Method for promoting generation of tea acid from Nemania sp YAFEF112

Abstract

The invention provides a method for promoting generation of tea acid from Nemania sp YAFEF112, and relates to the technical field of microbial culture. According to the method, mycelia of Nemania sp YAFEF112 are inoculated into a culture medium containing pruned walnut branches for culture, the culture medium is an MM culture medium, and the specific formula of the culture medium comprises 1.52 g / L of monopotassium phosphate, 0.52 g / L of magnesium sulfate, 6 g / L of sodium nitrate and 0.52 g / L of potassium chloride. According to the method, the defects in the prior art are overcome, and good conditions are provided for the production of the tea pickled acid by optimizing the formula of the fermentation culture medium, adding pruned walnut branches into the culture medium, improving the fermentation conditions and increasing the content of the tea pickled acid in the Nemania sp YAFEF112 hyphae.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and specifically to a method for promoting the production of tea-infused acid by Nemania sp YAFEF112. Background Technology

[0002] *Nemania* is a genus of endophytic fungi widely distributed in various plants, particularly belonging to the family Xylariaceae. These fungi possess a range of biological activities, including anticancer (Ramesh, Santosh et al., 2015; Solárová, Liskova et al., 2020; Varlı, Pham et al., 2022), anti-inflammatory (Varlı, Pham et al., 2022), antioxidant (Sarsaiya, Shi et al., 2019), antibacterial (Li, Chen et al., 2023), and cytotoxic activity (Kumarihamy, Ferreira et al., 2019). They have attracted attention due to their ability to produce structurally diverse and significantly bioactive secondary metabolites.

[0003] Nemania sp YAFEF112 is a fungus of the genus Nemania with anticancer activity, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20232696. This fungus has been disclosed in a patent application filed on February 19, 2024, with application number CN202410183485.6.

[0004] Theacid is a condensed phenolic acid compound, also known as lichen acid or red lichen acid. It has inhibitory potential against certain bacteria and fungi. Due to its phenolic hydroxyl structure, theacid is considered to have certain antioxidant capabilities. Theacid has been reported as a histidine decarboxylase inhibitor. Histidine decarboxylase is a key enzyme in the body that catalyzes the production of histamine, which is associated with various physiological and pathological processes such as allergies and inflammation. Therefore, this inhibitory activity suggests that theacid may have research value in anti-allergy or anti-inflammatory applications. Theacid shows certain application potential in the fields of medicine (such as developing anti-allergy drugs as a histidine decarboxylase inhibitor), cosmetics (utilizing its antioxidant properties), and the food industry (as a natural preservative). Theacid is a natural compound mainly extracted from lichens. Recent studies have found that theacid can also be isolated from some plant endophytic fungi. Based on this, how to increase the yield of theacid in these fungi is of high research value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for promoting the production of tannic acid by Nemania sp YAFEF112. By optimizing the fermentation medium formula and adding walnut pruning branches to the medium, fermentation conditions are improved, increasing the content of tannic acid in Nemania sp YAFEF112 mycelia, thus providing favorable conditions for the production of tannic acid.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for promoting the production of tannic acid by Nemania sp YAFEF112, wherein the method comprises inoculating Nemania sp YAFEF112 mycelium into a culture medium containing walnut pruning branches and culturing it.

[0007] Preferably, the culture medium containing walnut pruning branches consists of 15-25g of walnut pruning branches + 10-20mL of MM culture medium.

[0008] Preferably, the culture medium is MM medium, with a specific formula of 1.52 g / L potassium dihydrogen phosphate, 0.52 g / L magnesium sulfate, 6 g / L sodium nitrate, and 0.52 g / L potassium chloride.

[0009] Preferably, the culture method is to culture in a constant temperature incubator at 25°C for more than 20 days.

[0010] Preferably, the mycelium of Nemania sp YAFEF112 is obtained by inoculating the preserved Nemania sp YAFEF112 strain onto PDA medium and culturing it in a dark environment at a temperature of 26°C and a relative humidity of 50-80% for 10 days, during which mycelium forms next to the colony.

