Factors that influence barotolerance of Listeria monocytogenes and the mechanism of inactivation by high pressure processing

by Hayman, Melinda.

Listeria monocytogenes is a Gram-positive bacterium that causes the foodborne disease listeriosis. Although listeriosis is rare it is of concern to the food industry due to the severity of the disease and the high fatality rate. Listeriosis is commonly associated with ready-to-eat (RTE) foods, for example soft cheeses. As a result, there is a zerotolerance policy for the presence of L. monocytogenes in RTE foods and many costly food recalls are due to the presence of this pathogen. High pressure processing (HPP) is a non-thermal technology that can be used to pasteurize foods while maintaining their fresh-like qualities. Hydrostatic pressures of up to 700 MPa are applied to inactivate microorganisms in foods, thereby extending shelf life and improving food safety. The mechanism(s) of microbial inactivation by HPP are not understood, but are thought to involve the cell wall, cell membrane, DNA and/or proteins. The aim of this research was to elucidate the mechanism(s) of inactivation of L. monocytogenes by HPP in milk. Factors that influence barotolerance of L. monocytogenes were also investigated as a way of identifying potential mechanism(s) of inactivation. Initial experiments investigating various cell targets were unsuccessful at elucidating a mechanism of inactivation by HPP. Transmission electron microscopy and other experiments showed that the cell wall and cell membrane of L. monocytogenes were not affected by HPP. The effects of growth phase (mid-exponential, late-exponential or mid-stationary) and growth temperature (4, 15, 25, 35 and 43°C) on the inactivation of L. monocytogenes by HPP at 400 MPa were investigated. Stationary-phase cells were significantly more barotolerant than midexponential-phase cells. Growth temperature also had a significant effect on barotolerance, which generally increased with increasing growth temperature. Tailing iii inactivation kinetics were observed in stationary phase cells grown at 35 or 43°C, but not in stationary phase cells grown at 4, 15 or 25°C or exponential-phase cells grown at 4, 15, 25, 35 or 43°C. The effect of water activity on the barotolerance of L. monocytogenes was also investigated. Lyophilized cells (starting concentration 7.5 x 10 iv 7 CFU/g) were suspended in water/glycerol solutions or left dry and HP-processed at 600 MPa for 5 min. Dry cells or cells suspended in 100% glycerol showed no inactivation; however cells suspended in 100% water were completely inactivated. Glycerol concentrations greater than 40% (aw = 0.86) significantly increased barotolerance and there was a log-linear relationship between glycerol concentration and log10 CFU/ml. The effect of heat shock on barotolerance was also investigated. L. monocytogenes was grown to stationary-phase at 15°C and heat shocked at 48°C. Heat shock significantly enhanced the barotolerance of L. monocytogenes at 400 MPa, with 5 min of heat shock conferring maximal barotolerance. Addition of chloramphenicol (a protein synthesis inhibitor) prior to heat shock resulted in barotolerance similar to that of non-heat-shocked cells, indicating that synthesis of heat shock proteins (which are involved in stabilization and/or renaturation of proteins) was responsible for increased barotolerance. The above results indicated that proteins may play an important role in barotolerance. Therefore, differential scanning calorimetry (DSC) was employed to investigate the effect of HPP on proteins in whole cells. Thermograms of non-pressure-treated cells of L. monocytogenes showed that the largest peak, which is associated with cellular proteins, occurred at approximately 70°C and was irreversible. Lethal high pressure treatments significantly reduced this peak, indicating that HPP caused protein denaturation in whole cells. While inactivation of L. monocytogenes by HPP may be multi-factorial, results indicated that protein denaturation plays an important or even dominant role in inactivation of L. monocytogenes. Understanding the mechanism of inactivation by HPP and the factors that influence barotolerance will aid in the development of process guidelines for the manufacture of safe HP-processed foods. v