Bacillus-based product having virucide activity against enveloped virus and applications thereof

Abstract

The invention relates to new isolated strains of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, to a bacterial preparation comprising at least one of these strains, to their uses, in particular as active ingredient in a probiotic-based product having a virucidal activity against enveloped viruses, and to a process for cleaning and / or preventing from contamination and / or sanitizing a surface using at least one of these strains.

Description

[0001] BACILLUS-BASED PRODUCT HAVING VIRUCIDE ACTIVITY AGAINST ENVELOPED VIRUS AND APPLICATIONS THEREOF

[0002] FIELD OF THE INVENTION

[0003] The invention belongs to the field of probiotic products.

[0004] In particular, the invention relates to five new isolated strains of sporeforming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, to a bacterial preparation comprising at least one of these five strains of spore-forming bacteria of the Bacillus species, to their uses, in particular as active ingredient in a probiotic-based product having a virucidal activity against enveloped viruses, and to a process for cleaning and / or preventing from contamination and / or sanitizing a surface using at least one of these five strains.

[0005] TECHNICAL BACKGROUND

[0006] The detergent industry today faces a constant challenge to meet the requirements and standards relating to cleanliness and hygiene.

[0007] Current surface cleaning and disinfection products have shown their limits, particularly in the fight against the recontamination of surfaces by pathogens. In fact, the products traditionally used for surface disinfection only have a transient effect since they only ensure the elimination of pathogens already in place without preventing rapid recontamination of surfaces by pathogenic agents.

[0008] In addition, traditional disinfectant products have the disadvantage of being dangerous products (irritant, corrosive risk, etc.) containing molecules toxic to humans and the environment (chlorinated products, quaternary ammoniums, glutaraldehyde, etc.). In addition, these products are difficult to biodegrade.

[0009] Furthermore, the chemical agents contained in these disinfection products can react with other molecules used in cleaning, which results in an inhibition of their disinfectant activity and potentially an additional toxic risk for users.

[0010] Another disadvantage of using traditional chemical-based disinfectant compositions is the fact that they require a long contact time with the surface to be cleaned, up to 15 minutes. Thus, their effectiveness is compromised if this time is not respected. In practice, legislation is increasingly strict regarding the use of these disinfection products. There is therefore a growing demand from cleaning professionals and consumers to use products that are effective, but safer for humans and the environment.

[0011] In this context, alternative solutions have been sought.

[0012] Thus, cleaning products use a so-called "bio-control" approach, based on competitive antagonism between probiotic (non-pathogenic) microorganisms present in the product which colonize surfaces and other potentially pathogenic bacterial species.

[0013] There are products on the market used for cleaning and sanitizing surfaces in which spores of bacteria of the genus Bacillus are associated with a detergent formulation. As examples, one can mention the range of products PROBISANA ® sold by Chrisal N.V and the product PROBIO MULTI ® sold by Greenspeed, both containing a selection of natural probiotics as active ingredients.

[0014] International application WO 2016 / 170479 describes a product for cleaning, sanification and hygienization of surfaces or ambiants comprising a combination of non-ionic, cationic and amphoteric surfactant, in association with probiotic bacteria spores of the Bacillus genus B. subtilis, B. megaterium and B. pumilis species) and selected bacteriophages.

[0015] International application WO2021 / 243324 discloses an aqueous microbial cleaning composition comprising bacterial spores and a surfactant selected among polyalkylglucosides and sodium lauryl ether sulfates.

[0016] The detergent-based products on the marked or described in these prior art documents use a "bio-control" approach based on the fact that the probiotic (no pathogenic) microorganisms present therein can colonize the surfaces after said surfaces have been cleaned by the action of surfactants, thus competing with proliferation of other species potentially dangerous for people health.

[0017] However, the drawbacks of these products are that they involve the use of surfactants and detergents that have a non-negligible impact on the environment and health, that the microorganisms they comprise do not have a proven intrinsic virucidal activity and that their activity could be impacted by the presence of surfactants and detergents. With the recent appearance of pandemic caused by coronavirus (such as SAR.S-CoV-2), and more generally to fight against contaminations by enveloped viruses, there is a need for a probiotic product that, in addition to bactericide and cleaning properties, has also a virucidal activity against enveloped viruses.

[0018] OBJECTS AND SUMMARY

[0019] An object of the present invention is therefore to provide probiotic product that can be used to clean, and / or preventing from contamination and / or sanitizing surfaces without any human, animal and environmental toxicity.

[0020] The Applicants have isolated five new strains of spore-forming bacteria of the Bacillus species that have a virucidal activity against enveloped viruses.

[0021] A first object of the present invention are isolated strains of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, wherein said strains are selected from Bacillus altitudinis (P9), deposited under the number LMG P-33406 at the Belgian Coordinated Collections of Microorganisms (BCCM) on December 8, 2023, Bacillus subtilis (P13), deposited under the number LMG P-33407 at the BCCM on December 8, 2023, Bacillus pumilus (P14), deposited under the number LMG P-33408 at the BCCM on December 8, 2023, Bacillus nakamurai (P17), deposited under the number LMG P- 33409 at the BCCM on December 8, 2023, and Bacillus safensis (P18), deposited under the number LMG P-33410 at the BCCM on December 8, 2023.

[0022] A second object of the present invention is a bacterial preparation comprising at least one strain of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, wherein said at least one strain is selected in the group consisting of the five following strains: Bacillus altitudinis (P9), deposited under the number LMG P-33406 at the Belgian Coordinated Collections of Microorganisms (BCCM) on December 8, 2023, Bacillus subtilis (P13), deposited under the number LMG P-33407 at the BCCM on December 8, 2023, Bacillus pumilus (P14), deposited under the number LMG P- 33408 at the BCCM on December 8, 2023, Bacillus nakamurai (P17), deposited under the number LMG P-33409 at the BCCM on December 8, 2023, and Bacillus safensis (P18), deposited under the number LMG P-33410 at the BCCM on December 8, 2023 A third object of the present invention is the use of at least one of these five strains as defined according to the first object of the present invention, as active ingredient in a probiotic-based non therapeutical product having a virucidal activity against enveloped viruses.

