Method for determining selenium-rich shiitake mushrooms by using micro-area X-ray fluorescence spectrum

Abstract

The invention provides a method for judging selenium-enriched shiitake mushrooms by using a micro-area X-ray fluorescence spectrum, belongs to the technical field of food nutritional ingredient detection and identification, and aims to solve the technical problems of destructive sampling and non-visualized element space distribution in the existing selenium-enriched shiitake mushroom detection technology. The method comprises the following steps: freeze-drying shiitake mushrooms to fix element distribution; selecting a plurality of detection points on the freeze-dried lentinus edodes slices, performing single-point detection by adopting a micro-area X-ray fluorescence analyzer, and preliminarily judging that the lentinus edodes are selenium-rich lentinus edodes according to the condition that Se Kalpha signals of the selected detection points obviously exceed a background value; and carrying out element surface scanning on the surface of the preliminarily judged selenium-rich mushroom by adopting a micro-area X-ray fluorescence spectrum analyzer, and finally judging whether the mushroom is the selenium-rich mushroom or not according to the condition that delta capp of the whole pileus area (containing gill) is greater than or equal to 4counts and delta stipe of the stipe area is greater than or equal to 3counts. According to the method, a'rapid identification-comprehensive characterization 'dual-mode discrimination model is innovatively established, the selenium-rich shiitake mushrooms and common shiitake mushrooms can be effectively distinguished, and a visual and multi-dimensional basis is provided for quality evaluation of the selenium-rich shiitake mushrooms.

Description

Technical Field

[0001] This invention belongs to the technical field of food nutrient detection and identification, and particularly relates to the identification of selenium-enriched shiitake mushrooms. Background Technology

[0002] As the world's second most commercially cultivated edible fungus, shiitake mushroom (Lentinula edodes) has become a hot topic in functional food development due to its selenium-enriched varieties, which have physiological functions such as anti-oxidation and immune enhancement. The current detection standards for selenium-enriched agricultural products (GB1903.22-2016, etc.) mainly rely on hydride atomic fluorescence spectrometry (HG-AFS) and inductively coupled plasma optical emission spectrometry (ICP-OES). However, the current detection methods have many defects, as follows: (1) Hydride atomic fluorescence spectrometry (HG-AFS): The sample needs to be digested into a liquid state at high temperature (concentrated nitric acid-perchloric acid system, time ≥4h), which leads to the complete destruction of the shiitake mushroom tissue structure, making it impossible to trace the in-situ distribution of elements or conduct subsequent biochemical research. (2) Inductively coupled plasma optical emission spectrometry (ICP-OES): Although it can improve the efficiency of multi-element detection, it still requires sample crushing and acid hydrolysis, which introduces the risk of heavy metal pollution, and the detection results only reflect the total amount of elements. (3) Common limitations of HG-AFS / ICP-OES: It can only obtain total selenium data and cannot reveal the spatial distribution characteristics of selenium in key functional parts such as caps and gills (i.e., micro-area enrichment characteristics), resulting in blind spots in nutritional evaluation and safety control. (4) Bottleneck of synchrotron radiation technology: Although micro-area X-ray fluorescence spectroscopy (SR-μ-XRF) based on synchrotron radiation source can achieve a spatial resolution of ≤10μm, it requires more than 100 displacement splicing of centimeter-level samples, and the detection time of a single sample is too long. Moreover, it relies on large scientific facilities and is difficult to apply in routine detection. (5) Conventional X-ray fluorescence spectroscopy (XRF) technology: Although it can expand the detection area, the excitation efficiency and resolution drop sharply (e.g., the selenium detection limit needs to be higher than 5mg / kg), which cannot meet the requirements of selenium-enriched agricultural products (GHT 1135-2017) (0.1-5mg / kg).

[0003] In summary, existing detection technologies have significant limitations in detecting selenium in selenium-enriched shiitake mushrooms: ① Destructive sampling results in non-reusable samples; ② Pre-processing is complex and time-consuming (single-sample testing typically takes several hours); ③ It is difficult to achieve visual characterization of the spatial distribution of selenium, and it is even more impossible to simultaneously acquire information on associated elements. Developing rapid, non-destructive, and multi-dimensional detection technologies for selenium-enriched shiitake mushrooms has become an urgent need for current industrial upgrading.

