A strain of *Aspergillus cristatus* and its solid-state fermentation method for *Platycodon grandiflorus*

Abstract

This invention provides a strain of *Aspergillus cristatus* and a method for solid-state fermentation of *Platycodon grandiflorus*. The invention first isolates *Aspergillus cristatus* from Fu brick tea. Eurotium cristatum The strain NCPSec was deposited at the China Center for Type Culture Collection (CCTCC) on June 23, 2025, with accession number CCTCC NO: M 20251442. Solid-state fermentation of Platycodon grandiflorus using this strain not only improved the nutritional value of the Platycodon grandiflorus substrate but also significantly enhanced its antioxidant activity.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of *Aspergillus cristatus* and its method for solid-state fermentation of *Platycodon grandiflorus*. Background Technology

[0002] Platycodon grandiflorus ( Platycodon grandiflorum Platycodon grandiflorus (Jacq.) A.DC., a plant belonging to the genus Platycodon in the family Campanulaceae, is widely distributed in my country. Its underground rhizomes can be used both medicinally and as food. Due to its rich nutritional value and significant health benefits, it was included in the first batch of Chinese herbal medicines listed as "dual-purpose medicine and food." In traditional Chinese medicine, Platycodon grandiflorus is considered neutral in nature, with a sweet, bitter, and pungent taste, primarily affecting the lung and stomach meridians. As an important medicinal plant, Platycodon grandiflorus possesses diverse pharmacological activities. Its unique chemical components endow it with various potential therapeutic properties, including inhibiting inflammatory responses, relieving pain, suppressing cough and expectoration, anti-inflammatory and analgesic effects, vasodilating, protecting the gastric mucosa, regulating blood pressure, lowering blood sugar levels, and enhancing the body's immune function. These broad pharmacological effects make Platycodon grandiflorus of significant clinical application value.

[0003] In recent years, microbial solid-state fermentation has been widely applied and rapidly developed in the field of plant resource development and utilization. Microbial solid-state fermentation is a biotransformation process that utilizes screened beneficial bacteria to synthesize novel microbial material from medicinal herbs and nutritional supplements. This fermentation process not only does not destroy the effective components of the medicinal materials but may also generate new active ingredients or enhance existing medicinal effects. Through this technology, the function and composition of medicinal materials may undergo positive changes, and even generate new medicinal value. Microbial solid-state fermentation has the characteristics of relatively low production costs, simple and easy-to-control process, environmental friendliness, and sustainability, making it a promising area for the high-value utilization of plant resources.

[0004] Microbial-Platycodon grandiflorus two-way solid-state fermentation has become an important direction in the modernization research of traditional Chinese medicine in recent years. By utilizing specific microorganisms to ferment Platycodon grandiflorus, not only can the content of its medicinal components be increased, but its physicochemical properties can also be improved, and even new active substances can be generated. Currently, microbial-Platycodon grandiflorus two-way solid-state fermentation is still in the preliminary exploratory stage. Some researchers have used Ganoderma lucidum and the non-medicinal parts of Platycodon grandiflorus for solid-state two-way fermentation. The antioxidant capacity of the resulting Ganoderma lucidum-Platycodon grandiflorus mycelium was significantly improved, mainly due to the increased content of polysaccharides and total phenolic substances in the mycelium. Further pharmacodynamic evaluation showed that Ganoderma lucidum-Platycodon grandiflorus mycelium could significantly prolong the breathing time in mice with acute cerebral ischemia, indicating that it has a clear protective effect against cerebral ischemia and hypoxia. This research fully embodies the concept of comprehensive utilization of traditional Chinese medicine resources and is of great significance for promoting the modernization research of traditional Chinese medicine. However, there is still limited research on microbial-Platycodon grandiflorus two-way solid-state fermentation, and better microbial strains need to be screened to obtain better technical effects. Summary of the Invention

[0005] To address the above problems, this invention provides a strain of *Aspergillus cristatus* and a method for solid-state fermentation of *Platycodon grandiflorus*. This invention first isolates *Aspergillus cristatus* (…) from Fu brick tea. Eurotium cristatum NCPSec. Using this strain for solid-state fermentation of Platycodon grandiflorus not only improved the nutritional value of the Platycodon grandiflorus substrate but also significantly enhanced its antioxidant activity.

[0006] This invention first isolated *Aspergillus cristatus* from Fu brick tea. Eurotium cristatum The strain NCPSec was deposited on June 23, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251442. It exhibits the following microbiological characteristics: When cultured on malt extract agar plates, yellowish-white hyphae are observed on day 4; by day 6, the colony center gradually darkens and expands, with the central colony beginning to turn grayish-brown; on day 8, the central colony begins to age, producing numerous cleistothecia, and the grayish-brown area expands and further darkens (see [link to relevant documentation]). Figure 1 ).

[0007] The present invention also discloses the above-mentioned *Eurotium cristatum* ( Eurotium cristatum Application of NCPSec in solid-state fermentation of Platycodon grandiflorus.

[0008] This invention also discloses a method using *Aspergillus cristatus* (…). Eurotium cristatum A method for solid-state fermentation of Platycodon grandiflorus using NCPSec is characterized by inoculating a prepared *Aspergillus cristatus* spore suspension into a *Platycodon grandiflorus* substrate that has been autoclaved, followed by solid-state fermentation. After fermentation is completed, the fermentation product is collected, freeze-dried, sieved, and then frozen for storage. The fermentation conditions are: fermentation temperature 26-30℃, *Platycodon grandiflorus* substrate moisture content 40-50%, spore suspension inoculum 7-10%, and fermentation time 8-9 days. The preferred fermentation conditions are: fermentation temperature 28℃, *Platycodon grandiflorus* substrate moisture content 45%, spore suspension inoculum 9.5%, and fermentation time 8.5 days.

[0009] Platycodon grandiflorum matrix: Platycodon grandiflorum slices sterilized by high pressure.

[0010] Preparation of spore suspension: Frozen bacterial strains were inoculated onto malt extract solid medium for activation culture. Vigorous, morphologically typical single colonies were selected and passaged 2-3 times under the same conditions (7 days / generation at 28℃). Using aseptic technique, spores were gently scraped from the surface of the colonies with an inoculation loop, and the colonies were placed in a constant-temperature shaker to fully disperse the spores. The spore concentration was adjusted to (0.5-5) × 10⁻⁶ with sterile water. 6 The spores were prepared into a homogeneous suspension at a density of 1 spore per mL for later use.

[0011] The technical effects of this invention are:

[0012] 1. Fungi were isolated from Fu brick tea and, through colony morphology and molecular biological identification, were named *Aspergillus cristatus* (Eurotium cristatum). Eurotium cristatum NCPSec. The solid-state fermentation process of *Aspergillus cristatus* NCPSec onto *Platycodon grandiflorus* substrate was optimized through single-factor and response surface methodology. Using total antioxidant activity as the evaluation index, the optimal fermentation conditions were determined to be: fermentation temperature 28℃, *Platycodon grandiflorus* substrate moisture content 45%, spore suspension inoculum size 9.5%, and fermentation time 8.5 days.

[0013] 2. Under optimal fermentation conditions, the in vitro and in vivo antioxidant activities of the *Aureobasidium cristatum* NCPSec solid-state fermented platycodon extract were determined. In vitro results showed that the fermented extract increased DPPH, ABTS, and hydroxyl radical scavenging rates by 18.5%, 28.4%, and 82.5%, respectively, compared to the unfermented extract, with a total antioxidant capacity increase of 141%. In vivo experiments using the wild-type *Caenorhabditis elegans* N2 strain as the research subject showed that nematodes fed with the fermented extract had significantly lower levels of reactive oxygen species (ROS) than the unfermented group. At a feeding concentration of 275 μg / mL, ROS levels decreased by 15.6%, indicating that the *Aureobasidium cristatum* fermented platycodon extract effectively improved the excessive accumulation of ROS in nematodes, thereby reducing oxidative damage and enhancing antioxidant capacity. Furthermore, nematode lifespan testing results showed that, compared to the unfermented extract, the fermented extract effectively increased the average lifespan of nematodes, achieving a physiological protective effect of delaying aging.

[0014] 3. Experiments have shown that solid-state fermentation of Platycodon grandiflorus by Eurotium cristatum NCPSec not only significantly enhances the nutritional value of the Platycodon grandiflorus substrate, but also significantly increases the content of its bioactive components, providing a scientific basis for its application in functional foods and medicine.

[0015] 4. Sensory evaluation of the *Aspergillus cristatus* NCPSec solid-state fermented platycodon root extract revealed that fermentation alters the herbal flavor of platycodon root, restoring its unique "floral" aroma, and also improves the color and taste of the extract. As fermentation progresses, the extract color gradually changes from yellowish-green to reddish-brown, with a significant reduction in sourness and bitterness, and a significant increase in umami and saltiness.

[0016] 5. Experiments have shown that as the fermentation process proceeds, the content of toxic alkaloids such as aconitine and ricin continuously decreases; while the content of pharmacologically active components such as colchicine and its precursors, berberine, yohimbine, and papaverine is increased to varying degrees. Attached Figure Description

[0017] Figure 1 The growth of *Aspergillus cristatus* NCPSec on malt extract solid medium;

[0018] Figure 2 Phylogenetic tree of *Aspergillus cristatus* NCPSec constructed based on 18S rDNA sequence;

[0019] Figure 3 The effects of substrate moisture content on the appearance and total antioxidant activity of fermentation microorganisms were investigated. A: Growth of Platycodon grandiflorus microorganisms with different substrate moisture contents; B: Effect of different substrate moisture contents on the total antioxidant capacity of the microorganisms.

[0020] Figure 4 The effects of spore suspension inoculum amount on the appearance and total antioxidant activity of fermentation mycelium; where A: growth of Platycodon grandiflorus mycelium with different spore suspension inoculum amounts; B: effect of different spore suspension inoculum amounts on the total antioxidant capacity of the mycelium.

[0021] Figure 5 The effect of fermentation temperature on the appearance and total antioxidant activity of fermentation microorganisms; where A: growth of Platycodon grandiflorus microorganisms at different fermentation temperatures; B: effect of fermentation temperature on total antioxidant capacity.

[0022] Figure 6 The effects of fermentation time on the appearance and total antioxidant activity of fermentation microorganisms were investigated. A: Growth of Platycodon grandiflorus microorganisms at different fermentation times; B: Effect of fermentation time on total antioxidant capacity.

[0023] Figure 7 The effect of *Aurogonium cristatum* NCPSec solid-state fermentation of Platycodon grandiflorus on the in vitro antioxidant capacity of Platycodon grandiflorus mycelium; where A, B, C, and D represent DPPH radical scavenging capacity, ABTS radical scavenging capacity, hydroxyl radical scavenging capacity, and total antioxidant capacity, respectively.

[0024] Figure 8 The effect of Platycodon grandiflorum mycelium on ROS in N2 nematodes before and after fermentation;

[0025] Figure 9 The fluorescence of N2 type nematodes under a fluorescence microscope;

[0026] Figure 10 The effect of Platycodon grandiflorum mycelium on the survival rate of Caenorhabditis elegans before and after fermentation;

[0027] Figure 11 Nutrient content analysis of Platycodon grandiflorum mycelium fermented by Eurotium cristatum NCPSec, where A, B, C, D, E, and F are crude fiber, ash, moisture, fat, carbohydrate, and protein contents, respectively.

