Lucid ganoderma identification method based on time-of-flight mass spectrometry

Abstract

The invention discloses a ganoderma lucidum identification method based on time-of-flight mass spectrometry, and relates to the technical field of ganoderma lucidum identification, and the method comprises the following steps: obtaining a first to-be-identified protein fingerprint spectrum of a to-be-detected sporocarp; determining a first target protein fingerprint spectrum corresponding to the first to-be-identified protein fingerprint spectrum in a ganoderma lucidum protein fingerprint database; and determining the variety and growth time of the to-be-detected sporocarp according to the first target protein fingerprint spectrum. The method comprises the following steps: determining a first target protein fingerprint spectrum corresponding to a first to-be-identified protein fingerprint spectrum in a ganoderma lucidum protein fingerprint database on the basis of a corresponding relation among a sporocarp protein fingerprint spectrum, a variety and growth time in the ganoderma lucidum protein fingerprint database, the growth time of the to-be-detected sporocarp can be determined according to the variety and the growth time corresponding to the first target protein fingerprint spectrum, so that the growth time of the sporocarp is identified.

Description

Technical Field

[0001] The present application relates to the technical field of Ganoderma lucidum identification, and in particular to a Ganoderma lucidum identification method based on time-of-flight mass spectrometry. Background Art

[0002] Ganoderma lucidum includes different forms such as Ganoderma lucidum fruiting bodies, spores and mycelium. The growth time of Ganoderma lucidum fruiting bodies is different, and their market value is also different.

[0003] The most commonly used technologies for species identification are genome sequencing and fluorescent quantitative polymerase chain reaction (PCR) technology. Genome sequencing technology is used to identify at the deoxyribonucleic acid (DNA) level. For Ganoderma lucidum fruiting bodies of different growth times, the DNA test results are the same, and they can only be identified as Ganoderma lucidum samples. It is impossible to detect the growth period of the Ganoderma lucidum fruiting bodies. It is impossible to distinguish the Ganoderma lucidum with a short growth time on the market that is used to impersonate the Ganoderma lucidum with a long growth time. Fluorescent quantitative PCR technology also performs species analysis at the DNA level, and it is also unable to distinguish Ganoderma lucidum fruiting bodies with different growth times.

[0004] Therefore, how to identify the growth time of the fruiting body is a problem that those skilled in the art need to solve. Summary of the invention

[0005] The purpose of the present application is to provide a method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry, which is used to solve the problem that genome sequencing and fluorescent quantitative PCR technology cannot distinguish Ganoderma lucidum fruiting bodies of different growth times.

[0006] In order to solve the above technical problems, the present application provides a method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry, comprising:

[0007] Obtaining a first protein fingerprint to be identified of the fruiting body to be tested;

[0008] Determine a first target protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponding to the first protein fingerprint map to be identified;

[0009] Determining the variety and growth time of the fruiting body to be tested according to the first target protein fingerprint;

[0010] The Ganoderma lucidum protein fingerprint database includes the corresponding relationship between the fruiting body protein fingerprint map, varieties and growth time.

[0011] In a feasible embodiment, if the Ganoderma lucidum protein fingerprint database also includes the correspondence between mycelium protein fingerprints and varieties, and the correspondence between spore protein fingerprints and varieties, it also includes:

[0012] Obtaining a second protein fingerprint to be identified of the Ganoderma lucidum product to be tested;

[0013] Determine a second target protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponding to the second protein fingerprint map to be identified;

[0014] Determine the variety of the Ganoderma lucidum product to be tested according to the second target protein fingerprint;

[0015] Wherein, the Ganoderma lucidum product to be tested is any one of mycelium, spores and fruiting bodies.

[0016] In a feasible embodiment, if the Ganoderma lucidum protein fingerprint database further includes the correspondence between protein fingerprints and morphologies, the protein fingerprints include the mycelium protein fingerprint, the spore protein fingerprint and the fruiting body protein fingerprint, and further includes:

[0017] Determine a third target protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponding to the second protein fingerprint map to be identified;

[0018] The morphology of the Ganoderma lucidum product to be tested is determined according to the third target protein fingerprint.

[0019] In a feasible embodiment, if the Ganoderma lucidum protein fingerprint database includes a mycelium protein fingerprint database, a spore protein fingerprint database and a fruiting body protein fingerprint database, the mycelium protein fingerprint database includes the correspondence between the mycelium protein fingerprint map and the variety, the spore protein fingerprint database includes the correspondence between the spore protein fingerprint map and the variety, the fruiting body protein fingerprint database includes the correspondence between the fruiting body protein fingerprint map, the variety and the growth time, and further includes:

[0020] Determining the morphology of the Ganoderma lucidum product to be tested; wherein the morphology includes mycelium morphology, spore morphology and fruiting body morphology;

[0021] If the morphology is a mycelium morphology, calling the mycelium protein fingerprint database, determining a fourth target protein fingerprint map corresponding to the second protein fingerprint map to be identified in the mycelium protein fingerprint database, and determining the variety of mycelium according to the fourth target protein fingerprint map;

[0022] If the morphology is a spore morphology, calling the spore protein fingerprint database, determining a fifth target protein fingerprint map corresponding to the second protein fingerprint map to be identified in the spore protein fingerprint database, and determining the variety of the spore according to the fifth target protein fingerprint map;

[0023] If the morphology is a fruiting body morphology, the fruiting body protein fingerprint database is called to determine a sixth target protein fingerprint map in the fruiting body protein fingerprint database corresponding to the second protein fingerprint map to be identified, and the variety and growth time of the fruiting body are determined according to the sixth target protein fingerprint map.

[0024] In a feasible embodiment, it also includes:

[0025] Obtain mycelium samples, spore samples and fruiting body samples of different varieties; wherein the fruiting body samples include fruiting bodies of different growth times;

[0026] Pre-treating the mycelium sample, the spore sample and the fruiting body sample respectively;

[0027] The mycelium protein fingerprint of the pretreated mycelium sample, the spore protein fingerprint of the spore sample and the fruiting body protein fingerprint of the fruiting body sample are collected respectively;

[0028] The variety of each mycelium protein fingerprint map is recorded and the mycelium protein fingerprint database is constructed, the variety of each spore protein fingerprint map is recorded and the spore protein fingerprint database is constructed, and the variety and growth time of each fruiting body protein fingerprint map are recorded and the fruiting body protein fingerprint database is constructed.

