned fish products, improving the quality of conditioned products, and promoting the sustainable development of fish farming. However, the structural formation degree of "garlic clove meat" cannot be clearly determined at present, and there are few literature reports on the research on its influencing factors and evaluation methods. Therefore, determining the marker compounds for structure formation and establishing an evaluation method for the formation of garlic clove meat structure are of great guiding significance for determining the degree of garlic clove structure formation. Summary of the invention

[0004] In view of the above technical problems in the prior art, the present invention finds that the content of Telethonin protein on the myofibril Z disk is significantly correlated with the formation of garlic clove meat structure based on proteomics analysis, and its expression level has a certain positive correlation with the garlic clove meat structure score, which can be used as a biomarker indicating the formation of garlic clove meat structure, and is used to establish an evaluation method for the formation of garlic clove meat structure in conditioned fish. Further based on the correlation between the biomarker and other evaluation indicators, the present invention also establishes a comprehensive multi-indicator evaluation method for the formation of garlic clove meat structure in conditioned fish.

[0005] The present invention is specifically implemented through the following technical solutions:

[0006] The first aspect of the present invention provides the use of Telethonin protein as a biomarker for regulating the formation of fish garlic clove meat structure.

[0007] Furthermore, the amino terminal sequence of the Telethonin protein is as follows:

[0008] MPVCTVLEKKGGCVIRAELSCSVKEENNPNKRESYTADWRSINMKTQPEDRQSMLMSD DSRRETLSRYWQIRPLNQACPSGVLRVGTVDTGVREHQLLPYRNTLPLPIFKPAELGVRLGR GAPHTLEDLPPARVADGACPEKRPVEQIIRDLPPVKPMRMEFAKAPRTLGRSMSQEAQRG.

[0009] Furthermore, the use includes: detecting the content of Telethonin protein in fish meat before and after conditioning, when the difference multiple of the content of Telethonin protein after conditioning compared to the content of Telethonin protein before conditioning is greater than or equal to 3, the fish meat forms a garlic clove meat structure during the conditioning process.

[0010] The second aspect of the present invention provides the use of a reagent for detecting Telethonin protein in evaluating the formation of garlic clove structure in conditioned fish.

[0011] Furthermore, the Telethonin protein was determined by ultra-performance liquid chromatography-mass spectrometry.

[0012] Furthermore, the method for detecting the content of Telethonin protein comprises the following steps:

[0013] Weighing and crushing the prepared fish meat sample, adding a lysis buffer, and ultrasonically lysing the tissue cells, wherein the lysis buffer includes 1% (wt) SDS and 1% (wt) protease inhibitor;

[0014] The supernatant was centrifuged and added with acetone to precipitate the protein at low temperature. The protein precipitate was centrifuged and added with triethylammonium bicarbonate buffer (TEAB) at a final concentration of 200 mM. The precipitate was broken up by ultrasound. Trypsin was added at a ratio of 1:50 to the total protein mass. The solution was enzymatically hydrolyzed overnight. Then, dithiothreitol at a final concentration of 5 mM was added and reduced at 56°C for 30 min. Finally, iodoacetamide at a final concentration of 11 mM was added and the solution was incubated at room temperature in the dark for 15 min to obtain a trypsin-hydrolyzed peptide sample.

[0015] The trypsin enzymatic peptide samples were dissolved in mobile phase A and added to an ultra-high performance liquid chromatograph for separation. The ultra-high performance liquid chromatographic detection conditions were set as follows: mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile, mobile phase B was an acetonitrile solution containing 0.1% formic acid, and the liquid phase gradient was set at 0-14 min, 6%-24% B; 14-16 min, 24%-35% B; 16-18 min, 35%-80% B; 18-20 min, 80% B; the flow rate was maintained at 500 nl / min;

[0016] The peptides separated by ultra-high performance liquid chromatography were injected into the capillary ion source for ionization, and the data were collected by timsTOF Pro mass spectrometer; the mass spectrometry detection conditions were set as follows: the ion source voltage was 1.75 kV, the peptide parent ion and its secondary fragments were detected and analyzed by TOF, the data acquisition mode used the data-independent parallel accumulation serial fragmentation mode (dia-PASEF), the primary mass spectrometry scanning range was 300-1500 m / z, and after one primary mass spectrum was collected, 20 parallel accumulation continuous fragmentation mode collections were performed, and the secondary mass spectrometry scanning range was 400-850, and every 7 mass-to-charge ratios were used as an isolation window.

[0017] A third aspect of the present invention provides a method for evaluating the formation of garlic clove structure of conditioned fish, comprising the following steps:

[0018] The fish fillets are placed in a low-temperature environment for dry salting and conditioning, and samples are taken before and after conditioning to determine the content of Telethonin protein in the fish meat. The garlic meat structure score of the fish meat is evaluated based on the difference in the content of Telethonin protein.

[0019] Furthermore, the dry-salting and conditioning in a low-temperature environment includes: evenly applying 3% table salt on the surface of the fish fillet, and conditioning at 4°C for 0-168 hours.

[0020] Furthermore, the method for evaluating the formation of the structure of cooked fish garlic clove meat also includes: taking samples before and after cooking to measure the taste score, hardness, adhesion, elasticity, recovery, moisture content, TCA soluble peptide content, salt content, adhesiveness, and chewiness of the fish meat, and comprehensively evaluating the fish garlic clove meat structure score based on the Telethonin protein content, the taste score, the hardness, the adhesion, the elasticity, the recovery, the moisture content, the TCA soluble peptide content, the salt content, the adhesiveness, and the chewiness.

[0021] Furthermore, the fish garlic meat structure score is calculated using a first formula, the first formula comprising:

[0022] Y=-5.264095531943403+0.003258522322944499×X1 -0.9497588845442674×X 2 -0.0016894789348844697×X 3 -0.09880917548703838×X 4 -2.707318434401062×X 5 +80.6490049879852×X 6 +0.7005893931990175×X 7 -137.6445942889418×X 8 -15.041349663741006×X 9 +0.008477528239482557×X 10 -0.005420357697741518×X 11 ;

[0023] In the formula, X 1 is the content of Telethonin protein, X 2 Score for taste, X 3 is the hardness, X 4 is adhesion, X 5 is elasticity, X 6 For responsiveness, X 7 is the moisture content, X 8 is the TCA soluble peptide content, X9 is the salt content, X 10 For adhesion, X 11 is the chewiness, and Y is the garlic clove structure score.

