Decontamination surrogate microorganisms

Abstract

The invention relates to the validation of decontamination processes and in particular to new surrogate organisms and mixtures of said microorganisms used for validating the decontamination processes.

Claims

1. A process for controlling contamination of a medium the process comprising: a) adding at least one indicator microorganism to the medium; b) performing a decontamination process to the medium of step a); and c) determining the presence of the indicator microorganism at the end of the decontamination process; wherein the absence of the indicator microorganism at the end of the decontamination process confirms that contamination is controlled; wherein the at least one indicator microorganism is selected from Enterobacter hormaechei CNCM I-5058, Enterobacter mori CNCM I-5060, Pantoea calida CNCM I-5061, Envinia persicina CNCM I-5062, Envinia persicina CNCM I-5063, Pantoea calida CNCM I-5056, and mixtures thereof .

2. The process according to claim 1, wherein the indicator microorganisms are used in dry vegetative form.

3. The process according to claim 1, wherein the indicator microorganisms are used dry on an inert carrier.

4. The process according to claim 1, wherein the at least one indicator microorganism is Pantoea calida CNCM I-5056.

5. The process according to claim 1, wherein a mixture of at least 2 indicator microorganisms is used.

6. The process according to claim 1, wherein the decontamination process comprises one or more steps of pasteurization, drying, extrusion, roasting, cooking, sterilization, autoclaving and steam treatments.

7. The process according to claim 1, wherein the decontamination process is aimed at removing one or more target pathogenic microorganisms selected from Salmonella, Escherichia coli, Bacillus, Listeria, Campylobacter, Cronobacter sakazakii.

8. The process according to claim 1, wherein the indicator microorganism is Enterobacter hormaechei CNCM I-5058.

9. The process according to claim 1, wherein the indicator microorganism is Enterobacter mori CNCM I-5060.

10. The process according to claim 1, wherein the indicator microorganism is Pantoea calida CNCM I-5061.

11. The process according to claim 1, wherein the indicator microorganism is Erwinia persicina CNCM I-5062.

12. The process according to claim 1, wherein the indicator microorganism useful as surrogate is Erwinia persicina CNCM I-5063.

13. The process according to claim 1, wherein the indicator microorganism is Pantoea calida CNCM I-5056.

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

Description

DESCRIPTION OF THE FIGURES

(1) FIGS. 1 to 4 show the resistance curves of different surrogate microorganisms in comparison with the pathogens Salmonella and Cronobacter sakazakii.

(2) FIGS. 5 to 7 show the cell destruction dynamics of indicator microorganisms according to the invention, of the reference strain E. faecium ATCC 8459 and of four Salmonella serotypes on different products.

EXAMPLES

Example I. Isolation and Selection of Microorganisms

(3) 1. Isolation of Environmental Enterobacteriaceae

(4) A 0.5 g sample of dry products was placed in a 1.5 mL Eppendorf tube and heat treated in a dry bath for 15 min at 95° C. After cooling to room temperature, 1 mL of concentrated PBS (aw=0.950) was added before vortexing for 30 s. Successive 1/10 dilutions were prepared in PBS (aw=0.995) before spreading on Violet Red Bile Glucose Agar (VRBG) in an amount of 100 μL per plate. After incubation at 37° C. for 24-48 h, colonies were isolated on Tryptic Soya Agar (TSA) and incubated again at 37° C. for 24 h.

(5) 2. Identification of Environmental Enterobacteriaceae

(6) Amplifications of 16S rDNA of each isolate were performed directly by sub-culturing the colonies in the PCR mix. The primers used were: 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGH TAC CTT GTT ACG ACT T-3′). To perform the PCR, the “Taq Core Kit” (Quiagen, France) was used. Briefly, the PCR mix (50 μL per reaction) for a colony is composed of 0.5 μM of each primer, 0.2 mM dNTP mix, 0.75 U Taq polymerase and 1× buffer containing MgCl.sub.2. The amplification was verified by 1% agarose gel electrophoresis before sequencing the PCR products by the Sanger method. The sequences obtained were used to search the NCBI database (BLASTn) and in this way the isolates were identified. Twelve isolates were then selected.

(7) 3. Thermal Challenge

(8) a. Strain Culture Conditions

(9) All cultures were stored in Tryptic Soy Broth (TSB, Sigma-Aldrich) with 20% glycerol (Sigma-Aldrich) at −80° C. To restart the cultures, the bacteria were inoculated on TSA for 24 h at 37° C. and then five colonies of each bacterium were sub-cultured in 50 mL of TSB before being incubated at 37° C. for 8 h. These bacterial suspensions are then diluted in 50 mL of fresh TSB to achieve an optical density (OD) of 0.01 at 600 nm. Cultures in the stationary growth phase are thus obtained after 20 h at 37° C.

