ANTI-BIOFILM USE OF EXTRACELLULAR MEMBRANE VESCLELAE

Abstract

The present invention relates to the use of extracellular membrane vesicles of at least one probiotic to prevent or reduce biofilm formation on the surface of a material. The present invention also relates to a method for treating the surface of a material to prevent or reduce biofilm formation on said surface, said method comprising a step of contacting extracellular membrane vesicles from at least one probiotic with said surface. The present invention further relates to a material comprising on its surface extracellular membrane vesicles of at least one probiotic, or in which extracellular membrane vesicles of at least one probiotic are incorporated. Figure 2

Description

Title of the invention: ANTI-BIOFILM USE OF EXTRACELLULAR MEMBRANE VESICLES Technical field

[0001] The present invention relates to the use of biological tools to prevent or reduce the formation of a biofilm on the surface of a material.

[0002] The present invention finds applications in many fields of industry, such as for example in the food industry, the piping or surface treatment industry or even in the medical sector.

[0003] In the description below, the references in brackets ([ ]) refer to the list of references presented at the end of the text. State of the art

[0004] Biofilms appear as a viscous film made up of microorganisms, often bacteria, yeasts, fungi or algae.

[0005] They adhere to the surfaces of industrial equipment and colonize all industrial surfaces, such as pipes or membrane filters. Several studies have shown that during certain operations, particularly in plate heat exchangers, biofilms can form if manufacturers are not particularly vigilant. They are thus regularly responsible for serious contamination of finished products and numerous food poisonings. Biofilms are, for example, a major concern in the dairy processing industry. Bacterial biofilms can also develop on implants or during chronic infections; they constitute reservoirs of pathogens and can be the cause of nosocomial infections.

[0006] Biofilms are resistant to most conventional cleaning methods and tend to grow more in water or aqueous environments. However, other factors such as nutritional properties or temperature are also involved in the formation of biofilms.

[0007] Current strategies to limit biofilm formation are diverse (Rendueles O & Ghigo JM ([1]); Venkatesan N, Perumal G & Doble M ([2]); Rao PK & Sreenivasa MY ([3])). Some of these strategies are preventative and aim to prevent biofilm adhesion and formation. One such strategy is the use of synthetic signaling molecules, which disrupt the cell-to-cell communication system essential for biofilm formation. This latter application is still at the exploratory stage.

[0008] Prevention of biofilm formation currently involves nanotechnologies, with the incorporation of antibacterial agents into inert supports. The release of nanoparticles must nevertheless be taken into account depending on the applications.

[0009] Other strategies are curative and aim to eradicate biofilms. These are bactericidal methods, or methods of dispersing and disintegrating an already formed biofilm. Among the bactericidal methods, we can distinguish physical, chemical or biological approaches.

[0010] Physical and mechanical treatments, such as ionizing radiation, UV radiation and ultrasound, have been tested in the past. Their effectiveness is partial, but it is possible to combine these methods to potentiate the antibiofilm effect. In the case of ultrasound treatments, numerous deleterious effects have been reported on the quality of the food, its physical composition and its flavor.

[0011] The use of disinfectants, especially for fresh products, is common. However, these are much more active on planktonic cells. Due to their structure and in particular the EPS matrix (exopolymeric substances), the bacteria in the biofilm are more resistant. In the food industry, surfaces are cleaned with chlorinated derivatives, hydrogen peroxide, iodine, isothiazolinones, ozone, peracetic acid, acid compounds, aldehyde-based biocides, phenols, biguanides, surfactants, halogens and quaternary ammoniums. In general, these agents do not completely eradicate the biofilm, are not eco-responsible and are the cause in many cases of surface corrosion. The effect of essential oils on the destruction of biofilms is also being studied.

[0012] The use of bacteriophages is currently being developed for their bactericidal property.

[0013] Recently, the incorporation of biosurfactants into liposomes with antibiofilm activity has been proposed. In this case, these are reconstituted artificial vesicles. Effects have been demonstrated against a S. aureus biofilm, for potential applications relating to skin diseases.

