Method for simultaneously detecting six types of I-type ganoderic acid in ganoderma lucidum fermented mycelium

Abstract

The invention provides a method for simultaneously detecting six types of I-type ganoderic acid in ganoderma lucidum fermented mycelia. The method comprises the following steps: (1) pretreating the ganoderma lucidum fermented mycelia; (2) an ultra-high performance liquid chromatography detection step; (3) obtaining mass spectrum information of the compound; (4) establishing a dynamic multi-reaction monitoring DMRM analysis method; and (5) a data analysis step. According to the method for simultaneously detecting the six types of I-type ganoderic acid in the ganoderma lucidum fermented mycelium, accurate qualitative and quantitative analysis can be simultaneously carried out on the six types of I-type ganoderic acid compounds, so that an accurate and reliable detection means is provided for related basic research and product development and application of I-type triterpenes in ganoderma lucidum.

Description

Technical Field

[0001] This invention relates to the field of detection and analysis of active ingredients in medicinal fungi, specifically to a method for simultaneously detecting six type I ganoderic acids in Ganoderma lucidum fermentation mycelium. Background Technology

[0002] Ganoderma spp. is a well-known medicinal fungus in my country and East Asia. Triterpenes, especially ganoderic acids, are known to be characteristic components of Ganoderma and are also key active ingredients for developing drugs, health foods, or functional foods that enhance immunity, have anti-inflammatory, antiviral, and antitumor effects. Even during research on Ganoderma triterpenes, it was discovered that Ganoderma fermentation mycelium can produce high levels of ganoderic acids with structures different from those in the fruiting body. Ganoderic acids in Ganoderma fermentation mycelium can be divided into two types: type I ganoderic acids (without conjugated double bonds) and type II ganoderic acids (with conjugated double bonds). Type I ganoderic acids in Ganoderma are often present in low amounts and have fewer varieties. However, through gene editing to obtain engineered Ganoderma strains, it is possible to directionally produce high levels of new type I ganoderic acids. These ganoderic acids also possess strong anti-inflammatory activity, and their further biological activity and development value need to be explored based on the large-scale preparation of these compounds.

[0003] Therefore, it is necessary to establish a precise quantitative analysis method for this type of ganoderic acid to provide technical means for further high-yield production and development of these new type I ganoderic acids. Summary of the Invention

[0004] The purpose of this invention is to provide a method for simultaneously detecting six type I ganoderic acids in Ganoderma lucidum fermentation mycelium, the method comprising the following steps:

[0005] (1) Pretreatment steps of Ganoderma lucidum fermentation mycelium: Ganoderma lucidum mycelium is extracted with organic solvent, the supernatant is taken, filtered, and the filtrate is diluted to obtain the sample solution to be tested; a mixed standard solution of six types I ganoderic acids is prepared.

[0006] (2) Ultra-high performance liquid chromatography detection steps: The sample solution to be tested and the mixed standard solution of six type I ganoderic acids are separated by ultra-high performance liquid chromatography to obtain a better resolution;

[0007] (3) Steps for obtaining compound mass spectrometry information: Using mass spectrometry optimization software (Agilent Optimizer), the parent ion was scanned, daughter ion pairs were detected, and the optimal collision energy was found for the six type I ganoderic acid compounds;

[0008] (4) Steps for establishing the dynamic multiple reaction monitoring (DMRM) analysis method: In the data acquisition software (Agilent MassHunter Data Acquisition), based on the ultra-high performance liquid chromatography analysis conditions and triple quadrupole mass spectrometry detection conditions, the retention time, parent ion, daughter ion pairs and collision energy data were imported to establish a dynamic multiple reaction monitoring (DMRM) analysis method for six type I ganoderic acid compounds; at the same time, the mixed standard solution and sample solution of the six type I ganoderic acids were loaded and analyzed.

[0009] (5) Data analysis steps: Using quantitative analysis software (Agilent MassHunterQuantitativeAnalysis), standard curves for six compounds were created to validate the established method and to perform quantitative analysis on the Ganoderma lucidum mycelium sample solution.