[0011] This invention provides a method for promoting the production of tea-tainted acid from Nemania sp YAFEF112, which has the following advantages compared with the prior art: This invention uses MM medium as the basal medium and adds walnut pruning branches as the fermentation medium for Nemania sp YAFEF112 mycelia. This effectively increases the content of theopicin in the Nemania sp YAFEF112 mycelia, providing a new approach for developing the high-efficiency medicinal value of Nemania sp YAFEF112 and for the efficient production of theopicin. Furthermore, the process is simple and controllable, with a short cultivation cycle and low cost, enabling large-scale batch production. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating the growth of Nemania sp YAFEF112 mycelium cultured in MY liquid medium in an embodiment of the present invention. Figure 2 This is a schematic diagram illustrating the growth of Nemania sp YAFEF112 mycelia cultured on DM solid medium in an embodiment of the present invention. Figure 3 This is a schematic diagram illustrating the growth of Nemania sp YAFEF112 mycelium cultured on HTZ solid culture medium in an embodiment of the present invention. Figure 4 The HPLC detection results of peach pruning branch cultures obtained from HTZ solid culture medium in this embodiment of the invention are shown, where the peak indicated by the blue arrow is tea tartaric acid. Figure 5 The figures show the HPLC detection results of rice cultures obtained from DM solid culture medium in this embodiment of the invention, where the peak indicated by the blue arrow is tea-infused acid; Figure 6 The figures show the HPLC detection results of the MY liquid culture obtained from the MY liquid culture medium in this embodiment of the invention, wherein the peak indicated by the blue arrow is tea-infused acid. 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. Example 1

[0014] I. Fungal Culture First, the endophytic fungus *Nemania sp.* YAFEF112*, stored at -80°C, was removed from the freezer. After thawing naturally, it was inoculated onto PDA medium. The inoculated medium was then placed in a dark environment at 26°C and 50-80% relative humidity for 10 days. After incubation, it was stored at 4°C in a refrigerator (Haier, Qingdao, China). For fungal culture experiments, 0.5 cm² of mycelium was evenly taken from the edge of the colony and inoculated into different culture media, then placed in a constant temperature incubator at 28°C.

[0015] Culture medium setup and culture method: 1. MY liquid culture medium: The composition is 12.1 g / L malt yeast extract; the culture medium is placed in a 250 mL Erlenmeyer flask and sterilized by high temperature and high pressure steam treatment (121℃, 20 min) before use; Culture method: The fungal mycelium was cultured on a dark rotating shaker at 150 rpm for 20 days at 25℃. The culture results are as follows: Figure 1 As shown.

[0016] 2. The formula for DM solid culture medium is: 15g rice + 20 mL MM medium (potassium dihydrogen phosphate 1.52 g / L, magnesium sulfate 0.52 g / L, sodium nitrate 6 g / L, potassium chloride 0.52 g / L). Place the culture medium in a tissue culture flask and sterilize it by high-temperature, high-pressure steam (121℃, 20 min) before use.

[0017] Culture method: The fungal mycelium was harvested after 20 days in a constant temperature incubator at 25℃. The culture results are as follows: Figure 2 As shown.

[0018] 3. The HTZ solid culture medium formula is as follows: 20g of walnut pruning branches (dried to a moisture content ≤15%, then pulverized and passed through a 30-mesh sieve) + 20 mL of MM medium (potassium dihydrogen phosphate 1.52 g / L, magnesium sulfate 0.52 g / L, sodium nitrate 6 g / L, potassium chloride 0.52 g / L). Place the culture medium in a tissue culture flask and sterilize it under high temperature and high pressure (121 ℃, 20 min) before use.

[0019] Culture method: The fungal mycelium was harvested after 20 days in a constant temperature incubator at 25℃. The culture results are as follows: Figure 3 As shown.

[0020] II. Extraction of Fermentation Products: 1. Weigh 30 mg of fungal hyphae from different cultures into 2 mL centrifuge tubes respectively; 2. Add 1 mL of pre-cooled 50% methanol, add 2 glass beads, and vortex for 30 seconds; 3. Immerse in liquid nitrogen for rapid freezing for 5 minutes; 4. Remove the centrifuge tubes and thaw them at room temperature. Then place them in a high-throughput tissue homogenizer and homogenize at 55 Hz for 60 seconds. 5. Repeat step 2 twice; freeze at -20℃ and let stand for 30 minutes.

[0021] 6. Centrifuge at 12000 rpm and 4℃ for 20 min, and take 850 μL of the supernatant and concentrate it under vacuum until dry. 7. Add 150 μL of 50% methanol (containing 5 ppm of 2-chlorophenylalanine) and vortex for 30 seconds; 8. Centrifuge at 12000 rpm and 4℃ for 10 min, take the supernatant and filter it through a 0.22μm filter membrane, and add the filtrate to the test bottle.

[0022] III. Compound Detection: The detection method utilizes a combination of chromatography (LC-30A, Shimadzu, Japan) and mass spectrometry (TripleTOF 6600+, SCIEX, Foster City, CA, USA) to achieve the entire process from substance separation using chromatography to substance identification using mass spectrometry. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) enables accurate qualitative and quantitative analysis.

