Method for rapidly identifying grass carp variety and muscle characteristic flavor substance based on GC*GC-TOFMS technology

Abstract

The invention belongs to the technical field of aquatic product identification, and discloses a method for rapidly identifying grass carp varieties and muscle characteristic flavor substances based on a GC * GC-TOFMS (Gas Chromatography-Time of Flight Mass Spectrometry) technology, and the method adopts a solid phase microextraction technology and a comprehensive two-dimensional gas chromatography-time of flight mass spectrometry technology to carry out qualitative and semi-quantitative analysis on volatile substances in grass carp muscles. Establishing a muscle volatile flavor substance database of the disease-resistant grass carp and the common grass carp; through a multivariate statistical analysis method, a grass carp identification model based on volatile flavor substances is constructed, an OPLS-DA model is utilized to screen different volatile substances related to varieties, a visual heat map is generated, and accurate identification of disease-resistant grass carps and common grass carps and analysis of muscle characteristic flavor substances are realized. The method has the advantages of simplicity and convenience in operation, high detection speed, high accuracy, visual result and the like, and provides a new technical means and theoretical basis for variety identification and quality evaluation of disease-resistant grass carps.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic product identification, and in particular to a method for quickly identifying grass carp varieties and muscle characteristic flavor substances based on GC×GC-TOFMS technology. Background Art

[0002] grass carp( Ctenopharyngodon idellus ) is the main freshwater aquaculture economic fish in my country. Because of its tender meat, rapid growth and high yield, it occupies an important position in aquaculture waters such as ponds, lakes and reservoirs. In recent years, with the rapid expansion of grass carp farming, although the output has increased significantly, the general decline in its quality has become a key issue restricting the sustainable and healthy development of the grass carp industry. At present, under the background of consumers' increasing requirements for food safety and quality, the consumption demand for fish has shifted from simply pursuing quantity to pursuing quality and flavor. Therefore, grass carp products with excellent meat quality and high nutritional value are more popular with consumers.

[0003] Disease-resistant grass carp is a high-quality grass carp variety. This variety is obtained by backcrossing koi sperm-induced gynogenetic grass carp with selected male grass carp. It not only has the advantages of fast growth, high yield and strong disease resistance, but also has advantages in muscle quality, with unique flavor and taste. However, in order to make huge profits, some unscrupulous vendors sell ordinary grass carp farmed in ponds as disease-resistant grass carp, which seriously disrupts the market order. Due to the lack of effective standards for grass carp variety identification and fish meat quality evaluation, it is difficult for producers to bring farmed high-quality disease-resistant grass carp products to the market at a reasonable price, and it is also difficult for consumers to distinguish between grass carp products of different varieties and qualities. When regulatory authorities encounter complaints of violations such as selling inferior products as good ones, they are often helpless. This makes the market promotion and quality certification of disease-resistant grass carp face challenges. At present, the identification of fish meat varieties mainly adopts molecular biological identification methods. Although molecular biological identification methods have high accuracy and reliability, they are complicated to operate and are not suitable for real-time identification of large quantities of samples. They cannot obtain the flavor characteristics of fish meat and reflect the quality of fish meat. In order to solve the above problems, it is urgent to establish a method that can quickly and accurately identify disease-resistant grass carp and its muscle characteristic flavor substances to provide technical support for the market promotion and quality certification of disease-resistant grass carp.

[0004] In recent years, comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) has become a powerful tool for analyzing volatile substances in complex samples due to its advantages of high resolution, high sensitivity, large peak capacity and high acquisition frequency. This technology has shown broad application prospects in the fields of animal and plant variety identification, quality evaluation and resource identification. In view of this, the present invention proposes a method for rapid identification of grass carp varieties and muscle characteristic flavor substances based on GC×GC-TOFMS technology, which can achieve rapid and accurate identification of disease-resistant grass carp and screen out its differential volatile substances. This method provides an effective technical means for the market promotion and quality certification of disease-resistant grass carp, and has important practical significance and potential application value. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a method for quickly identifying grass carp varieties and muscle characteristic flavor substances based on GC×GC-TOFMS technology. The method extracts and analyzes volatile flavor substances in the muscles of disease-resistant grass carp and ordinary grass carp. Through multivariate statistical analysis methods, a grass carp identification model based on volatile flavor substances is constructed, thereby realizing rapid identification of disease-resistant grass carp and screening out its differential volatile substances.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is: A method for quickly identifying grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology, comprising the following steps: (1) Weigh the fish muscle sample to be tested, crush it and transfer it to a headspace bottle, and extract the volatile flavor compounds (VOCs) in the muscle sample by solid phase microextraction (SPME); (2) analyzing the volatile substances extracted in step (1) by comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOFMS); (3) Compare the mass spectrum data of the unknown volatile substance collected in step (2) with the mass spectrum information of the standard sample in the NIST2020 database to determine the type of the volatile substance; use deuterated n-hexanol solution as an internal standard and adopt the internal standard normalization method to quantitatively analyze the detected volatile substances; (4) The qualitative and quantitative data of volatile substances obtained in step (3) are compared with the differential identification analysis model and differential volatile substances of disease-resistant grass carp / common grass carp, so as to identify the varieties and muscle characteristic flavor substances of disease-resistant grass carp and common grass carp.