[0010] Preferably, the pretreatment step of Ganoderma lucidum fermentation mycelium in step (1) is as follows: the mycelium to be tested is frozen until completely dry, methanol is added at a material-to-liquid ratio of 1:20-40 (weight g: volume ml) (preferably 1:20), and ultrasonic extraction or heating extraction at 60℃ for 10-90 min (preferably ultrasonic extraction for 60 min) is performed. The extract is filtered through an organic phase microporous filter membrane with a pore size of 0.20 μm to obtain the sample solution to be tested.

[0011] Preferably, the method for preparing the mixed standard solution of the six compounds in step (1) is as follows: accurately weigh out astraodoric acid C (C 30 H 48 O4, compound 1), astraodoric acid B (C 30 H 46 O4, compound 2), astraeusins ​​G(C 32 H 50 O5, compound 3), astraodoric acid A (C 32 H 48 O5, compound 4), astraodoric acid D(C 32 H 50 O5, compound 5), 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid (C 34 H 52Six compounds (O6, compound 6) were prepared into a mixed standard solution with a concentration of 25 μg / mL using mass spectrometry grade methanol (each ganoderic acid compound had a concentration of 25 μg / mL). The solution was then serially diluted to obtain mixed standard solutions with concentrations of 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL.

[0012] Preferably, the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: Agilent EclipsePlus C18 column, 1.8 μm, 2.1 × 100 mm, detection wavelength: 210 nm; column temperature: 35℃; room temperature: 20℃; sample loading volume: 4 μL; flow rate: 0.4 mL / min; pressure: 800 bar; mobile phase A: 0.01% (volume percentage) glacial acetic acid aqueous solution; mobile phase B: methanol; elution program: 0 min, 24% A, 76% B; 15 min, 20% A, 80% B; 20 min, 10% A, 90% B; 21 min, 0% A, 100% B;

[0013] Preferably, the retention time, parent ion, daughter ion pair, and collision energy of each compound required for analysis in step (3) are obtained using mass spectrometry optimization software (Agilent Optimizer) as follows:

[0014] Astraodoric acid C: Retention time: 5.10 min; Precursor ion: 471.3; Quantitative ion: 98.9, Collision energy: 25; Qualitative ion: 55.2, Collision energy: 37;

[0015] Astraodoric acid B: Retention time: 6.65 min; Precursor ion: 469.3; Quantitative ion: 99.1, Collision energy: 17; Qualitative ion: 55.2, Collision energy: 37;

[0016] Astraeusins ​​G: Retention time: 7.62 min; Precursor ion: 513.3; Quantitative ion: 453.3, Collision energy: 29; Qualitative ion: 99.0, Collision energy: 45;

[0017] Astraodoric acid A: Retention time: 9.89 min; Precursor ion: 511.3; Quantitative ion: 451.3, Collision energy: 29; Qualitative ion: 99.0, Collision energy: 37;

[0018] Astraodoric acid D: Retention time: 12.19 min; Precursor ion: 513.3; Quantitative ion: 453.2, Collision energy: 25; Qualitative ion: 99.0, Collision energy: 45;

[0019] 3α,22α-Diacetoxylanosta-8,24-dien-26-oic acid: Retention time: 17.27 min; Precursor ion: 555.3; Quantitative ion: 495.4, Collision energy: 33; Qualitative ion: 99.0, Collision energy: 37;

[0020] Preferably, in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) is used as the analytical instrument. The chromatographic conditions for UHPLC separation (same as those in step (2)) are as follows: Agilent EclipsePlus C18 column, 1.8 μm, 2.1 × 100 mm, detection wavelength: 210 nm; column temperature: 35 °C; sample loading: 4 μL; flow rate: 0.4 mL / min; mobile phase A: 0.01% (volume percentage) glacial acetic acid aqueous solution; mobile phase B: methanol; elution program: 0 min, 24% A, 76% B; 15 min, 20% A, 80% B; 20 min, 10% A, 90% B; 21 min, 0% A, 100% B; and the mass spectrometry conditions are: electrospray ionization source (AJS). ESI was used as the ion source, and detection was performed in negative ion mode. Dynamic multiple reaction monitoring (DMRM) was selected. The capillary voltage was 3500V, the capillary outlet voltage was 380V, the drying gas flow rate was 16L / min, the drying gas temperature was 200℃, the sheath gas temperature was 320℃, the sheath gas flow rate was 12L / min, and the nozzle voltage was 2000V.

