A method for determining the harvesting time of *Mammillaria fasciata* using gas phase ion mobility spectrometry.

Abstract

This invention discloses a method for determining the harvest time of *Mammillaria rubra* using gas phase ion mobility spectrometry (GC-IMS). The method includes GC-IMS detection, volatile odor molecule data analysis and visualization, and PCA analysis. This invention employs a simple and rapid GC-IMS technique combined with chemical analysis to directly analyze *Mammillaria rubra* samples with different labeled harvest times. The method determines the characteristic information regions of the samples by using retention time and ion migration time, and identifies the samples through principal component analysis combined with GC-IMS fingerprint analysis. By combining morphological observation of *Mammillaria rubra* strains and the intensity changes of ion peaks of four specific compounds in each of two strains, the harvest time of *Mammillaria rubra* can be quickly and accurately determined. This method is simple to operate, has a short sample detection time, and does not require complex sample pretreatment, making it highly valuable for application.

Description

Technical Field

[0001] This invention belongs to the field of fungal volatile gas molecule detection technology, specifically relating to a method for determining the harvest time of *Agaricus bisporus* using gas phase ion mobility spectrometry. Background Technology

[0002] With advancements in technology and the continuous improvement of people's living standards, fungi have gradually become a top choice on modern dining tables, greatly promoting the development of the edible fungi industry. Volatile aroma compounds in edible fungi are one of the most significant factors affecting their flavor. Changes in the quality of edible fungi products during their development not only seriously affect their flavor and nutritional components but are also closely related to food safety. Therefore, it is essential to track, evaluate, and test the flavor and safety of edible fungi during their development.

[0003] The spotted jade mushroom has rich nutritional and medicinal value, exhibiting anti-tumor (dose-dependent inhibition of human liver and colon cancer), tumor cell proliferation inhibition, antifungal activity, and free radical scavenging activities. It also boasts a long shelf life and rich aroma. Industrialized cultivation of spotted jade mushrooms utilizes modern industrial facilities to artificially control environmental conditions, creating environments suitable for its different developmental stages. This allows for three-dimensional, large-scale, year-round cultivation, achieving high yields and high profits. The spotted jade mushroom has the longest known production cycle among edible fungi. Due to the long production cycle and numerous production stages, it not only increases production costs but also makes it highly susceptible to contamination by other microorganisms. Currently, industrialized production of spotted jade mushrooms takes approximately 7-20 days from bud formation to harvest. Existing technology typically uses measurements of the mushroom cap diameter and stem length to roughly determine harvesting suitability, generally harvesting when the stem length is 11-13 cm and the cap diameter is 1.5-3 cm. However, determining the optimal harvest time is influenced by various factors, such as inconsistent maturity times under different cultivation and environmental conditions, making it impossible to uniformly harvest according to a fixed growth period. Morphological observation methods can only judge the appearance of the *Pleurotus ostreatus* product; even when the strain's morphology meets standard values, it may not have reached its optimal flavor. Therefore, it is essential to establish a more precise method for determining the optimal harvest time of *Pleurotus ostreatus* to select the best harvest time in industrial production. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] Currently, the methods for determining the harvest time of edible fungi produced in industrial settings mainly involve measuring the external appearance of the fungi, such as the diameter of the cap and the length of the stem, or judging based on experience after the budding stage. These methods lack precision. With the development of methods for detecting volatile aroma compounds, this invention utilizes gas chromatography-ion mobility spectrometry (GC-IMS) to efficiently, rapidly, and in real-time detect volatile aroma molecules in multiple edible fungi samples. This allows for the acquisition of accurate flavor information and precise determination of the harvest time for commercially available edible fungi.

[0006] This invention primarily employs gas chromatography-ion mobility spectrometry (GC-IMS), an emerging comprehensive analytical technique for volatile organic compounds. This technique combines the high selectivity of chromatographic separation with the high sensitivity of IMS (detection limits can be as low as ppbv). It has advantages such as simple sample preparation, fast response speed, short analysis time, and high sensitivity. This technique can rapidly, non-destructively, and with high throughput detect and screen volatile components without the need for pretreatment and concentration. The content of volatile odor molecules in different samples can be compared using fingerprint spectra, making the flavor components intuitively visible.

