Sarocladium kiliense 19-11C and application thereof

Abstract

The application provides a strain of Gilmaniella vishniacov Sarocladium kiliense ) 19-11C and an application thereof, preservation number of the Gilmaniella vishniacov Sarocladium kiliense ) 19-11C is CGMCC No.41846. As a fungus, the Gilmaniella vishniacov 19-11C is naturally insensitive to tetracycline antibiotics, can survive and play a degradation function in a high-concentration drug environment. The strain does not carry horizontally transferable bacterial drug resistance genes, fundamentally avoiding the potential environmental risk of causing drug resistance gene spread in the repair process. The strain shows wide adaptability and stability to changes in temperature, pH and nutritional conditions in the environment, effectively decomposes doxycycline through various biochemical pathways such as removal and oxidation, significantly reduces the biological toxicity, and provides an environmentally friendly and ecologically safe biological solution for the residue treatment of tetracycline antibiotics in the environment.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a strain of *Cladosporium cleistoglossum* (…). Sarocladium kiliense 19-11C and its applications. Background Technology

[0002] Broad-spectrum antibacterial tetracycline antibiotics, such as doxycycline (DOX), are commonly used clinically to treat respiratory diseases. Antibiotics that cannot be completely absorbed by organisms are released into the environment. If not removed promptly, this can cause antibiotic contamination, leading to the emergence and spread of drug-resistant strains and resistance genes.

[0003] Currently, technologies for removing antibiotics from the environment mainly include physical, chemical, and biological methods. Physical methods primarily rely on adsorption and separation, while chemical methods degrade antibiotics through processes such as photolysis, hydrolysis, or advanced oxidation. For high concentrations of antibiotic pollution, advanced oxidation and other methods can achieve rapid removal. However, due to the complex composition of wastewater such as pharmaceutical wastewater, non-specific elimination methods such as advanced oxidation may produce undesirable byproducts. Compared to other methods, targeted degradation using specific microorganisms has attracted widespread attention due to its low cost and environmental friendliness, and is considered an effective method for degrading antibiotics in complex environments.

[0004] Numerous studies have reported on the microbial degradation of antibiotic residues, such as the degradation of tetracycline antibiotics using *Arthrobacter nigrum*, *Bacillus cereus*, and *Serratia marcescens*, and the degradation of β-lactam antibiotics using *Klebsiella pneumoniae*. However, these degrading bacteria often carry resistance genes, making them a potential source of resistance genes in the environment. Large-scale application may bring new ecological risks. Therefore, there is an urgent need to develop new strains that can efficiently degrade antibiotics without the risk of resistance transfer. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a strain of *Clerodendrum cyrtonema* (…). Sarocladium kiliense 19-11C and its applications enable the efficient and safe degradation of tetracycline antibiotics in the environment.

[0006] In a first aspect, the present invention provides a strain of *Clerodendrum cyrtonema* (… Sarocladium kiliense )19-11C, the *Killionella spp.* ( Sarocladium kilienseThe accession number for specimen 19-11C, CGMCC No. 41846, was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China (Postcode: 100101). Its taxonomic name is *Cladosporium cleistoglossum*. Sarocladium kiliense The collection name is 19-11C.

[0007] Secondly, the present invention provides a microbial inoculant containing the aforementioned *Cladosporium cleistoglossum* (…). Sarocladium kiliense 19-11C, or freeze-dried bacterial cells, or bacterial suspension, or supernatant or precipitate resuspension after centrifugation of the bacterial suspension.

[0008] According to the present invention, a microbial agent is preferably prepared by *Cladosporium cleistatum* (Kilionella spp.) in the microbial agent. Sarocladium kiliense The viable bacteria content at 19-11C is ≥1×10⁻⁶. 5 cfu / g.

[0009] Thirdly, the present invention provides a method for preparing the microbial agent, comprising the following steps: culturing the *Cladosporium cleistoglossum* (… Sarocladium kiliense 19-11C.

[0010] According to the method for preparing the microbial agent provided by the invention, preferably, the *Cladosporium cleistoglossum* (*Cladosporium cleistoglossum*) is cultured. Sarocladium kiliense The temperature of 19-11°C is 25°C to 30°C.

