Title: Use of agar diffusion assay to evaluate bactericidal activity of formulations of alkaline salts of fatty acids against bacteria associated with poultry processing Authors
Submitted to: Journal of Food Safety
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 12, 2011
Publication Date: July 4, 2011
Citation: Hinton Jr, A., Ingram, K.D. 2011. Use of agar diffusion assay to evaluate bactericidal activity of formulations of alkaline salts of fatty acids against bacteria associated with poultry processing. Journal of Food Safety. 31:357-364. Interpretive Summary: The agar diffusion method was used to examine the ability of fatty acids dissolved in potassium hydroxide (KOH) to kill bacteria associated with poultry processing. The experiments were performed by making small wells in solid agar media that had been inoculated with test bacteria. The empty wells were then filled with fatty acid-KOH solutions, and the plates were incubated to allow the bacteria to grow. Zones of inhibition of bacterial growth around the agar wells by the fatty acid-KOH solutions were measured after incubation. The first set of tests was performed to determine the ability of different concentrations of lauric acid-KOH to inhibit bacterial growth. Results indicated that higher concentrations of lauric acid-KOH produced larger zones of inhibition of bacterial growth. Additional test were then performed to compare the ability of several other fatty acid solutions, including lauric acid, to kill these bacteria. Caproic acid-KOH inhibited the growth of 1 of 8 bacteria; myristic acid inhibited 2 of 8 bacteria; caprylic acid inhibited 6 of 8; and capric and lauric acids inhibited all 8 bacteria. Adjusting the pH of these solutions from 9.5 to 12.5 produced little change in the ability of the solutions to kill the bacteria, but adjusting pH to 13.5 or higher increased the ability of the solutions to kill these bacteria. Other experiments showed that conditions created by KOH alone killed bacteria at pH 13.5 or higher, but fatty acids were primarily responsible for killing bacteria at pH 12.5 or lower. Findings from these experiments indicate that several fatty acids can be used to kill bacteria associated with poultry processing. These experiments also showed that the agar diffusion method can be used to quickly screen fatty acid formulations for their ability to kill bacteria.
Technical Abstract: The agar diffusion assay was used to examine antibacterial activity of alkaline salts of fatty acids (FA). Wells in agar media seeded with bacteria were filled with FA-potassium hydroxide (KOH) solutions, plates were incubated, and zones of inhibition were measured. The relationship between bactericidal activity and FA-KOH concentration was examined using 2% lauric acid-1% KOH, 5% lauric acid-2.5% KOH, 10% lauric acid-5% KOH, or 20% lauric acid-10% KOH. Significantly (P < 0.05) larger zones of inhibition were produced in wells with higher concentrations of lauric acid-KOH. Additional experiments examined effects of pH on inhibitory activity of 0.5 M caproic, caprylic, capric, lauric, or myristic acids acid in 1 M KOH adjusted to pH 9.5-13.5 with citric acid. Caproic acid-KOH inhibited C. jejuni, only; myristic-KOH inhibited Gram positive cocci, only; caprylic-KOH inhibited all isolates except Staphylococcus and Acinetobacter; while capric- and lauric-KOH inhibited all isolates. Results also indicated that there was generally no significant (P < 0.05) difference in the antibacterial activity of FA-KOH between pH 9.5-12.5; however, most FA-KOH solutions exhibited greater antibacterial activity at pH 13.5 or higher. Experiments were also conducted to determine the antibacterial activity of 1 M KOH solutions containing no added FA and adjusted to pH 9.5-13.5. Results indicated that 1 M KOH inhibited bacterial growth at pH 13.5 or higher. Findings demonstrated that alkaline salts of FA possess antibacterial activity towards bacteria associated with poultry processing. Findings also showed that the agar diffusion assay can be used to examine antibacterial activity of these solutions.