[0028] Figure 12Analysis of the content of active ingredients of *Aspergillus cristatus* NCPSec before and after fermentation; where A, B, C, and D are the changes in total saponin content, total polyphenol content, total flavonoid content, and sitosterol + spinachsterol content, respectively;

[0029] Figure 13 Color changes of Platycodon grandiflorum extract at different fermentation times;

[0030] Figure 14 Cluster heatmap of alkaloid samples with significant differences (fermentation days 0, 4, and 8.5). Detailed Implementation

[0031] The effects are illustrated below with reference to the embodiments and accompanying drawings.

[0032] Example 1: Isolation, purification and identification of strains

[0033] 1. Strain isolation

[0034] A tea sample containing abundant golden-yellow particles was selected from Fu brick tea and ground into a uniform powder. 5g of Fu brick tea powder was accurately weighed and added to 45mL of sterile physiological saline, along with an appropriate amount of sterile glass beads. The mixture was incubated at a constant temperature and shaken for 20 minutes to ensure the microorganisms in the tea leaves were evenly diluted in the sterile physiological saline. The tea powder suspension was then serially diluted to a final concentration of 10. -3 Up to 10 -6 Take 0.1 mL of each dilution and spread it evenly on malt extract agar plates. Incubate at 28°C for 5-7 days and observe colony growth.

[0035] Malt extract culture medium: 130 g / L malt extract, 0.1 g / L chloramphenicol, pH adjusted to 5.6±0.2.

[0036] 2. Strain purification

[0037] Select gradient plates with an appropriate number of colonies and observe and record the colony morphology. Use a sterile inoculation needle to pick up the hyphae of a single golden-yellow colony and inoculate it onto a blank malt extract medium. If contaminating bacteria are found on the medium, repeat the picking and inoculation process until a pure culture is obtained, thus obtaining strain NCPSec.

[0038] 3. Morphological identification of strains

[0039] The purified strain NCPSec was removed from cryopreservation and revived at room temperature. Then, using aseptic techniques in a clean bench, bacterial colonies were picked and transferred to pre-sterilized petri dishes, which were then incubated at 28°C. The morphology and growth of the colonies were observed daily.

[0040] When cultured on malt extract agar plates, yellowish-white mycelia were observed on day 4. By day 6, the color of the colony center gradually deepened and the colony area expanded, with the central colony beginning to turn grayish-brown. On day 8, the central colony began to age, producing numerous cleistothecia, and the grayish-brown area expanded and deepened further. Figure 1 As shown.

[0041] 4. PCR amplification of the 18S rDNA sequence of the strain

[0042] A small amount of mycelium was scraped from a fresh malt extract plate and sent to a sequencing company for sequencing. The obtained 18S rDNA sequence (shown in SEQ No. 1) was then compared with Blast similarity.

[0043] Download the relevant gene sequences from the GenBank database, and use them as... Neurospora crassa The strain was used as a foreign strain for comparative analysis. The obtained strain sequences were analyzed for homology using the BLAST tool in the NCBI database. The results showed that... Eurotium cristatum NCPSec sequence and Aspergillus cristatus The isolate *Fu Zhuan Chasubunit ribosomal* RNA gene (Sequence ID: MK386889.1) showed a similarity of 98%. A phylogenetic tree of *Chasubunit ribosomal* constructed based on the 18S rDNA sequence is shown below. Figure 2 As shown.

[0044] Based on the morphological identification results, the NCPSec strain can be confirmed as *Aspergillus cristatus*. Aspergillus cristatus, Coronavirus ( Eurotium cristatum (The asexual form of the strain). This strain was deposited on June 23, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20251442.

[0045] Example 2: Condition optimization of solid-state fermentation of Platycodon grandiflorus by Eurotium cristatum NCPSec

[0046] 1. Preparation of spore suspension

[0047] Frozen bacterial cultures were removed from a -80°C freezer and inoculated onto malt extract medium for activation culture. Vigorous, morphologically typical single colonies were selected and subcultured twice under the same conditions (7 days per generation at 28°C). Using aseptic techniques, spores were gently scraped from the colony surface with a disposable inoculation loop and transferred to a 250mL Erlenmeyer flask containing 100mL of sterile water and an appropriate amount of glass beads. The flask was placed in a shaker at 220 rpm for 20 minutes to fully disperse the spores. Spore counts were then performed using a hemocytometer, and the spore concentration was adjusted to 1×10⁻⁶ with sterile water. 6 The spores were prepared into a homogeneous suspension at a density of 1 spore per mL for later use.

[0048] 2. Inoculate with solid-state fermented bellflower

[0049] Preparation of Platycodon grandiflorus substrate: Dry the Platycodon grandiflorus slices to remove excess moisture, put 50g of the treated dried Platycodon grandiflorus slices into a 12×24cm cultivation bag, autoclave at 121℃ for 30min, and then let it stand at room temperature.

[0050] Using aseptic techniques, the prepared spore suspension of *Eurotium cristatum* NCPSec (1×10⁻⁶) was prepared. 6 A specific inoculum (N. serotonin / mL) was uniformly inoculated into autoclaved Platycodon grandiflorum substrate. The inoculated samples were then placed in a constant temperature incubator for solid-state fermentation. A sterilized Platycodon grandiflorum substrate without *Aspergillus cristatus* NCPSec was used as a blank control group, and parallel cultures were performed under the same conditions. Morphological changes during fermentation were observed periodically. After fermentation was completed, fermentation samples were collected, freeze-dried, passed through a 60-mesh sieve, and stored at -80℃ for later use.

[0051] 3. Single-factor optimization of solid-state fermentation of Platycodon grandiflorus by Eurotium cristatum NCPSec

[0052] Based on the results of preliminary experiments and after comprehensive evaluation considering factors such as practical operability, the conditions for fermenting Platycodon grandiflorus with Corynebacterium cristatum were determined. A single-factor experiment was designed based on the substrate moisture content of 50%, fermentation temperature of 28℃, inoculum size of 5%, and fermentation time of 7 days, with total antioxidant capacity as the optimization standard.

