<ol> <li>To evaluate and model the impact of high-pressure processing parameters (pressure, temperature and residence time) on the destruction, injury, recovery and growth of Listeria monocytogenes and other pathogens of concern in fluid milk.
<li>To evaluate ultrasound technology for destruction of vegetative and spore-forming pathogens in milk.
<li>To identify non-microbial indicators of pasteurization by high pressure processing and ultrasound and to develop biosensors from the best candidates.
<li>To develop a Multi-Locus Sequence Typing method for molecular tracking of Listeria spp. and use that method to identify and eliminate sources of post-pasteurization contamination in dairy plants.
<li>To develop and implement effective educational programs that can be used to a) increase awareness of biosecurity issues among players in the dairy production system, b) strengthen community readiness to handle a biosecurity event that might occur in a local dairy unit and c) encourage adoption of HACCP-based biosecurity measures in large and small dairy processing plants.</ol>
Non-thermal processes that promise to deliver fresh, high quality dairy foods are not well defined or validated. Post-pasteurization contamination with L. monocytogenes remains a significant risk factor. Dairy processors need to implement appropriate biosecurity measures to protect consumers. This project examines destruction of pathogens in dairy products by high pressure and ultra-sound and molecular tracking of L. monocytogenes to prevent recontamination. This project also will study how to best implement biosecurity training programs in the dairy industry.
Cells of L. monocytogenes and other pathogens will be mixed into raw milk and then subjected to HPP-treatments. Kinetics of destruction, injury and recovery of the organism will be studied. To assess damage and repair to the cytoplasmic membrane, cells will be HPP-injured and then exposed to dyes that typically do not penetrate normal cells with intact cytoplasmic membranes, but can penetrate cells whose cytoplasmic membranes are damaged. Dynamic equilibrium equations for the pressure field within the fluid milk will be solved using the finite element method. <p>
Objective 2. A number of parameters will be explored to define conditions at which microorganisms will be destroyed using high frequency ultrasound. These variables include, but are not limited to, the following: Ultrasound characteristics: Pulsed and continuous wave modes, sound frequency (60-200 kHz), bursts (1000 and 2000 per cycle), and pulse repetition frequency (50 and 100 ms), and exposure/contact time (10-300 sec). Suspending media: broth, milk (nonfat, 1% 2% and whole), volume levels, and initial temperature Objective 3. Effect of high pressure and ultrasound on enzyme inactivation. Alkaline phosphatase, lactic dehydrogenase, gamma-glutamyltransferase, aspartate, amino-transferase, glucose oxidase (GOX)and cholesterol oxidase (COX) will also be examined. The change in the structure or the extent of denaturation will be assessed by examining the spectra of these enzymes at various high pressure and ultrasound treatments proposed for pasteurization. Standard experiments for ALP activity will be conducted; parallel experiments will also be conducted for examination of the nature of denaturation by Raman spectroscopy.
Objective 4. A DNA microarray will be developed and used to identify genes that are common to all species and strains of Listeria. These genes will be amplified by PCR, sequenced at the PSU Nucleic Acid Facility, compared using appropriate software and a set of genes that give maximum discriminatory power will be selected to yield an optimum MLST method. Primer pairs for 3-6 genes identified above, which yield amplicons of similar size and Tm, will be identified and selected for multiplex PCR analysis. Listeria isolates from dairy plants will be tested using the multiplex PCR MLST method and the data analyzed to identify common sources of contamination and cost-effective intervention strategies.
Objective 5. Data on awareness of perceptions of risk and capacity for containment of bio-terrorism events would be gathered using key informant interviews with individuals representing a) four sectors in the dairy production system, and b) all five levels in the social ecological model (Gregson et al., 2001). The study team would identify 60-100 individuals to be interviewed, based on reputation, occupation, breadth of experience, and degree of representation of an informant group. Ultimately the results common across sectors and levels would produce a picture of the strengths and weaknesses in the dairy system for handling a bio-terrorism event and inform the production of educational materials.</p>
A literature review has been completed and specific experimental objectives have been established for studying the effect of high pressure processing (HPP) on Listeria monocytogenes in milk. A flow cytometry method has been established to investigate membrane damage of Listeria innocua after low-level thermal treatments. Preliminary data suggests that this method can be used to differentiate live cells from dead cells on the basis of membrane permeability. Collaborative research has been undertaken with Dr. Chen at the University of Delaware to investigate post-HPP recovery of L. monocytogenes at different stages of growth. Preliminary data has been collected and further HPP experiments will be conducted in the near future to determine the minimum process required to yield 100% injury. As a first step in examining the stability enzymes as indicators of pasteurization, two model systems, glucose oxidase and cholesterol oxidase were examined for their stability when binding with nanoparticles. Two different chemistries were developed for attachment of enzymes to inorganic materials, the foundation of biosensor construction. Fourier Transformed Infrared (FTIR) procedures and kinetic analysis protocols were finalized for assessment of enzyme stability. PCR conditions for Multi-Virulence-Locus Sequence Typing (MVLST) of Listeria monocytogenes were optimized by utilizing Touchdown PCR and Hot-Start and by designing new primers for clpP and dal. Sets of L. monocytogenes isolates associated with specific outbreaks were obtained from the Centers for Disease Control and Cornell University. MVLST and PFGE analysis of these sequences will be compared to determine which method yields better epidemiological relevance. Literature and database searches were conducted and the following were identified as the best candidates for a Multi-Locus-Sequence Typing approach for molecular subtyping of Listeria spp.: RecA, SigB, iap, ldh, 16S rDNA. Sequence analysis using these genes is ongoing.</p>
We anticipate that this project will result in the application of HPP for eliminating L. monocytogenes from raw milk through understanding the mechanisms of inactivation and the subsequent modeling of inactivation through combined heat and pressure processing. Understanding interactions between Penicillium and pressure-injured L. monocytogenes will help us improve the safety of soft cheeses. Research may result in the use of novel nonmicrobial markers as indicators of adequate pasteurization of milk. Optimizing MVLST subtyping for Listeria monocytogenes and development of a MLST scheme for subtyping Listeria spp. will help food processors identify and eliminate routes of Listeria contamination in dairy plants. </p>