[0024] The advantages and positive effects of the present invention are:

[0025] 1. The present invention uses fish meat conditioned with salt for different times as the research object, and uses ultra-high performance liquid chromatography-mass spectrometry to determine the protein types and contents of grass carp meat at different conditioning times, and screens differential proteins, and finds that Telethonin protein distributed on myofibril Z disks can be used as a differential protein indicating a garlic clove meat structure marker. By determining Telethonin protein before and after conditioning, based on the difference in its content, it is beneficial to quickly, reasonably and objectively evaluate the degree of garlic clove meat formation during the marinating process of fish meat, and provides an effective means and way to optimize conditioned fish products with typical garlic clove meat structure, which has positive significance for the development of high-quality conditioned fish products.

[0026] 2. The present invention uses Telethonin protein as an indicator protein for the formation of garlic clove structure, analyzes the correlation between the protein and other indicators, and establishes a multivariate linear regression equation with indicators with strong correlation to obtain a garlic clove formation evaluation model. The model can accurately evaluate the degree of garlic clove formation and characterize the quality of conditioned fish products, providing technical support and scientific guidance for the preparation of high-quality conditioned fish products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 The actual picture of the degree of formation of grass carp garlic clove meat under different conditioning time in the embodiment of the present invention;

[0029] Figure 2 This is a graph showing changes in TCA soluble peptide content in grass carp muscle at different conditioning times according to an embodiment of the present invention;

[0030] Figure 3 1 is a graph showing the characteristics of grass carp muscle tissue under different conditioning times according to an embodiment of the present invention, wherein FIG. AF is a graph showing the cross-sectional structure of grass carp muscle tissue of fresh grass carp, conditioned at 4°C for 0, 24, 72, 120, and 168 hours, FIG. GL is a graph showing the longitudinal structure of grass carp muscle tissue of fresh grass carp, conditioned at 4°C for 0, 24, 72, 120, and 168 hours, and FIG. MR is a graph showing the myoseptum structure of grass carp muscle tissue of fresh grass carp, conditioned at 4°C for 0, 24, 72, 120, and 168 hours;

[0031] Figure 4 The ultrastructure diagrams of grass carp muscle tissues at different conditioning times in the embodiments of the present invention are shown in Figures AF and AF, respectively, are muscle fiber structures of fresh grass carp muscle tissues, and grass carp muscle tissues conditioned at 4°C for 0, 24, 72, 120, and 168 hours; and Figures GL and GL, respectively, are muscle septum structures of fresh grass carp muscle tissues, and grass carp muscle tissues conditioned at 4°C for 0, 24, 72, 120, and 168 hours;

[0032] Figure 5It is a graph showing the quality evaluation and identification results of grass carp muscle proteomics at different conditioning times in an embodiment of the present invention; wherein, FIG. A is an SDS-PAGE electrophoresis graph, lanes 1-3, 4-6, 7-9, 10-12, and 13-15 are respectively grass carp samples conditioned at 4°C with 3% salt for 0, 24, 72, 120, and 168 hours, FIG. B is a graph showing the identification results of grass carp muscle protein extracts, and FIG. C is a graph showing the distribution of peptide lengths of each protein sample; FIG. D is a principal component analysis graph of the relative quantitative values ​​of protein components of each protein sample, and groups AE represent grass carp samples conditioned at 4°C with 3% salt for 0, 24, 72, 120, and 168 hours, respectively;

[0033] Figure 6 It is a screening and cross-relationship diagram of differentially expressed proteins in grass carp muscle at different conditioning times in the embodiment of the present invention; wherein, Figures A and B are differential protein volcano diagrams of B vs A, C vs A, D vs A, and E vs A, respectively, and Groups AE represent grass carp samples conditioned at 4°C with 3% salt for 0, 24, 72, 120, and 168 hours, respectively, and Figure e is a Venn diagram of differentially expressed proteins in different comparison groups;

[0034] Figure 7 This is a graph showing the results of correlation analysis between the potential marker Telethonin protein and the sensory evaluation indexes and physicochemical indexes of conditioned grass carp in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0037] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0038] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art. The meanings of the terms "comprise", "include", "contain", "have" and the like are non-restrictive, that is, other steps and other ingredients that do not affect the results can be added.

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below.

[0040] The unique "garlic meat" structure of prepared fish products was first used as an important quality evaluation indicator in the sensory evaluation of stinky mandarin fish, and has gradually been applied to the sensory evaluation of several other prepared fish products such as silver carp and grass carp. The "garlic meat" structure is an important sensory evaluation indicator, and it is also a common and widely recognized structural feature of prepared fish products in my country's catering industry. However, the evaluation standards for the "garlic meat" structure in related studies have remained at evaluation standards such as rubbing with chopsticks, separating fish meat from fish bones, and being in the shape of garlic cloves. It is greatly affected by human factors, and there are problems such as non-objective evaluation standards and the lack of unified standards.

[0041] The term "garlic meat" refers to a special structure formed by conditioning, processing and steaming fresh fish as raw material. The fish meat has the characteristics of separable slices, smooth and burr-free layers, bright color, and a refreshing, tender and smooth taste.

[0042] In order to solve the problem of difficulty in evaluating the structure of garlic clove meat in the process of fish meat conditioning, the present invention takes fish meat conditioned with salt for different times as the research object, and determines the protein types and contents of grass carp fish meat at different conditioning times by ultra-high performance liquid chromatography-mass spectrometry. Further, by comparing the differentially expressed proteins (DAPs) at different processing times, a common DAP-Telethonin protein that exists before and after processing at different time points is screened out. With the extension of the pickling time, the content of Telethonin protein shows an upward trend, and it shows a positive correlation with the formation degree of the "garlic clove meat" structure of fish meat. Telethonin (amino acid sequence, see NCBI protein number: XP_051742264.1) is a capping protein located on the myofibril Z disk, which anchors titin on the Z disk to maintain the orderly structure of myofibrils. In the process of "garlic clove meat" formation, the aforementioned protein macromolecules may be released due to the disintegration of the Z disk structure, which then shows a trend of increasing protein abundance. Further correlation analysis confirmed that Telethonin protein was significantly positively correlated with the garlic clove structure score and garlic clove taste score in the sensory evaluation, significantly positively correlated with elasticity and salt content, and significantly negatively correlated with hardness, adhesion, resilience, and adhesiveness. The above findings revealed that Telethonin protein can be used as a marker for the formation of garlic clove structure.

