Progress 12/02/10 to 10/27/15

Outputs
Progress Report 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. 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. Significant results were achieved over the life of this project, which will expire December 1, 2015. Safe processing parameters have been defined for refrigerated, acidified and fermented pickled vegetable products and dressings. Published data, including 12 peer-review journal articles and 3 book chapters, have been used by FDA and industry to define safe production practices for acidified vegetables in the US and abroad. The pH, acid concentration and temperature conditions required for thermal processing of acidified foods were determined for acidified vegetables with a pH of 4.6 or below. Requirements for the production of non-heated acidic products were also determined, including data for the safe production of dressings. For commercial vegetable fermentations, it was found that pH (rather than acid concentration) is the primary factor needed for predicting the killing of pathogenic bacteria that may be present on vegetables at the start of the fermentation. The relative effectiveness of food acids to cause a 100,000 reduction in cell numbers (a standard used by FDA for acidified foods process filings) of bacterial pathogens in acidified foods was reported, and was used to help develop a novel formulation to improve the safety of refrigerated pickled vegetables. The formulation may be adapted by industry as a food grade chemical (sodium fumarate) becomes available for commercial use. A study of the microbial ecology on the spoilage of fermented cucumbers in commercial scale (10,000 gallon) tanks and in laboratory fermentations has resulted in the identification of a new species of bacteria related to organisms that spoil beer. Understanding the microbial ecology of these spoilage fermentations is a critical first step in determining how to control and prevent the spoilage. Fundamental research also was done to determine mechanisms by which organic acids in foods kill pathogenic bacteria, principally Escherichia coli O157:H7, which has been responsible for deaths from consumption of acid beverages. A model was published detailing how external acid concentration and pH influences the internal cell physiology of bacterial pathogens. In the past year, data was published to show that production of ammonia by spoilage bacteria, which has previously been found to be a problem in acidic tomato based products (dressings, sauces) is not a problem for acidified vegetables. FDA had questioned the role of this type of spoilage in pickled vegetables, which may result in dangerously high pH. It was found that the acid present in acidified vegetables (principally vinegar) prevents this type of spoilage. These data have helped processors produce safe products without needlessly over-processing. In additional research, the presence of bacterial viruses, not active against humans but active against bacterial pathogens, was discovered in vegetable fermentations. One bacterial virus active against E. coli O157:H7 was found to specifically attack only this pathogen, while leaving other bacterial in the fermentation untouched. This bacterial virus remains under study and may have utility in the development of methods for targeted killing of the pathogen. Studies with kimchi, a fermented cabbage product that is imported as well as manufactured and sold in the US, have shown that processing conditions (including method of chopping) can play a significant role in the rate of die-off of undesirable bacteria during fermentation. A proposed NP108 project, 6070-41420-006-00D PrePlan (submitted for peer review) will continue the work of the current project. The planned research will help determine how different salts, salt concentrations, organic acids, pH, and other environmental variables affect the die-off of pathogenic bacteria in fermented and acidified foods. The proposed objectives include determining the safety of low and alternative salt fermentations, developing mathematical models for the reduction of pathogen cell populations in vegetable fermentations, and modeling buffer capacity to aid FDA in determining how small amounts of near neutral pH affect the final pH of acidified foods. The knowledge gained will be used to help processors and regulatory agencies assess and assure the safety of acidified and fermented food products. Accomplishments 01 Determination of conditions that prevent pH increase in acidified vegetables by ammonia based spoilage. FDA had proposed in a 2010 industry guidance that spores of Bacillus species need to be killed by heat processing for acidified vegetables. This would have been a significant burden to industry, because the times and temperatures needed to thermally process these products would negatively impact product texture and overall quality. Published research did not exist to show the significance of Bacillus spoilage of acidified vegetables, but the scientific literature has shown that the problem is a significant one for tomato processing. Research was done to show how the different acids present in tomato vs. acidified vegetables (such as cucumber pickles and peppers) affect the spoilage process. It was found that the acids added to preserve acidified vegetables (primarily vinegar) are sufficient to prevent this type of spoilage, while the acid naturally present in tomato products (malic acid) is not necessarily sufficient to prevent spoilage. These data were published and may be used by industry and FDA as the basis for safe production practices and science-based regulation. 02 Identification of a bacterial virus specific for a pathogenic Escherichia coli. A bacterial virus specifically active only against pathogenic strains of E. coli was isolated from a commercial vegetable fermentation. This virus was purified and found to attack only a single serotype of E. coli, a type which has caused a number of foodborne outbreaks in the U.S. and abroad, including fatalities. The virus may have application in food protection strategies that employ viruses to kill pathogens. This virus has the unique properties of being resistant to the harsh conditions of salt and acid that is present in vegetable fermentations. The data also show the ubiquity of the pathogenic E. coli in the environment, and that it may be present on vegetables used for commercial food fermentations. The data highlight the need, therefore, to follow guidelines for proper fermentation of vegetables to assure killing of bacterial pathogens.

