Cyanidin-3-glucoside and its application

Abstract

The application provides a high-yield Cangshan Lentinula edodes CSLe001 and application thereof, and relates to the technical field of Cangshan Lentinula edodes culture. The Cangshan Lentinula edodes CSLe001 has been preserved in the China Center for Type Culture Collection on June 16, 2025, and the preservation number is CCTCC NO: M 20251402. The strain is cultured in a walnut waste fungus bag, has a short harvesting time, high yield, and the Cangshan Lentinula edodes CSLe001 fruiting body and the mycelium obtained by culturing the strain in a culture medium added with walnut pruning branch powder have high melinoidin yield, thereby providing a new way for the production of melinoidin.

Description

Technical Field

[0001] The present invention relates to the technical field of Cangshan Lentinus edodes cultivation, and in particular to a high-yield Cangshan Lentinus edodes strain CSLe001 and application thereof. Background Art

[0002] Lentinus edodes ( Lentinula edodes ), also known as xiangsun or winter mushroom, belongs to Agaricales ( Agaricales ) Oudemansiella ( Omphalotaceae ) Craterellus ( Lentinula ) is an edible fungus. Lentinus edodes is rich in nutrients such as protein, polysaccharides, vitamins and trace elements. Lentinan has been proven to have effects such as antiviral activity, anti-aging, prevention of cardiovascular and cerebrovascular diseases, prevention of liver cirrhosis, and protection of renal function; the crude fiber in Lentinus edodes can help digestion, relieve constipation and assist weight loss; purine, choline and other components in Lentinus edodes can lower blood pressure and blood lipids, and prevent the occurrence of liver cirrhosis and arteriosclerosis; in recent years, studies have shown that eating Lentinus edodes has a certain therapeutic effect on reducing diabetes and its complications, such as sciatica and retinitis.

[0003] Cangshan Mountain is located in the central part of Dali Bai Autonomous Prefecture in Yunnan Province, west of Erhai Lake, between 100°55′00″-100°11′30″ east longitude and 25°34′30″-26°00′00″ north latitude. It is the boundary mountain between Dali City and Yangbi Yi Autonomous County, with a mountain range running from northwest to southeast. It starts from Guanglanyao Pass west of Huadianba in the north and ends at West Er River in the south, with a length of about 48 km and an east-west width of about 18 km. The connected ridges of Cangshan Mountain are arranged in rows, consisting of 19 peaks from north to south, with an altitude of 3074-4122 m. The highest peak, Malong Peak, has an altitude of 4122 m, with a relative height difference of about 2500 m from the valley on the west slope, and a relative height difference of about 2100 m from Erhai Lake on the east slope. There is a tradition of eating wild Lentinus edodes (xiangsun) in the Cangshan area, and wild Lentinus edodes from Cangshan has a strong fragrance and excellent taste.

[0004] In order to further develop and utilize Cangshan Lentinus edodes and meet people's demand for it, we have carried out works including preservation of Cangshan Lentinus edodes strains, resource evaluation, and screening of Cangshan Lentinus edodes strains suitable for producing Lentinus edodes from walnut industry waste, as well as technologies such as liquid spawn.

[0005] Mellein exhibits inhibitory activity against a variety of bacteria and fungi, especially has a strong inhibitory effect on bacteria of the genus Xanthomonas, and has potential for development as an antibacterial agent. Mellein is toxic to a variety of plants and algae, which gives it potential for development as a biological herbicide. In addition, mellein has been reported to have anti-hepatitis C virus (HCV) protease activity, as well as anti-tumor, anti-leukemia, antiviral and other activities.

