Construction method of crab metabolite and lipid fingerprint spectrum and crab quality evaluation method

Abstract

The invention discloses a crab metabolite and lipid fingerprint construction method and a crab quality evaluation method, and relates to the technical field of crab quality evaluation. The method comprises the following steps: extracting metabolites of to-be-detected crab tissues; and detecting the extracted metabolites and lipids by using a liquid chromatography-tandem mass spectrometry technology, and carrying out data processing, metabolite annotation and lipid annotation on the obtained mass spectrum data to obtain the fingerprints of the metabolites and lipids of the crabs. According to the method, rapid, sensitive and quantifiable global analysis on a large amount of endogenous metabolites and lipids in tissues such as edible parts of crabs with complex matrix characteristics is realized, and the method has outstanding advantages in the aspects of mining crab characteristic quality indexes and perfecting crab quality grade judgment standards.

Description

Technical Field

[0001] The present invention relates to the technical field of crab quality evaluation, and in particular to a method for constructing a metabolite and lipid fingerprint of a crab and a method for evaluating the quality of the crab. Background Art

[0002] Chinese mitten crab (Eriocheir sinensis), also known as river crab and hairy crab, is popular among consumers for its balanced amino acids, polyunsaturated fatty acids and trace elements, all of which contribute to its unique flavor and high economic value. Since the intrinsic characteristics of Chinese mitten crab are greatly affected by human factors such as breeding management and environmental factors such as geographical location, the quality of Chinese mitten crab in the crab market is inevitably uneven.

[0003] At present, the quality grade of Chinese mitten crab is mainly determined by external morphological characteristics such as weight, fatness and sex. The price of large crabs in the crab market is generally 2-5 times higher than that of small crabs. At present, the detection of the intrinsic quality characteristic components of Chinese mitten crab mainly focuses on the determination of nutrients such as amino acids, nucleotides, cholesterol and betaine. Most of them use a sample pretreatment technology and instrumental analysis method to target and analyze a specific type of nutrient in the crab sample, and it is impossible to conduct a comprehensive and systematic analysis of a large number of quality characteristic components in Chinese mitten crab at the same time. However, due to the complex and changeable biological characteristics of Chinese mitten crab itself, it is difficult for each type of nutrient information to accurately represent the intrinsic quality characteristics of the crab sample, resulting in the lack of application of intrinsic quality characteristic component indicators in the quality evaluation of Chinese mitten crab, and there is no report on the construction of metabolite and lipid fingerprints of Chinese mitten crab using metabolomics and lipidomics technologies.

[0004] Therefore, there is an urgent need for an analytical method that can systematically and comprehensively reflect the intrinsic quality components of Chinese mitten crab, which is particularly important for the subsequent mining of quality characteristic components and research on quality regulation mechanisms.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a metabolite and lipid fingerprint of crabs and a method for evaluating crab quality to solve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a method for constructing a metabolite fingerprint of crab, comprising the following steps:

[0009] S1: Extract metabolites from crab tissues to be tested;

[0010] S2: Use liquid chromatography-tandem mass spectrometry to detect the extracted metabolites, process the mass spectrometry data and annotate the metabolites to obtain the metabolite fingerprint of crab;

[0011] The liquid chromatography column used in the detection was ACQUITY UPLC BEH Amide (1.7 μm, 2.1*100 mm Column), and the column temperature was 40°C. The liquid chromatography conditions included: mobile phase A was acetonitrile-water (5:5, V / V) and 5-10 mM ammonium acetate, and mobile phase B was acetonitrile-water (95:5, V / V) and 5-10 mM ammonium acetate, with gradient elution, a flow rate of 0.2-0.4 min / L, and an injection volume of 2-4 μL.

[0012] The gradient elution program was as follows: 0 min, 95% to 98% mobile phase B; 0-2 min, 95% to 98% mobile phase B; 2-17 min, 50% mobile phase B; 17-21 min, 50% mobile phase B; 21-23 min, 95% to 98% mobile phase B; 23-25 ​​min, 95% to 98% mobile phase B;

[0013] Or, 0min, 95% to 98% mobile phase B; 0-1min, 95% to 98% mobile phase B; 1-17min, 50% mobile phase B; 17-21min, 50% mobile phase B; 21-22min, 95% to 98% mobile phase B; 22-25min, 95% to 98% mobile phase B;

[0014] The mass spectrometry parameters were: ESI as the ion source, full scan (Full MS) in positive and negative modes - ddMS 2 , Spray voltage: ±4000-4500 (V); Sheath gas pressure: 40-50 psi; Auxiliary gas pressure: 10-12 arbitrary units (Arb); Capillary temperature: 320-350 ° C; Full MS scan range: 100-1500 m / z, resolution: 70000, ddMS 2 The resolution was 17500 and the normalized collision energy was 30 Ev.

[0015] In a second aspect, the present invention provides a method for constructing a lipid fingerprint of crab, which comprises the following steps: S1: extracting lipids from crab tissue to be tested;

[0016] S2: Use liquid chromatography-tandem mass spectrometry to detect the extracted lipids, process the mass spectrometry data and annotate the lipids to obtain the lipid fingerprint of crabs;

[0017] The liquid chromatography column used in the detection was: xbridge C18 (3.5μm, 4.6*100mm Column), and the column temperature was 40°C; the liquid chromatography conditions included: mobile phase A was acetonitrile-water (6:4, V / V) and 5-10mM ammonium acetate, and mobile phase B was isopropanol-acetonitrile (9:1, V / V) and 5-10mM ammonium acetate, with gradient elution, a flow rate of 0.3-0.4min / L, and an injection volume of 2-4μL; the injection chamber temperature was set to 20°C;

[0018] The gradient elution program was 0 min, 20-30% mobile phase B; 0-2 min, 40-43% mobile phase B; 2-4 min, 50-60% mobile phase B; 4-8 min, 70-75% mobile phase B; 8-16 min, 80% mobile phase B; 16-18 min, 85% mobile phase B; 18-19 min, 85-95% mobile phase B; 19-19.1 min, 20-30% mobile phase B; 19.1-20 min, 20-30% mobile phase B;

[0019] The mass spectrometry parameters are as follows: ESI as the ion source, full scan (Full MS)-ddMS2 in positive and negative modes, spray voltage (Spray voltage): ±4000-4500 (V); sheath gas pressure (Sheath gas pressure): 40-50psi; auxiliary gas pressure (Auxiliary gas pressure): 10-12arbitrary units (Arb); capillary temperature (Capillary temperature): 320-350℃; Full MS scan range is 100-1500m / z, resolution is 70000, ddMS2 resolution is 17500, and standardized collision energy is 30Ev.

