Application of Litsea Cubeba Root Powder in Preparation of Activator of Antibacterial Type Secondary Metabolic Pathway of Litsea Cubeba Endophytic Fungus and Culture Medium

Abstract

The application belongs to the technical field of biology, and discloses application of Litsea cubeba root powder in preparation of an activator of Litsea cubeba endophytic fungus bacteriostatic type secondary metabolic pathway and a culture medium. The introduction of the Litsea cubeba root powder in the culture process of the Litsea cubeba endophyte can effectively activate the bacteriostatic type secondary metabolic pathway of the Litsea cubeba endophytic fungus, increase the synthesis of polyketide compounds, terpenoids and other substances in the fermentation culture process, thereby significantly improving the antibacterial activity of the obtained active substances, and having an excellent application prospect in the research and development of a new bacteriostatic substance with high efficiency, low toxicity and good environmental compatibility.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of Litsea cubeba root powder in the preparation of an activator for the antibacterial secondary metabolic pathway of Litsea cubeba endophytic fungi and the culture medium thereon. Background Technology

[0002] Anthrax bacteria ( Colletotrichum *Sp.* (spe.) infects host tissues and damages cell structure by producing capsules and exotoxins, leading to necrotic spots and rot in plant leaves, fruits, and other tissues; it is the pathogen of plant anthracnose. *Ustilago maydis* (spe.) Nigrospora oryzae Sclerotium truncatum ( ) reproduces through mycelium and secretes toxins that damage host cells, causing black mold disease in cereal crops such as rice and corn; Sclerotium truncatum ( ) Sclerotium rolfsii These pathogens invade the host through a combination of mechanical penetration and enzymatic corrosion, necrosis induced by toxins such as oxalic acid, and physical blockage of vascular bundles, causing white mold disease. This disease widely damages food and cash crops, causing significant losses to agricultural production year after year, and is a key pathogen requiring control in agricultural production. However, the extensive use of pesticides and their toxic side effects have not only led to drug resistance in these common plant pathogens, but also severely weakened control effects and disrupted ecological balance due to pesticide residues, the killing of natural enemies and beneficial microorganisms. Therefore, there is an urgent need to develop novel antibacterial substances that are highly effective, low in toxicity, and environmentally compatible.

[0003] Litsea cubeba[ Litsea cubeba [(Lour.) Pers.] Belonging to the genus Litsea, also known as mountain pepper, Litsea cubeba, etc., it is mainly distributed in China and Southeast Asian countries. The roots, stems, leaves, and fruits of Litsea cubeba can all be used medicinally. It is rich in volatile oils, alkaloids, lignans, flavonoids and other chemical components, and has anti-inflammatory, anti-tumor, antibacterial, antioxidant and analgesic pharmacological effects. At the same time, it can also be used as a raw material for the extraction and preparation of fragrances and flavorings, making it a plant resource with great development potential.

[0004] Plant endophytic fungi refer to a class of fungi that exist entirely or partially within healthy plant tissues throughout their life cycle and do not cause significant harm to the host. Plant endophytic fungi are found in almost all currently studied terrestrial and aquatic plants, exhibiting a wide variety of species and distribution. Through long-term co-evolution with plants, endophytic fungi have formed a mutually beneficial symbiotic relationship, helping the host resist abiotic stresses such as drought, salinity, and high temperatures. Simultaneously, they can defend against pests and diseases by secreting antibacterial substances or activating the plant's immune system. They are an important new source of bioactive antibacterial products, providing new insights for the development of novel antibacterial agents.

