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Enhancing Microbial Food Safety by Risk Analysis

D'Amico, Dennis
University of Connecticut
Start date
End date
Risk Assessment: Assess food safety risks in agriculture systems
Risk Management: Develop science-based interventions to prevent and mitigate food safety threats
Risk Communication: Communicate food safety messages to stakeholders
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NON-TECHNICAL SUMMARY: Unpasteurized milk and milk products can be a vehicle of foodborne illness and small-scale producers are often considered a higher risk. With consumer interest driving explosive growth of small-scale dairy manufacturing operations throughout the United States there is a need to better understand and identify the risks associated with this industry. Based on previous outbreak information and the lack of validated control strategies, there is a critical need for actionable, science-based interventions to mitigate these threats. In order to effect change and preserve this burgeoning industry, results of this basic and applied research must also reach the target populations including producers, regulators, and inspectors as well as those who advise them. This project integrates risk-based research and outreach to improve food safety. Through applied research we will determine the incidence of foodborne pathogens in raw milk and artisan cheese through microbiological sampling and testing. Utilizing standard microbiological techniques including checkerboard assays, we will screen natural antimicrobial compounds to identify synergistic interactions to formulate effective antimicrobial solutions for use in the production of artisan cheese. Risk-based management recommendations derived from the research aspects of this proposal will then be disseminated to stakeholders as well as to those who interact with stakeholders through an established national training program. Ultimately this work will improve the safety and quality of value-added dairy products thereby promoting their continuous production and ultimately preserving working landscapes and the sustainability of dairy farms.

APPROACH: Objective 1: Risk Assessment: Assess food safety risks in agriculture systems. To achieve this objective, raw milk and finished product samples will be obtained from producers located throughout the state of Connecticut that manufacture raw milk and raw milk cheese. Milk, filters, and milk products will be collected using standard methods, transported and stored under refrigeration until use. Time of sale will be considered and proper test and hold procedures will be followed, if applicable, to ensure that knowingly contaminated product is not released into commerce. Upon arrival, samples will be analyzed for indicator organisms (i.e. total aerobic bacteria, coliforms, where applicable) and select bacterial pathogens including (Salmonella spp., shiga-toxin producing Escherichia coli (STEC), Listeria spp., and Staphylococcus spp.) using standard methods (Compendium of Methods for the Microbiological Examination of Foods, the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM), or other applicable sources (AOAC, USDA, etc.)). Additionally, target pathogen population levels will be determined through surface plating serial dilutions of product samples onto chromogenic agar (Chromagar Microbiology, Paris, France) formulated and modified for each specific target pathogen. Results will be shared with producers in a timely manner so that corrective actions can be discussed and applied where applicable. Objective 2: Risk Management: Develop science-based interventions to prevent and mitigate food safety threats. To achieve this objective we propose to explore the use of natural antimicrobial compounds against Listeria monocytogenes inoculated into raw milk and onto fresh soft cheese. Based on the literature, greater antimicrobial effectiveness of antimicrobial pairings that exploit complementary antimicrobial mechanisms. To screen such combinations and to identify additive, synergistic or antagonistic mechanisms we will employ a "checkerboard" broth microdilution assay. Briefly, 60 μL of each antimicrobial solution at varying concentrations and 120 μL of bacterial culture (ca. 106 CFU) in tryptic soy broth and milk will be added to individual wells of a 96-well microtiter plate. The minimum inhibitory concentrations (MIC) of antimicrobials alone and in combination will be determined. Based on these results, select combinations will be chosen for further study in milk and cheese as antimicrobial wash and coating treatments. To test the inactivation kinetics of Listeriaon cheese samples, antimicrobials will be added alone or in combinations as determined above as antimicrobial dip and coating treatments. Raw milk samples will be obtained from the Kellog Dairy Center at the University of Connecticut. Fresh cheese samples will be produced from pasteurized milk at the University creamery following standard manufacturing procedures to achieve a final product within typical compositional ranges. Physicochemical analysis of finished product will include pH, total solids (TS), fat, and chloride. Surface and cheese interior pH measurements will also taken at each microbiological sampling interval. Cheese samples will be spot-inoculated with 100 µl of a 6-strain cocktail of L. monocytogenes to achieve a contamination level of ~1000 CFU/cm2. In order to evaluate the efficacy of antimicrobials as a final treatment to eliminate post-processing contamination with L. monocytogenes prior to packaging, cheese samples will be placed in a sterile Whirl-Pak bag containing sterile deionized water (control where appropriate), or antimicrobials alone or in combination as determined through the checkerboard assay. After treatment samples will be vacuum packed and incubated for varying times at various temperatures to mimic commercial practice. Following incubation, samples will be transferred to a second Whirl-Pak bag containing 50 ml of Dey-Engley neutralizing broth stomached for 2 min. Homogenized samples will be serially diluted and added to petri dishes followed by pour plating with CHROMagar Listeria to enumerate viable bacteria. This method has a limit of detection of 1 log CFU/mL The coating of cheese samples with antimicrobials, either alone or in combination will be conducted according to a well-established method using chitosan. Following production, antimicrobials will be added to the chitosan solution at the desired concentrations. Cheese samples will be separated into two groups: pre-inoculated and pre-coated, respectively. Cheeses from the pre-inoculated group will be inoculated as previously mentioned, followed by antimicrobial coating treatments, whereas the cheese samples in the pre-coated group will first receive an application of the antimicrobial coating and subsequently subjected to L. monocytogenes inoculation, again, as previously described. After coating treatment, the cheese samples will be vacuum packed and incubated for varying times at various temperatures to mimic commercial practice. The enumeration of L. monocytogenes on cheese samples will be conducted as previously described. Objective 3: Risk Communication: Communicate food safety messages to stakeholders. Communication and outreach will be achieved through the expansion of an already established and ongoing training program. Through the Project Leader's extension appointment, a harmonized training program will be expanded to disseminate basic cheese safety information including best practices for pathogen control and to provide training to attendees on areas such as GMPs, preventive controls, sanitation, microbial controls, testing, and environmental monitoring. Information pertaining to the risks associated with small-scale cheese production from raw ingredients to finished product will be updated and expanded. Deliverable interventions obtained during the course of this project will be incorporated as appropriate. It is expected that additional sections on control strategies will be included in addition to how such interventions can be incorporated into current operating procedures to comply with the FSMA. Artisan/Farmstead Cheesemaker Food Safety Workshops will be held in various locations around the country. Areas to be served immediately during the course of this project include New York, Connecticut, Tennessee, Southern California, Texas, Michigan, Missouri, and the Washington DC area. Additional locations will be added when and where appropriate. Members of the regional project in target areas will be notified and their participation sought. Additional one-on-one technical service in the state of Connecticut will be made available through the Project Leader's extension appointment. Evaluation: Overall, data will be collected concerning the incidence of pathogens and their indicator microorganisms in raw milk and cheese to assess the food safety risks associated with this agricultural system. Completion of this evaluation will constitute the milestone and will serve as the indication of a successful contribution to greater risk assessments. The identification of pathogens and subsequent corrective action will serve to reduce the incidence of foodborne illness associated with these products. The identification of at least one efficacious intervention will successfully increase the number of pathogen control strategies available to producers and further reduces the potential for foodborne illness. The success of educational and outreach efforts will be evaluated through the addition of pointed questions to workshop survey tools. For example, producers will be asked if their knowledge of food safety hazards has improved and if they plan to or have already implemented preventive controls based on the information provided. Other than those identified, the success of such preventive measures are difficult to evaluate.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Risk Assessment, Management, and Communication