[0007] In the above-mentioned method for rapidly identifying grass carp varieties and characteristic flavor substances in their muscles based on GC×GC-TOFMS technology, preferably, the disease-resistant grass carp is a high-quality grass carp variety, bred by backcrossing gynogenetic grass carp induced by koi sperm and selected male grass carp, while the common grass carp is the original species of grass carp from the Yangtze River waters.

[0008] Preferably, in step (1), the muscle sample is taken from the muscle above the lateral line on the back of the fish and below the dorsal fin; the solid phase microextraction conditions are: use an automatic sampler to process the sample, place the muscle sample in a headspace injection bottle, then add 10 μL of internal standard solution, and incubate at 70-90°C for 10-20 min; before adsorbing the sample, the SPME extraction head (DVB / CAR / PDMS extraction head, 50 / 30 μm×1 cm) is aged at 260-280°C for 10-15 min; then, at 70-90°C, the SPME extraction head adsorbs the sample for 20-30 min; after the adsorption is completed, the SPME extraction head is transferred to the GC injection port and desorbed at 240-260°C for 5-10 min.

[0009] Preferably, in step (2), the comprehensive two-dimensional gas chromatography conditions are: using high-purity helium as the carrier gas, with a constant flow rate of 1.0 mL / min; the temperature rise program of the one-dimensional chromatographic column DB-HeavyWax is: initial temperature 40-50°C, maintained for 2-5 min, increased to 220-250°C at a rate of 3-5°C / min, maintained for 4-6 min; the temperature rise program of the two-dimensional chromatographic column Rxi-5SilMS is 5°C higher than that of the one-dimensional chromatographic column, the modulator temperature is always 15°C higher than the column temperature of the two-dimensional chromatographic column, and the modulation period is 5-10 seconds; the injection port temperature is 240-270°C.

[0010] Preferably, in step (2), the time-of-flight mass spectrometry analysis conditions are: mass spectrometry transmission line temperature is 230-270°C, ion source temperature is 230-270°C, acquisition rate is 150-200 spectra / s, electron bombardment source is 70eV, detector voltage is 2021V, and mass spectrometry scanning range is m / z35-550.

[0011] Preferably, in step (3), the ChromaTOF software is used to compare the collected mass spectrometry data of the unknown volatile compound with the mass spectrometry information in the NIST2020 database, and a qualitative analysis is performed in combination with the compound mass spectrum, the compound retention index and the standard retention index to confirm the detected volatile compound; deuterated n-hexanol solution is used as the internal standard, and the internal standard normalization method is used to calculate the relative content of the detected volatile compound, and the ratio of the peak area of ​​the detected compound to the peak area of ​​the internal standard compound is multiplied by the known concentration of the internal standard compound to obtain the relative concentration of the detected compound.

[0012] Preferably, in step (4), the method for constructing the differential identification analysis model of disease-resistant grass carp / common grass carp comprises the following steps: (11) Muscle samples of disease-resistant grass carp and common grass carp were weighed, crushed and transferred to headspace vials, and volatile flavor compounds in the muscle samples were extracted by solid phase microextraction; (12) analyzing the volatile substances extracted in step (11) by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry; (13) comparing the mass spectrum data of the volatile substances collected in step (12) with the mass spectrum information of the standard samples in the NIST2020 database to determine the type of the volatile substances; using deuterated n-hexanol solution as an internal standard, the internal standard normalization method is used to quantitatively analyze the detected volatile substances; (14) The qualitative and quantitative data of volatile substances were preprocessed using SIMCA-P software, and then the principal component analysis (PCA) was used to achieve a preliminary distinction between disease-resistant grass carp and common grass carp, and the variability of samples within the group was evaluated. The qualitative and quantitative data of volatile substances were further processed using orthogonal partial least squares discriminant analysis (OPLS-DA), and an orthogonal partial least squares discriminant analysis model based on volatile flavor substances was constructed as a discrimination model for distinguishing disease-resistant grass carp from common grass carp.

