Product and method for removal of biofilms

Abstract

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

Claims

1. A method for removing a biofilm present on a substrate, comprising the steps of: (a) providing a detergent component comprising a sequestrant, a wetting agent and a dispersant, and providing an enzymatic component containing at least one protease, at least one laccase and at least one polysaccharidase; (b) placing the detergent component in solution in an aqueous phase; (c) placing the enzymatic component in solution in the solution formed at step (b) to form a solution comprising the sequestrant, the wetting agent, the dispersant, the at least one protease, the at least one laccase, and the at least one polysaccharidase; or (b′) placing the enzymatic component in solution in an aqueous phase; (c′) placing the detergent component in solution in the solution formed at step (b′) to form a solution comprising the sequestrant, the wetting agent, the dispersant, the at least one protease, the at least one laccase, and the at least one polysaccharidase, wherein the pH of the solution formed at step (c) or (c′) is between approximately 6.5 and 7.5; or optionally steps (b) and (c), or steps (b′) and (c′), are conducted simultaneously to provide a solution comprising the sequestrant, the wetting agent, the dispersant, the at least one protease, the at least one laccase, and the at least one polysaccharidase; (d) applying the solution formed at step (c) or (c′) to the substrate for a predetermined period of time to effect biofilm removal from the substrate; and (e) adding a basic solution after applying the solution formed at step (c) or (c′) to said substrate during said predetermined period of time so as to increase the pH to about 8 to 9.

2. The method according to claim 1, wherein biofilm removal from the substrate removes microorganisms from the substrate.

3. The method according to claim 1 further comprising applying a biocide to the substrate.

4. The method according to claim 1, wherein applying the solution formed at step (c) or (c′) to the substrate at step (d) comprises applying the solution to surface of a food production line.

5. The method according to claim 1, wherein applying the solution formed at step (c) or (c′) to the substrate at step (d) comprises circulating the solution within piping.

6. The method according to claim 1, wherein the substrate is a floor or surface.

7. The method according to claim 1, wherein the method is a clean-in-place method.

8. The method according to claim 1, wherein the method is an immersion method.

9. The method according to claim 1, wherein the biofilm is a food industry biofilm and the substrate is a floor or surface in a food production line.

10. The method according to claim 1, wherein the predetermined period of time is between 15 minutes and 4 hours.

11. The method according to claim 1, wherein the at least one enzymatic component of the solution formed at step (c) or (c′) comprises a proportion of the at least one protease of between 10 and 50%, a proportion of the at least one laccase of between 5 and 35%, and a proportion of the at least one polysaccharidase of between 5 and 20% by weight relative to the total weight of the enzymatic component.

12. The method according to claim 1, wherein the solution formed at step (c) or (c′) comprises about 1% by weight detergent component relative to the total weight of the solution and about 0.05% by weight enzymatic component relative to the total weight of the solution.

13. The method according to claim 1, wherein said at least one polysaccharidase is an alpha-amylase.

14. The method according to claim 1, wherein said at least one detergent component comprises a proportion of sequestrant of between 1 and 10% by weight relative to the total weight of the detergent component.

15. The method according to claim 1, wherein said at least one detergent component comprises a proportion of dispersant of between 1 and 10% by weight relative to the total weight of the detergent component.

16. The method according to claim 1, wherein said at least one detergent component comprises a proportion of wetting agent of between 1 and 15% by weight relative to the total weight of the detergent component.

17. The method according to claim 1, wherein said dispersant is a polymer soluble or partly soluble in water and is selected from the group consisting of polyethylene glycol, cellulose derivatives, a polymer comprising at least one acrylic acid or acrylic ester repeat unit, a polymer comprising at least one acrylic acid or acrylic ester repeat unit of general formula —(CH.sub.2—CH—COOR)— where R represents a hydrogen, alkyl or substituted alkyl, aryl or substituted aryl group, a polymer of acrylic acid, and a homopolymer of acrylic acid having a weight average molecular weight approximately of between 2000 and 6000.

18. The method according to claim 1, wherein the dispersant is a polymer comprising at least one acrylic acid or acrylic ester repeat unit.

19. The method according to claim 1, wherein said dispersant is a polymer having a weight average molecular weight of between approximately 500 and 10000.

