Metabolic marker for evaluating pork freshness and application thereof

Abstract

The invention relates to the technical field of food identification, and discloses a metabolic marker for evaluating pork freshness and application thereof. According to the invention, a metabolic marker for evaluating pork freshness is screened through metabolome analysis, and the metabolic marker comprises 6-methylquinoline and / or N-acetylputrescine. On the basis, the invention also provides a method for evaluating the pork freshness, which comprises the following steps: acquiring a pork sample to be detected, and detecting the content of metabolic markers (6-methylquinoline and / or N-acetylputrescine) in the pork sample to be detected, and evaluating the freshness of the pork sample to be detected according to the metabolic marker dynamic change trend line of the standard sample. The screened metabolic marker can be used for rapidly and accurately evaluating the pork freshness and effectively indicating the quality change of the pork in the storage process, and is beneficial to standardizing pre-management of food safety risks.

Description

Technical Field

[0001] The invention relates to the technical field of food identification, and in particular to metabolic markers for evaluating pork freshness and applications thereof. Background Art

[0002] Pork is an important animal food in people's daily life. my country is currently the country with the largest pork consumption, and its pig production and pork imports are among the highest in the world. The freshness of pork is an important indicator of pork quality. As pork deteriorates, its freshness decreases. Relevant studies have shown that pork deterioration begins after pigs are slaughtered. Active biological processes occur in the cells of pigs after slaughter. For example, the various proteases and lipases contained in pork still maintain a certain activity and continue to play a role in decomposing proteins and lipids in pork, while producing peptides, free amino acids and fatty acids, resulting in loss of quality and flavor. In addition, during the transportation, storage, processing and consumption of pork, environmental factors such as storage conditions and storage time, as well as its own factors, will also cause its freshness to decrease to varying degrees, and even cause corruption and deterioration, produce toxic and harmful chemical products, and increase safety risks.

[0003] Among the related technologies, how to quickly and accurately measure the freshness of pork has become an urgent problem to be solved. At present, traditional pork freshness evaluation is mostly concentrated on sensory testing, physical and chemical index determination, microbiological index testing and other methods. However, most of these methods have obvious shortcomings, such as strong subjectivity, long detection time, insufficient accuracy, and difficulty in measurement. Therefore, it is urgent to explore key metabolic markers related to fresh meat deterioration, and develop rapid, sensitive, and reliable real-time detection technologies to measure the deterioration of fresh meat, thereby improving the accuracy of individual selection, early identification and exclusion of high-risk pork, reducing possible product recall costs and brand reputation losses, and providing effective technical support and decision-making guidance for the pork industry chain in the fields of food safety management and risk prevention and control.

[0004] Based on this, the present invention has discovered an efficient metabolic marker through metabolome analysis, which can be used to quickly and accurately evaluate the freshness of pork, which has important theoretical significance and economic value for reducing the cost of pork freshness evaluation and improving the accuracy of evaluation. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a metabolic marker for evaluating the freshness of pork, which can quickly and accurately evaluate the freshness of pork, and the evaluation method is simple and low-cost.

[0006] The present invention also proposes an application of a product for detecting the above-mentioned metabolic markers for evaluating pork freshness in evaluating meat freshness or preparing a product for evaluating meat freshness.

[0007] The invention also provides a test kit for evaluating the freshness of pork.

[0008] The invention also provides a method for evaluating the freshness of pork.

[0009] The present invention also provides a method for screening metabolic markers for evaluating pork freshness as described above.

[0010] The present invention also proposes an application of the above-mentioned metabolic marker for evaluating pork freshness in constructing a pork freshness evaluation model, evaluating meat freshness or preparing a product for evaluating meat freshness.

[0011] In a first aspect of the present invention, a metabolic marker for evaluating the freshness of pork is provided, wherein the metabolic marker comprises 6-methylquinoline and / or N-acetylputrescine.

