Isolation and molecular identification of ethanol-tolerant Acetobacter species from Lansium domesticum (Dokong) and Nephelium lappaceum (Rambutan) vinegar

Abstract

Aims: Acetic acid bacteria (AAB) are a group of Gram-negative or Gram-variable bacteria that oxidise ethanol during the production of vinegar. The aim of this study was to isolate the AAB from both Lansium domesticum (Dokong) and Nephelium lappaceum (Rambutan), mother of vinegars (MV) and vinegars, as a potential starter culture for vinegar production.Methodology and results: The MV and vinegar samples were collected from six to eight weeks of fermented Dokong and Rambutan vinegar from the Food Laboratory of Universiti Malaysia Kelantan (UMK), Jeli. The enriched samples were inoculated on selective Carr and GYC solid media. From the Carr medium, thirty-seven isolates that showed a yellow clear zone and seventy-eight isolates that showed a halo clear zone on the GYC medium were selected. Sixty isolates that produced higher total acidity (>60%) were characterized by Gram staining. Sixteen Gram-negative and fourteen Gram-variable isolates were subjected to 2.0% ethanol Carr medium to select for ethanol tolerance. Five ethanol-tolerant isolates were suitable for 16S rRNA gene sequence analysis and molecular identification because they had 4% to 10% ethanol tolerance level utilisation on Carr solid medium. Based on the morphological and biochemical characteristics, isolates DV1 and RMV30 were Gram-variable. Meanwhile, RMV2, RMV19 and RMV37 were Gramnegative bacteria. RMV2, RMV19, RMV30 and RMV37 isolates were catalase-positive and oxidase negative. Only DV1 was catalase and oxidase positive. From the BLAST analysis, the obtained nucleotide sequences showed 100% homology, with RMV2, identified as Acetobacter fabarum, and DV1, RMV19, RMV30 and RMV37 were identified as A. pasteurianus.Conclusion, significance and impact of study: Based on 16S rRNA gene sequences, five isolates were identified as Acetobacter species: Four isolates, DV1, RMV19, RMV30 and RMV37 strains, were identified as A. pasteurianus and RMV2 was identified as A. fabarum. DV1, RMV2, RMV19, RMV30 and RMV37 showed significant differences at (p<0.05) for ethanol utilisation at 4% and the highest toleration up to an ethanol concentration of 10%. The ability to tolerate high ethanol concentration during vinegar fermentation is a desirable in producing high acetic acid for vinegar production.