METHOD FOR REMOVING BIOFILM AND USE OF A COMPOSITION COMPRISING A DETERGENT COMPONENT AND AN ENZYME COMPONENT

Description

[0001] The present invention relates to a method for removing biofilms and to the use of a composition comprising at least one enzymatic component and at least one detergent component.

[0002] Such a composition is known from document EP2243821, which discloses a composition for the removal of biofilms present on a substrate. This composition comprises (1) a detergent component simultaneously containing a wetting agent, a sequestering agent, and a dispersing agent, and (2) an enzymatic component simultaneously containing at least one protease, at least one laccase, and at least one polysaccharidase. This composition is described as being suitable for use in removing biofilms in facilities, for cleaning floors or surfaces in place or by soaking, for cleaning surgical equipment in place or by soaking, and for cleaning in the food industry.

[0003] Document WO2012 / 048757 A1 also discloses a composition for the removal of biofilms comprising at least one detergent component and at least one enzymatic component.

[0004] A biofilm is a viscous film that develops on all surfaces, resulting from the adhesion of microorganisms to these surfaces and their secretion of polymers that coat them and facilitate their adhesion. Biofilms thus form a protective layer around microorganisms and represent a recurring source of contamination of the surrounding environment, posing problems, for example, in the agri-food sector and in hospital settings.

[0005] More specifically, the accumulation of polymers secreted by bacteria creates a matrix composed primarily of polysaccharides, DNA, proteins, and lipids. This matrix protects these microorganisms from external aggressions and exhibits very high resistance to conventional cleaning and disinfection procedures. The microorganisms therefore thrive within this protective matrix and contaminate the surrounding environment, creating a particularly critical and difficult-to-eliminate reservoir.

[0006] It is recognized that the problem of biofilms is twofold. First, as mentioned above, they represent a permanent source of contamination that is very difficult to eliminate by conventional means, even the most aggressive ones. Indeed, conventional disinfectants are very often ineffective because they fail to reach the microorganisms protected by the biofilm matrix, which is composed of polysaccharides, DNA, proteins, and lipids.

[0007] Secondly, a biofilm is generally mixed, meaning that it is initially developed by certain bacterial strains but can harbor others, these strains living and developing in colonies. These colonies facilitate communication between bacteria and, among other things, the exchange and spread of resistance genes carried by certain bacteria. The biofilms resulting from these gene exchanges are then even more difficult to eliminate, requiring increasingly powerful disinfection or treatment methods, which, however, frequently encounter major resistance and / or tolerance problems.

[0008] The protective matrix of bacteria forming biofilms is so resistant that it constitutes a true barrier protecting bacteria from dehydration, the action of antibiotics and biocides (and more generally microbicidal molecules), phagocytosis and acids.

[0009] In hospitals, veterinary clinics, and the agri-food sector, the situation is particularly critical as numerous microorganisms responsible for biofilm formation are detected in many locations. In hospitals and veterinary clinics, biofilms are found on individuals (patients and animals), in the environment (operating rooms, surgical equipment, equipment used for maintaining this equipment, endoscopes, urinary catheters, medical equipment, dialysis or mechanical ventilation machines, etc.), and on surfaces (floors, walls, operating tables, etc.). In the agri-food sector, and especially in food processing plants, biofilms can be found on machinery, tables, packaging, and even on operators, despite the latter taking every possible precaution to avoid contaminating surfaces and tools.

[0010] From all this, it is clear that biofilms constitute a real problem, particularly in the healthcare sector (hospitals, dental practices, etc.), veterinary care, and the agri-food industry. This problem is all the more critical because biofilms can harbor bacteria responsible for potentially fatal infections in individuals, whether these bacteria are present in hospitals, veterinary practices, or food products. It is therefore essential to take all possible precautions to prevent the formation and development of biofilms.

[0011] Nowadays, to combat biofilms, enzymatic formulations including a detergent base are used, as proposed in document EP2243821. Most current formulations rely on a combination of several enzymes, these formulations generally always including at least one protease. However, even formulations (compositions) combining a detergent base and several enzymes have certain limitations in terms of biofilm removal from a substrate (a surface).

[0012] Unfortunately, it now appears that, even if a composition such as the one disclosed in document EP2243821 proves effective in removing biofilms, biofilm removal compositions very often rely on the use of an enzyme cocktail. More specifically, with regard to document EP2243821, the disclosed biofilm removal composition relies on the use of at least three enzymes (protease, laccase, and polysaccharidase) formulated in a detergent base that itself incorporates three different detergent agents (a sequestering, wetting, and dispersing agent). Therefore, formulating a biofilm removal composition according to document EP2243821 requires at least six different components, which is economically unfavorable since handling the different components to be formulated together is time-consuming and requires procuring all six components simultaneously.

[0013] Furthermore, it is recognized that in a formulation containing several enzymes, including a protease, the latter will inevitably and gradually digest the other enzymes present over time, thereby reducing their enzymatic activity. This, in turn, impacts the stability and efficacy of formulations containing a protease. In addition, current formulations have certain limitations, particularly when it comes to eliminating "old" biofilms—that is, biofilms that have been present for months or even years.

