A method for determining meat flavor compounds using TF-SPME

Abstract

This invention discloses a method for determining meat flavor compounds using TF-SPME. The method comprises the following steps: 1) weighing a fresh meat sample into a headspace vial and performing thin-film solid-phase microextraction (SPM) to obtain a TF-SPME sample; and 2) subjecting the TF-SPME sample to gas chromatography-mass spectrometry to separate and identify the meat flavor compounds. This method enables a more comprehensive and accurate determination of the composition and content of odor compounds in meat samples.

Description

Technical Field

[0001] The invention belongs to the technical field of mass analysis and relates to a method for measuring meat flavor compounds by adopting TF-SPME. Background Art

[0002] Meat is an essential component of the human diet, providing a variety of nutrients. Meat quality evaluation primarily encompasses four aspects: safety, nutritional quality, edible quality, and processing quality. Eating quality is a crucial indicator influencing consumer product selection. For example, Nachomkamon Saengsuk's research detailed the key differences in protein, fat, lipids, and flavor between five parts of lamb—shoulder, rib, loin, breast, and leg—as well as the potential impact of these factors on overall flavor, enabling consumers to select nutritious or appropriate cuts for cooking. Eating quality encompasses texture, flavor, and taste. In recent years, the widespread application of flavor detection and analysis technologies has enabled traditional sensory evaluation to be integrated with instrumental analysis and evaluation. This has driven the rapid development of technologies such as gas chromatography-mass spectrometry (GC-MS), gas chromatography ion mobility spectrometry (GC-IMS), and electronic nose (E-nose), leading to the development of gas chromatography-olfactory olfaction (GC-O) and comprehensive two-dimensional gas chromatography (GC*GC). Zhu Xijin et al. analyzed yaks from low and high altitudes using steaming, boiling, and roasting cooking methods. The measurement results of the electronic nose showed that the impact of altitude on flavor was greater than the cooking method. SPME-GC-MS was further used to analyze the composition and content of volatile compounds, and 36 compounds with odor activity values ​​OAV≥1 that had a significant impact on the overall flavor were identified.

[0003] With the increasing depth of meat flavor research, the enrichment and extraction of volatile compounds have become increasingly important, significantly impacting the final detection results. Depending on the enrichment stage, enrichment methods can be categorized as gas phase enrichment (static headspace (SHS) and dynamic headspace (DHS)), liquid phase enrichment (solvent extraction (SEP) and solvent-assisted evaporation extraction (SAFE)), and solid phase enrichment (solid phase microextraction (SPME), thin film solid phase microextraction (TF-SPME), and stir bar solid phase extraction (SBSE). Each of these enrichment methods has its own advantages and disadvantages, and is significantly affected by the state of the sample matrix. When testing different samples, the specific scope of application must be considered. For example, liquid samples such as beverages and alcohol are more suitable for enrichment using SBSE. The most widely used technologies for flavor detection of solid samples such as meat are SAFE and SPME. The advantage of SAFE technology is that it is not easy to produce excessive interference under low temperature and high pressure conditions and can more accurately reflect sample information. However, its disadvantages are also obvious. It requires the use of a large amount of organic reagents, the processing process is complicated, and it needs to be used together with other concentration methods, and may cause the loss of valuable compounds. SPME is a combination of collection, extraction, concentration and injection. Under headspace conditions, the adsorbent on the fiber surface can transfer volatile compounds to the GC injection system without direct contact with the sample, realizing trace-level compound detection. However, due to its manufacturing process, coating type and theoretical volume limitations, there is still room for further improvement. Summary of the Invention

[0004] The object of the present invention is to provide a method for determining meat flavor compounds using TF-SPME.

[0005] In the method of the present invention, three major influencing factors of pretreatment were investigated, namely, sample amount (1-5 g), extraction temperature (25-65 ° C) and extraction time (30-70 min). For some precious samples, a lower sample amount is expected, which means that the consumption of samples can be saved to achieve more repetitions or complete other research contents. The increase in extraction temperature will intensify molecular motion and increase the adsorption coefficient of volatile compounds, but too high a temperature may cause denaturation of analytes, affecting the authenticity of detection. Generally, the number and content of extracted compounds increase with time. After reaching the equilibrium of gas-solid phase, there will be no significant change. It is necessary to find the equilibrium time of extraction; three instrument conditions were optimized in the present invention, namely, the desorption temperature of the injection port (230-270 ° C), the desorption time (4-20 The initial temperature of the cold trap (-20 to 120°C) affects the desorption of compounds enriched by TF-SPME and is crucial for ensuring that all enriched compounds are fully transferred to the injection system. The initial temperature of the cold trap, the final step before compounds enter chromatographic analysis, is crucial for addressing asynchronous vaporization. By optimizing these parameters, we aim to achieve optimal TF-SPME detection conditions for meat flavor compounds, providing a foundation for subsequent flavor research.

