Relationship between Sublethal Injury and Inactivation of Yeast Cells by the Combination of Sorbic Acid and Pulsed Electric Fields

The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log₁₀ 5 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.     

Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral

Aims: The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral. Methods and Results: The occurrence of sublethal injury in the outer membrane was measured using selective recovery media containing bile salts. Loss of membrane integrity was measured by the increased uptake of the fluorescent dye propidium iodide (PI). PEF caused nonpermanent and permanent envelope permeabilization of Ent. sakazakii at pH 4·0. After PEF, most surviving cells showed transient cell permeabilization and sublethal injury in their outer membranes. The simultaneous application of a mild PEF treatment (100 pulses, 25 kV cm^?1) and 200 μl l^?1 of citral to cells suspended in pH 4·0 buffer at a final concentration of 10⁷ cells per ml showed an outstanding synergistic lethal effect, causing the inactivation of more than two extra log₁₀ cycles. Conclusions: Our results confirm that the detection of sublethal injury in the outer membrane after PEF may contribute to the identification of the treatment conditions under which PEF may act synergistically with hydrophobic compounds such as citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by PEF will aid the establishment of successful combined preservation treatments.     

Occurrence of sublethal injury after pulsed electric fields depending on the micro-organism, the treatment medium ph and the intensity of the treatment investigated

AIMS: The objective was to investigate the occurrence of sublethal injury after pulsed electric field (PEF) depending on the treatment time, the electric field strength and the pH of the treatment media in two Gram-positive (Bacillus subtilis ssp. niger, Listeria monocytogenes) and six Gram-negative (Escherichia coli, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella serotype Senftenberg 775W, Salmonella serotype Typhimurium, Yersinia enterocolitica) bacterial strains. METHODS AND RESULTS: A characteristic behaviour was observed for the Gram-positive and Gram-negative bacteria studied. Whereas Gram-positive bacteria showed a higher PEF resistance at pH 7.0, the Gram-negative were more resistant at pH 4.0. In these conditions, in which bacteria showed their maximum resistance, a large proportion of sublethally injured cells were detected. In most cases, the longer the treatment time and the higher the electric field applied, the greater the proportion of sublethally injured cells that were detected. No sublethal injury was detected when Gram-positive bacteria were treated at pH 4.0 and Gram-negative at pH 7.0. CONCLUSIONS: Sublethal injury was detected after PEF so, bacterial inactivation by PEF is not an 'all or nothing' event. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF.     

Relationship between Sublethal Injury and Microbial Inactivation by the Combination of High Hydrostatic Pressure and Citral or tert-Butyl Hydroquinone

The aim was to investigate (i) the occurrence of sublethal injury in Listeria monocytogenes, Escherichia coli, and Saccharomyces cerevisiae after high hydrostatic pressure (HHP) treatment as a function of the treatment medium pH and composition and (ii) the relationship between the occurrence of sublethal injury and the inactivating effect of a combination of HHP and two antimicrobial compounds, tert-butyl hydroquinone (TBHQ) and citral. The three microorganisms showed a high proportion of sublethally injured cells (up to 99.99% of the surviving population) after HHP. In E. coli and L. monocytogenes, the extent of inactivation and sublethal injury depended on the pH and the composition of the treatment medium, whereas in S. cerevisiae, inactivation and sublethal injury were independent of medium pH or composition under the conditions tested. TBHQ alone was not lethal to E. coli or L. monocytogenes but acted synergistically with HHP and 24-h refrigeration, resulting in a viability decrease of >5 log₁₀ cycles of both organisms. The antimicrobial effect of citral depended on the microorganism and the treatment medium pH. Acting alone for 24 h under refrigeration, 1,000 ppm of citral caused a reduction of 5 log₁₀ cycles of E. coli at pH 7.0 and almost 3 log₁₀ cycles of L. monocytogenes at pH 4.0. The combination of citral and HHP also showed a synergistic effect. Our results have confirmed that the detection of sublethal injury after HHP may contribute to the identification of those treatment conditions under which HHP may act synergistically with other preserving processes.     

