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Investigating the Antibacterial Effect of a Novel Gallic Acid-Based Green Sanitizer Formulation - PubMed

  • ️Mon Jan 01 2024

Investigating the Antibacterial Effect of a Novel Gallic Acid-Based Green Sanitizer Formulation

Esther W Mwangi et al. Foods. 2024.

Abstract

The purpose of the present study was to investigate the mechanism of action of our newly developed green sanitizer formulation comprising a natural phenolic compound, gallic acid (GA), strengthened by the Generally Recognized as Safe (GRAS) materials hydrogen peroxide (H2O2) and DL-lactic acid (LA). Combining 8 mM GA with 1 mM H2O2 resulted in an abundant generation of reactive oxygen species (ROS) and a bactericidal effect towards Gram-negative (Escherichia coli, Pseudomonas syringae, and Pectobacterium brasiliense) and Gram-positive (Bacillus subtilis) bacteria (4 to 8 log CFU mL-1 reduction). However, the exposure to this dual formulation (DF) caused only a modest 0.7 log CFU mL-1 reduction in the Gram-positive L. innocua population. Amending the DF with 20 mM LA to yield a triple formulation (TF) resulted in the efficient synergistic control of L. innocua proliferation without increasing ROS production. Despite the inability to grow on plates (>7 log CFU mL-1 population reduction), the TF-exposed L. innocua maintained high intracellular ATP pools and stable membrane integrity. The response of L. innocua to TF could be qualified as a "viable but nonculturable" (VBNC) phenomenon, while with the other species tested this formulation caused cell death. This research system may offer a platform for exploring the VBNC phenomenon, a critical food safety topic.

Keywords: 3,4,5-Trihydroxybenzoic acid; Escherichia coli; Listeria innocua; ROS; antimicrobial formulation; food safety; hydrogen peroxide; lactic acid; synergy; viable but nonculturable (VBNC).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1

Effects of DF (8 mM GA + 1 mM H2O2) and TF (8 mM GA + 1 mM H2O2 + 20 mM LA) against (A) Escherichia coli and (B) Listeria innocua compared with different concentrations of their individual ingredients, gallic acid (GA), hydrogen peroxide (H2O2), and lactic acid (LA), enumerated as logarithmic values of colony-forming units per milliliter (Log10 CFU mL−1) on LB or BHI plates, respectively. For GA, the highest concentration tested was limited by its water solubility. (The dashed lines show the method detection limits. The values not exceeding the limit indicate lack of visible bacterial growth). Each value indicates the means of triplicate tests. Error bars represent 95% confidence intervals (p < 0.05). The broken horizontal line indicates the limit of detection. MIC represents the minimum inhibitory concentration. Different letters above the bars indicate significant differences (p < 0.05).

Figure 1
Figure 1

Effects of DF (8 mM GA + 1 mM H2O2) and TF (8 mM GA + 1 mM H2O2 + 20 mM LA) against (A) Escherichia coli and (B) Listeria innocua compared with different concentrations of their individual ingredients, gallic acid (GA), hydrogen peroxide (H2O2), and lactic acid (LA), enumerated as logarithmic values of colony-forming units per milliliter (Log10 CFU mL−1) on LB or BHI plates, respectively. For GA, the highest concentration tested was limited by its water solubility. (The dashed lines show the method detection limits. The values not exceeding the limit indicate lack of visible bacterial growth). Each value indicates the means of triplicate tests. Error bars represent 95% confidence intervals (p < 0.05). The broken horizontal line indicates the limit of detection. MIC represents the minimum inhibitory concentration. Different letters above the bars indicate significant differences (p < 0.05).

Figure 2
Figure 2

Quantification of abiotic cell-free ROS generation by individual formulation ingredients and their combinations based on CellROX Deep Red reagent fluorescence. The fluorescence intensity data were collected every 10 min over a period of 1 h and the fluorescence change was calculated as added area under the curve (AAUC) compared to the blank (saline). Each value indicates the means of triplicate tests. Error bars represent 95% confidence intervals (p < 0.05). Different letters above the bars indicate significant differences (p < 0.05).

Figure 3
Figure 3

Effects of DF (GA + H2O2) and TF (GA + H2O2 + LA) formulations on viable counts of Gram-negative bacteria (E. coli (A), Pseudomonas syringae (B), and Pectobacterium brasiliense (C)) cells within 30 min incubation as determined by the plating test, with the logarithmic values of colony-forming units per milliliter (Log10 CFU mL−1) in blue box plots and the comparison to the ATP-based viability assay (ATP content % of positive control) in green box plots. Each value indicates the means of triplicate tests. The box plots represent the interquartile range of the 25th and 75th percentiles with the middle line as the median and whiskers extending to the minimum and maximum values of the data. X indicates the means of triplicate tests. Numbers in text boxes represent the ATP/CFU ratios. Asterisks indicate significant differences compared to control (p < 0.0001 for ****).

Figure 4
Figure 4

Effect of DF (GA + H2O2) and TF (GA + H2O2 + LA) formulations on viable counts of Gram-positive B. subtilis (A) and L. innocua (B) cells within 30 min incubation as determined by the plating test, with the logarithmic values of colony-forming units per milliliter (Log10 CFU mL−1) in blue box plots and the comparison to the ATP-based viability assay (ATP content % of positive control) in green box plots. Each value indicates the means of triplicate tests. Box plots represent the interquartile ranges of the 25th and 75th percentile with the middle line as the median and whiskers extending to the minimum and maximum values of the data. X indicates the means of triplicate tests. Numbers in text boxes represent the ATP/CFU ratios. Asterisks indicate significant differences compared to control (p < 0.05 for *, p < 0.001 for ***, p < 0.0001 for ****); ns means lack of significance.

Figure 5
Figure 5

Effect of TF (GA + H2O2 + LA) on viability (% of positive control) of L. innocua within 30 min incubation as determined by BacLight membrane integrity assay (fluorescence units). Each value indicates the means of triplicate tests. Box plots represent the interquartile ranges of the 25th and 75th percentile with the middle line as the median and whiskers extending to the minimum and maximum values of the data. X indicates the means of triplicate tests. Asterisks indicate significant differences compared to control (p < 0.0001 for ****); ns means lack of significance.

Figure 6
Figure 6

(A) Flow cytometry histogram profiles of L. innocua treated with TF (GA + H2O2 + LA) treated (blue) stained with PI shown as cell count distribution according to fluorescence intensities compared to the control (green) and heat-inactivated populations (red). (B) The graph summary representing the % frequency of the gated dead cells based on PI membrane extrusion. Each value indicates the means of triplicate tests. Box plots represent the interquartile ranges of the 25th and 75th percentile with the middle line as the median and whiskers extending to the minimum and maximum values of the data. X indicates the means of triplicate tests. Asterisks indicate significant differences compared to control (p < 0.01 for **, p < 0.0001 ****).

Figure 7
Figure 7

Normalized fluorescence curves of amplified DNA templates of non-treated (control), TF-treated, and heat-inactivated L. innocua by PMAxx-vqPCR assay targeting the 16S rRNA gene. The samples were incubated for 10 min, either with PMAxx (10 μM) or without it (0 μM), and photolysed for 15 min. Log values indicate viable cell counts derived from the standard curve ± standard deviation. Each value indicates the means of triplicate tests, the error bars represent 95% confidence intervals (p < 0.05), and Δ Rn represents fluorescence intensity. Negative control represents sample without DNA template.

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