[0023] A fourth object of the present invention is the use of a bacterial preparation according to the second object of the present invention, as active ingredient in a probiotic-based non therapeutical product having a virucidal activity against enveloped viruses.

[0024] A fifth object of the present invention is a process for cleaning and / or preventing from contamination and / or sanitizing a surface, said process comprising at least one step of applying on said surface, at least one strain as defined according to the first object of the present invention or at least one bacterial preparation according to the second object of the present invention.

[0025] A sixth object of the present invention is the strains as defined according to the first object of the present invention, for use as a medicament.

[0026] A seventh object of the present invention is the bacterial preparation as defined according to the second object of the present invention, for use as a medicament.

[0027] DEFINITIONS

[0028] The definitions below correspond to the meaning generally used in the context of the invention and are to be taken into account, unless another definition is explicitly indicated.

[0029] The terms "about", used in reference to a measurable value such as a quantity, a duration, and other similar values, must be understood as encompassing measurement uncertainties of ± 20% or ± 10%, preferably ± 5%, even more preferably ± 1% of the specified value.

[0030] The term "isolated" must be understood in the context of the invention as a synonym for removed or extracted from its environment or natural state. For example, a strain of isolated bacteria is a strain of bacteria extracted from the natural environment in which it is usually found, whether it is a surface or animal for example. Thus, a strain of bacteria naturally present on a surface or in a living animal is not a strain of bacteria within the meaning of the invention, while the same strain of bacteria, partially or completely separated from the others elements present in its natural context, is for its part "isolated" within the meaning of the invention.

[0031] In the context of the invention, the following abbreviations are used for the most common nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0032] The terms "homologous", "identical" and "identity" refer to sequence similarity or sequence identity between two nucleic acid molecules. When a position in each of the two compared sequences is occupied by the same base (for example, when a position in each of the two DNA molecules is occupied by an adenine), then the molecules are homologous or identical for this position. The percentage of identity between two sequences is a function of the number of corresponding positions shared by the two sequences, and corresponds to this number divided by the number of positions compared and multiplied by 100. For example, if 6 out of 10 of the positions in two matched sequences are identical, then the two sequences are 60% identical. Typically, the comparison is performed by aligning the two sequences to give maximum homology / identity.

[0033] DETAILED DESCRIPTION

[0034] As already stated the Applicants have identified new microorganism, in this case five new strains of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses. To the Applicant's knowledge, these are the first strains of the Bacillus species having a virucidal activity against enveloped viruses in addition to antagonist activity against several families of bacteria known to be pathogens likely to be present on surfaces to be cleaned.

[0035] Thus, according to a first object, the invention relates to isolated strains of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, wherein said strains are selected from Bacillus altitudinis (P9), deposited under the number LMG P-33406 at the Belgian Coordinated Collections of Microorganisms (BCCM) on December 8, 2023, Bacillus subtilis (P13), deposited under the number LMG P-33407 at the BCCM on December 8, 2023, Bacillus pumilus (P14), deposited under the number LMG P-33408 at the BCCM on December 8, 2023, Bacillus nakamurai (P17), deposited under the number LMG P- 33409 at the BCCM on December 8, 2023, and Bacillus safensis (P18), deposited under the number LMG P-33410 at the BCCM on December 8, 2023.

[0036] According to a particular embodiment of the present invention, said strains have a consensus nucleotidic sequence SEQ ID N°1 or a nucleotidic sequence having at least 95% of identity, preferably at least 96%, 97%, 98%, 99%, or even 99.5%, or even 99.9% of identity with said nucleotidic sequence SEQ ID N°l.

[0037] SEQ ID N°1 corresponds to the common partial sequence of the genes that share at least 95% of identity with the five strains according to the invention.

[0038] Advantageously, the strain Bacillus altitudinis, deposited under the number LMG P-33406 (strain P9) has the sequences SEQ ID N°2 to SEQ ID N° 9 or sequences having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with these sequences.

[0039] Advantageously, the strain Bacillus subtilis, deposited under the number LMG P-33407 (strain P13) has the sequences SEQ ID N°10 to SEQ ID N°12 or sequences having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with these sequences.

[0040] Advantageously, the strain Bacillus safensis, deposited under the number LMG P-33408 (strain P14) has the sequences SEQ ID N°13 to SEQ ID N°17 or sequences having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with these sequences.

[0041] Advantageously, the strain Bacillus nakamurai, deposited under the number LMG P-33409 (strain P17) has the sequences SEQ ID N°18 to SEQ ID N°90 or sequences having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with these sequences.

[0042] Advantageously, the strain Bacillus pumilus, deposited under the number LMG P-33410 (strain P18) has the sequence SEQ ID N°91 or sequences having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with these sequences.

[0043] The strains are considered as safe because they are free of virulence factors.

[0044] In according with the belonging of the strains of the invention to the Bacillus species, these are advantageously capable of growing in anaerobic and / or aerobic conditions, on classic rich media containing peptones and hydrolysates of animal or plant proteins, yeast extracts and salt, or on specific media or culture of Bacillus type strains well known to those skilled in the art.

[0045] As already mentioned, the strains according to the invention are capable of sporulating or forming spores, for example after cultivation in a medium promoting sporulation such as Difco Sporulation Medium (DSM).

[0046] The spore suspensions of these strains are stable between pH 4 and 10.

[0047] A first essential characteristic of the strains of the invention lies in their virucidal activity against enveloped-viruses, individually or in admixture.

[0048] A second essential characteristic of the strains of the invention lies in their virucidal activity against enveloped-viruses, individually or in admixture, in absence of additional substance like surfactants or detergents.

[0049] A third essential characteristic of the strains of the invention lies in their virucidal activity against enveloped-viruses, when they are used as spores. The spore germination corresponds to the virucidal activity observed.

[0050] The virucidal activity is dependent on the concentration of bacterial spores used.