[0011] More preferably, the *Clerodendrum trichotomum* ( Sarocladium kiliense The incubation temperature for 19-11C is 28℃.

[0012] Fourthly, the present invention provides a composition comprising the aforementioned microbial agent.

[0013] According to a composition provided by the present invention, preferably, the microbial agent contains *Cladosporium clematis* (…). Sarocladium kiliense The viable bacteria content at 19-11C is ≥1×10⁻⁶. 5 cfu / g.

[0014] The composition provided by the present invention preferably further comprises other active ingredients having the function of degrading tetracycline antibiotics.

[0015] Fifthly, the present invention provides the aforementioned *Clerodendrum trichotomum* (… Sarocladium kiliense 19-11C, the use of the microbial agent or the composition in products that degrade tetracycline antibiotics and / or reduce the toxicity of tetracycline antibiotics.

[0016] According to the present invention, the *Killingia spp.* ( Sarocladium kiliense 19-11C. The use of the microbial agent or the composition in products that degrade tetracycline antibiotics and / or reduce the toxicity of tetracycline antibiotics, preferably, the tetracycline antibiotic is doxycycline, doxycycline derivatives or doxycycline metabolites.

[0017] Sixthly, the present invention provides a method for degrading tetracycline antibiotics, comprising the following steps: culturing the *Cladosporium cleistoglossum* (*Cladosporium cleistoglossum*) in an environment containing the tetracycline antibiotics. Sarocladium kiliense 19-11C.

[0018] According to a method for degrading tetracycline antibiotics provided by the present invention, preferably, the culture of *Cladosporium cleistoglossum* (…) Sarocladium kiliense The conditions for 19-11C include any one or more of the following: (1) temperature of 15℃~40℃; (2) pH of 5~10; (3) organic nitrogen content in the culture medium is 0g / L~15g / L.

[0019] More preferably, the culture of the *Clerodendrum trichotomum* ( Sarocladium kiliense The conditions for 19-11C include any one or more of the following: (1) temperature of 25℃~40℃; (2) pH of 8~10; (3) organic nitrogen content of 2g / L~15g / L.

[0020] More preferably, the culture of the *Clerodendrum trichotomum* ( Sarocladium kiliense The conditions for 19-11C include any one or more of the following: (1) temperature is 30℃; (2) pH is 10; (3) the organic nitrogen content in the culture medium is 15g / L.

[0021] More preferably, the culture of the *Clerodendrum trichotomum* ( Sarocladium kiliense The conditions for 19-11C also include: the incubation time is at least 1 day.

[0022] More preferably, the culture time is at least 6 days.

[0023] More preferably, the organic nitrogen comprises tryptone.

[0024] More preferably, the culture of the *Clerodendrum trichotomum* ( Sarocladium kiliense The conditions for 19-11C also include: containing sugar sources and / or inorganic salts.

[0025] More preferably, the sugar source includes glucose.

[0026] More preferably, the glucose content is 2g / L to 3g / L.

[0027] More preferably, the glucose content is 2.5 g / L.

[0028] More preferably, the inorganic salt includes sodium salt, potassium salt and / or magnesium salt.

[0029] More preferably, the sodium salt comprises sodium chloride.

[0030] More preferably, the sodium chloride content is 4 g / L to 6 g / L. Even more preferably, the sodium chloride content is 5 g / L.

[0031] More preferably, the potassium salt comprises dipotassium hydrogen phosphate and / or potassium dihydrogen phosphate.

[0032] More preferably, the content of dipotassium hydrogen phosphate is 1 g / L to 2 g / L. Even more preferably, the content of dipotassium hydrogen phosphate is 1.5 g / L.

[0033] More preferably, the potassium dihydrogen phosphate content is 0.1 g / L to 1 g / L. Even more preferably, the potassium dihydrogen phosphate content is 0.5 g / L.

[0034] More preferably, the magnesium salt comprises magnesium sulfate heptahydrate.

[0035] More preferably, the content of magnesium sulfate heptahydrate is 0.1 g / L to 0.5 g / L. Even more preferably, the content of magnesium sulfate heptahydrate is 0.2 g / L.

