COMPOSITION OF MICROORGANISMS INDICATING DECONTAMINATION

Abstract

The invention relates to the validation of decontamination processes and in particular to a composition comprising new indicator organisms and a dye, said composition being used for validating the decontamination processes.

Claims

1. An indicator microorganism composition comprising at least one indicator microorganism and one, wherein said dye is selected from the group consisting of the dyes of the cyclohexadiene-2,5-ylidene family.

2. The composition according to claim 1, wherein the dye of the cyclohexadiene-2,5-ylidene family is selected from brilliant blue FCF, patent blue V and brilliant green BS.

3. The composition according to claim 1, wherein the dye is brilliant blue FCF.

4. The composition according to claim 1, wherein the indicator microorganism is selected from the non-pathogenic species Enterococcus faecium, Geobacillus stearothermophilus, Clostridium sporogenes, Staphylococcus carnosus, Enterobacter hormaechei, Erwinia persicina, Pantoea agglomerans, Pantoea calida, Pantoea dispersa and Pantoea gaviniae.

5. The composition according to claim 1, wherein the indicator microorganism is selected from the non-pathogenic species Enterococcus faecium, Enterobacter hormaechei, Erwinia persicina, Pantoea agglomerans, Pantoea calida, and Pantoea gaviniae.

6. The composition according to claim 1, wherein the indicator microorganism is selected from the strains Enterococcus faecium (ATCC 8459) and the strains deposited at the CNCM under the Budapest Treaty: Enterobacter hormaechei CNCM I-5058, Pantoea agglomerans CNCM I-5059, Pantoea calida CNCM I-5061, Erwinia persicina CNCM I-5062, Erwinia persicina CNCM I-5063, Pantoea agglomerans CNCM I-5054, Pantoea agglomerans CNCM I-5055, Pantoea calida CNCM I-5056.

7. The composition according to claim 1, wherein the indicator microorganism is selected from the strains Geobacillus stearothermophilus ATCC 12980, Enterococcus faecium NRRL B-2354, Pantoea agglomerans SPS 2F-1, Pantoea dispersa, Pediococcus spp. and Pediococcus acidilactici, Staphylococcus carnosus CS-299, Clostridium sporogenes PA3679, PA3676 and PA3678, Listeria innocua, and Escherichia coli K12.

8. The composition according to claim 1, wherein the weight ratio of indicator microorganism to dye ranges from 0.01 to 5.

9. The composition according to claim 1, wherein it comprises an inert support.

10. The composition according to claim 9, wherein the inert support is selected from maltodextrin, milk powder, talcum powder, silica, activated carbon and mixtures thereof in any proportion.

11. The composition according to claim 1, wherein the content of indicator microorganisms is at least 10.sup.9 CFU/g dry composition.

12. The composition according to claim 1, wherein the content of indicator microorganisms is at least 10.sup.10 CFU/g dry composition.

13. The composition according to claim 1, wherein it comprises from 1 to 5% by weight of dye from 85 to 98% by weight of inert support and from 1 to 10% by weight of indicator microorganisms in dry form.

14. The composition according to claim 1, wherein it is a powder which has a water activity equal to or less than 0.3.

15. A process for controlling a decontamination process, wherein said decontamination process is implemented in the presence of at least one indicator microorganism, said decontamination process comprises the step of determining the presence of the at least one indicator microorganism during or at the end of the decontamination process, and wherein the at least one indicator microorganism is comprised in a composition according to claim 1.

16. The process according to claim 15, wherein the decontamination process comprises one or more steps of pasteurization, drying, extrusion, roasting, cooking, sterilization, autoclaving and steam treatments.

17. The process according to claim 16, wherein the decontamination process comprises at least one step of pasteurization, drying, extrusion, roasting, cooking, sterilization, autoclaving, steam treatments, pulsed light, high-pressure treatments, or irradiation, sterilization by gases and by disinfectants.

18. The process according to claim 15, wherein the decontamination process is a process for decontaminating natural or manufactured products.

19. The process according to claim 15, wherein the decontamination process is a process for decontaminating nuts, herbs, seeds, spices, food powders, pet food and livestock feed, or cereals.