[0053] (1) Effect of substrate moisture content on solid-state fermentation of Platycodon grandiflorus mycelium: Substrate moisture contents of 40%, 45%, 50%, 55%, and 60% were set, with a bag weight of 50g and an inoculum weight of 5%. The substrates were placed in an incubator at 28℃ for 7 days for fermentation. After sampling, the total antioxidant capacity of the fermented mycelium was measured. A control group without inoculum was also set up with the same other conditions.

[0054] (2) Effect of fermentation temperature on solid-state fermentation of Platycodon grandiflorus mycelium: The incubator temperatures were set at 24℃, 26℃, 28℃, 30℃, and 32℃, with a bag weight of 50g, an inoculum weight of 7.5%, and a substrate moisture content of 45%. The total antioxidant capacity of the fermented mycelium was measured after sampling. A control group without inoculum was also set up with the same other conditions.

[0055] (3) Effect of inoculum size on solid-state fermentation of Platycodon grandiflorus mycelium: Fermentation substrate inoculum sizes of 2.5%, 5%, 7.5%, 10%, and 12.5% ​​were set, with a bag weight of 50g and a substrate moisture content of 45%. The substrates were placed in an incubator at 28℃ for 7 days for fermentation. After sampling, the total antioxidant capacity of the fermented mycelium was measured. A control group without inoculum was also set up, with other conditions being the same.

[0056] (4) Effect of fermentation time on solid-state fermented Platycodon grandiflorus mycelium: Fermentation time was calculated in days, with 4d, 6d, 8d, 10d and 12d respectively. The bag weight was 50g, the inoculum weight was 7.5%, the incubator temperature was 28℃, and the substrate moisture content was 45%. The total antioxidant capacity of the mycelium after fermentation was measured after sampling. At the same time, a control group without inoculum was set up with the same other conditions.

[0057] 4. Response surface optimization

[0058] After preliminary screening through single-factor experiments, the optimal addition ranges for each substance were integrated. Using the Box-Behnken experimental design method, three key factors were selected, each with three levels, to design a multi-factor experimental scheme. Different fermentation culture media were prepared according to the design scheme, and each group was fermented in triplicate. After fermentation, the total antioxidant capacity of the Platycodon grandiflorus mycelium in each group was measured. Statistical analysis of the experimental data was performed, a quadratic regression model was established, and a response surface methodology was plotted. The interaction effects of each factor were analyzed through the model to determine the optimal fermentation parameters, and validation experiments were conducted.

[0059] 5. Sample extraction

[0060] Take 1g of fermented Platycodon grandiflorus mycelium and extract it with 80% methanol at a material-to-liquid ratio of 1:15 (w / v) for 30 minutes using ultrasonic-assisted extraction at 200W. Centrifuge for 10 minutes to remove residue. Extract the precipitate a second time under the same conditions and centrifuge. Combine the two filtrates. Store at -20℃ for later use.

[0061] The total antioxidant capacity kit was used to detect the sample solution.

[0062] 6. Experimental Results

[0063] (1) Effect of substrate moisture content on solid-state fermentation of Platycodon grandiflorus by Eurotium cristatum NCPSec

[0064] As the substrate moisture content increases, brown pigment gradually accumulates on the surface of the fungus, and the brown portion increases with increasing moisture content, gradually changing color from brown to black. Figure 3 A). Meanwhile, the total antioxidant capacity first increases and then decreases with increasing matrix moisture content, reaching its highest level at a moisture content of 45%. Figure 3 B). Increased substrate moisture content is beneficial to the growth and reproduction of *Aspergillus cristatus*, but excessive moisture content will accelerate the aging of the strain and is not conducive to the accumulation of antioxidant active substances in the fermentation microbiota.

[0065] (2) Effect of spore suspension inoculum size on solid-state fermentation of Platycodon grandiflorus by Eurotium cristatum NCPSec

[0066] Depend on Figure 4 As shown in Figure A, when the spore suspension inoculum concentration was 7.5%, the Platycodon grandiflorus mycelium exhibited the best growth, with plump mycelium and no brown pigment accumulation. Simultaneously, under this inoculum concentration condition, the total antioxidant capacity of the mycelium was the highest. Figure 4 B). The total antioxidant capacity gradually increased with the increase of the spore suspension inoculum, and reached its peak at an inoculum of 7.5%, after which it showed a downward trend.

[0067] (3) Effect of fermentation temperature on solid-state fermentation of Platycodon grandiflorus by Eurotium cristatum NCPSec

[0068] Depend on Figure 5 As shown in Figure A, after 8 days of fermentation, the bacteria gradually covered the surface of the bellflower as the temperature rose. However, above 28°C, *Aspergillus cristatus* produced melanin, and the amount increased with rising temperature. Figure 5 As shown in Figure B, the total antioxidant capacity of the bacterial strain increases with increasing temperature, reaching a peak at 28℃. 28℃ is the optimal growth temperature for the strain in the Platycodon grandiflorus substrate. Above 28℃, the strain activity decreases and metabolic products decrease with increasing temperature, resulting in a decrease in total antioxidant capacity.

[0069] (4) Effect of fermentation time on solid-state fermentation of Platycodon grandiflorus by Eurotium cristatum NCPSec

[0070] Depend on Figure 6 As can be seen from Figure A, after 6 days of solid-state fermentation of Platycodon grandiflorus by *Eurotium cristatum* NCPSec, golden-yellow cleistothecia gradually appeared on the Platycodon grandiflorus substrate, giving the entire substrate a golden-yellow color, and by day 8, they covered the entire substrate. Meanwhile, as... Figure 6 As shown in Figure B, the total antioxidant capacity also showed a trend of first increasing and then decreasing with the increase of fermentation time, reaching a peak at 8 days. This result indicates that *Aspergillus cristatus* reaches its vigorous growth and metabolism period on day 8, and after 8 days, as the strain itself ages, the consumption of nutrients increases, leading to a decrease in metabolically active products. Therefore, 8 days was selected as the optimal fermentation time for *Aspergillus cristatus* fermentation of *Platycodon grandiflorus*.

