Enterobacter cloacae strain ecsf2508, bacterial inoculum, and preparation method and application thereof

Abstract

The application provides an enterobacter cloacae ECSF2508 strain, a bacterial agent, and a preparation method and application thereof, and belongs to the technical field of biotechnology.The classification and naming of the enterobacter cloacae ECSF2508 strain is enterobacter cloacae Enterobacter cloacae , the depositing unit is the China General Microbiological Culture Collection Center, the address is No. 3, Xibei Road, Chaoyang District, Beijing, the depositing date is December 15, 2025, and the depositing number is CGMCC No. NO37087.The strain of the application is an enterobacter cloacae, has the action of degrading butyl, is safe to the environment, and can be used for biodegradation of butyl and environmental safety.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a strain of Enterobacter cloacae ECSF2508, an inoculum agent, its preparation method, and its application. Background Technology

[0002] Butylene is a methoxylated hydroxamic acid, a compound widely found in gramineous crops such as corn and wheat, possessing antibacterial and insecticidal properties (Li Xiaohui et al., 2007; Argandona & Corvuera, 1985). Studies have found that butylene in four types of malt commonly consumed in daily diets has potential adverse effects on human health. It can affect the soil microbial community structure by altering the amount of rhizosphere fungi in wheat soil, and its residues in the soil can inhibit the germination of seeds in crops such as rice. Butylene and its metabolites in wheat have a significant inhibitory effect on the root and stem growth of herbaceous plants such as crabgrass, amaranth, wild oats, Kentucky bluegrass, and barnyard grass. When butylene remains in water bodies, it can inhibit marine bacteria, causing environmental pollution, which in turn contaminates agricultural products, posing a safety hazard. Furthermore, butylene exists in plants as glucosides, and when plants are damaged, it can be converted into more toxic glucosinolates under the action of glycosidases, thus becoming a stronger source of environmental pollution. Therefore, butadiene poses ecological risks. Consequently, the degradation pathways of butadiene have always been an important topic in crop production and environmental protection research.

[0003] Currently, the degradation techniques for DIMBOA (dimbolacrimal oleifera) are mainly divided into physical degradation, chemical degradation, and biodegradation. In chemical degradation, DIMBOA can undergo coordination reactions with some metal ions. For chemical control, DIMBOA can combine with iron atoms to form chelates, reducing DIMBOA content (Huang et al., 2022). Secondly, in non-biodegradation, hydrolysis is one of the main transformation pathways; however, its decomposition rate is highly dependent on pH and temperature (Woodward et al., 2022). Light can convert DIMBOA into other derivatives, thereby inducing and promoting degradation, but the instability of light under natural conditions leads to instability and incomplete degradation of DIMBOA. This indicates that existing DIMBOA degradation technologies have disadvantages such as high cost, secondary pollution, and demanding conditions; physical degradation has disadvantages such as low efficiency and difficulty in scaling up; and currently known microbial degradation strains are mostly derived from soil, with low degradation efficiency, poor specificity, susceptibility to contamination by other microorganisms, and no degradation technology specifically targeting the interaction between the fall armyworm gut and DIMBOA.

[0004] Chinese invention patent document CN108359622A discloses a maize continuous cropping obstacle repair agent based on allelopathic autotoxicity and its application. The repair agent is composed of *Pseudomonas oryzae* and *Burkholderia granatum* in a ratio of 1:3 to 3:1, with the concentration of *Pseudomonas oryzae* in the repair agent solution being 2.0-3.0 × 10⁻⁶. 8 The concentration of Burkholderia cfu / ml in the remedy culture was 2.0-3.0 × 10⁻⁶. 8 CFU / ml. Application method of the remediation agent: Use seed coating method. Before sowing, mix 50 g of corn seeds with 5 mL of fermentation liquid, stir thoroughly, and let stand for 3-5 hours before sowing. Chinese invention patent document CN117025462B discloses a *Enterococcus faecalis* strain that degrades insecticides and its uses. The preservation name of the *Enterococcus faecalis* strain is: EMBL-3, the depositary institution is: China Center for Type Culture Collection, the deposit date is: July 17, 2023, and the deposit number is: CCTCC M20231305. This strain was identified by gene sequence analysis as *Enterococcus faecalis*, named EMBL-3, and belongs to the Gram-positive bacteria. This strain can be used to degrade chlorantraniliprole and has prospects for further research and engineering applications in agricultural pest control, development of new green insecticides, and remediation of environmental insecticide pollutants. However, neither the above-mentioned remediation agent nor the strain can specifically degrade chlorantraniliprole, and the effect is poor. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Enterobacter cloacae ECSF2508, an inoculum agent, its preparation method and application, which has the function of degrading butyl cloth. This degradation is environmentally safe and can be used for the biodegradation of butyl cloth while ensuring environmental safety.