[0051] Another characteristic of the strains of the invention lied in their antagonistic activity against surface pathogens.

[0052] In the context of the invention, the terms "antagonistic activity", "antagonism" or "antagonistic power" have an equivalent meaning. They target the ability of the strains to reduce or even inhibit the growth and / or activity of different microorganisms, advantageously a surface pathogen.

[0053] This action can be carried out by production and secretion of inhibitory molecules (bacteriocins, antibiotics, toxins, etc.) and / or by competition.

[0054] By "competition", it is meant a competitive relationship between microorganisms when they depend on the use of the same resource (nutrients) and / or the same territory / space, thus reducing the availability of nutrients and / or space for each of the competitors, without there necessarily being a direct interaction between the competing microorganisms. In practice, the strains according to the invention will occupy the field by consuming the organic materials which will accumulate after cleaning and / or produce antagonistic molecules which will prevent "bad" bacteria from settling in, thus ensuring a long-term effect.

[0055] Surface pathogens are microorganisms in particular bacteria, advantageously belonging to the following families: Staphylococcaceae, Enterococcaceae, Enterobacteriaceae and Pseudomonadaceae.

[0056] Advantageously, the strains according to the invention exhibits antagonistic activity towards pathogenic bacteria of the following bacterial genus: Staphylococcus, Enterococcus, Escherichia and Pseudomonas.

[0057] According to a particular embodiment, the target pathogens are Staphylococcus aureus, Enterococcus hirae, Escherichia coli and Pseudomonas aeruginosa.

[0058] The antagonistic activity of a microorganism, in particular of strains according to the invention, can be demonstrated by different techniques known to those skilled in the art, in particular by a viability qPCR. test.

[0059] Finally, thanks to their enzymatic activities, the strains according to the invention are able to metabolize different substrates such as lipids, proteins, polysaccharides, etc., allowing their use of the cleaning of carious dirt on various surfaces.

[0060] A second object of the present invention is a bacterial preparation comprising a culture of at least one strain of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, wherein said at least one strain is selected in the group consisting of the five following strains: Bacillus altitudinis (P9), deposited under the number LMG P-33406 at the Belgian Coordinated Collections of Microorganisms (BCCM) on December 8, 2023, Bacillus subtilis (P13), deposited under the number LMG P-33407 at the BCCM on December 8, 2023, Bacillus pumilus (P14), deposited under the number LMG P- 33408 at the BCCM on December 8, 2023, Bacillus nakamurai (P17), deposited under the number LMG P-33409 at the BCCM on December 8, 2023, and Bacillus safensis (P18), deposited under the number LMG P-33410 at the BCCM on December 8, 2023.

[0061] According to a particular and preferred embodiment of the present invention, said bacterial preparation is free from any surface-active agent (or surfactant or detergent). Indeed, and as it is demonstrated in the following illustrative examples, the strains according to the invention does not need to be used in combination with a surfactant or a detergent to exhibit their virucidal and bactericidal activities.

[0062] The strains of the invention can be used individually or in admixture.

[0063] Therefore, according to an embodiment of the present invention, the bacterial preparation comprises a culture of at least one said five strains, at least two of said five strains, at least three of said five strains, at least four of said five strains or even more preferably said five strains.

[0064] In the bacterial preparation, said strains can be in the form of spores and / or of vegetative cells and / or of cells in the transitional sporulation phase.

[0065] According to a preferred embodiment of the invention, said strains are in the form of spores and are suspended in an aqueous medium, preferably an aqueous medium.

[0066] An aqueous medium is defined as a medium in which water constitutes the majority solvent. Such an aqueous medium may also contain a smaller quantity of a solvent other than water, capable of evaporating after application of the bacterial preparation to the surface to be cleaned, thus promoting its drying, advantageously a vegetable solvent such as for example isopropanol or ethanol.

[0067] The bacterial preparation according to the invention may further comprise one or several additional ingredients except surfactants, such as a preservative, a perfume, a dye, a thickening agent, an enzyme, a sequestering agent, an abrasive, an acidity corrector, etc. On this occasion, however, the person skilled in the art will ensure that the addition of these additional ingredients does not alter the intrinsic properties of the bacterial preparation, and in particular, the virucidal properties of the strain or strains it contains.

[0068] According to a particular embodiment, the preservative present in a bacterial preparation according to the invention, making it possible to stabilize the preparation without damaging the at least one strain contained therein, can be for example sodium benzoate, potassium sorbate, isothiazolinone type molecules, propylene glycol, phenoxyethanol or their mixtures. According to a particular embodiment, the sequestering agent used in a bacterial preparation according to the invention may be ethylene diamine tetraacetic acid (EDTA), sodium citrate or a mixture thereof.

[0069] According to a particular embodiment, the abrasive used in a bacterial preparation according to the invention may be calcium carbonate, magnesium carbonate or a mixture thereof.

[0070] According to another particular embodiment, the acidity corrector used in a bacterial preparation according to the invention may be lactic acid, malic acid, citric acid, sodium hydroxide or a mixture thereof.

[0071] According to another particular embodiment, a thickener such as xanthan or polyvinyl alcohol or any other stabilizing agent which prevents the redeposition of the bacterial strain can be added to the bacterial preparation according to the invention.

[0072] In the bacterial preparation of the invention, the concentration of said strain(s) is preferably from about 102to 1012CFU / mL, even more preferably from about 104to 108CFU / mL.

[0073] Throughout the rest of the description, the abbreviation CFU stands for "colony forming unit".

[0074] The terms "CFU" and "number of bacteria" can be used interchangeably.

[0075] According to a particular embodiment, the bacterial cells and / or spores, or even the extracellular extracts, present in the culture media can be used directly, concentrated or purified by conventional methods, such as centrifugation, filtration, or evaporation.

[0076] According to another embodiment, the bacterial preparation according to the invention comprises as the only microorganism, advantageously as the only probiotic strain, at least one of the five strains as defined according to the first object of the present invention.

[0077] In other words, a bacterial preparation according to the invention may not contain any other microorganisms, in particular no other bacteria.

