Method for identifying different grades of perigord truffles and application

Abstract

The invention relates to a method for identifying different grades of perigord truffles and application, and the method comprises the following steps: carrying out sample treatment on the perigord truffles, detecting by adopting gas chromatography-ion mobility spectrometry, and then identifying, the identification standard is as follows: when the odor activity value of the isoamyl acetate is greater than or equal to 1200 and the odor activity value of the 1-octylene-3-alcohol is greater than or equal to 3000, the truffle is judged as first-grade truffle; when the odor activity value of (E)-2-octylene-1-alcohol is greater than or equal to 100 and greater than or equal to 3000, and the odor activity value of ethyl enanthate is greater than or equal to 40 and greater than or equal to 20, judging that the truffle is second-grade truffle; when the detection value of the linalool is greater than or equal to 400 and greater than or equal to 800, and the odor activity value of the benzoin is greater than or equal to 1.3, judging that the truffle is third-grade truffle. According to the method, volatile aroma components of different grades of perigord truffles are detected and analyzed, the concept of volatile organic compounds (VOCs) is introduced, key indexes for judging the VOCs of the different grades of perigord truffles are established, and the three grades of perigord truffles can be effectively distinguished.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a method for identifying different grades of black truffles and an application thereof. Background Art

[0002] Black truffle (Tuber indicum) is a valuable edible fungus, highly prized for its unique flavor and nutritional value. In China, particularly in Yunnan Province, black truffle production is abundant, making it a significant economic resource. However, the quality and grading of black truffles are crucial for their market value and applications. Traditionally, grading of black truffles relies primarily on external characteristics, such as fruiting body size, weight, and surface damage. While this method is simple and intuitive, it lacks an in-depth assessment of the intrinsic quality of black truffles, particularly the analysis of their flavor components.

[0003] The flavor characteristics of black truffles are primarily determined by their volatile organic compounds (VOCs). The types and content of these compounds affect the aroma and taste of black truffles. The volatile composition of black truffles varies significantly between different grades. Using advanced analytical techniques to qualitatively and quantitatively analyze VOCs in black truffles not only helps to reveal the mechanism of flavor formation but also provides a scientific basis for quality evaluation and grading of black truffles, promoting the standardization and regularization of the black truffle industry. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method and application for identifying different grades of black truffles. The present invention uses the content of volatile organic compounds to identify the grade of black truffles, providing a scientific basis for the quality evaluation and grade classification of black truffles, and promoting the standardization and normalization of the black truffle industry.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for identifying different grades of black truffles, the method comprising: processing the black truffle sample and then identifying the sample by gas chromatography-ion mobility spectrometry;

[0007] The identification criteria are:

[0008] When the odor activity value of isoamyl acetate is ≥1200 and the odor activity value of 1-octen-3-ol is ≥3000, it is judged to be a first-grade black truffle;

[0009] When 3000 ≥ (E)-2-octen-1-ol odor activity value ≥ 100 and 40 ≥ ethyl heptanoate odor activity value ≥ 20, it is judged to be a second-level black truffle;

[0010] When the detection value of linalool is 800 ≥ 400 and the odor activity value of benzoin aldehyde is ≥ 1.3, it is judged to be a third-grade black truffle.

[0011] The present invention uses volatile organic compounds as standard substances to effectively identify black truffles, providing a simple, rapid, and accurate method for identifying different grades of black truffles. This method not only helps reveal the mechanism of flavor formation but also provides a scientific basis for quality evaluation and grading of black truffles, promoting the standardization and regularization of the black truffle industry.

[0012] Preferably, the calculation formula for the odor activity value is:

[0013]

[0014] Wherein, OAV is the odor activity value, C is the content detected by gas chromatography-ion mobility spectrometry, the unit is μg / kg, and T is the aroma threshold of volatile organic compounds in water.

[0015] Preferably, the sample processing comprises placing the sample in a headspace vial, adding an internal standard for incubation, and then performing headspace extraction.

