DECONTAMINATION METHOD

Abstract

A food composition including at least one strain of bacteria of the genus Bacillus, the strain being dispersed in the food composition, and a method for decontaminating or preventing surface contamination of a device for receiving food in which the food composition is brought into contact with the surface of the device.

Claims

1-10. (canceled)

11. A method for decontaminating or preventing contamination of surfaces of a device for receiving food, said method comprising a step of bringing a food composition into contact with the surfaces of said device, said food composition comprising at least one strain of bacteria of the genus Bacillus, said strain being dispersed in said food composition.

12. The method according to claim 11, said food composition further comprising one strain of lactic acid bacteria.

13. The method according to claim 11, where said strain of bacteria of the genus Bacillus is present at a rate of at least 10.sup.4 cfu per gram of the food composition.

14. The method according to claim 11, wherein said strain of bacteria of the genus Bacillus is one of the following strains: strain NOL01, deposited at the CNCM on Mar. 14, 2012 under number CNCM 1-4606, strain NOL02, filed at the CNCM on Jan. 21, 2016 under number CNCM 1-5043, and strain NOL03, deposited at the CNCM on Mar. 14, 2012 under number CNCM 1-4607, or any of the strains of bacteria of the genus Bacillus belonging to the same operational taxonomic unit, or OTU, as said NOL01 strain, or said NOL02 strain or said NOL03 strain, or a mixture of two or more of these strains.

15. The method according to claim 12, wherein said strain of lactic acid bacteria is one of the following strains: the NOL11 strain, deposited at the CNCM on Mar. 14, 2012 under number CNCM 1-4609, one of the strains of lactic acid bacteria belonging to the same operational taxonomic unit as said NOL11 strain, and a mixture of two or more of these strains.

16. The method according to claim 11, wherein said composition comprises at least one of the following combinations of bacteria: NOL01 and NOL11, NOL02 and NOL11, NOL03 and NOL11, NOL01, NOL02 and NOL11, NOL01, NOL03 and NOL11, NOL02, NOL03 and NOL11, and NOL01, NOL02, NOL03 and NOL11.

17. The method according to claim 11, wherein said surfaces are contaminated or contaminable by enterobacteriaceae.

18. A food composition, in which is dispersed at least one strain of bacteria of the genus Bacillus, notably Bacillus subtilis, and optionally comprising at least one strain of lactic acid bacteria.

19. The method according to claim 11, wherein the bacteria of genus Bacillus, is of the species Bacillus subtilis.

20. The method according to claim 17, wherein the Enterobacteriaceae are bacteria of the genus Salmonella, or Escherichia.

21. The method according to claim 17, wherein the Enterobacteriaceae are Salmonella enterica or Escherichia coli.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0122] FIG. 1 schematically shows a device after application of food compositions on contaminated boxes, with X the sampling zone and Y the sedimentation zone of the composition.

[0123] FIG. 2 is a graph showing the amount of Salmonella Typhimurium in Log.sub.10 CFU/g as a function of time in hours for a pig feed-type composition, comprising (B) or not comprising (A) the Bacillus and lactic acid bacteria (Test 1).

[0124] FIG. 3 is a graph showing the amount of E. coli in Log.sub.10 CFU/g as a function of time in hours for a pig feed-type composition, comprising (B) or not comprising (A) the Bacillus and lactic acid bacteria (Test 3).

[0125] FIG. 4 is a graph showing the amount of Salmonella Enteritidis in Log.sub.10 CFU/g as a function of time in hours for a ground corn-type composition, comprising (B) or not comprising (A) the Bacillus and lactic acid bacteria (Test 5).

[0126] FIG. 5 is a graph showing the amount of Salmonella Typhimurium in Log.sub.10 CFU/g as a function of time in hours for a soy meal-type composition, comprising (B) or not comprising (A) the Bacillus and lactic acid bacteria (Test 8).

[0127] FIG. 6 is a graph showing the evolution after 24 hours of the quantity of Salmonella Typhimurium in Log.sub.10 CFU/g as a function of time after the application of a food composition, comprising (1.) or not comprising (2.) the bacteria according to the invention (NOF01-03 and NOF11), over 6 series of tests: A and B crushed corn matrix, C and D sow feed matrix, or E and F without feed matrix.

EXAMPLES

Example 1: In Vitro Tests

[0128] The objective of these experiments was to test the ability of the food composition according to the invention to prevent, reduce or control the expansion of enterobacteriaceae in vitro.

