Weak-acid preservatives: pH and proton movements in the yeast Saccharomyces cerevisiae

Abstract

Weak-acid preservatives commonly used to prevent fungal spoilage of low pH foods include sorbic and acetic acids. The “classical weak-acid theory” proposes that weak acids inhibit spoilage organisms by diffusion of undissociated acids through the membrane, dissociation within the cell to protons and anions, and consequent acidification of the cytoplasm. Results from 25 strains of Saccharomyces cerevisiae confirmed inhibition by acetic acid at a molar concentration 42 times higher than sorbic acid, in contradiction of the weak-acid theory where all acids of equal pKa should inhibit at equimolar concentrations. Flow cytometry showed that the intracellular pH fell to pH4.7 at the growth-inhibitory concentration of acetic acid, whereas at the inhibitory concentration of sorbic acid, the pH only fell to pH6.3. The plasma membrane H+-ATPase proton pump (Pma1p) was strongly inhibited by sorbic acid at the growth-inhibitory concentration, but was stimulated by acetic acid. The H+-ATPase was also inhibited by lower sorbic acid concentrations, but later showed recovery and elevated activity if the sorbic acid was removed. Levels of PMA1 transcripts increased briefly following sorbic acid addition, but soon returned to normal levels. It was concluded that acetic acid inhibition of S. cerevisiae was due to intracellular acidification, in accord with the “classical weak-acid theory”. Sorbic acid, however, appeared to be a membrane-active antimicrobial compound, with the plasma membrane H+-ATPase proton pump being a primary target of inhibition. Understanding the mechanism of action of sorbic acid will hopefully lead to improved methods of food preservation.