Inactivation of Foodborne Pathogens with Non-Thermal Plasma Processing and Natural Antimicrobials and Genetic Mechanisms Underlying Microbial Inactivation or Adaption

Non-Technical Summary:<br/>
Escherichia coli O157:H7 and Salmonella spp. continue to plague the safety of our fresh fruits and vegetables, primarily because the food industry is lacking an effective means of inactivating these organisms. This project will seek to address these concerns by investigating application of plasma processing technologies, natural antimicrobials, and microbial ecology. Non-thermal plasma processing has the potential to be utilized as a means of inactivating pathogens on fresh fruits and vegetables while maintaining product quality; however, this technology is in its infancy and much research is still required to validate it prior to its application by the food industry. Natural antimicrobials will also be studied to determine their stability and efficacy in a produce rinse solution. These antimicrobials are gaining wider use by the food industry as consumers begin to demand more natural ingredients and food processing aids. We will seek to determine what processes affect their stability, and how their activity against foodborne pathogens can be improved. Gene expression profiles can give insights to microbial ecology and help researchers determine the best timing and application of interventions to help improve the overall safety of a food product. Through these studies, we will be able to determine how microorganisms adapt to interventions, like chlorine washes, and the best timing of interventions, such as when the cells are in distress, to improve the overall reduction of pathogens. This research will add to the scientific knowledge that is necessary to help improve the safety of fresh fruits and vegetables.
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Approach:<br/>
Intervention strategies will be evaluated amongst E. coli O157:H7 and Salmonella enterica serovars both in vivo and vitro to determine their application in the food industry. Reduction, inhibition, or growth will be compared to non-treated controls to determine the efficacy of these interventions. Gene expression profiles will be conducted by harvesting messenger RNA from control and treated pathogens and comparing the mRNA levels to give insights to mechanisms of adaptation or bacterial stress
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: Plant essential oils have been exploited over the millennia for their antimicrobial and preservative properties. Consumer demand for natural food products and additives is motivating research for industrial application, with the majority of research focus on pathogens to the exclusion of spoilage organisms. The potential utilization of plant essential oils and their constituents was examined against Gram positive lactic acid bacteria (LAB). While these organisms are beneficially used for many food fermentations, they are also a common causative agent in spoilage of various food products. Essential oils (EO) and EO components were incorporated into MRS agar plates (pH 6.5), which in turn were inoculated with nine individual LAB species and incubated for 96 h at recommended growth conditions, at which point minimum inhibitory concentrations were determined. All species exhibited sensitivity to the EO components with the exception of allyl isothiocyanate, which was ineffective at 0.75% (v/v). EO components thymol, carvacrol and cinnamaldehyde were found to be most inhibitory with MIC's of 0.025% to 0.2% while cinnamic acid was the least effective (MIC 0.5%). The EOs of cinnamon bark, clove bud, and thyme along with the EO component eugenol exhibited moderate inhibition with MICs of 0.1 to 0.2%. MIC's for LAB generally were found to be higher than those reported for most Gram positive and Gram negative pathogenic bacteria. Thus essential oils may have application for the food industry to limit spoilage and extend shelf-life but higher concentrations may be needed to inhibit LAB than foodborne pathogens. Numerous outbreaks of salmonellosis associated with low water activity foods have been reported in recent years. However, the adaptive mechanisms utilized by Salmonella to survive in low water activity foods for prolonged periods of time is not fully understood. The purpose of this study was to investigate the survival characteristics and changes in gene expression profiles associated with fatty acid biosynthesis of five serovars of Salmonella exposed to a low water activity food (sugar) over a 14-day period. Five serovars of stationary phase Salmonella were each inoculated into granular sugar (treatment; Aw=0.50) or sugar water (0.25% w/v; Aw= 0.99; control) and held aerobically over a 14-day period at 25C . After 14 days incubation at 25C, there was a 2.5 to 3.9 log reduction for all five strains. S. Tennessee ARI-33 and S. Tennessee K4643 displayed greater survival than S. Typhimurium, S. Enteritidis and S. Tennessee S13952, which showed no growth on XLT-4 after 14 days incubation. The fab A gene (unsaturated fatty acid biosynthesis) was observed to be up-regulated for all strains for at least one sampling time. S. Typhimurium and S. Enteritidis increased expression of the cfa gene (cyclopropane fatty acid biosynthesis) over 14 days and concurrently had a lower survival rate. The results suggested that low water activity environments might trigger unsaturated fatty acid biosynthesis of Salmonella, and cyclopropane fatty acid synthesis is not favorable for survival of the five serovars tested over 14 days.
<br/>PARTICIPANTS: These individuals contributed to the project: Laurel Gann, graduate student Wei Chen, graduate student.
<br/>TARGET AUDIENCES: Not relevant to this project.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: Research findings from this project will improve our ability to incorporate natural antimicrobials to prevent spoilage by lactic acid bacteria. Additionally, our understanding of fatty acid gene expression profiles will be improved to better understand the adaptation of Salmonella in low water activity foods.