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ARS Home » Southeast Area » Raleigh, North Carolina » Food Science and Market Quality and Handling Research Unit » Research » Research Project #440115

Research Project: Influence of Ingredients and Processing Methods on the Safety of Fermented and Acidified Foods

Location: Food Science and Market Quality and Handling Research Unit

2022 Annual Report

1. Determine how food components influence pathogen die-off in acidic food products. Modeling pH and acid effects on pathogen reduction in ready-to-eat vegetable fermentations. 2. Determine how processing conditions influence survival of fungal spores, toxins and vegetative cells in fermented or acidified vegetables. Supporting research to reduce food waste by fermentation.

Objective 1: Determine how food components influence pathogen die-off in acidic food products. Goal/hypothesis 1: Our hypothesis is that buffer capacity (BC) models can be used to link pH with acid accumulation and therefore log reduction times for bacterial pathogens in a binary (lactic and acetic) acid RTE vegetable fermentations. The goal is to develop methods for determining the safety of a variety of different RTE fermentations based on pH. Experimental design overview: 1. BC modeling of pH and lactic and acetic acid concentrations in a brined vegetable medium. BC models for pH and acid concentration will be developed using the combined buffering of CJ (or other vegetable brines) with the buffering of added acids. 2. What are the typical acid mixtures produced by heterolactic LAB? It is important to know the ratio of lactic and acetic acids typical of low salt fermentations because the acids have different antimicrobial effects. 3. How do acid mixtures affect pathogen (STEC) die-off in vegetable brines? Determination of log reduction times based on protonated lactic acid and acetic acid mixtures in ACJ. 4. Can the BC of unfermented brines be used to accurately model pH changes in fermentations? BC models will be used to assess pH changes resulting from acid production by LAB in CJ brine and cucumber and cabbage fermentations. 5. Validation of BC models for estimating pathogen die-off in fermentation based on pH. Reduction of bacterial pathogens will be estimated based on pH of cucumber and cabbage fermentation brines and data compared to CJ and cabbage or cucumber fermentations. 0bjective 2: Determine how processing conditions influence survival of fungal spores, toxins and vegetative cells in fermented or acidified vegetables. Goal/hypotheses: Discovery: Identify fungi that are present on spoiling cucumbers. Hypothesis 1: In addition to inhibition of bacterial pathogens (Objective 1) organic acids and pH will prevent growth or persistence of fungal cells and/or toxins in RTE vegetable fermentation brines. Hypothesis 2: LAB may produce acids and antifungal compounds active against fungal cells, and persistence of fungal toxins in fermentation brines may be reduced by LAB due to degradation or LAB binding. The goal is to develop recommendations for safe preservation of surplus vegetables by fermentation. Experimental design overview: 1. What fungi are present on spoiling cucumbers? Identification of the fungi typically present on spoiling cucumbers. 2. Do LAB present in cucumber fermentation produce antifungal compounds? LAB strains will be screened for antifungal compounds 3. Do fungal cells and/or toxins persist during fermentation of CJ by LAB? Fungi and/or toxins will be inoculated into ACJ brines or cucumber fermentations to measure die-off during fermentation due to acid accumulation and possibly antifungal compounds produced by LAB.

