A method for constructing metabolite and lipid fingerprints of crabs and a method for evaluating crab quality

Abstract

The present invention discloses a method for constructing a crab metabolite and lipid fingerprint and a crab quality evaluation method, relating to the technical field of crab quality evaluation. The method comprises extracting metabolites from the crab tissue to be tested; detecting the extracted metabolites and lipids using liquid chromatography-tandem mass spectrometry; and processing the mass spectrometry data, annotating metabolites, and annotating lipids to obtain the crab metabolite and lipid fingerprint. This method enables rapid, sensitive, and quantifiable global analysis of a large number of endogenous metabolites and lipids in crab tissues, such as the edible parts of the crab, which have complex matrix properties. It offers significant advantages in identifying characteristic crab quality indicators and improving crab quality grading criteria.

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 crab metabolite and lipid fingerprint and a crab quality evaluation method. Background Art

[0002] The Chinese mitten crab (Eriocheir sinensis), also known as river crab and hairy crab, is highly valued by consumers for its balanced nutritional profile, including amino acids, polyunsaturated fatty acids, and trace elements. These nutrients contribute to its unique flavor and high economic value. However, the inherent quality characteristics of Chinese mitten crab are significantly influenced by human factors, such as breeding management, and environmental factors, such as geographic location. This inevitably leads to varying quality within the crab market.

[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 the analysis of a specific type of nutrients 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. As a result, the application of intrinsic quality characteristic component indicators in the quality evaluation of Chinese mitten crab is not common. There are no reports on the construction of Chinese mitten crab metabolite and lipid fingerprints 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 exploration 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 crab metabolite and lipid fingerprint and a crab quality evaluation method 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: Liquid chromatography-tandem mass spectrometry was used to detect the extracted metabolites, and the obtained mass spectrometry data were processed and metabolite annotation was performed to obtain the metabolite fingerprint of crab;

[0011] The liquid chromatography column used for the test was an ACQUITY UPLC BEH Amide (1.7 μm, 2.1×100 mm column) at 40°C. The liquid chromatography conditions included: mobile phase A consisting of acetonitrile-water (5:5, v / v) and 5–10 mM ammonium acetate, and mobile phase B consisting of acetonitrile-water (95:5, v / v) and 5–10 mM ammonium acetate, with gradient elution at 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, 0 min, 95% to 98% mobile phase B; 0-1 min, 95% to 98% mobile phase B; 1-17 min, 50% mobile phase B; 17-21 min, 50% mobile phase B; 21-22 min, 95% to 98% mobile phase B; 22-25 min, 95% to 98% mobile phase B;

[0014] The mass spectrometry parameters were as follows: 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: 70,000, 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, comprising the following steps: S1: extracting lipids from crab tissue to be tested;

[0016] S2: Liquid chromatography-tandem mass spectrometry was used to detect the extracted lipids, and the mass spectrometry data were processed and lipid annotation was performed to obtain the lipid fingerprint of crab;

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

[0018] The gradient elution program was as follows: 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 were as follows: ESI as the ion source, full scan (Full MS)-ddMS2 in positive and negative modes, 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, ddMS2 resolution: 17500, normalized collision energy: 30 Ev.

[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 crabs; and classifying the crab species or breeding methods by comparing the metabolite fingerprints of multiple crabs obtained;

[0022] (2) Obtaining the lipid fingerprint of the target crab according to the above-mentioned crab lipid fingerprint construction method; 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 crab metabolite fingerprint construction method; then obtaining a metabolite fingerprint of the crab to be identified according to the above-mentioned crab metabolite fingerprint construction method; comparing the metabolite fingerprint of the identified crab with the metabolite fingerprints of multiple known crab species, 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 crab lipid fingerprint construction method; then obtain the lipid fingerprint of the crab to be identified according to the above-mentioned crab lipid fingerprint construction method; compare the lipid fingerprint of the identified crab with the lipid fingerprints of multiple crabs of known species or breeding methods, thereby identifying the crab species or breeding method.

[0026] In a fifth aspect, the present invention further 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 metabolite fingerprints of crabs, and / or obtaining lipid fingerprints of at least two crabs to be evaluated according to the above-mentioned method for constructing lipid fingerprints 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 crab. This method can construct a metabolite and lipid fingerprint of crab, which can be used to accurately characterize the intrinsic quality characteristics of crab, particularly Chinese mitten crab. Using multidimensional metabolite and lipid indicators, this method achieves a systematic, comprehensive, efficient, and accurate evaluation and determination of the intrinsic quality of crab, particularly Chinese mitten crab, and has important application value in both technical theory and practical application.