[0016] Preferably, the added amount of the sample is 0.5-3 g, for example, 0.5 g, 1 g, 1.5 g, 2 g, 2.5 g or 3 g.

[0017] Preferably, the internal standard comprises 2-methyl-3-heptanone.

[0018] Preferably, the mass ratio of the internal standard to the sample is (0.005-0.03) mg:1 g. The (0.005-0.03) mg can be, for example, 0.005 mg, 0.01 mg, 0.015 mg, 0.02 mg, 0.025 mg or 0.03, etc.

[0019] Preferably, the incubation temperature is 45-55°C and the incubation time is 10-20 minutes. The 45-55°C may be, for example, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C. The 10-20 minutes may be, for example, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, or 20 minutes.

[0020] Preferably, the headspace extraction temperature is 55-65°C, and the time is 15-25 minutes. The 55-65°C may be, for example, 55°C, 56°C, 58°C, 60°C, 62°C, 64°C, or 65°C. The 15-25 minutes may be, for example, 15 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, or 25 minutes.

[0021] Preferably, the injection volume of the headspace extraction is 150-250 μL, which can be, for example, 150 μL, 160 μL, 180 μL, 200 μL, 220 μL, 240 μL, or 250 μL.

[0022] Preferably, the rotation speed of the headspace extraction is 400-600 r / min, and the injection needle temperature is 80-90° C. The 400-600 r / min can be, for example, 400 r / min, 450 r / min, 500 r / min, 550 r / min or 600 r / min.

[0023] Preferably, the gas chromatograph column temperature is 55-65°C, and the run time is 25-35 minutes. The 55-65°C may be, for example, 55°C, 56°C, 58°C, 60°C, 62°C, 64°C, or 65°C. The 25-35 minutes may be, for example, 25 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, or 35 minutes.

[0024] Preferably, the carrier gas of the gas chromatography comprises nitrogen.

[0025] The nitrogen flow program of the gas chromatography was:

[0026] 0-2min, flow rate 1.5-2.5mL / min;

[0027] From 2 to 8 minutes, the flow rate changed evenly from 1.5 to 2.5 mL / min to 9 to 11 mL / min;

[0028] 8-10 min, the flow rate changes evenly from 9-11 mL / min to 90-110 mL / min;

[0029] 10-20min, flow rate is 90-110mL / min.

[0030] The 1.5-2.5 mL / min may be, for example, 1.5 mL / min, 1.6 mL / min, 1.8 mL / min, 2.0 mL / min, 2.2 mL / min, 2.4 mL / min, or 2.5 mL / min. The 9-11 mL / min may be, for example, 9 mL / min, 9.5 mL / min, 10 mL / min, 10.5 mL / min, or 11 mL / min. The 90-110 mL / min may be, for example, 90 mL / min, 95 mL / min, 100 mL / min, 105 mL / min, or 110 mL / min.

[0031] Preferably, the migration tube length of the ion mobility spectrometer is 50-55 mm, and the migration tube temperature is 40-50° C. The 50-55 mm may be, for example, 50 mm, 51 mm, 52 mm, 53 mm, 54 mm, or 55 mm. The 40-50° C. may be, for example, 40° C., 42° C., 44° C., 46° C., 48° C., or 50° C.

[0032] Preferably, the electric field strength of the ion mobility spectrometry is 450-550 V / cm, for example, 450 V / cm, 460 V / cm, 480 V / cm, 500 V / cm, 520 V / cm, 540 V / cm or 550 V / cm.

[0033] Preferably, the detection mode of the ion mobility spectrometry is a positive ion mode.

[0034] In a second aspect, the present invention provides an application of the method for identifying different grades of black truffles according to the first aspect in identifying the grades of black truffles.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] 1. The present invention uses a gas chromatography-ion mobility spectrometry detection method to gain an in-depth understanding of the volatile component composition and differences of black truffles of different grades, and to identify the grade of black truffles based on the odor activity values ​​of multiple volatile organic compounds. This method enables simple, rapid, and accurate identification, greatly reducing the difficulty of identification.