A. Preparation of the Inocula

[0129] Before use, the peptone water necessary for the preparation and counting of the cultures as well as the tap water used were sterilized at 121° C. for 20 minutes to avoid the presence of unwanted microorganisms.

[0130] Frozen isolates of pathogens, S. enterica or E. coli, were thawed and then dissolved in a culture medium for brain-heart infusion with yeast extracts (3.7% medium for brain-heart infusion (Merck, Darmstadt, Germany); 0.1% yeast extracts (Merck)) at 37° C. for 24 hours under aerobic conditions and without stirring. The inocula were then diluted in peptone water (0.85% sodium chloride (VWR, Langenfeld, Germany); 0.1% peptone (G-Science, Saint Louis, USA)) to reach an initial concentration of 1×10.sup.6 CFU/mL.

[0131] The initial concentrations in S. enterica or E. coli were measured by counting according to the Pasteurian method on agar for brain-heart infusion with yeast extracts, after incubation for 24 hours at 37° C.

A. Preparation of the Food Matrices

[0132] To prepare the samples comprising the NOL01, NOL02, NOL03 and NOL11 strains, the bacteria were mixed for 5 minutes (TopMix94323, Heidolph) in the food preparation so as to reach a minimum concentration of 1×10.sup.6 CFU/g in Bacillus subtilis (NOL01-NOL03) and minimum 1×10.sup.6 CFU/g in Lactococcus lactis (NOL11).

[0133] The different matrices were then incorporated or not in water corresponding to the recommendations of the feed manufacturers as to their use:

TABLE-US-00002 TABLE 2 Mass factor of water incorporation to obtain the Food preparation preparation, matrix base = 1 Pig feed 2.75 Crushed corn 0.00 Soy meal 0.00

[0134] The pig feed used in this example is a feed whose composition is given in the following table:

TABLE-US-00003 TABLE 3 Incorporation rate (by mass relative to Components the total mass of of the feed) Wheat 50.77% Peas 14.38% Wheat bran 12.92% Corn 9.85% Sunflower meal 5.13% Salt 2.00% Wheat gluten 2.00% Calcium carbonate 1.63% Water 0.50% Lysine Sulfate 70% 0.43% Vitamin and mineral premix 0.20% Sodium sulphate 0.10% L-threonine 0.07% DL-methionine 0.02%

[0135] For each sample, 2.5 g of food was weighed (Precision Series, Fisher

[0136] Scientific), in a sterile 120 mL jar (Sarstedt, Nümbreccht, Germany) from the same prepared pool. A jar corresponding to a time step for measuring the pathogens in a sample. The samples were then incorporated into sterilized water to model degraded process, storage and use conditions, at the rate of 2 mL of sterilized water per jar.

[0137] 250 μL of inoculum of S. enterica or E. coli was added to the treated samples and to the control samples using a pipette (Finnpipette F2, Thermoscientific). Each sample was vortexed for 1 minute to obtain a homogeneous mixture (TopMix94323, Heidolph). The samples were then incubated under aerobic conditions in an incubator (BE500, Memmert) for a period of up to 7 days at 25° C. to model the ambient temperature in animal husbandry, in a production plant, or in a storage and transport area.

[0138] For technical reasons related to the performance of part of the counts in the outside analysis laboratory, the quantities of each matrix were multiplied by 10 during the performance of some experiments. Therefore, for each sample, jars of 120 mL containing 25 g of food are used, to which we add 20 mL of sterilized water and 2.5 mL of inoculum of S. enterica or E. coli.

A. Pathogen Tracking

[0139] The counting of pathogenic bacteria was carried out at time 0 (T0), then at different time steps depending on the experiments carried out. At each time, the samples were analyzed to count S. enterica or E. coli according to at least one of the following reference methods: [0140] Standard ISO 21528-2:2017 adapted—Microbiology of the food chain—Horizontal method for the detection and enumeration of Enterobacteriaceae—Part 2: Colony-count technique [0141] Standard ISO 6579-1:2017 Microbiology of the food chain—Horizontal method for the detection, enumeration and serotyping of Salmonella—Part 1: Detection of Salmonella spp. [0142] Standard ISO 16649-2:2001 Microbiology of human and animal feeding stuffs—Horizontal method for enumeration of beta-glucuronidase-positive Escherichia coli—Part 2: Colony-count technique at 44° C. using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide.

