STRAIN OF BACILLUS PUMILUS PRESENTING STRONG ANTAGONISM TOWARDS SURFACE PATHOGENS

Abstract

The invention relates to a strain of Bacillus pumilus exhibiting antagonistic activity against surface pathogens. Abbreviated figure: Fig. 1

Description

Title of the invention: DEBACILLUS PUMILUS STRAIN EXHIBITING STRONG ANTAGONISM AGAINST SURFACE PATHOGENS Technical field of the invention

[0001] The invention relates to a strain of Bacilluspumilus which exhibits an antagonistic activity of interest towards pathogenic microorganisms, in particular surface pathogenic bacteria, a cleaning, sanitizing or detergent composition comprising said strain, and their use for cleaning and / or protecting surfaces from contamination. Technological background

[0002] The detergent industry today faces a constant challenge to meet the requirements and standards relating to cleanliness and hygiene.

[0003] Current products for cleaning and disinfecting surfaces have shown their limitations, particularly in the fight against recontamination of surfaces by pathogenic agents. Indeed, the products traditionally used for disinfecting surfaces only have a transient effect since they only ensure the elimination of bacteria already in place without preventing rapid recontamination of surfaces by pathogenic agents.

[0004] In addition, conventional disinfectant products have the disadvantage of being dangerous products (irritant, corrosive risk, etc.) containing molecules that are toxic to humans and the environment (chlorinated products, quaternary ammoniums, glutaraldehyde, etc.). In addition, these products are difficult to biodegrade.

[0005] 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.

[0006] Another disadvantage of using traditional chemical-based disinfectant compositions is 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.

[0007] In practice, legislation is becoming 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.

[0008] In this context, alternative solutions have been sought. 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.

[0009] 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. For example, several studies carried out in a hospital environment (Vandini A. et al., 2014, PLoS One, 9(9): el08598; Caselli et al., 2016, PLoS One, 11(2): e0148857) present results of inhibition of the development of pathogenic bacteria on surfaces thanks to the use of a product marketed by the company CHRISAL containing spores of B. subtilis, B. megaterium and B. pumilus. However, no data allows the identification of the bacterial strains used or reveals their effectiveness when each strain, taken in isolation, is used on the target surface pathogens.

[0010] Other documents describe the use of bacterial spores of the genus Bacillus in cleaning compositions, such as:

[0011] Document WO97 / 25865 describes the use of Bacillus spores (B. licheniformis, B. subtilis, B. polymyxa, B. amyloliquefaciens, B. pasteurii and / or B. laevolacticus) in a detergent formulation.

[0012] Documents WO2010 / 014715 and WO2010 / 006235 specifically relate to a strain of B. amyloliquefaciens and a strain of B. velezensis for preventing and / or reducing the formation of pathogenic biofilm on surfaces.

[0013] Document WO2017 / 074485 describes a detergent composition having the property of eliminating or preventing the presence of pathogens and biofilms on surfaces and which contains at least one bacterium of the genus Bacillus. In practice, mixtures of different species are tested.

[0014] Document WO2012 / 042220 describes a method for cleaning surfaces which comprises the use of an aqueous composition comprising spores of at least one strain of Bacillus of class 1 (in practice a mixture of B. circulons, B. megaterium and B. sphaericus), a terpene and one or more surfactants.

[0015] Document WO2019 / 110811 describes detergent compositions having the property of specific inhibition of the growth of pathogens or undesirable organisms, in which the bacteria used is B. amyloliquefaciens or B. velezensis.

[0016] Document WO2010 / 028460 describes a method for the preventive treatment of a abiotic surface in a domestic or medical environment, using a non-detergent aqueous solution containing a mixture of bacteria of the genus Bacillus.

[0017] It is clear from the above that there is a clear need for new technical solutions to ensure protection of surfaces against the development of pathogenic microorganisms present on these surfaces, i.e. to ensure effective cleaning and sanitation of these surfaces, while limiting or even preventing their recontamination. Description of the invention

[0018] The Applicant has isolated a new strain of Bacillus pumilus exhibiting numerous interesting properties in the context of surface cleaning, in particular a remarkable antagonistic activity, both in its intensity and its spectrum of action.

[0019] It should be noted that the use of B. pumilus strains as a probiotic ingredient has already been described in connection with the animal health feed industry. For example, WO2019 / 002476 describes a Bacillus pumilus DSM32539 strain and its use as a food probiotic to inhibit intestinal colonization by pathogenic bacteria in pig and poultry farms.

[0020] B. pumilus strains have also been studied to isolate antibacterial compounds (Chu et al., 2019, Applied Microbiology and Biotechnology, 103: 8375-81; Sagesse et al., 2018, Mar. Drugs, 16(6): 180). These studies revealed great heterogeneity in their inhibitory activity against pathogens.

[0021] Definitions

[0022] 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.

[0023] For the purposes of the invention, the articles "a" and "an" are used to refer to one or more (e.g., at least one) units of the grammatical object of the article. For example, "an element" designates at least one element, i.e., one or more elements.

[0024] The terms "about" or "approximately", used in reference to a measurable value such as a quantity, a time, and the like, should be understood to encompass measurement uncertainties of ± 20% or ± 10%, preferably ± 5%, even more preferably ± 1%, and particularly preferably ± 0.1% of the specified value.

[0025] Intervals: Throughout this description, the various features of the invention may be presented in the form of an interval of values. It should be understood that the description of values ​​in the form of an interval is solely for the purpose of making reading simpler and should not be interpreted as a rigid limitation of the scope of the invention. Accordingly, the description of a range of values ​​should be considered to specifically disclose all possible intermediate ranges and each of the values ​​within that range. For example, a description of an range from 1 to 6 should be considered to specifically describe each of the ranges it includes, such as the ranges 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and so on, and each of the values ​​within that range, for example, 1; 2; 2.7; 3; 4; 5; 5.3 and 6. This definition holds regardless of the scope of the range.

[0026] The term "isolated" should be understood in the context of the invention as synonymous with removed or extracted from its natural environment or state. For example, an isolated strain of bacteria or peptide is a strain of bacteria or peptide extracted from the natural environment in which it is usually found, whether a plant or a living animal for example. Thus, a strain of bacteria or peptide naturally present in a living animal is not an isolated strain of bacteria or peptide for the purposes of the invention, while the same strain of bacteria or peptide, partially or completely separated from other elements present in its natural context, is itself "isolated" for the purposes of the invention. An isolated strain of bacteria or peptide may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

[0027] The terms "coding" or "coding for", "codes" or "codes for" refer to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (e.g. rRNA, tRNA and mRNA), or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system.Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is generally described in sequence listings and databases, and the non-coding strand, used as a template for the transcription of a gene or cDNA, may be designated as coding for the protein or other product of that gene or cDNA.