[0007] The specific operating method for determining the harvest period of edible fungi by detecting volatile gas molecules as described in this invention includes the following steps:

[0008] (1) Step 1: Collection of *Mammillaria lobata* samples. Samples of *Mammillaria lobata* were collected and tested at four time points: 15, 17, 18, and 20 days after scratching and continued growth. After collection, the edible fungi products were wrapped in modified air packaging (MAP) at 4℃ and tested immediately. For the white strain of *Mammillaria lobata*, the 15-day sample was an immature *Mammillaria lobata* with a cap diameter less than 1 cm. The 20-day sample was a fully mature *Mammillaria lobata* with a cap diameter of 1.5-2 cm and not yet flattened. The 17- and 18-day samples were nearly mature *Mammillaria lobata* with a cap diameter of 1 cm and similar morphology. For the brown strain of *Mammillaria lobata*, the 15-17 day samples were immature *Mammillaria lobata* with a cap diameter less than 0.5 cm and similar morphology. The 18-day sample was nearly mature *Mammillaria lobata* with a cap diameter greater than 1 cm. The 20-day sample was tested and found to be a fully mature spotted mushroom sample, with a cap diameter of 1.5-2 cm and not yet flattened. After scratching for 20 days, the flavor of the spotted mushroom strain no longer showed significant changes.

[0009] (2) Step 2, Gas-phase ion mobility spectrometry detection: GC-IMS ( Sample analysis was performed using a combined platform (Gesellschaftfür Analytische Sensorsysteme mbH, Dortmund, Germany). A 490 gas chromatograph (Agilent, USA) equipped with an MXT-WAX capillary column (30m × 0.53mm), an autosampler (SolidPhase Micro Extraction, 57330-U, Supelco, USA), and a headspace sampling device. Before the experiment, the modified air packaging (MAP) was removed, and the entire *Mammillaria lobata* sample was separated into caps and stipes for separate analysis. 1.2g of *Mammillaria lobata* caps or stipes were weighed, chopped, and placed in a 20mL headspace vial. After incubation at 55°C for 20 minutes, the sample was injected. Then, the autosampler (CTC Analytics AG, Zwingen, Switzerland) used the instrument's built-in 1mL sealed heated syringe (80°C, non-split mode) to directly sample from the 500μL headspace vial at 55°C. GC-IMS program settings: The following program was followed: 2 mL / min, duration 0–2 min; 100 mL / min, duration 2–45 min; elution and separation of analytes at 60 °C. The analytes were driven to the ionization chamber and ionized in positive ion mode by a 3H ionization source with 300 MBq activity. The generated ions were driven to a drift tube (9.8 cm long), which operated at a constant temperature (45 °C) and voltage (5 kV). Both the carrier gas and purge gas were 99.999% high-purity nitrogen, and the drift gas (nitrogen) was set to 150 mL / min. Each spectrum report is the average of 12 scans. All analyses were performed in triplicate. The syringe was automatically flushed with a nitrogen stream 5 min before and after each analysis to avoid cross-contamination. Three-dimensional fingerprint information spectra of the *Mammillaria fasciata* samples were obtained by GC-IMS detection. The instrument's accompanying analytical software includes LAV (Laboratory Analytical Viewer) and three plugins: Reporter, Gallery Plot, and Dynamic PCA, as well as GC×IMS Library Search. It enables qualitative and relative quantification of volatile odor molecules. The application software's built-in NIST and IMS databases allow for qualitative analysis of substances. Using n-ketone C4-C9 (China National Pharmaceutical Group Chemical Reagent Beijing Co., Ltd.) as an external reference, the retention index (RI) of volatile compounds is calculated, and the RI is also calculated using an automated mass spectrometry deconvolution and identification system. Volatile compounds are identified by comparing the RI with the drift time (the time required for ions to reach the collector through the drift tube, in milliseconds) of standards in the GC-IMS library, and the content of volatile compounds is quantified based on the peak intensity in HS-GC-IMS.

[0010] (3) Step 3, Selection of Spectral Feature Regions: Extract feature value regions based on the three-dimensional fingerprint information spectrum of the samples obtained in Step 2. Based on the color difference or peak intensity signal of the characteristic substances in the fingerprint information spectrum, select spectral feature regions where the color change of substances is obvious or the peak intensity difference is large between samples of *Mammillaria fasciata* at different development times.

[0011] (4) Step four: Analysis and visualization of volatile odor molecule data. SPSS software was used to standardize the HS-GC-IMS data for heatmap clustering, and principal component analysis (PCA) was used with the SIMCA 14.1 software package (Umetrics) to assess similarities and differences. One-way ANOVA was performed using Origin version 9.0 software (OriginLab Co., Northampton, Massachusetts) to determine significant differences between samples. LAV software (GAS, Dortmund, Germany) was used to view and process the TP-HS-GC-IMS data. PCA and PLS-DA were performed using SIMCA version 14.0 (Umetrics). The study was conducted in Sweden. Heatmap analysis and hierarchical clustering analysis were performed using Ihanbo bioinformatics. Principal component analysis was used to differentiate *Mammillaria fasciata* samples at different developmental stages.

