Extreme resistance to weak-acid preservatives in the spoilage yeast Zygosaccharomyces bailii

Weak-acid preservatives, such as sorbic acid and acetic acid, are used in many low pH foods to prevent spoilage by fungi. The spoilage yeast Zygosaccharomyces bailii is notorious for its extreme resistance to preservatives and ability to grow in excess of legally-permitted concentrations of preservatives. Extreme resistance was confirmed in 38 strains of Z. bailii to several weak-acid preservatives. Using the brewing yeast Saccharomyces cerevisiae as a control, tests showed that Z. bailii was ~ 3-fold more resistant to a variety of weak-acids but was not more resistant to alcohols, aldehydes, esters, ethers, ketones, or hydrophilic chelating acids. The weak acids were chemically very diverse in structure, making it improbable that the universal resistance was caused by degradation or metabolism. Examination of Z. bailii cell populations showed that extreme resistance to sorbic acid, benzoic acid and acetic acid was limited to a few cells within the population, numbers decreasing with concentration of weak acid to < 1 in 1000. Re-inoculation of resistant sub-populations into weak-acid-containing media showed that all cells now possessed extreme resistance. Resistant sub-populations grown in any weak-acid preservative also showed ~ 100% cross-resistance to other weak-acid preservatives. Tests using 14C-acetic acid showed that weak-acid accumulation was much lower in the resistant sub-populations. Acid accumulation is caused by acid dissociation in the higher pH of the cytoplasm. Tests on intracellular pH (pHi) in the resistant sub-population showed that the pH was much lower, ~ pH 5.6, than in the sensitive bulk population. The hypothesis is proposed that extreme resistance to weak-acid preservatives in Z. bailii is due to population heterogeneity, with a small proportion of cells having a lower intracellular pH. This reduces the level of accumulation of any weak acid in the cytoplasm, thus conferring resistance to all weak acids, but not to other inhibitors.