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J. LEE, Dept. of Botany, Miami Univ., Pearson Hall, 352, Oxford, OH 45056 and G. Kaletunç, Dept. of Food, Agricultural & Biological Engineering, Ohio State Univ., 590 Woody Hayes Dr., 210 Agricultural Engineering Bldg., Columbus, OH 43210-1058. DSC thermograms of bacteria display several overlapping endothermic peaks associated with various cellular components. Some peaks were assigned to particular cellular components by comparison to the transition temperatures of isolated cell components. Use of thermal data on isolated components to interpret complex systems, such as bacteria, ignores interactions between the components and the potential alteration of thermal properties as a result of the separation process. This study aims to evaluate bisbenzimide as a probe to identify the cellular DNA transition in vivo from whole cell DSC thermogram. Bisbenzimide is a membrane permeable stain which binds A-T regions of double stranded DNA and known to increase the thermal stability of DNA molecule. Two bisbenzimide molecules (Hoechst 33258 and 33342) were used to prepare solutions at concentrations of 0.005, 0.01, 0.05, 0.25 or 0.5 mg/ml-1. Bisbenzimide was used to detect DNA transition in control, pressure treated, and pressure-nisin treated cells of Escherichia coli and Salmonella Enteritidis. A portion of bacterial cell pellet was suspended in bisbenzimide solution at 1:100 (wt/vol) ratio, incubated at 37 oC for 30 min, and centrifuged to obtain cells pellets. Pellets were transferred to DSC crucibles and thermograms were recorded from 1 to 150 oC at 4 oC/min. The optimum bisbenzimide concentration was determined to be 0.005 mg/ml-1 above which the ribosomal peak transition temperature increased suggesting DNA site was saturated. Bisbenzimide increased the transition temperature of putative DNA peak by 6~7 oC in whole cell DSC thermogram of control and pressure treated cells thereby confirming the endothermic peak corresponding to DNA melting transiton in vivo. Using bisbenzimide as a probe in DSC thermogram enables us to determine the thermal stability of DNA in a bacterial cell in vivo as well as to monitor the bacterial DNA damage as a result of physical or chemical treatments used during food preservation
Session 33, Food Microbiology: General
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |