EP2627752A1

EP2627752A1 - Product and method for the removal of biofilms

Info

Publication number: EP2627752A1
Application number: EP10765641.5A
Authority: EP
Inventors: Gauthier Boels; Gordon Blackman; Sébastien FASTREZ
Original assignee: Realco SA
Current assignee: Realco SA
Priority date: 2010-10-15
Filing date: 2010-10-15
Publication date: 2013-08-21
Also published as: WO2012048757A1; CA2817105A1

Abstract

Composition for the removal of biofilms present on a substrate, characterized in that it comprises at least one detergent component comprising at least one sequestrant and one agent that is simultaneously a wetting agent and a dispersant, and at least one enzymatic component containing at least one protease, at least one laccase and at least one polysaccharidase, method thereof for the removal of biofilms and use thereof.

Description

"PRODUCT AND METHOD FOR REMOVING BIOFILMS"

The invention relates to the field of biofilm removal. More particularly, the invention relates to a composition and a method for removing biofilms.

Hygiene is of increasing importance in the food industry, in hospitals, and particularly in the surgical sphere, in water purification and desalination plants, in the treatment of process water, and in particular in wastewater treatment plants. water used in cooling towers and in the necessities of everyday life, for example contact lenses. It is frequently observed that, during the circulation of water on a support, microorganisms circulating freely in the water can adhere to the surface. These microorganisms can then develop an adhesive extracellular matrix composed of polymeric substances. A community of microorganisms adhered to a surface and encompassed in such a matrix is called a biofilm. Generally these biofilms are composed of bacteria.

It is unfortunately observed that this matrix is very resistant, and may constitute a barrier for agents that act against microorganisms. Conventional treatments based on soda and / or with different biocides do not act sufficiently effectively because they do not penetrate the entire thickness of the biofilm or are inhibited by certain molecules making up this matrix. The treatment is then only partially effective on the upper surface of the biofilm. In addition, the latter can also trap other microorganisms, including pathogens, than the one that was initially installed.

A process for the enzymatic treatment of a biological film is known from WO98 / 26807. In this process, the biofilm with a cleaning composition comprising one or more hydrolases for removing or releasing the biofilm layer from the surface. In a second step, the biofilm is brought into contact with a bactericidal disinfecting composition to destroy the bacterial cells present in the film. However, the simultaneous use of these two compositions is at the origin of a degree of inactivation of certain enzymes in the final mixture. The speed and effectiveness of the cleaning can therefore be improved by using a composition in which the inactivation of the enzymes is absent.

Document US2003 / 0205247 also discloses the use of aqueous solutions containing enzymes for cleaning storage or fermentation tanks, containing one or more enzymes selected from the following: laccases, peroxidases, oxidoreductases, transferases, isomerases, lyases or ligases and a thickening agent with a foam booster. The field of action of the aforementioned solutions is relatively narrow, specific to the brewery, since the enzymes chosen and illustrated are particularly known to be active on polyphenols which mainly constitute fermentation residues, tannins and the like. The thickening agent is an agent for modifying the viscosity and the thixotropy of the solution used in order to allow better adhesion of the solution and / or the foam on the surfaces to be treated of the tanks. These solutions are not suitable for cleaning different plants of tanks or tanks (including for example many tubings) and are not suitable for the removal of other types of microorganisms. and therefore not for a wide range of biofilms.

WO 92/13807 discloses the use of a composition for the removal of biomass and biofilm from substrates in aqueous systems, i.e. in which water is circulated or stored. This type of system suffers from the presence of alkaline or acidic biomass and biofilms due to the type of organism generally present in this type of installation.

To solve this problem, the document WO 92/13807 provides for the use of polysaccharidases and / or proteases and anionic surfactants such as SDS or DBS (respectively sodium dodecyl sulphate and dodecylbenzene sulphonic acid) for the elimination of biomass and biofilms.

Unfortunately, this type of composition will not be effective for a wide range of biofilms caused by various microorganisms and will have limited efficacy for biofilm removal.

As can be seen from the foregoing, these compositions are always targeted for a targeted type of biofilm or microorganism and / or for a particular application.

There is therefore a need for a composition and method capable of eliminating biofilms, which are effective in a reasonable time, to act on a broad class of biofilms produced by a broad class of microorganisms or groups of microorganisms, which are not harmful for the support of the biofilm, and act both to prevent the development of biofilms and biofilms long established, and having reached a stage of cohesion and significant resistance.

