A strain of camphor type cinnamomum burmanii endophytic fungus and a method for producing polysaccharide and application thereof

Abstract

The application belongs to the technical field of microbial fermentation, and discloses a strain of camphor type yinxiang tree endophytic fungus and a method and application thereof for producing polysaccharide. The strain is named Diaporthe LY-10, has a preservation number of GDMCC No. 63754, and was preserved in the Guangdong Microbial Culture Collection Center, located at No. 59, Building 5, Guangzhou City, on August 28, 2023. The fungus has the ability to produce biological polysaccharide, the polysaccharide produced by the fungus has good hypoglycemic activity and antioxidant activity, and has an inhibitory effect on a plurality of food-borne pathogenic bacteria, and can be used for preparing hypoglycemic products, bacteriostatic drugs and antioxidant preparations, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to an endophytic fungus of the Dipterocarpus tinctoria type and its method and application for producing polysaccharides. Background Technology

[0002] Plant endophytic fungi are a class of fungi that colonize healthy plant tissues at a certain stage, reproduce normally, and do not cause significant toxicity to the host plant. Endophytic fungi and host plants live in a mutually beneficial symbiotic relationship. While obtaining nutrients from the host for growth, they also produce secondary metabolites that indirectly promote the growth and development of the host plant. Studies have found that plant endophytic fungi not only metabolize chemical components identical or similar to those found in the host plant, but also produce many bioactive chemical components, making them an important resource for various novel bioactive compounds. Compared with plant extraction, obtaining these compounds through plant endophytic fungi has significant advantages, such as rapid cell growth, ease of cultivation, convenient downstream processing, cost savings, and green sustainability. Currently, various endophytic fungal secondary metabolites have been applied in various fields.

[0003] Polysaccharides are a class of unique biomolecules produced by organisms and widely found in nature. They possess diverse and, in most cases, complex chemical structures, exhibiting a variety of biological activities such as antitumor, antioxidant, hypoglycemic, antibacterial, and antiviral activity. They also possess advantages such as high bioactivity, low cytotoxicity, and high safety, making them suitable for a wide range of (potential) applications. In recent years, polysaccharides, microbial metabolites, have emerged as a novel fermentation product with unique physicochemical properties and have been applied as emulsifiers, thickeners, stabilizers, gelling agents, suspending agents, and lubricants in various fields including petroleum, chemical, food, pharmaceutical, environmental protection, and cosmetics.

[0004] Cinnamomum burmannii is an evergreen tree belonging to the genus Cinnamomum in the Lauraceae family. It is one of the tree species with strong bactericidal properties, making it an ideal tree for pollution control, landscaping, and street planting. It is also a valuable medicinal tree. The bark, leaves, and roots of Cinnamomum burmannii can be used as medicinal materials. The *Lingnan Caiyao Lu* records: "Cinnamomum burmannii leaves, also known as fragrant cinnamon leaves or cinnamon-scented cinnamon leaves; pungent in taste and fragrant in aroma; the leaves are decocted and used by women to wash their hair, which can dispel wind." It has a beautiful and neat appearance, with evergreen branches and leaves year-round, and a cinnamon-like fragrance. Borneol-type Cinnamomum burmannii (Dryobalanopsaromatica), also known as camphor tree, is rich in dextrorotatory borneol in its branches and leaves. It is a physiological or chemical type of Cinnamomum burmannii, with no difference in external morphology from the original Cinnamomum burmannii. Natural borneol can be obtained from its branches and leaves through water distillation and cooling crystallization. Natural borneol, also known as dextrorotatory borneol, was first recorded in the "Mingyi Bielu". It has the effects of clearing blockages and dispersing stagnant heat. It is often used for symptoms such as delirium due to fever, stroke with phlegm syncope, and chest pain. It is also found in formulas such as compound Danshen dripping pills, Angong Niuhuang pills, and Bingpeng powder.

