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ARS Home » Southeast Area » Raleigh, North Carolina » Food Science Research » Research » Research Project #430619

Research Project: Intervention Strategies for Controlling Human Pathogens Associated with Fermented and Acidified Vegetables

Location: Food Science Research

2018 Annual Report

1a. Objectives (from AD-416):
1. Determining the safety of low and alternative salt fermentations, produced nationally and internationally. 2. Develop predictive models for 5-log reduction times for pathogenic Escherichia coli in fermented and acidified vegetable products. 3. Enhance buffer capacity models for predicting pH changes in acidified foods with low acid ingredients.

1b. Approach (from AD-416):
The experimental approaches that will be use to achieve the objectives will include mathematical modeling, molecular ecology studies, and biochemical analysis of fermentation brines. Specifically, for Objective 1, to determine the effects of salts on pathogen reduction in fermentations, growth and death of bacterial pathogen cocktails (strain mixtures) will be measured in fermentations by conventional bacterial plating methods using automated plating equipment. Log reduction times for pathogens will be calculated using linear or nonlinear (Weibull) models. Biochemical analysis for salts, organic acids and sugars, will be done by titration (for salts), and high performance liquid chromatography (for acids and sugars). A matrix of salt types and concentrations will be tested to determine how salt effects pathogen die-off. For Objective 2, mathematical modeling approaches to determine the reduction in pathogen populations during fermentation will utilize non-linear systems of ordinary differential equations (rate equations) using Matlab computer software. In addition computer simulation models will be developed using the C++ programming language. Data for these models will be obtained from the experiments in Objective 1. Model results will be compared to data generated under a variety of conditions to determine if the models accurately describe the data. To accomplish Objective 3, predicting pH of buffered acidified foods with low acid additives, mathematical models will be based on published ionic equilibria equations for buffered acid and base solutions. Novel methods for numerical solutions to these equations will be implemented with Matlab software. An automated titrator will be used to confirm predicted buffer capacity curve data. To fit data to the models, several optimization algorithms will be used from the Matlab Optimization Toolkit, or independently programmed in Matlab or C++. The knowledge gained will be used to help processors and regulatory agencies assess and assure the safety of acidified and fermented food products.

3. Progress Report:
The effects of salt on the competitive growth of pathogenic and lactic acid producing bacteria in a cucumber juice medium were determined. These data included three salt conditions: the concentration and type used in typical commercial cucumber fermentations (6% sodium chloride, NaCl), the experimental low salt cucumber fermentation currently undergoing trials at commercial fermentation plants (1.1% calcium chloride), and kimchi, sauerkraut and other ready-to-eat fermented vegetables (2% NaCl). The data show that regardless of salt concentration, the time to pathogens die off (pathogenic Escherichia coli strains) in the presence of lactic acid bacteria was similar, with 1 to 3 log reduction occurring within a 2-3 hours window in the cucumber juice model system. These data are consistent with previous results showing that the salt type or concentration did not greatly alter the death kinetics of E. coli O157:H7 in pure culture experiments with fermentation acids. Cucumber fermentations in 1.1% calcium chloride or 6% NaCl showed similar patterns in the reduction in naturally occurring Enterobacteriacea during fermentation, demonstrating that the findings in model systems are applicable to whole cucumber fermentations. These results are significant because they contradict the widely held assumption by the food safety community that salt concentration is a primary safety factor in vegetable fermentations. Now, additional factors, such as differences in growth rates and acid resistance will have added importance and be the subject of future investigations. These data directly support research project plan to discover how different salts affect the growth and survival of pathogenic E. coli in vegetable fermentations. Development has continued on the computer simulation model for competitive growth of bacteria in vegetable fermentations. The model used realistic parameters for controlling cell growth, including sugar utilization rates (allowing cell division), and inhibition of metabolism by organic acids. The model has currently been tested using parameters derived from laboratory measurements of the biochemistry of vegetable broth (cucumber juice) fermentation by Lactobacillus plantarum, which is the most acid resistant bacterium that usually ends up dominating most vegetable fermentations. Two key parameters for the model have been identified; the per-cell rate of sugar utilization, and the amount of sugar that needs to be processed by a cell to allow cell division. These parameters were used to generate model growth curves that accurately predict the growth and acid production in laboratory fermentations. This novel cell-based simulation approach has proved to be flexible and to closely reflect the actual mechanisms controlling bacterial growth. Continued work will include incorporating data to expand the model and allow effects of bacterial competition to be predicted for complex multi-cell fermentations. Significant progress was made fitting buffer capacity models using a novel Fourier series approach to rapidly fit buffer capacity curves. Using this method, the fitted model can be further analyzed to pick out a set of buffers, based on peaks in the buffer capacity curves, that allow predicting the pH of the solution. The model was validated using acid ingredients representative of acid food products. This approach has been used to demonstrate that the pH of solutions with complex (or unknown) buffering components can be accurately predicted for both acid foods and low acid (high pH) foods that are typically ingredients in acid or acidified foods. These results build on previous results showing pH can be accurately predicted for defined mixtures of laboratory acid and base solutions. These data will aid both industry and FDA for clarifying regulatory questions about how to define acid foods (naturally below pH 4.6) and acidified foods (high pH foods to which acid is added to bring the pH down below 4.6). Research supported by a research agreement with Pickle Packers International was done to determine whether the application of a brief blanching step could improve the safety of non-pasteurized acidified vegetable products (including refrigerated pickles). Previous research has shown that pathogenic bacteria such as Escherichia coli O157:H7 could survive for > 25% of the shelf life of refrigerated pickles, which are not heat processed. The current data support the hypothesis that blanching may be a useful method for improving safety of refrigerated pickles without negatively impacting quality. A reassessment of the cucumber fermentation microbiota using culture independent and dependent techniques was performed. The presence of opportunistic bacterial pathogens such as Citrobacter freundii, Citrobacter brakii, Enterobacter spp., Pseudomonas fluorescens, Stenotrophomonas maltophilia and Kluyvera cryocrescens, and the antibiotic resistant pathogen Serratia marcescens in the initial stage of commercial cucumber fermentations was recognized. Salt in fermentations of at least 2% salt (NaCl) or higher and decreasing pH (below pH 5.2) was inhibitory for these populations. The population density corresponding to these organisms reached undetectable levels by DNA sequence analysis in commercial cucumber fermentations by day 10, presumably due to the developing acidity from the conversion of sugars to acids.

