BE1030646A1 - SAMPLING KIT INCLUDING A PRESERVATION SOLUTION FOR THE COLLECTION OF MICROORGANISMS - Google Patents

Abstract

Kit comprising a solution for preserving microorganisms taken from surfaces, in a living or revivable state, for subsequent detection and laboratory analysis comprising: at least one nutrient extract, at least one polyoxyethylene sorbitan derivative and/or at least one phosphoglyceride derivative, sampling kit comprising this conservation composition and uses of this kit.

Description

SAMPLING KIT INCLUDING A PRESERVATION SOLUTION FOR THE

COLLECTION OF MICROORGANISMS

Technical area

The present invention relates to a sampling kit comprising a solution for stabilizing (preserving) microorganisms taken from surfaces (industrial and/or agri-food and/or communities), in the living or revivable state, for detection and detection. subsequent laboratory analysis, a kit and a method for sampling microorganisms from surfaces and a use of the kit according to the invention for the detection of contamination.

Prior art

In many areas of activity such as the industrial, agri-food sectors, communities, medical and veterinary sectors, and particularly on the surfaces of these areas of activity, problematic contaminations due to the presence of microorganisms are observed very frequently. .

In hospitals and in medical or veterinary practices, the presence of microorganisms is often responsible for nosocomial diseases while in the food industry and in communities, Mmicroorganisms are responsible for the degradation of perishable foodstuffs but also for the synthesis of contaminants towards products, for example from a meat, fruit, vegetable or other production chain, which makes them unfit for consumption and/or toxic.

Nowadays, strict hygiene standards are imposed and it is therefore necessary to ensure that the number of microorganisms responsible for such contamination is kept below an acceptable threshold value, this acceptable threshold value being specific to each area of activity and for each contaminant.

Planktonic bacteria are a first example of contaminating microorganisms, these bacteria are free on a liquid or solid substrate and pose a problem in themselves because they are able to directly contaminate all types of surfaces such as, for example, foodstuffs. , medical tools, conveyor belts, storage tanks or even human patients or animals themselves.

However, today, it is widely recognized that the problems linked to contamination by microorganisms are all the more important as the latter form biofilms.

Indeed, in any type of industry and more particularly in the field of the food industry, biofilms form inevitably, given the richness of the surrounding environment.

Biofilms are defined as a consortium of

Mmicroorganisms embedded in a matrix of extracellular polymeric substances. In other words, biofilms are viscous and/or sticky films which develop on all surfaces, following the adhesion of microorganisms to these surfaces and the secretion by them of polymers covering them, facilitating their adhesion to the surface and thus forming the extracellular matrix. Biofilms thus constitute a protective layer around microorganisms which is also very resistant to chemical and thermal attacks, which makes their elimination particularly difficult using conventional biocides and also represents a recurring and significant source of contamination of the surrounding environment.

This resistance is explained on the one hand by the matrix of extracellular polymeric substances which forms a physical barrier to the diffusion of biocidal molecules in the biofilm and, on the other hand, by the sessile state of the microorganisms and their capacity to very quickly exchange genes responsible for certain biocide resistance mechanisms with each other.

From then on, we easily understand that biofilms are very resistant and extremely varied structures, for example different strains of the same bacteria can form different biofilms, these biofilms being even more varied as they are composed of several different bacteria. In addition, bacteria exchange resistance genes with each other and the environment also influences the biofilm, this further accentuates their complexity and resistance.

Additionally, the extracellular matrix of biofilms can be identified when it is strongly developed but, in the majority of cases, the biofilm grows insidiously in the facilities and its presence will only be detected when it has reached a problematic size, or again when analyzing the quality of the finished product.

Concerning the growth of the biofilm, this took place in a cyclical manner, comprising a growth phase during which the accumulation of microorganisms occurs and a detachment phase during which entire pieces of biofilms and microorganisms are detached by erosion and under the effect of their weight to contaminate surfaces, foodstuffs, medical tools, conveyor belts, storage tanks or even human patients or animals themselves.

This should result in the industrialist stopping his production line and carrying out a cleaning cycle to eliminate the biofilm, representing many hours of work and loss of output.

From all this, it appears that contaminating microorganisms and/or biofims constitute a real problem, particularly in the industrial field, for example health care (hospitals, dental or medical practices), veterinary care and the agri-food industry. and communities.

This problem is all the more critical since microorganisms and/or biofilms can involve bacteria responsible for potentially fatal infections in individuals, whether these bacteria are present in hospitals, veterinary practices or even in the food and beverage industry. are ultimately found on food products intended for consumption.

As indicated above, the problem of microorganisms and more particularly biofilms is twofold. On the one hand, conventional disinfectants and biocides are very often ineffective because they do not succeed in reaching the microorganisms protected by the extracellular matrix of the biofilm, this matrix being of complex and very variable structure and composition. On the other hand, a biofilm is generally mixed, that is to say it contains a multitude of different bacteria or the same bacteria but of different strains, which favors the propagation of resistance genes between the bacteria of the biofilm. and thus makes their treatment very difficult or even ineffective.