[0014] Furthermore, enzymes are used to disperse the biofilm. These are mainly hydrolases (α-amylases, proteases, ribonucleases for example), oxidoreductases (such as glucose oxidases or haloperoxidases), transferases (such as transaminase) or lyases (such as alginate lyase). Since these enzymes do not kill bacteria, they are generally combined with bactericidal methods.

[0015] There is therefore a real need for tools that overcome these defects, drawbacks and obstacles of the prior art, in particular a tool that makes it possible to prevent or reduce the formation of a biofilm on the surface of a material.

[0016] Description of the invention

[0017] Following extensive research, the Applicant has succeeded in demonstrating that the use of membrane vesicles, naturally produced by probiotics, prevents the formation of biofilm by pathogenic or undesirable microorganisms on biotic and abiotic surfaces.

[0018] Very advantageously, the vesicles are derived from probiotics, which provides an advantage for their use, particularly for health. This origin guarantees a safe, natural, eco-responsible, non-polluting product, with a broad spectrum of action, which can even constitute a protective film, with a preventive action.

[0019] Advantageously, several effects are possible, including the antibiofilm and immunomodulatory effect, depending on the probiotic strain used to isolate the vesicles.

[0020] Thus, a first object of the invention relates to the use of extracellular membrane vesicles of at least one probiotic to prevent or reduce the formation of a biofilm on the surface of a material.

[0021] For the purposes of the present invention, the term "extracellular membrane vesicles" means any vesicle of a lipid nature, released spontaneously or induced (by culture conditions or by physicochemical treatments) into the medium by the probiotic, and containing at least one active principle belonging to this producing bacterium. Advantageously, it is possible to produce vesicles loaded with active principles which may be lipids, proteins, nucleic acids or exopolysaccharides.

[0022] For the purposes of the present invention, the term "probiotic" means any living microorganism which, when ingested in sufficient quantity, has a beneficial effect on the health of the host. This may include probiotic bacteria or yeasts, including a bacterium such as a lactobacillus, a bifidobacterium, an enterococcus, a propionibacterium, a streptococcus and a bacterium of the genus Bacillus, or a yeast such as Saccharomyces cerevisiae and Saccharomyces boulardi or a mixture thereof. The probiotic bacteria can be chosen from: L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, L. buchneri, L. fermentum, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, or a mixture thereof, preferably the probiotic bacteria are L. casei, L. paracasei and L.plantarum or one of their mixtures. The probiotic yeasts suitable for the present invention may be chosen from: Saccharomyces cerevisiae and Saccharomyces boulardii or one of their mixtures.

[0023] According to the invention, combinations of different types of vesicles, for example from different types of probiotic bacteria, can be made. For example, vesicles from one or more bacterial species can be used. different, the number of different species not being limited. It may be, for example, a mixture of E. casei and L. paracasei. Optionally, the vesicles may be used in combination with at least one antimicrobial, which the person skilled in the art may choose from known antimicrobials depending on the intended application.

[0024] For the purposes of the present invention, the term “biofilm” means a multicellular community of microorganisms adhering to each other and to a surface, and secreting an adhesive and protective matrix.

[0025] According to the invention, the biofilm may be a bacterial biofilm, a yeast biofilm or a mixed biofilm. For the purposes of the present invention, the term "bacterial biofilm" means a biofilm whose multicellular community of microorganisms consists essentially of bacteria. For the purposes of the present invention, the term "yeast biofilm" means a biofilm whose multicellular community of microorganisms consists essentially of yeasts. For example, the bacterial biofilm may be formed by at least one bacterial species selected from the family Enterobacteriaceae, including Salmonella enterica Enteritidis, Hafnia alvei and / or Citrobacter freundii, the genus Staphylococcus, including Staphylococcus aureus or Staphylococcus epidermidis, the genus Bacillus, including Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, including Pseudomonas aeruginosa and the genus Enterococcus, including Enterococcus faecalis.For example, the yeast biofilm may be formed by the yeast species Candida albicans. For the purposes of the present invention, the term "mixed biofilm" means a biofilm composed of a community of different types of microorganisms, which may include in particular bacteria, yeasts and / or phages. For example, the mixed biofilm may comprise a mixture of at least one bacterium chosen from the family of enterobacteria, in particular Salmonella enterica Enteritidis, Hafnia alvei and / or Citrobacter freundii, the genus Staphylococcus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the genus Bacillus, in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis, and the yeast species Candida albicans.

