Synergistic Antimicrobial Combinations Inhibit and Inactivate Listeria monocytogenes in Neutral and Acidic Broth Systems

Abstract

The use of antimicrobial compounds can be an effective approach to control Listeria monocytogenes in ready-to-eat foods, but it can also be limited by cost, restrictions on concentrations in foods, and potential changes to organoleptic properties. Combinatorial approaches that produce additive or synergistic effects allow for reductions in individual antimicrobial concentrations while achieving the same level of control. The present study determined the MIC and MBC of an antimicrobial compound when used alone or in binary combinations against L. monocytogenes in growth media adjusted to pH values 7.4 and 5.5 and characterized interactions as synergistic, additive, or antagonistic. Inhibitory and bactericidal concentrations were defined as changes in L. monocytogenes counts of ≤1.0 or ≥3.0 log CFU/mL compared with the starting inoculum, respectively. Individually, lauric arginate (LAE), hydrogen peroxide (HP), and ε-polylysine (EPL) inhibited L. monocytogenes growth at the lowest concentrations when applied alone in broth adjusted to pH 7.4. Similarly, LAE, EPL, and HP had the lowest MBCs in broth adjusted to both pH levels. The inhibitory efficacy of both caprylic acid and sodium caprylate (SC) increased at the lower pH, with reductions in MICs of >98%. In total, 35 and 19 additive or synergistic inhibitory and bactericidal combinations were identified at pH values 7.4 and 5.5, respectively. Combinations of acidified calcium sulfate with lactic acid (ACSL) and SC were among the most synergistic inhibitory groupings at both pH levels, whereas EPL+LAE were the most effective bactericides at pH 7.4. Combinations of SC with EPL or ACSL were also among the most effective bactericides at pH 5.5. These data serve as a foundation for developing more effective antimicrobial approaches for the control of L. monocytogenes in foods with different pH levels.

Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination

“…According to US Food and Drug Administration (FDA) guidance on the control of L. monocytogenes in ready-to-eat foods, a listeristatic formulation that combines one or more antimicrobial substances is generally considered to be effective as a process control if growth studies that include samples at various points during product shelf life show an increase of <1 log 10 cycle over 2 or more time intervals (e.g., days, weeks, and so on, based on sampling plan) in the number of L. monocytogenes during replicate trials with the food of interest (FDA, 2017b). Several antimicrobials generally recognized as safe (GRAS) by the FDA including caprylic acid (CA; FDA, 2015), hydrogen peroxide (HP; FDA, 2015), lauric arginate (LAE;FDA, 2005a,b), and ε-polylysine (EPL; FDA, 2011) have demonstrated efficacy in controlling L. monocytogenes under varying experimental conditions (Luchansky et al, 2005;Amrouche et al, 2010;Kozak et al, 2017), including applications in milk (Romanova et al, 2002;Geornaras et al, 2007;Ma et al, 2013) and cheese (Soni et al, 2010;Gadotti et al, 2014). Limited data are available on the efficacy of sodium caprylate (SC) against L. monocytogenes (Kozak et al, 2017), but several fatty acid derivatives including SC have demonstrated antimicrobial effects against gram-positive bacteria (Kabara et al, 1972;Nair et al, 2004).…”

Applications of Edible Coatings Formulated with Antimicrobials Inhibit Listeria monocytogenes Growth on Queso Fresco

“…Bacterial strains, growth conditions, and inoculum Preparation A cocktail was prepared as previously described (Kozak et al, 2017) using eight L. monocytogenes strains associated with outbreaks linked to soft cheeses or isolated from cheese processing environments ( Table 1). The cocktail was serially diluted in Butterfield's phosphate buffer (BPB), pelleted through centrifugation (30 min, 4,000 g at 4°C) (Thermo Scientific Sorvall Legend X1R, ThermoFisher Scientific, Waltham, MA, USA), and resuspended in BPB to attain ~7 log CFU/mL.…”

“…LAE can bind to anionic biopolymers naturally present within the mouth leading to a perceived bitterness or astringency, but the impact of complexation interactions (i.e., LAE and chitosan) on the sensory attributes of LAE (i.e., ability to cause astringency) are also not known (Bonnaud et al, 2010). As an alternative, the use of combinations of antimicrobials that work additively or synergistically to inhibit or inactivate L. monocytogenes can potentially enhance antimicrobial efficacy without increasing individual usage concentrations (Kozak et al, 2017).…”

“…Antimicrobial treatments for incorporation in chitosan coatings were selected based on previous work (Kozak et al, 2017(Kozak et al, , 2018a and included: 25% ACSL, 5% HP, 5% LAE, 25% ACSL + 10% SC, and 5% LAE + 10% SC. Preparation and application of chitosan coating solutions were adapted from previously published methods Chen et al, 2012 were added directly to mixed chitosan solutions on a v/v basis.…”

“…), can further enhance their efficacy (Pranoto et al, 2005;Zivanovic et al, 2005;Duan et al, 2007;Beverlya et al, 2008;Jiang et al, 2011;Petrou et al, 2012;Guo et al, 2014;Shekarforoush et al, 2015;Paparella et al, 2016). Previous research has identified effective antimicrobial treatments for the control of L. monocytogenes in broth and whole milk including acidified calcium sulfate with lactic acid (ACSL), hydrogen peroxide (HP), lauric arginate (LAE), and sodium caprylate (SC) (Kozak et al, 2017(Kozak et al, , 2018a. However, application of these antimicrobials to the surface of QF as aqueous dips were comparably less effective in inactivating L. monocytogenes and inhibiting growth during storage (Kozak et al, 2018b).…”