[0005] Borneol-type Cinnamomum has high medicinal and economic value, but there are very few research reports on its endophytic fungi. Exploring the endophytic fungi of Borneol-type Cinnamomum and conducting in-depth research on its secondary metabolites, developing and utilizing its endophytic fungal resource bank, and discovering low-toxicity, high-efficiency, and inexpensive anticancer drugs, antibacterial drugs, insecticides, antioxidants, plant hormones, immunosuppressants, etc. in the metabolites are of great significance. Summary of the Invention

[0006] The primary objective of this invention is to provide an endophytic fungus of the borneol-type Cinnamomum camphora, thereby enriching the variety of endophytic fungi of the borneol-type Cinnamomum camphora.

[0007] Another object of the present invention is to provide a method for producing polysaccharides by the above-mentioned endophytic fungi of the borneol-type Cinnamomum camphora.

[0008] Another object of the present invention is to provide a polysaccharide obtained by the above method.

[0009] Another object of the present invention is to provide the application of the above-mentioned endophytic fungi and / or the above-mentioned polysaccharides.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] An endophytic fungus from a Dipterocarpus stenoptera species, named Diaporthe sp. LY-10, with accession number GDMCC No: 63754, was deposited on August 28, 2023, at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0012] The above-mentioned method for producing polysaccharides from endophytic fungi of *Cinnamomum camphora* includes the following steps:

[0013] (1) The above-mentioned endophytic fungi of the borneol-type Cinnamomum camphora were inoculated into a liquid fermentation medium and fermented to obtain a fermentation broth;

[0014] (2) The fermentation broth was subjected to solid-liquid separation. The liquid was first added to trichloroacetic acid solution to remove protein, resulting in solution A.

[0015] (3) After letting solution A stand overnight, solid-liquid separation was performed. The obtained liquid was added to anhydrous ethanol and left to stand overnight again to obtain solution B. After solid-liquid separation of solution B, the solid was taken, and water was added to redissolve the solid to obtain crude polysaccharide solution.

[0016] (4) The crude polysaccharide solution obtained in step (3) was dialyzed with deionized water, then preliminarily purified by cellulose column, further separated and purified by dextran gel column, and freeze-dried to obtain pure polysaccharide.

[0017] The endophytic fungus of the camphor-type Cinnamomum camphora mentioned in step (1) is preferably the camphor-type Cinnamomum camphora endophytic fungus obtained after activation.

[0018] The preferred activation conditions are: culturing at 25–30°C and 50–150 rpm on PDA medium for 3–7 days; more preferably, culturing at 28°C and 110 rpm on PDA medium for 4 days.

[0019] The inoculation amount described in step (1) is preferably calculated as 1 to 10% by volume; more preferably as 2 to 6% by volume.

[0020] The liquid fermentation medium mentioned in step (1) is preferably modified Martin's medium or potato glucose medium (PDA).

[0021] The fermentation conditions described in step (1) are: fermentation at 25-30℃ and 50-150 rpm for 5-15 days; preferably: fermentation at 28℃ and 110 rpm for 10 days.

[0022] The preferred method for solid-liquid separation in step (2) is filtration using a Buchner funnel.

[0023] The concentration of the trichloroacetic acid solution mentioned in step (2) is 60-90% (v / v); more preferably 80% (v / v).

[0024] The amount of trichloroacetic acid solution added in step (2) is preferably based on a concentration of 3-5% (v / v) in solution A; more preferably based on a concentration of 4% (v / v) in solution A.

[0025] The preferred method for solid-liquid separation in step (3) is centrifugation.

[0026] The preferred centrifugation conditions are centrifugation at 6000-10000 rpm for 5-15 minutes; more preferably, centrifugation at 8000 rpm for 10 minutes.

[0027] The amount of anhydrous ethanol added in step (3) is preferably 1 to 5 times the liquid volume; more preferably 3 times the liquid volume.

[0028] The dialysis bag used in step (4) has a specification of 1000-3000 Da; preferably 2000 Da.

[0029] The dialysis time in step (4) is 60-84 hours; preferably 72 hours.

[0030] The cellulose mentioned in step (4) is preferably DEAE-52 cellulose.

[0031] The preliminary purification steps described in step (4) are preferably as follows:

[0032] 1) Add the crude polysaccharide solution to the cellulose column;

[0033] 2) Elute with elution buffer;

[0034] 3) Collect the eluent to obtain a single polysaccharide component, and freeze-dry it for preservation.