[0078] According to an advantageous embodiment, a bacterial preparation according to the invention does not contain any other bacteria of the Bacillus species. According to an alternative embodiment, said bacterial preparation comprises, in addition to the at least one strain according to the invention, at least one other microorganism, for example another probiotic strain. In particular, another probiotic strain may be a Bacillus type strain different from that of the invention advantageously presented in sporulated form. Among these additional strains, one can mention a strain of Bifidobacterium, Carnobacterium, Corynebacterium, Gluconobacter, Lactococcus, Lactobacillus (L.), Leuconostoc, Oenococcus, Pasteuria, Pediococcus (P.), Propionibacterium, Streptococcus, Xanthomonas and their mixtures.

[0079] The pH of the bacterial preparation according to the invention is preferably from about 4 to 10, even more preferably from about 5 to 9.

[0080] A third object of the present invention is the use of at least one strain as defined according to the first object of the present invention, as active ingredient in a probiotic-based non therapeutical product having a virucidal activity against enveloped viruses.

[0081] A fourth object of the present invention is the use of a bacterial preparation according to the second object of the present invention, as active ingredient in a probiotic-based non therapeutical product having a virucidal activity against enveloped viruses.

[0082] According to a preferred embodiment of the invention, said enveloped viruses include influenza, human cytomegalovirus (HCMV), HIV, respiratory syncytial virus (RSV), vaccinia virus, arbovirus and human coronaviruses such as NL63, 229E, OC43, HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2, but not limited to.

[0083] According to these uses, said product further has bactericide properties and / or cleaning properties.

[0084] A fifth object of the invention is a process for cleaning and / or preventing from contamination and / or sanitizing a surface, said process comprising at least one step of applying on said surface, at least one strain as defined according to the first object of the present invention or at least one bacterial preparation according to the second object of the present invention. The term "cleaning" refers to the use of such a composition for cleaning surfaces, particularly inert surfaces. The term cleaning also encompasses metabolization of organic substrates (dirt) present on a surface.

[0085] The terms "preventing from contamination" means avoiding or diminishing the risk of contamination of surfaces, particularly inert surfaces, by a pathogen, in particular by pathogenic bacteria belonging to at least the families Staphylococcaceae, Enterococcaceae, Enterobacteriaceae and Pseudomonaceae.

[0086] The term "sanitizing" refers to the fact that due to the presence of the strains according to the invention in the product, the application of such a product makes it possible to avoid and / or reduce the proliferation of pathogens on a surface and eliminate pathogens present on a surface, in particular pathogenic bacteria belonging to at least the families Staphylococcaceae, Enterococcaceae, Enterobacteriaceae and Pseudomonaceae.

[0087] According to a preferred embodiment of the invention, said strain or said bacterial preparation is applied on said surface in an amount of about 4.103to 4.1011CFU / m2of surface, even more preferably in an amount of about 4.106to 4.108CFU / m2.

[0088] According to a particular embodiment, the inert surface is a community surface, for example hospitals, nurseries or even accommodation establishments for the elderly, or a surface of a public or private establishment, for example kitchens.

[0089] According to the process of the invention, said strains or said bacterial preparation can be applied according to the usual procedures for cleaning and / or sanitizing and / or preventing from contamination, inert surfaces in addition to or replacing disinfection procedures known to those skilled in the art.

[0090] The process according to the invention, and therefore the use of the strains or of the bacterial preparation according to the invention, makes it possible to protect inert surfaces from contamination by enveloped-viruses pathogenic bacteria, with a lasting effect up to at least 1 day, 2 days, 3 days, 4 days or even at least 5 to 7 days.

[0091] A sixth object of the present invention is the strains as defined according to the first object of the present invention, for use as a medicament. A seventh object of the present invention is the bacterial preparation as defined according to the second object of the present invention, for use as a medicament.

[0092] According to a particular embodiment of the present invention, said medicament is for the treatment of diseases caused by enveloped-viruses, such as influenza, human cytomegalovirus (HCMV), HIV, respiratory syncytial virus (RSV), vaccinia virus, arbovirus and human coronaviruses such as NL63, 229E, OC43, HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2, but not limited to.

[0093] The manner in which the invention can be carried out and the advantages which result from it will become clearer from the examples of implementation which follow, given for informational and non-limiting purposes, in support of the appended figures.

[0094] BRIEF DESCRIPTION OF THE FIGURES

[0095] - Figure 1 is a graph representing virucidal activity of strains P9, P13, P14, P17 and P18 individually or in admixture on phi6 bacteriophages comparatively to reference strains DSM 31005, DSM 361, DSM 108493 and DSM 26896. On this graph, the bacteriophage phi6 concentration (log PFU / mL) after 24 hours of contact with bacterial strains is given for each tested strain. The grey dot line represents the initial concentration of bacteriophages. The dark broken line represents the bacteriophage concentration after 24h at 30°C in PBS (negative control).

[0096] - Figure 2 is a graph representing virucidal activity of strains P9, P13, P14, P17 and P18 individually or in admixture on MS2 bacteriophages (non-enveloped bacteriophage) comparatively to a commercial Bacillus biomass. On this graph, the MS2 concentration (log PFU / mL) after 24 hours of contact with bacterial strains is given for each tested strain. The dark broken line represents the bacteriophage concentration after 24h at 30°C in PBS.

[0097] - Figure 3 is a graph representing the results of the bactericide activity of the strains P13, P14, P17 and P18 individually or in admixture comparatively to a commercial Bacillus biomass and to bleach against S. aureus. On this graph, the S. aureus concentration (log CFU / mL) is given for each tested strain / product. Figure 3A presents results of simultaneous co-culture respectively with S. aureus and Figure 3B presents results of a 3-hour pretreatment with the strains assessed. The grey dot line represents the initial concentration of S. aureus. The dark broken line represents the S. aureus concentration after 24h at 30°C in PBS.