A. Statistical Analysis of Results

[0143] In view of the small number of samples during each test, the effect of the treatment with the food according to the invention on the quantity of pathogen at each time step and for each test is measured by the Wilcoxon test with a significance threshold set at 5% (p value less than or equal to 0.05), and a trend threshold set at 10% (p-value less than or equal to 0.1).

A. Summary of the Tests Carried Out

[0144]

TABLE-US-00004 TABLE 4 Samples Count per Test Matrix Pathogen standard treatment 1 Pig feed Salmonella ISO 5 Typhimurium 6579-1: 2017 2 Pig feed Salmonella ISO 3 Typhimurium 21528-2: 2017 3 Pig feed Escherichia ISO 5 coli 16649-2: 2001 4 Pig feed Escherichia ISO 3 coli 21528-2: 2017 5 Crushed corn Salmonella ISO 3 Enteritidis 21528-2: 2017 6 Soy meal Salmonella ISO 6 Typhimurium 21528-2: 2017

A. Summary of Test Results

[0145]

TABLE-US-00005 TABLE 5 Effect produced at the Mean Final final time difference time (test Control − Test Test Matrix Pathogen (hours) p-value) (Log.sub.10 CFU/g) 1 Pig feed Salmonella 120 0.010 3.9 Typhimurium 2 Pig feed Salmonella 120 0.100 2.1 Typhimurium 3 Pig feed Escherichia 48 0.060 3.1 coli 4 Pig feed Escherichia 120 0.100 1.3 coli 5 Crushed corn Salmonella 48 0.100 3.6 Enteritidis 6 Soy meal Salmonella 120 0.026 1.9 Typhimurium

[0146] The results are presented in FIGS. 2 to 5.

[0147] These different results in vitro highlight a difference in the development of pathogens greater than 1 Log.sub.10 CFU/g owing to the use of Bacillus and lactic acid bacteria. This effect is also present in matrices favorable to the development of enterobacteriaceae, which in practice are often the source of new contaminations in cascade.

[0148] The industrial use of Bacillus and lactic acid bacteria in food compositions is therefore promising. Indeed, the contact of these Bacillus and lactic acid bacteria with surfaces of devices for preparing, storing and conveying food via food compositions as defined in the invention will allow these Bacillus and lactic acid bacteria to spread on said surfaces in a preferred manner, thus limiting the proliferation of enterobacteria and therefore reducing the risk of subsequent recontamination.

Example 2: Surface Test

[0149] Objective: demonstrate an effect of a food composition containing at least one Bacillus on controlling surface contamination. For practical reasons, the performance of this protocol can be adapted.

A. Preparation of the Inocula

[0150] Before use, the peptone water necessary for the preparation and counting of the cultures as well as the tap water used are sterilized at 121° C. for 20 minutes to avoid the presence of unwanted microorganisms.

[0151] Frozen isolates of pathogens, S. enterica or E. coli, are thawed and then dissolved in a culture medium for brain-heart infusion with yeast extracts (3.7% medium for brain-heart infusion (Merck, Darmstadt, Germany); 0.1% yeast extracts (Merck)) at 37° C. for 24 hours under aerobic conditions and without stirring. The inocula are then diluted in peptone water (0.85% sodium chloride (VWR, Langenfeld, Germany); 0.1% peptone (G-Science, Saint Louis, USA)) to reach an initial concentration of 1×10.sup.2 CFU/mL.

[0152] The initial concentrations in S. enterica or E. coli are checked by counting according to the Pasteurian method on agar for brain-heart infusion with yeast extracts, after incubation for 24 hours at 37° C.

A. Preparation of the Food Matrices

[0153] To prepare the test samples, that is to say, the food compositions according to the invention, the mix of NOL bacteria (NOL01, NOL02, NOL03 and NOL11) is mixed manually for 5 minutes in the food composition to be tested so as to achieve a minimum concentration of 1×10.sup.6 CFU/g in Bacillus subtilis (NOL01-NOL03) and minimum 1×10.sup.6 CFU/g in Lactococcus lactis (NOL11). For each sample, 15 g of raw food matrix is weighed (Precision Series, Fisher Scientific), in a sterile 40 mL jar (Sarstedt, Nümbreccht, Germany).

[0154] To prepare the control samples, 15 g of raw food composition to be tested is weighed (Precision Series, Fisher Scientific), in a sterile 40 mL jar (Sarstedt, Nümbreccht, Germany).

A. Surface Preparation

[0155] Flat, sterile petri dishes made of smooth polystyrene, 14 cm in diameter (Sarstedt, Nümbreccht, Germany), considered representative of the surfaces encountered in cattle feed factories, are used to prepare the test surfaces.

