“Escherichia coli‐milk” biofilm removal from stainless steel surfaces: Synergism between ultrasonic waves and enzymes

Three different methods to standardize biofilm removal for in situ sanitary control of closed surfaces in the food industry have been developed and compared, i.e. sonication, enzymatic treatment and a combined treatment which involved the application of ultrasound to enzyme preparations. The biofilm studied was an Escherichia coli model biofilm, made with milk on stainless steel sheets. Plate counting and epifluorescence microscopy were used to assess the efficiency of each treatment. The results are expressed in percentages, 100% denoting total removal, obtained with a flat ultrasonic transducer (T1) developed and presented in a previous study. The application of ultrasound by a patented curved transducer, T2 (10 s, 40 kHz), specifically devised for closed surfaces, was not sufficient to completely remove the biofilm (30 ± 7%). This biofilm was dislodged by two proteolytic enzyme preparations tested by immersion, viz. a 15‐min application of protease (84±1%) and a 30‐min trypsin application (95±8%). Using a combined treatment, the results showed a synergism between ultrasonic waves and proteolytic or glycolytic enzyme preparations, with removal of a significant amount of biofilm, i.e. 61–96% depending on the conditions tested, i.e. two to three times greater compared to sonication alone (30%). This application was in agreement with an industrial control, i.e. a good reproducible recovery of the biofilm in 10 s compared with 30 or 15 min with the enzyme alone.     

In situ rheology of yeast biofilms

The aim of the present work was to investigate the in situ rheological behavior of yeast biofilms growing on stainless steel under static and turbulent flow. The species used (Rhodototula mucilaginosa, Candida krusei, Candida kefyr and Candida tropicalis) were isolated from a clarified apple juice industry. The flow conditions impacted biofilm composition over time, with a predominance of C. krusei under static and turbulent flow. Likewise, structural variations occurred, with a tighter appearance under dynamic flow. Under turbulent flow there was an increase of 112 μm in biofilm thickness at 11 weeks (p < 0.001) and cell morphology was governed by hyphal structures and rounded cells. Using the in situ growth method introduced here, yeast biofilms were determined to be viscoelastic materials with a predominantly solid-like behavior, and neither this nor the G’₀ values were significantly affected by the flow conditions or the growth time, and at large deformations their weak structure collapsed beyond a critical strain of about 1.5–5%. The present work could represent a starting point for developing in situ measurements of yeast rheology and contribute to a thin body of knowledge about fungal biofilm formation.