[0071] (5) Response surface methodology optimization of solid-state fermentation conditions for Platycodon grandiflorus by Eurotium cristatum NCPSec

[0072] A response surface methodology experiment was conducted with substrate moisture content (A), inoculum size (B), and fermentation time (C) as independent variables and the total antioxidant capacity (Y) of Platycodon grandiflorum NCPSec fermentation as the dependent variable.

[0073] Based on the experimental results, the optimal fermentation conditions were a substrate moisture content of 45.7%, a spore suspension inoculum size of 9.4%, and a fermentation time of 8.74 days. The model predicted a total antioxidant capacity of 21 μmol / g under these fermentation conditions. To facilitate experimental operation, the optimal fermentation conditions were adjusted to a fermentation temperature of 28 ℃, a substrate moisture content of 45%, a spore suspension inoculum size of 9.5%, and a fermentation time of 8.5 days.

[0074] Example 3: Detection of in vitro antioxidant capacity of Platycodon grandiflorum mycelium before and after fermentation

[0075] Sample preparation: Under optimized fermentation conditions, 1g of Platycodon grandiflorus mycelium was fermented. Extraction was performed with 80% methanol at a material-to-liquid ratio of 1:15 (w / v) under ultrasonic-assisted extraction at 200W for 30 minutes. The sample was centrifuged for 10 minutes to remove residue. The precipitate was then extracted a second time under the same conditions and centrifuged again. The two filtrates were combined and stored at -20℃ for later use. The preparation methods for the sample solutions before and after fermentation were the same. The raw material for the sample solution before fermentation was unfermented Platycodon grandiflorus substrate (the same Platycodon grandiflorus was used for both fermentation and unfermentation).

[0076] 1. Determination of DPPH free radical scavenging activity

[0077] This experiment should be conducted entirely under light-protected conditions. Add 0.2 mL of the pre- and post-fermentation extract of *Platycodon grandiflorus* (sample solution) and 0.2 mL of DPPH solution to the test tubes, respectively. Shake well and incubate in a 37°C water bath for 30 min. Observe the reaction at OD... 517nm For A1, 0.2 mL of anhydrous ethanol was added to the bacterial extract before and after fermentation instead of DPPH solution. This operation was repeated and recorded as A2. 0.2 mL of distilled water was added to 0.2 mL of DPPH solution instead of the bacterial extract. This operation was repeated and recorded as A0. Ascorbic acid (Vc) was set up as a positive control group.

[0078] DPPH clearance rate (%) = [A0 - (A1 - A2)] / A0 × 100%;

[0079] In the formula: A0: OD of the blank solution 517nm Absorbance;

[0080] A1: OD of the sample solution or vitamin C517nm Absorbance;

[0081] A2: OD of the sample solution itself 517nm Absorbance value.

[0082] The scavenging effect of Platycodon grandiflorum microbiota on DPPH free radicals before and after fermentation, such as Figure 7 As shown in Figure A, the DPPH free radical scavenging ability of the platycodon mycelium gradually increased with the extension of fermentation time, reaching the highest level of 82.83% at 8.5 days, which was 18.5% higher than that of unfermented platycodon.

[0083] 2. Determination of ABTS free radical scavenging activity

[0084] The sample solution was tested using the ABTS free radical scavenging ability kit. The results showed that: Figure 7 As shown in Figure B, the ABTS free radical scavenging ability increased with prolonged fermentation time, reaching 79.05% at 8.5 days, representing a 28.4% increase in ABTS free radical scavenging ability compared to before fermentation.

[0085] 3. Determination of hydroxyl radical scavenging ability (·OH)

[0086] Mix 1 mL of 9 mmol / L FeSO4 solution with 1 mL of 9 mmol / L salicylic acid solution (CH3CH2OH), then add 1 mL of the pre- and post-fermentation extract of Platycodon grandiflorum (sample solution), mix thoroughly, add 1 mL of 8.8 mmol / L H2O2, and incubate in a 25°C water bath for 30 min. Observe the OD value... 510nm The absorbance was measured at a certain point, with distilled water and vitamin C used as controls under the same procedure.

[0087] Hydroxyl radical scavenging rate (%) = (A0-A1) / A0 × 100%;

[0088] In the formula: A0 represents the OD value in the blank solution. 510nm The absorbance value;

[0089] A1 is the absorbance value of the sample liquid to be tested.

[0090] This invention tested the hydroxyl radical scavenging ability of the extracts from Platycodon grandiflorus before and after solid-state fermentation, and the results are as follows: Figure 7 As shown in Figure C, both the Platycodon grandiflorum mycelium samples before and after fermentation exhibited hydroxyl radical scavenging activity. The sample fermented for 8.5 days showed a scavenging capacity of 36.99%, representing an 82.5% increase compared to the Platycodon grandiflorum matrix before fermentation. This further confirms that solid-state fermentation can significantly enhance the hydroxyl radical scavenging ability of Platycodon grandiflorum mycelium.

[0091] 4. Determination of FRAP's total antioxidant capacity and free radical scavenging ability

[0092] The total antioxidant capacity of the sample solution was measured using a reference total antioxidant capacity kit. The results showed that the total antioxidant capacity of the Platycodon grandiflorum mycelium significantly increased with prolonged fermentation time. At 8.5 days of fermentation, the total antioxidant capacity of the sample reached a maximum of 22.6 μmol / g, representing a 141% increase compared to the antioxidant capacity of the Platycodon grandiflorum substrate before fermentation. Figure 7 D). This indicates that solid-state fermentation of *Aspergillus cristatus* can significantly enhance the antioxidant activity of *Platycodon grandiflorus* mycelium.

