Enterococcus casei and application thereof

Abstract

The invention discloses enterococcus casei EC, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.32874. The strain has an obvious degradation effect on polyethylene plastics.

Description

Technical Field

[0001] The present invention relates to microbial strains, and specifically to a strain of Enterococcus casseliflavus and its application. Background Art

[0002] Polyethylene (PE) is a widely used polymer synthetic material in daily life, often used to make garbage bags, food packaging bags, plastic bottles, and agricultural mulch films, etc. PE has good stability, is resistant to acid and alkali corrosion, is insoluble in common solvents at room temperature, has low water absorption, and has excellent electrical insulation performance and low-temperature resistance. PE has a large molecular weight, strong surface hydrophobicity, and low surface energy, resulting in a very slow degradation rate in the natural environment and being prone to causing "white pollution". Traditional methods such as landfill, incineration, and recycling have played a certain role in reducing polyethylene pollution, but they are also prone to causing secondary pollution. Using microorganisms to degrade polyethylene is one of the relatively green and environmentally friendly methods at present, but the degradation effect often depends on the performance of the selected microbial strains. Summary of the Invention

[0003] The object of the present invention is to provide a strain of Enterococcus casseliflavus EC, with the preservation number of CGMCC No. 32874, which was preserved in the China General Microbiological Culture Collection Center on December 02, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. This strain was isolated from the intestine of Anoplophora glabripennis larvae and has a good effect on degrading polyethylene.

[0004] The microbiological characteristics of strain EC are as follows: it is round, milky white, convex on the LB solid medium, with a smooth, complete, opaque edge and a slightly raised center, and the diameter is 0.5 - 1 mm. The 16S rDNA sequence alignment results show that it is in the same branch as Enterococcus casseliflavus and has a similarity of 99.72% with E. casseliflavus EGM182.

[0005] Another object of the present invention is to provide a bacterial suspension or bacterial agent containing Enterococcus casseliflavus EC.

[0006] The third object of the present invention is to provide the application of Enterococcus casseliflavus EC or its bacterial suspension or bacterial agent in degrading polyethylene.

[0007] Advantages of the present invention: After the polyethylene plastic sheet was degraded by Enterococcus casei EC for 8 weeks, the average mass of the treatment group decreased by 0.088 g, and the mass loss ratio was 52.38%. There was an obvious weight change compared with the control treatment group, indicating that Enterococcus casei EC has a degradation effect on the polyethylene plastic sheet. At the same time, when observing the samples of the treatment group and the control group under a scanning electron microscope, the surface of the samples in the treatment group was rougher and obvious microbial erosion holes and grooves appeared. And there were obvious differences in the contact angles of the liquid in the treatment group and the control group on the PE plastic surface. The contact angle of the treatment group was smaller and the surface hydrophobicity was weaker. Description of the Drawings

[0008] Figure 1 It is a colony morphology photo of strain EC;

[0009] Figure 2 It is the phylogenetic relationship between strain EC and other Enterococci;

[0010] Figure 3 It is a comparison picture of the plastic surfaces of the treatment group and the control group;

[0011] Figure 4 It is a comparison picture of the contact angles of the liquids in the treatment group and the control group on the plastic surface. Detailed Embodiments

[0012] Example 1 Isolation, Screening, Identification and Preservation of Enterococcus casei

[0013] 1. Isolation and Screening

[0014] After the Anoplophora glabripennis larvae collected from the willow trees on the East Second Ring Road in Baoding City, Hebei Province were starved for 24 h, in a laminar flow hood, the insect bodies were soaked in 75% alcohol for 90 s, rinsed 3 times with sterile water for 30 s each time, the intestines were dissected in 1× phosphate buffered saline (PBS), and placed in a sterilized 1.5 mL centrifuge tube containing 200 μL of PBS solution. They were ground into a homogenate using a disposable grinding pestle, and the original solution was diluted into 1×10 -3 、1×10 -5 、1×10 -7 -fold dilutions. 100 μL of each diluted bacterial solution was evenly spread on the LB medium, with the PBS solution as the control, and 3 parallel experiments were set for each concentration. After incubation at 37 °C for 24 h, different single colonies were selected according to the color, morphology, size, etc. of the colonies using a sterile inoculation loop and inoculated onto the corresponding medium for purification culture until monoclonal colonies were obtained. Then different colonies were picked and inoculated into the LB liquid medium, and shaken overnight at 200 rpm and 37 °C. The cultured bacterial solution was stored in 50% sterile glycerol and placed in a -80 °C ultra-low temperature refrigerator for subsequent use. Finally, an endophytic bacterium was selected, labeled as EC, for plastic degradation experiments.