[0043] Based on the above research findings, one embodiment of the present invention provides the use of Telethonin protein as a biomarker for regulating the formation of fish garlic clove meat structure.

[0044] The present invention has found that the Telethonin protein distributed on the myofibril Z disk can be used as a differential protein indicating the garlic clove structure marker. By measuring the Telethonin protein before and after conditioning, based on the difference in its content, it is helpful to quickly, reasonably and objectively evaluate the garlic clove formation degree in the fish pickling process, which provides an effective means and way to optimize the conditioned fish products with typical garlic clove structure, and has positive significance for the development of high-quality conditioned fish products.

[0045] Optionally, the amino terminal sequence of the Telethonin protein is as follows:

[0046] MPVCTVLEKKGGCVIRAELSCSVKEENNPNKRESYTADWRSINMKTQPEDRQSMLMSD DSRRETLSRYWQIRPLNQACPSGVLRVGTVDTGVREHQLLPYRNTLPLPIFKPAELGVRLGR GAPHTLEDLPPARVADGACPEKRPVEQIIRDLPPVKPMRMEFAKAPRTLGRSMSQEAQRG.

[0047] The use includes: detecting the content of Telethonin protein in fish meat before and after conditioning; when the difference multiple of the content of Telethonin protein after conditioning compared with the content of Telethonin protein before conditioning is greater than or equal to 3, that is, the ratio of the content of Telethonin protein after conditioning to the content of Telethonin protein before conditioning (or conditioning 0h) is greater than or equal to 3, the fish meat forms a garlic clove meat structure during the conditioning process.

[0048] Another embodiment of the present invention provides the use of a reagent for detecting Telethonin protein in evaluating the formation of garlic clove structure in conditioned fish.

[0049] The present invention is based on ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) technology to determine Telethonin protein. The method for detecting the content of Telethonin protein comprises the following steps:

[0050] Weighing and crushing the prepared fish meat sample, adding a lysis buffer, and ultrasonically lysing the tissue cells, wherein the lysis buffer includes 1% (wt) SDS and 1% (wt) protease inhibitor;

[0051] The supernatant was centrifuged and added with acetone to precipitate the protein at low temperature. The protein precipitate was centrifuged and added with triethylammonium bicarbonate buffer (TEAB) at a final concentration of 200 mM. The precipitate was broken up by ultrasound. Trypsin was added at a ratio of 1:50 to the total protein mass. The solution was enzymatically hydrolyzed overnight. Then, dithiothreitol at a final concentration of 5 mM was added and reduced at 56°C for 30 min. Finally, iodoacetamide at a final concentration of 11 mM was added and the solution was incubated at room temperature in the dark for 15 min to obtain a trypsin-hydrolyzed peptide sample.

[0052] The trypsin-digested peptide samples were dissolved in mobile phase A and added to an ultra-high performance liquid chromatograph NanoElute UHPLC system (Bruker Daltonics) for separation. The ultra-high performance liquid chromatography detection conditions were set as follows: mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile, mobile phase B was an acetonitrile solution containing 0.1% formic acid, and the liquid phase gradient was set at 0-14 min, 6%-24% B; 14-16 min, 24%-35% B; 16-18 min, 35%-80% B; 18-20 min, 80% B; the flow rate was maintained at 500 nl / min;

[0053] The peptides separated by ultra-high performance liquid chromatography were injected into the capillary ion source for ionization, and the data were collected by timsTOF Pro mass spectrometer; the mass spectrometry detection conditions were set as follows: the ion source voltage was 1.75 kV, the peptide parent ion and its secondary fragments were detected and analyzed by TOF, the data acquisition mode used the data-independent parallel accumulation serial fragmentation mode (dia-PASEF), the primary mass spectrometry scanning range was 300-1500 m / z, and after one primary mass spectrum was collected, 20 parallel accumulation continuous fragmentation mode collections were performed, and the secondary mass spectrometry scanning range was 400-850, and every 7 mass-to-charge ratios were used as an isolation window.

[0054] According to Telethonin protein as a biomarker for the formation of garlic clove meat structure, another embodiment of the present invention provides a method for evaluating the formation of garlic clove meat structure in conditioned fish, comprising the following steps:

[0055] The fish fillets are placed in a low-temperature environment for dry salting and conditioning, and samples are taken before and after conditioning to determine the content of Telethonin protein in the fish meat. The garlic meat structure score of the fish meat is evaluated according to the difference in the content of Telethonin protein.

[0056] Generally speaking, when the content of the Telethonin protein after conditioning is greater than or equal to 3 compared to the content of the Telethonin protein before conditioning, that is, the difference multiple of the Telethonin protein content is greater than or equal to 3, the fish meat forms a good garlic clove meat structure during the conditioning process. The content of the Telethonin protein is measured by UHPLC-MS / MS technology.

[0057] Optionally, the low temperature environment dry salting includes: applying 3% salt evenly on the surface of the fish fillet, and standing and conditioning at 4° C. for 0-168 hours. 3% (w / w) salt is based on the weight of the fish, and the amount of salt accounts for 3% of the total weight of the fish.

[0058] Optionally, the fish fillet is a grass carp fillet.

[0059] Optionally, the method for evaluating the formation of the structure of garlic clove meat in cooked fish further includes: taking samples before and after cooking to measure the taste score, hardness, adhesion, elasticity, recovery, moisture content, TCA soluble peptide content, salt content, adhesiveness, and chewiness of the fish meat, and comprehensively evaluating the fish garlic clove meat structure score based on the Telethonin protein content, the taste score, the hardness, the adhesion, the elasticity, the recovery, the moisture content, the TCA soluble peptide content, the salt content, the adhesiveness, and the chewiness.

