Interaction effects of fumaric acid, pH and ethanol on the growth of lactic and acetic acid bacteria in planktonic and biofilm states.

The microbial stability of wine can be compromised by the presence of lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which can cause spoilage via off flavour production, increased acetic acid production, or biofilm formation. To manage the growth of LAB in winemaking, fumaric acid (FA) has been proposed as an alternative to traditional antimicrobial agents, such as sulfur dioxide (SO2). This study aimed to evaluate the inhibitory effects of FA on the growth of LAB and AAB based on the influence of pH and ethanol in a synthetic wine-like medium. The research involved the determination of the individual, 2 × 2 combined, and combined minimum inhibitory concentrations (MICs) of fumaric acid, pH, and ethanol. Specifically, the MIC90 was defined as the concentration required to inhibit the growth of more than 90 % of the initial population, and the MIC50 was defined as the concentration required to inhibit the growth of more than 50 % of the initial population. These thresholds were assessed in 19 bacterial strains (13 LAB and 6 AAB strains) at pH values of 3.5 and 4.0 and ethanol concentrations of 0, 4, 8 and 12 % v/v. Additionally, the impact of FA on biofilm formation was evaluated in the ten bacterial strains that were observed to be most resistant to FA. The results revealed that the inhibitory effects of FA were enhanced at lower pH values and at higher ethanol concentrations. LAB strains (such as Oenococcus oeni) were particularly sensitive to FA, whereas non-Oenococcus LAB strains demonstrated resistance to concentrations exceeding 2 g/L under the tested pH (3.5-4.0) and ethanol (0-12 % v/v) conditions. AAB strains (such as Acetobacter aceti) tolerated FA concentrations greater than 2 g/L at pH 4.0 in the absence of ethanol; however, the susceptibility increased with increasing ethanol concentrations and decreasing pH. Furthermore, FA significantly inhibited biofilm formation (particularly at a pH of 3.5 and ethanol concentrations greater than 8 % v/v). In conclusion, when combined with low pH and high ethanol concentrations, FA offers a promising strategy for controlling bacterial growth and biofilm formation in winemaking. This approach has the potential to complement or replace the use of traditional chemical preservatives, such as SO2.