Phaeoacremonium aleophilum agb-001 and its fermentation product, preparation method and application

Abstract

The present application belongs to the technical field of plant endophytic fungus separation culture and microbial fermentation product research, and discloses an endophytic fungus from goodyera repanda, i.e., botryobasidium pilatum AGB-001, a fermentation product, a preparation method and application. After liquid fermentation, the mycelium and insoluble solids of the strain are removed by filtration and sterilized by high-pressure steam to obtain a fermentation crude product; the results of examples show that active related components such as crude polysaccharide, total polyphenol and total flavonoid can be detected in the fermentation product and the freeze-dried powder thereof; further in-vitro activity evaluation is carried out on the fermentation product, and the results show that the fermentation product has good scavenging activity on DPPH free radicals and ABTS free radicals, and shows good cell compatibility to HaCaT cells in a low to moderate concentration range. The present application can provide a technical basis for the development and utilization of endophytic fungus resources from goodyera repanda and functional products from fermentation sources.

Description

Technical Field

[0001] This invention belongs to the technical field of plant endophytic fungi isolation and culture and microbial fermentation product research, specifically involving an endophytic fungus derived from *Anoectochilus roxburghii*—*Carbonobacter chamaejasminoides* (…). Daldinia eschscholtzii AGB-001 and its fermentation products, preparation methods and applications. Background Technology

[0002] Circular carbonaceous fungus ( Daldinia eschscholtzii *Carya*, belonging to the genus *Carya* in the family Caryaceae, is a widely distributed wood-dwelling fungus, mostly isolated from environmental substrates such as decaying wood, rotten branches, and soil. This fungus produces a rich variety of secondary metabolites, including polysaccharides, polyphenols, flavonoids, naphthalene derivatives, and polyketides. Some of these metabolites have been shown to possess antioxidant, anti-inflammatory, and antibacterial activities. Therefore, *Carya* is an important fungal resource for mining natural bioactive metabolites and has attracted considerable attention in the field of natural product development.

[0003] *Carya spp.* can be isolated from a variety of environmental substrates, with most reported strains originating from saprophytic environments such as decaying wood, rotten branches, and soil. The growth environment and host substrate significantly influence the physiological metabolism of *Carya spp.*, and strains from different sources exhibit varying characteristics in colony morphology, metabolite composition, and biological activity. Therefore, identifying *Carya spp.* strains from different habitats is beneficial for obtaining strains with diverse traits and expanding the scope for the development and utilization of this type of fungus. Summary of the Invention

[0004] This invention provides an endophytic fungus of Anoectochilus roxburghii AGB-001, its fermentation products, preparation methods, and applications.

[0005] Golden Thread Lotus ( Anoectochilus roxburghii *Anoectochilus roxburghii*, a rare medicinal plant belonging to the genus *Anoectochilus* of the Orchidaceae family, can colonize various endophytic fungi within its tissues. These endophytic fungi form a symbiotic relationship with the host plant, and through long-term domestication by the host's microenvironment, they often produce unique metabolites, making them an important source of novel natural active substances. This invention isolates an endophytic *Anoectochilus roxburghii* strain from the tissues of a healthy *Anoectochilus roxburghii* plant. Daldinia eschscholtzii )AGB-001.

[0006] Although there have been reports on *Carya chamaejas*, most are strains from saprophytic environments; however, no strain of this endophytic *Carya chamaejas* originating from the host *Anoectochilus roxburghii* has been publicly disclosed. Due to the difference in the habitat of the strain, the *Carya chamaejas* AGB-001 isolated in this invention exhibits metabolic characteristics distinct from similar strains from saprophytic sources. Its fermentation products contain active components such as crude polysaccharides, total polyphenols, and total flavonoids, possessing free radical scavenging and antioxidant activity, and also showing good compatibility with skin keratinocytes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention discloses a strain of *Carya spp.* AGB-001, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 27, 2026, with the accession number GDMCC NO.68910.

[0008] The ITS sequence of the *Carya spp.* AGB-001 is shown in SEQ ID NO.1. The ITS sequence has been submitted to the NCBI database with accession number PX806354.

[0009] Secondly, the present invention also discloses the fermentation product of the above-mentioned *Carya spp.* AGB-001.

