WO2022079318A1

WO2022079318A1 - Detergent composition for eliminating biofilms on industrial equipment

Info

Publication number: WO2022079318A1
Application number: PCT/EP2021/078834
Authority: WO
Inventors: Sébastien FASTREZ; Laurent Delhalle; Laetitia Fontaine
Original assignee: Realco
Priority date: 2020-10-16
Filing date: 2021-10-18
Publication date: 2022-04-21
Also published as: BE1028711B1; BE1028711A1

Abstract

The present invention relates to a detergent composition for eliminating biofilms on industrial equipment, in particular on surfaces or in closed circuits which are typically found in institutional or industrial environments, or even in households, the composition comprising a surfactant component and an enzymatic component, and to a method for eliminating biofilms on industrial surfaces.

Description

DETERGENT COMPOSITION FOR THE ELIMINATION OF BIOFILMS IN INDUSTRIAL INSTALLATIONS

The present invention relates to a detergent composition for the elimination of biofilms on industrial installations, in particular on surfaces or in closed circuits, which are typically found in institutional or industrial environments or even in communities. The industrial installations according to the present invention are typically industrial installations in the agrifood field, such as for example production lines, community kitchens, without however being limited thereto.

Hygiene is becoming increasingly important in industry, particularly in the food industry, in water purification and desalination plants, in the treatment of process water, and in particular in the water used in cooling towers. It is frequently observed that, during the circulation of water or organic or food matter on a surface (open or of a circuit), microorganisms circulating freely in the water or in the organic matter (for example food) can adhere to the surface. These microorganisms can then develop an adhesive extracellular matrix composed of polymeric substances and hosting a community of microorganisms which is called biofilm.

A biofilm is a viscous film which develops on all surfaces, following the adhesion of microorganisms to these surfaces and the secretion by them of polymers which cover them and facilitate their adhesion. Biofilms thus constitute a protective layer around microorganisms and represent a recurring source of contamination of the surrounding environment which poses major problems in terms of health, for example in the food industry. More specifically, the accumulation of polymers secreted by bacteria creates a matrix composed essentially of polysaccharides, DNA, proteins as well as lipids which protects these microorganisms from external attacks and which has a very strong resistance to cleaning and cleaning procedures. conventional disinfection. These matrices also include, in the case of the food industry, food residues. Microorganisms therefore easily develop within this protective matrix and contaminate the surrounding environment by constituting a particularly critical reservoir that is difficult to eliminate.

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

Second, a biofilm is mixed, in the sense that it is initially developed by certain bacterial strains but can harbor others, these strains living and growing in colonies. However, these colonies promote communication between bacteria and, among other things, the exchange and propagation of resistance genes carried by certain bacteria. The biofilms resulting from these exchanges of genes are then even more difficult to eliminate and it is necessary to resort to increasingly powerful means of disinfection or treatment which, however, frequently come up against major problems of resistance and/or tolerance.

The protective matrix of the bacteria forming the biofilms is so resistant that it constitutes a real barrier protecting the bacteria from the disinfectants which could act against the microorganisms and therefore against the contaminations linked to the presence of biofilms. Conventional treatments based on various disinfectants, even when they are, in certain cases, in combination with other compounds derived from chemistry (formulated detergents and/or sequestrants and/or dispersants and/or surfactants, soda), n do not act sufficiently effectively because they do not penetrate or only to a limited extent the biofilm in its thickness.

Furthermore, disinfectants can be inhibited by certain molecules making up this matrix. Therefore, current treatments are only partially effective, this only on the surface of the biofilm, the biofilm matrix effectively protecting bacteria from dehydration phenomena, the action of antibiotics and biocides (and more generally microbicidal molecules) , phagocytosis and acids. In this sense, it is also generally accepted that biofilms have a resistance up to 1000 times greater to biocides compared to a planktonic bacterium (not protected by a biofilm).

It is therefore understood that the treatment and/or prevention of bacterial infections involving the formation of biofilms is a real challenge because the bacteria are protected from biocidal agents and external aggressions.

In addition, a biofilm can also trap other microorganisms, pathogens in particular, than the one that settled initially.

Document WO98/26807 discloses a process for the enzymatic treatment of a biological film. In this method, the biofilm is contacted with a cleaning composition comprising one or more hydrolases to remove or release the biofilm layer from the surface. In a second step, the biofilm is brought into contact with a bactericidal disinfectant 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 mix. The speed and efficiency of cleaning can therefore be improved by using a composition in which the inactivation of enzymes is absent.

Also known from document US2003/0205247 is a use of aqueous solutions containing enzymes for cleaning storage or fermentation tanks, containing one or more enzymes chosen 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 brewing, 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 modifying the viscosity and the thixotropy of the solution used in order to allow better adhesion of the solution and/or of the foam, on the surfaces to be treated of the tanks. These solutions are not suitable for cleaning different installations of tanks or reservoirs (comprising for example many pipes) and are not suitable for the elimination of other types of microorganisms and therefore not for a wide range of biofilms.

Document WO 92/13807 discloses the use of a composition allowing the elimination of biomass and biofilm on substrates in aqueous systems, that is to say in systems in which water is put in circulation or in storage. This type of system suffers from the presence of biomass and alkaline or acidic biofilms due to the type of organism generally present in this type of installation. 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 exhibit limited effectiveness for biofilm removal.

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

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

Nowadays, in order to fight against biofilms, enzymatic formulations comprising a detergent base are used, which document EP2243821 moreover proposes. This enzymatic detergent formulation necessarily contains 3 enzymes and shows adequate efficacy in disintegration by the combined action of enzymes, dispersant and surfactants present.

