Attachment and removal of biofilm of Salmonella enterica subsp. enterica serovar Typhimurium 53647 embedded in liquid whole egg on stainless steel, silicone, and nylon

Authors: Bermudez-Aguirre, Luz; Uknalis, Joseph; Niemira, Brendan

Submitted to: Food Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2024
Publication Date: 12/12/2024
Citation: Bermudez-Aguirre, L.D., Uknalis, J., Niemira, B.A. 2024. Attachment and removal of biofilm of Salmonella enterica subsp. enterica serovar Typhimurium 53647 embedded in liquid whole egg on stainless steel, silicone, and nylon. Food Control. 171:111104. https://doi.org/10.1016/j.foodcont.2024.111104.
DOI: https://doi.org/10.1016/j.foodcont.2024.111104

Interpretive Summary: Food residues and microorganisms in the processing line and equipment represent a food safety risk. Food is an excellent medium to promote microbial growth, and the presence of pathogens can promote the development of biofilms. These complex structures are difficult to remove from the food processing environment and offer an excellent niche for microorganisms to grow and hide from sanitation processes. This research studied the growth and attachment of a pathogenic microorganism, Salmonella Typhimurium, in whole liquid egg. Three contact surfaces were evaluated, representing some of the most common in the egg industry: stainless steel, silicone, and nylon. The conventional sanitation methods using bleach and peracetic acid (200 ppm) were used for comparison. A two-step method using enzymes and organic acid at lower concentrations was evaluated. The biofilms were allowed to grow and attached at 37°C, and the number of microorganisms was assessed after 1, 24, and 48 h. Cells were firmly attached to the three materials after 24 h, reaching a maximum growth after 48 h, mainly in nylon. The standard use of bleach allowed for the faster reduction of the biofilm in stainless steel. After 5 minutes, all the cells in the three materials were inactivated using bleach. Peracetic acid (200 ppm) also inactivated the cells after 10 min of contact. However, using the pre-treatment with an enzyme (ficin), the biofilm network opened and allowed better contact of the microbial cells with the sanitizer. Using ficin reduced the required contact time (1 min) and the concentration (120 ppm) of peracetic acid. Combining enzymes and organic acids represents a green solution for removing biofilms and inactivating pathogens in the food industry without using chemicals.

Technical Abstract: Biofilms are an ongoing safety risk in the egg industry. Egg white and yolk are rich in protein, fat and nutrients that allow rapid microbial growth. Different points of the processing lines of the egg industry present materials and surfaces that can harbor the growth of pathogens in embedded egg residues and/or in bacterial biofilms. The aim of this work was to study the biofilm formation, attachment, and removal of Salmonella Typhimurium inoculated in liquid whole egg (LWE, 10^3 cfu/ml) on three surfaces: stainless steel (SS), silicone (S), and nylon (N). Cells were allowed to grow and attach at 37°C for 1, 24, and 48 h (static conditions). Biofilm removal and sanitization were conducted with conventional sodium hypochlorite treatment (200 ppm) and peracetic acid (PAA, 200 ppm). The use of enzymatic treatment ficin (6.25 U/ml) was studied before the application of PAA at lower concentrations (90 and 120 ppm). Contact time for all PAA treatments was 1-, 5- and 10-min. Scanning Electron Microscope (SEM) was used to assess the biofilm attachment in the different materials. Results showed a quick growth and biofilm formation of Salmonella in the three working materials after 24 h, reaching more than 7 log. Nylon presented the highest cell attachment after 48 h. SEM images confirmed these findings showing a thick biofilm with many cells embedded in the egg matrix. Chorine was able to remove and sanitize better stainless steel after 1 min (p < 0.05), but all surfaces were Salmonella-free after 5 min; similar results were observed for PAA after 10 min. The enzymatic pre-treatment with ficin was able to open the egg biofilm matrix and reduce the contact time with the sanitizer showing the best results for PAA at 120 ppm and 1 min of treatment with no viable cells detected in any of the three materials. The use of a two-step sanitization process, using an enzymatic treatment followed by contact with organic acids, such as PAA, represents a viable option for the removal of LWE biofilms and inactivation of Salmonella cells in stainless steel, silicone, and nylon. This can be an option for the egg industry to reduce the use of chlorine in the processing lines and minimize food safety risks. Further research is required to optimize the best sanitation conditions and explore the use of additional enzymes.