20. The method according to claim 1, wherein said wetting agent is selected from the group consisting of C.sub.6 to C.sub.10 sodium alkyl sulphates, C.sub.6 to C.sub.10 alcohol ether sulphates, and C.sub.6 to C.sub.10 alkyl aryl sulphonates.

21. The method according to claim 1, wherein the sequestrant is selected from the group consisting of a phosphonate, a phosphinate, a phosphate, a salt thereof, and a mixture thereof.

22. The method according to claim 1, wherein the enzymatic component is in the form of a dry solid, a powder, or a lyophilizate.

23. A method for removing a biofilm present on a substrate, comprising the steps of: (a) providing a detergent component comprising a sequestrant, a wetting agent and a dispersant, and providing an enzymatic component containing at least one protease, at least one laccase and at least one polysaccharidase; (b) placing the detergent component in solution in an aqueous phase; (c) placing the enzymatic component in solution in the solution formed at step (b) to form a solution comprising the sequestrant, the wetting agent, the dispersant, the at least one protease, the at least one laccase, and the at least one polysaccharidase; or (b′) placing the enzymatic component in solution in an aqueous phase; (c′) placing the detergent component in solution in the solution formed at step (b′) to form a solution comprising the sequestrant, the wetting agent, the dispersant, the at least one protease, the at least one laccase, and the at least one polysaccharidase, wherein the pH of the solution formed at step (c) or (c′) is between approximately 6.5 and 7.5; or optionally steps (b) and (c), or steps (b′) and (c′), are conducted simultaneously to provide a solution comprising the sequestrant, the wetting agent, the dispersant, the at least one protease, the at least one laccase, and the at least one polysaccharidase; (d) applying the solution formed at step (c) or (c′) to the substrate for a predetermined period of time to effect biofilm removal from the substrate; and (e) adding a basic solution after applying the solution formed at step (c) or (c′) to said substrate during said predetermined period of time so as to increase the pH to obtain an alkaline pH between 8 and 11.

Description

DESCRIPTION OF THE DRAWINGS

(1) The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

(2) FIG. 1 is a graph showing the cell quantity of a Pseudomonas fluorescens strain on a substrate after application of different treatment solutions.

(3) FIG. 2 is a graph showing the cell quantity of a Bacillus mycoides strain on a substrate after application of different treatment solutions.

(4) FIG. 3 is a graph showing the cell quantity of a Bacillus cereus strain on a substrate after application of different treatment solutions.

(5) FIG. 4 gives images of the surface of a substrate, taken under epifluorescence microscopy, after application of different treatment solutions to said substrate coated with a biofilm of the strain Pseudomonas fluorescens.

(6) FIG. 5 gives images of the surface of a substrate taken under epifluorescence microscopy after application of different treatment solutions to said substrate coated with a biofilm of the strain Bacillus mycoides.

(7) FIG. 6 gives images of the surface of a substrate taken under epifluorescence microscopy after application of different treatment solutions to said substrate coated with a biofilm of the strain Bacillus cereus.

(8) FIG. 7 gives the results of staining of biofilms before cleaning with cleaning solutions (A) and after cleaning with said solutions (B).

(9) FIG. 8 is a graph showing the efficacy of four enzymes against biofilms derived from the strain Chryseobacterium meningosepticum.

(10) FIG. 9 is a graph showing the efficacy of four enzymes against biofilms derived from the strain Bacillus Cereus.

(11) FIG. 10 is a graph showing the efficacy of four enzymes against biofilms derived from the strain Pseudomonas fluorescens.

DETAILED DESCRIPTION

(12) While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

(13) In the Figures, identical or similar elements carry the same references.

Example 1

(14) Description of chosen test conditions: the tests were conducted as part of Clean-In-Place (CIP) procedure in a pilot industrial plant. The biofilms were developed on a planar face of a stainless steel cylinder perforated in its centre. This cylinder, once placed in a pipe having the same inner diameter as the outer diameter of the cylinder, causes sudden narrowing of the diameter thereof forcing liquid to pass through the central perforation. This stress leads to flow perturbations thereby creating dead zones on the surface of the cylinder. These dead zones promote the formation of a biofilm and hinder its mechanical detachment by the flow. They therefore typically represent zones that are difficult for biofilm removal.