[0060] Optionally, the fish garlic meat structure score is calculated using a first formula, the first formula comprising:

[0061] Y=-5.264095531943403+0.003258522322944499×X 1 -0.9497588845442674×X 2 -0.0016894789348844697×X 3 -0.09880917548703838×X 4 -2.707318434401062×X 5 +80.6490049879852×X 6 +0.7005893931990175×X 7 -137.6445942889418×X 8 -15.041349663741006×X 9 +0.008477528239482557×X 10 -0.005420357697741518×X 11 ;

[0062] Where, X 1 is the content of Telethonin protein, X 2 Score for taste, X 3 is the hardness, X 4 is adhesion, X 5 is elasticity, X 6 For responsiveness, X 7 is the moisture content, X 8 is the TCA soluble peptide content, X9 is the salt content, X 10 For adhesion, X 11 is the chewiness, and Y is the garlic clove structure score.

[0063] Taking Telethonin protein as the indicator protein, the correlation between this protein and other indicators was analyzed, and the indicators with strong correlation (taste score, hardness, adhesion, elasticity, recovery, moisture content, TCA soluble peptide content, salt content, adhesiveness, and chewiness) were established together to establish a multivariate linear regression equation, and the above-mentioned quantitative relationship evaluation model was obtained, which is conducive to more accurate evaluation of garlic clove meat formation and characterization of the quality of prepared fish products, providing technical support and scientific guidance for the preparation of high-quality prepared fish products.

[0064] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions.

[0065] The grass carp (2.0-2.5 kg) used in the experiments in the following examples were purchased from the farmers' market of Huazhong Agricultural University.

[0066] The screening of biomarkers for the formation of garlic clove structure in conditioned fish in the following embodiments comprises the following steps:

[0067] Raw material pretreatment: slaughter, skin and bone the fresh grass carp, and cut the fish into two pieces in the middle;

[0068] Normal pressure static conditioning: Apply 3% of the total weight of the fish fillet with salt evenly on the surface of the fish fillet, and let it stand at 4°C for 0-168h;

[0069] Quality appraisal of prepared fish meat: including (1) determination of sensory evaluation index of garlic clove structure: steam-heat the prepared fish fillets for 15 minutes, and evaluate and score the garlic clove structure, color, taste and flavor of the prepared grass carp after heating according to the sensory evaluation standard; (2) determination of physical and chemical indexes: texture, Masson staining, transmission electron microscopy observation, whole texture TPA determination, pickling yield, centrifugal loss and water binding index, salt content, TCA soluble peptide content determination of the prepared grass carp;

[0070] Differentially expressed proteins (DAPs) and biomarker screening: UPLC-MS / MS was used to determine the protein content, and the DIA data (data collected by dia-PASEF) were processed using the DIA-NN search engine (v.1.8). The differentially expressed proteins were screened based on the protein abundance ratio before and after conditioning > 1.5 or < 0.66, and the corresponding T-test p < 0.05. The cross-correlations of DAPs sets at different time points after conditioning were further compared, and common DAPs were screened to obtain telethonin protein as a potential biomarker related to the changes in fish protein at different time points of conditioning and pickling and the structural characteristics of garlic clove meat.

[0071] Correlation analysis between DAP and other indicators: The screened biomarkers of garlic clove structure formation were subjected to correlation analysis with the data of sensory evaluation indicators and physicochemical indicators.

[0072] Based on the screened biomarkers of garlic clove structure formation in prepared fish products and their highly correlated indicators, a garlic clove structure score structural evaluation model was established.

[0073] 1. Preparation of samples with different degrees of garlic clove structure formation

[0074] Fresh grass carp was slaughtered, scaled, gilled, and visceral removed, the head was cut off, and the spine was removed from the back. The meat slices on both sides were taken, rinsed with clean water, and then the surface moisture was absorbed with absorbent paper. 3% salt was evenly applied to the surface of the grass carp slices based on the meat weight, and the slices were conditioned at 4°C for 0, 24, 72, 120, and 168 hours. Grass carp samples with different garlic clove meat structure formation degrees were prepared by controlling the salting time.

[0075] Table 1 Preparation conditions of grass carp samples with different garlic clove meat formation degrees

[0076]

[0077] 2. Determination of sensory evaluation indexes of garlic clove structure

[0078] Grass carp that had been conditioned at 4°C for 0, 24, 72, 120, and 168 hours were steamed for 15 minutes and placed on a black bottom plate. Digital images in JPEG format were taken with a NIKON D7000 digital camera at a resolution of 4928×3264 pixels. The color was in RGB mode, and the camera was located directly above the plane where the sample was located. In addition, the garlic meat score, taste, flavor, and color were sensory evaluated. Fifteen sensory personnel, seven of whom were male and eight were female, all had professional backgrounds in the food field and had a certain level of expertise in sensory evaluation. The evaluation criteria are shown in Table 2.

[0079] Table 2 Evaluation criteria for garlic clove samples with different structural formation degrees

[0080]

[0081] The image collection and sensory evaluation scores of the prepared fish meat are as follows: Figure 1 As shown in Table 3, Figure 1 From left to right in the figure, the garlic was cured with salt for 0, 168, 24, 72, and 120 hours. It can be seen that as the curing time increases, the scores of garlic clove structure and color increase, and the scores of flavor and mouthfeel increase first and then decrease. Finally, the overall score of 72-168 hours of curing with 3% salt is better, and a good garlic clove structure can be obtained, among which the garlic clove structure cured for 168 hours is the best.

[0082] Table 3 Sensory evaluation results of grass carp garlic meat

[0083]

[0084] 3. Determination of physical and chemical indicators of conditioned grass carp meat

[0085] The grass carp conditioned at 4°C for 0, 24, 72, 120, and 168 h were subjected to texture, Masson staining, transmission electron microscopy observation, whole texture TPA determination, pickling yield, centrifugal loss and water binding index, salt content, and TCA soluble peptide content determination.

[0086] The moisture content determination refers to the first method direct drying method in GB 5009.3-2016 National Food Safety Standard Determination of Moisture in Food.

[0087] The pickling yield, centrifugal loss and water binding index were calculated by the following formula:

[0088] Yield(%)=(W 0 / W 1 )×100;

[0089] Centrifuging loss(CL,%)=(W 2 -W 3 ) / W 2 ×100;

[0090]

[0091] Note: 0 , W 1 , W 2 , W 3 Respectively represent the weight before pickling, after pickling, before centrifugation and after centrifugation at 3000r / min at 4℃ for 20 minutes. 0 The moisture content of fish meat before centrifugation (%), ω 1 The moisture content of fish meat after centrifugation.

