CN115436510B - Method for carrying out differential identification on flavor compounds in livestock and poultry meat based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrum - Google Patents

Abstract

The invention discloses a method for identifying the difference of flavor compounds in livestock and poultry meat based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrum. It comprises the following steps: 1) Separating, namely grinding the livestock and poultry meat sample to be detected to obtain a chopped sample; 2) Performing headspace solid-phase microextraction on the chopped sample to obtain a mixed unknown compound; 3) Identifying, namely performing gas chromatography-electrostatic field orbitrap high-resolution mass spectrometry on the extracted mixed unknown compound to obtain GC-MS data; the GC-MS data are identified through MS modes, retention indexes and HRF processing in NIST and a homeflag library, and the compound components contained in the livestock and poultry meat sample to be detected are obtained; 4) And (3) carrying out statistical analysis on the compound components obtained in the step (3) to obtain the flavor compound corresponding to the livestock and poultry meat sample to be detected. The invention combines gas chromatography with high-resolution orbitrap mass spectrometry, and is used for distinguishing various meat types and identifying the authenticity of livestock and poultry meat through a statistical analysis tool.

Description

Method for carrying out differential identification on flavor compounds in livestock and poultry meat based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrum

Technical Field

The invention belongs to the field of livestock and poultry meat identification and analysis, and relates to a method for carrying out flavor compound difference identification in livestock and poultry meat based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrometry.

Background

Meat quality includes safety, nutritional and organoleptic aspects, which can be affected to a large extent by many factors, such as genetics, breeding and the environment. With the improvement of living standard, consumers increasingly pay attention to nutrition and flavor. Flavor has become one of the important determinants influencing consumer purchase options as an important component of sensory evaluation.

The chemical composition is the basis for the flavor of the food. With the rapid development and perfection of analytical instrumentation, research into flavor compounds is gradually systemized, and more flavor compounds are found. Flavor consists of aroma and taste. The aromatic compounds have complex components, mainly including alcohols, esters, organic acids, ketones, aldehydes, furans, pyrazines, hydrocarbons, sulfur compounds, and the like. The different components constitute a unique fragrance style. Aromatic compounds have a very broad range of polarity, solubility, volatility and thermal stability. This would present a great challenge for non-targeted analysis of aromatic compounds. Basically, the analysis of aromatic compounds comprises three steps: extracting, separating and identifying.

As extraction methods, headspace solid phase microextraction (HS-SPME), solvent Assisted Flavor Evaporation (SAFE), stirring rod adsorption extraction (SBSE) and Dynamic Headspace (DHS) are the dominant methods. HS-SPME is a solvent-free micro-extraction technique, integrating sampling, extraction, concentration and sample injection. The method can directly extract the analyte from the complex matrix and is widely used for the differential analysis of the aroma compounds. The extraction efficiency is affected by factors such as fiber type, extraction time and extraction temperature. Because of the small size of the fibers, the adsorption capacity is relatively low. Compared with SPME, the stirring rod in SBSE has larger surface area and more adsorption sites, thereby improving adsorption capacity. However, there is competitive adsorption of both SPME and SBSE, and the profile of volatile compounds may depend largely on the coating material. SAFE is a mild solvent extraction technique that does not differentiate between volatile compounds. Therefore, SAFE is commonly used for analysis and accurate quantification of volatile compounds. However, some of the procedures in SAFE and SBSE are not automated, limiting analysis throughput.

As for separation of aromatic compounds, gas chromatography-ion mobility spectrometry (GC-IMS) and gas chromatography-mass spectrometry (GC-MS) are mainly used. Mass spectrometry can provide structural information making it a dominant technique in the field of flavor research. The introduction of high resolution mass spectrometry improves the mass accuracy of the fragments and improves the identification capacity. In addition, the introduction of two-dimensional gas chromatography can increase peak capacity and reduce co-elution phenomena, significantly increasing the number of identified compounds. So far, the identification of aromatic compounds is based mainly on MS spectra and Retention Index (RI), depending on NIST and Wiley libraries. However, different specifications of columns and different types of analytical instruments may cause the MS spectrum and RI to be different, thereby affecting the accuracy of the identification and resulting in false positive or false negative results.