[0005] As an important aromatic medicinal plant, Litsea cubeba's endophytic fungi, through long-term co-evolution with their host, can synthesize novel and uniquely active bioactive substances, making it an important source for discovering new natural antibacterial agents. However, the secondary metabolites extracted from Litsea cubeba endophytic fungal cultures currently available exhibit low antibacterial activity and are not ideally effective against plant pathogens such as Anthracnose, Ulva oryzae, and Sclerotium truncatum, thus having significant limitations. Summary of the Invention

[0006] In the process of developing novel antibacterial agents, the inventors of this invention discovered that under natural pure culture conditions, the antibacterial secondary metabolic pathways of the endophytic fungi of Litsea cubeba are mostly silent or poorly expressed, maintaining only basal growth metabolism, resulting in low antibacterial activity of the obtained secondary metabolites. In order to effectively activate the antibacterial secondary metabolic pathways of the endophytic fungi of Litsea cubeba during the culture process and improve the antibacterial activity of the obtained secondary metabolites, the inventors, through in-depth and extensive research and numerous experiments, creatively discovered that introducing Litsea cubeba root powder during the fermentation culture of the endophytic fungi of Litsea cubeba can significantly improve the antibacterial activity of the final obtained secondary metabolites. This has excellent application prospects in the development of novel antibacterial substances that are highly efficient, low in toxicity, and environmentally compatible.

[0007] The reasons why Litsea cubeba root powder can effectively activate the antibacterial secondary metabolic pathways of Litsea cubeba endophytic fungi are speculated to be as follows: Litsea cubeba roots are rich in conserved root signaling molecules such as terpenes and flavonoids, which can be recognized by endophytic fungi as symbiotic stress signals. The introduction of Litsea cubeba root powder will change the culture nutrient conditions such as carbon-nitrogen ratio and the culture microenvironment, forcing endophytic fungi to shift to antibacterial and stress-resistant secondary metabolism. At the same time, the active substances contained in Litsea cubeba root powder will change the chromatin state of endophytic fungi and release gene silencing in the pathway. Through multiple mechanisms of action such as homology signal simulation, microenvironmental stress regulation and epigenetic regulation, Litsea cubeba can effectively activate antibacterial secondary metabolic pathways such as polyketide synthesis pathway and terpene synthesis pathway in endophytic fungi, improve the expression level of antibacterial active substances in endophytic fungi, and thus significantly enhance the antibacterial activity of the obtained secondary metabolites.

[0008] More importantly, using Litsea cubeba root powder as an activator demonstrates a consistent antibacterial enhancement effect on endophytic fungi extracted from different regions, climates, plants, and tissues. This represents a universal activation strategy. First, Litsea cubeba endophytic fungi have long co-evolved with their hosts, developing conserved host signal recognition and metabolic regulation systems. Regardless of differences in the origin or growth environment of Litsea cubeba plants, the core receptor proteins, pathway regulatory signal genes, and signal transduction pathways responsible for recognizing plant root signals and regulating antibacterial secondary metabolism within the endophytic fungi exhibit high homology. Second, the pathway gene cluster structures and catalytic enzymes of different Litsea cubeba endophytic fungi... Although there are certain differences in function and metabolic regulation mechanisms, the framework of antimicrobial secondary metabolic pathways in endophytic fungi is conserved. Antimicrobial metabolic pathways such as polyketides and terpenes are core conserved metabolic pathways in endophytic fungi and are not unique to a single strain. Therefore, the activation effect of Litsea cubeba root powder can be adapted to Litsea cubeba endophytic fungi from different sources, showing universal activation of Litsea cubeba endophytic fungi. Furthermore, the microenvironmental stress regulation effect of Litsea cubeba root powder is not strain-specific and does not depend on the specific genetic characteristics of the strain. Endophytic fungi initiate compensatory activation of antimicrobial secondary metabolic pathways in response to the changes in culture environment brought about by Litsea cubeba root powder, ultimately showing the commonality of activation effect.

[0009] Furthermore, the purification of secondary metabolites of endophytic fungi from Litsea cubeba using a combination of ethyl acetate extraction and 40%~60% methanol aqueous solution ODS reverse-phase column elution can effectively enrich antibacterial components such as polyketides and terpenes, further improving the antibacterial activity of the obtained active substances. Based on this, the technical solution of the present invention is obtained.

[0010] In a first aspect, the present invention provides the application of Litsea cubeba root powder in the preparation of an activator for the antibacterial secondary metabolic pathway of Litsea cubeba endophytic fungi.