[0010] In one embodiment, the fermentation product is a liquid fermentation crude product obtained by culturing and fermenting *Carya spp.* AGB-001 and then removing the mycelium and insoluble solids, or a freeze-dried powder obtained by freeze-drying the liquid fermentation crude product.

[0011] Preferably, the fermentation product is not further separated and purified by organic solvent extraction, microporous membrane filtration, or concentration treatment.

[0012] Thirdly, the present invention also discloses a method for preparing the fermentation product of the above-mentioned *Caryota buergerianum* AGB-001, comprising the following steps: (1) Inoculate AGB-001 of the charcoal shell bacteria into a liquid culture medium and carry out liquid shaking fermentation culture; (2) After fermentation, the mycelium and insoluble solids were removed by filtration to obtain the crude product of liquid fermentation; (3) The filtered liquid fermentation crude product is sterilized by high pressure steam to obtain sterilized liquid fermentation crude product; (4) Optionally, the sterilized crude fermentation product is freeze-dried to obtain freeze-dried fermentation product powder.

[0013] In one embodiment, the liquid culture medium in step (1) is PDB culture medium; the fermentation culture conditions are: temperature 25~32 ℃, rotation speed 100~160 rpm, fermentation time 5~9 days.

[0014] In one implementation, the filtration in step (2) is carried out by filter paper filtration, the filter paper is 1~8μm filter paper, and the filtrate obtained by filtration is sterilized by high pressure steam at 115~125 ℃ for 12~20 min to obtain sterilized liquid fermentation crude product.

[0015] As one implementation method, the freeze-drying conditions in step (4) are as follows: the sample is pre-cooled at -80~-40 ℃ for 12~24h, and then freeze-dried at a cold trap temperature of -89 ℃ and a vacuum degree of ≤ 1.0 Pa for 48~72h.

[0016] Fourthly, the present invention also discloses the application of the above-mentioned *Caryota buergerianum* AGB-001 or its fermentation products in the preparation of antioxidant active products or the extraction of antioxidant compounds.

[0017] As one implementation method, it is used in the preparation of topical skin products, such as skin care products.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention is the first to isolate endophytic charcoal-shelling bacteria AGB-001 from Anoectochilus roxburghii, providing a new microbial raw material for the preparation of antioxidant products or the extraction of antioxidant compounds; (2) The fermentation product of AGB-001 of the charcoal shell obtained in this invention has excellent DPPH and ABTS free radical scavenging ability and exhibits good antioxidant activity. It can be used directly as an antioxidant raw material and can meet the efficacy development needs of antioxidant products. (3) This invention investigates the processing stability of fermentation products of AGB-001 of *Carya spp.*, clarifies the applicable processing conditions for crude fermentation products, and suggests that complex extraction and repeated precision filtration of fermentation products should be avoided as much as possible. The crude fermentation products after coarse filtration with filter paper or their freeze-dried powder should be selected as raw materials to reduce the risk of decreased activity of fermentation products. (4) The present invention completes the in vitro compatibility evaluation through the HaCaT cell model, and provides a reference for the safe use concentration of the fermentation product in vitro. The fermentation product has good compatibility with skin keratinocytes at an appropriate addition concentration, which makes it easier for formulation developers to determine the product addition range, reduce the formulation screening cost in the development process of skin topical products, and facilitate the practical application of the raw material in skin care products. Attached Figure Description

[0019] Figure 1 A schematic diagram of the pretreatment process for tissue samples from *Anoectochilus roxburghii* and the isolation process for endophytic fungi. Figure 2The colony morphology of AGB-001 on PDA medium (cultured for 1-7 days); Figure 3 Microscopic images of the hyphae and spores of AGB-001; Figure 4 The phylogenetic tree of AGB-001 was constructed as follows; Figure 5 The graph shows the results of the detection of the DPPH free radical scavenging ability of AGB-001 fermentation products; Figure 6 The graph shows the results of the detection of the scavenging ability of AGB-001 fermentation products against ABTS free radicals. Figure 7 The graph shows the results of detecting the effect of AGB-001 fermentation products on HaCaT cell viability. Figure 8 The results of Calcein-AM / PI live / dead staining of HaCaT cells after treatment with AGB-001 fermentation products (Merge in the figure refers to the combined display of Calcein-AM and PI staining results).