[0013] Preferably, the method for screening differential volatile substances between disease-resistant grass carp and common grass carp comprises the following steps: on the basis of steps (11)-(14), the differential volatile substances related to the species are screened by an orthogonal partial least squares discriminant analysis model and a visual heat map is generated, wherein the specific screening conditions are VIP>1.75, FC≥2 or ≤0.5, and T test p<0.05, and the screened differential volatile substances are used as muscle characteristic flavor substances for distinguishing disease-resistant grass carp from common grass carp.

[0014] More preferably, a total of 28 differential volatile substances were screened out, among which: The volatile substances that were upregulated in the disease-resistant grass carp compared with the common grass carp included (8 kinds): any one or more of 5-methyl-5-nonanol, 3,5-di-tert-butyl-2-hydroxybenzonitrile, 7-methylpentadecane, 3,5-dimethylundecane, 3-acetylheptane-2,6-dione, nonanamide, 2,3-dimethyl-2-(propan-2-yl)butanoic acid, 1H-Isoindole, 3-methoxy-4,7-dimethyl-; The volatile substances that are downregulated in the disease-resistant grass carp compared with the common grass carp include (20 kinds): any one or more of 9,12,15-octadecatriene, 2,4-hexadienal, butyraldehyde, undecylbenzene, 4-ethylbenzaldehyde, 1-methyl-1,2,3,4-tetralin, nonanehydrazine, ethyl propionate, oct-1,4-diene, 2-methyl-1-octen-3-yne, 1-octene, 1,2-vinylcyclohexane, 5-methylnonane, 2-pentyl-2-cyclopentene-1-one, (3E,5E)-nona-3,5-dien-2-one, 1-methyl-1H-pyrazole-3-carboxylic acid methyl ester, Dihexyverine, 4-methoxybutan-2-one, 2,5-dimethyl-1,5-hexadien-3-ol, and Zonarene.

[0015] The beneficial effects of the present invention are: 1. The present invention combines solid phase microextraction technology with comprehensive two-dimensional gas chromatography-time of flight mass spectrometry technology (GC×GC-TOFMS), and is applied for the first time to grass carp variety identification and flavor substance analysis; solid phase microextraction technology is used to extract and enrich volatile substances in grass carp muscle, and has the advantages of no need for complex sample pretreatment, simple operation, short time consumption, high sensitivity and good reproducibility, and can extract more types and quantities of volatile substances; compared with traditional gas chromatography-mass spectrometry (GC-MS), GC×GC-TOFMS technology shows higher peak capacity, separation ability and sensitivity, significantly improves the accuracy of volatile substance detection, and obtains more comprehensive flavor substance information, which is particularly suitable for rapid identification and detection of batch samples.

[0016] 2. The present invention constructs a disease-resistant grass carp identification model through multivariate statistical analysis, and screens out differential volatile substances as potential biomarkers through the OPLS-DA model to generate a visual heat map. This method can not only distinguish the differences between disease-resistant grass carp and common grass carp varieties, but also realizes the volatile flavor substance analysis and quality evaluation of disease-resistant grass carp, providing an efficient and reliable method for grass carp variety identification, breeding and quality evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 GC×GC-TOF-MS three-dimensional total ion current chromatogram of volatile substances in common grass carp muscle in the embodiment; Figure 2GC×GC-TOF-MS three-dimensional total ion current chromatogram of volatile substances in the disease-resistant grass carp muscle in the embodiment; Figure 3 The figure is a Venn diagram of the amount of volatile substances in the muscles of two grass carps in the embodiment; Figure 4 It is a graph showing the types and relative contents of different volatile substances in the muscles of two grass carps in the embodiment; Figure 5 : is the PCA score diagram of volatile substances in the muscles of two grass carps in the embodiment; Figure 6 OPLS-DA score diagram of volatile substances in the muscles of two grass carps in the embodiment; Figure 7 This is a clustering heat map of 28 differentially volatile substances screened out from the muscles of two grass carps in the example. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0020] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0021] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0022] Example 1 A method for quickly identifying grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology, comprising the following steps: 1. Randomly select 8 healthy grass carps and 8 disease-resistant grass carps weighing about 500g; take appropriate amount of muscle tissue above the lateral line on the back and below the dorsal fin of each fish, and mince the muscle tissue; mix every two grass carp muscle samples of the same species into one sample, place it in a cryopreservation tube, and store it in a -80℃ refrigerator for subsequent analysis.