[0015] 2. Colony characteristics and colony morphology

[0016] The strain EC was circular, milky white, convex on the LB solid medium, with a smooth, complete, opaque edge and a slightly raised center, and the diameter was 0.5 - 1 mm( Figure 1 ).

[0017] 3. 16S rDNA sequence analysis

[0018] Pick the isolated and purified single colony EC into the LB liquid medium and culture it at 37°C and 150 r / min until the OD 600 is 0.6 - 0.8. Dilute the bacterial solution and take 1 μL as a template. Use the universal primers 27F (5′AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) of the bacterial 16S rRNA gene to amplify the 16S rRNA gene of the purified isolate. The PCR reaction system is as follows: Taq enzyme 25 μL, DNA template 3 μL, upstream primer 1 μL, downstream primer 1 μL, ddH 2 O 20 μL, a total of 50 μL, and add it to a 0.2 mL centrifuge tube.

[0019] PCR reaction conditions: Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min 30 s, a total of 35 cycles; final extension at 72°C for 7 min, and store at 4°C. The PCR product was detected by 1% agarose gel electrophoresis. The target band was about 1500 bp. The qualified PCR products were sent to Beijing Tsingke Biotechnology Co., Ltd. for DNA sequencing work.

[0020] The 16S rDNA sequence of the strain EC was BLAST aligned in NCBI. The results showed that the similarity of its 16S rDNA sequence with E.casseliflavus EGM182 reached 99.72%. Combining the characteristics such as colony morphology and 16S rDNA sequence analysis, the strain EC was preliminarily identified as Enterococcus casseliflavus.

[0021] 4. Construction of phylogenetic tree

[0022] Download the gene sequences of 13 complete Enterococcus casseliflavus strains from GenBank. The GenBank accession numbers are Enterococcus casseliflavus ECB140 (GCA_022870765.1), Enterococcus casseliflavus FDAARGOS_1121 (GCA_016727325.1), Enterococcus casseliflavus FDAARGOS_998 (GCA_016127635.1), Enterococcus casseliflavus FDAARGOS_1122

[0023] (GCA_016727305.1), Enterococcus casseliflavus EC291 (GCA_009707345.1), Enterococcus casseliflavus EC369 (GCA_003641225.1), Enterococcus casseliflavus ASE4

[0024] (GCA_029215545.1), Enterococcus casseliflavus FDAARGOS_1120

[0025] (GCA_016727345.1), Enterococcus casseliflavus SP11 (GCA_023523715.1), Enterococcus casseliflavus ASE2 (GCA_029201225.1), Enterococcus casseliflavus CQFYY22 - 063 (GCA_027944555.1), Enterococcus casseliflavus EC20 (GCA_000157355.2), Enterococcus casseliflavus EGM182 (GCA_014844215.1). Use the MEGA7 software together with an outgroup genome, E. gilvus ATCC BAA - 350 (GCA_000407545.1), to determine the classification of the sequenced strain EC( Figure 2 ) and construct a phylogenetic tree for strain EC. Finally, identify strain EC as Enterococcus casseliflavus.

[0026] 5. Strain preservation

[0027] Preserve strain EC, classified and named as Enterococcus casseliflavus. The preservation date is December 2, 2024. The preservation unit is the China General Microbiological Culture Collection Center, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. The preservation number is CGMCC No: 32874.

[0028] Example 2 Preparation of Enterococcus casseliflavus EC bacterial suspension

[0029] Activate Enterococcus casei EC on an LB slant, pick up a loop and inoculate it into an LB medium (3 g beef extract, 10 g tryptone, 5 g NaCl, 1000 mL sterile water, pH = 7.4), and incubate it at 37 °C and 180 r / min with constant shaking for 16 h. Under sterile conditions, transfer the bacterial liquid to a sterile centrifuge tube and centrifuge it at 3500 r / min for 5 min. Pour off the supernatant, and wash it three times with a sterilized inorganic salt medium (1.0 g K 2 HPO 4 , 0.3 g KH 2 PO 4 , 0.1 g MgSO 4 ·7H 2 O, 1.0 g NaCl, 1.0 g NH 4 NO 3 , 1000 mL sterile water) to make a bacterial suspension.

[0030] Example 3 Degradation test of Enterococcus casei EC on polyethylene (PE) plastic

[0031] 1. Experimental preparation

[0032] Soak the polyethylene plastic in 75% ethanol for 4 h, then rinse it thoroughly with sterile water, dry it in an oven and cut it. Based on the weight of 30 mL of the inorganic salt medium, use sterilized scissors in a laminar flow hood to cut the polyethylene plastic into rectangular plastic sheets at a weight ratio of 0.5% of the medium weight, weigh and record it as the weight before treatment.