[0004] Micro-X-ray fluorescence spectrometry (μ-XRF) combined with high-resolution elemental mapping enables non-destructive and rapid surface analysis at the micrometer scale, making it suitable for studying the elemental distribution of complex samples. For example, patent publication number CN 118937381A discloses a method and related apparatus for determining representative volumetric units of heterogeneous shale. Multiple locations are selected on the vertical surface of the heterogeneous shale sample. The paleoenvironmental index of each location is calculated based on the elemental distribution map. Based on the paleoenvironmental index and location information of each location, the vertical surface is divided into several sedimentary facies zones. The proportion of each sedimentary facies zone is calculated. Based on the proportion of each sedimentary facies zone, a scanning area in the vertical surface is determined, and the corresponding mineral distribution map is obtained. An initial region in the scanning area is determined based on the proportion of each sedimentary facies zone, and the initial region is adjusted based on the mineral distribution map to determine the representative volumetric unit of the heterogeneous shale sample, thereby efficiently determining the representative volumetric unit of strongly heterogeneous shale. However, there are currently no reports on the application of μ-XRF technology combined with high-resolution elemental surface scanning (mapping) to the detection of selenium-enriched shiitake mushrooms. As an agricultural product with special nutritional and commercial value, the micro-regional distribution characteristics of selenium in different tissue parts such as the cap, gills, and stipe of selenium-enriched shiitake mushrooms, as well as the elemental enrichment patterns under different growth stages and cultivation conditions, are of great significance for product quality assessment, functional development, and safety control. Due to the significant differences in the tissue structure, elemental content levels, and matrix materials of selenium-enriched shiitake mushrooms, directly applying existing μ-XRF detection methods is difficult to accurately meet the detection needs of selenium-enriched shiitake mushrooms. It cannot effectively solve key problems such as quantitative analysis of selenium in micro-regions, non-destructive rapid detection, and visualization of the distribution in different parts of selenium-enriched shiitake mushrooms. Therefore, there is an urgent need to explore innovative application solutions of this technology in the field of selenium-enriched shiitake mushroom detection. Summary of the Invention

[0005] To address the technical problems of destructive sampling and lack of visualization of elemental distribution in existing selenium-enriched shiitake mushroom detection technologies, this invention proposes a method for identifying selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy. This method innovatively establishes a dual-mode discrimination model of "rapid identification-comprehensive characterization," which can effectively distinguish between selenium-enriched shiitake mushrooms and ordinary shiitake mushrooms, and provide more information for the quality evaluation of selenium-enriched shiitake mushrooms.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A method for identifying selenium-enriched shiitake mushrooms using μ-XRF includes the following steps:

[0008] (1) Sample preparation: Cut 5-10 mm thick complete slices along the longitudinal direction of the stipe, freeze them in liquid nitrogen for 10 min and then freeze-dry them (-80℃, 8Pa, 24h) to maintain the internal element distribution characteristics and ensure the slice shape is flat. The remaining mushroom body is reserved for other uses.

[0009] (2) Multiple detection points were selected in the cap area (including gills) and base of the stipe of freeze-dried shiitake mushroom slices. Single-point detection was performed using a μ-XRF analyzer. Based on whether the selected detection points contained selenium, it was preliminarily determined whether the mushroom was a selenium-rich shiitake mushroom.

[0010] (3) The surface of the mushroom initially identified as selenium-rich in step (2) was scanned using a μ-XRF analyzer. The final determination of whether the mushroom is selenium-rich was based on whether there were selenium-rich areas in the scan results.

[0011] The number of detection points in step (2) shall not be less than 3.

[0012] The location of the detection point includes the cap area (including gills) and the base of the stipe.

[0013] The dwell time for a single point during single-point detection is 300ms.

[0014] The method for determining whether a sample contains selenium in step (2) is as follows: if the net intensity of the Se Kα characteristic peaks at three or more detection points exceeds the bremsstrahlung background, the sample is marked as a potential selenium-rich sample and proceeds to the subsequent scanning steps.

[0015] The full scan in step (3) adopts a regional dynamic scanning strategy.

[0016] The scanning area of ​​the regional dynamic scanning strategy includes the cap, gills, and stipe.

[0017] The proposed regional dynamic scanning strategy has a scanning step size of 60μm, a single-point dwell time of 300ms, and a total scanning time of ≤20h.

