Method for determining meat flavor compounds by adopting TF-SPME

Abstract

The invention discloses a method for determining a meat flavor compound by adopting TF-SPME. The method for determining the meat flavor compounds by adopting TF-SPME comprises the following steps: 1) weighing meat paste of a collected fresh meat sample, putting the meat paste into a headspace bottle, and carrying out thin-film solid-phase microextraction to obtain a TF-SPME sample; and 2) carrying out gas chromatography-mass spectrometry determination on the TF-SPME sample so as to separate and identify the meat flavor compound. According to the invention, more comprehensive components and more accurate contents of odor compounds in meat samples can be realized.

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 important part of human food, providing a variety of nutrients for the human body. Meat quality evaluation mainly includes four aspects: safety quality, nutritional quality, edible quality and processing quality. Among them, edible quality is an important indicator that affects consumers' product selection. For example, in the study of Nachomkamon Saengsuk, the main differences in protein, fat, lipid and flavor between the five parts of sheep shoulders, ribs, waists, breasts and legs were discussed in detail, as well as the potential impact of these indicators on the overall flavor, so that consumers can choose nutritious or appropriate parts for cooking. The edible quality of food includes texture characteristics, flavor quality and taste quality. In recent years, the widespread application of flavor detection and analysis technology has enabled traditional sensory evaluation to enter a stage of combining with instrumental analysis and evaluation, promoting the rapid development of gas chromatography-mass spectrometry (GC-MS), gas chromatography ion mobility spectrometry (GC-IMS), electronic nose technology (E-nose) and other technologies, and derived gas chromatography odor olfactory combination technology (GC-O), comprehensive two-dimensional gas chromatography technology (GC*GC), etc. 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 influence 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 greater impact on the overall flavor were identified.

[0003] With the gradual deepening of meat flavor research, the enrichment and extraction of volatile compounds have become particularly important, which has a significant impact on the final test results. According to the different enrichment stages, the enrichment methods can be divided into gas phase enrichment (static headspace SHS and dynamic headspace DHS), liquid phase enrichment (solvent extraction SEP, solvent-assisted evaporation extraction SAFE) and solid phase enrichment (solid phase microextraction SPME, thin film solid phase microextraction TF-SPME, stirring rod solid phase extraction SBSE). These enrichment methods have their own advantages and disadvantages, and are greatly affected by the state of the sample matrix. In the detection of different samples, their specific scope of use needs to be considered. For example, liquid samples such as beverages and wines are more suitable for enrichment with 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 too much interference under low temperature and high pressure conditions and can reflect sample information more accurately, but its disadvantages are also obvious. It requires the use of a large amount of organic reagents, the processing process is complicated and 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, thereby realizing trace-level compound detection. However, due to the limitations of its manufacturing process, coating type and theoretical volume, 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 main influencing factors of pretreatment are 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 of extraction temperature will intensify molecular motion and increase the adsorption coefficient of volatile compounds, but too high temperature may cause denaturation of analytes and affect the authenticity of detection. Usually, 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 are optimized in the present invention, namely, injection port desorption temperature (230-270°C), desorption time (4-20 min) and the initial temperature of the cold trap (-20~120℃). The first two conditions will affect the desorption of TF-SPME enriched compounds and are important conditions for whether all enriched compounds can be transferred to the injection system. The initial temperature of the cold trap is the last step before the compound enters the chromatographic analysis and is an important part of solving the asynchronous vaporization of the compound. By optimizing these parameters, we hope to obtain the best detection conditions of TF-SPME in meat flavor compounds, providing experimental basic conditions for subsequent flavor research.

[0006] The present invention provides 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 a TF-SPME sample; 2) subjecting the TF-SPME sample to gas chromatography-mass spectrometry to separate and identify meat flavor compounds.

[0007] 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.

[0008] 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.

[0009] In the above method, in step 1), the process of the thin film solid phase microextraction is as follows: the collected fresh meat sample is made into a meat paste sample and weighed into a headspace bottle, heated in a 25~165℃ metal bath for 1~40 min (specifically, it can be heated in a 55±1℃ metal bath 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) is added, and the thin film solid phase microextraction material is fixed in the form of headspace using a fixture, the lid is tightened, and incubated in a 25~165℃ (specifically, it can be 55±1℃) metal bath for 1~80 min (specifically, it can be 55~65 min or 60min), and after cooling, it is loaded into a TDU thermal desorption tube for use.