[0098] - Figure 4 is a graph representing the results of the bactericide activity of the strains P13, P14, P17 and P18 individually or in admixture comparatively to a commercial Bacillus biomass and to bleach against E. hirae. On this graph, the E. hirae concentration (log CFU / mL) is given for each tested strain / product. Figure 4A presents results of simultaneous co-culture respectively with E. hirae and Figure 4B presents results of a 3-hour pre-treatment with the strains assessed. The grey dot line represents the initial concentration of S. aureus. The dark broken line represents the S. aureus concentration after 24h at 30°C in PBS.

[0099] - Figure 5 is a graph representing the results of the bactericide activity of the strains P13, P14, P17 and P18 individually or in admixture comparatively to a commercial Bacillus biomass and to bleach against P. aeruginosa. On this graph, the P. aeruginosa concentration (log CFU / mL) is given for each tested strain / product. Figure 5A presents results of simultaneous co-culture respectively with P. aeruginosa and Figure 5B presents results of a 3-hour pre-treatment with the strains assessed. The grey dot line represents the initial concentration of S. aureus. The dark broken line represents the S. aureus concentration after 24h at 30°C in PBS.

[0100] - Figure 6 is a graph representing the results of the bactericide activity of the strains P13, P14, P17 and P18 individually or in admixture comparatively to a commercial Bacillus biomass and to bleach against E. coll. On this graph, the E. coli concentration (log CFU / mL) is given for each tested strain / product. Figure 6A presents results of simultaneous co-culture respectively with E. coli and Figure 6B presents results of a 3-hour pre-treatment with the strains assessed. The grey dot line represents the initial concentration of S. aureus. The dark broken line represents the S. aureus concentration after 24h at 30°C in PBS.

[0101] - Figure 7 is a graph representing virucidal activity of strains P9, P13, P14, P17 and P18 on Influenza virus H3N2 comparatively to reference strains DSM31005 and DSM361 and to the admixture of the five strains. On this graph, the log reduction of TCID50 of Influenza after 24 hours of contact with bacterial strains is given for each tested strain. Bacterial strains are tested as spores (hatched bars) or vegetative cells in exponential (white bars) or stationary phase (black bars). - Figure 8 is a graph representing virucidal activity of strain P9 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid line). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0102] - Figure 9 is a graph representing virucidal activity of strain P13 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0103] - Figure 10 is a graph representing virucidal activity of strain P14 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0104] - Figure 11 is a graph representing virucidal activity of strain P17 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0105] - Figure 12 is a graph representing virucidal activity of strain P18 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0106] - Figure 13 is a graph representing virucidal activity of two examples of PCHS products on phi6 bacteriophages according to comparative example 6. On this graph, the bacteriophage phi6 concentration (log PFU / mL) after 24 hours of contact with bacterial strains is given for each product. A control consists of a suspension of phi6 in TSB. EXAMPLES

[0107] EXAMPLE 1: Bacterial isolation. and bioinformatic a is of the five strains of the invention

[0108] 1.1. Material and methods

[0109] Surfaces from various surfaces (kitchen swab, cat and dog skins were swabbed, and bacteria were isolated on tryptone soya agar (TSA) plate. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, 51306) according to the manufacturer's instructions. DNA was quantified with the Qubit® 2.0 Fluorometer (Invitrogen, Q32866) and the Qubit® dsDNA HS Assay kit (Life Technologies, Q32851). The DNA concentration was adjusted to be within the range of three and 17 ng / pL for subsequent sequencing. Libraries for the short reads sequencing were prepared using the Nextera™ DNA Flex Library Prep Kit (Illumina) and sequenced on the Miseq® platform (Illumina) achieving 250-bp paired-end reads. The quality of the raw reads was checked with FastQC ® software before and after processing the sequences with Trimmomatic ® software. The DNA extracted was also sequenced in long reads by using the Oxford Nanopore MinlON Mklc. Long reads were de novo assembled by QIAGEN CLC Genomics software and polished with short reads. The genome annotation was done by Prokka ® software. The antibiotic resistance genes were determined by CARD (Comprehensive Antibiotic Resistance Database). Core genes shared by the five bacteria have been determined by the high-speed stand-alone pan genome pipeline Roary ® software, running with standard parameters.

[0110] 1.2. Results

[0111] Genomic characterization of bacteria

[0112] The strain P9 was isolated on a kitchen swab, strains P13 and P14 on dog skin and strains P17 and P18 on cat skin.

[0113] All strains selected were considered risk group 1 and P18 was classified as QPS (Qualified Presumption of Safety) strain. All strains were free of virulence factors, intrinsic resistance to phenicol was observed in P9 and P18. Nevertheless, multiple antibiotic resistance genes were detected in P13 and P14.

[0114] Genome characterization of bacteria for each strain is detailed in Table 1 below: TABLE 1

[0115] (*): MDR = Multi-drug resistance: aminoglycoside, cephalosporin, fluoroquinolone, lincosamide, macrolide, nucleoside, oxazolidinone, peptide antibiotic, phenicol, pleuromutilin, streptogramin and tetracycline. (**): MDR. = Multi-drug resistance: carbapenem, cephalosphorin, cephamyicin, fluoroquinolone, glycopeptide, lincosamide, macrolide, monobactam, nucleoside, oxazolidinone, phenicol, pleuromutilin, streptogramin and tetracycline.

[0116] The five strains P9, P13, P14, P17 and P18 share 71 genes with an identity above 95 %. The consensus sequence (common sequence of the five strains) corresponds to SEQ ID n°l.