[0035] The eluent mentioned in step 2) is preferably a NaCl solution with a concentration of 0.1 mol / L.

[0036] The elution flow rate in step 2) is 0.6–0.8 mL / min; preferably 0.7 mL / min.

[0037] The collection time mentioned in step 3) is 2 to 8 minutes; preferably 5 minutes.

[0038] The dextran gel mentioned in step (4) is preferably at least one of Sephadex G-75 dextran gel, Sephadex G-100 dextran gel and Sephadex G-200 dextran gel.

[0039] The separation and purification steps described in step (4) are preferably as follows:

[0040] A) Dissolve the preliminarily purified polysaccharide component in distilled water, and then add it to the dextran gel column;

[0041] B) Elute with elution solution;

[0042] C) Collect the eluent to obtain a single polysaccharide component, which is then freeze-dried and stored.

[0043] The eluent used in step B) is preferably distilled water.

[0044] The elution flow rate described in step B) is 0.2–0.4 mL / min; preferably 0.3 mL / min.

[0045] The collection time described in step C) is 5 to 15 minutes; preferably 10 minutes.

[0046] A polysaccharide from an endophytic fungus of the borneol-type Cinnamomum camphora tree was prepared by the above method.

[0047] The application of the above-mentioned endophytic fungi of *Cinnamomum camphora* in the preparation of polysaccharides from endophytic fungi of *Cinnamomum camphora*.

[0048] The above-mentioned polysaccharides from endophytic fungi of the borneol-type Cinnamomum camphora tree are used in the preparation of antibacterial agents, antioxidants, and / or hypoglycemic agents.

[0049] Applications of the above-mentioned polysaccharides from endophytic fungi of the Dipterocarpus tinctoria type in food, pharmaceuticals, daily chemical products, or feed.

[0050] The present invention has the following advantages and effects compared with the prior art:

[0051] This invention isolates a borneol-type endophytic fungus from *Cinnamomum camphora*, enriching the variety of endophytic fungi in this species. This endophytic fungus, *Diaporthe* sp. LY-10, exhibits a strong ability to produce biopolysaccharides. Optimization of its liquid fermentation medium further increases the yield of endophytic fungal polysaccharides, laying the foundation for future industrial production. Its fermentation product—borneol-type *Cinnamomum camphora* endophytic fungal polysaccharide—possesses good hypoglycemic and antioxidant activities, and also shows antibacterial effects against various foodborne pathogens. It can be used to prepare hypoglycemic products, antibacterial drugs, and antioxidant agents, demonstrating promising application prospects. Attached Figure Description

[0052] Figure 1 This is a morphological image of Diaporthe sp. LY-10 on a PDA medium plate.

[0053] Figure 2 This is the phylogenetic tree of Diaporthe sp.LY-10.

[0054] Figure 3 This is the elution curve of crude polysaccharide obtained by fermentation of Diaporthe sp. LY-10 purified by DEAE-52 cellulose anion exchange column.

[0055] Figure 4 This is the elution curve of crude polysaccharide obtained from the fermentation of Diaporthe sp. LY-10 purified by Sephadex G200 glucose gel column.

[0056] Figure 5 These are the antioxidant experimental results of Diaporthe sp. LY-10 polysaccharide D1; among them, the curve with values ​​represented by dots represents vitamin C, and the curve with values ​​represented by squares represents polysaccharide D1.

[0057] Figure 6 These are the hypoglycemic results of Diaporthe sp. LY-10 polysaccharide D1; among them, the curve with values ​​represented by triangles represents acarbose; the curve with values ​​represented by dots represents polysaccharide D1. Detailed Implementation

[0058] The present invention will now be described in further detail, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0059] Example 1

[0060] This example illustrates the isolation and purification process of endophytic fungi.