[0029] In a feasible embodiment, the mycelium sample is pretreated, including:

[0030] Pick the mycelium from the seed culture medium into a centrifuge tube filled with 75% alcohol;

[0031] The centrifuge tube was shaken and centrifuged to obtain precipitated mycelium;

[0032] After centrifugation, remove the supernatant from the centrifuge tube and dry it at room temperature for a set time;

[0033] After drying, add a certain amount of 70% formic acid solution into the centrifuge tube and mix the formic acid solution and mycelium;

[0034] After the mycelium is completely dissolved, an equal volume of acetonitrile solution is added to the solution in the centrifuge tube, mixed evenly, and centrifuged to obtain a supernatant;

[0035] The supernatant is dripped onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, the target point is covered with a matrix solution, and after drying, the mycelium sampling chip is obtained.

[0036] In a feasible embodiment, the spore sample is pre-treated, including:

[0037] Spores were collected from mature fruiting bodies;

[0038] Use crushing equipment to break the spores;

[0039] Take the broken spore powder into a centrifuge tube and add 75% alcohol into the centrifuge tube;

[0040] The centrifuge tube was shaken and centrifuged, the supernatant in the centrifuge tube was removed, and the tube was dried at room temperature for a set time;

[0041] After drying, add a certain amount of 70% formic acid solution to the centrifuge tube and vortex for a set time;

[0042] Add an equal volume of acetonitrile solution to the solution in the centrifuge tube and shake again for a set time;

[0043] The supernatant is dripped onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, the target point is covered with a matrix solution, and after drying, the spore sampling chip is obtained.

[0044] In a feasible embodiment, the fruiting body sample is pretreated, comprising:

[0045] Sampling starts from the fruiting of the fruiting body, and sampling is done every other day;

[0046] The sampled fruiting bodies are crushed respectively, and the crushed fruiting bodies are decomposed according to the fiber texture;

[0047] The decomposed fruit body fiber strips are placed in a sample tube of a focused ultrasonic instrument, acid-washed glass beads and lysis solution are added in sequence, and the fruit body fiber strips are subjected to protein-assisted extraction using a focused ultrasonic disruptor;

[0048] After sonication, an equal volume of acetonitrile solution was added to the sample tube, and a focused ultrasonicator was used for secondary sonication;

[0049] The sample tube after the second ultrasound is centrifuged for a set time to obtain a supernatant;

[0050] The supernatant is dripped onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, the target point is covered with a matrix solution, and after drying, the mycelium sampling chip is obtained.

[0051] In a feasible embodiment, adding the supernatant dropwise to the target spot of the mass spectrometry sample plate comprises:

[0052] The calibrant solution and the supernatant in the centrifuge tube were added dropwise onto the target spot of the mass spectrometry sample plate in a nine-square grid pattern;

[0053] Among them, the calibration solution is added to the central target of the nine targets, and the clear solution is added to the remaining eight targets. The central target is used for instrument calibration before spectrum acquisition.

[0054] In a feasible embodiment, collecting the mycelium protein fingerprint of the mycelium sampling chip includes:

[0055] A mass spectrometer is used to collect multiple mycelial protein fingerprints for each target point to which the supernatant is added;

[0056] Among them, each mycelium protein fingerprint spectrum is obtained by multiple acquisitions and accumulation by a mass spectrometer, and the accumulated spectrum peak intensity of each mycelium protein fingerprint spectrum is greater than 10,000 and the baseline is less than 3,000.

[0057] In a feasible embodiment, the variety and growth time of each fruiting body protein fingerprint are recorded and the fruiting body protein fingerprint database is constructed, including:

[0058] Analyze and classify all fruiting body protein fingerprints of the same variety at different growth times to obtain multiple groups of spectrum groups, and record the time characteristics of each group of the spectrum groups; wherein the time characteristics include the growth time corresponding to all fruiting body protein fingerprints in the spectrum group;

[0059] Eliminating the fruiting body protein fingerprints with a deviation exceeding a preset value in the spectrum group to obtain a processed spectrum group;

[0060] Extracting characteristic values ​​of all fruiting body protein fingerprints in the treated pattern group to establish a super fingerprint of the treated pattern group, and adding the time characteristics and varieties of the treated pattern group to the naming of the super fingerprint corresponding to the treated pattern group;

[0061] The fruiting body protein fingerprint database is constructed based on the named super fingerprint map.

[0062] In a feasible embodiment, determining the target protein fingerprint map corresponding to the protein fingerprint map to be identified in the Ganoderma lucidum protein fingerprint database includes:

[0063] Extracting features from the fingerprint of the protein to be identified;

[0064] Using a neural network model, the features in the protein fingerprint to be identified are compared with each protein fingerprint in the Ganoderma lucidum protein fingerprint database to determine the similarity between the protein fingerprint to be identified and each protein fingerprint;

[0065] The protein fingerprint corresponding to the highest similarity among all the similarities is used as the target protein fingerprint.