Impacts
(N/A)

Publications

• Yang, Z., Meng, X., Breidt, F., Dean, L.L., Arritt, F.M. 2015. Effects of acetic acid and arginine on pH elevation and growth of Bacillus licheniformis in an acidified cucumber juice medium. Journal of Food Protection. 78(4):728-737.
• Lu, Z., Breidt, F. 2015. Escherichia coli O157:H7 bacteriophage (phi)241 isolated from an industrial cucumber fermentation at high acidity and salinity. Frontiers in Microbiology. 6:67. doi: 10.3389/fmicb.2015.00067.
• Kyung, K., Medina-Pradas, E., Kim, S., Lee, Y., Kim, K., Choi, J., Cho, J., Chung, C., Barrangou, R., Breidt, F. 2015. Microbial ecology of watery kimchi. Journal of Food Science. 80(5):M1031-M1038.

Progress 10/01/13 to 09/30/14

Outputs
Progress Report 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. 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. To meet the requirements of the Food Safety Modernization Act, data was urgently needed by industry (particularly small processors) to determine safe processing parameters for acidified foods. To respond to this need, research was carried out to adequately support required industry process filings for thermal processing conditions that will achieve required reductions in bacterial pathogens of concern for fresh vegetable products, including Escherichia coli O157:H7, Salmonella, and Listeria. These data are now used in FDA process filings by most producers of acidified vegetables for commercial sale in the U.S. To address industry and FDA concerns about pH rise in acidified foods as a consequence of spoilage microorganisms, research was conducted to determine how the natural buffering of acidified foods can affect pH changes. Computer software was written to implement new models from previous research on buffering, to be relevant for acidified food acids. Experimental validation was carried out and showed the relative ability of commonly used food acids (acetic acid, lactic acid, citric acid and others) to prevent pH change in these food products. These models are useful for determining which spoilage microorganisms should be considered to be potentially hazardous, because increased pH may occur during growth of these organisms, which could allow concomitant growth of pathogenic bacteria normally inhibited by low pH. Accomplishments 01 Determination of thermal processing for acidified foods with a pH above 4.1. Published research did not exist to support required FDA process filings that showed a safe reduction of bacterial pathogens during thermal processing of acidified foods with a pH above 4.1. Using groups of strains of the bacteria of concern, including Escherichia coli O157:H7, Salmonella, and Listeria, researchers in Raleigh, North Carolina determined the times and temperatures of processing needed to assure safety. This research addressed critical needs for the acidified foods industry, especially small processors.

Impacts
(N/A)

Publications

• Breidt, F., Kay, K.L., Osborne, J., Ingham, B., Arritt, F. 2014. Thermal processing of acidified foods with pH 4.1 to pH 4.6. Food Protection Trends. 34(3):132-138.