[0026] For the purposes of the present invention, the term "preventing the formation of a biofilm" means the action of completely preventing the formation of a biofilm on a surface devoid of biofilm. In particular, the vesicles prevent the adhesion of bacteria to the treated surface. The preventive effect of the extracellular membrane vesicles can take place for a period of up to several weeks to several months after treatment of the surface, in particular if the surface is conditioned with the vesicles and the material is stabilized, for example by drying.

[0027] For the purposes of the present invention, the term "reducing the formation of a biofilm" means the action of partially preventing the formation of a biofilm on a surface free of biofilm. The reduction may be a reduction of at least 20% in the formation of a biofilm, compared to an identical surface kept under the same conditions, in the absence of treatment. The effect of reducing the formation of a biofilm of the extracellular membrane vesicles may take place for a period of up to several weeks to several months after treatment of the surface, in particular if the surface is conditioned with the vesicles and the material is stabilized, for example by drying.

[0028] The extracellular membrane vesicles may be produced according to any method known to those skilled in the art. The production method may, for example, comprise the following steps:

[0029] (a) cultivation of at least one probiotic under conditions suitable for the production of extracellular membrane vesicles,

[0030] (b) separation of the at least one probiotic and the membrane vesicles extracellular produced in step (a) and,

[0031] (c) purification and concentration of extracellular membrane vesicles.

[0032] The culture step (a) can be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic. For example, in the case of lactobacilli, the culture can be carried out in MRS medium, at 37°C, for 24 h.

[0033] The separation step (b) can also be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic. It may, for example, be a filtration step. For example, in the case of lactobacilli, centrifugation can be carried out at 4000 g for 20 min and filtration can be carried out with a filter having a pore size of approximately 0.22 μm.

[0034] The purification and concentration step (c) may be carried out under standard conditions known to those skilled in the art, depending on the nature of the probiotic. The purification and concentration step may comprise at least one technique known to those skilled in the art, such as differential centrifugation, illustrated by Zaborowska et al. ([4]), density gradient, illustrated by Kim et al. ([5]) or Dean et al. ([6]), size exclusion chromatography, illustrated by Kuhn et al. ([7]), ultrafiltration, illustrated by Mata Forsberg et al. ([8]), Dommguez Rubio et al. ([9]), Choi et al., 2020 (

[10] ) or Kim et al. ([5]), immunocapture (IC), illustrated by Wubbolts et al. (

[11] ), such as PIC on a column, by magnetic beads coupled to antibodies or any other surface coupled to a specific antibody, or even precipitation, as illustrated by Bâuerl et al. (

[12] ) this list is not exhaustive.In the case of lactobacilli, ultrafiltration can be a filtration. exclusion at approximately 100 kDa. Advantageously, step (c) can make it possible to obtain a solution of vesicles with a concentration of approximately 1011 particles / mL, this number being given for information purposes and being able to vary according to the conditions of implementation of the different steps of protocol c).

[0035] The stabilization and preservation of the extracellular membrane vesicles can be carried out under standard conditions known to those skilled in the art. For example, the stabilization and preservation step can comprise a drying and / or freezing step.

[0036] The material on which the extracellular membrane vesicles are used may be any material on which a biofilm is likely to form. It may in particular be a material chosen from metals, metal alloys, polymers, glass, ceramics, and foods.