[0033] 2. Degradation test of polyethylene plastic

[0034] Add 30 mL of the inorganic salt medium to a 50 mL conical flask, add the disinfected and cleaned polyethylene plastic sheets to the conical flask, inoculate 1 mL of the bacterial suspension into each flask, and incubate it with shaking in a shaker at 180 rpm and 37 °C for 8 weeks. Use the inorganic salt medium without inoculating the bacterial suspension as the control group, and set 3 parallel samples for each treatment. After 8 weeks, sample and analyze the mass loss, surface morphology change and surface hydrophobicity change of the plastic sheets, etc.

[0035] 3. Determination of the weight loss rate of the plastic

[0036] Soak the rectangular plastic sheets after 8 weeks of liquid culture in a 2% SDS solution overnight, wash it 3 times with sterile water, rinse the reagents on the plastic surface thoroughly, dry it, soak the plastic sheets in 75% ethanol for 4 h before weighing, then rinse it thoroughly with sterile water and dry it for weighing, which is used as the weight after treatment, and calculate the weight loss rate of the plastic sheets. The results are shown in Table 1.

[0037] Weight loss rate (%) = (weight of plastic sheet before liquid culture - weight of plastic sheet after liquid culture) / weight of plastic sheet before liquid culture × 100%

[0038] Plastic mass loss = weight of plastic sheet before liquid culture - weight of plastic sheet after liquid culture

[0039] Table 1 Effect of Enterococcus casei on mass loss of PE plastic sheets

[0040] PE (control group) PE (treatment group) Weight before treatment (g) 0.164 0.168 Weight after treatment (g) 0.084 0.080 Mass loss ratio (%) 48.78 52.38

[0041] As can be seen from Table 1, after the plastic sheets were cultured in the above-mentioned culture method for 8 weeks, the average mass of the treatment group decreased by 0.088 g, and the mass loss ratio was 52.38%. There was an obvious weight change compared with the control treatment group, indicating that Enterococcus casei EC played a role in degrading the plastic sheets.

[0042] 4. Determination of changes in microscopic morphological characteristics of plastic surface

[0043] The rectangular plastic sheets after 8 weeks of liquid culture were immersed in 2% SDS solution overnight, washed 3 times with sterile water to rinse off the reagents on the plastic surface, air-dried and then cut into 10 mm × 10 mm square plastic sheets, and the microscopic characteristics changes on the plastic surface were observed with a scanning electron microscope (SEM). The results are as Figure 3 shown.

[0044] As Figure 3 can be seen, after 8 weeks of culture, it was observed under the scanning electron microscope that the surface of the treatment group was rough compared with the control group, and obvious microbial erosion holes and grooves appeared on the surface, while the surface of the control group was relatively smooth and intact.

[0045] 5. Detection of plastic surface hydrophobicity

[0046] Common methods for detecting changes in plastic surface hydrophobicity include the method for measuring surface tension, the method for measuring contact angle, and the method for measuring wettability.

[0047] Method for measuring surface tension: The hydrophobicity of the surface can be judged according to the magnitude of the surface tension. A small surface tension indicates strong surface hydrophobicity; a large surface tension indicates weak surface hydrophobicity.

[0048] Method for measuring contact angle: The hydrophobicity of the surface can be judged according to the contact angle of the liquid on the surface. A large contact angle indicates strong surface hydrophobicity; a small contact angle indicates weak surface hydrophobicity.

[0049] Method for measuring wettability: The hydrophobicity of the surface can be judged according to the wettability of the liquid on the surface. Strong wettability indicates weak surface hydrophobicity; weak wettability indicates strong surface hydrophobicity.

[0050] On the rectangular plastic sheets treated in the control group and the experimental group respectively, 10 μL of sterile water was added dropwise using a pipette, and photographs were taken. The contact angle was measured based on the photographs, and the results are as Figure 4 shown. It can be seen from Figure 4 that after 8 weeks of cultivation, there is a significant difference in the contact angle of the liquid between the treatment group and the control group on the PE plastic surface. The contact angle of the treatment group is smaller and the surface hydrophobicity is weaker, indicating that the plastic has been degraded.

[0051] The above embodiments are illustrative of the present invention rather than limiting it. Technical solutions without substantial transformation under the concept of the present invention are within the scope of protection.

Claims

1. Enterococcus casseliflavus EC, deposit number is CGMCC No.32874.

2. A bacterial suspension or bacterial agent containing the Enterococcus casei EC according to claim 1.

3. Use of the Enterococcus casei EC according to claim 1 or the bacterial suspension or bacterial agent according to claim 2 in degrading polyethylene plastics.

Images