[0037] 2. The flavor characteristics of black truffles are primarily determined by their volatile organic compounds (VOCs). The types and content of these compounds affect the aroma and taste of black truffles. Therefore, the present invention conducts qualitative and quantitative analysis of the odor activity values ​​of VOCs in black truffles. This not only helps to reveal the mechanism of flavor formation but also provides a scientific basis for quality evaluation and grading of black truffles, promoting the standardization and regularization of the black truffle industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 HS-GC-IMS three-dimensional spectra of three grades of black truffles.

[0039] Figure 2 HS-GC-IMS two-dimensional top-down images of three grades of black truffles.

[0040] Figure 3 These are the ion mobility spectra of three grades of black truffle samples.

[0041] Figure 4 These are the volatile fingerprints of three grades of black truffle samples. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0043] Example 1

[0044] In this example, gas chromatography-ion mobility spectrometry was performed on black truffles of different grades.

[0045] First, three different grades of black truffles, Grade 1 (G1), Grade 2 (G2), and Grade 3 (G3), harvested in 2024, were collected for testing. 1.0 g of minced black truffle pulp from each grade was placed in a 20 mL headspace vial, and 10 μL of 0.01 g of the internal standard 2-methyl-3-heptanone was added for detection. The samples were then incubated at 50°C for 15 minutes.

[0046] The incubated samples were subjected to headspace extraction, wherein the incubation temperature for the headspace extraction was 60°C, the incubation time was 20 min, the injection volume was 200 μL, non-split injection was used, the incubation speed was 500 r / min, and the injection needle temperature was 85°C.

[0047] Headspace extracted samples were subjected to gas chromatography-ion mobility spectrometry (GC-IMS) analysis. GC-IMS conditions were as follows: column temperature: 60°C; carrier gas: high-purity nitrogen (≥99.999%); chromatographic run time: 30 min; inlet temperature: 80°C; ionization source: tritium (3H); migration tube length: 53 mm; electric field strength: 500 V / cm; migration tube temperature: 45°C; flow rate: 75.0 mL / min; positive ion mode.

[0048] The nitrogen flow program for gas chromatography was:

[0049] 0-2min, flow rate 2mL / min;

[0050] From 2 to 8 minutes, the flow rate was changed evenly from 2 mL / min to 10 mL / min;

[0051] From 8 to 10 minutes, the flow rate was uniformly changed from 10 mL / min to 100 mL / min.

[0052] 10-20 min, flow rate is 100 mL / min.

[0053] Example 2

[0054] This embodiment performs classification analysis on the detection results.

[0055] The samples tested in Example 1 were classified and statistically analyzed, and the target substances were qualitatively analyzed by retrieval and comparison using the built-in GC retention index (NIST 2020) database and IMS migration time database in the VOCal software; the area normalization method was used for quantification; the Reporter and GalleryPlot plug-ins in the VOCal data processing software supporting the GC-IMS instrument were used to generate two-dimensional spectra, three-dimensional spectra, difference spectra and fingerprint spectra of the volatile components, respectively, for comparing the differences in volatile organic compounds between samples; the Dynamic PCA plug-in was used for principal component analysis (PCA) and drawing of its charts.

[0056] The specific analysis results are as follows:

[0057] The Reporter plug-in was used to perform three-dimensional spectral analysis on the differences in volatile flavor compounds of black truffles of different grades. The results are as follows: Figure 1 The two-dimensional top view produced by the Reporter plug-in is shown as follows. Figure 2 As shown in Figure 1, the three-dimensional spectra directly reflect the differences in volatile organic compounds (VOCs) among the four samples. The X, Y, and Z axes represent ion migration time (ms), retention time, and ion peak intensity, respectively. In terms of the detected volatile organic compound (VOC) content, G1 > G2 > G3.

