Auricularia auricula-judae geographical characteristic molecular marker and application thereof

Abstract

The application provides a black fungus provenance characteristic molecular marker and application thereof, and the molecular marker comprises the following: the molecular marker 2-acetyl furan, beta-pinene and 2-pinene of Shaanxi black fungus; the molecular marker 2-propanol and 2, 6-dimethyl pyrazine of Heilongjiang black fungus; the molecular marker pentanal and 2-butanone of Jilin black fungus; the molecular marker n-hexanal and acetic acid of Zhejiang black fungus; and the molecular marker 2-methyl propionaldehyde and hexanoic acid of Fujian black fungus. The molecular marker can be used for identifying the provenance of black fungus. The application first provides the provenance characteristic molecular marker of black fungus, the molecular marker can replace all chemical components of black fungus and serve as a detection basis of the provenance of black fungus; compared with existing black fungus physical and chemical indexes and artificial sensory recognition analysis, the provenance characteristic molecular marker of black fungus provided by the application improves the accuracy and recognition rate of detection, and provides a simple, rapid and effective scheme for provenance identification of black fungus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food identification, and relates to black fungus origin identification, in particular to a black fungus origin characteristic molecular marker and application thereof. BACKGROUND

[0002] Black fungus (Auricularia auricula) belongs to Auriculariaceae and Auricularia, is a precious medicinal and edible fungus in China, and is deeply loved by consumers. In China, the cultivation yield of black fungus is only less than that of shiitake mushroom, and is the second largest variety of edible fungus cultivation in China. Artificial cultivation and natural growth of black fungus exist in many provinces. However, due to the influence of natural geographical environment such as water and soil, climate and sunshine, the relative contents of substances contained in black fungus from different origins are different, and therefore, it is necessary to identify the origin of black fungus. At present, simple physicochemical analysis indexes including water content, salt content, vitamin content, protein content and color cannot completely identify the origin of black fungus. As for flavor, artificial sensory recognition analysis is currently mainly used, which is difficult to guarantee accuracy, has low recognition efficiency, and has great difference.

[0003] In recent years, gas chromatography-ion mobility spectrometry (GC-IMS) technology has developed rapidly and is widely used in food quality and safety detection and origin tracing. GC-IMS technology has many advantages such as convenient sample pretreatment, simple operation, high accuracy and sensitivity, and there is no literature report on identification of black fungus origin by using GC-IMS technology. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide a black fungus origin characteristic molecular marker and application thereof, and to solve the technical problem that the accuracy and recognition rate of black fungus origin identification in the prior art need to be further improved.

[0005] In order to solve the above technical problems, the technical scheme is adopted as follows:

[0006] A black fungus origin characteristic molecular marker comprises a molecular marker of Shaanxi black fungus, a molecular marker of Heilongjiang black fungus, a molecular marker of Jilin black fungus, a molecular marker of Zhejiang black fungus and a molecular marker of Fujian black fungus. The molecular marker of Shaanxi black fungus is 2-acetylfuran, beta-pinene and 2-pinene; the molecular marker of Heilongjiang black fungus is 2-propanol and 2,6-dimethylpyrazine; the molecular marker of Jilin black fungus is pentanal and 2-butanone; the molecular marker of Zhejiang black fungus is n-hexanal and acetic acid; and the molecular marker of Fujian black fungus is 2-methylpropanal and hexanoic acid.

[0007] The present application also has the following technical features:

[0008] The application also protects the use of the Auricularia auricular-judicia characteristic molecular marker as described above for identifying the origin of Auricularia auricular.

[0009] The method of the application specifically comprises the following steps:

[0010] Step one, obtaining the Auricularia auricular-judicia characteristic molecular marker.

[0011] Step two, preparing the sample of the Auricularia auricular to be tested.

[0012] Step three, GC-IMS detection of the Auricularia auricular to be tested:

[0013] The sample of the Auricularia auricular to be tested prepared in step two is subjected to gas chromatography-ion mobility detection, and the GC-IMS data of the sample of the Auricularia auricular to be tested is obtained after the detection is completed.

[0014] Step four, analyzing and determining the origin of the Auricularia auricular:

[0015] The GC-IMS data of the sample of the Auricularia auricular to be tested in step three is analyzed, the volatile organic matter composition of the Auricularia auricular to be tested is confirmed, and whether the migration spectrum characteristic peak of the Auricularia auricular-judicia characteristic molecular marker is observed in the GC-IMS spectrum is observed, so as to determine the origin of the Auricularia auricular.