[0010] 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; 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; 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.

[0011] 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 of 240°C is maintained for 8 min, and there is no splitting mode; 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 min, and the split ratio can be 5:1.

[0012] In the above method, 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 is helium, and the carrier gas flow rate can be 1 mL / min.

[0013] In the above method, in step 2), 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. The full scan mode was adopted, and the mass scan range was 30-400 m / z.

[0014] The present invention also provides a method for identifying characteristic flavor substances of flaxseed pork based on the determination of meat flavor compounds by TF-SPME, comprising the following steps: 1) The meat pastes of the flaxseed pork 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 flaxseed pork and ordinary white pork to be tested; 2) subjecting the flaxseed pork and common white pork TF-SPME samples to be tested to gas chromatography-mass spectrometry to separate and identify the flavor compounds therein; 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 to be tested; 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.

[0015] In the above method, the major difference compounds include up-regulated compounds and down-regulated compounds; The up-regulated 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.

[0016] In the above method, in step 4), the mass percentage of each up-regulated compound in the main difference compounds in the livestock and poultry meat sample to be tested is increased by 1.99~16.45 times compared with the main difference compounds in the ordinary white pork, and the total mass percentage of the up-regulated compounds is increased by 3.5~4.25 times, and the mass percentage of each down-regulated compound is decreased by 23%~79%, and the total mass percentage of the down-regulated compounds is decreased by 40%~60%.

[0017] The present invention further provides a method for authenticating flaxseed pork based on the determination of meat flavor compounds using TF-SPME, comprising the following steps: 1) The fresh meat sample puree and ordinary white pork to be tested are weighed into headspace bottles respectively, and thin film solid phase microextraction is performed to obtain TF-SPME samples of the fresh meat sample puree and ordinary white pork to be tested; 2) subjecting the fresh meat sample to be tested and the common white pork TF-SPME sample to gas chromatography-mass spectrometry, respectively, to separate and identify the flavor compounds in the fresh meat sample to be tested and the common white pork; 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 difference compounds in the livestock and poultry meat samples to be tested and ordinary white pork; 4) Compare the contents of the main differential compounds in the livestock and poultry meat samples to be tested with the contents of the main differential compounds in known ordinary white pork to identify the authenticity of the flaxseed pork.

[0018] In the above method, the major difference compounds include up-regulated compounds and down-regulated compounds; The up-regulated 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.

[0019] In the above method, in step 4), compared with the main difference compounds in the common white pork, the mass percentage of each up-regulated compound in the livestock and poultry meat sample to be tested is increased by 1.99~16.45 times, the total mass percentage of the up-regulated compounds is increased by 3.5~4.25 times, and the mass percentage of each down-regulated compound is decreased by 23%~79%, and the total mass percentage of the down-regulated compounds is decreased by 40%~60%, then the livestock and poultry meat sample to be tested is flaxseed pork; otherwise, it is common white pork.

[0020] The present invention has the following beneficial effects: The present invention establishes a odor compound enrichment and desorption technology based on TF-SPME. Compared with the traditional SPME technology, this technology has a larger theoretical volume of the adsorption material, which reduces competitive adsorption. In the study, a variety of esters and lactone compounds that SPME failed to detect were extracted, making it possible to achieve a more comprehensive composition of odor compounds in meat samples and more accurate content. In the enrichment stage, multiple groups of samples can be operated simultaneously to achieve high throughput and greatly save time; in the thermal desorption process, the "thermal desorption-cold enrichment" two-step method is applied, which makes the detection of compounds more stable and reproducible. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the effect of sample amount on volatile compounds in pretreatment.

[0022] Figure 2 This is the effect of extraction temperature on volatile compounds during pretreatment.

[0023] Figure 3 This is the effect of extraction time on volatile compounds in pretreatment.

[0024] Figure 4 This is the effect of thermal desorption temperature on volatile compounds in instrumental conditions.

[0025] Figure 5 This is the effect of thermal desorption time on volatile compounds in instrument conditions.