Disclosure of Invention

The invention aims to provide a method for identifying the difference of flavor compounds in livestock and poultry meat based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrometry.

The accurate aroma analysis of the invention is very important for meat quality research and establishing micro-correlation between meat flavor and aroma compounds. In this study, a volatile compound analysis method was developed that uses gas chromatography in combination with high resolution orbitrap mass spectrometry and was used to distinguish between various meat categories such as pork, beef, mutton, chicken and duck breast by means of statistical analysis tools.

The invention provides a method for identifying flavor compounds in livestock and poultry meat based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrometry, which comprises the following steps:

1) Separation

Grinding the livestock and poultry meat sample to be detected to obtain a chopped sample;

2) Headspace solid phase microextraction

Performing headspace solid-phase microextraction on the chopped sample to obtain a mixed unknown compound;

3) Authentication

Carrying out gas chromatography-electrostatic field orbit trap high-resolution mass spectrometry on the extracted mixed unknown compound to obtain GC-MS data;

the GC-MS data are identified through MS modes, retention indexes and HRF processing in NIST and a homeflag library, and the compound components contained in the livestock and poultry meat sample to be detected are obtained;

4) Statistical analysis

And 3) carrying out statistical analysis on the compound components obtained in the step 3) to obtain the flavor compound corresponding to the livestock and poultry meat sample to be detected.

In the method, the livestock and poultry meat sample to be detected comprises at least one of duck meat, chicken meat, beef, mutton and pork;

the treatment of the livestock and poultry meat sample to be detected is as follows: removing fascia from livestock and poultry meat to be tested, mincing, placing into a stewing bag, stewing in a water bath at 80 ℃ for 30 minutes, cooling, and grinding in liquid nitrogen for later use.

In the above method, the fiber head type used in the headspace solid-phase microextraction comprises at least one of 85 μm Polyacrylate (PA), 50 μm/30 μm divinylbenzene/Carboxen/polydimethylsiloxane (abbreviated as CAR/DVB/PDMS), 95 μm Carbon WR and 30 μm PDMS; preferably DVB/CAR/PDMS and Carbon WR, more preferably CAR/DVB/PDMS.

In the above method, the flow of the headspace solid-phase microextraction is as follows: adding 2-methyl-3 heptanone solution into the chopped sample, placing the chopped sample into a container sealed by a magnetic cover of a PTFE-silica gel diaphragm, incubating for 10-30 min at 40-60 ℃, and then extracting the chopped sample by using fibers, wherein the extracted fibers are desorbed at a sample inlet. Between successive analyses, the extracted fibers were aged under the post-injection port.

The flow of the headspace solid-phase microextraction is specifically as follows: the minced sample was introduced into a 20mL glass vial, followed by 5. Mu.L of 2-methyl-3 heptanone solution (50. Mu.g/mL) and the vial was immediately sealed with a magnetic cap fitted with a PTFE-silica gel septum; the sample bottle is incubated for 10-20 min at 55 ℃, and is extracted by using a fiber head, the extracted fiber is desorbed at a sample inlet, the quality is checked in continuous analysis, and the extracted fiber is aged at 270 ℃ at a rear sample inlet.

In the method, the temperature of the extraction of the fiber head can be 37-65 ℃, specifically can be 55 ℃, and the time can be 10-59 min, specifically can be 50min;

the desorption temperature in the sample inlet can be 230-270 ℃, specifically 250 ℃ or 250-260 ℃, and the time can be 1-8 min, specifically 3min or 2-5 min;

the aging temperature of the post-injection port is 230-270 ℃, specifically 270 ℃ or 260-280 ℃ for 8-12 min, specifically 10min.

In the above method, the working conditions of the gas chromatography-electrostatic field orbitrap high-resolution mass spectrum are as follows:

automatic sample injector: triPlus RSH;

mass analyzer: trace 1310GC, Q-Exactive Orbitrap;

film thickness: an inner diameter of 60m 0.25mm 0.25 μm;

chromatographic column: VF-WAX ms (Agilent, santa Clara, calif.);

helium with a constant flow rate of 1mL/min (99.9999%) was used as carrier gas;

the temperature programming conditions are as follows: maintaining the temperature at 40 ℃ for 2 minutes, then heating to 230 ℃ at a rate of 4 ℃/minute, and maintaining the temperature for 5 minutes;

transmission line 1 and transmission line 2 were each set at 250 ℃;

ionization mode: electron bombardment ionization of 70eV;

scanning mode: full scanning;

resolution ratio: 60,000FWHM;

scanning range: 30-400m/z, and the automatic gain control target value is 1E6;

the MS ion source and transmission line temperatures were set to 280 ℃ and 250 ℃, respectively.