[0018] The method for determining selenium-enriched shiitake mushrooms in step (3) is as follows: The selenium element surface scan results are analyzed in ImageJ, and the total area of ​​the cap (including gills) has a selenium element signal Δ_cap ≥ 4 counts and the stipe area has a selenium element signal Δ_stipe ≥ 3 counts. Here, Δ_cap is the average selenium element signal of the total area of ​​the cap (including gills) minus the background signal, and Δ_stipe is the selenium element signal of the stipe area minus the background signal.

[0019] In step (3), while detecting selenium, one or more of copper, zinc, iron and manganese are also detected.

[0020] The beneficial effects of this invention are:

[0021] (1) This invention proposes a rapid, non-destructive and comprehensive method for identifying selenium-enriched shiitake mushrooms. It can accurately determine whether a shiitake mushroom is selenium-enriched in a short time, and comprehensively characterize the distribution of selenium and other trace elements in the entire freeze-dried shiitake mushroom sample. This provides strong technical support for the development, production and quality control of selenium-enriched shiitake mushrooms.

[0022] (2) Non-destructive analysis: Non-contact micro-area X-ray excitation technology is used to directly perform in-situ non-destructive analysis on freeze-dried shiitake mushroom slices. The internal structure of the cap, gills and stipe can be completely exposed without grinding or digestion. The freeze-dried slices retain their morphology after the test and can be reused. This supports the reuse of the same sample in identification, retention, retesting and in-depth research, and does not affect subsequent analysis of metabolite composition, avoiding sample resource loss and data chain breakage caused by digestion in traditional methods.

[0023] (3) Rapid identification: This invention utilizes μ-XRF technology to preliminarily determine whether shiitake mushrooms are selenium-enriched shiitake mushrooms in a short time simply by analyzing the energy at the sampling points. This method can determine the selenium enrichment attribute of shiitake mushrooms in an extremely short time (seconds), with initial screening requiring only ≤5 minutes per sample, greatly improving identification efficiency. It is particularly suitable for rapid detection scenarios, such as real-time quality monitoring on shiitake mushroom production lines.

[0024] (4) Comprehensive Characterization: Unlike traditional methods that can only obtain the average content of elements, this invention uses high-resolution elemental surface scanning (mapping) technology to intuitively display the distribution characteristics of each element in shiitake mushrooms in the form of two-dimensional images. It can generate multi-element distribution heatmaps for samples up to 15cm×15cm, achieving distribution analysis: accurately locating selenium-rich areas (such as the transition zone between the gills and the main body of the cap), and combining the co-distribution maps of elements such as Fe and Zn, it can provide spatial omics basis for the breeding of selenium-rich shiitake mushroom varieties and subsequent product processing. This multi-element synergistic analysis capability helps to gain a deeper understanding of the nutritional composition of shiitake mushrooms and provides more comprehensive information.

[0025] (5) Ultra-large field of view coverage: Through multi-focal spot X-ray tube synergistic excitation technology and detector adaptive gain, ultra-large field of view coverage is achieved. While maintaining 20μm resolution, the single scan area reaches 15cm×15cm, covering typical selenium-rich shiitake mushroom fruiting bodies (cap diameter ≤8cm), supporting non-destructive analysis of whole mushroom surface, and significantly shortening the detection time to 18.5h.

[0026] (6) Easy to operate: Based on commercial μ-XRF devices (such as Bruker M4 TORNADO), the operation is simple and can be started after 1 hour of training. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Comparison of μ-XRF characteristic energy spectra of the cap region.

[0029] Figure 2 The images show μ-XRF selenium elemental scanning spectra of common shiitake mushrooms and selenium-enriched shiitake mushrooms.

[0030] Figure 3 This is a screenshot of the ImageJ window and an illustration of the ROI annotation.

[0031] Figure 4 The images show μ-XRF multi-element full-scan energy dispersive spectra of common shiitake mushrooms and selenium-enriched shiitake mushrooms.

[0032] Figure 5 This is a superimposed scanning energy spectrum of Se-Zn and Se-Fe.

[0033] Figure 6 This is a flowchart of the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Sample preparation

[0036] Select fresh shiitake mushrooms of uniform size and without mechanical damage. After gently wiping the surface with lint-free paper, use a ceramic blade to cut continuous longitudinal slices of 5-10 mm thickness along the stem, perpendicular to the cap (the slices must completely include the cap, gills, and stem structure). Immediately lay the slices flat on a freeze-drying tray, pre-freeze with liquid nitrogen for 10 minutes, and then freeze-dry (cold trap temperature -80℃, vacuum degree 8Pa, 24 hours) to ensure that the freeze-dried slices are flat and free of curling.