[0011] In this invention, the particle size of the Litsea cubeba root powder is 100 μm to 200 μm. More specifically, the particle size of the Litsea cubeba root powder can be 100 μm, 110 μm, 125 μm, 150 μm, 160 μm, 180 μm, 200 μm, or any value between them. In this case, the Litsea cubeba root powder has ideal specific surface area, mycelial contact efficiency, and conserved root signaling molecule release rate, enabling it to better activate the antibacterial secondary metabolic pathways of the endophytic fungi of Litsea cubeba.

[0012] In this invention, the preparation of the Litsea cubeba root powder is a conventional technique for pre-processing existing plant raw materials, limited to the ability to produce powder of a specific particle size from the Litsea cubeba root. This invention does not impose any particular limitations on this method. More specifically, the preparation of the Litsea cubeba root powder can be, but is not limited to, taking fresh Litsea cubeba roots, washing, drying, and pulverizing them to obtain the Litsea cubeba root powder.

[0013] Secondly, the present invention provides a composite culture medium comprising Litsea cubeba root powder and rice in a mass ratio of 1:(10~40). More specifically, the mass ratio of Litsea cubeba root powder to rice can be 1:10, 1:15, 1:20, 1:30, 1:40 or any value therebetween; more preferably 1:(20~30).

[0014] In this invention, the particle size of the Litsea cubeba root powder is 100μm to 200μm. More specifically, the particle size of the Litsea cubeba root powder can be 100μm, 110μm, 125μm, 150μm, 160μm, 180μm, 200μm or any value between them.

[0015] In this invention, the preparation of the composite culture medium includes: mixing the Litsea cubeba root powder and rice to obtain a mixture; mixing the mixture with water and subjecting it to high-pressure sterilization to obtain the composite culture medium.

[0016] In this invention, the ratio of the mixture to water in the preparation of the composite culture medium is (0.5~2) g:1 mL. More specifically, the ratio of the mixture to water can be 0.5 g:1 mL, 0.8 g:1 mL, 1 g:1 mL, 1.3 g:1 mL, 1.5 g:1 mL, 2 g:1 mL, or any value between them.

[0017] In this invention, during the preparation of the composite culture medium, the autoclaving temperature is 110℃~130℃, and the time is 15min~30min. More specifically, the autoclaving temperature can be 110℃, 112℃, 115℃, 118℃, 120℃, 125℃, 130℃, or any value between them; the autoclaving time can be 15min, 20min, 25min, 30min, or any value between them.

[0018] Thirdly, the present invention provides a method for improving the antibacterial activity of secondary metabolites of Litsea cubeba endophytic fungi, the method comprising: fermenting the Litsea cubeba endophytic fungi using the above-mentioned composite culture medium to obtain a fermentation culture.

[0019] In this invention, the method for preparing the Litsea cubeba endophytic fungus includes: taking parts of Litsea cubeba plant for surface disinfection and tissue cutting to obtain tissue blocks to be separated; taking the tissue blocks to be separated and culturing them in a separation culture medium until hyphae grow around the tissue blocks; taking the hyphae and culturing them in a purification culture medium for tip purification to obtain the Litsea cubeba endophytic fungus.

[0020] In this invention, the parts of the Litsea cubeba plant include one or more of the following: roots, stems, leaves, and fruits.

[0021] In this invention, the surface disinfection treatment in the preparation of the Litsea cubeba endophytic fungus is a conventional technique for pretreatment of existing plant raw materials, and this invention does not impose any particular limitations on it. More specifically, the surface disinfection treatment includes: immersing the Litsea cubeba plant part in 70-78% ethanol for 1-5 minutes, followed by immersion in 5-6% sodium hypochlorite solution for 2-5 seconds to complete the surface disinfection treatment.

[0022] In this invention, the tissue cutting in the preparation of the Litsea cubeba endophytic fungus is a conventional technique for pretreatment of existing plant raw materials, and this invention does not impose any particular limitations on it. More specifically, the size of the tissue block to be separated specifically includes a length of 1mm~4mm, a width of 1mm~4mm, and a height of 1mm~4mm.

[0023] In this invention, the isolation culture medium used in the preparation of the Litsea cubeba endophytic fungus is a conventional technique for culturing existing plant endophytic fungi, and this invention does not impose any particular limitations on it. More specifically, specific examples of the isolation culture medium include, but are not limited to, Bengal red medium.

