Hurdle Technology Approach to Control Listeria monocytogenes Using Rhamnolipid Biosurfactant

Abstract

This study evaluates the combination of mild heat with a natural surfactant for the inactivation of L. monocytogenes Scott A in low-water-activity (a_w) model systems. Glycerol or NaCl was used to reduce the a_w to 0.92, and different concentrations of rhamnolipid (RL) biosurfactant were added before heat treatment (60 °C, 5 min). Using glycerol, RL treatment (50-250 µg/mL) reduced bacterial population by less than 0.2 log and heat treatment up to 1.5 log, while the combination of both hurdles reached around 5.0 log reduction. In the NaCl medium, RL treatment displayed higher inactivation than in the glycerol medium at the same a_w level and a larger synergistic lethal effect when combined with heat, achieving ≥ 6.0 log reduction at 10-250 µg/mL RL concentrations. The growth inhibition activity of RL was enhanced by the presence of the monovalent salts NaCl and KCl, reducing MIC values from >2500 µg/mL (without salt) to 39 µg/mL (with 7.5% salt). The enhanced antimicrobial activity of RL promoted by the presence of salts was shown to be pH-dependent and more effective under neutral conditions. Overall, results demonstrate that RL can be exploited to design novel strategies based on hurdle approaches aiming to control L. monocytogenes.

Keywords: Listeria monocytogenes; antimicrobial activity; biosurfactant; hurdle technology; low moisture foods; rhamnolipid.

Figure 1

Logarithmic reduction factor (LRF; log(N₀/N)) of L. monocytogenes Scott A by heat treatment (60 °C, 5 min) in sodium phosphate buffer (pH 6.7) at a_w > 0.99 (control, white bars), 0.98 or 0.92, adjusted with glycerol (gray bars) or NaCl (black bars). Error bars represent the standard deviation of the means of triplicates. Different letters indicate statistically significant differences (p ≤ 0.05) in the inactivation at different a_w levels and using different solutes. The dashed line indicates the LRF corresponding to the quantification limit of surviving cells (1000 CFU/mL).

Figure 2

Logarithmic reduction factor (LRF; log(N₀/N)) of L. monocytogenes Scott A in sodium phosphate buffer (pH 6.7) of a_w 0.92 with (A) glycerol or (B) NaCl by heat, RL at different concentrations and the combination of heat and RL. The white bar represents heat inactivation alone (60 °C, 5 min; without RL added). For each RL concentration, the gray bar presents inactivation by RL at room temperature (~20 °C, 5 min), while the black bar indicates inactivation by the combination of RL and heat (60 °C, 5 min). The dashed line indicates the maximum detectable LRF corresponding to the quantification limit (1000 CFU/mL). Error bars represent the standard deviations of the means of triplicates. Asterisk indicates statistically significant differences (p ≤ 0.05) between the theoretical additive inactivation by heat and RL and the experimental inactivation obtained by the combined treatment, and therefore the occurrence of a synergistic lethal effect. Lowercase and capital letters indicate statistically significant differences (p ≤ 0.05) in the inactivation by RL and by the combination of heat and RL treatment, respectively, among the different RL concentrations tested.

Figure 3

Growth kinetics of L. monocytogenes Scott A in BHI medium supplemented with (a) different concentrations of NaCl, without RL and (b) in combination with 500 µg/mL of RL at 30 °C. Data points represent the mean values of three replicates. Error bars have been omitted for clarity.

Figure 4

Fluorescence microscopy images of L. monocytogenes Scott A after incubation in BHI medium supplemented with NaCl (5%), rhamnolipids (500 µg/mL) and the combination of both for (a) 4 h and (b) 24 h at 30 °C. Green and red cells correspond to live and dead cells, respectively. Scale bar 10 µm.

Figure 5

Growth kinetics of L. monocytogenes Scott A in BHI medium supplemented with (a) different concentrations of KCl, without RL and (b) in combination with 500 µg/mL of RL at 30 °C. Data points represent the mean values of three replicates. Error bars have been omitted for clarity.

Figure 6

Growth kinetics of L. monocytogenes Scott A in BHI medium at pH 5.0 with and without the addition of 500 µg/mL of RL and 5% NaCl at 30 °C. Data points represent the mean values of three replicates. Error bars have been omitted for clarity.