[0020] In a third aspect, the present invention further provides a method for classifying crab varieties or crab farming methods, which comprises at least one of the following methods (1)-(2):

[0021] (1) Obtaining a metabolite fingerprint of a target crab according to the above-mentioned method for constructing a metabolite fingerprint of a crab; and classifying the crab species or breeding methods by comparing the metabolite fingerprints of multiple crabs measured;

[0022] (2) Obtaining the lipid fingerprint of the target crab according to the above-mentioned method for constructing the lipid fingerprint of crab; and classifying the crab species or breeding methods by comparing the lipid fingerprints of multiple crabs measured.

[0023] In a fourth aspect, the present invention further provides a method for identifying crab species or breeding methods, which comprises at least one of the following methods (1)-(2):

[0024] (1) Obtaining metabolite fingerprints of multiple crabs of known species or breeding methods according to the above-mentioned method for constructing metabolite fingerprints of crabs; then obtaining a metabolite fingerprint of a crab to be identified according to the above-mentioned method for constructing metabolite fingerprints of crabs; comparing the metabolite fingerprint of the identified crab with the metabolite fingerprints of multiple known species of crabs, thereby identifying the crab species or breeding method;

[0025] (2) Obtain lipid fingerprints of multiple crabs of known species or breeding methods according to the above-mentioned method for constructing the lipid fingerprint of crabs; then obtain the lipid fingerprint of the crab to be identified according to the above-mentioned method for constructing the lipid fingerprint of crabs; compare the lipid fingerprint of the identified crab with the lipid fingerprints of multiple crabs of known species or breeding methods, so as to identify the crab species or breeding method.

[0026] In a fifth aspect, the present invention also provides a method for evaluating crab quality, comprising the following steps:

[0027] (1) Obtaining metabolite fingerprints of at least two crabs to be evaluated according to the above-mentioned method for constructing the metabolite fingerprint of crabs, and / or, obtaining lipid fingerprints of at least two crabs to be evaluated according to the above-mentioned method for constructing the lipid fingerprint of crabs;

[0028] (2) The annotated metabolites and / or lipid components were subjected to OPLS-DA analysis, and quality characteristic components were screened based on the OPLS-DA model. The differential metabolites and / or lipid components were screened with P < 0.05 and VIP > 1 as the screening conditions, and the crab quality was evaluated based on the differential metabolites and / or lipid components obtained by screening.

[0029] The present invention has the following beneficial effects:

[0030] The present invention provides a method for constructing a metabolite and lipid fingerprint of crabs, which can construct a metabolite and lipid fingerprint of crabs, and the metabolite and lipid fingerprint can be used to accurately characterize the intrinsic quality characteristics of crabs (especially Chinese mitten crabs). The metabolite and lipid multidimensional indicators are used to achieve the effect of systematically, comprehensively, efficiently and accurately evaluating and determining the intrinsic quality of crabs (especially Chinese mitten crabs), which has important application value in technical theory and practical promotion.

[0031] The invention overcomes the shortcoming that the current quality component analysis methods are unable to conduct a comprehensive and systematic analysis of a large number of quality characteristic metabolites in Chinese mitten crabs, and realizes a rapid, sensitive and quantifiable global analysis of a large number of endogenous metabolites and lipids in tissues such as the edible parts of crabs with complex matrix characteristics. It has outstanding advantages in exploring crab characteristic quality indicators and improving crab quality grade judgment standards.

[0032] The fingerprint map construction method provided by the present invention is also of reference significance for the mining of other aquatic product quality evaluation indicators. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 This is the metabolic fingerprint information of Chinese mitten crab in positive ion mode in one embodiment of the present invention;

[0035] Figure 2 This is the lipid fingerprint information of Chinese mitten crab in positive ion mode in one embodiment of the present invention;

[0036] Figure 3 This is a statistical diagram of lipid classification of Chinese mitten crab in one embodiment of the present invention;

[0037] Figure 4 This is a classification diagram of metabolites of Chinese mitten crab in one embodiment of the present invention;

[0038] Figure 5 The PCA model discrimination result of the quality of Chinese mitten crab in one embodiment of the present invention;

[0039] Figure 6 This is a heat map of the components of the quality characteristics of Chinese mitten crab with significant differences in one embodiment of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0041] In a first aspect, the present invention provides a method for constructing a metabolite fingerprint of crab, comprising the following steps:

[0042] S1: Extract metabolites from crab tissues to be tested;

[0043] S2: Use liquid chromatography-tandem mass spectrometry to detect the extracted metabolites, process the mass spectrometry data and annotate the metabolites to obtain the metabolite fingerprint of crab;

[0044] The liquid chromatography column used in the detection was ACQUITY UPLC BEH Amide (1.7 μm, 2.1*100 mm Column), and the column temperature was 40°C. The liquid chromatography conditions included: mobile phase A was acetonitrile-water (5:5, V / V) and 5-10 mM ammonium acetate, and mobile phase B was acetonitrile-water (95:5, V / V) and 5-10 mM ammonium acetate, with gradient elution, a flow rate of 0.2-0.4 min / L, and an injection volume of 2-4 μL.

[0045] The gradient elution program was as follows: 0 min, 95% to 98% mobile phase B; 0-2 min, 95% to 98% mobile phase B; 2-17 min, 50% mobile phase B; 17-21 min, 50% mobile phase B; 21-23 min, 95% to 98% mobile phase B; 23-25 ​​min, 95% to 98% mobile phase B;

[0046] Or, 0min, 95% to 98% mobile phase B; 0-1min, 95% to 98% mobile phase B; 1-17min, 50% mobile phase B; 17-21min, 50% mobile phase B; 21-22min, 95% to 98% mobile phase B; 22-25min, 95% to 98% mobile phase B;

[0047] The mass spectrometry parameters were: ESI as the ion source, full scan (Full MS) in positive and negative modes - ddMS 2, Spray voltage: ±4000-4500 (V); Sheath gas pressure: 40-50 psi; Auxiliary gas pressure: 10-12 arbitrary units (Arb); Capillary temperature: 320-350 ° C; Full MS scan range: 100-1500 m / z, resolution: 70000, ddMS 2 The resolution was 17500 and the normalized collision energy was 30 Ev.