[0012] The metabolic markers according to the embodiments of the present invention have at least the following beneficial effects:

[0013] (1) The present invention uses ultra-performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) to separate and qualitatively and quantitatively analyze the total metabolites of pork, and then uses principal component analysis (PCA) to perform discriminant analysis based on the metabolites to screen differential metabolites, and then uses orthogonal partial least squares-discriminant analysis (OPLS-DA) to discriminate the metabolites obtained, and selects metabolites with variable importance in projection (VIP) greater than or equal to 1. At the same time, the statistical significance of the difference is evaluated by T test (p<0.05), and the final key metabolites (6-methylquinoline and N-acetylputrescine) are confirmed in combination with fold change analysis (FC>2 or FC<0.5). These two metabolites are closely related to microbial activity and putrescine metabolism, respectively, and can effectively indicate the quality changes of pork during storage.

[0014] (2) Assessing pork freshness based on the metabolic markers of the present invention helps to improve the accuracy of individual selection and provides an effective technical solution for improving the efficiency of pork freshness detection in the market. At the same time, it can also provide a scientific judgment basis for the pork industry chain in raw material procurement, processing and quality control, and help standardize the pre-emptive management of food safety risks.

[0015] In some embodiments of the present invention, the metabolic markers include 6-methylquinoline and N-acetylputrescine.

[0016] In some embodiments of the present invention, the metabolic marker further comprises at least one of isopentylamine, DL-isoleucine, formylhydantoin, acetophenone, 1,3-propylenediamine, N,N-dimethylacrylamide, and N-acetyl-L-phenylpropionic acid ethyl ester.

[0017] The second aspect of the present invention provides a use of a product for detecting the metabolic markers for evaluating pork freshness described in the first aspect in evaluating meat freshness or preparing a product for evaluating meat freshness.

[0018] The application according to the embodiment of the present invention has at least the following beneficial effects: the metabolic markers screened by the present invention for evaluating the freshness of pork have good identification accuracy,

[0019] In some embodiments of the invention, the meat comprises pork.

[0020] In some embodiments of the present invention, the pork includes pork tenderloin, pork belly or pork hind leg.

[0021] The third aspect of the present invention provides a kit for evaluating the freshness of pork, the kit comprising a reagent for detecting the metabolic marker for evaluating the freshness of pork as described in the first aspect.

[0022] A fourth aspect of the present invention provides a method for evaluating the freshness of pork, comprising:

[0023] Obtain a pork sample to be tested, detect the content of the metabolic marker for evaluating the freshness of pork as described in the first aspect in the pork sample to be tested, and evaluate the freshness of the pork sample to be tested based on the dynamic change trend line of the metabolic marker of the standard sample.

[0024] In some embodiments of the present invention, the method for detecting the content of metabolic markers for evaluating the freshness of pork includes ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry detection.

[0025] In some embodiments of the present invention, the pork sample to be tested includes pork tenderloin, pork belly or pork hind leg.

[0026] The fifth aspect of the present invention provides a method for screening metabolic markers for evaluating pork freshness according to the first aspect, comprising:

[0027] S1. Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry was used to separate and qualitatively and quantitatively analyze the total metabolites of pork;

[0028] S2. The principal component analysis method was used to screen the differential metabolites, and then the orthogonal partial least squares method was used to analyze and select the differential metabolites with variable importance projection values ​​greater than or equal to 1. The statistical significance of the differences was evaluated by T test.

[0029] S3. Combine fold change analysis to obtain metabolic markers for evaluating pork freshness.

[0030] The screening according to the embodiment of the present invention has at least the following beneficial effects: the metabolic markers (6-methylquinoline and N-acetylputrescine) for evaluating the freshness of pork obtained by the screening method of the present invention are closely related to microbial activity and putrescine metabolism, and can effectively indicate the quality changes of pork during storage.

[0031] In some embodiments of the present invention, the T-test p<0.05.

[0032] In some embodiments of the invention, the fold change analysis is FC>2 or FC<0.5.

[0033] The sixth aspect of the present invention provides the use of the metabolic marker for evaluating pork freshness as described in the first aspect in constructing a pork freshness evaluation model, evaluating meat freshness, or preparing a product for evaluating meat freshness.