[0014] On the other hand, although it is not its purpose, WO2008 / 157350 incidentally indicates that Dispersin B can cut the β-1,6 bonds of poly-N-acetyl glucosamine that may be present in a biofilm.

[0015] The invention aims to overcome the drawbacks of the prior art by providing a biofilm removal process with a composition that is easier to formulate (to implement), whose stability and effectiveness over time is maintained and which allows effective action against all types of biofilms, including "old" biofilms.

[0016] To resolve at least part of the problems of the prior art, it is provided according to the invention, the use of a composition for the elimination of biofilms by cleaning in place, by soaking or by spraying of said surface, characterized in that said at least one enzymatic component comprises β-1,6-N-acetylglucosaminidase and in that said detergent component contains at least one wetting agent, at least one sequestering agent and at least one dispersing agent, said wetting agent representing between 1 and 30% of the detergent component, said sequestering agent representing between 1 and 10% of the detergent component and said dispersing agent representing between 1 and 10% of the detergent component.

[0017] For the purposes of the present invention, the enzymatic component may comprise β-1,6-N-acetylglucosaminidase as the sole enzyme or this same enzyme associated with others.

[0018] In particular, β-1,6-N-acetylglucosaminidase cleaves the poly-β-1,6-N-acetyl-D-glucosamide polymer (PNAG), which is an exopolysaccharide.

[0019] Quite surprisingly and contrary to expectations, it has been demonstrated within the scope of the present invention that such a composition comprising at least β-N-acetylglucosaminidase in at least one detergent component, which itself comprises at least one wetting agent, at least one sequestering agent, and at least one dispersing agent, is at least as effective for biofilm removal as a composition according to document EP2243821. This is because, as mentioned above, the prior art composition relies on the combined use and implementation of three enzymes in a single detergent component. It follows that, according to the present invention, a composition highly effective in removing biofilms from a surface can be prepared using only β-1,6-N-acetylglucosaminidase instead of an enzymatic cocktail based on the simultaneous presence of three different enzymes.Formulating such a composition is therefore easier since it requires fewer different compounds, which is more economically advantageous.

[0020] Even more surprisingly and unexpectedly, it has been determined, within the framework of the present invention, that the three agents of the detergent component exhibit a synergistic effect that optimizes the efficiency of β-1,6-N-acetylglucosaminidase in terms of biofilm removal, as well as in terms of its enzymatic activity over time. On the one hand, it has been demonstrated that this particular combination of three detergent agents (at least one wetting agent + at least one sequestering agent + at least one dispersing agent) ensures a significantly higher enzymatic activity of β-1,6-N-acetylglucosaminidase over time compared to the enzymatic activities observed for this same enzyme when the detergent component comprises, for example, only two of these three detergent agents.On the other hand, it has been pointed out that this particular combination of three detergent agents increases the effectiveness of the composition: optimal biofilm removal is observed when β-1,6-N-acetylglucosaminidase is formulated in a detergent component comprising simultaneously at least one wetting agent, at least one sequestering agent and at least one dispersing agent rather than in a detergent component comprising, for example, only two of these three detergent agents.

[0021] Furthermore, with such a composition and unlike a composition according to document EP2243821, the presence of a protease is not essential to obtain adequate elimination of biofilms on surfaces, thus eliminating the problem explained above and linked to the presence of this enzyme in a formulation where several enzymes are present.

[0022] Furthermore, contrary to the known prior art biofilm removal compositions, it has been shown that a composition comprising at least β-1,6-N-acetylglucosaminidase in at least one detergent component containing at least one wetting agent and at least one sequestering agent and at least one dispersing agent, makes it possible to act effectively even on "old" biofilms that are particularly difficult to remove.

[0023] Within the framework of the present invention, it has been shown that, following possible detection of the presence of biofilms, for example using a detection kit as described in document EP2537601, treatment with a composition according to the invention allows β-1,6-N-acetylglucosaminidase to efficiently and versatilityally degrade organic polymers of different natures constituting the matrix of biofilms formed by a multitude of different microorganisms.

[0024] Under the action of β-1,6-N-acetylglucosaminidase and in conjunction with the action of the detergent component, the biofilm matrix is ​​weakened and swollen, allowing it to be removed from the treated surface. Furthermore, and surprisingly, it has also been shown that the use of the composition according to the invention is not specific to a particular microorganism and therefore to a particular type of biofilm, but is suitable for numerous bacterial strains.

[0025] The detergent action of the composition further ensures the effectiveness of the composition used in the process according to the invention. To this end, the invention provides a detergent base (detergent component) that is compatible with and can act synergistically with the enzymatic activity of the enzyme component. In addition, the invention provides a detergent base that significantly improves the speed and efficiency of biofilm removal. For these reasons, the present invention combines a wetting agent, a dispersing agent, and a sequestering agent in the proportions described above.The combined actions of these three agents in the detergent component of the composition allow the superficial part of the biofilm to be removed, the organic structures of the biofilm to be wetted and swollen, thus promoting the accessibility of the enzymatic component which in turn weakens and degrades the biofilm matrix.