[0036] According to the present invention, a method for degrading tetracycline antibiotics preferably includes the following steps: culturing the *Cladosporium cleistoglossum* (… Sarocladium kiliense 19-11°C, to obtain seed culture; inoculate the seed culture into an environment containing the tetracycline antibiotic; the inoculation amount of the seed culture is at least 0.1% v / v.

[0037] More preferably, the inoculation amount of the seed liquid is 0.1% v / v to 0.3% v / v.

[0038] More preferably, the inoculation amount of the seed liquid is 0.2% v / v.

[0039] According to the method for degrading tetracycline antibiotics provided by the present invention, preferably, the concentration of the tetracycline antibiotics in the environment is 0.1 mg / mL to 10 mg / mL.

[0040] More preferably, the tetracycline antibiotic is present in the environment at a concentration of 4 mg / mL to 6 mg / mL.

[0041] Furthermore, the concentration of the tetracycline antibiotic in the environment is 5 mg / mL.

[0042] According to the present invention, a method for degrading tetracycline antibiotics is provided, wherein the tetracycline antibiotic is doxycycline, doxycycline derivatives, or doxycycline metabolites.

[0043] The present invention has the following beneficial effects:

[0044] This invention isolates *Cladosporium cleistoglossum* (a type of mold) from compost soil in a livestock farm. Sarocladium kiliense This fungus, aged 19-11°C, possesses both high efficiency and safety. As a fungus, it is naturally insensitive to tetracycline antibiotics and can survive and degrade drugs even in high-concentration environments. Compared to common bacterial degrading bacteria, this strain does not carry horizontally transferable bacterial resistance genes, fundamentally avoiding the potential environmental risk of resistance gene diffusion during the remediation process. Furthermore, this strain exhibits broad adaptability and stability to changes in environmental temperature, pH, and nutrient conditions, making it more advantageous in practical applications. *Clerodendrum cyrtonema* ( Sarocladium kiliense 19-11C effectively decomposes doxycycline through multiple biochemical pathways such as removal and oxidation, significantly reducing its biotoxicity and providing an environmentally friendly and ecologically safe biological solution for the remediation of tetracycline antibiotic residues in the environment. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0046] Figure 1 This is a colony image of strain 19-11C provided by the present invention.

[0047] Figure 2 This is an optical microscope image of strain 19-11C provided by the present invention.

[0048] Figure 3 This is the molecular identification result of strain 19-11C provided by the present invention.

[0049] Figure 4 The graph shows the degradation effect of strain 19-11C provided by this invention on doxycycline under different temperature conditions.

[0050] Figure 5 The graph shows the degradation effect of strain 19-11C provided by this invention on doxycycline under different pH conditions.

[0051] Figure 6This is a graph showing the degradation effect of strain 19-11C provided by this invention on doxycycline under different organic nitrogen contents.

[0052] Figure 7 This is an analysis diagram of the degradation products of doxycycline by strain 19-11C provided by the present invention. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0055] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0056] Example 1: Isolation, screening, identification and preservation of strain 19-11C

[0057] 1. Isolation and screening of strain 19-11C

[0058] This invention uses TSA solid culture medium to screen doxycycline-degrading strains from compost soil (i.e., compost yard soil) in a livestock farm in Pinggu District, Beijing.

[0059] Weigh 1.5g of tryptone, 0.5g of soybean peptone, 1.5g of sodium chloride, and 0.5g of agar powder, add them to 1000ml of distilled water, sterilize at 121℃ for 15 minutes, pour into plates and cool to obtain a 1 / 10 concentration TSA solid medium. Subsequently, add sterile doxycycline solution to the medium to a final concentration of 50mg / L to obtain a 1 / 10 concentration TSA solid plate.

[0060] Take 1g of soil from the stockpile and add 9mL of sterile water. Shake at 200rpm for 1 hour at 30℃ to allow microorganisms to dissolve in the water. After standing for a period of time, wait for the solution to separate into layers. Take 1mL of the upper suspension and add it to 9mL of PBS buffer to obtain a 10% concentration. -1 Bacterial suspension.

[0061] The bacterial suspension was sequentially diluted to a concentration of 10. -4 10 -5 and 10 -6Then, 100 μL of bacterial suspensions of various concentrations were spread on 1 / 10 concentration TSA solid plates and incubated at 28°C for 3–5 days. The bacterial strains growing on the plates were picked and the above method was repeated for isolation and purification. The purified strains obtained after three consecutive purifications were retained and recorded as strain 19-11C.

