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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
The overall objective of this research is to develop methods to prevent the growth of pathogenic and spoilage microorganisms in minimally preserved, brined, and fresh-cut foods and optimizing safety, quality, and sensory attributes specifically through:.
1)development of fundamental knowledge of the biochemistry of bacterial adaptation to acidified environments;.
2)determination, through analysis of gene/protein expression profiles, the responses to intrinsic and extrinsic stressors and, in particular, the effect of oxygen imposed on pathogenic bacteria during production, processing, and storage of acid and acidified foods;.
3)development of data and its use for the development of mechanistic models for growth, survival and inactivation of pathogens.

1b.Approach (from AD-416)
The effect of common food acids and acid preservatives will be evaluated for their relative ability to enhance killing of acid-tolerant food pathogens, particularly Escherichia coli O157:H7, in the absence of oxygen and independent of pH. Work will be carried out at biosafety level 2 (BSL-2) due to the organisms under investigation. Selected acid/pathogen strain combinations will be analyzed using genetic and biochemical analyses to determine the mechanisms by which acids are responsible for killing E. coli and other pathogens. This information will be utilized to identify common metabolic targets for the killing effects of acids and acid preservatives, as well metabolic targets unique to particular acids. Since we have found that oxygen increases the killing rates of acid-tolerant pathogens at low pH, similar investigations will be done to determine the genetic and metabolic responses of acid-tolerant pathogens to acids in the presence of molecular oxygen and oxygen radicals. These results will be used to determine the mechanisms by which oxygen species enhance killing of pathogens in acid and acidified foods. Data from genetic and metabolic experiments will be used to develop mechanistic mathematical models and validate the models that are developed in order to test hypotheses developed from genetic and metabolic investigations of acid-killing and acid resistance of food pathogens.

3.Progress Report
Fresh fruits and vegetables typically harbor up to a million bacteria per gram. A very small percentage of these may be disease causing bacteria, such as E. coli. Although outbreaks of pathogens have not occurred in acidified vegetables, fatalities have resulted from bacterial contamination in fruit juices, which have similar acid conditions (similar pH values). Up to half of the $1.5 billion per year pickled vegetable market consists of products which are preserved and sold without heat processing. Methods for killing bacterial pathogens that might be on fruits and vegetables using food grade acids were developed. We found that under the conditions of acidified vegetables, there was over a 100 fold difference in acid killing of pathogenic E. coli for different food grade acids, and that fumaric acid, benzoic and sorbic acids were among the most efficient acids for killing acid resistant pathogenic E. coli strains under conditions typical of acidified vegetables. We also investigated how the internal cell physiology of pathogenic E. coli responds to acids by measuring the internal cell acid concentrations, pH and electrical charge difference between the internal cell and external environments. Understanding how E. coli responds to acids, and how different acids kill the organism, may lead to better approaches to ensuring safety for a large number of acid and acidified foods.

1. Mathematical models for acid killing of pathogenic E. coli. ARS researchers in Raleigh, North Carolina developed mathematical models of how the internal bacterial cell acid conditions correspond to the external cell acid and pH conditions for disease causing E. coli strains. The results indicate how decreases in intracellular pH were correlated with the corresponding times required to achieve a significant reduction in cell numbers. Modeling the killing of cells as a function of external acid and salt concentrations under conditions typical of acid and acidified foods revealed a protective effect of salt for selected conditions. Novel acid formulations were developed. The results of this study may help manufacturers adjust product formulations to improve the safety of a variety of acid and acidified foods made with fresh vegetables, including salsas, dressings, and pickled vegetable products.

Review Publications
Pan Y, Breidt F, Gorski LA. 2010. Synergistic effects of sodium chloride, Glucose, and temperature on biofilm formation by Listeria monocytogenes serotype 1/2a and 4b strains. Appl Environ Microbiol. 76(5):1433-1441.

Breidt, F., Sandeep, K.P., Arritt, F. 2010. Use of Linear Models for Thermal Processing Acidified Foods. Food Protection Trends. 30(5):268-272.

Pan, Y., Breidt, F., Kathariou, S. 2009. Competition of Listeria monocytogenes Serotype 1/2a and 4b strains in mixed culture biofilms. Applied and Environmental Microbiology. 75(18):5846-5852.

Last Modified: 11/28/2015
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