To solve this problem, the present invention provides a composition for the removal of biofilms present on a substrate characterized in that it comprises:

at least one detergent component comprising a sequestering agent and a simultaneously wetting and dispersing agent, at least one enzymatic component containing at least one protease, at least one laccase and at least one polysaccharidase.

The constitution of a composition comprising a detergent component and an enzymatic component containing at least one protease, at least one laccase and at least one polysaccharidase makes it possible, surprisingly, to significantly improve the speed and the effectiveness of the elimination of a biofilm while being able to attack various types of biofilms. This composition makes it possible to eliminate all or almost all of the biofilm and can act on even mature biofilms or having an earlier development cycle and developed by multiple species or different microorganisms.

To date, it must be known that no effective composition can ensure the complete elimination of biofilms present in the facilities. For example, in the field of the food industry, biofilms inevitably form (given the richness of the surrounding environment). Biofilms exhibit cyclic growth activity including a growth phase during which microorganism accumulation occurs and a detachment phase during which whole pieces of biofilms break off under erosion and under their own weight. When an industrialist is confronted with this phenomenon, he should actually stop his production line and carry out different alternating washing cycles with sodium hydroxide, and with many detergents and or chemical and / or enzymatic cleaners since he does not exist. of versatile composition. This represents many hours of work and loss of efficiency of the installation.

Therefore, in practice, the production is not stopped and when the biofilm is in breakage, the batches produced are contaminated and discarded until the level of contamination microorganisms of the foods produced are again acceptable in view of the standards in force. In addition, it is not feasible for manufacturers to have in stock a particular detergent or enzymatic solution for each microorganism likely to contaminate its production chain.

It is thus very surprising that the present invention makes it possible to provide a perfectly polyvalent detergent composition that makes it possible to eliminate a wide spectrum of biofilms, in various types of installation and that does not require any particular precautions to be taken. employment. The detergent removes a superficial portion of the biofilm and wets and swells the organic structures of the biofilm through the dispersancy and wetting of the simultaneously wetting and dispersing agent present in the detergent component. This therefore promotes the accessibility of the enzymatic component which weakens and degrades the matrix of the biofilm. This combined action of the three types of enzyme and detergent component promotes accessibility to the composition of deeper layers and allows optimal detachment of any type of biofilm while preserving the substrate.

This type of simultaneously wetting and dispersing agent further reduces the supply of external compounds in the installations during the cleaning step and thus simplifies the validation procedures of the cleaning steps.

In a particular embodiment according to the invention, the composition is an aqueous cleaning solution having a pH of between approximately 8 and 1 1, preferably approximately between 9.5 and 10.5 and more preferably between approximately 9.5. and 10. The pH value of said composition significantly influences its effectiveness vis-à-vis the biofilm. A solution of said composition whose pH is approximately between 8 and 1 1 therefore makes it possible to eliminate surprisingly all or almost all of the biofilm.

Alternatively, said composition may be in solid form and then dissolved in a solvent before use to obtain, by dilution in an aqueous phase, a cleaning solution whose pH is approximately between 8 and 11.

In an advantageous variant according to the invention, the composition is a liquid solution which is then diluted in an aqueous phase to obtain an aqueous cleaning solution having a pH of between 6.5 and 7.5, more particularly around 7. From in this way, the pH of the composition is particularly suitable for the action of the enzymatic component, in particular laccase. In addition, it is provided according to the invention to increase the pH in a subsequent phase of application of the composition according to the invention so as to obtain an alkaline pH particularly suitable for the removal of the biofilm as mentioned above while keeping the optimum efficiency of the enzymatic component.

Alternatively, said composition can be in solid form and then dissolved in a solvent before use before being diluted in an aqueous phase to obtain a cleaning solution whose pH is between approximately 6.5 and 7.5.

Preferably, said at least one enzymatic component comprises a proportion of protease (s) of between 10 and 50%, a proportion of laccase (s) of between 5 and 35% and a proportion of polysaccharidase (s) of between 5 and 20%. % by weight relative to the weight of the enzymatic component, a conventional excipient or solvent being optionally added to reach 100% by weight of the enzymatic component, for example an alcohol.

According to a preferred embodiment of the invention, the enzymatic component may contain between 1 and 10 proteases, preferably between 1 and 5 proteases, more preferably contain 2, 3, 4 or 5 proteases.