(15) Three different strains were used: Pseudomonas fluorescens (supplied by University of Cornell, Department of Food Science, Ithaca, N.Y., 14853, USA (Kathryn J. Boor, kjb4@cornell.edu); Bacilfus mycoides (supplied by the AFSSA laboratory in Maison-Alfort, France (Brigitte Carpentier, b.carpentier@lerpac.afssa.fr); Bacillus cereus (supplied by INRA, UR638, Processus aux Interfaces et Hygiene des Materiaux, Villeneuve d'Ascq, France (Christine Faille, Christine.faille@lille.inra.fr).

(16) A growth medium was prepared as follows: powder meat extracts (Biokar) were dissolved in 0.1% distilled water and sterilized. 20 ml of inoculum with 5.10.sup.7 CFU/ml in a solution of Trypticase Soy Broth (TSB, Biokar) were deposited on the surface of the cylinders. These cylinders were incubated in a moist chamber at 30° C. for 2 hours to allow adhesion of the cells. The TSB solution was then removed and replaced by 20 ml of the meat growth medium and the cylinders were incubated 24 h at 30° C. The growth medium was then replaced by fresh meat medium and the cylinders were again incubated for 24 h.

(17) Cleaning procedure comprised placing the cylinders contaminated with the biofilm in straight pipes of a pilot industrial plant cleaned by circulation (CIP cleaning). The treatment solutions were circulated for 30 minutes at 45° C. at a flow rate of 300 l/h. For two of the strains, tests were also performed at a flow rate of 600 l/h. Each test was repeated three times and a mean was determined.

(18) The development and removal of the biofilms were monitored by microscope observation and by quantification of viable cells on the cylinders via a count on Trypticase Soy Broth medium after detachment of the cells.

(19) Description of the Protocol for Preparation of the Composition

(20) The detergent component was prepared by mixing in a determined volume of water: a phosphonate, a polyacrylate and an anionic wetting agent. The respective proportions in the detergent component were 3%, 4% and 3%. The pH of the solution was brought to 13.3 by dilution. A solution of the enzymatic component was prepared. This comprised a proportion of 30% proteases (EC 3.4.21), 30% laccase (EC 1.10.3.2) and 10% alpha-amylase (EC 3.2.1.1) and a conventional excipient.

(21) The pH of the solution of the enzymatic component was brought to 9 by progressively adding a solution of potassium hydroxide. The solution of the composition according to the invention was prepared by adding the detergent component and enzymatic component to water. The solution of the said composition of the invention comprised 1% detergent component and 0.05% enzymatic component. The pH of the solution of said composition according to the invention was approximately 10.

(22) Description of the Tests and Test Results

(23) Test with the Pseudomonas fluorescens Strain

(24) The graph given in FIG. 1 shows the quantity of biofilm present on the wall of the cylinder after application of treatment solutions following the conditions set forth above. The surfaces of the substrate were coated with a biofilm formed by the Pseudomonas fluorescens strain. The initial quantity of biofilm present before treatment is denoted A1 in FIG. 1.

(25) Three solutions were compared: a solution solely containing water (B1); 0.5% sodium hydroxide solution (C1) and a solution of the composition of the invention (D1). The solutions were applied at a rate of 300 l/h and temperature of 45° C. Solution (B1) comprising water allowed evaluation of flow-induced mechanical detachment of the biofilms.

(26) It was observed that the biofilm resisted mechanical detachment since the quantities of biofilm are identical (curves A1 and B1). The application of a solution of the composition of the invention allowed a major reduction in the biofilm (curve D1, 10.sup.3 CFU) compared with a sodium hydroxide solution (curve C1, 10.sup.5 CFU). The composition of the invention is therefore more efficient than a reference treatment (sodium hydroxide solution) on a biofilm resistant to mechanical detachment.

(27) FIG. 4 shows several images of the surface of a substrate, taken under epifluorescence microscopy, after application of the different treatment solutions at 300 l/h. Image (B4) shows the surface of the substrate subjected to a water solution, image (C4) shows the surface of the substrate subjected to a 0.5% sodium hydroxide solution and image (D4) shows the surface of the substrate subjected to a solution of the composition of the invention.