[0006] Mellein was first isolated from Aspergillus apis and subsequently from various fungi. Recently, Cabudaje et al. (Cabutaje, EM, Bungihan, ME, De Leon, AM, Pecundo, MH, Osaki-Oka, K., Kido, K., ...&Ishihara, A. (2025). Mellein and its derivatives isolated from the basidiomycete mushroom Lentinus tigrinus exhibit inhibitory activity against plant pathogenic microorganisms. Journal of Applied Microbiology, 136(11), lxaf270.) reported that Mellein and its derivatives isolated from basidiomycete mushrooms exhibit inhibitory activity against plant pathogenic microorganisms. Journal of Applied Microbiology, 136(11), lxaf270. Xu et al. (Xu, X., Yu, C., Liu, Z., Cui, X., Guo, X., & Wang, H. (2024). Chemical composition, antioxidant and anti-inflammatory activity of shiitake mushrooms (Lentinus edodes). Journal of Fungi, 10(8), 552. ) used rice to cultivate shiitake mushrooms. Although no keratocycline was detected, 6-hydroxykeratocycline and 5-hydroxykeratocycline were isolated. Based on this, further research on the yield of keratocycline in shiitake mushrooms is promising. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a high-yield Cangshan shiitake mushroom strain CSLe001 and its applications. A new Cangshan shiitake mushroom strain CSLe001 is isolated and preserved, exhibiting high yield and a short growth time. Furthermore, this Cangshan shiitake mushroom, when cultured in a specific culture medium, also demonstrates a high yield of apigenin, providing a new pathway for apigenin production.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A high-yielding Cangshan shiitake mushroom strain CSLe001, the proposed classification name of which is Cangshan shiitake mushroom CSLe001. Lentinula edodes var Cangshan CSLe001 was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC NO: M 20251402.

[0009] The aforementioned Cangshan shiitake mushroom CSLe001 can be used to produce honey koji.

[0010] The method to enhance the production of acetoxin by *Lentinula cylindrica* CSLe001 is to inoculate *Lentinula cylindrica* CSLe001 liquid seeds into HTZM medium and culture statically at 25°C to obtain mycelium, or to inoculate *Lentinula cylindrica* CSLe001 liquid seeds into walnut waste substrate bags to obtain fruiting bodies; the formula of the HTZM medium is 100g of walnut pruning branch powder and 50ml of MS solution, and the mass concentration of the MS solution is 4.79g / L.

[0011] Preferably, the preparation method of the liquid seed of *Vitex negundo* CSLe001 includes the following steps: (1) The mycelium of *Lentinula edodes* CSLe001 was inoculated onto a PDA culture medium plate and cultured to obtain a solid culture. (2) Take a 1cm×1cm mycelium from the solid culture, put it into a 2ml sterile centrifuge tube, add 700ul sterile water and 2 steel balls, grind it at 180rpm for 2min to obtain a culture suspension, and then inoculate the culture suspension into MY medium to obtain Cangshan shiitake mushroom CSLe001 liquid seed.

[0012] Preferably, the culture conditions in step (1) are dark culture at 28°C for 15 days.

[0013] Preferably, the culture method for inoculating the bacterial suspension into MY culture medium in step (2) is to first culture it statically in the dark at 28°C for 3 days, and then transfer it to a shaker for dark culture at 28°C and 150 rpm for 20 days.

[0014] Preferably, the method for preparing the walnut pruning branch powder is as follows: first, the walnut pruning branches pruned from the walnut tree are crushed into particles of about 5cm, then baked in an oven at 60℃ for 2 hours, and then crushed into powder with a diameter of 0.6-1.0 mm, which is the walnut pruning branch powder.