[0058] In order to further compare the differences in volatiles among the three grades of black truffle samples, the ion migration time and reaction ion peak (RIP) position were normalized. Figure 2 The figure shows a two-dimensional top view of the four samples. The background is blue. The red vertical line at the horizontal axis 1.0 is the reactive ion peak (RIP peak). The vertical axis is the gas chromatography retention time (s). Each point on the right side of the RIP peak represents a volatile organic compound. Taking the spectrum of the G1 sample as a reference, the color depth reflects the intensity of its ion peak. From white to red, the darker the color, the higher the ion peak intensity. The results show that the content of VOCs is G1>G2>G3. The signals of most volatile compounds are between 10-600s, which is consistent with the Figure 3 The results are consistent.

[0059] In order to make the differences between samples more obvious, the spectrum of sample G1 was selected as the reference, and the spectra of samples G2 and G3 with the same concentration were subtracted from the reference. The difference comparison chart of different samples was obtained, as shown in Figure 2. Figure 3As shown. If the content of volatile organic compounds in the target sample is the same as that in the reference sample, the background after subtraction is white. Red indicates that the concentration of the substance in the target sample is higher than that in the reference sample. Conversely, blue indicates that the concentration of the substance in the target sample is lower than that in the reference sample. Compared with other grades of black truffle samples, there are more red spots in the G1 sample, indicating that the concentration of volatile components in the G1 sample is higher than that in other samples. The content of VOCs is G1>G2>G3, which is consistent with the results of the previous analysis. Figure 1 、 Figure 2 consistent.

[0060] Example 3

[0061] This example constructs the identification method

[0062] In order to further compare the differences in volatile flavor compounds in black truffle samples of different grades, the GalleryPlot plug-in was used to create fingerprint maps. The results are shown in Figure 2. Figure 4 As shown. In the fingerprint, each row represents all the signal peaks selected in a sample, and each column represents the signal peaks of the same volatile organic compound in different samples. The numbers in the figure are separated but cannot be qualitatively identified compounds. From the figure, you can see the complete information of the volatile organic compounds in each sample, as well as the differences between different samples. Some volatile compounds may produce multiple signals (monomers and dimers) due to different properties and concentrations. The M and D after the names of some substances represent their monomers and dimers, respectively. By comparing and analyzing the volatile substances in G1, G2, and G3 samples,

[0063] The results showed that the volatile substances in G1 sample mainly included acetic acid, linalool, 1-pentanol, Z-2-penten-1-ol, 1-penten-3-ol, 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, ethanol, 2-methyl-2-pentenal, 3-methyl-2-butenal, 2-methyl-2-propenal, butyraldehyde, propionaldehyde, acetaldehyde, 3-hydroxy-2-butanone, 2-heptanone, 2,3-pentanedione, 2-methyl-2-propenal, acetaldehyde, 2-heptanone, 1-pentanedione, 2-methyl-2-pentenal, 2-methyl-2-propenal, acetaldehyde, 3-hydroxy-2-butanone, 2-heptanone, 1-pentanedione, 2-methyl-2-propenal, acetaldehyde, 2-heptanone, 1-pentanedione, 2-methyl-2-pentenal, 1-propanol, ethanol, 2-methyl-2-pentenal, 3-methyl-2-butenal, 2-methyl-2-propenal, acetaldehyde, 2-heptanone, 1-pentanedione, 2-methyl-2-propenal, 1-pentanol, 1-propanol, 1-propanol, 1-pentanedione, 2-methyl-2-propenal, 1-pentanone, 1-propanol, 1-propanol, 1-pentanedione, 1-methyl-2-pentenal, 1-propenal, 1-propenal, 1-pentanone, 1-propenal, 1-propenal, 1-propenal, 1-pentanone, 1-propenal, 1-prop -Butanone, octyl acetate, ethyl heptanoate, ethyl valerate, isoamyl acetate, ethyl E-2-butenoate, isobutyl isobutyrate, ethyl 3-methylbutyrate, butyl acetate, ethyl isobutyrate, isobutyl acetate, ethyl formate, 2-acetylfuran, 2-heptylfuran, 2-ethylfuran, 2-ethyl-3,4-dimethylpyrazine, 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, 4,5-dimethylthiazole, α-terpinene, etc. The volatile substances in sample G2 mainly include E-2-octen-1-ol, 1-octanol, 1-octen-3-ol, 1-hexanol, 1-butanol, E-2-octenal, E-2-heptenal, nonanal, octanal, heptanal, hexanal, 3-methylbutanal, 5-methyl-2(3H)-furanone, 1-octen-3-one, acetone, methyl 3-methylbutyrate, hexyl propionate, and ethyl acetate. The volatile substances in sample G2 mainly include acetaldehyde, benzaldehyde, methylthiopropionaldehyde, 3-octanol, 2-butanol, 3-octanone, cyclopentanone, 2-pentanone, isobutyl 3-methylbutyrate, butyl 2-methylbutyrate, propyl acetate, and p-xylene.