[0034] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0036] Figure 1 This is a schematic diagram of pork sample processing according to the present invention;

[0037] Figure 2 This is a schematic diagram of the lipid extraction process of pork samples of the present invention;

[0038] Figure 3 Schematic diagram of PCA and OPLS-DA analysis results of pork tenderloin samples under different storage conditions of the present invention, wherein A is the PCA analysis result, B is the OPLS-DA analysis result, and C is the PCA cross-validation result;

[0039] Figure 4 Schematic diagram of PCA and OPLS-DA analysis results of pork belly samples under different storage conditions of the present invention, wherein A is the PCA analysis result, B is the OPLS-DA analysis result, and C is the PCA cross-validation result;

[0040] Figure 5 Schematic diagram of PCA and OPLS-DA analysis results of pork hind leg samples under different storage conditions of the present invention, wherein A is the PCA analysis result, B is the OPLS-DA analysis result, and C is the PCA cross-validation result;

[0041] Figure 6 VIP value screening and differential metabolite distribution of pork samples from different parts of the present invention, wherein A is the VIP value screening result, and B is the Venn diagram;

[0042] Figure 7 This is a schematic diagram of the analysis of the effects of different storage times and temperatures on changes in pork metabolites according to the present invention;

[0043] Figure 8 The secondary fragment information of the key differential metabolite 6-methylquinoline of the present invention;

[0044] Fig. 9 The secondary fragment information of the key differential metabolite N-acetylputrescine of the present invention;

[0045] Fig.10 Schematic diagram of the dynamic change of differential metabolism of the markers of the present invention, wherein A is 6-methylquinoline and B is N-acetylputrescine;

[0046] Fig.11 The freshness identification results of blind sample 1 and blind sample 2 of the present invention are shown in FIG. 1 , wherein a is 6-methylquinoline and b is N-acetylputrescine. DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0048] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0049] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0050] Example 1: Screening of metabolic markers related to pork freshness

[0051] In this embodiment, ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry is used to separate and qualitatively and quantitatively analyze the total metabolites of pork, and the principal component analysis (PCA) method and orthogonal partial least squares method (OPLS-DA) are combined to perform metabolite screening and discriminant analysis to obtain metabolic markers related to pork freshness. The specific method includes the following content.

[0052] 1. Experimental Methods

[0053] 1. Sample collection and metabolomics pre-treatment:

[0054] (1) Sample collection:

[0055] Fresh pork samples with different fat contents (including back fat, leg meat and pork belly) were purchased from the local pork market. The fat content of back fat was 8%, the fat content of leg meat was 30%, and the fat content of pork belly was 60%. After being cut in the laboratory, they were placed in polyethylene self-sealing bags and stored at -20℃ (frozen), 4℃ (refrigerated), and 40℃ (high temperature storage). The specific process diagram is shown in the figure below. Figure 1 shown.

[0056] (2) Lipid extraction pretreatment:

[0057] Weigh 5 g of sample, grind it by freezing, and then extract it with 20 mL of methanol-acetonitrile-water (4:1:1, v:v:v). Perform low-temperature ultrasonic extraction for 30 min. Centrifuge for 10 min, take the supernatant and blow it to dryness with nitrogen. Redissolve it with 1 mL of acetonitrile-water (1:1, v:v) solution, centrifuge it for 5 min at low temperature (10000 r / min, 4°C), take the supernatant and filter it through a 0.22 μm organic needle filter membrane for testing. The specific process diagram is as follows Figure 2 shown.

[0058] 2. Metabolome mass spectrometry analysis:

[0059] The samples after the lipid extraction pretreatment were subjected to mass spectrometry analysis, specifically using a data acquisition instrument system, ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to achieve metabolite separation and data acquisition, and the specific relevant parameter settings are shown below.

[0060] (1) Setting chromatographic conditions:

[0061] 1) Chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm);

[0062] 2) Mobile phase: Phase A is ultrapure water (0.1% formic acid (V / V), phase B is methanol (0.1% formic acid (V / V));

[0063] 3) Flow rate 0.4 ml / min, column temperature 40 °C, injection volume 5 μl;

[0064] 4) Needle washing solution: isopropyl alcohol: water (1:9, V / V);

[0065] 5) Switching valve: between 0.5 and 12 minutes, the eluent is switched to the mass spectrometer detector, and before 0.5 minutes and after 12 minutes, the eluent is switched to waste liquid;