[0006] The technical solution of this invention is implemented as follows: This invention provides a strain of Enterobacter cloacae ECSF2508, which is classified as Enterobacter cloacae. The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is December 15, 2025; and the accession number is CGMCC No. NO37087.

[0007] As a further improvement of the present invention, the 16S rRNA gene sequence of the *Enterobacter cloacae* ECSF2508 strain is shown in SEQ ID No. 1. Sequencing analysis confirmed that this strain is classified as *Enterobacter cloacae*.

[0008] As a further improvement of the present invention, the Enterobacter cloacae ECSF2508 strain was purified and cultured by streak plating on LB agar. The colonies were milky white, round and smooth with slightly raised edges, Gram-negative, and the cell morphology was a short and stout rod without spores.

[0009] This invention further protects the use of the above-mentioned Enterobacter cloacae ECSF2508 strain in the preparation of a bacterial agent for degrading butylated filaments.

[0010] This invention further protects a butylated filtrate degrading agent, the active ingredient of which includes at least one of (a), (b), and (c): (a) Fermentation broth of the above-mentioned Enterobacter cloacae ECSF2508 strain; (b) The supernatant of sonicated lysis of the above-mentioned Enterobacter cloacae ECSF2508 strain cells; (c) Ultrasonic lysis precipitate of the above-mentioned Enterobacter cloacae ECSF2508 strain cells.

[0011] This invention further protects a method for preparing the above-mentioned butyl degrading bacterial agent, comprising the following steps: (1) Enterobacter cloacae strain ECSF2508 was isolated from the midgut of fall armyworm larvae and cultured on LB medium; (2) Then, the purified Enterobacter cloacae ECSF2508 was obtained by streaking purification on LB medium. (3) Mix at least one of the fermentation broth obtained by fermenting Enterobacter cloacae ECSF2508 strain, the ultrasonic lysis supernatant of Enterobacter cloacae ECSF2508 strain cells, and the ultrasonic lysis precipitate of Enterobacter cloacae ECSF2508 strain cells to obtain the butyl degrading agent.

[0012] This invention further protects the use of the above-mentioned butyl degrading bacterial agent in the preparation of butyl degrading agents.

[0013] The present invention has the following beneficial effects: The Enterobacter spp. ECSF2508 provided by the present invention is a strain that can efficiently degrade the maize defensive secondary metabolite butyl. It has the characteristics of strong degradation ability, convenient culture, environmental safety and non-toxicity, and can be applied to the study of butyl biodegradation and the intestinal detoxification mechanism of pests.

[0014] Compared with the prior art, the present invention has the following advantages: (1) The Enterobacter cloacae ECSF2508 isolated by this invention has a strong specific degradation ability against maize's typical defense substance, butylene, and the degradation effect is significant, which makes up for the problem of the scarcity of existing butylene-degrading strains. (2) The Enterobacter cloacae ECSF2508 strain has simple culture conditions, fast growth and reproduction rate, strong tolerance, and is easy to culture and propagate on a large scale in the laboratory, and has high potential for industrial application. (3) The strain of this invention is derived from the midgut of the fall armyworm larvae. It is a natural intestinal symbiotic bacterium with good biocompatibility and no secondary pollution to the environment. This strain can help the fall armyworm detoxify the host plant defense substance dimbut, improve the pest's adaptability to the corn host, and can also be used as a functional strain for related research such as plant insect resistance interaction and secondary metabolite degradation. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present 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 only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a colony morphology diagram of the Enterobacter cloacae ECSF2508 strain of the present invention.

