Natural Antimicrobials Suitable for Combating Desiccation-Resistant Salmonella enterica in Milk Powder

Abstract

Some Salmonella enterica strains survive well in low-water activity (low-aw) foods and cause frequent salmonellosis outbreaks in these products. Methods are needed to overcome such desiccation-resistant Salmonella and to improve the safety of low-aw foods. Building on a recent finding, we hypothesized that natural antimicrobial food additives, which are active against cytoplasmic membrane, could overcome this desiccation resistance phenomenon, and thus, sensitize the pathogen to drying and mild processing. Food additives were screened for the ability to cause leakage of intracellular potassium ions; retention of these ions is vital for protecting Salmonella against desiccation. Two antimicrobial food additives, carvacrol and thymol, caused considerable potassium leakage from the desiccation-resistant S. enterica serovars, Tennessee and Livingstone. Thus, carvacrol and thymol were investigated for their ability to sensitize the desiccation-adapted S. enterica to heat treatment. The combined use of food additives, at their minimum inhibitory concentrations, with heat treatment at 55 °C for 15 min caused 3.1 ± 0.21 to more than 5.5 log colony forming unit (CFU)/mL reduction in desiccation-adapted S. enterica, compared to 2.4 ± 0.53–3.2 ± 0.11 log CFU/mL reduction by sole heat treatment. Carvacrol was the additive that caused the greatest potassium leakage and sensitization of Salmonella to heat; hence, the application of this compound was investigated in a food model against Salmonella Typhimurium ASD200. Addition of carvacrol at 200 or 500 ppm into liquid milk followed by spray-drying reduced the strain’s population by 0.9 ± 0.02 and 1.3 ± 0.1 log CFU/g, respectively, compared to 0.6 ± 0.02 log CFU/g reduction for non-treated spray-dried milk. Additionally, freeze-drying of milk treated with high levels of carvacrol (5000 ppm) reduced the population of Salmonella Typhimurium ASD200 by more than 4.5 log CFU/g, compared to 1.1 ± 0.4 log CFU/g reduction for the freeze-dried untreated milk. These findings suggest that carvacrol can combat desiccation-resistant S. enterica, and thus, potentially improve the safety of low-aw foods.

Carvacrol and Thymol Combat Desiccation Resistance Mechanisms in Salmonella enterica Serovar Tennessee

“…The preparation of desiccation-adapted dry cells of Salmonella Tennessee was performed as reported in previous studies [9,11], described in the materials and methods section, and demonstrated in Figure 1. The desiccation-adapted cells were used in various experiments after resuspension in different media as required in each experiment.…”

“…We hypothesized that carvacrol and thymol are candidate food additives to combat desiccation-resistant Salmonella, in view of their activity against bacterial cytoplasmic membranes. In a previous study, the two additives sensitized desiccation-adapted Salmonella to heating at 55 • C for 15 min [11]. Furthermore, the anti-Salmonella effect of carvacrol was apparent during sprayor freeze-drying of liquid milk, and this effect was noticeable during storage of the dried milk at room temperature [11].…”

“…In a previous study, the two additives sensitized desiccation-adapted Salmonella to heating at 55 • C for 15 min [11]. Furthermore, the anti-Salmonella effect of carvacrol was apparent during sprayor freeze-drying of liquid milk, and this effect was noticeable during storage of the dried milk at room temperature [11]. These findings prompted us to explore the mechanisms by which carvacrol and thymol impair desiccation resistance in Salmonella.…”

“…The potassium-sensitive benzofuran isophthalate probe (PBFI; Invitrogen, Carlsbad, CA, USA) was used to determine the release of potassium ions from desiccation-adapted Salmonella Tennessee as described previously [9,11]. Briefly, aliquots (1 mL, each) of desiccation-adapted cells in saline (Figure 1) were harvested by centrifugation at 8000× g for 5 min, and cell pellets were resuspended in 5 mM HEPES buffer (Sigma Aldrich) containing 5 mM glucose (Fisher Scientific, Fair Lawn, NJ, USA).…”

“…However, paenibacterin has not been approved for use in food, and producing the compound in large quantities necessitates laborious purification procedures [10]. Alternatively, membrane-active food ingredients and additives that can combat desiccation-adapted Salmonella, in similar fashion to paenibacterin, were investigated [11]. Of these tested compounds, carvacrol and thymol were the most capable of causing the leakage of potassium ions from the cells of selected desiccation-resistant Salmonella serovars.…”