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Assessment of Salmonella inactivation in lab scale

Contaminated feed represents one of the first introduction pathways for Salmonella and other Enterobacteriaceae into the food value chain. One potential measure to reduce this risk is targeted heat treatment of the feedstuff. But for the implementation of heat treatment as a validated “kill-step” systematic studies are still lacking.

Dr. Edyta MARGAS
Food and Feed Safety Leader
Bühler AG
In the previous article influence factors and challenges of Enterobacteriaceae reduction with focus on Salmonella by heat treatment as well as extracts of the existing literature were discussed. As the literature does not provide sufficient information to design a reliable “kill-step” to ensure Salmonella free feed, a laboratory study was carried out to define a target organism and to evaluate the inactivation kinetics under different process conditions. The basis for the study was the guidelines of the Grocery Manufacturers Association for “Validating the reduction of Salmonella and other pathogen in heat processed low-moisture foods”.

One of the main issues in previous studies is the variations of evaluated strains or that strains are not defined at all. To perform a validation, the most practicable way is to analyze the worst case scenario. This means that the most heat resistant strain which is relevant for the specific product is used for the validation of the process. Therefore, in a first step, Salmonella strains commonly associated with feed were pre-defined in a literature study. Li et al (2012), for example, found that the top 6 most commonly isolated serotypes from animal feeds, feed ingredients, pet foods, pet treats and supplements for pets in the USA were S. Senftenberg (8.9% of isolates), S. Montevideo (8.9%), S. Mbandaka (8.6%), S. Tennessee (6.2%) and S. Typhimurium (5.4%). A review of Salmonella prevalence in worldwide animal feed production carried out by Maciorowski et al. (2006) found S. Agona, S. Livingstone and S. Mbandaka to be the most prevalent serotypes associated with complete feeds.

The analysis of also further studies revealed that the most common Salmonella serotypes that are consistently found as contaminants of both animal feed ingredients and finished feeds are S. Senftenberg, S. Mbandaka, S. Montevideo and S. Agona. These serotypes, and additionally S. Tennessee, were therefore selected and further tested to assess the most heat resistant Salmonella serotype: By artificially contaminating broiler feed with the selected serotypes and exposing it to steam for a specific time under controlled conditions, a serotype of S. Agona which showed the lowest reduction rate – and therefore the highest heat resistance – was defined as target organism for the inactivation trials.

In the next step, this organism was used for the evaluation of the inactivation kinetics under different controlled process conditions in broiler feed. Broiler feed was artificially contaminated feed with the highly heat resistant Salmonella strain and treated under controlled conditions in a lab scale set-up at 65°C, 70°C, 75°C, 80°C and 85°C at 12% and 19% moisture for different times. These process conditions represent the “worst case scenario” (low moisture – low temperature) as well as optimal conditions for inactivation (high moisture – high temperature). The aim was to define the time that is needed to achieve a reduction of 90% (1 log) of the initial cell count, also named “D-value”, under defined process conditions. These times can be used as a basis for the definition of the required process parameters in the production process.

Adjusted to 12% moisture, the D-value (time for 90% reduction) was 178.2 seconds at 65°C and 3.1 seconds at 85°C, respectively. This demonstrates the significant influence of temperature on the inactivation: With each increase of 5°C at a constant moisture level, the time needed to achieve a 1 log reduction decreased between 2 to 3 times at 12% moisture. The importance of moisture becomes obvious when comparing the D-values at 19% moisture: at 65°C 81.1 seconds were needed to achieve a 1 log reduction, at 85°C just 0.7 seconds. The increase in moisture significantly reduces the time needed for inactivation. The time to achieve a 90% reduction (D-value) at 12% moisture was approximately 2-4 times longer than the corresponding D-values determined at 19% moisture across the whole temperature range assessed.

The outstanding characteristic of this study is that the “worst-case scenario” was assessed under controlled conditions. This means that the most heat resistant Salmonella serotype was tested under the most critical process conditions (low moisture – low temperature) with a systematic approach. The collected data can be used as a basis for up-scaling.

However, as laboratory data are a first indication to design the “kill-step” but are not sufficient to design the process, pilot and industrial scale studies are necessary. For these trials it is not possible to work with pathogens like it was done in the laboratory study. Therefore, a surrogate, a bacterium with the same properties as the target organism but without pathogenic potential, has to be defined. For the validation of processes in food production in low moisture conditions the most commonly used surrogate for Salmonella is a specific strain of Enterococcus faecium. This strain was compared to Salmonella Agona regarding heat resistant and assessed as conservative surrogate. This means that this surrogate is slightly more heat resistant than the target organism, represents therefore again the worst case scenario and is thus suitable for the validation in pilot and industrial scale.

The described study is one of the first that identified the most resistant Salmonella strain as member of the family of Enterobacteriaceae associated with dry materials and provided basis information about Salmonella inactivation in broiler feed under controlled conditions. The collected data and information about a suitable surrogate can be used as a basis for upscaling of kill-step validation tests into pilot and industrial scale. Pilot and industrial scale studies are still necessary as effects of the equipment and the environment have to be included into the evaluation. The up-scaling into pilot scale will be described in the following article of this series.

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