Fusarium and application thereof in degradation of ginkgolic acid
By using liquid and solid-state fermentation technologies with Fusarium laieritium DLT-118, the problems of low degradation efficiency and safety risks of ginkgolic acid have been solved, achieving efficient and green degradation of ginkgolic acid and improving the safety and industrialization potential of ginkgo extract.
Patent Information
- Application Number
- CN202610034302.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-12
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies are inefficient in degrading ginkgolic acid, result in significant loss of active ingredients, are complex in process, and pose potential safety risks, making it difficult to achieve efficient and green industrialization of ginkgo extract.
Liquid fermentation of Fusarium laieritium DLT-118 was used to utilize its specific degradation ability of ginkgolic acid. Combined with solid-state fermentation, the structure of ginkgo leaf powder was improved, and the contact area between mycelium and substrate was increased to achieve efficient degradation.
A 96.47% degradation rate of ginkgolic acid was achieved, reducing the cytotoxicity of the extract and improving the safety and environmental friendliness of the ginkgo extract, making it suitable for industrial application.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to a strain of Fusarium and its application in the degradation of ginkgolic acid. Background Technology
[0002] ginkgo( Ginkgo biloba L. (Ginkgo biloba) is a relict plant whose leaves are rich in flavonoids and terpene lactones, and are widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases. However, it contains ginkgolic acids (GAs), the structural formula of which and the HPLC analysis of ginkgolic acid at 310 nm wavelength are shown in the figure. Figure 1 (As shown) It has risks such as sensitization and cytotoxicity, and its content is strictly limited in both domestic and international pharmacopoeias.
[0003] To reduce ginkgolic acid content, existing technologies mainly include physical, chemical, and biological methods, such as organic solvent extraction, macroporous resin adsorption, enzymatic hydrolysis, coordination-solvent synergistic methods, ultrasound-assisted extraction, or cyclodextrin inclusion. However, these methods generally suffer from problems such as the risk of organic solvent residue, significant loss of effective components, complex processes, high costs, or limitations in large-scale application. While microbial degradation strategies in biological methods have the potential to be green and highly specific, current research mainly focuses on removal processes, and in-depth exploration of the specific mechanisms of ginkgolic acid degradation by endophytic fungi in Ginkgo biloba and the development of highly efficient strains is still lacking.
[0004] Therefore, developing a biodegradation technology that can efficiently degrade ginkgolic acid, retain active ingredients to the maximum extent, and is suitable for industrialization, thereby further improving the safety of ginkgo extract and promoting the sustainable development of the industry, is an urgent problem to be solved by this invention. Summary of the Invention
[0005] In view of the above-mentioned prior art, the purpose of this invention is to overcome the problems of low degradation efficiency, large loss of effective components, complex process and potential safety risks in existing ginkgo acid degradation technology, and to provide a highly efficient, green microbial degradation method based on Fusarium, which can retain the active components of ginkgo leaves to the maximum extent.
[0006] To achieve the above objectives, the present invention provides a strain of Fusarium, wherein the Fusarium is a bacterial strain. Fusarium laieritium DLT-118, with accession number CCTCC NO: M 20252976, was deposited at the China Center for Type Culture Collection on December 19, 2025.
[0007] This strain was isolated from ginkgo trees and belongs to the endophytic fungi family, exhibiting natural adaptation to its host environment. Colony morphology (fluffy white, producing brick-red myxospore masses) and ITS rDNA sequence identification both support its taxonomic position and reliable origin.
[0008] The present invention also provides the application of Fusarium as described above in the degradation of ginkgolic acid.
[0009] Preferably, the process of Fusarium degrading ginkgolic acid is liquid fermentation.
[0010] Preferably, the temperature of the liquid fermentation process is 20℃-35℃.