20. The process according to claim 15, to validate the decontamination of pathogens selected from Salmonella, Escherichia coli, Bacillus, Listeria, Campylobacter and Cronobacter sakazakii.

21.-24. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0064] FIG. 1 shows the log loss curves of Enterococcus faecium as a function of the dyes after heat treatment at 100° C.

[0065] FIG. 2 shows the log loss curves of Enterococcus faecium with and without the dye brilliant blue FCF after heat treatment at 100° C. on an inert support.

[0066] “Log loss” refers to the difference between initial viability and viability at a given time.

EXAMPLES

Example 1: Test of Dyes: Chlorophyllin and Brilliant Blue FCF

[0067] Method: The Enterococcus faecium strain in dry form was mixed with an inert support (maltodextrin) and with each dye individually. The subsequent mixture is placed in an Eppendorf tube in a dry bath at a temperature of 100° C. Samples were taken at 2, 5 and 10 min and cell counts were performed on TSA (Trypticase Soy Agar) medium for 24 h at 37° C.

[0068] The results are shown in FIG. 1.

Example 2: Comparison of E. faecium Compositions at 100° C.

[0069] The heat resistance of E. faecium with or without the dye brilliant blue FCF is compared in three forms of compositions: fermentation must (Must), without support (Pure) and on inert support (Support).

[0070] Must: addition of 3% of blue dye powder to 5 mL of fermentation must in a sterile jar. Vortexing for homogenization, distribution in tubes and heat treatment.

[0071] Pure: addition of 3% of dye to 5 g of pure lyophilisate in a sterile jar. Vortexing for homogenization, distribution in tubes and heat treatment.

[0072] Support: addition of 1% of pure lyophilisate to 5 g of maltodextrin in a sterile jar (1/100 dilution). Vortexing for homogenization, then addition of 3% of dye. Vortexing then distribution in tubes and heat treatment.

[0073] The log loss results in each case are given in Table 1.

TABLE-US-00001 TABLE 1 Log loss at 100° C. 2 5 10 min min min Must With Dye 1.23 1.44 2.16 Without Dye 1.57 2.27 4.03 Pure With Dye 0.02 0.17 0.50 Without Dye 0.01 0.03 0.93 Support With Dye 0.13 0.94 1.97 Without Dye 0.76 2.89 3.48

[0074] The log loss curves for the composition on maltodextrin support, with or without dye, are shown in FIG. 2.

Example 3: Test on Nonfat Dried Milk NFDM for 3 Strains at 90 and 100° C.

[0075] The test is performed for the following 3 strains: E. faecium ATCC 8459, P. agglomerans CNCM I-5055 and A. pascens CNCM I-5181.

[0076] Addition of 10% of pure lyophilisate of the strains to 5 g of maltodextrin. Vortexing for homogenization. Addition of 3% brilliant blue FCF dye. Vortexing. Addition of 1% of the dyed strain mixture diluted 1/10 to 5 g NFDM. Vortexing. Deposition of 0.1 g in Eppendorf tubes then heat treatment of the tubes in a dry bath at 90 and 100° C. for 2, 5 and 10 minutes

[0077] The results are given in Table 2.

TABLE-US-00002 TABLE 2 Log loss at 90° C. Log loss at 100° C. 2 5 10 2 5 10 Strain min min min min min min E. faecium With Dye 0.51 0.36 0.46 0.36 0.59 1.29 ATCC 8459 Without Dye 0.4 1.65 2.65 0.11 1.6 3.1 P. agglomerans With Dye 0.07 0.07 0.37 0.13 0.67 0.72 CNCM I-5055 Without Dye 0.68 1.28 1.18 0.54 1.32 1.53 A. pascens With Dye 0.06 0.46 0.62 0.26 0.35 1.82 CNCM I-5181 Without Dye 0.67 0.84 0.97 0.8 1.89 2.75

Example 4: Test on Black Pepper for 3 Strains at 90 and 100° C.

