Comparison of Antimicrobials and Delivery Methods on the Inactivation of Listeria monocytogenes on Apples

Comparison of Antimicrobials and Delivery Methods on the Inactivation of Listeria monocytogenes on Apples Rebecca B. Stearns The United States Department of Agriculture-National Institute of Food and Agriculture recognizes Listeria Monocytogenes as an emerging and under researched pathogen. Due to this recognition for more research and the numerous multistate listeriosis outbreaks originating from apples (2014-2015,2017, and 2019), experiments in this paper were focused on inactivating Listeria Monocytogenes from apples. Contamination of Listeria on apples can occur at the harvest and/or packing process of apple production. Some strategies that producers utilize to reduce this risk are by applying antimicrobials via garden spray, electrostatic spray, and dipping methods. There are also several antimicrobials claimed to kill microbes on produce surfaces that are on the market for farmers and apple producers. However, the wholesale distributor Appalachian Harvest requires their local West Virginia suppliers to only utilize a H2O2-PAA mixer known as SaniDate-5.0®. Therefore, as described in chapter two, experiments were conducted to analyze which delivery method and concentration of SaniDate-5.0® is the most effective at reducing Listeria monocytogenes on fresh apple surfaces. In order to compare these processes, three organic apple types (Honey Crisp, Pink Lady, and Fuji) were dip inoculated with Listeria monocytogenes (2strain, serotype 1/2b). Apples were either not treated (control) or treated with water only, or with the H2O2-PAA mixer (0.0064, 0.1, 0.25 and 0.50%) for 20 s via garden spray, electrostatic spray, or dip. The initial microbial load of the control groups was 6.80-6.90 log/CFU. Overall, the dip method (3.33 log/CFU) was more effective than garden spray (2.5 log/CFU) and electrostatic spray methods (2.0 log/ CFU) at inactivating Listeria monocytogenes, regardless of the concentration of antimicrobial (P<0.05). On average, the garden spray methods were more effective than the electrostatic spray methods for most SaniDate-5.0® concentrations (P<0.05). Reductions of Listeria monocytogenes were greatest (P<0.05) when 0.25% and 0.5% of the antimicrobial was applied to the apple by all of the delivery methods. To confirm results, the method of action of SaniDate 5.0® on Listeria monocytogenes inactivation was analyzed using atomic force microscopy. Samples were sent to the University of Connecticut where Dr. Luo performed Atomic Force Microscopy analysis. The topographical observation under atomic force microscopy revealed changes in membrane permeability that led to extracellular leakage and eventually cytolysis when introduced to 0.5% of the antimicrobial solution. There was not a significant change in cell morphology until 0.1% of SaniDate 5.0 was added and more drastic changes of the cell were noticed when Listeria cells were subjected to 0.25 and 0.5% antimicrobial. Overall findings of this research indicated that 0.5% delivered through the dip method was most effective in inactivating Listeria monocytogenes. Atomic force microscopy revealed the possible mechanism of action of this concentration. That is to say that 0.5% SaniDate 5.0® causes oxidation and cytolysis of the bacterial cell, thus leading to cell death. It was hypothesized that the reason the dip method was more effective than either spray method at inactivating Listeria, despite not having the most residual water on the apples, was due to the apple being fully submerged in the washing solution. Although there are other aspects to consider such as cost of treatment and cross-contamination occurrences in wash methods, this research can be utilized as a tool to help local apple farmers prevent the spread of Listeria monocytogenes to consumers.