[0048] The present invention provides a method for constructing a metabolite fingerprint of crabs, which can construct a metabolite fingerprint of crabs, and the metabolite fingerprint can be used to accurately characterize the intrinsic quality characteristics of crabs (especially Chinese mitten crabs). By using multidimensional indicators of metabolites, the effect of systematically, comprehensively, efficiently and accurately evaluating and determining the intrinsic quality of crabs (especially Chinese mitten crabs) can be achieved, which has important application value in technical theory and practical promotion.

[0049] The invention overcomes the shortcoming that the current quality component analysis methods are unable to conduct a comprehensive and systematic analysis of a large number of quality characteristic metabolites in Chinese mitten crabs, and realizes a rapid, sensitive and quantifiable global analysis of a large number of endogenous metabolites and lipids in tissues such as the edible parts of crabs with complex matrix characteristics. It has outstanding advantages in exploring crab characteristic quality indicators and improving crab quality grade judgment standards.

[0050] The method for extracting metabolites from the crab tissue to be tested can use organic and / or inorganic reagents to extract metabolites, organic reagents such as alcohols, chlorinated alkanes, etc., alcohols include but are not limited to monohydric alcohols, dihydric alcohols, trihydric alcohols or polyhydric alcohols, monohydric alcohols include but are not limited to methanol, ethanol, propanol or butanol. Chlorinated alkanes include but are not limited to chloroform, dichloromethane, etc.

[0051] The obtained fingerprint spectrum includes but is not limited to: the ordinate is the relative abundance of the metabolite, and the abscissa is time. In other embodiments, the ordinate or abscissa of the above fingerprint spectrum can also be other values ​​after mathematical conversion (such as taking an absolute constant). As long as it can reflect the relationship between the peak area of ​​the metabolite and the type of metabolite, it belongs to the fingerprint spectrum.

[0052] In a preferred embodiment of the present invention, the crab belongs to the family Sphaerocephala or the family Archaeocephala; the method for constructing the metabolite fingerprint provided by the present invention has reference value for constructing the metabolite fingerprint of various aquatic animals, such as other crustaceans, such as shrimp, lobster, crayfish, krill, copepods, barnacles, or hybrids thereof.

[0053] The crustacean is selected from the group consisting of Chinese white shrimp, pink shrimp, black tiger shrimp, freshwater shrimp, Gulf shrimp, Pacific white shrimp, whiteleg shrimp, giant tiger shrimp, rock shrimp, Akiama paste shrimp, Southern rough shrimp, fleshy prawn, banana prawn, Northern prawn, blue crab, peekytoe crab, frog crab, northern yellow crab, snow crab, king crab, stone crab, Dungeness crab, soft shell crab, Cromer crab, American lobster, spiny lobster, squat lobster, gooseneck barnacle, picoroco barnacle, or hybrids thereof.

[0054] In a preferred embodiment of the present invention, the crab is the Chinese mitten crab of the family Archaeochaetidae;

[0055] In a preferred embodiment of the present invention, step S1 comprises: first mixing a mixture of dichloromethane and ethanol with the crab tissue to be tested, grinding and centrifuging to obtain a bottom solid, taking the bottom solid and mixing it with a methanol aqueous solution; grinding, centrifuging, taking the supernatant to dry, then re-dissolving in a methanol aqueous solution, centrifuging, taking the supernatant for liquid chromatography-tandem mass spectrometry detection;

[0056] In a preferred embodiment of the present invention, the mixing volume ratio of dichloromethane and methanol is 2:1 or 3:1, and the mixing ratio of the mixed solution of dichloromethane and methanol to the crab tissue to be tested is 1.6ml-2.1ml:50mg; under the above mixing ratio, metabolites can be efficiently extracted and obtained.

[0057] In a preferred embodiment of the present invention, the volume ratio of methanol to water in the methanol aqueous solution is 1:1-1.5. In a preferred embodiment of the present invention, the amount of methanol aqueous solution added is 1.5-2 ml;

[0058] In a preferred embodiment of the present invention, the grinding and centrifugation conditions are: grinding on ice, centrifugation at 12000-20000 rpm, 0-5°C for 8-15 min; grinding on ice to prevent metabolites from being degraded.

[0059] In a preferred embodiment of the present invention, the centrifugation conditions after reconstitution are 12000-16000 rpm, 0-5°C, and centrifugation for 15-20 min; the purpose of reconstitution is to improve the purity of metabolites and remove the interference of insoluble matter on metabolite detection.

[0060] In a preferred embodiment of the present invention, data processing of the acquired mass spectrum data includes at least one of the following processing methods: peak alignment, peak extraction, noise reduction, normalization and null value filling of the mass spectrum data.

[0061] In a preferred embodiment of the present invention, metabolite annotation is performed using the Compound Discover 3.3 software built-in database mz cloud and the public database HMDB for metabolite identification. In other embodiments, the above metabolite annotation can also be performed using other annotation software.