Nonlimiting examples of protease enzymes belonging to class EC 3.4 and capable of being used in the invention are amino peptidases (EC 3.4.1 1), dipeptidases (EC 3.4.13), dipeptidyl peptidases and tripeptidyl peptidases (EC 3.4.14), peptidyl dipeptidases (EC 3.4.15), serine carboxypeptidases (EC 3.4.16), metallo carboxypeptidases (EC 3.4.17), cysteine carboxypeptidases (EC 3.4.18). ), omega peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metalloendopeptidases (EC 3.4.24) ), threonine endopeptidases ((EC 3.4.25), and endopeptidases belonging to class EC 3.4.99.

Preferably, the proteases belong to the class EC 3.4.21. Proteases are available commercially and in various forms including powders, granules, suspensions, liquid solutions.

The laccases used in the invention belong to the class EC 1 .10.3.2. Laccases are copper-containing enzymes whose function is to oxidize a substrate in the presence of oxygen. More specifically, laccases are oxidoreductases that work with molecular oxygen as an electron acceptor.

The at least one polysaccharidase used in the invention is an enzyme whose function is to break bonds within the polysaccharides. Preferably, the at least one polysaccharidase may be an alpha-amylase, cellulase, hemi-cellulase, glucosidase, beta-glucanase or pectinase.

More preferably, the at least one polysaccharidase may be an alpha-amylase belonging to the class EC 3.2.1.1, whose function is to break (1 -4) -alpha-glycosidic bonds in polysaccharides containing three or more units alpha - (1 -4) -D-glucose. Preferably, the enzymatic component may comprise a proportion of laccase (s) of approximately 30%, a proportion of protease (s) of approximately 30%, a proportion of alpha-amylase (s) of approximately 10% by weight. relative to the weight of the enzymatic component, a conventional excipient or solvent being optionally added to achieve 100% by weight of the enzymatic component.

According to another preferred embodiment, if the enzymatic component comprises 2 proteases, the proportion of laccase (s) can be approximately 30%, the total proportion of proteases approximately 30%, the proportion of alpha-amylase ( s) of approximately 10% by weight relative to the weight of the enzymatic component, a conventional excipient or solvent being optionally added to reach 100% by weight of the enzymatic component.

According to another preferred embodiment, if the enzyme component comprises 2 proteases, the proportion of laccase may be 30%, the total proportion of proteases 30%, the proportion of alpha-amylase 10% by weight relative to the weight of the enzymatic component, a conventional excipient or solvent being optionally added to reach 100% by weight of the enzymatic component.

For example, the ratio between each protease may be between 1: 2 and 2: 1, preferably the ratio between each protease may be 1: 1. The enzymes present in the enzymatic component have a complementary action on the biofilm. For example, laccase is highly effective on soils not attacked by alpha-amylase or proteases.

According to a preferred embodiment of the invention, the enzymatic component may be a solution or in solid form. Preferably, the enzymatic component is a solution whose pH may be between 8 and 10. Preferably, the enzymatic component is an aqueous solution whose pH may be between 8.5 and 9.5; more preferably the pH may be approximately 9.0.

Alternatively, the enzymatic component may be in solid form such as, for example, in the form of a lyophilizate, powders, granules or in any other form permitting the solubilization of said component in a solvent, then it will be subsequently dissolved in a solvent. The solvent may be water or an aqueous, acidic, alcoholic, basic, buffered or neutral solution. The solubilized enzymatic component can then in this case be further diluted in an aqueous solution optionally containing one or more compounds such as for example detergents to form the cleaning solution.

In an advantageous embodiment according to the invention, said at least one detergent component comprises a proportion of sequestering agent of between 1 and 10% by weight relative to the total weight of the detergent component, which represents an optimum between efficiency, stability and cost.

The sequestering agent is a chemical substance having the ability to form complexes with mineral ions which it fixes in a form preventing their precipitation by the usual reactions. By way of example, the sequestering agent may be ethylene-diamine tetraacetic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate, calcium gluconate, citric acid, phosphoric acid, tartaric acid, sodium acetate, sorbitol, a compound having 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, a amine or an amine oxide carrying at least, in its structure, a functional group phosphine, phosphine oxide, phophinite, phosphonite, phosphite, phosphonate, phosphinate or phosphate, alone or in combination, or a salt thereof.