[0066] The present application provides a method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry, comprising: obtaining a first protein fingerprint to be identified of a fruiting body to be identified; determining a first target protein fingerprint corresponding to the first protein fingerprint to be identified in a Ganoderma lucidum protein fingerprint database; and determining the variety and growth time of the fruiting body to be identified based on the first target protein fingerprint. Based on the correspondence between the fruiting body protein fingerprint database, including the fruiting body protein fingerprint, variety, and growth time, when the first target protein fingerprint corresponding to the first protein fingerprint to be identified is determined in the Ganoderma lucidum protein fingerprint database, the growth time of the fruiting body to be identified can be determined based on the variety and growth time corresponding to the first target protein fingerprint, thereby achieving identification of the growth time of the fruiting body. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0068] FIG1 is a flow chart of a method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry provided in an embodiment of the present application;

[0069] FIG2 is a schematic diagram of a nine-grid patterning method provided in an embodiment of the present application;

[0070] FIG3 is a schematic diagram showing a comparison between a super map provided in an embodiment of the present application and a fingerprint map of a protein to be identified;

[0071] FIG4 is a protein fingerprint of a fruiting body with a growth period of 27 to 55 days provided in an embodiment of the present application;

[0072] FIG5 is a first identification comparison diagram provided in an embodiment of the present application;

[0073] FIG6 is a second identification comparison diagram provided in an embodiment of the present application;

[0074] FIG7 is a third identification comparison diagram provided in an embodiment of the present application;

[0075] FIG8 is a fourth identification comparison diagram provided in an embodiment of the present application;

[0076] FIG. 9 is a fifth identification comparison diagram provided in an embodiment of the present application. DETAILED DESCRIPTION

[0077] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0078] The core of this application is to provide a Ganoderma lucidum identification method based on time-of-flight mass spectrometry, which is used to identify the growth time of the fruiting body.

[0079] The present application does not make any specific limitation on time-of-flight mass spectrometry, which may be matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is a mass spectrometry technology used to analyze macromolecular biological samples, such as proteins, peptides, nucleic acids, etc., by establishing a known protein fingerprint database, and then identifying the results by comparing the collected protein fingerprint spectrum to be identified through the database; surface-enhanced laser desorption protein time-of-flight mass spectrometry; Laser Desorption Ionization Mass Spectrometry (LDI-MS); VUVDI-RTOF-MS (Vacuum Ultraviolet Laser Desorption / Ionization Reflectron Time-of-Flight Mass Spectrometry), which is a mass spectrometry imaging technology that combines the techniques of vacuum ultraviolet laser desorption / ionization (VUVDI) and reflectron time-of-flight mass analysis (RTOF-MS); matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS).

[0080] In order to enable those skilled in the art to better understand the present application, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0081] FIG1 is a flow chart of a method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry provided in an embodiment of the present application. As shown in FIG1 , the method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry includes:

[0082] S10: Obtaining a first protein fingerprint to be identified of the fruiting body to be tested.

[0083] S11: Determine a first target protein fingerprint map in the Ganoderma lucidum protein fingerprint database that corresponds to the first protein fingerprint map to be identified.

[0084] S12: Determine the variety and growth time of the fruiting body to be tested according to the first target protein fingerprint.

[0085] Among them, the Ganoderma lucidum protein fingerprint database includes the correspondence between the fruiting body protein fingerprint map, variety and growth time.

[0086] Regarding how to determine the first target protein fingerprint map corresponding to the first protein fingerprint map to be identified in the Ganoderma lucidum protein fingerprint database, the first protein fingerprint map to be identified can be compared one by one with the fruiting body protein fingerprint map in the database. The comparison between the two maps can include the comparison of the number of characteristic peaks in the map, the position of characteristic peaks, and the height of characteristic peaks. Of course, the similarity between the first protein fingerprint map to be identified and each fruiting body protein fingerprint map can also be calculated, and the first target protein fingerprint map corresponding to the first protein fingerprint map to be identified can be determined based on the similarity. The variety and growth time corresponding to the first target protein fingerprint map are the variety and growth time of the fruiting body to be tested.

[0087] The embodiment of the present application provides a method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry, including: obtaining a first protein fingerprint to be identified of a fruiting body to be tested; determining a first target protein fingerprint corresponding to the first protein fingerprint in a Ganoderma lucidum protein fingerprint database; and determining the variety and growth time of the fruiting body to be tested according to the first target protein fingerprint. Based on the correspondence between the fruiting body protein fingerprint database, including the fruiting body protein fingerprint, variety, and growth time, when the first target protein fingerprint corresponding to the first protein fingerprint to be identified is determined in the Ganoderma lucidum protein fingerprint database, the growth time of the fruiting body to be tested can be determined according to the variety and growth time corresponding to the first target protein fingerprint, thereby achieving identification of the growth time of the fruiting body.

[0088] Based on the above embodiments, if the Ganoderma lucidum protein fingerprint database also includes the correspondence between mycelium protein fingerprint maps and varieties, and the correspondence between spore protein fingerprint maps and varieties, the embodiments of the present application also include: obtaining a second protein fingerprint map to be identified of the Ganoderma lucidum product to be tested; determining a second target protein fingerprint map corresponding to the second protein fingerprint map to be identified in the Ganoderma lucidum protein fingerprint database; determining the variety of the Ganoderma lucidum product to be tested according to the second target protein fingerprint map; wherein the Ganoderma lucidum product to be tested is any one of mycelium, spores and fruiting bodies.

[0089] In the embodiment of the present application, the second protein fingerprint of the Ganoderma lucidum product to be tested is obtained by: taking about 1g of the Ganoderma lucidum product and crushing it thoroughly; taking a small amount of the crushed sample and adding it to a centrifuge tube; adding 50-100uL of lysis solution (60%-70% formic acid solution) to the centrifuge tube and fully shaking and mixing; standing for 5 minutes, taking 1uL of the supernatant and spotting it on the target point of the specimen plate; covering the target point with 1uL of matrix solution after drying at room temperature, and continuing to dry; after complete drying, using a mass spectrometer to collect the second protein fingerprint of the Ganoderma lucidum product to be tested. Since the Ganoderma lucidum protein fingerprint database in the embodiment of the present application also includes the correspondence between the mycelium protein fingerprint and the variety, and the correspondence between the spore protein fingerprint and the variety, the variety of the mycelium and the variety of the spore can also be identified on the basis of identifying the variety and growth time of the fruiting body. The variety here refers to the Ganoderma lucidum variety corresponding to the fruiting body, mycelium or spore.