[0064] Calculation of relative odor activity value (OAV): The relative odor activity value (OAV) method is used to evaluate the contribution of each volatile component to the overall flavor of the sample. The calculation formula is:

[0065]

[0066] Where: OAV is the odor activity value of a particular aroma component; C is the content of a particular aroma component, expressed in μg / kg; and T is the aroma threshold of the compound in water. Components with larger OAV values ​​contribute more to the overall flavor of the sample. Components with an OAV of 1 ≤ ≤ 100 are considered key aroma components, those with an OAV of 0.1 ≤ < 1 have significant flavor-modifying effects, and those with an OAV of < 0.1 have potential flavor-modifying effects. Table 1 shows the corresponding OAV and VIP values ​​for key aroma components in different grades of black truffle.

[0067] Table 1

[0068]

[0069]

[0070] The results in Table 1 show that the contents of isoamyl acetate, 1-octen-3-ol, (E)-2-octen-1-ol, ethyl heptanoate, linalool and benzoic aldehyde vary greatly in black truffles of different grades. An identification method was constructed based on the results, and the specific method is shown in Table 2.

[0071] Table 2

[0072]

[0073] The results show that the criteria for constructing the identification method are:

[0074] When the odor activity value of isoamyl acetate is ≥1200 and the odor activity value of 1-octen-3-ol is ≥3000, it is judged to be a first-grade black truffle;

[0075] When 3000 ≥ (E)-2-octen-1-ol odor activity value ≥ 100 and 40 ≥ ethyl heptanoate odor activity value ≥ 20, it is judged to be a second-level black truffle;

[0076] When the detection value of linalool is 800 ≥ 400 and the odor activity value of benzoin aldehyde is ≥ 1.3, it is judged to be a third-grade black truffle.

[0077] Example 4

[0078] This example conducts a repeatability test to detect the accuracy

[0079] Thirty groups of black truffle samples (10 each for G1, G2, and G3) of known grade were randomly selected as the validation set. These samples were tested according to the method in Example 1, and the odor activity values ​​of various volatile organic compounds were calculated using the method in Example 3. The specific test results are shown in Table 3 (for G1 samples), Table 4 (for G2 samples), and Table 5 (for G3 samples).

[0080] Table 3

[0081]

[0082] Table 4

[0083]

[0084]

[0085] Table 5

[0086]

[0087] The above results show that the mean OAV of isoamyl acetate in G1 samples was 1280±85 (range, 1205-1350), and the mean OAV of 1-octen-3-ol was 3450±210. In G2 samples, the mean OAV of (E)-2-octen-1-ol was 115±15, and the mean OAV of ethyl heptanoate was 22±3. In G3 samples, the mean OAV of linalool was 420±50, and the mean OAV of benzoic aldehyde was 1.38±0.2. Accuracy statistics showed that based on OAV threshold determination, the graded discrimination accuracy of the 30 sample groups reached 96.7% (with only one G3 sample misclassified as G2).