[0023] 2. Weigh 0.6 g of muscle samples from disease-resistant grass carp and common grass carp respectively, transfer them to headspace bottles after uniform crushing, and immediately seal the headspace bottles with screw caps for solid phase microextraction; set up 4 biological replicates for each species of grass carp.

[0024] Solid phase microextraction (SPME) conditions were as follows: Samples were processed using an automatic sampler. The muscle samples were placed in a headspace injection bottle, followed by the addition of 10 μL of 10 mg / L deuterated n-hexanol solution and incubation at 80°C for 10 min. Before adsorbing the samples, the SPME extraction head (DVB / CAR / PDMS extraction head, 50 / 30 μm×1 cm) was aged at 270°C for 10 min. Subsequently, the SPME extraction head adsorbed the samples at 80°C for 25 min. After adsorption, the SPME extraction head was transferred to the GC injection port and desorbed at 250°C for 5 min.

[0025] 3. The samples obtained after solid phase microextraction were analyzed by comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOFMS). The GC×GC-TOF-MS three-dimensional total ion current chromatograms of volatile substances in the muscles of common grass carp and disease-resistant grass carp are shown in Figure 1 and Figure 2 .

[0026] The analysis conditions of comprehensive two-dimensional gas chromatography are as follows: The LECO Gasus BT4D (LECO, St. Joseph, MI, USA) GC×GC-TOFMS chromatographic system consists of an Agilent 8890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA), a two-stage jet modulator and a split / splitless injection module, and the mass spectrometry system is a high-resolution TOF mass spectrometer. The first-dimensional chromatographic column is a DB-HeavyWax column (30m×250μm×0.5μm); the second-dimensional chromatographic column is an Rxi-5SilMS column (2m×150μm×0.15μm). High-purity helium is used as the carrier gas with a constant flow rate of 1.0mL / min. The temperature rise program of the first-dimensional column DB-HeavyWax is as follows: the initial temperature is 50℃, maintained for 2min, and then increased to 230℃ at a rate of 5℃ / min and maintained for 5min. The temperature program of the two-dimensional column Rxi-5SilMS is 5°C higher than that of the one-dimensional column, the modulator temperature is always 15°C higher than the column temperature of the two-dimensional column, the modulation cycle is 6 seconds, and the injection port temperature is 250°C.

[0027] The analysis conditions of time-of-flight mass spectrometry were as follows: The detector voltage was 2021 V and the mass spectrometer scanning range was m / z 35-550.

[0028] 4. ChromaTOF software was used to compare the collected mass spectrometric data of the unknown volatile compounds with the mass spectrometric information in the NIST2020 database, and qualitative analysis was performed in combination with the compound mass spectrum, compound retention index and standard retention index to confirm the detected volatile compounds; the internal standard normalization method was used for relative quantitative analysis, with 10 μL of 10 mg / L deuterated n-hexanol solution as the internal standard, and the ratio of the peak area of ​​the detected compound to the peak area of ​​the internal standard compound was multiplied by the known concentration of the internal standard compound to obtain the relative concentration of the detected compound.

