Alkaline stress led to an increase in the lag phase at pH 11. Alkaline solutions such as NaOH are generally used in detergents to eliminate carbonized sediment, oil or grease. They facilitate protein denaturation, fats saponification, and have a bactericidal activity. Rowbury & Hussain (1996), in studies on Escherichia coli cells exposed to alkaline pH (pH 8·8 to pH 10), reported damage to the outer membrane, ribosomes, proteins and DNA. In addition, a total dissociation of NaOH into Na+ and OH– ions disturbed the energetic metabolism of the bacteria. An Na+ excess in the cell would modify the transmembrane Na+ gradient maintained through an Na+/H+ antiporter, and when the extracellular pH increased, the antiporter activity would increase too ( Karpel et al. 1991 ). Two hypotheses are then proposed. (i) At high alkaline pH, the antiporters might be saturated with the excess of Na+ entering the cell; consequently, the ion gradients might not be able to be maintained and membrane proteins, implicated in the production of energy, would be inhibited. (ii) Such high pH values could lead to the saponification of membrane lipids and destabilization of those proteins whose activity depended on the integrity of the lipid bilayer. In addition to the loss of energy production, these phenomena would rapidly inhibit growth.
The experiments performed in this study emphasized that the antilisterial activity of acetic acid was greater than that of lactic or hydrochloric acid, for all five strains. The lag and generation times observed with acetic acid were longer than for the other acids.
These results were consistent with those of other authors on different strains of L. monocytogenes. At the same pH, Sorrells et al. (1989) established that at 10, 25 and 35 °C, acetic and lactic acids were more inhibitory against L. monocytogenes than citric and hydrochloric acids. Conner et al. (1990) also reported that acetic and lactic acids were the most inhibitory. In contrast, Sorrells et al. (1989) observed that with an equimolar concentration of acid, the order of activity was lactic acid, followed by acetic and then hydrochloric acid at 25 and 35 °C.
These results, as well as the findings of El-Shenawy & Marth (1989), highlight the varying influence of pH and temperature, depending on the organic acid used.
A number of investigators have reported the inhibitory effects of low pH and organic acids on L. monocytogenes ( Adams & Hall 1988; Conner et al. 1990 ; Ita & Hutkins 1991). Two inhibitory mechanisms have been proposed: (i) an intracellular acidification (lost of homeostasis) and (ii) a specific effect of the acid (non-dissociated form) on metabolic activities.
Ita & Hutkins (1991) observed that low intracellular pH was not the major factor in the inhibition of L. monocytogenes at acid pH; indeed, cells treated with organic acids or HCl at pH values as low as 3·5 were able to maintain their cytoplasmic pH at a value near 5. Consequently, the efficiency of the treatments using organic acids would be due to the non-dissociated fraction rather than to proton toxicity.
The inhibitory effect of these acids can be correlated with their dissociation constant (pKa value) and with the greater permeability of the cell membrane to weak acids in their undissociated form. Among the acids we have tested, hydrochloric acid is totally dissociated in aqueous environments whereas acetic acid (pKa = 4·76) has the highest concentration of undissociated acid at pH 5·4 (14·3 mmol l−1), and lactic acid (pKa = 3·86) has the lowest (2·4 mmol l−1). Also, acetic acid, a weak acid with the highest pKa value, is more efficient against L. monocytogenes than a stronger hydrochloric acid used at the same pH. These data are consistent with the results obtained by Sorrells et al. (1989) .
The highest inhibitory effect of acetic acid can be explained by its ability to diffuse through the cell membrane which is permeable to non-dissociated, non-protonated and lipophilic weak acids. This leads to an accumulation of the acid within the cell cytoplasm, acidification of the cytoplasm, disruption of the proton-motive force and inhibition of substrate transport.
Lactic acid may be less inhibitory as it cannot passively penetrate the cell membrane.
Treatments based on organic acids seem to be the most efficient (against Gram-negative bacteria) and are widely used in decontamination processes such as meat (carcass) decontamination. Lactic acid is suitable for this purpose because it is a natural constituent of meat and is ‘generally accepted as safe’ ( Van Netten 1996). The intrinsic lactic acid content of meat, together with the buffering capacity, determine the resulting meat surface pH and thus, the bactericidal effectiveness of the agent applied.
Due to its higher pKa, acetic acid is theoretically a better antimicrobial agent than lactic acid. In practice, the lactic acid appeared to be the better meat decontamination agent ( Van Netten 1996). Mixtures of both were also tested on E. coli and Salmonella enteritidis strains ( Adams & Hall 1988) and results confirmed that the undissociated acid was the active antimicrobial species.