[0066] 6) Elution gradient: 0 min mobile phase A% / mobile phase B% (95:5, V / V), 0.5 min 95:5 (V / V), 10 min 0:100 (V / V), 12 min 0:100 (V / V), 12.5 min 95:5 (V / V), 15 min 95:5 (V / V);

[0067] (2) Set mass spectrometry conditions:

[0068] The Q Exactive series mass spectrometer Full MS-ddms2 mode was used to collect the primary and secondary spectra of the samples. Electrospray ionization (ESI) positive ion and negative ion modes were used for detection. Full MS-ddms2 data scanning mode was used, and the positive / negative modes were scanned once each to obtain the total ion current chromatogram of each mixed standard working solution.

[0069] Mass scan range m / z: 100-1500, ion spray voltage ±3500V, sheath gas 45psi, auxiliary heating gas 15psi, auxiliary heating temperature 350℃, ion source heating temperature 350℃, 10-20-30V cycle collision energy, MS1 resolution 70000, MS2 resolution 17500, vertex excitation 1-4s, target ion number (AGC target) 1e6 .

[0070] 3. Multidimensional data analysis and differential metabolite screening:

[0071] Xcalibur 3.0 software was used to perform chromatographic peak extraction and peak alignment on the mass spectrometry data collected above, and then the mzCloud database was used for metabolite identification. 8928 and 7165 metabolites were identified in the positive ion mode and negative ion mode, respectively.

[0072] The data were then imported into SIMCA17.1 for chemometric analysis, and principal component analysis (PCA) was used for dimensionality reduction to explore the distribution characteristics between samples, and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to screen out differential metabolites with VIP (variable importance in projection) values ​​greater than 1.0. The statistical significance of the differences was further evaluated by T test (p<0.05), and the final key metabolites were confirmed by combining fold change analysis (FC>2 or FC<0.5).

[0073] 2. Experimental Results

[0074] 1. PCA and OPLS-DA model analysis results:

[0075] The analysis results based on PCA and OPLS-DA models are as follows Figures 3 to 5 As shown, Figure 3 This is the analysis result of pork tenderloin. Figure 4 This is the analysis result of pork belly. Figure 5 The results of the analysis of hind leg meat show that there is a certain separation between the groups of samples in the PCA analysis, but there is some overlap, indicating that its distinguishing ability is limited; after further optimization through OPLS-DA, the groups of samples are completely separated, and the model stability and predictive ability are significantly enhanced. Cross-validation (Permutation Test) shows that the model does not have overfitting phenomenon and can be used to screen differential metabolites.

[0076] 2. Differential metabolite screening results:

[0077] Based on the above model analysis results, the metabolites of tenderloin, streaky pork and hind leg meat were further analyzed. The results are as follows Figure 6 As shown in A, metabolites in different parts have significant specificity and intersection. The VIP value distribution results show that the number of metabolites with VIP>1 varies between different parts. Among them, the pork belly sample has the most VIP>1 metabolites, which is 1237; followed by the hind leg meat, which is 1196; and the tenderloin has the least, which is 1106. The above results show that metabolites in different parts have significant specificity.

[0078] Furthermore, the metabolite intersection of the three samples of tenderloin, streaky pork and hind leg meat was statistically analyzed, and the Venn diagram is as follows: Figure 6 As shown in Figure B, there are 732 common differential metabolites between the three parts (tenderloin, pork belly, and hind leg meat), which are possible stability markers. In addition, pork belly has 247 specific metabolites, tenderloin and hind leg meat have 161 and 241, respectively. The site-specific distribution shows that the metabolic characteristics of different parts are obvious, which helps to distinguish the specific parts of pork during storage through metabolite analysis.

[0079] 3. Screening results of pork freshness metabolic markers:

[0080] PCA-X and OPLS-DA models were used to distinguish samples under different storage conditions (D0: initial sample; T4; T25; T40) and QC samples. Figure 7 As shown, the PCA results show ( Figure 7 A in the figure), the storage temperature significantly affects the metabolite distribution of the samples, and the samples at different temperatures are well separated in the principal component space (R 2 =0.861, Q2=0.478). The OPLS-DA model further optimized the separation of differences between groups (R 2 =0.811, Q2 =0.653), the effect of storage temperature on the abundance and type of metabolites is clearer (e.g. Figure 7 (as shown in B in the figure).