In the above method, in step 3), the compound component contained in the livestock and poultry meat sample to be detected can be determined according to any one of the following data:

a) HRF score higher than 95;

b) A matching factor based on MS mode higher than 750;

c) The retention index difference based on the homeflag library is less than 20;

d) The retention index difference based on NIST pool was within 50.

In the above method, the statistical analysis is verified by the following method:

principal Component Analysis (PCA), t-test and partial least squares discriminant analysis (PLS-DA analysis for short).

In the method, the analysis is carried out by the method: the flavor compounds in the duck meat are 2-amyl furan, 2-butyl furan, 2-hexyl furan, 1-octen-3-ol (1-octen-3-ol) and 1-octen-3-one.

The flavor compounds in the beef are carbon disulfide, dimethyl sulfoxide and fatty acid methyl esters, wherein the fatty acid methyl esters comprise methyl butyrate, methyl octanoate and methyl nonanoate.

The invention also provides a method for identifying the authenticity of the livestock and poultry meat based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrum, which comprises the following steps:

1) Separation

Grinding the livestock and poultry meat sample to be detected to obtain a chopped sample;

2) Headspace solid phase microextraction

Performing headspace solid-phase microextraction on the chopped sample to obtain a mixed unknown compound;

3) Authentication

Carrying out gas chromatography-electrostatic field orbit trap high-resolution mass spectrometry on the extracted mixed unknown compound to obtain GC-MS data;

the GC-MS data are identified through MS modes, retention indexes and HRF processing in NIST and a homeflag library, and the compound components contained in the livestock and poultry meat sample to be detected are obtained;

4) Statistical analysis

Carrying out statistical analysis on the compound components obtained in the step 3) to obtain flavor compounds corresponding to the livestock and poultry meat samples to be detected;

5) And comparing the flavor compound corresponding to the livestock and poultry meat sample to be detected with the flavor compound of the known sample to identify the authenticity of the livestock and poultry meat.

In the above method, in step 5), when the beef authenticity is identified, the flavor compound corresponding to the livestock and poultry meat sample to be detected is compared with the flavor compound of the known beef sample as follows A1) -H1) the difference compound, when the abundance ratio of the difference compound satisfies the following range and the p value is less than 0.01, the livestock and poultry meat sample to be detected may be duck meat, and as a result, it is determined that the livestock and poultry meat sample to be detected is not beef and/or is not pure beef:

a1 2E, 4E-Decadienal (2, 4-Decadienal, (E, E) -) 19.20 to 27.32.

B1 2E-Heptenal (2-Heptal) (E) -) 6.91-9.77;

c1 2-pentylfuran (Furan, 2-pentayl-) 3.67-5.28;

d1 1-OCTEN-3-OL (1-OCTEN-3-OL) 4.34-6.36;

e1 2-n-octyl furan (2-n-octyfuran) 17.41 to 25.62;

f1 1-Octen-3-one (1-Octen-3-one) 2.92 to 4.38;

g1 2.92 to 4.38 of 2-n-Butyl furan;

h1 2E, 4E-NONADIENAL (2, 4-NONADIENAL) (E, E) -) 7.16 to 10.37;

when the mutton is identified as true or false, comparing the flavor compound corresponding to the livestock and poultry meat sample to be detected with the flavor compound of the known mutton sample as follows A2) -F2) a difference compound, and when the abundance ratio of the difference compound satisfies the following range, the livestock and poultry meat sample to be detected may be duck meat, and judging that the livestock and poultry meat sample to be detected is not mutton and/or is not pure mutton as a result:

a2 2-pentylfuran (Furan, 2-pentayl-) 1.74-2.60;

b2 2.10 to 3.16 portions of 2-n-Butyl furan;

c2 2-n-octyl furan (2-n-Octyl furan) 5.50-7.96

D2 1.86 to 2.78 of 1-octen-3-ol;

e2 2E, 4E-Decadienal (2, 4-Decadienal) (E, E) -) 11 to 16.5;

f2 2E, 4E-NONADIENAL (2, 4-NONADIENAL) (E, E) -) 6.36-9.37.