[0021] The identification basis of the method for simultaneous detection of six type I ganoderic acids in Ganoderma lucidum fermentation mycelium of the present invention is as follows: when using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry for detection, the ganoderic acid compounds in Ganoderma lucidum mycelium must not only meet the requirement of consistent retention time, but also meet the requirement that the primary parent ion and secondary daughter ion pairs meet the detection parameters under specific mass spectrometry conditions in order to accurately quantify the compound.

[0022] The beneficial effects of this invention are as follows: The technical solution of this invention establishes a dynamic multiple reaction monitoring (DMRM) analysis method by using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-DMRM). This method can simultaneously and accurately perform qualitative and quantitative analysis on six specific type I ganoderic acid compounds in Ganoderma lucidum fermentation mycelium (especially Ganoderma lucidum engineered strain fermentation mycelium), thereby providing a precise and reliable technical means for related research and product development of Ganoderma lucidum fermentation mycelium. Attached Figure Description

[0023] Figure 1 Structural formulas of six type I ganoderic acids

[0024] Figure 2Ultra-high performance liquid chromatography (210 nm) of six type I ganoderic acids

[0025] Figure 3 Total ion chromatogram of six type I ganoderic acids

[0026] Compound 1: Astraodoric acid C;

[0027] Compound 2: Astraodoric acid B;

[0028] Compound 3: Astraeusins ​​G;

[0029] Compound 4: Astraodoric acid A;

[0030] Compound 5: Astraodoric acid D;

[0031] Compound 6: 3α,22α-Diacetoxylanosta-8,24-dien-26-oic acid; Detailed Implementation

[0032] The technical solution of the present invention is further illustrated below through specific embodiments, but the scope of protection of the present invention is not limited to the content described. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are conventional commercial reagents or prepared according to conventional methods unless otherwise specified.

[0033] Ganoderma lucidum engineered strain ΔW2: This strain is preserved at the School of Life Science and Technology, Kunming University of Science and Technology. The construction method is described in the invention patent (A type I ganoderic acid and its preparation method and application, publication number: CN120624504A).

[0034] Strain 1: G. lingzhi, strain number: Cui9166;

[0035] Strain 2: G. sessile, strain number: Dai16403;

[0036] Strain 3: G. gibbosum, strain number: Cui16209;

[0037] Strain 4: G. angustisporum, strain number: Dai19889;

[0038] Strain 5: G. sichuanense, strain number: Dai19651;

[0039] Strain 6: G. leucocontextum, strain number: Dai12418;

[0040] Strain 7: G. sinense, strain number: Dai20076;

[0041] Strain 8: G. orbiforme, strain number: Dai20550;

[0042] Strain 9: G. shanxingense, strain number: Dai20225A;

[0043] Strain 10: G. mastoporum, strain number: Dai20635;

[0044] Strain 11: G. tsugae, strain number: Dai 20238;

[0045] The above strains 1-11 are preserved at the College of Ecology and Nature Conservation, Chinese Forestry University.

[0046] Strain 12: G. resinaceum, strain number: 5412, this strain is preserved at the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences.

[0047] Example 1: Isolation and preparation of six type I ganoderic acids from Ganoderma lucidum engineered strain ΔW2

[0048] (1) Fermentation of Ganoderma lucidum engineered strain ΔW2: Ganoderma lucidum engineered strain ΔW2 was inoculated into a primary shake flask and cultured in the dark at 150 r / min and 25℃ for 7 days with shaking. It was then transferred to a secondary shake flask at a volume percentage of 10% and cultured in the dark at 150 r / min and 25℃ for 3 days with shaking. It was then cultured statically for 21 days. The upper mycelium was collected and freeze-dried.