Consequently, from one environment to another or from one sector of activity to another, it is quite common for the biofilms detected to be completely different. In order to detect contaminants and/or biofilms, there are kits for detecting the presence of contaminants and/or biofilms on surfaces or in more specific installations (water circuits, etc.), these kits (such as disclosed in the document

EP2537601) make it possible to carry out selective coloring of contaminants and more particularly biofilms. It is therefore currently possible to determine areas where contaminants and/or biofilms are present in order to eliminate the latter without however knowing the precise nature of the microorganisms (bacteria) at the origin of the targeted biofilm. 5 | as a result, compositions for eliminating contaminants and/or biofilms comprising several enzymes are used without however knowing whether the enzymes used and possibly formulated in a detergent base (see for example the document

EP2243821) are really adequate to act on a given biofilm.

For this reason, current treatments are rather random and non-specific, which is economically unprofitable and can result in wasted time and considerable plant downtime.

Furthermore, techniques for sampling microorganisms from surfaces have been developed in order to characterize and/or quantify the populations of microorganisms present on surfaces and responsible for contamination. These sampling methods therefore make it possible to determine, on a surface, the different types (of strains) of bacteria and microorganisms present.

Among the methods for sampling microorganisms present on a surface, one of the references in this area is the Standard

ISO 18593:2004({F) which provides and defines horizontal methods for sampling techniques on surfaces (more specifically on surfaces encountered in the environment of the agri-food industries). The “cloth/sponge” method is an example of sampling techniques cited in this Standard, this method making it possible to search for and/or count microorganisms present on a surface. Briefly, according to this sampling method, it involves wetting the cloth/sponge with a quantity of diluent which is sterile physiological serum, sampling the surface in two perpendicular directions before introducing the cloth/sponge into a sterile container with the diluent. Subsequently, after possible storage of the sample, it is analyzed quantitatively and/or qualitatively.

If such a method makes it possible to ensure a sampling of free microorganisms from the surface of a substrate, that is to say a sampling of planktonic microorganisms, it is found to be ineffective when the surface is contaminated by microorganisms having formed a biofilm. Indeed, as explained above, the microorganisms protected by the extracellular matrix of the biofilm, this matrix being of complex and very variable structure and composition and the biofilms being mixed, they are even more varied and complex as they are composed of several different bacteria or different strains.

In addition, it turns out that sampling techniques based on a support impregnated with water can have a beneficial effect on the development of microorganisms, even when taking a sample from a surface. Indeed, it is recognized that certain bacteria exhibit increased growth in the presence of water, the support soaked in water therefore promoting this growth during sampling but also after it since a thin film of water remains on the support treaty where the sampling took place. This can therefore promote the development of certain microorganisms that are not collected or because they are protected by a biofilm. | Detergent and enzymatic compositions have therefore been developed which are used for the removal of contaminants and microorganisms from surfaces. These compositions are particularly advantageous in that they make it possible to effectively degrade and destructure the extracellular matrix of the biofilms, thus making it possible to release the bacteria or contaminants in order to sample them for their subsequent analysis in the laboratory to determine which treatment for eliminating these contaminants will be appropriate and applied to the surface.

Unfortunately, if such compositions make it possible to ensure sampling of microorganisms and contaminants in a representative manner, it turns out that on the one hand, culturing the samples taken and therefore the microorganisms is difficult, even impractical, and On the other hand, the samples taken are difficult to analyze using the different laboratory analysis methods. The samples taken being only slightly or not compatible with laboratory analysis methods and processes, this leads to a difficult and insufficient characterization of the biofilms, and therefore to poorly or not specific elimination treatments of the contaminating microorganisms of a surface.

It is thus easy to understand that an elimination treatment which is determined after a process of sampling, culturing and laboratory analysis of the contaminants, and which is only slightly or not effective is really problematic for manufacturers, leading to a loss of time, recurring contaminations and ultimately a real shortfall in production.

In order to overcome the disadvantages cited above, we know from the state of the art the document EP3366756 which discloses a solution for neutralizing the biocidal effect of the different products which could have been used to disinfect the surfaces to be sampled.

We also know the document US2019071708 which discloses a method for collecting microorganisms from a surface comprising a system for neutralizing an acid disinfectant, the latter comprising dibasic sodium phosphate, monobasic potassium phosphate, sodium pyruvate , polysorbate 80 and lecithin from sunflower.

Unfortunately, although the solutions provided by documents EP3366756 and US2019071708 make it possible to neutralize a disinfectant or a biocide, it appears that these solutions are insufficiently compatible with subsequent laboratory analyzes and that they do not make it possible to overcome the disadvantages mentioned above. Before.

