Anti-paratuberculosis mycorrhizal extract and application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YANGTZE UNIVERSITY
- Filing Date
- 2026-04-30
- Publication Date
- 2026-07-24
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of microbial technology and animal pathogen control technology, specifically to an endophytic fungal extract and its application against *Glassonella parasuis*, and more particularly to the application of an endophytic fungus and its metabolites in inhibiting *Glassonella parasuis*. Background Technology
[0002] Parasporum glaucus ( Glaesserella parasuis *Glassie hygroscopicus* is one of the main pathogens causing Glasger's disease in pigs, often leading to symptoms such as polyserositis, arthritis, and meningitis, seriously affecting the healthy development of the pig farming industry. Currently, the control of this pathogen mainly relies on antibiotics and vaccines, but these methods suffer from problems such as increased drug resistance, significant serotype differences, and unstable control effects. Therefore, there is an urgent need to develop safe and efficient new alternative control methods.
[0003] Endophytic fungi, as symbiotic microbial resources within plants, can produce a variety of bioactive secondary metabolites, showing great potential for application in antibacterial and antifungal applications. However, systematic research on endophytic fungi derived from Fritillaria species and their application in the control of Glassiella parasuis remains lacking. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides an anti-Glasser bacteria (anti- G. parasuis The study aims to explore the extracts and applications of endophytic fungi to address the issues of strong antibiotic dependence and high risk of drug resistance in existing prevention and control technologies.
[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses an endophytic fungus, Talaromyces sp. SQ3, which was deposited at the China Center for Type Culture Collection on March 10, 2026, with accession number CCTCC M 2026406.
[0006] Correspondingly, an endophytic fungus, Talaromyces sp. SQ3, has an ITS sequence as shown in SEQ ID NO: 1.
[0007] Correspondingly, an antibacterial agent prepared using endophytic fungi.
[0008] Preferably, the active ingredient of the antibacterial agent is an ethyl acetate extract of endophytic fungi.
[0009] Correspondingly, an antibacterial agent prepared from endophytic fungi.
[0010] Preferably, the active ingredient of the antibacterial agent includes an ethyl acetate extract of endophytic fungi.
[0011] Correspondingly, the application of an endophytic fungus or antimicrobial agent in the inhibition of Gracilaria parasuis.
[0012] The present invention has the following beneficial effects: 1. The endophytic fungal strain disclosed in this invention is derived from Fritillaria cirrhosa in Hubei Province, identified as a fungus of the genus *Talaromyces* by molecular biological methods, and deposited at the China Center for Type Culture Collection (CCTCC). Studies have shown that the fermentation products of this strain and the extract obtained by ethyl acetate extraction have inhibitory effects on *Glassella parasuis* (…). Glaesserella parasuis It exhibits significant inhibitory effects, effectively suppressing its growth and reducing its pathogenic risk. The endophytic fungal strains and their extracts described in this invention can be used to prepare antibacterial agents against *Glassonella parasuis*, showing promising application prospects in the field of livestock disease control.
[0013] 2. The endophytic fungi disclosed in this invention have mild culture conditions, mature extraction technology, and are easy to scale up for production, thus showing good application prospects. Attached Figure Description
[0014] Figure 1 A represents endophytic fungi. Talaromyces Colony morphology of sp. SQ3, B represents endophytic fungi. Talaromyces Microstructure of sp. SQ3, C represents endophytic fungi. Talaromyces Phylogenetic tree of sp.SQ3; Figure 2 In the table, A represents ABTS radical scavenging activity (2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity), B represents DPPH radical scavenging activity (1,1-diphenyl-2-trinitrophenylhydrazine radical scavenging activity), and C represents superoxide anion radical scavenging activity. Figure 3 A represents different concentrations Talaromyces Growth curve of *Glassonella parasuis* treated with sp. SQ3 extract, B is... Talaromyces The effect of sp.SQ3 extract on biofilm formation of *Glassonella parasuis*, C is... Talaromyces Effect of sp. SQ3 extract on alkaline phosphatase (ALP) content in *Glassie parasuis* cultures, D = Talaromyces The effect of sp. SQ3 extract on the total protein content of bacterial supernatant. E and F are scanning electron micrographs, showing the effect of the extract on the total protein content of bacterial supernatant. Talaromyces Morphological changes of *Glassella parasuis* before and after treatment with sp.SQ3 extract. Detailed Implementation
[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0016] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.