[0037] The extracellular membrane vesicles can be incorporated into products for treating these materials. This can be, for example, a spray, or a covering product such as paint, lacquer or varnish.

[0038] In the context of the treatment of a surface according to the invention, the use means an ex vivo, non-theraeutic use.

[0039] Another subject of the invention relates to a method for treating a surface of a material to prevent or reduce the formation of a biofilm on said surface, said method comprising a step of bringing extracellular membrane vesicles originating from at least one probiotic into contact with said surface.

[0040] For the purposes of the present invention, the term "treatment" means the application of a layer of extracellular membrane vesicles to the surface of the material. The application takes place in particular under normal conditions of use of the material, for example at room temperature and atmospheric pressure. The quantity of vesicles applied to the surface can be determined by a person skilled in the art, depending on the material and the type of vesicle.

[0041] Another subject of the invention relates to a material comprising on its surface extracellular membrane vesicles of at least one probiotic.

[0042] According to the invention, the vesicles cover at least partially, and preferably completely, the surface of the material. They can thus form a layer, with a thickness which can be between 10 and 500 nm. The thickness will be determined by a person skilled in the art depending on the applications.

[0043] The material may be a packaging material, especially food packaging, a water pipe, a heat exchanger, a pipeline, or a catheter. The material may also be a material used in the medical sector, because in addition to the anti-biofilm activity, the vesicles may provide anti-inflammatory activity (Mata Forsberg et al. ([8]), Kim et al. (

[13] ), Nahui Palomino et al. (

[14] ), Yamasaki- Yashiki et al. (

[12] ), (Bauerl et al. (

[12] ), Choi et al. (

[10] ), Kuhn et al. ([7]). Thus, the vesicles can be incorporated into dressings, creams or cover certain medical devices.

[0044] Other advantages may also become apparent to those skilled in the art upon reading the examples below, illustrated by the appended figures, given for illustrative purposes. Brief description of the figures

[0045] [Fig.l] represents the diagram illustrating the steps of the protocol carried out in examples 1 and 2.

[0046] Figure 2 represents a part of the action spectrum of the antibiofilm effect of the vesicles of L. casei BL23 and L. paracasei ATCC334. The pathogens S aureus ([Fig.2A]), S.epidermis ([Fig.2A]), H. alvei ([Fig.2B]), S. enterica ([Fig.2B]), E. faecalis ([Fig.2C]), P. Aeruginosa ([Fig.2C]) and B. subtilis ([Fig.2D]) were treated with 0.04 pg / pl of vesicles. A control is carried out with the vesicles purified from the MRS culture medium. The formation of the biofilm by the pathogens after 24 h at 37°C is quantified by staining with Crystal Violet.

[0047] EXAMPLES

[0048] Example 1: Isolation of membrane vesicles

[0049] Material:

[0050] -MRS culture medium (Man, Rogosa, Sharpe)

[0051] -0.22 pm PES (Polyethersulfone) filters

[0052] - 10 ml syringes

[0053] - 250 ml Pyrex bottles (sterile)

[0054] - 15 ml and 50 ml centrifuge tube (sterile)

[0055] -Ultrafiltration system (100-kDa-exclusion filter):

[0056] Amicon® Ultra (ref. UFC510008)

[0057] Centricon Plus-70 (ref. UFC710008)

[0058] -Heraeus Multifuge X3R Centrifuge

[0059] - BIOLiner swing-out rotor adapter (11646190)

[0060] Adapter for 50 ml centrifuge tubes

[0061] Adapter for 250 ml bottles

[0062] - Ultracentrifuge OPTIMA L series 90K (NS COL 96J32)

[0063] -Beckman SW41 Ti Swinging-Bucket Rotor (ref. 331362)

[0064] -Thin-wall polypropylene tube 13.2 mL, 14 x 89 mm (ref. 33137)

[0065] -Phosphate Buffered Saline (sterile)

[0066] Protocol

[0067] Step 1: Cultivation of lactobacilli

[0068] A protocol for culturing lactobacilli with standard parameters is proposed below.