[0011] The strain provided by this invention Fusarium laieritium DLT-118 exhibits a 96.47% degradation rate of ginkgolic acid. This high efficiency likely stems from the strain's unique metabolic mechanism, evolved through long-term symbiosis with the host as an endophytic fungus of Ginkgo biloba, enabling it to specifically degrade ginkgolic acid. It can degrade ginkgolic acid via liquid fermentation, and the solid-state fermentation makes the Ginkgo leaf powder structure loose and porous (it should be noted that the fermentation process for Ginkgo leaf powder is solid-state, while the degradation of ginkgolic acid is a liquid-state process; the two fermentation methods differ), further increasing the contact area between the mycelium and the substrate, thus promoting degradation efficiency. Based on this, the Ginkgo endophytic fungus provided by this invention... Fusarium laieritium DLT-118 degradation of ginkgolic acid is an excellent technical strategy that combines high efficiency, specificity, safety, and environmental friendliness. Attached Figure Description
[0012] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is the structural formula of ginkgolic acid and the HPLC analysis chromatogram of ginkgolic acid components at a wavelength of 310 nm; among them, peaks 1-5 correspond to the identified ginkgolic acid monomers, namely: GA1, C13:0; GA2, C15:1; GA3, C17:2; GA4, C15:0; GA5, C17:1. Figure 2 The test results were obtained from the strains treated with Ginkgo biloba extract and the untreated strains in Example 1 and Comparative Example 2. Fusarium lateritium Comparison of mycelial morphology of DLT-118; Figure 3 The test results showed that the unfermented ginkgo leaves in Example 2 and Comparative Example 3 were compared with those processed by the bacterial strain. Fusariumlateritium Scanning electron microscope image of ginkgo leaves after solid-state fermentation with DLT-118; Figure 4 The effects of Ginkgo biloba extract treatment on bacterial strains were detected in Example 3 and Comparative Example 4. Fusariumlateritium The effect of DLT-118 on oxidative stress was shown in the figure; compared with the CK group, p<0.05, p<0.01, **p<0.001; Figure 5 This is a graph showing the cytotoxicity assessment results of the degraded Ginkgo biloba extract in Example 4; different letters indicate significant differences as identified by one-way ANOVA (p<0.05). Detailed Implementation
[0013] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.
[0014] In this invention, the human gastric mucosal epithelial cell line GES-1 was purchased from Wuhan Pronosei Life Sciences Co., Ltd.; the human normal lung epithelial cell line Beas-2B was obtained from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. The technical solution of this invention will be further described below through specific embodiments. Application examples
[0015] 4g of Ginkgo biloba leaf powder was weighed and added to 100mL of ethanol. The mixture was ultrasonically extracted for 2 hours at 160W power and 40kHz frequency. After ultrasonic extraction, the solid residue was filtered off, and the filtrate was concentrated by rotary evaporation to obtain the Ginkgo biloba leaf extract. The ginkgolic acid content in the obtained Ginkgo biloba leaf extract was determined by high-performance liquid chromatography (HPLC). The specific method was as follows: an Agilent 1260 system equipped with an RP-C188 column (4.6×250mm, 5μm), a detection wavelength of 310nm, a column temperature of 40°C, and a flow rate of 1.0mL / min; mobile phase A was 0.4% (v / v) phosphoric acid solution, and mobile phase B was acetonitrile. The gradient elution program was 0–30 min (75–90% B), 30–45 min (90% B), and 45–50 min (90–95% B).
[0016] The fungal strain cultured on PDA plates (i.e., the Fusarium strain preserved in this invention) Fusarium lateritium DLT-118 was inoculated into 200 mL of sterile PDB liquid medium containing the prepared Ginkgo biloba extract (final concentration 1 mg / mL) dissolved in methanol. After culturing at 28°C and 120 rpm for 7 days with shaking, 200 mL of n-butanol was added to the fermentation flask, and the mixture was allowed to stand for phase separation. After removing the culture medium and mycelial residue, the supernatant organic phase was collected, concentrated under reduced pressure, and filtered through a 0.22 μm organic filter membrane to obtain the sample after ginkgolic acid degradation treatment. The peak area of ginkgolic acid in the sample was measured.
[0017] The detection of ginkgolic acid content in the sample should be performed following the same procedure as described above for detecting ginkgolic acid content in ginkgo leaf extract. Comparative Example 1
[0018] The procedure was followed as in the application example, except that the step of inoculating 200 mL of sterile PDB liquid medium with fungal strains was omitted to obtain the control sample (denoted as the GE group). The peak area of ginkgolic acid in the control sample was measured.
[0019] The degradation rate of ginkgolic acid was calculated by comparing the content of the application example and the comparative example. The formula is: degradation rate (%) = (AB) / A × 100, where A is the peak area of total ginkgolic acid measured in the comparative example and B is the peak area of total ginkgolic acid measured in the application example.
[0020] The detection results of the application example and comparative example 1 are as follows: Figure 1 As shown, that is Figure 1 Of the two chromatographic curves, the upper curve corresponds to Example 1, and the lower curve corresponds to Application Example. Through... Figure 1 It can be seen that, Fusariumlateritium DLT-118 exhibits a significant degradation ability for ginkgolic acid, with a degradation rate of 96.47%.