[0031] The invention overcomes the shortcomings of the current quality component analysis methods that 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 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 following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any 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 This is 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 Chinese mitten crab with significant differences in quality characteristics in one embodiment of the present invention. DETAILED DESCRIPTION

[0040] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are 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: Liquid chromatography-tandem mass spectrometry was used to detect the extracted metabolites, and the obtained mass spectrometry data were processed and metabolite annotation was performed to obtain the metabolite fingerprint of crab;

[0044] The liquid chromatography column used for the test was an ACQUITY UPLC BEH Amide (1.7 μm, 2.1×100 mm column) at 40°C. The liquid chromatography conditions included: mobile phase A consisting of acetonitrile-water (5:5, v / v) and 5–10 mM ammonium acetate, and mobile phase B consisting of acetonitrile-water (95:5, v / v) and 5–10 mM ammonium acetate, with gradient elution at 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, 0 min, 95% to 98% mobile phase B; 0-1 min, 95% to 98% mobile phase B; 1-17 min, 50% mobile phase B; 17-21 min, 50% mobile phase B; 21-22 min, 95% to 98% mobile phase B; 22-25 min, 95% to 98% mobile phase B;

[0047] The mass spectrometry parameters were as follows: ESI as the ion source, full scan (Full MS) in positive and negative modes - ddMS 2Spray 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: 70,000, 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 crab. This method can construct a metabolite fingerprint of crab, which can be used to accurately characterize the intrinsic quality characteristics of crab, particularly Chinese mitten crab. By utilizing multidimensional metabolite indicators, a systematic, comprehensive, efficient, and accurate evaluation and determination of the intrinsic quality of crab, particularly Chinese mitten crab, is achieved, with significant application value in both technical theory and practical application.

[0049] The invention overcomes the shortcomings of the current quality component analysis methods that 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 the metabolites. Organic reagents include 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 fingerprint spectrum obtained 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 value). 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 Scyphopodidae or the family Archaeocaridae. The metabolite fingerprint construction method provided by the present invention has reference value for constructing metabolite fingerprints of various aquatic animals. For example, it can be applied to 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 Cynomoridae;

[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 solid at the bottom, and mixing the solid at the bottom with a methanol-water solution; grinding and centrifuging, taking the supernatant, blowing it dry, then re-dissolving it in a methanol-water solution, centrifuging, and 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 dichloromethane and methanol mixture to the crab tissue to be tested is 1.6 ml-2.1 ml: 50 mg; 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, centrifuging 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 obtained 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's own database mz cloud and the public database HMDB for metabolite identification. In other embodiments, the 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, comprising the following steps: S1: extracting lipids from crab tissue to be tested;

[0063] S2: Liquid chromatography-tandem mass spectrometry was used to detect the extracted lipids, and the mass spectrometry data were processed and lipid annotation was performed to obtain the lipid fingerprint of crab;

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

[0065] The gradient elution program was as follows: 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 were as follows: ESI as the ion source, full scan (Full MS)-ddMS2 in positive and negative modes, 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, ddMS2 resolution: 17500, normalized collision energy: 30 Ev.

[0067] The method for extracting lipids can use organic and / or inorganic reagents to extract metabolites. Organic reagents include 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 fingerprint obtained 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 can also be other values ​​after mathematical conversion (such as taking an absolute constant value). As long as it can reflect the relationship between the peak area of ​​lipids and the type of lipids, it belongs to the fingerprint.

[0069] In a preferred embodiment of the present invention, step S1 comprises: mixing the crab tissue to be tested with a mixture of dichloromethane and methanol, grinding and centrifuging to obtain a supernatant, air-drying the supernatant, re-dissolving it in an isopropanol-acetonitrile solution supplemented with 5mM ammonium acetate, centrifuging, and obtaining 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. This extraction method can extract a wider variety and number of lipids, facilitating 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 include but are 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 rich information and poor useful information is 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 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 crabs; and classifying the crab species or breeding methods by comparing the metabolite fingerprints of multiple crabs obtained;

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

[0078] This method includes but is not limited to classifying multiple unknown crabs into species, such as tracing their origin; and classifying multiple crabs into breeding methods, such as classifying different feeds, feed dosages, and lake water breeding.

[0079] In a preferred embodiment of the present invention, the classification method further comprises extracting and analyzing features of 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 the crabs are determined to be of the same species or cultured using the same method. Those skilled in the art can compare the peak areas and peak profiles to assess 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 crab metabolite fingerprint construction method; then obtaining a metabolite fingerprint of the crab to be identified according to the above-mentioned crab metabolite fingerprint construction method; comparing the metabolite fingerprint of the identified crab with the metabolite fingerprints of multiple known crab species, 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 crab lipid fingerprint construction method; then obtain the lipid fingerprint of the crab to be identified according to the above-mentioned crab lipid fingerprint construction method; compare the lipid fingerprint of the identified crab with the lipid fingerprints of multiple crabs of known species or breeding methods, thereby identifying 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 the metabolite fingerprints of the crab to be identified, the method further 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 rule includes: if the metabolite data of the identified crab can be clearly 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 clearly 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 further 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 metabolite fingerprints of crabs, and / or obtaining lipid fingerprints of at least two crabs to be evaluated according to the above-mentioned method for constructing lipid fingerprints of crabs;

[0096] (2) The obtained metabolites and / or lipid components were annotated and subjected to PLS-DA analysis or OPLS-DA analysis. Quality characteristic components were screened based on the PLS-DA or OPLS-DA model. Differential metabolites and / or lipid components were screened with P < 0.05 and VIP > 1 as the screening conditions. 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 with reference to 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) Collection of edible tissues of Chinese mitten crab

[0101] Male and female Chinese mitten crabs (Eriocheir sinensis) were collected from a breeding base in Chongming District, a representative breeding area in Shanghai, following three different feeding regimes: normal aquatic feed (CON), aquatic feed supplemented with 0.1% plant activated carbon (AC1), and aquatic feed supplemented with 2.5% plant activated carbon (AC2). Edible tissues (hepatopancreas, gonads, and muscle) of Chinese mitten crabs were collected and randomly mixed as one replicate sample from one female and one male crab under the same feeding regime. Five replicate samples were prepared under the same feeding regime, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

[0102] (2) Extraction of metabolites

[0103] After thawing edible tissue (mixed sample of hepatopancreas, gonads, and muscle tissue) on ice, 50 mg of 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 15,000 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] To the solids at the bottom, 1.5 mL of pre-chilled methanol / water (1:1, v / v) was added. The sample mixture was ground on ice and refrigerated at -18°C for 2 h. Subsequently, the sample was centrifuged at 15,000 rpm and 4°C for 10 min. The supernatant was collected and nitrogen-purged to dryness. The sample was reconstituted in 120 μL of methanol / water (1:1, v / v) and vortexed until completely reconstituted. The sample was then centrifuged at 15,000 rpm and 4°C for 20 min. 50 μL of the supernatant was transferred to a Q-Exactive Orbitrap mass spectrometer for untargeted metabolomics data acquisition. Equal amounts of the supernatants from all samples used for untargeted metabolomics analysis were combined 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 selected was ACQUITY UPLC BEH Amide (1.7 μm, 2.1*100 mm Column). The specific instrument mass spectrometry parameters were: ESI as the ion source, full scan (Full MS)-ddMS in positive and negative modes 2Spray 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: 70,000, ddMS 2 The resolution was 17500 and the normalized collision energy was 30 Ev.

[0108] The column temperature was set at 40°C. Liquid phase conditions were gradient elution with mobile phase A consisting of acetonitrile-water (5:5, v / v) + 5 mM ammonium acetate and mobile phase B consisting of acetonitrile-water (95:5, v / v) + 5 mM ammonium acetate. The flow rate was 0.3 min / L, the injection volume was 2 μL, and the injection chamber temperature was set at 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 the metabolite fingerprint 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 at 10,000 rpm for 20 min at room temperature. Aspirate 50 μL of the supernatant and place it on 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 as follows: ESI as the ion source, full scan (Full MS)-ddMS2 in positive and negative modes, 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, ddMS2 resolution: 17500, normalized collision energy: 30 Ev.

[0120] The liquid phase conditions were gradient elution with mobile phase A consisting of acetonitrile-water (6:4, V / V) + 5 mM ammonium acetate and mobile phase B consisting of isopropanol-acetonitrile (9:1, V / V) + 5 mM ammonium acetate. 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. The raw lipid fingerprint was processed using Compound Discover 3.3 software.

[0124] Lipid compound identification and analysis were performed 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 clearly clustered into the same class, 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. 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 could be determined as a quality characteristic component that significantly distinguished 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 the 38 quality characteristic metabolites were mainly organic acid and its derivative metabolites (amino acids, peptides and their analogues and carbohydrates), and the 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 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) were 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 was obtained; the metabolite fingerprint of the identified crab was compared with the metabolite fingerprints of multiple known varieties of crab, and the crab variety was identified based on the similarity of the spectra.

[0137] In other embodiments, this 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 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) were 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 was obtained; the lipid fingerprint of the identified crab was compared with the lipid fingerprints of multiple known varieties of crab, and the crab variety was identified based on the similarity of the patterns.

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

[0142] The invention overcomes the shortcomings of the current quality component analysis methods that 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 foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for constructing a metabolite fingerprint of crab, characterized in that: It includes the following steps: S1: extracting metabolites from the crab tissue to be tested; the S1 step comprises: first 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-water solution; after grinding and centrifugation, taking the supernatant, blowing it dry, and then re-dissolving it in a methanol-water solution, centrifuging and taking the supernatant for liquid chromatography-tandem mass spectrometry detection; the 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; the volume ratio of methanol to water in the methanol-water solution is 1:1-1.5; S2: Liquid chromatography-tandem mass spectrometry was used to detect the extracted metabolites, and the obtained mass spectrometry data were processed and metabolite annotation was performed to obtain the metabolite fingerprint of crab; The liquid chromatography column used for the test was an ACQUITY UPLC BEH Amide, 1.7 μm, 2.1 x 100 mm column, and the column temperature was 40°C. The liquid chromatography conditions included: mobile phase A consisting of acetonitrile-water and 5 mM ammonium acetate in a volume ratio of 5:5, and mobile phase B consisting of acetonitrile-water and 5 mM ammonium acetate in a volume ratio of 95:5, with gradient elution at 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, 98% mobile phase B; 0-1 min, 98% mobile phase B; 1-17 min, 50% mobile phase B; 17-21 min, 50% mobile phase B; 21-22 min, 98% mobile phase B; 22-25 min, 98% mobile phase B; The mass spectrometry parameters were: ESI as the ion source, full scan 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 Archaeocaridae.

3. The method for constructing the metabolite fingerprint of crab according to claim 2, characterized in that: The crab is the Chinese mitten crab of the family Cynomoridae.

4. The method for constructing a metabolite fingerprint of crab according to claim 1, characterized in that: The amount of the methanol aqueous solution added is 1.5-2 ml.

5. The method for constructing a metabolite fingerprint of crab according to claim 1, characterized in that: The grinding and centrifugation conditions are as follows: grinding on ice, centrifuging at 12000-20000 rpm, 0-5°C for 8-15 min; The centrifugation conditions after reconstitution are 12000-16000 rpm, 0-5°C, and centrifugation for 15-20 min.

6. The method for constructing a metabolite fingerprint of crab according to claim 1, characterized in that: The data processing performed on the obtained 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; The metabolite annotation was performed using the Compound Discover 3.3 software's own database mz cloud and the public database HMDB for metabolite identification.