[0062] 2. Biological identification of strain 19-11C

[0063] (1) Colony morphology identification

[0064] Strain strain 19-11C was cultured on potato dextrose agar medium (12.0 g potato extract, 20.0 g glucose, 15.0 g agar, 1 L water). Figure 1 As shown, the colony surface of this strain is wrinkled, fluffy or cottony, and relatively loose in texture. The colony edge has filamentous hyphae and is relatively neat. No pigment diffusion was observed, and the colony is milky white.

[0065] (2) Microscopic morphological identification

[0066] The microstructure of strain 19-11C was observed under an optical microscope, such as... Figure 2 As shown, its fungal body is composed of hyphae and spores. Numerous branched hyphae are visible. The hyphae are relatively slender and flexible, and are interwoven. There are elliptical spore structures. The spores are relatively uniform in size and accompany the hyphae. They are distributed around or on the hyphae, exhibiting the structural characteristic of hyphae and spores coexisting.

[0067] (3) Molecular biological identification

[0068] Hyphae of colonies of strain 19-11C were picked and placed in sterile water, frozen at -80°C for 5 minutes, boiled at 100°C for 2 minutes, centrifuged at 10,000 rpm for 5 minutes, and the supernatant was used as a template. Colony PCR amplification was performed using the primer combination for the fungal ITS region: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO:1)) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO:2)).

[0069] The PCR amplification system is as follows: 12.5 μL of 2×PCR Mix (containing Taq enzyme, dNTPs, and buffer); 1 μL of ITS1 primer (SEQ ID NO:1) (10 μmol / L); 1 μL of ITS4 primer (SEQ ID NO:2) (10 μmol / L); 1 μL of template DNA (20–50 ng / μL); and 9.5 μL of sterile deionized water. After gentle mixing, the mixture is briefly centrifuged.

[0070] The PCR program is as follows: Pre-denaturation: 94℃ for 5 min; Cyclic amplification (35 cycles): 94℃ for 30 s (denaturation), 55~58℃ for 30 s (annealing, adjust according to primer Tm value), 72℃ for 1 min; Final extension: 72℃ for 10 min, ensuring complete extension of the product; Incubation: 4℃.

[0071] Electrophoresis detection: Take 5 μL of PCR product, mix with 1 μL of Loading Buffer, spot on a 1.5% agarose gel, electrophoresis at 120V for 20-30 min, observe with a UV gel imager, obvious product in the target product region, and obtain the sequence of the ITS gene of the bacterium after full-length sequencing.

[0072] Through comparative analysis, such as Figure 3 As shown, strains 19-11C and Sarocladium kiliense MUCL 9724 (NR130684.1) showed the highest similarity (99.63%), therefore strain 19-11C belongs to *Cladosporium cleistoglossum*. Sarocladium kiliense .

[0073] 3. Preservation of strain 19-11C

[0074] Strain 19-11C was deposited on March 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41846, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China (Postcode: 100101). Its taxonomic name is *Cladosporium cleistoglossum*. Sarocladium kiliense The collection name is 19-11C.

[0075] Example 2: Degradation effect of strain 19-11C on doxycycline under different temperature conditions

[0076] 1. Activation of strain 19-11C and culture of seed culture

[0077] Weigh 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride and 15g of agar powder, add them to 1000ml of distilled water, sterilize at 121℃ for 15 minutes, pour into plates and cool to obtain TSA solid culture medium.

[0078] Weigh 17g of tryptone, 3g of soybean peptone, 5g of sodium chloride, 2.5g of dipotassium hydrogen phosphate and 2.5g of glucose, add them to 1000ml of distilled water, and sterilize at 121℃ for 15 minutes to obtain TSB liquid culture medium.

[0079] Strain strain 19-11C was activated using TSA solid medium and cultured at 28°C for 48 hours. It was then inoculated into TSB liquid medium and cultured at 28°C for 24 hours to obtain the seed culture.

