RESUMO
The production of vinegar on an industrial scale from different raw materials is subject to constraints, notably the low tolerance of acetic acid bacteria (AAB) to high temperatures and high ethanol concentrations. In this study, we used 25 samples of different fruits from seven Moroccan biotopes with arid and semi-arid environmental conditions as a basic substrate to isolate thermo- and ethanol-tolerant AAB strains. The isolation and morphological, biochemical and metabolic characterization of these bacteria allowed us to isolate a total number of 400 strains with characters similar to AAB, of which six strains (FAGD1, FAGD10, FAGD18 and GCM2, GCM4, GCM15) were found to be mobile and immobile Gram-negative bacteria with ellipsoidal rod-shaped colonies that clustered in pairs and in isolated chains. These strains are capable of producing acetic acid from ethanol, growing on peptone and oxidizing acetate to CO2 and H2O. Strains FAGD1, FAGD10 and FAGD18 show negative growth on YPG medium containing D-glucose > 30%, while strains GCM2, GCM4 and GCM15 show positive growth. These six strains stand out on CARR indicator medium as isolates of the genus Acetobacter ssp. Analysis of 16S rDNA gene sequencing allowed us to differentiate these strains as Acetobacter fabarum and Acetobacter pasteurianus. The study of the tolerance of these six isolates towards pH showed that most of the six strains are unable to grow at pH 3 and pH 9, with an ideal pH of 5. The behavior of the six strains at different concentrations of ethanol shows an optimal production of acetic acid after incubation at concentrations between 6% and 8% (v/v) of ethanol. All six strains tolerated an ethanol concentration of 16% (v/v). The resistance of the strains to acetic acid differs between the species of AAB. The optimum acetic acid production is obtained at a concentration of 1% (v/v) for the strains of FAGD1, FAGD10 and FAGD18, and 3% (v/v) for GCM2, GCM4 and GCM15. These strains are able to tolerate an acetic acid concentration of up to 6% (v/v). The production kinetics of the six strains show the highest levels of growth and acetic acid production at 30 °C. This rate of growth and acetic acid production is high at 35 °C, 37 °C and 40 °C. Above 40 °C, the production of acid is reduced. All six strains continue to produce acetic acid, even at high temperatures up to 48 °C. These strains can be used in the vinegar production industry to minimize the load on cooling systems, especially in countries with high summer temperatures.
RESUMO
Industrially, the sensitivity of acetic acid bacteria (AAB) to the high temperatures and the high ethanol concentrations is the major concerns for manufacturers. This study was conceived and designed to isolate and identify new thermo- and ethanol-tolerant AAB from Opuntia ficus-indica L. fruits. As a result, among 140 isolated bacterial strains, five selected strains (CR1, CR5, CR23, CZ2, and CZ15) exhibited important acetic acid production until 40 °C. The use of 16S rDNA gene analysis was insufficient to identify selected bacteria. Indeed, except CR5 that presented 100% similarity to A. cerevisiae, the other strains presented similar homology rates simultaneously to the 16S rDNA sequences of A. cerevisiae and A. malorum. The reidentification by 16S-23S rDNA gene sequencing showed that CR1, CR23, and CZ15 were A. malorum, which were shown tolerance to the highest concentration of ethanol (12%) and produced elevated amount (40 g/L) of acetic acid at 37 °C. In summary, we showed the thermotolerance and ethanol tolerant character of new A. malorum strains, which can be used as a starter for vinegar production. Furthermore, during the molecular characterization of the isolated strains, we concluded that 16S-23S rDNA internal transcribed spacer sequence is of great importance for discriminating between AAB species as a complement to the identification by 16S rDNA sequencing.