[0020] Preservation Information Circular carbonaceous fungus ( Daldinia eschscholtzii AGB-001 was deposited on August 27, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.68910. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments, but the present invention is not limited to the embodiments described herein. Conventional improvements and equivalent substitutions made by those skilled in the art without departing from the spirit and essence of the present invention should fall within the protection scope of the present invention.

[0022] Terminology Explanation: DPPH: 1,1-Diphenyl-2-trinitrophenylhydrazine, a stable nitrogen-centered organic free radical; when antioxidants are present in the system, DPPH free radicals are scavenged or reduced, the purple color of the solution weakens and the absorbance decreases. Therefore, the DPPH free radical scavenging rate can be calculated by the change in absorbance, which can be used to evaluate the antioxidant capacity of a sample to provide hydrogen atoms / electrons and scavenge free radicals. ABTS: 2,2'-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, which generates blue-green ABTS cationic free radicals after oxidation with potassium sulfate, etc.; after the addition of antioxidant samples, the free radicals are scavenged or reduced, the system color becomes lighter and the absorbance decreases. Based on this, the ABTS free radical scavenging rate is calculated to evaluate the antioxidant capacity of water-soluble and lipid-soluble samples. HaCaT: Human immortalized keratinocyte cell line, derived from adult skin epidermis, has the basic morphology and differentiation characteristics of normal keratinocytes. It can be stably passaged in vitro and is therefore widely used as a cell model for skin biology, skin toxicity, irritation and safety evaluation of cosmetics / skincare ingredients. Calcein-AM / PI dual-fluorescence staining: A commonly used method for detecting cell viability / dead state using dual-fluorescence staining. Calcein-AM (calcein acetoxymethyl ester) itself does not fluoresce, but it can penetrate the cell membrane of living cells and enter the cell. It is hydrolyzed by intracellular esterases into calcein, emitting green fluorescence. Therefore, green fluorescence primarily represents living cells with intact cell membranes and esterase activity. PI (propidium iodide) cannot penetrate intact living cell membranes and can only enter dead cells with damaged cell membranes or late-stage apoptotic cells, binding to nucleic acids and emitting red fluorescence. When used together, the green and red fluorescence signals can be used under a fluorescence microscope to distinguish living cells from dead / damaged cells, thus providing a direct evaluation of the sample's impact on cell viability and cell membrane integrity.

[0023] The following embodiments involve the following material descriptions: PDB medium: For every 1000 mL of distilled water, use 200 g of potato and 20 g of glucose. Specifically, cut 200 g of peeled potato into chunks, add an appropriate amount of distilled water, boil for 20–30 minutes, filter, add 20 g of glucose to the filtrate, and bring the volume to 1000 mL with distilled water. Sterilize before use. For solid medium, add an additional 15 g of agar. PDB medium is a liquid medium, while PDA medium is a solid medium.

[0024] Complete culture medium: Gibco 11995065 DMEM high glucose medium was used as the basal medium, with an additional 1 wt% Gibco 15140122 antibiotic and 10 wt% Morase Biotech FBS-P100 premium fetal bovine serum.

[0025] Filter paper: Beekman medium-speed qualitative filter paper.

[0026] CCK-8 Reagent Kit: Dongren Chemical CCK-8 Cell Viability Assay Reagent.

[0027] Microplate reader: 870C full-wavelength microplate reader, Guangzhou Bixiluo Technology Co., Ltd.

[0028] Example 1: Isolation and purification of endophytic fungi derived from Anoectochilus roxburghii Select healthy *Anoectochilus roxburghii* plants without obvious diseases or mechanical damage, take tissue samples, and rinse them with running water to remove surface soil and attached impurities. Then, perform surface disinfection in a clean bench, followed by repeated rinsing with sterile water and blotting dry with sterile filter paper.