[0026] For the purposes of this invention, the detergent component comprises at least one wetting agent, at least one dispersing agent, and at least one sequestering agent in the proportions described above. As stated above, this detergent component first acts by removing a surface portion of the biofilm, thereby wetting and / or swelling the organic structures of the biofilm. In this way, the detergent component facilitates the accessibility of the enzymatic component by disrupting the biofilm matrix. The enzymatic component then acts synergistically with the detergent component, further weakening and degrading the biofilm matrix.This combined action of the enzymatic and detergent components, perfectly compatible with the proper function of β-1,6-N-acetylglucosaminidase, promotes the accessibility of the composition to deeper layers of biofilms and allows for rapid and optimal detachment of all types of biofilm while preserving the treated substrate (the treated surface). Thus, the detergent component enables the enzymes to act quickly on all biofilm structures.

[0027] The dispersing agent of the detergent component improves the separation of particles in a suspension to prevent clumping, aggregation, and / or settling. This dispersing agent may be a water-soluble or partially water-soluble polymer such as polyethylene glycol, cellulose derivatives, or a polymer comprising at least one acrylic acid or acrylic ester unit. Preferably, the dispersing agent is a polymer comprising at least one acrylic acid or acrylic ester unit with the general formula -(CH₂-CH-COOR)-, where R represents a hydrogen, alkyl, or substituted alkyl, aryl, or substituted aryl group. For example, the dispersing agent is a polymer with an average molecular weight (Mw) of approximately 500 to 10,000.

[0028] Preferably, the dispersing agent is a polymer of acrylic acid. For example, the dispersing agent may be a homopolymer of acrylic acid having an average molecular weight of approximately between 2000 and 6000.

[0029] For example, according to the present invention, said dispersing agent of said detergent component is the C6 alkylglucoside.

[0030] The presence of a dispersing agent in the formula prevents bacterial particle aggregation during surface cleaning, ensuring optimal removal of biofilm particles detached from the surface by enzymes. Indeed, rather than aggregating, these particles remain separated in a suspension, do not redeposit, and do not re-adhere to the cleaned surface.

[0031] The wetting agent in a detergent component is an amphiphilic chemical substance, or a composition containing such a chemical substance, which modifies the surface tension between two surfaces. The wetting agent has the advantage of facilitating the spreading of a liquid on a solid and also enhancing contact between two surfaces. More specifically, the wetting agent promotes contact between the detergent component and a surface, and consequently, between enzymes and their substrate. For example, on stainless steel surfaces frequently encountered in the food processing industry, as well as in hospital and veterinary settings, the wetting agent ensures a homogeneous spreading of the composition and thus its perfect distribution on the surfaces to be decontaminated, such as production tools, work surfaces, floors, operating tables, and medical instruments.

[0032] The wetting agent can be anionic, cationic, nonionic, or zwitterionic. Preferably, the wetting agent can be anionic or nonionic wetter, meaning that the hydrophilic portion is negatively charged or has no net charge, or it can be a composition comprising an anionic wetter. More specifically, the wetting agent can be a sucrose ester or a composition comprising a sodium alkyl sulfate and an alcohol.

[0033] The wetting agent may be foaming or non-foaming. Preferably, in the detergent component according to the invention, said wetting agent is non-foaming when hot and is preferably selected from the group of sodium alkyl sulfates in C6 to C10, ether alcohol sulfates in C6 to C10 and alkylearyl sulfonates in C6 to C10.

[0034] A sequestering agent is a chemical substance capable of forming complexes with mineral ions, which it binds in a form that prevents their precipitation through normal reactions. Examples of sequestering agents include ethylenediaminetetraacetic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate, calcium gluconate, citric acid, phosphoric acid, tartaric acid, sodium acetate, sorbitol, and compounds containing a phosphorus atom. Preferably, the sequestering agent may be a phosphorus oxide such as a phosphonate, a phosphinate or a phosphate or a mixture thereof, or a salt thereof, an amine or an amine oxide bearing at least one phosphine, phosphine oxide, phophinite, phosphonite, phosphite, phosphonate, phosphinate or phosphate functional group, alone or in combination, or a salt thereof.

[0035] More preferably, the sequestering agent may be a phosphonate or a salt thereof, an amine or an amine oxide comprising at least one phosphine, phosphine oxide, phosphinite, phosphonite, phosphite, phosphonate, phosphinate, or phosphate functional group, alone or in combination, or a salt thereof. By way of non-limiting example, the phosphonate may have the general formula R1<(R2<O)(R3<O)P=O in which R1<, R2<, and R3< independently represent a hydrogen, alkyl, substituted alkyl, substituted or unsubstituted alkyl-amino, substituted or unsubstituted aminoalkyl, aryl, or substituted aryl group.By way of non-limiting example, the amine or amine oxide may have one, two, or three substituents of the general formula CR4<R5<W, where R4< and R5< independently represent a hydrogen, alkyl, substituted alkyl, substituted or unsubstituted alkyl-amino, substituted or unsubstituted aminoalkyl, aryl, or substituted aryl group, and W represents a phosphonate, phosphinate, or phosphate group. The sequestering agent may be in the form of a sodium, calcium, lithium, magnesium, or potassium salt; preferably, the sequestering agent may be in the form of a sodium, calcium, or potassium salt.

[0036] Preferably, the sequestering agent is an agent that can be used safely in the food sector, i.e. that the sequestering agent presents no health risks, alone or in combination with other components.