[0016] Specifically, in step three, when the gas chromatography-ion mobility detection is performed, the parameter settings of the gas chromatography-ion mobility spectrum unit are as follows: the column temperature is 60℃; the carrier gas / drift gas is nitrogen; the IMS detector temperature is 45℃; and the analysis time is 30min.

[0017] Specifically, in step three, when the gas chromatography-ion mobility detection is performed, the parameter settings of the automatic headspace sampling unit are as follows: the sampling volume is 500μL, the incubation time is 20min, the incubation temperature is 60℃, the sampling needle temperature is 65℃, and the incubation rotation speed is 500r / min.

[0018] Specifically, in step three, when the gas chromatography-ion mobility detection is performed, the parameter settings of the gas chromatography are as follows: the drift gas flow rate is 150mL / min; the gas chromatography carrier gas flow rate is 0-2min, 2mL / min, 2-10min, 2-20mL / min, 10-20min, 20-100mL / min, 20-30min, and 100-150mL / min.

[0019] Compared with the prior art, the application has the following technical effects:

[0020] The application first provides a characteristic molecular marker of Auricularia auricula-judae origin, which can replace all chemical components of Auricularia auricula-judae and serve as a detection basis for Auricularia auricula-judae origin; compared with existing Auricularia auricula-judae physical and chemical indexes and artificial sensory recognition analysis, the characteristic molecular marker of Auricularia auricula-judae origin improves the accuracy and recognition rate of detection, and provides a simple, rapid and effective scheme for Auricularia auricula-judae origin identification; meanwhile, the application also provides a new idea for edible fungus origin detection in a broad sense, and makes it possible to popularize and apply edible fungus origin analysis and detection. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a morphological diagram of Auricularia auricula-judae samples from different origins.

[0022] Figure 2 It is a Library Search qualitative analysis of volatile substances in Auricularia auricula-judae samples from different origins. Figure 2 In the figure, the red vertical line at the horizontal coordinate 1.0 is a reaction ion peak (RIP), and each point to the right of the RIP represents a volatile organic compound. The component and concentration difference between different samples are represented by the presence or absence of the peak (color point) or the color depth. The vertical coordinate represents the retention time (s) of gas chromatography, and the horizontal coordinate represents the ion migration time.

[0023] Figure 3 It is a three-dimensional spectrum of volatile substance components in Auricularia auricula-judae samples from different origins. Figure 3 In the figure, the vertical coordinate represents the retention time of gas chromatography, the horizontal coordinate represents the ion migration time of volatile compounds, and the third coordinate represents the peak intensity.

[0024] Figure 4 It is a top view of the spectrum of volatile substance components in Auricularia auricula-judae samples from different origins. Figure 4 In the figure, the vertical line at the horizontal coordinate 1.0 represents a reaction ion peak (RIP). Each point to the right of the RIP represents a volatile compound extracted from the sample.

[0025] Figure 5 It is a difference spectrum of volatile substance components in Auricularia auricula-judae samples from Shaanxi and other five origins. Figure 5 In the figure, the vertical line at the horizontal coordinate 1.0 represents a reaction ion peak (RIP). Each point to the right of the RIP represents a volatile compound extracted from the sample.

[0026] Figure 6 It is a Gallery Plot fingerprint spectrum of Auricularia auricula-judae samples from different origins. Figure 6 In the figure, the substances shown in the box are volatile components unique to each sample.

[0027] Figure 7 It is a PCA analysis diagram of volatile substances in Auricularia auricula-judae from different origins.Figure 7 In the diagram, the horizontal axis represents principal component 1 with a contribution rate of 59%, and the vertical axis represents principal component 2 with a contribution rate of 25%. Each point represents a sample, and each sample has three parallel samples.

[0028] The specific content of the present invention will be further explained in detail below with reference to the embodiments. Detailed Implementation

[0029] It should be noted that, unless otherwise specified, all testing instruments and consumables used in this invention are those known in the art. For example:

[0030] Gas chromatography-ion migration detection uses existing technologies known to Flavor analyzer, purchased from Zhejiang Keruida Scientific Instruments Co., Ltd.

[0031] Gas chromatography-ion migration detection uses a column known in existing technology, model and specifications FS-SE-54-CB-1, 15m, ID: 0.53mm, manufactured by Shimadzu Corporation, Japan.

[0032] The following are specific embodiments of the present invention. It should be noted that the present invention is not limited to the following specific embodiments. All equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0033] Example 1:

[0034] This embodiment provides molecular markers for the geographical characteristics of black fungus, including: 2-acetylfuran, β-pinene, and 2-pinene for Shaanxi black fungus; 2-propanol and 2,6-dimethylpyrazine for Heilongjiang black fungus; pentanal and 2-butanone for Jilin black fungus; hexanal and acetic acid for Zhejiang black fungus; and 2-methylpropanal and hexanoic acid for Fujian black fungus.