[0049] (2) Extraction and extraction of mycelium: freeze-dried Ganoderma lucidum mycelium was soaked in 95% ethanol aqueous solution at room temperature for 24 hours each time. The extracts were combined and concentrated to obtain Ganoderma lucidum mycelium extract. The Ganoderma lucidum mycelium extract was further extracted with an equal volume of petroleum ether three times. The residue was then extracted with an equal volume of ethyl acetate three times. The ethyl acetate extracts were combined and concentrated under reduced pressure to obtain ethyl acetate extract.

[0050] (3) Preparation of six type I ganoderic acids (the structural formulas of the six type I ganoderic acids are shown in the figure) Figure 1Ethyl acetate extract was separated using YMC ODS C18 (50 μm particle size, 12 nm pore size) packing material, with a gradient elution of 0.01% (v / v) glacial acetic acid aqueous solution as phase A and acetonitrile as phase B. The fractions were: 0-5 min, B: 55%; 5-10 min, B: 55%-60%; 10-30 min, B: 60%-70%; 30-35 min, B: 70%-75%; 35-60 min, B: 75%-80%; 60-80 min, B: 80%-100%; 80-100 min, B: 100%-100%. The flow rate was 200 mL / min. One fraction was collected every 400 mL, and similar fractions were combined to obtain 24 fractions: Fr1-Fr24.

[0051] Fr12 fraction was repeatedly recrystallized to obtain compound 1 (astraodoric acid C); Fr15 fraction was subjected to medium-high pressure reversed-phase chromatography using an Agilent Zorbax Eclipse Plus-C18 PrepHT column (5 μm, 2.12 × 250 mm), with acetonitrile (A)-water (B) as the mobile phase and gradient elution (0-25 min, A: 55%-65%; 25-75 min, 65%-85%), collecting the peak at 28.1 min to obtain compound 2 (astraodoric acid B); Fr18 fraction was subjected to medium-high pressure reversed-phase chromatography using an Agilent Zorbax Eclipse Plus-C18 PrepHT column (5 μm, 2.12 × 250 mm), with acetonitrile (A)-water (B) as the mobile phase and gradient elution (0-50 min, A: 65%-85%), collecting the peak at 25.6 min to obtain compound 3 (astraeusins). G); Fr19 fraction was repeatedly recrystallized, and the recrystallized crystals were prepared into thin layers. The mixture was developed with petroleum ether:ethyl acetate = 7:3 (v / v) to obtain compound 4 (astraodoric acid A) (Rf = 0.32) and compound 5 (astraodoric acid D) (Rf = 0.44), respectively. Fr21 was subjected to medium-high pressure reversed-phase chromatography using an Agilent Zorbax Eclipse Plus-C18 PrepHT column (5 μm, 2.12 × 250 mm), with acetonitrile (A)-water (B) as the mobile phase. Gradient elution was performed (0-15 min, A: 90%-100%; 15-30 min, A: 100%). The peak at 16.9 min was collected to obtain compound 6 (3α,22α-diacetoxylanosta-8,24-dien-26-oic acid). The NMR data of the six type I ganoderic acid compounds are shown in Tables 1 and 2.

[0052] Example 2: Establishment of the detection method

[0053] (1) Pretreatment steps of fermentation mycelium of Ganoderma lucidum engineered strain ΔW2: Take 5.0g of dried fermentation mycelium of Ganoderma lucidum engineered strain in Example 1, add 100mL of ethanol at a material-to-liquid ratio of 1:20 (weight-to-volume ratio, weight g: volume ml), extract by ultrasonication for 60min, and filter the supernatant through an organic phase microporous filter membrane with a pore size of 0.20μm to obtain the sample solution to be tested.

[0054] Preparation of mixed standard solutions: Take the six type I ganoderic acid compounds (compounds 1-6) prepared in Example 1 and prepare mixed standard solutions with a concentration of 25 μg / mL using mass spectrometry grade methanol (where the concentration of each standard is 25 μg / mL). Dilute them stepwise with mass spectrometry grade methanol to prepare mixed standard solutions of 10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL, and store them in a refrigerator at 4°C for later use.