[0015] This invention provides a high-yield Cangshan shiitake mushroom strain CSLe001 and its applications, with advantages over existing technologies as follows: This invention screens for a strain of *Lentinula chansonii* capable of efficiently decomposing lignin by adding guaiacol to the culture medium, and names it *Lentinula chansonii* CSLe001. Lentinula edodes var Cangshan CSLe001, this strain, uses walnut waste substrate bags for inoculation to produce fruiting bodies, effectively shortening the fruiting time and increasing yield. The fruiting bodies of CSLe001 Shiitake mushrooms from Cangshan Mountain and the mycelium obtained by cultivating this strain on a culture medium supplemented with walnut pruning powder have a high yield of arganin, providing a new approach for the production of arganin. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the growth of the PDKASY strain that efficiently decomposes lignin, as described in this invention. Figure 2 Phylogenetic tree of *Lentinula edodes* CSLe001; Figure 3 A schematic diagram illustrating the color change of the Cangshan shiitake mushroom CSLe001; Figure 4 A schematic diagram showing the fruiting bodies of the Cangshan shiitake mushroom CSLe001; Figure 5 A schematic diagram showing the difference in aspergillin content in honey across different samples; Figure 6 This is a schematic diagram of HPLC detection of the fruiting body (CSF) of *Lentinula edodes* CSLe001 from Cangshan Mountain. The peak indicated by the blue arrow represents apigenin. Figure 7 This is a schematic diagram of HPLC detection of L808 fruiting body (L808F), where the peak indicated by the blue arrow is honey aspergillin; Figure 8 This is a schematic diagram of HPLC detection of mycelium of *Lentinula edodes* CSLe001 in HTZM medium, where the peak indicated by the blue arrow is apigenin. Figure 9 This is a schematic diagram of HPLC detection of mycelium of *Lentinula edodes* CSLe001 in MEY medium, where the peak indicated by the blue arrow represents apigenin. Figure 10 This is a schematic diagram of HPLC detection of mycelium of *Lentinula edodes* CSLe001 in DM medium, where the peak indicated by the blue arrow represents apigenin. Figure 11 The growth of *Lentinula edodes* CSLe001 in MEY medium; Figure 12 The growth of *Lentinula edodes* CSLe001 in HTZM medium; Figure 13 The growth of *Lentinula edodes* CSLe001 in DM medium. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] The mushroom CSLe001 used below is proposed to be classified and named as *Lentinulaedodes var. Cangshan* CSLe001. It was deposited on June 16, 2025 at the China Center for Type Culture Collection (depository address: Wuhan University, China) with accession number CCTCC NO: M 20251402.

[0019] The purchase link for the Shiitake Mushroom 808 (Experiment No. L808) spawn is: https: / / item.taobao.com / item.htm?abbucket=15&id=979555710779&mi_id=0000EEcnvyPfpSvAnAx1w3EKRJEZulIq0tQA7OV4XSzrNTY&ns=1&priceTId=2150448 817749208933813243e0fe3&skuId=5937748633643&spm=a21n57.1.hoverItem.6&utparam=%7B %22aplus_abtest%22%3A%2288a262a6ee3925590b3cc1b3f315b8a1%22%7D&xxc=taobaoSearch; The purchase link for potato starch is: https: / / item.jd.com / 10205449045834.html?spmTag=YTAyMTkuYjAwMjM1Ni5jMDAwMDQ2ODkuc2VhcmNoX2NvbmZpcm0lMkNhMDI0MC5iMDAyNDkzLmMwMDAwNDAyNy4xJTIzc2t1X2NhcmQ&pvid=1207af171d1f4c74b56c56ad5a001413; The purchase link for PDA culture medium is: https: / / item.jd.com / 10127969630452.html?spmTag=YTAyMTkuYjAwMjM1Ni5jMDAwMDQ2ODkuc2VhcmNoX2NvbmZpcm0lMkNhMDI0MC5iMDAyNDkzLmMwMDAwNDAyNy4xJTIzc2t1X2NhcmQ&pvid=ddea80d748e142429584b4bf88ea0906; The purchase link for Malt Extract is: https: / / item.jd.com / 10210912458789.html?spmTag=YTAyMTkuYjAwMjM1Ni5jMDAwMDcyMTAua2V5d29yZF9lbnRlciUyQ2EwMjQwLmIwMDI0OTMuYzAwMDA0MDI3LjQlMjNza3VfY2FyZA; The purchase link for MS medium (containing sucrose and agar) is: https: / / item.jd.com / 10070722079686.html?spmTag=YTAyMTkuYjAwMjM1Ni5jMDAwMDcyMTAua2V5d29yZF9lbnRlciUyQ2EwMjQwLmIwMDI0OTMuYzAwMDA0MDI3LjIlMjNza3VfY2FyZA; The walnut waste mushroom bag was prepared according to the invention patent CN117814070A "A method for preparing edible fungi bags based on walnut waste". The components are as follows by weight: 40 parts walnut oil cake, 50 parts walnut branches, 15 parts wheat bran, 30 parts Fagaceae plant sawdust, 1.5 parts gypsum powder, 1.5 parts lime, 2 parts brown sugar, and 25 parts corn cob (particle size 0.1-2cm).