[0006] The present invention provides a method for determining meat flavor compounds using TF-SPME, comprising the following steps:

[0007] 1) The collected fresh meat sample paste was weighed into a headspace bottle and subjected to thin film solid phase microextraction to obtain a TF-SPME sample;

[0008] 2) subjecting the TF-SPME sample to gas chromatography-mass spectrometry to separate and identify meat flavor compounds.

[0009] In the above method, in step 1), the extraction amount of the thin film solid phase microextraction can be 1-5 g, the temperature can be 25-165° C., specifically 25-65° C., and the time can be 1-70 min, specifically 30-70 min.

[0010] In the above method, in step 1), the extraction amount of the thin film solid phase microextraction is preferably 3 g, the temperature is preferably 55° C., and the time is preferably 60 min.

[0011] In the above method, in step 1), the thin film solid phase microextraction process is as follows: the collected fresh meat sample is made into a meat paste sample and weighed into a headspace bottle, heated in a metal bath at 25~165℃ for 1~40 min (specifically, it can be heated in a metal bath at 55±1℃ for 30 min or 25~40 min), and after cooling, 1~50 μg / mL 2-methyl-3-heptanone methanol solution (specifically, 5 μg / mL 2-methyl-3-heptanone methanol solution can be used), and the thin film solid phase microextraction material is fixed in the form of a headspace using a holder, the lid is tightened, and the mixture is incubated in a metal bath at 25~165℃ (specifically, it can be 55±1℃) for 1~80 min (specifically, it can be 55~65 min or 60 min). After cooling, the mixture is loaded into a TDU thermal desorption tube for standby use.

[0012] In the above method, the gas chromatography uses a gas chromatograph equipped with an automatic sampling unit, a thermal desorption system and a cold trap system;

[0013] The desorption temperature of the thermal desorption system may be 10-350°C (specifically 40-350°C), the heating rate may be 1-720°C / min, the desorption time may be 1-20 min, and the split ratio may be splitless to 100:1;

[0014] The enrichment temperature of the cold trap system can be -150~0°C, specifically -140~0°C, and the heating rate can be 1~12°C / s.

[0015] In the above method, the initial temperature of the thermal desorption system may be 30°C, the heating rate may be 300°C / min, the desorption temperature may be 240°C, the temperature may be maintained at 240°C for 8 min, and the flow may not be split;

[0016] The initial temperature of the cold trap system can be -40°C, the heating rate can be 10°C / s, the desorption temperature can be 240°C, the final temperature can be maintained for 8 minutes, and the split ratio can be 5:1.

[0017] In the above method, in step 2), the gas chromatography separation conditions are:

[0018] Chromatographic column: VF-WAXms capillary column, 60 m×0.25 mm×0.25 μm; temperature program: initial temperature 40°C, hold for 2.0 min, increase to 230°C at 4°C / min, hold for 5 min; transfer line temperature 250°C; carrier gas is helium, and the carrier gas flow rate can be 1 mL / min.

[0019] In the above method, in step 2), the mass spectrometry conditions are as follows:

[0020] The electron bombardment ion source had an electron energy of 70 eV and an ion source temperature of 280°C; the transfer line temperature was 250°C; and the full scan mode was used with a mass scan range of 30–400 m / z.

[0021] The present invention also provides a method for identifying characteristic flavor substances of flaxseed pork based on the determination of meat flavor compounds using TF-SPME, comprising the following steps:

[0022] 1) The flaxseed pork and regular white pork pastes were weighed into headspace vials, and thin film solid phase microextraction was performed to obtain TF-SPME samples of the flaxseed pork and regular white pork.

[0023] 2) subjecting the flaxseed pork and conventional white pork TF-SPME samples to gas chromatography-mass spectrometry to separate and identify the flavor compounds therein;

[0024] 3) performing a major difference compound analysis on the flavor compounds to obtain the major difference compounds between the flaxseed pork and ordinary white pork;

[0025] 4) Comparing the contents of the main different compounds in the flaxseed pork and ordinary white pork to confirm the differences in the characteristic flavor substances of the flaxseed pork.

[0026] In the above method, the major differential compounds include up-regulated compounds and down-regulated compounds;

[0027] The up-regulating compounds include ethyl acetate, 2-ethyl-furan, 2,3-pentanedione, (E)-2-pentenal, 1-butanol, 1-penten-3-ol, 2-ethylthiophene, 2-ethyl-2-butenal, (E)-2-hexenal, styrene, 2-ethyl-pyridine, (E)-2-(2-pentenyl)furan, (E)-2-heptenal, indane, 3-ethyl-pyridine, 3-methyl-1-hexanol, 2-pentenal, Thiophene, (E,E)-2,4-heptadienal, 3,5-octadien-2-one, benzofuran, 5-vinyl-4-methyl-thiazole, 5-ethyl-2-furanaldehyde, ethyl benzoate, 4-isopropylbenzaldehyde, naphthalene, 2-phenyl-propenal, 2-hydroxyacetophenone, (E)-2-tridecanal, 1-indanone, 2-methyl-3-phenyl-2-propenal, 4-ethyl-phenol, 3-ethyl-phenol, coumarin;

[0028] The down-regulating compounds include 2-propylfuran, camphene, 1-methyl-pyrrole, 2-hexyl-furan, 3-methyl-2-furanone, (E)-2-dodecanal, 2-pyrrolecarboxaldehyde, octanoic acid, and 5-methyl-2-pyrrolecarboxaldehyde.

