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ARS Home Southeast Area Raleigh, North Carolina Food Science and Market Quality and Handling Research Unit Research





- Breidt, Frederick
- Johanningsmeier, Suzanne
- Perez Diaz, Ilenys
- 2021
- 2020
- 2019
- 2018
- 2017
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Intervention Strategies for Controlling Human Pathogens Associated with Fermented and Acidified Vegetables **Location:
                            Food Science and Market Quality and Handling Research Unit** 2021 Annual Report **Objectives**
                                

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. **Approach**
                                

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. **Progress Report**
                                

This is the final report for the Project 6070-41420-008-00D terminated in April
2021,which has been replaced by new Project 6070-41420-009-00D.
Buffer capacity modeling. ARS scientists at Raleigh, North Carolina suggests buffer capacity is a critical factor for determining how pH may change with the addition of ingredients to acid or acidified foods, which are made safe by low pH. While models have been published in the scientific literature for predicting pH in water-based solutions, ARS scientists at Raleigh, North Carolina have developed a method for identifying buffers in undefined food ingredient solutions and using the data to predict how pH may change with added food ingredients for acidic foods. The models were used by ARS scientists at Raleigh, North Carolina for determining the buffer capacity of typical ingredients in these food products, and calculating how these ingredients may change pH, a vital safety factor for foods primarily protected from disease causing bacteria by pH. ARS scientists at Raleigh, North Carolina have developed mathematical models and methods for the analysis of food ingredients to allow pH predictions in a final product with mixed ingredients and acids typical of salad dressing products. The models have been shown to accurately predict pH acid-base solutions and can predict relative amount of buffering present in different food ingredients. These models have broad applicability with many acid or acidified foods, and aid producers and regulatory agencies in determining the safety of acidified foods products.  

Bacterial competition in vegetable fermentations. The acid resistance of pathogenic Escherichia coli strains likely explains the pathogen’s ability to transit the human stomach, which makes these organisms a concern in acid or acidified foods. ARS scientists at Raleigh, North Carolina screened multiple E. coli strains to assess their acid resistance in simulated stomach acid and vegetable fermentation acids. These strains were separated into two groups that differed in acid resistance, measured their growth rates, and ability to survive in competition with lactic acid bacteria in model vegetable fermentations. Genome sequence data showed that one of the acid sensitive strains was missing two acid resistance genes. Unexpectedly, ARS scientists at Raleigh, North Carolina found that the acid sensitive strain was as competitive or more competitive than the acid resistant strains in model vegetable fermentations. These results indicate that the resistance to acids in laboratory tests may not be an accurate predictor of E. coli survival in vegetable fermentations and have triggered new research on efforts to understand how bacterial competition works in fermented foods.

Viruses active against enteric bacteria in vegetable fermentations. Bacteriophage are viruses that specifically attack bacteria and are widely present in the natural environment and in food fermentations. The diversity and functional role that these phages play in infecting Enterobacteriaceae, which are spoilage and potentially pathogenic bacteria that can grow in the early stages of vegetable fermentations, was investigated. In samples from the first few days of a commercial vegetable fermentation 26 independent bacteriophage were isolated, along with 39 Enterobacteriaceae strains that were tested to see if the phage would infect them. Two-thirds of the phage were active against these cultures. This study by ARS scientists at Raleigh, North Carolina, combined with previous similar studies of sauerkraut fermentations, revealed the abundance and variety of phages infecting Enterobacteriaceae bacteria in the early stages of vegetable fermentations. The data show that both bacteriophage infection and inhibition by lactic acid both play a role in maintaining the safety of vegetable fermentations by reducing Enterobacteriaceae populations. 

Critical controls for fermented foods. Under the Food Safety Modernization Act, a business that manufactures fermented foods may be required to conduct a risk analysis and establish pertinent preventive controls. Retail food establishments operating under the Food and Drug Administration (FDA) Food Code must often seek a variance for manufacture of fermented foods and beverages. Developing food safety programs can be a challenge for small-scale producers with little access to training and resources, especially as manufacture of fermented products involves microbiologically complex systems that may not be effectively or appropriately managed by standard time-temperature controls. Working with university extension faculty, the science behind traditional vegetable fermentation processes, e.g., cabbage, cucumbers and peppers, was reviewed by ARS scientists at Raleigh, North Carolina to identify relevant hazards based on intrinsic and extrinsic factors inherent in the fermentation systems that influence microbial survival. It was determined by ARS scientists at Raleigh, North Carolina that one Critical Control Point in the manufacture of traditionally fermented vegetable products, namely a steady and sustained pH decline to < 4.6, was important for food safety. Additional Control Points at key steps, i.e., vegetable preparation and salt addition; fermentation time and temperature; refrigerated storage; and/or packaging for shelf stability were also identified by ARS scientists at Raleigh, North Carolina, and an example for safe manufacture of fermented vegetables was developed, based on the fermentation of kimchi.

Microbiota in vegetable fermentations with varying salt levels. The influence of salt type used in cover brines on the microbiota of laboratory and commercial scale cucumber fermentations was investigated by ARS scientists at Raleigh, North Carolina. Laboratory fermentation cover brines with calcium chloride (low salt brining technology) induced faster microbial growth as compared to cover brines with no salt or 6% sodium chloride typical of traditional commercial fermentations. In the initial days of fermentation Enterobacteriaceae such as Citrobacter and Enterobacter was favored in fermentations brined with sodium chloride, in which it took longer for the lactic acid bacteria to grow compared to other salt conditions. Lactobacilli dominated all fermentation brines by the third day, regardless of salt type or content, and 80 to 88% of the population was composed of lactobacilli by the seventh day of fermentation, except in fermentations without salt, in which a mixed population of lactic acid bacteria were still prevalent. In general, the population of lactic acid bacteria found in commercial cucumber fermentations brined with 1.1% calcium chloride were similar to the bacteria found in laboratory fermentations. Understanding how salt affects the microbiota of commercial fermentations will facilitate optimization of low salt fermentation technology. 