The invention has the following beneficial effects:

a gas chromatograph-quadrupole orbitrap high-resolution mass spectrometer was used to determine the volatility characteristics of five of the most commonly consumed meats (chicken breast, duck breast, mutton, pork and beef). According to the research, compounds with larger influence on meat flavor, such as sulfur compounds, aldehydes and ketones, are used as research models, and key parameters, such as fiber head type, extraction temperature, extraction time and desorption time, in a headspace solid-phase microextraction (HS-SPME) sampling method are systematically optimized. And the different meat flavor profiles are distinguished by combining statistical means such as Principal Component Analysis (PCA), t-test, partial least squares discriminant analysis (PLS-DA) and the like. For example, 1-octen-3-ol, 1-octen-3-one and 2-amyl furan are derived from the oxidative degradation of n-6 fatty acid and can be used as key flavor compounds of duck meat; fatty acid methyl ester odor compounds such as methyl pelargonate come from oleic acid oxidative degradation, dimethyl sulfoxide and carbon disulfide are generated by sulfur-containing amino acids, and are both in strong positive correlation with beef.

Drawings

FIG. 1 is a graph showing the effect of different solid phase microextraction fibers on flavor compound analysis.

FIG. 2 shows the effect of extraction temperature and extraction time on volatile compounds such as aldehydes, ketones, sulfur compounds, furans, and the like.

FIG. 3 is a graph showing the effect of desorption time on a portion of typical volatile compounds.

Fig. 4 is a principal component analysis of five different meat volatile compounds.

Fig. 5 is a volatile compound variable projection importance analysis (VIP).

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1,

1 reagents and materials

1.1. Food products

Pig ridges, niu Xiaohuang melon strips, sheep legs, chicken breasts and duck breasts were all purchased from local markets. The meat was deproteinized, mashed, put into a retort pouch, and steamed in a water bath at 80℃for 30 minutes, cooled and ground in liquid nitrogen, and the following analysis was carried out.

1.2. Reagent(s)

Acetoin, butyraldehyde, hexanal, nonanal, (E, E) -2, 4-decenal, (E) -2-nonenal, dimethyl trithio, ethyl acetate, 1-octen-3-ol, 1-octen-3-one, 2-acetyl-2-thiazoline, (Z) -5-octen-1-ol, decanal, dimethyl sulfone, hexyl acetate, methyl mercaptan, methyl butyrate, methyl caproate, amyl acetate, phenylacetaldehyde, 2, 3-pentanedione, 2-acetylthiazole, 2-ethylfuran, 2-methylthien, 3-octen-2-one, 4-isopropyltoluene, acetonate and benzaldehyde were purchased from Ala Ding Shenghua technologies, inc. (Shanghai China). Heptanal, octanal, 2-heptanone, 2-octanone, 3-octanone, 4-heptanone, dimethyl sulfide, valeraldehyde, 2-methyl-3-heptanone, and n-alkane (C7-C40) were purchased from Sigma-Aldrich (Shanghai, china). 2-pentylfuran, 2-heptylfuran, 2-hexylfuran and 2-butylfuran were purchased from Alfa Aesar (China, shanghai). Methanol (HPLC grade) was purchased from Merck (dammstatt, germany).

SPME flow

The study was performed in a TriPlus RSH autosampler (Thermo Fisher Scientific (delavay, germany)) using HS-SPME trapping technology. To ensure faster extraction, the vials were kept in a stirred state during extraction.

The sample extraction procedure was as follows: 3g of the minced sample was introduced into a 20mL glass vial, followed by 5. Mu.L of 2-methyl-3 heptanone solution (50. Mu.g/mL) and the vial was immediately sealed with a magnetic cap fitted with a PTFE-silica gel septum. The sample bottles were incubated at 55℃for 20 minutes and extracted at 55℃for 40 minutes using 50/30 μm DVB/CAR/PDMS fibers (Supelco, inc., bellefonte, pa., USA). The extracted fibers were then desorbed in a 250 ℃ sample inlet for 3 minutes. Between consecutive analyses, the extracted fibers were aged at 270 ℃ for 10 minutes at the post-injection port.

1.3. GC-HRMS analysis of volatile Compounds

All analyses were performed on a Q-Exactive Orbitrap mass analyzer equipped with a Triplus RSH autosampler and Trace 1310GC (Thermo Fisher Scientific, bremen, germany). A VF-WAX ms (Agilent, santa Clara, calif.) column was used with an inner diameter of 60m 0.25mm 0.25 μm. Helium (99.9999%) at a constant flow rate of 1mL/min was used as carrier gas. The temperature programming conditions are as follows: the temperature was maintained at 40℃for 2 minutes, then raised to 230℃at a rate of 4℃per minute, and maintained for 5 minutes. The transmission line 1 and the transmission line 2 were each set at 250 ℃. Ionization mode: electron bombardment ionization (70 eV); scanning mode: full scanning; resolution ratio: 60,000 (FWHM). Scanning range: 30-400m/z, the automatic gain control target value is 1E6. The MS ion source and transmission line temperatures were set to 280 ℃ and 250 ℃, respectively.

GC-MS data were collected and processed using the traceFinder 4.0 and Xcalibur 4.1 software (Thermo Scientific). Identification of volatile compounds was performed using NIST17 (v 2.3), wiley9 and self-built homeflag libraries, in combination with Linear Retention Index (LRI). The self-built homeflag database is built using standards. In addition, a High Resolution Filter (HRF) tool in Tracefinder software was used to annotate each measured m/z peak and evaluate the mass accuracy of these ions. A series of normal paraffins (C7-C40) were run under the same chromatographic conditions to calculate the LRI. The concentration of volatile compounds was assessed using the internal standard (2-methyl-3-heptanone) equivalent.

1.4. Statistical analysis

Peak extraction and peak alignment were performed using a Tracefinder deconvolution plug-in, where S/N.gtoreq.10, response threshold 500 000, retention time offset window 3 seconds, and blank filling was performed. For statistical analysis, after log 10 transformation and automatic scaling of samples using metaanalysis 5.0, clustering and differential analysis were performed using Principal Component Analysis (PCA), volcanic plot analysis, and partial least squares discriminant analysis (PLS-DA). The chart in the method optimization was then generated using Microsoft Excel 16.30.

2. Results and discussion

2.1 Separation optimization

Hydrocarbons, alcohols, aldehydes, ketones, furans, sulfur compounds, pyridines, pyrazines and other hybrid compounds are the main components of volatile compounds in foods such as meat. These compounds range in polarity from non-polar to highly polar, which places high demands on non-targeted analysis of volatile compounds. The study compares the effect of non-polar 5% phenyl methyl polysiloxane (DB-5) and polar polyethylene glycol (WAX) immobilization relative to volatile compound separation analysis. By taking mutton as a research model, the research shows that the polar solid phase of polyethylene glycol has better advantage in the identification quantity of volatile compounds, and the polar solid phase is increased by more than 50% compared with a nonpolar solid phase.

2.2 solid-phase microextraction optimization

SPME is the most widely used extraction method for volatile compounds analysis. Among the influencing parameters, the fiber type is the most critical parameter, which can significantly influence the retention behavior of the compound. Depending on the type of fiber, the type of compound and its strength may vary greatly. The extraction efficiency of volatile compounds was evaluated by screening 85 μm Polyacrylate (PA), 50 μm/30 μm divinylbenzene/Carboxen/polydimethylsiloxane (abbreviated as CAR/DVB/PDMS), 95 μm carbonWR, 30 μm PDMS extraction fibers. Figure 1 shows the response intensity trend of volatile compounds after four fiber extractions. Clearly, DVB/CAR/PDMS and Carbon WR show better performance and higher response strength for most compounds. However, carbon WR exhibits poor performance in terms of sulfur-containing compounds. Given that sulfur-containing compounds play a key role in meat flavor, DVB/CAR/PDMS was selected for the next optimization.