[0080] 2. Degradation of doxycycline by strain 19-11C at different temperatures

[0081] Weigh out 3.4g of tryptone, 0.6g of soybean peptone, 1g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, and 0.5g of glucose. Add them to 1000ml of distilled water, adjust the pH to 7, and sterilize at 121℃ for 15 minutes to obtain a 20% TSB liquid medium. Dispense the 20% TSB liquid medium into culture flasks at 40% of their volume, i.e., pour 100mL of the 20% TSB liquid medium into a 250mL Erlenmeyer flask.

[0082] Add 50 mg of doxycycline to 10 mL of methanol and dissolve it completely to obtain a doxycycline solution with a concentration of 5 mg / mL.

[0083] After 100 mL of 20% TSB liquid medium has cooled to room temperature, add doxycycline solution with a concentration of 5 mg / mL at a volume ratio of 10 mL: 100 μL. The final concentration of doxycycline in the medium is 50 mg / L, and this medium is called doxycycline medium.

[0084] In a clean bench, 200 μL of seed culture was inoculated into doxycycline medium using a sterile pipette tip and cultured at different temperatures. Six temperature gradients were set up: 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃. A total of 18 flasks were inoculated (three replicates for each temperature gradient, for a total of six temperature gradients), with doxycycline medium without seed culture serving as a blank control. The Erlenmeyer flasks were placed in a shaker and incubated at 100 rpm in the dark.

[0085] 3. Extraction and detection of doxycycline in culture medium

[0086] (1) Sampling: On the 6th day of culture, samples were taken from each culture bottle in a clean bench. 2 mL of bacterial solution was taken into a sterile tube using a sterile pipette tip, filtered with a disposable syringe and filter (aqueous phase), and the filtrate was collected and placed in a -20℃ refrigerator.

[0087] (2) Extraction: Take 1 mL of filtrate and add 4 mL of Na4EDTA solution (195 mL of ultrapure water + 0.4 g Na4EDTA) for extraction. Collect the extract for subsequent sample loading and detection.

[0088] (3) Activation: Activate the HLB column and SAX column with 5 mL of methanol and 5 mL of 0.1% H3PO4 in sequence.

[0089] (4) Adsorption: Connect the SAX (upper) and HLB (lower) columns in series using a solid phase extraction adapter, load the sample, turn on the vacuum pump, control the vacuum pump pressure to be below -50 kPa, and use the solid phase extraction port knob to adjust the solution flow rate to 5 mL / min.

[0090] (5) Elution: After adsorption is complete, wash the column with 10 mL of ultrapure water, dry it under vacuum for 10 min, remove the SAX column, add 10 mL of methanol, and collect the eluent in a 15 mL centrifuge tube.

[0091] (6) Detection: The content of doxycycline in the eluent was detected by liquid chromatography-mass spectrometry, and the content of doxycycline in the bacterial culture was calculated. The degradation rate of doxycycline was also statistically analyzed.

[0092] like Figure 4 As shown, the degradation rate of doxycycline was higher than 40% under the culture temperature range of 15℃ to 40℃, with the highest degradation rate (70.57%) at a culture temperature of 30℃. In the range of 15℃ to 30℃, the degradation rate of doxycycline gradually increased with the increase of temperature, and reached a peak at 30℃.

[0093] Example 3: Degradation effect of strain 19-11C on doxycycline under different pH conditions

[0094] 1. Activation of strain 19-11C and culture of seed culture

[0095] Same as Example 2.

[0096] 2. Degradation of doxycycline by strains at 19-11C under different pH conditions

[0097] Following the method in Example 2, a 20% concentration of TSB liquid culture medium was prepared. The pH was adjusted using sterile NaOH and HCl (pH was adjusted to 5 or 6 using 1 mol / L HCl solution, and pH was adjusted to 8, 9 or 10 using 1 mol / L NaOH solution) to obtain 20% concentration TSB liquid culture media with pH values ​​of 5, 6, 7, 8, 9 and 10.

[0098] Then, a doxycycline solution with a concentration of 5 mg / mL was added to obtain doxycycline culture media with pH values ​​of 5, 6, 7, 8, 9 and 10 (the final concentration of doxycycline was 50 mg / L).

[0099] In a clean bench, 200 μL of seed culture was aspirated using a sterile pipette tip and inoculated into doxycycline medium at various pH values, for a total of 18 flasks (3 replicates for each pH gradient, for a total of 6 pH gradients). Doxycycline medium without seed culture (pH 7) served as a blank control. The flasks were placed in a shaker at 30°C and 100 rpm in the dark.