Brief summary of the invention

There is therefore a real need to provide a sampling kit comprising a solution for preserving (stabilizing) microorganisms taken from industrial agri-food surfaces, in a living or revivable state, for subsequent detection and laboratory analysis which has the effect of aim of overcoming the disadvantages of the state of the art by allowing the samples of planktonic microorganisms, contaminants, biofilms taken to be able to be adequately and efficiently cultured and these same samples can then be analyzed in the laboratory, for example in microbiological analyses, molecular biology, flow cytometry and even immunological tests, in a reliable, efficient and reproducible manner.

In addition, this preservation (stabilization) solution incorporated into the sampling kit prevents a detergent and enzymatic solution used in the sample as well as the microorganisms sampled from interfering with the culturing and laboratory analyzes and therefore therefore are compatible. In this way, laboratory analyzes of microorganisms can be representative and reliable in terms of characterization of microorganisms to determine the appropriate and effective elimination treatment.

To solve this problem, according to the invention a sampling kit is provided comprising a preservation solution allowing the stabilization of microorganisms taken from industrial agri-food surfaces, in the living or revivable state, for detection and analysis in the laboratory. subsequent applications comprising two of the three compounds below, or the three compounds below: - A nutrient, preferably a protein hydrolyzate or a mixture of peptides, - optionally a protease inhibitor, - a preservative inhibitor chosen from a polyoxyethylene sorbitan derivative and/or a phosphoglyceride derivative.

This conservation solution is designed in relation and/or synergy with a sampling solution (described below) and is advantageously combined with it in this kit.

The characteristics of the conservation solution according to the present invention have the advantage of providing a stabilization solution for the collection and conservation of microorganisms in the living or revivable state, making it possible to cultivate them in an efficient and effective manner. adequately, the samples taken using detergent and enzymatic sampling compositions, and that these same samples can then, after being cultured, be analyzed in the laboratory, for example by different methods such as microbiological, molecular biology, cytometry analyses. flow or even immunological tests etc.

As explained above, in order to be able to effectively and representatively sample microorganisms, contaminants and more particularly when they are in the form of a biofilm, it is necessary to destructure and degrade their extracellular matrix to release the bacteria of the biofilm and thus be able to sample them. It is therefore necessary to use detergent and enzymatic compositions for sampling; these compositions must be effective for all types of biofilms encountered in industry and more particularly in the food industry. These compositions generally include several types of enzymes including proteases which are very advantageous for destructuring biofilms.

However, the enzymes used in the sampling compositions and more particularly the proteases can interact and hydrolyze the enzymes used in laboratory analysis reactions, such as for example alkaline phosphatases, tag polymerases, luciferases, etc. Additionally, enzymes and proteases can also interfere with antigen-antibody complexes during immunological-type reactions. These disruptions lead to laboratory analyzes that lack reliability and representativeness for the microorganisms analyzed. has appeared that the incorporation, in the preservation solution according to the present invention, of at least one protease inhibitor makes it possible, to a certain extent, to overcome this drawback, to inhibit the action of the protease after sampling and therefore make it possible to provide samples of microorganisms, contaminants and biofilms taken which are compatible with subsequent laboratory analyses.

The protease inhibitors of the present invention, when incorporated, are preferably chosen from sulfonylation agents (e.g. AEBSF, PMSF, DFP), chelating agents (e.g. EDTA), alkylating agents (e.g. . fluoromethyl, chloromethyl and acyloxymethyl ketones, epoxides, aziridines, vinyl sulfones, and others Acceptor

Michael), acylating agents (e.g. B-lactams, lactones, aza-peptides, heterocyclic derivatives), phosphonylation agents (e.g. DFP), phosphonyl or sulfonyl fluorides, thiadiazoles, sulfate salts amino acids and tissue inhibitors of matrix metalloproteinases (TIMP). PMSF is preferred, although it is difficult to apply due to its toxicity and the difficulty in putting it into solution.

In addition, detergent and enzymatic compositions for collecting microorganisms are made by mixing various commercial compounds such as surfactants, enzymes or even viscosity or stabilizing agents. These different commercial compounds include preservatives to prevent the growth of microorganisms and to guarantee long shelf life of the products. These preservatives, which are thus found in detergent and enzymatic compositions, seem a priori to pose no problem, or even to be advantageous for increasing the duration of use of the compositions. However, the inventors have noticed that these compounds, even diluted, have a disruptive effect in the case of laboratory analyzes and more particularly in the case of microbiology analyses. Indeed, certain laboratory techniques and analyses, to demonstrate pathogenic microorganisms, include an enrichment step allowing the growth of microorganisms present in low concentration during sampling and, unfortunately, the inventors have noticed that the presence of preservatives disrupts or even prevents the growth of microorganisms, at least certain microorganisms, during this step and therefore leading to laboratory analyzes which lack reliability and representativeness for the microorganisms sampled. has also surprisingly appeared that the presence of at least one polyoxyethylene sorbitan derivative and/or at least one phosphoglyceride derivative makes it possible to partially overcome this drawback, to inhibit the action of preservatives and therefore to allow the implementation culture and growth of microorganisms or contaminants taken, even in small quantities, which can ultimately be detected and analyzed in the laboratory subsequently, in a reliable and representative manner, without interference.

