Biofilm detection solution and method of application to surfaces

Abstract

The invention provides a biofilm detection product comprising an optical brightener revealed by fluorescence under UV exposure. The product contains sodium 2,2'-([1,1'-biphenyl]-4,4'-diyldi-2,1-ethendiyl)bis-benzenesulfonate. It can be used on various surfaces, in particular surfaces of agro-industrial equipment, equipment in hospitals or the paper industry, but also on surfaces in food-related services.

Description

Description Title of the invention: Biofilm detection solution and method of application to surfaces Technical Field

[0001] The invention relates to the detection of microorganism biofilms and is particularly suitable for the detection of thin biofilms. The invention finds application in many fields, in particular for the detection of biofilms which form on the surfaces of equipment in the agro-industry and food service. Prior art

[0002] Despite the attention paid to equipment design and hygiene programs, the presence of biofilms is a persistent problem in many sectors, particularly the agribusiness and food service sectors. Food contact surfaces are prone to the development of biofilms when they are not sufficiently cleaned: microorganisms take advantage of the presence of nutrient residues to develop a colony adhering to the surfaces, which consolidates over time and becomes very difficult to remove.

[0003] Biofilms are composed of microorganisms, extracellular polymeric substances secreted by these microorganisms called exopolysaccharides (EPS), DNA, proteins, as well as organic and inorganic residues. These residues are also an interesting anchor point for microorganisms during the initial formation of the biofilm. When surfaces are cleaned and sanitized, biofilms are more or less removed depending on their degree of maturity and level of concretion. The microorganisms nesting there are therefore less exposed and can survive more easily. As the biofilm matures, portions of it detach and release microorganisms into its environment. This cascade thus leads to a reduction in the shelf life of food products prepared in an environment containing biofilms, in addition to potentially exposing consumers to pathogens present in the biofilm.It is therefore relevant to develop a preventive biofilm detection system that would allow us to contain their spread more effectively. Indeed, the earlier a biofilm is detected, the easier it is to eliminate it.

[0004] There are currently two main biofilm detection systems applicable in the agro-industry and food service fields: stain detection systems and catalase detection systems.

[0005] Dye detection systems are simple to use and relatively reliable. However, they can only be used on steel and aluminum. Porous surfaces such as rubber and other plastics are, however, welcoming substrates for microorganisms, which can then develop a biofilm. The ability to detect biofilms on these surfaces is therefore crucial. Moreover, even when these Detection systems are used on non-porous surfaces, they have the unfortunate tendency to stain surrounding equipment and the user themselves. These stains are sometimes difficult to remove, especially when the use of an oxidizing agent is not possible due to the nature of the substrate.

[0006] This is the case, for example, of the Coomassie blue-based biofilm detection kit proposed in application WO2012175671. This kit contains a blue dye that stains heavily and must be handled with care to avoid staining surrounding surfaces. This kit is therefore only applicable to steel or aluminum surfaces. It stains hands, clothing, and any other slightly porous surfaces. In addition, the use of an oxidizing agent to remove the coloring is not possible when the surface is sensitive to it.

[0007] Applications W02013030419, JP2005210997, EP1536225 and FR2928457 also describe stain detection products which cannot be used on less porous surfaces, which are however the most prone to the development of biofilms.

[0008] Some biofilm detection systems use a chemical matrix that is not compatible with the agro-industry and food service, or at least less preferable. The kit described in application WO2012175671 proposes a concentration of 10% unneutralized acetic acid. It goes without saying that such a matrix gives off a very strong odor that is unpleasant for users, not to mention that this acid concentration contributes to denaturing and precipitating protein residues, making them even more difficult to remove from surfaces, thus risking denaturing the organoleptic properties of food.

[0009] It is therefore desirable to have a detection system that does not generate odors for the user, and which is compatible with use in the food industry.

[0010] The second type of commonly used biofilm detection system contains hydrogen peroxide, which reacts with the catalases of aerobic bacteria present on surfaces. It is therefore not a direct measure of the presence of biofilms. US10927397, EP 2902497, and US 20180105861 illustrate this technology and present a biofilm detection system where hydrogen peroxide reacts with catalases naturally present in aerobic and facultative anaerobic bacteria. This is therefore a system that detects metabolically active microorganisms rather than the biofilm matrix. Furthermore, the metabolic activity of bacteria and catalases are transiently attenuated after cleaning and sanitizing operations, which necessarily leads to false negatives when the catalase detection system is applied before the bacteria have had time to recover.Indeed, most cleaning and sanitation operations act in the surface layer of the biofilm by reducing the microbial population and inactivating their enzymes, sometimes generating false negatives when the biofilm has not been completely eliminated by cleaning operations. This is why it is important to detect the biofilm matrix or the. microorganisms integrated into this matrix, rather than their metabolic or enzymatic activity.

