A Klebsiella mutans BS2309 strain for degrading dincloth and its application

Abstract

The present invention relates to the field of microbial technology, and discloses a Klebsiella variegata BS2309 strain for degrading dinclot and its application. The strain is classified and named as: Klebsiella variegata Klebsiella smallpox ; Deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: March 25, 2024; deposit number: CGMCC No. 30141. The strain of the present invention is a degrading bacterium that can be used to degrade the toxic substance DIMB, has the characteristics of strong degradation ability and environmental safety, and can be used for biodegradation of DIMB.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Klebsiella mutans BS2309 strain capable of degrading DIMB and application thereof. Background Art

[0002] Dimb, also known as hydroxamic acid, is a benzoxazinone compound with a structure of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one. It is a plant secondary metabolite. Dimb exhibits antibacterial activity against a variety of pathogenic fungi, such as Septoria spp. and Corn leaf blight pathogen; Dimb and its degradation products are resistant to a variety of insects, especially aphids and corn borers. Therefore, Dimb is widely used in the agricultural field. Some studies have shown that Dimb has the potential to cause mutations and teratogenesis, which means that in some cases, Dimb may pose a risk to human health, especially when exposed to higher concentrations; In addition, studies have shown that high concentrations of Dimb are cytotoxic to plant cells and can cause cytoplasmic dissolution, cell collapse and cell rupture. Therefore, it is necessary to provide a microorganism with degradation ability and environmental friendliness to alleviate the harm caused by excessive use of Dimb to plants and the environment. Summary of the invention

[0003] In view of the problems in the background technology, the present invention provides a Klebsiella mutans that can safely degrade dincloth. Klebsiella variicola BS2309 strain and its application.

[0004] Specifically, the present invention provides the following technical solutions:

[0005] The first aspect of the present application provides a Klebsiella mutans strain that degrades tinfoil Klebsiella variicola BS2309 strain, which is classified as Klebsiella mutans ( Klebsiella variicola ); deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: March 25, 2024; deposit number: CGMCC No. 30141.

[0006] Furthermore, the 16S rDNA gene sequence of the Klebsiella varicose bacteria BS2309 strain is the nucleotide sequence shown in SEQ ID No.1.

[0007] In another aspect, the present invention provides a bacterial liquid containing the Klebsiella mutans BS2309 strain.

[0008] On the other hand, the present invention provides an extracellular enzyme extract of a BS2309 strain, wherein the extracellular enzyme extract is prepared by inoculating the BS2309 strain into a liquid culture medium, adding an inducer, DIBU, to the fermented bacterial solution, and continuing to culture the fermented bacterial solution. The supernatant obtained by centrifuging the induced bacterial solution is the extracellular enzyme extract of the BS2309 strain.

[0009] Furthermore, the extract of the present invention, the liquid culture medium is LB liquid culture medium (NaCl 10.0 g, tryptone 10.0 g, yeast extract 5 g, distilled water to 1000 mL, pH = 7.5 ± 0.1, sterilized at 121 ° C for 20 min).

[0010] Furthermore, the extract of the present invention is fermented and cultured under shaking conditions at 25° C. and 180 rpm.

[0011] In another aspect, the present invention provides a bacterial agent, which contains an extracellular enzyme extract of any of the BS2309 strains.

[0012] Furthermore, the present invention provides the use of the Klebsiella mutans BS2309 strain, or the bacterial solution, or the extracellular enzyme extract of the BS2309 strain, or the bacterial agent in the preparation of a DIBU degradation preparation.

[0013] Furthermore, the present invention provides a method for preparing an extracellular enzyme extract of BS2309 strain, comprising the following steps:

[0014] (1) Bacterial culture: The Klebsiella mutans BS2309 strain was inoculated into a pre-prepared LB liquid medium (10.0 g NaCl, 10.0 g tryptone, 5 g yeast extract, distilled water to 1000 mL, pH = 7.5 ± 0.1, sterilized at 121 °C for 20 min), and cultured at 25 °C and 180 rpm with shaking until the OD reached 0. 600 nm =1.0;

[0015] (2) Induction: Dilute the cultured bacterial solution to a final OD 600 nm =0.1; add DIBU to a concentration of 5 µg / mL, and culture at 180 rpm and 25°C for 20 h to obtain the induced bacterial solution;

[0016] (3) Centrifugation: The induced bacterial solution is centrifuged and the supernatant is the crude extract of Klebsiella mutans exocellular enzymes.

