Combined enzymes and Aleppo pine essential oil to control Cronobacter sakazakii biofilms on stainless steel and plastic surfaces

This study aimed to investigate the antibiofilm activity of Aleppo pine essential oil (APEO); hydrolytic enzyme mixtures or their combination in two sequential washing steps against C. sakazakii on plastic and stainless steel surfaces. The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of APEO against C. sakazakii strains were 500–1,000 µg/ml, and 1,000–4,000 µg/ml, respectively. Further, APEO showed antibiofilm activity where 4 × MIC APEO at 25 °C for 30 min reduced C. sakazakii cells by 1.8 and 1.6 log CFU/coupon on plastic and stainless steel, respectively. Similarly, both enzyme mixtures reduced the C. sakazakii cells attached to both surfaces by 1.7–2.2 log CFU/coupon. However, the two-step sequential cleaning regime with enzyme mixture of 10% protease, 5% α-amylase, and 1% lipase at 50 °C for 30 min followed by 4 × MIC APEO for 30 min reduced C. sakazakii biofilm on both surfaces by 4.4–4.5 log CFU/coupon compared to the control.