[0012] (5) Step 5: Selection of marker compounds for the harvesting period of white strains of *Mammillaria rubra*: Based on the differences in peak intensity signals, aroma compounds that can significantly distinguish white strains of *Mammillaria rubra* at different harvesting times are selected from the characteristic regions of the fingerprint spectrum of the cap and stipe of white strains of *Mammillaria rubra* in Step 3. The optimal harvesting time of *Mammillaria rubra* is accurately determined by using marker aroma compounds. Based on the differences in peak intensity signals and the corresponding retention and migration times of the substances, four substances that can significantly distinguish mature white strains of *Mammillaria rubra* are selected from the characteristic regions in Step 3: Among them, substance 1 ((E)-2-heptenal) has a gas phase retention time of 579.021 sec and an ion drift time of 1.26 ms, which is used to further confirm the mature and immature white strains of *Mammillaria rubra* in the principal component analysis. When the peak intensity of this substance in the stipe is 2993.13±134.53V, it is a fully mature white *Mammillaria rubra*; substance 2 (3-octanone) has a gas phase retention time of 471.824 sec and an ion drift time of 1.26 ms. The ion drift time of substance 3 (3-octanol) was 1.3 ms, and the peak intensity of this substance in the cap was 9208.46±32.69 V, indicating that the white spotted mushroom was fully mature. The gas phase retention time of substance 3 (3-octanol) was 757.96 sec, and the ion drift time was 1.78 ms. The peak intensity of this substance in the cap was 12684.47±395.76 V, indicating that the white spotted mushroom was fully mature. The gas phase retention time of substance 4 (2-pentylfuran) was 440.9 sec, and the ion drift time was 1.26 ms. The peak intensity of this substance in the cap was 1023.32±78.95 V, indicating that the white spotted mushroom was fully mature.

[0013] (6) Step six, selection of characteristic compounds for the harvesting period of brown strain of spotted mushroom: Based on the difference in the intensity signal of the substance peak, select aroma compounds that can significantly distinguish brown strains of spotted mushroom at different harvesting times from the fingerprint spectrum characteristic region of brown strain of spotted mushroom in step three, and use the characteristic aroma compounds to accurately determine the optimal harvesting time of spotted mushroom. Based on the differences in peak intensity signals and the corresponding retention and migration times of the substances, three substances that can significantly distinguish mature brown strains of *Mammillaria rubra* were selected from the characteristic region in step three: Substance 1 (tert-butanol) had a gas phase retention time of 349.13 sec and an ion drift time of 1.18 ms, and was used to further confirm mature brown strains of *Mammillaria rubra* in principal component analysis. When the peak intensity of this substance in the cap was 132.42 ± 5.86 V, it indicated that the brown strain was mature. Substance 2 (diisobutanol) had a gas phase retention time of 307.27 sec and an ion drift time of 1.17 ms. In overly mature or immature brown strains of *Mammillaria rubra*, the peak intensity of this substance in the cap was less than 2160.04 ± 63.32 V. Substance 3 (diisobutanol) in the stipe of overly mature or immature brown strains of *Mammillaria rubra* had a peak intensity less than 4381.42 ± 68.24 V.

[0014] This invention relates to the application of gas chromatography-ion mobility spectrometry (GC-IMS) technology for identifying the optimal harvest time of edible fungi in the industrial production of *Mammillaria lobata*. A difference map is obtained by subtracting from a topographic map, which visually displays the differences in test data. If the volatile compounds are consistent, the subtracted background is white; red indicates a concentration higher than the reference, and blue indicates a concentration lower than the reference. The 3D topographic view after background subtraction clearly identifies the changes in VOCs during the development of *Mammillaria lobata* samples.

[0015] This invention is achieved through GC-IMS was used to analyze the volatile organic compound (VOC) fingerprints of edible fungi samples from different harvest time points. The compounds were characterized by comparing their IMS drift times and retention indices with those of real reference compounds. Due to differences in concentration, some individual compounds were observed to produce signals or spots with varying brightness. Comparison of VOC fingerprints allows for a more refined analysis of the changes in individual compounds during the storage of edible fungi samples.

[0016] Based on GC-IMS data, this invention uses principal component analysis (PCA) based on the signal intensity of compounds to highlight the differences in the overall composition of volatile compounds in *Mammillaria fasciata* samples from different harvest times. This can effectively distinguish the quality differences between white and brown strains of *Mammillaria fasciata* from different harvest times.