[0092] Determination of salt (NaCl) content: weigh about 1.00 g of the prepared fish sample into a 50 mL centrifuge tube, add 10 mL of distilled water to homogenize, and place at room temperature for 1 hour. Use a salinometer to measure the NaCl concentration and record the reading as X. 0 The calculation formula of NaCl content in the sample is as follows:

[0093] NaCl(%)=X 0 ×(m 1 +10);

[0094] Note: m 1 Meat weight, 1.00g.

[0095] Texture measurement: The fish meat was cut into samples of 2 cm × 2 cm × 1 cm in size and placed at the center of the probe base of the texture analyzer (TA-XT plus, Stable MicroSystems, UK). The probe used was P / 36R, the trigger value was 5 g, the compression deformation was 40%, the pre-test rate was 2.0 mm / s, the test rate was 1 mm / s, and the post-test rate was 2.0 mm / s. Each sample was measured 6 times in parallel.

[0096] TCA soluble peptide determination: Weigh 3.00g of ground fish sample, add 27mL 5% TCA (w / v), homogenize at 6000r / min for 2min, place at 4℃ for 1h, centrifuge at 10000×g, 4℃ for 10min, and the TCA-soluble peptide content in the supernatant is determined by Lowry method, expressed as μmolTyr / g fish. Take 1mL of sample dilution, add 5mL 0.4mol / L sodium carbonate solution and 1mL folin phenol in turn, shake well, then react in a 40℃ water bath for 20min, and measure the corresponding absorbance at a wavelength of 680nm.

[0097] Texture characteristics, water binding index, pickling rate, moisture content, etc. are all important indicators for evaluating fish quality. The texture characteristics of fish are shown in Table 4. As shown in Table 4, compared with the conditioning time of 0h, with the increase of pickling time, the hardness, adhesiveness, chewiness and resilience of the fish gradually decreased, while the adhesion and elasticity increased. Among them, the hardness, elasticity, adhesiveness and chewiness were between 1889.46-1981.50g, 0.96-0.97, 1082.58-1118.46, 1025.39-1074.20, and remained stable during the pickling period of 120-168h.

[0098] Table 4 Texture characteristics of grass carp muscle during conditioning

[0099]

[0100]

[0101] The results of physical and chemical properties are shown in Table 5. As shown in Table 5, with the extension of pickling time, the salt content and water binding index increase; the centrifugal loss rate decreases. For the samples with 3% salt added, the yield range is 94.78-96.25% during pickling for 120-168h, the salt content range is 2.90-2.93%, the centrifugal loss range is 10.54-11.45% and the water binding index range is 87.51-87.88%. Figure 2 (The horizontal axis is the conditioning time, and the vertical axis is the TCA soluble peptide content) It can be seen that as the conditioning time increases, the TCA soluble peptide content first increases and then decreases.

[0102] Table 5 Physicochemical properties of grass carp muscle during conditioning

[0103]

[0104] 4. Observation of muscle tissue structure of grass carp meat

[0105] The fish meat was cut into small pieces of 8mm×8mm×3mm, fixed with 10% formaldehyde solution at room temperature for 24h, then dehydrated with 50%, 70%, 80%, 95%, 100% ethanol gradient for 15min, and then replaced by dimethylbenzene immersion. The immersed samples were embedded in paraffin, cut into 4μm slices with a slicer, then unfolded in water, transferred to a slide, dried in a 60℃ oven for 1h, dewaxed with dimethylbenzene, and then eluted twice with 100%, 95%, 80% ethanol gradient, rinsed with water for 5min, and then stained with Masson's trichrome (MT). Finally, the slides were sealed with neutral gum, observed and photographed under an optical microscope, and the magnification was 100 times.

[0106] Figure 3 To show the histological characteristics of grass carp muscle conditioned at different times, Figure AF shows the cross-sectional structure of fresh grass carp muscle tissue and grass carp conditioned at 4°C for 0, 24, 72, 120, and 168 hours, Figure GL shows the longitudinal structure of fresh grass carp muscle tissue and grass carp conditioned at 4°C for 0, 24, 72, 120, and 168 hours, and Figure MR shows the myoseptum structure of fresh grass carp muscle tissue and grass carp conditioned at 4°C for 0, 24, 72, 120, and 168 hours. In the figure, mf stands for muscle fiber, e stands for endomysium, p stands for perimysium, and mc stands for myocommata. The yellow arrow, green arrow, and pink arrow indicate the degradation of endomysium, perimysium, and myoseptum, respectively. Myofiber is the basic structural unit of muscle. Figure 3It can be seen that the muscle fibers in the cross-section of fresh grass carp muscle are full and have complete boundaries. The outside is wrapped by the connective tissue endomysium, and the arrangement is tight and orderly. During the 0-24h of conditioning, the muscle fibers and the endomysium and perimysium remain intact. During the 72-168h of conditioning, the muscle fibers contract and aggregate, the adhesion between fibers is significantly reduced, and the distance between muscle fibers gradually increases; the muscle fibers are separated from the endomysium, the muscle fiber bundles are separated from the perimysium, and the collagen fibers and proteoglycans in the connective tissue endomysium, perimysium and their matrix are reduced. At the same time, the muscle fibers in the longitudinal sections of fresh fish muscle and conditioning 0h samples are closely arranged. As the conditioning time increases, the endomysium and perimysium covering the muscle fibers separate and degrade from the muscle fibers, and the gaps between muscle fibers increase. In addition, the septum of fresh fish and conditioning 0h muscle is intact. As the conditioning time increases, holes and cracks appear in the collagen fiber layer in the septum, which gradually increase with the extension of pickling time. The penetration of sodium chloride increases the electrostatic repulsion between myofibrillar proteins, thereby promoting protein denaturation and aggregation, while reducing the hydrodynamic radius of protein molecules. The connection between the sarcomere and the endomysium is broken, and the collagen fibers and proteoglycans in the connective tissue are degraded, resulting in the loss of adhesion between muscle fibers and muscle fibers, and between muscle fibers and septa, and the expansion of the extracellular and septal gaps. Endogenous enzymes such as matrix metalloproteinases and collagenases degrade the collagen fiber network, promoting the increase of cavities and spacing within the septa.

