preparation method of Listeria monocytogenes biofilm:

1. Inoculate the strain frozen at -8℃ into solid BHI culture medium, and culture at 37℃ for 18 hours to activate. Single colonies were selected and cultured in 5mL liquid BHI medium at 37℃ and 2r/min overnight. The next day, according to the ratio of 1:1, it was transferred to 1L fresh liquid BHI medium to continue the culture until the OD6 was 1..

2. 2.2 Extraction of membrane vesicles by ultrafiltration concentration method When bacteria are cultured to an OD6 of 1., the supernatant is collected by centrifugation at 4℃ and 6r/min for 2min, filtered by a .45μm or .22μm filter, and then transferred to an ultrafiltration concentration tube with a molecular weight of 1kD, and centrifuged at 4℃ and 4r/min for 2min. Take the concentrated solution, centrifuge at 4℃ and 31,174 r/min for 3 hours, then discard the supernatant, suspend and precipitate with 1mL of phosphate buffer, repeat centrifugation once, resuspend the precipitate in 1mL of phosphate buffer, filter it with a .22μm filter, check its sterility, and store it at -8℃, which is the extracted membrane vesicle. The extracted membrane vesicles of EGDe, EGDE δ PRFA and EGDE δ PRFA+Perl3-PRFA * were expressed as EGDe-MVs, δ ΔprfA-MVs and prfA*-MVs, respectively.

3. Extraction of membrane vesicles by Optiprep density gradient centrifugation; Optiprep gradient centrifugation solution was prepared with DMEM with concentrations of 25%, 3%, 35%, 4% and 45% respectively. Suck 1mL of product extracted by ultrafiltration concentration method (same as 1.2.2) and transfer it to Beckman centrifuge tube, then add 2mL of 45%, 4%, 35%, 3% and 25%Optiprep in turn, and perform ultracentrifugation at 4℃ and 31,174 r/min for 15 hours. After centrifugation, carefully collect 3%-35% of the components in Beckman centrifuge tube, add 1mL of phosphate buffer, and perform ultracentrifugation again. After repeated centrifugation for three times, the supernatant was discarded, and the precipitate was resuspended in 1mL of phosphate buffer, filtered by a .22μm filter, and then tested for sterility, and then stored at -8℃, which was the extracted membrane vesicle.

4. BCA method is used to determine the protein concentration of membrane vesicles and calculate the yield of membrane vesicles. For the specific determination method of MVs protein concentration, please refer to the instructions of BCA protein concentration determination kit. The number of colonies when OD6 was 1. was counted by dilution plate coating method, and the yield of membrane vesicles was determined by the amount of protein obtained per 113 CFU (μg).

5. Observe the morphology of membrane vesicles by negative staining electron microscope. Soak the copper mesh in the MVs sample and absorb it for about 2min, then take it out. Use filter paper to contact the copper mesh vertically to remove excess liquid. Then soak the copper mesh in PTA dye solution for 5 minutes, take it out and naturally dry it in the shade for 2-3 days, then it can be used for electron microscope observation.

6. Influence on the formation of bacterial biofilm. The culture and detection of bacterial biofilm refer to the method of Feng Feifei et al. [1]. After the MVs of three strains of bacteria were diluted to the same concentration (.2mg/mL), 1μL of the corresponding MVs were added to a 96-well plate containing 1μL of bacterial liquid (the same strain or different strains) according to the ratio of 1:1. In addition, two groups of MVs-free references were set: one group added the same amount of bacterial liquid, and the other group added the same amount of phosphate buffer. After the above operations, the 96-well plate was cultured at 37℃ for 24, 48 and 72 hours respectively. After the corresponding culture time, the OD6 value was measured by enzyme-labeled instrument to detect its growth. Subsequently, the culture medium of each hole was discarded, and the generated biofilm was washed with distilled water, dried at room temperature, dyed with 1% ammonium oxalate crystal violet, and its optical absorption value of OD595 was determined. The experimental data obtained were analyzed by Origin8 and SPSS22. The biofilm stained with crystal violet was directly observed under an inverted microscope, and photographed and recorded.

7. Detection of hemolytic activity of membrane vesicles The measurement of hemolytic activity refers to the method of Xinhui et al. [12]. After the MVs from different strains were diluted to the same concentration (.2mg/mL), a certain amount of MVs was added to the centrifuge tube containing 1mL of red blood cell suspension, the same amount of phosphate buffer was added to the negative control group, and the same amount of sterile water was added to the positive control group. In addition, the same amount of MVs was added to 1mL of red blood cell suspension, and then 2mmol/LDTT was added. Incubate the centrifuge tube at 37℃ for 3 hours, then centrifuge at room temperature and 26r/5min for 5 minutes, and suck 8μL of supernatant to determine the value of OD543 in a disposable cuvette, which is the hemolytic activity value of MVs.

8. Effects of membrane vesicles on the weight, survival rate, pupation rate and development time of the larvae of Helicoverpa armigera [13]. After the MVs from different strains were diluted to the same concentration (.2mg/mL), 5μLLm-MVs was absorbed and injected under the first gastropod of larvae, while the control group was injected with the same amount of phosphate buffer, with 18 larvae in each group. After the injection, the larvae were weighed every 24 hours, and the survival rate, pupation rate and larval development time (from the date of purchase to the last age) were counted. The calculation method of the data was: pupation rate = pupation number/total number of test insects × 1%; Survival rate = survival number/total number of trial insects ×1%.