However, no data is present on the reduction of the biomass present in the biofilm although the formulation is versatile and attacks the biofilm quickly by attacking it in different ways.

There is therefore a need to have alternative formulations which are versatile, namely active against a multitude of biofilms, since it is only rarely known in advance which strain(s) are at the origin of the formation of these biofilms, and which are rapidly active without necessarily requiring an additional stage of mechanical abrasion to eliminate the biofilm, in particular in the case of aged biofilms. To solve this problem, the present invention provides a composition for the elimination of biofilms on industrial installations, in particular on surfaces in contact with foodstuffs and more particularly on open surfaces or in closed circuits, which are typically found in professional, institutional or industrial environments or even in communities, characterized in that it comprises a surfactant component and an enzymatic component, said surfactant component being packaged with the enzymatic component or separately, said surfactant component has one or more agents wetting and/or detergent, and one or more sequestering agents, said enzymatic component comprising at least one endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1.30 and EC 3.1.31, preferably at a content of 10 to 1000 U/ml, preferably 50 to 500 U/ml, preferably this from 100 to 500 U/ml, more particularly from 150, or even 200 to 400 U/ml.

The concentration is expressed according to the present invention in terms of U/ml. One unit (U) is equal to the amount of protein that induces a change in absorbance at 260nm of 1.0 in 30 minutes at 37°C.

By “versatile”, it is meant that the composition is active against a wide range of strains of bacteria and for a diversity of applications.

Indeed, for it to be possible to industrially exploit a composition for eliminating biofilms on industrial installations, it is advantageous to have an active composition against biofilms on industrial installations generated by several bacterial strains and for various applications so as not to have to formulate a multitude of different formulations which require a logistical complexity penalizing an industrial exploitation of such a composition. The possibility of obtaining a formulation which is active against biofilms on professional, industrial installations generated by several bacterial strains and intended for different applications allows at least the manufacture of a concentrate which can then be diversified for certain applications or to formulate a versatile composition.

Said at least one endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1.30 and EC 3.1.31 of the composition used according to the present invention has an activity on RNA and on DNA. Its enzymatic activity hydrolyzes DNA and RNA substrates into 5'-phospho(mono/oligo)nucleotides (EC 3.1.30) or 3'-phospho(mono/oligo)nucleotides (EC 3.1.31) products.

In addition, and surprisingly, the use of an endoribonuclease (RNase) chosen from the EC 3.1.30 or EC 3.1.31 classes allows a reduction in the biomass of biofilms originating from numerous bacterial strains and to rapidly disintegrate the biofilm. in cooperation with the wetting agent, the sequestering agent and, preferably with the dispersing agent, which allows a rapid elimination of the latter.

Indeed, while other RNases belonging to the EC4.6.1 classification, such as RNase I and RNase A have no effect on biofilms or a limited effect on certain types of biofilms only, it has been demonstrated , surprisingly that an endoribonuclease chosen specifically from the EC 3.1.30 or EC 3.1.31 class makes it possible to drastically reduce the biomass of the biofilms and thus to potentiate the disinfectant agent which, without this composition, is not capable of enter the biofilm and treat the contamination, this for biofilms of varied strains of bacteria and with a non-specific use of the application, and this in combination with the action of the detergent component of the composition according to the present invention.

The constitution of a composition comprising a surfactant component and an enzymatic component containing at least endoribonuclease chosen specifically from the EC 3.1.30 or EC 3.1.31 class makes it possible, surprisingly, to significantly improve the speed and efficiency 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 those having an earlier development cycle and developed by multiple different species or microorganisms.

To date, apart from some effective enzymatic detergent formulations, there is no composition allowing the complete elimination of biofilms present in industrial installations. For example, in the field of the food industry, and more particularly concerning surfaces in contact with foodstuffs, biofilms inevitably form (given the richness of the surrounding environment). Biofilms exhibit a cyclical growth activity comprising a growth phase during which the accumulation of microorganisms occurs and a detachment phase during which whole pieces of biofilm detach by erosion and under the effect of their own weight. When a manufacturer is confronted with this phenomenon, he should actually stop his production line and carry out different washing cycles alternated with soda, and with many detergents and/or chemical and/or enzymatic cleaners since there is no of versatile composition. However, this represents many hours of work and loss of performance of the installation.

Therefore, in practice, production is not stopped and when the biofilm is in the breaking phase, the batches produced are contaminated and discarded until the level of contamination by the microorganisms of the food produced is again acceptable to the given the standards in force. In addition, it is not possible for manufacturers to have in stock a specific detergent or enzymatic solution for each microorganism likely to contaminate their production line.

It is therefore very surprisingly that the present invention makes it possible to provide a perfectly versatile detergent composition which makes it possible to eliminate a broad spectrum of biofilms, in various types of industrial installation and requiring no special precautions for use. The detergent eliminates a superficial part of the biofilm and wets and swells the organic structures of the biofilm thanks to the wetting and/or detergent character, and preferably and/or to the dispersing character of the surfactant component. This therefore promotes the accessibility of the enzymatic component which weakens and degrades the matrix of the biofilm. This combined action of the enzymatic component and the surfactant component promotes accessibility to the composition of the deeper layers and allows optimal detachment of any type of biofilm while preserving the industrial surface.

The dependent claims refer to other advantageous forms of use.

In an advantageous embodiment, in the detergent composition according to the present invention, the enzymatic component additionally comprises a second enzyme chosen from the group of glycosidases, in particular polysaccharidases, oxidoreductases, lyases, transferases, proteases and peptidases, lipases and esterases.

More particularly, the second enzyme of the enzymatic component is chosen from the group of glycosidases, in particular polysaccharidases, and/or proteases.