(28) These data clearly show that the composition of the invention removed the entirety of the matrix of the biofilms (diffuse staining) leaving only isolated cells on the surfaces or small groups of cells distributed over a single layer unprotected by a matrix. The sodium hydroxide solution on the other hand did not allow complete removal of the matrix (diffuse staining) which still partly protects residual cells. A subsequent disinfection phase will therefore be much more efficient after application of a solution of the composition according to the invention.

(29) Test with the Bacillus mycoides Strain

(30) The graph given in FIG. 2 shows the quantity of biofilm present on the wall of the cylinder after applications of treatment solutions following the conditions set forth above. The surfaces of the substrate were coated with a biofilm formed by the Bacillus mycoides strain. The amount of biofilm present before treatment is shown by curve (A2).

(31) Five solutions were compared: a solution solely containing water (B2), 0.5% sodium hydroxide solution (C2), a solution of the composition according to the invention (D2 and F2) and a 2% sodium hydroxide solution (E2). The solutions A2-D2 were applied at a flow rate of 300 l/h whilst the solutions E2-F2 were applied at a flow rate of 600 l/h.

(32) The biofilm of Bacillus mycoides is relatively sensitive to mechanical detachment its quantity being reduced by 90% with the application of solution (B2). Application of the solution of the composition of the invention (D2 and F2) showed equivalent performance to the sodium hydroxide solution (C2 and E2) whether the flow rate was 300 l/h or 600 l/h.

(33) FIG. 5 shows several images of the surface of the substrate taken under epifluorescence microscopy after application of the different treatment solutions at 300 l/h. Image (BS) shows the surface of the substrate subjected to a water solution, image (C5) shows the surface of the substrate subjected to a 0.5% sodium hydroxide solution, and image (D5) shows the surface of the substrate subjected to a solution of the composition of the invention.

(34) These clearly show that the composition of the invention removed most of the matrix of the biofilms, only leaving isolated cells or small groups of cells distributed over a single layer unprotected by a matrix. The sodium hydroxide solution did not allow more efficient removal of the biofilm. The subsequent action of a disinfectant solution will therefore be more efficient if a solution of the composition of the invention is previously applied.

(35) Test with the Bacillus cereus Strain

(36) The graph give in FIG. 3 shows the amount of biofilm present on the wall of the cylinder after application of treatment solutions following the conditions set forth above. The surfaces of the substrate were coated with a biofilm formed by the Bacillus cereus strain. The amount of biofilm present before treatment is shown by curve A3.

(37) Five solutions were compared: a solution solely containing water (B3), 0.5% sodium hydroxide solution (C3), a solution of the composition of the invention (D3 and F3) and a 2% sodium hydroxide solution (E3). Solutions B3, C3, D3 were applied at a flow rate of 300 l/h, whilst solutions E3 and F3 were applied at a faster rate (600 l/h). A faster flow rate has an influence on mechanical detachment.

(38) The biofilm of Bacillus cereus fully resists mechanical detachment (curve A3 and B3). The composition of the invention showed greater efficacy at a flow rate of 300 l/h compared with a 0.5% sodium hydroxide solution (curve C3) and even at a flow rate of 600 l/h compared with a more concentrated sodium hydroxide solution (curve E3).

(39) FIG. 6 shows several images of the surface of a substrate, taken under epifluorescence microscopy, after application of the different treatment solutions. Image (B6) shows the surface of the substrate subjected to a solution of water, image (C6) shows the surface of the substrate subjected to a 0.5% sodium hydroxide solution and image (D6) shows the surface of the substrate subjected to a solution of the composition according to the invention.

(40) These images again show that the composition of the invention removes almost all the matrix of the biofilms, only leaving isolated cells or small groups of cells distributed over a single layer unprotected by a matrix.

(41) The sodium hydroxide solution does not allow efficient removal of the biofilm. The disinfection phase of the substrate after the removal phase of the biofilm will therefore be more efficient after application of a solution of the composition of the invention for this removal phase.