(10) b. Inoculation of Milk Powder

(11) For each bacterium, the 50 mL cultures are centrifuged (3400 g, 10 min at 25° C.) then washed twice in 25 mL of PBS. Finally, a final centrifugation is performed, the supernatant is removed, and the pellets are weighed. Milk powder (26% fat) is added to each pellet with a ratio of 1:20 (m.sub.pellet:m.sub.powder) and the whole is homogenized using a mortar. Inoculated milk powder is thus obtained.

(12) c. Drying Process

(13) For the inoculated milk powder, airtight containers, containing saturated salt solutions for controlling water activity and thus the relative humidity of the atmosphere, are used. Lithium chloride, potassium acetate, potassium carbonate and sodium bromide were used to obtain water activity of 0.11, 0.25, 0.44 and 0.58. The atmospheres thus obtained are maintained under convection using a fan. For each strain, the inoculated powder is spread on Petri dishes (about 5 g per dish). These dishes, without lids, are then placed in the airtight containers for 16 h to reach equilibrium water activity. All drying was carried out at room temperature.

(14) d. Heat Treatment

(15) 0.1 g of dried inoculated milk powder is placed in a 0.2 mL tube and treated at different temperatures (85° C., 90° C., 95° C. and 100° C.) for a given time (0 s, 30 s, 60 s, 90 s, 120 s, 150 s and 180 s) using a thermocycler before being cooled to 4° C. The samples are rehydrated by adding 1 mL of PBS before vortexing for 30 s. A CFU count was performed after incubation on TSA for 24 h at 37° C. The results are expressed as log.sub.10(N/N.sub.0), where N is the CFU count after treatment and N.sub.0 is the initial CFU count of the milk powder before treatment (t=0 s).

(16) TABLE-US-00001 Species Deposit No. D-value Enterococcus faecium ATCC 8459 23.69 Enterobacter hormaechei CNCM I-5058 4.36 Pantoea agglomerans CNCM I-5059 1.81 Enterobacter mori CNCM I-5060 3.95 Pantoea calida CNCM I-5061 2.03 Erwinia persicina CNCM I-5062 1.74 Erwinia persicina CNCM I-5063 1.96 Pantoea agglomerans CNCM I-5054 1.27 Pantoea agglomerans CNCM I-5055 1.33 Pantoea calida CNCM I-5056 1.14 Salmonella Typhimurium DSM 10506 1.21 Cronobacter sakazakii PAC 103183T 1.13

Example II. Validation of a Decontamination Process

(17) The technology most commonly used in the decontamination sector, in both food-processing and pharmaceutical industries, remains autoclaves. It is thus possible to heat the product in a chamber, while either static or in motion, simply by condensation of steam on said product. The product can then be dried by a combination of heating and vacuum treatment. There are hundreds of autoclave manufacturers worldwide, a certain number of which work on pasteurization of dry food products.

(18) The classic pasteurization cycle for these devices consists of the following steps: Phase 1: Air removal. Several cycles are performed to remove as much air as possible. This step is necessary to allow steam to penetrate through the product. Phase 2: Heating. Steam is injected in order to heat the product. The chamber enclosure is also heated by electrical resistance to prevent condensation. Phase 3: Pasteurization. Once the product has reached a target temperature, there is a holding time at that temperature. The treatment time-temperature pair is defined upstream of the validation work. The time-temperature pair is crucial for the efficacy of the treatment. Phase 4: Drying. The steam is removed by vacuum drying. Phase 5: Aeration. The chamber is ventilated by a stream of filtered air at atmospheric pressure

(19) The product is then discharged from the chamber in the direction of production in order to prevent contact with untreated material. Depending on the chosen cycle, its temperature at the end of the process varies from 30 to 50° C. The product is not packaged until it has returned to room temperature because bagging when too hot could cause germs to develop.

(20) In situ validation of a decontamination process generally comprises three main steps: Preparatory phase: process evaluation, risk assessment, model germ qualification, development of the in situ validation protocol Execution phase: inoculation of the product to be tested, execution of validation “batches”, sample recovery Synthesis phase: counting of the model germs, writing of the analysis report and/or validation report

(21) During the development of the validation protocol, the following are defined, among other things: Whether or not it is necessary to pre-treat the product to be tested (for example: irradiation) The number of validation batches and the duration of each validation batch to be prepared The amount of product to be inoculated (from 25 g to >10 t depending on the decontamination processes and the validation method selected) The desired level of inoculation and the amount of model germ to be used The method for inoculating the product with the model germ (there are various possibilities, including inoculation in the laboratory, directly in the factory, at a service provider, etc.) The sampling method at the end of the production line (including, among other things, sample number and size) The method for counting the model germ (among other things: selective or non-selective medium)

(22) The answers to these various questions depend essentially on three parameters: type of process to be validated, target pathogen, product to be inoculated.