[0037] Advantageously, according to the invention, said at least one enzymatic component comprises at least one additional enzyme selected from the group consisting of α-polysaccharidoses (lactase, amylase, α-glucosidase, ...), β-polysaccharidases (cellulase, hemicellulase, β-glucanase, arabanase, pectinase, chitinase, xylanase, dextranase, lysozyme, pullulanase, β-glucisidase, mannanase, ...), oxidoreductases (laccase, ...), lyases (pectate lyase, ...), transferases, proteases and peptidases (metalloprotease, serine proteases, exopeptidase, endoprotease, cystine protease, ...), lipases and esterases (lysophospholipase, phospholipase, ...).

[0038] Preferably, the composition for biofilm removal has a pH between 5 and 11.

[0039] As described above, said at least one detergent component comprises a sequestering agent proportion of between 1 and 10%, a dispersing agent proportion of between 1 and 10%, and a wetting agent proportion of between 1 and 30% by weight relative to the total weight of the detergent component. Preferably, the wetting agent proportion is between 5 and 20% by weight relative to the total weight of the detergent component. Preferably, the wetting agent proportion is 15% by weight relative to the total weight of the detergent component.

[0040] Advantageously, according to the invention, said at least one enzymatic component and said at least one detergent component are in solution in a solvent, for example in solution in a solvent in such a way as to form a vaporizable solution.

[0041] Preferably, according to the invention, said at least one enzymatic component and said at least one detergent component are in solid form, for example in the form of a lyophilized powder, granules or any other form soluble in a solvent.

[0042] Preferably, said at least one enzymatic component is a solution whose pH can be approximately between 8 and 10.

[0043] Preferably, said at least one enzymatic component is an aqueous solution whose pH can be approximately between 5 and 11; more preferably the pH can be approximately between 7 and 10, and this to preserve the maximum integrity of the enzymes.

[0044] Alternatively, the at least one enzymatic component may be in solid form, such as a lyophilized powder, granules, or any other form that allows the component to be solubilized in a solvent and subsequently dissolved in that solvent. The solvent may be water or an aqueous solution that is acidic, basic, alcoholic, buffered, or neutral. The solubilized at least one enzymatic component may then be further diluted in an aqueous solution that may contain one or more compounds, such as detergents, to form the cleaning solution.

[0045] As for the said at least one enzymatic component, the said at least one detergent component may be in solid form to be dissolved in a solvent and / or in an aqueous phase or in liquid form.

[0046] When in solid form, it can either be dissolved directly in the solution formed by the enzymatic component possibly already diluted in the aqueous phase, or be dissolved in a solvent, prior to its dilution in the solution formed by the enzymatic component and the aqueous phase, or in the aqueous phase directly, before the dilution of the enzymatic component.

[0047] When said at least one detergent component is in liquid form, 100% of the detergent component is eventually achieved generally with the aid of water and, prior to application to the biofilm, it will be diluted in an aqueous phase, possibly already containing the enzymatic component.

[0048] Other embodiments of a composition according to the invention are indicated in the attached claims.

[0049] The invention also relates to a method for removing biofilms present on a surface, said method comprising the following steps: a) making available at least one detergent component containing at least one sequestering agent and at least one dispersing agent and at least one wetting agent in the proportions described above, and at least one enzymatic component comprising β-1,6-N-acetylglucosaminidase; b) dissolving or diluting said at least one detergent component in a solvent; c) dissolving said at least one enzymatic component in the solution formed in step b) to form a composition; or b') dissolving or diluting said at least one enzymatic component in a solvent, c') dissolving said at least one detergent component in the solution formed in step b') to form a composition, d) applying said composition formed in step c) or c') to said surface for a predetermined period of time.

[0050] Preferably, according to the process of the invention, the application step of the composition is combined with a mechanical abrasion step of the surface with the composition, for example, by mechanized or manual brushing, or by medium- or high-pressure application. An additional mechanical abrasion step allows the aqueous-phase solution of the composition to act on the different layers of the biofilms and also to mechanically contribute to the breakdown of the polymer matrix, thereby removing layers from the surface of the biofilms so that the enzyme and other components of the composition can reach the different layers of the biofilms more effectively, ensuring optimal surface treatment for the efficient elimination of biofilms.

[0051] Advantageously, the process according to the invention includes a subsequent step of applying a biocide to said surface. An additional disinfectant biocide treatment, following the action of the enzymatic solution during the surface treatment step by soaking, ensures the destruction of the bacteria released at the end of the surface treatment.

[0052] Preferably, according to the process according to the invention, said step of applying the solution of said composition, is a step taking place during a predetermined period of time of between 1 minute and 1 hour, preferably between 3 minutes and 30 minutes, of a solution comprising said composition and a previously formed aqueous dilution phase.

[0053] Preferably, the process according to the invention makes it possible to ensure the elimination of at least 75%, preferably at least 90%, of microorganisms present on a surface and protected by a biofilm.

[0054] This rate of elimination of microorganisms, of at least 75%, preferably of at least 90%, is calculated on the basis of the microorganisms present in a given environment (for example on the surface of a medical tool or on the surface of soil) before and after a surface treatment step according to the invention.

