Combined enzymes and Aleppo pine essential oil to control <i>Cronobacter sakazakii</i> 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 <i>C. sakazakii</i> on plastic and stainless steel surfaces. The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of APEO against <i>C. sakazakii</i> 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 <i>C. sakazakii</i> cells by 1.8 and 1.6 log CFU/coupon on plastic and stainless steel, respectively. Similarly, both enzyme mixtures reduced the <i>C. sakazakii</i> 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 <i>C. sakazakii</i> biofilm on both surfaces by 4.4-4.5 log CFU/coupon compared to the control.