[0028] Unless otherwise indicated, for the purposes of the invention, a "nucleotide sequence encoding an amino acid sequence" designates all nucleotide sequences which encode the amino acid sequence, including degenerate nucleotide sequences making it possible to obtain said amino acid sequence. This particularly concerns so-called optimized sequences in which the modifications are intended to improve the stability and transcription of the RNA or to allow the production of the protein in a specific host. The nucleotide sequence which codes for a protein or RNA or cDNA may optionally include introns.

[0029] The term "polynucleotide" as used in the context of the invention is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, the terms nucleic acids and polynucleotides as used in the context of the invention are interchangeable. It is well known in the field of molecular biology and genetic engineering that nucleic acids are polynucleotides, which can be hydrolyzed into monomers. Nucleotides in monomeric form can be hydrolyzed into nucleosides.As used in the context of the invention, the term polynucleotide refers, without limitation, to any type of nucleic acid molecule, i.e., nucleic acid molecules obtainable by any means available in the art, including by recombinant means, namely the cloning of nucleic acid sequences from a recombinant library or the genome of a cell, using ordinary cloning technologies such as PCR, or by synthesis.

[0030] 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.

[0031] For the purposes of the invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein by definition contains at least two amino acids, without limitation as to the maximum number of amino acids. Polypeptides include indifferently several peptides and / or proteins, which themselves comprise two or more amino acids linked to each other by peptide bonds. As used herein, the term refers both to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers for example, and to longer chains, which are generally referred to in the art as proteins, of which there are many types.“Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homomers, heteromers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

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

[0033] As already stated, the Applicant has identified a microorganism, in this case a new strain of bacteria of the species Bacillus pumilus, which exhibits antagonistic activity at least against pathogenic bacteria belonging to the families Staphylococcaceae, Enterobacteriaceae and Listeriaceae. To the Applicant's knowledge, this is the first strain of B. pumilus having a broad spectrum of antagonism against at least these three families of bacteria known to be pathogens likely to be present on surfaces to be cleaned.

[0034] Thus and according to a first aspect, the present invention relates to a strain of Bacillus pumilus exhibiting antagonistic activity against pathogenic bacteria belonging to at least the following three families: Staphylococcaceae, Enterobacteriaceae and Listeriaceae, advantageously belonging to at least the following three genera: Staphylococcus, Escherichia and Listeria.

[0035] According to a particular embodiment, this is the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on July 1, 2020, under number 1-5531.

[0036] The present invention also relates to strains derived from strain 1-5531. In the context of the invention, the term "derivative" or "mutant" or "variant" means any strain which diverges from strain 1-5531 from a genomic point of view due to mutation(s) which may result from spontaneous mutations or random or site-directed mutagenesis. Such mutations may correspond to deletions, point modifications (one or more bases) or the introduction of exogenous sequences by genetic engineering. Such mutations may or may not be silent. In the case of non-silent mutations, a derived strain according to the invention exhibits at least one of the characteristics of strain 1-5531 as described below, advantageously all of these characteristics. Consequently, a derived strain advantageously exhibits the same phenotype as strain 1-5531. Preferably according to the invention, a derived strain exhibits an activity antagonistic to pathogenic bacteria belonging to at least the following three families: Staphylococcaceae, Enterobacteriaceae and Listeriaceae.

[0037] The strains of B. pumilus according to the invention belong to class 1 according to European directive 2000 / 54 / EC targeting microorganisms which have never been described as causative agents of diseases in humans and which do not present any danger to the environment. They also have QPS status (presumption of recognized safety) according to EFSA (European Food Safety Authority) and GRAS status ("Generally Recognized As Safe") according to the FDA (Food and Drug Administration).

[0038] The belonging of the strains of the invention to the species B. pumilus is apparent for example from the sequencing of characteristic phylogenetic markers, advantageously of the 16S ribosomal DNA, of the gyrB and groEL genes. Thus and according to a particular embodiment, a strain according to the invention has: - the sequence SEQ ID NO: 1 corresponding to the partial sequence of 16S rDNA, or a sequence having at least 95%, 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with SEQ ID NO: 1, advantageously 100% identity; and / or - the sequence SEQ ID NO: 2 corresponding to the partial sequence of the gyrB gene, or a sequence having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with SEQ ID NO: 2; and / or - the sequence SEQ ID NO: 3 corresponding to the partial sequence of the groEE gene or a sequence having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with SEQ ID NO: 3.

[0039] Advantageously, a strain according to the invention has the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, or sequences having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with these.

[0040] According to another advantageous embodiment, a strain according to the invention has a genomic sequence having at least 95%, advantageously at least 96%, 97%, 98%, 99% or even 99.5% or even 99.9% identity with the genomic sequence of strain 1-5531.

[0041] In accordance with the fact that the strains of the invention belong to the species B. pumilus, they are advantageously capable of growing under anaerobic and aerobic conditions, on conventional rich media containing peptones and hydrolysates of animal or vegetable proteins, yeast extracts and salt, or on specific media for the culture of strains of the Bacillus type well known to those skilled in the art.

[0042] According to another particular embodiment, a strain according to the invention is capable of sporulating or forming spores, for example after culture in a medium promoting sporulation such as Difco Sporulation Medium (DSM).

[0043] An essential characteristic of a B. pumilus strain according to the invention lies in its antagonistic activity towards surface pathogens. In the context of the invention, the terms "antagonistic activity", "antagonism" or "antagonistic power" have an equivalent meaning. They refer to the capacity of the strain to reduce or even inhibit the growth and / or activity of a different microorganism, advantageously a surface pathogen. This action can be exerted by production and secretion of inhibitory molecules (bacteriocins, antibiotics, toxins, etc.) and / or by competition. By "competition" 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 bacterial strain according to the invention will occupy the terrain by consuming the organic matter which will accumulate after cleaning and / or produce antagonistic molecules which will prevent the "bad" bacteria from settling, thus ensuring a long-term effect.

[0044] In the context of the invention, the surfaces to be cleaned are advantageously inert surfaces, for example comprising or consisting of a material chosen from the following list: a polymer or copolymer such as polyvinyl chloride (PVC), earthenware, stainless steel, glass.

[0045] Surface pathogens are microorganisms, in particular bacteria, advantageously belonging to the following families: Staphylococcaceae, Enterobacteriaceae and Listeriaceae.

[0046] Advantageously, the strain of B. pumilus according to the invention exhibits antagonistic activity against pathogenic bacteria of the following bacterial genus: Staphylococcus, Escherichia and Eisteria.

