FACTORS THAT INFLUENCE BAROTOLERANCE OF LISTERIA MONOCYTOGENES AND THE MECHANISM OF INACTIVATION BY HIGH PRESSURE PROCESSING
Open Access
- Author:
- Hayman, Melinda
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 18, 2007
- Committee Members:
- Stephen John Knabel, Committee Chair/Co-Chair
Ramaswamy C Anantheswaran, Committee Chair/Co-Chair
John Floros, Committee Member
Allen T Phillips, Committee Member
Hassan Gourama, Committee Member
Maryann Victoria Bruns, Committee Member - Keywords:
- injury
high pressure processing
Listeria monocytogenes
differential scanning calorimetry - Abstract:
- 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-milk cheeses. There is a zero-tolerance policy for the presence of L. monocytogenes in RTE foods and many 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 may not be affected by HPP. The effects of growth phase (exponential, late-exponential or mid-stationary) and growth temperature (4, 15, 25, 35 and 43°C) on inactivation of L. monocytogenes by HPP at 400 MPa were investigated. Stationary phase cells were significantly more barotolerant than exponential phase cells. Growth temperature also had a significant effect on barotolerance, which generally increased with increasing growth temperature. Tailing 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 107 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, but cells suspended in 100% water were completely inactivated. Glycerol concentrations greater than 40% (aw = 0.80) significantly increased barotolerance, there was a log-linear relationship between glycerol concentration and log CFU/ml of survivors. 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 reduced barotolerance to the level of non-heat-shocked cells, indicating that synthesis of heat shock proteins (which are involved in stabilization and/or renaturation of proteins) were 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 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, our results indicate that protein denaturation plays an important or even dominant role. Understanding the mechanism of microbial inactivation by HPP and the factors that influence barotolerance will aid in the development of process guidelines for the manufacture of HP-processed foods.