Roger F. McFeeters

Independent effects of acetic acid and pH on survival of Escherichia coli in simulated acidified pickle products.

Our objective was to determine the effects of organic acids and pH on the rate at which selected strains of Escherichia coli O157:H7 die in acid solutions representative of acidified pickle products (pH < 4.6). We used gluconic acid/sodium gluconate (pKa = 3.7) as a noninhibitory buffer to maintain pH at selected values in the absence of other organic acids. This was possible because we found that the inhibitory effects of this acid on E. coli strains at pH 3.1 were independent of acid concentration over a range of 2 to 200 mM. By this method, the lethal effects of acetic acid solutions (100 to 400 mM) at selected pH values between 3.1 and 4.1 were compared with the effects of pH alone (as determined using gluconate buffer). We found D-values were two- to fourfold lower with acetic acid compared with the effect of pH alone for simulated pickle brines in this pH range. Glutamic acid, an amino acid that is known to enhance acid resistance in E. coli and is a component of pickle brines, protected the E. coli strains from the specific effects of acetic acid.

Utilization of electron acceptors for anaerobic mannitol metabolism by Lactobacillus plantarum. Compounds which serve as electron acceptors

In a chemically defined medium under anerobic conditions Lactobacillus plantarum grew on glucose, but was unable to grow with mannitol as the carbon source. Mannitol was a suitable carbon source, provided 1 of 11 compounds (pyruvic acid, α -ketobutyric acid, α -ketovaleric acid, α -ketocaprylic acid, acetic acid, acetyl phosphate, acetaldehyde, citric acid, axoloacetic, malic acid, and fumaric acid) was added to the medium as an electron acceptor. The extent of mannitol fermentation was proportional to the concentration of available electron acceptor. The reduced products of the electron acceptor compounds were identified and possible pathways for the metabolism of electron acceptors were proposed. Strains from two other species of lactobacilli which ferment mannitol appear to use different electron acceptor pathways.

Fermentation of cucumbers brined with calcium chloride instead of sodium chloride.

Waste water containing high levels of NaCl from cucumber fermentation tank yards is a continuing problem for the pickled vegetable industry. A major reduction in waste salt could be achieved if NaCl were eliminated from the cucumber fermentation process. The objectives of this project were to ferment cucumbers in brine containing CaCl(2) as the only salt, to determine the course of fermentation metabolism in the absence of NaCl, and to compare firmness retention of cucumbers fermented in CaCl(2) brine during subsequent storage compared to cucumbers fermented in brines containing both NaCl and CaCl(2) at concentrations typically used in commercial fermentations. The major metabolite changes during fermentation without NaCl were conversion of sugars in the fresh cucumbers primarily to lactic acid which caused pH to decrease to less than 3.5. This is the same pattern that occurs when cucumbers are fermented with NaCl as the major brining salt. Lactic acid concentration and pH were stable during storage and there was no detectable production of propionic acid or butyric acid that would indicate growth of spoilage bacteria. Firmness retention in cucumbers fermented with 100 to 300 mM CaCl(2) during storage at a high temperature (45 degrees C) was not significantly different from that obtained in fermented cucumbers with 1.03 M NaCl and 40 mM CaCl(2). In closed jars, cucumber fermentations with and without NaCl in the fermentation brine were similar both in the chemical changes caused by the fermentative microorganisms and in the retention of firmness in the fermented cucumbers.

Characteristics of Spoilage-Associated Secondary Cucumber Fermentation

ABSTRACT Secondary fermentations during the bulk storage of fermented cucumbers can result in spoilage that causes a total loss of the fermented product, at an estimated cost of

Determination of 5-Log Reduction Times for Food Pathogens in Acidified Cucumbers during Storage at 10 and 25°C†‡

6,000 to

Determination of 5-Log Pathogen Reduction Times for Heat-Processed, Acidified Vegetable Brines †‡

15,000 per affected tank. Previous research has suggested that such fermentations are the result of microbiological utilization of lactic acid and the formation of acetic, butyric, and propionic acids. The objectives of this study were to characterize the chemical and environmental conditions associated with secondary cucumber fermentations and to isolate and characterize potential causative microorganisms. Both commercial spoilage samples and laboratory-reproduced secondary fermentations were evaluated. Potential causative agents were isolated based on morphological characteristics. Two yeasts, Pichia manshurica and Issatchenkia occidentalis, were identified and detected most commonly concomitantly with lactic acid utilization. In the presence of oxygen, yeast metabolic activities lead to lactic acid degradation, a small decline in the redox potential (Eh, Ag/AgCl, 3 M KCl) of the fermentation brines, and an increase in pH to levels at which bacteria other than the lactic acid bacteria responsible for the primary fermentation can grow and produce acetic, butyric, and propionic acids. Inhibition of these yeasts by allyl isothiocyanate (AITC) resulted in stabilization of the fermented medium, while the absence of the preservative resulted in the disappearance of lactic and acetic acids in a model system. Additionally, three Gram-positive bacteria, Lactobacillus buchneri, a Clostridium sp., and Pediococcus ethanolidurans, were identified as potentially relevant to different stages of the secondary fermentation. The unique opportunity to study commercial spoilage samples generated a better understanding of the microbiota and environmental conditions associated with secondary cucumber fermentations.