[0017] Beneficial effects: The Klebsiella mutans provided by the present invention Klebsiella variicolaBS2309 is a kind of degrading bacteria that can be used to degrade diaper. It has the advantages of strong degradation ability and environmental safety, and can be used for biodegradable diaper.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) Highly efficient degradation ability: Klebsiella mutans provided by the present invention Klebsiella variicola BS2309 strain has a strong ability to degrade dinbu, with a degradation efficiency of up to 94.5%, and can effectively convert toxic dinbu into non-toxic substances. Klebsiella variicola The BS2309 strain is simple to culture, grows quickly, and is easy to culture and reproduce in large quantities indoors, which is conducive to industrial production and application.

[0020] (2) Environmental safety: This strain will not produce any substances harmful to the environment during the degradation of dincloth, and has the characteristics of being non-polluting to the environment. It is a green and safe biodegradation method. Food safety guarantee: By using Klebsiella mutans Klebsiella variicola The BS2309 strain can degrade DIMB, which can ensure the safety of foods developed using DIMB-containing materials as raw materials and reduce the potential risks of DIMB to human health.

[0021] (3) Broad application prospects: This strain can efficiently degrade DIBU and can be widely used in the biodegradation of DIBU, with broad application prospects.

[0022] (3) Promoting sustainable agricultural development: by using Klebsiella mutans Klebsiella variicola BS2309 strain biodegrades dincloth, which can reduce the use of chemical substances, promote the sustainable development of agriculture, and reduce the impact of agricultural production on the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a culture colony diagram of Klebsiella mutans BS2309 of the present invention;

[0024] Figure 2 This is a cell morphology diagram of the Klebsiella mutans BS2309 strain of the present invention;

[0025] Figure 3 It is a growth curve diagram of Klebsiella mutans BS2309 of the present invention. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0027] In a first aspect, the present invention provides a Klebsiella mutans strain capable of degrading tinfoil. Klebsiella variicola BS2309 strain, which is a variant Klebsiella, and its deposited name is variant Klebsiella ( Klebsiella variicola ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: March 25, 2024; deposit number: CGMCC No.30141.

[0028] Klebsiella mutans Klebsiella variicola The BS2309 strain is a Gram-negative bacterium with a short rod-shaped cell morphology and a size of 0.5-0.8×1-2um. After streak purification and culture on LB solid medium (NaCl 10.0 g, tryptone 10.0 g, yeast extract 5 g, agar 15.0 g, distilled water to 1 000 mL, pH = 7.5±0.1, sterilized at 121 ℃ for 20 min), the BS2309 strain colonies were nearly circular, with neat edges, orange color, smooth and moist surface, slightly convex in the center, and relatively flat around.

[0029] The following are examples of the Klebsiella mutans BS2309 provided by the present invention and its application in DIMB degradation.

[0030] Example 1 Isolation and identification of Klebsiella mutans BS2309

[0031] 1.1 Materials

[0032] 1.1.1 Test strains

[0033] The strain was isolated on June 20, 2023 from the midgut of the third instar larvae of Spodoptera frugiperda in the corn field in Wangtouzhai Village, Pupiao Town, Longyang District, Baoshan City, Yunnan Province (N 25°0'17", E 99°0' 21", 1370 m above sea level).

[0034] 1.1.2 Isolation and culture of strains

[0035] Clean the clean bench with 75% alcohol, disinfect all dissection instruments with 75% alcohol, and then irradiate with ultraviolet light for 45 minutes to complete the disinfection of the clean bench. Take 50 healthy third-instar larvae of fall armyworm of the same size, repeat 3 times, a total of 150, and starve them in a sterilized culture dish for 8 hours. Then freeze the starved fall armyworm larvae at -20℃ for 2-3 minutes. After paralysis, rinse with sterile water for surface disinfection twice, 1 minute each time, and then disinfect with 75% alcohol for 90 seconds, and finally rinse with sterile water twice, 1 minute each time. After taking it out, place it in a clean and sterile culture dish, use a sterilized scalpel to dissect the fall armyworm from the ventral surface, use sterile tweezers to remove the midgut from the tail of the dissected insect body, take out the contents and dissolve them in a centrifuge tube containing 1 mL sterile PBS buffer, and fully homogenize. The purified strains were stored in LB solid medium in a 4°C refrigerator.

