High hydrostatic pressure effects on bacterial bioluminescence
Abstract
Consumers' demands for fresh-like food products that are free of microbial contamination have provided opportunities for high pressure processing applications. This technology can inactivate microbial vegetative cells while preserving food sensory and nutritional qualities. However, the mechanisms that promote this means of microbial inactivation have not been fully understood. In this study, a high-pressure system was developed to monitor cellular metabolism using in situ bioluminescence. Preliminary characterization of the high pressure system was performed using Pseudomonas fluorescens 5RL expressing the lux genes from Vibrio fischeri. Initial stepwise increases in pressure below 34 MPa resulted in a slight increase in luminescence while pressures at 34 MPa and above resulted in decreased bioluminescence. Bioluminescence from square wave exposure to pressures of 69, 103 and 138 MPa showed reductions greater than 95%, however, when cells were returned to ambient pressure light values returned to 50, 39, and 4% of the initial light values, respectively. One possible explanation for these results is the reversible denaturation of the lux enzymes. Since previous reports have suggested a correlation between temperature and pressure on protein structure, two strains of E. coli were constructed expressing lux proteins from Photorhabdus luminescens or Vibrio fischeri which have different thermal stabilities. The strains were exposed to square wave perturbations as described above. The E. coli strain expressing the lux proteins from V. fischeri showed similar results to P. fluorescens 5RL. The E. coli strain expressing the lux proteins from P. luminescens showed similar luminescent recovery values to the strain expressing the V. fischeri lux proteins, however the difference in recovery rate was 33 fold. These results show a correlation between thermal and pressure stability of the lux proteins. The kinetics of the luminescence suggest that square wave exposure to pressure induces protein denaturation/renaturation. The rapid recovery of the E. coli strain containing the lux proteins from P. luminescens might also suggest that the proteins did not denature but other aspects of cell metabolism were affected.
Degree
M.S.
Advisors
Applegate, Purdue University.
Subject Area
Food Science
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.