[0090] Based on the above embodiments, in the embodiments of the present application, if the Ganoderma lucidum protein fingerprint database also includes the correspondence between protein fingerprint maps and morphologies, the protein fingerprint maps include mycelium protein fingerprint maps, spore protein fingerprint maps and fruiting body protein fingerprint maps, and also include: determining a third target protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponding to the second protein fingerprint map to be identified; and determining the morphology of the Ganoderma lucidum product to be tested according to the third target protein fingerprint map.

[0091] That is to say, each mycelium protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponds to information on the variety and morphology, each spore protein fingerprint map corresponds to information on the variety and morphology, and each fruiting body protein fingerprint map corresponds to information on the variety, morphology, and growth time. With the marketization of Ganoderma lucidum products, whole Ganoderma lucidum rarely appears, and most of them are processed Ganoderma lucidum products, such as powdered Ganoderma lucidum. Therefore, it is difficult to identify the Ganoderma lucidum morphology (mycelium, fruiting body, and spores) of the processed Ganoderma lucidum products themselves. Therefore, based on the previous embodiment, the embodiment of the present application can further identify the morphology of the Ganoderma lucidum product to be tested.

[0092] Based on the above embodiments, in the embodiments of the present application, if the Ganoderma lucidum protein fingerprint database includes a mycelium protein fingerprint database, a spore protein fingerprint database and a fruiting body protein fingerprint database, the mycelium protein fingerprint database includes the correspondence between the mycelium protein fingerprint map and the variety, the spore protein fingerprint database includes the correspondence between the spore protein fingerprint map and the variety, and the fruiting body protein fingerprint database includes the correspondence between the fruiting body protein fingerprint map, the variety and the growth time, it also includes: determining the morphology of the Ganoderma lucidum product to be tested; wherein the morphology includes mycelium morphology, spore morphology and fruiting body morphology; if the morphology is mycelium morphology, calling the mycelium protein fingerprint database to determine the morphology The fourth target protein fingerprint map in the mycelium protein fingerprint database corresponds to the second protein fingerprint map to be identified, and the variety of the mycelium is determined according to the fourth target protein fingerprint map; if the morphology is spore morphology, the spore protein fingerprint database is called to determine the fifth target protein fingerprint map in the spore protein fingerprint database corresponding to the second protein fingerprint map to be identified, and the variety of the spore is determined according to the fifth target protein fingerprint map; if the morphology is fruiting body morphology, the fruiting body protein fingerprint database is called to determine the sixth target protein fingerprint map in the fruiting body protein fingerprint database corresponding to the second protein fingerprint map to be identified, and the variety and growth time of the fruiting body are determined according to the sixth target protein fingerprint map.

[0093] The embodiment of the present application divides the Ganoderma lucidum protein fingerprint database into three small databases: mycelium protein fingerprint database, spore protein fingerprint database and fruiting body protein fingerprint database. When testing the Ganoderma lucidum product to be tested, if the form of the Ganoderma lucidum product to be tested can be identified by the naked eye, or for the Ganoderma lucidum product to be tested with a known form, the small database corresponding to the form of the Ganoderma lucidum product to be tested can be directly called to determine the variety and / or growth time of the Ganoderma lucidum product to be tested with a known form through the small database. The advantage of dividing the Ganoderma lucidum protein fingerprint database into small databases of different forms is that it can improve the efficiency of the identification of the Ganoderma lucidum product to be tested, and can also improve the accuracy of the identification.

[0094] Based on the above embodiments, the embodiments of the present application also include: obtaining mycelium samples, spore samples and fruiting body samples of different varieties; wherein the fruiting body samples include fruiting bodies of different growth times; pre-treating the mycelium samples, spore samples and fruiting body samples respectively; collecting the mycelium protein fingerprint map of the pretreated mycelium sample, the spore protein fingerprint map of the spore sample and the fruiting body protein fingerprint map of the fruiting body sample respectively; recording the variety of each mycelium protein fingerprint map and constructing a mycelium protein fingerprint database, recording the variety of each spore protein fingerprint map and constructing a spore protein fingerprint database, recording the variety and growth time of each fruiting body protein fingerprint map and constructing a fruiting body protein fingerprint database.

[0095] In the implementation of the present application, a mycelium sampling chip is prepared for the mycelium of each Ganoderma lucidum variety; a spore sampling chip is prepared for the spores of each Ganoderma lucidum variety; and a fruiting body sampling chip is prepared for the fruiting body of each growth time of each Ganoderma lucidum variety. The growth time is calculated from the fruiting of the fruiting body, and sampling is performed every other day from the start of fruiting. Therefore, for one Ganoderma lucidum variety, there are: a fruiting body sampling chip with a growth time of 1 day, a fruiting body sampling chip with a growth time of 3 days, a fruiting body sampling chip with a growth time of 5 days, and so on, thereby ensuring that a comprehensive situation is obtained during database data analysis and improving the accuracy of database identification.

[0096] In the embodiment of the present application, the mycelium sample is pretreated to obtain a mycelium sampling chip, which specifically includes: picking mycelium from the seed culture medium and putting it into a centrifuge tube filled with 75% alcohol; shaking the centrifuge tube and centrifuging to obtain precipitated mycelium; after centrifugation, removing the supernatant in the centrifuge tube, and drying at room temperature for a set time; after drying, adding a certain amount of 70% formic acid solution to the centrifuge tube, and mixing the formic acid solution and mycelium; after the mycelium is completely dissolved, adding an equal volume of acetonitrile solution to the solution in the centrifuge tube, mixing, and centrifuging to obtain a supernatant; dropping the supernatant onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, covering the target point with the matrix solution, and obtaining a mycelium sampling chip after drying.

[0097] To make the preparation process of mycelium sampling chip clearer, the details are as follows:

[0098] (1) Pick up Ganoderma lucidum mycelium from the seed culture medium and place it in a 1.5 mL centrifuge tube. Add 1 mL of 75% alcohol to the centrifuge tube in advance.

[0099] (2) Wash the mycelium by shaking for 1 minute, and then centrifuge at 12000 rpm for 2 minutes to precipitate the Ganoderma mycelium.

[0100] (3) Completely remove the supernatant and dry at room temperature for 5 minutes.