[0069] - Inoculate 15 ml of MRS with approximately 50 μl of a lactobacillus preserved in 20% glycerol at 80°C (Cryotube outlet)

[0070] - Incubate at 37°C, 24 h

[0071] - Inoculate 15 ml of MRS with the cryotube outlet. Dilute to obtain an optical density at 600 nm of 0.05 (D0600nm=0.05) (Pre-culture)

[0072] - Incubate at 37°C, 24 h

[0073] -Inoculate 250 ml of MRS at D0600nm=0.05 with the pre-culture (Working culture)

[0074] - Incubate at 37°C for 24 h

[0075] Step 2: Concentration and isolation of vesicles by filtration and ultrafiltration

[0076] The biological material is maintained at 4°C under aseptic conditions during step 2

[0077] -Centrifuge the 250 ml at 4000 g for 20 min to precipitate the cells

[0078] -Collect the clarified supernatant in a sterile 250 ml bottle

[0079] -Filter the clarified supernatant through a 0.22 pm filter

[0080] -Concentrate the supernatant using an ultrafiltration system (100-kDa-exclusion filter) (ref. UFC710008)

[0081] -Concentrate the 250 ml until you obtain a volume of 10-15 ml of liquid

[0082] - Filter through a 0.22 pm filter (to remove aggregated matter)

[0083] -Ultracentrifuge the concentrated supernatant at 110,000g for 2 h at 4°C

[0084] -Remove the supernatant and resuspend the pellet with 500 µl of PBS at 4°C (fraction of concentrated vesicles)

[0085] - Carry out a protein assay (Bradford assay) of the concentrated vesicular fraction thus obtained.

[0086] - Keep the vesicular fraction cold.

[0087] Example 2: Antibiofilm activity of the vesicular fraction of lactobacilli

[0088] Material

[0089] -TSB culture medium

[0090] - 96-well culture plate flat bottom with lid 1 GREINER 2515432

[0091] -Pro-Lab Diagnostics™ Crystal Violet Solution (ref. 12926287)

[0092] Protocol

[0093] Step 1: Formation of a biofilm on polystyrene microplates and treatment with the concentrated vesicle fraction

[0094] - Inoculate 15 ml of TSB with approximately 50 μl of bacteria stored in 20% glycerol at 80°C (Cryotube outlet)

[0095] - Incubate at 37°C, 24 h (± agitation, ± aerobic depending on the bacteria studied)

[0096] - Inoculate 15 ml of TSB with the cryotube outlet. Dilute to obtain an optical density at 600nm of 0.05 (OD600nm=0.05) (Pre-culture)

[0097] - Incubate at 37°C, 24 h (± agitation, ± aerobic depending on the bacteria studied)

[0098] -Inoculate 20 ml of TSB at D0600nm=0.05 with the pre-culture (Working culture)

[0099] -Distribute the working culture into the wells of a polystyrene plate and add the vesicular fraction at a final concentration of 0.04 pg / pl in 100 pl of final volume.

[0100] - Incubate at 37°C, 24 h (± shaking, ± aerobic depending on the bacteria studied)

[0101] Step 2: Quantification of biofilm formation by crystal violet staining

[0102] -Remove the suspended bacteria present in each of the wells of the polystyrene plate

[0103] -Wash each well of the polystyrene plate twice with 200 μl of distilled water

[0104] -Add 150 μl of 0.5% crystal violet (dilution in distilled water) to each well

[0105] - Incubate for 1 hour without shaking

[0106] - Wash each well of the polystyrene plate twice with 200 μl of distilled water

[0107] - Add 150 μl of 95% ethanol (dilution in distilled water) to each well

[0108] - Measure the absorbance at 595 nm

[0109] LIST OF REFERENCES

[0110] 1. Rendueles O & Ghigo JM, Multi-species biofilms: how to avoid unfriendly neighbors FEMS Microbiol Rev 2012; 36:972-989. DOI: 10.1111 / d. 1574-6976.2012.00328.x