[0037] Example 1

[0038] A method for identifying selenium-enriched shiitake mushrooms using μ-XRF, the procedure is as follows: Figure 6 As shown, the specific steps include:

[0039] Quick identification of selenium-enriched shiitake mushrooms:

[0040] One group of commercially available common shiitake mushrooms and three groups of selenium-enriched shiitake mushrooms were selected as test samples. After freeze-drying (cold trap temperature -80℃, vacuum degree 8Pa, drying time 24h), the surface of the freeze-dried slices was polished to a smooth surface to avoid physical grinding. They were then fixed to a special sample holder with carbon adhesive and kept horizontal. Several detection points were selected on the freeze-dried shiitake mushroom samples, and single-point detection was performed using a Bruker M4 TORNADO μ-XRF analyzer. When the intensity of the Se-Kα characteristic peak at the detection point significantly exceeded the bremsstrahlung background, and the characteristic peak was detected at all detection points, it was preliminarily identified as selenium-enriched shiitake mushroom; the absence of a Se-Kα characteristic peak or the peak intensity not exceeding the background threshold indicated common shiitake mushrooms.

[0041] Specific steps for single-point detection: ① Place the freeze-dried shiitake mushroom slices (the sample surface must be flat) on the sample holder, ensuring the upper surface is level; ② Instrument parameters: X-ray tube: Rh target, voltage 50±1kV, current 500±10μA, detector: silicon drift detector (SDD), energy resolution ≤145eV; measurement mode: single point, spot size 20μm; environment: sample chamber vacuum ≤2kPa, automatic helium purging; ③ Using the instrument's built-in high-resolution CCD microscope, accurately locate and mark 3 detection points ≥10mm apart on the surface of the freeze-dried slice. Using the real-time image as a reference, adjust the XYZ axes of the sample stage to make the center of the spot coincide with the marked points. After ensuring that the microbeam accurately falls on the target area, start the acquisition; ④ Acquire the 0-40keV energy spectrum, focusing on reading the net peak area of ​​Se-Kα at 11.22keV. Note: Five repeated tests were performed on the same test point in the middle of the cap. The Se-Kα intensity RSD was ≤10% (meeting the accuracy requirements of ISO 3497:2000 nondestructive testing equipment).

[0042] The results show ( Figure 1 The ordinary shiitake mushroom sample only showed the matrix background (bremsstrahlung background) at 11.22 keV (Se Kα characteristic peak energy), and no Se Kα characteristic peak appeared, so it was determined that selenium was not detected. However, the selenium-enriched shiitake mushroom sample clearly showed the Se Kα characteristic peak on the background. Subsequently, the selenium enrichment area was further confirmed by multi-element spatial distribution characterization.

[0043] Multi-element spatial distribution characterization:

[0044] After single-point testing is completed, the following operations are performed on the same lyophilized sheet:

[0045] 1 Sample Placement

[0046] ① After single-point detection, move the lyophilized slice (still fixed in the original sample holder) to the sample stage of the μ-XRF analyzer; ② Use a high-precision CCD to confirm that the slice is still horizontal; ③ Adjust the XYZ axes of the sample stage so that the area to be imaged falls completely into the center of the scanning field of view; ④ Close the sample chamber, evacuate to ≤2kPa and turn on the automatic helium purging.

[0047] 2 Instrument Parameters

[0048] X-ray source: Rh target X-ray tube, 50kV / 600μA, with excitation energy covering Se-Kα (11.22keV) and trace elements such as Cu (8.04keV), Zn (8.63keV), Fe (6.40keV), and Mn (6.40keV).

[0049] Detector: Silicon Drift Detector (SDD), energy resolution <140eV.

[0050] Scanning mode: Area scanning (Mapping): Step size 50-100μm (60μm preferred), single point dwell time 200-400ms (300ms preferred), total scan time ≤20h (dynamically adjusted according to the actual sample area).

[0051] 3. Scanning and Data Acquisition

[0052] ① Start the Bruker M4 TORNADO program and first perform a full spectrum acquisition from 0 to 40 keV; ② Monitor the Se-Kα and Cu-Kα, Zn-Kα, Fe-Kα, and Mn-Kα characteristic lines in real time.