Progress 10/01/12 to 09/30/13

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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.

Impacts
(N/A)

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.

Progress 10/01/11 to 09/30/12

Outputs
Progress Report 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. 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. Safe processing of acidified vegetables currently requires a 5-log reduction of bacterial food pathogens. Some acidified foods, such as refrigerate pickles, can�t meet the 5-log reduction standard with the current technology. Fumaric acid, which we have previously found to accelerate acid killing, was added to a refrigerated pickle formulation and found to effectively kill Escherichia coli O157:H7 within 9 days at 50�F and did not affect the acceptability of the flavor or texture. Fumaric acid was also used in semi-commercial scale trials with the natural preservative allyl isothiocyanate to preserve acidified cucumbers in a reduced salt brine. It was observed that fumaric acid is more effective than other traditional preservatives to prevent bacterial growth. The assessment of the use of lauric arginate to aid in the microbial stabilization of acidified cucumbers showed that this natural preservative is not robust enough to prevent the proliferation of microbes long term. Combinations of lauric arginate with natural preservatives for environmentally friendly preservation of cucumbers and for reduction in pathogen numbers in vegetable brines (without fermentation) are currently being evaluated. For fermented products we have previously shown that the killing of E. coli depends on pH. We, therefore, have investigated how chemical buffering changes during fermentation. A mathematical model for buffering was developed using Matlab software. The model was validated and the results from the model predicted the buffer components present in a complex acid solution. The model will have use in determining how pH changes during fermentation and competitive growth of bacteria. In addition to this work, preliminary studies to determine how cells of pathogenic E. coli strains are affected by organic acid solutions were done. A microbial metabolomics approach was devised to study the intracellular metabolite pools of E. coli O157:H7 which had been exposed to neutral pH, acid pH, or acid pH plus acetic acid. Cells exposed to acid pH utilize glutamate, supporting current theories for the acid resistance of E. coli O157:H7. Furthermore, 27 metabolites (including amino acids and nitrogenous metabolites) were found to decrease, and 20 to increase in response to acetic acid stress. The sources of experimental variation in the data were determined and the work provided a promising first look into the metabolism of acid-stressed E. coli O157:H7, which may be useful in developing enhanced preservation treatments for fruits and vegetables. Accomplishments 01 Development of a buffer capacity model to predict pH changes in acid and acidified foods. The growth of spoilage bacterial may increase pH in acidified foods, resulting in unsafe products. Conversely, pH reduction fermented foods is important for assuring safety. However, there is currently no accurate method for predicting pH changes in acid and acidified foods because of the unknown buffering compounds which affect changes. To address this problem, ARS researchers at Raleigh, NC develop a buffer capacity model that can be used to determine pH changes in acid and acidified foods as acids and bases are added for processing, or are produced by the growth of bacteria. The model showed that the complex ac buffering present in commercial vegetable fermentation brines can be predicted even in the presence of unknown buffer compounds. Research to validate the model showed that pH changes in commercial fermented vegetables can be predicted. Spoilage related pH increase in acidified foods and beverages may be similarly modeled. By determining the buffer capacity of acid and acidified foods using the model, the potential heal hazards that may occur (or be prevented) due to microbial activity could be predicted during production of acid and acidified foods, improving safety.

Impacts
(N/A)

Publications

Progress 10/01/10 to 09/30/11

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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 f extended periods (up to one month or more) in some common pickled vegetable products, have caused disease outbreaks in acidic juice produc 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), th common acid in acidified vegetables, is one of the less effective acids. However, supplementation of vinegar with small amounts of fumaric acid ( 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 effe on flavor or character of these products. The data from these studies a currently being used by the food industry for trials with new product formulations. Additional research has shown the time and acid condition needed in vegetable fermentations to kill acid resistant E. coli strains These data have been used by industry and the Food and Drug Administrati to assure the safety of acidified foods.

Impacts
(N/A)

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.