[0029] In the above method, in step 4), the mass percentage of each up-regulated compound in the main differential compounds in the livestock and poultry meat sample to be tested is increased by 1.99 to 16.45 times compared with the main differential compounds in the ordinary white pork, and the total mass percentage of the up-regulated compounds is increased by 3.5 to 4.25 times, and the mass percentage of each down-regulated compound is decreased by 23% to 79%, and the total mass percentage of the down-regulated compounds is decreased by 40% to 60%.

[0030] The present invention further provides a method for authenticating flaxseed pork by measuring meat flavor compounds using TF-SPME, comprising the following steps:

[0031] 1) The fresh meat sample paste and ordinary white pork to be tested were weighed into headspace bottles respectively, and thin film solid phase microextraction was performed to obtain TF-SPME samples of the fresh meat sample paste and ordinary white pork to be tested;

[0032] 2) subjecting the fresh meat sample puree and the common white pork TF-SPME sample to gas chromatography-mass spectrometry, respectively, to separate and identify flavor compounds in the fresh meat sample and the common white pork;

[0033] 3) performing principal component analysis on the flavor compounds in the livestock and poultry meat samples to be tested and ordinary white pork, respectively, to obtain the main differential compounds between the livestock and poultry meat samples to be tested and ordinary white pork;

[0034] 4) Comparing the contents of the major differential compounds in the livestock and poultry meat samples to be tested with the contents of the major differential compounds in known ordinary white pork to identify the authenticity of the flaxseed pork.

[0035] In the above method, the major differential compounds include up-regulated compounds and down-regulated compounds;

[0036] The up-regulating compounds include ethyl acetate, 2-ethyl-furan, 2,3-pentanedione, (E)-2-pentenal, 1-butanol, 1-penten-3-ol, 2-ethylthiophene, 2-ethyl-2-butenal, (E)-2-hexenal, styrene, 2-ethyl-pyridine, (E)-2-(2-pentenyl)furan, (E)-2-heptenal, indane, 3-ethyl-pyridine, 3-methyl-1-hexanol, 2-pentyl -thiophene, (E,E)-2,4-heptadienal, 3,5-octadien-2-one, benzofuran, 5-vinyl-4-methyl-thiazole, 5-ethyl-2-furanaldehyde, ethyl benzoate, 4-isopropylbenzaldehyde, naphthalene, 2-phenyl-propenal, 2-hydroxyacetophenone, (E)-2-tridecanal, 1-indanone, 2-methyl-3-phenyl-2-propenal, 4-ethyl-phenol, 3-ethyl-phenol, coumarin;

[0037] The down-regulating compounds include 2-propylfuran, camphene, 1-methyl-pyrrole, 2-hexyl-furan, 3-methyl-2-furanone, (E)-2-dodecanal, 2-pyrrolecarboxaldehyde, octanoic acid, and 5-methyl-2-pyrrolecarboxaldehyde.

[0038] In the above method, in step 4), if the mass percentage of each up-regulated compound in the tested livestock and poultry meat sample is increased by 1.99 to 16.45 times compared with the main different compounds in the ordinary white pork, the mass percentage of the up-regulated compounds is increased by 3.5 to 4.25 times, and the mass percentage of each down-regulated compound is decreased by 23% to 79%, and the mass percentage of the total down-regulated compounds is decreased by 40% to 60%, then the tested livestock and poultry meat sample is flaxseed pork; otherwise, it is ordinary white pork.

[0039] The present invention has the following beneficial effects:

[0040] This study establishes a odor compound enrichment and desorption technique based on TF-SPME. Compared to traditional SPME, this technique boasts a larger theoretical volume of adsorbent material, reducing competitive adsorption. In this study, multiple ester and lactone compounds, not detected by SPME, were extracted, enabling a more comprehensive and accurate characterization of the odor compound composition in meat samples. Simultaneous processing of multiple sample groups during the enrichment phase enables high throughput and significant time savings. The two-step "thermal desorption-cold enrichment" process for thermal desorption results in more stable and reproducible compound detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the effect of sample amount on volatile compounds in pretreatment.

[0042] Figure 2 This is the effect of extraction temperature on volatile compounds during pretreatment.