[0117] The sequences of strains P9, P13, P14, P17 and P18 correspond to SEQ ID n°2 to SEQ ID N°9 for strain P9, to SEQ ID n°10 to SEQ ID N°12 for strain P13, to SEQ ID n°13 to SEQ ID N°17 for strain P14, to SEQ ID n°18 to SEQ ID N°90 for strain P17 and to SEQ ID n°91 for strain P18. EXAMPLE 2: Study of the virucidal activity of the five strains of the invention

[0118] 2.1. Material and methods

[0119] The virucidal activity of the five strains according to the invention was tested on bacteriophages MS2 and phi6 representing non-enveloped and enveloped viruses, respectively. First, viruses were put in contact: 3 mL of each strain P9, P13, P14, P17 or P18, diluted in tryptone soya broth (TSB) to reach a 106spores / mL concentration were added with 3 mL of each virus diluted in TSB to reach a 106plaque-forming unit (PFU) / mL concentration into a 6 wells-plate. Two controls to assess the stability of bacteriophages and the growth of tested strains were made by adding 3 mL of virus and 3 mL of each strain, respectively, to 3 mL of TSB. Plates were incubated at 30°C with an agitation at 80 rpm during 24 h. The virucidal activity of the five strains according to the invention on phi6 bacteriophages was compared to the virucidal activity of an admixture of the five strains P9, P13, P14, P17 or P18 each at a concentration of 2.105CFU / mL in the mixture and to 4 reference strains:

[0120] - Bacillus pumilus DSM 31005, denoted DSM 31005

[0121] - Bacillus pumilus DSM 361, denoted DSM 361

[0122] - Bacillus altitudinis DSM 108493, denoted DSM 108493 and

[0123] - Bacillus altitudinis DSM 26896, denoted DSM 26896.

[0124] The virucidal activity of the strains P9, P14, P17 and P18 according to the invention on MS2 bacteriophages was compared to the virucidal activity of the admixture of the five strains P9, P13, P14, P17, P18 and the Bacillus biomass BEBS2 found in the products of the brand Provilan ® sold by Probiotic Group Luxembourg (BEBS2 biomass).

[0125] Then, each of the cultures was filtrated to remove the strains before titration of lysis plaques on E. coli K12 (bacteriophage MS2) and Pseudomonas syringae DSM 21482 (bacteriophage phi6).

[0126] 2.2. Results

[0127] The results are given on figures 1 and 2 annexed.

[0128] Figure 1 is a graph representing virucidal activity of strains P9, P13, P14, P17 and P18 on phi6 bacteriophages comparatively to the admixture of the five strains and to reference strains DSM 31005, DSM 361, DSM 108493 and DSM 26896. On this graph, the bacteriophage phi6 concentration (log PFU / mL) is given for each tested strain. The grey dot line represents the initial concentration of bacteriophages. The dark broken line represents the bacteriophage concentration after 24h at 30°C in PBS (negative control). Figure 2 is a graph representing virucidal activity of strains P9, P13, P14, P17 and P18 on MS2 bacteriophages comparatively to BEBS2 biomass and to the admixture of the five strains. On this graph, the MS2 concentration (log PFU / mL) after 24 hours of contact with bacterial strains is given for each tested strain. The dark broken line represents the bacteriophage concentration after 24h at 30°C in PBS.

[0129] These results show that the admixture of the five strains of the invention, and the strains P9, P14, P17 and P18 alone, don't have virucidal activity on the non-enveloped bacteriophage MS2 (Fig. 2). On the contrary, results presented on Fig. 1 show that the five strains of the invention have a high virucidal activity on the enveloped bacteriophage phi6.

[0130] EXAMPLE 3: Study of the enzymatic activity of the five strains of the invention

[0131] 3.1. Material and methods

[0132] The enzymatic activity of the five strains P9, P13, P14, P17 and P18 was evaluated on different media: i) Lipid degradation:

[0133] Tributyrin agar medium had the following composition: tributyrin 10 g.L’1, yeast extract 3 g.L’1, peptone 5 g.L’1, agar 15 g.L’1. ii) Casein degradation

[0134] Skim milk agar medium has the following composition: skim milk 28 g.L-1, casein enzymatic hydrolysate 5 g.L’1, yeast extract 2.5 g.L’1, glucose 1 mL.L’1, agar 15 g.L’1. iii) Lecithinase activity:

[0135] Egg yolk agar medium had the following composition: pancreatic digest of casein 10 g.L’1, meat extract 3 g.L’1, yeast extract 1 g.L’1, NaCI 5 g.L’1, egg yolk emulsion 100 mL.L’1, agar 15 g.L’1. iv) Starch hydrolysis:

[0136] Starch agar medium has the following composition: peptone 5 g.L’1, starch 10 g.L’1, agar 10 g.L’1. v) Haemolysis (ThermoFisher):

[0137] Columbia sheep blood agar medium has the following composition: Columbia agar + 7% sheep blood. vi) Urease activity:

[0138] Urea agar medium has the following composition: urea agar base 24 g.L’1, 40% urea solution 50 mL.L’1. vii) Cellulolytic activity:

[0139] Carboxymethylcellulose agar medium had the following composition: carboxymethylcellulose 26 g.L-1, yeast extract 1 g.L’1, agar 15 g.L’1).

[0140] Each strain was resuspended in PBS to obtain an OD600nm (optical density measured at a wavelength of 600 nm in 1 cm light path) between 0.5 and 0.6 and 10 pL were dropped in the middle of a Petri dish. Plates were incubated one week at 20°C. The starch hydrolysis and cellulolytic activity were revealed with lugol and Congo red / acridine orange, respectively. The list of enzymatic activities of the five strains according to the invention was completed by API ZYM® (Biomerieux 25200) according to the manufacturer recommendations: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, o-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, o-galactosidase, p-galactosidase, p- glucuronidase, o-glucosidase, p-glucosidase, N-acetyl-p-glucosaminidase, o- mannosidase, and o-fucosidase.