[0088] When analyzing the aromatic substances of black truffles of different grades, multiple samples were randomly selected for sampling, and after verification, the OAV and VIP values ​​obtained for the tested grades were all consistent. This shows that the identification method of the present application can accurately distinguish between domestic black truffle grades one, two, and three, and is a simple, fast, and accurate method for identifying different grades of black truffles.

[0089] In summary, the present invention provides a method and application for identifying different grades of black truffles. The present invention uses the content of volatile organic compounds to identify the grade of black truffles, providing a scientific basis for the quality evaluation and grading of black truffles, and promoting the standardization and normalization of the black truffle industry.

[0090] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for identifying different grades of black truffles, characterized in that: The method comprises the steps of processing a black truffle sample and then performing identification by gas chromatography-ion mobility spectrometry detection; The identification criteria are: When the odor activity value of isoamyl acetate is ≥1200 and the odor activity value of 1-octen-3-ol is ≥3000, it is judged to be a first-grade black truffle; When 3000 ≥ (E)-2-octen-1-ol odor activity value ≥ 100 and 40 ≥ ethyl heptanoate odor activity value ≥ 20, it is judged to be a second-level black truffle; When the detection value of linalool is 800 ≥ 400 and the odor activity value of benzoin aldehyde is ≥ 1.3, it is judged to be a third-grade black truffle.

2. The method for identifying different grades of black truffles according to claim 1, wherein: The calculation formula of the odor activity value is: Wherein, OAV is the odor activity value, C is the content detected by gas chromatography-ion mobility spectrometry, and T is the aroma threshold of volatile organic compounds in water.

3. The method for identifying different grades of black truffles according to claim 2, wherein: The sample processing includes placing the sample in a headspace vial, adding an internal standard for incubation, and then performing headspace extraction; Preferably, the amount of the sample added is 0.5-3 g; Preferably, the internal standard comprises 2-methyl-3-heptanone; Preferably, the mass ratio of the internal standard to the sample is (0.005-0.03) mg:1 g.

4. The method for identifying different grades of black truffles according to claim 2 or 3, characterized in that: The incubation temperature is 45-55° C. and the incubation time is 10-20 minutes.

5. The method for identifying different grades of black truffles according to any one of claims 2 to 4, characterized in that: The headspace extraction temperature is 55-65°C and the time is 15-25 minutes; Preferably, the injection volume of the headspace extraction is 150-250 μL; Preferably, the rotation speed of the headspace extraction is 400-600 r / min, and the injection needle temperature is 80-90°C.

6. The method for identifying different grades of black truffles according to any one of claims 1 to 5, characterized in that: The gas chromatographic column temperature is 55-65° C., and the running time is 25-35 minutes.

7. The method for identifying different grades of black truffles according to any one of claims 1 to 6, characterized in that: The carrier gas of the gas chromatography comprises nitrogen; The nitrogen flow program of the gas chromatography was: 0-2min, flow rate 1.5-2.5mL / min; From 2 to 8 minutes, the flow rate changed evenly from 1.5 to 2.5 mL / min to 9 to 11 mL / min; 8-10 min, the flow rate changes evenly from 9-11 mL / min to 90-110 mL / min; 10-20min, flow rate is 90-110mL / min.

8. The method for identifying different grades of black truffles according to any one of claims 1 to 7, characterized in that: The migration tube length of the ion mobility spectrometer is 50-55 mm, and the migration tube temperature is 40-50° C.

9. The method for identifying different grades of black truffles according to any one of claims 1 to 8, characterized in that: The electric field strength of the ion mobility spectrometer is 450-550 V / cm; Preferably, the detection mode of the ion mobility spectrometry is a positive ion mode.

10. Use of the method for identifying different grades of black truffles according to any one of claims 1 to 9 in identifying the grades of black truffles.

Images