In addition, the preservation solution according to the present invention, by comprising at least one nutrient extract, ensures that the microorganisms, contaminants and biofilms taken remain in a living or revivable state for cultivation and laboratory analyzes subsequent ones.

The dependent claims refer to other advantageous embodiments.

Preferably, the protease inhibitor is chosen from the group comprising benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride (PMSF).

Preferably, at least one polyoxyethylene sorbitan derivative is polyoxyethylene sorbitan monooleate.

Indeed, the inventors have identified that polyoxyethylene sorbitan monooleate is particularly effective in inhibiting the action of preservatives in samples taken by detergent and enzymatic sampling compositions.

Preferably, the polyoxyethylene sorbitan derivative is present at a content of at least 4 g/L, preferably at a content of at least 4.3 g/L, preferably at a content of at least 4.6 g /L, advantageously at a content of at least 4.9 g/L.

Preferably, the phosphoglyceride derivative is phosphatidylcholine.

Advantageously, the phosphoglyceride is present at a content of between 0.5 and 3 g/L, preferably at a content of between 05 and 25 g/L, preferably between 05 and 2 g/L, advantageously between 0.5 and 1 .5 g/L.

Advantageously, the nutrient extract is present at a content of at least 1 g/L, preferably at least 2 g/L, preferably at least 3 g/L, advantageously at least 4 g/L. L and particularly advantageously at least 5 g/L.

Preferably, the at least one nutrient extract is enriched in peptone (or is the peptone) at a content of at least 2 g/L, preferably at least 4 g/L, preferably at least 6 g/L, preferably at least 8 g/L, advantageously at least 9 g/L, particularly advantageously at least 10 g/L.

Preferably, the preservation solution according to the invention further comprises at least one osmotic agent chosen from the group comprising sodium chloride, potassium chloride, potassium sulfate, tribasic sodium phosphate, dibasic sodium phosphate. , monobasic sodium phosphate, preferably said at least one osmotic agent is sodium chloride.

Advantageously, the osmotic agent is present at a content of at least 3 g/L, preferably at least 4 g/L, advantageously at least 5 g/L.

Other embodiments of the preservation solution according to the invention are indicated in the appended claims.

The invention mainly relates to a kit for sampling microorganisms on industrial agri-food surfaces, in the living or revivable state, for subsequent detection and laboratory analysis.

As explained above, the advantage of the detergent and enzymatic compositions associated with the present conservation composition is that they allow on the one hand to destructure and degrade the extracellular matrix of the biofilms in order to release the bacteria and allow sampling and on the other hand after sampling to be able to determine precisely which treatment for eliminating these contaminants will be appropriate and to be applied to the surface, after laboratory analyzes have been carried out to precisely identify and characterize the contaminants.

Therefore, it is important that the solutions for collecting and storing microorganisms are compatible with laboratory analyzes so that the characterization of the microorganisms is reliable, efficient and representative of the population sampled.

To solve this problem, the present invention provides a kit for sampling microorganisms on industrial agri-food surfaces, in the living or revivable state, for subsequent detection and laboratory analysis comprising: - a sampling composition and 'sampling of microorganisms comprising one or more surfactant(s), at least one protease and at least one enzyme chosen from the group comprising a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase, - QU less a sterile harvesting means of said microorganisms, - At least one sterile container, for example a sterile sachet, comprising the solution according to the present invention, the present preservation composition comprising two of the three compounds below or the three compounds below: - a nutrient, preferably a protein hydrolyzate or a mixture of peptides, - optionally a protease inhibitor,

- A preservative inhibitor chosen from a polyoxyethylene sorbitan derivative and/or a phosphoglyceride derivative.

This has the advantage of allowing efficient harvesting of microorganisms, and ensuring their survival while awaiting tests, then ensuring that there will be no interference with the tests to be carried out, that there This involves the cultivation of microorganisms, a measurement of ATP consumption, genetic analysis tests (e.g. PCR, sequencing) or immunological analysis (e.g. FACS, ELISA) and a combination of these. this.

Preferably, the sampling solution of the kit according to the invention comprises sodium laureth sulfate (as the sole surfactant).

This has the advantage of not disturbing too much the metabolism of the microorganisms to be sampled, including the most fragile microorganisms.

In one embodiment of the sampling kit according to the invention, the surfactants are present at a content of at least 0.01% (preferably at least 0.02%) by weight relative to the weight of said composition, of preferably at a content of at most 5% (preferably at most 1%, or even at most 0.5%, or even at most 0.1%) by weight.