[0062] In a second aspect, the present invention provides a method for constructing a lipid fingerprint of crab, which comprises the following steps: S1: extracting lipids from crab tissue to be tested;

[0063] S2: Use liquid chromatography-tandem mass spectrometry to detect the extracted lipids, process the mass spectrometry data and annotate the lipids to obtain the lipid fingerprint of crabs;

[0064] The liquid chromatography column used in the detection was: xbridge C18 (3.5μm, 4.6*100mm Column), and the column temperature was 40°C; the liquid chromatography conditions included: mobile phase A was acetonitrile-water (6:4, V / V) and 5-10mM ammonium acetate, and mobile phase B was isopropanol-acetonitrile (9:1, V / V) and 5-10mM ammonium acetate, with gradient elution, a flow rate of 0.3-0.4min / L, and an injection volume of 2-4μL; the injection chamber temperature was set to 20°C;

[0065] The gradient elution program was 0 min, 20-30% mobile phase B; 0-2 min, 40-43% mobile phase B; 2-4 min, 50-60% mobile phase B; 4-8 min, 70-75% mobile phase B; 8-16 min, 80% mobile phase B; 16-18 min, 85% mobile phase B; 18-19 min, 85-95% mobile phase B; 19-19.1 min, 20-30% mobile phase B; 19.1-20 min, 20-30% mobile phase B;

[0066] The mass spectrometry parameters are as follows: ESI as the ion source, full scan (Full MS)-ddMS2 in positive and negative modes, spray voltage (Spray voltage): ±4000-4500 (V); sheath gas pressure (Sheath gas pressure): 40-50psi; auxiliary gas pressure (Auxiliary gas pressure): 10-12arbitrary units (Arb); capillary temperature (Capillary temperature): 320-350℃; Full MS scan range is 100-1500m / z, resolution is 70000, ddMS2 resolution is 17500, and standardized collision energy is 30Ev.

[0067] The method for extracting lipids can use organic and / or inorganic reagents to extract metabolites, organic reagents such as alcohols, chlorinated alkanes, acetonitrile, etc., alcohols include but are not limited to monohydric alcohols, dihydric alcohols, trihydric alcohols or polyhydric alcohols, monohydric alcohols include but are not limited to methanol, ethanol, propanol or butanol. Chlorinated alkanes include but are not limited to chloroform, dichloromethane, etc.

[0068] The obtained fingerprint spectrum includes but is not limited to: the ordinate is the relative abundance of lipids, and the abscissa is time. In other embodiments, the ordinate or abscissa of the above fingerprint spectrum can also be other values ​​after mathematical conversion (such as taking an absolute constant). As long as it can reflect the relationship between the peak area of ​​lipids and the type of lipids, it belongs to the fingerprint spectrum.

[0069] In a preferred embodiment of the present invention, step S1 includes: first mixing a mixture of dichloromethane and methanol with the crab tissue to be tested, grinding and centrifuging to obtain a supernatant, taking the supernatant and blowing it dry, then re-dissolving it in an isopropanol-acetonitrile solution added with 5mM ammonium acetate, centrifuging and taking the supernatant for liquid chromatography-tandem mass spectrometry detection; the volume ratio of isopropanol to acetonitrile in the isopropanol-acetonitrile solution is 1 to 8:2. According to the above extraction method, more types and numbers of lipids can be extracted to facilitate subsequent lipid identification.

[0070] In a preferred embodiment of the present invention, the volume ratio of dichloromethane to methanol in the mixture of dichloromethane and methanol is 3:1 to 2:1, and the volume ratio of the supernatant obtained by grinding and centrifugation to the isopropanol-acetonitrile solution of 5 mM ammonium acetate is 4 to 9:1;

[0071] In a preferred embodiment of the present invention, the grinding and centrifugation conditions are: grinding on ice, centrifuging at 12000-20000 rpm, 0-5°C for 8-15 min;

[0072] In a preferred embodiment of the present invention, the centrifugation conditions after reconstitution are: 8000-12000 rpm, centrifugation for 15-25 min. The centrifugation conditions after reconstitution are: including but not limited to: 8000 rpm, 10000 rpm, 10500 rpm, 12000 rpm, centrifugation for 15-20 min, 18-23 min, 22-25 min.

[0073] In a preferred embodiment of the present invention, data processing of the obtained mass spectrometry data includes at least one of the following processing methods: peak alignment, peak extraction, noise reduction, normalization and null value filling of the mass spectrometry data; through the above data processing, the current situation of abundant information and scarce useful information can be alleviated.

[0074] In a preferred embodiment of the present invention, lipid annotation is performed using Compound Discover 3.3 software, using the public lipid library LipidBank and Lipidmaps to perform lipid compound identification and analysis.

[0075] In a third aspect, the present invention further provides a method for classifying crab varieties or crab farming methods, which comprises at least one of the following methods (1)-(2):

[0076] (1) Obtaining a metabolite fingerprint of a target crab according to the above-mentioned method for constructing a metabolite fingerprint of a crab; and classifying the crab species or breeding methods by comparing the metabolite fingerprints of multiple crabs measured;

[0077] (2) Obtaining the lipid fingerprint of the target crab according to the above-mentioned method for constructing the lipid fingerprint of crab; and classifying the crab species or breeding methods by comparing the lipid fingerprints of multiple crabs measured.

[0078] The method includes but is not limited to classifying the species of multiple unknown crabs, such as tracing the origin, etc.; classifying the breeding methods of multiple crabs, such as classifying different feed feeding methods, feed dosage classification, lake water breeding classification, etc.

[0079] In a preferred embodiment of the present invention, the classification method further comprises extracting features and analyzing the metabolite data obtained after obtaining the metabolite fingerprint of the target crab;

[0080] In a preferred embodiment of the present invention, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis;

[0081] In a preferred embodiment of the present invention, the classification rule includes: if the metabolite data of multiple target crabs can be clearly clustered into the same category, then they are judged to be crabs of the same species or the same breeding method. A person skilled in the art can compare the peak area and peak graph to evaluate whether they are clustered into the same category.

[0082] In a preferred embodiment of the present invention, the classification method further comprises extracting and analyzing features of the lipid data obtained after obtaining the lipid fingerprint of the target crab;

[0083] In a preferred embodiment of the present invention, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis;

[0084] In a preferred embodiment of the present invention, the classification rule includes: if the lipid data of multiple target crabs can be obviously clustered into the same category, they are judged to be crabs of the same species or the same breeding method.