(23) Thus, the validation “kit” provided may vary in particular in: Mixing level of the model germ with the product to be tested (the model germ may be supplied in concentrated form to be inoculated or in pre-mixed form with the product) Concentration level of model germ Amount supplied (from several kgs/tens of kgs for the concentrated form to several tons for the pre-mixed version)

Example III. Production of Indicator Microorganisms by Fermentation

(24) Pre-Culture

(25) Pre-culture of the surrogate microorganism should be started between 16 h and 24 h before fermentation. The Erlenmeyer flask containing the culture medium is inoculated with surrogate microorganism at a ratio of 1:5. The pre-culture is incubated at 37° C. with shaking at 150 rpm.

(26) Fermentation Process

(27) Culture begins when the entire pre-culture has been inoculated in the fermenter. The shaking, aeration and substrate addition conditions are as follows: pH maintained by a base throughout the culture Temperature maintained at 37° C. by the heating double-walled enclosure and/or jacket, and throughout the culture Oxygen saturation of the initial medium before inoculation (pO2>90%) Shaking (rpm): 200-500. Aeration (L/min): 1-3.

(28) At the end of the culture, the fermenter culture medium is transferred to sterile bottles in order to recover all biomass.

(29) Biomass Recovery and Preparation in Dry Form

(30) Biomass is recovered by centrifugation or by another technique such as ultrafiltration that allows the cells to be separated from the culture medium.

(31) Cryoprotectant, once sterilized, is added at a volume ratio of 1:1 to the biomass and the whole is frozen at −80° C. for at least 24 h with a view to potential lyophilization.

Example IV. Use of Indicator Microorganisms on Different Dry Products

(32) The objective of these examples is to show the thermal destruction kinetics of different indicator microorganisms and to compare them to that of Salmonella in different products with low water activity.

(33) Microorganisms Tested

(34) Four different Salmonella serotypes (Senftenberg, Enteridis, Typhimurium and Mbandaka), inoculated individually or in cocktail form, are used as control strains for comparison with the model microorganisms tested.

(35) The thermal resistance of two different indicator microorganisms is tested: Enterobacter hormaechei CNCM I-5058, dry preparation. Pantoea agglomerans CNCM I-5055, dry preparation.

(36) The strain Enterococcus faecium (ATCC® 8459™) was used as reference strain because it is widely used as a biological tracer in validation of dry food treatment processes.

(37) Inoculation Protocols

(38) Two different methods are used to inoculate the different products: Liquid inoculation of the pathogens: broth cultures of the four Salmonella serotypes prepared the day before are used independently to inoculate the products produced (paprika powder, milk powder and macadamia nuts). After inoculation the product is placed under a type II biological safety cabinet in order to equilibrate its water activity. Dry inoculation of the indicator microorganisms: the different products (paprika powder, milk powder and macadamia nuts) were inoculated independently with the surrogate microorganisms in dry form following production by fermentation and stabilization by lyophilization. No resting time is required after inoculation with the model microorganisms in dry form, which results in very little modification of the product's properties (aw, moisture percentage, etc.) and allows faster use of the inoculated matrices.

(39) Results

(40) The cell destruction dynamics at 90° C. and 100° C. of strain E. hormaechei CNCM I-5058, reference strain E. faecium ATCC 8459 and the 4 Salmonella serotypes on paprika powder are shown in FIGS. 5A (treatment at 90° C.) and 5B (treatment at 100° C.). The results show in all cases that the behaviour of the model microorganism E. hormaechei CNCM I-5058 is always closer to all Salmonella serotypes, thereby confirming its nature as a surrogate germ well suited to the target pathogen on the product in question.

(41) The cell destruction dynamics at 100° C. and 110° C. of strain P. agglomerans CNCM I-5055, reference strain E. faecium ATCC 8459 and the 4 Salmonella serotypes in cocktail form on macadamia nuts are shown in FIGS. 6A (treatment at 100° C.) and 6B (treatment at 110° C.). The results show that the behaviour of the model microorganism P. agglomerans CNCM I-5055 is closer to all Salmonella serotypes, thereby confirming its nature as a surrogate germ well suited to the target pathogen on the product in question.

(42) The cell destruction dynamics at 85° C. and 100° C. of strain P. agglomerans CNCM I-5055, reference strain E. faecium ATCC 8459 and the 4 Salmonella serotypes in cocktail form on skim milk powder are shown in FIGS. 7A (treatment at 85° C.) and 7B (treatment at 100° C.). The results show that the behaviour of the model microorganism P. agglomerans CNCM I-5055 is closer to all Salmonella serotypes, thereby confirming its nature as a surrogate germ well suited to the target pathogen on the product in question.

REFERENCES

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