[0017] This invention provides an endophytic fungus that was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC M 2026406. The ITS sequence of the endophytic fungus is shown in SEQ ID NO: 1. The extract obtained by ethyl acetate extraction of the fermentation broth of this endophytic fungus can effectively inhibit *Glassie parasuis*.
[0018] The ITS sequence is: .
[0019] The present invention will be further described below with reference to specific embodiments.
[0020] Example 1: Isolation and Identification of Endophytic Fungi The screening and isolation of the strains were carried out according to the following steps: *Fritillaria hupehensis* (from Enshi Tujia and Miao Autonomous Prefecture, Hubei Province) was used as the starting material. Fritillaria hupehensis The bulbs, being plant material, were first thoroughly rinsed with tap water to remove surface impurities. Then, they underwent surface disinfection in a clean bench by sequentially immersing them in a 75% ethanol solution for 30 seconds and a 2% sodium hypochlorite solution for 2 minutes. Afterward, they were rinsed three times with sterile distilled water to remove residual disinfectant. The disinfected bulbs were then placed on sterile filter paper to absorb surface moisture and cut into small pieces approximately 5mm × 5mm. These treated bulb pieces were inoculated onto potato dextrose agar (PDA) plates and cultured at 28°C in the dark for 7 days. Once colonies had formed, newly formed mycelia at the colony edges were selected and purified three times using the streak plating method to obtain a pure culture strain.
[0021] After isolation and purification, this strain grew well on solid culture medium, with colonies exhibiting a typical cottony appearance, an orange-yellow to red surface, and indistinct alternating orange-yellow and red concentric rings (e.g., Figure 1 As shown in Figure A). Microscopic observation reveals that its hyphae are well-developed and have typical morphology, consistent with the characteristics of filamentous fungi (e.g., Figure 1 As shown in B).
[0022] Preliminary screening of endophytic fungi was conducted using a plate confrontation double culture method. Bipolaris maydis and Phytophthora infestans To identify the pathogen, antagonistic activity was evaluated using the inhibition rate [inhibition rate (%) = (colon radius of control group - colony radius of confrontation group) / colony radius of control group × 100]. Results showed that the strains... Talaromyces sp.SQ3 showed the highest inhibition rates against the two pathogenic fungi, at 53.12% and 46.87%, respectively, and was therefore selected as the target strain for subsequent research.
[0023] Molecular biological identification of the strain was performed using ITS sequence analysis. Colonies cultured for 7 days were used to extract total DNA using a fungal genomic DNA extraction kit. The extracted genomic DNA was then used as a template for PCR amplification of the ITS region. The amplification primers were ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The total volume of the PCR reaction system was 20 μL, including 10 μL of 2×Taq Master Mix, 1 μL of DNA template, 1 μL each of forward and reverse primers (10 μM), and 8 μL of ddH2O. The amplification program was set as follows: 94℃ pre-denaturation for 5 min; followed by 35 cycles, each cycle consisting of 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 45 s; finally, a final extension at 72℃ for 10 min. The PCR product was purified and sent to Shanghai Sangon Biotech Co., Ltd. for bidirectional Sanger sequencing. The obtained sequence is shown in SEQ ID NO: 1.
[0024] The obtained ITS sequence was analyzed for homology using NCBI BLAST, and the results showed that this strain was similar to... Talaromyces The sequence homology of strain CBS 286.36 (GenBank accession number: JX315671.1) was 99.24%. Further, using MEGA X software, a phylogenetic tree was constructed based on the neighbor-joining method (e.g., ...). Figure 1 As shown in C), based on the comprehensive molecular phylogenetic results, this strain was identified as... Talaromyces sp.SQ3 It was deposited on March 10, 2026, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The accession number is CCTCC M 2026406.
[0025] Example 2: Fermentation of Endophytic Fungi and Preparation of Extracts The preparation of the ethyl acetate extract and the evaluation of its antioxidant activity were carried out as follows.
[0026] Several endophytic fungal blocks, approximately 6 mm in diameter, were inoculated into 500 mL of potato dextrose liquid medium (PDB) and cultured at 28 °C and 180 rpm for 8 days with shaking. After fermentation, the fermentation broth was filtered through double-layered sterile gauze to separate the mycelium from the fermentation supernatant. The supernatant was extracted with an equal volume of ethyl acetate, repeated three times, with shaking for 30 min each time and allowing to stand for separation. The resulting organic phases were combined and concentrated under reduced pressure using a rotary evaporator to remove the solvent. The residue was dissolved in distilled water and then freeze-dried under vacuum for 72 h to obtain the ethyl acetate extract.