Progress Report
Progress was made on all two objectives, which fall under NP108. Under Objective 1, buffer models were developed for determining pH of buffered solutions with added acids. This novel technology was applied to vegetable fermentation brines. The pH changes that occur during the fermentation of vegetables by lactic acid bacteria depend on the production of weak acids and on the buffering of the fermentation medium. Undefined buffering components of fermentation media make estimates of pH changes from acid production difficult. Buffer models were, therefore, developed for a model cucumber fermentation brine system linking pH changes to acid concentrations. A novel titration method was used to measure buffer capacity (BC) in cucumber juice medium made from three grades of pickling cucumbers based on diameter. Fermentation of juice made from cucumbers of different sizes resulted in differences in fermentation biochemistry. The results of modeling indicated that the pH of the medium after 24 and 48 hours of fermentation by selected lactic acid bacteria could be predicted from acid concentrations based on the measured BC of the corresponding unfermented medium. The differences for all observed and predicted pH values of the fermentation samples, based on measured acid concentrations, had an error (root mean square error) of 0.064 pH units. The buffer models included a quantitative measure of the effect on pH of the malolactic reaction caused by the lactic acid bacteria. These models have application for assessing the influence of a variety of lactic acid bacteria on pH changes in food fermentations. Linking pH to fermentation acid concentrations may aid manufacturers and regulatory agencies in assessing the safety of fermented foods. A computer program has been developed by ARS scientists at Raleigh, North Carolina, to process titrator data and generate buffer models of aqueous food ingredients or formulations for acid and acidified foods. The program was configured for processing output files with acid and base titration data from a commercial titrator. Data was automatically extracted from titrator files and converted into BC values for the pH range of pH 2 to 12 (typical for food ingredients). A model of the BC data was then generated using a curve fitting algorithm. The output from the program included data for a series of buffers that essentially define all the buffering of the food ingredient and, therefore, how the ingredient may influence the pH of a food product. A novel method was developed that utilized a numerical integration procedure to determine the area under the BC curve. This method resulted in a single quantitative measure to define the total buffering of a food ingredient. The total buffering metric is a novel and simple way to compare the relative pH impact of low acid (high pH) ingredients in acid and acidified foods. The output data from the program may be useful for product development by manufacturers of acid and acidified foods, as well as regulatory agencies for assessing how food ingredients may influence product pH. Under Objective 2, the identification of mold isolates previously obtained from spoiling cucumbers was confirmed through a Material Transfer Research Agreement with a collaborator. Seventeen isolates were identified by the collaborator using both classical and molecular techniques. The results confirmed earlier conclusions that the majority of the isolates were Fusarium species, which are common on spoiling fruits and vegetables. Salmonella is one of the leading causes of foodborne illnesses worldwide. The rapid emergence of multidrug-resistant Salmonella strains has increased global concern of salmonellosis. Bacteriophages (phage) that selectively kill Salmonella may be used as biocontrol agents in foods because: they don’t attack other bacteria or animals; do not alter sensory nutritional quality of foods; and are safe to eat and environmentally friendly. Due to the large variation of Salmonella serotypes, Salmonella phages with broad host ranges are needed if biocontrol applications of phage will be successful. A collaborative effort involving researchers from Kennesaw State University, Kennesaw, Georgia; North Carolina State University, Raleigh, North Carolina; and ARS researchers from Raleigh, North Carolina, resulted in the isolation and characterization of 14 Salmonella phages from turkey fecal and cecal samples. Selected phages were characterized and were found to have unique structural protein profiles. Six phages shared eight hosts including seven different Salmonella serovars and one Shigella sonnei strain. One phage, phiEnt, displayed the broadest host range infecting 11 Salmonella strains from nine serovars and three Shigella strains from two species. In the model food system (cucumber juice or beef broth), phiEnt infection had high lytic activity and also had good thermal stability. These results indicate that phiEnt has potential to be used as a biocontrol agent against diverse Salmonella serovars in foods.

1. Development of a software program for buffer modeling of food ingredients. For many acidic foods, including fermented and acidified vegetables, salsas, salad dressings, and others, maintaining a pH below 4.6 is a critical control to prevent botulism. The pH of acidic foods is controlled by acid content, low acid ingredients and buffering, however, buffering of foods remains largely uncharacterized. An ARS researcher in Raleigh, North Carolina, has developed an easy-to-use Matlab graphical user interface program, BufferCapacity3, to automate the process of quantifying the buffers present in foods by utilizing acid/base titration data. A novel feature of the BufferCapacity3 program is to quantify the total buffer capacity of a food ingredient and, therefore, indicate how it may alter the pH of acid or acidified food formulations. Buffer capacity data and technology have been used by the salad dressing industry trade association and member companies in assessments of the safety and regulatory status of acid food products. The Graphical User Interface program has been made available for download on the USDA-ARS software download website, along with instructions and sample data.

Review Publications
LaFountain, L.J., Johanningsmeier, S.D., Breidt, F., Stoforos, G.N., Price, R.E. 2022. Effects of a brief blanching process on quality, safety, and shelf life of refrigerated cucumber pickles. Journal of Food Science. 87(4):1475-1488.
Lu, Z., Marchant, J., Thompson, S., Melgarejo, H., Ignatova, D., Kopic, S., Damaj, R., Paramo, R., Reed, A., Breidt, F., Kathariou, S. 2022. Bacteriophages isolated from turkeys infecting diverse Salmonella serovars. Frontiers in Microbiology. 13:933751.