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Interactions of Microorganisms with Electricity

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2000, Doctor of Philosophy, Ohio State University, Food Science and Nutrition.
Consumers’ demand for minimally processed foods with limited use of chemical additives led to the recent advances in nonthermal food preservation technologies. Pulsed electric field (PEF), high hydrostatic pressure, intense pulsed-light, ultraviolet light, and ultrasound are some of the newly developed technologies. The food industry is also interested in potent antimicrobials (e.g., ozone) as alternatives to conventional sanitizers. PEF is one of the emerging technologies in the food industry. PEF inactivates microorganisms in liquid foods such as orange juice, milk and liquid egg. The mechanism of microbial inactivation by PEF is not well understood. Further research is needed to confirm the mechanisms of microbial inactivation, identify the pathogens of most resistant to PEF, develop validation methods to ensure microbiological effectiveness, and standardize and develop effective methods for monitoring treatment. The goals of this study were (a) to verify the association between cell injury and death by PEF and the increase in membrane porosity, (b) to investigate the role of the cell envelope in the resistance to PEF, (c) to explore the factors that weaken cell envelope and sensitize cells to PEF, and (d) to investigate effect of sublethal ohmic electrical treatment on the growth beneficial bacteria. The association between cell injury and death by PEF and the increase in membrane porosity was verified by using a fluorescent nucleotide-binding probe, Propidium Iodide (PI) to quantify the membrane damage to PEF. Cell suspensions of Lactobacillus leichmannii ATCC 4797 Listeria monocytogenes Scott A and Escherichia coli O157:H7 were subjected to PEF. Cells treated or untreated with PEF were stained with PI, and changes in fluorescence intensities were measured by a spectrofluorometer. Increase in field strength decreased the count of survivors and proportionally increased the fluorescence intensity; this observation indicated that cell inactivation by PEF is caused by membrane damage. Cell envelope of a gram-negative bacterium (E. coli O157:H7) was modified by EDTA, lysozyme or their combination before applying the PEF treatment. The combination of lysozyme with PEF treatment did not significantly increase the inactivation of E. coli O157:H7 when compared to PEF treated cells (p >0.05). When cells were pre-treated with EDTA, followed by a PEF treatment, significantly higher inactivation (p < 0.05) was observed (2.1 log10 CFU/ml) than when PEF treatment was applied alone (1.8 log10 CFU/ml). More microbial inactivation (2.5 log10 CFU/ml) was obtained when PEF was applied to EDTA plus lysozyme treated E. coli O157:H7 cells (p <0.05). Fluorescence staining technique showed that pre-treatment with lysozyme and EDTA increased the PI uptake by the PEF-treated cells. Selected physical and chemical factors affecting cytoplasmic membranes (i.e., incubation temperature and ozone) were investigated for possible sensitization of cells to PEF. Ozone attack unsaturated fattys acids in membranes. Incubation temperature may alter cell membrane structure and fluidity. In this study, L. monocytogenes Scott A was grown at 7, 22 and 37°C and treated with PEF at 20 and 25 kV/cm. PEF treatment decreased the population of L. monocytogenes, which was grown at 7°C by 1.4 and 5.4 log10 CFU/ml, respectively. Cells grown at 22°C were inactivated 1.2 and 2.0 log10 CFU/ml by 20 and 25 kV/cm electric field strength The greater inactivation (3.3 and 6.1 log10 CFU/ml) was obtained when L. monocytogenes Scott A cells were grown at 37°C and treated with 20 and 25 kV/cm electric field intensities, respectively. Relative fluorescence intensities significantly increased by PEF treated (20 kV/cm) cells when incubation temperature was increased. The higher the incubation temperature, the higher the inactivation and relative fluorescence intensity were determined. Lb. leichmannii, E. coli, and L. monocytogenes were suspended in 0.1% NaCl and treated with ozone, PEF and ozone plus PEF. Cells were treated with 0.25 to 1.00 µg ozone/ml of cell suspension, PEF at field strengths of 10 to 30 kV/cm; and selected combinations of ozone and PEF. Synergy between ozone and PEF varied with the treatment level and the bacterium treated. Lb. leichmannii, treated with PEF (20 kV/cm) after exposure to 0.75 and 1.00 µg/ml ozone, was inactivated by 7.1, and 7.2 log10 CFU/ml, respectively. However, ozone at 0.75 and 1.00 µg/ml and PEF at 20 kV/cm inactivated 2.2, 3.6 and 1.3 log10 CFU/ml, respectively. Ozone at 0.5 and 0.75 µg/ml inactivated 0.5 and 1.8 log10 E. coli CFU/ml, respectively, and PEF at 15 kV/cm inactivated 1.8 log10 CFU/ml. Ozone (0.5 and 0.75 µg/ml) followed by PEF (15 kV/cm) inactivated 2.9 and 3.6 log10 CFU/ml, respectively. Population of L. monocytogenes decreased 0.1, 0.5, 3.0, and 0.8 log10 CFU/ml when treated with 0.25, 0.5, and 0.75 µg ozone/ml, and PEF (15 kV/cm), respectively. However, when the bacterium was treated with 15 kV/cm, after exposure to 0.25, 0.5, and 0.75 µg ozone/ml, 1.7, 2.0, and 3.9 log10 CFU/ml were killed, respectively. Exposure of microorganisms to ozone followed by the PEF treatment showed a synergistic bactericidal effect. Synergy was more apparent at mild than severe doses of ozone, and when the combination treatment was applied to Lb. leichmannii than to E. coli or L. monocytogenes. Lag period of Lactococcus lactis subsp. lactis ATCC 11454 decreased 2.5, 1.8, and 1.56 hours by low-voltage ohmic heating when compared with conventional heating at 25, 30, and 37°C, respectively. Sublethal ohmic heating resulted in lower nisin activity in the fermented medium than conventional heating. Electrical current increased during the fermentation probably due to the production of lactic acid and other polar components. In conclusion, a fluorescence staining technique was useful in accessing membrane damage associated with PEF treatments and in determining relative sensitivities of microorganisms to PEF. The efficacy of PEF against E. coli O157:H7 increased when the cell envelope was altered by the combination of EDTA and lysozyme. L. monocytogenes Scott A grown at 37°C were easily inactivated by PEF, when compared to those grown at 7 and 22°C. Exposure of cells to ozone sensitized them to the action of PEF. It might be useful in food fermentations to apply electricity to decrease the lag period of cultures at the initial stage of growth, and then apply conventional heating at the later stages. The measurement of electrical current when ohmic heating is applied at a constant voltage may be used to monitor the growth of culture instead of plate counting or measuring absorbance.
Ahmed E. Yousef (Advisor)
David Min (Committee Member)
Sudhir K. Sastry (Committee Member)
Thomas H. Shellhammer (Committee Member)
156 p.

Recommended Citations

Citations

  • Unal, R. (2000). Interactions of Microorganisms with Electricity [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1392802714

    APA Style (7th edition)

  • Unal, Ragip. Interactions of Microorganisms with Electricity. 2000. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1392802714.

    MLA Style (8th edition)

  • Unal, Ragip. "Interactions of Microorganisms with Electricity." Doctoral dissertation, Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu1392802714

    Chicago Manual of Style (17th edition)