In the static headspace solid-phase microextraction process, the volatile compounds reach dynamic balance among the fiber, the sample matrix and the gas phase. On the premise of specific fiber types, the extraction temperature and the extraction time greatly contribute to the extraction efficiency. The invention carries out comprehensive optimization on the two parameters. FIG. 2 shows the effect of extraction temperature and extraction time on the response intensity of hydrocarbons, aldehydes, ketones, esters, furans, sulfur compounds and other heterocyclic compounds. As the extraction time increases, the intensity of typical volatile compounds such as aldehydes, furans, and sulfur compounds increases. However, ketones, alcohols, hydrocarbons, heterocyclic compounds, and the like may be different. Due to the dynamic equilibrium, the prolonged extraction time causes desorption of these compounds. Thus, 50min was chosen as the optimal extraction time for volatile compounds. In the optimization of the extraction temperature, as the temperature is continuously increased, desorption gradually becomes the main action of volatile compounds, and the response intensity of typical volatile compounds reaches the highest at 55 ℃ during 50min of extraction. Finally, the desorption time of the SPME fiber at the sample inlet is closely related to the response intensity of the volatile compounds. The study optimized the study of desorption time, as shown in fig. 3, as the desorption time increased from 1min to 7min, the intensity of most of the compounds increased steadily from 1min to 4 min, and as desorption progressed, the intensity decreased sharply. Thus, incubation at 55℃for 10min, extraction at 55℃for 50min, and desorption at 250℃for 3min were considered optimal conditions SPME.

2.3 identification of flavour compounds

The study was based on NIST and self-built homeflag spectrum libraries, with identification of volatile compounds by mass spectrogram, retention index and HRF tools. When matching using a conventional low resolution NIST spectral library, it is necessary to incorporate HRF tools to provide accurate mass numbers of ions to improve qualitative accuracy. The identification principle of the flavor compound is as follows: the HRF score is higher than 95, and the positive and negative matching degree of the mass spectrogram is higher than 750. In addition, the retention index difference of the homeflag spectrum library is less than 20, and the retention index difference of the nist library is less than 50. Under the optimal analysis conditions, based on the above identification rules, five different varieties of meat were analyzed to identify 148 volatile compounds in total, as shown in table 1. The volatile compound list is used for statistical analysis, evaluating the difference of different varieties of meat, and determining the difference marking compounds of different varieties of meat. Hydrocarbons, aldehydes, ketones, esters, acids, furans, sulfur compounds and furanones are the major components of volatile compounds in meat. Among hydrocarbons, aromatic hydrocarbons are the main hydrocarbons, mainly from feeds. Fatty aldehydes and enals, such as hexanal, octanal, 2-decenal, etc., are mainly produced by thermal oxidative degradation of lipids, while acetaldehyde and aromatic aldehydes are produced by Strecker degradation of amino acids and their derivatives, such as glycine, phenylalanine. Furans, alcohols, acids and ketones also result from the thermal oxidative degradation of lipid unsaturated fatty acids. For sulfur-containing compounds, thiamine degradation, maillard reactions, strecker degradation and further oxidation thereof may be the primary formation pathways. Furanones, furfurals and derivatives thereof are produced by Maillard reactions.

2.4 statistical analysis

The profile analysis of the volatile compounds is mainly based on a metabonomics method, various volatile compounds with mass less than 550Da are comprehensively characterized, global distribution diagrams of different varieties of meat volatile compounds are provided, and the different compounds are effectively identified. The volatility characterization differences between the five different meats were visualized by PCA analysis (fig. 4). Based on all the identified volatile compounds, the first two principal components account for more than 60% of the total difference between samples and indicate that the meat differences of the five different varieties are evident and can be effectively separated. Clustering of five replicate samples indicated that the applied analysis method was of good reproducibility and robustness, which is important in non-targeted differentiation studies.