[0093] In summary, under optimal fermentation conditions, the DPPH, ABTS, and hydroxyl radical scavenging rates of the *Aureobasidium cristatum* NCPSec-fermented *Platycodon grandiflorus* extract were increased by 18.5%, 28.4%, and 82.5%, respectively, compared to the unfermented *Platycodon grandiflorus* substrate. Simultaneously, the total antioxidant capacity was also significantly enhanced, increasing by 141% compared to the unfermented *Platycodon grandiflorus* substrate. This indicates that *Aureobasidium cristatum* NCPSec fermentation of *Platycodon grandiflorus* can effectively improve the in vitro antioxidant capacity of *Platycodon grandiflorus* mycelium.

[0094] Example 4: Effects of Platycodon grandiflorum mycelium on the antioxidant activity of Caenorhabditis elegans before and after solid-state fermentation

[0095] 1. Preparation of culture reagent for Caenorhabditis elegans

[0096] M9 buffer: Weigh 15g NaH2PO4·12H2O, 3g KH2PO4, and 5g NaCl, add an appropriate amount of deionized water to dissolve, and then bring the volume to 1000mL. Place the solution in an autoclave and sterilize at 121℃ for 30min.

[0097] Potassium phosphate buffer solution: Weigh 136g KH2PO4, add 900mL deionized water and mix well. Adjust the pH to 6.0, then add deionized water to bring the volume to 1L. Sterilize in an autoclave at 121℃ for 30min.

[0098] S-buffer: Add 0.5mol / L KH2PO4, 0.5mol / L K2HPO4, and 0.1mol / L NaCl to a final volume of 100mL, then sterilize in an autoclave at 121℃ for 30min.

[0099] NGM medium: NaCl 3g, peptone 2.5g, agar powder 17.5g, add deionized water 975 mL, sterilize at 121℃ for 30 min, and when the temperature drops to 65℃, add the following solutions (all sterilized): 0.5 mL of 1 mol / L CaCl2, 1 mL of 1 mol / L anhydrous magnesium sulfate, 25 mL of potassium phosphate buffer, and 1 mL of 5 mg / mL anhydrous cholesterol ethanol solution.

[0100] LB medium: 2g peptone, 2g NaCl, 1g yeast extract, add deionized water to bring the volume to 100 mL, adjust the pH to 7.0, and sterilize at 121℃ for 30 min.

[0101] 2. Escherichia coli OP50 ( E.coil Cultivation and preservation of OP50

[0102] E.coil OP50 is a uracil auxotrophic strain, commonly used as food for *C. elegans*. Removed from storage at -80°C. E.coil The OP50 bacterial strain was placed at room temperature until it thawed, then inoculated into LB liquid medium at a 2% inoculum. The culture was then incubated overnight at 37°C with shaking at 180 rpm. This process was repeated until the absorbance at 600 nm reached approximately 1. The culture was then aliquoted and stored at 4°C.

[0103] 3. Selection and culture of *Caenorhabditis elegans*

[0104] Take 200 μL of OP50 bacterial suspension and drop it into NGM solid medium, spread it evenly on the surface of the medium, let it dry naturally, and then incubate it upside down in a 37℃ constant temperature incubator for 24 hours.

[0105] Caenorhabditis elegans, which are at the same stage, were inoculated into cultured plants. E.coil After standing in OP50-NGM plates until the moisture evaporates, transfer them to a 20℃ constant temperature incubator for incubation.

[0106] 4. Synchronization of Caenorhabditis elegans

[0107] First, observation under a stereomicroscope confirmed that most nematodes in the culture dish were in the oviposition stage. The NGM plate surface was rinsed with sterile ultrapure water, and the eluent was collected into a sterile 15 mL centrifuge tube. Centrifugation was performed at 1500 rpm for 2 minutes. The supernatant was collected, and sterile ultrapure water was added again and mixed thoroughly. This process was repeated twice. Once no obvious culture medium impurities were observed under a stereomicroscope, 5 mL of Bleach solution (Bleach solution: 6.5 mL sterile ultrapure water, 2.5 mL NaClO, 1 mL 10 mol / L NaOH mixed) was added to the centrifuge tube, and the mixture was shaken intermittently for 3 minutes. Lysis was stopped when no obviously broken nematodes were observed floating in the centrifuge tube under a stereomicroscope. Add 5 mL of LM9 buffer to the centrifuge tube to terminate lysis, centrifuge at 2500 rpm for 2 min, aspirate the supernatant, add 10 mL of LM9 buffer to rinse the eggs and remove impurities from the solution, centrifuge at 2500 rpm for 2 min, repeat this step three times, then add 10 mL of S-buffer and incubate on a rotating shaker at 20℃ for 12 h, then add dropwise... E.coilAfter drying, the OP50-NGM plates were placed in an incubator at 20℃ for later use.

[0108] 5. Effects of solid-state fermentation of Platycodon grandiflorum mycelium on reactive oxygen species (ROS) in Caenorhabditis elegans cells

[0109] Weigh 1g of Platycodon grandiflorum mycelium powder (both before and after fermentation), add 15mL of 80% methanol, and extract by sonication at 40℃ for 20min. Collect the supernatant, concentrate under reduced pressure to a paste-like consistency, dissolve in 1mL of DMSO, reconstitute, and filter through a 0.22μm filter membrane. Add 0.5% of the solution to NGM plates and incubate at 37℃ for 12h. Then store at 4℃ for later use. Add the synchronized M9 buffer containing eggs to... E.coil 48 hours after incubation, nematode growth was observed on OP50-NGM plates. Before oviposition, 70 nematodes were picked into a culture medium containing platycodon mycelium extract, with three replicates per group. A positive control group containing only DMSO was used. The plates were cultured for four days, with the NGM plates coated with the extract changed daily. Nematodes from each group were then transferred to 1 ml of LM9 buffer, washed three times, centrifuged at 4°C for 10 min, and the supernatant was discarded. The remaining approximately 100 μL was transferred to sterile NGM medium and allowed to crawl for 10 min. Using black 96-well plates, 50 μL of LM9 buffer and 50 μL of LDCFH-DA (50 μmol / L) were added. Twenty nematodes were picked from each treatment group, with three replicates. Untreated nematodes served as the control group, and DMSO as the positive control.