[0107] 5. Ultrastructural observation of conditioned grass carp meat

[0108] The ultrastructure of muscle fibers and septa was observed by transmission electron microscopy (TEM). The fish meat was cut into 1mm×1mm×1mm slices longitudinally with a scalpel and fixed with 2.5% (v / v) glutaraldehyde solution at 4°C for 48h. The fixative was discarded and rinsed with 0.1mol / L phosphate buffer for 3 times, 10min each time, and then fixed with 1% osmium acid solution for 1h, then dehydrated with 70%, 80%, 95%, 100% (v / v) ethanol gradient for 10min, and finally dehydrated with pure acetone for 20min. Soaked in a mixture of acetone and epoxy resin (v / v=1 / 1) at room temperature for 1h, and then embedded with epoxy resin at 60°C for 48h. The above samples were sliced ​​with an ultrathin slicer and stained with uranyl acetate and lead acetate citrate for 15min, and then photographed with a TEM microscope.

[0109] Figure 4The ultrastructure of grass carp muscle tissue conditioned at different times is shown, where Figure AF is the muscle fiber structure of fresh grass carp muscle tissue conditioned at 4°C for 0, 24, 72, 120, and 168 hours, and Figure GL is the myoseptum structure of fresh grass carp muscle tissue conditioned at 4°C for 0, 24, 72, 120, and 168 hours, respectively. In the figure, Sar stands for Sarcomere, Sr stands for Sarcoplasmic reticulum, and CF stands for Collagen Fibers. The basic building block of muscle fiber is myofibril, and myofibril is composed of numerous sarcomeres. Sarcomeres are composed of actin filaments, myosin filaments, Z-disks, H-bands, and other components. The endomysium, perimysium, and septum are the connective tissues of the muscle, and their components are collagen fibers (main component), elastic fibers, and proteoglycans. From Figure 4 It can be seen that the Z-disk, Z-line, H-band, and M-line components of the sarcomere in the myofibrils of fresh fish and grass carp muscle cured for 0 hours have complete structures, the sarcoplasmic reticulum is complete, and the gaps between myofibrils are small. When the conditioning is 24-72 hours, the H-band in the sarcomere gradually narrows to completely disappear, the gaps between myofibrils gradually increase, and the Z-disk gradually degrades; after conditioning for 120 hours, the myofibril gaps gradually narrow, and after conditioning for 168 hours, the M-line and Z-line on the H-band completely disappear, and the Z-disk is vaguely visible. The mutual connection between adjacent myofibrils fills the gaps between myofibrils, so that all myofibrils in each muscle fiber are connected as a whole. The septum of fresh fish muscle and the sample with a pickling time of 0 hours is tightly attached to the muscle fiber, and the collagen fibers in the septum are complete and evenly distributed. After pickling for 24 hours, the muscle fiber is separated from the septum. After 72 hours of pickling, the gaps in the septum enlarged and the collagen fibers were partially degraded. After 120-168 hours of pickling, the fragmentation of collagen fibers intensified. The degradation of Z discs, H bands, Z lines and M lines and the degradation of collagen fibers and interfibrous proteoglycan bridges in connective tissue can be attributed to the action of endogenous proteases (such as protease B+L, calpain and serine protease). The disintegration of connective tissue structure promotes the separation between muscle fibers and muscle fibers, and between muscle fibers and septum, resulting in reduced hardness and increased muscle tenderness; in addition, structural proteins such as Z discs and M lines in myofibrils make myofibrils easier to cross-link, promote the increase of elasticity, and change the muscle structure and texture. This may be the key reason why garlic cloves are tender, juicy, elastic and smooth in taste.

[0110] 6. Identification of protein peptides in grass carp muscle after conditioning for different periods of time

[0111] Protein extraction: Weigh appropriate amount of fish meat conditioned at 4°C for different time periods into a mortar pre-cooled with liquid nitrogen, add liquid nitrogen to fully grind into powder, add lysis buffer (1% SDS + 1% protease inhibitor) 4 times the volume of the powder, and ultrasonically lyse. Centrifuge at 4°C and 12000g for 10 min, take the supernatant and use the BCA kit to determine the protein concentration. Each group of samples has 3 parallels. In addition, the extracted grass carp muscle protein samples were tested by SDS-PAGE method, and the results (see Figure 5 Figure A in the middle shows that the protein bands are clear and proteins of different molecular weights can be effectively separated. The size and number of protein bands in each treatment group are consistent, and there are differences in proteins between groups. The protein abundance meets the experimental requirements, and subsequent trypsin hydrolysis and protein separation and identification can be performed.

[0112] Trypsin hydrolysis: adjust the volume of the supernatant of the lysed protein to the same level with lysis buffer, add 1 volume of pre-cooled acetone, vortex mix, then add 4 volumes of pre-cooled acetone, precipitate at -20℃ for 2h, centrifuge at 4500g for 5min, discard the supernatant, wash the precipitate with pre-cooled acetone 2-3 times; after drying the precipitate, add triethylammonium bicarbonate buffer (TEAB) with a final concentration of 200mM, ultrasonically break up the precipitate, add trypsin at a ratio of 1:50 (protease: protein, m / m), and hydrolyze overnight; then add dithiothreitol (DTT) with a final concentration of 5mM, reduce at 56℃ for 30min; finally, add iodoacetamide (IAA) with a final concentration of 11mM, and incubate at room temperature in the dark for 15min.

[0113] LC-MS / MS identification of grass carp muscle protein peptides: The trypsin enzymatic peptides were dissolved in liquid chromatography (LC) mobile phase A and separated using the NanoElute ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile solution containing 0.1% formic acid, and the liquid phase gradient was set as follows: 0-14min, 6%-24% B; 14-16min, 24%-35% B; 16-18min, 35%-80% B; 18-20min, 80% B, and the flow rate was maintained at 500nl / min. After being separated by the ultra-high performance liquid phase (UHPLC) system, the peptides were injected into the capillary ion source for ionization and then entered the timsTOF Pro mass spectrometer for data acquisition. The ion source voltage was set to 1.75kV, and the peptide parent ions and their secondary fragments were detected and analyzed using the TOF detector. The data acquisition mode used was data-independent parallel accumulation serial fragmentation mode (dia-PASEF). The primary mass spectrometry (MS) scan range was set to 300-1500 m / z. After one primary mass spectrum acquisition, 20 parallel accumulation serial fragmentation (PASEF) mode acquisitions were performed. The secondary mass spectrometry (MS) scan was in the range of 400-850, and every 7 mass-to-charge ratios (m / z) was used as an isolation window.