[0011] Finally, common biofilm detection systems sometimes lack sensitivity towards biofilms, which can also lead to false negatives.

[0012] There is therefore a need to provide detection systems with greater sensitivity and specificity in order to limit false negatives. Statement of the invention

[0013] The invention addresses these various issues by providing a biofilm detection product comprising an optical brightener that can be revealed by fluorescence under ultraviolet (UV) ray exposure. The optical brightener may also be referred to in the present description as a “fluorescent agent” or “marker” or “tracer”.

[0014] The invention also provides a detection product with multiple advantages particularly suited to the detection of biofilms on surfaces which are regularly in contact with food or foodstuffs.

[0015] The inventor has indeed surprisingly discovered that a particular optical brightener, Fluorescent Brightener 351 (FB351) or Fluorescent Brightener 87 (FB87), detects food biofilms with very high sensitivity. Indeed, the quantity required for detection is much lower (approximately 1000 times less) compared to the visible dye-based markers traditionally used for this application. This is particularly important for the detection of young or immature biofilms that are very thin and thus undetectable by touch or the naked eye.

[0016] In addition, the molecule being colorless, it does not visibly stain the surfaces on which the solution is applied.

[0017] Finally, the FB351 marker and the FB87 marker bind specifically to the constituents of the biofilm matrix, which reduces the risk of false negatives. Indeed, if a facility cleaning procedure has not completely removed the biofilm, the test will be positive.

[0018] In a particular embodiment, the invention proposes a means of detecting biofilms that simultaneously provides several advantages. The detection solution comprising the optical brightener FB 351 or FB87 has a high sensitivity threshold after a relatively short exposure time. It does not stain the surfaces on which it is applied, in particular porous surfaces. Finally, it meets the constraints linked to use on surfaces in contact with foodstuffs or medical equipment, such as in particular odor and toxicity. Brief description of the drawings

[0019] [Fig. 1] Figure 1A shows residual coloration after application of the prior art product, while in Figure 1B the product of the invention leaves no detectable trace.

[0020] [Fig. 2] Figure 2A highlights the very low detection of a biofilm of B. su bt il is pa a product of the prior art in comparison with Figure 2B where we see that the kit of the invention allows obvious detection by exposure to UV light.

[0021] [Fig. 3] Figure 3A shows that the E. co / i biofilm is not detected by a prior art product compared to Figure 3B where detection is evident by exposure to UV light.

[0022] [Fig. 4] Figure 4A highlights the very low detection of a L. innocua biofilm by a prior art product in comparison with Figure 4B where it is seen that the kit of the invention allows obvious detection by exposure to UV light. Description of the embodiments A first subject of the invention relates to a solution for detecting a biofilm comprising from 10% to 90% by mass of a volatile alcohol, from 0.1% to 20% by mass of a carboxylic acid comprising from 2 to 6 carbon atoms, and from 0.01 ppm to 10,000 ppm (equivalent to 10 -6 % to 1% by mass) of an optical brightener, the optical brightener being sodium 2,2'-([l,l'-biphenyl]-4,4'-diyldi-2,l-ethenediyl)bis-benzenesulfonate. The sum of the mass proportions of volatile alcohol, carboxylic acid and optical brightener may not exceed 100% by mass.

[0023] A "biofilm" is a matrix of microorganisms, such as bacteria, yeasts or fungi, in which the cells adhere to each other and / or adhere to a surface by means of extracellular polysaccharides that they secrete, proteins, DNA and inorganic materials, including minerals that contribute to the concretion of the biofilm. Biofilm cells are physiologically distinct from cells of the same organism, which are individual cells suspended in a liquid medium (called planktonic). Biofilms are therefore composed of an amalgam of microorganisms, extracellular polymeric substances secreted by these microorganisms and other constituents secreted by the bacteria or resulting from their lysis (DNA), as well as organic and inorganic compounds originating from the environment surrounding the biofilm.

