A culture method for promoting arbuscular fungus arbuscular fungus arbuscular fungus arbuscular fungus arbuscular fungus arbuscular fungus arbuscular fungus arbuscular fungus ar

Abstract

The application provides a culture method for promoting Armillaria gallica to produce orpinel, and relates to the technical field of Armillaria gallica culture. The culture method is that liquid spawn of Armillaria gallica is inoculated into XM liquid culture medium for culture, and the specific formula of the XM liquid culture medium is as follows: millet powder 2 g / L, sodium butyrate 1 g / L, potassium nitrate 1 g / L, sodium chloride 0.5 g / L, magnesium sulfate 0.52 g / L, potassium dihydrogen phosphate 1.52 g / L, magnesium acetate 1 g / L, and the rest is water. The application overcomes the defects of the prior art, optimizes the culture medium formula of Armillaria gallica, adds millet powder as an exogenous component to improve the yield of orpinel of Armillaria gallica, and provides a new direction for the production of orpinel.

Description

Technical Field

[0001] This invention relates to the field of Armillaria galbana culture technology, specifically to a culture method for promoting the production of red fucoidic acid by Armillaria galbana. Background Technology

[0002] Lecanoric acid is a phenolic acid condensate dimer of sphagnum mossic acid, first discovered in lichens and widely found in various lichen species. Recent studies have also isolated lecanoric acid and its derivatives from several other fungal species. Lecanoric acid possesses biological activities including anticancer toxicity, antibacterial activity, antimycobacterial activity, and antiviral activity. Recent research has found that lecanoric acid induces M-phase arrest in colon cancer cells, exerting an antiproliferative effect. A 2023 study found that lecanoric acid has an inhibitory effect on tea tree pathogenic fungi, and is superior to some commercially available chemical and plant-derived fungicides.

[0003] Armillaria gallica is a fungus of significant ecological and economic value. Rich in nutrients and possessing a unique flavor, it is highly regarded as an edible fungus. It is rich in various amino acids, polysaccharides, and trace elements, exhibiting high nutritional value. Armillaria gallica is not only an important wood decomposer in forest ecosystems but also an indispensable symbiotic partner in the cultivation of the traditional Chinese medicine Gastrodia elata. Current research on Armillaria gallica mainly focuses on its symbiotic relationship with Gastrodia elata. In recent years, there have also been reports on its biological activities, such as anti-cancer, antidepressant, and antioxidant effects. However, there are currently no reports on the high production of fucoidic acid by Armillaria gallica. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a cultivation method for promoting the production of red fucoidic acid by Armillaria galbana. By optimizing the culture medium formula of Armillaria galbana and adding millet powder as an exogenous component, the yield of red fucoidic acid by Armillaria galbana is increased, providing a new direction for the production of red fucoidic acid.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for promoting the production of red fucoidic acid by Armillaria galbana, wherein the method involves inoculating a liquid strain of Armillaria galbana into XM liquid medium for cultivation. The specific formulation of the XM liquid medium is as follows: 2 g / L millet powder, 1 g / L sodium butyrate, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, 1 g / L magnesium acetate, and the remainder is water.

[0006] Preferably, the inoculation and culture in XM liquid medium is carried out under dark culture conditions at 25°C.

[0007] Preferably, the method for preparing the liquid culture of Armillaria mellea includes the following steps: (1) Inoculate Armillaria galbana into MY medium and incubate in the dark at 25°C for 10 days to obtain solid culture; (2) Take mycelium from solid inoculum, mix it with sterile water and grind it to obtain inoculum suspension; (3) The bacterial suspension was inoculated into GKNM medium and cultured on a shaker for 10 days to obtain liquid bacterial culture.

[0008] Preferably, the specific preparation method of the bacterial suspension is as follows: take a 1cm×1cm mycelium, put it into a 2ml sterile centrifuge tube, add 700ul of sterile water, add 2 steel balls, grind it with a grinder at 180rpm for 2min to obtain the bacterial suspension.

[0009] Preferably, the GKNM culture medium is formulated with 10 g / L glucose, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, and the remainder being water.

[0010] Preferably, the shaking incubation conditions are 150 rpm, 25°C, and dark incubation.