This has the advantage of ensuring good sampling while preserving the metabolism of the microorganisms sampled.

Preferably, the protease of the sampling composition of the kit according to the invention is a serine protease (e.g. a subtilisin) and the conservation composition of the kit of the present invention advantageously comprises a (water-soluble) serine protease inhibitor.

This has the advantage of blogging any interfering proteolytic activity for downstream testing.

Advantageously, the sampling composition of the kit further comprises at least one stabilizing agent present at a content of at least 15% relative to the weight of said composition, and chosen from the group comprising polyols, propane 1,2-diol and propane 1,3-diol, preferably the polyol is glycerol.

Preferably, the sampling composition of the kit according to the invention further comprises at least one sequestering agent (e.g. carboxy methyl inulin, a phosphonate, sodium glycolate and mixtures thereof) at a content of at least 0.3%. in weight relative to the weight of said composition.

Advantageously, the sampling composition and/or the conservation composition of the kit according to the invention has (have) been sterilized by physical means, preferably chosen from gamma radiation, electron beam irradiation and sterilizing filtration.

Preferably, the sampling composition of the kit according to the invention is packaged in a container allowing colbered application.

This has the advantage of ensuring that a known quantity of the sampling composition has been applied to the surface to be analyzed, and that this quantity is sufficiently homogeneous depending on the different locations of the surface and/or that a subsequent application will be substantially identical. Thus the sample is reliable and representative of the microorganisms present.

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

The invention further relates to a method for sampling microorganisms from industrial agri-food surfaces, in the living or revivable state, for subsequent detection and laboratory analysis comprising the following steps: a) at least one application, preferably a spraying of a composition for sampling microorganisms comprising one or more surfactants, at least one protease and at least one enzyme chosen from the group comprising a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase, on a surface to be sampled, b) allowing said composition to act on said surface to be sampled, c) depositing at least one sterile harvesting means of said microorganisms on said surface to be sampled, d) allowing said at least one sterile harvesting means to be soaked with said composition, e) sterilely take said at least one means for harvesting soaked microorganisms and place it in at least one sterile container, for example a sterile bag, comprising the preservation solution according to the invention.

Advantageously, the at least one application, preferably a spray, of a sampling composition is at least one calibrated application, preferably a calibrated spray.

Advantageously, step b) takes place for a duration of at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 1 minute, preferably at least 2 minutes, preferably at least 3 minutes , advantageously at least 4 minutes, particularly advantageously at least 5 minutes.

Optionally, the method according to the invention further comprises at least one preliminary step of preheating said sampling composition, preferably at a temperature between 20 and 60 °C, preferably between 30 and 50 °C, advantageously between 40 and 50 °C.

Preferably, the method according to the invention further comprises at least one step of storing said sterile container containing said soaked harvesting means, at a temperature lower than 15°C, preferably lower than 10°C, advantageously lower than 5°C. vs.

In one embodiment of the method according to the invention, the at least one sterile harvesting means is a tissue, a wipe, a sponge.

Other embodiments of the sampling method according to the invention are indicated in the appended claims.

The invention finally relates to a use of the kit according to the invention for the detection of contamination of the Salmonella genus on agri-food industrial surfaces, preferably of the genus Salmonella.

Saimonella enterica and/or Salmonella bongori.

Another aspect of the invention relates to a use of the kit according to the invention for the detection of contamination of the Listeria genus on industrial agri-food surfaces, preferably of the genus

Listeria monocytogenes.

Another aspect of the invention relates to a use of the kit according to the invention for the detection of contamination of the type

Pseudomonas on food-processing industrial surfaces, preferably of the genus Pseudomonas aeruginosa.

Another aspect of the invention relates to a use of the kit according to the invention for the detection of contamination of the type

Staphylococcus on food industrial surfaces, preferably of the Staphylococcus aureus genus.

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

Detailed description of an embodiment of the invention

Other characteristics and advantages of the present invention will be drawn from the non-limiting description which follows, and with reference to the drawings and examples.

Examples.

The first obstacles that the inventors had to overcome relate to the lack of representativeness of the biofilms generated in the laboratory, compared to the situation on site. This particularly complicates the choice of enzymatic activities (nature of enzymes, their abundances, presence of potentially deleterious compounds). On the basis of these preliminary tests, the inventors therefore decided that all the compositions which would be formulated in the laboratory should be tested in real conditions: many promising formulations on the laboratory scale are unusable, in practice, and the inventors have not found no published guidelines, even using their expertise, that could have helped them identify functional compositions.

However, once a complex formulation has been identified, such as those described below, certain compounds in this formulation can be replaced by others, which is easily testable in the laboratory (effectiveness, absence of toxicity for microorganisms). , then on site, or directly on site. Thus, to a certain extent, one protease is substitutable for another, in particular when these are serine proteases (subtilisins). However, when commercial enzymatic compositions are used, the inventors have noticed that, depending on the suppliers, and even the brands, different additives risk interfering with microbiology analyses, thus variations in the composition of the present invention are possible, thanks to the teaching of the present invention, but require following the rules of caution described in the present invention.

