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United States Department of Agriculture

Agricultural Research Service

Research Project: Control of Human Pathogens Associated with Acidified Produce Foods

Location: Food Science Research

2011 Annual Report


1a.Objectives (from AD-416)
1. To define conditions to assure a 5 log reduction of acid tolerant pathogens in refrigerated or bulk stored acidified vegetables.

2. To determine how the metabolism of Escherichia coli O157:H7 (internal pH, membrane potential, ion concentrations, and cell metabolites) are affected as cells are exposed to organic acid and salt conditions typical of acidified foods.

3. To determine the survival of E. coli O157:H7 in commercial fermentation brines, with and without competing microflora, and under a variety of extrinsic and intrinsic conditions.


1b.Approach (from AD-416)
A cocktail of five or more pathogenic Escherichia coli O157:H7 strains from the USDA/ARS Food Science Research Unit culture collection will be used for these studies. While our previous work has focused on E. coli O157:H7 (from human, food, animal, and environmental sources) additional serotypes, including O145 strains obtained from ARS sources will also be used in this research. Previous research on acidified vegetable brines has shown that E. coli O157:H7 is the most acid resistant vegetative pathogen of concern for acidified vegetable products. E. coli O157:H7 and related serotypes can’t grow in most acidified vegetable products, the objective is to prevent bacterial pathogens from surviving long enough in non-heat treated acid and acidified foods to cause disease. Bacterial strains will be grown statically for 15 h at 37°C in non-selective broth (Luria broth) supplemented with 1 g/L glucose to induce acid resistance. Cell viability before, during and after acid treatments will be determined by plating on non-selective media to allow enumeration of injured cells with a spiral plater and an automated plate reader (Spiral Biotech). Samples from acid treatment of bacterial cells will be diluted in MOPS buffer at neutral pH prior to plating. The lower limit for detection is 10^2 to 10^3 CFU/mL for this method. In addition to standard plating, an MPN method done with microtiter plates, custom MatlabTM software, and a microtiter plate reader has been developed in our laboratory. This method can be used to determine log number for a range of cell concentrations from 10^8 to <30 CFU/mL, and will supplement spiral or standard plating techniques when cell numbers are lower than 10^3 CFU/mL. Most acid solutions will be prepared based on the protonated acid concentration. The acid concentration required to achieve specific protonated concentration for a given pH and ionic strength will be determined using a Matlab computer program (pHTools ) developed in our laboratory, or custom Matlab functions. Sodium gluconate will be used as a non-inhibitory buffer in acid solutions to allow comparisons of the effects of organic acids with the effect of pH alone. Cucumber juice medium or brined cucumbers will be used for these studies as representative of brined vegetable products, because these media do not contain inhibitors of microbial survival or growth, but do contain amino acids and other compounds that may aid in survival of the pathogens. Acid concentrations will be confirmed by HPLC using a Thermo Separation Products HPLC system with a Bio-Rad HPX-87H column and UV detector. For acid challenge experiments requiring anaerobic conditions, a Coy anaerobic chamber will be used and media or acid solutions allowed to equilibrate in the chamber for 24 h to remove dissolved oxygen.


3.Progress Report
Fresh fruits and vegetables typically have up to a million bacteria per gram, with no ill effect on human health. However, a very small percentage of the bacteria present may cause disease, such as the Escherichia coli strains that recently caused 18 deaths in Germany. Although outbreaks of pathogens have not occurred in acidified vegetables, research has shown that these disease causing Escherichia coli (E. coli) strains are unusually acid resistant, and fatalities have resulted due to contamination by E. coli in acidic fruit juices. Up to half of the $1.5 billion per year pickled vegetable market in the United States consists of products which are preserved and sold without heat processing, and these products have similar acid conditions as acidic fruit juices. We found that disease causing E. coli strains can survive for up to one month or more in some common pickled vegetable products. We found that the addition of small amounts of fumaric acid, a food grade additive, increases by more than 10 fold the killing of E. coli in typical pickled vegetable product formulations, without altering flavor or product character. These data are currently being used by manufacturers in product trials. In addition, we have done basic research on the nature of acid resistance of the disease causing E. coli strains. The acid resistance that helps survival in acidified foods also helps the organism survive the stomach acid. We are exploring the internal cell biochemical pathways using a small molecule fingerprinting technique based on gas chromatography (2-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry). We have also investigated how the acid resistant E. coli is killed during the fermentation of vegetables. Published data from this project has been used by the Food and Drug Administration and manufacturers to assure the safety of fermentations made from imported vegetables.


4.Accomplishments
1. The safety of acidified foods. Comparison of the bacterial killing effects of a variety of food acids has shown that fumaric acid is an effective additive that is specifically active in killing acid resistant disease causing Escherichia coli (E. coli) strains in acidified foods. These acid resistant E. coli strains, which have been shown to survive for extended periods (up to one month or more) in some common pickled vegetable products, have caused disease outbreaks in acidic juice products, as well as a variety of meats and vegetables. A survey of food grade acids has shown that specific acids have very different killing effects (over 100 fold different) on E. coli, and that vinegar (acetic acid), the common acid in acidified vegetables, is one of the less effective acids. However, supplementation of vinegar with small amounts of fumaric acid (a food grade acid) increases acid killing of E. coli over 10 fold. This acid can be added to acidified vegetable products with little or no effect on flavor or character of these products. The data from these studies are currently being used by the food industry for trials with new product formulations. Additional research has shown the time and acid conditions needed in vegetable fermentations to kill acid resistant E. coli strains. These data have been used by industry and the Food and Drug Administration to assure the safety of acidified foods.


Review Publications
Lu, H.J., Breidt, F., Perez-Diaz, I.M., Osborne, J.A. 2011. Antimicrobial effects of weak acids on the survival of Escherichia coli O157:H7 under anaerobic conditions. Journal of Food Protection. 6:893-898.

Hosein, A.M., Breidt, F., Smith, C.E. 2011. Modeling the effects of sodium chloride, acetic acid and intracellular pH on the survival of Escherichia coli O157:H7. Applied and Environmental Microbiology. 77(3):889-895.

Breidt, F., Caldwell, J.M. 2011. Survival of Escherichia coli O157:H7 in cucumber fermentation brines. Journal of Food Science. 76(3):M198-M203.

Last Modified: 7/23/2014
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