In another advantageous embodiment, in the detergent composition according to the present invention, the enzymatic component further comprises one or more additional enzymes, for example a third, and/or a fourth, and/or a fifth, and/or a sixth, and/or a seventh, and/or an eighth enzyme chosen from the group of glycosidases, in particular polysaccharidases, oxidoreductases, lyases, transferases, proteases and peptidases, lipases and esterases.

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

Non-limiting examples of protease enzymes belonging to class EC 3.4 and capable of being used in the invention are amino-peptidases (EC 3.4.11), 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), metallo endopeptidases (EC 3.4.24 ), threonine endopeptidases ((EC 3.4.25), and endopeptidases belonging to the class (EC 3.4.99)).

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

The laccases used in the invention belong to class EC 1.10.3.2. Laccases are copper-containing enzymes and function 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 polysaccharides. Preferably, the at least one polysaccharidase can 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 class EC 3.2.1.1, having the function of breaking (1-4)-alpha-glycosidic bonds in polysaccharides containing three or more alpha-( 1-4)-D-glucose. Preferably, according to the present invention, one or each wetting agent of said one or more wetting agents of the surfactant component is chosen from the group of anionic, cationic, non-ionic, amphoteric or zwitterionic wetting agents, more particularly an anionic or non-anionic wetting agent -ionic.

Preferably, according to the present invention, one or each detergent agent of said one or more detergent agents of the surfactant component is chosen from the group of anionic, cationic, nonionic, amphoteric or zwitterionic wetting agents, more particularly a cationic, amphoteric detergent agent or zwitterionic.

In particular, one or each wetting agent of said one or more wetting agents is a sucrose ester or a composition comprising a C4 to C12 sodium alkyl sulphate, more particularly C4 to Cio, preferably C4 to Cs alcohol and an alcohol , preferably an alkoxylated fatty alcohol with a Cs to CM variable chain, for example ethoxylated and/or propoxylated and/or butoxylated, an ethereal alcohol sulphate Cs to C12, more particularly Cs to Cio, preferably Cs, Cs to C12, more particularly Cs to C10, preferably Cs, alkylearyl sulfonates, a C4 to C12, more particularly C4 to C10, preferably C4 to Cs, alkylpolyglucoside.

The wetting agent of the detergent component is an amphiphilic chemical substance, or a composition comprising said amphiphilic chemical substance, which modifies the surface tension between two surfaces. The wetting agent has the advantage of promoting the spreading of a liquid on a solid but also of accentuating the contact between two surfaces. More particularly, the wetting agent promotes contact between the detergent component and a surface and, therefore, between the enzymes and their substrate. Now, in an industrial environment, the surfaces to be treated are often made of stainless steel or of a material on which the application of a liquid gives rise to the formation of droplets. This characteristic of the surfaces to be treated makes it difficult to apply a composition in liquid form evenly. to fight against the presence of biofilms. This is why the wetting agent is a particularly advantageous constituent of said composition according to the invention since it allows, even on stainless steel type surfaces, a homogeneous spreading of the composition and thus its perfect distribution on the surfaces to be decontaminated. , for example on industrial machinery, especially in the food industry.

Advantageously, according to the present invention, one or each wetting agent of said one or more wetting agents is a non-hot foaming agent.

The fact that the wetting agent is non-foaming when hot allows the composition according to the invention to be used for the treatment of industrial machines having pipes, such as for example extruders, but also water circulation circuits. Indeed, the wetting agent being non-foaming, this makes it possible to avoid the formation of foam, without altering, quite the contrary, the surfactant performance of the composition according to the invention. It is understood that the supply of an effective surfactant solution without generating foam limits the rinsing steps, which is particularly desirable, especially for industrial machines having pipes which can thus be reused quickly for a new production.

More preferably, said wetting agent of said detergent component is ethoxylated 2-ethylhexanol.

In particular, one or each detergent agent of said one or more detergent agents is an alkyl polyglucoside, an alkyl amine oxide, betaines, for example an alkyl betaine.

For applications on open surfaces of industrial installations, one or each detergent agent of said one or more detergent agents is a foaming agent, which makes it possible to improve the adhesion of the formulation to the surfaces. More preferably, said wetting agent of said detergent component is the alkyl polyglucoside and/or the alkyl betaine and/or an alkyl amine oxide.

In yet another embodiment of the composition according to the present invention, one or each sequestering agent of said one or more sequestering agents is chosen from the group comprising ethylene-diamine-tetraacetic acid, glucono-delta-lactone, gluconate sodium, potassium gluconate, calcium gluconate, citric acid, phosphoric acid, tartaric acid, sodium acetate, sorbitol, a compound comprising 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 carrying at least, in its structure , a phosphine, phosphine oxide, phophinite, phosphonite, phosphite, phosphonate, phosphinate or phosphate functional group, 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 comprising at least, in its structure, one functional group phosphine, phosphine oxide, phosphinite, phosphonite, phosphite, phosphonate , phosphinate or phosphate, alone or in combination or a salt thereof. By way of non-limiting example, the phosphonate may be of general formula RI (R2 O)(R3 O)P=O in which RI, R2 and R3 independently represent a hydrogen, alkyl, substituted alkyl, substituted alkyl-amino or no, substituted or unsubstituted aminoalkyl, aryl or substituted aryl. By way of non-limiting example, the amine or the amine oxide may comprise one, two or three substituent(s) of general formula (CR4R5W) in which R4 and R5 independently represent 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 can be in the form of a sodium, calcium, lithium, magnesium or potassium; preferentially, the sequestering agent can be in the form of a sodium, calcium or potassium salt.