[0017] One application of the aforementioned method involves using gas phase ion mobility spectrometry to obtain four specific compounds each from the white and brown strains of *Mammillaria fasciata* to be tested, thereby identifying the optimal harvest time for the white and brown strains of *Mammillaria fasciata*.

[0018] The beneficial effects of this invention are as follows: This invention employs a simple and rapid gas phase-ion mobility spectrometry (GC-IMS) technique combined with chemical analysis to directly analyze *Mammillaria lobata* samples with different labeled harvest times. The characteristic information regions of the samples are determined by retention time and ion migration time, and identification is performed through principal component analysis combined with GC-IMS fingerprint analysis. By combining morphological observation of *Mammillaria lobata* strains and the changes in ion peak intensity of four specific compounds in each of the two strains, the harvest time of *Mammillaria lobata* can be quickly and accurately determined. This method is simple to operate, has a short sample detection time, and does not require complex sample pretreatment, making it highly valuable for application.

[0019] This invention can quickly and effectively determine the flavor changes of *Mammillaria fasciata* samples during their development by analyzing 3D topographic views of volatile compounds, volatile organic compound fingerprints, and principal component analysis diagrams. It also identifies the marker compounds of the *Mammillaria fasciata* maturation stage, providing a direct basis for determining the harvesting period of *Mammillaria fasciata* products, and has important value in industrial production. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments 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. Wherein:

[0021] Figure 1 Three-dimensional topographic map of volatile compounds in a white strain of Hypsizygus mamoreus.

[0022] Figure 2 Fingerprint of volatile organic compounds for white strains of *Mammillaria fasciata*

[0023] Figure 3 PCA analysis of volatile organic compounds in the cap and stipe of white strains of *Mammillaria fasciata* harvested at different times.

[0024] Figure 4 Three-dimensional topographic map of volatile compounds in a brown strain of *Mammillaria rubra*.

[0025] Figure 5 Fingerprint of volatile organic compounds of brown strain of *Mammillaria rubra*

[0026] Figure 6 PCA analysis of volatile organic compounds (VOCs) in the caps and stipes of brown strains of *Mammillaria rubra* harvested at different times. Detailed Implementation

[0027] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0028] Example 1:

[0029] A method for rapidly determining the harvest time of *Mammillaria rubra* using gas phase ion mobility spectrometry: In this embodiment, the caps of white *Mammillaria rubra* strains at 15, 17, 18, and 20 days after scratching were used as the detection objects. Gas phase-ion mobility spectrometry was employed, and four samples were tested under identical conditions. Each group of samples was tested three times, resulting in a total of 12 *Mammillaria rubra* samples with three-dimensional fingerprint spectra. The 15-18 day samples were from immature white *Mammillaria rubra* strains, while the 20 day samples were from mature white *Mammillaria rubra* strains.

[0030] Step (1): Preparation of *Mammillaria fasciata* samples: White strains of *Mammillaria fasciata* were cultivated by Shanghai Fengke Biotechnology Co., Ltd., China. Samples were collected 15, 17, 18, and 20 days after scratching and wrapped in modified air packaging (MAP). The samples were then refrigerated at 4°C and transported to the laboratory for immediate testing. Before the experiment, the packaging was removed, and the samples were divided into cap and stipe parts. The cap part was then tested.

[0031] Step (2), GC-IMS detection and three-dimensional spectral analysis. The white variety of *Mammillaria leucocephala* was unpacked and separated into caps and stems. 1.2g of cap or stem was weighed, chopped, and placed in a 20mL headspace vial. After incubation at 55℃ for 20 minutes, the sample was injected. The gas chromatography-ion mobility spectrometry (GC-IMS) instrument used an integrated capillary column, MXT-WAX (30m × 0.53mm), and the ionization source was tritium (…). 3 The carrier gas and purge gas were both high-purity nitrogen (99.999%). Before the detection began, the gas chromatography-ion mobility spectrometry (GC-IMS) instrument was purged for 3 hours to remove any residual components from the previous detection. After purging, 100 μL of gas was extracted from the headspace vial for detection. The instrument's carrier gas flow rate was set to 2.0 mL / min, the drift tube temperature to 45℃, the injector temperature to 80℃, and the analysis time for each sample was 45 min, thus obtaining the three-dimensional fingerprint information spectrum of the sample.

[0032] Step (3), selection of three-dimensional fingerprint information spectrum and spectrum feature region: the feature value region on the fingerprint information spectrum obtained in step two is extracted by comparison method. The comparison method selects the spectrum feature region where the color change of the characteristic compound of the spotted mushroom is obvious or the peak intensity difference is large at different harvesting times based on the color difference or peak intensity signal of the characteristic compound of the spotted mushroom in the fingerprint information spectrum.