[0043] Figure 3 This is the effect of extraction time on volatile compounds in pretreatment.

[0044] Figure 4 This is the effect of thermal desorption temperature on volatile compounds in instrumental conditions.

[0045] Figure 5 This is the effect of thermal desorption time on volatile compounds under instrument conditions.

[0046] Figure 6 The effect of cold trap temperature on volatile compounds.

[0047] Figure 7 The heat map and volcano map of the differences between SPME and TF-SPME are shown. Figure 7(a) is a comparison chart of total peak areas, (b) is a heat map of compound differences, and (c) is a volcano map.

[0048] Figure 8 It is the principal component analysis PCA of volatile compounds and Volcano Plot. Figure 8 (a) Principal component analysis PCA, (b) LNA / White difference volcano plot, (c) LNA / White up-regulated compound comparison graph, and (d) LNA / White down-regulated compound comparison graph.

[0049] Figure 9 To test the stability of the method. DETAILED DESCRIPTION

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0051] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0052] Example 1

[0053] 1. Materials and Reagents

[0054] Chromatographic-grade methanol, acetonitrile, and ultrapure water were used. 2-Methyl-3-heptanone and n-alkanes (C7-C40) were purchased from Sigma (Shanghai). TF-SPME membrane (PDMS / DVB) (thickness: 450 μm) and Tenax TA™ liners were purchased from Zest, Germany; SPME extraction needles (DVB / CAR / PDMS) were purchased from Supelco.

[0055] 2. Instruments and equipment

[0056] A Trace 1310 gas chromatograph coupled with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Germany) was used. The automated sample injection unit (MPS), thermal desorption system (TDU-2), and cold trap system (CIS-4) were all from ZESTE, Germany. A laboratory-built metal bath was used.

[0057] 3. Sample pretreatment

[0058] Pork samples from flaxseed pigs (denoted as the LNA group) and white pigs (denoted as the white group) were provided by the production company and complied with national regulations for pig slaughter. Fresh meat samples were pre-chilled with liquid nitrogen, transported on dry ice, and stored in a -80°C freezer in the laboratory. Prior to the experiment, they were thawed at 4°C for 2 h, and after removing surface fat and intermuscular fascia, the meat was minced into a puree.

[0059] 3.1 TF-SPME sample pretreatment and extraction single factor experiment

[0060] The sample pretreatment method optimized three experimental conditions: sample mass (1, 2, 3, 4, 5 g), extraction temperature (25, 35, 45, 55, 65℃), and extraction time (30, 40, 50, 60, 70 min). When investigating the effect of sample mass, the extraction temperature and extraction time were selected as 55℃ and 60 min, respectively. When investigating the effect of extraction temperature, the sample mass and extraction time were selected as 3 g and 60 min, respectively. When investigating the effect of extraction time, the sample mass and extraction temperature were selected as 3 g and 55℃, respectively.

[0061] 3.2 Single-factor experiment on instrumental conditions for volatile compounds using TF-SPME

[0062] The instrument detection conditions optimized three experimental conditions: TDU desorption temperature (230, 240, 250, 260, 270℃), TDU desorption time (4, 8, 12, 16, 20 min), and CIS enrichment temperature (-20, -40, -60, -80, -100, -120℃). When investigating the TDU desorption temperature, the TDU desorption time and CIS enrichment temperature were selected as 8 min and -60℃ respectively. When investigating the TDU desorption time, the TDU desorption temperature and CIS were selected as 250℃ and -60℃ respectively. When investigating the CIS enrichment temperature, the TDU desorption temperature and TDU desorption time were selected as 250℃ and 8 min respectively.

[0063] 3.3 Comparison of SPME and TF-SPME extraction methods

[0064] The experimental conditions finally determined based on TF-SPME sample pretreatment and instrument conditions were compared with the SPME method.

[0065] 4. GC-MS experimental method

[0066] 4.1 SPME sample pretreatment and extraction conditions

[0067] Fresh meat puree samples were placed in a retort bag in an 80°C water bath for 30 minutes. After cooling naturally, 3.000 ± 0.0001 g of homogenized sample was weighed and placed in a brown headspace vial. 10 μL of a 5 μg / mL 2-methyl-3-heptanone methanol solution was added. The sample was incubated at 55°C for 20 minutes. Extraction was performed using a DVB / CAR / PDMS (50 / 30 μm, Supelco) fiber tip at 55°C for 40 minutes. Desorption was then performed at the autosampler inlet for 3 minutes.