In addition, the sequestering agent can also be sodium citrate, gluconate, iminodisuccinate, methylglycine-diacetic acid, or even glutamic diacetic acid.

According to a preferred embodiment of the present invention, the composition according to the present invention comprises one or more dispersing agents.

More particularly, according to the present invention, one or each dispersing agent of said one or more dispersing agents is chosen from the group comprising a soluble or partially water-soluble polymer such as for example polyethylene glycol, cellulose derivatives or a polymer comprising at least one acrylic acid or acrylic ester unit or a polyphosphate.

The dispersing agent of the surfactant component improves the separation of particles from a suspension in order to prevent clumping, aggregation and/or settling.

Preferably, the dispersing agent is a polymer comprising at least one acrylic acid or acrylic ester unit of general formula —(CH2 —CH—COOR)— in which R represents a hydrogen, alkyl or substituted alkyl, aryl or substituted aryl group.

Advantageously, said one or each dispersing agent of said one or more dispersing agents is a polymer having an average molecular weight Mw of approximately between 500 and 10,000, preferably between 2,000 and 6,000.

More preferably, the dispersing agent is a polymer of acrylic acid. In particular, the dispersing agent can be a homopolymer of acrylic acid having an average molecular weight of approximately between 2000 and 6000. Advantageously, said dispersing agent of said surfactant component is C6 alkylglucoside.

In an advantageous variant of the present invention, in which the surfactant component is a composition in solid form, containing from 1 to 50%, preferably from 1 to 30% by weight, more particularly from 1 to 10% by weight of agents wetting agents and/or detergents, relative to the total weight of the surfactant component.

In a preferred embodiment, in the surfactant composition according to the present invention, the detergent component is a composition in solid form, containing from 1 to 10% of sequestering agent.

Preferably, according to the present invention, the surfactant component is a composition in solid form, containing from 1 to 10% of dispersing agent.

More preferably, the surfactant component is a composition in solid form, having a weight ratio of wetting agents and/or detergents relative to the weight of dispersing agents of between 0.7 and 1.3, more particularly between 0.8 and 1 ,2, more particularly around 1 +_0,L

In a variant according to the present invention, the surfactant component is a composition in the form of a liquid concentrate to be diluted in water, containing from 1 to 50%, preferably from 1 to 30% by weight, more particularly from 1 to 10% by weight of wetting agents and/or detergents, relative to the total weight of the surfactant component.

Advantageously, the surfactant component is a composition in the form of a liquid concentrate, containing from 1 to 10% of sequestering agent.

Preferably, the surfactant component is a composition in the form of a liquid concentrate, containing from 1 to 10% of dispersing agent. More particularly, according to the present invention, the surfactant component is a composition in the form of a liquid concentrate, having a weight ratio of wetting and/or detergent agents relative to the weight of dispersing agents of between 0.7 and 1.3, more particularly between 0.8 and 1.2, more particularly around 1+.0.1.

Even more preferentially, the surfactant component is a composition in the form of a liquid concentrate containing water at a content of between 30 and 85% by weight relative to the total weight of the composition.

Advantageously, according to the present invention, which the surfactant component is a composition in the form of a liquid concentrate having a pH of between 5 and 8, for example having a pH of 5.2, 5.4, 5.6, 5 .8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6 or even 7.8.

Advantageously, according to the present invention, which enzymatic component is a composition in the form of a liquid concentrate having a pH of between 6 and 9, for example having a pH of 6.0, 6.2, 6.4, 6, 6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8, 6 or even 8.8.

In a particular embodiment according to the invention, the composition is an aqueous cleaning solution having a pH of between approximately 7 and 10, for example a pH of 7.2, of

7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2,

9.4, 9.6, 9.8.

Indeed, the pH value of said composition significantly influences its effectiveness with respect to the biofilm, in particular because the pH of the composition influences the proper functioning of the enzymes. It is easily understood that the composition according to the present invention, having an optimal operating pH of the enzymes, further promotes the embrittlement and degradation of the matrix of the biofilm and therefore optimal detachment of any type of biofilm while preserving the industrial surface.

In yet another advantageous embodiment, in the detergent composition according to the present invention, the enzymatic component is in solid form, in the form of a lyophilisate, powders, granules or in any other solid or pseudo-solid form allowing the solubilization said enzymatic component in a solvent.

Indeed, alternatively, said composition can be in solid or pseudo-solid form and then be dissolved in a solvent before use in order to obtain, by dilution in an aqueous phase, a cleaning solution whose pH is between approximately 7 and 10.

In an advantageous variant according to the invention, the detergent composition is a liquid solution which is then diluted in an aqueous phase to obtain an aqueous cleaning solution having a pH of between 7 and 10, for example a pH of 7.2, of 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8. In this way, the pH of the composition is particularly suitable for the action of the enzymatic component, and in particular, for example, if the endoribonuclease according to the invention is combined with 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 elimination of the biofilm as mentioned above while maintaining the optimum efficiency of the enzymatic component.

Alternatively, said composition may be in solid form and then be dissolved in a solvent before use before being diluted in an aqueous phase in order to obtain a cleaning solution whose pH is between approximately 9 and 12, more particularly between 10 and 11.5.

More particularly, the detergent composition according to the present invention comprises from 1 to 100% by weight, more particularly from 1 to 50% by weight, for example from 5 to 20% by weight of said endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1.30 and EC 3.1.31 relative to the total weight of said enzymatic component.

More particularly, the detergent composition according to the present invention comprises from 1 to 50% by weight, more particularly from 10 to 35% by weight of protease relative to the total weight of said enzymatic component.

More particularly, the detergent composition according to the present invention comprises from 5 to 50% by weight, more particularly from 5 to 35% by weight of glycosidase relative to the total weight of said enzymatic component.