[0047] According to a particular embodiment, targeted pathogenic bacterial species are: Staphylococcus wameri (for example ATCC 27836), Staphylococcus epidermis (for example ATCC 12228), Staphylococcus hominis (for example ATCC 700236), Staphylococcus aureus (for example SA 1199), Escherichia coli (for example W3110 or DSM1058) and Eisteria monocytogenes (for example CIP 82.110).

[0048] Such microorganisms are called pathogens insofar as they are potentially dangerous to human health in the event of inhalation, contact or ingestion, for example. Thus, a strain of B. pumilus according to the invention can be considered a probiota or probiotic strain, advantageously a surface probiotic strain.

[0049] The antagonistic activity of a microorganism, in particular of the B. pumilus strain according to the invention, can be demonstrated by different techniques known to those skilled in the art.

[0050] As is evident from the examples below (example 1.6.1), this activity can be demonstrated by the formation of a halo of antagonism around the bacterial colonies tested in solid medium. Thus and preferably, a halo of at least 2 mm, or even at least 5 mm, advantageously at least 10 mm, even more advantageously at least 15 mm is observed in a diffusion antagonism test on paper discs in TSA (Trypto-Casein Soy Agar) agar medium comprising the pathogenic strain, in particular the SA 1199 strain of Staphylococcus aureus, the ATTCC 27836 strain of Staphylococcus wameri, the ATCC 12228 strain of Staphylococcus epidermis, the ATCC 700236 strain of Staphylococcus hominis, the W3110 or DSM1058 strain of Escherichia coli, or the CIP 82.110 strain of Listeria monocytogenes.According to a particular embodiment, the strain according to the invention exhibits an antagonistic activity, as evaluated by this test on agar medium, at least equal to that of the strain 1-5531 and / or greater than that of the strains of B. pumilus DSM27T and DSM361.

[0051] Alternatively and as illustrated in example 1.6.2, the antagonistic activity of the strain according to the invention can be tested by competition tests in co-culture on liquid medium, by monitoring the growth of the strain of the invention and of the pathogen.

[0052] According to one embodiment, the B. pumilus strain according to the invention has antagonistic, antibacterial, antibiotic, bacteriostatic, bactericidal and / or sporicidal properties with respect to pathogenic bacteria from the bacterial families Staphylococcaceae and / or Enterobacteriaceae and / or Listeriaceae, advantageously Staphylococcaceae, Enterobacteriaceae and Listeriaceae.

[0053] According to another embodiment, the B. pumilus strain according to the invention has an antagonistic activity towards at least one pathogenic bacterium from the families Staphylococcaceae and / or Enterobacteriaceae and / or Listeriaceae, advantageously towards at least one pathogenic bacterium from the bacterial genera Staphylococcus and / or Escherichia and / or Listeria, preferably towards the species Staphylococcus wameri and / or Staphylococcus epidermis and / or Staphylococcus hominis and / or Staphylococcus aureus and / or Escherichia coli and / or Listeria monocytogenes.

[0054] According to other embodiments, a strain of B. pumilus according to the invention, in particular strain 1-5531 or a strain derived therefrom, has at least one of the following characteristics:

[0055] A sensitivity to antibiotics or antimicrobials compatible with the requirements of EFSA (Guide for the assessment of bacterial sensitivity to antimicrobials EFSA Journal 2012; 10 (6): 2740 4) in relation to Bacilli. This makes it possible to reduce the risk of propagation of antibiotic resistance genes to surface pathogens. Thus, advantageously and as demonstrated for strain 1-5531 (example 1.3), the B. pumilus strain according to the invention is sensitive to the following antibiotics: ampicillin, vancomycin, gentamicin, erythromycin, clindamycin, kanamycin, streptomycin, chloramphenicol and tetracycline. Even more advantageously, it has a minimum inhibitory concentration (MIC) less than or equal to, advantageously less than 4 mg / L for vancomycin, gentamicin, erythromycin and clindamycin, and 8 mg / L for kanamycin, streptomycin, chloramphenicol and tetracycline.Thus and according to a particular embodiment, the strain according to the invention has a sensitivity to antibiotics, in particular to those listed above, at least equal to that of the strain 1-5531 and / or greater than that of the strain of B. pumilus DSM361, in particular with respect to chloramphenicol.

[0056] A capacity to grow in a saline medium. As demonstrated in the examples below (example 1.4), strain 1-5531 and therefore the B. pumilus strains according to the invention are capable of growing in the presence of high concentrations of salts, in particular NaCl, in this case capable of reaching 100 g / L (10%), in particular in modified Difco Sporulation Medium (DSM + 5 g / L glucose). According to a particular embodiment, the strain according to the invention exhibits growth in the presence of high concentrations of salts, in particular in the presence of at least 10 g / L (1%), for example 50 g / L (5%) or even 100 g / L (10%) of NaCl, unaffected (comparable to that observed in the absence of salt) and / or at least equal to that of strain 1-5531 and / or greater than that of the B. pumilus strain DSM27T.

[0057] A capacity to adhere to an inert surface and to invade it. As can be tested under the conditions of Example 1.5, the B. pumilus strains according to the invention advantageously have a capacity to adhere to an inert surface, for example polyvinyl chloride (PVC) or earthenware, at least equal to that of the strain 1-5531 and / or greater than that of the B. pumilus strain DSM27T. Furthermore, this adhesion is observed for at least 24 hours, or even for several days. A strain according to the invention is furthermore capable of developing and surviving on inert surfaces for several days. This property can be demonstrated in a TSA agar test, namely by measuring the extent of colony development outside a paper disc inoculated with said strain. Thus and advantageously, the B. pumilus strain according to the invention has the ability to invade an agar surface around a previously inoculated paper disc of at least 2 mm, or even at least 5 mm, advantageously at least 10 mm, even more advantageously at least 15 mm in a TSA agar test. Preferably, this ability is at least equal to that of strain 1-5531 and / or greater than that of B. pumilus strains DSM27T and DSM361.

[0058] According to another aspect, the invention relates to an extracellular preparation obtained from a culture of the B. pumilus strain according to the invention, which may correspond to a crude culture supernatant, a filtered culture supernatant, molecules obtained from these supernatants such as peptides and / or lipopeptides, advantageously purified according to techniques known to those skilled in the art.

[0059] As demonstrated in the examples, such a strain can be used for the preparation of a cleaning, sanitizing and / or detergent composition.

[0060] Thus and according to another aspect, the invention relates to a composition comprising a strain of B. pumilus as described previously, in particular: - a strain of B. pumilus exhibiting antagonistic activity against pathogenic bacteria belonging to at least the families Staphylococcaceae, Enterobacteriaceae and Listeriaceae; or - strain 1-5531; or - a strain derived from strain 1-5531 and having retained antagonistic activity against pathogenic bacteria belonging to at least the families Staphylococcaceae, Enterobacteriaceae and Listeriaceae.