[0111] 2. Venkatesan N, Perumal G & Doble M, Bacterial resistance in biofilm-associated Bacteria Future Microbiol 2015; 10(11): 1743-50. DOI: 10.2217 / fmb. 15.69

[0112] 3. Rao PK & Sreenivasa MY, Probiotic Lactobacillus Strains and Their Antimicrobial Peptides to Counteract Biofilm- Associated Infections- A Promising Biological Approach SM J Bioinform Proteomics. 2016; 1(2): 1009.

[0113] 4. Zaborowska, M., Taulé Flores, C., Vazirisani, F., Shah, F.A., Thomsen, P., Trobos, M., 2020. Extracellular Vesicles Influence the Growth and Adhesion of Staphylococcus epidermidis Under Antimicrobial Sélective Pressure. Front. Microbiol. 11, 1132.

[0114] 5 . Kim, H., Kim, M., Myoung, K., Kim, W., Ko, J., Kim, K.P., Cho, E.-G., 2020. Comparative Lipidomic Analysis of Extracellular Vesicles Derived from Lactobacillus plantarum APsulloc 331261 Living in Green Tea Leaves Using Liquid Chromatography-Mass Spectrometry. Int. J. Mol. Sci. 21, 8076.

[0115] 6 . Dean, S.N., Rimmer, M.A., Turner, K.B., Phillips, D.A., Caruana, J.C., Hervey, W.J., Leary, D.H., Walper, S.A., 2020. Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery. Front. Microbiol. 11, 710.

[0116] 7 . Kuhn, T., Koch, M., Fuhrmann, G., 2020. Probiomimetics—Novel Lactobacillus -Mimicking Microparticles Show Anti-Inflammatory and Barrier-Protecting Effects in Gastrointestinal Models. Small 16, 2003158.

[0117] 8 . Mata Forsberg, M., Bjôrkander, S., Pang, Y., Lundqvist, L., Ndi, M., Ott, M., Escribâ, I.B., Jaeger, M.-C., Roos, S., Sverremark-Ekstrôm, E., 2019. Extracellular Membrane Vesicles from Lactobacilli Dampen IFN-y Responses in a Monocyte-Dependent Manner. Sci. Rep. 9, 17109.

[0118] 9 . Dominguez Rubio, A.P., Martinez, J.H., Martinez Casillas, D.C., Coluccio Leskow, F., Piuri, M., Pérez, O.E., 2017. Lactobacillus casei BL23 Produces Microvesicles Carrying Proteins That Hâve Been Associated with Its Probiotic Effect. Front. Microbiol. 8, 1783.

[0119] 10 . Choi, J.H., Moon, C.M., Shin, T.-S., Kim, E.K., McDowell, A., Jo, M.- K„ Joo, Y.H., Kim, S.-E., Jung, H.-K., Shim, K.-N., Jung, S.-A., Kim, Y.-K., 2020. Lactobacillus paracasei-derived extracellular vesicles attenuate the intestinal inflammatory response by augmenting the endoplasmic réticulum stress pathway. Exp. Mol. Med. 52, 423-437.

[0120] 11. Wubbolts, R., Leckie, R.S., Veenhuizen, P.T.M., Schwarzmann, G., Môbius, W., Hoernschemeyer, J., Slot, J.-W., Geuze, H.J., Stoorvogel, W., 2003. Proteomic and Biochemical Analyses of Human B Cell-derived Exosomes: POTENTIAL IMPLICATIONS FOR THEIR FUNCTION AND MULTIVESICULAR BODY FORMATION. J. Biol. Chem. 278, 10963-10972.