Advantageously, the composition according to the invention comprises from 5 to 50% by weight, more particularly from 5 to 35% by weight of lipase relative to the total weight of said enzymatic component.

Also advantageously, the enzymatic component is in liquid form comprising a phase containing an alcohol, more particularly a glycol.

In an advantageous embodiment, in the detergent composition according to the present invention, the enzymatic component comprises from 1 to 50% by weight, more particularly from 1 to 40% by weight, for example from 5 to 35% by weight of said enzyme endoribonuclease chosen from the group consisting of enzymes belonging to classes EC 3.1.30 and EC 3.1.31 relative to the total weight of said enzymatic component.

In another advantageous embodiment, in the detergent composition according to the present invention, the enzymatic component comprises from 1 to 25% by weight, more particularly from 10 to 20% by weight of protease relative to the total weight of said enzymatic component. In an advantageous embodiment, in the detergent composition according to the present invention, the enzymatic component comprises from 5 to 20% by weight, more particularly from 5 to 10% by weight of glycosidase relative to the total weight of said enzymatic component.

The detergent composition according to the present invention is advantageously used for the elimination of biofilms on professional installations, in particular on surfaces or in closed circuits of professional environments.

Further embodiments of the detergent composition according to the present invention are found in the appended claims.

The present invention further relates to a process for the elimination of biofilms on industrial surfaces, in particular on surfaces or in closed circuits, which are typically found in professional, institutional or industrial environments or even in communities. The industrial installations according to the present invention are typically industrial installations in the agri-food field, such as for example production lines, community kitchens, or even any surface in contact with agri-foodstuffs, without however being limited thereto.

More particularly, the present invention relates to a process for the elimination of biofilms on industrial surfaces comprising the steps of: providing a detergent composition according to the present invention comprising a surfactant component and an enzymatic component, said surfactant component being packaged with the enzymatic component or separately, said surfactant component has one or more wetting and/or detergent agents and one or more sequestering agents, said enzymatic component comprising at least an endoribonuclease enzyme selected from the group consisting of enzymes belonging to classes EC 3.1.30 and EC 3.1.31, preferably at a content of 10 to 1000U/ml, preferably 50 to 500U/ml, preferably 100 to 500U/ ml, more particularly from 150, or even 200 to 400 U/ml, applying said detergent composition to an industrial surface for a predetermined period of time for the elimination of biofilms on said surface.

In an advantageous embodiment of the method according to the invention, the elimination of the biofilms on said surface is an elimination of the microorganisms on said surface.

In an advantageous embodiment of the method according to the invention, said industrial surface is chosen from the group comprising the production lines of the agro-food industry, community kitchens, floors, walls, ceilings, appliances household appliances, worktops, cooking surfaces, sinks, pipes, closed circuits, communities, conveyor belts, food industry surfaces.

In an advantageous embodiment of the method according to the invention, said elimination of biofilms on said surface is a cleaning-in-place method.

In one embodiment of the process according to the invention, the detergent composition according to the invention is in liquid concentrated form, of liquid solution or else in solid form, preferably to be diluted in an aqueous phase.

In one embodiment of the method according to the invention, the application of the detergent composition to an industrial surface is an application, for example by vaporization. Further embodiments of the method for the removal of biofilms from industrial surfaces according to the present invention are found in the appended claims.

Other characteristics, details and advantages of the invention will emerge from the description given below, on a non-limiting basis and with reference to the appended drawings.

FIG. 1 illustrates the reduction in the percentage of biomass of different biofilms of bacteria of multiple strains of P. aeruginosa, following contact with a composition comprising 400 U/ml of denarase

Efficacy of a composition according to the invention for reducing the biomass of biofilms involved in the contamination of agri-foodstuffs

In order to test the effectiveness of a composition according to the invention, comprising at least one endoribonuclease enzyme, several experiments were carried out on different bacterial strains (isolates and ATCC collection strains) (Table 1).

Table 1.-

The effect of a treatment with the composition according to the invention on the biomass of the biofilms was studied in a static model in 96-well plates. The curative enzymatic treatment is carried out on a preformed 24-hour biofilm. To test its effectiveness in reducing the biomass of biofilms, the composition according to the invention is brought into contact with the biofilm for 1 hour at 37°C. The enzyme solution is then removed, and the Loose biomass is removed by washing steps. Crystal violet (CV) dye is then used to quantify residual biomass. The latter interacts with dead and living cells, and the macromolecules of the extracellular matrix of the biofilm (such as DNA and exopolysaccharides). The CV labeling is quantified by spectrophotometry by measuring the absorbance at 570 nm.

1.1 method of culture, enzymatic treatment, quantification of biomass and analysis of results on P. aeruginosa

P. aeruginosa strains (Table 1) were inoculated in 5ml of TGN culture medium (Tryptic Soy Broth VWR + 1% glucose + 2% NaCI) from 20 .1 of a glycerol stock stored at -80° vs. The strains were then incubated at 130 rpm aerobically at 37°C. After 18 h of growth, the precultures were diluted in TGN to reach an optical density at 620 nm (OD620) of 0.05, which corresponds to +/- 5*10 ⁶ CFU/ml. Two hundred microliters of each culture (n=4 for each condition tested) were placed in the wells of a 96-well plate and incubated at 37° C. without shaking for 24 hours to allow the formation of the biofilm. The biofilms are then washed 2× with 200 μl PBS pH 7.5 (Sigma) in order to eliminate the planktonic cells (ie non-adherent to the biofilm). The composition according to the invention or the control compositions CT- (negative control) and CT+ (positive control) (200.1) are added to the washed biofilm and the biofilm is incubated for I h at 37° C. (n=4 for each condition). The plates are then emptied, the biofilm is washed 2× with 200 J PBS pH 7.5 (Sigma) and dried at 60° C. for 18 h before analyzing the biomass.