[0027] The antioxidant activity of the extract was determined at a series of concentrations ranging from 187.5 to 6000 μg / mL, with ascorbic acid (VC) used as a positive control. DPPH and ABTS were employed. + The in vitro antioxidant capacity was evaluated using a superoxide anion free radical scavenging assay. The free radical scavenging rate was calculated using the formula [(control group absorbance - sample group absorbance) / control group absorbance × 100%]. The results showed that the extract at a concentration of 187.5 μg / mL effectively combated ABTS. + The scavenging rates of DPPH free radicals reached 92.51% and 88.14%, respectively (e.g. Figure 2 As shown in A and B), this extract exhibits significant antioxidant activity. Its scavenging activity against superoxide anion free radicals is concentration-dependent, with the scavenging rate decreasing significantly with decreasing concentration (e.g., ...). Figure 2 (As shown in C). At all tested concentrations, the scavenging activity of TSEA was significantly lower than that of the positive control VC.
[0028] Example 3 Evaluation of the control effect against Gracie parasuis In the in vitro antibacterial experiment, the microdilution method was first used to determine the effect of the extract on... Glaesserella parasuis To determine the minimum inhibitory concentration (MIC), extracts were prepared in BHI medium at concentration gradients of 25, 50, 75, and 100 mg / mL, and compared with logarithmic growth phase bacterial suspensions (OD). 600 =0.6) co-incubated at 37℃ for 24h, and the lowest concentration at which no visible bacterial growth was observed was taken as the MIC. The results showed that the MIC of the extract was 25mg / mL.
[0029] Further analysis of its antibacterial kinetics was conducted using bacterial growth curves. G. parasuis Inoculated into BHI medium containing extracts at concentrations ranging from 1 / 8 MIC to 1 MIC, and cultured with shaking at 37°C and 180 rpm. OD was measured every 2 hours. 600 Values and plot growth curves (e.g.) Figure 3 (As shown in A). Furthermore, the inhibitory effect of the extract on bacterial biofilm formation was evaluated using crystal violet staining; OD was measured after co-incubating the bacterial culture and extract for 24 hours. 600 Values reflecting biofilm formation capacity (e.g.) Figure 3 As shown in B).
[0030] To investigate its effects on bacterial cell structure and detect changes in cell wall integrity, alkaline phosphatase (ALP) activity and protein leakage levels in bacterial culture supernatants were measured. ALP content was determined using appropriate kits, and protein concentration was determined using the BCA method (e.g., Figure 3(As shown in C and D); simultaneously, bacterial samples were fixed with 2.5% glutaraldehyde, lyophilized, and sputter-coated with gold. Cell morphology changes were then observed using a scanning electron microscope, compared with a morphologically intact control group (e.g., ...). Figure 3 Compared to E), after treatment with extracts at 1 / 4 MIC concentration, G. parasuis Significant morphological changes occur, manifested as roughened and broken bacterial edges and irregular arrangement, with marked damage to the cell structure (e.g. Figure 3 As shown in F), this indicates that the extract may inhibit bacterial growth by disrupting bacterial cell structure.
[0031] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An endophytic fungus, Talaromyces sp. SQ3, characterized by: It was deposited at the China Center for Type Culture Collection on March 10, 2026, with accession number CCTCC M 2026406.
2. An endophytic fungus, Talaromyces sp. SQ3, characterized by: The ITS sequence of the endophytic fungus is shown in SEQ ID NO:
1.
3. An antibacterial preparation made using the endophytic fungus described in claim 1 or 2.
4. The antibacterial agent according to claim 3, characterized in that: The active ingredient of the antibacterial agent is an ethyl acetate extract of endophytic fungi.
5. An antimicrobial preparation comprising the endophytic fungus of claim 1 or 2.
6. The antibacterial agent according to claim 4, characterized in that: The active ingredient of the antibacterial agent includes an ethyl acetate extract of endophytic fungi.
7. The use of an endophytic fungus as described in claim 1 or 2, or an antimicrobial agent as described in any one of claims 3-6, in inhibiting *Glassonella parasuis*.