[0029] In this example, the types and contents of volatile substances in the above-mentioned grass carp muscle samples were obtained under the analytical conditions. A total of 2230 compounds were detected in the muscles of disease-resistant grass carp and common grass carp, including 1392 compounds in disease-resistant grass carp and 1708 compounds in common grass carp. The two had a total of 870 compounds, such as Figure 3 The identified volatile flavor substances were classified into alcohols, aldehydes, carboxylic acids, esters, heterocyclic compounds, hydrocarbons, ketones, and unclassified flavor substances, e.g. Figure 4 , some of which cannot be clearly classified. The variety of volatile flavor substances found in fish meat may be responsible for its complex flavor characteristics. The volatile flavor substances detected in grass carp muscle with the highest relative content are alcohols, followed by hydrocarbons, esters and ketones. The relative contents of carboxylic acids, hydrocarbons and ketones in the muscle of disease-resistant grass carp are higher than those of ordinary grass carp, while the relative contents of alcohols and heterocyclic compounds are lower than those of ordinary grass carp. Alcohols are mainly derived from the oxidation of unsaturated fatty acids and amino acid metabolism. Among them, the threshold of unsaturated alcohols is low, and they usually show mushroom and grass aromas; the production of ketone substances is related to the oxidation of fatty acids and the degradation of amino acids, and generally presents floral and fruity aromas in aquatic products; hydrocarbons are generated by the self-oxidation of fatty acids, among which olefin compounds have a lower threshold and mostly have fruity aromas; heterocyclic compounds such as pyrrole and pyrazine compounds have nutty and roasted aromas; the aroma threshold of carboxylic acids is generally high, and their direct contribution to meat flavor is not significant. Comparative analysis revealed differences in the types and relative contents of the main volatile flavor substances between disease-resistant grass carp and common grass carp, indicating that the flavor of disease-resistant grass carp muscle had changed.

[0030] 5. Use SIMCA-P (v13.0) software to preprocess the obtained volatile substance data. Then, the obtained volatile substances in the muscles of disease-resistant grass carp and common grass carp were subjected to principal component analysis (PCA). Figure 5 The PCA results showed that there was an obvious separation trend between disease-resistant grass carp and common grass carp. The variance contribution rate of PC1 was 34.9%, and the variance contribution rate of PC2 was 15.4%. The first two principal components reflected the characteristics of 50.3% of all volatile substances, which basically reflected the overall information of the samples and accurately described the differences between disease-resistant grass carp and common grass carp.

[0031] In order to further distinguish the differences between different groups and reduce the intra-group errors, the overall differences of volatile substances between groups were distinguished by supervised orthogonal partial least squares discriminant analysis (OPLS-DA), and the OPLS-DA model was obtained, as shown in Figure 6 It shows that disease-resistant grass carp and common grass carp are clearly divided into two categories, showing obvious separation. The OPLS-DA discriminant model consists of 1 prediction component and 2 orthogonal components. The R2X, R2Y and Q2 values ​​are 0.498, 0.999 and 0.883, respectively. R2X and R2Y represent the explanation rate of the model for the X and Y matrices, respectively, and Q2 represents the prediction ability of the model. In theory, the closer the R2 and Q2 values ​​are to 1, the better the model is, and the lower the R2 and Q2 values ​​are, the worse the fitting accuracy of the model is. This shows that the discriminant analysis results of the model are highly accurate.

[0032] The OPLS-DA model was used to screen the differential volatile substances related to varieties. The specific screening conditions were VIP>1.75, FC≥2 or ≤0.5 and T test p<0.05. Based on the above conditions, 28 differential volatile substances that can distinguish between disease-resistant grass carp and common grass carp were identified as potential biomarkers: 5-methyl-5-nonanol, 3,5-di-tert-butyl-2-hydroxybenzonitrile, 7-methylpentadecane, 3,5-dimethylundecane, 3-acetylheptane-2,6-dione, nonanamide, 2,3-dimethyl-2-(propan-2-yl)butyric acid, 1H-Isoindole, 3-methoxy-4,7-dimethyl-, 9,12,15-octadecadiene, 2,4-hexadienal, butyraldehyde, decadecane, 1,2-dimethyl-2-(propan-2-yl)butyric acid ... Alkylbenzene, 4-ethylbenzaldehyde, 1-methyl-1,2,3,4-tetrahydronaphthalene, nonanehydrazine, ethyl propionate, oct-1,4-diene, 2-methyl-1-octen-3-yne, 1-octene, 1,2-vinylcyclohexane, 5-methylnonane, 2-pentyl-2-cyclopentene-1-one, (3E,5E)-nona-3,5-dien-2-one, 1-methyl-1H-pyrazole-3-carboxylic acid methyl ester, Dihexyverine, 4-methoxybutan-2-one, 2,5-dimethyl-1,5-hexadien-3-ol, Zonarene. According to the above-screened differential volatile substance composition and its relative content, a visual heat map is generated, such as Figure 7 The changes and differences in the content of differentially volatile substances in the muscles of disease-resistant grass carp and common grass carp are displayed for subsequent query and comparison in the identification of unknown grass carp.