[0061] Endophytic fungal isolation: Leaf sections of healthy, pest-free *Cinnamomum camphora* plants grown in Guangdong were selected. After sampling, the samples were immediately placed in sealed bags, labeled, and brought back to the laboratory. They were stored at 4°C and processed within 24 hours. Freshly collected *Cinnamomum camphora* leaf tissue was rinsed thoroughly with running water, then rinsed again with sterile water, and finally blotted dry with clean filter paper. The leaves were cut into 1cm × 1cm pieces, each serving as a sample. On a sterile laminar flow hood, the samples were first disinfected by immersion in 75% ethanol for 30 seconds, followed by rinsing three times with sterile water. Then, they were disinfected by immersion in 3% sodium hypochlorite for 3 minutes, followed by rinsing three times with sterile water. 100 μL of the final rinse solution was spread onto PDA medium as a control. After the samples have been completely sterilized, use sterile filter paper to absorb the surface moisture. Place the samples on PDA medium and modified Martin's agar medium, with 3 samples arranged in an approximately equilateral triangle on each medium. Incubate in a 28°C constant temperature incubator for 4-7 days.

[0062] Purification of endophytic fungi: Observe the growth of the strain, pick the tips of hyphae from the tissue edge and transfer them to fresh PDA medium and modified Martin's agar medium. Incubate at 28°C for 4-7 days. Then, pick the tips of hyphae from the tissue edge from the previous medium and transfer them to fresh medium, incubating at 28°C for another 4-7 days. After multiple purifications, single colonies are obtained.

[0063] Example 2

[0064] This example illustrates the screening and identification process for endophytic fungi, using the phenol-sulfuric acid method to detect polysaccharide yield for screening.

[0065] The single colony purified in Example 1 was activated and cultured in PDA medium at 28°C for 4 days. Three colonies were inoculated into 500 mL Erlenmeyer flasks using a 7 mm perforator, with each flask containing 200 mL of PDB medium. Fermentation was carried out at 28°C and 110 rpm for 10 days. After fermentation, the mixture was filtered using a Buchner funnel. The filtrate was collected and 80% (v / v) trichloroacetic acid was added to bring the final concentration of trichloroacetic acid in the solution to 4% (v / v). After standing overnight, the mixture was centrifuged (8000 rpm for 10 min). The supernatant was collected and three volumes of anhydrous ethanol were added. After standing overnight, the resulting suspension was centrifuged at 8000 rpm for 10 min. The centrifuged precipitate was collected and dissolved in distilled water. The polysaccharide yield was determined by the phenol-sulfuric acid method.

[0066] Using polysaccharide yield as the screening criterion, a fungus LY-10 with a crude polysaccharide yield of 3.12±0.21 g / L was obtained.

[0067] Morphological characteristics: such as Figure 1 As shown, after 7 days of cultivation on solid PDA medium at 28°C, the colonies of strain LY-10 were observed to be pale yellow, with underdeveloped aerial hyphae, a felt-like texture, and irregularly spread radial hyphae with irregular colony edges. After 30 days of cultivation, scattered conidiophores were produced on the medium, and transparent to yellow spore horns were secreted from the conidiophores.

[0068] The rDNA ITS region of fungus LY-10 was amplified and sequenced, yielding a nucleotide sequence of 555 bp. Blast and pairwise sequence alignment analyses showed that fungus LY-10 shared 99–100% sequence homology with Diaporthe sp. (See phylogenetic tree). Figure 2 .

[0069] The ITS sequence of LY-10 (also shown in SEQ ID NO.1):

[0070] .

[0071] Example 3

[0072] The endophytic fungus *Diaporthe* sp. LY-10 of the camphor type was activated and cultured in PDA solid medium for 4 days. Three mycelial cakes were inoculated into 500 mL Erlenmeyer flasks using a 7 mm mycelial cake punch, with each flask containing 200 mL of PDB medium. Fermentation was carried out at 28℃ and 110 rpm for 10 days. After fermentation, the mixture was filtered, and the concentrated liquid was collected. 80% (v / v) trichloroacetic acid was added to bring the final trichloroacetic acid concentration in the solution to 4% (v / v). After standing overnight, the mixture was centrifuged, and the supernatant was added to 3 times the volume of anhydrous ethanol. After standing overnight, the resulting suspension was centrifuged at 8000 rpm for 10 minutes. The precipitate was collected and dissolved in distilled water. The yield was determined to be 3.12 ± 0.21 g / L by the phenol-sulfuric acid method. The solution was dialyzed against flowing distilled water for 72 h (dialysis bag molecular weight cutoff 2000 Da), and concentrated under reduced pressure to obtain a crude polysaccharide solution sample.