[0061] Advantageously, the composition according to the invention is a cleaning, sanitizing and / or detergent composition. The term "cleaning" refers to the use of such a composition for cleaning surfaces, in particular inert surfaces. The term "sanitizing" refers to the fact that due to the presence of the strain according to the invention in the composition, the application of such a composition makes it possible to protect the surfaces from the development of surface pathogens, in particular pathogenic bacteria belonging at least to the families Staphylococcaceae, Enterobacteriaceae and Listeriaceae. The term "detergent" refers to the fact that the composition comprises, in addition to the strain according to the invention, at least one detergent agent having surfactant properties.

[0062] According to a particular embodiment, the composition according to the invention comprises the strain of B. pumilus in the form of spores and / or vegetative cells and / or cells which are in the transitional phase of sporulation. According to an advantageous embodiment, the strain is in the form of spores.

[0063] Advantageously, the strain of B. pumilus of the invention represents between 105 CFU / ml and 1012 CFU / ml of said composition, advantageously between 106 and 109 CFU / ml, of preferably between 107 and 108 CFU / ml, for example from 106 to 108 CFU / ml. In the remainder of the description, the abbreviation CFU means "colony forming unit". The terms "CFU" and "bacteria" may be used interchangeably.

[0064] The B. pumilus strain or the extracellular preparation according to the invention can be obtained by cultivating the strains of the present invention according to methods well known to those skilled in the art, for example by liquid fermentation in batch mode, fed-batch or continuous culture or by solid-state fermentation using conventional culture media, sporulating or not, well known to those skilled in the art such as for example the following medium: peptone at 5 g / L, meat extract at 3 g / L and manganese sulfate (MnSO4, H2O) at 10 mg / L.

[0065] 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, evaporation or by precipitation using for example solvents or ammonium sulfate.

[0066] Thus and in a particular embodiment, to eliminate the residual culture medium, the bacterial cells, advantageously sporulated, of the B. pumilus strain according to the invention are washed in more or less purified water or in a neutral buffer well known to those skilled in the art, such as for example a phosphate buffer of the saline phosphate buffer type (or PBS comprising: NaCl 8 g / l, KCl 0.2 g / L, Disodium phosphate 1.44 g / L and monopotassium phosphate 0.24 g / L) or in a 9 g / L saline solution.

[0067] According to another embodiment, the composition according to the invention comprises as the only microorganism, advantageously as the only probiotic strain, advantageously as the only surface probiotic strain, the strain of B. pumilus as defined above. In other words, a composition according to the invention may not contain any other microorganism, in particular any other bacteria. According to an advantageous embodiment, a composition according to the invention does not contain other bacteria of the genus Bacillus.

[0068] According to an alternative embodiment, the composition comprises, in addition to the strain according to the invention, at least one other microorganism, for example another probiotic strain, in particular a surface and / or food strain, in particular a bacterium. In particular, another probiotic strain may be a strain of Bacillus type different from that of the invention, advantageously in sporulated form, a strain of Lactobacillus (L.), Pediococcus (P.), Enterococcus (E.) or mixtures thereof.

[0069] As an example, another probiotic strain may be chosen from the following list: B. licheniformis, B. amyloliquefaciens, B. megaterium, B. subtilis, B. polymyxa, B. lentus, B. pumilus different from that of the invention, B. velezensis, B. mojavensis, B. sphaericus, B. smithii, B. laterosporus, B. coagulans, B. alevi, B. cereus, B. badins, B. thuringiensis, B. pasteurii, B. laevolacticus, B. circulans, L. plantarum, L. reuteri, L. rhamnosus, L. casei, P. acidilactici, P. pentosaceus, E. faecium.

[0070] According to a particular embodiment, such a strain is in a free or encapsulated form to be more resistant, in particular to lactic acid bacteria of the genus Eactobacillus, Pediococcus and / or Enterococcus.

[0071] Preferably, the composition according to the invention further comprises at least one compound compatible with the other components of the composition, advantageously at least with the strain according to the invention. Such a compound may be selected from the following list: a surfactant (or surfactant), a preservative, a perfume, a sequestrant, a texturizer, a wax, a gelling agent, an abrasive, a colorant, an acidity regulator, a base, a polymer, a resin, an anti-foaming agent, a solvent, a water, a stabilizing agent, a thickener and an enzyme, for example a lipase or a protease.

[0072] In relation to the preparation of detergent compositions, the composition according to the invention may comprise one or more surfactants chosen from: - a surfactant of microbial origin, for example a biosurfactant preferably of the rhamnolipid, surfactin, sophorolipid type, a mannosylerythritol lipid, a trehalose lipid or any other surfactant of microbiological origin obtained by a biotechnological process; - a surfactant of plant or petrochemical origin such as an alkylpolyglycoside, an ethoxylated fatty alcohol, a fatty acid ester of glycerol, a sodium lauryl sulfate, a linear alkylbenzene sulfonate, a carboxylic acid, or a soap, preferably but not necessarily chosen on the basis of its low toxicity to humans and its low impact on the environment; or - mixtures thereof.

[0073] The surfactants used in the context of the invention may be surfactants of anionic, cationic, amphoteric or non-ionic type.

[0074] According to a particular embodiment, the preservative present in a composition according to the invention, making it possible to stabilize the composition without damaging the probiotic bacteria, may be sodium benzoate, potassium sorbate, molecules of the isothiazolinone type, propylene glycol, phenoxyethanol or their mixtures.

[0075] According to a particular embodiment, the sequestrant used in a composition according to the invention is EDTA, sodium citrate or their mixture.

[0076] According to another particular embodiment, the abrasive used in a composition according to the invention is calcium carbonate, magnesium carbonate or their mixture.

[0077] According to another particular embodiment, the acidity corrector used in a composition according to the invention is lactic acid, malic acid, citric acid, sodium hydroxide or their mixtures.

[0078] 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 a composition according to the invention.

[0079] The composition according to the invention may be in any form normally used for application to an inert surface, for example in concentrated or diluted form, liquid or powder. Thus, the composition according to the invention may be in the form of a solution, soaking bath, steam, spray for spraying, projection, impregnated cloth or pad or wipe...

[0080] Advantageously, the composition according to the invention is in the form of an aqueous solution, advantageously a ready-to-use solution or a concentrated solution to be diluted. An aqueous solution is defined as a solution in which water constitutes the majority solvent. Such a composition may also contain a smaller quantity of a solvent other than water, capable of evaporating after application of the composition to the surface to be cleaned, thus promoting its drying, advantageously a plant-based solvent such as, for example, isopropanol or ethanol.