To quantify the residual biomass, a 1% CV solution (Sigma) is added to the biofilms at ambient temperature for 15'. The unfixed dye is then washed off with distilled water. The dye attached to the biofilm is then detached and dissolved with 66% peracetic acid and the absorbance at 570 nm (A570) is measured in a SpectraMax M3 multimode reader. This absorbance value corresponds to the biomass of the biofilm. To analyze the effect of the compositions according to the invention, a statistical test (one-way ANOVA and Tukey's post-hoc test) is carried out by comparing the absorbance values of the untreated biofilms (negative control, CT-=treatment with buffer without enzyme) and biofilms treated with the enzyme compositions. The difference is significant when the pvalue of the statistical test is less than or equal to 0.05 (* character shown on the graphs). When this is specified in the comparative examples, the activity of the enzymatic compositions are compared with each other (one-way ANOVA and post-hoc Tukey test).

A composition comprising denarase (c-Lecta), which is an endoribonuclease from S. marc escens belonging to the enzymatic class EC 3.1.30, was tested at a concentration of 400 U/ml in 20 mM Tris-HCl. The negative control (CT-) is composed of 20 mM Tris-HCl only. The Aszo absorbance of the CV measured on the CT- corresponds to 100% biomass. The positive control (CT+) contains 1% Enzyme ^R in 20 mM Tris-HCl; OneLife sa),

Results: Disruptive activity on P. aeruginosa

The disruptive activity of the biofilm of denarase according to example 1 (400 U/ml) was tested on 3 strains of P. aeruginosa (PAO1, ATCC27853 and 618). In this experiment, the CT- is 20 mM Tris-HCl pH 7 without enzyme and its biomass corresponds to 100%. The CT+ is composed of 1% enziGure ^R in this same buffer.

The compositions tested are listed in Table 2.

Table 2.-

The results presented in Figure 1 show that denarase (Ex.1) is very effective in reducing the biomass of P. aeruginosa biofilms.

Different compositions according to the invention have been formulated, these different compositions according to the invention make it possible, advantageously and particularly surprisingly, to eliminate biofilms on industrial installations, these biofilms being complex and generated by several bacterial strains. In addition, these different compositions according to the invention are versatile and have improved biofilm elimination speed and efficiency, making it possible to eliminate all or almost all of the biofilm, whether they are mature or in a cycle of earlier development.

Example 2 - Detergent composition according to the invention for the elimination of biofilms on an open surface:

A detergent composition according to the invention was prepared for the elimination of biofilms on an open surface, was added to water: between 1 and 20% of alkylpolyglucoside and / or alkyl betaine and / or alkyl amine oxide as detergent agent, between 0.5 and 5% phosphonate and/or sodium citrate and/or gluconate and/or iminodisuccinate and/or methylglycine-diacetic acid and/or glutamic diacetic acid as agent sequestrant, an endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1 .30 and EC 3.1 .31 at a content of 400 U/ml, between 0.05 and 5% of protease, between 0.05 and 2% polysaccharidase a and between 0.01 and 2% laccase as enzymatic component.

Example 2-3 - Detergent composition according to the invention for the elimination of biofilms on an open surface: A detergent composition according to the invention was prepared for the elimination of biofilms on an open surface, was added to water: between 1 and 20% of alkylpolyglucoside and / or alkyl betaine and / or alkyl amine oxide as detergent agent, between 0.5 and 5% phosphonate and/or sodium citrate and/or gluconate and/or iminodisuccinate and/or methylglycine-diacetic acid and/or glutamic diacetic acid as agent sequestrant, between 0.5 and 5% polyacrylate as dispersing agent, an endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1 .30 and EC 3.1 .31 at a content of 400 U/ml, between 0.05 and 5% protease, between 0.05 and 2% polysaccharidase a and between 0.01 and 2% laccase as enzymatic component.

Example 4 - Detergent composition according to the invention for the elimination of biofilms on a surface of closed circuits:

A detergent composition according to the invention was prepared for the elimination of biofilms on a surface of a closed circuit, the following were added to the water: between 5 and 30% of alkoxylated fatty alcohol (for example ethoxylated, propoxylated , butoxylated) variable chain (C8-C14) and/or short chain (C4-C8) alkyl polyglucoside and/or short chain (C4-C8) alkyl sulphate (for example functionalized or non-functionalized) as agent wetting, between 5 and 20% of phosphonate and / or sodium citrate and / or gluconate and / or iminodisuccinate and / or methylglycine-diacetic acid and / or glutamic diacetic acid as sequestering agent, an endoribonuclease enzyme selected from the group consisting of enzymes belonging to classes EC 3.1 .30 and EC 3.1 .31 at a content of 400 U/ml, between 0.05 and 5% protease, between 0.05 and 2% polysaccharidase a and between 0.01 and 2% laccase as enzymatic component.

Example 5 - Detergent composition according to the invention for the elimination of biofilms on a surface of closed circuits:

A detergent composition according to the invention was prepared for the elimination of biofilms on a surface of a closed circuit, the following were added to the water: between 5 and 30% of alkoxylated fatty alcohol (for example ethoxylated, propoxylated , butoxylated) variable chain (C8-C14) and/or short chain (C4-C8) alkyl polyglucoside and/or short chain (C4-C8) alkyl sulphate (for example functionalized or non-functionalized) as agent wetting agent, between 5 and 20% of phosphonate and/or sodium citrate and/or gluconate and/or iminodisuccinate and/or methylglycine-diacetic acid and/or glutamic-diacetic acid as sequestering agent, between 0.5 and 5% polyacrylate as dispersing agent, an endoribonuclease enzyme selected from the group consisting of enzymes belonging to classes EC 3.1 .30 and EC 3.1 .31 at a content of 400 U/ml, between 0.05 and 5% protease, between 0.05 and 2% polysaccharidase a and between 0.01 and 2% laccase as enzymatic component.