[0033] 6. Referring to the operating methods of steps 2-4, weigh the fish muscle sample to be tested, transfer it to a headspace bottle after crushing, extract the volatile flavor substances in the muscle sample by solid phase microextraction, and analyze the volatile substances by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry. Compare the obtained mass spectrometry data with the mass spectrometry information of the standard samples in the NIST2020 database to determine the type of volatile substances; use deuterated n-hexanol solution as the internal standard and use the internal standard normalization method to quantitatively analyze the detected volatile substances; finally, compare the qualitative and quantitative data of volatile substances with the aforementioned OPLS-DA model and 28 kinds of differential volatile substances to achieve the identification of the varieties and muscle characteristic flavor substances of disease-resistant grass carp and common grass carp.

[0034] In summary, the present invention provides a method for quickly identifying grass carp varieties and muscle characteristic flavor substances based on GC×GC-TOFMS technology. This method collects and analyzes volatile substances through SPME-GC×GC-TOFMS technology, and can obtain more comprehensive volatile flavor substance information compared to traditional gas chromatography-mass spectrometry. Subsequently, a grass carp identification model based on volatile flavor substances is constructed through multivariate statistical analysis methods, and the OPLS-DA model is used to screen related differential volatile substances to generate a visual heat map. The types and contents of volatile substances in the muscles of disease-resistant grass carp and ordinary grass carp are significantly different. Based on the above technology, rapid identification of disease-resistant grass carp and differential volatile flavor substance analysis can be achieved. Therefore, this method has good performance for quickly distinguishing disease-resistant grass carp and ordinary grass carp products, and can provide a fast, accurate and practical method for quality evaluation and market promotion of disease-resistant grass carp.

Claims

1. A method for rapidly identifying grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology, characterized in that: The following steps are involved: (1) Weigh the fish muscle sample to be tested, transfer it to a headspace bottle after crushing, and extract the volatile flavor substances in the muscle sample by solid phase microextraction; (2) analyzing the volatile substances extracted in step (1) by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry; (3) comparing the mass spectrum data of the volatile substances collected in step (2) with the mass spectrum information of the standard samples in the NIST2020 database to determine the type of the volatile substances; Deuterated n-hexanol solution was used as the internal standard and the internal standard normalization method was used to quantitatively analyze the detected volatile substances; (4) The qualitative and quantitative data of volatile substances obtained in step (3) are compared with the differential identification analysis model and differential volatile substances of disease-resistant grass carp / common grass carp, so as to identify the varieties and muscle characteristic flavor substances of disease-resistant grass carp and common grass carp.

2. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 1, characterized in that: The disease-resistant grass carp is bred by backcrossing gynogenetic grass carp induced by koi sperm with male grass carp, and the common grass carp is the original species of grass carp in the Yangtze River waters.

3. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 1, characterized in that: In step (1), the muscle sample is taken from the muscle above the lateral line on the back of the fish and below the dorsal fin; the solid phase microextraction conditions are: using an automatic sampler to process the sample, placing the muscle sample in a headspace injection bottle, then adding an internal standard solution, incubating at 70-90°C for 10-20 minutes, and aging the SPME extraction head at 260-280°C for 10-15 minutes before adsorbing the sample; Subsequently, the SPME extraction head adsorbed the sample at 70-90°C for 20-30 minutes. After the adsorption, the SPME extraction head was transferred to the GC injection port and desorbed at 240-260°C for 5-10 minutes.

4. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 1, characterized in that: In step (2), the chromatographic column of the comprehensive two-dimensional gas chromatography adopts a GC×GC column system: the one-dimensional chromatographic column is a DB-HeavyWax type chromatographic column; the two-dimensional chromatographic column is: Rxi-5SilMS type chromatographic column.

5. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 1, characterized in that: In step (2), the comprehensive two-dimensional gas chromatography conditions are: using high-purity helium as the carrier gas; the temperature program of the one-dimensional chromatographic column DB-HeavyWax is: initial temperature 40-50°C, maintained for 2-5 minutes, increased to 220-250°C at a rate of 3-5°C / min, and maintained for 4-6 minutes; the temperature program of the two-dimensional chromatographic column Rxi-5SilMS is 5°C higher than that of the one-dimensional chromatographic column, the modulator temperature is always 15°C higher than the column temperature of the two-dimensional chromatographic column, the modulation period is 5-10 seconds, and the injection port temperature is 240-270°C.

6. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 1, characterized in that: In step (2), the time-of-flight mass spectrometry analysis conditions are: mass spectrometry transmission line temperature is 230-270°C, ion source temperature is 230-270°C, acquisition rate is 150-200 spectra / s, and mass spectrometry scanning range is m / z35-550.

7. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 1, characterized in that: In step (3), the ChromaTOF software is used to compare the collected mass spectrometry data of volatile substances with the mass spectrometry information of the NIST2020 database, and a qualitative analysis is performed in combination with the compound mass spectrum, compound retention index, and standard retention index to confirm the detected volatile compounds; Deuterated n-hexanol solution was used as the internal standard and the internal standard normalization method was used to calculate the relative content of the detected volatile compounds. The relative concentration of the detected compound was obtained by multiplying the ratio of the peak area of ​​the detected compound to the peak area of ​​the internal standard compound by the known concentration of the internal standard compound.

8. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 1, characterized in that: In step (4), the method for constructing the differential identification analysis model of disease-resistant grass carp / common grass carp comprises the following steps: (11) Muscle samples of disease-resistant grass carp and common grass carp were weighed, crushed and transferred to headspace vials, and volatile flavor compounds in the muscle samples were extracted by solid phase microextraction; (12) analyzing the volatile substances extracted in step (11) by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry; (13) comparing the mass spectrum data of the volatile substances collected in step (12) with the mass spectrum information of the standard samples in the NIST2020 database to determine the type of the volatile substances; Deuterated n-hexanol solution was used as the internal standard and the internal standard normalization method was used to quantitatively analyze the detected volatile substances; (14) The qualitative and quantitative data of volatile substances were preprocessed using SIMCA-P software. Then, principal component analysis was used to achieve a preliminary distinction between disease-resistant grass carp and common grass carp, and the variability of samples within the group was evaluated. Orthogonal partial least squares discriminant analysis was used to further process the qualitative and quantitative data of volatile substances, and an orthogonal partial least squares discriminant analysis model based on volatile flavor substances was constructed as a discrimination model for distinguishing disease-resistant grass carp from common grass carp.

9. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 8, characterized in that: The method for screening differential volatile substances between disease-resistant grass carp and common grass carp comprises the following steps: on the basis of steps (11) to (14), the differential volatile substances related to varieties are screened by an orthogonal partial least squares discriminant analysis model and a visualized heat map is generated, wherein the specific screening conditions are VIP>1.75, FC≥2 or ≤0.5, and T test p<0.05, and the screened differential volatile substances are used as muscle characteristic flavor substances for distinguishing disease-resistant grass carp from common grass carp.

10. The method for rapid identification of grass carp species and muscle characteristic flavor substances based on GC×GC-TOFMS technology according to claim 9, characterized in that: Among the screened differential volatile substances, the volatile substances that were upregulated in the disease-resistant grass carp compared with the common grass carp included: 5-methyl-5-nonanol, 3,5-di-tert-butyl-2-hydroxybenzonitrile, 7-methylpentadecane, 3,5-dimethylundecane, 3-acetylheptane-2,6-dione, nonanamide, 2,3-dimethyl-2-(propan-2-yl)butanoic acid, any one or more of 1H-Isoindole, 3-methoxy-4,7-dimethyl; Among the screened differential volatile substances, the volatile substances that are downregulated in disease-resistant grass carp compared with common grass carp include: any one or more of 9,12,15-octadecatriene, 2,4-hexadienal, butyraldehyde, undecylbenzene, 4-ethylbenzaldehyde, 1-methyl-1,2,3,4-tetralin, nonanehydrazine, ethyl propionate, oct-1,4-diene, 2-methyl-1-octen-3-yne, 1-octene, 1,2-vinylcyclohexane, 5-methylnonane, 2-pentyl-2-cyclopentene-1-one, (3E,5E)-nona-3,5-dien-2-one, 1-methyl-1H-pyrazole-3-carboxylic acid methyl ester, Dihexyverine, 4-methoxybutan-2-one, 2,5-dimethyl-1,5-hexadien-3-ol, and Zonarene.

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