[0029] The treated tissue was cut into appropriately sized pieces and inoculated onto PDA agar plates, then cultured at 28 °C. In this example, 28 °C represents the actual culture conditions used and is not intended to limit the entire range of culture temperatures applicable to this invention. After fungal hyphae grew around the tissue pieces, vigorous hyphae without obvious contamination at the edge of the target colony were selected and transferred to new PDA plates for further culture. After repeated transfers and purification, a pure culture strain was obtained. (See below) Figure 1 .

[0030] Example 2: Strain Identification The purified AGB-001 was inoculated onto PDA medium and incubated at 28 °C. After incubation, the colonies were nearly circular with well-developed aerial hyphae. The center of the colony was grayish-green to grayish-black, while the outer edge was dark gray to black. Yellowish-green to yellowish-brown soluble pigments were visible diffusing around the culture medium. Figure 2 Simultaneously, mycelial sections or sporulation structures were prepared and stained with lactic acid cotton blue. Microscopic observation revealed that the target strain's hyphae were slender, branched, septate, and transparent to light brown, with nearly round to elliptical spores visible. The spores were mostly clustered or distributed along the hyphae. Figure 3 .

[0031] Genomic DNA of the target strain was further extracted and amplified by PCR using universal primers for the fungal ITS region. The primer sequences were: ITS1: TCCGTAGGTGAACCTGCGG (SEQ ID NO.2); ITS4: TCCTCGCTTATTGATATGC (SEQ ID NO.3). A phylogenetic tree was constructed based on the ITS sequence of AGB-001, and the results showed that AGB-001 and... D. eschscholtzii The reference strains clustered into the same branch. Based on colony morphology, hyphae, and spore morphology characteristics, AGB-001 was identified as... Daldinia eschscholtzii ,See Figure 4 The strain AGB-001 of *Carya spp.* has been deposited with the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO.68910. The ITS sequence of *Carya spp.* AGB-001 has been submitted to the NCBI database, accession number PX806354, and is designated as SEQ ID NO.1. The ITS sequence of AGB-001 is as follows: .

[0032] Example 3: Preparation of AGB-001 fermentation product and preparation of freeze-dried powder *Carya spp.* AGB-001 was inoculated into PDB liquid medium. A mycelial cake with a diameter of approximately 5 mm was taken and inoculated at a rate of one mycelial cake per 150 mL of PDB liquid medium. Fermentation was carried out at 28 ℃ and 130 rpm for 7 days. The 28 ℃, 130 rpm, and 7 days mentioned in this embodiment are specific implementation conditions used to illustrate the specific implementation of the technical solution of this invention, but are not the only limitation on the fermentation process parameters of this invention. Those skilled in the art can make routine adjustments to the process parameters within the range that enables the strain to ferment normally and produce active products. After fermentation, the mycelium and insoluble solids were removed by medium-speed qualitative filter paper filtration. The product was then sterilized by high-pressure steam at 121 ℃ for 16 min to obtain the crude liquid fermentation product.

[0033] Multiple batches of 300 mL fermentation products were lyophilized, with each batch yielding 1.28–1.43 g of lyophilized powder. Specifically, the samples were pre-frozen at -80 °C for 12 h, then placed in a vacuum freeze dryer and freeze-dried for 72 h at a cold trap temperature of -89 °C and a vacuum degree ≤ 1.0 Pa to obtain lyophilized samples. Based on the actual lyophilized mass, the lyophilized powder equivalent of each 1 mL of fermentation product was approximately 4.27–4.77 mg. To facilitate dose-response comparison between different detection systems and to account for some losses, the lyophilized powder mass corresponding to 300 mL of fermentation product was converted to an equivalent of 1.50 g. Based on this calculation, the lyophilized powder equivalent concentration of the fermentation product was 5.0 mg / mL, meaning that each 1 mL of fermentation product was equivalent to 5.0 mg of lyophilized powder.

[0034] Example 4: Detection of AGB-001 Fermentation Products and Lyophilized Powder Components The fermentation products and freeze-dried powder were analyzed for crude polysaccharide, total polyphenols, total flavonoids, and amino acids. Specifically, the crude polysaccharide content was determined using the phenol-sulfuric acid colorimetric method in SN / T 4260-2015 "Determination of Crude Polysaccharides in Exported Plant-Derived Foods - Phenol-Sulfuric Acid Method"; the total polyphenol content was determined using the Folin-Ciocalteu colorimetric method, referring to the tea polyphenol detection method in GB / T 8313-2018 "Detection Method for Tea Polyphenols and Catechins in Tea"; the total flavonoid content was determined using the method for "Determination of Total Flavonoids in Health Foods" in the "Technical Guidelines for the Inspection and Evaluation of Physicochemical and Hygienic Indicators of Health Foods (2020 Edition)"; and the amino acid content was determined according to QB / T 2409-1998 "Determination of Amino Acid Content in Cosmetics".