[0101] (4) Add 50-100uL of 70% formic acid solution to the precipitate and use a pipette to mix thoroughly to completely dissolve the mycelium.

[0102] (5) Add an equal volume of acetonitrile solution to the solution and mix again using a pipette tip.

[0103] (6) Centrifuge at 12000 rpm for 2 minutes and keep the supernatant for later use.

[0104] (7) FIG. 2 is a schematic diagram of a nine-grid spotting method provided in an embodiment of the present application. As shown in FIG. 2 , the calibration solution and the supernatant in the centrifuge tube are added to the target points of the mass spectrometry sample plate in a nine-grid spotting method; wherein, 1uL of the calibration solution for time-of-flight mass spectrometry is added to the central target point of the nine target points, and 1uL of the supernatant is added to the remaining eight target points. The central target point is used for instrument calibration before spectrum acquisition.

[0105] (8) Wait for the sample to completely dry at room temperature, then cover each target point with 1uL of matrix solution and completely dry again at room temperature. The mycelium sampling chip is now ready.

[0106] The embodiment of the present application pre-treats the spore sample to obtain the spore sampling chip, which specifically includes: collecting spores from mature fruiting bodies; using a crushing device to break the spore wall; taking the broken spore powder into a centrifuge tube, and adding 75% alcohol to the centrifuge tube; shaking the centrifuge tube and centrifuging it, removing the supernatant in the centrifuge tube, and drying it at room temperature for a set time; after drying, adding a certain amount of 70% formic acid solution to the centrifuge tube, and vortexing it for a set time; adding an equal volume of acetonitrile solution to the solution in the centrifuge tube, and shaking it again for a set time; dropping the supernatant onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, covering the target point with a matrix solution, and obtaining the spore sampling chip after drying.

[0107] To make the spore sampling chip preparation process clearer, the details are as follows:

[0108] (1) Ganoderma lucidum spore powder was collected from mature fruiting bodies.

[0109] (2) Use special Ganoderma lucidum spore crushing equipment to break the Ganoderma lucidum spore wall.

[0110] (3) After breaking the spore wall, take a small amount of spore powder into a 1.5 mL centrifuge tube and add 1 mL of 75% alcohol.

[0111] (4) Wash by shaking for 1 minute, and then centrifuge at 12000 rpm for 2 minutes.

[0112] (5) Completely remove the supernatant and dry at room temperature for 5 minutes.

[0113] (6) Add 50-100uL 70% formic acid solution to the precipitate and vortex for 2 minutes.

[0114] (7) Add an equal volume of acetonitrile solution and shake again for 2 minutes.

[0115] (8) As shown in FIG2 , the calibrant solution and the supernatant in the centrifuge tube are added to the target points of the mass spectrometer sample plate in a nine-square grid pattern; 1uL of the calibrant solution for time-of-flight mass spectrometry is added to the center target point of the nine target points, and 1uL of the supernatant is added to the remaining eight target points. The center target point is used for instrument calibration before spectrum acquisition.

[0116] (9) Wait for the sample to completely dry at room temperature, then cover each target spot with 1uL of matrix solution and completely dry again at room temperature. The spore sampling chip is now ready.

[0117] The present application embodiment pre-treats the fruiting body sample to obtain the fruiting body sampling chip, which specifically includes: sampling from the beginning of fruiting of the fruiting body, and sampling once every other day; crushing the sampled fruiting bodies respectively, and decomposing the crushed fruiting bodies according to the fiber texture; taking the decomposed fruiting body fiber strips into the sample tube of the focused ultrasonic instrument, adding acid-washed glass beads and lysis solution in sequence, and using a focused ultrasonic crusher to perform protein-assisted extraction on the fruiting body fiber strips; after ultrasonication, adding an equal volume of acetonitrile solution to the sample tube, and performing secondary ultrasonication using a focused ultrasonic crusher; centrifuging the sample tube after the secondary ultrasonication for a set time to obtain a supernatant; dropping the supernatant onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, covering the target point with a matrix solution, and obtaining a mycelium sampling chip after drying.

[0118] To make the preparation process of mycelium sampling chip clearer, the details are as follows:

[0119] (1) Sampling of fruiting bodies begins when mushrooms begin to grow (generally starting in April each year), and sampling is carried out every other day to ensure that a comprehensive picture is obtained during database data analysis and to improve the accuracy of database identification.

[0120] (2) After sampling, the fruiting bodies are crushed with scissors, and then crushed again according to the fiber texture with pointed tweezers.

[0121] (3) Take 3-5 decomposed fruiting body fiber strips and put them into the sample tube of the focused ultrasonic instrument, add 30-50 500um acid-washed glass beads, and then add 50uL of lysis solution (60-70% formic acid solution), and use a focused ultrasonic disruptor for protein-assisted extraction. The disruption parameters are: pulse 1000ms, interval 150ms, power 90%, time 300s, temperature 15℃.

[0122] (4) After sonication, add an equal volume of acetonitrile to the sample tube and repeat the sonication.

[0123] (5) Take out the sample tube after ultrasound, centrifuge at 12000 rpm for 2 minutes, and keep the supernatant for later use.

[0124] (6) As shown in Figure 2, the calibrant solution and the supernatant in the centrifuge tube are added to the target points of the mass spectrometry sample plate in a nine-square grid pattern; among them, 1uL of the calibrant solution for time-of-flight mass spectrometry is added to the central target point of the nine target points, and 1uL of the supernatant is added to the remaining eight target points. The central target point is used for instrument calibration before spectrum acquisition.

[0125] (8) Wait for the sample to completely dry at room temperature, then cover each target spot with 1uL of matrix solution and completely dry again at room temperature. The spore sampling chip is now ready.