[0020] Example 1: Isolation and identification of Cangshan shiitake mushroom CSLe001: 1. Collection of microbial strains, Wild Cangshan shiitake mushrooms were collected from various locations including Heiduizi Village in Shahe Village, Taiping Township, Qingkou Village in Qingkou Village, Pingcun Village in Xiangyang Village, Pingpo Town, Taoshuping Village in Maidi Village, Yangjiang Town, and Kucao'ao Village in Xiuling Village, Cangshan Town. The collected mushrooms were then placed in foam boxes with ice packs for low-temperature preservation.

[0021] 2. Isolation and identification of highly efficient lignin-decomposing strains The ability of guaiacol to detect laccase, a key enzyme in lignin decomposition, was used to evaluate the ability of strains to produce laccase, thereby screening strains that can efficiently decompose lignin.

[0022] PDKASY medium preparation PDKASY medium formula: 0.25 g / L guaiacol, 2.78 g / L WPM medium, 7 g / L potato extract, 20 g / L glucose, 100 μg / mL ampicillin, 100 μg / mL streptomycin, 100 μg / mL gentamicin, 15 g / L agar, balance water.

[0023] The specific process is as follows: (1) After blowing away the debris from the wild-collected shiitake mushrooms with a hair dryer, use sterilized tweezers next to an alcohol lamp in a clean bench to separate the cap and stem. Take the mycelium (about 0.2 cm) at the junction of the cap and stem, and inoculate the mycelium onto PDKASY solid medium. Observe the results after dark incubation at 28℃ for 8 days. Figure 1 ).

[0024] (2) Preservation and identification of strains The mycelium with the largest red circle was inoculated onto a new PDA medium plate and cultured for 4 days. A portion of the inoculum was then transferred to test tubes for later use. The remaining mycelium was collected for DNA extraction.

[0025] (3) Strain identification ① Morphological identification: This strain grew well on PDA solid medium, and a large number of white aerial mycelia formed on the surface of the colony.

[0026] ② DNA Extraction: Genomic DNA was extracted from the mycelium using the CTAB method. The simplified procedure includes: grinding the mycelium with liquid nitrogen, treating with CTAB and PVP, cleaving with β-mercaptoethanol, and incubating in a 65°C water bath; removing proteins by phenol-chloroform extraction, purifying the DNA by ethanol precipitation, and finally dissolving the DNA sample in EB buffer.

[0027] ③ ITS sequence amplification and sequencing The ITS region (including the ITS1, 5.8S rRNA, and ITS2 regions) in the rDNA was amplified using the fungal universal primers ITS1F and LR3 to obtain the sequence shown in SEQ ID No. 1.

[0028] SEQ ID No. 1:

[0029] ④ Phylogenetic analysis Based on ITS sequences, a phylogenetic tree was constructed using MEGA software. Figure 2 Based on morphological characteristics and molecular phylogenetic results, this strain was identified as... Lentinula edodes The strain was named: *Lentinula spp.* CSLe001.

[0030] Example 2: Preparation of Shiitake Mushroom CSLe001 Liquid Spawn: 1. Activation of the microbial strain: After identifying CSLe001 as *Lentinula edodes*, a 0.4 cm mycelium was picked from a preserved test tube and inoculated onto a PDA medium plate. The plate was then incubated in the dark at 28°C for 15 days to obtain a solid culture.

[0031] Take a 1cm × 1cm mycelium from the cultured solid inoculum and place it in a 2ml sterile centrifuge tube. Add 700ul of sterile water and two steel balls. Grind the mixture at 180rpm for 2 minutes to obtain a mycorrhizal suspension. Then, inoculate the suspension into 100ml of different liquid culture media. First, incubate statically in the dark at 28℃ for 3 days, then transfer to a shaker for dark incubation at 28℃ and 150rpm for 20 days. This method facilitates the rapid acquisition of a large number of mycorrhizal pellets, which is beneficial for subsequent inoculation of mycorrhizal logs.