[0035] The test results show that, under the detection method and result expression method used in this embodiment, the contents of crude polysaccharide, total polyphenols, and total flavonoids in the freeze-dried powder sample were 39.49±0.82, 4.10±0.12, and 3.23±0.19, respectively; and the contents of crude polysaccharide, total polyphenols, and total flavonoids in the liquid fermentation product sample were 51.66±1.02, 7.14±0.10, and 11.23±0.77, respectively; the amino acid content did not reach the detection limit. The above test results are used to characterize the composition of the fermentation product and freeze-dried powder of *Carya carnivora* AGB-001 in this embodiment.

[0036] Table 1. Component analysis results of fermentation products and lyophilized powder of *Carya paliurus* AGB-001.

[0037] Example 5: Determination of the DPPH and ABTS free radical scavenging ability of AGB-001 fermentation products. DPPH free radical scavenging rate determination method: The fermentation product was a crude liquid fermentation product obtained after AGB-001 fermentation, in which mycelia and insoluble solids were removed by filtration through filter paper, and then sterilized by autoclaving at 121 °C for 16 min. Before experimental determination, the product was serially diluted with distilled water according to lyophilized powder equivalents, resulting in concentrations of 25, 50, 100, 200, 400, and 800 μg / mL. 100 μL of 0.2 mmol / L DPPH radical ethanol solution was accurately pipetted into a 96-well plate, followed by 100 μL of sample solutions at concentrations of 25, 50, 100, 200, 400, and 800 μg / mL, respectively. The plate was incubated in the dark at room temperature for 30 min, and the absorbance (A2) was measured at 517 nm. Simultaneously, the absorbance (A1) of 100 μL sample solution + 100 μL anhydrous ethanol was measured at 517 nm, and the absorbance (A0) of the blank control (100 μL 0.2 mmol / L DPPH radical ethanol solution + 100 μL anhydrous ethanol) was measured at 517 nm. Vitamin C of the same concentration was used as a positive control, and all samples were tested in triplicate. The DPPH radical scavenging rate (%) was calculated using the following formula:

[0038] Method for determining ABTS free radical scavenging rate: Equal volumes of 7.4 mmol / L ABTS and 2.6 mmol / L K₂S₂O₈ were reacted in the dark for 12 h, and then diluted with 95% ethanol to an absorbance of 0.68–0.72 at 734 nm to obtain the ABTS free radical working solution. The solution was then serially diluted with distilled water according to lyophilized powder equivalents, resulting in fermentation product concentrations of 5, 10, 20, 40, 80, and 160 μg / mL, respectively. Add 160 μL of ABTS free radical working solution to a 96-well plate, then add 40 μL of sample solutions at concentrations of 5, 10, 20, 40, 80, and 160 μg / mL, respectively. Vortex to mix, let stand for 6 min, and then measure the absorbance (A2) at 734 nm. Use 160 μL of ABTS free radical working solution + 40 μL of anhydrous ethanol as a blank absorbance (A0), and measure the absorbance (A1) of 40 μL of sample solution + 160 μL of anhydrous ethanol. Use vitamin C of the same concentration as a positive control. Perform three parallel experiments for all samples. The formula for calculating the ABTS cationic free radical scavenging rate (%) is:

[0039] Determination of DPPH and ABTS free radical scavenging capacity of fermentation products from *Carya spp.* AGB-001: Fermentation products of different concentrations were subjected to DPPH and ABTS free radical scavenging experiments, with vitamin C as a positive control. The results are shown in the figure. Figure 5 , Figure 6 The results showed that the fermentation products exhibited scavenging ability against both DPPH and ABTS free radicals, and the scavenging rate increased with increasing sample concentration. Specifically, within the concentration range of 25–800 μg / mL, the scavenging rate of the fermentation products against DPPH free radicals increased from 14.61±1.12% to 95.49±0.30%; and within the concentration range of 5–160 μg / mL, the scavenging rate of the fermentation products against ABTS free radicals increased from 19.36±0.95% to 99.03±0.16%. The vitamin C positive control showed the expected free radical scavenging activity in the corresponding detection system.