[0055] (2) Ultra-high performance liquid chromatography detection steps: The sample solution to be tested and the mixed standard solution of six type I ganoderic acids are separated by ultra-high performance liquid chromatography to obtain a better resolution;

[0056] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: An Agilent Eclipse Plus C18 column (1.8 μm, 2.1 × 100 mm) was used; detection wavelength: 210 nm; column temperature: 35℃; sample loading volume: 4 μL; flow rate: 0.4 mL / min; mobile phase A: 0.01% glacial acetic acid aqueous solution; mobile phase B: methanol; elution program: 0 min, 24% A, 76% B; 15 min, 20% A, 80% B; 20 min, 10% A, 90% B; 21 min, 0% A, 100% B. The HPLC results of the mixed standard solution of six type I ganoderic acids are shown below. Figure 2 .

[0057] (3) Steps for obtaining compound mass spectrometry information: Prepare 5 ppm methanol solutions of six type I ganoderic acid compounds respectively. Determine the parent ion information of the six type I ganoderic acid compounds in negative ion mode. Then, use Agilent Optimizer software to automatically optimize the daughter ions and collision energies after determining the parent ions.

[0058] Astraodoric acid C: Retention time: 5.10 min; Precursor ion: 471.3; Quantitative ion: 98.9, Collision energy: 25; Qualitative ion: 55.2, Collision energy: 37;

[0059] Astraodoric acid B: Retention time: 6.65 min; Precursor ion: 469.3; Quantitative ion: 99.1, Collision energy: 17; Qualitative ion: 55.2, Collision energy: 37;

[0060] Astraeusins ​​G: Retention time: 7.62 min; Precursor ion: 513.3; Quantitative ion: 453.3, Collision energy: 29; Qualitative ion: 99.0, Collision energy: 45;

[0061] Astraodoric acid A: Retention time: 9.89 min; Precursor ion: 511.3; Quantitative ion: 451.3, Collision energy: 29; Qualitative ion: 99.0, Collision energy: 37;

[0062] Astraodoric acid D: Retention time: 12.19 min; Precursor ion: 513.3; Quantitative ion: 453.2, Collision energy: 25; Qualitative ion: 99.0, Collision energy: 45;

[0063] 3α,22α-Diacetoxylanosta-8,24-dien-26-oic acid: Retention time: 17.27 min; Precursor ion: 555.3; Quantitative ion: 495.4, Collision energy: 33; Qualitative ion: 99.0, Collision energy: 37;

[0064] (4) Establishment of the Dynamic Multiple Reaction Monitoring (DMRM) Quantitative Analysis Method: Under the parameter settings of ultra-high performance liquid chromatography and triple quadrupole mass spectrometry in the data acquisition software (Agilent MassHunter Data Acquisition), the mass spectrometry acquisition mode was changed to multiple reaction monitoring (MRM). Information such as the parent ion, quantitative and qualitative daughter ion pairs, and collision energies of the compounds were then imported into the detection method. A mixed standard solution of six type I ganoderic acids was loaded, and after running the program, the acquisition mode was updated to dynamic multiple reaction monitoring (DMRM). The total ion current results of the mixed standard solution of the six type I ganoderic acids under this mode are shown in [Figure 1]. Figure 3 .

[0065] The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) separation were as follows: an Agilent Eclipse Plus C18 column (1.8 μm, 2.1 × 100 mm) was used; the detection wavelength was 210 nm; the column temperature was 35℃; the room temperature was 20℃; the sample loading volume was 4 μL; the flow rate was 0.4 mL / min; the pressure was 800 bar; mobile phase A was 0.01% glacial acetic acid aqueous solution; mobile phase B was methanol; and the elution program was: 0 min, 24% A, 76% B; 15 min, 20% A, 80% B; 20 min, 10% A, 90% B; 21 min, 0% A, 100% B.

[0066] The triple quadrupole mass spectrometry analysis conditions were as follows: an electrospray ionization source (AJS ESI) was used as the ion source; detection was performed in negative ion mode; dynamic multiple reaction monitoring (DMRM) was used; capillary voltage: 3500V; capillary outlet voltage: 380V; drying gas flow rate: 16L / min; drying gas temperature: 200℃; sheath gas temperature: 320℃; sheath gas flow rate: 12L / min; nozzle voltage: 2000V.