(42) The different tests performed in this Example demonstrate the efficacy of the composition of the invention in removing a biofilm from a substrate. The composition of the invention proves to be more efficient than a sodium hydroxide solution routinely used in the prior art. The composition of the invention also exhibits high efficacy against several types of biofilm derived from different bacteria. The composition of the invention therefore brings an efficient solution to the problems raised in the prior art and known to those skilled in the art.

Example 2

(43) Tests were also performed on two industrial production lines (butter and margarine production). Stainless steel test coupons (8*1 cm) were placed for 15 days in production circuits having sporadic contamination. These coupons were therefore subjected to cycles of production and standard cleaning of the installations.

(44) The presence of biofilm in the plant was determined by the development of a biofilm on the coupons. Specific Clean-In-Place procedure using the composition of the invention was applied in the plant in the presence of the contaminated coupons, and whose efficacy was demonstrated through the removal of the biofilms formed on the coupons.

(45) The presence and size of the biofilms on the coupons were determined in a laboratory by staining the coupons and comparing the staining obtained with a visual scale (HYDROBIO®, BKG) and by observation under an optical microscope (40× and 100×).

(46) Two cleaning protocols were tested: the first protocol was used as reference and corresponded to the application of solutions known in the prior art, the second protocol included the addition of a step to apply a solution of the composition according to the invention.

(47) In particular Protocol I comprised the following steps: cleaning with water, alkaline cleaning, rinsing, sanitization (or disinfection) and final rinsing. Protocol 1 was applied every 32 hours for one week. Cleaning with water was applied for 15 minutes at a temperature of between 60 and 65° C. Alkaline cleaning allows pipes to be cleaned of organic matter present after a production cycle. This step was conducted using a 3.5% sodium hydroxide solution applied for 2*15 minutes at a temperature of between 70-75° C.

(48) The sanitization step allows the removal of germs which may still be present but are unprotected by the matrix of the biofilm. This step entailed the application of a 1.5% solution of Deptil OX (HYPRED) for 2*15 minutes at a temperature of between 20 and 25° C. The Deptil OX solution contains a mixture of peracetic acid and hydrogen peroxide. The rinsing steps were performed for 15 minutes at a temperature of between 20 and 25° C.

(49) Protocol II differed from Protocol I in that it comprised a specific cleaning step before the sanitization (or disinfection) step. In addition, Protocol II was only applied once.

(50) The specific cleaning step consisted of the application of a solution of the composition according to the invention. This mixture was applied for 30 minutes at a temperature of between 40 and 45° C. The results obtained are given in Table 1.

(51) TABLE-US-00001 TABLE 1 Quantity of biofilm Quantity of biofilm after Protocol I (g/m.sup.2) after Protocol II (g/m.sup.2) Production line 1 25 <5 Production line 2 25 to 35 <5

(52) Protocol I did not allow the limiting and prevention of growth of a biofilm despite the sanitization (disinfection) step. The biofilm which had formed on the stainless steel coupons at the heart of the installation was therefore very resistant. The application of Protocol II including a specific cleaning step with a solution of the composition of the invention allowed much more efficient removal of this biofilm (below the detection limit). Greater efficacy of the specific cleaning was therefore demonstrated which therefore provides an innovative solution to prior art problems.

Example 3

(53) Tests allowing a comparison between optimal pH values were conducted.

(54) Biofilms were developed on stainless steel coupons previously washed and sterilized. An inoculum of bacterium (Pseudomonas aeruginosa) was prepared by culture for 16 h in a 10% TSB medium (Tryptone Soy Broth). This pre-culture was diluted to obtain an optical density (OD) of 0.05 to 600 nm, and 500 μl of this solution were spread on each coupon. The coupons were incubated 48 h in Petri dishes held under moist conditions and placed in an oven at 30° C. After 2 hours, the bacterial solution was replaced by 500 μl of fresh 10% TSB medium. After 24 h, this culture medium was renewed.

(55) The coupons were then cleaned and placed on metal rods separated by nuts. They were then placed in the cleaning solutions. The coupons were left immersed in the above-mentioned cleaning solutions for a total time of 30 minutes.