Example 1

A meat processing company located in Belgium was selected for this study. All facilities were previously cleaned and disinfected before taking samples.

The dropout protocol is applied with a limitation of mechanical actions that could introduce a reproducibility error; it includes the following steps:

Quickly scan (screen) the areas to be sampled with the UV lamp and BDK detection kit (Realco);

Preheat the sampling solution to 45°C;

Place two templates on the surfaces to be sampled (in order to delimit an area of 10cm by 10cm): physiologist water vs sampling composition (14 sprays to cover the area; 1.2 g/spray);

Leave the sampling solution to act for 5 minutes;

Place the sterile wipe on the two sampling areas and leave to soak;

Take sterilely using forceps and place in a sterile transport bag;

Store the samples in refrigerated containers at 4°C;

Afterwards evaluate the two zones with the lamp and the BDK.

A total of 6 sampling zones were included in this study: Zone 1 round table; Zone 2 lid for trash table; Zone 3 stainless steel tank; Zone 4 table saw; Zone 5 plastic surface; Zone 6 derinding.

In these 6 zones, the sample composition comprising enzymes (including a subtilisin, activity equivalent to 10

Anson units/kg) and detergents [approximately 10% by weight) allows a much better recovery of microorganisms, both in classic microbiology and by ATP measurement (2G) or by culture. Conversely, there were no more microorganisms remaining on the surfaces where the sampling solution was placed, unlike the surface treated with physiological water. The difference is especially marked at the level of classic microbiology, compared to ATP 2G or by test involving a culture step (little difference noticed).

The inventors reproduced the example, to include metagenomic analyses, while varying the contact time of the sample composition on the surface (30 seconds and 5 minutes). Again, the enzymatic composition allows for much better sampling (e.g. 100 times better in classic microbiology and 14 times better in aPCR). The bacteria found show an increased proportion in

Pseudomonas.

Example 2 - addition of a preservation solution;

Salmonella

The inventors, suspecting deleterious effects associated with the sample composition, sought to determine to what extent an adjuvant solution could counteract these, in the context of tests aimed at determining the presence of Salmonella and/or

Listeria, either by PCR or OPCR, or by using commercial detection kits, for example those from BioMérieux. To do this, a so-called preservation solution was tested (in g/l): Proteins (beef extract; 5); Peptone, 10; Polysorbate 80 (Tween 80; 5); Lecithin 0.7; NaCl, 5. In addition, various serine protease inhibitors have been used, with the aim of neutralizing the subtilisin activity of the sample solution.

The first series of tests was carried out in the laboratory, where known quantities of the bacteria (0.1 ml of 107 CFU/mI of Salmonella) were incubated either in a physiological solution (100 mI) or in the sample composition of example 1 ($ml + 94 ml physiological water}, i.e. in the composition of example 1, before the preservation solution is added (6 ml sampling composition, 90 ml water, 4 ml of the solution of conservation).

When 107 CFU/ml of Salmonella are analyzed (therefore 104

CFU/ml in the final mixture), the different situations are compatible with the identification of the microorganism by means of PCR analyzes or the Vidas® Salmonella test. However, evidence by the test

Vidas SLM® (broth culture, then Specific measurement by antigen test and fluorescence; this test provides information on the absence or presence of Salmonella) fails when the sample composition is present, whereas it works when the bacteria are diluted in physiological water or when the preservative solution has been added.

Furthermore, in this example, the inventors consider that the concentrations of Salmonella are high, which does not necessarily, or even rarely, represent the situation in the field, where even 1 CFU must be detected.

Example 3 - addition of a preservation solution; Listeria

In this case, the enzymatic solution causes problems at the detection level (enumeration and qPCR); These problems are reduced with the preservation solution, however the Vidas® commercial kit (LMX) still does not work.

In addition, when the quantity of Listeria inoculated is reduced, the microorganism is generally no longer detected following mixing (Vidas LMX®, never; Vidas LMO2®, not every time) with the sampling composition, while detection remains robust when the mixing was carried out only in physiological water. In this case (deliberately extreme), the conservation composition did not improve detection. As a precaution, the tests were reproduced and three strains of Listeria were tested with always the same results; these are strains ATCC 19114, NCTC 11994 and ATCC 13932.

By comparing all the results, the inventors conclude that the sample composition slows down the growth of microorganisms (Listeria), a problem which is partially resolved by the preservation composition. On the other hand, the sample composition interferes with certain downstream microbiological tests. View that the nature and abundance of microorganisms on a surface to be tested is unknown in advance, the inventors therefore conclude that the compositions of examples 1 to 3 are useful, but would benefit from being further improved.