[0068] 4.2 TF-SPME sample pretreatment and extraction conditions

[0069] 3.000±0.0001 g of uniform sample from the fresh meat paste was weighed and placed in a brown headspace bottle. 10 μL of 5 μg / mL 2-methyl-3-heptanone methanol solution was added and heated in a 55°C metal bath for 30 min. After cooling, the TF-SPME was fixed in the form of a headspace using a holder, the lid was tightened, and the sample was incubated in a 55°C metal bath for 60 min. After cooling, the TF-SPME was removed, washed with 35°C warm water, and the surface was wiped dry with dust-free paper. It was then placed in a sample injection tube for detection on the machine.

[0070] 4.3 Thermal Desorption System TDU Conditions

[0071] The initial temperature of TDU was 30°C, the heating rate was 300°C / min, the desorption temperature was 240°C, the final temperature was maintained for 8 minutes, and there was no split mode. The initial temperature of the cold trap system CIS was -40°C, the heating rate was 10°C / s, the desorption temperature was 240°C, the final temperature was maintained for 8 minutes, and the split ratio was 5:1.

[0072] 4.4 GC conditions

[0073] Gas chromatography separation conditions: chromatographic column: VF-WAXms capillary column (60 m × 0.25 mm × 0.25 μm); heating program: initial temperature 40°C, hold for 2.0 min, increase to 230°C at 4°C / min, hold for 5 min; transfer line temperature 250°C; carrier gas: high-purity helium (purity >99.999%), carrier gas flow rate 1 mL / min.

[0074] 4.5 MS conditions

[0075] An electron impact (EI) ion source with an electron energy of 70 eV and an ion source temperature of 280°C was used; the transfer line temperature was 250°C; and the full scan mode was used with a mass scan range of 30–400 m / z.

[0076] 5. Results and Analysis

[0077] 5.1 Single-factor experiment for sample pretreatment

[0078] 5.1.1 Effect of sample amount on extracted compounds

[0079] like Figure 1As shown, the total peak area of ​​detected compounds reached its highest value at a sample weight of 3 g. Further increases in sample weight had no significant effect on the total peak area. In fact, the high water content affected the adsorption efficiency of some compounds. Aldehydes, ketones, alcohols, and S- and N-containing compounds all showed an increasing trend with increasing sample weight, but sample weights of 4 g and 5 g did not show significant differences from 3 g. Ester compounds achieved their maximum peak area at 3 g. Taking all factors into consideration, a 3 g sample weight ensures high efficiency in compound detection while also conserving sample volume.

[0080] 5.1.2 Effect of extraction temperature on extracted compounds

[0081] Based on previous experience, a single factor experiment with a 10°C difference in extraction temperature from 25°C to 65°C was set. Figure 2 As shown in the figure, in this set of experiments, it can be observed that the total peak area reaches a maximum value at 55°C, and is significantly different from other temperatures. Aldehydes, alcohols, esters, S-containing and N-containing compounds also show the same trend, which means that temperature has a greater effect on the adsorption of compounds; only ketone compounds are not sensitive to temperature, and there is no significant difference in the range of 25°C-65°C.

[0082] 5.1.3 Effect of extraction time on extracted compounds

[0083] The time of solid phase extraction has a great influence on the enrichment of compounds in the sample. In this group of experiments, five time gradients of 30, 40, 50, 60, and 70 min were set to determine the effect of time changes on the results. Figure 3 As shown in the figure, extending the enrichment of the compound from 30 min to 60 min will gradually increase the enrichment of the compound, but after reaching 70 min, it will not continue to increase. This shows that too long extraction time not only takes up a lot of resources, but also does not produce better experimental results.

[0084] In this set of experiments, three factors that have a significant impact on the experimental results during sample pretreatment were investigated, namely sample amount, extraction temperature, and extraction time. Each factor showed different patterns. By integrating the experimental results of each factor, a TF-SPME experimental method was established with a meat product sample amount of 3 g, an extraction temperature of 55°C, and an extraction time of 60 min.

[0085] 5.2 Single-factor experiment with instrument conditions

[0086] 5.2.1 TDU desorption temperature

[0087] As the sampling device for TF-SPME adsorption materials after enriching the sample volatile compounds, TDU plays an important role in the test. According to the material characteristics of TF-SPME, the highest desorption temperature it can reach is 270℃. Below this temperature, finding a suitable desorption temperature can not only obtain reasonable test results but also extend the service life of the material. Therefore, 5 desorption temperatures were set from 230-270℃. Figure 4 It can be observed that under the conditions of 240-270℃, there is no significant difference in the total peak area of ​​the compounds. In other compounds such as aldehydes, the changes are also small. The total peak area at 240℃ is significantly higher than that at 230℃. Based on stability and economy considerations, 240℃ is a better choice in this set of experiments.

[0088] 5.2.2 TDU desorption time

[0089] exist Figure 5 Figure 2 shows the effect of the desorption time of the TF film in a high-temperature chamber on various compound types. In terms of total peak area, there were no significant differences between the groups, with the 8-minute group showing the highest peak area. Aldehydes and alcohols also showed the highest peaks in the 8-minute group. In groups with a time greater than 8 minutes, significant differences were observed between the other groups, such as aldehydes at 16 minutes, alcohols at 20 minutes, SN-containing compounds at 20 minutes, and esters at 12, 16, and 20 minutes, respectively. In this set of experiments, 8 minutes was a preferred choice.

