Determining conditions for inactivation of multispecies biofilm cells by peracetic acid applying response surface methodology

Abstract

Biofilms can form on various surfaces and are typically associated with multiple microbial species. Particularly in the food industry, biofilms are often a source of contamination, necessitating mitigation strategies. In this study, Response Surface Methodology (RSM) was utilized to optimize conditions for the inactivation of multispecies biofilm cells, with peracetic acid (PAA) (0.05%–0.5%), treatment time (5–30 min), and temperature (25–60 °C) as independent variables. The multispecies biofilm consisted of Gram-positive and Gram-negative bacteria previously isolated from biofilms formed in the presence of raw milk. Stainless steel coupons were immersed in reconstituted whole milk inoculated with Pseudomonas fluorescens, Rahnella inusitata, Staphylococcus aureus, and Micrococcus aloeverae. Sessile cell counts of approximately 10⁸ CFU/cm² were obtained after 10 days of incubation at 4 °C. The response surface model exhibited a good fit, with R² of 0.949 and adjR² of 0.883. All independent variables had a positive effect on the inactivation of biofilm cells. The maximum inactivation achieved was approximately 7 log (CFU/cm²), with the highest values observed at 0.5% PAA, for 30 min at 60 °C. Epifluorescence microscopy revealed that many cells in the biofilm were dead or injured after treatment at the central point (PAA = 0.275%; Time = 17.5 min; Temperature = 42.5 °C). RSM helps to predict better conditions for maximum biofilm eradication and proves to be a promising approach for monitoring the inactivation of multispecies biofilm cells, which warrants further exploration.