More preferably the sequestering agent may be a phosphonate or a salt thereof, an amine or an amine oxide having at least, in its structure, a functional group phosphine, phosphine oxide, phosphinite, phosphonite, phosphite, phosphonate, phosphinate or phosphate, alone or in combination or a salt thereof. By way of nonlimiting example, the phosphonate may be of general formula R ¹ (R ² O) (R ³ O) P = O in which R ¹ , R ² and R ³ independently represent a hydrogen, alkyl or substituted alkyl group substituted or unsubstituted alkyl-amino, substituted or unsubstituted aminoalkyl, aryl or substituted aryl. By way of nonlimiting example, the amine or the amine oxide may comprise one, two or three substituents of general formula CR ⁴ R ⁵ W in which R ⁴ and R ⁵ independently represent one of the other a hydrogen, alkyl, substituted alkyl, substituted or unsubstituted alkylamino, 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 salt of sodium, calcium, lithium, magnesium or potassium; preferably, the sequestering agent may be in the form of a salt of sodium, calcium, or potassium.

In an advantageous embodiment according to the invention, the proportion of agent simultaneously wetting and dispersing between 15 and 50% by weight relative to the total weight of the detergent component, preferably from 20 to 30%, which represents a compromise between efficiency, stability and price.

A simultaneously wetting and dispersing agent is an amphiphilic chemical substance which modifies the surface tension between two surfaces which has the advantage of promoting the spread of a liquid on a solid (anionic, cationic, nonionic or zwitterionic) while having a dispersing nature, that is to say having the ability to improve the separating the particles from a suspension to prevent agglutination, aggregation and / or settling.

Advantageously, said simultaneously wetting and dispersing agent is chosen from the group consisting of non-hot foaming wetting agents and preferably comprises a C ₆ to C 12, preferably C ₈ , alkylglucoside derived from renewable raw materials and optionally an ethoxylated alcohol.

The wetting and dispersing agent is preferably a preparation comprising an ethoxylated alcohol and an alkylpolyglucoside (in particular an octyl glucoside) derived from renewable raw materials. As can be seen, this simultaneously wetting and dispersing agent allows particularly high surfactant and emulsifying performance without destabilizing the enzymes present in the composition according to the invention since this simultaneously wetting and dispersing agent is an anionic agent which has a low overall net charge. so as not to interfere with the ion exchange between the enzymes and substrates of the latter. Simultaneously wetting and dispersing agents derived from renewable raw materials are particularly suitable and act synergistically with the enzymes by not destabilizing them compared to current surfactants of petroleum origin (longer carbon chain). In addition, according to the present invention, the simultaneously wetting and dispersing agent may be anionic, cationic or nonionic, but in any case it will have a low overall ionic charge to be compatible and not destabilize the enzymes.

In addition, the simultaneously wetting and dispersing agent according to the invention comprising an ethoxylated alcohol and an alkylpolyglucoside is particularly versatile in that it is compatible with a wide pH range, with the presence of many acids or bases and builders, while being non-foaming hot.

The fact that the simultaneously wetting and dispersing agent is a wetting agent, not hot foaming allows versatile use, whether in tanks and surfaces or in facilities with many pipes and tubes, thereby avoiding the formation of without altering, on the contrary, the surfactant and / or emulsifying performance of the composition according to the invention. In addition, as already mentioned above, the presence of a simultaneously dispersing and wetting agent limits the supply of polymeric substance in the installations, for example, in circulation or water treatment installations. It is understood that the provision of an effective detergent solution without foam generation limits the rinsing steps, which is particularly desirable, especially in multiple pipe or pipe installations.

Other embodiments of the composition according to the invention are mentioned in the appended claims.

The subject of the invention is also a method for eliminating biofilms present on a substrate, characterized in that it comprises the following steps of:

a) providing a detergent component comprising a sequestering agent as well as a wetting agent and a dispersing agent and an enzymatic component containing at least one protease, at least one laccase, and at least one polysaccharidase,

b) dissolving the detergent component in an aqueous phase,

c) dissolving the enzymatic component in the solution formed in step b) to form the solution of said composition according to the invention or b ') dissolving the enzymatic component in an aqueous phase, - c ') dissolving the detergent component in the solution formed in step b') to form the solution of said composition according to the invention

- d) applying the solution of said composition formed in step c) or c ') on the substrate for a predetermined period of time, in particular between 15 minutes and 4 hours.

For the purposes of the present invention, the term "solution" is used to dissolve a solid compound in a liquid phase or to dilute a liquid compound in a liquid phase in which the liquid compound is miscible.

Alternatively, steps b) and c), or b ') and c'), can be performed simultaneously to form a solution of said composition according to the invention.