It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made thereto without departing from the scope of the appended claims. For example, the enzyme denarase was used in the examples, it goes without saying that the examples could have illustrated the activity of the benzonase endonuclease® available from the company Millipore Sigma. Furthermore, the compositions of examples 1 to 4 could also comprise additional enzymes such as for example a lipase and/or a mannanase and/or a polysaccharidase P and/or a poly-N-acetyl glucosaminidase.

Claims

28 CLAIMS

1. Detergent composition for the elimination of biofilms on industrial or institutional installations, comprising a surfactant component and an enzymatic component, said surfactant component being packaged with the enzymatic component or separately, said surfactant component has one or more wetting agents and/or detergents, and one or more sequestering agents, said enzymatic component comprising at least one endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1.30 and EC 3.1.31, preferably at a content of 10 to 1000U/ml, preferably 50 to 500 U/ml, preferably 100 to 500 U/ml, more particularly 150, or even 200 to 400 U/ml.

2. Detergent composition for the elimination of biofilms on industrial installations according to claim 1, in which the enzymatic component further comprises a second enzyme chosen from the group of glycosidases, in particular polysaccharidases, oxido-reductases, lyases, transferases, proteases and peptidases, lipases and esterases.

3. Detergent composition for the elimination of biofilms on industrial plants according to claim 2, in which the second enzyme of the enzymatic component is chosen from the group of glycosidases, in particular polysaccharidases and/or proteases.

4. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 3, in which the enzymatic component further comprises one or more additional enzymes, for example a third, and/or a fourth, and/or a fifth, and/or a sixth, and/or a seventh, and/or an eighth enzyme chosen from the group of glycosidases, in particular polysaccharidases, oxidoreductases, lyases, transferases, proteases and peptidases, lipases and esterases.

5. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 4, in which one or each wetting agent of said one or more wetting agents of the surfactant component is chosen from the group of wetting agents anionic, cationic, nonionic, amphoteric or zwitterionic, more particularly an anionic or nonionic wetting agent.

6. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 5, in which one or each detergent agent of said one or more detergent agents of the surfactant component is chosen from the group of wetting agents anionic, cationic, nonionic, amphoteric or zwitterionic, more particularly a cationic, amphoteric or zwitterionic detergent agent.

7. A detergent composition for the removal of biofilms on industrial plants according to claim 5 or 6, in which one or each wetting agent of said one or more wetting agents is a sucrose ester or a composition comprising a sodium alkyl sulphate in C4 to C12, more particularly C4 to C10, preferably C4 to Cs and an alcohol, preferably a Cs to CM variable chain alkoxylated fatty alcohol, for example ethoxylated, propoxylated, butoxylated, an ethereal Cs alcohol sulphate C12, more particularly C6 to Cio, preferably Cs, Cs to C12, more particularly Cs to C10, preferably Cs, alkylpolyglucoside C4 to C12, more particularly C4 to C10 alkylpolyglucoside , preferably C4 to Cs.

8. Detergent composition for eliminating biofilms on industrial installations according to one of claims 6 or 7, in which one or each detergent agent of said one or more detergent agents is an alkyl polyglucoside, an alkyl betaine, an alkyl amine oxide.

9. A detergent composition for the removal of biofilms on industrial plants according to any one of claims 5 to 8, in which one or each wetting agent of said one or more wetting agents is a non-hot foaming agent.

10. A detergent composition for the removal of biofilms on industrial plants according to any one of claims 6 to 9, in which one or each detergent agent of said one or more detergent agents is a foaming agent.

1 1. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 10, in which one or each sequestering agent of said one or more sequestering agents is chosen from the group comprising ethylene-acid diamine-tetraacetic acid, glucono-delta-lactone, sodium gluconate, potassium gluconate, calcium gluconate, citric acid, phosphoric acid, tartaric acid, sodium acetate, sorbitol, a compound containing a phosphorus atom.

12. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 1 1, comprising one or more dispersing agents.

13. Detergent composition for the elimination of biofilms on industrial installations according to claim 12, in which one or each dispersing agent of said one or more dispersing agents is chosen from the group comprising a polymer soluble or partially soluble in water such as for example polyethylene glycol, cellulose derivatives or a polymer comprising at least one acrylic acid or acrylic ester unit or a polyphosphate.

14. Detergent composition for the elimination of biofilms on industrial plants according to any one of claims 12 or 13, in which said one or each dispersing agent of said one or more dispersing agents is a polymer having an average molecular weight Mw comprised approximately between 500 and 10000, preferably between 2000 and 6000.

15. Detergent composition for the elimination of biofilms on industrial installations according to any one of the preceding claims, in which the surfactant component is a composition in solid form, containing from 1 to 50%, preferably from 1 to 30% by weight, more particularly from 1 to 10% by weight of wetting agents and/or detergents, relative to the total weight of the surfactant component.

16. Detergent composition for the elimination of biofilms on industrial installations according to any one of the preceding claims, in which the surfactant component is a composition in solid form, containing from 1 to 10% of sequestering agent.

17. Detergent composition for the elimination of biofilms on industrial installations according to any one of the preceding claims, in which the surfactant component is a composition in solid form, containing from 1 to 10% of dispersing agent.