Influence of Sodium Chloride, pH, and Lactic Acid Bacteria on Anaerobic Lactic Acid Utilization during Fermented Cucumber Spoilage

Outbreaks of acid-resistant foodborne pathogens in acid foods with pH values below 4.0, including apple cider and orange juice, have raised concerns about the safety of acidified vegetable products. For acidified vegetable products with pH values between 3.3 and 4.6, previous research has demonstrated that thermal treatments are needed to achieve a 5-log reduction in the numbers of Escherichia coli O157:H7, Listeria monocytogenes, or Salmonella enterica. For some acidified vegetable products with a pH of 3.3 or below, heat processing can result in unacceptable product quality. The purpose of this study was to determine the holding times needed to achieve a 5-log reduction in E. coli O157:H7, L. monocytogenes, and S. enterica strains in acidified vegetable products with acetic acid as the primary acidulant, a pH of 3.3 or below, and a minimum equilibrated temperature of 10 degrees C. We found E. coli O157:H7 to be the most acid-resistant microorganism for the conditions tested, with a predicted time to achieve a 5-log reduction in cell numbers at 10 degrees C of 5.7 days, compared with 2.1 days (51 h) for Salmonella or 0.5 days (11.2 h) for Listeria. At 25 degrees C, the E. coli O157:H7 population achieved a 5-log reduction in 1.4 days (34.3 h).

Metabolism of lactic acid in fermented cucumbers by Lactobacillus buchneri and related species, potential spoilage organisms in reduced salt fermentations ☆

Recent outbreaks of acid-resistant food pathogens in acid foods, including apple cider and orange juice, have raised concerns about the safety of acidified vegetable products. We determined pasteurization times and temperatures needed to assure a 5-log reduction in the numbers of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella strains in acidified cucumber pickle brines. Cocktails of five strains of each pathogen were (separately) used for heat-inactivation studies between 50 and 60 degrees C in brines that had an equilibrated pH value of 4.1. Salmonella strains were found to be less heat resistant than E. coli O157:H7 or L. monocytogenes strains. The nonlinear killing curves generated during these studies were modeled using a Weibull function. We found no significant difference in the heat-killing data for E. coli O157:H7 and L. monocytogenes (P = 0.9709). The predicted 5-log reduction times for E. coli O157:H7 and L. monocytogenes were found to fit an exponential decay function. These data were used to estimate minimum pasteurization times and temperatures needed to ensure safe processing of acidified pickle products and show that current industry pasteurization practices offer a significant margin of safety.

Effects of pH Adjustment and Sodium Ions on Sour Taste Intensity of Organic Acids

UNLABELLED Cucumbers are preserved commercially by natural fermentations in 5% to 8% sodium chloride (NaCl) brines. Occasionally, fermented cucumbers spoil after the primary fermentation is complete. This spoilage has been characterized by decreases in lactic acid and a rise in brine pH caused by microbial instability. Objectives of this study were to determine the combined effects of NaCl and pH on fermented cucumber spoilage and to determine the ability of lactic acid bacteria (LAB) spoilage isolates to initiate lactic acid degradation in fermented cucumbers. Cucumbers fermented with 0%, 2%, 4%, and 6% NaCl were blended into slurries (FCS) and adjusted to pH 3.2, 3.8, 4.3, and 5.0 prior to centrifugation, sterile-filtration, and inoculation with spoilage organisms. Organic acids and pH were measured initially and after 3 wk, 2, 6, 12, and 18 mo anaerobic incubation at 25 °C. Anaerobic lactic acid degradation occurred in FCS at pH 3.8, 4.3, and 5.0 regardless of NaCl concentration. At pH 3.2, reduced NaCl concentrations resulted in increased susceptibility to spoilage, indicating that the pH limit for lactic acid utilization in reduced NaCl fermented cucumbers is 3.2 or lower. Over 18 mo incubation, only cucumbers fermented with 6% NaCl to pH 3.2 prevented anaerobic lactic acid degradation by spoilage bacteria. Among several LAB species isolated from fermented cucumber spoilage, Lactobacillus buchneri was unique in its ability to metabolize lactic acid in FCS with concurrent increases in acetic acid and 1,2-propanediol. Therefore, L. buchneri may be one of multiple organisms that contribute to development of fermented cucumber spoilage. PRACTICAL APPLICATION Microbial spoilage of fermented cucumbers during bulk storage causes economic losses for producers. Current knowledge is insufficient to predict or control these losses. This study demonstrated that in the absence of oxygen, cucumbers fermented with 6% sodium chloride to pH 3.2 were not subject to spoilage. However, lactic acid was degraded by spoilage microorganisms in reduced salt, even with pH as low as 3.2. Efforts to reduce salt in commercial brining operations will need to include control measures for this increased susceptibility to spoilage. Lactobacillus buchneri was identified as a potential causative agent and could be used as a target in development of such control measures.

Novel Quantitative Assays for Estimating the Antimicrobial Activity of Fresh Garlic Juice

Recent evidence suggests that Lactobacillus buchneri may play an important role in spoilage-associated secondary fermentation of cucumbers. Lactic acid degradation during fermented cucumber spoilage is influenced by sodium chloride (NaCl) concentration, pH, and presence of oxygen. Objectives were to evaluate these factors on lactic acid utilization by L. buchneri, and to compare the biochemical changes to those which occur during fermented cucumber spoilage. Effects of NaCl (0, 2, 4, and 6% w/w), pH (3.8 vs 5.0), and aerobic environment were investigated using fermented cucumber media (FC) inoculated with spoilage microorganisms. At pH 3.8, L. buchneri degraded lactic acid in all NaCl concentrations. The highest rate of lactic acid utilization occurred in FC with 2% NaCl (P < 0.05). Lactic acid utilization was nearly identical under aerobic and anaerobic conditions, indicating that oxygen does not influence lactate metabolism by L. buchneri. Lactic acid utilization was accompanied by increases in acetic acid and 1,2-propanediol, and Lactobacillus rapi was able to convert 1,2-propanediol to propionic acid and propanol. L. buchneri initiated spoilage in a wide range of environmental conditions that may be present in commercial cucumber fermentations, and L. rapi may act syntrophically with L. buchneri to produce the commonly observed spoilage metabolites.