[0026] Figure 6 This is the effect of cold trap temperature on volatile compounds.

[0027] Figure 7 Heat map and volcano map of the differences between SPME and TF-SPME. Figure 7 (a) is a total peak area comparison chart, (b) is a compound difference heat map, and (c) is a volcano map.

[0028] Figure 8 It is the principal component analysis PCA of volatile compounds and the volcano plot. Figure 8(a) is principal component analysis PCA, (b) is LNA / White difference volcano plot, (c) is LNA / White up-regulated compound comparison chart, and (d) is LNA / White down-regulated compound comparison chart.

[0029] Fig. 9 To test the stability of the method. DETAILED DESCRIPTION

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1 1. Materials and reagents Chromatographic grade methanol, acetonitrile and ultrapure water. 2-Methyl-3-heptanone and normal alkanes (C7-C40) were purchased from Shanghai Sigma. TF-SPME film (PDMS / DVB) (film thickness: 450 μm) and Tenax TA™ liner were purchased from Zestek, Germany; SPME extraction needle (DVB / CAR / PDMS) was purchased from Supelco.

[0033] 2. Instruments and equipment Trace 1310 gas chromatograph combined with Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Germany). The automatic injection unit (MPS), thermal desorption system (TDU-2) and cold trap system (CIS-4) were from Zestek, Germany. The metal bath equipment was made in the laboratory.

[0034] 3. Sample pretreatment The pork samples collected from the flaxseed pig group (denoted as the LNA group) and the common white pig (denoted as the white group) were provided by the production enterprises and met the national standards for pig slaughtering. The collected fresh meat samples were pre-cooled with liquid nitrogen, transported with dry ice, and stored in a -80℃ ultra-low temperature refrigerator in the laboratory. They were thawed at 4℃ for 2 h before the experiment, and after removing the surface fat and intermuscular fascia, they were broken into meat paste.

[0035] 3.1 TF-SPME sample pretreatment and extraction single factor experiment The sample pretreatment method optimized the three experimental conditions of 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 55℃ and 60 min respectively. When investigating the effect of extraction temperature, the sample mass and extraction time were 3 g and 60 min respectively. When investigating the effect of extraction time, the sample mass and extraction temperature were 3 g and 55℃ respectively.

[0036] 3.2 Single factor test of TF-SPME volatile compounds instrument conditions 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 the 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 the TDU desorption time were selected as 250℃ and 8 min respectively.

[0037] 3.3 Comparison of SPME and TF-SPME extraction methods The experimental conditions finally determined based on TF-SPME sample pretreatment and instrument conditions were compared with the SPME method.

[0038] 4. GC-MS experimental method 4.1 SPME sample pretreatment and extraction conditions The fresh meat paste sample was placed in a cooking bag in an 80℃ water bath for 30 min, then taken out and cooled naturally, 3.000±0.0001 g of uniform sample was weighed and placed in a brown headspace bottle, and 10 μL of 5 μg / mL 2-methyl-3-heptanone methanol solution was added. The sample was incubated at 55℃ for 20 min, extracted with a DVB / CAR / PDMS (50 / 30 μm, Supelco) fiber head at 55℃ for 40 min, and desorbed at the injection port of the autosampler for 3 min.

[0039] 4.2 TF-SPME sample pretreatment and extraction conditions Weigh 3.000±0.0001 g of uniform sample from the fresh meat paste sample and place it in a brown headspace bottle, add 10 μL of 5 μg / mL 2-methyl-3-heptanone methanol solution, heat it in a 55°C metal bath for 30 min, and after cooling, use a fixture to fix the TF-SPME in the form of headspace, tighten the lid and incubate it in a 55°C metal bath for 60 min. After cooling, take out the TF-SPME, wash it with 35°C warm water, wipe the surface with dust-free paper, and put it into the sample injection tube for detection on the machine.

[0040] 4.3 Thermal desorption system TDU conditions 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 min, 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 min, and the split ratio was 5:1.

[0041] 4.4 GC conditions 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 is high-purity helium (purity >99.999%), and the carrier gas flow rate is 1 mL / min.