[0073] The crude polysaccharide solution sample was initially purified using a cellulose chromatography column (DEAE-52 cellulose anion exchange resin as the matrix). The specific steps were as follows: 5 mL of crude polysaccharide solution sample was added to the pre-treated DEAE-52 cellulose column. The purification eluent was sequentially obtained from NaCl solutions of concentrations of 0, 0.1, 0.3, and 0.5 mol / L, and the eluents of different concentrations were collected. The elution flow rate was 0.7 mL / min, and each tube was collected for 5 min. The fraction collected with 0.1 mol / L NaCl as the eluent was named the single polysaccharide fraction A0, and was freeze-dried and stored. Finally, further purification was performed using a glucose gel chromatography column (Sephadex G200 glucose gel as the matrix). A0 was dissolved in distilled water to prepare a 5 mg / mL solution. 10 mL of this solution was added to a pre-treated Sephadex G200 glucose gel column. Distilled water was used as the eluent. The eluent was collected using an automated fraction collector at a flow rate of 0.3 mL / min for 10 min per column, yielding a single polysaccharide fraction, D1. This fraction was then freeze-dried and stored as a powder. The phenol-sulfuric acid method was used to determine the polysaccharide concentration in the eluent throughout the purification process, and elution curves were plotted. The preliminary purification elution curve is shown below. Figure 3 As shown, the purified product is a single polysaccharide fraction eluted with 0.1 mol / L NaCl solution; the separation and purification elution curve is shown below. Figure 4 As shown, this further illustrates that the dextran obtained after gel column purification is a single polysaccharide component.

[0074] Example 4

[0075] This embodiment tests the antibacterial effect of the obtained polysaccharide D1, including the following steps:

[0076] The antibacterial activity of polysaccharide D1 against various foodborne pathogens was detected using the filter paper disc method. 6mm diameter circular filter paper discs were placed in small beakers, sterilized at 121℃ for 30 min, and then immersed in D1 solution (prepared with sterile water, concentration 5 mg / mL) for 10 min under aseptic conditions. The discs were then placed on LB agar plates containing bacteria using sterile forceps. The culture medium was incubated at 37℃ for 12 h. The appearance of inhibition zones was observed, and their diameter was measured using the cross-hatching method. All bacteria used were purchased from the Institute of Microbiology, Guangdong Academy of Sciences. The antibacterial activity results of polysaccharide D1 are shown in Table 1. It can be seen that polysaccharide D1 has certain antibacterial effects against Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella, with better antibacterial effects against Escherichia coli and Staphylococcus aureus.

[0077] Table 1. Diameter of the inhibition zone of polysaccharide D1 against different pathogenic bacteria.

[0078]

[0079] Example 5

[0080] This embodiment describes the determination of the DPPH free radical scavenging ability of the obtained polysaccharide D1, including the following steps:

[0081] Preparation of DPPH solution and polysaccharide sample solution: Prepare a 0.02 mmol / L DPPH solution with anhydrous ethanol and store in a cool, dark place. Set the concentration of D1 polysaccharide solution to 2, 4, 6, 8, and 10 mg / mL.

[0082] DPPH scavenging ability determination: Take a 96-well plate, add 100 μL of polysaccharide samples of different concentrations and 100 μL of DPPH solution, mix them evenly, react under light-protected conditions for 30 min, and measure the absorbance A at 517 nm. X Simultaneously, the absorbance A1 of the control group (anhydrous ethanol plus polysaccharide solution) and the absorbance A0 of the blank group (distilled water plus DPPH solution) were measured. C As a positive control, the DPPH free radical scavenging rate (X, %) was calculated using the following formula:

[0083] X = [1-(A x -A1) / A0]×100

[0084] The results of the DPPH free radical scavenging ability of polysaccharide D1 are as follows: Figure 5 As shown, compared with the DPPH free radical scavenging ability of the control group Vc, polysaccharide D1 has a certain DPPH free radical scavenging ability, but it is significantly weaker than that of Vc. The DPPH scavenging ability of polysaccharide D1 is dose-dependent, increasing with increasing concentration.

