Prevention of microbes-induced spoilage in sodium chloride-free cucumber fermentations employing preservatives.

This study evaluated preservatives to stabilize sodium chloride (NaCl)-free-cucumber fermentations. The brining of air-purged laboratory cucumber fermentations with 100.0 mM calcium chloride (CaCl2 ) and 25.0 mM acetic acid resulted in immediate rises in pH, the chemical reduction of the medium, and malodors. Supplementation with 3.0 mM sodium benzoate or 3.0 mM potassium sorbate enabled a decline in pH, a continuous oxidative state of the medium, and delayed rising pH spoilage. However, lactic and acetic acids eventually disappeared in fermentations supplemented with preservatives. The amount of preservatives needed to suppress growth of brined-cucumber-spoilage microbes was determined in Fermented Cucumber Juice Medium (FCJM). Supplementation of FCJM with 10.0 mM sodium benzoate was inhibitory for the spoilage yeasts, Issatchenkia occidentalis and Pichia manshurica, and the lactobacilli, Lentilactobacillus buchneri and Lentilactobacillus parafarraginis, but not of Zygosaccharomyces globiformis. Potassium sorbate inhibited the spoilage yeasts at 15.0 mM in FCJM but not the lactobacilli. Supplementation of FCJM with 20.0 mM fumaric acid had a bactericidal effect on the spoilage-associated lactobacilli. As expected, NaCl-free-commercial cucumber fermentations brined with 100 mM CaCl2 , no acetic acid, and 6 mM potassium sorbate resulted in complete fermentations, but supported rising pH, microbially induced spoilage during long-term storage. Post-fermentation supplementation with 12 mM sodium benzoate, 10 mM fumaric acid, a combination of the two, or 10 mM fumaric acid and 2 mM AITC prevented microbial activity during long-term bulk storage. PRACTICAL APPLICATION: Several preservative-based strategies for stabilizing NaCl-free cucumber fermentation in a commercial production setting were developed, enabling the implementation of a processing technology that reduces wastewater volumes and environmental impact.

Genotypic and phenotypic diversity among Lactobacillus plantarum and Lactobacillus pentosus isolated from industrial scale cucumber fermentations.

The Lactobacillus plantarum and Lactobacillus pentosus genotypes existing in industrial-scale cucumber fermentations were defined using rep-PCR-(GTG)5. The ability of each genotype to ferment cucumbers under various conditions was evaluated. Rep-PCR-(GTG)5 was the technique capable of illustrating the most intraspecies discrimination compared to the sequencing of housekeeping genes (recA, dnaK, pheS and rpoA), MLST and RAPD with primers LP1, OPL5, M14 and COC. Ten genotypic clusters were defined for the 199 L. pentosus tested and three for the 17 L. plantarum clones. The ability of the 216 clones genotyped and 37 additional cucumber fermentation isolates, of the same species, to rapidly decrease the pH of cucumber juice medium under various combinations of sodium chloride (0 or 6%), initial pH (4.0 or 5.2) and temperatures (15 or 30 °C) was determined using a fractional factorial screening design. A reduced fermentation ability was observed for the L. plantarum strains as compared to L. pentosus, except for clone 3.2.8, which had a ropy phenotype and aligned to genotypic cluster A. L. pentosus strains belonging to three genotypic clusters (B, D and J) were more efficient in cucumber juice fermentation as compared to most L. plantarum strains. This research identified three genetically diverse L. pentosus strains and one L. plantarum as candidates for starter cultures for commercial cucumber fermentations.

Comparative genomics of eight Lactobacillus buchneri strains isolated from food spoilage.

BACKGROUND: Lactobacillus buchneri is a lactic acid bacterium frequently associated with food bioprocessing and fermentation and has been found to be either beneficial or detrimental to industrial food processes depending on the application. The ability to metabolize lactic acid into acetic acid and 1,2-propandiol makes L. buchneri invaluable to the ensiling process, however, this metabolic activity leads to spoilage in other applications, and is especially damaging to the cucumber fermentation industry. This study aims to augment our genomic understanding of L. buchneri in order to make better use of the species in a wide range of applicable industrial settings. RESULTS: Whole-genome sequencing (WGS) was performed on seven phenotypically diverse strains isolated from spoiled, fermented cucumber and the ATCC type strain for L. buchneri, ATCC 4005. Here, we present our findings from the comparison of eight newly-sequenced and assembled genomes against two publicly available closed reference genomes, L. buchneri CD034 and NRRL B-30929. Overall, we see ~ 50% of all coding sequences are conserved across these ten strains. When these coding sequences are clustered by functional description, the strains appear to be enriched in mobile genetic elements, namely transposons. All isolates harbor at least one CRISPR-Cas system, and many contain putative prophage regions, some of which are targeted by the host's own DNA-encoded spacer sequences. CONCLUSIONS: Our findings provide new insights into the genomics of L. buchneri through whole genome sequencing and subsequent characterization of genomic features, building a platform for future studies and identifying elements for potential strain manipulation or engineering.

Phenotypic and genotypic diversity of Lactobacillus buchneri strains isolated from spoiled, fermented cucumber.

Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab® rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm-11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting >97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this species to enable control of lactic acid degradation in fermented plant materials.