Pulsed electric fields cause sublethal injury in Escherichia coli

AIMS: The objective was to investigate the occurrence of sublethal injury in Escherichia coli by pulsed electric fields (PEF) at different pH values. METHODS AND RESULTS: The occurrence of sublethal injury in PEF-treated E. coli cells depended on the pH of the treatment medium. Whereas a slight sublethal injury was detected at pH 7, 99.95% of survivors were injured when cells were treated at pH 4 for 400 micros at 19 kV. The PEF-injured cells were progressively inactivated by a subsequent holding at pH 4. CONCLUSIONS: PEF cause sublethal injury in E. coli. The measurement of sublethal injury using a selective medium plating technique allowed prediction of the number of cells that would be inactivated by subsequent storage in acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF technology and contributes to the knowledge of the mechanism of microbial inactivation by PEF.     

Effects of pulsed electric fields on inactivation and metabolic activity of Lactobacillus plantarum in model beer

AIMS: Inactivation and sublethal injury of Lactobacillus plantarum at different pulsed electric field (PEF) strengths and total energy inputs were investigated to differentiate reversible and irreversible impacts on cell functionality. METHODS AND RESULTS: Lactobacillus plantarum was treated with PEF in model beer (MB) to determine critical values of field strength and energy input for cell inactivation. Below critical values, metabolic activity and membrane integrity were initially reduced without loss of viability. Above critical values, however, irreversible cell damage occurred. Presence of nisin or hop extract, during PEF treatment, resulted in an additional reduction of cell viability by 1;5 log cycles. Also, addition of the hop extract resulted in an additional two log cycles of sublethal injury. Partial reversibility of membrane damage was observed using propidium iodide (PI) uptake and staining. Inoculated MB containing hops was stored after PEF to evaluate the efficacy of such treatment for beer preservation. CONCLUSION: Cells were inactivated only above critical values of 13 kV x cm(-1) and 64 kJ x kg(-1); below these values cell damage was reversible. Storage experiments revealed that surviving cells were killed after 15 h storage in MB containing hops. SIGNIFICANCE AND IMPACT OF THE STUDY: Both reversible and irreversible cell damage due to PEF treatment was detected, depending on specific treatment conditions. The combination of PEF and hop addition is a promising nonthermal method of preservation for beer.     

Biosynthetic requirements for the repair of sublethal membrane damage in Escherichia coli cells after pulsed electric fields

AIMS: The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Escherichia coli cells after exposure to pulsed electric fields (PEF). METHODS AND RESULTS: The partial loss of the barrier and homeostatic functions of the cytoplasmic membrane was examined by adding sodium chloride to the recovery media. More than 4 log10 cycles of survivors were sublethally injured after PEF. Repair of such sublethal membrane damages occurred when survivors to PEF were incubated in peptone water for 2 h. Two different types of sublethally injured cells were detected. Whereas a small proportion (<5%) repaired after PEF in less than 2 min, the repair of the remaining 95% injured cells lasted 2 h and was dependent on biosynthetic requirements. The addition of inhibitors such as chloramphenicol, cerulenin, penicillin G, rifampicin and sodium azide to the liquid repair medium showed that the repair required energy and lipid synthesis, and was not dependent on protein, peptidoglican or RNA synthesis. CONCLUSIONS: Cell survival after PEF is dependent on the repair of the cytoplasmic membrane. Requirement of lipid synthesis for the repair of sublethally injured cells confirms that the cytoplasmic membrane is a target directly involved in the mechanism of inactivation by PEF. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge about the damages inflicted by PEF might help in the design of more efficient treatments.     

Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields

Two pathogenic microorganisms Escherichia coli O157:H7 and Staphylococcus aureus, suspended in peptone solution (0.1% w/v) were treated with 12, 14, 16 and 20 kV/cm electric field strengths with different pulse numbers up to 60 pulses. Pulsed electric field (PEF) treatment at 20 kV/cm with 60 pulses provided nearly 2 log reduction in viable cell counts of E. coli O157:H7 and S. aureus. S. aureus cells were slightly more resistant than E.coli O157:H7 cells. The results related to the effect of initial cell concentration of E. coli O157:H7 on the PEF inactivation showed that more inactivation was obtained by decreasing initial cell concentration. Any possible injury by PEF was also investigated after applying 20 kV/cm electric field to the microorganisms. As a result, it was determined that there was 35.92 to 43.36% injury in E. coli O157:H7 cells, and 17.26 to 30.86% injury in S. aureus cells depending on pulse number. The inactivation results were also described by a kinetic model.     