[0110] This invention uses the 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent probe method to determine the reactive oxygen species (ROS) levels in wild-type Caenorhabditis elegans (N2), and acquires images of the nematodes labeled with the fluorescent probe using a fluorescence microscope. Figure 8 As shown, wild-type *C. elegans* fed with different concentrations (27.5 μg / mL, 55 μg / mL, and 275 μg / mL) of *Eurotium cristatum* NCPSec solid-state fermented methanol extract of *Platycodon grandiflorus* showed significantly lower ROS fluorescence intensity levels in vivo compared to the unfermented group, with decreases of 7.1%, 6%, and 15.6%, respectively. Fluorescence microscopy observations showed ( Figure 9 The fluorescence intensity of nematodes in the treated group was significantly reduced. These results indicate that Platycodon grandiflorum mycelium can effectively alleviate the excessive accumulation of ROS in nematodes, thereby reducing oxidative damage. The solid-state fermentation process of *Aspergillus cristatus* NCPSec can efficiently utilize the nutrients in the Platycodon grandiflorum substrate, converting them into functional components with enhanced antioxidant activity.

[0111] 6. Effects of solid-state fermentation of Platycodon grandiflorum mycelium on the lifespan of Caenorhabditis elegans

[0112] Transmit synchronized L4 late-stage nematodes to E.coilOP50-NGM plates were used for culture. Plates containing extracts of Platycodon grandiflorus mycelium before and after fermentation served as the control and experimental groups, while plates containing DMSO served as the blank control group. Three replicates were set up for each experimental and control group, with at least 30 nematodes picked from each replicate. The time the nematodes were picked onto the plate was recorded as day 0. Every 24 hours, the nematodes were re-cultured on different plates, and their survival was recorded (death was defined as the nematode not wriggling when gently touched with a platinum needle and the pharyngeal pump ceasing). Nematodes that crawled to the culture medium wall and died during transfer were not included in the total survival count.

[0113] At 20℃, the effect of *Aspergillus cristatus* NCPSec fermentation on the lifespan of nematodes was as follows: Figure 10 As shown in Table 1, compared with the DMSO control group, both fermentation and solid-state fermentation of Platycodon grandiflorus significantly prolonged the average lifespan of nematodes in a concentration-dependent manner. The solid-state fermented Platycodon grandiflorus extract (FPG) produced by *Aspergillus cristatus* significantly increased the average lifespan of nematodes by 5.7% at a concentration of 275 μg / mL compared to the unfermented group (NFPG). This result indicates that solid-state fermentation of Platycodon grandiflorus by *Aspergillus cristatus* NCPSec can reduce the concentration of free radicals in nematodes, thereby mitigating free radical damage and thus delaying the aging process.

[0114] Table 1. Effects of Platycodon grandiflorum mycelium on the average lifespan of Caenorhabditis elegans before and after fermentation.

[0115]

[0116] In summary, the ROS fluorescence intensity in wild-type *C. elegans* fed with fermented mycelial extract was significantly lower than that in the unfermented group, with decreases of 7.1%, 6%, and 15.6% at feeding concentrations of 27.5 μg / mL, 55 μg / mL, and 275 μg / mL, respectively. Further research revealed that the fermented mycelial extract also prolonged the average lifespan of the nematodes, further demonstrating that *Eurotium cristatum* NCPSec fermentation of *Platycodon grandiflorus* mycelium can enhance the nematodes' ability to resist oxidative damage, effectively improve ROS accumulation in *C. elegans*, and thus delay aging.

[0117] Example 5: Analysis of plasminoides composition from solid-state fermentation of Platycodon grandiflorus by *Aspergillus cristatus* NCPSec

[0118] 1. Nutritional composition analysis

[0119] like Figure 11 As shown, the protein content in the mycelium plasminogen lysate (FPG) increased significantly from 6.54 g / 100 g to 7.45 g / 100 g after fermentation, an increase of 14% compared to before fermentation (see [link]). Figure 11 F); the crude fiber and ash content increased by 17% and 21% respectively compared to before fermentation (see F); Figure 11 A and Figure 11 D). This phenomenon may be related to the degradation of macromolecules by extracellular proteases secreted by *Aspergillus cristatus* during fermentation, as well as the synthesis of proteins within the bacteria themselves. Other nutrients, such as carbohydrate and fat content (see [reference needed]). Figure 11 C and Figure 11 E) remains essentially unchanged before and after fermentation. P >0.05). This indicates that *Aspergillus cristatus* effectively biotransformed the nutrients in the *Platycodon grandiflorus* substrate during the two-way solid-state fermentation process, significantly increasing the content of some key nutrients and thus enhancing the nutritional value of the *Platycodon grandiflorus* mycelium.

[0120] 2. Analysis of active ingredients

[0121] like Figure 12 As shown, this invention determined the changes in the content of sterols, saponins, polyphenols, and flavonoids—active components with antioxidant, anti-inflammatory, and antitumor effects—in the solid-state fermented platycodon mycelium of *Aspergillus cristatus* NCPSec. Compared with unfermented platycodon, the total flavonoid content, relative content of sitosterol, and spinasterol in the platycodon mycelium increased by 26.5%, 46.8%, and 115.8%, respectively (see [reference]). Figure 12 C and Figure 12 (D) The relative contents of total saponins and total polyphenols increased by 250% and 43.6%, respectively (see D). Figure 12 A and Figure 12 B).