[0156] The dishes are manually swabbed (Sodibox, Névez, France) with 1 mL of solution containing S. enterica or E. coli at 2×10.sup.4 CFU/mL, to reach a target concentration of approximately 1.3×10.sup.6 CFU/m.sup.2 of surface, thus representing a surface tested for contamination by pathogens. Between each passage of the swab, the dish is left to dry under the Microbiological Safety Station (MSS), then swabbed again by turning it a quarter turn clockwise.

[0157] The operation is repeated until the 1 mL of solution is completely spread over the dish.

[0158] In the context of tests E and F without food composition, the bacterial composition according to the invention was dissolved in 1 mL and applied according to the same experimental protocol, namely swabbing until the 1 mL of solution corresponding to the dose was exhausted of the composition of bacteria according to the invention present in the 15 g of food matrix (tests A, B, C, D)

A. Application of Food Matrices and Measurements

[0159] Each contaminated surface is subject to an application of 15 g of food matrix (control or test) distributed over the dish by horizontal stirring in order to obtain a homogeneous layer over the entire surface. The dishes will then be sprayed manually (Style 1.5, Matabi) with 8 mL of physiological water, thus modeling the incorporation of water during the manufacturing method. Then, the dishes are placed in a drying oven at 25° C. for a period of 4 hours in order to model the action of the food composition containing at least one Bacillus at a retention area recognized by the person skilled in the art as being conducive to pathogen development.

[0160] To represent the effect of a food composition containing at least one Bacillus on the control of surface contamination using a sandblaster in an industrial environment, the petri dish closed by a lid and a paraffin seal (Parafilm, Sigma-Aldricht) is then tilted vertically until the complete elimination of the food composition at the sampling zone on the surface, i.e. the upper 75% of the total surface of the petri dish in the vertical position), i.e. up to 4.2 cm in height. FIG. 1 shows the device used.

[0161] For each control group and treatment with the composition according to the invention, produced in pairs, 8 dishes are produced in order to be able to measure the evolution of the pathogens immediately after application (4 dishes) and 24 hours after application (4 dishes).

[0162] A surface swab (Sodibox, Névez, France) is taken at the sampling surface, i.e.

[0163] the upper 75% of the total surface of the petri dish in the vertical position, for each dish according to standard NF EN ISO 18593, before being stored at 4° C. for analysis.

A. Pathogen Tracking

[0164] At each time step, the samples are analyzed in order to count S. enterica or E. coli according to at least one of the following reference methods: [0165] Standard ISO 21528-2:2017 adapted—Microbiology of the food chain—Horizontal method for the detection and enumeration of Enterobacteriaceae—Part 2: Colony-count technique, [0166] Standard ISO 6579-1:2017 Microbiology of the food chain—Horizontal method for the detection, enumeration and serotyping of Salmonella—Part 1: Detection of Salmonella spp., and [0167] Standard ISO 16649-2:2001 Microbiology of human and animal feeding stuffs—Horizontal method for enumeration of beta-glucuronidase-positive Escherichia coli—Part 2: Colony-count technique at 44° C. using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide.

A. Summary of Results

[0168] All of the test results are presented in the table below and [FIG. 6].

TABLE-US-00006 TABLE 6 Mean change difference Final time Control − Test Matrix Pathogen (hours) (Log 10 CFU/g) Crushed corn Salmonella 24 1.2 Typhimurium Sow feed Salmonella 24 3.0 (see Example 1 for Typhimurium composition) No food matrix Salmonella 24 0 Typhimurium

[0169] In view of the small number of samples during each test, the effect of the treatment with the food according to the invention on the difference in evolution of the quantity of pathogen after 24 h is measured by the Wilcoxon test with a significance threshold set at 5% (p value less than or equal to 0.05), and a trend threshold set at 10% (p-value less than or equal to 0.1). The treatment effect is significant for the 4 tests comprising a food matrix (p-value=0.02857)

[0170] From these experiments, the following conclusions can be reached: [0171] regardless of the food matrix used, whether simple (crushed corn) or complex (pig/sow feed), it is found that the composition according to the invention has a significant effect on reducing pathogenic bacteria present on a surface. It even seems notable that the more complex the feed, the greater the effectiveness; [0172] simply spraying the bacteria according to the invention, without food matrix, has no effect. This is due in particular to the fact that without a nutrient supply, the bacteria are not able to multiply in order to exert their inhibiting effect on the growth of pathogenic bacteria.