Preferably, the method comprises the following steps of:

a) providing a detergent component containing a sequestering agent as well as a wetting agent and a dispersing agent; and an enzymatic component containing at least one protease, at least one laccase, and at least one polysaccharidase,

b) dissolving the detergent component in the water,

c) dissolving the enzymatic component in the solution formed in step b) to form the solution of said composition according to the invention, d) applying the solution of said composition formed in step c) on the substrate for a predetermined period of time, preferably between 15 minutes and 4 hours.

Advantageously, the pH of the solution of said composition formed in step c) or c ') is between 8 and 1 1, preferably approximately between 9.5 and 10.5 and more preferably between 9.5 and 10. .

According to a preferred embodiment of the invention, the pH of the solution formed during step b) is approximately between 1, 0 and 14.0, preferably between approximately 12.0 and 14.0 and more preferably between 12.8 and 13.8.

In a variant according to the invention, the pH of the solution of said composition formed in step c) or c ') is between 6.5 and 7.5 and a basic solution is added after said application d) of the solution of said composition on said substrate during said predetermined period of time, so as to effect a pH jump of up to about 8 to 9.

In this way, when the composition according to the invention is applied to the substrate, the prevailing pH is between 6.5 and 7.5, which allows the laccase to be in optimal conditions of activity. Then, making a pH jump of up to about 8 to 9 allows the other enzymes to have their optimal efficiency. In this way, they will in turn have optimal activity conditions, which results in the biofilm being removed in a particularly advantageous manner since each enzyme present will have optimal conditions to perform its action and the biofilm will be detached and eliminated. completely. In addition, since the basic components are generally used in installations in which a biofilm can be formed, there is no addition of exogenous component that could be problematic to validate the cleaning step. For example, to sanitize an installation, it is common to apply soda and therefore, the supply of substance of different nature is limited in the context of the present invention.

Preferably, the temperature of the solution of the detergent component formed in step b) or c ') may be between approximately 35 ° C and 50 ° C.

According to a preferred embodiment of the invention, the composition according to the invention is applied to a substrate covered with a biofilm for approximately 30 to 50 minutes, which represents a relatively short application time for such efficiency. Preferably, the detergent component comprises a sequestering ratio of between 1 and 10%, a proportion of simultaneously wetting dispersant of between 15 and 50%, a conventional excipient such as water is optionally added to reach the 100% by weight of the component. detergent.

Preferably, the enzymatic component comprises a proportion of protease (s) of between 10 and 50%, a proportion of laccase (s) of between 5 and 35% and a proportion of polysaccharidase (s) of between 5 and 20%. More preferably, the at least one polysaccharidase may be an alpha-amylase. A conventional excipient or solvent is optionally added to achieve 100% by weight of the enzymatic component, such as an alcohol.

The present method effectively eliminates all or almost all of the biofilm, leaving then on the substrate only isolated cells without protection of the matrix. The posterior action of a biocide destroys the microbial strain. A subsequent disinfection phase will therefore be much more effective following the application of a solution of the composition according to the invention than following the application of a conventional cleaning phase does not allow this total elimination of the matrix.

Thus, according to a preferred embodiment of the invention, the method further comprises a subsequent step of applying a biocide. For example, the biocides may be, without limitation, of the oxidizing type such as peracetic acid, hydrogen peroxide, potassium monopersulfate, sodium hypochlorite. According to the invention, the application of a biocide must be subsequent to the application of the composition according to the invention to avoid the deactivation of the enzymes present in said composition by the biocides.

Other embodiments of the process according to the invention are mentioned in the appended claims. The invention also relates to a use of a composition according to the invention for the removal of biofilms present on a substrate, in particular for cleaning floors and surfaces, for cleaning in place or soaking. The composition can be used in closed or soaking facilities. Soaking cleaning is used in particular to clean surgical equipment, contact lenses. The composition according to the invention can be used for the cleaning of technical and process water circuits, air conditioning exchanger systems or in the food industry.

Other forms of use according to the invention are mentioned in the appended claims.

The present invention also relates to a kit for the elimination of biofilms present on a substrate characterized in that it comprises

at least one sample of detergent component in solution or in solid form, containing a sequestering agent, a simultaneously wetting and dispersing agent, and

at least one sample of enzymatic component in solution or in solid form containing at least one protease, at least one laccase and at least one polysaccharidase.

Preferably, the at least one polysaccharidase may be an alpha-amylase.

According to a preferred embodiment of the invention, the sample of the enzymatic component may contain between 1 and 10 proteases, preferably between 1 and 5 proteases, more preferably may contain 2, 3, 4 or 5 proteases.