[0114] A total of 12,836 peptides were identified from the grass carp muscle protein samples. After searching the database (Ctenopharyngodon_idellus_7959_NCBI_seqkit.fasta), 12,030 of them were unique peptide sequences. By analyzing the specific peptides, 2,122 protein components were identified, of which 2,092 protein components could be quantitatively compared (see Figure 5 The peptide lengths are mainly distributed between 7 and 17 amino acids (see Figure 5 Figure C in the middle), indicating that the distribution of peptide lengths identified by mass spectrometry meets the quality control requirements. PCA analysis results (see Figure 5 Figure D in the middle shows that the repeated samples of each group tend to aggregate, indicating good repeatability within the sample group. At the same time, there are significant differences in the protein expression of fish muscle in different pickling time groups. The protein expression of the pickling 0h treatment group and the pickling 24, 72, 120, and 168h treatment groups is significantly different. The pickling 24h group is significantly different from the other four groups, and the sample groups pickled for 72, 120, and 168h tend to aggregate, indicating that the protein expression is similar in the later stage of pickling.

[0115] 7. Screening of differentially expressed proteins

[0116] The raw data files of LC-MS / MS (data collected by dia-PASEF) were analyzed by DIA-NN software (v1.8). The reference database of grass carp genome in NCBI was used for retrieval, and the database was Ctenopharyngodon_idellus_7959_NCBI_seqkit.fasta (48945 sequences). Peptide digestion was performed with trypsin, and up to 1 amino acid deletion was allowed. The removal of N-terminal Met and carbamidomethyl on Cys was designated as fixed modification. The final results were screened at the parent ion and protein levels with 1% FDR. The quantitative information of the screened proteome was used for bioinformatics analysis. The paired sample T test was used to screen the differentially abundant proteins, and p < 0.05 and protein abundance ratio Fold Change (FC) > 1.5 or < 0.66 were set to be statistically significant. The differentially abundant proteins were subjected to in-depth analysis such as GO enrichment analysis, KEGG pathway enrichment analysis, COG / KOG functional annotation, cluster analysis, protein interaction analysis and subcellular localization analysis. The data were analyzed using SPSS2021 and Microsoft Excel 2019. Graphics were drawn using software such as Origin 2021, R language, and Adobe Illustrator2021.

[0117] Compared with the conditioning 0h group, 59 (26 up-and 33 down-regulated), 74 (46 up-and 28 down-regulated), 56 (33 up-and 23 down-regulated), and 77 (33 up-and 44 down-regulated) DAPs were identified in the grass carp muscle protein groups of the conditioning 24, 72, 120, and 168h sample groups, respectively. Figure 6 In the figure, groups AE represent grass carp samples conditioned at 4℃ with 3% salt for 0, 24, 72, 120, and 168 hours, respectively. B vs A, C vs A, D vs A, and E vs A represent the comparison between grass carp samples conditioned at 24, 72, 120, and 168 hours and grass carp samples conditioned at 0 hours, respectively. The Venn diagram is further used to understand the cross-relationship of the DAPs sets of each comparison group. Figure 6 As shown in Figure e, there is a common DAP (protein number: XP_051742264.1; protein description: Telethonin) in the muscle protein groups of the four comparison groups. This common differential protein can be used as a potential biomarker for the changes in fish protein at different pickling time points and the structural characteristics of garlic cloves. The content of Telethonin protein and the difference fold relative to the conditioning 0h group are shown in Table 6.

[0118] Table 6 Telethonin protein content and protein content difference fold

[0119]

[0120] Telethonin (XP_051742264.1) is a capping protein located on the sarcomere Z-disk. It anchors titin to the Z-disk to maintain the ordered structure of myofibrils. Telethonin is very important for the assembly of titin and the anchoring of the Z-disk. Proteomic data showed that the abundance of Z-disk-related DAPs was high, and the abundance of titin isoform X1 (XP_051762790.1), titin (XP_051762794.1) and Telethonin proteins increased significantly after 168 hours of conditioning. The reason for this result may be due to the disintegration of the Z-disk structure, releasing the aforementioned protein macromolecules, which is consistent with Figure 4 The results observed by TEM are consistent with those shown above. Telethonin is the only protein with common differential abundance in all comparison groups, so it can be used as a potential indicator protein for the formation of garlic clove-like structures.

[0121] 7. Correlation between potential markers and sensory evaluation, texture characteristics, and physicochemical properties of conditioned grass carp

[0122] The correlation analysis was conducted on the data of Telethonin, a marker of garlic clove structure formation, sensory evaluation scores, and physical and chemical indicators, including taste, flavor, color, hardness, adhesion, elasticity, resilience, moisture content, water binding index, salt content, centrifugal loss, cohesion, adhesiveness, chewiness, etc.; it showed that Telethonin protein content showed a significant correlation with multiple indicators (see Figure 7 Telethonin was significantly positively correlated with the garlic clove structure score and garlic clove taste score in the sensory evaluation, significantly positively correlated with elasticity, salt content, and TCA soluble peptide content, and significantly negatively correlated with hardness, adhesion, resilience, adhesiveness, chewiness, and moisture content.

[0123] 8. Establishment of a comprehensive evaluation model for the structure of garlic cloves in conditioned grass carp meat

[0124] Multiple indicators with significant correlation with Telethonin protein, a marker of garlic clove structure formation, were selected (including taste score, hardness, adhesion, elasticity, recovery, moisture content, TCA soluble peptide content, salt content, adhesiveness, and chewiness), and a quantitative relationship was established using the multivariate linear regression method. The resulting multivariate linear regression equation (R2 = 0.9996) was as follows:

[0125] Y=-5.264095531943403+0.003258522322944499×X 1 -0.9497588845442674×X 2 -0.0016894789348844697×X 3 -0.09880917548703838×X 4 -2.707318434401062×X 5 +80.6490049879852×X 6 +0.7005893931990175×X 7 -137.6445942889418×X 8 -15.041349663741006×X 9 +0.008477528239482557×X 10 -0.005420357697741518×X 11 ;

[0126] In the formula, X 1 is the content of Telethonin protein, X 2 Score for taste, X 3 is the hardness, X 4 is adhesion, X5 is elasticity, X 6 For responsiveness, X 7 is the moisture content, X 8 is the TCA soluble peptide content, X9 is the salt content, X 10 For adhesion, X 11 is the chewiness, and Y is the garlic clove structure score.