[0035] Example 2:

[0036] This embodiment provides a method for obtaining molecular markers characteristic of black fungus origin as described in Example 1. The method first establishes a GC-IMS database of black fungus from different origins; then, it analyzes the GC-IMS database of black fungus from different origins to obtain molecular markers characteristic of black fungus origin. The method specifically includes the following steps:

[0037] Step 1.1, Prepare experimental materials:

[0038] Black fungus samples from Shaanxi, Jilin, Heilongjiang, Zhejiang, and Fujian were collected and grouped as shown in Table 1; morphological images of the black fungus samples were photographed and retained, as shown in Table 1. Figure 1As shown, black fungus from different origins was pulverized through a 100-mesh sieve to obtain black fungus powder from different origins.

[0039] Table 1. Information on Black Fungus Samples

[0040] Serial number Number Sample source 1 Q1 Shaanxi Province 2 Q2 Shaanxi Province 3 Q3 Shaanxi Province 4 H1 Heilongjiang Province 5 H2 Heilongjiang Province 6 H3 Heilongjiang Province 7 J1 Jilin Province 8 J2 Jilin Province 9 J3 Jilin Province 10 Z1 Zhejiang Province 11 Z2 Zhejiang Province 12 Z3 Zhejiang Province 13 F1 Fujian Province 14 F2 Fujian Province 15 F3 Fujian Province

[0041] Step 1.2: Obtain GC-IMS data of black fungus samples from different origins:

[0042] use A flavor analyzer was used to perform gas chromatography-ion migration (GC-IMS) analysis on the black fungus powders from different origins obtained in step 1.1. After the analysis, GC-IMS data of black fungus samples from different origins were obtained, which included information on the types and contents of volatile substances in the black fungus samples.

[0043] In this embodiment, the analytical conditions used for gas chromatography-ion migration detection are as follows:

[0044] The conditions for the gas phase-ion mobility spectrometry unit were as follows: column temperature 60℃; carrier gas / drift gas high-purity nitrogen (purity ≥99.999%); IMS detector temperature 45℃; analysis time 30 min.

[0045] The conditions for the automated headspace sampling unit are as follows: injection volume of 500 μL, incubation time of 20 min, incubation temperature of 60℃, injection needle temperature of 65℃, and incubation rotation speed of 500 r / min.

[0046] The gas chromatography conditions were as follows: drift gas flow rate (E1) was 150 mL / min; carrier gas flow rate (E2) was 0–2 min, 2 mL / min, 2–10 min, 2–20 mL / min, 10–20 min, 20–100 mL / min, 20–30 min, and 100–150 mL / min.

[0047] Step 1.3, Preliminary Data Analysis:

[0048] The adopted The flavor analyzer's accompanying software, VOCal, and plugins analyze the GC-IMS data of black fungus samples from different origins obtained in step 1.2 from different perspectives. They obtain and observe GC-IMS three-dimensional spectra, GC-IMS spectra, and GC-IMS differential spectra, and conduct preliminary analysis to determine whether there are significant differences in volatile organic compounds among black fungus samples from different origins, in order to decide whether to continue the analysis.

[0049] In this embodiment, the data analysis process includes:

[0050] The VOCal software was used to analyze the GC-IMS data qualitatively and quantitatively, and the results are shown in Figure 2 、 Figure 3 and Figure 4 . Figure 2 、 Figure 3 and Figure 4 The vertical line at the horizontal coordinate 1.0 in each of the figures represents a reaction ion peak (RIP), and each point on the right side represents a volatile organic compound. The differences in components and concentrations between different samples are represented by the presence or absence of peaks (color points) or the color depth. The vertical coordinate represents the retention time (s) of gas chromatography, and the horizontal coordinate represents the ion mobility time (normalized). Each point on both sides of the RIP peak represents a volatile organic compound, and the darker the color, the greater the concentration.

[0051] Figure 3 and Figure 4 are the GC-IMS three-dimensional spectrum (retention time, mobility time, and peak intensity) and two-dimensional overhead view (retention time and mobility time) of the volatile substances in the Auricularia auricular sample from Wudi, respectively. As can be seen from Figure 3 , the peak signal distribution of all samples is similar, but the peak signal intensity of each sample is different, that is, the differences in volatile organic compounds in different samples can be directly observed. By combining the corresponding two-dimensional overhead view, the differences and changes in volatile substances can be more obviously reflected. Through the GC-IMS spectrum of the Auricularia auricular sample from Wudi, it can be clearly seen that the volatile components of Auricularia auricular produced in different regions are significantly different.