[0078] The test is performed for the following 3 strains: E. faecium ATCC 8459, P. agglomerans CNCM I-5055 and A. pascens CNCM I-5181

[0079] Addition of 10% of pure lyophilisate of the strains to 5 g of maltodextrin. Vortexing for homogenization. Addition of 3% of brilliant blue FCF dye. Vortexing. Addition of 1% of the dyed strain mixture diluted 1/10 to 5 g of black pepper followed by a spray of wet fixing agent for good adhesion. Mixing with a sterile spatula. Drying 2 h. Deposition of 0.2 g in Eppendorf tubes then heat treatment of the tubes in a dry bath at 90 and 100° C. for 2, 5 and 10 minutes

[0080] The results are given in Table 3.

TABLE-US-00003 TABLE 3 Log loss at 90° C. Log loss at 100° C. 2 5 10 2 5 10 Strain min min min min min min E. faecium With Dye 0.31 0.82 1.46 1.35 1.37 2.19 ATCC 8459 Without Dye 0.91 1.43 3.39 1.05 0.91 5.6 P. agglomerans With Dye 1.12 1.6 1.77 1.26 1.45 2.14 CNCM I-5055 Without Dye 1.15 1.75 2.88 1.84 3.41 3.95 A. pascens With Dye 0.59 0.76 0.85 0.27 0.64 0.85 CNCM I-5181 Without Dye 0.91 1.09 1.67 1.09 1.79 2.69

Example 5: Test on Macadamia for 3 Strains at 90 and 100° C.

[0081] The test is performed for the following 3 strains: E. faecium ATCC 8459, P. agglomerans CNCM I-5055 and A. pascens CNCM I-5181.

[0082] Addition of 10% pure lyophilisate of the strains to 5 g of maltodextrin. Vortexing for homogenization. Addition of 3% of dye. Vortexing. Addition of 1% of the dyed strain mixture diluted 1/10 to 5 g of macadamia. Mixing with a sterile spatula. Drying 2 h. Deposition of 0.3 g in Eppendorf tubes then heat treatment of the tubes in a dry bath at 90 and 100° C. for 0, 2, 5, 10 minutes

[0083] The results are given in Table 4.

TABLE-US-00004 TABLE 4 Log loss at 90° C. Log loss at 100° C. 2 5 10 2 5 10 Strain min min min min min min E. faecium With Dye 0.2 0.53 1.2 0.24 0.47 1.24 ATCC 8459 Without Dye 0.58 0.69 0.54 0.23 0.94 1.24 P. agglomerans With Dye 0.3 0.6 0.14 0.14 0.15 1.36 CNCM I-5055 Without Dye 0.31 2.01 2.46 0.74 1.1 2.31 A. pascens With Dye 0.25 0.18 0.38 0.05 0.12 0.6 CNCM I-5181 Without Dye 0.27 0.19 1.24 0.65 0.8 1.54