Example 4 - Identifications of deleterious compounds

The inventors searched the various commercial enzymes to see if certain compounds interfered with downstream microbiological tests. The inventors have noticed that the presence of compounds such as phenoxyethanol, potassium sorbate or thiazolinone derivatives is quite widespread. Thus, the inventors have selected enzymatic compositions which are devoid of such compounds, or as poor as possible, even if the inventors have found significant concentrations of 2-phenoxyethanol in numerous commercial enzymatic compositions. Among the compounds sought are Bronopol, triclosan, idocarb, chlorhexidine, DCOIT, BIT, OIT, CIT, MIT, ethyl-paraben, methylparaben, butythylparaben, phenylparaben, isoproylparaben, isobutylparaben, propylparaben, salisylic acid, 4-hydroxybenzoic acid, 4-methylbenzoic acid, benzoic acid, sorbic acid, dehydroacetic acid, 1-phenoxypropane-20l , 1,2-dioromo-2,4-dicyanobutane, 2-hydroxybiphenyl, 2-phenoxyethanol (the most abundantly found, sometimes up to 0.4% by weight), bensyl alcohol and chlorophenesin.

Comparative example 1

Suspecting an interference between the proteases of the sample composition and the enzymes or antibodies of the downstream tests, the inventors tested the effect of the addition of protease inhibitors in the preservation solution (see example 3), while avoiding as much as possible commercial enzymatic compositions comprising compounds identified as deleterious (see example 4).

Different ways of applying the protease inhibitor were tested. The chosen protease being a subtilisin or trypsin, two serine protease inhibitors were tested: PMSF and AEBSF.

PMSF provided good inhibition of the proteolytic activities of the serine proteases tested. On the other hand, AEBSF only inhibited trypsin. Measuring tests where Listeria (the 3 strains above were tested) is brought into contact with a composition comprising a subtilisin, then AEBSF in sufficient concentration to cause complete inhibition of the subtilisin have shown that different microbiological tests gave lower values, or even did not detect Listeria under the test conditions, unlike the control without enzymes or when PMSF is added.

Thus, the inventors chose to incorporate PMSF into the preservation solution to be tested, despite the disadvantages of this molecule.

Unfortunately, even under these conditions, the Vidas® test

LMX does not demonstrate the presence of Listeria monocytogenes.

Example 5 – Optimization of the sample composition

The inventors then decided to test the effectiveness of a highly diluted sampling composition and avoiding as much as possible the compounds identified as deleterious. Additionally, enzymatic activities are reduced, as is detergent content. Detergents are chosen after laboratory testing for their compatibility with microorganism life, including Listeria, which has been identified as more sensitive.

First of all, with detergent concentrations of 10% by weight, even in the presence of low enzymatic activity, in the presence of minimal preservative agent contents (e.g. non-detectable for almost all; phenoxyethanol less than 4 ppm ) and in the absence of

PMSF, Listeria detection tests, such as Vidas®, do not work well (massive underquantification), or not in a reproducible manner.

Even less specific tests do not work when a Listeria culture is subjected to these different treatments.

Thus, all the parameters of the composition must be checked, with the problem that too high a dilution of the enzymatic activities risks no longer allowing the microorganisms to drop out, while too high a dilution of the detergents risks no longer allowing application. homogeneous composition or good contact with the biofilm. Thus, the inventors noticed that, in addition to the quantity of the components, their nature had to be reconsidered.

A diluted formulation was tested comprising approximately 20% by weight of glycerol, approximately 0.03% by weight of an anionic detergent of the (sodium) laureth sulfate type, 0.4% by weight of sequestrant (carboxymethylinulin); 0.4% by weight of sodium glycolate; 0.04% by weight of a subtilisin (activity 1 Anson Unit], as well as a lipase; an amylase, a cellulase and a mannanase. The inventors have selected enzymes, here commercial enzymes, devoid of preservative agents such such as potassium sorbate, MIT, OIT, BIT and CIT.

When possible, the inventors used commercial enzymes devoid of phenoxyethanol (cellulase and mannanase). or with a low content of this molecule (subtilisin; 4 g/kg of the enzyme formulation, lipase; 0.249 g/kg and amylase; 0.233 g/kg). Enzymes other than subtilisin were added in weight contents of between 0.1 and 0.5% (weight of the enzyme composition: total weight) and the quantity of enzyme is typically 1 to 10% by weight relative to to commercial formulation.

Following the use of this composition, the presence of

Listeria is detected, and the Vidas® LMX test for Listeria is, finally, positive and reproducible. The inventors further noted that, under these diluted conditions, the addition of the protease inhibitor PMSF is not necessary, and even reduces the spread of Listeria.

The inventors consider that the concentration of enzymes other than proteases (here, a subtilisin) can still be reduced somewhat, e.g. the lowest concentration which allows detachment, just like those of the sequestering agent. In light of the present invention, this can be tested in the laboratory on the 3 strains of Listerig or in other models, including, preferably, more complex ones.