[0081] Examples of formulation of a composition according to the invention are presented in Tables 6 and 7 below. As demonstrated in the examples, such compositions ensure the durability over time (at least 1 year) of a strain according to the invention (Example 2.2), while preserving its ability to adhere to an inert surface for several days (Example 2.3) and to protect such a surface from contamination by pathogens (Example 2.4).

[0082] According to another aspect, the invention relates to the use of the B. pumilus strain according to the invention or of the composition according to the invention for controlling, inhibiting and / or preventing the proliferation, on an inert surface, of pathogenic bacteria belonging in particular to the families Staphylococcaceae, Enterobacteriaceae or Listeriaceae, advantageously of pathogenic bacteria belonging to the genera Staphylococcus and / or Escherichia and / or Eisteria, preferably of pathogenic bacteria belonging to the species Staphylococcus warneri and / or Staphylococcus epidermis and / or Staphylococcus hominis and / or Staphylococcus aureus and / or Escherichia coli and / or Listeria monocytogenes.

[0083] Advantageously, the invention relates to the use of the strain of B. pumilus according to the invention or of the composition of the invention for controlling, inhibiting and / or preventing the proliferation, on an inert surface, of at least pathogenic bacteria belonging to the families Staphylococcaceae, Enterobacteriaceae and Listeriaceae, advantageously belonging to the genus Staphylococcus, Escherichia and Listeria, preferably Staphylococcus wameri, Staphylococcus epidermis, Staphylococcus hominis, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes.

[0084] 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 a supermarket or even a company.

[0085] According to another aspect, the invention relates to a method for cleaning and / or protecting a surface from contamination, advantageously an inert surface, in particular with respect to pathogenic bacteria belonging to the Staphylococcaceae, Enterobacteriaceae and Listeriaceae families. In practice, such a method comprises the application to the surface of the strain of B. pumilus according to the invention or of the composition according to the invention, advantageously at a level of 105 to 107 CFU / m2 of surface.

[0086] According to the invention, the strain of B. pumilus or the composition can be applied according to the usual procedures for sanitizing inert surfaces in addition to or as a replacement for disinfection procedures known to those skilled in the art. According to a particular embodiment, this application is carried out using non-woven wipes or microfibers.

[0087] As demonstrated in the examples, the method according to the invention, and therefore the use of the B. pumilus strain or the composition according to the invention, makes it possible to protect inert surfaces from contamination by pathogenic bacteria, with a lasting effect of up to at least 1 day, 2 days, 3 days, 4 days or even at least 5 days.

[0088] The manner in which the invention can be implemented and the advantages which result therefrom will become more apparent from the following examples of implementation, given for informational and non-limiting purposes, in support of the appended figure. Description of the figures

[0089] [Fig.l] shows the results of a comparative antagonism test on solid agar medium of the strains DSM27T, DSM361 and CNCM 1-5531 against the strain S. wameri (ATCC 27836). Examples of achievements Example 1: Probiotic strain according to the invention 1. Strain

[0090] The present study was carried out using a strain isolated from marine sediments and deposited with the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) under number 1-5531 dated July 1, 2020. 2. Sequencing

[0091] Partial sequencing of phylogenic markers, namely 16S ribosomal DNA (SEQ ID NO: 1), and the gyrB (SEQ ID NO: 2) and groEL (SEQ ID NO: 3) genes, made it possible to determine that strain 1-5531 belongs to the species Bacillus pumilus. 3. Antibiotic sensitivity 3.1. Purpose of the study

[0092] The European Food Safety Authority (EFSA) via the Scientific Panel on Additives used in Animal Feed (FEEDAP Panel) has defined for Bacillus strains, the minimum inhibitory concentrations (MIC) for certain antibiotic substances, also called antimicrobials, such as: ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol (Guide for the assessment of bacterial susceptibility to antimicrobials, EFSA Journal 2012; 10(6): 2740). The MIC corresponds to the smallest concentration of antibiotic capable of inhibiting bacterial growth. For each bacterial strain / antibiotic pair there is a threshold from which the bacteria is said to be resistant (see Table 1).

[0093] These antimicrobials correspond to a relevant range of antimicrobials of human and veterinary importance. To be used as a surface probiotic, it is therefore advisable to comply with the EFSA requirements in terms of antibiotic resistance. Knowing that certain strains of Bacillus pumilus do not meet the EFSA requirements, such as for example the strain Bacillus pumilus DSM361 having a MIC of 16 mg / L with respect to chloramphenicol, the sensitivity of a probiotic strain according to the invention, namely the strain CNCM 1-5531, with respect to the antibiotics defined by the FEEDAP group was therefore tested. 3.2. Cultivation conditions

[0094] For antibiotic sensitivity tests, strain 1-5531 is first cultured in liquid on Mueller-Hinton II (MHII) medium for 18 hours. The culture obtained is then diluted to 1 / 100 or 1 / 20 and incubated at 37°C with shaking for at least 3 hours. When the optical density (OD) of the culture measured at 600 nm reaches 0.5-0.7, a bacterial suspension at 1.106 CFU / ml is made and 100 μl of this suspension are deposited in the wells of a 96-well plate containing 100 μl of an antibiotic concentration gradient (0.25-64 mg / L). The final concentration of bacteria per well is 5.105 CFU / ml. The concentration gradient is obtained by making successive dilutions (1 / 2). The plate is then incubated at 37°C for 18 to 24 hours.

[0095] After incubation, 50 μl of a 0.2 mg / ml iodonitrotetrazolium chloride (INT) solution is placed in each well. The reading is taken after 30 minutes of incubation at room temperature. LTNT is reduced by bacterial dehydrogenases, giving a red color in the wells containing live bacteria. The absorbance of each well is then measured using an INFINITE PRO 200 microplate reader (TECAN). An absorbance threshold is calculated which makes it possible to determine the OD value from which there is bacterial growth. 3.3. Results

[0096] The results are shown in Table 1 below.

[0097] [Tables 1] Antibiotic MIC for strains of the genus Bacillus (FEEDAP values) MIC for strain 1-5531 (invention) Vancomycin 4 mg / L 0.25 mg / L Gentamicin 4 mg / L 1 mg / L Kanamycin 8 mg / L 2 mg / L Streptomycin 8 mg / L 8 mg / L Erythromycin 4 mg / L 1 mg / L Chloramphenicol 8 mg / L 8 mg / L Tetracycin 8 mg / L 0.5 mg / L Clindamycin 4 mg / L 4 mg / L Ampicillin Not required Not measured

[0098] These data show that strain 1-5531 meets EFSA requirements (FEEDAP values) in terms of antibiotic sensitivity.

[0099] 4. Growth in the presence of a high concentration of NaCl

[0100] It has been found that strain 1-5531 is capable of growing under anaerobic and aerobic conditions, on conventional rich media containing peptones and hydrolysates of animal or vegetable proteins, yeast extracts or on specific media for the culture of Bacillus type strains.