[0024] Microorganisms may in particular be microorganisms naturally present in agro-industrial processes, such as, for example, Bacillus spp., Listeria spp., Streptococcus spp., Eschericia spp., Pseudomonas spp. and Staphylococcus spp.

[0025] The quantity of volatile alcohol preferably ranges from 15% to 90%, from 20% to 80%, from 30% to 70% or from 40 to 60% by mass relative to the mass of the detection solution. The volatile alcohol can be, for example, ethanol, methanol, isopropanol or a mixture thereof. Volatile alcohol advantageously increases the wetting of the solution, which thus covers surfaces more easily. In addition, it allows the removal of soiling that is not biofilms, particularly fatty substances, which reduces the risk of false positives. An alcohol compatible with the intended industrial use should be chosen. For the agro-industry and food service, it is preferable to use ethanol.

[0026] The carboxylic acid comprising from 2 to 6 carbon atoms may be a monocarboxylic acid comprising from 2 to 6 carbon atoms, preferably comprising from 3 to 4 carbon atoms, and more preferably 3 carbon atoms.

[0027] The carboxylic acid comprising from 2 to 6 carbon atoms may independently comprise at least one hydroxyl group.

[0028] Examples of carboxylic acids include acetic acid, citric acid, lactic acid, or glycolic acid. The choice of carboxylic acid will be primarily driven by cost and compatibility with the intended industrial application.

[0029] The concentration of carboxylic acid in the solution can vary to some extent. The chosen concentration is the result of a compromise taking into account the optimal acidity level for biofilm labeling, cost, and user and equipment safety. Thus, the amount of carboxylic acid can range from 1% to 10% by mass relative to the mass of the detection solution.

[0030] It is preferable to use lactic acid, which has a less unpleasant odor than acetic acid, particularly for compatible use in the agro-industry and food service.

[0031] The detection solution may also include water, for example 10% to 90% by mass, 30% to 70% by mass, or 40% to 60% by mass of the mass of the solution.

[0032] In a particular embodiment, the detection solution contains from 10% to 90% by mass of water, from 15% to 90% by mass of volatile alcohol, from 0.1% to 20% of carboxylic acid comprising from 2 to 6 carbon atoms, and from 0.01 ppm to 10,000 ppm of optical brightener, the sum of the proportions of water, alcohol, carboxylic acid and optical brightener not being able to exceed 100% by mass. In this embodiment, the concentration of optical brightener is advantageously from 0.01 ppm to 100 ppm, preferably from 0.1 ppm to 10 ppm, and more preferably from 0.5 ppm to 1.5 ppm.

[0033] In a particular embodiment, the detection solution contains from 40% to 60% by mass of water, from 40% to 60% by mass of volatile alcohol and from 1 to 5% of carboxylic acid comprising from 2 to 6 carbon atoms, and from 0.01 ppm to 10,000 ppm of optical brightener, the sum of the proportions of water, alcohol, carboxylic acid and optical brightener not being able to exceed 100% by mass.

[0034] The detection solution may contain a surfactant, in particular in a concentration of less than 10% by mass, or even less than 5% by mass or 1% by mass. In a particular embodiment, the detection solution is free of it.

[0035] The optical brightener used in the detection solution of the invention is sodium 2,2'-([l,l'-biphenyl]-4,4'-diyldi-2,l-ethenediyl)bis-benzenesulfonate. Its chemical name according to IUPAC nomenclature is benzenesulfonic acid, 2,2'-([l,l'-biphenyl]-4,4'-diyldi-2,l-ethenediyl)bis-, disodium salt (CAS number: 27344-41-8). This compound, also known as FB 351, is commercially available under the trade name Tinopal® CBS-X supplied by BASF.

[0036] The optical brightener used in the detection solution of the invention may alternatively be the compound with the chemical name according to the IUPAC nomenclature tetrasodium; 5-[[4-[bis(2-hydroxyethyl)amino]-6-(3-sulfonatoanilino)-l,3,5-triazin-2-yl]amino]-2-[(E)-2-[4-[[4-[bis(2-hydroxyethyl)amino]-6-(3-sulfonatoanilino)-l,3,5-triazin-2-yl]amino]-2-sulfonatophenyl]ethenyl]benzenesulfonate (CAS number: 12768-91-1). This compound is also designated as FB 87. A person skilled in the art will choose a concentration of optical brightener high enough to detect thin biofilms. For certain fields of application, the skilled person will take care to limit the concentration of optical brightener in order to avoid staining sensitive surfaces when exposed to UV radiation. Sensitive surfaces encountered in the agro-industry and food service are, for example, rubber and other porous plastics.