[0017] Figure 2 This is a morphological diagram of the Enterobacter cloacae ECSF2508 strain of the present invention. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0019] The strain of Enterobacter cloacae, ECSF2508, is classified as Enterobacter cloacae. The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 15, 2025, and the accession number is CGMCC No. NO37087.

[0020] Example 1: Isolation and identification of Enterobacter cloacae ECSF2508 On July 10, 2025, the inventors of this invention isolated Enterobacter cloacae ECSF2508 from the midgut of fall armyworm larvae. The inventors discovered that this strain exhibits strong specific degradation activity against DIMBOA, a defensive secondary metabolite of maize.

[0021] (1) Screening and identification of gut degrading bacteria in fall armyworm larvae The test insect source was fall armyworms that had been fed with corn seedlings in the laboratory for a long time (environmental conditions: 16L:8D light, 25±1℃ temperature, 60±5% relative humidity). Twenty healthy 2nd and 4th instar larvae each were selected (3 replicates, 120 larvae in total). After starvation for 8 hours, they were dissected in a clean bench: the larvae were paralyzed by freezing at -20℃, rinsed with sterile water, and disinfected with 75% alcohol. The intestines were removed, and the contents were homogenized with sterile PBS buffer to a final volume of 1 mL.

[0022] Gradient dilution of homogenate (10³, 10³) 4 10 5 After being diluted (by multiples), the cultures were spread onto inorganic salt medium containing 10 mg / L butylated oxaliplatin (experimental group) and inorganic salt medium without butylated oxaliplatin (control group), respectively, and incubated at 25°C. Colony growth was observed every 24 hours. Specific strains that grew only in the experimental group were screened, purified by streak plating on LB medium (2-3 times), and then stored on LB slant agar at 4°C.

[0023] Morphological identification: Single colonies of the above-screened strains were streaked onto LB agar and incubated at 25°C for 48 h. Following the identification methods in *Manual of Common Bacterial Systematic Identification* and *Bergey's Manual of Bacteriological Identification*, the morphology, color, elevation, edge shape, and wetness of each colony were observed and recorded. Gram staining was used to stain the strain, observing cell morphology and color; purple represented Gram-positive bacteria, and red represented Gram-negative bacteria. Results were as follows: After purification culture on LB agar, the colonies of this strain were milky white, round, smooth, and slightly raised at the edges. Figure 1 Gram staining was negative; the cells were short, stout rods, and there were no spores. Figure 2 ).

[0024] Molecular identification: Single colonies of the above bacteria were picked and inoculated into liquid LB medium and cultured at 25°C and 180 rpm for 24 h. 2 mL of bacterial culture from each strain was transferred to a centrifuge tube and centrifuged at 12000 rpm for 2 min, discarding the supernatant. 500 μL of sterile water was added to the precipitate, and the mixture was vortexed for 1 min to ensure complete resuscitation. The mixture was then frozen in liquid nitrogen for 10 min, boiled in water for 5 min, and centrifuged at 12000 rpm for 2 min. The supernatant was used as the PCR template DNA. The 16S rRNA gene was amplified using the universal bacterial primers 27f and 1492r. The reaction system consisted of: 12.5 μL of 2×Rapid Taq Master Mix; 1 μL of 10 μM 27f; 1 μL of 10 μM 1492r; and 100 ng μL of... -1Take 1 μL of template and add ddH2O to a final volume of 25 μL. After adding the reaction mixture to a centrifuge tube, briefly centrifuge to remove any droplets suspended on the tube wall, then place the tube in a PCR instrument for amplification. Take 3 μL of the PCR product for gel electrophoresis. The identified PCR product is sent to Sangon Biotech (Kunming) Co., Ltd. for sequencing. The sequencing results are assembled using Contigexpress software. The assembled 16S rRNA sequence is compared with data in the NCBI database using the NCBIBlast program to identify the most similar typical strain and download the sequence (as shown below). A phylogenetic tree is constructed using MEGA7 software, employing the internationally recognized Neighbor-Joining method and the Kimura two-parameter correction model. Bootstrap validation is performed with 1000 replicate samplings to analyze and evaluate the stability of the phylogenetic tree's topological structure.

