MODE OF ACTION

The mode of action of organic acids in inhibiting microbial growth appears to be related to maintenance of acid–base equilibrium, proton donation, and the production of energy by the cells. It is thus essential to understand each of these concepts before consideration of the actual mode of action of the organic acids. Biological and chemical systems depend on an interaction between acid and base systems. The microbial cell normally reflects this equilibrium by attempting to maintain an internal pH near neutrality (Baird-Parker, 1980). Homeostasis is the tendency of a cell to sustain chemical equilibrium despite a fluctuation in the acid–base environment. Through the interaction of a series of chemical mechanisms, this delicate balance is maintained and alteration of this balance causes destruction of the microbial cell. Proteins, nucleic acids, and phospholipids can be structurally altered by pH changes. The availability of metallic ions to the organism is also altered and becomes a function of membrane permeability because membranes are less permeable to charged molecules than to uncharged molecules. These changes in membrane permeability can exert a dual effect by impairing transport of nutrients into the cell or by causing the leakage of internal metabolites to the outside. Changes resulting from pH can destroy bacteria, molds, and yeasts, although some microorganisms such as Acetobacter can exist at and even require extremes of acidity (Langworthy, 1978).

The terms strong or weak are used to describe acids and reflect the degree with which acids readily donate a proton or dissociate in aqueous solutions. Inorganic acids, such as hydrochloric,

Organic Acids 125

because of their low pKa almost entirely dissociate in solution. Acetic acid and other organic acids only slightly ionize and do not readily give up their proton(s) to water. When an acid enters and ionizes within the cell, the problem becomes one of elimination of the excess protons. This ejection process becomes a fundamental issue in how a cell produces energy.

Energy produced through chemical reactions is essential for cellular processes. Synthesis of macromolecules, maintenance of osmotic gradients, and active transport of molecules across the membrane depend on energy generated by the cell in the form of adenosine triphosphate (ATP). In anaerobic microorganisms, the glycolytic pathways inefficiently generate ATP. In aerobic organ- isms, the electron transport system (ETS) principally generates ATP with oxygen as the terminal electron acceptor (Gould et al., 1983).

Before energy can be produced, materials must be transported into the cell. One means for substrate entry into the cell is active transport, which also allows greater internal concentration of solute than external concentration. To maintain this unequal gradient, energy must be expended. For this reason, active transport is coupled with energy-yielding processes. The energy evolves from the oxidation of the substrates and the respiratory chain. The respiratory chain or ETS transverses the membrane, which does not permit H+ or OH- ions to penetrate and ejects accumu- lated protons to the external environment. This process generates a chemical and electric gradient capable of driving metabolic reactions (Dawes and Sutherland, 1992).

Early experiments by Levine and Fellers (1940) demonstrated that acetic acid was more lethal at a higher pH than hydrochloric acid or lactic acid. They concluded that this toxicity was not the result of hydrogen ion concentration alone, but seemed to be a function of the undissociated molecule. With acetic acid, lowering the pH increased the inhibitory activity, confirming that the undissociated molecule was the effective inhibitor. Thus, the inhibition by extracellular fatty acids used as antimicrobial agents would increase with decreasing pH, in agreement with pKa values (Freese et al., 1973).

Sheu and Freese (1972) determined that short-chain fatty acids reversibly reacted with the cell membrane and altered its structure. It was postulated that this interfered with the regeneration of ATP by uncoupling the ETS, or the transport of metabolites into the cell was altered. Further studies by Sheu and coworkers (1972) indicated that acetate uncoupled the amino acid carrier protein from the ETS and inhibited amino acid transport noncompetitively. Serine uptake was inhibited in membrane vesicles of B. subtilis when exposed to fatty acids; using the same system, L-leucine

and L-malate were shown to uncouple both substrate transport and oxidative phosphorylation from

the ETS (Freese et al., 1973).

In a later study, Sheu et al. (1975) recognized that if reducing compounds were no longer available to the cell because of transport inhibition by the fatty acids, oxygen consumption would be reduced. To understand these processes, the bacterial cell was converted to a spheroplast under isotonic conditions and these membrane vesicles were used to study uptake of a substrate against a gradient. Because oxygen consumption was observed in the presence of membrane preparations and an energy source, inhibition transport would not necessarily be linked with ATP generation. The active transport of a molecule would depend on the proton gradient generated during the oxidation of a substrate. Lipophilic agents, such as the short-chain fatty acids, would shuttle protons through the membrane until the proton motive force had been destroyed and transport thus elimi- nated.

Freese and Levine (1978) further postulated that the most effective antimicrobial agents would be lipophilic enough to attach to microbial membranes yet be soluble in the aqueous phase. This is because they can approach the membrane from the aqueous medium, yet easily and without requiring energy penetrate the membrane lipid bilayer. Undissociated acids of short chain length can penetrate the cell more easily because they possess these characteristics.

Not all acids are effective against all microorganisms. E. coli and B. subtilis are equally inhibited by equal concentrations of compounds containing up to six carbons. However, twice the amount of a C8 (caprylic) acid is needed to inhibit the growth of E. coli compared to B. subtilis. The

difference between the two organisms could be because of the lipopolysaccharide layer that sur- rounds the Gram-negative E. coli, thus acting as a mechanical barrier and preventing passage of the acid into the cell. Another theory proposes that Gram-negative organisms can rapidly metabolize the acidulant and therefore not allow it to accumulate within the cell (Freese et al., 1973; Kabara et al., 1972). Furthermore, some acids, lactic and citric, reduced the internal pH of the cell more than acetic acid; however, other changes associated with metabolic or physiologic processes may be similarly affected (Ita and Hutkins, 1991).

Hunter and Segal (1973) in studies using Penicillium chrysogenum suggest that weak acids at or below their pKa could discharge the proton gradient and ionize within the cell to acidify the interior. It was postulated that the rate of proton leakage into the cell versus proton ejection would determine the inhibition of the cell (Freese et al., 1973). Eklund (1980) has questioned the mode of action of the weak lipophilic acids. He states that although inhibition of uptake of nutrients contributes to growth inhibition, it does not seem to be the sole cause of the static action.

In summary, the current data suggest that the mode of action of short-chain lipophilic acids requires the destruction of the proton motive force, thereby limiting substrate transport. It is further speculated that acids, which possess both lipoidal and aqueous solubilities, are the most effective antimicrobial agents and that some sort of membrane attachment of the acid is involved. Studies have indicated that cells, when placed in fresh medium devoid of the inhibitor, can reinitiate transport, suggesting a static response rather than a killing effect (Freese et al., 1973). Obviously, there is need for further research on the mode of action of these antimicrobial agents.