[0141] 3.2. Results

[0142] The results are reported in table 2 below in which the sign "+" means that the strain presents the indicated enzymatic activity while the sign means that the strain does not present the indicated enzymatic activity: TABLE 2

[0143] These results show that the five strains demonstrate interesting enzymatic activities allowing the cleaning of various dirt: milk, blood, starch or cellulose. EXAMPLE 4: Study of the bactericidal activity of the five strains of the invention

[0144] 4.1. Material and methods

[0145] The biocidal activity of the five strains according to the invention, individually and in admixture was tested against Enterococcus hirae (ATCC10541), Escherichia coli (NCTC10536,) Staphylococcus aureus (ATCC6538), and Pseudomonas aeruginosa (ATCC15442) was quantified by viability qPCR with primers listed in the following Table 3:

[0146] TABLE 3

[0147] The viability qPCR can only detect DNA from live cells thanks to a pretreatment with a photoreactive membrane-impermeant DNA binding dye (PMAxx ®, Biotium). Because the dye is cell membrane-impermeable, when a sample containing both live and dead bacteria is treated, only dead bacteria with compromised cell membranes are susceptible to DNA modification. The dye intercalates into dsDNA and forms a covalent linkage upon exposure to intense visible light, inhibits PCR amplification.

[0148] 1 mL of target pathogen at 107CFU / mL were cocultured with 1 mL of strain (P9, P13, P14, P17 and P18 individually or in admixture) ( 107spores / mL) in 8 mL liquid culture (TSB), meaning an initial concentration of both target pathogens and strains at 106CFU / mL or spores / mL, respectively. Each co-culture was incubated for 24 hours at 30°C (110 rpm). To test the preventive action of the strains according to the invention, the culture was pre-treating with Bacillus strains three hours before adding the pathogen. After the co-culture, samples were treated with the PMAxx® dye, DNA was extracted (Qiagen QIAamp Mini Kit, 51306), and qPCR were performed. Bleach treatment was used as positive control for the biocide and culture in PBS is used as negative control showing the normal growth of pathogen. All experiments were done in triplicate. Biocide activity of the strains P9, P13, P14, P17 and P18 individually and in admixture were quantified by viability qPCR and compared with biocide activity of biomass BEBS2 and bleach.

[0149] 4.2. Results

[0150] The results are given on figures 3 to 6 annexed.

[0151] On these figures, the concentration of the target pathogen (in log CFU / mL) is given for each tested coculture. Strains assessed are P9, P13, P14, P17, P18 independently and in admixture, in comparison with biomass BEBS2 and with bleach as positive control for biocide. Figures 3A, 4A, 5A and 6A present results of simultaneous co-culture respectively with S. aureus, E. hirae, P. aeruginosa and E. coli while figures 3B, 4B, 5B and 6B present results of the 3-hour pre-treatment with the strains assessed. The grey dot line represents the initial concentration of the target pathogen and the dark broken line represents the S. aureus concentration after 24h at 30°C in PBS.

[0152] The presence in co-culture of the strains P9 to P18 (alone or in admixture) significantly decreases the number of S. aureus. Strains P9, P13, P14 and P17 reduce the S. aureus concentration by more than 5 log (minimal reduction required to be a biocide according to EU standards). In case of a pre-treatment with strains 3 hours before the contamination with S. aureus, the treatment also significantly reduces the pathogen growth. The presence of the strains according to the invention reduces the population of S. aureus in comparison to a pathogen monoculture but also in comparison to the initial contamination of pathogen. Concerning the other pathogens, the presence of the strains according to the invention does not allow to reduce the pathogen concentration above the initial contamination except in the case of a strain pre-treatment three hours before the E. hirae contamination. EXAMPLE 5: Virucidal activity of the five strains of the invention on Influenza virus

[0153] 5.1. Material and methods

[0154] The virucidal activity of the strain according to the invention was confirmed on Influenza A virus H3N2. The method is based on standard ISO 21702 and ISO 18184. Viral inoculum was composed of Influenza A virus H3N2 A / Hong Kong / 8 / 68 TC adapted ATCC VR-1679 propagated in Minimum Essential Medium (MEM) lx (ThermoFisher, 31095052) and adjusted to a concentration between 106and 5 106Tissue Culture Infectious Dose / mL (TCID50 / mL). 3 mL of this viral inoculum were got in contact with 3 mL of a suspension of the Bacillus strains (spores, exponential or stationary phases) in 6-well microplates and incubated at 30°C under agitation 80 rpm and a relative humidity of not less than 90%. After 24h, all the Bacillus / v rus mixtures were filtered on a 0.2 / 0.8 pm syringe filter and the infectivity titer of virus was measured by the TCID50 method. The suspension is 10-times diluted in MEM IX with 1% Antibiotic - Antimycotic (ATBF) solution. The confluent host cell cultures, MDK (dog kidney origin) cells ATCC CCL-34 were washed three times with MEM IX with 1% ATBF and inoculate with 100 pL of the suspension and its 10-time dilution. Cultures were incubated one hour at 34°C. The supernatant was drained, and culture was washed with MEM IX with 1% of ATBF and then MEM IX with 1% ATBF and trypsin (3 pL / mL) was added to cultivate cells during seven days at 34°C. The TCID50 was calculated by the Behrens and Karber method. The reduction of TCID50 is evaluated by comparison with the TCID50 obtained by contact between virus and TSB.

[0155] The virucidal activity of the five strains according to the invention were evaluated at different steps of the bacterial growth: (a) a spore suspension adjusted at a concentration between 106and 5 106spores / mL in Tryptone Soya Broth (TSB, Oxoid, CM0129); (b) a suspension of the strains in exponential phase obtained by 2 h of culture in TSB at 30°C and 150 rpm; (c) a suspension of the strains in stationary phase obtained by 24 h of culture in TSB at 30°C and 150 rpm.

[0156] 5.2. Results

[0157] The results are presented on Figure 7 annexed. Figure 7 is a graph representing virucidal activity of strains P9, P13, P14, P17 and P18 on Influenza virus H3N2 comparatively to reference strains DSM31005 and DSM361 and to the admixture of the five strains. On this graph, the log reduction of TCID50 of Influenza after 24 hours of contact with bacterial strains is given for each tested strain.

[0158] These results show that the admixture of the five strains of the invention, and the strains P9, P13, P14, P17 and P18 alone, have a virucidal activity on Influenza. This activity is higher when spores of the strains were in contact with viruses.