[0033] Step (4), volatile odor molecule data analysis and visualization. Qualitative and relative quantification of volatile odor molecules. Using n-ketone C4-C9 (China National Pharmaceutical Group Chemical Reagent Beijing Co., Ltd.) as an external reference, the retention index (RI) of volatile compounds was calculated, and the RI was also calculated using an automated mass spectrometry deconvolution and identification system. Qualitative analysis of substances was performed using the NIST and IMS databases built into the application software. Volatile compounds were identified by comparing the RI with the drift time (the time required for ions to reach the collector through the drift tube, in milliseconds) of standards in the GC-IMS library.

[0034] Step (5) uses SPSS 19.0 data processing software to perform orthogonal transformation and dimensionality reduction on the peak intensity data of all feature regions selected in Step 3, enabling rapid classification of cap samples of white *Mammillaria fasciata* at 12 different developmental stages. The cumulative variance contribution rates of the cap samples in PCA analysis of the white strains were 35% and 52%, respectively. As shown in the PCA diagram, the cap samples of white strains in the early growth stage (15d) clustered in the lower left corner, the samples in the later growth stage (20d) clustered in the lower right corner, and the samples in the middle growth stage (17-18d) were located in the middle region.

[0035] Step (6) Establishment of a method for detecting the harvest time of white strains of *Mammillaria rubra*: Based on the differences in peak intensity signals and the corresponding retention and migration times of the substances, significantly different compounds that can distinguish *Mammillaria rubra* from different harvest times are selected from the fingerprint spectrum feature region of step (3). Combined with the qualitative and quantitative analysis of compounds in step (4), the harvest time can be accurately and quickly determined. Specific analysis is as follows:

[0036] The gas phase retention time of substance 1 ((E)-2-heptenal) was 579.021 sec, and the ion drift time was 1.26 ms. Its peak intensity was lowest (744.96 V) in the stipe samples of the white strain of *Mammillaria rubra* harvested at 15 days, while it increased to 1798.42 V and 2404.9 V in samples harvested at 17 and 18 days, respectively. Subsequently, the peak intensity of (E)-2-heptenal in the stipes of 20 days was 2913.69 V, which was 2.41, 3.23, and 3.91 times that of the immature samples harvested at 15 days. This indicates that (E)-2-heptenal can clearly distinguish between mature and immature white strains of *Mammillaria rubra*, and that this substance had the highest peak intensity and content in the stipes of the white strain of *Mammillaria rubra* harvested at 20 days. See Tables 1 and 2 for details.

[0037] The gas phase retention time of substance 2 (3-octanone) was 471.824 sec, and the ion drift time was 1.3 ms. In the cap samples of immature white strain of *Mammillaria rubra* at 15 days, the peak intensity of this substance was 2333.22 V. In the samples at 17-18 days, the peak intensity of this substance did not change much, at 5026.78 V and 5258.88 V respectively. In the caps of fully mature white *Mammillaria rubra*, the peak intensity of this substance increased sharply, reaching 9246.02 V.

[0038] The gas phase retention time of substance 3 (3-octanol) was 757.96 sec, and the ion drift time was 1.78 ms. In the cap samples of the immature white strain of *Mammillaria fasciata* at 15 days, the peak intensity of this substance was 320.94 V. In the samples at 17 days and 18 days, the peak intensity of this substance did not change much, at 362.49 V and 402.99 V respectively. However, in the cap samples at 20 days, the peak intensity of this substance increased significantly to 13080.22 V, which was 40.76 times that of the immature 15-day sample. This clearly distinguishes the mature white strain of *Mammillaria fasciata*.

[0039] The gas phase retention time of substance 4 (2-pentylfuran) was 440.9 sec, and the ion drift time was 1.26 ms. In the cap of the immature white strain of *Mammillaria fasciata* at 15 days, the peak intensity of this substance was 110.41 V. In the samples at 17 days and 18 days, the peak intensity of this substance did not change much, at 463.95 V and 366.84 V respectively. In the cap of the fully mature white strain of *Mammillaria fasciata* at 20 days, the peak intensity of this substance increased to 1120.02 V.

[0040] In summary, the present invention utilizes GC-IMS technology, combined with volatile organic compound fingerprinting and principal component analysis, to rapidly and effectively determine the optimal harvest time for the white strain of *Mucuna macrocarpa* by detecting the peak intensity changes of four differentially expressed volatile substances at different harvest times.