[0042] 4.5 MS conditions Electron impact (EI) ion source, electron energy 70 eV, ion source temperature 280 °C; transfer line temperature 250 °C; full scan mode, mass scan range 30-400 m / z.

[0043] 5. Results and Analysis 5.1 Single factor experiment of sample pretreatment 5.1.1 Effect of sample amount on extracted compounds like Figure 1 As shown in the figure, when the sample weight is 3 g, the total peak area of ​​the detected compounds reaches the highest value. Further increasing the sample weight has no significant difference on the total peak area. On the contrary, due to its high water content, the adsorption efficiency of some compounds is affected. Aldehydes, ketones, alcohols, S-containing and N-containing compounds all show an increasing trend with the increase of sample weight, but the sample weights of 4 g and 5 g do not show significant differences from 3 g. The peak area of ​​ester compounds is the largest at 3 g. Taking all factors into consideration, the sample weight of 3 g can not only ensure the high efficiency of sample detection of compounds, but also save some samples.

[0044] 5.1.2 Effect of extraction temperature on extracted compounds The extraction temperature was set from 25℃ to 65℃ in a single factor test with a 10℃ difference based on previous experience. Figure 2 As shown, 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.

[0045] 5.1.3 Effect of extraction time on extracted compounds 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.

[0046] In this group of experiments, three factors that have a greater impact on the experimental results during sample pretreatment were investigated, namely sample amount, extraction temperature and extraction time. Each of them showed different rules. By integrating their respective experimental results, a TF-SPME experimental method with a meat sample amount of 3 g, an extraction temperature of 55°C and an extraction time of 60 min was established.

[0047] 5.2 Single-factor experiment with instrument conditions 5.2.1 TDU desorption temperature As the sampling device for TF-SPME adsorption materials to enrich the volatile compounds in the sample, 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°C. 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, five desorption temperatures were set from 230-270°C. 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.

[0048] 5.2.2 TDU desorption time exist Figure 5The figure shows the effect of the desorption time of the TF film in the high temperature chamber on various types of compounds. In terms of total peak area, there is no significant difference between the groups. The 8 min group has the highest peak area, and the aldehyde and alcohol compounds are also the highest in the 8 min group. In the groups longer than 8 min, such as aldehydes at 16 min, alcohols at 20 min, SN-containing compounds at 20 min, and esters at 12, 16, and 20 min, they are significantly different from other groups. In this group of experiments, 8 min is a better choice.

[0049] 5.2.3 CIS cold trap temperature 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 this group of experiments, the total peak area at -40℃ and the peak areas of other types of compounds are significantly different from those at other temperatures. Therefore, -40℃ is a better condition for selecting the cold trap temperature.

[0050] 5.3 Comparison of SPME and TF-SPME techniques 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 it, indicating that under their own optimal experimental conditions, different pretreatment technologies have different enrichment efficiencies for volatile compounds in the same sample. TF-SPME has a higher compound solvent due to its larger surface area, which also enables it to have a detection capability far beyond that of traditional SPME. However, due to the imperfect way of bonding 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 in the upper right part, nearly 2 / 3 of the compounds show that TF-SPME technology is better than SPME. 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. The compounds with more TF-SPME include 2-acetyl-thiazole, dimethyl sulfoxide, formic acid and other compounds, and the compounds with more SPME include 3-methyl-butyric acid, 2-octanone, indane and other compounds, which can be used as differential compounds to distinguish the two technologies.

[0051] In order to determine the stability of the pretreatment technology and the instrument, the internal standard added to the sample was tested, and the overall RSD was 6%, which is generally better than the results of using SPME. In addition, the detection time of the two technologies, SPME and TF-SPME, was also investigated. 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 balanced in the incubator for 20 minutes, and then inserted into the extraction head for extraction 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.

[0052] Example 2 Comparison between normal white pigs and flaxseed pigs 1. Volatile compounds In the present 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.

[0053]

[0054] 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.

[0055] 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 derivatives, 21 furans, 4 terpenes, and 2 others.

[0056] 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%, PC2 was 14%, and the flavor composition of the two groups of pork was effectively distinguished. Figure 8The 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, and 3-hydroxy-1-methyl-2-thiophene. ,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, and coumarin, a total of 33 compounds. The compounds downregulated by LNA relative to the white group 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. 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 8 In (d), the total content of the highest compound in the white group is 0.954 μg / kg, while that in the LNA group is 0.333 μg / kg, with 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.

