A culture method for improving the production of rhein by talaromyces purpureus

Abstract

The application provides a culture method for improving the production yield of rheum emodin by Talaromyces purpureogenus, and relates to the technical field of medicinal fungus culture. The culture method is that mycelium of Talaromyces purpureogenus is inoculated into a liquid culture medium containing walnut oil residue and walnut green peel powder, and is cultured in a constant-temperature shaking table at a temperature of 28 DEG C and a rotating speed of 150 r / min. The application overcomes the defects of the prior art, optimizes the culture medium formula by adding walnut oil residue and walnut green peel powder, improves the fermentation condition, and improves the content of rheum emodin after the culture of Talaromyces purpureogenus, thereby providing a new direction for the production of rheum emodin.

Description

Technical Field

[0001] This invention relates to the field of medicinal fungi cultivation technology, specifically to a cultivation method for increasing the yield of rhein produced by *Pleurotus erythrorhizon*. Background Technology

[0002] Emodic acid (also known as dihydrooxyanthraquinone) is an anthraquinone bioactive compound with the following structure:

[0003] .

[0004] Emodic acid is found in some plants, such as rhubarb (Rheum species) and Phellodendron amurense (Phellodendron bark). It can also be isolated from some endophytic fungi. Emodic acid possesses various biological activities, including antioxidant, anti-inflammatory, antitumor, and antibacterial effects. It can alleviate inflammatory responses by inhibiting the production of inflammatory mediators or reducing the activation of inflammatory signaling pathways. It plays an anticancer role by inducing apoptosis in cancer cells and inhibiting tumor cell proliferation. Some studies suggest that it may exert its anticancer effects by regulating multiple signaling pathways, such as p53 and MAPK. Emodic acid can help improve liver health by inhibiting oxidative damage and inflammatory responses in hepatocytes, and exhibits potential antidiabetic activity by improving glucose metabolism or reducing oxidative damage caused by diabetes.

[0005] *Talaromyces purpurogenus* is a fungus widely distributed in nature, especially in plant tissues, soil, and certain specific environments. It has attracted considerable attention due to its ability to produce abundant secondary metabolites. Researchers are increasingly interested in it because of the diverse biological activities exhibited by its metabolites and its potential applications in agriculture and industry.

[0006] *Plasmodium* species can produce diverse and bioactive secondary metabolites, including anthraquinones, alkaloids, polyketides, and non-adrenergic derivatives. These compounds exhibit various pharmacological activities such as antibacterial, antitumor, anti-inflammatory, and antioxidant effects. Recent research has also discovered that *Plasmodium* species can produce rhein. Therefore, increasing the production of rhein from *Plasmodium* species is a crucial direction for current rhein production. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a cultivation method for increasing the yield of rhein produced by *Pleurotus erythrorhizon*. By adding walnut oil residue and walnut green husk powder to optimize the culture medium formula and improve fermentation conditions, the rhein content of *Pleurotus erythrorhizon* after cultivation is increased, providing a new direction for the production of rhein.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A cultivation method for increasing the yield of rhein produced by *Pleurotus erythrorhizon*, wherein the cultivation method involves inoculating the mycelium of *Pleurotus erythrorhizon* into a liquid culture medium containing walnut oil residue and walnut husk powder, wherein the specific formula of the liquid culture medium is: 4 g / L walnut oil residue + 2 g / L walnut husk powder + MS 4.74 g / L + magnesium acetate 4 g / L + potassium propionate 10 g / L + water.

[0010] Preferably, the walnut green husk powder is prepared by immediately baking the walnut green husk in an oven at 60°C for 3-5 hours after separating it from the walnut kernel, and then pulverizing the dried walnut green husk to a particle size of less than 50μm.

[0011] Preferably, the conditions for culturing in the liquid culture medium are constant temperature shaking culture, and the culture temperature is controlled at 28°C and the rotation speed is 150 r / min.

[0012] Preferably, the method for obtaining the mycelium of the *Purple Basket Fungus* includes the following steps:

[0013] Preferably, *Basilus purpureus* is inoculated onto potato dextrose agar medium and cultured in a dark environment at 28°C for 5 days to obtain the mycelium of *Basilus purpureus*.

[0014] Preferably, the mycelium is added to a centrifuge tube containing 0.7 ml of sterile water before inoculation, and the mycelium is broken up using a tissue disruptor before inoculation.

[0015] This invention provides a cultivation method for increasing the yield of rhein produced by *Pleurotus erythrorhizon* fungi, which has the following advantages compared to existing technologies:

[0016] This invention uses a culture medium supplemented with walnut oil residue and walnut green husk powder to cultivate the mycelium of *Pleurotus erythrorhizon*, effectively increasing the final rhein content of *Pleurotus erythrorhizon*. Compared with the addition of other substances such as black beans and walnut pruning powder, the final rhein content is significantly increased, providing a new technical direction for the industrial production of rhein. Furthermore, the overall cultivation method is simple and suitable for widespread application. Attached Figure Description

[0017] Figure 1 This is a diagram showing the growth of *Purple Basketella* in HTQP liquid medium in an embodiment of the present invention.