4. Accomplishments
1. Establishment of standards for challenge studies for processing cold-filled acidified food products. To file a scheduled process for acidified foods producers must cite or carry out a scientific study to determine if the product meets federal food safety standards. ARS researchers at Raleigh, North Carolina had a leading role in the development of a protocol (and a webinar) detailing the appropriate scientific methods for challenge studies for the assurance of safety of cold filled acidified foods that do not receive a heat process. The webinar was developed and presented in collaboration with scientists from the University of Georgia, Athens, Georgia and the University of Wisconsin, Madison, Wisconsin. The webinar was hosted by the ‘Beverage and Acid/Acidified Foods Professional Development Group’ of the International Association for Food Protection. Over 200 people registered as attendees. This webinar is now freely available on the IAFP website. The protocol details the scientific considerations needed to conduct such a study, including the methods for growing cells, conducting an acid challenge, and analyzing the data. Many factors can influence the survival of bacterial pathogens in acid and acidified foods, and researchers may not be aware of some of them. We reviewed these factors, primarily based on previous publications from the ARS, Raleigh, North Carolina laboratory, and described how to appropriately control them. The webinar will be useful to researchers, industry stakeholders, and aid FDA to help assure that challenge studies are done with consideration of details that can assure safety.

2. Determining the presence of nitrate and nitrite in fermented and acidified vegetables. The influence of nitrate and nitrite in foods on human health has been controversial, with literature citing both positive and negative health effects. ARS researchers at Raleigh, North Carolina, measured the concentration of these compounds in a wide variety of acidified vegetables (made by adding vinegar or other acids to fresh fruits and vegetables), as well as some fermented foods currently available in the U.S. market. This was done in collaboration with a researcher from Jiangnan University, Wuxi, China, who was a visiting scientist at the Raleigh, North Carolina location. The naturally present antioxidants in foods were also of interest in the study of nitrite and nitrate levels in foods due to interactions between these compounds, so antioxidant levels were also measured. We found that nitrite was relatively rare in acidified vegetables, but was present in some of the fermented foods tested. Nitrate, on the other hand, was found to be present at varying levels in many acidified products. These results provide new information for evaluating nitrate and nitrite content in pickled fruit and vegetable products, and may be used to help assess the potential health consequences of these compounds in US consumer diets.

Review Publications
Ding, Z., Johanningsmeier, S.D., Price, R.E., Reynolds, R., Truong, V., Conley Payton, S.B., Breidt, F. 2018. Evaluation of nitrate and nitrite contents in pickled fruit and vegetable products. Food Control. 90:304-311.
Kay, K., Breidt, F., Fratamico, P.M., Baranzoni, G., Kim, G., Grunden, A., Oh, D. 2017. Escherichia coli O157:H7 acid sensitivity correlates with flocculation phenotype during nutrient limitation. Frontiers in Microbiology. 8:1404.