7. 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; the S1 step comprises: 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 drying it, then re-dissolving it in an isopropanol-acetonitrile solution supplemented with 5mM ammonium acetate, centrifuging and obtaining the supernatant for liquid chromatography-tandem mass spectrometry detection; the volume ratio of isopropanol to acetonitrile in the isopropanol-acetonitrile solution is 9:1-8:2; the volume ratio of dichloromethane to methanol in the mixture of dichloromethane and methanol is 3:1-2:1, and the volume ratio of the supernatant obtained by grinding and centrifuging to the isopropanol-acetonitrile solution containing 5mM ammonium acetate is 4-9:1; S2: Liquid chromatography-tandem mass spectrometry was used to detect the extracted lipids, and the mass spectrometry data were processed and lipid annotation was performed to obtain the lipid fingerprint of crab; The liquid chromatography column used for the test was an xbridge C18 column (3.5 μm, 4.6 x 100 mm column) at 40°C. The liquid chromatography conditions included: mobile phase A consisting of acetonitrile-water and 5 mM ammonium acetate in a volume ratio of 6:4, and mobile phase B consisting of isopropanol-acetonitrile and 5 mM ammonium acetate in a volume ratio of 9:1, with gradient elution at a flow rate of 0.3-0.4 min / L and an injection volume of 2-4 μL. The injection chamber temperature was set at 20°C. The gradient elution program was as follows: 0 min, 30% mobile phase B; 0-2 min, 40% mobile phase B; 2-4 min, 60% mobile phase B; 4-8 min, 75% mobile phase B; 8-16 min, 80% mobile phase B; 16-18 min, 85% mobile phase B; 18-19 min, 95% mobile phase B; 19-19.1 min, 30% mobile phase B; 19.1-20 min, 30% mobile phase B; The mass spectrometry parameters were as follows: ESI as the ion source, full scan-ddMS2 in positive and negative modes, 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, ddMS2 resolution: 17500, and normalized collision energy: 30 Ev.

8. The method for constructing the lipid fingerprint of crab according to claim 7, characterized in that: The grinding and centrifugation conditions are as follows: grinding on ice, centrifuging at 12000-20000 rpm, 0-5°C for 8-15 min; The centrifugation conditions after reconstitution are: 8000-12000 rpm, centrifugation for 15-25 min.

9. The method for constructing the lipid fingerprint of crab according to claim 7, characterized in that: The data processing performed on the obtained 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; The lipid annotation was performed using Compound Discover 3.3 software, and the public lipid library LipidBank and Lipidmaps were used to perform lipid compound identification analysis.

10. A method for classifying crab varieties or crab farming methods, characterized in that: The method includes 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 6; and classifying the crab species or breeding methods by comparing the metabolite fingerprints of multiple crabs obtained; (2) Obtaining a lipid fingerprint of a target crab according to the method for constructing a lipid fingerprint of crab according to any one of claims 7 to 9; and classifying the crab species or breeding methods by comparing the lipid fingerprints of multiple crabs obtained.

11. The method for classifying crab species according to claim 10, characterized in that: The classification method further comprises extracting and analyzing features of the metabolite data obtained after obtaining the metabolite fingerprint of the target crab.

12. The crab species classification method according to claim 11, characterized in that: The feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; The classification rules include: if the metabolite data of multiple target crabs can be clearly clustered into the same class, they are judged to be crabs of the same species or the same breeding method; The classification method further comprises extracting and analyzing features of the lipid data obtained after obtaining the lipid fingerprint of the target crab; The feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; The classification rules include: if the lipid data of multiple target crabs can be clearly clustered into the same class, they are judged to be crabs of the same species or the same breeding method.

13. A method for identifying crab species or breeding methods, characterized in that: The method includes 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 a metabolite fingerprint of crabs according to any one of claims 1 to 6; then obtaining a metabolite fingerprint of a crab to be identified according to the method for constructing a metabolite fingerprint of crabs according to any one of claims 1 to 6; 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 lipid fingerprints of crabs as described in any one of claims 7 to 9; then obtain the lipid fingerprint of the crab to be identified according to the method for constructing lipid fingerprints of crabs as described in any one of claims 7 to 9; 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.

14. The method for identifying crab species according to claim 13, characterized in that: After obtaining metabolite fingerprints of multiple crabs of known species or breeding methods and the metabolite fingerprints of the crab to be identified, the identification method further includes feature extraction and analysis of the metabolite data obtained by the detection.

15. The method for identifying crab species according to claim 14, characterized in that: The feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; The identification rules include: if the metabolite data of the identified crab can be clearly 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 the crab of the known species or the breeding method.

16. The method for identifying crab species according to claim 13, characterized in that: The identification method further comprises extracting and analyzing features of the lipid data obtained after obtaining the lipid fingerprint of the target crab.

17. The method for identifying crab species according to claim 16, characterized in that: The feature extraction method is selected from at least one of cluster analysis and PCA principal component analysis; 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 the crab of the known species or the breeding method.

18. 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 to 6, 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 7 to 9; (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. Differential metabolites and / or lipid components were screened with P < 0.05 and VIP > 1 as the screening conditions. The crab quality was evaluated based on the differential metabolites and / or lipid components obtained by screening.

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