[0032] 2. Different liquid culture medium formulations: WPGY: WPM: 2.78g / L, potato flour: 7g / L, glucose: 20g / L, yeast powder: 2g / L; MYWM: MY: 7.5g / L, Mannitol: 20g / L, WPM: 2.78g / L; MYG: Maltose: 7g / L, Yeast powder: 5g / L, Glucose: 10g / L; GNMK: Mannitol: 20 g / L, (NH4)2SO4: 0.5 g / L, MgSO4: 1 g / L, KH2PO4: 1 g / L.

[0033] By comparing the growth of *Viola yedoensis* CSLe001 culture media after cultivation using the four culture media mentioned above, the GNMK formula, which exhibits low fermentation viscosity and a high number of mycelial pellets, was selected for subsequent inoculation of spawn.

[0034] 3. Comparison of fruiting performance of Cangshan shiitake mushrooms CSLe001 and L808 on walnut waste substrate bags L808 liquid culture preparation method: After activating the L808 culture, inoculate it into GNMK liquid culture medium. First, incubate statically in the dark at 28℃ for 3 days, then transfer it to a shaker for dark incubation at 28℃ and 150 rpm for 20 days. The liquid culture is then obtained. Liquid spawn of shiitake mushroom 808 and CSLe001 was inoculated onto walnut waste substrate bags. The inoculated substrate bags were then placed in a cultivation room with the same environment, and the temperature in the cultivation room was maintained within the range of 20-25℃, and the humidity was controlled within the range of 75-85%. The mycelial growth of each group of spawn bags was observed and recorded. After the color change period, the spawn bags were removed and cultured again. The color change period and the time to fruiting period for spawn bags L808 and CSLe001 were recorded. The specific results are shown in Table 1 below: Table 1

[0035] As can be seen from Table 1, the color-changing time and fruiting time of Cangshan shiitake mushroom CSLe001 are significantly shorter than those of L808.

[0036] To compare yields, the average yield and average mushroom weight of L808 and CSLe001 were measured. As shown in Table 2, the average yield and average mushroom weight of Cangshan shiitake mushroom CSLe001 were superior to L808 in both indicators. This indicates that Cangshan shiitake mushroom CSLe001 is more suitable for subsequent production using mushroom bags made from walnut waste.

[0037] Table 2

[0038] Example 3: I. Cultivation of Aspergillus oryzae from Shiitake Mushroom CSLe001 and L808 of Cangshan: 1. Liquid seed culture Activation of microbial strains: A 0.4 cm mycelium of *Viola yedoensis* CSLe001 was picked from a preserved test tube and inoculated onto a PDA medium plate. The plate was then incubated in the dark at 28°C for 15 days to obtain a solid culture.

[0039] Take a 1cm × 1cm mycelium from the cultured solid spawn and place it in a 2ml sterile centrifuge tube. Add 700ul of sterile water and two steel columns. Grind the mixture at 180rpm for 2 minutes to obtain a spawn suspension. Inoculate this suspension into MY culture medium. First, incubate statically in the dark at 28℃ for 3 days, then transfer to a shaker for dark incubation at 28℃ and 150rpm for 20 days. This yields the liquid seed culture of *Lentinula edodes*.

[0040] 2. Cultivation of Cangshan shiitake mushrooms in different culture media Formulas for different culture media: MEY medium: 15 g / L Malt Extract, the remainder being water. Prepare the liquid medium by dispensing 100 portions into 250 ml Erlenmeyer flasks and autoclaving (121 ℃, 30 min) for later use. DM medium: Put 100 g of rice and 100 ml of water into a tissue culture bottle, autoclave (121 ℃, 30 min) and set aside. HTZM medium: 100g of walnut pruning branch powder and 50ml of MS solution (4.79g / L) were placed in a tissue culture bottle and sterilized in an autoclave (121 ℃, 30 min) for later use.