[0037] FB351 or FB87 will preferably be used at a concentration ranging from 0.01 ppm to 10,000 ppm (equivalent to 10 -7 % to 1% by mass), from 0.1 ppm to 500 ppm, from 0.1 ppm to 100 ppm, from 0.1 ppm to 50 ppm, and more preferably ranging from 0.1 ppm to 10 ppm (equivalent to 10 -5 % to W 3 % by mass of the mass of the solution).

[0038] The pH of the detection solution is preferably less than 7.0. It is for example less than a value chosen from 6.5, 6.0, 5.5, 5.0, 4.5, 4.0 or 3.0. The pH of the detection solution is greater than 1.0, preferably greater than 1.5 or 2.0. In a particular embodiment, the pH of the detection solution is between 2.0 and 3.0.

[0039] The detection solution of the invention can be applied to any type of surface likely to carry a biofilm, in particular, a surface in the pharmaceutical environment, in the medical environment, in a water treatment installation, in a paper production unit.

[0040] The surface on which the detection solution is applied can be open or closed.

[0041] In a particular embodiment, the surface is a surface of equipment in the agro-industry or in food service. In the field of food service, the detection solution of the invention may be used, for example, on the surface of door handles and knobs, water dispensers including sinks, water fountains, kiosks, touch screens, keyboards, escalators, elevator buttons, beverage serving pitchers, tables, condiment dispensers, and food service utensils. The detection solution of the invention may also be used on the surface of conveyor belts, transport carts or molds, but also on food cutting surfaces, or production line surfaces.

[0042] The biofilm detection solution of the invention can thus be used in closed systems, for example in a system cleaned by cleaning-in-place where the fluorescent agent binds to the biofilms and is then released by a product which degrades the biofilms, the concentration of fluorescent agent finally harvested being proportional to the quantity of biofilms removed. A person skilled in the art will know, based on his knowledge general, use the appropriate biofilm degradation product depending on the geometry and chemical nature of the surface.

[0043] A second subject of the invention is a kit for detecting a biofilm comprising a first bottle containing the solution described above, and a second bottle containing a rinsing solution which makes it possible to eliminate the tracer which has not reacted with the biofilm and thus to reveal the fluorescence only when the tracer has attached to a biofilm. This kit has a certain advantage compared to biofilm detection systems which must be carried out in a laboratory or at least using specialized equipment and which thus have the disadvantage of not being easily transposed to the agro-industry and food service where an instant and on-site result is necessary while avoiding the use of complex and / or expensive equipment.

[0044] In the kit of the invention, the rinsing solution advantageously comprises a solvent base identical to that of the detection solution. In a particular embodiment, the rinsing solution therefore comprises from 10% to 90% by mass of a volatile alcohol and from 0.1% to 20% by mass of a carboxylic acid comprising from 2 to 6 carbon atoms, the volatile alcohol and the carboxylic acid possibly being in accordance with the description given above.

[0045] The rinse solution may also comprise water, for example from 10% to 90% by mass or from 30% to 70% by mass, of the mass of the rinse solution.

[0046] In a particular embodiment, the rinsing solution contains from 40% to 60% by mass of water, from 40% to 60% by mass of volatile alcohol and from 1 to 5% of carboxylic acid comprising from 2 to 6 carbon atoms, the sum of the proportions of water, alcohol and carboxylic acid not being able to exceed 100% by mass.

[0047] In one embodiment, the rinsing solution is devoid of an optical brightener, such as FB351 or FB87, in order to avoid the revelation by UV fluorescence of optical brightener molecules which would not be bound to the biofilm. In an advantageous embodiment, the composition of the rinsing solution is identical to that of the detection solution except that it does not contain an optical brightener.

[0048] The biofilm detection kit of the invention may be used on the surfaces mentioned in particular in the context of the description of the detection solution constituting the first subject of the invention.

[0049] A third subject of the invention is a method for detecting a biofilm on a surface of industrial equipment or a surface of community equipment, said method comprising - a step of applying to said surface a sufficient quantity of sodium 2,2'-([l,l'-biphenyl]-4,4'-diyldi-2,l-ethenediyl)bis-benzenesulfonate, and - a step of exposing the surface to UV radiation.