[0100] 3. Extraction and detection of doxycycline in culture medium

[0101] On day 6 of cultivation, samples were taken according to the method in Example 2, the content of doxycycline was determined, and the degradation rate of doxycycline was statistically analyzed.

[0102] like Figure 5 As shown, the degradation rate of doxycycline was higher than 46% under pH conditions of 5–10, with the highest degradation rate (87.71%) at pH 10. In the pH gradient range of 6–10, the degradation rate of doxycycline increased with increasing pH value.

[0103] Example 4: Degradation effect of strain 19-11C on doxycycline under different organic nitrogen contents

[0104] 1. Activation of strain 19-11C and culture of seed culture

[0105] Same as Example 2.

[0106] 2. Degradation of doxycycline by strain 19-11C under different organic nitrogen contents

[0107] Weigh 1.5g K2HPO4, 1.0g NaCl, 0.5g KH2PO4 and 0.2g MgSO4·7H2O, add them to 1000ml distilled water, adjust the pH to 7.0, sterilize at 121℃ for 15 minutes to obtain an inorganic salt culture medium with an organic nitrogen content of 0g / L.

[0108] Weigh 1.5g K2HPO4, 1.0g NaCl, 0.5g KH2PO4, 0.2g MgSO4·7H2O and 2g / L tryptone, add them to 1000ml distilled water, adjust the pH to 7.0, sterilize at 121℃ for 15 minutes to obtain an inorganic salt culture medium with an organic nitrogen content of 2g / L.

[0109] Weigh 1.5g K2HPO4, 1.0g NaCl, 0.5g KH2PO4, 0.2g MgSO4·7H2O and 5g / L tryptone, add them to 1000ml distilled water, adjust the pH to 7.0, sterilize at 121℃ for 15 minutes to obtain an inorganic salt culture medium with an organic nitrogen content of 5g / L.

[0110] Weigh 1.5g K2HPO4, 1.0g NaCl, 0.5g KH2PO4, 0.2g MgSO4·7H2O and 10g / L tryptone, add them to 1000ml distilled water, adjust the pH to 7.0, sterilize at 121℃ for 15 minutes to obtain an inorganic salt culture medium with an organic nitrogen content of 10g / L.

[0111] Weigh 1.5g K2HPO4, 1.0g NaCl, 0.5g KH2PO4, 0.2g MgSO4·7H2O and 15g / L tryptone, add them to 1000ml distilled water, adjust the pH to 7.0, sterilize at 121℃ for 15 minutes to obtain an inorganic salt culture medium with an organic nitrogen content of 15g / L.

[0112] The inorganic salt culture media with different organic nitrogen contents were dispensed into 250 mL Erlenmeyer flasks at 100 mL each, and then doxycycline solution with a concentration of 5 mg / mL was added to obtain doxycycline culture media with different organic nitrogen contents (the final concentration of doxycycline was 50 mg / L).

[0113] In a clean bench, 200 μL of seed culture was aspirated with a sterile pipette tip and inoculated into 20% TSB medium. A total of 15 bottles were inoculated and cultured at 28°C for 48 h. After centrifugation to retain the bacterial cells, the cells were washed once with 35 mL of sterile physiological saline and then resuspended in 200 μL of doxycycline medium with different organic nitrogen contents. All cells were then transferred to doxycycline medium with corresponding organic nitrogen contents and incubated at 30°C and 100 rpm in the dark. Three replicates were performed for each organic nitrogen content, for a total of five peptone gradients. Doxycycline medium without seed culture was used as a blank control.

[0114] 3. Extraction and detection of doxycycline in culture medium

[0115] On day 6 of cultivation, samples were taken according to the method in Example 2, the content of doxycycline was determined, and the degradation rate of doxycycline was statistically analyzed.

[0116] like Figure 6 As shown, the degradation rate of doxycycline was higher than 48% under organic nitrogen content ranging from 0 g / L to 15 g / L. The highest degradation rate (70.30%) was observed when the organic nitrogen content was 15 g / L. Within the range of 2 g / L to 10 g / L, the degradation rate of doxycycline decreased with increasing organic nitrogen content, but remained higher than 55%.