It is another object of the present invention to identify specific compounds that are critical to distinguishing between different meat samples. Compounds that caused differences between the sample groups were found by unpaired t-test, as shown in table 2. By comparison of every two meats, the fold change in abundance of tens of compounds >2 or the fold change <0.5. Wherein, the number of different compounds of mutton and beef is less, and the number of the different compounds is only 38. In the pathway of formation of meat flavor, lipid oxidation is one of the major pathways. Saturated Fatty Acids (SFA) and monounsaturated fatty acids (MUSFA) are the main fatty acids, which are similar in content in mutton and beef. In addition, polyunsaturated fatty acid (PUSFA) levels in mutton and beef are low relative to pork and duck. This will therefore lead to a close profile of volatile compounds produced by oxidative degradation of the lipids. However, when pork, duck and chicken are compared, the amount of the volatile compounds is obviously increased, and the reason is that polyunsaturated fatty acids such as linoleic acid, linolenic acid, arachidonic acid and the like are more easily oxidized in the reheating process, and the polyunsaturated fatty acids have relatively complex volatile compound compositions generated by oxidative degradation due to more sites where oxidation can occur.

Based on PLS-DA analysis results of five meats, 58 discrimination compounds (VIP. Gtoreq.1) were screened out in total. Figure 5 shows the 15 most important discriminant compounds. The following data were obtained: the content of 2-amyl furan, 2-butyl furan, 2-hexyl furan, 1-ocent-3-ol and 1-ocent-3-one in duck meat is obviously higher than that of other four kinds of meat. The above compounds are mainly produced by oxidative degradation of n-6 fatty acids such as linoleic acid and arachidonic acid, and the content of the fatty acids in duck meat is obviously higher than that of other four kinds of meat, so that the flavor compounds are main characteristic compounds of the duck meat, which are different from other meat. In addition, carbon disulfide, dimethyl sulfoxide, and fatty acid methyl esters including methyl butyrate, methyl octanoate, and methyl nonanoate are the most important discriminators of beef. Carbon disulphide and dimethylsulfoxide should be derived from sulphur containing amino acids such as methionine and cysteine. In addition, more medium chain fatty acid methyl esters are produced when large amounts of oleic acid are present.

3. Summary

The invention develops an analysis strategy for non-targeted analysis of volatile compounds in different meat categories. Therefore, the method based on HS-SPME-GC-MS is studied in detail, and the influence parameters such as the type of the extracted fiber, the extraction temperature, the extraction time and the like are comprehensively optimized. For compound validation, retention index, self-built high-resolution mass spectrometry based flavor compound database, low-resolution mass spectrometry NIST database matching, and high-resolution mass spectrometry based HRF score tools were combined. The developed non-target flavor compound analysis method can be used for meat category identification, and the volatility profiles of duck meat, chicken, beef, mutton and pork can be effectively distinguished by combining a statistical analysis tool. In addition, a portion of aldehydes, furans, sulfur compounds, esters, alcohols, and ketones, and other volatile compounds can be used as biomarkers for identification. 2-pentylfuran, 2-n-butylfuran, 2-hexylfuran, 1-octen-3-ol and 1-octen-3-one were positively correlated with duck meat. Fatty acid methyl esters will be the primary identification biomarker for beef. This phenomenon is mainly caused by the difference in fatty acid composition in different meats.

TABLE 1 volatile compounds identified in five different meats

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TABLE 2 amounts of different meat difference compounds analyzed based on volcanic diagrams (fold. Gtoreq.2, p < 0.05)

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Claims (4)

1. A method for identifying the authenticity of beef or mutton based on gas chromatography-electrostatic field orbit trap high-resolution mass spectrum comprises the following steps:

1) Separation

Grinding the livestock and poultry meat sample to be detected to obtain a chopped sample;

2) Headspace solid phase microextraction

Performing headspace solid-phase microextraction on the chopped sample to obtain a mixed unknown compound;

3) Authentication

Carrying out gas chromatography-electrostatic field orbit trap high-resolution mass spectrometry on the extracted mixed unknown compound to obtain GC-MS data;

the GC-MS data are identified through MS modes, retention indexes and HRF processing in NIST and a homeflag library, and the compound components contained in the livestock and poultry meat sample to be detected are obtained;

4) Statistical analysis

Carrying out statistical analysis on the compound components obtained in the step 3) to obtain flavor compounds corresponding to the livestock and poultry meat samples to be detected;