[0126] In the embodiment of the present application, the mycelium protein fingerprint of the mycelium sampling chip is collected specifically including: using a mass spectrometer to collect multiple mycelium protein fingerprints for each target point to which the supernatant is added; wherein each mycelium protein fingerprint is obtained by multiple acquisitions and accumulations by the mass spectrometer, and the accumulated spectrum peak intensity of each mycelium protein fingerprint is greater than 10000, and the baseline is less than 3000. Among them, the parameters of the mass spectrometer are: delay stage voltage 20Kv, extraction stage voltage 18.1Kv, focusing stage voltage 7Kv, delay time 230ns, laser 60HZ, single acquisition excitation 100 times. If 3 spectra are collected for each target point, that is, a mycelium sampling chip can obtain 24 spectra, and the 24 spectra can be analyzed and feature extracted to obtain a summary spectrum covering all features of the 24 spectra. The collection of spore protein fingerprints and the collection of fruiting body protein fingerprints refer to the collection of mycelium protein fingerprints.

[0127] In the embodiment of the present application, multiple spectra are collected for each target to avoid the characteristic signal not being collected. Each mycelium protein fingerprint spectrum is accumulated by multiple collections by a mass spectrometer, which has the following effects: it can improve the signal-to-noise ratio, significantly improve the signal intensity, and more clearly distinguish the signal from the noise in the spectrum, thereby improving the accuracy of the analysis; reduce the impact of background interference on the analysis results, making the spectrum cleaner and convenient for identifying and analyzing peaks; enhance characteristic signals, making the peaks more prominent, and facilitating subsequent peak identification and quantitative analysis; reduce random errors, and improve the stability and repeatability of the measurement results. Peak intensity refers to the intensity of the signal at a specific wavelength or mass number in the spectrum. A peak intensity greater than 10,000 means that the signal is strong enough to be clearly identified and analyzed; the baseline refers to the background intensity when there is no signal in the spectrum. A baseline below 3,000 means that the background noise level is low, which makes it easier to distinguish the real signal.

[0128] Based on the above embodiments, the embodiments of the present application record the variety and fruiting body growth time of each fruiting body protein fingerprint map and construct a fruiting body protein fingerprint database, specifically including: analyzing and classifying all fruiting body protein fingerprint maps of the same variety at different growth times to obtain multiple groups of map groups, and recording the time characteristics of each map group; wherein the time characteristics include the fruiting body growth time corresponding to all fruiting body protein fingerprint maps in the map group; eliminating the fruiting body protein fingerprint maps in the map group whose deviation exceeds a preset value to obtain a processed map group; extracting the characteristic values ​​of all fruiting body protein fingerprint maps in the processed map group to establish a super fingerprint map of the processed map group, and adding the time characteristics and Ganoderma lucidum variety of the processed map group to the naming of the super fingerprint map corresponding to the processed map group; constructing a fruiting body protein fingerprint database according to the named super fingerprint map.

[0129] In the embodiment of the present application, for a Ganoderma lucidum variety, all fruiting body protein fingerprints of different growth times are analyzed and classified, the purpose of which is to classify fruiting body protein fingerprints with similarities into one category, which can reduce the amount of data in the fruiting body protein fingerprint database. The present application embodiment does not specifically limit how to analyze and classify all fruiting body protein fingerprints, and the protein fingerprints can be classified using decision trees, random forests, xgboost (eXtreme Gradient Boosting, an integrated learning algorithm based on a gradient boosting framework), etc.

[0130] The following is a method for classifying protein fingerprints using decision trees. Data preprocessing: Clean the protein fingerprint data, including noise removal, standardization and other steps, so that the model can better understand and process the data. Feature selection: Use machine learning methods to screen the test set for characteristic peaks, and select differential characteristic peaks as classification features; statistical methods or prior knowledge can be used to determine the features most relevant to protein classification. Model training: Use the decision tree algorithm to train the model. The decision tree recursively selects the optimal features for segmentation and builds a tree model; each time the segmentation occurs, the algorithm selects an optimal feature to split the data set until the leaf node contains only instances of a single category. Model evaluation: Use the test set to evaluate the model and check the accuracy and generalization ability of the model. Model optimization: According to the results of the model evaluation, the model may need to be tuned, such as adjusting the depth of the decision tree, the minimum number of segmentation samples and other parameters to prevent overfitting or underfitting. Apply the model: Apply the trained model to the fruiting body protein fingerprint to be classified for classification and grouping.

[0131] Regarding recording the time characteristics of each group of atlas groups, for example, the growth time of the fruiting body protein fingerprints in a group of atlas groups after classification is 1 day, 3 days, 5 days and 29 days, and the time characteristics of this group of atlas groups are 1 day to 29 days, and the time characteristics are from the minimum time to the maximum time in the atlas group. In the embodiment of the present application, the fruiting body protein fingerprints with large deviations in the atlas group are eliminated to obtain the atlas group after processing, and the time characteristics of the atlas group after processing remain unchanged, that is, they are still the time characteristics of the atlas group before processing.

[0132] Regarding extracting the characteristic values ​​of all the fruiting body protein fingerprints in the processed spectrum group to establish a super fingerprint of the processed spectrum group, it can specifically include: extracting the characteristic values ​​of each fruiting body protein fingerprint in the processed spectrum group, the characteristic values ​​include characteristic peak intensity, position, height, width, and area, etc., merging the characteristic values ​​of all fruiting body protein fingerprints into a data set to form a super fingerprint. Each super fingerprint in the fruiting body protein fingerprint database corresponds to a time feature and a variety. Furthermore, after the fruiting body protein fingerprint database is constructed according to the named super fingerprint, it also includes: obtaining a fruiting body verification sample, and using the fruiting body protein fingerprint database to identify the variety and growth time of the fruiting body verification sample; comparing the identified variety and growth time with the actual variety and actual growth time of the fruiting body verification sample; if the comparison result does not meet the preset requirements, rebuilding the fruiting body protein fingerprint database.

[0133] Based on the above embodiment, the embodiment of the present application determines the target protein fingerprint map corresponding to the protein fingerprint map to be identified in the Ganoderma lucidum protein fingerprint database, specifically including: extracting features in the protein fingerprint map to be identified; using a neural network model to compare the features in the protein fingerprint map to be identified with each protein fingerprint map in the Ganoderma lucidum protein fingerprint database, and determining the similarity between the protein fingerprint map to be identified and each protein fingerprint map; and taking the protein fingerprint map corresponding to the highest similarity among all similarities as the target protein fingerprint map. Among them, the protein fingerprint map to be identified can be the first protein fingerprint map to be identified and the second protein fingerprint map to be identified mentioned above.