[0021] Example 1: Detection of Ginkgo biloba extract against bacterial strains Fusarium lateritium The influence of DLT-118 micromorphology (denoted as GE group) First, the strain Fusarium lateritium 1 cm² mycelial blocks of DLT-118 were transferred from PDA plates to 200 mL of sterile PDB liquid medium. Ginkgo biloba extract dissolved in methanol was added to the PDB liquid medium containing the mycelial blocks to bring the final concentration of the extract to 1 mg / mL. The inoculated fermentation medium was cultured at 28°C and 120 rpm for 3, 5, and 7 days, respectively. Fresh mycelia were then rapidly immersed in electron microscopy fixative and fixed at room temperature for 30 minutes, followed by storage at 4°C. Next, the mycelia were washed three times with 0.1 M PBS to remove residual reagents, and then fixed in the dark with 1% osmium tetroxide solution (prepared with 0.1 M PBS) at room temperature for 1–2 hours, followed by three more washes with 0.1 M PBS. Next, a gradient dehydration process was performed: the sample was successively immersed in 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100% ethanol solutions for 15 minutes each, and finally treated with isoamyl acetate for 15 minutes to complete the dehydration. The dehydrated sample was then completely dried in a critical point desiccator. After drying, the sample was firmly fixed onto a conductive carbon film double-sided adhesive on the sample stage and subjected to gold sputtering for approximately 30 seconds. Finally, the sample was observed using a Hitachi SU8100 scanning electron microscope (SEM). The obtained SEM images are shown below.Figure 2 As shown. Microstructure of DLT-118 (denoted as CK group)
[0022] The procedure was performed according to the method in Example 1, except that the supplemented Ginkgo biloba extract dissolved in methanol was replaced with an equal volume of methanol. The resulting electron micrograph is shown below. Figure 2 As shown.
[0023] pass Figure 2 It can be seen that after 3 days of treatment, compared with the control group, the mycelia in the GE-treated group showed partial shrinkage, indicating that the extract has an inhibitory effect on the normal growth of the strain. By day 5, the degree of shrinkage in the treated group was significantly reduced compared with the control group. On day 7, no obvious shrinkage was observed in the GE-treated group. These phenotypic changes may be due to... Fusarium lateritium DLT-118 gradually adapted to the GE-containing environment. Meanwhile, over time, ginkgolic acid was progressively degraded, significantly reducing the toxin concentration in the culture medium. This decrease in toxin levels alleviated the stress on the fungus, leading to the disappearance of the hyphal shrinkage phenotype.
[0024] Detection Example 2, bacterial strain Fusarium lateritium Analysis of Ginkgo Leaf Morphology by DLT-118 Solid-State Fermentation Completely wet 1g of ginkgo leaf powder with 1mL of sterile water and sterilize. Under sterile conditions, prepare the bacterial strain. Fusarium lateritium DLT-118 was inoculated into the above-mentioned sterile ginkgo leaf powder and fermented statically at 28°C for 7 days. The ginkgo leaf powder samples were processed using scanning electron microscopy (SEM) sample preparation procedures. The fermentation process was analyzed using SEM. Fusarium lateritium The structure and morphological characteristics of Ginkgo leaf powder after 7 days of solid-state fermentation with DLT-118 are shown in the scanning electron microscope image below. Figure 3 As shown. Comparative Example 3: Morphological Analysis of Untreated Ginkgo Leaves
[0025] The procedure was performed according to the method used in Case 2, except that no bacterial strain was inoculated. Fusarium lateritium DLT-118. Scanning electron micrograph of unfermented ginkgo leaf powder as shown. Figure 3 As shown.
[0026] pass Figure 3 It can be seen that the fermentation process significantly altered the physical structure of ginkgo leaf powder. Unfermented leaf powder exhibited a dense structure, smooth surface, and clear edges. Figure 3 (Figure A, left side of the middle section); and through Fusarium lateritium After fermentation with DLT-118, the samples exhibited a distinctly loose and porous morphological characteristic. Figure 3(Figure B on the right). Hyphae colonization and the accompanying bioerosion lead to the formation of grooves and pores. This porous structure effectively increases the contact area between the hyphae and the substrate, thus creating a more favorable interfacial environment for the degradation of ginkgolic acid.