[0121] 12 . Bâuerl, C., Coll-Marqués, J.M., Tarazona-Gonzâlez, C., Pérez-Martinez, G., 2020. Lactobacillus casei extracellular vesicles stimulate the EGFR pathway likely due to the presence of proteins P40 and P75 bound to their surface. Sci. Rep. 10 , 19237 .

[0122] 13 . Kim , W. , Lee , EJ , Bae , I.-H. , Myoung , K. , Kim , ST , Park , PJ , Lee , K.-H. Pham, AVQ, Ko, J., Oh, SH, Cho, E.-G., 2020. Lactobacillus plantarum -derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization in vitro. J. Extracell. Vesicles 9 , 1793514 .

[0123] 14 . Nahui Palomino , RA , Vanpouille , C. , Laghi , L. , Parolin , C. , Melikov , K. , Backlund, P., Vitali, B., Margolis, L., 2019. Extracellular vesicles from symbiotic vaginal lactobacilli inhibit HIV-1 infection of human tissues. Nat. Commun. 10 , 5656 .

[0124] 15 . Yamasaki-Yashiki , S. , Miyoshi , Y. , Nakayama , T. , Kunisawa , J. , Katakura , Y. , 2019. IgA-enhancing effects of membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC15893. Biosci. Microbiota Food Health 38, 23-29.

Claims

Claims

1. Use of extracellular membrane vesicles of at least one probiotic for preventing or reducing the formation of a biofilm on the surface of a material.

2. Use according to claim 1, wherein the at least one probiotic is selected from a probiotic bacterium such as lactobacillus, bifidobacterium, enterococcus, propionibacterium, streptococcus and a bacterium of the genus Bacillus, or a yeast such as Saccharomyces cerevisiae and Saccharomyces boulardi or a mixture thereof.

3. Use according to claim 1 or 2, wherein the biofilm is a bacterial biofilm, a yeast biofilm or a mixed biofilm.

4. Use according to claim 3, wherein: - said bacterial biofilm comprises at least one bacterium chosen from the family of enterobacteria, in particular Salmonella enterica Ente ritidis, Hafnia alvei and / or Citrobacter freundii, the genus Staphylococcus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the genus Bacillus, in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis;- said yeast biofilm comprises the yeast species Candida albicans - said mixed biofilm comprises a mixture of at least one bacterium chosen from the family of enterobacteria, in particular Salmonella enterica Ente rit idis, Hafnia alvei and / or Citrobacter freundii, the genus Staphylococcus, in particular Staphylococcus aureus or Staphylococcus epidermidis, the genus Bacillus, in particular Bacillus cereus or Bacillus subtilis, the genus Pseudomonas, in particular Pseudomonas aeruginosa and the genus Enterococcus, in particular Enterococcus faecalis, and the yeast species Candida albicans.;

5. Use according to any one of the preceding claims, wherein the extracellular membrane vesicles are produced by a method comprising the following steps: (a) culturing at least one probiotic under conditions suitable for the production of extracellular membrane vesicles, (b) separation of the at least one probiotic and the extracellular membrane vesicles produced in step (a) and, (c) purification and concentration of the extracellular membrane vesicles.

6. Use according to claim 5, wherein said purification and concentration step comprises at least one step selected from differential centrifugation, density gradient, size exclusion chromatography, ultrafiltration, immunocapture and precipitation.

7. Use according to any one of the preceding claims, wherein said material is selected from metals, metal alloys, polymers, glass, ceramics, and foods.

8. A method of treating a surface of a material to prevent or reduce the formation of a biofilm on said surface, said method comprising a step of bringing extracellular membrane vesicles from at least one probiotic into contact with said surface.

9. Material comprising on its surface extracellular membrane vesicles of at least one probiotic, or in which extracellular membrane vesicles of at least one probiotic are incorporated, in which said vesicles form a layer with a thickness of between 10 and 500 nm on said surface.

10. Material according to claim 9, selected from a packaging material, especially food packaging, a water pipe, a heat exchanger, a pipeline, a catheter, a dressing, a cream and a medical device.

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