[0085] In a fourth aspect, the present invention further provides a method for identifying crab species or breeding methods, which comprises at least one of the following methods (1)-(2):

[0086] (1) Obtaining metabolite fingerprints of multiple crabs of known species or breeding methods according to the above-mentioned method for constructing metabolite fingerprints of crabs; then obtaining a metabolite fingerprint of a crab to be identified according to the above-mentioned method for constructing metabolite fingerprints of crabs; comparing the metabolite fingerprint of the identified crab with the metabolite fingerprints of multiple known species of crabs, thereby identifying the crab species or breeding method;

[0087] (2) Obtain lipid fingerprints of multiple crabs of known species or breeding methods according to the above-mentioned method for constructing the lipid fingerprint of crabs; then obtain the lipid fingerprint of the crab to be identified according to the above-mentioned method for constructing the lipid fingerprint of crabs; compare the lipid fingerprint of the identified crab with the lipid fingerprints of multiple crabs of known species or breeding methods, so as to identify the crab species or breeding method.

[0088] In a preferred embodiment of the present invention, after obtaining metabolite fingerprints of multiple crabs of known species or breeding methods and metabolite fingerprints of crabs to be identified, it also includes feature extraction and analysis of the metabolite data obtained by the detection;

[0089] In a preferred embodiment of the present invention, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis;

[0090] In a preferred embodiment of the present invention, the identification rules include: if the metabolite data of the identified crab can be obviously clustered into the same category as the metabolite data of crabs of any known species or known breeding method, then the crab is identified as a crab of the known species or the breeding method.

[0091] In a preferred embodiment of the present invention, after obtaining the lipid fingerprint of the target crab, the method further includes extracting and analyzing the lipid data obtained by the detection;

[0092] In a preferred embodiment of the present invention, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis;

[0093] In a preferred embodiment of the present invention, the identification rules include: if the lipid data of the identified crab can be obviously clustered into the same category as the lipid data of crabs of any known species or known breeding method, then the crab is identified as a crab of the known species or the breeding method.

[0094] In a fifth aspect, the present invention also provides a method for evaluating crab quality, comprising the following steps:

[0095] (1) Obtaining metabolite fingerprints of at least two crabs to be evaluated according to the above-mentioned method for constructing the metabolite fingerprint of crabs, and / or, obtaining lipid fingerprints of at least two crabs to be evaluated according to the above-mentioned method for constructing the lipid fingerprint of crabs;

[0096] (2) The annotated metabolites and / or lipid components were subjected to PLS-DA analysis or OPLS-DA analysis, and quality characteristic components were screened based on the PLS-DA or OPLS-DA model. The differential metabolites and / or lipid components were screened with P < 0.05 and VIP > 1 as the screening conditions, and the crab quality was evaluated based on the differential metabolites and / or lipid components obtained by screening.

[0097] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0098] Example 1

[0099] This embodiment provides a method for constructing a metabolite fingerprint of Chinese mitten crab, which comprises the following steps:

[0100] (1) Collecting edible tissues of Chinese mitten crab

[0101] Male and female Chinese mitten crabs fed with three different feeding methods (normal aquatic feed group (CON), aquatic feed group supplemented with 0.1% plant activated carbon as a food additive (AC1), and aquatic feed group supplemented with 2.5% plant activated carbon as a food additive (AC2)) were collected from a breeding base in Chongming District, a representative breeding area in Shanghai. After collecting the edible tissues (hepatopancreas, gonads, and muscles) of the Chinese mitten crabs, the edible tissues of one female and one male crab under the same feeding method were randomly mixed as one parallel sample. Five parallel samples were set up under the same feeding method, which were quick-frozen in liquid nitrogen and then stored at -80°C for later use.

[0102] (2) Extraction of metabolites

[0103] After thawing the edible tissue (mixed sample of hepatopancreas, gonad and muscle tissue) on ice, 50 mg of the sample was weighed and placed in a 2 mL centrifuge tube. 1.6 mL of pre-cooled dichloromethane / methanol (3:1, V / V) mixed extract was added. The sample mixture was placed on ice for tissue grinding and centrifuged at 15000 rpm at 4 °C for 10 min. The supernatant (for lipid analysis) and the solids at the bottom (for non-targeted metabolomics analysis) were collected after centrifugation.

[0104] Add 1.5 mL of pre-cooled methanol / water (1:1, V / V) mixed extract to the solids at the bottom, place the sample mixture on ice for tissue grinding and refrigerate at -18°C for 2 hours. Subsequently, centrifuge at 15000 rpm and 4°C for 10 minutes, collect the supernatant after centrifugation, blow it to dryness on a nitrogen blower, redissolve it in 120 μL of methanol / water (1:1, V / V) solution, vortex until completely redissolved, centrifuge it at 15000 rpm and 4°C for 20 minutes, and take 50 μL of supernatant and place it on the Q-Exactive Orbitrap mass spectrometer for non-targeted metabolomics data acquisition. All sample supernatants used for non-targeted metabolomics analysis were mixed in equal amounts to prepare quality control samples.

[0105] (3) Metabolite Detection by Liquid Chromatography-Mass Spectrometry

[0106] The metabolomics analysis of the samples was performed using Q-Exactive Orbitrap mass spectrometry to obtain the metabolite fingerprint of edible tissues ( Figure 1 ).

[0107] When constructing the metabolite fingerprint, the chromatographic column was selected as ACQUITY UPLC BEH Amide (1.7 μm, 2.1*100 mm Column), and the specific instrument mass spectrometry parameters were: ESI was used as the ion source, and full scan (Full MS)-ddMS was performed in positive and negative modes 2, Spray voltage: ±4000-4500 (V); Sheath gas pressure: 40-50 psi; Auxiliary gas pressure: 10-12 arbitrary units (Arb); Capillary temperature: 320-350 ° C; Full MS scan range: 100-1500 m / z, resolution: 70000, ddMS 2 The resolution was 17500 and the normalized collision energy was 30 Ev.

[0108] The column temperature was set at 40°C. The liquid phase conditions were acetonitrile-water (5:5, V / V) + 5mM ammonium acetate as mobile phase A and acetonitrile-water (95:5, V / V) + 5mM ammonium acetate as mobile phase B for gradient elution, with a flow rate of 0.3 min / L, an injection volume of 2 μL, and an injection chamber temperature of 4°C. The gradient elution program was 0 min, 98% B; 0-1 min, 98% B; 1-17 min, 50% B; 17-21 min, 50% B; 21-22 min, 98% B; 22-25 min, 98% B.