[0101] The present test aims to show the behavior of this strain in a saline medium. It compares the growth of the 1-5531 strain and the DSM27T strain (type strain of Bacillus p umilus) on the modified Difco Sporulation Medium (DSM + 5 g / L glucose) in the presence of 100 g / L NaCl (10%). The measurement of the OD at 600 nm (OD600) makes it possible to quantify the growth of the microorganisms after 24 h of culture.

[0102] The results of the test are shown in Table 2 below.

[0103] [Tables2] Strains Medium DO600 1-5531 Modified DSM 8.58 Modified DSM + 10% NaCl 8.85 DSM27T Modified DSM 11.84 Modified DSM + 10% NaCl 3.48

[0104] It emerges from these results that, on the modified DSM medium, in the presence of 10% NaCl, the growth of strain 1-5531 in 24 h is identical to that on medium without NaCl. For strain DSM27T, growth is greatly slowed down in the presence of 10% NaCl and after 24 h reaches an optical density approximately 3 times lower than on the medium without NaCl. 5. Adhesion capacity on inert surfaces

[0105] All tests are carried out under sterile conditions on 5x5 cm surfaces sterilized by autoclaving (15 min at 121°C) and placed in sterile 90 mm Petri dishes. Strain 1-5531 is compared to the type strain of Bacillus pumilus DSM27T.

[0106] The sporulated probiotic bacterial suspensions are prepared from a 4-day culture at 30°C with shaking in a sporulation-promoting medium (DSM).

[0107] 5 mL of bacterial suspension at a given concentration (1.104 bacteria probiotics / mL) are placed on the PVC or earthenware surface. These are incubated for 30 minutes to allow time for the bacteria to settle and adhere to the surfaces. The excess bacterial solution is then removed to obtain a completely dry surface.

[0108] The surface is then incubated for 24 hours at room temperature, under microbiological safety conditions.

[0109] Contact boxes (TSA medium) are then applied after 24 hours to the surfaces to be tested. They are held in place for 10 seconds with constant pressure.

[0110] The adhesion of each strain on the different plates is evaluated after counting the colonies present on the contact plates. The results obtained are grouped in Table 3 below. [YES] [Tables3] Adhesion to surfaces (Bacterial count of contact plates) Strain Concentration of the applied solution CFU / mL on PVC plates on earthenware plates 1-5531 1.104 153 colonies 157 colonies DSM27T 1.104 12 colonies 63 colonies

[0112] The results show that strain 1-5531 is capable of adhering to both PVC and earthenware plates. In addition, it reveals that the strain of the invention adheres more effectively to these two types of surface than the reference strain DSM27T (2 to 12 times more bacteria adhered to the surface). 6. Antagonism effectiveness 6.1. Antagonism test on solid agar medium

[0113] The antagonism efficacy of strain 1-5531 against several target surface pathogens (Eschericha coli, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus epidermis and Staphylococcus hominis) was evaluated on agar surface and compared to that of the type strains of Bacillus pumilus DSM27t and DSM361.

[0114] An overnight culture of the pathogenic strain is diluted in Trypto-Casein Soy Broth (TSB) medium and placed on a 90 mm Petri dish containing Trypto-Casein Soy Agar (TSA) medium so as to obtain cell lawns per dish of 105 CFU of E. coli (W3110 or DSM1058), 5.105 CFU of Listeria monocytogenes (CIP 82.110), 106 CFU of S. aureus (SA 1199), S. warneri (ATCC 27836), S. epidermis (ATCC 12228) or S. hominis (ATCC 700236).

[0115] The agar plates are then left to dry under a hood at room temperature for 1 hour.

[0116] Paper discs are then placed sterilely on the agar. 15 μl of an overnight culture of the probiotic strain (1-5531, DSM27T or DSM361) are then deposited on the discs. The bacterial concentration of the cultures is adjusted by dilution in TSB medium so that each deposit contains exactly the same quantity of bacteria: 107 of probiotic microorganisms per paper disc (1-5531, DSM27T or DSM361).

[0117] After 24 hours of incubation at the optimal temperature for culturing the pathogen (30°C for Staphylococcus; 37°C for E. coli; 30°C for Listeria monocytogenes), the presence of a halo of growth antagonism of at least 2 mm around the strains probiotics (1-5531, DSM Tl1 and DSM361) attests to the surface antagonistic effect on the tested pathogens.

[0118] As an example, the results obtained for strains DSM27T, DSM361 and 1-5531 against strain S. wameri (ATCC 27836) are shown in [Fig.l]. The zone of antagonism observed around strain 1-5531 demonstrates that pathogenic bacteria cannot grow around colonies of this strain. The results also confirm the ability of strain 1-5531 to invade an agar surface around a previously inoculated paper disc, much greater than what is observed with strains DSM27T and DSM361.

[0119] Table 4 below summarizes the measurements of halos of growth antagonism of different pathogens observed around the probiotic strains on agar surfaces.

[0120] [Tables4] Pathogenic strain Halo of antagonism 1-5531 Halo of antagonism DSM27T Halo of antagonism DSM361 E. coli DSM 1058 14.5mm E. coli W3110 5.5mm 0mm 0mm S. wameri 18.85mm 0mm 1.9mm S. aureus 17mm Listeria monocytogenes 5.3mm S. epidermis 11mm 0mm 8mm S. hominis 14mm 0mm 7mm

[0121] The results show that strain 1-5531 is capable of inhibiting very effectively, on an agar surface, the growth of both S. aureus, S. wameri, S. epidermis, S. hominis, E. coli and Listeria monocytogenes. These efficacy results are not found for the other two strains of B. pumilus DSM27T and DSM361 which have a much lower antagonism spectrum and / or antagonism efficacy.

[0122] 6 .2. Competition tests in co-culture on liquid medium

[0123] The antagonism efficacy of strain 1-5531 was evaluated against strain pathogenic S. wameri (ATCC 27836) using liquid co-culture assays.

[0124] Overnight precultures on Luria-Bertani (LB) medium of the B. pumilus 1-5531 and S. wameri strains are used to inoculate pure culture or co-culture tests on the same medium (dilution respectively to 1 / 1000th and 1 / 100th). The count carried out at the start of the tests shows that the 20 ml LB vials contain 1.6.104 CFU / ml of the probiotic strain and 2.6.105 CFU / ml of the S warneri strain.

[0125] After growth for 24 h at 30°C with stirring, the test and control flasks are counted on LB-agar. The results obtained are grouped in Table 5 below.