[0018] Figure 2 This is a diagram showing the growth of *Purple Basketella* in HTYZ liquid medium in an embodiment of the present invention.

[0019] Figure 3 This is a diagram showing the growth of *Purple Basketella* in HTZT liquid medium in an embodiment of the present invention.

[0020] Figure 4 This is a diagram showing the growth of *Purple Basketella* in HD liquid culture medium in an embodiment of the present invention.

[0021] Figure 5 In the HPLC detection of HTQP culture in this embodiment of the invention, the peak indicated by the red arrow is rhein;

[0022] Figure 6 In the HPLC detection of HTYZ culture in this embodiment of the invention, the peak indicated by the red arrow is rhein;

[0023] Figure 7 For HPLC detection of HTZT culture in this embodiment of the invention, the peak indicated by the red arrow is rhein;

[0024] Figure 8 In the HPLC detection of HD liquid culture in this embodiment of the invention, the peak indicated by the red arrow is rhein. Detailed Implementation

[0025] 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.

[0026] The *Purple* genus *Basilaria* used in the following examples is the endophytic fungus YAFEF086 (T) from the roots of *Polygonatum yunnanense*. alaromyces purpurogenus ); the preservation name is *Purple Basket-like Fungus* ( Talaromyces purpurogenus The fungus is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China; deposit date: May 19, 2022; accession number: CCTCCM 2022612. Furthermore, this fungus has been disclosed in the prior patent application with publication number "CN116144504A" entitled "Endophytic fungus YAFEF086 strain in the roots of Polygonatum odoratum and its application".

[0027] The MS purchase link used below is: https: / / item.jd.com / 10100248006811.html;

[0028] The link to purchase a sample of rhein is: https: / / www.abmole.cn / products / emodic-acid.html. Example 1:

[0029] Culture of *Purple Basket Fungi*:

[0030] 1. Preparation of culture medium:

[0031] Potato glucose agar (PDA) medium: 200 g / L peeled potatoes + 20 g / L glucose + 20 g / L agar + water;

[0032] HD liquid culture medium: 4 g / L black bean powder + 20 g / L glucose + water;

[0033] HTYZ liquid culture medium: 4 g / L walnut oil residue + 4.74 g / L MS + 4 g / L magnesium acetate + 10 g / L potassium propionate + water;

[0034] HTQP liquid culture medium: 4 g / L walnut oil residue + 2 g / L walnut green husk powder + MS 4.74 g / L + magnesium acetate 4 g / L + potassium propionate 10 g / L + water

[0035] HTZT liquid culture medium: 4 g / L walnut oil residue + 2 g / L walnut pruning branch powder + 4.74 g / L MS + 4 g / L magnesium acetate + 10 g / L potassium propionate + water.

[0036] The preparation of walnut green husk powder is as follows: after separating the walnut green husk from the walnut kernel, bake it in an oven for 4 hours (60℃). Then, grind the dried walnut green husk continuously for 5 minutes using an ultrafine grinder. The walnut green husk powder particles are less than 50 micrometers. Collect the powder for later use.

[0037] Preparation of walnut pruning branch powder: After pruning the walnut branches, bake them in an oven for 4 hours (60℃). Then, grind the dried walnut pruning branches continuously for 5 minutes using an ultrafine grinder. The resulting walnut pruning branch powder particles are less than 50 micrometers. Collect the powder for later use.

[0038] All culture media were sterilized by high temperature and high pressure steam treatment (121℃, 20min).

[0039] 2. Inoculation and culture of *Purple Basketformes*:

[0040] Remove *Basilaria purpureus* bacteria stored at -80°C. After thawing naturally, inoculate them onto PDA medium. Then, incubate the inoculated medium in the dark at 28°C for 5 days. Afterward, evenly collect 0.5 cm samples from the edge of each colony. 2The mycelium was added to a centrifuge tube containing 0.7 ml of sterile water. The mycelium was then broken up using a tissue homogenizer (180 rpm, 1 min). Afterward, it was inoculated into liquid media of HD, HTYZ, HTQP, and HTZT, respectively, and cultured on a constant-temperature shaker (temperature 28℃, rotation speed 150 rpm, culture time 5 days) (specific culture details are as follows). Figure 1-4 (As shown).

[0041] 3. Extraction methods for fermentation products after culture:

[0042] (1) Weigh 30 mg of fungal hyphae from different culture media into 2 mL centrifuge tubes respectively;

[0043] (2) Add 1 mL of pre-cooled 50% methanol, add 2 glass beads, and vortex for 30 s;

[0044] (3) Immerse in liquid nitrogen for rapid freezing for 5 minutes;

[0045] (4) Remove the centrifuge tubes and thaw them at room temperature. Place them in a high-throughput tissue homogenizer and homogenize at 55 Hz for 60 seconds.