[0109] (4) Metabolite fingerprint processing:

[0110] The obtained non-targeted metabolomics raw data files were imported into Compound Discover 3.3 software for data preprocessing including peak alignment, peak extraction, noise reduction and normalization. Metabolites were identified using the Compound Discover 3.3 software's own database mz cloud and the public database HMDB. The corresponding two-dimensional data matrix was annotated and generated, and metabolite peak identification, peak area integration and absolute value processing were performed on the matrix.

[0111] The statistical diagram of the metabolite classification obtained by identification shows ( Figure 3 ), HMDB annotated 852 metabolites in 12 major categories, including organic acids and derivatives (258), lipids and lipid-like molecules (121), organic heterocyclic compounds (99), organic oxygen compounds (94), polyketides (56) and nucleotides and their analogs (53).

[0112] Reference for constructing metabolite fingerprints of edible tissues Figure 1 shown.

[0113] Example 2

[0114] This embodiment provides a method for constructing a metabolite fingerprint of Chinese mitten crab, which comprises the following steps:

[0115] (1) Take about 1.5 mL of the supernatant after centrifugation for lipid analysis in Example 1, blow it to dryness on a nitrogen blower, and redissolve it in 120 μL of isopropanol-acetonitrile (9:1, V / V) + 5 mM ammonium acetate solution, vortex until completely redissolved, and centrifuge it at 10000 rpm for 20 minutes at room temperature. Take 50 μL of the supernatant and place it in a QE high-resolution mass spectrometer for non-targeted lipidomics data acquisition. All sample supernatants used for non-targeted lipidomics analysis were mixed in equal amounts to prepare quality control samples.

[0116] (2) Liquid chromatography-mass spectrometry detection of lipids

[0117] The samples were analyzed for lipidomics using Q-Exactive Orbitrap mass spectrometry, and the lipid fingerprints were referenced Figure 2 shown.

[0118] When constructing the lipid fingerprint, the chromatographic column selected was xbridge C18 (3.5 μm, 4.6*100 mm Column).

[0119] The specific instrument mass spectrometry parameters are: ESI as the ion source, full scan (Full MS)-ddMS2 in positive and negative modes, spray voltage (Spray voltage): ±4000-4500 (V); sheath gas pressure (Sheath gas pressure): 40-50psi; auxiliary gas pressure (Auxiliary gas pressure): 10-12arbitrary units (Arb); capillary temperature (Capillary temperature): 320-350℃; Full MS scan range is 100-1500m / z, resolution is 70000, ddMS2 resolution is 17500, and standardized collision energy is 30Ev.

[0120] The liquid phase conditions were as follows: mobile phase A was acetonitrile-water (6:4, V / V) + 5 mM ammonium acetate and mobile phase B was isopropanol-acetonitrile (9:1, V / V) + 5 mM ammonium acetate for gradient elution, the flow rate was 0.4 min / L, the injection volume was 4 μL, and the injection chamber temperature was set to 20°C. The column temperature was 40°C.

[0121] The gradient elution program was as follows: 0 min, 30% B; 0-2 min, 40% B; 2-4 min, 60% B; 4-8 min, 75% B; 8-16 min, 80% B; 16-18 min, 85% B; 18-19 min, 95% B; 19-19.1 min, 30% B; 19.1-20 min, 30% B.

[0122] (3) Construction of lipid fingerprint.

[0123] The obtained non-targeted lipidomics raw data files were imported into Compound Discover 3.3 software for data preprocessing such as peak alignment, peak extraction, noise reduction, and normalization, and the raw lipid fingerprint was processed with Compound Discover 3.3 software.

[0124] Lipid compounds were identified and analyzed based on the public lipid libraries LipidBank and Lipidmaps. The corresponding two-dimensional data matrix was annotated and generated, and metabolite peak identification, peak area integration and absolute value processing were performed on the matrix.

[0125] A total of 3059 lipids in 5 major categories and 54 subcategories were annotated. The five major categories mainly include glycerophospholipids (GP) (1785), glycerides (GL) (749), sphingolipids (SP) (437), fatty acids (FA) (81) and sterol lipids (ST) (7) Figure 4 ).

[0126] Example 3

[0127] This embodiment provides a method for classifying Chinese mitten crab varieties:

[0128] The data matrix obtained by combining the 3059 lipid compounds identified in Example 2 and the 852 metabolic compounds identified in Example 1 was imported into MetaboAnalyst software and PCA principal component analysis was performed using the PCA model.

[0129] The analysis results are shown in Figure 5 The Chinese mitten crab samples under the same feeding method can be obviously clustered into the same category, and the Chinese mitten crab samples under three different feeding methods can also be clearly distinguished, indicating that the metabolite and lipid fingerprint data in Examples 1 and 2 have a good clustering trend, and the PCA model constructed based on metabolite and lipid fingerprint technology can effectively distinguish the quality differences of Chinese mitten crab.

[0130] Example 4

[0131] This embodiment provides a method for evaluating the quality of Chinese mitten crab.

[0132] For the metabolite and lipid component data obtained by analysis and identification in Example 1 and Example 2, the OPLS-DA model was used to screen differential metabolites and lipids. When VIP>1 and P<0.05, it can be determined as a quality characteristic component that significantly distinguishes the quality grades.

[0133] The quality characteristic components were screened based on the OPLS-DA model. 38 differential metabolites and 42 differential lipids were selected as the quality characteristic identification components of Chinese mitten crab with P < 0.05 and VIP > 1 as the screening conditions. Figure 6 The results showed that 38 quality characteristic metabolites were mainly organic acid and its derivative metabolites (amino acids, peptides and their analogs and carbohydrates), and 42 quality characteristic lipids were mainly SP lipids (ceramide (Cer) and hexosylceramide (HexCer)) and GP lipid phosphatidylethanolamine (PE).

[0134] Example 5

[0135] This embodiment provides a method for identifying the species of Chinese mitten crab, which comprises the following steps:

[0136] According to the method for constructing the metabolite fingerprint of Chinese mitten crab provided in Example 1, metabolite fingerprints of multiple known varieties of Chinese mitten crab (Yangcheng Lake hairy crab, Chongming clear water crab, Hongze Lake hairy crab and Taihu hairy crab) are obtained; then according to the method for constructing the metabolite fingerprint of crab provided in Example 1, the metabolite fingerprint of the crab to be identified is obtained; the metabolite fingerprint of the identified crab is compared with the metabolite fingerprints of multiple known varieties of crabs, and the crab variety is identified according to the similarity of the spectra.