[0055] For example, this microorganism elimination rate can be calculated using ATP-metric luminometric analysis, which determines, through bioluminescence, the quantity of microorganisms present on a surface based on Adenosine Triphosphate molecules previously collected from it, for example, by swabbing. Since this ATP-metric analysis yields results expressed in relative light units (URLs), the microorganism elimination rate is defined according to the following formula: 100 - [(URL value measured after treatment / URL value measured before treatment) x 100].

[0056] This microorganism elimination rate can also be established by a bacteriological analysis consisting of counting the bacterial colonies on the same surface before and after a surface treatment carried out according to the invention. In the case of bacteriological analyses, the microorganism elimination rate is calculated according to the following formula: 100 - [(CFU value after treatment / CFU value before treatment) x 100].

[0057] It is understood that any other technique well known to a person skilled in the art and allowing the quantities of microorganisms to be determined before and after treatment of a surface can also be used within the framework of the present invention.

[0058] Other embodiments of the process according to the invention are indicated in the attached claims.

[0059] Other forms of use of a composition according to the invention are indicated in the attached claims.

[0060] Other features, details and advantages of the invention will become apparent from the description given below, by way of non-limiting reference and with reference to the attached figures. There figure 1 illustrates the results obtained in terms of biofilm elimination (biofilm reduction %) formed by Staphyloccocus epidermis (EP = composition according to document EP2243821; disp. = dispersant; seq. = sequestering agent; mou. = wetting agent; β-1,6-N- = β-1,6-N-acetylglucosaminidase). The figure 2 illustrates the results obtained in terms of biofilm elimination (biofilm reduction %) formed by Escherichia coli (EP = composition according to document EP2243821; disp. = dispersant; seq. = sequestering agent; mou. = wetting agent; β-1,6-N- = β-1,6-N-acetylglucosaminidase). The figure 3 illustrates the results obtained in terms of biofilm elimination (biofilm reduction %) formed by Pseudomonas fluorescens (EP = composition according to document EP2243821; disp. = dispersant; seq. = sequestering agent; mou. = wetting agent; β-1,6-N- = β-1,6-N-acetylglucosaminidase). The figure 4 illustrates the results of measurements of β-1,6-N-acetylglucosaminidase enzymatic activity over time depending on whether this enzyme is formulated in the presence of three detergent agents (wetting agent + dispersing agent + sequestering agent), or in the presence of two of these three detergent agents, or whether this enzyme is not formulated in a detergent component. figure 5 illustrates the results of measurements of enzymatic activities of three cellulases over time depending on whether these enzymes are formulated or not in the presence of three detergent agents (wetting agent + dispersing agent + sequestering agent) or depending on whether this enzyme is not formulated in a detergent component. Exemples Example 1 - comparative tests: effectiveness of a composition used in a process according to the invention and effectiveness of a composition according to document EP2243821

[0061] Comparative tests were carried out to verify that a composition used in a process according to the invention is at least as effective in terms of biofilm removal as a composition as disclosed in document EP2243821 (cocktail of 3 enzymes in a detergent component comprising 3 detergent agents).

[0062] The composition used in a process according to the invention is as follows: Enzymatic component: β-1,6-N-acetylglucosaminidase at a rate of 0.005% by volume relative to the total volume of the composition; Detergent component: Biorem ®< A1 at a rate of 0.25% by volume relative to the total volume of the composition.

[0063] The composition according to document EP2243821 is as follows: Enzymatic component: Biorem ®< 10 at a rate of 0.01% by volume relative to the total volume of the composition; Detergent component: Biorem ®< A1 at a rate of 0.25% by volume relative to the total volume of the composition.

[0064] These components are prepared at room temperature by diluting them in water before conducting comparative tests on a biofilm developed by Pseudomonas fluorescens .

[0065] The initial steps of the procedure are similar to those in the protocol described in Annex F of ISO 15883. Indeed, the biofilm culture and cleaning protocol corresponds to that described in this standard. The quantity of biofilms present was assessed using the detection kit as per document EP2243821.

[0066] Practically speaking, a biofilm was developed by Pseudomonas fluorescens in a PTFE (polytetrafluoroethylene) tube with an inner diameter of 6 mm. The tube was filled with a liquid preculture undergoing exponential growth. The preculture was prepared 24 hours before the test: TSB culture medium was inoculated with the bacterial strain. Pseudomonas fluorescens at 30°C for 12 hours. This preculture was then diluted in 10% TSB culture medium to obtain an optical density between 300 nm and 600 nm. The inoculated culture medium was then circulated through the PTFE tube for 3 days. At the end of this period, the PTFE tube was cut into 5-10 cm sections, on which the different compositions were tested. These different compositions were circulated for 15 minutes at a flow rate of 100 ml / min at a temperature of 45°C. Finally, the amount of biofilm present was assessed by staining each tube section with the detection kit disclosed in document EP2537601.

