UDC 663.12:576.314:577.171.54ISSN 1330-9862 original scientific paper(FTB-998)The Effect of Ethanol on the Plasma MembranePermeability of Spoilage YeastsCélia Quintas1,2*, Emília Lima-Costa1and Maria C. Loureiro-Dias1,31Unidade de Ciências e Tecnologias Agrárias, Campus de Gambelas2Escola Superior de Tecnologia, Campus da Penha,Universidade do Algarve, 8000 Faro, Portugal3Departamento de Botânica e Engenharia BiológicaInstituto Superior de Agronomia, 1349-017 Lisboa, PortugalReceived: November 10, 1999Accepted: January 21, 2000SummaryThe effect of ethanol on the passive proton influx and on leakage of compounds absor-bing at 260 nm, as representatives of intracellular content, was studied on food spoilageyeasts such as Saccharomyces cerevisiae, Zygosaccharomyces bailii, Pichia sp. and Debaryomyceshansenii. For volume fraction below 10 %, the effect of ethanol on the proton influx was ingeneral weak, but above a certain treshold of ethanol high values were observed. In Z. bai-lii ethanol had no effect up to 20 % (volume fraction). Previous growth of the cells in thepresence of benzoic acid or ethanol did not affect the influx of protons in the presence ofethanol. Leakage of compounds absorbing at 260 nm was not observed at 25 °C and oc-curred at 30 °C only after a rather long incubation in high concentrations of ethanol,which induced cell death. This suggests that in the yeasts this process does not control theleakage of compounds in the presence of ethanol.Key words: food spoilage yeasts, proton influx, plasma membrane permeabilityIntroductionThe yeast plasma membrane is one of the main tar-gets for ethanol. It has been shown that ethanol inhibitsprocesses of mediated transport (1,2) and stimulates thetraffic of compounds that cross the membrane by simplediffusion (3–5). This alteration in membrane permeabil-ity may be deleterious to the cells promoting the leakageof intracellular constituents or the entry of toxic extra-cellular substances which disturb the composition of thecytoplasm. Protons are usually in higher concentrationsin the extracellular environment and yeast cells use apowerful H+-pump ATPase that keeps the intracellularpH at physiological values, suitable for enzyme functi-on, and generates a proton-motive force across the plas-ma membrane. Disturbing the homeostasis of the mem-brane to H+, ethanol may induce intracellular acidifica-tion and degradation of ATP by the ATPase.The objective of our work was to study the effect ofethanol on the leakage of protons and of intracellularcompounds which absorb at 260 nm in yeasts involvedin food spoilage. Saccharomyces cerevisiae was chosen forbeing resistant to ethanol; Zygosaccharomyces bailii andPichia sp. were chosen for their resistance to weak acidpreservatives and Debaryomyces hansenii was includedas a spoilage yeast particularly resistant to salt. The ef-fect of previous adaptation of cells to ethanol and tobenzoic acid, which induce probable alterations in thecomposition of the plasma membrane, was also consid-ered.47CÉLIA QUINTAS et al.: Plasma Membrane Permeability of Spoilage Yeasts, Food technol. biotechnol. 38 (1) 47–51 (2000)* Corresponding authorMaterials and MethodsStrains and culture conditionsFour strains representative of species associated withfood spoilage were used: Saccharomyces cerevisiae IGC 3507,Zygosaccharomyces bailii IGC 4806, Debaryomyces hanseniiIGC 2968 and Pichia sp. IGC 4595, kindly provided bythe Portuguese Yeast Culture Collection. Inocula wereprepared on agar YPD slants (2 % (w/v) glucose, 2 %(w/v) peptone, 0,5 % yeast extract, 2 % (w/v) agar),grown for 24 h at 25 °C. Cultures were prepared in 300–500 mL of mineral medium (6) with vitamins and 2 %(w/v) glucose, in 1 L Erlenmeyer flasks. Cultures werestirred magnetically and incubated aerobically at 26 °Cin a water bath. For adaptation to benzoic acid, the acidwas incorporated in the medium to a final concentrationof1mM(Sacch. cerevisiae, Z. bailii and Pichia sp.) or 0.5 mM(D. hansenii). For adaptation to ethanol, the alcohol wasadded to the medium to a final volume fraction of 8 %(Sacch. cerevisiae, Z. bailii and Pichia sp.) or 2 % (D. han-senii). Exponential phase cells (A260 nm@ 1) were harves-ted by centrifugation at 4 °C, washed twice with colddistilled water, for the passive proton influx assays, orwith phosphate buffer (50 mM, pH=5) for measuring theleakage of compounds absorbing at 260 nm.Passive proton influxCell suspensions were prepared in water at a con-centration of 60–70 mg/mL and kept on ice. Protonmovements were measured at 30 °C with a standard pHmeter, Radiometer PHM62, connected to a recorder. In awater-jacked cell of 10 mL capacity, with magnetic stir-ring, 0.5 mL of cell suspension, distilled water and etha-nol were mixed to obtain a final volume of 5 mL. ThepH value of these suspensions was 5–6. The pH wasquickly adjusted to 4.0 by the addition of HCl (100 mM)and the subsequent pH recovery was recorded, during 1min. After each assay, the curve was calibrated using 10mM HCl. The rate of proton influx was calculated as therate of decrease of the concentration of extracellular pro-tons (3). All the experiments were performed in the pre-sence of 2-deoxy-D-glucose (1 mM) and antimycin (2mg/mL), to minimise H+movements created by theplasma membrane ATPase activity.Leakage of compounds absorbing at 260 nmCell suspensions (1–2 mg/mL) were prepared inphosphate buffer (50 mM, pH=5) with ethanol in the de-sired concentrations in tightly closed flasks. These sus-pensions were incubated in a shaking water bath at 30°C for 4 h. At regular time intervals, samples (5 mL)were harvested, centrifuged for 5 min (6000 rpm) at 4 °Cand the supernatants were separated immediately. Leak-age was evaluated by measuring the absorbance of thesupernatants at 260 nm in a spectrophotometer (Shimad-zu UV-160-A). The content in compounds absorbing at260 nm was estimated by using NAD+as a standard.Evaluation of cell viabilityThe criterion of ability to form colonies was used totest viability of cells in which leakage of compounds ab-sorbing at 260 nm was measured. Appropriate dilutionsof cell suspensions, prepared at the beginning of experi-ments, were used in order to obtain a suitable numberof colonies in plates. Cell suspensions were incubated asdescribed in the previous paragraph. Samples (100mL)removed, when the loss of compounds absorbing at 260nm was significantly high, were plated on YPD agar.Plates were incubated