[0011] This invention provides a cultivation method for promoting the production of red fucoidic acid by Armillaria mellea, which has the following advantages compared with the prior art: This invention describes a liquid strain of Armillaria mellea cultured in GKNM medium with millet powder added. Compared to ordinary GKNM medium, GKNM medium with added rice powder, GKNM medium with added corn powder, and GKNM medium with added potato powder, this effectively increases the yield of red fusinate, providing a new direction for the production of red fusinate. Attached Figure Description

[0012] Figure 1 A schematic diagram showing the difference in red fusinate content in Armillaria galbana AG36 mycelium under different culture medium conditions; Figure 2 This is a schematic diagram of HPLC detection of Armillaria galbana AG36 culture in XM liquid medium, where the peak indicated by the green arrow is erythropoietin. Figure 3 This is a schematic diagram of HPLC detection of Armillaria galbana AG36 culture in YM liquid medium, where the peak indicated by the green arrow is erythropoietin. Figure 4 This is a schematic diagram of HPLC detection of Armillaria galbana AG36 culture in TD liquid medium, where the peak indicated by the green arrow is erythropoietin. Figure 5This is a schematic diagram of HPLC detection of Armillaria galbana AG36 culture in DM liquid medium, where the peak indicated by the green arrow is erythropoietin. Figure 6 This is a schematic diagram of HPLC detection of Armillaria galbana AG36 culture in GKNM liquid medium, where the peak indicated by the green arrow is erythropoietin. Figure 7 The growth of Armillaria galbana AG36 in GKNM liquid medium; Figure 8 The growth of Armillaria galbana AG36 in TD liquid medium; Figure 9 The growth of Armillaria galbana AG36 in DM liquid medium; Figure 10 The growth of Armillaria galbana AG36 in YM liquid medium; Figure 11 The growth of Armillaria galbana AG36 in XM liquid medium. Detailed Implementation

[0013] 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.

[0014] Example 1: 1. Formulations for different culture media: DM liquid culture medium: 2 g / L rice powder, 1 g / L sodium butyrate, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, and the remainder is water.

[0015] YM liquid culture medium: 2 g / L corn powder, 1 g / L sodium butyrate, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, 1 g / L magnesium acetate, and the remainder is water.

[0016] XM liquid culture medium: 2 g / L millet powder, 1 g / L sodium butyrate, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, 1 g / L magnesium acetate, and the remainder is water.

[0017] TD liquid culture medium: potato powder 2g / L, sodium butyrate 1g / L, potassium nitrate 1g / L, sodium chloride 0.5g / L, magnesium sulfate 0.52g / L, potassium dihydrogen phosphate 1.52g / L, magnesium acetate 1g / L, and the remainder is water.

[0018] GKNM: 10 g / L glucose, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, and the remainder is water.

[0019] All culture media were sterilized by high-temperature and high-pressure steam sterilization (121°C, 30 min) before use.

[0020] 2. Preparation of solid microbial cultures: The Armillaria galbana (purchase link: https: / / www.biobw.org / China-strain / bio-33036.html, platform number: bio-33036, named Armillaria galbana AG36) was removed from a 4°C freezer and inoculated onto Malt-Yeast (MY) medium (Difco, Lawrence, USA). The inoculated medium was then incubated in the dark at 25°C for 10 days before harvesting for later use.

[0021] 3. Preparation of liquid bacterial cultures: Take a 1cm × 1cm mycelium from the cultured solid culture 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 culture suspension. Then, inoculate the culture suspension into 100ml of liquid GKNM medium. After culturing on a shaker for 10 days (150rpm, 25℃, dark), obtain the liquid culture.

[0022] 4. Production of red rosinase: Take 2 ml of liquid bacterial culture and inoculate it into DM liquid medium, YM liquid medium, XM liquid medium, TD liquid medium and GKNM medium respectively, and incubate in the dark at 25℃ for 20 days to obtain cultures in different media.

[0023] 5. Extraction of culture: The cultures from the different culture media were extracted using the following methods. (1) Sampling: Accurately weigh 20 mg of fungal hyphae from different culture media and place them in a 2 mL centrifuge tube.