[0050] In this process, sodium 2,2'-([l,l'-biphenyl]-4,4'-diyldi-2,l-ethenediyl)bis-benzenesulfonate can be replaced by 5-[[4-[bis(2-hydroxyethyl)amino]-6- (3-sulfonatoanilino)-l,3,5-triazin-2-yl]amino]-2-[(E)-2-[4-[[4-[bis(2-hydroxyethyl)amino]-6- Sodium (3-sulfonatoanilino)-l,3,5-triazin-2-yl]amino]-2-sulfonatophenyl]ethenyl]benzenesulfonate. Industrial equipment can be equipment from the agro-industry, the paper industry, the pharmaceutical industry or a water treatment plant.

[0051] The surface of an agro-industrial equipment can be located in a dairy industry, in a food manufacturing workshop, in a slaughterhouse or in a meat processing workshop for example.

[0052] The surface of a community facility can be a food service surface and can be any surface of the service tables of a restaurant, a cafeteria, or a hospital room, as well as the surfaces of kitchens.

[0053] The biofilm detection method preferably comprises a step of cleaning the surfaces to be tested prior to the step of applying the optical brightener, which may be formulated in the form of the detection solution described above. It is indeed preferable to ensure that the surfaces to be tested have been previously cleaned in order to remove less stubborn soils (which are not biofilms) which may possibly cause false positives. In general, routine cleaning aims to remove lipid, starch or protein residues. However, stubborn soils comprising organic matter may remain following this routine cleaning. A stubborn soil is not a biofilm in itself, but can become a biofilm if bacteria proliferate there.

[0054] The detection solution of the invention constitutes a tool for detecting biofilms, but also a tool for the preventive detection of stubborn soiling that causes the formation of biofilms, in particular residues containing whey proteins and starch. In addition, more specific protocols can be used when necessary to discriminate between stubborn soiling and biofilms, before applying the detection solution to the surfaces to be tested. The solution of the invention is not sensitive to fats since the ethanol and carboxylic acid used as solvents eliminate greasy residues on the surfaces due to their detergent properties.

[0055] In a particular embodiment of the detection method of the invention, the optical brightener is formulated in a detection solution in accordance with the description given above. The detection solution can be packaged in a bottle equipped with a sprayer and be sprayed onto the surface to be tested, allowing sufficient contact time. The contact time of the detection solution may be between 1 minute and 5 minutes, preferably between 1 minute 30 seconds and 3 minutes.

[0056] The step of exposing the surface of the biofilm to UV radiation, after application of the optical brightener, makes it possible to reveal the biofilms by illumination with a UV lamp, ideally at a wavelength of 365 nm since it offers optimal sensitivity.

[0057] The biofilm detection method of the invention may comprise the use of a detection kit as described with reference to the second subject of the invention and in accordance with the preceding description. In this embodiment, the rinsing solution is sprayed onto the surface after the detection solution application step, in order to rinse off excess detection solution not bound to biofilms. This step also allows the removal of soiling of other types than biofilms, if applicable, thanks to the detergent and descaling properties of the product. The contact time of the rinsing solution may be between 1 minute and 5 minutes, preferably between 1 minute 30 seconds and 3 minutes. The biofilms revealed can then be removed by a procedure known to those skilled in the art.

[0058] The method of the invention may comprise at least one step of rinsing the surface, said rinsing step being subsequent to the application step and prior to the step of exposure to UV radiation. The rinsing step may be carried out with water or with the rinsing solution described above.

[0059] According to one embodiment, the method of the invention may further comprise at least one water rinsing step, the surface being able to be rinsed with water after application of the detection solution and / or after application of the rinsing solution. This proves particularly useful on a horizontal surface.

[0060] .