[0024] In this invention, the isolation and culture of the endophytic fungus from Litsea cubeba is carried out under dark conditions at a temperature of 25°C to 30°C. More specifically, the isolation and culture temperature can be 25°C, 26°C, 27°C, 28°C, 30°C, or any value between them.

[0025] In this invention, the purification culture medium used in the preparation of the Litsea cubeba endophytic fungus is a conventional technique for culturing existing plant endophytic fungi, and this invention does not impose any particular limitations on it. More specifically, specific examples of the purification culture medium include, but are not limited to, PDA medium.

[0026] In this invention, the number of repetitions of the tip purification culture is 2 to 4. More specifically, the number of repetitions of the tip purification culture can be 2, 3, or 4. In this way, while achieving effective purification of endophytic fungi, the ability of endophytic fungi to synthesize antibacterial active substances, their responsiveness to Litsea cubeba root powder, and their growth vitality can be well preserved.

[0027] In this invention, the preferred method includes: extracting the fermentation culture with ethyl acetate and eluting it using an ODS reversed-phase column to obtain the active substance; wherein the eluent used in the ODS reversed-phase column elution is a 40%~60% methanol aqueous solution. More specifically, the ethyl acetate extraction and ODS reversed-phase column elution are conventional techniques for the separation and purification of natural products, and this invention does not impose any particular limitations on them.

[0028] Fourthly, the present invention provides the application of the above-mentioned composite culture medium or the method for improving the antibacterial activity of secondary metabolites of Litsea cubeba endophytic fungi in the preparation of antibacterial reagents.

[0029] In this invention, the antibacterial agent has an inhibitory effect on plant pathogens such as anthracnose, rice nigricans, and *Sclerotium sclerotiorum*. More specifically, specific examples of the antibacterial agent include, but are not limited to, one or more of the following: antibacterial drugs, disinfectants, preservatives, and water quality conditioners. Detailed Implementation

[0030] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0031] Example 1 This embodiment illustrates a method for preparing an active substance derived from endophytic fungi of Litsea cubeba, specifically including the following steps: S1. Fresh roots, stems, leaves and fruit tissues of Litsea cubeba purchased from Pingnan, Fujian were soaked in 75% ethanol for 2 minutes, then soaked in 5.25% sodium hypochlorite solution for 3 seconds. After that, they were taken out, rinsed 3 times with sterile water and dried. The tissues were cut with a sterile knife to obtain tissue blocks of 3mm×3mm×2mm to be separated.

[0032] S2. The tissue block to be separated was picked up with forceps and inserted into Bengal red medium. It was cultured upright in a constant temperature dark environment at 28℃ until hyphae grew around the tissue. The hyphal tips were purified three times on PDA medium using the hyphal tip inoculation method. The endophytic fungal cake of Litsea cubeba (5 mm in diameter) was cut from the PDA medium obtained from the last culture using a sterile punch.

[0033] S3. Take one piece of endophytic fungal cake extracted from the root, stem, leaf and fruit tissues of Litsea cubeba and inoculate it onto compound rice culture medium I. Ferment and culture at room temperature in the dark for 35 days to obtain fermented culture.

[0034] S4. Mix the fermentation culture and ethyl acetate at a volume ratio of 1:1, soak and extract at room temperature for 12 hours, then filter. Repeat the above soaking and extraction twice with ethyl acetate on the obtained filter residue. Combine the organic phases and concentrate and evaporate to dryness until the residual volume of the solution is about 5 mL to obtain the crude extract of secondary metabolites.

[0035] S5. Dissolve the crude extract of secondary metabolites in an appropriate amount of methanol, and load it onto an ODS reversed-phase silica column (Beijing Lvbaicao Technology Development Co., Ltd., catalog number R10030B) using a wet loading method. Then, elute with a 50% methanol aqueous solution as the eluent, and freeze-dry after rotary evaporation to obtain the active substance.