[0085] Example 6

[0086] This embodiment describes the determination of the inhibitory ability of the obtained polysaccharide D1 against α-amylase, including the following steps:

[0087] Sample solution preparation: Prepare α-amylase solution with a concentration of 1 U / mL and D1 solutions with concentrations of 0.125, 0.25, 0.5, 1, 2, 4, and 8 mg / mL using distilled water. The concentration of soluble starch solution is 1% (mass fraction).

[0088] Mix 50 μL of α-amylase solution with 50 μL of polysaccharide D1 solution and react at 37 °C for 15 min. Add 50 μL of starch solution and react for 10 min. Then add 100 μL of DNS reagent and heat in a boiling water bath for 15 min. After cooling, measure the absorbance A at 540 nm. XAcarbose at the same concentration was used as a control. The measured value using distilled water instead of the sample was recorded as A1; the measured value using distilled water instead of the sample and α-amylase solution was recorded as A0; the measured value using distilled water instead of α-amylase solution was recorded as A2. The inhibition rate (X, %) was calculated using the following formula:

[0089]

[0090] The results of the inhibitory effect of polysaccharide D1 on α-amylase are as follows: Figure 6 As shown, compared with the inhibitory ability of acarbose in the control group against α-amylase, polysaccharide D1 has a better inhibitory ability against α-amylase, but it is weaker than that of acarbose.

[0091] Comparative Example 1

[0092] This comparative example measures the yield of LY-10 polysaccharides by adjusting the fermentation time of Example 3.

[0093] The endophytic fungus *Diaporthe* sp. LY-10 of the camphor type was activated and cultured in PDA medium at 28℃ for 4 days. Three mycelial cakes were inoculated into 500mL Erlenmeyer flasks using a 7mm mycelial cake punch, with each flask containing 200mL of mycelium. Fermentation was carried out at 28℃ and 110rpm for 6 days. After fermentation, the mixture was filtered, and the filtrate was collected. 80% (v / v) trichloroacetic acid was added to bring the final trichloroacetic acid concentration to 4% (v / v). After standing overnight, the mixture was centrifuged, and the supernatant was added to three times its volume of anhydrous ethanol. After standing overnight, the resulting suspension was centrifuged at 8000rpm for 10 minutes. The precipitate was collected and dissolved in distilled water. The yield was determined to be 1.41±0.15g / L using the phenol-sulfuric acid method. The polysaccharide yield was lower than the yield after 10 days of fermentation (3.12±0.21g / L), therefore the fermentation time should be extended to 10 days.

[0094] Comparative Example 2

[0095] This comparative example measures the polysaccharide production of another endophytic fungus, LY-4, which was screened simultaneously with LY-10 and is of the borneol type.

[0096] The endophytic fungus LY-4, a type of camphor-type *Cinnamomum camphora*, was activated and cultured on solid medium at 28℃ for 4 days. Three mycelial cakes were inoculated into 500mL Erlenmeyer flasks using a 7mm mycelial cake punch, with each flask containing 200mL of mycelium. Fermentation was carried out at 28℃ and 110rpm for 10 days. After fermentation, the mixture was filtered, and the filtrate was collected. 80% (v / v) trichloroacetic acid was added to bring the final trichloroacetic acid concentration to 4% (v / v). After standing overnight, the mixture was centrifuged, and the supernatant was added to three times its volume of anhydrous ethanol. After standing overnight, the resulting suspension was centrifuged at 8000rpm for 10 minutes. The precipitate was collected, dissolved in distilled water, and the yield was determined to be 0.51±0.11g / L using the phenol-sulfuric acid method. It is evident that the crude polysaccharide yield of strain LY-4 was significantly lower than that of *Diaporthe* sp. LY-10.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An endophytic fungus of a camphor-type Cinnamomum camphora tree, characterized by: The endophytic fungi of the camphor-type Cinnamomum camphora are named *Cinnamomum* genus (…). Diaporthe sp.) LY-10, with accession number GDMCC No: 63754, was deposited on August 28, 2023, at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A method for producing polysaccharides from endophytic fungi of *Cinnamomum camphora*, characterized in that... Includes the following steps: (1) The endophytic fungi of the camphor-type Cinnamomum camphora described in claim 1 are inoculated into a liquid fermentation medium for fermentation to obtain a fermentation broth; (2) The fermentation broth was subjected to solid-liquid separation. The liquid was first added to trichloroacetic acid solution to remove protein, resulting in solution A; (3) After letting solution A stand overnight, solid-liquid separation was performed. The obtained liquid was added to anhydrous ethanol and left to stand overnight again to obtain solution B. After solid-liquid separation of solution B, the solid was taken and dissolved in water to obtain crude polysaccharide solution. (4) The crude polysaccharide solution obtained in step (3) was dialyzed with deionized water, then preliminarily purified by cellulose column, further separated and purified by dextran gel column, and freeze-dried to obtain pure polysaccharide.