[0023] HPLC detection parameters and conditions: The analytical procedures described by Qiao et al. were performed using an LC-30A system (LC-30A, Shimadzu, Japan) connected to a mass spectrometer (TripleTOF 6600+) (SCIEX, Foster City, CA, USA). Chromatographic separations were performed using a Waters ACQUITY Premier HSS T3 column (1.8µm, 2.1 mm, 100 mm, Waters, Milford, MA, USA). The mobile phases were A: H₂O (containing 0.1% formic acid) and B: acetonitrile (containing 0.1% formic acid). The flow rate was 0.4 mL / min. The mobile phase gradients were: 0 min, 5% B; 2 min, 20% B; 5 min, 60% B; 6–7.5 min, 99% B; and 7.6–10 min, 5% B. The injection volume was 4 μL.

[0024] Mass spectrometry detection parameters and conditions: Data acquisition was performed using Information Dependent Acquisition (IDA) mode, via Analyst TF 1.7.1 software (Sciex, Concord, Canada). Ion source parameters were set as follows: Ion source gas 1 (GAS1) 50 psi; Ion source gas 2 (GAS2) 50 psi; Curtain gas (CUR) 25 psi; Temperature (TEM) 550°C; Declustering voltage (DP) 60V and -60V in both positive and negative modes; Ion spray voltage fluctuation (ISVF) 5000V and -4000V in both positive and negative modes.

[0025] Time-of-flight mass spectrometry (TOF MS) scanning parameters were set as follows: mass range 50-1000 Da; cumulative time 200 ms; dynamic background subtraction enabled. Product ion scanning parameters were set as follows: mass range 25-1000 Da; cumulative time 40 ms; collision energy 30 V and -30 V in positive and negative modes, respectively; collision energy diffusion value 15; resolution set to UNIT units; charge state 1 to 1; intensity threshold 100 cps; exclusion of isotopic ions in the 4 Da range; mass tolerance 50 ppm; maximum number of candidate ions monitored per cycle 18.

[0026] (Note: psi is the pressure unit, pounds per square inch; Da is the atomic mass unit; ms is the millisecond; V is the voltage unit, volt; °C is the temperature unit, degree Celsius; cps is the count per second; ppm is the part per million.) Compound identification methods The search was conducted using the PerSonalbio Next-Generation Metabolomics Database (PSNGM), which includes a self-built standard database, the mzCloud database (https: / / www.mzcloud.org / ), LIPIDMAPS (https: / / www.lipidmaps.org / ), HMDB (https: / / hmdb.ca / ), MoNA (https: / / mona.fiehnlab.ucdavis.edu / ), NIST_2020_MSMS, and an AI-predicted MSMS map database. The main search parameters were as follows: MS1 tolerance for identification 0.01, MS2 tolerance for identification 0.05, smoothing level 3, minimum peak height 10000, minimum peak width 5, massslice width 0.05, and identification score cut-off 70.

[0027] By comparing with database data, the compound with a retention time of 3.036 minutes was identified as tea extract acid.

[0028] The specific HPLC detection results for each culture medium are as follows: Figure 4-6 As shown, the walnut pruning branch culture was a product of HTZ solid medium, the rice solid culture was a product of DM solid medium, and the MY liquid culture was a product of MY liquid medium.

[0029] After determining the peak pattern of tannic acid, the relative ratios of its peak areas were calculated to compare the differences in tannic acid content in Nemania sp YAFEF112 mycelia cultured on different culture media. The results are shown in the table below:

[0030] The above test results show that the content of tea-infused acid in the mycelium of Nemania sp YAFEF112 varies greatly under different culture conditions. Compared with other culture methods, HTC solid fermentation medium greatly increases the content of tea-infused acid, providing a new and effective way for the large-scale production of tea-infused acid.

[0031] 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 promoting the production of tea-infused acid from Nemania sp YAFEF112, characterized in that, The method involves inoculating Nemania sp YAFEF112 mycelium into a culture medium containing walnut pruning branches and culturing it.

2. The method according to claim 1, characterized in that: The culture medium containing walnut pruning branches consists of 15-25g of walnut pruning branches + 10-20mL of MM culture medium.

3. The method according to claim 2, characterized in that: The specific formulation of the MM culture medium is: 1.52 g / L potassium dihydrogen phosphate, 0.52 g / L magnesium sulfate, 6 g / L sodium nitrate, and 0.52 g / L potassium chloride.

4. The method according to claim 1, characterized in that: The culture method involves culturing in a constant temperature incubator at 25°C for more than 20 days.

5. The method according to claim 1, characterized in that, The method for obtaining the mycelium of Nemania sp YAFEF112 is as follows: The preserved Nemania sp YAFEF112 strain was inoculated onto PDA medium and cultured for 10 days in a dark environment at 26°C and 50-80% relative humidity. Mycelium formed next to the colonies.

Images