[0122] 3. Sensory evaluation

[0123] Sensory evaluation of the *Platycodon grandiflorus* mycelium extract from solid-state fermentation (3g of *Platycodon grandiflorus* mycelium sample before and after fermentation was added to boiling water at a material-to-liquid ratio of 1:50 and soaked for 10 minutes, then filtered through filter paper to obtain a clear extract, which was cooled at 4℃ for later use) revealed that fermentation alters the herbal flavor of *Platycodon grandiflorus*, restoring its unique "mycelium flower" aroma, and improving both the color and taste of the mycelium extract. As fermentation progressed, the color of the extract gradually changed from yellowish-green to reddish-brown (see [link to relevant documentation]). Figure 13 The sour and bitter tastes were significantly reduced, while the umami and salty tastes were significantly increased.

[0124] 4. Analysis of alkaloid content in metabolites

[0125] This invention employs ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) to construct a metabolic map of non-volatile substances in Platycodon grandiflorum during fermentation.

[0126] Sample preparation: Weigh 100 mg of lyophilized powder before and after fermentation into a 2 mL centrifuge tube. Add 600 µL of methanol solution containing 2-chloro-L-phenylalanine to the sample, and add a steel ball. Vortex for 30 seconds to mix thoroughly. Then, place the sample in a tissue homogenizer for disruption. Place the sample in an ice bath for ultrasonic extraction for 15 minutes. Centrifuge at 12000 rpm for 10 minutes at 4°C. Collect the supernatant and filter it through a 0.22 μm filter membrane. Transfer the filtrate to a liquid chromatography bottle for LC-MS analysis.

[0127] Chromatographic conditions: ACQUITY UPLC HSS T3 (2.1 × 100 mm, 1.8 µm) column, flow rate 0.3 mL / min, column oven set at 40℃, injection volume 2 μL. In positive ion mode, the mobile phase (A) was 0.1% formic acid acetonitrile and (B) 0.1% formic acid aqueous solution, with gradient elution. In negative ion mode, the mobile phase was acetonitrile (A) and 5 mmol formic acid ammonium aqueous solution (B), with gradient elution. The gradient elution program is shown in Table 2.

[0128] Table 2 Gradient elution program

[0129]

[0130] Mass spectrometry conditions: A high-resolution mass spectrometer detector was used, equipped with an electrospray ionization (ESI) source, and data were acquired in both positive and negative ion modes. Detailed mass spectrometry parameters are shown in Table 3. The first-stage full scan was performed at a resolution of 70,000, with a scan range of 100-1000 m / z. Second-stage fragmentation was then performed using high-energy collisional dissociation (HCD) at a collision energy of 30 eV and a second-stage resolution of 17,500. During data acquisition, the top three ions with the highest signal intensity were selected for fragmentation analysis, and dynamic exclusion was enabled to remove redundant MS / MS information, thereby improving data acquisition efficiency and analytical accuracy.

[0131] Table 3 Mass Spectrometry Parameters

[0132]

[0133] Cluster heatmap analysis was used to analyze alkaloid samples. In this study, cluster heatmap analysis was employed, with each row corresponding to a metabolite; light red represented high relative content, and dark blue represented low relative content.

[0134] Cluster heatmap of alkaloid samples with significant differences as follows Figure 14As shown, solid-state fermentation of *Aurorus cristatus* plays a crucial role in the content of related alkaloids in the platycodon root matrix. As fermentation progresses, the content of toxic alkaloids such as aconitine and ricin in the platycodon root matrix continuously decreases, and the content of toxic metabolites also decreases. This demonstrates that *Aurorus cristatus* NCPSec fermentation can alter the non-volatile components of the platycodon root matrix, achieving a reduction in toxicity and enhancement of efficacy. The content of some alkaloids, such as colchicine and its precursors, shows a significant upward trend. Colchicine, an extract from autumn crocus, has become a drug used to treat cardiovascular diseases. In addition, some pharmacologically active alkaloids, such as berberine, yohimbine, and papaverine, show a significant upward trend during fermentation.

Claims

1. A strain of *Eurotium cristatum* ( Eurotium cristatum The strain is registered under NCPSec and its preservation number is CCTCC NO: M20251442.

2. The application of *Aspergillus cristatus* NCPSec as described in claim 1 in solid-state fermentation of *Platycodon grandiflorus*.

3. A method for solid-state fermentation of Platycodon grandiflorus using *Aspergillus cristatus* NCPSec as described in claim 1, characterized in that, The prepared *Eurotium cristatum* NCPSec spore suspension was inoculated into a *Platycodon grandiflorus* substrate that had been autoclaved, and then solid-state fermentation was carried out. After the fermentation was terminated, the fermentation product was collected. The fermentation conditions were: fermentation temperature 26-30℃, *Platycodon grandiflorus* substrate moisture content 40-50%, spore suspension inoculation amount 7-10%, and fermentation time 8-9 days.

4. The method for solid-state fermentation of Platycodon grandiflorus using *Aspergillus cristatus* NCPSec as described in claim 3, characterized in that, The platycodon matrix is ​​platycodon slices that have been sterilized by high pressure.

5. The method for solid-state fermentation of Platycodon grandiflorus using *Aspergillus cristatus* NCPSec as described in claim 3, characterized in that, The fermentation conditions were as follows: fermentation temperature 28 ℃, water content of Platycodon grandiflorus substrate 45%, spore suspension inoculum 9.5%, and fermentation time 8.5 days.

6. The method for solid-state fermentation of Platycodon grandiflorus using *Aspergillus cristatus* NCPSec as described in claim 3, characterized in that, The preparation method of the *Aurogonium cristatum* NCPSec spore suspension is as follows: Frozen *Aurogonium cristatum* NCPSec strain is inoculated onto malt extract solid medium for activation culture. Vigorous, morphologically typical single colonies are selected and passaged 2-3 times under the same conditions. Using aseptic technique, spores are gently scraped from the surface of the colonies with an inoculation loop, and the colonies are placed in a constant-temperature shaker to fully disperse the spores. The spore concentration is adjusted to (0.5-5) × 10⁻⁶ using sterile water. 6 The spores were prepared at a density of 1 spore per mL to form a homogeneous spore suspension for later use.

Images