[0067] The detection kit consists of two products: a staining reagent and a cleaning reagent. The blue dye in the staining reagent adheres specifically to the EPS (extracellular polymeric substances) that form the protective matrix of the biofilm. The surface to be analyzed was sprayed with the staining solution. After 5 minutes of incubation on the surface, the excess solution was absorbed. The surface was then rinsed using the cleaning solution from the detection kit, which requires a 5-minute contact time. Finally, the surface was rinsed with water and analyzed. The presence or absence of biofilm was demonstrated by a blue color, the intensity of which varied depending on the amount of biofilm present on the surface. An intense blue coloration indicates a significant presence of biofilm.

[0068] The results of these tests are presented in Table 1 below. Table 1: Nettoyage Coloration Avant nettoyage Coloration bleu intense Eau Coloration bleu intense Composition selon le document EP2243821 Absence de coloration bleu Composition selon l'invention Absence de coloration bleu

[0069] As can be seen from these comparative tests, for portions of tubes initially heavily contaminated by biofilm (intense blue colour before cleaning), cleaning with water had no effect on the biofilm and the portions of tubes initially heavily contaminated by biofilm therefore retained their intense blue colour.

[0070] However, whether cleaning with the composition according to document EP2243821 or with the composition used in the process according to the invention, complete biofilm removal was ensured (absence of blue staining) after cleaning with both compositions. This therefore indicates that a composition used in the process according to the invention allows for the removal of biofilms at least equivalent to the composition according to document EP2243821, but using only one enzyme, namely β-1,6-N-acetylglucosaminidase, instead of three enzymes. Example 2 - Efficacy tests (in terms of biofilm removal) of a composition used in the process according to the invention, depending on whether the detergent component simultaneously comprises a wetting agent, a dispersing agent and a sequestering agent, or a combination of two of these three detergent agents.

[0071] Efficacy tests were carried out on biofilm developed by different bacterial strains, namely the strains Staphyloccocus epidermis , Escherichia coli And Pseudomonas fluorescens Biofilms were developed in the laboratory under static conditions in multiwell plates, and the effectiveness of β-1,6-N-acetylglucosaminidase (12.5 ppm active enzyme in the composition, corresponding to 0.00125% by volume relative to the total volume of the composition) was evaluated on these biofilms following its formulation in the following detergent components: wetting agent + sequestering agent + dispersing agent = Biorem ®< A1 at 1% by volume relative to the total volume of the composition; wetting agent of Biorem ®< A1 at 0.22% by volume relative to the total volume of the composition + sequestering agent of Biorem ®< A1 at 0.25% by volume relative to the total volume of the composition; wetting agent of Biorem ®< A1 at 0.22% by volume relative to the total volume of the composition + dispersing agent of Biorem ®< A1 at 0.085% by volume relative to the total volume of the composition; sequestering agent of Biorem ®< A1 at 0.25% by volume relative to the total volume of the composition + dispersing agent of Biorem ®< A1 at 0.085% by volume relative to the total volume of the composition.

[0072] In parallel, β-1,6-N-acetylglucosaminidase (12.5 ppm active enzyme in the composition, which corresponds to 0.00125% by volume relative to the total volume of the composition) was tested alone (not formulated in a detergent component but in aqueous solution) and the effectiveness of a composition according to document EP2243821 (BioremA1 ®< 1% by volume relative to the total volume of the composition and Biorem10 ®< 0.025% by volume relative to the total volume of the composition). A. Biofilm preparation

[0073] Bacterial colonies were obtained by separate inoculations of each of the strains ( Staphyloccocus epidermis , Escherichia coli Or Pseudomonas fluorescens ) on a general-purpose solid agar (PCA) and then incubated for 18 h at 37°C. Next, a few bacterial colonies (approximately 20 colonies) were suspended in their respective appropriate culture media. Specifically, the bacterial colonies were suspended in the following culture media: Staphyloccocus epidermis : suspension of approximately 20 colonies in 2 mL of TGN (30 g / L of Tryptic Soy Broth + 1 g / L of glucose + 2 g / L of NaCl); Escherichia coli : suspension of approximately 20 colonies in 2 mL of LBG (15 g / L peptone water + 5 g / L yeast extract + 10 g / L glucose); Pseudomonas fluorescens : suspension of approximately 20 colonies in 2 mL of TSB (30 g / L of Tryptic Soy Broth).

[0074] Each suspension was adjusted by adding culture medium (TGN, LBG, or TSB) to achieve an optical density (OD) of 0.05 at 620 nm, and then the bacterial suspensions were placed in the wells of multiwell plates at a rate of 100 µL per well. Staphyloccocus epidermis 220 µL per well for Escherichia coli and 220 µL per well for Pseudomonas fluorescens before incubation for 24 hours at 37°C for Staphyloccocus epidermis , for 48 hours at 30°C to Escherichia coli and for 48 hours at 30°C to Pseudomonas fluorescens Of course, for each of the bacterial strains tested, wells were filled only with culture medium to constitute negative controls. B. Enzymatic treatment of formed biofilms and quantification of biofilm removal

[0075] Before treating the biofilms formed with various compositions, including one used in the process according to the invention, the wells of the multi-well plates were emptied of the culture media that had allowed the establishment and development of the different biofilms. The biofilm treatment itself was carried out by filling the wells with 1 mL of the cleaning solutions (compositions), with softened water being added only to the two wells of the positive control, while the two wells of the negative control remained empty. Following these fillings, an incubation at 45°C for 15 min was performed (the cleaning time required to eliminate the biofilms).