Pulsed electric field alters molecular chaperone expression and sensitizes Listeria monocytogenes to heat

Pulsed electric field (PEF)-resistant and PEF-sensitive Listeria monocytogenes strains were sublethally treated with electric pulses at 15 kV/cm for 29 micro s and held at 25 degrees C for 5 to 30 min prior to protein extraction. The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting. After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A). The levels of GroEL and DnaJ increased back to the basal expression level within 30 min. A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment. Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578. Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 micro s) and heat (55 degrees C, 10 min) were applied in sequence. When PEF and heat treatments were applied separately, the populations of L. monocytogenes Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml. Cells treated first with PEF and incubated at 25 degrees C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively. The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates. It is concluded that PEF sensitized L. monocytogenes to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level.     

Pulsed Electric Field Alters Molecular Chaperone Expression and Sensitizes Listeria monocytogenes to Heat

Pulsed electric field (PEF)-resistant and PEF-sensitive Listeria monocytogenes strains were sublethally treated with electric pulses at 15 kV/cm for 29 μs and held at 25°C for 5 to 30 min prior to protein extraction. The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting. After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A). The levels of GroEL and DnaJ increased back to the basal expression level within 30 min. A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment. Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578. Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 μs) and heat (55°C, 10 min) were applied in sequence. When PEF and heat treatments were applied separately, the populations of L. monocytogenes Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml. Cells treated first with PEF and incubated at 25°C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively. The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates. It is concluded that PEF sensitized L. monocytogenes to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level.     

Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF). METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli. This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water. However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators. PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods. Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.     

Sensitivity of heat-stressed yeasts to essential oils of plants.

Eight strains of yeasts (Candida lipolytica, Debaryomyces hansenii, Hansenula anomala, Kloeckera apiculata, Lodderomyces elongisporus, Rhodotorula rubra, Saccharomyces cerevisiae, and Torulopsis glabrata) were examined for changes in sensitivity to eight essential oils of plants (allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme) after being sublethally heat stressed. With the exception of garlic oil for all test yeasts, onion oil for S. cerevisiae, and oregano oil for R. rubra, the essential oils at concentrations of up to 200 ppm in recovery media did not interfere with colony formation by unheated cells. However, some oils, at concentrations as low as 25 ppm in recovery media, reduced populations of sublethally heat-stressed cells compared to populations recovered in media containing no test oils. This demonstrates that the yeasts were either metabolically or structurally damaged as a result of being exposed to elevated temperatures and that essential oils prohibited repair of injury. The size (diameter) of colonies produced on oil-supplemented recovery agar by heat-stressed cells was reduced compared to that observed on unsupplemented agar. Pigment production by heated R. rubra was inhibited by oils of oregano, savory, and thyme, but enhanced by garlic and onion oils. The influence of essential oils on survival of yeasts in thermally processed foods and in the enumeration of stressed cells in these foods should not be minimized.     

Sensitivities of Germinating Spores and Carvacrol-Adapted Vegetative Cells and Spores of Bacillus cereus to Nisin and Pulsed-Electric-Field Treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.     

Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10⁶ cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.     

Sensitivities of germinating spores and carvacrol-adapted vegetative cells and spores of Bacillus cereus to nisin and pulsed-electric-field treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

Environmental factors influencing the inactivation of Listeria monocytogenes by pulsed electric fields

AIMS: To investigate the influence of the growth phase, growth temperature, storage time, pH and aw of the treatment medium on the resistance of Listeria monocytogenes to pulsed electric fields (PEF). METHODS AND RESULTS: Square wave pulses of 2 micros at a frequency of 1 Hz and 25 and 28 kV cm(-1) were used. Cells were more PEF resistant in the stationary than in the exponential phase at both incubation temperatures investigated (4 and 35 degrees C). Cells grown at 4 degrees C were more PEF sensitive than cells grown at 35 degrees C independent of the growth phase. After a treatment of 25 kV cm(-1) and 800 micros, 1.48, 3.86 and 5.09 log10 cycles of inactivation were obtained at pH 7.0, 5.4 and 3.8, respectively. A reduction in the aw of the treatment medium protected cells against PEF treatments. CONCLUSIONS: The PEF resistance of L. monocytogenes depended on different environmental factors. The influence of growth conditions and treatment medium characteristics should be known and controlled to obtain reproducible and reliable PEF inactivation data. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous conclusions and misinterpretation of results are possible if factors affecting the PEF resistance of L. monocytogenes are not considered during PEF inactivation studies.     

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.