[0081] In addition, a permutation test was performed based on the above results, and the results are as follows Figure 7 As shown in C, the OPLS-DA model has strong predictive ability and no overfitting. 2 The values ​​of VIP>1, P<0.05, FC>2 or FC<0.5 were significantly higher than those of the replacement model, which verified the reliability of the analysis results. This result shows that storage temperature significantly affects the change pattern of pork metabolites, and the metabolic characteristics of samples at different temperatures can be effectively distinguished. By screening conditions (VIP>1, P<0.05, FC>2 or FC<0.5), several significantly different metabolites (such as N-acetylputrescine and 6-methylquinoline) were finally identified from samples under various storage conditions, as shown in Table 1:

[0082] Table 1: Differential metabolites

[0083]

[0084] In addition, the distribution of VIP values ​​showed that the contribution of differential metabolites was mainly concentrated in specific temperature groups (e.g. Figure 7 The changes in metabolite specificity and abundance at different temperatures further support the correlation between metabolite signatures and storage conditions (freshness).

[0085] Furthermore, based on the identification of multiple significant differential metabolites from the above screening, two key differential metabolites, N-acetylputrescine and 6-methylquinoline, were further screened, among which Figure 8 and Fig. 9 The mass spectrometric characteristics and chemical structures of the key differential metabolites N-acetylputrescine and 6-methylquinoline screened out in pork samples are shown.

[0086] N-acetylputrescine is a putrescine derivative, and its production process is related to protein degradation and amino acid decarboxylation. During pork storage, putrescine can be converted into N-acetylputrescine through transaminase reaction, and this metabolic process is closely related to the activity of microorganisms. The high abundance of N-acetylputrescine indicates that the sample has significant protein degradation and increased microbial activity under storage conditions. Through mass spectrometry detection, its ionization mode is [M+H] + , with a molecular weight of 131.11766 and clear fragment ion characteristics, supporting its potential as a spoilage marker.

[0087] 6-Methylquinoline is a biologically active metabolite with multiple effects such as antibacterial and antiviral effects. During storage, its generation is related to fat oxidation and amino acid degradation. Mass spectrometry results show that the ionization mode of 6-methylquinoline is [M+H]+, the molecular weight is 144.08043, and it has stable secondary fragment characteristics. This indicates that 6-methylquinoline may reflect the cumulative effects of fat oxidation and microbial activity during storage, and its concentration changes can be used as an indicator of storage temperature and time.

[0088] In addition, it can be analyzed from the above results that the retention time and secondary fragmentation spectra of the two metabolites show consistency, which further verifies their reliability as identification of pork freshness. In addition, in high-resolution mass spectrometry analysis, N-acetylputrescine and 6-methylquinoline as differential metabolites have clear detection signals and good repeatability, so they can be used as preferred marker candidates.

[0089] Example 2: Detection of the correlation between storage temperature and metabolic markers

[0090] This example detects the correlation between different storage temperatures and the content of freshness-related metabolic markers (N-acetylputrescine and 6-methylquinoline), and the specific experimental method is as follows:

[0091] Referring to the sample collection, metabolome pretreatment and detection methods of Example 1 above, different tenderloin, streaky pork and hind leg meat samples with different storage temperatures and storage times were tested, wherein the storage temperatures were set to 4°C (T4), 25°C (T25) and 40°C (T40), which corresponded to the low temperature group, medium temperature group and high temperature group, respectively; the storage time was set to 0 to 15 days. Samples were taken for testing every day during the experiment, with 3 replicates per group.

[0092] Test results such as Fig.10 As shown in the figure, it shows the concentration change rules of two key metabolic markers (6-methylquinoline and N-acetylputrescine) under different storage temperatures and times. Among them, the concentration of 6-methylquinoline shows a continuous increase under different temperature conditions with the increase of storage days, especially in the high temperature (T40) group. The concentration in the low temperature group (T4) changes little and increases slowly. The concentration of N-acetylputrescine increases rapidly in the early storage period (D0-D3), and then shows a continuous growth trend under high temperature conditions (T40), while the concentration changes tend to be stable under low temperature conditions (T4).

