Abstract
We developed a top-down strategy to characterize an antimicrobial, oxidizing sanitizer, which has diverse proposed applications including surface-sanitization of fresh foods, and with benefits for water resilience. The strategy involved finding quenchers of antimicrobial activity then antimicrobial mode of action, by identifying key chemical reaction partners starting from complex matrices, narrowing down reactivity to specific organic molecules within cells. The sanitizer electrolyzed-water (EW) retained partial fungicidal activity against the food-spoilage fungus Aspergillus niger at high levels of added soils (30–750 mg mL–1), commonly associated with harvested produce. Soil with high organic load (98 mg g–1) gave stronger EW inactivation. Marked inactivation by a complex organics mix (YEPD medium) was linked to its protein-rich components. Addition of pure proteins or amino acids (≤1 mg mL–1) fully suppressed EW activity. Mechanism was interrogated further with the yeast model, corroborating marked suppression of EW action by the amino acid methionine. Pre-culture with methionine increased resistance to EW, sodium hypochlorite, or chlorine-free ozonated water. Overexpression of methionine sulfoxide reductases (which reduce oxidized methionine) protected against EW. Fluoroprobe-based analyses indicated that methionine and cysteine inactivate free chlorine species in EW. Intracellular methionine oxidation can disturb cellular FeS-clusters and we showed that EW treatment impairs FeS-enzyme activity. The study establishes the value of a top-down approach for multi-level characterization of sanitizer efficacy and action. The results reveal proteins and amino acids as key quenchers of EW activity and, among the amino acids, the importance of methionine oxidation and FeS-cluster damage for antimicrobial mode-of-action.
Highlights
Chemical sanitizers and disinfectants have applications for control of microbial contamination and growth in diverse settings, including healthcare and food industries as well as domestic use, with a global market approaching USD 20 billion
Adding soil to electrolyzed water (EW) treatments (360–400 mg L−1 free available chlorine (FAC)) inactivated EWdependent killing only at high soil levels (Figure 1A); the soil concentration (750 mg mL−1) shown in the figure was chosen after preliminary tests with soil “8” showed that lower soil additions were insufficient for suppressing EW activity
Our findings that incidental protein and particular amino acids strongly affect the fungicidal EW activity led us to new understanding of the oxidizing mode of EW action, in which reduced-methionine of cells plays a critical role
Summary
Chemical sanitizers and disinfectants have applications for control of microbial contamination and growth in diverse settings, including healthcare and food industries as well as domestic use, with a global market approaching USD 20 billion. More recent applications have been reported in the food industry, including processing-water sanitization and surface sanitization of factory surfaces and equipment, and of fresh produce (Gil et al, 2015; Kaczmarek et al, 2019). One major contributor to this loss is microbial spoilage, including by molds such as Aspergillus niger, a common food spoilage mold found on diverse fruits and vegetables (Taniwaki et al, 2018). The guidelines for sanitizers to achieve > 5 log reduction (Block, 2001) are usually met with EW for microorganisms in suspension, but lower efficiencies have been reported for certain species or conditions
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