[0024] (2) Extraction solution treatment: Add 1 mL of 50% methanol solution pre-cooled to 4°C, and add 2 glass beads, and vortex for 30 seconds.

[0025] (3) Freezing treatment: Immerse the centrifuge tubes in liquid nitrogen for 5 minutes to freeze rapidly.

[0026] (4) Grinding treatment: After thawing at room temperature, grind at 55 Hz for 60 seconds using a high-throughput tissue homogenizer.

[0027] (5) Repeat the process: Repeat steps (2)-(4) twice, and then let it stand at -20°C for 30 minutes.

[0028] (6) Concentration treatment: Centrifuge at 12000 rpm and 4°C for 20 minutes, and take 850 μL of supernatant to concentrate under vacuum until completely dry.

[0029] (7) Resolution treatment: Add 150 μL of 50% methanol solution containing 5 ppm 2-chlorophenylalanine and vortex for 30 seconds.

[0030] (8) Filtration treatment: Centrifuge at 12000 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.

[0031] 6. Compound detection methods (1) A high-performance liquid chromatography-tandem mass spectrometry 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) Chromatographic conditions Column: Waters ACQUITY Premier HSS T3 (1.8 μm, 2.1 × 100 mm) Mobile phase: Phase A: Ultrapure water (containing 0.1% formic acid) Phase B: Acetonitrile (containing 0.1% formic acid) Flow rate: 0.3 mL / min Gradient procedure: 0 min: 5% B 2 min: 30% B 5 min: 70% 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).

[0032] (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 7. Compound Identification Methods Based on the PSNGM metabolomics database integration platform of Pasenno, it includes: 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 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: 80 points All data analysis was performed using the Analyst TF 1.7.1 software platform.

[0033] 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 and quality control. Differential compounds were screened using multivariate statistical analysis, and compounds were identified by comparing with mass spectrometry data in the database. The compound with a retention time of 3.087 minutes was identified as erythropoietin, and the peak areas of erythropoietin in different samples were obtained to compare the differences in erythropoietin content in different samples (Table 1). Figure 1 (As shown). The test results showed that Armillaria mellea AG36 had the highest content of fucoidic acid in its mycelium under XM liquid culture conditions, which was 2.38 times that under YM liquid culture conditions, 3.15 times that under TD liquid culture conditions, 15.26 times that under GKNM liquid culture conditions, and 17.33 times that under DM liquid culture conditions.

[0034] Table 1

[0035] As can be seen from the table above, the XM medium liquid fermentation medium significantly increases the content of fucoidic acid in red powder compared to other culture methods, providing a new and effective approach for the large-scale production of fucoidic acid.

[0036] 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 method for cultivating Armillaria mellea to promote the production of fucoidic acid, characterized in that, The cultivation method involves inoculating a liquid culture of Armillaria mellea into XM liquid medium for cultivation. The specific formula of the XM liquid medium is as follows: 2 g / L millet powder, 1 g / L sodium butyrate, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, 1 g / L magnesium acetate, and the remainder is water. The method for preparing the liquid culture of Armillaria mellea includes the following steps: (1) Inoculate Armillaria galbana into MY medium and incubate in the dark at 25°C for 10 days to obtain solid culture; (2) Take mycelium from solid inoculum, mix it with sterile water and grind it to obtain inoculum suspension; (3) The bacterial suspension was inoculated into GKNM medium and cultured on a shaker for 10 days to obtain liquid bacterial culture; the formula of the GKNM medium was 10 g / L glucose, 1 g / L potassium nitrate, 0.5 g / L sodium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, and the remainder was water; the conditions for the shaker culture were 150 rpm, 25 ℃, and dark culture.

2. The cultivation method according to claim 1, characterized in that: The inoculation and culture conditions in XM liquid medium were as follows: dark culture at 25°C.

3. The cultivation method according to claim 1, characterized in that: The specific preparation method of the bacterial suspension is as follows: take a 1cm×1cm mycelium, put it into a 2ml sterile centrifuge tube, add 700ul of sterile water, add 2 steel balls, grind it with a grinder at 180rpm for 2min to obtain the bacterial suspension.

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