[0041] Table 1. Changes of four differentially volatile substances in *Mucuna macrocarpa* white strain at different harvest times.

[0042]

[0043] Example 2:

[0044] Analysis of the differences in volatile aroma components of brown strains of *Mammillaria rubra* at different harvest times:

[0045] The specific steps are as follows:

[0046] (1) Step 1: Samples of the brown strain of *Imperata cylindrica* were collected at 15, 17, 18, and 20 days after cultivation by Shanghai Fengke Biotechnology Co., Ltd., China. The samples were wrapped in modified air-packed containers, refrigerated at 4℃, and transported to the laboratory for immediate testing. Before the experiment, the packaging was removed, and the samples were separated into cap and stipe parts for separate testing. Samples of the brown strain of *Imperata cylindrica* at 15-17 days were immature *Imperata cylindrica*, with cap diameters less than 0.5 cm and similar morphology. Samples at 18 days were nearly mature *Imperata cylindrica*, with cap diameters greater than 1 cm. Samples at 20 days were fully mature *Imperata cylindrica*, with cap diameters reaching 1.5-2 cm and not yet flattened. Three parallel samples were set up for each group, for a total of 12 samples. 1.2 g of *Imperata cylindrica* cap or stipe was weighed, chopped, and placed in a 20 mL headspace vial. The vials were incubated at 55℃ for 20 minutes before injection.

[0047] (2) Step Two: GC-IMS Detection and Three-Dimensional Spectrum Analysis. After harvesting, the brown strain of *Mammillaria rubra* was unpacked at 4℃ using modified atmosphere packaging technology, separating the caps and stipes. The samples were then analyzed using a GC-IMS platform. GC-IMS detection conditions: The gas chromatography-ion mobility spectrometry (GC-IMS) system used an integrated capillary column, MXT-WAX (30m × 0.53mm), and the ionization source was tritium (…). 3 The carrier gas and purge gas were both high-purity nitrogen (99.999%). Before the detection began, the gas chromatography-ion mobility spectrometry (GC-IMS) instrument was purged for 3 hours to remove any residual components from the previous detection. After purging, 100 μL of gas was extracted from the headspace vial for analysis. The instrument's carrier gas flow rate was set to 2.0 mL / min, drift tube temperature to 45 °C, and injector temperature to 80 °C. The analysis time for each sample was 45 min, thus obtaining the three-dimensional fingerprint information spectrum of the tested samples. Most of the red signals from the brown strains of *Mammillaria rubra* (cap and stipe) retained in the range of 200-800 s, with a drift time of 1.0-1.5 s.

[0048] (3) Step 3: Construct and analyze the IMS fingerprints of volatile compounds in the cap and stipe of the brown variety of *Mammillaria rubra*. Three-dimensional fingerprint information spectrum and selection of spectral feature regions: A comparative method is used to extract the feature value regions from the fingerprint information spectrum obtained in Step 2. This comparative method selects spectral feature regions where the color changes or peak intensity differences of the characteristic compounds of *Mammillaria rubra* are significant at different harvesting times, based on the color variations or peak intensity signals in the fingerprint information spectrum.

[0049] (4) Step Four: Data Analysis and Visualization of Volatile Odor Molecules. Qualitative and relative quantitative analysis of volatile odor molecules. Using n-ketone C4-C9 (China National Pharmaceutical Group Chemical Reagent Beijing Co., Ltd.) as an external reference, the retention index (RI) of volatile compounds was calculated, and the RI was also calculated using an automated mass spectrometry deconvolution and identification system. Qualitative analysis of substances was performed using the NIST and IMS databases built into the application software. Volatile compounds were identified by comparing the RI with the drift time (the time required for ions to reach the collector through the drift tube, in milliseconds) of standards in the GC-IMS library.

[0050] (5) In step five, PCA analysis was performed on the volatile organic compounds (including 3 parallels) of the cap and stipe of the brown variety of Mushroom Species at four different growth stages. The cumulative variance contribution rates were 52% and 21% (cap) and 68% and 17% (stipe), respectively.

[0051] (6) Establishment of a method for detecting the harvest time of brown strains of *Mammillaria rubra*: Based on the differences in peak intensity signals and the corresponding retention and migration times of the substances, two significantly different compounds that can distinguish *Mammillaria rubra* from different harvest times were selected from the fingerprint spectral feature region in step three. Combined with the qualitative and quantitative analysis of the compounds in step four, a precise and rapid determination of the harvest time was achieved. Specific analysis is as follows:

[0052] The gas phase retention time of substance one (tert-butanol) was 349.13 sec, and the ion drift time was 1.18 ms. It was used to further confirm the mature and immature brown strains of *Mammillaria fasciata* in the principal component analysis. In the cap samples of immature brown strains of *Mammillaria fasciata* at 15 days, the peak intensity of this substance was 70.94 V. In the samples at 17 days and 18 days, the peak intensity of this substance did not change much, at 92.28 V and 108.39 V respectively. However, in the cap samples at 20 days, the peak intensity of this substance increased significantly to 131.89 V, which can clearly distinguish the mature white strains of *Mammillaria fasciata*.