18. Detergent composition for eliminating biofilms on industrial installations according to any one of the preceding claims, in which the surfactant component is a composition in solid form, having a weight ratio of wetting and/or detergent agents with respect to the weight of dispersing agents comprised between 0.7 and 1.3, more particularly between 0.8 and 1.2, more particularly around 1+.0.1.

19. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 14, in which the surfactant component is a composition in the form of a liquid concentrate to be diluted in water, containing 1 to 50%, preferably from 1 to 30% by weight, more particularly from 1 to 10% by weight of wetting agents and/or detergents, relative to the total weight of the surfactant component.

20. Detergent composition for the elimination of biofilms on industrial installations according to any one of 32 claims 1 to 14 and 19, in which the surfactant component is a composition in the form of a liquid concentrate, containing from 1 to 10% of sequestering agent.

21. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 14 or 19 to 20, in which the surfactant component is a composition in the form of a liquid concentrate, containing from 1 to 10% of dispersing agent.

22. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 14 or 19 to 21, in which the surfactant component is a composition in the form of a liquid concentrate, having a weight ratio of agents wetting agent and/or detergent with respect to the weight of dispersing agents comprised between 0.7 and 1.3, more particularly between 0.8 and 1.2, more particularly around 1+0.1.

23. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 14 or 19 to 22, in which the surfactant component is a composition in the form of a liquid concentrate contains water at a content between 30 and 85% by weight relative to the total weight of the composition.

24. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 14 or 19 to 23, in which the surfactant component is a composition in the form of a liquid concentrate having a pH of between 5 and 8 .

25. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 14 or 19 to 24, in which the enzymatic component is a composition in the form of a liquid concentrate having a pH of between 6 and 9 .

26. Detergent composition for the elimination of biofilms on industrial installations according to any one of 33 preceding claims, in which the enzymatic component is in solid form, in the form of a lyophilisate, of powders, of granules or in any other solid or pseudo-solid form allowing the solubilization of said component in a solvent.

27. Detergent composition for the elimination of biofilms on industrial installations according to claim 26, comprising from 1 to 100% by weight, more particularly from 1 to 50% by weight, for example from 5 to 20% by weight of said enzyme endoribonuclease chosen from the group consisting of enzymes belonging to classes EC 3.1.30 and EC 3.1.31 relative to the total weight of said enzymatic component.

28. Detergent composition for the elimination of biofilms on industrial installations according to claim 26 or claim 27, comprising from 1 to 50% by weight, more particularly from 10 to 35% by weight of protease relative to the total weight of said enzymatic component.

29. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 26 to 28 comprising from 5 to 50% by weight, more particularly from 5 to 35% by weight of glycosidase relative to the total weight of said enzymatic component.

30. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 26 to 29 comprising from 5 to 50% by weight, more particularly from 5 to 35% by weight of lipase relative to the total weight of said enzymatic component.

31. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 1 to 25, in which the enzymatic component is in liquid form comprising a phase containing an alcohol, more particularly a glycol.

32. Detergent composition for the elimination of biofilms on industrial installations according to claim 31, 34 comprising from 1 to 50% by weight, more particularly from 1 to 40% by weight, for example from 5 to 35% by weight of said endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1 .30 and EC 3.1 .31 relative to the total weight of said enzymatic component.

33. Detergent composition for the elimination of biofilms on industrial installations according to claim 31 or claim 32, comprising from 1 to 25% by weight, more particularly from 10 to 20% by weight of protease relative to the total weight of said enzymatic component.

34. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 31 to 33, comprising from 5 to 30% by weight, more particularly from 5 to 20% by weight of glycosidase relative to the total weight of said enzymatic component.

35. Detergent composition for the elimination of biofilms on industrial installations according to any one of claims 31 to 34, comprising from 5 to 20% by weight, more particularly from 5 to 10% by weight of glycosidase relative to the total weight of said enzymatic component.

36. Process for the elimination of biofilms on industrial surfaces comprising the steps of:

- providing a detergent composition according to any one of the preceding claims, comprising a surfactant component and an enzymatic component, said surfactant component being packaged with the enzymatic component or separately, said surfactant component has one or more wetting agents and/or detergents, and one or more sequestering agents, said enzymatic component comprising at least one endoribonuclease enzyme chosen from the group consisting of enzymes belonging to classes EC 3.1 .30 and EC 3.1 .31 , of preferably at a content of 10 to 1000 U/ml, preferably 50 to 500 U/ml, preferably 100 to 500 U/ml, more particularly 150, or even 200 to 400 U/ml,

- applying said detergent composition to an industrial surface for a predetermined period of time for the elimination of biofilms on said surface.

37. A method for the removal of biofilms from industrial surfaces according to claim 36, wherein said removal of biofilms from said surface is removal of microorganisms from said surface.

38. Process for the elimination of biofilms on industrial surfaces according to claim 36 or 37, in which said surface is chosen from the group comprising production lines of the food industry, community kitchens, floors, walls, ceilings, household appliances, worktops, cooking surfaces, sinks, pipes, closed circuits, communities, conveyor belts, food processing industry surfaces.

39. A method for the removal of biofilms from industrial surfaces according to any one of claims 36 to 38, wherein said removal of biofilms from said surface is a clean-in-place method.

40. Process for the elimination of biofilms on industrial surfaces according to any one of claims 36 to 39, in which the said detergent composition is in liquid concentrated form, of liquid solution, in solid form, preferably to be diluted in a phase watery.

41. A method for the removal of biofilms from industrial surfaces according to any one of claims 36 to 40, wherein said application of said detergent composition to an industrial surface is spray application.