A total of 32 lactic acid bacteria (LAB) were isolated from 13 honey samples commercially marketed in Malaysia, 6 strains identified as Lactobacillus acidophilus by API CHL50. The isolates had antibacterial activities against multiple antibiotic resistant's Staphylococcus aureus (25 to 32 mm), Staphylococcus epidermis (14 to 22 mm) and Bacillus subtilis (12 to 19 mm) in the agar overlay method after 24 h incubation at 30 °C. The crude supernatant was heat stable at 90 °C and 121 °C for 1 h. Treatment with proteinase K and RNase II maintained the antimicrobial activity of all the supernatants except sample H006-A and H010-G. All the supernatants showed antimicrobial activities against target bacteria at pH 3 and pH 5 but not at pH 6 within 72 h incubation at 30 °C. S. aureus was not inhibited by sample H006-A isolated from Libyan honey and sample H008-D isolated from Malaysian honey at pH 5, compared to supernatants from other L. acidophilus isolates. The presence of different strains of L. acidophilus in honey obtained from different sources may contribute to the differences in the antimicrobial properties of honey.

Problem statement: This study reports the isolation of lactic acid bacteria from 13 honey samples produced in Malaysia, Libya and Saudi Arabia and their antibacterial activity against three Gram negative pathogenic bacteria. Approach: A modified MRS agar with 0.8% CaCO3 and MRS with 1% glucose was found to facilitate isolation of LAB compared to MRS, tomato juice agar and modified tomato juice agar. 32 isolates were confirmed LAB by catalase test and Gram staining. Six isolates were screened for antibacterial activity and identified as strains of Lactobacillus acidophilus 1 by API CH50. Results: All the isolates showed very good inhibitory activity against target Gram negative bacteria as indicated by the diameter of inhibition zone: Salmonella Typhimurium (23-30 mm), Escherichia coli (7-18 mm) and Enterobacter aerogenes (10-18 mm) after 24 h incubation at 30�C. Supernatants of L. acidophilus 1 strains showed good antibacterial activity against all target bacteria. Heating the supernatants at 90 and 121�C for 1 h enhanced the antibacterial activity against all target bacteria except supernatants H006-A and H010-G against S. Typhimurium. Antibacterial activity of supernatants were maintained after pH adjustment to 3, but at pH5 supernatants H006-A, H008-D and H010-G lost the activity against S. Typhimurium and E. coli within 48 h of incubation while at pH 6 all supernatants lost activity except against E. aerogenes. Enzymes treatments of supernatants with RNase II and Proteinase K for 1 h inhibited all target bacteria except supernatants H008-D, H008-E and H006-A which were relatively sensitive to both enzymes against S. Typhimurium and E. coli. Conclusion/Recommendations: In conclusion, honey from different sources contains strains of L. acidophilus 1 that produced compounds with good antibacterial activity which may be responsible for the antibacterial properties of honey. � 2012 Science Publications.

Honey is rich with complex natural components which could be useful as antibacterial agents or as preservative. Honey contains high concentration of sugars, low amount of water, high osmolality and often dark colour which influence its antibacterial activity. Disc diffusion, well method, micro dilution assay are methods commonly used to determine the antibacterial activity of honey. In this study, microtiter and microbial plate count were included to ascertain the potency of honey as antibacterial agent against multiple antibiotic resistant pathogenic bacteria (Staphylococcus aureus, Salmonella Typhimurium, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa) with concentration of 0.2 g mL-1. Results found that well diffusion method tends to give higher inhibitory zone than disc diffusion method but there was no correlation among the bacteria was observed except for S. Typhimurium, E. coli (R = 0.310, 0.505 and 0.316, respectively). Nan photometer assay and microtiter plates assay showed comparable results with moderately strong correlation (R2 = 0.681 and 0.767, respectively) for S. aureus and S. typhimurium, but poor correlation was found for E. coli, B. subtilis and P. aeruginosa (R2 = 0.441, 0.308 and 0.383, respectively). Determining the number of survivors by plating on agar after nanophotometer assay or microtiter plate assay had confirmed the effectiveness of honey as antimicrobial agent against target bacteria; which confirmed that honey has the potency to inhibit pathogens even at low concentration. � 2014 M.M. Aween et al.