[0061] An oxidizing agent can also be sprayed to neutralize the detection solution. However, it is preferable not to use such an oxidizing agent, especially on fragile surfaces that risk being damaged. The invention is illustrated by the following examples. Unless otherwise stated, the temperature is 20°C ± 5°C and the pressure is atmospheric pressure. EXAMPLES

[0062] Example 1: Preparation of the invention kit and comparative kits

[0063] Biofilm detection kits were prepared: kits according to the invention and comparative kits each comprising the detection solution and the rinsing solution, the compositions of which are detailed in Table 1 below:

[0064] [Table 1] Table 1: Composition of detection kits The ingredients were mixed at room temperature in the order they appear in the table. The pH of the detection solution is between 2.5 and 3.0. Each solution was placed in a Twist Grip® brand sprayer ovals bottle, supplied by Plastic Bottle Corporation, equipped with an HT-17710 Jet Spray® brand sprayer, supplied by Emballages Richard Inc. The active compound used in the kit of the invention is: - Fluorescent Brightener 351 (FB 351), chemical name benzenesulfonic acid, 2,2'-([l,l'- biphenyl]-4,4'-diyldi-2,l-ethenediyl)bis-, disodium salt (CAS number: 27344-41-8) commercially available under the trade name Tinopal® CBS-X supplied by BASF, or - Fluorescent Brightener 87 (FB 87) with CAS number: 12768-91-1 and IUPAC name tetrasodium; 5-[[4-[bis(2-hydroxyethyl)amino]-6-(3-sulfonatoanilino)-1,3,5-triazin-2-yl]amino]-2-[(E)-2-[4-[[4-[bis(2-hydroxyethyl)amino]-6-(3-sulfonatoanilino)-1,3,5-triazin-2- yl]amino]-2-sulfonatophenyl]ethenyl]benzenesulfonate. The active compounds used for comparison are: - 4,4'-bis((4-anilino-6-((2-hydroxyethyl)methylamino)-s-triazin-2-yl)amino)-, disodium salt (CAS number: 13863-31-5), such as the commercial product branded Tinopal® 5BM sold by the company BASF, - 4,4'-Diamino-2,2'-stilbenedisulfonic acid (also called "amsonic acid" in English) with CAS number: 81-11-8. Example 2: Evaluation of the kit on a porous surface: observation of the coloration in comparison with a kit of the prior art

[0065] Methodology:

[0066] The kit of Example 1 containing FB351 and a kit in accordance with the teaching of application WO2012175671, known as a kit containing a blue dye, Coomassie blue, were applied to a porous surface commonly used in the agro-industry field and subject to contamination by biofilms, in this case food grade rubber (butadiene-acrylonitrile tiles, supplied by E.JAMES & CO).

[0067] 100 μL of the detection solutions were sprayed onto the substrate. After an appropriate contact time, the substrate was rinsed with 1.5 mL of the appropriate rinsing solution. After an appropriate contact time, the substrate was rinsed with water and the excess was blotted with absorbent paper.

[0068] Different contact times were evaluated to compare the different kits. It was found that the prior art kit containing a blue dye had a greater tendency to stain the rubber than the other kits, even when the contact times were reduced to two minutes instead of the five minutes recommended by the manufacturer.

[0069] Results : Two photographs of the surfaces treated by the kit of the invention with the optical brightener FB351 and by the kit of the prior art containing the blue dye are shown in Figure 1A (under visible light) and Figure 1B (under UV) respectively. They were taken with a Samsung Galaxy S22 mobile phone, in automatic mode, without magnification. The photos were taken under conditions where the photos best represented the observation at the naked eye. The AloneFire® SV18 brand UV lamp, UV 365 nm, 12 watts, was located approximately 20 cm from the biofilm. In Figure 1A, a residual blue coloration is observed on the rubber after application of the prior art kit. In Figure 1B, it is observed that the use of the FB351 kit left no trace on the rubber, even under UV illumination.

[0070] Example 3: Application of kits of the invention and comparative kits, on biofilms

[0071] The kits of Example 1 and the prior art kit mentioned in Example 2 were applied to single-strain biofilms.

[0072] Methodology:

[0073] Single-strain biofilms were grown on stainless steel coupons, using a method described by Gamble ("Food Grade Dye for Assessment of Biofilm Removal from Stainless Steel by Cleaning and Sanitizing Agents", Food Protection Trends, Vol 39, No. 6, pp. 442-448) and adapted to the specifics of the experiment.

[0074] The three bacterial species used for the tests (Bacillus sz / / sCHOOOOl, Escherichia coii ATCC 8739 and Listeria innocua HPB13) are well documented in the literature for their role in the formation of biofilms in the agro-industry. It was observed that biofilms formed generally uniformly on the different coupons. The different replicates also confirmed the excellent reproducibility of the experiment.