[0090] 5.2.3 CIS cold trap temperature

[0091] Cold trap is the step before volatile compounds enter the chromatographic column. The refrigerant is liquid nitrogen, which plays the role of enriching compounds under low temperature conditions, such as Figure 6 As shown in the figure, in this group of experiments, the total peak area at -40℃ and the peak areas of other compounds are significantly different from those at other temperatures. Therefore, -40℃ is a better condition for selecting the cold trap temperature.

[0092] 5.3 Comparison of SPME and TF-SPME Techniques

[0093] This group of experiments compared the differences between SPME and TF-SPME, such as Figure 7 As shown in (a), the total peak area of ​​TF-SPME reached 1.22×10 9, while SPME is only half of that, indicating that under their respective optimal experimental conditions, different pretreatment technologies have significantly different enrichment efficiencies for volatile compounds in the same sample. TF-SPME, due to its larger surface area, has a higher compound solvent, which also enables it to have a detection capability far beyond that of traditional SPME. However, due to the imperfect bonding method between the extraction phase and the material in the TF film, there is currently a lack of more adsorbent extraction materials, such as the most common PDMS / DVB / CAR three-in-one fiber in SPME technology, which may also lead to differences between different technologies; Figure 7 It can be clearly observed from the heat map in (b) that TF-SPME is superior to SPME in nearly 2 / 3 of the compounds in the upper right part. These compounds include a large number of main meat flavor compounds, such as aldehydes, furans, esters, etc.; in the lower left 1 / 3, the detection of these compounds is better than TF-SPME. Figure 7 The volcano plot in (c) can be used to obtain the differential compounds between the two technologies. TF-SPME has more compounds such as 2-acetyl-thiazole, dimethyl sulfoxide, and formic acid, while SPME has more compounds such as 3-methyl-butyric acid, 2-octanone, and indane, which can be used as differential compounds to distinguish the two technologies.

[0094] To determine the stability of the pretreatment technology and instrument, the internal standard added to the sample was tested, and the overall RSD was 6%, which is generally better than the results of SPME. In addition, the detection time of the two technologies, SPME and TF-SPME, was also examined. Since the same gas chromatography heating program was used in this application, the chromatographic detection time was fixed; in the automatic injection program of SPME, the sample needs to be equilibrated in the incubator for 20 minutes, and then inserted into the extraction head for 40 minutes, which takes a total of 1 hour, and only one sample can be pretreated at the same time; in the TF-SPME technology, since the TF extraction process is independent of the incubation extraction, multiple samples can be processed simultaneously, and a single TF film can be directly injected. This operation method greatly saves time and improves the detection throughput of volatile compounds in the laboratory.

[0095] Example 2 Comparison between ordinary white pigs and flaxseed pigs

[0096] 1. Volatile compounds

[0097] In this application, the TF-SPME pretreatment technology established in Example 1 was used to detect volatile compounds in common white pigs and flaxseed pigs, and the concentration of each compound was calculated using the following internal standard method.

[0098]

[0099] Where: Ca is the content of volatile compounds in the sample, in micrograms per kilogram (μg / kg); Sa is the peak area of ​​volatile flavor compounds in the sample; Sis is the peak area of ​​the internal standard 2-methyl-3-heptanone in the sample; Cis is the concentration of the internal standard, in micrograms per milliliter (μg / mL); Vis is the volume of the internal standard, in milliliters (mL); m is the mass of the sample, in grams (g); 1000 is the coefficient for converting μg / g to μg / kg.

[0100] A total of 196 compounds were detected in the two groups of samples, including 41 aldehydes, 23 ketones, 16 alcohols, 11 phenols, 7 acids, 18 esters, 8 lactones, 36 S- and N-containing compounds, 9 benzene series, 21 furans, 4 terpenes, and 2 others.