[0126] [Tables5] Duration of co-culture Control (pure culture) Co-culture B. pumüus 1-5531 0 h 1.6.104 1.6.104 24 h 1.7.1010 2.6.10® S. wameri 0 h 2.6.105 2.6.105 24 h 1.9.109 <1.105

[0127] The results show that after 24 h of incubation at 30°C with shaking of the coculture, the S. wameri strain did not start its growth when it was in the presence of the 1-5531 strain (<1.105 CFU / ml instead of 1.9.109 CFU / ml in the absence of the 1-5531 strain). The presence of the B. pumilus strain according to the invention therefore prevents the pathogenic S. wameri strain from developing on rich medium at its optimal growth temperature.

[0128] Example 2: Cleaning formulation according to the invention 1. Examples of formulation

[0129] The invention also relates to the use of strain 1-5531 as described in Example 1 in a cleaning, sanitizing and / or detergent formulation for protecting surfaces from the development of pathogenic bacteria. Two examples of ready-to-use cleaning formulations are presented in Tables 6 and 7 below.

[0130] [Tableauxô] Designation Concentration (% by mass) Anionic surfactant 1-5% Non-ionic surfactant 1-5% Perfume 0.01-0.2% Vegetable solvent 1-5% Acidity regulator 0.01-0.1% Strain 1-5531 10® -108UFC / ml Water 75-97%

[0131] [Tables?] Designation Concentration (% by mass) Non-ionic surfactant 5-10% Perfume 0.01-0.2% Sequestrant 0.2-2% Thickener 0.05-0.2% Preservative 0.01-0.5% Strain 1-5531 106 -10s CFU / ml Water 87-95%

[0132] 2. Holding the strain in a detergent formulation

[0133] To guarantee the presence of bacteria over the long term and therefore their resistance in a detergent formulation according to the invention, a count in such a formulation (according to Table 6) ready to use (PAE) stored at 20°C is carried out after 1 year of storage.

[0134] The stability results over time are presented in Table 8 below and confirm the resistance of strain 1-5531 in the detergent formulation.

[0135] [Tables8] To T12 months PAE formula stored at 20°C 2.8.107 CFU / ml 2.6.107 CFU / ml

[0136] 3. Adhesion and survival of the probiotic on PVC and earthenware surfaces after cleaning with cleaning formulations of the invention using a microfiber.

[0137] All tests are carried out under sterile conditions in a microbiological safety cabinet. The microfibers used are sterilized by autoclaving at 121°C for 15 min.

[0138] Two cleaning formulations are used for these tests: a ready-to-use cleaning formulation (RTU; see Table 6) containing 1.107 probiotic bacteria / mL and a concentrated equivalent cleaning formulation containing 1.108 probiotic bacteria / mL and diluted to 1% for this test (i.e. 1.106 probiotic bacteria / mL).

[0139] The tests are carried out on 5x5 cm surfaces sterilized by autoclaving (15 min at 121°C) and placed in sterile 90 mm Petri dishes.

[0140] The surfaces (earthenware tiles or PVC slabs) are cleaned (5 successive passes) using a microfibre soaked in a PAE probiotic solution or a concentrated solution to be diluted to 1% comprising the strain 1-5531.

[0141] After 30 min of drying, the surface is incubated for 24 h or 5 days at room temperature under a microbiological safety cabinet.

[0142] Contact plates are then applied to the surfaces and incubated in an oven for 24 hours at the optimal temperature for probiotic growth.

[0143] The bacterial count obtained on the contact boxes makes it possible to highlight the level of adhesion of bacteria to surfaces.

[0144] A neutralizer (based on soy lecithin) is systematically used for the preparation of contact boxes in order to neutralize the possible inhibitory action of the surfactants present in the cleaning formulation.

[0145] The results obtained for counting contact boxes on PVC and earthenware plates are grouped in table 9 below.

[0146] [Tables9] Final concentration of the preparation used Quantity of bacteria detected on the contact box (25cm2) Cleaning formulation Earthenware PVC Ready to use 24 h or 5 days 1.107UFC / ml >300 >300 Concentrate diluted to 1% 24 h or 5 days 1.106 UFC / ml > 300 >300

[0147] These results show that cleaning several target surfaces (PVC or earthenware), using a microfiber, with compositions of the invention, in the form ready to use or concentrated (diluted to 1%), allows more than 300 probiotic bacteria to be deposited per 25 cm2 of surface.

[0148] 4. Capacity of the cleaning formulations of the invention to protect surfaces contamination by target pathogens

[0149] The tests are carried out under the same conditions as previously (example 2.3).

[0150] A ready-to-use (RTU) cleaning formulation containing 1.107 probiotic bacteria / mL (1-5531) is used for these tests.

[0151] The tests are carried out with pathogenic strains resistant to certain antibiotics in order to be able to distinguish them, on surfaces, from non-resistant probiotic bacteria (1-5531). The Staphylococcus aureus strain used (SA 1199) is resistant to 34 mg / L of streptomycin and the Escherichia coli strain used (W3110) is resistant to 68 mg / L of vancomycin.

[0152] Bacterial suspensions of the pathogenic strains are prepared from cells obtained from an overnight culture diluted in TSB medium.

[0153] 30 min, 24 h or 5 days after cleaning the surfaces with the formulation cleaning agent of the invention, 5 mL of pathogenic bacterial suspension at a given concentration (105 to 108 pathogenic bacteria / mL depending on the surface to be tested) are deposited on the PVC or earthenware surface.

[0154] These surfaces are incubated for an additional 30 min to allow time for the bacteria to sediment and adhere to the surfaces. Excess pathogen solution is removed using a micropipette to obtain a completely dry surface.

[0155] Contact plates are then applied after 24 h to the surfaces and are incubated in an oven for 24 h at the optimum temperature for growth of the pathogenic bacteria.

[0156] The bacterial count obtained on the contact boxes highlights the level of bacterial contamination on the surfaces.

[0157] A neutralizer (based on soy lecithin) is systematically used for the preparation of contact boxes in order to neutralize any possible inhibitory action of the surfactants present in the cleaning formulation.

[0158] Depending on the pathogen tested, an antibiotic (streptomycin or vancomycin) is added to the contact dishes.

[0159] To validate the results obtained, three “control” tests are carried out: - Witness 1: pathogenic bacterial suspension on the surface without application of the cleaning formulation; - Witness 2: cleaning of the surface with the cleaning formulation without application of the pathogenic bacterial suspension; and - Control 3: application on the surface of probiotic bacteria (1-5531) resuspended in water without detergent formulation, then addition of a pathogenic bacterial suspension. 4.1. Test on PVC plates

[0160] The results obtained for counting contact boxes on PVC plates in the presence of Staphylococcus aureus SA 1199 are presented in Table 10 below. Note that for this experiment, the quantity of pathogens (S. aureus SA 1199) deposited on the surface (5X5cm) is 5.105 CFU.