[0036] 1.1.3 Species identification

[0037] Morphological identification: Pick a single colony of the highly efficient degradation strain obtained in the above screening and streak it on LB solid medium (NaCl 10.0g, tryptone 10.0g, yeast extract 5g, agar 15.0g, distilled water to 1000mL, pH=7.5±0.1, sterilized at 121℃ for 20min), culture it at 25℃ for 72h, observe and record the single colony morphology, color, ridge, edge shape, dryness and wetness and other characteristics. Stain the strain with Gram staining method to observe the cell morphology and color, purple represents positive bacteria, and red represents negative bacteria. Refer to the methods of "Common Bacterial System Identification Manual" and "Berger's Bacterial Identification Manual" for identification.

[0038] Molecular identification: First, the bacterial DNA was extracted by freeze-thaw method. The specific operation steps are as follows: the strains were inoculated into LB liquid medium (NaCl 10.0 g, tryptone 10.0 g, yeast extract 5 g, distilled water added to 1 000 mL, pH = 7.5 ± 0.1, sterilized at 121 ° C for 20 min), and placed in a shaking incubator at 25 ° C and 180 r / min for 48 h. Take 2 mL of bacterial solution and place it in a centrifuge tube, centrifuge it at 12000 r / min for 2 min, and discard the supernatant. Add 500 μL of sterile water to the above precipitate, vortex and oscillate for 1 min to completely suspend and mix the bacteria. Then freeze it with liquid nitrogen for 10 min, boil it in a water bath for 5 min, centrifuge it at 12000 r / min for 2 min, and take the supernatant as PCR template DNA for use. Second, use bacterial universal primers 27f and 1492r to amplify the 16S rRNA gene. The reaction system was 25 μL: 1 μL of each primer, 1 μL of template DNA, 12.5 μL of 2ÍTaq PCR master mix, and 9.5 μL of dd H2O. Reaction conditions: 94℃ heat denaturation for 5 min; 94℃ denaturation for 1 min, 53℃ annealing for 1 min, 72℃ extension for 2 min, 32 cycles, and 72℃ extension for 10 min. Take 3 μL of PCR product for gel electrophoresis detection. Third, send the identified PCR product to Sangon Biotech (Kunming) Co., Ltd. for sequencing. Fourth, use Contigexpress software to splice the sequencing results, and compare the spliced ​​16S rDNA sequence with the data in the NCBI database through the NCBI Blast program to find the most similar typical strains and download the sequence (as shown below). Fifth, use MEGA7 software to construct a phylogenetic tree using the internationally accepted neighbor-joining method and Kimura two-parameter correction model. Bootstrap validation was performed with 1000 repeated samplings to analyze and evaluate the stability of the topological structure of the phylogenetic tree.

[0039] The gene sequence of 16S rDNA of Klebsiella mutans strain BS2309 (SEQ ID No. 1):

[0040]

[0041] 1.2 Results

[0042] After streaking purification culture on LB solid medium (NaCl 10.0 g, tryptone 10.0 g, yeast extract 5 g, agar 15.0 g, distilled water to 1 000 mL, pH = 7.5 ± 0.1, sterilized at 121 ° C for 20 min), the BS2309 colony was found to be nearly circular, with neat edges, orange, smooth and moist surface, slightly convex in the center, and relatively flat around. Gram staining was red, Gram-negative bacteria, and the cell morphology was short rod-shaped ( Figure 1 ).

[0043] Klebsiella mutans Klebsiella variicola BS2309 strain is a Gram-negative bacterium with a short rod-shaped cell morphology ( Figure 2 ), with a size of 0.5-0.8×1-2um. After streaking and purification on LB solid culture medium plates, the colonies of strain BS2309 were nearly circular, with neat edges, orange color, smooth and moist surface, slightly convex in the center, and relatively flat around.

[0044] According to the growth curve of the Klebsiella var. mutans BS2309 strain culture, no slow growth period was found. 0-6 h was the logarithmic growth period, with the fastest growth at 2-4 h; 6-28 h was the stable period, with the maximum growth reaching 24 h and then slowly declining; 28-48 h was the decay period ( Figure 3 is the growth curve of Klebsiella mutans BS2309 of the present invention).