[0067] (5) Limit of detection and limit of quantitation: The limit of detection (LOD) and limit of quantitation (LOQ) are calculated based on the standard deviation of the response value and the slope of the standard curve. Where: LOD = 3σ / S, LOQ = 10σ / S, σ: standard deviation of the response value, S: slope of the standard curve, and the standard deviation of the response value is the residual standard deviation of the standard curve.

[0068] (6) Standard curve preparation: Take the prepared mixed standard working solutions of 5μg / mL, 2μg / mL, 1μg / mL, 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL and 20ng / mL and load them according to the ultra-high performance liquid chromatography and mass spectrometry analysis conditions in step (4) above. Plot the concentration of the compound on the horizontal axis and the quantitative ion response value of the compound on the vertical axis to prepare the quantitative standard curve equation, as shown in Table 3.

[0069] Example 3: Methodological Validation and Sample Testing

[0070] Methodological validation and testing for Example 2: The methodological validation was conducted in accordance with laboratory quality control standards for food physicochemical testing and relevant pharmacopoeia regulations.

[0071] (1) Precision: The mixed standard solution was injected 6 times on the same day. The concentrations of the six type I ganoderic acids obtained from the 6 experiments were calculated based on the standard curve, and the intra-day precision was calculated. The mixed standard solution was injected twice a day for three consecutive days, and the inter-day precision was calculated based on the results of the 6 experiments.

[0072] The results showed that the RSDs for the intra-day precision determination of the six type I ganoderic acids were 3.08%, 2.69%, 3.15%, 4.01%, 3.43%, and 3.24%, respectively; the RSDs for the inter-day precision determination were 5.59%, 7.36%, 8.77%, 5.56%, 6.00%, and 6.67%, respectively, all less than 15.00%, indicating that the method has good intra-day and inter-day precision. Specific results are shown in Tables 4 and 5.

[0073] (2) Stability: The sample solution from Example 2 was injected at 0h, 2h, 4h, 6h, 8h, 12h, and 24h, respectively. The stability of the sample was calculated based on the results of the seven experiments. The results showed that the RSDs of the six type I ganoderic acids were 3.07%, 2.90%, 2.94%, 2.76%, 3.61%, and 2.94%, respectively, all less than 15.00%, indicating that the sample properties were stable within 24 hours. Specific results are shown in Table 6.

[0074] (3) Repeatability: Six parallel samples of the fermentation mycelium of the Ganoderma lucidum engineered strain ΔW2 from Example 1 were weighed and treated according to the pretreatment steps in Example 2. The samples were then injected for testing, and the repeatability was calculated based on the results of the six experiments. The results showed that the RSDs of the six type I ganoderic acids were 2.44%, 2.28%, 2.42%, 2.57%, 2.89%, and 1.79%, respectively, all less than 15.00%, indicating good repeatability. Specific results are shown in Table 7.

[0075] (4) Recovery rate: Take three portions of ethanol extract of fermented mycelium of Ganoderma lucidum strain ΔW2 with known concentration (according to the extraction method in Example 2), 200 μL for each portion, and add 80%, 100% and 120% of the content of each compound as reference standard respectively. Each sample is injected three times, and the recovery rate is calculated based on the experimental results.

[0076] Recovery rate % = (Measured value - Amount of analyte in the test sample) / Amount of reference standard added × 100%

[0077] The specific recovery results are shown in Table 8. The results confirmed that the 80% recoveries of the six ganoderic acids were 100.43%, 92.17%, 99.01%, 113.42%, 113.67%, and 94.39%, respectively; the 100% recoveries were 107.32%, 104.78%, 101.22%, 96.38%, 101.52%, and 97.59%, respectively; and the 120% recoveries were 94.62%, 101.95%, 91.19%, 90.49%, 99.21%, and 92.70%, respectively. The RSDs of all the above recoveries were within 8.0%, meeting the method requirements.