[0127] The above formula predicts that the garlic clove structure scores Y at 0, 24, 72, 120, and 168 hours are 1.004, 2.992, 7.003, 7.317, and 8.351, respectively, which are close to the actual values ​​of 1, 3.2, 6.6, 7.5, and 8.5. It can be seen that the evaluation model provided by the present invention can accurately evaluate the degree of formation of the garlic clove meat structure, and is used to guide the fish meat conditioning process, so as to prepare high-quality conditioned fish products with firm fish meat texture, tender and smooth taste, and better formation of the garlic clove meat structure.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of Telethonin protein as a biomarker for regulating the formation of fish garlic clove structure.

2. The use of Telethonin protein as a biomarker for regulating the formation of fish garlic clove structure according to claim 1, characterized in that: The amino terminal sequence of the Telethonin protein is as follows: MPVCTVLEKKGGCVIRAELSCSVKEENPNKRESYTADWRSINMKTQPEDRQSMLMSD DSRRETLSRYWQIRPLNQACPSGVLRVGTVDTGVREHQLLPYRNTLPLPIFKPAELGVRLGR GAPHTLEDLPPARVADGACPEKRPVEQIIRDLPPVKPMRMEFAKAPRTLGRSMSQEAQRG.

3. The use of Telethonin protein as a biomarker for regulating the formation of fish garlic clove structure according to claim 1, characterized in that: The use includes: detecting the content of Telethonin protein in fish meat before and after conditioning, and when the difference multiple of the content of Telethonin protein after conditioning compared with the content of Telethonin protein before conditioning is greater than or equal to 3, the fish meat forms a garlic clove meat structure during the conditioning process.

4. Application of reagents for detecting Telethonin protein in evaluating the structural formation of garlic clove meat in cooked fish.

5. Use of the reagent for detecting Telethonin protein according to claim 4 in evaluating the formation of the structure of garlic clove meat in conditioned fish, characterized in that: The content of Telethonin protein was determined by ultra-performance liquid chromatography-mass spectrometry.

6. Use of the reagent for detecting Telethonin protein according to claim 5 in evaluating the formation of garlic clove structure in conditioned fish, characterized in that: The method for detecting the content of Telethonin protein comprises the following steps: Weigh the prepared fish meat sample, grind it, add lysis buffer, and ultrasonically lyse the tissue cells, wherein the lysis buffer includes 1% SDS and 1% protease inhibitor; The supernatant was centrifuged and added with acetone to precipitate the protein at low temperature. The protein precipitate was centrifuged and added with a final concentration of 200 mM triethylammonium bicarbonate buffer. The precipitate was ultrasonically broken up. Trypsin was added at a ratio of 1:50 to the total protein mass. The solution was enzymatically hydrolyzed overnight. Then, dithiothreitol was added at a final concentration of 5 mM and reduced at 56°C for 30 min. Finally, iodoacetamide was added at a final concentration of 11 mM and incubated at room temperature in the dark for 15 min to obtain a trypsin-hydrolyzed peptide sample. The trypsin enzymatic peptide samples were dissolved in mobile phase A and added to an ultra-high performance liquid chromatograph for separation. The ultra-high performance liquid chromatographic detection conditions were set as follows: mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile, mobile phase B was an acetonitrile solution containing 0.1% formic acid, and the liquid phase gradient was set at 0-14 min, 6%-24% B; 14-16 min, 24%-35% B; 16-18 min, 35%-80% B; 18-20 min, 80% B; The peptides separated by ultra-high performance liquid chromatography were injected into the capillary ion source for ionization, and the data were collected by the tims TOFPro mass spectrometer. The mass spectrometry detection conditions were set as follows: the ion source voltage was 1.75 kV, the peptide parent ion and its secondary fragments were detected and analyzed using a TOF detector, the data acquisition mode used a data-independent parallel accumulation serial fragmentation mode, the primary mass spectrometry scanning range was 300-1500 m / z, and after one primary mass spectrum was acquired, 20 parallel accumulation continuous fragmentation mode acquisitions were performed, and the secondary mass spectrometry scanning range was 400-850, with every 7 mass-to-charge ratios as an isolation window.

7. A method for evaluating the structural formation of cooked fish garlic cloves, characterized in that: The following steps are involved: The fish fillets are placed in a low-temperature environment for dry salting and conditioning, and samples are taken before and after conditioning to determine the content of Telethonin protein in the fish meat. The garlic meat structure score of the fish meat is evaluated based on the difference in the content of Telethonin protein.

8. The method for evaluating the formation of garlic clove structure of cooked fish according to claim 7, characterized in that: The low-temperature dry-salting conditioning comprises: evenly applying 3% table salt on the surface of the fish fillet, and conditioning at 4°C for 0-168 hours.

9. The method for evaluating the formation of garlic clove structure of cooked fish according to claim 7, characterized in that: The method for evaluating the formation of the structure of garlic clove meat of conditioned fish also includes: The fish meat is sampled before and after conditioning to measure the taste score, hardness, adhesion, elasticity, recovery, moisture content, TCA soluble peptide content, salt content, adhesiveness, and chewiness. The fish meat garlic clove structure score is comprehensively evaluated based on the Telethonin protein content, the taste score, the hardness, the adhesion, the elasticity, the recovery, the moisture content, the TCA soluble peptide content, the salt content, the adhesiveness, and the chewiness.

10. The method for evaluating the formation of garlic clove structure of cooked fish according to claim 9, characterized in that: The fish garlic meat structure score is calculated using a first formula, wherein the first formula includes: Y=-5.264095531943403+0.003258522322944499×X1-0.9497588845442674×X2- 0.0016894789348844697×X3-0.09880917548703838×X4-2.707318434401062×X5+80.6490049879852×X6+0.7005893931990175×X7-137.6445942889418×X8-15.041349663741006×X9+0.008477528239482557×X 10 -0.005420357697741518×X 11 ; Wherein, X1 is the content of Telethonin protein, X2 is the taste score, X3 is the hardness, X4 is the adhesion, X5 is the elasticity, X6 is the recovery, X7 is the moisture content, X8 is the TCA soluble peptide content, X9 is the salt content, X 10 For adhesion, X 11 is the chewiness, and Y is the garlic clove structure score.

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