[0040] The above results indicate that, under the detection conditions of this embodiment, the fermentation product of *Carya spp.* AGB-001 exhibits DPPH and ABTS free radical scavenging activities, which show an increasing trend with increasing sample concentration.

[0041] Example 6: Stability Observation and Liveness Analysis of AGB-001 Fermentation Products Using the fermentation product of *Carya paliurus* AGB-001 prepared in Example 4 as the research object, the stability of the sample under different treatment conditions was observed. The sample was concentrated under vacuum rotary evaporation at 40 °C. Free radical scavenging activity was detected in the recovered liquid, suggesting that components in the fermentation system may have undergone transfer, volatilization, or activity changes during the concentration process. Further observation revealed that this concentrated sample was initially colorless, gradually turning yellow after exposure to air for approximately 30 minutes, while its scavenging activity significantly decreased, indicating that this part of the system had reduced stability under air exposure conditions.

[0042] Extraction of the fermentation product with ethyl acetate resulted in changes in the system's properties and a decrease in activity. Treatment with petroleum ether failed to induce stable stratification, and subsequent concentration also revealed a decline in system stability. Filtration through a 0.22 μm membrane to remove a large amount of dark-colored substances also led to a rapid decrease in the activity of the filtrate. These observations suggest that microporous membrane filtration and other further separation processes may cause loss of components related to activity retention or changes in system composition. Under the experimental conditions of this example, the crude fermentation product without further separation and purification exhibited better activity retention compared to the samples subjected to the aforementioned further treatments.

[0043] Based on the above observations, the subsequent activity evaluation of this invention mainly uses the crude fermentation product after necessary filter paper filtration as the evaluation object, and combines the freeze-dried mass for equivalent conversion, so as to take into account both sample operability and activity retention.

[0044] In addition, the results showed that the antioxidant active components in the crude fermentation product of this strain are quite sensitive to processing methods such as air exposure, organic solvent extraction, precision membrane filtration, and high-temperature rotary evaporation concentration. Excessive separation and purification can lead to loss of activity. Therefore, in the development of cosmetics and skincare products, complex extraction and repeated precision filtration of fermentation products should be avoided as much as possible. The crude fermentation product after coarse filtration with filter paper or its freeze-dried powder should be preferred as the raw material. At the same time, during the production and storage of the product, the long-term contact between the raw material and air should be minimized to maximize the preservation of the antioxidant activity of the fermentation product.

[0045] Example 7 Effect of AGB-001 fermentation product on HaCaT cell viability To evaluate the in vitro cytocompatibility of the fermentation product of *A. coli* AGB-001, the CCK-8 cell viability assay was used to investigate its effect on HaCaT cell viability. In this embodiment, the cytotoxicity evaluation was used to characterize the in vitro safety of the fermentation product against normal skin keratinocytes. HaCaT cells were cultured in DMEM high-glucose medium (hereinafter referred to as complete medium) containing fetal bovine serum and antibiotics at 37 ℃ and 5% CO2 until the logarithmic growth phase. Cell suspensions were prepared by trypsin digestion and seeded into 96-well plates. After cell adhesion and stabilization, the original culture medium was discarded, and different concentrations of the fermentation product were added for 24 h and 48 h, respectively. The fermentation product was the crude liquid fermentation product obtained after AGB-001 fermentation, filtered through filter paper to remove mycelia and insoluble solids, and then autoclaved at 121 ℃ for 16 min. Before cell experiments, the product was serially diluted with complete medium according to lyophilized powder equivalents. The concentrations of the fermentation product, calculated as lyophilized powder equivalents, were 0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 μg / mL, respectively. After treatment for 24 h and 48 h, CCK-8 cell viability assay reagent was added to each well. After incubation at 37 ℃ and 5% CO2 for 1–2 h, absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated for each group. Parallel wells were set up for each experimental group, and the cell viability was normalized to 100% for the control group (without fermentation product treatment). Results are as follows: Figure 7 As shown, within the concentration range of 3.9–125 μg / mL, cell viability remained at a high level after 24 h and 48 h of fermentation product treatment, with no obvious cytotoxicity observed. However, cell viability decreased to some extent with further increases in sample concentration. Specifically, the cell viability after 24 h and 48 h of treatment with 500 μg / mL was approximately 86.1% and 83.9%, respectively.

