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 Food Science and Market Quality and Handling Research Unit: Raleigh, NC
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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** 2019 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**
                                

ARS researchers have data for delta psi measurements of acid stressed pathogenic Escherichia coli (STEC), however, continued progress on the internal pH, delta psi and internal salts/metabolites of STEC strains has been suspended because of the loss of a scintillation counter at our location that is needed for quantifying radionuclide activity in samples. Related work on growth and survival of STEC in model vegetable fermentation brines was completed, showing the growth and death rates of STEC strains in both calcium chloride and traditional sodium chloride fermentation brines with a variety of salt levels selectively favored natural fermentative lactic acid bacteria as salt increased. Data from commercial fermentations showed that higher salt levels favored maximum growth levels of normal fermentative bacteria over potential pathogens, although pre-acidification of brines prior to fermentation with acetic acid could reduce survival of enteric (potentially pathogenic) bacteria. This is important because low salt calcium chloride fermentation technology is being developed to reduce waste salt in commercial vegetable fermentations.
 
A computer simulation model to be used for the competitive growth of bacteria was developed using C++ programming language. This mechanistic model has parameters previous determined to control growth at a cell-based level, including per-cell rates of sugar utilization, and the amount of sugar needed for cell division. A multi-threaded program was implemented on a North Carolina State University High Performance Computing (HPC) System server, in collaboration with NC State HPC personnel. The model has been transferred to the ARS CERES computer system via Scinet for continued development by our research group. High throughput processing is required for predicting bacterial growth for up to 1 billion cells. Parameters for acid inhibition of cell death depend on the evolving pH in fermentation brines, and therefore required development of buffer capacity models (BC models, see below) for fermentation brines. Vegetable broths were fermented to various stages and titrated to determine BC by titration and then estimating pH by the BC model. Preliminary data has shown that systematic changes in BC of the fermentations may allow pH prediction over time, based on production of fermentation acids and cell growth. This has been a major stumbling block with previous models because only empirical methods were available for pH prediction.    
Buffer capacity (BC) models were developed to predict the stability of pH changes in acid and acidified food systems. The models were validated with food industry samples. These models consist of two components: first is the determination of a matrix of weak acids and bases present in foods with chemically undefined food ingredients, and second is the pH prediction from these matrices. The BC model process that has been developed requires both laboratory methods and computer models. The first step was titration of food ingredients between pH 2 and pH 12 using an automated titrator. While not designed for generating BC data, the dynamic dosing feature of the titrator was used to generate smooth curves for both acid and base titrations. Data from the titrator was imported into the Matlab computer program using custom (Python) computer code. Once in Matlab, a novel curve fitting method using trigonometric regression was used to analyze the data. This was followed by an optimization algorithm to determine a matrix of buffer values consisting of concentration and equilibrium constant values for weak acids and bases in solution. Finally, pH prediction was done based on the buffer values. These modeling steps were implemented using a series of custom programs that are controlled by a Matlab “live-script” file. The model has been successfully validated and used to examine salad dressing products, as well as fermentation brines from vegetable fermentations. **Accomplishments**
                                

1. Determining the effects of Sodium Chloride or Calcium Chloride concentration on the growth and death of bacterial pathogens in vegetable fermentations. Salt concentration has long been considered an important factor for the quality of fermented vegetable products, but the role of salts in bacterial growth and death during vegetable fermentation remains unclear. ARS scientists in Raleigh, North Carolina, compared the effects of sodium chloride and calcium chloride used in commercial cucumber fermentations on the growth and death of the normal fermentation lactic acid bacteria (LAB) and pathogenic bacterial strains that could be in the brines. The data showed that low salt concentrations had a stimulatory effect on the growth rates of bacteria compared with a no-salt control, but higher salt concentrations decreased growth rates for pathogens; to a lesser extent, LAB growth rates were also reduced. However, no consistent pattern was observed when comparing pathogen death rates with salt type or concentration. For vegetable fermentation safety concerns, the results suggest that an important effect of salt addition is enhancement of the growth of LAB compared to pathogenic Escherichia coli strains. 2. Effects of brine acidification on cucumber fermentation bacteria in calcium or sodium chloride brines. Commercial fermentation for bulk preservation of cucumbers typically relies on natural microbiota and high salt sodium chloride (NaCl) brines. An alternative process utilizing low salt calcium chloride brines was previously developed to eliminate NaCl from fermentation brines for reduced environmental impact. ARS researchers in Raleigh, North Carolina, found that potentially pathogenic enteric bacteria survived the longer in the cucumbers in 6% NaCl brines compared to calcium brines with no initial brine acidification, likely due to slower fermentation under these conditions. However, the addition of acetic acid as a pre-treatment to fermentation brines significantly reduced the survival of these bacteria in both calcium and sodium salt treatments. These data show that pre-acidification of fermentation brines can improve fermentation safety. 3. Development of hot-fill pasteurization of cucumber pickle spears as an alternative to tunnel pasteurization. For commercial production of acidified vegetable products, a tunnel pasteurizer is typically used for thermal processes. To help reduce energy costs and use of water, ARS researchers in Raleigh, North Carolina, developed a hot-fill method for pasteurization of cucumber pickle spears in 24 oz pickle jars. The method required refilling jars multiple times with a hot brine (around 175oF). The data showed that for cucumber spears a hot fill method could achieve or exceed temperatures typically used for commercial pasteurization of pickle by most manufacturers. These conditions exceed published values needed for the required reduction of bacterial pathogens in acid and acidified vegetable products and were sufficient to meet typical industry processing conditions to assure good quality texture and sensory properties. Although further development of processing equipment may be needed for inverting and refilling jars, the in-jar pasteurization process has potential application for cucumber spears and related products and may be used to save on the water usage and costs of currently used tunnel pasteurizers. **Review Publications**
                            *Breidt, F., Andress, E., Ingham, B. 2018. Recommendations for designing and conducting cold-fill hold challenge studies for acidified food products. Food Protection Trends. 38(5):322-328.*
                                *Dupree, D., Price, R.E., Burgess, B., Andress, E., Breidt, F. 2019. Effects of sodium chloride or calcium chloride concentration on the growth and survival Escherichia coli O157:H7 in model vegetable fermentations. Journal of Food Protection. 82(4):570-578. https://doi.org/10.4315/0362-028X.JFP-18-468.*
                                *Yavuz, N., Foster, L., Sharma, T., Patel, K., Stoforos, G., Sandeep, K.P., Planitkar, P., Breidt, F. 2019. Hot-fill pasteurization of cucumber pickle spears: An alternative to tunnel pasteurization. Food Protection Trends. 38(4):258-265. http://staging.nxtbook.com/nxtbooks/trilix/fpt_20180708/stage.php.*
                                *McMurtrie, E.K., Johanningsmeier, S.D., Price, R.E., Breidt, F. 2019. Effect of brine acidification on fermentation microbiota and texture quality of cucumbers fermented in calcium chloride brines. Journal of Food Science. 84(5):1129-1137.  https://doi.org/10.1111/1750-3841.14600.*


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