[0076] After this incubation time, the wells were emptied of the cleaning solutions and rinsed with sterile 0.9% saline before drying the plates for 1 hour at 60°C. Following this drying, 0.5 mL of 0.1% crystal violet was added to each well before incubation for 15 minutes at room temperature. After the 15-minute incubation at room temperature, the wells were emptied of the crystal violet solution, rinsed twice with distilled water, and then dried on absorbent paper if necessary. 1 mL of 33% acetic acid was then added to each well before incubation for 1 hour at room temperature. The plates were then read at a wavelength of 570 nm (absorbent measurement by spectrophotometry - Thermo Spectronic UV1).The reduction in biofilm quantity is calculated relative to an uncleaned control and a control containing no biofilm: crystal violet (CV) is a cationic dye that binds nonspecifically to negatively charged biofilm components and stains them blue. The absorbance measurements of crystal violet thus allow for the quantification of the biofilm remaining after cleaning.

[0077] The results obtained are presented to figures 1 , 2 And 3 , respectively for biofilms formed by Staphyloccocus epidermis, Escherichia coli Or Pseudomonas fluorescens.

[0078] As can be seen, whatever the biofilm considered (formed by S . epidermis or by E. coli or by P . fluorescensA composition according to the invention (β-1,6-N- + disp. + seq. + mou.) significantly improves biofilm removal compared to other tested compositions. This demonstrates that the three detergent agents exhibit an unexpected synergistic effect, optimizing the action of β-1,6-N-acetylglucosaminidase in terms of biofilm removal. Indeed, when β-1,6-N-acetylglucosaminidase is formulated in a detergent component containing only two of the three detergent agents, biofilm removal is reduced. The same observation can be made with a composition according to document EP2243821, which does not remove biofilms as effectively compared to a composition used in the process according to the invention. Example 3 - Enzymatic activity of β-1,6-N-acetylglucosaminidase depending on whether the detergent component simultaneously comprises a wetting agent, a dispersing agent and a sequestering agent or a combination of two of these three detergent agents

[0079] The enzyme activity of β-1,6-N-acetylglucosaminidase over time was measured using the substrate 4-nitrophenyl-N-acetyl-βD-glucosaminide. This substrate releases a yellow compound when hydrolyzed by β-1,6-N-acetylglucosaminidase. The greater the degree of hydrolysis, the higher the concentration of the yellow compound and the more active the β-1,6-N-acetylglucosaminidase. The degree of hydrolysis is quantified by measuring the absorbance of the solution at 405 nm and allows for the assessment of the enzymatic activity of β-1,6-N-acetylglucosaminidase.

[0080] In general, the enzymatic activity of β-1,6-N-acetylglucosaminidase over time was measured according to the following steps: a) β-1,6-N-acetylglucosaminidase was mixed in the different compositions to be tested (see below) and the solutions thus obtained were maintained at a temperature of 40°C for 2 h in order to simulate the cleaning conditions; b) samples of these solutions were taken at t = 0 (t 0 ) and t = 2 h (t 2h ) in order to measure the enzymatic activity over time of β-1,6-N-acetylglucosaminidase.

[0081] More specifically, the following protocol was followed: a) A buffered solution at pH 5.9 was prepared: 50 mM sodium phosphate + 5 mM 4-nitrophenyl-N-acetyl-β-D-glucosaminide + 100 mM NaCl; b) The solution obtained in a) was preheated to 37°C and dispensed 1 mL into Eppendorf tubes; c) 100 µL of the compositions whose enzymatic activity over time is to be measured (including one composition according to the invention) were placed in the Eppendorf tubes of point b); d) The concentration of β-1,6-N-acetylglucosaminidase was adjusted by dilution with phosphate buffer if necessary to obtain a concentration of approximately 3.7 µg / mL of β-1,6-N-acetylglucosaminidase; e) the reaction was stopped by adding 5 µL of 10N NaOH after 5 min; f) the absorbance was measured at 405 nm (- Thermo Spectronic UV1 after 5 minutes (at t 0 + 5 min and t 2h + 5 min), a negative control (blank) being prepared with only water.

[0082] More specifically, the enzymatic activity of β-1,6-N-acetylglucosaminidase was measured over time in the following compositions: wetting agent + sequestering agent + dispersing agent (= Biorem ®< A1 at 0.25% by volume relative to the total volume of the composition) + β-1,6-N-acetylglucosaminidase at 7.4 µg / mL; wetting agent of Biorem ®< A1 at a rate of 0.055% by volume relative to the total volume of the composition + sequestering agent of Biorem ®< A1 at a rate of 0.0625% by volume relative to the total volume of the composition + β-1,6-N-acetylglucosaminidase at 7.4 µg / mL; Biorem®< A1 wetting agent at a rate of 0.055% by volume relative to the total volume of the composition + Biorem®< A1 dispersing agent at a rate of 0.021% by volume relative to the total volume of the composition + β-1,6-N-acetylglucosaminidase at 7.4 µg / mL; Biorem®< A1 sequestering agent at a rate of 0.0625% by volume relative to the total volume of the composition + Biorem®< A1 dispersing agent at a rate of 0.021% by volume relative to the total volume of the composition + β-1,6-N-acetylglucosaminidase at 7.4 µg / mL;β-1,6-N-acetylglucosaminidase alone at 7.4 µg / mL in aqueous solution. ;

