Location: Food Science and Market Quality and Handling Research Unit
2024 Annual Report
Objectives
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.
Approach
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
Natural media prepared from plant materials are often used as a model system for microbial studies of fermented vegetables. Bacterial pathogen survival in cucumber brines has been used to generate food safety data for regulatory purposes. However, the use of natural vegetable broths, such as cucumber juice, for food safety research may result in variability in microbial growth or pathogen survival patterns due to inconsistent chemical constituents, confounding the interpretation of results from food safety studies. An artificial medium was developed (artificial cucumber juice (ACJ)) that has the same buffering as cucumber juice (CJ), so that both media have similar pH with addition of acid or base. Because the components of ACJ are known, the medium can be consistently reproduced in the laboratory, and the effects of individual chemical constituents on microbial growth or pathogen survival can be studied. Using the artificial medium, the influence of metal ions (magnesium and manganese), amino acids, peptide concentrations, and vitamins on growth of lactic acid bacteria were investigated. The ACJ medium was optimized to equal or exceed the bacterial growth rates observed with CJ. Ongoing studies have also used ACJ to study factors influencing survival of pathogenic bacteria (Escherichia coli) in response to acid addition. The method of matching the buffer capacity between different growth media is novel and allows a new way to optimize and study the influence of media components on bacterial growth and survival.
Software to process optical density data for microbial growth studies using 96 well microtiter plates has been developed (ProcessMicroplate.mlx). The software was developed using the Matlab programming environment. While the method of using 96 well microtiter plates for microbial growth studies has been commonly used by microbiologists, it has recently been shown that the results for the growth curves from these studies are subject to significant bias and error based on the initial cell concentration and optical density (OD). The estimated growth rates can vary as much as 50% as initial OD is varied. Similarly, lag time and maximum OD achieved can vary as well with initial OD. To highlight this issue and to help determine how growth parameters can be measured accurately, a method for preparing 96 well microtiter plates was developed and published on the USDA protocols.io website. The method includes the use of the ProcessMicroplate.mlx software, which can be accessed through the protocols.io site or directly from the USDA software download website. The method and software may have broad application for microbial growth studies which have previously suffered from reproducibility problems.
Buffer models for individual acid and low-acid food ingredients have been used to estimate the pH of ingredient mixtures in silico. Using newly developed buffer modeling software (BufferCapacity3 and IngredientDB), the total buffering (tBeta) for ingredients or mixtures and the influence of ingredients on pH and pH stability of an acidified elderberry syrup were determined. The predicted pH from the software was found to closely match experimentally determined values (mostly within 0.1 pH units). The data clearly demonstrated how the buffer models and software can be used for product development by manufacturers of acid and acidified foods.
The safety of fermented vegetables depends on acid penetration into the vegetable material. The acidification rates in baby carrot and cut asparagus during lactic acid fermentation or direct acidification was measured in low salt (< 2%) brines. Controlled fermentations were conducted with a lactic acid bacteria starter culture (Lactiplantibacillus species). Initial measurements showed that the pH of the carrot and asparagus material (as determined by blending) were reduced to levels that inhibit pathogenic bacteria (pH 4.6 or below) within 2 days of fermentation, which was similar to the more commonly fermented vegetable – pickling cucumbers. Corresponding studies with asparagus that was directly acidified achieved safe pH values within 1 day. Tests of core (center) samples for the pH of carrots and other vegetables are ongoing. These studies will inform safe fermentation/acidification practices for novel pickled vegetable products.
Fungal cultures (17 isolates) isolated from cucumbers used for commercial fermentations have been identified by molecular methods. The identity of the cultures was confirmed by collaboration with a commercial company that specializes in identification of food spoilage fungi. Most cultures were Fusarium species, common to vegetable spoilage. To investigate survival of fungi in cucumber fermentation brines, methods for growing cultures, inoculating brines and selectively plating fungi and lactic acid bacteria from mixed cultures have been developed. Preliminary studies have shown (as expected) that fungi rapidly loose viability in anaerobic vegetable brine fermentations in competition with a Lactiplantibacillus species; however, in fermentation brines exposed to air the fungal species formed surface films. These studies will be used to support further research on spore and/or toxin persistence in vegetable fermentation brines.
Accomplishments
1. Use of pH modeling to assure safety of acidified foods. ARS scientists in Raleigh, North Carolina have developed methods to assess how individual ingredients affect the final product pH of an acidified elderberry syrup. These studies used publicly available software previously developed by the Raleigh laboratory. Results from the studies included a measure of total ingredient buffering (defined as tBeta) for the ingredients in the syrup product, which included: citric acid, elderberry juice, malic acid, ascorbic acid, lemon juice, spices and others. These data showed that the buffer modeling methods may be used to guide manufacturers when adjusting formulations, so that a desired pH may be stably achieved for safety and/or product quality. To validate the results, a total of 16 different formulations having pH values between pH 3 and pH 4 were prepared. The estimated pH values of the syrup formulations from buffer modeling software were found to match measurements typically within 0.1 pH units. Several large food companies that supply ingredients or manufacture acid and acidified foods have subsequently adapted the technology for use with product development and to help meet regulatory compliance for pH and pH stability. The measurement of tBeta, or total buffering developed for this work has been applied to over 40 ingredients common to other acid and acidified foods and the data have been made available in a public database file on the USDA software download website. The software and data for buffer modeling have also been presented to the Food and Drug Administration for use as a scientific tool that can help clarify acid and acidified food regulations.