[0134] In the traditional database, all the fruiting body maps collected at different growth times are synthesized into a super map representing the fruiting body through analysis software, and then the synthesized super map is used to detect and analyze the collected protein fingerprint map to be identified. Figure 3 is a schematic diagram of a comparison between a super map and a protein fingerprint map to be identified provided in an embodiment of the present application. As shown in Figure 3, the super map in the traditional database can only detect about 40% of the fingerprint maps, and 60% of the fingerprint maps cannot be detected.

[0135] When constructing a fruiting body protein fingerprint database, the present application found that the spectra of fruiting bodies with different growth times were quite different. The characteristic spectra established cannot be replaced by a total synthetic map. It is necessary to build a library for fruiting bodies in different time periods and establish a super fingerprint spectrum suitable for fruiting body identification in different time periods. Figure 4 is a fruiting body protein fingerprint spectrum with a fruiting body growth time of 27 to 55 days provided by an embodiment of the present application. As shown in Figure 4, the spectrum similarity between 27 days and 55 days is relatively high. All similar fingerprint spectra with a growth time of 27 to 55 days are synthesized into a super fingerprint spectrum for detecting fruiting body samples in this time period. During identification, not only can variety identification be performed, but also the growth time of the fruiting body can be judged.

[0136] The sample map is identified using the super fingerprint map of the fruiting body growth time of 15-55 days. Figure 5 is the first identification comparison diagram provided in the embodiment of the present application. The lower half of Figure 5 is the super fingerprint map of the fruiting body for 15-55 days, and the upper half of Figure 5 is the fingerprint map of Ganoderma lucidum spores; Figure 6 is the second identification comparison diagram provided in the embodiment of the present application. The lower half of Figure 6 is the super fingerprint map of the fruiting body for 15-55 days, and the upper half of Figure 6 is the fingerprint map of Ganoderma lucidum mycelium; Figure 7 is the third identification comparison diagram provided in the embodiment of the present application. The lower half of Figure 7 is the super fingerprint map of the fruiting body for 15-55 days. The upper half of Figure 7 is the fingerprint of the Ganoderma lucidum fruiting body growing for 19 days; Figure 8 is the fourth identification comparison diagram provided in the embodiment of the present application, the lower half of Figure 8 is the super fingerprint of the fruiting body for 15 days to 55 days, and the upper half of Figure 8 is the fingerprint of the Ganoderma lucidum fruiting body growing for 34 days; Figure 9 is the fifth identification comparison diagram provided in the embodiment of the present application, the lower half of Figure 9 is the super fingerprint of the fruiting body for 15 days to 55 days, and the upper half of Figure 9 is the fingerprint of the Ganoderma lucidum fruiting body growing for 74 days. As shown in Figures 5 to 9, when the growth time of the fruiting body is within the time interval covered by the super fingerprint, the upper and lower maps have a high degree of similarity; when the growth time of the fruiting body is not within the time interval covered by the super fingerprint or is not the map of the fruiting body, the upper and lower maps have a low degree of similarity. Therefore, the fruiting body protein fingerprint database established by this method can relatively accurately analyze the growth time of the fruiting body, and then accurately evaluate the corresponding value of the product.

[0137] The above is a detailed introduction to a method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry provided by the present application. The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same and similar parts between the various embodiments can be referred to each other. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

[0138] It should also be noted that, in this specification, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprises a ..." does not exclude the presence of other identical elements in the process, method, article or device including the element.

Claims

1. A method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry, characterized in that: include: Obtaining a first protein fingerprint to be identified of the fruiting body to be tested; Determine a first target protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponding to the first protein fingerprint map to be identified; Determining the variety and growth time of the fruiting body to be tested according to the first target protein fingerprint; The Ganoderma lucidum protein fingerprint database includes the corresponding relationship between the fruiting body protein fingerprint map, varieties and growth time.

2. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 1, characterized in that: If the Ganoderma lucidum protein fingerprint database also includes the correspondence between mycelium protein fingerprints and varieties, and the correspondence between spore protein fingerprints and varieties, it also includes: Obtaining a second protein fingerprint to be identified of the Ganoderma lucidum product to be tested; Determine a second target protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponding to the second protein fingerprint map to be identified; Determine the variety of the Ganoderma lucidum product to be tested according to the second target protein fingerprint; Wherein, the Ganoderma lucidum product to be tested is any one of mycelium, spores and fruiting bodies.

3. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 2, characterized in that: If the Ganoderma lucidum protein fingerprint database further includes the correspondence between protein fingerprints and morphologies, the protein fingerprints include the mycelium protein fingerprint, the spore protein fingerprint and the fruiting body protein fingerprint, and further includes: Determine a third target protein fingerprint map in the Ganoderma lucidum protein fingerprint database corresponding to the second protein fingerprint map to be identified; The morphology of the Ganoderma lucidum product to be tested is determined according to the third target protein fingerprint.

4. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 2, characterized in that: If the Ganoderma lucidum protein fingerprint database includes a mycelium protein fingerprint database, a spore protein fingerprint database and a fruiting body protein fingerprint database, the mycelium protein fingerprint database includes the correspondence between the mycelium protein fingerprint map and the variety, the spore protein fingerprint database includes the correspondence between the spore protein fingerprint map and the variety, and the fruiting body protein fingerprint database includes the correspondence between the fruiting body protein fingerprint map, the variety and the growth time, it also includes: Determining the morphology of the Ganoderma lucidum product to be tested; wherein the morphology includes mycelium morphology, spore morphology and fruiting body morphology; If the morphology is a mycelium morphology, calling the mycelium protein fingerprint database, determining a fourth target protein fingerprint map corresponding to the second protein fingerprint map to be identified in the mycelium protein fingerprint database, and determining the variety of mycelium according to the fourth target protein fingerprint map; If the morphology is a spore morphology, calling the spore protein fingerprint database, determining a fifth target protein fingerprint map corresponding to the second protein fingerprint map to be identified in the spore protein fingerprint database, and determining the variety of the spore according to the fifth target protein fingerprint map; If the morphology is a fruiting body morphology, the fruiting body protein fingerprint database is called to determine a sixth target protein fingerprint map in the fruiting body protein fingerprint database corresponding to the second protein fingerprint map to be identified, and the variety and growth time of the fruiting body are determined according to the sixth target protein fingerprint map.

5. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 4, characterized in that: Also includes: Obtain mycelium samples, spore samples and fruiting body samples of different varieties; wherein the fruiting body samples include fruiting bodies of different growth times; Pre-treating the mycelium sample, the spore sample and the fruiting body sample respectively; The mycelium protein fingerprint of the pretreated mycelium sample, the spore protein fingerprint of the spore sample and the fruiting body protein fingerprint of the fruiting body sample are collected respectively; The variety of each mycelium protein fingerprint map is recorded and the mycelium protein fingerprint database is constructed, the variety of each spore protein fingerprint map is recorded and the spore protein fingerprint database is constructed, and the variety and growth time of each fruiting body protein fingerprint map are recorded and the fruiting body protein fingerprint database is constructed.

6. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 5, characterized in that: The mycelium sample is pretreated, comprising: Pick the mycelium from the seed culture medium into a centrifuge tube filled with 75% alcohol; The centrifuge tube was shaken and centrifuged to obtain precipitated mycelium; After centrifugation, remove the supernatant from the centrifuge tube and dry it at room temperature for a set time; After drying, add a certain amount of 70% formic acid solution into the centrifuge tube and mix the formic acid solution and mycelium; After the mycelium is completely dissolved, an equal volume of acetonitrile solution is added to the solution in the centrifuge tube, mixed evenly, and centrifuged to obtain a supernatant; The supernatant is dripped onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, the target point is covered with a matrix solution, and after drying, the mycelium sampling chip is obtained.

7. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 5, characterized in that: The spore sample is pre-treated, comprising: Spores were collected from mature fruiting bodies; Use crushing equipment to break the spores; Take the broken spore powder into a centrifuge tube and add 75% alcohol into the centrifuge tube; The centrifuge tube was shaken and centrifuged, the supernatant in the centrifuge tube was removed, and the tube was dried at room temperature for a set time; After drying, add a certain amount of 70% formic acid solution to the centrifuge tube and vortex for a set time; Add an equal volume of acetonitrile solution to the solution in the centrifuge tube and shake again for a set time; The supernatant is dripped onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, the target point is covered with a matrix solution, and after drying, the spore sampling chip is obtained.

8. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 5, characterized in that: The fruiting body sample is pretreated, comprising: Sampling starts from the fruiting of the fruiting body, and is done every other day; The sampled fruiting bodies are crushed respectively, and the crushed fruiting bodies are decomposed according to the fiber texture; The decomposed fruit body fiber strips are placed in a sample tube of a focused ultrasonic instrument, acid-washed glass beads and lysis solution are added in sequence, and the fruit body fiber strips are subjected to protein-assisted extraction using a focused ultrasonic disruptor; After sonication, an equal volume of acetonitrile solution was added to the sample tube, and a focused ultrasonicator was used for secondary sonication; The sample tube after the second ultrasound is centrifuged for a set time to obtain a supernatant; The supernatant is dripped onto the target point of the mass spectrometry sample plate, and after the liquid on the target point is dried, the target point is covered with a matrix solution, and after drying, the mycelium sampling chip is obtained.

9. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to any one of claims 6 to 8, characterized in that: Add the supernatant onto the target spot on the mass spectrometry sample plate, including: The calibrant solution and the supernatant in the centrifuge tube were added dropwise onto the target spots of the mass spectrometry sample plate in a nine-square grid pattern; Among them, the calibration solution is added to the central target of the nine targets, and the clear solution is added to the remaining eight targets. The central target is used for instrument calibration before spectrum acquisition.

10. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 6, characterized in that: Collecting the mycelium protein fingerprint of the mycelium sampling chip includes: A mass spectrometer is used to collect multiple mycelial protein fingerprints for each target point to which the supernatant is added; Among them, each mycelium protein fingerprint spectrum is obtained by multiple acquisitions and accumulation by a mass spectrometer, and the accumulated spectrum peak intensity of each mycelium protein fingerprint spectrum is greater than 10,000 and the baseline is less than 3,000.

11. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to claim 5, characterized in that: Record the variety and growth time of each fruiting body protein fingerprint map and construct the fruiting body protein fingerprint database, including: Analyze and classify all fruiting body protein fingerprints of the same variety at different growth times to obtain multiple groups of spectrum groups, and record the time characteristics of each group of the spectrum groups; wherein the time characteristics include the growth time corresponding to all fruiting body protein fingerprints in the spectrum group; Eliminating the fruiting body protein fingerprints with a deviation exceeding a preset value in the spectrum group to obtain a processed spectrum group; Extracting characteristic values ​​of all fruiting body protein fingerprints in the treated pattern group to establish a super fingerprint of the treated pattern group, and adding the time characteristics and varieties of the treated pattern group to the naming of the super fingerprint corresponding to the treated pattern group; The fruiting body protein fingerprint database is constructed based on the named super fingerprint map.

12. The method for identifying Ganoderma lucidum based on time-of-flight mass spectrometry according to any one of claims 1 to 3, characterized in that: Determine the target protein fingerprint map corresponding to the protein fingerprint map to be identified in the Ganoderma lucidum protein fingerprint database, including: Extracting features from the fingerprint of the protein to be identified; Using a neural network model, the features in the protein fingerprint to be identified are compared with each protein fingerprint in the Ganoderma lucidum protein fingerprint database to determine the similarity between the protein fingerprint to be identified and each protein fingerprint; The protein fingerprint corresponding to the highest similarity among all the similarities is used as the target protein fingerprint.

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