According to another preferred embodiment, the sample of the enzymatic component contained in the kit may comprise 2 proteases. Preferably, if the sample of the enzymatic component comprises 2 proteases, the proportion in laccase can be approximately 30%, the total proportion of proteases may be approximately 30%, the proportion of alpha-amylase may be approximately 10% by weight relative to the total weight of the sample of the enzymatic component, a conventional excipient or sovlant being added to reach 100% by weight of the enzymatic component.

Preferably, if the sample of the enzymatic component comprises 2 proteases, the proportion of laccase (s) may be 30%, the total proportion of proteases may be 30%, the proportion of alpha-amylase (s) may be 10%. %. For example, the ratio between each protease can be between 1: 2 and 2: 1, preferably the ratio between each protease can be 1: 1.

If the sample of the enzymatic component contained in the kit is a solution, the pH thereof may be between 8 and 10, preferably the pH may be between 8.5 and 9.5, more preferably the pH may be to be approximately 9.0.

Preferably, the sample of the detergent component comprises a sequestering ratio of approximately between 1 and 10%, a proportion of simultaneously wetting and dispersing agent of 15 to 50% by weight relative to the total weight of the detergent component, the 100% being possibly reached using a conventional excipient, such as water.

By way of example, the sequestering agent may be ethylene diamine tetraacetic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate, calcium gluconate, citric acid, phosphoric acid, tartaric acid, sodium acetate, sorbitol, a compound having a phosphorus atom. Preferably, the sequestering agent may be a phosphorus oxide such as phosphonate, phosphinate or phosphate, or a salt thereof, an amine or an amine oxide, or a salt thereof bearing at least, in its structure, a functional group phosphine, phosphine oxide, phophinite, phosphonite, phosphite, phosphonate, phosphinate or phosphate.

More preferably, the sequestering agent may be a phosphonate or a salt thereof, an amine or an amine oxide, or a salt thereof, comprising at least, in its structure, a functional group phosphine, oxide phosphine, phosphinite, phosphonite, phosphite, phosphonate, phosphinate or phosphate. By way of non-limiting example, the phosphonate may be of general formula R ¹ (R ² O) (R ³ O) P = O in which R ¹ , R ² and R ³ are independently selected from the group hydrogen, alkyl, substituted alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted aminoalkyl, aryl or substituted aryl. By way of nonlimiting example, the amine or the amine oxide may comprise one, two or three substituents of general formula CR ⁴ R ⁵ W in which R ⁴ and R ⁵ are independently selected from the group hydrogen, alkyl, substituted alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted aminoalkyl, aryl or substituted aryl, and W is selected from the phosphonate, phosphinate or phosphate group. The sequestering agent may be in the form of a salt of sodium, calcium, lithium, magnesium or potassium; preferably, the sequestering agent may be in the form of a salt of sodium, calcium, or potassium.

Advantageously, said simultaneously wetting and dispersing agent is selected from the group consisting of non-hot foaming wetting agents and preferably comprises an alkylglucoside C ₆ to C, preferably C ₈ , derived from renewable raw materials and optionally an ethoxylated alcohol.

In addition, the simultaneously wetting and dispersing agent according to the invention comprising an ethoxylated alcohol and an alkylpolyglucoside is particularly polyvalent in that it is compatible with a wide pH range, with the presence of numerous acids or bases and builders, while being non-foaming hot.

Advantageously, in the kit according to the invention, the sample of the enzymatic component is an aqueous solution whose pH is between 8 and 10.

If the sample of the detergent component is a solution, the pH thereof is between 8.5 and 14.0.

If the cleaning solution formed by dilution in an aqueous phase of the detergent component and the enzymatic component should have a pH between 6.5 and 7.5, then the detergent component, if in the form of a solution, will have a pH between 9 and 10. The dissolution in the aqueous phase of the two compounds, by For example, the presence of buffers in one or the other component or the dilution effect will have the effect of reducing the pH of the cleaning solution so that it is optimal for the operation of laccases.

If the cleaning solution formed by dilution in an aqueous phase of the detergent component and the enzymatic component should have a pH of about 10, then the detergent component, if it is in the form of a solution will have a pH preferably between 12.0 and 14.0, more preferably between 12.8 and 13.8. Dissolving the two compounds in the aqueous phase will bring the pH of the cleaning solution to about 9.5 to 10.5. For example, the components may further contain alkaline compounds for this purpose.

Preferably, the kit according to the invention further comprises a biocide.

Other embodiments of the kit according to the invention are mentioned in the appended claims.