[0075] For each kit, 10 μL of the detection solution was deposited on each of the three biofilms. The contact time of the detection solution was five minutes for the prior art kit and two minutes for the other kits.

[0076] At the end of the contact time of the detection solution, rinsing was carried out with the appropriate rinsing solution for each of the kits. After the appropriate contact times (two minutes for the optical brightener-based kits and five minutes for the prior art kit), the coupons were rinsed with water. It was validated that the matrices of the detection and rinsing solutions of the kit of the invention did not remove the biofilms significantly. The biofilms are firmly anchored on the stainless steel coupons and are difficult to remove, similar to the biofilms detected in the agro-industry and food service.

[0077] Results :

[0078] The results are essentially the same for all three biofilms.

[0079] The UV exposure photographs were taken under the same conditions as in Example 2 and in a dark environment to facilitate visualization, but the biofilms were also clearly visible under ordinary lighting conditions. The FB351 and FB87 kits were more sensitive than the prior art kit. The comparative kits based on Tinopal® 5BM and 4,4'-Diamino-2,2'-stilbenedisulfonic acid (amsonic acid) did not reveal any biofilms.

[0080] B. subtüis biofilm: the prior art kit only weakly stained the biofilm (Figure 2A), while detection using the inventive kit comprising FB 351, by UV light, is evident (Figure 2B).

[0081] E. coh' biofilm The prior art kit did not detect the biofilm (Figure 3A), while detection using the inventive kit comprising FB 351, by UV light is evident (Figure 3B).

[0082] L. innocua biofilm: the prior art kit only weakly stained the biofilm (Figure 4A), while detection using the inventive kit comprising FB 351, by UV light, is evident (Figure 4B).

[0083] In conclusion, the FB351 and FB87-based kits were the most sensitive, followed by the prior art kit. However, the Amsonic acid and Tinopal®5BM-based kits did not reveal biofilms.

[0084] Furthermore, it was noted that the odor of the FB351 kit was much less bothersome than that of the prior art kit. It was also noted that the FB351 kit was more pleasant to use since it did not leave residual stains on equipment or users.

Claims

Claims

1. A biofilm detection solution comprising from 10% to 90% by mass of a volatile alcohol, preferably from 30% to 90% by mass of a volatile alcohol, from 0.1% to 20% by mass of a carboxylic acid comprising from 2 to 6 carbon atoms, and from 0.01 ppm to 10,000 ppm of an optical brightener, preferably from 0.01 ppm to 10 ppm of an optical brightener, the optical brightener being sodium 2,2'-([1,1'-biphenyl]-4,4'-diyldi-2,1-ethenediyl)bis-benzenesulfonate.

2. A biofilm detection solution according to claim 1, characterized in that the volatile alcohol is ethanol, methanol, isopropanol or a mixture thereof.

3. A biofilm detection solution according to claim 1 or 2, characterized in that the carboxylic acid comprising from 2 to 6 carbon atoms is acetic acid, citric acid, lactic acid or glycolic acid.

4. A biofilm detection kit comprising a first bottle containing the detection solution according to one of the preceding claims, and a second bottle containing a rinsing solution.

5. Biofilm detection kit according to the preceding claim, characterized in that the rinsing solution comprises from 10% to 90% by mass of a volatile alcohol and from 0.1% to 20% by mass of a carboxylic acid comprising from 2 to 6 carbon atoms.

6. A method of detecting a biofilm on a surface of industrial equipment or a surface of community equipment, said method comprising - a step of applying to said surface a sufficient quantity of sodium 2,2'-([l,l'-biphenyl]-4,4'-diyldi-2,l-ethenediyl)bis-benzenesulfonate, and - a step of exposing the surface to UV radiation.

7. Method for detecting a biofilm according to the preceding claim, characterized in that it further comprises: - a rinsing step, said rinsing step being subsequent to the application step and prior to the step of exposure to UV radiation.

8. Method for detecting a biofilm according to claim 6 or 7, characterized in that the industrial equipment is equipment from the agro-industry, the paper industry, the pharmaceutical industry, or a water treatment installation.

9. Method for detecting a biofilm according to claim 6 or 7, characterized in that the surface of the community equipment is a surface of a food service.

10. Method for detecting a biofilm according to one of claims 6 to 9, characterized in that the surface is open or closed.

11. Method for detecting a biofilm according to one of claims 6 to 10, characterized in that the surface is a steel surface or a plastic surface.

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