REFERENCES

[0084] Annous B A, Kozempel M F. 1998. Influence of growth medium on thermal resistance of Pediococcus sp. NRRL B-2354 (formerly Micrococcus freudenreichii) in liquid foods. J Food Prot. 61(5):578-81. [0085] Bianchini & al., Use of Enterococcus faecium as a Surrogate for Salmonella enterica during Extrusion of a Balanced Carbohydrate-Protein Meal. J. Food Prot., Vol. 77, No. 1 [0086] Borowski, A. G., S. C. Ingham, and B. H. Ingham, 2009. Validation of ground- and formed beef jerky processes using commercial lactic acid bacteria starter cultures as pathogen surrogates. Journal of Food Protection 72: 1234-1247 [0087] Cheriaa & al., Removal of Triphenylmethane Dyes by Bacterial Consortium, The Scientific World Journal, Vol. 2012, p. 9, 2012. [0088] Enache & al., Development of a Dry Inoculation Method for Thermal Challenge Studies in Low-Moisture Foods by Using Talc as a Carrier for Salmonella and a Surrogate (Enterococcus faecium). Journal of Food Protection, 2015. 78: 1106-1112 [0089] Erdogan & al., Evaluating Pediococcus acidilactici and Enterococcus faecium NRRL B-2354 as Thermal Surrogate Microorganisms for Salmonella for In-Plant Validation Studies of Low-Moisture Pet Food Products. Journal of Food Protection, Vol. 78, No. 5, 2015, Pages 934-939. [0090] Fudge & al., The Isolation and Identification of Pantoea dispersa strain JFS as a Non-Pathogenic Surrogate for Salmonella Typhimurium Phage Type 42 in Flour, International Journal of Food Microbiology 219 (2016) 1-6 [0091] Garcia-Hernandez R, McMullen L, Gänzle M G. 2015. Development and validation of a surrogate strain cocktail to evaluate bactericidal effects of pressure on verotoxigenic Escherichia coli. Int J Food Microbiol. 205:16-22. [0092] Guidelines for Using Enterococcus faecium NRRL B-2354 as a Surrogate Microorganism in Almond Process Validation. Almond Board of California Guideline, October 2007 (ABC, 2007). [0093] Gurtler & al., Selection of surrogate bacteria in place of E. coli O157:H7 and Salmonella Typhimurium for pulsed electric field treatment of orange juice. International Journal of Food Microbiology 139 (2010) 1-8 [0094] Kopit. B. Kim, R. J. Siezen, L. J. Harris, and M. Marco. & al., Safety of the Surrogate Microorganism Enterococcus faecium NRRL B-2354 for Use in Thermal Process Validation, Appl. Environ. Microbiol. 2014, 80(6):1899. DOI:10.1128/AEM.03859-13. [0095] Niebuhr & al., Evaluation of non-pathogenic surrogate bacteria as process validation indicators for Salmonella enteric for selected antimicrobial treatments, cold storage and fermentation in meat, J Food Prot. 2008 April; 71(4):714-8. [0096] Okelo, P. O., D. D. Wagner, L. E. Carr, F. W. Wheaton, L. W. Douglass, S. W. Joseph. 2006. Optimization of extrusion conditions for elimination of mesophilic bacteria during thermal processing of animal feed mash. Animal Feed Science and Technology 129:116-137. [0097] Okelo, P. O., S. W. Joseph, D. D. Wagner, F. W. Wheaton, L. W. Douglass, and L. E. Carr, 2008. Improvements in Reduction of Feed Contamination: An Alternative Monitor of Bacterial Killing During Feed Extrusion. Journal Applied Poultry Research 17: 219-228. [0098] Olukanni & al, Biodegradation of Malachite Green by Extracellular Lacase Producing Bacillus thurigensis RUN1, Journal of Basic & Applied Sciences, 2013, 9, 543-549. [0099] Rodriguez et al., Surrogates for validation of electron beam irradiation of foods, International Journal of FDood Microbiology, 110 (2006) 117-122 [0100] Sommers C H, Geveke D J and, Fan X. Inactivation of Listeria Innocua on Frankfurters That Contain Potassium Lactate and Sodium Diacetate by Flash Pasteurization. 2008. J Food Sci 73 (2), M72-M74. 3 2008 [0101] Vasan, A., R. Geier, S. C. Ingham, and B. H. Ingham. 2014. Thermal tolerance of 0157 and non-0157 Shiga toxigenic strains of Escherichia coli, Salmonella, and potential pathogen surrogates, in frankfurter batter and ground beef of varying fat levels. Journal of Food Protection. 77:1501-11. [0102] Wallace M, Larson K, Wolf I, Thompson D and Zottola E. Thermal inactivation of Clostridium sporogenes PA 3679 and Bacillus stearothermophilus 1518 in low-acid home-canned foods. 2006 Journal of Food Science 43(6):1738-1740. [0103] Williams, 2010 Williams, P., W. M. Leong, B. H. Ingham, S. C. Ingham, 2010. Lethality of Small-Scale Commercial Dehydrator and Smokehouse/Oven Drying Processes Against Escherichia coli O157:H7-, Salmonella spp.-, Listeria monocytogenes-, and Staphylococcus aureus-inoculated Turkey Jerky and the Ability of a Lactic Acid Bacterium to Serve as a Pathogen Surrogate. Poster presented at the annual meeting of the Institute of Food Technologists. Chicago, Ill. July 2010. [0104] WO 2017/186907, WO 2008/026104, WO 2009/027855