3. The method for producing polysaccharides from endophytic fungi of *Cinnamomum camphora* according to claim 2, characterized in that: The endophytic fungi of the camphor-type Cinnamomum camphora mentioned in step (1) are camphor-type Cinnamomum camphora endophytic fungi obtained after activation; The inoculation amount mentioned in step (1) is calculated as 1 to 10% by volume; The liquid fermentation medium mentioned in step (1) is a modified Martin's medium or potato dextrose medium; The fermentation conditions described in step (1) are: fermentation at 25-30℃ and 50-150 rpm for 5-15 days.

4. The method for producing polysaccharides from endophytic fungi of *Cinnamomum camphora* according to claim 3, characterized in that: The activation conditions are: cultured on PDA medium at 25-30°C and 50-150 rpm for 3-7 days; The fermentation conditions described in step (1) are: fermentation at 28℃ and 110 rpm for 10 days.

5. The method for producing polysaccharides from endophytic fungi of *Cinnamomum camphora* according to claim 2, characterized in that: The solid-liquid separation method described in step (2) is filtration using a Buchner funnel; The concentration of the trichloroacetic acid solution mentioned in step (2) is 60-90% (v / v); The amount of trichloroacetic acid solution added in step (2) is calculated based on its concentration in solution A being 3 to 5% (v / v).

6. The method for producing polysaccharides from endophytic fungi of *Cinnamomum camphora* according to claim 2, characterized in that: The solid-liquid separation method described in step (3) is centrifugation; The amount of anhydrous ethanol added in step (3) is 1 to 5 times the volume of the liquid. The dialysis bag used in step (4) has a capacity of 1000-3000 Da; The dialysis time mentioned in step (4) is 60-84 hours; The cellulose mentioned in step (4) is DEAE-52 cellulose; The dextran gel mentioned in step (4) is at least one of Sephadex G-75 dextran gel, Sephadex G-100 dextran gel and Sephadex G-200 dextran gel; The preliminary purification steps described in step (4) are as follows: 1) Add the crude polysaccharide solution to the cellulose column; 2) Elute with elution buffer; 3) Collect the eluent to obtain a single polysaccharide component, and freeze-dry it for storage; The separation and purification steps described in step (4) are as follows: A) Dissolve the preliminarily purified polysaccharide component in distilled water, and then add it to the dextran gel column; B) Elute with elution buffer; C) Collect the eluent to obtain a single polysaccharide component, and freeze-dry it for preservation.

7. The method for producing polysaccharides from endophytic fungi of *Cinnamomum camphora* according to claim 6, characterized in that: The eluent mentioned in step 2) is a 0.1 mol / L NaCl solution; The elution flow rate described in step 2) is 0.6–0.8 mL / min; The collection time mentioned in step 3) is 2–8 minutes; The eluent mentioned in step B) is distilled water; The elution flow rate described in step B) is 0.2–0.4 mL / min; The collection time described in step C) is 5 to 15 minutes.

8. A polysaccharide from an endophytic fungus of the camphor-type Cinnamomum camphora, characterized in that: It is prepared by the method described in any one of claims 2 to 7.

9. The application of the endophytic fungus of *Cinnamomum camphora* as described in claim 1 in the preparation of polysaccharides from the endophytic fungus of *Cinnamomum camphora*.

10. The use of the polysaccharide of endophytic fungi of the camphor-type Cinnamomum camphora as described in claim 8 in the preparation of antibacterial agents, antioxidants and / or hypoglycemic agents.

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