The inventors then tested a possible synergy with the preservation solution (see examples 2 and 3): given that the results are already very good, the improvement associated with the addition of the preservation solution is marginal, but it has was however interesting in the case of a test which includes propagation followed by detection by PCR.

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

Claims (19)

CLAIMS 1, Kit for sampling microorganisms potentially present on a surface, in a living or revivable state, for subsequent detection and laboratory analysis comprising: - a composition for sampling microorganisms comprising a surfactant, at least one protease and at least one enzyme chosen from the group comprising a polysaccharidase, a laccase, a lipase, a cellulase and a mannanase, - QU less a means for sterile harvesting of said microorganisms, - QU less a sterile container, for example a sterile bag, comprising a solution for preserving microorganisms taken from surfaces, in a living or revivable state, for subsequent detection and laboratory analysis comprising: - At least one nutrient extract, preferably a mixture of peptides or a protein hydrolyzate ; - At least one polyoxyethylene sorbitan derivative and/or at least one phosphoglyceride derivative. 2. Microorganism sampling kit according to claim 1, wherein the surfactant of the sampling composition comprises at least one anionic surfactant. 3. Microorganism sampling kit according to claim 1 or 2, in which one or more surfactants of the sampling composition are present at a content of at least 0.01%, preferably at least 0.02% by weight relative to the weight of said composition, preferably at a content of at most 5% by weight, preferably at most 1%, preferably at most 0.5%, or even at most 0.1% (sum surfactants present: total weight of the composition). 4. Microorganism sampling kit according to one of claims 1 to 3, in which the sampling composition comprises at least one serine protease. 5. Microorganism sampling kit according to one of claims 1 to 4, in which the sampling composition further comprises a stabilizing agent chosen from a polyol (glycerol), propane 1,2 diol and propane 1,3 -diol. 6. Microorganism sampling kit according to claim 5, in which the stabilizing agent of the sampling composition is present at a content of at least 15% relative to the weight of said composition. 7. Microorganism sampling kit according to any one of claims 1 to 6, in which the sampling composition further comprises at least one sequestering agent at a content of at least 0.3% by weight relative to the weight of said composition. 8. Microorganism sampling kit according to any one of claims 1 to 7, wherein said at least one means for sterile harvesting of said microorganisms is a tissue, a wipe, a swap, a sponge, a scraper, a swab. 9. Microorganism sampling kit according to any one of claims 1 to 8, in which said sampling composition is a sterile sampling composition, preferably sterilized by physical means, such as by electron beam, by gamma radiation or by sterilizing filtration. 10. Microorganism sampling kit according to any one of claims 1 to 9, wherein said sampling composition and/or said preservation solution is packaged in a calibrated container, preferably in a colored spray container. 11. Sampling kit according to any one of the preceding claims, in which the preservation solution comprises a protease inhibitor, preferably a serine protease inhibitor, preferably a sulfonyl fluoride derivative substituted by a group comprising at least one aromatic radical in C, preferably chosen from the group comprising benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride, preferably in which said at least one sulfonyl fluoride derivative substituted by a group comprising at least one aromatic radical in C is phenylmethylsulfonyl fluoride . 12. Sampling kit according to any one of the preceding claims, in which said at least one polyoxyethylene sorbitan derivative present in the preservation solution is polyoxyethylene sorbitan monooleate. 13. Sampling kit according to any one of the preceding claims, in which said at least one polyoxyethylene sorbitan derivative is present in the preservation solution at a content of at least 4 g/L, preferably at a content of at least 4.3 g/L, preferably at a content of at least 46 g/L, advantageously at a content of at least 4.9 g/L. 14. Sampling kit according to any one of the preceding claims, wherein said at least one phosphoglyceride derivative of the preservation solution is phosphatidylcholine. 15. Sampling kit according to any one of the preceding claims, in which said at least one phosphoglyceride derivative is present in the preservation solution at a content of between 0.5 and 3 g/L, preferably at a content of between 0. .5 and 25 g/L, preferably between 0.5 and 2 g/L, advantageously between 0.5 and 1.5 g/L. 16. Use of the kit according to any one of the preceding claims for the detection of contamination of the Salmonella genus on industrial agri-food surfaces, preferably of the Salmonella enterica and/or Salmonella bongori genus. 17. Use of the kit according to any one of preceding claims 1 to 15 for the detection of contamination of the Listeria genus on industrial agri-food surfaces, preferably of the Listeria monocytogenes genus. 18. Use of the kit according to any one of preceding claims 1 to 15 for the detection of contamination of the Pseudomonas genus on food-processing industrial surfaces, preferably of the Pseudomonas aeruginosa genus. 19, Use of the kit according to any one of claims 1 to 15 for the detection of contamination of the Staphylococcus genus on industrial food surfaces, preferably of the Staphylococcus aureus genus.