[0053] 4 Image Processing and Pseudo-Color Overlay

[0054] ① Use ESPRIT software to subtract the background and perform dead-time correction; ② Using the lowest background value as the baseline, map Se counts to a magenta color level, Zn counts to a cyan color level, and other elements as needed; ③ Perform pseudo-color overlay on Se and Zn to generate a Se-Zn co-distribution map; ④ Output Figure 2 (Se distribution) Figure 4 (Distribution of Se, Cu, Zn, Fe, Mn) Figure 5 (Se-Zn co-distribution).

[0055] 5ImageJ Semi-Quantitative Judgment Steps (1) Data Import

[0056] Save the Se-Kα count matrix output from the μ-XRF analyzer as a semicolon-separated text file named Se.txt. Replace all semicolons with spaces in Excel, then in ImageJ, execute File→Import→Text Image to generate a single-channel 32-bit grayscale image, named "Se".

[0057] (2) ROI drawing and naming

[0058] On SeMap:

[0059] Use the Polygon Selection tool to draw a closed region along the outermost edge of the cap (including the gill structure), such as... Figure 3 As shown, add an ROI Manager and name it "cap"; draw a pattern in the stipe region using the same method and name it "stipe"; draw patterns in the background region that is far from the fruiting body and on the same plane as the sample, add them to the ROI Manager and name them "background".

[0060] (3) Counting extraction

[0061] In the ROI Manager, select cap, stipe, and background in sequence, then execute Measure to obtain the Mean value in the Results table (unit: counts / pixel, already calibrated by the instrument's energy channel).

[0062] (4)Judgment

[0063] a) Measured in ImageJ

[0064] Δ_cap=Mean_cap-Mean_background

[0065] Δ_stipe=Mean_stipe-Mean_background

[0066] b) Criteria

[0067] If Δ_cap≥4counts and Δ_stipe≥3counts, then it is determined to be a selenium-enriched shiitake mushroom; otherwise, it is determined to be a regular shiitake mushroom.

[0068] 6. Limitations on Result Interpretation

[0069] Due to matrix effects and geometric differences between samples, this method is mainly used to compare the relative intensity and analyze the distribution characteristics (such as the signal intensity ratio between regions) of different regions within the same scanning field of view; direct and absolute content comparison cannot be made between slices from different batches or on different trays using μ-XRF signal intensity.

[0070] Using μ-XRF technology, element distribution maps are displayed in the form of two-dimensional color images, such as... Figure 2 As shown in Table 1, selenium is marked in purplish-red; the darker the color and the sharper the outline, the higher the degree of selenium enrichment, indicating a higher selenium content in the shiitake mushroom. Figure 2It can be seen that the distribution of selenium in selenium-enriched shiitake mushrooms and ordinary shiitake mushrooms is significantly different: ordinary shiitake mushrooms ( Figure 2 Selenium content in samples A (<0.1 mg / kg) was generally low, with the selenium element exhibiting a random, discrete distribution indistinguishable from the background. In contrast, selenium-enriched shiitake mushrooms (Figures B / C / D, selenium content ≥0.1 mg / kg) showed distinct high-selenium areas in regions such as the gills, and the distribution of selenium became increasingly clear as the selenium content in the mushroom increased. This method yielded consistent results in all three groups of commercially available shiitake mushroom samples, demonstrating good repeatability. Further comparison of the results with the total selenium content trend measured by ICP-OES showed a high degree of consistency, thus verifying the accuracy and reliability of this method.

[0071] Table 1 Figure 2 ROI count of *Lentinula edodes* and determination results based on the original criterion

[0072]

[0073]

[0074] *The selenium content is the actual value measured by ICP-MS in the same batch, used for method cross-validation.

[0075] Elemental distribution analysis:

[0076] In addition to selenium, distribution images of other trace elements such as copper, zinc, iron, and manganese were also generated, represented by blue, cyan, orange, and bright yellow, respectively. Figure 4 As shown, this study helps to further investigate the potential relationship between selenium enrichment and the spatial distribution of other nutrients (such as zinc, iron, and manganese) in selenium-enriched shiitake mushrooms, and is of great significance for a comprehensive understanding of the nutritional balance of shiitake mushrooms.

