Increasing the Safety and Functionality of Foods by the Use of Novel Interventions, Detection and Probiotic Bacteria

NON-TECHNICAL SUMMARY: Food safety and functional foods are important not only to the agriculture and economy of the state of Missouri, but they also affect consumers directly. This project will examine rapid methods for detection of pathogens in foods, elucidate how pathogens are transmitted during food processing and develop functional soyfoods containing probiotics.

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APPROACH: PCR- and biosensor-based techniques will be improved upon as techniques for rapid detection of pathogens. DNA and RNA sequences that would make suitable primers for simultaneous detection of target pathogens will be continually searched in the literature. Other biosensor-based rapid methods will also be explored to achieve the objectives. Food samples will be artificially inoculated with increasing concentrations of pathogens, DNA isolated and PCR ran. Once a standard curve has been established, naturally contaminated samples will be tested to optimize our PCR-based methods, specifically by removing food inhibitors that typically limit the sensitivity of the technique. Probiotic bacteria will be screened for long-term viability, acid and bile tolerance, and antagonistic effects against pathogens by in vitro well diffusion or disk assays. Selected strains with the best characteristics will be used in in vitro and in vivo animal studies for determining their health benefits when ingested in foods. Novel probiotic foods will be developed and the long term storage viability of the cultures determined. Chemical, physical and sensory analyses of the developed products will be performed. In vitro and in vivo studies will focus on specific end points that are indicators of health benefits. We will utilize strains of L. monocytogenes, E. coli O157:H7 and Salmonella that have been genetically engineered to carry the green fluorescent protein (GFP) gene. These strains will be introduced into a simulated food processing/retail environment at various potential points of contamination and their survival and transmission pattern monitored by way of the GFP trait to pin-point specific critical control points that may exist. We will sample various areas on contaminated equipment by swabbing and plating to determine how well these pathogens survive and contaminate food. The bacterial marker can easily be monitored quantitatively, by conventional culture-based methods, or qualitatively, as individual cells via fluorescence microscopy. Foodborne pathogens can be induced to the viable-but-nonculturable (VBNC) state when in the presence of commonly used antimicrobial preservatives. We will investigate the VBNC phenomenon in E. coli O157:H7, Salmonella and L. monocytogenes in meat products and meat processing equipment following routine cleaning and sanitizing protocols and subsequent storage in various packaging and refrigeration conditions. The meat products will be artificially inoculated with varying concentrations of the pathogens and exposed to different intervention steps, while equipment will be artificially contaminated with the pathogens and the product processed as routinely done, followed by typical cleaning and sanitizing steps. VBNC cells will be monitored by the use of ethidium monoazide (EMA), a stain that can selectively penetrate dead cells because of their compromised membrane/cell wall systems and bind to the intracellular DNA. We will use EMA coupled with PCR to monitor for the presence and recovery of VBNC cells.