[0137] In other embodiments, the method can also be used to identify the breeding method of Chinese mitten crab.

[0138] Example 6

[0139] This embodiment provides a method for identifying the species of Chinese mitten crab, which comprises the following steps:

[0140] According to the method for constructing the lipid fingerprint of Chinese mitten crab provided in Example 2, lipid fingerprints of multiple known varieties of Chinese mitten crab (Yangcheng Lake hairy crab, Chongming clear water crab, Hongze Lake hairy crab and Taihu hairy crab) are obtained; then according to the method for constructing the lipid fingerprint of crab provided in Example 2, the lipid fingerprint of the crab to be identified is obtained; the lipid fingerprint of the identified crab is compared with the lipid fingerprints of multiple known varieties of crabs, and the crab variety is identified according to the similarity of the maps.

[0141] In other embodiments, the method can also be used to identify the breeding method of Chinese mitten crab.

[0142] The invention overcomes the shortcoming that the current quality component analysis methods are unable to conduct a comprehensive and systematic analysis of a large number of quality characteristic metabolites in Chinese mitten crabs, and realizes a rapid, sensitive and quantifiable global analysis of a large number of endogenous metabolites and lipids in tissues such as the edible parts of crabs with complex matrix characteristics. It has outstanding advantages in exploring crab characteristic quality indicators and improving crab quality grade judgment standards.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a metabolite fingerprint of crab, characterized in that: It includes the following steps: S1: Extract metabolites from crab tissues to be tested; S2: Use liquid chromatography-tandem mass spectrometry to detect the extracted metabolites, process the mass spectrometry data and annotate the metabolites to obtain the metabolite fingerprint of crab; The liquid chromatography column used in the detection was ACQUITY UPLC BEH Amide (1.7 μm, 2.1*100 mm Column), and the column temperature was 40°C. The liquid chromatography conditions included: mobile phase A was acetonitrile-water (5:5, V / V) and 5-10 mM ammonium acetate, and mobile phase B was acetonitrile-water (95:5, V / V) and 5-10 mM ammonium acetate, with gradient elution, a flow rate of 0.2-0.4 min / L, and an injection volume of 2-4 μL. The gradient elution program was as follows: 0 min, 95% to 98% mobile phase B; 0-2 min, 95% to 98% mobile phase B; 2-17 min, 50% mobile phase B; 17-21 min, 50% mobile phase B; 21-23 min, 95% to 98% mobile phase B; 23-25 ​​min, 95% to 98% mobile phase B; Or, 0min, 95% to 98% mobile phase B; 0-1min, 95% to 98% mobile phase B; 1-17min, 50% mobile phase B; 17-21min, 50% mobile phase B; 21-22min, 95% to 98% mobile phase B; 22-25min, 95% to 98% mobile phase B; The mass spectrometry parameters were: ESI as the ion source, full scan (Full MS) in positive and negative modes - ddMS 2 , Spray voltage: ±4000-4500 (V); Sheath gas pressure: 40-50 psi; Auxiliary gas pressure: 10-12 arbitrary units (Arb); Capillary temperature: 320-350 ° C; Full MS scan range: 100-1500 m / z, resolution: 70000, ddMS 2 The resolution was 17500 and the normalized collision energy was 30 Ev.

2. The method for constructing the metabolite fingerprint of crab according to claim 1, characterized in that: The crab belongs to the family Scutellaria or the family Archaeocarpus; Preferably, the crab is the Chinese mitten crab of the family Cynomoridae; Preferably, the step S1 comprises: firstly mixing a mixture of dichloromethane and methanol with the crab tissue to be tested, grinding and centrifuging to obtain a bottom solid, and mixing the bottom solid with a methanol aqueous solution; grinding, centrifuging, taking the supernatant to dry, then re-dissolving in a methanol aqueous solution, centrifuging, taking the supernatant for liquid chromatography-tandem mass spectrometry detection; Preferably, the mixing volume ratio of the dichloromethane and methanol is 2:1 or 3:1, and the mixing ratio of the dichloromethane and methanol mixture to the crab tissue to be tested is 1.6-2.1 ml: 50 mg; Preferably, the volume ratio of methanol to water in the methanol aqueous solution is 1:1-1.5; Preferably, the amount of methanol aqueous solution added is 1.5-2 ml; Preferably, the grinding and centrifugation conditions are: grinding on ice, centrifuging at 12000-20000 rpm, 0-5°C for 8-15 min; Preferably, the centrifugation conditions after reconstitution are 12000-16000 rpm, 0-5°C, and centrifugation for 15-20 min; Preferably, the data processing performed on the acquired mass spectrum data includes at least one of the following processing methods: peak alignment, peak extraction, noise reduction, normalization processing and null value filling of the mass spectrum data; Preferably, the metabolite annotation is performed using the Compound Discover 3.3 software's own database mz cloud and the public database HMDB for metabolite identification.

3. A method for constructing a lipid fingerprint of crab, characterized in that: The method comprises the following steps: S1: extracting lipids from crab tissue to be tested; S2: Use liquid chromatography-tandem mass spectrometry to detect the extracted lipids, process the mass spectrometry data and annotate the lipids to obtain the lipid fingerprint of crabs; The liquid chromatography column used in the detection was: xbridge C18 (3.5μm, 4.6*100mm Column), and the column temperature was 40°C; the liquid chromatography conditions included: mobile phase A was acetonitrile-water (6:4, V / V) and 5-10mM ammonium acetate, and mobile phase B was isopropanol-acetonitrile (9:1, V / V) and 5-10mM ammonium acetate, with gradient elution, a flow rate of 0.3-0.4min / L, and an injection volume of 2-4μL; the injection chamber temperature was set to 20°C; The gradient elution program was 0 min, 20-30% mobile phase B; 0-2 min, 40-43% mobile phase B; 2-4 min, 50-60% mobile phase B; 4-8 min, 70-75% mobile phase B; 8-16 min, 80% mobile phase B; 16-18 min, 85% mobile phase B; 18-19 min, 85-95% mobile phase B; 19-19.1 min, 20-30% mobile phase B; 19.1-20 min, 20-30% mobile phase B; The mass spectrometry parameters are as follows: ESI as the ion source, full scan (Full MS)-ddMS2 in positive and negative modes, spray voltage (Spray voltage): ±4000-4500 (V); sheath gas pressure (Sheath gas pressure): 40-50psi; auxiliary gas pressure (Auxiliary gas pressure): 10-12arbitrary units (Arb); capillary temperature (Capillary temperature): 320-350℃; Full MS scanning range is 100-1500m / z, resolution is 70000, ddMS2 resolution is 17500, and standardized collision energy is 30Ev.