[0078] (5) Sample detection: To investigate the effect of different static culture times on the yield of type I ganoderic acids, the Ganoderma lucidum engineered strain ΔW2 was fermented according to the method in Example 1, but different static culture time points were set, namely, samples were taken and freeze-dried on the 14th, 21st, 28th, 35th and 42nd days of static culture. After drying, the mycelium was treated according to the pretreatment steps in Example 2, and the content of ganoderic acids was determined. The results are shown in Table 9. It was found that the total accumulation of the six type I ganoderic acids reached the highest level of 69384.66 μg / g on the 35th day of mycelial fermentation. Among them, 3α,22α-Diacetoxylanosta-8,24-dien-26-oic acid (6) was the ganoderic acid compound with the highest content at different static culture fermentation periods.

[0079] Fermentation culture of strains 1-12 was carried out completely according to the method of Example 1. The mycelium of each strain was freeze-dried and treated according to the pretreatment steps of Example 2. The contents of the six types of type I ganoderic acids were determined, and the results are shown in Table 10 and Table 10 (continued). The fermentation mycelium of G. leucocontextum and G. resinaceum contained all six types of type I ganoderic acids, while the fermentation mycelium of the other 10 Ganoderma species contained only some types of type I ganoderic acids. Overall, the six types of type I ganoderic acids are expressed at low levels in multiple species of Ganoderma.

[0080] Table 1. Six Type I Ganoderic Acids 13 C10 NMR data (125MHz, CDCl3)

[0081]

[0082]

[0083] Table 2 Six Type I Ganoderic Acids 1 H NMR data (500MHz, CDCl3)

[0084]

[0085] Table 3. Standard curves and parameters for quantitative analysis of six types I ganoderic acids

[0086]

[0087] Table 4. Intraday Precision Results

[0088]

[0089] Table 5. Daytime Precision Results

[0090]

[0091] Table 6. Sample stability results

[0092]

[0093]

[0094] Table 7. Sample repeatability results

[0095]

[0096] Table 8. Sample Recovery Results

[0097]

[0098] Table 9. Measurement results (μg / g) of engineered strain ΔW2 samples at different static culture periods.

[0099]

[0100]

[0101] Table 10. Results of mycelial sample determination (μg / g) of different Ganoderma lucidum strains

[0102]

[0103] Table 10 (continued): Results of mycelial sample determination (μg / g) of different Ganoderma lucidum strains

[0104]

Claims

1. A method for simultaneously detecting six type I ganoderic acids in Ganoderma lucidum fermentation mycelium, characterized in that... The method includes the following steps: (1) Pretreatment steps of Ganoderma lucidum fermentation mycelium: Add organic solvent to extract the mycelium, take the supernatant, filter, and dilute the filtrate to obtain the sample solution to be tested; prepare a mixed standard solution of six types I ganoderic acids; (2) Ultra-high performance liquid chromatography detection steps: The sample solution to be tested and the mixed standard solution of six types I ganoderic acids are separated by ultra-high performance liquid chromatography; (3) Steps for obtaining compound mass spectrometry information: Using the mass spectrometry optimization software Agilent Optimizer, the parent ion was scanned, daughter ion pairs were detected, and the optimal collision energy was found for the six types of type I ganoderic acids. (4) Steps for establishing the dynamic multiple reaction monitoring (DMRM) analysis method: In the data acquisition software Agilent MassHunterData Acquisition, under the conditions of ultra-high performance liquid chromatography and triple quadrupole mass spectrometry, the retention time, parent ion, daughter ion pairs and collision energy data were imported to establish the dynamic multiple reaction monitoring (DMRM) analysis method for six type I ganoderic acid compounds; at the same time, the mixed standard solution and sample solution of the six type I ganoderic acid compounds were loaded and analyzed. (5) Data analysis steps: Using the quantitative analysis software Agilent MassHunter Quantitative Analysis, standard curves for six type I ganoderic acid compounds were created to validate the established method and to perform quantitative analysis on the ganoderic fermentation mycelium sample solution.

2. The method for simultaneous detection of six type I ganoderic acids in Ganoderma lucidum fermentation mycelium according to claim 1, wherein the pretreatment step of Ganoderma lucidum mycelium in step (1) is as follows: the mycelium to be tested is frozen until completely dry, methanol is added at a material-to-liquid ratio of 1:20-1:40 (g weight: ml volume), and ultrasonic extraction or heating extraction at 60℃ for 10-90 min is performed. The extract is filtered through an organic phase microporous filter membrane with a pore size of 0.20 μm to obtain the sample solution to be tested.