[0025] 16S rRNA identification results of Enterobacter cloacae strain ECSF2508 (SEQ ID NO1): Example 2: Determination of the degradation effect of Enterobacter cloacae ECSF2508 on butylated ... The potential degrading bacteria were activated and inoculated at a 10% inoculum into an inorganic salt medium containing 200 μg of butylated gluten (10 mg / L). Uninoculated butylated gluten medium was used as a control. The culture was incubated at 25℃ and 180 rpm for 72 h with shaking. The supernatant was collected by centrifugation, and the butylated gluten content was determined by LC-MS (Shim-pack XR-ODS III column, mobile phase: 0.05% formic acid aqueous solution-acetonitrile, flow rate: 0.3 mL / min). The degradation rate was calculated as (degradation rate = (1 - butylated gluten content after reaction / control butylated gluten content) × 100%).

[0026] The results are shown in Table 1.

[0027] Table 1. Degradation efficiency of Enterobacter cloacae ECSF2508 on butylated ...

[0028] According to the degradation ability test of Enterobacter cloacae strain ECSF2508 on butyl cloth, the degradation rate of butyl cloth by Enterobacter cloacae strain ECSF2508 reached 94.34% after 12 hours and 99.97% after 72 hours. This indicates that Enterobacter cloacae strain ECSF2508 has the ability to degrade butyl cloth.

[0029] In summary, the Enterobacter cloacae ECSF2508 strain of the present invention is an insect gut symbiotic bacterium that can efficiently degrade butyl cloth. The strain has excellent degradation performance, is easy to cultivate and does not cause environmental pollution. It can be widely used in the microbial degradation of butyl cloth and in the development of functional microbial agents.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Enterobacter cloacae ECSF2508, characterized in that, The strain of Enterobacter cloacae ECSF2508 is classified as Enterobacter cloacae. Enterobacter cloacae The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is December 15, 2025; and the accession number is CGMCC No. 37087.

2. The Enterobacter cloacae ECSF2508 strain according to claim 1, characterized in that, The 16S rRNA gene sequence of the Enterobacter cloacae ECSF2508 strain is shown in SEQ ID No.

1.

3. The Enterobacter cloacae ECSF2508 strain according to claim 1, characterized in that, The Enterobacter cloacae ECSF2508 strain was purified and cultured by streak plating on LB agar. The colonies were milky white, round and smooth with slightly raised edges, Gram-negative, and the cell morphology was a short, stout rod without spores.

4. The use of the Enterobacter cloacae ECSF2508 strain according to any one of claims 1-3 in the preparation of a microbial agent for degrading butyl cloth.

5. A butyl cloth degrading microbial agent, characterized in that, Its active ingredients include at least one of (a), (b), and (c): (a) Fermentation broth of the Enterobacter cloacae ECSF2508 strain culture as described in any one of claims 1-3; (b) The supernatant of sonicated lysis of Enterobacter cloacae ECSF2508 strain cells as described in any one of claims 1-3; (c) The ultrasonic lysis precipitate of Enterobacter cloacae ECSF2508 strain cells as described in any one of claims 1-3.

6. A method for preparing the butyl degrading bacterial agent as described in claim 5, characterized in that, Includes the following steps: (1) Enterobacter cloacae strain ECSF2508 was isolated from the midgut of fall armyworm larvae and cultured on LB medium; (2) Then, the purified Enterobacter cloacae ECSF2508 was obtained by streaking purification on LB medium. (3) Mix at least one of the fermentation broth obtained by fermenting Enterobacter cloacae ECSF2508 strain, the ultrasonic lysis supernatant of Enterobacter cloacae ECSF2508 strain cells, and the ultrasonic lysis precipitate of Enterobacter cloacae ECSF2508 strain cells to obtain the butyl degrading agent.

7. The use of the butadiene-degrading bacterial agent as described in claim 5 in the preparation of butadiene-degrading agents.

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