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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

2013 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:
With the advent of the Food Safety Modernization Act and updated guidance from FDA on the production of acidified foods, data in the scientific literature is needed to support process filings for acidified foods and the determination of critical controls for safe manufacture of a variety of acidified vegetables, including refrigerated products, as well as shelf stable acidified products that are not heat processed. These products use fresh vegetable ingredients that may contain acid resistant pathogens such as Escherichia coli O157:H7. Currently a 5-log reduction of Escherichia coli and acid resistant pathogens (including Salmonella and Listeria) is required under the Food Safety Modernization Act for safe production practices and for filing processes with FDA. Therefore, we have determined how selected preservative acids (acetate, benzoate, fumarate and others) can be used to achieve a 5-log reduction in pathogen numbers in refrigerated and shelf stable products for which heating will destroy product quality. For many types of acidified foods that have pH values between 3.3 and 3.8, we determined hold times need to allow a 5-log reduction, a standard accepted by FDA. Data for combined acids, including acetic acid and benzoate showed that preservative acids such as benzoate can reduce hold times required to allow a 5-log pathogen reduction from more than 10 days to less than 3 days, resulting in major time and cost reduction for industry.


4.Accomplishments
1. Safety of refrigerated acidified foods. Refrigerated cucumber pickle products cannot be heat processed due to the loss of characteristic sensory attributes. Typically brined refrigerated pickles contain relatively low concentrations of acetic acid with pH values of 3.7 to 4.0. Refrigeration (4 to 10C) helps to inhibit the growth of spoilage bacteria and maintain flavor, texture, and appearance of the pickles. Previous research has shown that pathogenic Escherichia coli strains are unusually acid resistant and survive better in refrigerated acid solutions than at higher temperatures. We found that E. coli O157:H7, which can have an infectious dose of 1 to 10 organisms, can survive for 1 month or longer in brines typical of commercial refrigerated pickles. Our objective was to develop methods to assure a 5-log reduction of pathogenic E. coli in these types of products, while maintaining the sensory characteristics. A novel brine formulation was developed, based on current commercial refrigerated pickle brines, which contained fumaric acid, benzoic acid, acetic acid, and sodium chloride. Sensory data indicate that this formulation did not affect ¿avor or other sensory attributes typical of the product, compared to traditional formulations. We achieved a 5-log reduction of E. coli O157:H7 during holding for 2 to 9 days, depending on holding temperature. Growth of spoilage lactic acid bacteria was also inhibited during holding. These results can be used by manufacturers to assure a 5-log reduction in cell numbers of E. coli O157:H7, Salmonella and Listeria without a heat process during the manufacture of refrigerated pickle products. Currently producers rely on good agricultural practices for the prevention of contamination to produce safe products. The brine formulation developed can allow producers of refrigerated pickle products to use fumaric acid as a post-harvest method for assuring safety.

2. Safety of dressings and related products. For shelf stable (room temperature) acidified food products that are not thermally processed, we developed acid and pH processing conditions that are based on the concentration of acetic acid and benzoic acid. Data from these experiments showed that benzoic acid can be used to significantly accelerate acid killing of vegetative bacterial pathogens, and helps establish a scientific basis for using preservative to meet food safety requirement. To meet FSMA requirements producers of dressings and related products must demonstrate a 5-log reduction in bacterial pathogens. This research shows how a 5-log reduction can be achieved due to common ingredients in a variety of products, preventing the need for extensive testing of many different products.


Review Publications
Lu, H.J., Breidt, F., Perez-Diaz, I.M. 2013. Development of an effective treatment for a 5-log reduction of Escherichia coli in refrigerated pickle products. Journal of Food Science. 78(2):M264-M269.

Breidt, F., Kay, K., Cook, J., Osborne, J., Ingham, B., Arritt, F. 2013. Determination of 5-log reduction times for Escherichia coli O157:H7, Salmonella enterica, or Listeria monocytogenes in acidified foods with pH 3.5 or 3.8. Journal of Food Protection. 76(7):1245-1249.

Last Modified: 4/23/2014
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