[0093] Specifically, the correlation test results between different storage temperatures and freshness-related metabolic markers showed that: under high temperature conditions (T40), in the three parts of tenderloin, pork belly and hind leg meat, the concentrations of metabolic markers increased rapidly and fluctuated greatly, indicating that high-temperature storage aggravated lipid oxidation and protein degradation reactions, leading to the massive production of 6-methylquinoline and N-acetylputrescine; under medium temperature conditions (T25), the growth trend of the concentration of metabolic markers was between T40 and T4, indicating that medium temperature storage had a certain promoting effect on the metabolic process, but did not reach the intensity of high temperature conditions; under low temperature conditions (T4), the concentration of metabolic markers changed little, indicating that low temperature can effectively inhibit the occurrence of deterioration metabolic reactions.

[0094] The results of correlation test between different pork parts and freshness-related metabolite markers showed that: in tenderloin, the production rate of 6-methylquinoline and N-acetylputrescine was slow and the concentration was low, which may be related to its low fat content and limited degradation substrates; in pork belly, the concentration of metabolite markers increased significantly, indicating that its higher fat content has a significant promoting effect on lipid oxidation and amino acid degradation reactions; in hind leg meat, the change in concentration of metabolite markers was between tenderloin and pork belly, reflecting the influence of moderate fat and protein content on metabolite production.

[0095] The correlation test results of different storage times and freshness-related metabolic markers showed that: in the early storage period (D0-D3), the concentration of metabolic markers increased rapidly, especially N-acetylputrescine, which may be related to the accelerated initial protein degradation and active amino acid decarboxylation reaction; in the middle and late storage period (D5-D15), the concentration of 6-methylquinoline increased significantly, indicating that lipid oxidation became the main metabolic pathway in the late storage period.

[0096] The above results show that 6-methylquinoline and N-acetylputrescine show obvious temperature and time dependence during pork storage, and are sensitive indicators of pork quality deterioration. Among them, low temperature can significantly inhibit the generation of deterioration-related metabolites (6-methylquinoline and N-acetylputrescine), indicating that their freshness is relatively higher (consistent with the expected results); secondly, with the extension of storage time, 6-methylquinoline and N-acetylputrescine increase to varying degrees, indicating that their freshness decreases (consistent with the expected results). It can be seen that the relevant metabolic markers screened by the present invention can be used for pork freshness evaluation. In addition, it can be analyzed from the above test results that due to certain differences in 6-methylquinoline and N-acetylputrescine in meat with different fat contents, when managing pork with different fat contents, a freshness evaluation system can be formulated according to the differences in the generation of metabolites of meat with different fat contents to achieve more accurate distinction.

[0097] Example 3: Real sample verification

[0098] In order to verify the feasibility of 6-methylquinoline and N-acetylputrescine as freshness-related metabolic markers, this example uses pork tenderloin as the sample to be tested, and the specific method is as follows:

[0099] Pork tenderloin samples (blind sample 1 and blind sample 2) were purchased from the market, wherein blind sample 1 was pork stored at a relatively high temperature (40°C), and blind sample 2 was pork stored under refrigeration (4°C). The pretreatment steps of Example 1, as well as the chromatographic and mass spectrometric identification conditions, were used, and mass spectrometric data were collected to perform peak detection, peak alignment, peak area normalization, and filtering operations. The pretreated data was imported into the dynamic change trend lines (such as those of Example 1) of 6-methylquinoline (a) and N-acetylputrescine (b) in pork tenderloin at different temperatures (40°C, 25°C, and 4°C). Fig.10 The trend line was used to identify the freshness of the blind sample 1 and the blind sample 2.

[0100] The identification results are as follows Fig.11 As shown, the dynamic change of 6-methylquinoline shows that the concentration range of blind sample 1 matches 40℃ and 25℃ days 1-2, and it is judged as a stale sample; the concentration range of blind sample 2 matches 40℃ and 25℃ days 0-1, and 4℃ days 3-5, which is at the critical point between fresh samples and stale samples. The dynamic change trend of N-acetylputrescine further verifies the above results, where the concentration of blind sample 1 matches 40℃ days 2-3 and 25℃ days 5-7, indicating that the sample is pork stored at 40℃ for about 2 days; blind sample 2 matches 40℃ days 0-1, 25℃ days 2, and 4℃ days 3-5, which confirms again that it is at the critical state between fresh samples and stale samples (i.e. pork stored at 4℃ for 3-5 days).