[0052] Using the Reporter plug-in, the differences between the spectra of the samples (two-dimensional overhead view, three-dimensional spectrum, and difference spectrum) were directly compared, and the results are shown in Figure 5 . As can be seen from Figure 5 , the S1 sample was used as a reference. When the retention time is 100-600 seconds and the drift time is between 1.0 and 1.5, most of the signals appear. When comparing the volatile substance composition spectrum of Auricularia auricular from different regions with Auricularia auricular from Shaanxi (S1) as a reference, the concentrations of the corresponding volatile substances in the other five samples are immediately apparent.

[0053] Step 1.4, determine the candidate molecular marker:

[0054] Select the ion mobility spectrum characteristic peak position point in the GC-IMS spectrum obtained in step 1.3, and determine the corresponding volatile organic compound as a candidate molecular marker. The information of the candidate molecular marker is shown in Table 2.

[0055] Table 2, list of volatile compounds in Auricularia auricular

[0056]

[0057]

[0058]

[0059] Step 1.5, Preliminary screening of molecular markers:

[0060] Based on the gallery plot fingerprints of black fungus samples from different origins, a very intuitive comparison and analysis was performed on all samples to preliminarily determine the differences in candidate molecular markers among black fungus samples from different origins. The results are as follows: Figure 6 As shown, Figure 6 In the middle of the image, one row represents the volatile composition of a sample, and one column represents the signal peaks of a certain volatile substance in different samples. The brightness of the signal peaks indicates the concentration of that substance. Figure 6 The following information was obtained: 2-acetylfuran, β-pinene, and 2-pinene were mainly found in black fungus samples from Shaanxi Province; 2-propanol and 2,6-dimethylpyrazine were mainly found in black fungus samples from Heilongjiang Province; pentanal and 2-butanone were mainly found in black fungus samples from Jilin Province; n-hexanal and acetic acid were mainly found in black fungus samples from Zhejiang Province; and 2-methylpropionaldehyde and hexanoic acid were mainly found in black fungus samples from Fujian Province. Therefore, the above volatile organic compounds were initially screened as molecular markers.

[0061] Step 1.6: Verify the effectiveness of the screened molecular markers:

[0062] For the molecular markers initially screened in step 1.5, VOC cluster analysis was performed on black fungus samples from different origins using a dynamic principal component analysis (PCA) plugin. Figure 7 As shown. Figure 7 The results further supported the findings of the cluster heatmap analysis and the correlation analysis between samples, thus confirming the effectiveness of the screened molecular markers.

[0063] Example 3:

[0064] This embodiment describes the application of the black fungus origin-specific molecular markers from Example 1 for identifying the origin of black fungus. This application uses GC-IMS technology to detect the black fungus samples and obtain GC-IMS data. By analyzing the presence of black fungus origin-specific molecular markers in the GC-IMS data, the origin of the black fungus samples can be determined. The specific steps of this application include the following:

[0065] Step 1: Obtain molecular markers characteristic of black fungus origin:

[0066] In this embodiment, the method for obtaining molecular markers characteristic of the black fungus origin is exactly the same as in Example 2.

[0067] Step 2, Preparation of the black fungus sample to be tested:

[0068] First, the appearance of the test Auricularia auricular-judae was observed, and the Auricularia auricular-judae with abnormal color, mold, etc. was removed. If the appearance of the test Auricularia auricular-judae was normal, the Auricularia auricular-judae was crushed and passed through a 100-mesh screen to obtain the test Auricularia auricular-judae sample.

[0069] Step three, GC-IMS detection of the test Auricularia auricular-judae:

[0070] Using The flavor analyzer was used to detect the test Auricularia auricular-judae sample obtained in step two by gas chromatography-ion mobility (GC-IMS). After the detection, the GC-IMS data of the test Auricularia auricular-judae sample were obtained, which included the types and contents of volatile substances in the Auricularia auricular-judae sample.

[0071] In this embodiment, the analysis conditions of the gas chromatography-ion mobility detection were exactly the same as those in step 1.2 of Example 2.

[0072] Step four, analysis and determination of the origin of Auricularia auricular-judae:

[0073] The GC-IMS data of the test Auricularia auricular-judae sample obtained in step three were subjected to qualitative analysis to confirm the volatile organic components of the test Auricularia auricular-judae and observe whether the GC-IMS spectrum had the migration spectrum characteristic peak of the characteristic molecular marker of the Auricularia auricular-judae origin, so as to determine the origin of the Auricularia auricular-judae.