[0083] The results obtained are illustrated in the figure 4and are expressed as a percentage of the β-1,6-N-acetylglucosaminidase activity measured for a composition not including a detergent component (relative enzymatic activity). As can be seen, from the outset at t0, i.e., from the moment β-1,6-N-acetylglucosaminidase is formulated in a detergent component simultaneously containing a wetting agent, a sequestering agent, and a dispersing agent, its enzymatic activity is strongly and significantly potentiated / increased compared to the enzymatic activities measured for β-1,6-N-acetylglucosaminidase when formulated in a detergent component containing only two of the three detergent agents.Subsequently, over time and in particular after 2 h, a significantly higher enzymatic activity of β-1,6-N-acetylglucosaminidase is observed for a composition used in the process according to the invention compared to the enzymatic activities measured for β-1,6-N-acetylglucosaminidase when formulated in a detergent component comprising only two of the three detergent agents.

[0084] This clearly demonstrates the synergistic effect of the three detergent agents which together significantly potentiate / increase the enzymatic activity of β-1,6-N-acetylglucosaminidase, which is consequently more effective over time.

[0085] For comparison with β-1,6-N-acetylglucosaminidase, three cellulases (other enzymes from a different family – Celluclast® < 1.5L, Carezyme® < 4500L, Viscozyme® < 120L) were also tested in terms of enzymatic activity over time after formulation in Biorem® < A1 as a detergent component at a concentration of 0.025% by volume relative to the total volume of the composition. Specifically, the enzymatic activity of the three cellulases was measured according to the endo-cellulase protocol (CELLG5® method) of the Megazyme® kit, following the manufacturer's recommended protocol. The enzymatic activities of cellulases alone and those formulated in a detergent component according to the invention were thus compared.

[0086] The amount of cellulase in the composition was determined to be within a concentration range that falls within the measurement range of the commercial kit. Preliminary tests were performed on each cellulase tested, and an appropriate concentration for measurement was selected for each cellulase.

[0087] There figure 5This illustrates the results obtained, and it can be seen that, unlike the results obtained for β-1,6-N-acetylglucosaminidase, the same detergent component (wetting agent + dispersing agent + sequestering agent) does not have the same effect on the cellulases. Indeed, no potentiation / increase in enzymatic activity is observed directly following the formulation of each of the cellulases in this detergent component. Furthermore, for these three cellulases, no potentiation / increase in enzymatic activity is maintained over time by this detergent component. On the contrary, for Cellulase 2, its formulation in a detergent component identical to that of a composition used in the process according to the invention significantly reduces its enzymatic activity.This further highlights the surprising effect observed with a composition used in the process according to the invention where β-1,6-N-acetylglucosaminidase, unlike these three cellulases, has a significantly increased enzymatic activity when in the presence of the three detergent agents (wetting agent + dispersing agent + sequestering agent).

[0088] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made to it without departing from the scope of the attached claims.

Claims

1. Method for removing biofilms present on a surface, said method comprising the following steps: a) providing - at least one detergent component containing at least one sequestering agent, at least one dispersing agent, and at least one wetting agent, and - at least one enzymatic component comprising β1,6-N-acetylglucosaminidase; b) dissolving or diluting said at least one detergent component in a solvent; c) dissolving said at least one enzymatic component in the solution formed in step (b) to form a composition in which the proportion of sequestering agent is between 1 and 10%, the proportion of dispersing agent is between 1 and 10%, and the proportion of wetting agent is between 1 and 30% by weight relative to the total weight of the detergent component; or b') dissolving or diluting said at least one enzymatic component in a solvent, c') dissolving said at least one detergent component in the solution formed in step b') to form a composition in which the proportion of sequestering agent is between 1 and 10%, the proportion of dispersing agent is between 1 and 10%, and the proportion of wetting agent is between 1 and 30% by weight relative to the total weight of the detergent component, d) applying said composition formed in step c) or c') to said surface for a predetermined period of time.

2. Method according to claim 1, characterised in that said step of applying said composition is associated with a step of mechanically abrading said surface using said composition, for example by mechanised or manual brushing, or alternatively by medium- or high-pressure application.

3. Method according to claim 1 or 2, characterised in that it comprises a subsequent step of applying a biocide to said surface.

4. Method according to any one of the preceding claims, wherein the enzymatic component comprises at least one additional enzyme selected from the group consisting of α-polysaccharidases, β-polysaccharidases, oxidoreductases, lyases, transferases, proteases, peptidases, lipases, and esterases.

5. Use of a composition comprising at least one enzymatic component comprising β-1,6-N-acetylglucosaminidase and at least one detergent component containing at least one wetting agent, at least one sequestering agent and at least one dispersing agent, for the removal of biofilms present on a surface by clean-in-place cleaning, soaking or spraying of said surface, wherein the proportion of sequestering agent in said detergent component is between 1 and 10%, the proportion of dispersing agent is between 1 and 10%, and the proportion of wetting agent is between 1 and 30% by weight relative to the total weight of the detergent component.