[0041] Method for making walnut pruning branch powder: After pruning walnut branches from walnut trees, first use a gasoline-powered branch crusher to crush the branches into particles of about 5cm. After bringing them back to the laboratory, bake them in an oven at 60 degrees Celsius for 2 hours, and then use an electric grinder to crush the walnut branches into powder of about 0.8 mm in diameter for later use.

[0042] 3. Inoculation and Culture 10 ml of the cultured liquid seed of *Lentinula edodes* was inoculated into MEY, DM, and HTZM mediums, respectively. After inoculation, the mycelium was harvested after static culture at 25°C for 45 days.

[0043] II. Extraction and Compound Detection Analysis Protocol for Crude Samples 1. Sample crude extract extraction process (1) Sampling process: Accurately weigh 30 mg of mycelium of MEY, DM, HTZM and fruiting bodies of CSLe001 and L808 and place them in 2 mL centrifuge tubes.

[0044] (2) Extraction treatment: Add 1 mL of 50% methanol solution pre-cooled to 4°C, and add 2 glass beads, and vortex for 30 seconds.

[0045] (3) Freezing treatment: Immerse the centrifuge tubes in liquid nitrogen for 5 minutes to freeze rapidly.

[0046] (4) Grinding treatment: After thawing at room temperature, grind at 55 Hz for 60 seconds using a high-throughput tissue homogenizer.

[0047] (5) Repeat the process: Repeat steps (2)-(4) twice, and then let it stand at -20°C for 30 minutes after completion.

[0048] (6) Concentration treatment: Centrifuge at 12,000 rpm and 4°C for 20 minutes, and take 850 μL of supernatant to concentrate under vacuum until completely dry.

[0049] (7) Resolution treatment: Add 150 μL of 50% methanol solution containing 5 ppm 2-chlorophenylalanine and vortex for 30 seconds.

[0050] (8) Filtration treatment: Centrifuge at 12,000 rpm and 4°C for 10 minutes, take the supernatant and filter it through a 0.22 μm filter membrane, and collect the filtrate into the test bottle.

[0051] 2. Compound detection methods A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) system was used, wherein: Chromatography system: LC-30A ultra-high performance liquid chromatography system (Shimadzu, Japan) Mass spectrometry system: TripleTOF 6600+ high-resolution mass spectrometer (SCIEX, USA) (1) Chromatographic conditions Column: Waters ACQUITY Premier HSS T3 (1.8 μm, 2.1 × 100 mm) Mobile phase: Phase A: Ultrapure water (containing 0.2% formic acid) Phase B: Acetonitrile (containing 0.2% formic acid) Flow rate: 0.4 mL / min Gradient procedure: 0 min: 5% B 2 min: 15% B 5 min: 75% B 6-7.5 min: 95% B 7.6-10 min: 5% B Injection volume: 4 μL Analysis mode: Positive / negative ion mode (the same gradient is used in negative ion mode); (2) Mass spectrometry conditions Acquisition Mode: Information Dependency Acquisition (IDA) Ion source parameters: Ion source gas 1 / 2: 50 psi Curtain air: 25 psi Temperature: 550°C Declustering voltage: ±60 V (positive / negative mode) Ion spray voltage: ±5000 / 4000 V (positive / negative mode) Level 1 mass spectrometry parameters: Quality range: 50-1000 Da Cumulative time: 200 ms Dynamic background removal: Enabled Secondary mass spectrometry parameters: Quality range: 25-1000 Da Cumulative time: 40 ms Collision energy: ±30 V (positive / negative mode) Collision energy diffusion: 15 Intensity threshold: 100 cps Maximum number of candidate ions: 18 / cycle.

[0052] 3. Compound identification methods (1) Based on the PSNGM metabolomics database integration platform of Pasenno, including: Self-built standard product database mzCloud Mass Spectrometry Database LIPID MAPS lipid database HMDB Human Metabolome Database MoNA Metabolite and Chemical Entity Database NIST 2020 MSMS Standard Mass Spectrometry Library AI predicts MSMS graph library (2) Main identification parameters: Level 1 mass spectrometry tolerance: 0.01 Da Secondary mass spectrometry tolerance: 0.05 Da Smoothing level: 3 Minimum peak height: 10,000 Minimum peak width: 5 Quality slice width: 0.05 Assessment score threshold: 70 points All data analysis was performed using the Analyst TF 1.7.1 software platform.

