[5-1130-P] Other Categories (5th)

[5-1130-P-29] Isolation and Identification of Acetic Acid Bacteria from Philippine Fermented Rice Cake Batters by 16S rRNA Gene Sequence Analysis

Audrey Mae Villamin Orillaza¹, Honey Bhabes R Iñigo¹, *Baby Richard Ragudo Navarro¹ (1. Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los Baños(Philippines))

Keywords:Acetic acid bacteria, fermented rice cake, 16S rRNA sequence analysis, phylogenetics

As part of our work to study the microflora of Philippine traditional fermented foods, batters from fermented rice cakes, or puto in the vernacular, from different parts of the Philippines were sampled and used for the isolation, screening and purification of acetic acid bacteria (AAB) by culture-based methods. Pure AAB isolates were then identified by DNA-based methods [i.e., cetyl trimethylammonium bromide (CTAB) DNA extraction, polymerase chain reaction (PCR), and 16S rRNA gene sequence analysis], DNA base composition determination, phenotypic characterization, and phylogenetic analysis. Six isolates were obtained from three types of rice cake batter: puto Calasiao, puto Lanson, and puto Boac batters. The AAB isolates were identified to belong to the genera Acetobacter at 94-99% homology with DNA base compositions ranging from 54.40-55.74 mol% GC content. The isolates were Gram-negative, catalase-positive rods that oxidize ethanol to acetic acid and grow in mannitol agar and in most sugars. None of them were cellulose producer or motile. 02CPPu1-2 produced a water-soluble brown pigment in glucose-yeast extract-peptone (GYP) medium and 24BMTa2-3 yielded γ-pyrones from D-glucose. From the phylogenetic tree deduced from the 16S rRNA gene sequence analysis results, the isolates clearly formed an independent clade distinct from the type strains of other genera of acetic acid bacteria. The puto Lanson and puto Boac batter isolates were closely related to A. pasteurianus and A. lovaniensis, respectively. On the other hand, the puto Calasiao isolates were associated with none of the type species of AAB. Overall, our data suggest that the fermented rice cake batter isolates comprise a possibly newspecies of acetic acid bacteria under the genus Acetobacter. This is very interesting considering that all the isolates were sourced from batters of only traditionally fermented rice cakes. DNA-DNA hybridization and detailed phenotypic characterization are recommended to verify this new species possibility, which may be linked the difference in geographical location, raw material and processing technique employed in traditional rice cake making in the Philippines.