[0027] Example 3: Oxidative stress effect strain Fusarium lateritium DLT-118 was cultured in PDB medium with 1 mg / mL Ginkgo biloba extract dissolved in methanol added (designated as GE group). The culture was carried out at 28℃ and 120 rpm for 7 days, yielding the strain. Fusarium lateritium DLT-118 mycelium. The activities of CAT and SOD, as well as the content of MDA in the mycelium, were determined using detection kits (product numbers: BC0205, BC5165, BC6415) from Beijing Solarbio Science & Technology Co., Ltd., to evaluate the effects of Ginkgo biloba extract treatment on the strain. Fusarium lateritium The effect of DLT-118 on oxidative stress. Specifically, 0.1 g of the strain was taken. Fusariumlateritium DLT-118 mycelium was mixed with 1 mL of protein extraction buffer from the kit (provided with the kit). The mixture was further homogenized and then centrifuged at 8000 rpm for 5 minutes. The supernatant was retained for subsequent analysis; specifically, the retained supernatant was used to detect the corresponding CAT and SOD activities and MDA content using the aforementioned detection kit from Beijing Solarbio Science & Technology Co., Ltd. The results are as follows: Figure 4 As shown. Comparative Example 4
[0028] The procedure was performed according to the method in Example 3, except that the 1 mg / mL Ginkgo biloba extract dissolved in methanol was replaced with methanol only (designated as the CK group). The results are as follows. Figure 4 As shown, where, Figure 4 Figure A corresponds to CAT activity, Figure B corresponds to SOD activity, and Figure C corresponds to MDA content.
[0029] pass Figure 4 It can be seen that, compared with the CK group, the CAT activity in the GE-treated group was significantly reduced; at the same time, the MDA content and SOD activity in its hyphae were higher than those in the control group. These results indicate that GE-induced MDA accumulation exacerbates cellular lipid peroxidation, thereby causing oxidative damage to the strain. Fusarium lateritium DLT-118 responds to GE stress by initiating ginkgolic acid degradation, while oxidative damage is a concomitant phenomenon in this detoxification process. This complementary relationship between active response and passive damage reflects a survival strategy adopted by fungi under stress conditions.
[0030] Example 4: Cytotoxicity assessment of ginkgolic acid degradation products Cell viability was assessed using the MTT assay to evaluate the strain. Fusariumlateritium DLT-118 treatment was used to degrade the cytotoxicity of Ginkgo biloba extract after ginkgolic acid degradation. The specific procedure is as follows: Human normal lung epithelial cells (Beas-2B) and gastric mucosal cells (GES-1) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and the cells were incubated at 37°C with 5%... Cells were kept in a humidified incubator to maintain growth. Cells were then cultured in individual wells. Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours. Subsequently, cells were treated with different concentrations (12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL, 200 mg / mL) of the sample for 48 hours. After treatment, 20 μL of MTT solution was added to each well. The cells were incubated for another 4 hours. After removing the MTT solution, the formazan crystals in each well were dissolved in 150 μL of dimethyl sulfoxide (DMSO). Absorbance was measured at 490 nm using a microplate reader. Cell viability was expressed as a percentage relative to the control group (untreated). The results are shown below. Figure 5 As shown.
[0031] pass Figure 5 As can be seen, within the concentration range of 25-200 mg / mL, the treated Ginkgo biloba extract did not exhibit significant toxicity to either type of cell, and cell viability remained at a high level in all concentration groups. Therefore, this biodegradation process effectively reduced the potential toxicity risks associated with high ginkgolic acid content.
[0032] The strains of the present invention Fusariumlateritium Although DLT-118 initially exhibited stress responses such as hyphal contraction upon contact with Ginkgo extract, it gradually adapted, demonstrating good environmental adaptability. During fermentation, the strain counteracted oxidative stress by regulating the activity of antioxidant enzymes such as superoxide dismutase (SOD). The degraded Ginkgo extract showed significantly reduced toxicity to human lung epithelial cells (Beas-2B) and gastric mucosal cells (GES-1), with high cell survival rates, indicating a significant improvement in its biosafety. This degradation process is a biotransformation, conducted under mild conditions, requiring no toxic chemical reagents, and is environmentally friendly. This technology provides a new strategy for the high-value utilization of Ginkgo resources, helps improve the quality and safety of Ginkgo-related products (such as pharmaceuticals and health supplements), and can provide a reference for the biodetoxification technology of other natural products.
[0033] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
[0034] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.
[0035] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.
Claims
1. A strain of Fusarium, characterized in that, The Fusarium is Fusarium laieritium DLT-118, with accession number CCTCC NO: M 20252976, was deposited at the China Center for Type Culture Collection on December 19, 2025.
2. The application of Fusarium in the degradation of ginkgolic acid as described in claim 1.
3. The application according to claim 2, characterized in that, The process by which Fusarium degrades ginkgolic acid is a liquid fermentation.
4. The application according to claim 3, characterized in that, The temperature for liquid fermentation is 20℃-35℃.