[0045] Example 2 Determination of the degradation effect of Klebsiella mutans BS2309 on DIMB

[0046] 1 Materials

[0047] The BS2309 strain of Klebsiella mutans was used as the material. 5 mL of a single colony of Klebsiella mutans was selected and inoculated into the LB liquid medium prepared in advance (10.0 g NaCl, 10.0 g tryptone, 5 g yeast extract, distilled water to 1 000 mL, pH = 7.5 ± 0.1, sterilized at 121 °C for 20 min), and cultured at 25 °C and 180 rpm with shaking until the OD 600 nm =1.0.

[0048] Induction: Take 1 mL of Klebsiella variotii culture medium into 9 mL of LB liquid medium in a 100 mL Erlenmeyer flask to make the final OD 600 nm= 0.1; supplement with Klebsiella variabilis buffer (control) or 5 µg / mL DIBU, and culture at 180 rpm and 25°C for 20 h. Centrifuge the Klebsiella variabilis bacterial suspension at 4000×g for 20 min at 25°C, and the supernatant is the Klebsiella variabilis extracellular enzyme extract. Repeat this process 3 times.

[0049] Extracellular enzyme degradation system: Take 100 µL of 50 mM sodium acetate buffer at pH 5.5, 400 µL of 100 mM sodium acetate buffer at pH 5.0 (0.2 mM d-butyl) substrate, and 450 µL of extracellular enzyme extract, a total of 950 µL, and incubate at 25°C for 35 h. Heat at 90°C for 10 min to terminate the enzyme reaction, then centrifuge at 17,000 × g for 10 min and transfer the supernatant. Use it for LC-MS analysis of intermediate metabolites produced during the degradation process.

[0050] 2. Results

[0051] The degradation capacity of DIMB was tested by using the extracellular enzyme of the efficient degradation strain. The results showed that the degradation rate of DIMB by the extracellular enzyme of Klebsiella mutans BS2309 strain reached 94.5%, indicating that Klebsiella mutans BS2309 strain has a strong ability to degrade DIMB.

[0052] In summary, the Klebsiella mutans BS2309 strain of the present invention is an insect intestinal symbiotic bacterium that can be used to degrade DIMB, has the advantages of strong degradation effect and no environmental pollution, and can be used in the practice of biodegradation of DIMB.

[0053] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A Klebsiella mutans BS2309 strain, characterized in that The strain is classified and named as: Klebsiella mutans Klebsiella variicola ; Deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Deposit date: March 25, 2024; Deposit number: CGMCC No.30141.

2. A bacterial liquid, characterized in that: The bacterial liquid contains the Klebsiella mutans BS2309 strain according to claim 1.

3. An extracellular enzyme extract of BS2309 strain, characterized in that: The extracellular enzyme extract is prepared by inoculating the BS2309 strain described in claim 1 into a liquid culture medium, adding an inducer, DIBU, to the fermented bacterial solution, and then continuing to culture the induced bacterial solution by centrifugation, and the supernatant obtained is the extracellular enzyme extract of the BS2309 strain.

4. The extract according to claim 3, characterized in that The liquid culture medium is LB liquid culture medium.

5. The extract according to claim 3, characterized in that The culture conditions are shaking culture at 25° C. and 180 rpm.

6. A bacterial agent, characterized in that: The bacterial agent contains the extracellular enzyme extract of the BS2309 strain according to any one of claims 3-5.

7. Use of the Klebsiella mutans BS2309 strain according to claim 1, the bacterial solution according to claim 2, the extracellular enzyme extract of the BS2309 strain according to any one of claims 3 to 5, or the bacterial agent according to claim 6 in the preparation of a DIBU degradation preparation.

8. A method for preparing an extracellular enzyme extract of BS2309 strain, characterized in that: The following steps are involved: (1) Bacterial liquid culture: The Klebsiella mutans BS2309 strain of claim 1 was inoculated into a pre-prepared LB liquid culture medium and cultured at 25°C and 180 rpm with shaking until the OD 600 nm =1.0; (2) Induction: Dilute the cultured bacterial solution to a final OD 600 nm =0.1; add DIBU to a concentration of 5 µg / mL, and culture at 180 rpm and 25°C for 20 h to obtain the induced bacterial solution; (3) Centrifugation: The induced bacterial solution is centrifuged and the supernatant is the extracellular enzyme extract of Klebsiella mutans.

Images