Ilenys M. Pérez-Díaz

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

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

6,000 to

Role of selected oxidative yeasts and bacteria in cucumber secondary fermentation associated with spoilage of the fermented fruit.

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.

Bacteriophage Ecology in a Commercial Cucumber Fermentation

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.

Characterization of Cucumber Fermentation Spoilage Bacteria by Enrichment Culture and 16S rDNA Cloning

Changes during the spoilage of fermented cucumber pickles have been attributed to the metabolism of different yeasts and bacteria. In this study six organisms isolated from commercial spoiled cucumber pickles were evaluated for their possible role in primary and secondary cucumber fermentations. The ability of the yeasts Issatchenkia occidentalis and Pichia manshurica to utilize lactic and acetic acids during aerobic metabolism was confirmed and associated with increases in brine pH and the chemical reduction of the fermentation matrix. Lactobacillus buchneri and Pediococcus ethanolidurans were able to produce lactic acid from sugars, but only L. buchneri produced acetic acid at the expense of lactic acid under both aerobic and anaerobic conditions regardless of the initial acidic pH of 3.2 in the medium. The formation of secondary products was associated with the metabolism of Clostridium bifermentans and Enterobacter cloacae, which metabolic activity was observed at medium pH above 4.5. Individually, the selected spoilage microorganisms were found to be able to produce changes associated with secondary cucumber fermentations. The fact that oxidative yeasts and L. buchneri were able to produce chemical changes associated with the initiation of the spoilage process indicates that prevention of the secondary fermentation could be achieved by inhibiting these organisms.

Preservation of Acidified Cucumbers with a Natural Preservative Combination of Fumaric Acid and Allyl Isothiocyanate that Target Lactic Acid Bacteria and Yeasts

ABSTRACT To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts include Lactobacillus brevis (67% of all hosts), Lactobacillus plantarum (21%), Weissella paramesenteroides, Weissella cibaria, and Pediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attacked L. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations.

Influence of Microbial Growth on the Redox Potential of Fermented Cucumbers

UNLABELLED Commercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fermentations can result in significant economic loss for industrial producers. The microbiota that result in spoilage remain incompletely defined. Previous studies have implicated yeasts, lactic acid bacteria, enterobacteriaceae, and Clostridia as having a role in spoilage fermentations. We report that Propionibacterium and Pectinatus isolates from cucumber fermentation spoilage converted lactic acid to propionic acid, increasing pH. The analysis of 16S rDNA cloning libraries confirmed and expanded the knowledge gained from previous studies using classical microbiological methods. Our data show that Gram-negative anaerobic bacteria supersede Gram-positive Fermincutes species after the pH rises from around 3.2 to pH 5, and propionic and butyric acids are produced. Characterization of the spoilage microbiota is an important first step in efforts to prevent cucumber fermentation spoilage. PRACTICAL APPLICATION An understanding of the microorganisms that cause commercial cucumber fermentation spoilage may aid in developing methods to prevent the spoilage from occurring.

Preparation of a Lactobacillus Plantarum Starter Culture for Cucumber Fermentations That Can Meet Kosher Guidelines

Without the addition of preservative compounds cucumbers acidified with 150 mM acetic acid with pH adjusted to 3.5 typically undergo fermentation by lactic acid bacteria. Fumaric acid (20 mM) inhibited growth of Lactobacillus plantarum and the lactic acid bacteria present on fresh cucumbers, but spoilage then occurred due to growth of fermentative yeasts, which produced ethanol in the cucumbers. Allyl isothiocyanate (2 mM) prevented growth of Zygosaccharomyces globiformis, which has been responsible for commercial pickle spoilage, as well as the yeasts that were present on fresh cucumbers. However, allyl isothiocyanate did not prevent growth of Lactobacillus plantarum. When these compounds were added in combination to acidified cucumbers, the cucumbers were successfully preserved as indicated by the fact that neither yeasts or lactic acid bacteria increased in numbers nor were lactic acid or ethanol produced by microorganisms when cucumbers were stored at 30 degrees C for at least 2 mo. This combination of 2 naturally occurring preservative compounds may serve as an alternative approach to the use of sodium benzoate or sodium metabisulfite for preservation of acidified vegetables without a thermal process.

Preservation of Acidified Cucumbers with a Combination of Fumaric Acid and Cinnamaldehyde That Target Lactic Acid Bacteria and Yeasts

Commonly, pH measurements are used during the production of fermented cucumbers to indirectly monitor growth of lactic acid bacteria (LAB) and acid production. Redox potential (E(h)) measurements, which are determined by the potential of an electron to reduce an acceptor, could serve as an alternative tool to monitor the progress of fermentation allowing the detection of the metabolic activity and/or growth of LAB and other microorganisms. Pasteurized and inoculated jars of cucumbers were observed to better understand how the E(h) changes during the cucumber fermentation and how it could be used as a monitoring tool. Jars of diced, brined cucumbers were pasteurized and inoculated with microbes previously isolated from fermented cucumbers including Lactobacillus plantarum, Zygosaccharomyces globiformis, and Enterobacter aerogenes. Although an initial decrease in E(h) was observed for all microorganisms, distinctive trends in E(h) occurred when these organisms were inoculated. After a 2-wk fermentation period, the E(h) (Ag/AgCl, 3 M KCl) in jars inoculated with L. plantarum, Z. globiformis, and E. aerogenes was at +453 +/- 55, +104 +/- 5, and -156 +/- 73 mV, respectively. Cucumbers inoculated with a mixture of L. plantarum and Z. globiformis had a terminal E(h) value of +202 +/- 24 mV, which was between that found for the individual microorganisms. L. plantarum dominated the E(h) trend when inoculated along with E. aerogenes with a final E(h) of +411 +/- 72 mV. The results showed that changes in E(h) continued after pH measurements became stable. Thus E(h) measurement can provide a tool to continuously monitor microbial growth during the course of cucumber fermentations.