[0057] 2. Method stability 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. Fig. 9 As shown, the average peak area of ​​the internal standard in the sample is 1944092694, and the relative standard deviation (RSD) is 6%, indicating good method stability.

Claims

1. A method for determining meat flavor compounds using TF-SPME, comprising the following steps: 1) Weigh the collected fresh meat sample paste into a headspace bottle, perform thin film solid phase microextraction, and obtain the TF-SPME sample corresponding to the sample; 2) subjecting the TF-SPME sample to gas chromatography-mass spectrometry to separate and identify meat flavor compounds.

2. The method according to claim 1, characterized in that In step 1), the thin film solid phase microextraction has an extraction amount of 1 to 5 g, a temperature of 25 to 165° C., and a time of 1 to 70 min; 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 to 165° C. for 1 to 40 min, and after cooling, 1 to 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 headspace using a fixture, the lid is tightened and incubated in a metal bath at 25 to 165° C. for 1 to 80 min, and after cooling, the surface is rinsed with 35° C. warm water and wiped clean with dust-free paper, and the TF-SPME is loaded into the TDU thermal desorption tube for standby use.

3. The method according to claim 1 or 2, characterized in that: The gas chromatography uses a gas chromatograph equipped with an automatic sampling unit, a thermal desorption system and a cold trap system; The desorption temperature of the thermal desorption system is 10 to 350° C., the heating rate is 1 to 720° C. / min, the desorption time is 1 to 20 min, and the split ratio is splitless to 100:1; The enrichment temperature of the cold trap system is -150 to 0°C, and the heating rate is 1 to 12°C / s; The initial temperature of the thermal desorption system is 30°C, the heating rate is 300°C / min, the desorption temperature is 240°C, the temperature is maintained at 240°C for 8 minutes, and there is no split mode; The initial temperature of the cold trap system 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.

4. The method according to any one of claims 1 to 3, characterized in that In step 2), the gas chromatography separation conditions are: Chromatographic column: VF-WAXms capillary column, 60m×0.25mm×0.25μm; temperature program: initial temperature 40℃, hold for 2.0min, increase to 230℃ at 4℃ / min, hold for 5min; transfer line temperature 250℃; carrier gas is helium, carrier gas flow rate is 1mL / 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; the full scan mode was adopted, and the mass scan range was 30-400 m / z.

5. 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: 1) The flaxseed pork and ordinary white pork pastes to be tested were weighed into headspace bottles respectively, and thin film solid phase microextraction was performed to obtain TF-SPME samples of the flaxseed pork and ordinary white pork to be tested; 2) subjecting the flaxseed pork and common white pork TF-SPME samples to be tested to gas chromatography-mass spectrometry to separate and identify the flavor compounds therein; 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 to be tested; 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.

6. The method according to claim 5, characterized in that The major differential compounds include up-regulated compounds and down-regulated compounds; The up-regulated 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.

7. The method according to claim 6, characterized in that In step 4), compared with the main difference compounds in the livestock and poultry meat samples to be tested and the main difference 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%.

8. A method for authenticating flaxseed pork based on the determination of meat flavor compounds using TF-SPME, comprising the following steps: 1) The fresh meat sample paste and ordinary white pork to be tested are weighed into headspace bottles respectively, and thin film solid phase microextraction is 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 to be tested and the common white pork TF-SPME sample to gas chromatography-mass spectrometry, respectively, to separate and identify the flavor compounds in the fresh meat sample to be tested and the common white pork; 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 difference compounds in the livestock and poultry meat samples to be tested and ordinary white pork; 4) Compare the contents of the main differential compounds in the livestock and poultry meat samples to be tested with the contents of the main differential compounds in known ordinary white pork to identify the authenticity of the flaxseed pork.

9. The method according to claim 8, characterized in that The major differential compounds include up-regulated compounds and down-regulated compounds; The up-regulated 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.

10. The method according to claim 9, 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 difference compounds in the ordinary white pork, 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%, then the tested livestock and poultry meat sample is flaxseed pork; otherwise, it is ordinary white pork.

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