[0074] In this embodiment, five test Auricularia auricular-judae samples were respectively from Shaanxi, Heilongjiang, Jilin, Zhejiang and Fujian, and were respectively recorded as sample 1, sample 2, sample 3, sample 4 and sample 5. The detection results were as follows: the molecular markers in the sample 1 were 2-acetylfuran, β-pinene and 2-pinene, which completely matched the molecular markers of the Auricularia auricular-judae from Shaanxi; the molecular markers in the sample 2 were 2-propanol and 2,6-dimethylpyrazine, which completely matched the molecular markers of the Auricularia auricular-judae from Heilongjiang; the molecular markers in the sample 3 were pentanal and 2-butanone, which completely matched the molecular markers of the Auricularia auricular-judae from Jilin; the molecular markers in the sample 4 were n-hexanal and acetic acid, which completely matched the molecular markers of the Auricularia auricular-judae from Zhejiang; and the molecular markers in the sample 5 were 2-methylpropanal and hexanoic acid, which completely matched the molecular markers of the Auricularia auricular-judae from Fujian. The above results showed that the characteristic molecular markers of the Auricularia auricular-judae origin according to the present application could accurately identify the actual origin of the Auricularia auricular-judae.

Claims

1. A geographical characteristic molecular marker of Auricularia auricular, which is used for identifying Auricularia auricular of Shaanxi, Heilongjiang, Jilin, Zhejiang and Fujian, characterized in that, The molecular marker of Shanxi Auricularia auricular, the molecular marker of Heilongjiang Auricularia auricular, the molecular marker of Jilin Auricularia auricular, the molecular marker of Zhejiang Auricularia auricular and the molecular marker of Fujian Auricularia auricular. The molecular marker of Shanxi Auricularia auricular is 2-acetyl furan, beta-pinene and 2-pinene; the molecular marker of Heilongjiang Auricularia auricular is 2-propanol and 2,6-dimethylpyrazine; the molecular marker of Jilin Auricularia auricular is pentanal and 2-butanone; the molecular marker of Zhejiang Auricularia auricular is n-hexanal and acetic acid; and the molecular marker of Fujian Auricularia auricular is 2-methylpropanal and hexanoic acid.

2. The use of the Auricularia auricular mushroom geographical characteristic molecular marker according to claim 1 for identifying the geographical origin of Auricularia auricular mushrooms, characterized in that, The producing areas are Shanxi, Heilongjiang, Jilin, Zhejiang and Fujian.

3. Use according to claim 2, wherein the compound is ###0002### The method of the application specifically comprises the following steps: Step one, obtaining the molecular marker of the producing area of Auricularia auricular; Step two, preparing the sample of the Auricularia auricular to be tested; Step three, detecting the Auricularia auricular to be tested by GC-IMS: The sample of the Auricularia auricular to be tested prepared in step two is detected by gas chromatography-ion mobility detection, and the GC-IMS data of the sample of the Auricularia auricular to be tested is obtained after the detection is completed; Step four, analyzing and determining the producing area of Auricularia auricular: The GC-IMS data of the sample of the Auricularia auricular to be tested in step three is analyzed, the volatile organic matter composition of the Auricularia auricular to be tested is confirmed, and whether the GC-IMS spectrum has the migration spectrum characteristic peak of the molecular marker of the producing area of Auricularia auricular is observed, so as to determine the producing area of Auricularia auricular.

4. Use according to claim 3, wherein the compound is ###0002### In step three, when the gas chromatography-ion mobility detection is performed, the parameter settings of the gas chromatography-ion mobility spectrum unit are as follows: the column temperature is 60℃; the carrier gas / drift gas is nitrogen; the IMS detector temperature is 45℃; and the analysis time is 30min.

5. The use according to claim 3, wherein the compound is ###0002### In step three, when the gas chromatography-ion mobility detection is performed, the parameter settings of the automatic headspace sampling unit are as follows: the sampling volume is 500μL, the incubation time is 20min, the incubation temperature is 60℃, the sampling needle temperature is 65℃, and the incubation rotation speed is 500r / min.

6. The use according to claim 3, wherein the compound is ###0002### In step three, when the gas chromatography-ion mobility detection is performed, the parameter settings of the gas chromatography are as follows: the drift gas flow rate is 150mL / min; the gas chromatography carrier gas flow rate is 0-2min, 2mL / min, 2-10min, 2-20mL / min, 10-20min, 20-100mL / min, 20-30min, 100-150mL / min.

Images