Biochemical Characterization of Fermented Cucumber Spoilage using Nontargeted, Comprehensive, Two-dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry: Anaerobic Lactic Acid Utilization by Lactic Acid Bacteria

Cucumbers are preserved commercially by fermentation in brine with approximately 6% sodium chloride. Occasionally, fermented cucumbers spoil after the primary fermentation is completed. This spoilage is characterized by a decrease in lactic acid concentration and rise in brine pH caused by unidentified microorganisms. It was hypothesized that 1) Lactic acid degradation in fermented cucumbers can be initiated by lactic acid bacteria (LAB) that are able to maintain metabolic activity in the presence of sodium chloride and acid pH, and 2) A metabolomic approach to studying fermented cucumber spoilage will provide insight into biochemical changes that are yet unknown. Objectives were to 1) Determine the effects of NaCl and pH on anaerobic lactic acid utilization by spoilage microorganisms; 2) Determine the ability of LAB isolated from spoiled fermentations to initiate lactic acid degradation in fermented cucumbers; and 3) Develop and apply non-targeted, comprehensive, two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-ToFMS) to detect biochemical changes during fermented cucumber spoilage.;Mixed cultures obtained from spoiled reduced NaCl and commercial cucumber fermentations as well as LAB isolated from those mixed cultures were evaluated for their ability to metabolize lactic acid in filter-sterilized fermented cucumber slurry (FCS). Loss of lactic acid and formation of major metabolites were measured using high performance liquid chromatography. Non-targeted GCxGC-ToFMS of volatile components and trimethylsilyl (TMS) derivatives of non-volatile components was used to detect other metabolite changes that occurred during spoilage.;Spoilage organisms from reduced NaCl and commercial cucumber fermentations degraded lactic acid anaerobically in FCS in the range of pH 3.2 to 5.0 at NaCl concentrations up to 6% NaCl. Over 18 months incubation, only cucumbers fermented with 6% NaCl to a pH of 3.2 completely prevented anaerobic lactic acid degradation. A clear association between lactic acid utilization and increases in acetic acid, propionic acid and n-propanol was evident across treatments.;Among 13 LAB species isolated from spoilage, only Lactobacillus buchneri and Lactobacillus parafarraginis were able to metabolize lactic acid in FCS. L. buchneri was able to degrade lactic acid under both aerobic and anaerobic atmospheres in pH 3.8 FCS with up to 6% NaCl. L. buchneri was able to degrade both D- and L-lactic acid, and decreases in lactic acid were accompanied by increases in acetic acid, 1,2-propanediol, and ethanol. Another spoilage isolate, Lactobacillus rapi, converted 1,2-propanediol added to FCS to propionic acid and propanol.;Among 314 volatile components detected in fermented cucumber brine, 199 had peak areas with coefficients of variation below 30%. Peak identifications (214/314) established by mass spectral library matching were 92% accurate based on 63 authentic standards. In contrast, only 21% of TMS-metabolite peaks were identified by mass spectral match, and many of these identifications were incorrect. Analysis of variance of log2 peak areas combined with hierarchial clustering analysis revealed 62 volatile and 30 non-volatile metabolites that changed in concentration during spoilage with mixed cultures and isolated L. buchneri (P < 0.01). These changes included decreases in sugars, amino acids, long chain fatty acids, aldehydes, and ketones and increases in several alcohols, butanoic and pentanoic acids. Most of the detected changes preceded lactic acid utilization.;This is the first documented evidence of the role of lactic acid bacteria in fermented cucumber spoilage. L. buchneri was capable of initiating lactic acid degradation under a wide range of environmental conditions that may be present in commercial cucumber fermentations. L. rapi may act syntrophically with L. buchneri to convert lactic acid to acetic acid, propionic acid and n-propanol, three of the major components found in spoiled commercial cucumber fermentations. Biochemical profiling using non-targeted GCxGC-ToFMS led to discovery of changes in several metabolites during spoilage that were previously unknown.