[0161] [Tables 10] Condition Medium contact boxes Count contact boxes Test 1 Count contact boxes Test 2 Pathogens (S. aureus) Probiotics (1-5531) Pathogens (S. aureus) Probiotics (1-5531) SA 1199 without application formulation (control 1) TSA + neutralizer + streptomycin 34mg / L 110 130 Formulation without application SA 1199 (control 2) TSA + neutralizer > 300 > 300 Probiotic without detergent + SA 1199 after 30 min. 24 h or 5 days (control 3) TSA + neutralizer + streptomycin 34mg / L 0 0 0 0 Formulation + SA 1199 after 30 min (trial 1) TSA + neutralizer + streptomycin 34mg / L 0 0 Formulation + SA 1199 after 24 h (trial 2) TSA + neutralizer + streptomycin 34mg / L 0 0 Formulation + SA 1199 after 5 days (trial 3) TSA + neutralizer + streptomycin 34mg / L 0

[0162] These results show that the application of the cleaning formulation according to the invention makes it possible to protect the surface of the PVC plates from contamination by Staphylococcus aureus SA 1199.

[0163] The results obtained for counting contact boxes on PVC plates in the presence of Escherichia coli W3110 are presented in Table 11 below. Note that for this experiment, the quantity of pathogens (E. coli W3110) deposited on the surface (5X5cm) is 2.5.106 CFU.

[0164] [Tables 11] Conditions Environment Contact boxes Count contact boxes Test 1 Count contact boxes Test 2 Pathogens (E. coli) Probiotics (1-5531) Pathogens (E. coli) Probiotics (1-5531) W3110 without application formulation (control) TSA + neutralizer + vancomycin 68mg / L 249 242 Formulation without application W3110 (control 2) TSA + neutralizer > 300 > 300 Probiotic without detergent + W3110 after 30 min (control 3) TSA + neutralizer + vancomycin 68mg / L 0 17 Formulation + W3110 after 30 min (test TSA + neutralizer + vancomycin 68mg / L 0 0 Formulation + W3110 after 24h (test 2) TSA + neutralizer + vancomycin 68mg / L 0 0 Formulation + W3110 after 5 days (trial 3) TSA + neutralizer + vancomycin 68mg / L 0 0

[0165] These results show that the application of a cleaning formulation according to the invention makes it possible to protect the surface of PVC plates from contamination by Escherichia coli W3110. 4.2. Test on earthenware plates

[0166] The results obtained for counting contact boxes on earthenware plates in the presence of Staphylococcus aureus SA 1199 are presented in Table 12 below. Note that for this experiment, the quantity of pathogens (S. aureus SA 1199) deposited on the surface (5X5cm) is 5.108 CFU.

[0167] [Tablesl2] Conditions Environment boots contacts Count boxes contacts Pathogens (S. aureus) Probiotics (1-5531) SA 1199 without application formulation (control 1) TSA + neutralizer + streptomycin 34mg / L 95 Formulation without application SA 1199 (control 2) TSA + neutralizer > 300 Probiotic without detergent + SA 1199 after 30 min (control 3) TSA + neutralizer + streptomycin 34mg / L 0 Formulation + SA 1199 after 30 min (trial 1) TSA + neutralizer + streptomycin 34mg / L 0 Formulation + SA 1199 after 24 h (trial 2) TSA + neutralizer + streptomycin 34mg / L 0

[0168] These results show that the application of a cleaning formulation according to the invention makes it possible to protect the surface of the earthenware plates from contamination by Staphylococcus aureus SA 1199.

[0169] The results obtained for counting contact plates on earthenware plates in the presence of Escherichia coli W3110 are presented in Table 13 below. Note that for this experiment, the quantity of pathogens (E. coli W3110) deposited on the surface (5X5cm) is 4.108 CFU.

[0170] [Tablesl3] Counting contact boxes Conditions Environment boots contacts Pathogens (E. coli) Probiotics (1-5531) W3110 without application formulation (control 1) TSA + neutralizer + vancomycin 68mg / L 136 Formulation without application W3110 (control 2) TSA + neutralizer > 300 Probiotic without detergent + W3110 after 30 min (control 3) TSA + neutralizer + vancomycin 68mg / L 0 Formulation + W3110 after 30 min (trial 1) TSA + neutralizer + vancomycin 68mg / L 0 Formulation + W3110 after 24h (trial 2) TSA + neutralizer + vancomycin 68mg / L 0 Formulation + W3110 after 5 days (trial 3) TSA + neutralizer + vancomycin 68mg / L 0

[0171] These results show that the application of a cleaning formulation according to the invention makes it possible to protect the surface of the earthenware plates from contamination by Escherichia coli W3110.

[0172] In conclusion, the application of a cleaning composition according to the invention (detergent formulation or aqueous solution) comprising the probiotic strain of Bacillus pumilus 1-5531 makes it possible to protect the 2 types of surface (PVC and earthenware) from contamination by 2 types of pathogens (S. aureus and E. coli).

Claims

Claims

1. Strain of Bacillus pumilus exhibiting antagonistic activity against pathogenic bacteria belonging to at least the following three families: Staphylococcaceae, Enterobacteriaceae and Listeriaceae, advantageously belonging to at least the following three genera: Staphylococcus, Escherichia and Listeria, said strain being the strain deposited at the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on July 1, 2020, under number 1-5531.

2. Use of the B. pumilus strain according to claim 1 in a cleaning, sanitizing and / or detergent composition.

3. A composition comprising the B. pumilus strain of claim 1.

4. Composition according to claim 3, characterized in that the strain represents from 106 to 108 CFU / mL of the composition.

5. Composition according to claim 3 or 4, characterized in that it does not comprise any other microorganism, in particular any other probiotic.

6. Composition according to one of claims 3 to 5, characterized in that the strain is in the form of spores and / or vegetative cells and / or cells which are in the transitional phase of sporulation, advantageously in the form of spores.

7. Composition according to one of claims 3 to 6, characterized in that it further comprises at least one compound selected from the group consisting of: a surfactant, a preservative, a perfume, a sequestrant, a texturizer, a wax, a gelling agent, an abrasive, a colorant, an acidity regulator, a base, a polymer, a resin, an anti-foaming agent, a solvent, water, a stabilizing agent, a thickener and an enzyme.

8. Composition according to one of claims 3 to 7, characterized in that it is in the form of an aqueous solution, advantageously a ready-to-use solution or a concentrated solution to be diluted.

9. A method for cleaning and / or protecting a surface from contamination comprising applying the B. pumilus strain according to claim 1 or 2 or the composition according to one of claim 3 to 8, advantageously by applying 105 to 107 UFC / m2 of surface.

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