[0046] (5) Repeat steps (2)-(3) twice; freeze at -20℃ and let stand for 30 minutes;

[0047] (6) Centrifuge at 12,000 rpm and 4℃ for 20 min, and take 850 μL of the supernatant and concentrate it under vacuum until dry;

[0048] (7) Add 150 μL of 50% methanol (containing 5 ppm of 2-chlorophenylalanine) and vortex for 30 s;

[0049] (8) Centrifuge at 12,000 rpm and 4℃ for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane, and add the filtrate to the test bottle.

[0050] 4. Rhein sample detection:

[0051] 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.

[0052] HPLC detection parameters and conditions

[0053] According to the procedures described in Qiao, X., He, WN, Xiang, C., Han, J., Wu, LJ, Guo, DA, & Ye, M. (2011). Qualitative and quantitative analyses of flavonoids in Spirodela polyrrhiza by high‐performance liquid chromatography coupled with mass spectrometry. Phytochemical analysis, 22(6), 475-483., all analyses were performed using an LC-30A system (LC-30A, Shimadzu, Japan) connected to a mass spectrometer (TripleTOF 6600+). (SCIEX, Foster City, CA, USA). Chromatographic separation was performed using a Waters ACQUITY Premier HSS T3 column (1.8µm, 2.1 mm, 100 mm, Waters, Milford, MA, USA). The mobile phase consisted of 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 gradient was: 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, with an injection volume of 4 μL. Alternatively, the elution gradient was the same as in the positive mode under negative ion conditions.

[0054] Mass spectrometry detection parameters and conditions

[0055] 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.

[0056] 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.

[0057] Compound identification methods

[0058] 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 are 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.

[0059] By comparing the data with the database, the compound with a retention time of 4.189 minutes was identified as rhein.

[0060] The content of rhein in each culture medium was detected using the method described above, and the results were obtained by analyzing the peak chromatograms of each culture medium (e.g., Figure 5-8 As shown in the table below, the relative ratios of corresponding peak areas in cultures of different media were calculated to compare the differences in rhein content in *Pachyphytum purpureus* mycelia cultured in different media.

[0061]

[0062] As shown in the table above, HTQP liquid culture medium significantly increases the content of rhein compared to other culture methods. This invention utilizes walnut oil residue and green walnut husks from walnut industry waste as the main culture medium to obtain rhein through liquid fermentation of *Pleurotus erythrorhizon*, providing a new and effective approach for the large-scale production of rhein.

[0063] 5. Extraction method of rhein after culture in culture medium:

[0064] (1) After culturing the culture medium, the mycelium in the fermentation broth was filtered to obtain the bacterial solution. Ethyl acetate was added to the bacterial solution at a volume ratio of 1:1.5. After sonication for 1 hour, the solution was allowed to stand to separate into layers and the supernatant was extracted. This operation was repeated 3 times. The supernatants were combined and refluxed using a rotary evaporator and dried to obtain the crude extract of the fermentation broth.

[0065] (2) Purify the crude extract to obtain high-purity rhein:

[0066] A. First, the fermentation broth extract was coarsely fractionated using AB-8 type (weakly polar) macroporous resin. The column was flushed sequentially with pure water, 25% ethanol, 50% ethanol, 80% ethanol and ethyl acetate (3 column volumes of each eluent) to separate the fractions. Then, TLC was used to determine the fraction containing rhein and the fractions containing rhein were merged.

[0067] B. Then, gradient elution was performed using an LC3050 preparative chromatograph. A preliminary experiment was conducted using a 0.2 ml sample loop to collect the independent peaks. The position of the peak containing rhein was determined by TLC combined with the standard, and the UV absorption characteristics of the peak were recorded.

[0068] C. Replace the 20ml sample loop, repeat the elution conditions of the preliminary experiment, collect the eluent of the target peak, and obtain pure rhein using a rotary evaporator.

[0069] 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 cultivation method for increasing the yield of rhein produced by *Pleurotus erythrorhizon*, characterized in that, The cultivation method involves inoculating the mycelium of *Pleurotus erythrorhizon* into a liquid culture medium containing walnut oil residue and walnut husk powder. The specific formula of the liquid culture medium is: 4 g / L walnut oil residue + 2 g / L walnut husk powder + MS 4.74 g / L + magnesium acetate 4 g / L + potassium propionate 10 g / L + water.

2. The cultivation method according to claim 1, characterized in that: The walnut green husk powder is prepared by immediately baking the walnut green husk in an oven at 60°C for 3-5 hours after separating it from the walnut kernel, and then pulverizing the dried walnut green husk to a particle size of less than 50μm.

3. The cultivation method according to claim 1, characterized in that: The liquid culture medium is cultured in a constant temperature shaker, with the culture temperature controlled at 28°C and the rotation speed at 150 r / min.

4. The cultivation method according to claim 1, characterized in that: The method for obtaining the mycelium of the *Purple Basket-like Fungus* includes the following steps: The mycelium of *Basilaria purpurea* was obtained by inoculating *Basilaria purpurea* onto potato dextrose agar medium and culturing it in the dark at 28°C for 5 days.

5. The cultivation method according to claim 1, characterized in that: Before inoculation, the mycelium is added to a centrifuge tube containing 0.7 ml of sterile water, and the mycelium is broken up using a tissue disruptor before inoculation.

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