Other features, details and advantages of the invention will emerge from the description given below, without limitation and with reference to the accompanying drawings and examples.

FIG. 1 illustrates the results of tests comparing the effectiveness of the composition according to the invention with sodium hydroxide

FIG. 2 illustrates the results of tests comparing the effectiveness of the composition according to the invention with other detergent compositions

FIG. 3 illustrates the results of tests comparing the effectiveness of the composition according to the invention with other detergent compositions by carrying out a pH jump.

Example 1.-

An attempt to compare the effectiveness of the composition according to the invention with a rinsing with water and with NaOH was made.

Biofilms are developed on reverse osmosis membranes used in desalination plants. A bacterial inoculum (Pseudomonas aeruginosa) is prepared by culture for 16 hours in a 10% TSB (tryptone soy broth) medium. This preculture is diluted to obtain an optical density (OD) of 0.05 to 600 nm and 500 μΙ of this solution were spread on each membrane. The coupons are incubated for 48 hours in petri dishes kept moist and placed in an oven at 30 ° C. After 2 hours, the bacterial solution is replaced by 500 μl of fresh medium, TSB 10%. After 24 hours, this culture medium is renewed.

The coupons are then cleaned and placed in the cleaning solutions. The membranes are allowed to soak in the above cleaning solutions for a total of 120 minutes at 200 rpm at 35 ° C.

Cleaning solutions are as follows:

1. Water

2. 0.5% liquid NaOH

3. Composition according to the invention at pH 7.

The results of this test are presented in Table 1 .- Table 1.-

These results are also presented in FIG. 1 which illustrates, from left to right, the membrane after treatment with water, Liquid NaOH and the composition according to the invention at pH 7.

As can be seen, only the composition according to the invention allows a substantial elimination of the biofilm.

Example 2 An experiment comparing the effectiveness of the composition according to the invention with other detergent compositions was carried out. The procedure is identical to that of Example 1 with the exception of the cleaning solutions which are as follows:

1. Composition containing 3 different agents, namely a sequestering agent, a wetting agent, and a dispersing agent and an enzymatic component containing at least one laccase, a protease and a polysaccharidase.

2. Composition containing two different agents, namely a sequestering agent and a simultaneously wetting and dispersing agent and an enzymatic component similar to point 1 at pH 7

3. Composition containing two different agents, namely a sequestering agent and a simultaneously wetting and dispersing agent and an enzymatic component similar to item 1 at pH 10.

The results are shown in Table 2 and Figure 2.

Table 2.-

These results are also presented in Figure 2 which illustrates from left to right the membrane after treatment with solution 1, solution 3 and solution 2.

As can be seen, only the composition according to the invention at pH 7 or 10 allows substantial removal of the biofilm.

Example 3.-

Steel coupons were treated with a composition similar to Composition 1 of Example 2 and a composition according to the invention initially at pH 7 with a pH jump to 10 after 15 minutes, following the protocol below.

Biofilms are developed on stainless steel coupons previously washed and sterilized. A bacterial inoculum (Pseudomonas aeruginosa) is prepared by culturing for 16 hours in 10% TSB (tryptone soy broth) medium. This preculture is diluted to obtain an optical density (OD) of 0.05 to 600 nm and 500 μΙ of this solution were spread on each coupon. The coupons are incubated for 48 hours in petri dishes kept moist and placed in an oven at 30 ° C. After 2 hours, the bacterial solution is replaced by 500 μl of fresh medium, TSB 10%. After 24 hours, this culture medium is renewed.

The coupons were then placed in test tubes containing 10 ml of a TSB + 0.5% Tween 80 solution for counting the bacteria. The test tubes were incubated for 5 minutes on the table before being sonicated for 2 minutes and vortexed for 30 seconds. The incubation, sonication and vortex steps were repeated.

The coupons were then recovered and the medium comprising the TSB solution + 0.5% tween was subjected to serial dilutions using peptone water (peptone water without indole: prepare 10 ml of a solution at 15g / l, then dilute 1 ml of this solution in 1 liter of sterile water) and was then spread on a petri dish and incubated at 30 ° C overnight prior to counting. The biofilm was also stained as follows: the coupons were drained and immersed in the staining solution for 10 minutes selective for the biofilm matrix proteins. Then, the coupons are immersed in the various cleaning solutions 1 and 2 mentioned below for two times 10 minutes (the cleaning solutions are replaced between the two cleaning brackets). before drying in the open air.