[0036] In step S3, the preparation of compound rice culture medium I includes: taking fresh roots of Litsea cubeba purchased from Pingnan, Fujian (from the same batch as in step S1 but different plants), washing and drying them, then crushing them through a 100-mesh sieve to obtain Litsea cubeba root powder; mixing rice and Litsea cubeba root powder evenly at a mass ratio of 20:1 to obtain a mixture; mixing the mixture with sterile water evenly at a solid-liquid ratio of 1g:1mL, autoclaving at 121℃ for 20min, and cooling to room temperature to obtain compound rice culture medium I.

[0037] Example 2 This embodiment uses the method provided in Example 1 to prepare the active substance. The difference is that in step S1, fresh root, stem, leaf and fruit tissues of Litsea cubeba purchased from Yongzhou, Hunan Province are used instead of root, stem, leaf and fruit tissues of Litsea cubeba from Pingnan, Fujian Province. Other conditions are kept the same to obtain the active substance.

[0038] Example 3 This embodiment uses the method provided in Example 1 to prepare the active substance. The difference is that in step S1, fresh roots, stems, leaves and fruit tissues of wild Litsea cubeba purchased from Liping, Guizhou Province are used instead of the roots, stems, leaves and fruit tissues of Litsea cubeba from Pingnan, Fujian Province. Other conditions are kept the same to obtain the active substance.

[0039] Example 4 This embodiment uses the method provided in Example 1 to prepare the active substance. The difference is that in step S1, fresh root, stem, leaf and fruit tissues of Litsea cubeba purchased from Honghe, Yunnan, are used instead of root, stem, leaf and fruit tissues of Litsea cubeba from Pingnan, Fujian. Other conditions are kept the same to obtain the active substance.

[0040] Example 5 This embodiment uses the method provided in Example 1 to prepare the active substance. The difference is that in step S1, fresh root, stem, leaf and fruit tissues of Litsea cubeba purchased from Sanming, Fujian, are used instead of root, stem, leaf and fruit tissues of Litsea cubeba from Pingnan, Fujian. Other conditions are kept the same to obtain the active substance.

[0041] Comparative Example 1 The comparative example prepared the active substance according to the method provided in Example 1, except that in step S3, rice culture medium was used instead of compound rice culture medium I, while other conditions remained the same (including the Litsea cubeba endophytic fungal cakes used, all of which were taken from the same PDA culture medium in step S2 of Example 1) to obtain the active substance.

[0042] In this comparative example, the preparation of the rice culture medium includes: mixing rice and sterile water evenly according to a solid-liquid ratio of 1:1, autoclaving at 121°C for 20 min, and cooling to room temperature to obtain the rice culture medium.

[0043] Comparative Example 2 The comparative example prepared the active substance according to the method provided in Example 2, except that in step S3, the rice culture medium provided in Comparative Example 1 was used instead of the compound rice culture medium I, while other conditions remained the same (including the Litsea cubeba endophytic fungal cakes used, all of which were taken from the same PDA culture medium in step S2 of Example 2) to obtain the active substance.

[0044] Comparative Example 3 The comparative example prepared the active substance according to the method provided in Example 3. The difference was that in step S3, the rice culture medium provided in Comparative Example 1 was used instead of the compound rice culture medium I, while other conditions remained the same (including the Litsea cubeba endophytic fungal cakes used, all of which were taken from the same PDA culture medium in step S2 of Example 3) to obtain the active substance.

[0045] Comparative Example 4 The comparative example prepared the active substance according to the method provided in Example 4, except that in step S3, the rice culture medium provided in Comparative Example 1 was used instead of the compound rice culture medium I, while other conditions remained the same (including the Litsea cubeba endophytic fungal cakes used, all of which were taken from the same PDA culture medium in step S2 of Example 4) to obtain the active substance.

[0046] Comparative Example 5 The comparative example prepared the active substance according to the method provided in Example 5, except that in step S3, the rice culture medium provided in Comparative Example 1 was used instead of the compound rice culture medium I, while other conditions remained the same (including the Litsea cubeba endophytic fungal cakes used, all of which were taken from the same PDA culture medium in step S2 of Example 5) to obtain the active substance.

[0047] Example 6 This embodiment uses the method provided in Example 1 to prepare the active substance. The difference is that in step S3, compound rice culture medium II is used instead of compound rice culture medium I, while other conditions remain the same (including the Litsea cubeba endophytic fungal cakes used, all taken from the same PDA plate in step S2 of Example 1), and the active substance is obtained.

