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United States Department of Agriculture

Agricultural Research Service

2008 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
The safety of acidified foods, which are foods that have acid or acid food ingredients added to fresh vegetables was investigated. These foods include pickled vegetables that are not heat treated and are solely protected by the added acid. Disease causing bacteria can possibly survive for extended periods under the conditions of packing and sale of these food products. Survival of bacteria can depend on how acid the product is and the temperature at which the product is stored. The holding time needed for acid killing of disease causing bacteria was determined for a variety of conditions typical of these types of food products. The recommendations from this study were reported to the industry and to the Food and Drug Administration (FDA). These safety guidelines are now used by industry and form the scientific basis for the commercial process filings required by FDA. This research addresses National Program 108 (Food Safety) Component 1 (Pathogens, Toxins and Chemical Contaminants) problem 1.2 (Pathogens, Toxins and Chemical Contaminants Postharvest) sub-problem 1.2.3 (Production and Processing Ecology).

Research has shown that dissolved oxygen plays a key role in the destruction of bacteria in acid solutions. Acid solutions with and without dissolved oxygen were tested for the ability to kill bacteria. It was found that the trace amounts of dissolved oxygen was enough to greatly accelerate killing of bacteria by acid. This research may be used to accelerate acid killing of bacteria in acid and acidified foods. This research addresses National Program 108 (Food Safety) Component 1 (Pathogens, Toxins and Chemical Contaminants) problem 1.2 (Pathogens, Toxins and Chemical Contaminants Postharvest) sub-problem 1.2.3 (Production and Processing Ecology).

A method for determining the numbers of live bacteria in liquids or removed from surface films was investigated. The method does not require traditional plating techniques that are used for counting the bacteria present in a sample, which could consist of a food or food extract. The method under investigation uses a gene amplification technique called Polymerase Chain Reaction (PCR) (used to amplify DNA), that allows a quantitative measure of how much genetic material is being amplified and can be used to determine how many bacteria were present in a sample being tested. A major problem with this technique is that genetic material from both live cells and dead cells can be amplified and can contribute to the estimated count of bacteria present. In this research, it was found that a special dye treatment can be applied to the sample containing bacterial cells prior to isolating the DNA, and this will only allow the amplification of genes from live bacteria, not dead cells. Although this technique had been previously reported, this research refined the method and has demonstrated accurate counting of live cells in samples that had a high concentrations of dead cells. This research addresses National Program 108 (Food Safety) Component 1 (Pathogens, Toxins and Chemical Contaminants) problem 1.2 (Pathogens, Toxins and Chemical Contaminants Postharvest) sub-problem 1.2.1 (Detection and Validation).

1. Title: The safety of acidified foods.

This research directly addresses the National Program Action Plan for Food Safety 108, Component 1 (Pathogens, Toxins and Chemical Contaminants) section 1.2.3, to understand the physiological status of bacteria and survival in niches. Aspects of this work may also lead to process intervention strategies (section 1.2.4).

In response to concerns by the United States Food and Drug Administration (FDA), and in conjunction with the principal trade association for the acidified foods industry (Pickle Packers International), research was carried out to determine the conditions needed to assure the destruction of disease causing bacteria in acidified foods. Acidified foods are foods to which acid or acid ingredients are added to preserve the food. The results of this project showed the holding times and temperatures needed to assure the safety of acidified foods that are not typically heat treated, and basic research was also carried out to determine the effects of organic acids on the survival of bacteria in acid and acidified foods. Safe production practices and industry process filings required for the production and sale of acidified foods in the United States are now based on this research. The FDA now uses the research and data as the basis for determining safe production practices for acidified foods in the United States.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer


Review Publications
Pan, Y. and Breidt, F. 2007. Enumeration of Viable Listeria monocytogenes Cells by Real-Time PCR with Propidium Monoazide and Ethidium Monoazide in the Presence of Dead Cells. Appl. Environ. Microbiol. 73(24):8028-8031

Breidt, F., Hayes, J.S., Mcfeeters, R.F. 2007. Determination of 5-log reduction times for food pathogens in acidified cucumbers during storage at 10 and 25°C. Journal of Food Protection. 70(11):2638-2641.

Last Modified: 7/27/2015
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