Cleaning composition:

2. Composition containing two different agents, namely a sequestering agent and a simultaneously wetting and dispersing agent and an enzymatic component similar to point 1 at pH 7 and then at pH 10

Results:

In 1/3 of the petri dishes (4 of 12) corresponding to the coupons cleaned with the composition No. 1, bacteria grew (average> 10 ⁹ CFU). The bacteria did not develop on the petri dishes corresponding to the coupons cleaned with the composition according to the invention.

Figure 3 illustrates the results of staining of biofilms. FIG. 3A illustrates the indicators before cleaning while FIG. B illustrates the steel coupons after cleaning with the solution 1 at different pHs and FIG. C illustrates the steel coupons after cleaning with the composition according to the invention. As can be seen, the coupons after cleaning with the composition according to the invention have a cleaner appearance than those cleaned with the composition No. 1, and not have no bacteria attached to their surfaces while biofilm is detected for 1/3 of the coupons cleaned with the composition No. 1.

Claims

1. Composition for the elimination of biofilms present on a substrate characterized in that it comprises:

at least one detergent component comprising at least one sequestering agent and a simultaneously wetting and dispersing agent,

at least one enzymatic component containing at least one protease, at least one laccase and at least one polysaccharidase.

2. Composition according to claim 1, characterized in that the composition is a solution having a pH of between approximately 8 and 1 1.

3. Composition according to claim 1, characterized in that the composition is a solution having a pH of between 6.5 and 7.5, more particularly around 7.

4. Composition according to any one of claims 1 to 3, wherein said at least one enzymatic component comprises a proportion of protease (s) of between 10 and 50%, a proportion of laccase (s) of between 5 and 35% and a proportion of polysaccharidase (s) of between 5 and 20% by weight relative to the weight of the enzymatic component, a conventional excipient or solvent being optionally added to reach 100%.

5. Composition according to any one of claims 1 to 4, characterized in that said at least one polysaccharidase comprises an alpha-amylase.

6. Composition according to any one of claims 1 to 5 ,, characterized in that said at least one detergent component comprises a sequestering agent proportion of between 1 and 10% by weight relative to the total weight of the detergent component.

7. Composition according to any one of claims 1 to 6, comprising a proportion of simultaneously wetting agent and dispersant between 15 and 50% by weight, based on the total weight of the detergent component

The composition of any one of claims 1 to 7, wherein said simultaneously wetting and dispersing agent is selected from the group consisting of non-hot foaming wetting agents and preferably comprises a C ₆ to C ₁₂ alkyl preferentially ₈ , derived from renewable raw materials and optionally an ethoxylated alcohol.

9. Method for the elimination of biofilms present on a substrate characterized in that it comprises the following steps of:

a) providing a detergent component containing at least one sequestering agent and a simultaneously wetting and dispersing agent and an enzymatic component containing at least one protease, at least one laccase, and at least one polysaccharidase, b) dissolving the detergent component in an aqueous phase,

c) dissolving the enzymatic component in the solution formed in step b) to form the solution of said composition according to any one of claims 1 to 8, or b ') dissolving the enzymatic component in an aqueous phase ,

c ') dissolving the detergent component in the solution formed in step b') to form the solution of said composition according to any one of claims 1 to 8, d) applying the solution of said composition formed to step c) or c ') on the substrate for a predetermined period of time, in particular between 15 minutes and 4 hours

The method according to claim 9, characterized in that the method further comprises a subsequent step of applying a biocide on the substrate.

11. The method of claim 9 or 10, characterized in that the pH of the solution of said composition is between 8 and 1 1.

12. The method of claim 9 or 10, wherein the pH of the solution of said composition is between 6.5 and 7.5 and wherein a basic solution is added after said application d) of the solution of said composition on said substrate during said predetermined period of time, so as to effect a pH jump of up to about 8 to 9.

13. Use of a composition according to claims 1 to 8 for the removal of biofilms present on a substrate.

14. Use according to claim 13, a composition according to claims 1 to 8 for cleaning floors and surfaces, for cleaning in place or soaking.

15. Kit for the removal of biofilms on a substrate characterized in that it comprises

at least one sample of detergent component in solution or in solid form comprising at least a first sequestering agent and at least one simultaneously wetting and dispersing agent,

16. Kit according to claim 15, characterized in that the sample of the enzymatic component is an aqueous solution whose pH is between 8 and 10.

17. Kit according to claim 15 or 16, characterized in that the sample of the detergent component is an aqueous solution whose pH is between 8.5 and 14.

Kit according to any one of claims 15 to 18, further comprising a biocide.