[0046] The above results indicate that, under the detection conditions of this embodiment, the effect of AGB-001 fermentation product on HaCaT cell viability exhibits a certain concentration- and time-dependent relationship. Within the tested low to moderate concentration range of 3.9–125 μg / mL, cell viability remained at a high level; only when the fermentation product was at a higher effective concentration did cell viability show a decreasing trend. Therefore, the results of this embodiment support the existence of a certain degree of in vitro cell compatibility for the AGB-001 fermentation product under the corresponding detection conditions.

[0047] The in vitro toxicity data of keratinocytes in this embodiment provide a safe dosage reference for the use of this fermentation product in topical skin products. Experiments confirmed that the fermentation product is safe for keratinocytes at low to moderate concentrations, while high concentrations lead to decreased cell viability with prolonged exposure. When developing skincare products, the actual concentration of the fermentation product can be controlled within a safe range to maximize its antioxidant effects while avoiding the potential skin irritation risks associated with high concentrations.

[0048] Example 8: Effects of AGB-001 fermentation products on cell viability / death status To further verify the effect of the fermentation products on cell viability in Example 7, the live / dead status of the treated cells was observed using Calcein-AM / PI dual fluorescence staining. HaCaT cells were seeded in culture plates containing complete culture medium at a concentration of 8 x 10⁻⁶. 4100 μL of cells were inoculated into each well of a 96-well plate at 37 °C and 5% CO2 until the cells adhered and reached approximately 60%–80% confluence. The original culture medium was then discarded, and different concentrations of fermentation product were added for 24 h and 48 h, respectively. The fermentation product was a crude liquid fermentation product obtained after AGB-001 fermentation, filtered through filter paper to remove mycelia and insoluble solids, and then autoclaved at 121 °C for 16 min. Before cell experiments, the product was serially diluted with complete culture medium according to lyophilized powder equivalents. The concentrations of the fermentation product, based on lyophilized powder equivalents, were 0, 62.5, 250, and 500 μg / mL. After treatment, the culture medium was slowly discarded, and the cells were gently washed 2–3 times with PBS buffer to remove residual culture medium and serum. Next, prepare the live / dead cell staining working solution: Take 1 mL of PBS buffer, add 12 μL of Calcein-AM stock solution and 35 μL of PI stock solution, mix well, and use in the dark. Add an appropriate amount of staining working solution to each well and incubate for 15-30 min in the dark. After staining, discard the staining solution and gently wash once with PBS buffer. Then, add an appropriate amount of PBS to cover the cells for microscopic observation. Images are acquired using a fluorescence microscope, where green fluorescence indicates live cells and red fluorescence indicates dead cells or cells with damaged cell membrane integrity. The results are shown below. Figure 8 As shown.

[0049] The observation results showed that the cells in the blank control group mainly exhibited uniform green fluorescence, with good cell adhesion and no obvious red fluorescence signal. Under low to medium concentrations of fermentation product treatment, the cells still mainly showed green fluorescence, with only a small number of red fluorescent cells, indicating that the fermentation product had little effect on cell membrane integrity and cell viability within this concentration range. As the concentration of fermentation product further increased, at a concentration of 250 μg / mL and a culture time of 48 h, the number of red fluorescent cells gradually increased, and some cells were observed to shrink, become rounded, and have weakened adhesion ability, suggesting that cell viability was affected to some extent under high concentration conditions.

[0050] The above results are generally consistent with the cell viability test results in Example 7, further supporting that the AGB-001 fermentation product has certain in vitro cell compatibility under the tested low to medium concentration conditions; when the concentration of the fermentation product is further increased, its impact on cell viability increases.