[0101] Use Log to store data 10 After calculation, the data were normalized using Pareto scaling and PCA analysis was performed. Figure 8 In (a), PC1 reached 34.8% and PC2 reached 14%, and the flavor composition of the two groups of pork was effectively distinguished. Figure 8 The analysis results of the volcano plot in (b) showed that there were 42 compounds as differential compounds between the two groups of pork, among which the compounds upregulated in the LNA group relative to the white group included ethyl acetate, 2-ethyl-furan, 2,3-pentanedione, (E)-2-pentenal, 1-butanol, 1-penten-3-ol, 2-ethylthiophene, 2-ethyl-2-butenal, (E)-2-hexenal, styrene, 2-ethyl-pyridine, (E)-2-(2-pentenyl)furan, (E)-2-heptenal, indane, 3-ethyl-pyridine, 3-methyl-1-hexanol, 2-pentyl-thiophene, (E,E)-2,4-heptadienal, 3-hydroxy-1-methyl-2-thiazolinone, 1-hydroxy-2-methyl-3 ... , 5-octadien-2-one, benzofuran, 5-vinyl-4-methyl-thiazole, 5-ethyl-2-furanaldehyde, ethyl benzoate, 4-isopropylbenzaldehyde, naphthalene, 2-phenyl-propenal, 2-hydroxyacetophenone, (E)-2-tridecanal, 1-indanone, 2-methyl-3-phenyl-2-propenal, 4-ethyl-phenol, 3-ethyl-phenol, coumarin, a total of 33 compounds, LNA relative to the white group down-regulated compounds include 2-propylfuran, camphene, 1-methyl-pyrrole, 2-hexyl-furan, 3-methyl-2 furanone, (E)-2-dodecanal, 2-pyrrolecarboxaldehyde, octanoic acid, 5-methyl-2-pyrrolecarboxaldehyde. In Figure 8 In (c), the total content of 33 upregulated compounds in the LNA group was 22.674 μg / kg, while that in the white group was 6.086 μg / kg, which was only 1 / 4 of that in the LNA group; Figure 8In (d), the white group had a higher total compound content of 0.954 μg / kg, while the LNA group had a content of 0.333 μg / kg, a difference of only 0.6 μg / kg. The difference in the content of volatile compounds detected by TF-SPME technology can prove the difference between the two groups of samples.

[0102] 2. Method stability

[0103] After the method was established, an internal standard compound (2-methyl-3-heptanone) was added to the sample as a basis for determining the stability of the method, e.g. Figure 9 As shown in the figure, the average peak area of ​​the internal standard in the samples is 1944092694, and the relative standard deviation (RSD) is 6%, indicating that the method is stable.

Claims

1. A method for determining meat flavor compounds using TF-SPME, comprising the following steps: 1) The collected fresh meat sample paste was weighed into a headspace bottle and subjected to thin film solid phase microextraction to obtain the corresponding TF-SPME sample; The extraction volume of the thin film solid phase microextraction is 1-5g, the temperature is 25-65°C, and the time is 60-70min; In step 1), the thin film solid phase microextraction process is as follows: the collected fresh meat sample is made into meat paste and weighed into a headspace bottle, heated in a metal bath at 25-165°C for 1-40 minutes, and after cooling, 1-50 μg / mL 2-methyl-3-heptanone methanol solution is added, and the thin film solid phase microextraction material is fixed in the form of a headspace using a holder, the lid is tightened, and incubated in a metal bath at 25-65°C for 60-70 minutes. After cooling, the surface is rinsed with 35°C warm water and wiped clean with lint-free paper, and the TF-SPME is loaded into a TDU thermal desorption tube for standby use; the material used for the thin film solid phase microextraction is PDMS / DVB; 2) subjecting the TF-SPME sample to gas chromatography-mass spectrometry to separate and identify meat flavor compounds; The gas chromatography uses a gas chromatograph equipped with an automatic sampling unit, a thermal desorption system and a cold trap system; The initial temperature of the thermal desorption system was 30°C, the heating rate was 300°C / min, the desorption temperature reached 240°C, and the temperature was maintained at 240°C for 8 minutes without splitting; The initial temperature of the cold trap system was -40°C, the heating rate was 10°C / s, the desorption temperature reached 240°C, the temperature was maintained at 240°C for 8 min, and the split ratio was 5:

1.

2. The method according to claim 1, characterized in that In step 2), the gas chromatography separation conditions are: Chromatographic column: VF-WAXms capillary column, 60 m × 0.25 mm × 0.25 μm; temperature program: initial temperature 40°C, hold for 2.0 min, increase to 230°C at 4°C / min, hold for 5 min; transfer line temperature 250°C; carrier gas: helium, carrier gas flow rate 1 mL / min; The mass spectrometry conditions are as follows: The electron bombardment ion source had an electron energy of 70 eV and an ion source temperature of 280°C; the transfer line temperature was 250°C; and the full scan mode was used with a mass scan range of 30–400 m / z.