[0159] EXAMPLE 6: Virucidal activity of the five strains of the invention during time and in comparison with bacterial growth

[0160] 6.1. Materials and methods

[0161] The virucidal activity of the five strains according to the invention were evaluated on bacteriophage phi6 as in example 2 but samples were taken after 2, 6, 24 and 48h. The concentration of total bacterial cells was evaluated by growth on TSA. The concentration of spores was evaluated by growth on TSA after pasteurization (80°C, 20 min) of the samples.

[0162] 6.2. Results

[0163] The results are presented on Figures 8 to 13 annexed.

[0164] Figure 8 is a graph representing virucidal activity of strain P9 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid line). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0165] Figure 9 is a graph representing virucidal activity of strain P13 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0166] Figure 10 is a graph representing virucidal activity of strain P14 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0167] Figure 11 is a graph representing virucidal activity of strain P17 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0168] Figure 12 is a graph representing virucidal activity of strain P18 on phi6 bacteriophage at different time after contact between bacteria and viruses. On this graph, the virucidal activity on phi6 bacteriophages is given in log reduction at different time of contact (0, 6, 24 and 48h) (solid lines). For each time, the ratio between spores and vegetative cells are also represented (dash line).

[0169] These results show that the germination of Bacillus spores corresponds to the virucidal activity, consequently the spores need to germinate for virucidal activity.

[0170] EXAMPLE 7: Virucidal activity of detergent compositions containing spores of selected probiotics

[0171] 7.1 Material and method

[0172] The virucidal activity of two commercial Probiotics In Progress (PIP) products of Chrisal (Lommel, Belgium) (PIP Pruex AHS Stabilizer and PIP Pruex Water Plus) as examples of Probiotic Cleaning Hygiene System (PCHS) have been evaluated on bacteriophage phi6. These two PIPs of Chrisal corresponds to the commercial products mentioned in the article by D'Accolti Maria et al. (Viruses, 2021, 13(11), page 2227) and in the article by A. Vendini et al. (Pios One, 2014, 9(9), page el08598). These PIP products are EU Ecolabel detergents containing 107CFU / mL spores of selected probiotics belonging to the Bacillus genus (namely B. subtilis, B. pumilus, and B. megaterium species).

[0173] The same protocol than in example 2 was applied. PCHS products were diluted 10-times before use according to the recommendations contained in the article by D'Accolti Maria et al. article.

[0174] 7.2. Results

[0175] The results are given on Figure 13 annexed. The figure 13 shows that PCHS products diluted 10-times have no virucidal effects on bacteriophage phi6.

[0176]

[0177] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICRO-ORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

[0178]

[0179] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICRO-ORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

[0180]

[0181] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICRO-ORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

[0182]

[0183] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICRO-ORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

[0184]

[0185] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICRO-ORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE

Claims

CLAIMS1. Isolated strains of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, wherein said strains are selected from Bacillus altitudinis (P9), deposited under the number LMG P-33406 at the Belgian Coordinated Collections of Microorganisms (BCCM) on December 8, 2023, Bacillus subtilis (P13), deposited under the number LMG P-33407 at the BCCM on December 8, 2023, Bacillus pumilus (P14), deposited under the number LMG P-33408 at the BCCM on December 8, 2023, Bacillus nakamurai (P17), deposited under the number LMG P-33409 at the BCCM on December 8, 2023, and Bacillus safensis (P18), deposited under the number LMG P-33410 at the BCCM on December 8, 2023.

2. Isolated strains according to claim 1, wherein said strains have a consensus nucleotidic sequence SEQ ID N°1 or a nucleotidic sequence having at least 95% of identity with said nucleotidic sequence SEQ ID N°l.

3. A bacterial preparation comprising a culture of at least one strain of spore-forming bacteria of the Bacillus species having a virucidal activity against enveloped viruses, wherein said at least one strain is selected in the group consisting of the five following strains: Bacillus altitudinis (P9), deposited under the number LMG P-33406 at the Belgian Coordinated Collections of Microorganisms (BCCM) on December s, 2023, Bacillus subtilis (P13), deposited under the number LMG P-33407 at the BCCM on December 8, 2023, Bacillus pumilus (P14), deposited under the number LMG P-33408 at the BCCM on December 8, 2023, Bacillus nakamurai (P17), deposited under the number LMG P-33409 at the BCCM on December 8, 2023, and Bacillus safensis (P18), deposited under the number LMG P-33410 at the BCCM on December 8, 2023.

4. The bacterial preparation according to claim 3, wherein said preparation is free from any surface-active agent.

5. The bacterial preparation according to claim 3 or 4, wherein said preparation comprises said five strains.

6. The bacterial preparation according to any one of claims 3 to 5, wherein said strains are in the form of spores and / or of vegetative cells and / or of cells in the transitional sporulation phase.

7. The bacterial preparation according to claim 6, wherein said strains are in the form of spores and further wherein said spores are suspended in an aqueous medium.

8. The bacterial preparation according to any one of claims 3 to 7, wherein the concentration of said strain is from 102CFU / mL to 1012CFU / mL.

9. Use of at least one strain as defined in claim 1 or 2, as active ingredient in a probiotic-based non-therapeutical product having a virucidal activity against enveloped viruses.

10. Use of a bacterial preparation according to any one of claims 3 to 8, as active ingredient in a probiotic-based non-therapeutical product having a virucidal activity against enveloped viruses.

11. Use according to claim 9 or 10, wherein said product further has bactericide properties and / or cleaning properties.

12. A process for cleaning and / or preventing from contamination and / or sanitizing a surface, said process comprising at least one step of applying on said surface, at least one strain as defined in claim 1 or 2 or at least one bacterial preparation as defined in any one of claims 3 to 8.

13. The process according to claim 12, wherein said strain or said bacterial preparation is applied on said surface in an amount of 4.103to 4.1011CFU / m2of surface.

14. The strains as defined in claim 1 or 2, for use as a medicament.

15. The bacterial preparation as defined in any one of claims 3 to 8, for use as a medicament.

16. The strain for use according to claim 14 or the bacterial preparation for use according to claim 15, wherein said medicament is for the treatment of diseases caused by enveloped-viruses.

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