Identification of lactic acid bacteria, physicochemical properties and antibacterial activities of Malaysian heterotrigona itama honey

Abstract

Honey produced by Malaysian stingless bee, Heterotrigona itama (H. itama) is highly valued for its numerous therapeutic benefits. Despite the extensive studies on lactic acid bacteria (LAB), data on LAB from raw Malaysian H. itama honey is unavailable. Moreover, there is a huge demand for novel antibacterial agents as the issue of multi-drug resistant bacteria is on the rise. Natural compounds such as honey may serve as a possible source of reliable solutions to this problem. Herein, this study reports the isolation and identification of LAB as well as physicochemical properties, antioxidant and antibacterial activities of H. itama honey against pathogenic bacteria. This study also reports the changes in the nutritional properties of H. itama honey by assessing the changes in LAB population, physicochemical aspects and antioxidant activity. Four LAB strains, Sy-1, Sy-2, Sy-3 and Sy-4 were successfully isolated from raw H. itama honey. Based on 16S rRNA analysis, strain Sy-1 is closely related with the Lactobacillus genus (>91%) while strains Sy-2, Sy-3 and Sy-4 are phylogenetically grouped in the Fructobacillus fructosus subcluster with sequence similarities of 98%, 96% and 95%, respectively. API 50 CHL test showed that these LAB strains preferred fructose and glucose as substrates. The LABs showed good antibacterial activities against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Physicochemical analysis demonstrated that H. itama honey has lower pH (2.7 ± 0.19) but higher in moisture (30.3 ± 0.56%) and protein content (15156 ± 2 mg/kg), than the honey of sting bee Apis mellifera. HPLC data showed H. itama honey has low glucose (12.05 ± 0.54 g/100 g) and fructose contents (9.94 ± 0.43 g/100 g), high in maltose (25.7 ± 1.66 g/100g) and absent of sucrose. Antioxidant activities of the honey were significantly correlated to phenolic contents (p < 0.01) but were moderately associated to colour and flavonoid contents. Agar well diffusion assay showed H. itama honey are more potent against Gram-positive bacteria (31.38 ± 3.12 mm) than Gram-negative bacteria (23.75 ± 5.13 mm). Importantly, honey Sy-1 exhibited the strongest antioxidant and antibacterial activities amongst the honey samples. A 28-day storage duration test of the H. itama honey showed that viability of LAB population in honey reduced significantly (p < 0.05), meanwhile pH, moisture content, phenolic and flavonoid contents as well as antioxidant capacity remained relatively stable (p > 0.05). This study demonstrated that Malaysian H. itama honey is not only a valuable reservoir for new LAB, but it also preserves probiotic properties and is rich in nutritious elements. Thus, the findings support the potential therapeutic use of LAB isolated from raw Malaysian H. itama honey in treating various ailments.