5) Comparing the corresponding flavor compound of the livestock and poultry meat sample to be detected with the flavor compound of the known beef or mutton sample to obtain the abundance times of the difference compound;

when the beef is identified as true or false, comparing the flavor compound corresponding to the livestock and poultry meat sample to be detected with the flavor compound of the known beef sample as follows A1) -H1), and when the abundance times of the difference compound meet the following range and the p value is smaller than 0.01, the livestock and poultry meat sample to be detected is possibly duck meat, and the result is judged that the livestock and poultry meat sample to be detected is not beef and/or is not pure beef:

a1 2E, 4E-decadienal 19.20-27.32;

b1 6.91-9.77 of 2E-heptenal;

c1 3.67-5.28 parts of 2-amyl furan;

d1 1-octen-3-ol 4.34 to 6.36;

e1 17.41-25.62 of 2-n-octyl furan;

f1 2.92-4.38 parts of 1-octene-3-ketone;

g1 2.92-4.38 parts of 2-n-butyl furan;

h1 2E, 4E-nonadienal 7.16-10.37;

when the mutton is identified as true or false, comparing the flavor compound corresponding to the livestock and poultry meat sample to be detected with the flavor compound of the known mutton sample as follows A2) -F2) a difference compound, and when the abundance ratio of the difference compound satisfies the following range, the livestock and poultry meat sample to be detected may be duck meat, and judging that the livestock and poultry meat sample to be detected is not mutton and/or is not pure mutton as a result:

a2 1.74-2.60 parts of 2-amyl furan;

b2 2.10-3.16 parts of 2-n-butyl furan;

c2 5.50 to 7.96 of 2-n-octyl furan

D2 1.86 to 2.78 of 1-octen-3-ol;

e2 2E, 4E-decadienal 11-16.5;

f2 2E, 4E-nonadienal 6.36-9.37.

2. The method according to claim 1, characterized in that: the treatment of the livestock and poultry meat sample to be detected is as follows: removing fascia from livestock and poultry meat to be tested, mincing, placing into a stewing bag, stewing in a water bath at 80 ℃ for 30 minutes, cooling, and grinding in liquid nitrogen for later use;

the flow of the headspace solid-phase microextraction is as follows: adding 2-methyl-3 heptanone solution into the chopped sample, and filling the chopped sample into a container sealed by a magnetic cover of a PTFE-silica gel diaphragm for 40-60 percent ^o Incubating for 10-30 min, extracting by using a fiber head, desorbing the extracted fiber at a sample inlet, and aging the extracted fiber at a rear sample inlet between continuous analysis;

the temperature of the fiber head extraction is 37-65 DEG C ^o C, the time is 10-59 min;

the desorption temperature in the sample inlet is 230-270 DEG C ^o C, the time is 1-8 min;

aging the rear sample inlet at 230-270 ℃ for 10-30 min;

the working conditions of the gas chromatography-electrostatic field orbitrap high-resolution mass spectrum are as follows:

automatic sample injector: triPlus RSH;

mass analyzer: trace 1310GC Q-Exactive Orbitrap;

film thickness: inner diameter 60m ×0.25× 0.25mm ×0.25 μm;

chromatographic column: VF-WAX ms;

helium with constant flow rate of 1mL/min is used as carrier gas;

the temperature programming conditions are as follows: maintaining at 40 ℃ for 2 minutes, then raising the temperature to 230 ℃ at a rate of 4 ℃ per minute, and maintaining for 5 minutes;

the transmission line 1 and the transmission line 2 are both set at 250 ℃;

ionization mode: electron bombardment ionization 70eV;

scanning mode: full scanning;

resolution ratio: 60,000FWHM;

scanning range: 30-400m/z, the automatic gain control target value is 1E6;

the MS ion source and transmission line temperatures were set to 280 ℃ and 250 ℃, respectively;

the statistical analysis is verified by principal component analysis, t-test and partial least squares discriminant analysis PLS-DA analysis.

3. The method according to claim 1 or 2, characterized in that: the fiber head type used for the headspace solid phase microextraction comprises at least one of 85 μm polyacrylate, 50 μm/30 μm divinylbenzene/Carboxen/polydimethylsiloxane, 95 μm Carbon WR and 30 μm PDMS.

4. The method according to claim 1 or 2, characterized in that: in the step 3), the compound components contained in the livestock and poultry meat sample to be detected can be judged according to any one of the following data:

a) HRF score higher than 95;

b) A matching factor based on MS mode higher than 750;

c) The retention index difference of the homeflavor library is less than 20;

d) The retention index difference of NIST pool is within 50.

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