[0053] The gas phase retention time of substance 2 (isobutanol) was 307.27 sec, and the ion drift time was 1.17 ms. In the cap samples of brown strains of *Isobutanol* at 15d and 17d, the peak intensity of this substance did not change much, at 1122.1V and 1321.45V respectively. In the cap samples of immature brown strains at 18d, the peak intensity of this substance was 1794.9V. However, in the cap samples at 20d, the peak intensity of this substance increased significantly to 2192.12V, which can clearly distinguish the mature brown strains of *Isobutanol*.

[0054] The peak intensity of substance 2 (isobutanol) in the stipe of brown strain of Mushroom flounder harvested at 15 days was 736.98V, the peak intensity of the substance in the stipe of 17 days was 857.69V, and the peak intensity of the substance in the stipe of mature brown strain of Mushroom flounder harvested at 18-20 days increased sharply to 2844.02V-4334.15V.

[0055] Table 2. Peak intensity variations of four differentially volatile substances in the brown variety of *Mucuna macrocarpa* at different harvest times.

[0056]

[0057]

[0058] Table 3 Analysis Conditions

[0059]

[0060] Table 4 Gas Chromatography Conditions

[0061] Time E1 E2 R 00:00,000 150 mL / min 2ml / min rec 02:00,000 150 mL / min 2ml / min - 20:00,000 150 mL / min 100ml / min 45:00,000 150 mL / min 100mL / min stop

[0062] Figure 1 Three-dimensional topographic map of volatile compounds in a white strain of Hypsizygus marmoreus.

[0063] Among them: (A) Three-dimensional topographic maps of volatile compounds in the cap samples of white strains at different harvesting times; (B) Three-dimensional topographic maps of volatile compounds in the stipe samples of white strains at different harvesting times. Sample harvesting time: 15, 17, 18, and 20 days after scratching.

[0064] Figure 2 Fingerprint of volatile organic compounds from white strains of *Mammillaria fasciata*

[0065] The comparison included: (A) a comparison of fingerprint spectra of volatile compounds in cap samples of white strains collected at different harvest times; and (B) a comparison of fingerprint spectra of volatile compounds in stipe samples of white strains collected at different harvest times. Sample harvest times: 15, 17, 18, and 20 days after scratching.

[0066] Figure 3 PCA analysis of volatile organic compounds in the caps and stipes of white strains of *Mammillaria fasciata* harvested at different times.

[0067] Among them: (A) PCA analysis diagram of volatile compounds in cap samples of white strains at different harvesting times; (B) PCA analysis diagram of volatile compounds in stipe samples of white strains at different harvesting times. Sample harvesting time: 15, 17, 18, and 20 days after scratching.

[0068] Figure 4Three-dimensional topographic map of volatile compounds in a brown strain of *Mammillaria fasciata*.

[0069] Among them: (A) Three-dimensional topographic maps of volatile compounds in cap samples of brown strains at different harvesting times; (B) Three-dimensional topographic maps of volatile compounds in stipe samples of brown strains at different harvesting times. Sample harvesting times: 15, 17, 18, and 20 days after scratching.

[0070] Figure 5 Fingerprint of volatile organic compounds from brown strains of *Mammillaria fasciata*

[0071] The comparison included: (A) a comparison of fingerprint spectra of volatile compounds in cap samples of brown strains collected at different harvest times; and (B) a comparison of fingerprint spectra of volatile compounds in stipe samples of brown strains collected at different harvest times. Sample harvest times: 15, 17, 18, and 20 days after scratching.

[0072] Figure 6 PCA analysis of volatile organic compounds in the cap and stipe of brown strains of *Mammillaria fasciata* at different harvest times.

[0073] Among them: (A) PCA analysis diagram of volatile compounds in cap samples of brown strains from different harvesting times; (B) PCA analysis diagram of volatile compounds in stipe samples of brown strains from different harvesting times. Sample harvesting time: 15, 17, 18, and 20 days after scratching.