4. The method for constructing the lipid fingerprint of crab according to claim 3, characterized in that: The step S1 comprises: firstly mixing a mixture of dichloromethane and methanol with the crab tissue to be tested, grinding and centrifuging to obtain a supernatant, taking the supernatant and drying it, then re-dissolving it in an isopropanol-acetonitrile solution added with 5 mM ammonium acetate, centrifuging and taking the supernatant for liquid chromatography-tandem mass spectrometry detection; the volume ratio of isopropanol to acetonitrile in the isopropanol-acetonitrile solution is 9:1 to 8:2; Preferably, the mixed volume ratio of dichloromethane and methanol in the mixed solution of dichloromethane and methanol is 3:1 to 2:1, and the mixed volume ratio of the supernatant obtained by grinding and centrifugation and the 5 mM ammonium acetate isopropanol-acetonitrile solution is 4 to 9:1; Preferably, the grinding and centrifugation conditions are: grinding on ice, centrifuging at 12000-20000 rpm, 0-5°C for 8-15 min; Preferably, the centrifugation conditions after reconstitution are: 8000-12000 rpm, centrifugation for 15-25 min; Preferably, the data processing performed on the acquired mass spectrum data includes at least one of the following processing methods: peak alignment, peak extraction, noise reduction, normalization processing and null value filling of the mass spectrum data; Preferably, the lipid annotation is performed using Compound Discover 3.3 software, using the public lipid library LipidBank and Lipidmaps to perform lipid compound identification analysis.

5. A method for classifying crab varieties or crab farming methods, characterized in that: It includes the method shown in at least one of the following (1)-(2): (1) Obtaining a metabolite fingerprint of a target crab according to the method for constructing a metabolite fingerprint of a crab according to any one of claims 1 to 2; and classifying the crab species or breeding methods by comparing the metabolite fingerprints of multiple crabs measured; (2) Obtaining a lipid fingerprint of a target crab according to the method for constructing a lipid fingerprint of a crab as described in any one of claims 3 to 4; and classifying the crab species or breeding methods by comparing the lipid fingerprints of multiple crabs measured.

6. The method for classifying crab species according to claim 5, characterized in that: The classification method further comprises extracting features and analyzing metabolite data obtained by detection after obtaining the metabolite fingerprint of the target crab; Preferably, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; Preferably, the classification rule includes: if the metabolite data of multiple target crabs can be obviously clustered into the same category, then they are judged to be crabs of the same species or the same breeding method; Preferably, the classification method further comprises extracting features and analyzing lipid data obtained from the detection after obtaining the lipid fingerprint of the target crab; Preferably, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; Preferably, the classification rule includes: if the lipid data of multiple target crabs can be obviously clustered into the same category, they are judged to be crabs of the same species or the same breeding method.

7. A method for identifying crab species or breeding methods, characterized in that: It includes the method shown in at least one of the following (1)-(2): (1) Obtaining metabolite fingerprints of multiple crabs of known species or breeding methods according to the method for constructing metabolite fingerprints of crabs as described in any one of claims 1 to 2; then obtaining a metabolite fingerprint of a crab to be identified according to the method for constructing metabolite fingerprints of crabs as described in any one of claims 1 to 2; comparing the metabolite fingerprint of the identified crab with the metabolite fingerprints of the multiple known species of crabs, thereby identifying the crab species or breeding method; (2) Obtain lipid fingerprints of multiple crabs of known species or breeding methods according to the method for constructing the lipid fingerprint of crabs as described in any one of claims 3-4; then obtain the lipid fingerprint of the crab to be identified according to the method for constructing the lipid fingerprint of crabs as described in any one of claims 3-4; compare the lipid fingerprint of the identified crab with the lipid fingerprints of the multiple known species or breeding methods, thereby identifying the crab species or breeding method.

8. The method for identifying crab species according to claim 7, characterized in that: The identification method further comprises extracting features and analyzing metabolite data obtained from the detection after obtaining metabolite fingerprints of multiple crabs of known species or breeding methods and metabolite fingerprints of crabs to be identified; Preferably, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; Preferably, the identification rule includes: if the metabolite data of the identified crab can be obviously clustered into the same category as the metabolite data of crabs of any known species or known breeding method, then the crab is identified as a crab of the known species or the breeding method.

9. The method for identifying crab species according to claim 7, characterized in that: The identification method further comprises extracting features and analyzing lipid data obtained from the detection after obtaining the lipid fingerprint of the target crab; Preferably, the feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; Preferably, the identification rule includes: if the lipid data of the identified crab can be obviously clustered into the same category as the lipid data of crabs of any known species or known breeding method, then the crab is identified as a crab of the known species or the breeding method.

10. A method for evaluating crab quality, characterized in that: It includes the following steps: (1) Obtaining metabolite fingerprints of at least two crabs to be evaluated according to the method for constructing a metabolite fingerprint of crabs according to any one of claims 1-2, and / or obtaining lipid fingerprints of at least two crabs to be evaluated according to the method for constructing a lipid fingerprint of crabs according to any one of claims 3-4; (2) The annotated metabolites and / or lipid components were subjected to OPLS-DA analysis, and quality characteristic components were screened based on the OPLS-DA model. The differential metabolites and / or lipid components were screened with P < 0.05 and VIP > 1 as the screening conditions, and the crab quality was evaluated based on the differential metabolites and / or lipid components obtained by screening.

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