[0117] 4. Analysis of doxycycline degradation products by strain 19-11C

[0118] Mass spectrometry was used to detect and analyze intermediate metabolites produced during the degradation of doxycycline by strain 19-11C, such as... Figure 7 As shown, the results indicate that doxycycline is effective against Cladosporium clethroides (Cladosporium clethroides). Sarocladium kiliense Degradation under the influence of 19-11C mainly occurs through carbonyl and amino group removal reactions, but it can also be degraded through oxidation, desaturation, and other pathways, involving a total of 5 metabolic pathways, as follows:

[0119] Pathway 1: Deamination and degradation

[0120] Doxycycline undergoes a direct deamination reaction, losing its functional group and generating an amino-containing degradation product (molecular formula C). 20 H 19 O8 + );

[0121] Pathway 2: Demethylation

[0122] Doxycycline undergoes a direct demethylation reaction, losing a group and removing the methyl structure from the molecule to generate a degradation product (molecular formula C). 21 H 23 N2O8 + );

[0123] Pathway 3: Decarboxylation degradation

[0124] Doxycycline undergoes a direct decarboxylation reaction, losing a group and removing the carboxyl group from the molecule to generate an amino-containing degradation product (molecular formula C). 21 H 24 NO7 + );

[0125] Pathway 4: Oxidative Degradation

[0126] Doxycycline undergoes direct oxidative modification, introducing new oxygen-containing functional groups into its molecular structure. This is accompanied by loss and desaturation processes, generating degradation products containing double bonds (molecular formula C). 22 H 25 N2O9 + );

[0127] Path 5: Desaturation degradation

[0128] Doxycycline first forms a double bond through a desaturation reaction (while simultaneously losing the bond), and then further loses functional groups, ultimately generating a nitrogen-free degradation product (molecular formula C). 20 H 17 O8 + ).

[0129] The above-mentioned biochemical reactions significantly reduced the toxicity of doxycycline.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Clerodendrum cyrtonema* ( Sarocladium kiliense 19-11C, characterized in that, The Kirlian broom fungus ( Sarocladium kiliense The accession number for 19-11C is CGMCC No. 41846.

2. A microbial inoculant, characterized in that, Contains the *Killingia spp.* as described in claim 1 (… Sarocladium kiliense 19-11C.

3. The microbial agent according to claim 2, characterized in that, The microbial agent contains *Clerodendrum kilianum* (…). Sarocladium kiliense 19-11°C freeze-dried mycelium, or the aforementioned *Cladosporium cleistoglossum* ( Sarocladium kiliense ) 19-11C bacterial solution.

4. The method for preparing the microbial inoculant according to claim 2, characterized in that, Includes the following steps: culturing the *Clerodendrum trichotomum* as described in claim 1 (… Sarocladium kiliense 19-11C.

5. A composition, characterized in that, It includes the microbial agent as described in claim 2 or 3.

6. The *Cladosporium clethroides* as described in claim 1 (… Sarocladium kiliense 19-11C. The use of the microbial agent of claim 2 or 3 or the composition of claim 5 in the preparation of products that degrade doxycycline and / or reduce the toxicity of doxycycline.

7. A method for degrading doxycycline, characterized in that, Includes the following steps: Culture the *Clerodendrum trichotomum* of claim 1 in an environment containing the doxycycline. Sarocladium kiliense 19-11C.

8. The method according to claim 7, characterized in that, The culture of *Clerodendrum cyrtomata* as described in claim 1 (… Sarocladium kiliense The conditions for 19-11C include any one or more of the following: (1) The temperature is 15℃~40℃; (2) pH is 5-10; (3) The content of organic nitrogen is 0 g / L to 15 g / L.

9. The method according to claim 7 or 8, characterized in that, Includes the following steps: Culture of the aforementioned Cleopatra clematis ( Sarocladium kiliense 19-11°C, to obtain seed solution; The seed culture was inoculated in an environment containing the doxycycline; The inoculation amount of the seed solution is at least 0.1% v / v.

10. The method according to claim 7 or 8, characterized in that, The concentration of doxycycline in the environment is 0.1 mg / mL to 10 mg / mL.

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