[0051] This embodiment visually demonstrates through live and dead cell staining that, at appropriate concentrations, fermentation products can effectively maintain the cell membrane integrity and cell activity of keratinocytes in the skin; only at higher concentrations do the fermentation products affect the cell membrane and cell activity. These results further corroborate the applicable concentration range of fermentation products for topical skin application from the perspective of cell membrane integrity, providing intuitive cellular experimental evidence for skincare product formulation development and the determination of appropriate addition amounts.

[0052] The technical solution provided by the invention has been described in detail above. Specific examples have been used to illustrate the principle and implementation of the invention. The description of the above embodiments is only for the purpose of helping to understand the method and core idea of ​​the invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the invention. Therefore, the content of this specification should not be construed as a limitation of the invention.

Claims

1. *Carya chapensis* AGB-001, characterized in that, The accession number is: GDMCC NO.68910.

2. A fermentation product of *Carya spp.*, characterized in that, The described *Caryophyllum pulveratum* is *Caryophyllum pulveratum* AGB-001, with accession number GDMCC NO.68910.

3. The fermentation product according to claim 2, characterized in that, The fermentation product is a liquid fermentation crude product obtained by culturing and fermenting *Carya spp.* AGB-001 and removing mycelium and insoluble solids, or a freeze-dried powder obtained by freeze-drying the liquid fermentation crude product.

4. The fermentation product according to claim 3, characterized in that, It meets at least one of the following conditions: A through E A. The crude polysaccharide content in the liquid fermentation crude product is 50-60%, the total polyphenol content is 7-8%, and the total flavonoid content is 10-13%. B. The freeze-dried powder contains 35-45% crude polysaccharide, 3.5-4.5% total polyphenols, and 3-4% total flavonoids by mass. C. The lyophilized powder equivalent concentration of the fermentation product is 5.0 mg / mL. Based on the lyophilized powder equivalent, the scavenging rate of the fermentation product against DPPH free radicals ranges from 13.49% to 95.79% within a concentration range of 25–800 μg / mL. D. The lyophilized powder equivalent concentration of the fermentation product is 5.0 mg / mL. Based on the lyophilized powder equivalent, the scavenging rate of the fermentation product against ABTS free radicals ranges from 18.41% to 99.19% within a concentration range of 5–160 μg / mL. E. The lyophilized powder equivalent concentration of the fermentation product is 5.0 mg / mL, and the fermentation product, based on lyophilized powder equivalent, exhibits in vitro cell compatibility in the concentration range of 3.9 to 62.5 μg / mL.

5. A method for preparing the fermentation product according to any one of claims 2-4, characterized in that, Includes the following steps: S1. Inoculate *Carya spp.* AGB-001 into liquid culture medium and carry out liquid shaking fermentation culture; S2. After fermentation, filter to remove mycelium and insoluble solids to obtain liquid fermentation crude product; S3. The filtered liquid fermentation crude product is sterilized by high-pressure steam to obtain sterilized liquid fermentation crude product; S4. If the fermentation product is a freeze-dried powder, the sterilized liquid fermentation crude product is freeze-dried to obtain the freeze-dried fermentation product powder.

6. The preparation method according to claim 5, characterized in that, The liquid culture medium of S1 is PDB medium; the fermentation culture conditions are: temperature 25~32 ℃, rotation speed 100~160 rpm, fermentation time 5~9 days.

7. The preparation method according to claim 5, characterized in that, The S2 filtration is performed using filter paper filtration. The filtrate obtained is then sterilized by high-pressure steam at 115~125 ℃ for 12~20 min to obtain the sterilized liquid fermentation crude product.

8. The preparation method according to claim 5, characterized in that, The freeze-drying conditions for S4 are as follows: pre-cool the sample at -80 to -40 ℃ for 12 to 24 h, and then freeze-dry for 48 to 72 h.

9. The use of the fermentation product of *Carya carnivora* AGB-001 as described in claim 1, or the fermentation product obtained by any one of claims 2-4, or the fermentation product obtained by any one of claims 5-8 in the preparation of antioxidant active products or the extraction of antioxidant compounds.

10. The application according to claim 9, characterized in that, It is used in the preparation of topical skin products.

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