3. The method for simultaneous detection of six type I ganoderic acids in Ganoderma lucidum fermentation mycelium according to claim 1, wherein the preparation method of the mixed standard solution of the six type I ganoderic acids in step (1) is as follows: take six type I ganoderic acid standards, prepare a mixed standard solution with a concentration of 25 μg / mL using mass spectrometry grade methanol, and then dilute stepwise to obtain mixed standard solutions of 5 μg / mL, 2 μg / mL, 1 μg / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 20 ng / mL.

4. The method for simultaneous detection of six type I ganoderic acids in Ganoderma lucidum fermentation mycelium according to claim 1, wherein the chromatographic conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: Agilent Eclipse Plus C18 column, 1.8 μm, 2.1 × 100 mm, detection wavelength: 210 nm; column temperature: 35 °C; sample loading: 4 μL; flow rate: 0.4 mL / min; mobile phase A: 0.01% (v / v) glacial acetic acid aqueous solution; mobile phase B: methanol; elution program: 0 min, 24% A, 76% B; 15 min, 20% A, 80% B; 20 min, 10% A, 90% B; 21 min, 0% A, 100% B.

5. The method for simultaneous detection of six type I ganoderic acids in Ganoderma lucidum fermentation mycelium according to claim 1, wherein the retention time, parent ion, daughter ion pair and collision energy information of each compound required for analysis in step (3) are obtained by using the mass spectrometry optimization software Agilent Optimizer as follows: Astraodoric acid C: Retention time: 5.10 min; Precursor ion: 471.3; Quantitative ion: 98.9, Collision energy: 25; Qualitative ion: 55.2, Collision energy: 37; Astraodoric acid B: Retention time: 6.65 min; Precursor ion: 469.3; Quantitative ion: 99.1, Collision energy: 17; Qualitative ion: 55.2, Collision energy: 37; Astraeusins ​​G: Retention time: 7.62 min; Precursor ion: 513.3; Quantitative ion: 453.3, Collision energy: 29; Qualitative ion: 99.0, Collision energy: 45; Astraodoric acid A: Retention time: 9.89 min; Precursor ion: 511.3; Quantitative ion: 451.3, Collision energy: 29; Qualitative ion: 99.0, Collision energy: 37; Astraodoric acid D: Retention time: 12.19 min; Precursor ion: 513.3; Quantitative ions: 453.2, collision energy: 25; qualitative ions: 99.0, collision energy: 45; 3α,22α-Diacetoxylanosta-8,24-dien-26-oic acid: Retention time: 17.27 min; Precursor ion: 555.3; Quantitative ion: 495.4, Collision energy: 33; Qualitative ion: 99.0, Collision energy:

37.

6. The method for simultaneous detection of six type I ganoderic acids in Ganoderma lucidum fermentation mycelium according to claim 1, wherein in step (4), ultra-high performance liquid chromatography-triple quadrupole mass spectrometry is used as the analytical instrument, wherein the chromatographic conditions for ultra-high performance liquid chromatography separation are as follows: Agilent Eclipse Plus C18 column, 1.8 μm, 2.1 × 100 mm, detection wavelength: 210 nm; column temperature: 35 ℃; sample loading: 4 μL; flow rate: 0.4 mL / min; mobile phase A: 0.01% v / v glacial acetic acid aqueous solution; mobile phase B: methanol; elution program: 0 min, 24% A, 76% B; 15 min, 20% A, 80% B; 20 min, 10% A, 90% B; 21 min, 0% A, 100% B; mass spectrometry conditions are: electrospray ionization source AJS ESI was used as the ion source, and detection was performed in negative ion mode. Dynamic multiple reaction monitoring (DMRM) was selected, with capillary voltage of 3500V and capillary outlet voltage of 380V; drying gas flow rate of 16L / min, drying gas temperature of 200℃, sheath gas temperature of 320℃, sheath gas flow rate of 12L / min, and nozzle voltage of 2000V.

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