3. A method for identifying characteristic flavor substances of flaxseed pork by measuring meat flavor compounds using the TF-SPME method of claim 1 or 2, comprising the following steps: 1) The flaxseed pork and regular white pork pastes were weighed into headspace vials, and thin film solid phase microextraction was performed to obtain TF-SPME samples of the flaxseed pork and regular white pork. 2) subjecting the flaxseed pork and conventional white pork TF-SPME samples to gas chromatography-mass spectrometry to separate and identify the flavor compounds therein; 3) The above flavor compounds were analyzed for major differential compounds to obtain the major differential compounds between the flaxseed pork and ordinary white pork; The major differential compounds include up-regulated compounds and down-regulated compounds; The up-regulating compounds include ethyl acetate, 2-ethyl-furan, 2,3-pentanedione, (E)-2-pentenal, 1-butanol, 1-penten-3-ol, 2-ethylthiophene, 2-ethyl-2-butenal, (E)-2-hexenal, styrene, 2-ethyl-pyridine, (E)-2-(2-pentenyl)furan, (E)-2-heptenal, indane, 3-ethyl-pyridine, 3-methyl-1-hexanol, 2-pentenal, Thiophene, (E,E)-2,4-heptadienal, 3,5-octadien-2-one, benzofuran, 5-vinyl-4-methyl-thiazole, 5-ethyl-2-furanaldehyde, ethyl benzoate, 4-isopropylbenzaldehyde, naphthalene, 2-phenyl-propenal, 2-hydroxyacetophenone, (E)-2-tridecanal, 1-indanone, 2-methyl-3-phenyl-2-propenal, 4-ethyl-phenol, 3-ethyl-phenol, coumarin; The down-regulating compounds include 2-propylfuran, camphene, 1-methyl-pyrrole, 2-hexyl-furan, 3-methyl-2-furanone, (E)-2-dodecanal, 2-pyrrolecarboxaldehyde, octanoic acid, and 5-methyl-2-pyrrolecarboxaldehyde; 4) Comparing the contents of the main different compounds in the flaxseed pork and ordinary white pork to confirm the differences in the characteristic flavor substances of the flaxseed pork.

4. The method according to claim 3, characterized in that In step 4), compared with the main different compounds in the flaxseed pork to be tested and the main different compounds in the ordinary white pork, the mass percentage of each up-regulated compound is increased by 1.99% to 16.45 times, the total mass percentage of the up-regulated compounds is increased by 3.5% to 4.25 times, and the mass percentage of each down-regulated compound is decreased by 23% to 79%, and the total mass percentage of the down-regulated compounds is decreased by 40% to 60%.

5. A method for authenticating flaxseed pork by measuring meat flavor compounds using the TF-SPME method of claim 1 or 2, comprising the following steps: 1) The fresh meat sample paste and ordinary white pork to be tested were weighed into headspace bottles respectively, and thin film solid phase microextraction was performed to obtain TF-SPME samples of the fresh meat sample paste and ordinary white pork to be tested; 2) subjecting the fresh meat sample puree and the common white pork TF-SPME sample to gas chromatography-mass spectrometry, respectively, to separate and identify flavor compounds in the fresh meat sample and the common white pork; 3) Principal component analysis was performed on the flavor compounds in the livestock and poultry meat samples and ordinary white pork to obtain the main differential compounds between the livestock and poultry meat samples and ordinary white pork; The major differential compounds include up-regulated compounds and down-regulated compounds; The up-regulating compounds include ethyl acetate, 2-ethyl-furan, 2,3-pentanedione, (E)-2-pentenal, 1-butanol, 1-penten-3-ol, 2-ethylthiophene, 2-ethyl-2-butenal, (E)-2-hexenal, styrene, 2-ethyl-pyridine, (E)-2-(2-pentenyl)furan, (E)-2-heptenal, indane, 3-ethyl-pyridine, 3-methyl-1-hexanol, 2-pentyl -thiophene, (E,E)-2,4-heptadienal, 3,5-octadien-2-one, benzofuran, 5-vinyl-4-methyl-thiazole, 5-ethyl-2-furanaldehyde, ethyl benzoate, 4-isopropylbenzaldehyde, naphthalene, 2-phenyl-propenal, 2-hydroxyacetophenone, (E)-2-tridecanal, 1-indanone, 2-methyl-3-phenyl-2-propenal, 4-ethyl-phenol, 3-ethyl-phenol, coumarin; The down-regulating compounds include 2-propylfuran, camphene, 1-methyl-pyrrole, 2-hexyl-furan, 3-methyl-2-furanone, (E)-2-dodecanal, 2-pyrrolecarboxaldehyde, octanoic acid, and 5-methyl-2-pyrrolecarboxaldehyde; 4) Comparing the contents of the major differential compounds in the livestock and poultry meat samples to be tested with the contents of the major differential compounds in known ordinary white pork to identify the authenticity of the flaxseed pork.

6. The method according to claim 5, characterized in that In step 4), if the mass percentage of each up-regulated compound in the tested livestock and poultry meat sample is increased by 1.99 to 16.45 times compared with the main different compounds in the ordinary white pork, the mass percentage of the up-regulated compounds is increased by 3.5 to 4.25 times, and the mass percentage of each down-regulated compound is decreased by 23% to 79%, and the total mass percentage of the down-regulated compounds is decreased by 40% to 60%, then the tested livestock and poultry meat sample is flaxseed pork; otherwise, it is ordinary white pork.

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