Location: Food Science and Market Quality and Handling Research Unit
2025 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
Growth and acid production by lactic acid bacteria are critical to the safety of vegetable fermentations, but little data has been published about factors controlling growth rates, lag times, and maximum growth in these food fermentations. Using a laboratory medium (artificial cucumber juice, ACJ) that was developed to have similar fermentation properties to cucumber juice (CJ), biochemical factors that influence the growth of lactic acid bacteria (LAB) were discovered. It was found that the principal buffer controlling pH in cucumber juice fermentations was malic acid. Reducing malic acid concentrations in ACJ resulted in lower final cell counts and less acid produced, even if residual sugar for growth was available. There are two principal factors that prevent growth of LAB in vegetable fermentations pH and the total acid concentrations for lactic and/or acetic acids. Data from ACJ studies with an LAB strain that predominates in cucumber fermentations showed that pH, rather than the accumulation acid, was more influential in stopping growth of LAB. Additional data showed how other factors, including a specific metal ion in cucumber fermentation brines, manganese, influenced both growth rate and maximum cell density, supporting previously published results. Lipid compounds, either from cells of the vegetable material being fermented or added in ACJ were critical to LAB growth. This work, for the first time, has allowed research to be done to identify how individual chemical components, naturally present in vegetable fermentation brines, can impact the growth rates of the fermentation bacteria and the completeness of sugar utilization. The data indicate that the growth medium which has been developed may be easily modified for the study of a variety of vegetable and other food fermentations to control pH evolution and acid production, which influence quality and safety.
Results from a Cooperative Research and Development Agreement project have shown that a natural antimicrobial of plant origin can be used to replace sodium benzoate in pickled vegetable products. Inoculated pack experiments were carried out using spoilage lactic acid bacteria purposely added to brined pickles in jars containing the novel antimicrobial. The bacteria were added to the extent that jars protected by sodium benzoate were overwhelmed with the spoilage bacteria and became turbid and commercially unacceptable, however, jars protected by the antimicrobial were not. Preservation of the jars was achieved to the extent that no growth of the spoilage bacteria could be observed. Microbial and biochemical analysis of the preserved jars showed the natural antimicrobial allowed either no growth or only very limited growth of spoilage microbes. Preliminary sensory tests of the natural antimicrobial used in the tests indicated that, depending on concentration, little or no impact on flavor was observed using a typical industrial brine formula. Currently, limited commercial trials are being prepared to gauge consumer acceptance.
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 and compared to that of pickled cucumber. Controlled fermentations were conducted with a lactic acid bacteria starter culture (Lactiplantibacillus species). Vegetable and brine pHs were measured separately during the initial stage of fermentation to determine acid penetration into the vegetables. In addition, carrot core samples (3 x 3 mm diameter) were collected to assess acid penetration to the center of the vegetable. Across all salt levels, the pH of the fermented vegetables steadily decreased to below 4.6 within 2 days of fermentation. Complete acid equilibration required 3, 4, and 7 days for cut asparagus, baby carrot, and whole cucumber, respectively. Corresponding studies with carrot and asparagus that were directly acidified achieved safe pH values within 1 day. These studies will inform fermentation/acidification practices for the safe manufacturing of novel pickled vegetable products.
An integral solution for vegetable waste lies in the development of extended shelf-life fermented or acidified vegetable blends produced with low salt. ARS scientists in Raliegh,North Carolina developed compositions and methods for the preparation of five brightly colored, fermented vegetable blend prototypes to be tested in a human clinical trial by a sister Unit in Davis, California for health benefits. Because of the low salt content in such vegetable blends and/or their acidification with inorganic acids instead of organic acids, a series of tests were conducted to assure their safety. It was determined that a preservative, such as sorbic acid or benzoic acid, is needed on the low salt blends to assure safety and that pH must be maintained at 3.40 ± 0.1 to attain a reasonable hold time. It was additionally confirmed that the preservation of the vegetable blends by acidification with an organic acid is more efficient than when using an inorganic acid. The safety of innovative products derived from such preserved vegetable blends, including bavarois, coulis, spreads, custards and other culinary presentations, is the target of ongoing studies.
Accomplishments
1. Identification of chemical factors influencing fermentation safety. ARS scientists in Raleigh, North Carolina applied a novel technique called buffer modeling to develop a synthetic bacterial growth medium. The medium (ACJ) was designed to give similar results as natural vegetable fermentations when fermented with lactic acid bacteria. In laboratory studies fermentations with this medium resulted in similar changes in bacterial cell counts, acid levels, pH and other biochemical changes as would be found in natural fermentations. ACJ medium was then used to investigate how the biochemical components that are naturally present in commercial cucumber fermentations influence the extent of fermentation and fermentation safety. The results showed that specific chemical constituents of cucumber fermentation brines including malic acid manganese ions, and lipids from the cucumber material have strong influences on the growth rates of the natural fermentation bacteria and sugar utilization. Because these components could be adjusted at will in ACJ medium, specific effects could individual biochemical components be determined for the first time. This study demonstrated that buffer modeling technique works, and that these methods can be similarly applied to a variety of food fermentations. The results and data are useful for making food safety recommendations.