[0074] In summary, the GC-IMS technology described in this invention, combined with the volatile organic compound fingerprint spectrum and principal component analysis, can rapidly and effectively determine the optimal harvest time for the brown strain of *Mucuna macrocarpa* by detecting the changes in peak intensity of four differentially expressed volatile substances at different harvest times. This technology holds promise for large-scale application in determining the optimal harvesting time during *Mucuna macrocarpa* production and has significant value in industrial applications.

[0075] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for determining the harvesting time of *Mammillaria rubra* using gas phase ion mobility spectrometry, characterized in that: include, Gas phase-ion mobility spectrometry (GC-IMS) detection: The cap and stipe samples of white or brown spores of *Mammillaria fasciata* were subjected to GC-IMS detection to obtain fingerprint information spectra of the samples; based on the peak intensity signals of characteristic compounds of *Mammillaria fasciata* in the fingerprint information spectra, spectral feature regions with large differences in peak intensity at different harvesting times were selected. Volatile odor molecule data analysis and visualization: Calculate the retention index of volatile compounds, perform qualitative analysis of volatile compounds, and identify volatile compounds; PCA analysis was performed. Based on the differences in peak intensity signals and the corresponding retention and migration times of the substances, significantly different compounds that could distinguish *Mammillaria fasciata* from different harvest times were selected. Among them, the gas phase retention time of (E)-2-heptenal in the stipe of the white *Mammillaria fasciata* species was 579.021 sec, and the ion drift time was 1.26 ms; the gas phase retention time of 3-octanone in the cap of the white *Mammillaria fasciata* species was 471.824 sec, and the ion drift time was 1.3 ms; the gas phase retention time of 3-octanol in the cap of the white *Mammillaria fasciata* species was 757.96 sec, and the ion drift time was 1.78 ms; the gas phase retention time of 2-pentylfuran in the cap of the white *Mammillaria fasciata* species was 440.9 sec, and the ion drift time was 1.26 ms. The gas phase retention time of tert-butanol in the cap of the brown spore of *Mammillaria rubra* was 349.13 sec, and the ion drift time was 1.18 ms. The gas phase retention time of isobutanol in the brown spore of *Mammillaria rubra* was 307.27 sec, and the ion drift time was 1.17 ms. The gas chromatography-ion mobility spectrometry (GC-IMS) detection was performed using a GC-IMS combined platform. The 490 gas chromatograph was equipped with an MXT-WAX capillary column, an autosampler, and a headspace sampling device. 1.2 g of the cap or stipe of the *Mammillaria fasciata* was weighed, chopped, and placed in a headspace vial. After incubation at 55 °C for 20 min, the sample was injected, and then the autosampler directly extracted the sample from the headspace vial at 55 °C. The gas phase-ion mobility spectrometry (GC-IMS) detection was programmed as follows: 2 mL / min for 0-2 min; 150 mL / min for 2-45 min; elution and separation of the compound were performed at 60 °C, and the compound was driven into the ionization chamber by a ionization agent with an activity of 300 MBq. 3 The H ionization source ionizes in positive ion mode, and the generated ions are driven to the drift tube, which operates at a constant temperature of 45 °C and a voltage of 5 kV. The drift gas is nitrogen gas with a flow rate of 150 mL / min. When the peak intensity of (E)-2-heptenal in the stipe of the white spore of *Mammillaria fasciata* reaches 2856.6 V, the peak intensity of 3-octanone in the cap reaches 9186.299 V, the peak intensity of 3-octanol in the cap reaches 12377.61 V, and the peak intensity of 2-pentylfuran in the cap reaches 944.37 V, it indicates that the white spore of *Mammillaria fasciata* is mature and ready for harvesting. When the peak intensity of tert-butanol in the cap of the brown spore-bearing mushroom reaches 126.56 V, the peak intensity of isobutanol in the cap reaches 2096.72 V, and the peak intensity of isobutanol in the stipe reaches 4313.18 V, it indicates that the brown spore-bearing mushroom is mature and ready for harvesting.

2. The method for determining the harvesting time of *Mammillaria pulcherrima* by gas phase ion mobility spectrometry according to claim 1, characterized in that: The retention index of volatile compounds is calculated using n-ketone C4-C9 as an external reference.

3. The method for determining the harvesting time of *Mammillaria lobata* by gas phase ion migration spectroscopy according to claim 1 or 2, characterized in that: The PCA analysis includes standardizing the data using SPSS software for heatmap clustering.

4. The method for determining the harvesting time of *Mammillaria pulcherrima* by gas phase ion mobility spectrometry according to claim 1 or 2, characterized in that: The PCA analysis included using Origin version 9.0 software to determine significant differences between different samples through one-way ANOVA.

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