Identification of Lactobacillus spp. and Fructobacillus spp. isolated from fresh Heterotrigona itama honey and their antagonistic activities against clinical pathogenic bacteria

Using an anaerobic and culture enrichment method, four strains of lactic acid bacteria (LAB) viz. Sy-1, Sy-2, Sy-3 and Sy-4 were isolated from fresh stingless bee Heterotrigona itama (Family: Apidae, Tribe: Meliponini, Genus: Trigona) honey collected from four geographical localities of Malaysia. Phylogenetic and comparative analyses of conserved region by 16S rRNA gene profiling identified strain Sy-1 being closely related to the genus Lactobacillus (>91%) but exhibited low level strains kinship to other known species within the genus. It is probable that strain Sy-1 is a novel species of the genus Lactobacillus. Conversely, strains Sy-2, Sy-3 and Sy-4 were phylogenetically located in the Fructobacillus fructosus subcluster with sequence similarities of 98, 96 and 95%, respectively. Fructobacillus fructosus was the dominant LAB following its isolation in three out of the four honey samples despite their different geographical origins. API 50 CHL test revealed the strains could proficiently utilize a wide range of carbohydrates, with fructose and glucose being the preferred substrates. All LAB strains possess excellent antagonistic activity against five different clinically pathogenic bacteria, as observed from the zones of inhibition. This study demonstrates the H. itama honey as a reservoir for novel LAB with probiotic properties, envisaging its potential therapeutic role in food.