[0077] In addition to selenium, two-dimensional distribution maps of copper (blue), zinc (cyan), iron (orange), and manganese (bright yellow) were also collected simultaneously. Figure 4 As shown in the figure, the overall content and distribution pattern of copper in the two groups of samples are basically the same; the enrichment areas of selenium and zinc highly overlap, and the zinc signal is significantly enhanced in selenium-enriched shiitake mushrooms; as the selenium content increases, the iron signal weakens and the orange area shrinks significantly, suggesting that selenium may inhibit iron transport; the bright yellow area of ​​manganese also darkens, indicating that the manganese content decreases synchronously.

[0078] The above research provides a more comprehensive understanding of the distribution patterns of various elements in selenium-enriched shiitake mushrooms, offering a more accurate basis for quality evaluation and nutritional analysis.

[0079] Element co-distribution characterization

[0080] Figure 5A shows the co-distribution of Se and Zn in selenium-enriched shiitake mushrooms. It can be seen that the gill area shows a large area of ​​Se (purple-red) and Zn (blue-green) overlapping, and large areas of blue-purple patches in the gill area; Se is evenly distributed in the cap, while Zn is mainly concentrated in the gills and surface. Figure 5 B shows that after the addition of exogenous selenium, the co-distribution of Se-Zn in the cap area was significantly enhanced, and the overall bright blue-green area was significantly expanded.

[0081] Figure 5 CD represents the Se-Fe co-distribution map. The results show that regardless of the addition of exogenous selenium, Fe (orange) is consistently highly enriched on the sample surface, while the overlap with Se (purple-red) is less than 7%, indicating that selenium and iron are spatially independent. These results not only demonstrate that this method can effectively identify selenium-enriched shiitake mushrooms but also reveal the synergistic or independent distribution characteristics of multiple elements, providing a basis for further research on the interactions between nutrient elements.

[0082] In this embodiment of the invention, one group of commercially available common shiitake mushrooms and three groups of selenium-enriched shiitake mushrooms were used as representative samples for verification. It should be understood that the invention is not limited to the above-mentioned quantities. Those skilled in the art can select common shiitake mushrooms and selenium-enriched shiitake mushroom samples from different sources and in different quantities according to actual needs, all of which fall within the protection scope of this invention.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy, characterized in that, Includes the following steps: (1) Cut thin slices of shiitake mushrooms along the stipe-cap axis and freeze-dry them; (2) Select several detection points on freeze-dried shiitake mushroom slices and perform single-point detection using a micro-area X-ray fluorescence analyzer. Based on the intensity of the characteristic peak of selenium at the selected detection points, preliminarily determine whether it is a selenium-enriched shiitake mushroom. (3) Use a micro-area X-ray fluorescence spectrometer to perform elemental surface scanning on the surface of the mushroom initially identified as selenium-rich in step (2). Based on the spatial distribution characteristics of selenium in the scanning results, determine whether it is a selenium-rich mushroom.

2. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 1, characterized in that, The number of detection points in step (2) shall not be less than 3.

3. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 2, characterized in that, The selection of the detection point location includes the cap area and the base of the stipe, and the cap area contains gill structures.

4. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 3, characterized in that, The dwell time for a single point during single-point detection is 200-400ms.

5. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 4, characterized in that, The preliminary judgment method in step (2) is: the intensity of the Se Kα characteristic peak at all detection points exceeds the bremsstrahlung background.

6. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to any one of claims 1-5, characterized in that, The surface scanning in step (3) adopts a regional dynamic scanning strategy.

7. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 6, characterized in that, The scanning area of ​​the regional dynamic scanning strategy includes the cap and stipe.

8. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 7, characterized in that, The scanning step size of the regional dynamic scanning strategy is 50-100μm, the single-point dwell time is 200-400ms, and the total scanning time is ≤20h.

9. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 8, characterized in that, The method for determining selenium-enriched shiitake mushrooms in step (3) is as follows: the surface scan results of selenium elements are analyzed in ImageJ, and Δ_cap≥4counts and Δ_stipe≥3counts are obtained. Δ_cap is the selenium element signal of the entire cap area (including gills) minus the background signal, and Δ_stipe is the selenium element signal of the stipe area minus the background signal.

10. The method for determining selenium-enriched shiitake mushrooms using micro-area X-ray fluorescence spectroscopy according to claim 1, characterized in that, In step (3), while performing surface scanning detection on selenium, one or more elements among copper, zinc, iron and manganese are simultaneously detected, with a scanning energy range of 0-40 keV.

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