[0053] Data Analysis: Based on the above detection results, metabolomics data analysis was performed on the tested samples. Specific steps are as follows: Data preprocessing was performed, including format conversion, peak identification, filtering, alignment, and normalization. Data was checked using chromatograms (HPLC chromatograms) and quality control. Differential compounds were screened using multivariate statistical analysis, and compounds were identified by comparing them with mass spectrometry data in the database. The peak areas of arbuscular mycotoxin in different samples were obtained to compare the differences in arbuscular mycotoxin content in different samples (Table 3 and...). Figure 5(As shown). The test results showed that the fruiting bodies (CSF) of CSLe001 cultivated using walnut pruning branches as the main substrate had the highest content of melanoicin, which was 2.3 times that of the mycelium (HTZM) in the solid culture medium of CSLe001 walnut pruning branches. This was significantly higher than the content of melanoicin in the fruiting bodies (L808F) of L808 (the content of melanoicin in the fruiting bodies (CSF) of CSLe001 was 3.86 times that in the fruiting bodies (L808F) of L808), and also higher than that in rice medium (DM) and malt extract medium (MEY).

[0054] Table 3

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-yield Cangshan shiitake mushroom ( Lentinula edodes var Cangshan CSLe001, characterized in that, The proposed classification name for *Viola yedoensis* CSLe001 is *Viola yedoensis* CSLe001. Lentinula edodes var Cangshan CSLe001 was deposited at the China Center for Type Culture Collection on June 16, 2025, with accession number CCTCC NO: M20251402.

2. The application of the *Vitex negundo* CSLe001 as described in claim 1 in the production of honey aspergillin.

3. A method for enhancing the production of acetamiprid from *Viburnum sibiricum* CSLe001, characterized in that: The method involves inoculating the liquid seed of *Lentinula edodes* CSLe001 (preservation number CCTCC NO: M 20251402) into HTZM medium and statically culturing at 25°C to obtain mycelium, or inoculating the liquid seed of *Lentinula edodes* CSLe001 into walnut waste substrate bags to obtain fruiting bodies; the formulation of the HTZM medium is 100g of walnut pruning branch powder and 50ml of MS solution, wherein the mass concentration of the MS solution is 4.79g / L. The walnut waste inoculum bag is composed of the following components by weight: 40 parts walnut oil cake, 50 parts walnut branches, 15 parts wheat bran, 30 parts Fagaceae plant sawdust, 1.5 parts gypsum powder, 1.5 parts lime, 2 parts brown sugar, and 25 parts corn cob, wherein the corn cob has a particle size of 0.1-2 cm.

4. The method according to claim 3, characterized in that: The preparation method of the liquid seed of Cangshan shiitake mushroom CSLe001 includes the following steps: (1) The mycelium of CSLe001 shiitake mushroom from Cangshan was inoculated onto a PDA medium plate and cultured to obtain a solid culture; (2) Take a 1cm×1cm mycelium from the solid culture, put it into a 2ml sterile centrifuge tube, add 700μl of sterile water, and add 2 steel balls. Grind it at 180rpm for 2min to obtain a culture suspension. Then inoculate the culture suspension into MY medium to obtain the liquid seed of Cangshan shiitake mushroom CSLe001.

5. The method according to claim 4, characterized in that: The culture conditions in step (1) are dark culture at 28°C for 15 days.

6. The method according to claim 4, characterized in that: The culture method for inoculating the bacterial suspension into MY culture medium in step (2) is to first culture it statically in the dark at 28°C for 3 days, and then transfer it to a shaker for dark culture at 28°C and 150 rpm for 20 days.

7. The method according to claim 3, characterized in that: The method for preparing the walnut pruning branch powder is as follows: first, crush the walnut pruning branches cut from the walnut tree into particles of about 5cm, then bake them in an oven at 60℃ for 2 hours, and then crush them into powder with a diameter of 0.6-1.0 mm, which is the walnut pruning branch powder.

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