[0048] In this embodiment, the preparation of compound rice culture medium II includes: taking fresh Litsea cubeba roots, washing and drying them, then pulverizing them through a 100-mesh sieve to obtain Litsea cubeba root powder; mixing rice and Litsea cubeba root powder evenly at a mass ratio of 10:1 to obtain a mixture; mixing the mixture with sterile water evenly at a solid-liquid ratio of 1:1, autoclaving at 121°C for 20 minutes, and cooling to room temperature to obtain compound rice culture medium II.

[0049] Example 7 This embodiment uses the method provided in Example 1 to prepare the active substance. The difference is that in step S3, compound rice culture medium III is used instead of compound rice culture medium I, while other conditions remain the same (including the Litsea cubeba endophytic fungal cakes used, all of which are taken from the same PDA culture medium in step S2 of Example 1) to obtain the active substance.

[0050] In this embodiment, the preparation of compound rice culture medium III includes: taking fresh Litsea cubeba roots, washing and drying them, then pulverizing them through a 100-mesh sieve to obtain Litsea cubeba root powder; mixing rice and Litsea cubeba root powder evenly at a mass ratio of 30:1 to obtain a mixture; mixing the mixture with sterile water evenly at a solid-liquid ratio of 1:1, sterilizing at 121°C for 20 minutes, and cooling to room temperature to obtain compound rice culture medium III.

[0051] Example 8 This embodiment uses the method provided in Example 1 to prepare the active substance. The difference is that in step S3, compound rice culture medium IV is used instead of compound rice culture medium I, while other conditions remain the same (including the Litsea cubeba endophytic fungal cakes used, all of which are taken from the same PDA culture medium in step S2 of Example 1) to obtain the active substance.

[0052] In this embodiment, the preparation of compound rice culture medium IV includes: taking fresh Litsea cubeba roots, washing and drying them, then pulverizing them through a 100-mesh sieve to obtain Litsea cubeba root powder; mixing rice and Litsea cubeba root powder evenly at a mass ratio of 40:1 to obtain a mixture; mixing the mixture with sterile water evenly at a solid-liquid ratio of 1:1, autoclaving at 121°C for 20 minutes, and cooling to room temperature to obtain compound rice culture medium IV.

[0053] Example 9 This embodiment uses the method provided in Example 1 to prepare active substances. The difference is that in step S3, only the endophytic fungal cake extracted from the root of Litsea cubeba is fermented and cultured, while other conditions remain the same (including the Litsea cubeba endophytic fungal cake used, which is taken from the same PDA medium in step S2 of Example 1) to obtain active substances.

[0054] Comparative Example 6 The comparative example prepared the active substance according to the method provided in Example 1, except that in step S3, the comparative compound rice culture medium I was used instead of the compound rice culture medium I, while other conditions remained the same (including the Litsea cubeba endophytic fungal cakes used, all of which were taken from the same PDA culture medium in step S2 of Example 1) to obtain the active substance.

[0055] In this comparative example, the preparation of the comparative compound rice culture medium I included: taking fresh leaves of Litsea cubeba purchased from Pingnan, Fujian, washing and drying them, then pulverizing them through a 100-mesh sieve to obtain Litsea cubeba leaf powder; mixing rice and Litsea cubeba leaf powder evenly at a mass ratio of 20:1 to obtain a mixture; mixing the mixture with sterile water evenly at a solid-liquid ratio of 1:1, and autoclaving at 121℃ and 105kPa for 20 minutes, then cooling to room temperature to obtain the comparative compound rice culture medium I.

[0056] Comparative Example 7 The comparative example prepared the active substance according to the method provided in Example 1, except that in step S3, the comparative compound rice culture medium II was used instead of compound rice culture medium I, while other conditions remained the same (including the Litsea cubeba endophytic fungal cakes used, all of which were taken from the same PDA culture medium in step S2 of Example 1) to obtain the active substance.