[0101] Further, the accuracy of the above verification results was judged, and the results were consistent with expectations, that is, blind sample 1 was pork that had been placed at a higher temperature (40°C) for about 48 hours without opening; blind sample 2 was fresh pork tenderloin purchased from a supermarket, which was pork that had been refrigerated at 4°C for about 96 hours without opening.

[0102] In summary, the present invention provides a metabolic marker for evaluating the freshness of pork and its application. First, the present invention uses ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to separate and qualitatively and quantitatively analyze the total metabolites of pork, and then uses principal component analysis (PCA) to perform discriminant analysis based on the metabolites to screen differential metabolites, and then uses orthogonal partial least squares method (OPLS-DA) to discriminate the metabolites obtained, select metabolites with variable importance projection values ​​greater than or equal to 1, and evaluate the statistical significance of the difference by T test (p<0.05), and confirm the final key metabolites (6-methylquinoline and N-acetylputrescine) in combination with fold change analysis (FC>2 or FC<0.5). These two metabolites are closely related to microbial activity and putrescine metabolism, respectively, and can effectively indicate the quality changes of pork during storage. Specific analysis shows that 6-methylquinoline has antibacterial and antiviral activities, and may have a certain inhibitory effect on microorganisms; while N-acetylputrescine is a key product of putrescine metabolism, which is closely related to amino acid metabolism and amine production. In the actual pork freshness assessment work, only one or two of the two metabolites of 6-methylquinoline and N-acetylputrescine need to be tested by the above metabolomics to quickly and efficiently indicate the freshness of pork, providing a scientific basis for pork quality evaluation and storage optimization.

[0103] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A metabolic marker for evaluating pork freshness, characterized in that: The metabolic markers include 6-methylquinoline and / or N-acetylputrescine.

2. The metabolic marker according to claim 1, characterized in that The metabolic marker further comprises at least one of isopentylamine, DL-isoleucine, formylhydantoin, acetophenone, 1,3-propylenediamine, N,N-dimethylacrylamide, and N-acetyl-L-phenylpropionic acid ethyl ester.

3. Use of a product for detecting the metabolic marker for evaluating pork freshness according to claim 1 or 2 in evaluating meat freshness or preparing a product for evaluating meat freshness.

4. The use according to claim 3, characterized in that: The meat includes pork.

5. A kit for evaluating the freshness of pork, characterized in that: The kit comprises a reagent for detecting the metabolic marker for evaluating the freshness of pork according to claim 1 or 2.

6. A method for evaluating the freshness of pork, characterized in that: include: Obtain a pork sample to be tested, detect the content of the metabolite marker for evaluating the freshness of pork as described in claim 1 or 2 in the pork sample to be tested, and evaluate the freshness of the pork sample to be tested based on the dynamic change trend line of the metabolite marker of the standard sample.

7. The method according to claim 6, characterized in that The method for detecting the content of metabolic markers for evaluating pork freshness comprises ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry detection.

8. The method according to claim 6, characterized in that The pork sample to be tested includes pork tenderloin, pork belly or pork hind leg meat.

9. A method for screening metabolic markers for evaluating pork freshness according to claim 1 or 2, characterized in that: include: S1. Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry was used to separate and qualitatively and quantitatively analyze the total metabolites of pork; S2. The principal component analysis method was used to screen the differential metabolites, and then the orthogonal partial least squares method was used to analyze and select the differential metabolites with variable importance projection values ​​greater than or equal to 1. The statistical significance of the differences was evaluated by T test. S3. Combine fold change analysis to obtain metabolic markers for evaluating pork freshness.

10. Use of the metabolic marker for evaluating pork freshness as claimed in claim 1 or 2 in constructing a pork freshness evaluation model, evaluating meat freshness or preparing a product for evaluating meat freshness.

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