(56) The cleaning solutions were the following:

(57) 1. Composition of the invention at pH 4.5, immersion for 30 minutes

(58) 2. Composition of the invention at pH7, immersion for 30 minutes

(59) 3. Composition of the invention at pH 9.7, immersion for 30 minutes

(60) The coupons were subsequently placed in test tubes containing 10 ml of TSB solution+0.5 Tween 80 for bacterial count The test tubes were incubated for 5 minutes on the table before being sonicated for 2 and a half minutes and vortexed for 30 seconds. The incubation, sonication and vortex steps were repeated.

(61) The coupons were afterwards collected and the medium comprising the TSB solution+0.5 Tween was subjected to serial dilutions using peptone water (peptone water free of indole: preparation of 10 ml of 15 g/l dissolution. Dilution of 1 ml of this solution in 1 litre of sterile water); this medium was then spread on Petri dishes and incubated at 30° C. overnight before counting.

(62) The biofilm was also stained as follows: the test coupons were drained and immersed in the staining solution for 10 minutes, selective for the proteins of the biofilm matrix. The coupons were then immersed in the different above-mentioned cleaning solutions 1 to 3, for a time of twice 10 minutes (the cleaning solutions were replaced between the two cleaning steps) before being left to dry in open air.

(63) Results

(64) At acid pH (4.5): a mat of bacteria developed on the surface of the Petri dishes, which indicated the presence of a large quantity of residual biofilm on the coupons.

(65) At neutral pH (7): a mat of bacteria developed on the surface of the Petri dishes.

(66) At alkaline pH (9.7): no bacteria present in the Petri dishes.

(67) FIG. 7 gives the results of the staining of the biofilms. FIG. 7A illustrates the references before cleaning with treatment solutions 1 to 3, whilst FIG. 7B illustrates the steel coupons after cleaning with solutions 1 to 3 from left to right. As can be seen, cleaning at alkaline pH with the composition of the invention is clearly more efficient than at acid pH or at neutral pH, the biofilm developed on the coupon being fully detached with alkaline pH whereas it persists at other pH values.

Comparative Example

(68) A test intended to determine the efficacy of four enzymes against three bacterial strains was conducted.

(69) The results of this test are given in FIGS. 8 to 10. Biofilms were formed on steel coupons (12) as in Example 3.

(70) 4 coupons were therefore produced for each bacterial strain (Pseudomonas fluorescens, Bacillus cereus and Chryseobacterium meningosepticum). The coupons were then rinsed with water before each being incubated with a cleaning solution respectively containing a first, a second protease, a laccase and a polysaccharidase for 30 minutes.

(71) Tables 2 gives the results of the bacterial counts whilst FIGS. 8 to 10 show the logarithm of the results of bacterial count along the Y-axis and the type of enzyme used along the X-axis.

(72) TABLE-US-00002 TABLE 2 Chryseobacterium Bacillus Pseudomonas meningosepticum cereus fluorescens Initial contamination 2.0 × 10.sup.8 7.0 × 10.sup.6 2.0 × 10.sup.8 level First protease 4.0 × 10.sup.5 7.0 .Math. 10.sup.2 1.0 × 10.sup.2 Second protease 2.0 × 10.sup.6 9.0 × 10.sup.3 3.8 × 10.sup.4 Laccase 8.0 × 10.sup.3 1.3 × 10.sup.5 1.6 × 10.sup.2 Amylase 1.0 × 10.sup.5 9.6 × 10.sup.4 3.9 × 10.sup.6

(73) As can be seen, some bacterial strains are more sensitive to the action of the polysaccharidase and laccase, as is the case for Chryseobacterium meningosepticum (Gram−). As for Bacillus Cereus (Gram+), this is more sensitive to the combination of the two proteases whereas Pseudomonas fluorescens (Gram−) is more sensitive to the combination of the first protease and the laccase.

(74) Evidently, the present invention is in no way limited to the above-described embodiments and numerous modifications can be made thereto without departing from the scope of the appended claims. For example, the fields of application of the compositions of the invention have proved to have particular use for cleaning by immersion e.g. in the hospital sector (cleaning laboratory equipment, surgical equipment), or by circulation within piping e.g. in the agri-food industry, in air conditioning installations, in water purifying and desalinating plants, for sailing vessel hulls and any type of immersed equipment, in washing machine, dishwasher circuits etc., or for soaking and circulation in piping.