[0057] In this comparative example, the preparation of the comparative compound rice culture medium II included: taking fresh Litsea cubeba fruit purchased from Pingnan, Fujian, washing and drying it, then pulverizing it through a 100-mesh sieve to obtain Litsea cubeba fruit powder; mixing rice and Litsea cubeba fruit powder evenly at a mass ratio of 20:1 to obtain a mixture; mixing the mixture with sterile water evenly at a solid-liquid ratio of 1:1, and autoclaving it at 121℃ and 105kPa for 20 minutes, then cooling it to room temperature to obtain the comparative compound rice culture medium II.

[0058] Comparative Example 8 The comparative example prepared the active substance according to the method provided in Example 1, except that in step S5, the eluent was methanol, and other conditions were kept the same (including the Litsea cubeba endophytic fungal cake used, which was taken from the same PDA medium in step S2 of Example 1) to obtain the active substance.

[0059] Test case This test example illustrates the relevant performance of the active substances provided in the above embodiments, and uses a comparative example as a reference. Specifically, it includes: 1. Test of antibacterial rate: (1) According to the addition amount of active substance with a concentration of 60 mg / mL, take active substance and 50% methanol aqueous solution and mix them evenly to obtain the sample solution to be tested; (2) The sample solution to be tested was added during the preparation of PDA culture medium at a concentration of 1 mg / mL to obtain the drug-containing culture medium; (3) Take anthrax bacteria preserved in the laboratory ( Colletotrichum sp.), *Ustilago maydis* Black spore rice ) and neatly arranged small sclerotia ( Sclerotium rolfsii The pathogen was inoculated onto PDA medium and activated in a constant temperature incubator at 28℃ for 5 days. Then, pathogenic bacterial discs (0.5 cm in diameter) were cut from the PDA medium using a sterile punch and inoculated into the center of each drug-containing medium. The medium was then incubated upside down in a constant temperature incubator at 28℃ for 72 hours. The mycelial diameter was measured using the cross-cross method, and the inhibition rate was calculated according to the following formula. A medium prepared with an equal volume of 50% methanol aqueous solution instead of the test sample solution was used as the control group, and a PDA medium containing 60 mg / mL nystatin was used as the positive control group. Three replicates were taken for each group, and the average value was taken. The results are shown in Table 1.

[0060] Equation (1) 2. Minimum inhibitory concentration (MIC) [2] Test: (1) According to the addition amount of active substance with a concentration of 10.24 mg / mL, take active substance and methanol and mix them evenly to obtain mother liquor; take PDB culture medium to dilute the mother liquor by 2 times to obtain a series of test solutions of different concentrations; (2) The anthrax bacteria, rice black spores and neat sclerotium preserved in the laboratory were inoculated into PDB medium, activated in a constant temperature incubator at 28℃ for 2 days, and the concentration of the obtained bacterial solution was diluted 50 times to obtain the pathogenic bacterial solution to be tested. (3) Add the test pathogen solution to a 96-well plate at a rate of 10 μL / well; then add the test solution of each series concentration to a 96-well plate at a rate of 90 μL / well and incubate at 28℃ for 24 h. The lowest concentration of the well in which no visible mycelial clusters are found is the minimum inhibitory concentration. PDA medium containing 10.24 mg / mL nystatin is used instead of the drug-containing medium as a positive control. Three replicates are taken for each group and the average value is taken. The results are shown in Table 1.

[0061] Table 1.

[0062] As shown in Table 1, compared to Comparative Examples 1-5, which did not include Litsea cubeba root powder as an antibacterial secondary metabolic pathway activator during fermentation, the antibacterial rate of the active substances prepared by the methods provided in Examples 1-5 of this invention, when tested on solid plates, was 65.81%-100%, demonstrating a good inhibitory effect on mycelial growth. Furthermore, when tested on liquid culture medium, the minimum inhibitory concentration (MIC) of the active substances prepared by the methods provided in Examples 1-5 of this invention was 1 μg / mL-8 μg / mL, indicating a significant improvement in antibacterial activity. This demonstrates that the introduction of Litsea cubeba root powder can effectively activate the antibacterial secondary metabolic pathway of the endophytic fungi in Litsea cubeba, increasing the synthesis of polyketides, terpenes, and other substances during fermentation, thereby significantly improving the antibacterial activity of the obtained active substances. Meanwhile, the conservation of Litsea cubeba and its endophytic fungi determines the commonality of pathway responses. Regardless of the origin or growth environment of the Litsea cubeba plants from which the endophytic fungi originate, the core receptor proteins, pathway regulatory genes, and signal transduction pathways responsible for recognizing root signals and regulating antibacterial secondary metabolism within them are highly homologous. Furthermore, the fungal secondary metabolic pathway framework is species-conserved, which means that Litsea cubeba root powder has the same antibacterial enhancement effect on Litsea cubeba endophytic fungi from different regions, climates, plants, and tissues.