Summary report for 2015-2021. 

Significant results were achieved by ARS scientists at Raleigh, North Carolina over the life of the project, which was completed in 2021. Results from research on Objective 1 have shown that fermentation salts (sodium and calcium chlorides) primarily enhance the growth of lactic acid bacteria and therefore are only indirectly inhibitory to vegetative bacterial pathogens such as Escherichia coli, Salmonella enterica and Listeria monocytogenes. The most acid resistant pathogen of concern (E. coli) was found to grow in the early stages of vegetable fermentations using a model system with no plant inhibitors of bacterial growth, even if the salt concentration was high (6%). However, these organisms rapidly died off as organic acid increased, and pH dropped. Surprisingly, data showed that one of the most acid resistant E. coli strains may not survive as long in fermentations as a more acid sensitive resistant strain. This was likely due to the affect the acid resistant E. coli strain had on buffering the pH of the medium, aiding growth of competing lactic acid bacteria. The published data shed new light on the complex interactions that occur during bacterial competition in fermented foods. 

Results from competitive growth modeling efforts for Objective 2 showed that a key factor, predicting pH in the fermentation medium, was needed for these models to be widely applicable to fermented vegetables. To address this research need, buffer models developed by ARS scientists at Raleigh, North Carolina for Objective 3 (see below) were adapted to a model vegetable fermentation system. Using in silico models, it was found that pH predictions based on the initial buffering of the medium were useful for predicting the extent of fermentation pH changes. Further development of this technology is an important objective in our subsequent food safety research plans. 

Buffer capacity (BC) models to predict the changes in pH due to low acid ingredients in acid and acidified foods were successfully developed for Objective 3. The modeling effort included the development of a series of algorithms that can be used to: 1) generate BC models from titration data; 2) model BC curves to generate data on buffers in a food ingredient that control pH; 3) Allow comparison of relative buffering of different food ingredients; 4) predict pH for a given set of ingredients. These algorithms were encoded using Matlab modeling software and made available for public download on the ARS software website. A stand-alone Windows 10 program that may be used for comparison of ingredient buffering was also developed and similarly published. Model results have been used by industry trade associations and made available to FDA to aid in the regulatory process of differentiating different types of acidic food products. **Accomplishments**
                                

1. Determination of the buffer capacity of ingredients in acidic foods. ARS researchers in Raleigh, North Carolina, used recently developed buffer capacity models to analyze food ingredients commonly used in salad dressings and related pickled vegetable products. This was done by combining classical physical chemistry concepts of pH modeling with modern numerical computing methods. For 24 food ingredients commonly used in acidic food products like salad dressings, including acids as well as flavoring ingredients and spices that have a pH close to neutral, a model of buffering was developed. These models were then used to estimate how each ingredient, if added to an acidic food product, would (or would not) change pH of the product. Compared to the acids typically added in these acidic foods (usually acetic acid) most other salad dressing ingredients tested had very little influence on pH change at concentrations typically used for commercial products. This was found to be due to very weak buffering activity. For acidic food products where pH is a critical control factor for food safety, these data can be used to quantify safe concentration limits. The results may be useful to both manufacturers and regulatory agencies for defining safe production practices and to assure product stability. 2. Determining how bacterial competition influences the rate of reduction of pathogenic bacteria in vegetable fermentations with different salt concentrations. ARS scientists at Raleigh, North Carolina, believe the effects of microbial competition on the survival of pathogenic bacteria in vegetable fermentations has previously been demonstrated, but quantitative data on pathogen die-off is lacking. To investigate the effects of fermentation conditions on the survival of acid resistant Escherichia coli strains, ARS scientists measured the die-off of different pathogenic E. coli strains in laboratory vegetable fermentations. Unexpectedly one of the most acid resistant strains of E. coli did not survive as long as a less acid resistant strain in competition with lactic acid bacteria (LAB). The data indicated that competition with LAB is dependent on complex factors that include buffering of the fermentation medium. This buffering can alter expected pH changes and influence all bacteria in the fermentation (including E. coli). Methods to measure buffering have been developed for use in future studies. These data aid both manufacturers and regulatory agencies concerned with assuring safety of fermented vegetable foods. 3. Enhanced safety of refrigerated cucumber pickles. A brief blanching procedure was developed by ARS scientists at Raleigh, North Carolina, to improve the safety of refrigerated cucumber pickles. The traditional manufacturing process for these products has limited efficacy against pathogenic bacteria which may be on cucumbers from environmental sources. The brief blanching procedure for raw cucumbers was investigated to determine the reduction in bacterial cell counts as well as the effect of blanching on finished product quality. The data were used to model pathogen kill at varying depths within the cucumber for an optimized procedure of 90 seconds of blanching in 80°C (176°F) water. The modeling was done in collaboration with North Carolina State University research engineers. The resulting model can show the predicted death of acid resistant strains of Escherichia coli, which is the most acid resistant pathogen of concern for these products. Unexpectedly, the data also showed that the blanching procedure was effective in improving some quality aspects of refrigerated pickle products made with cucumbers subjected to the blanch treatment. These data have been presented to industry stakeholders and may be of general use to the pickled vegetable industry for improving refrigerated product safety.


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