[0063] Furthermore, a comparison of the data from Comparative Examples 6 and 7, in which other tissues of Litsea cubeba were introduced as inducers during fermentation culture, with that of Example 1 shows that only Litsea cubeba root powder possesses the bioactivity to activate the antibacterial secondary metabolic pathway of Litsea cubeba endophytic fungi. Litsea cubeba root powder is rich in conserved root signaling molecules such as terpenes and flavonoids, which can be recognized by its endophytic fungi as symbiotic stress signals, forcing the endophytic fungi to shift to antibacterial and stress-resistant secondary metabolism, relieving the silencing of pathway genes, thereby activating the silenced antibacterial metabolic pathway and significantly improving the expression level of antibacterial metabolic genes.

[0064] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. Application of Litsea cubeba root powder in the preparation of activators for antibacterial secondary metabolic pathways of Litsea cubeba endophytic fungi.

2. The application according to claim 1, characterized in that, The particle size of the Litsea cubeba root powder is 100μm~200μm.

3. A composite culture medium, characterized in that, The composite culture medium consists of Litsea cubeba root powder and rice in a mass ratio of 1:(10~40).

4. The composite culture medium according to claim 3, characterized in that, The particle size of the Litsea cubeba root powder is 100μm~200μm.

5. The composite culture medium according to claim 3, characterized in that, The preparation of the composite culture medium includes: mixing the Litsea cubeba root powder and rice to obtain a mixture; mixing the mixture with water and subjecting it to high-pressure sterilization to obtain the composite culture medium.

6. The composite culture medium according to claim 5, characterized in that, The ratio of the mixture to water is (0.5~2)g:1mL; Optionally, the autoclaving temperature is 110℃~130℃ and the time is 15min~30min.

7. A method for improving the antibacterial activity of secondary metabolites of endophytic fungi in Litsea cubeba, characterized in that, The method includes: fermenting the endophytic fungi of Litsea cubeba using the composite culture medium described in claim 3 to obtain a fermentation culture.

8. The method for improving the antibacterial activity of secondary metabolites of endophytic fungi in Litsea cubeba according to claim 7, characterized in that, The method for preparing the Litsea cubeba endophytic fungus includes: taking parts of Litsea cubeba plant for surface disinfection and tissue cutting to obtain tissue blocks to be separated; taking the tissue blocks to be separated and culturing them in a separation medium until hyphae grow around the tissue blocks; taking the hyphae and culturing them in a purification medium for tip purification to obtain the Litsea cubeba endophytic fungus. Optionally, the parts of the Litsea cubeba plant include one or more of the following: roots, stems, leaves, and fruits; Optionally, the isolation medium is Bengal red medium; Optionally, the isolation and culture are carried out in the dark, and the temperature of the isolation and culture is 25°C to 30°C. Optionally, the purification medium is PDA medium; Optionally, the tip purification culture is repeated 2 to 4 times.

9. The method for improving the antibacterial activity of secondary metabolites of endophytic fungi in Litsea cubeba according to claim 7, characterized in that, The method includes: extracting the fermentation culture with ethyl acetate and eluting it with an ODS reverse-phase column to obtain the active substance; wherein the eluent used in the ODS reverse-phase column elution is a 40%~60% methanol aqueous solution.

10. The application of the composite culture medium of claim 3 or the method of improving the antibacterial activity of secondary metabolites of Litsea cubeba endophytic fungi of claim 7 in the preparation of antibacterial reagents.

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