Commercial Cucumber Fermentations Research Articles

Annotated whole-genome sequences of fermentative and spoilage associated Bacilli and proteobacteria autochthonous to commercial cucumber fermentation.

We report 36 whole-genome sequences, along with annotations, of fermentative (n = 12) and spoilage associated (n = 6) lactic acid bacteria, Lysinibacillus (n = 3), Streptococcus (n = 1), and Proteobacteria (n = 14) isolated from commercial cucumber fermentations. Fifty-three percent of the genome sequence assemblies consist of 1-4 contigs, and the remainder have fewer than 16.

Whole-genome sequencing and annotation of selected Enterobacteriaceae derived from commercial cucumber fermentation.

We report five whole-genome sequences, along with annotations, representing Enterobacteriaceae from the genera Enterobacter (n = 1), Pantoea (n = 1), and Leclercia (n = 3) isolated from commercial cucumber fermentations performed in North Carolina and Minnesota, USA.

Genome-Wide Comparative Analysis of Lactiplantibacillus pentosus Isolates Autochthonous to Cucumber Fermentation Reveals Subclades of Divergent Ancestry.

Lactiplantibacillus pentosus, commonly isolated from commercial cucumber fermentation, is a promising candidate for starter culture formulation due to its ability to achieve complete sugar utilization to an end pH of 3.3. In this study, we conducted a comparative genomic analysis encompassing 24 L. pentosus and 3 Lactiplantibacillus plantarum isolates autochthonous to commercial cucumber fermentation and 47 lactobacillales reference genomes to determine species specificity and provide insights into niche adaptation. Results showed that metrics such as average nucleotide identity score, emulated Rep-PCR-(GTG)5, computed multi-locus sequence typing (MLST), and multiple open reading frame (ORF)-based phylogenetic trees can robustly and consistently distinguish the two closely related species. Phylogenetic trees based on the alignment of 587 common ORFs separated the L. pentosus autochthonous cucumber isolates from olive fermentation isolates into clade A and B, respectively. The L. pentosus autochthonous clade partitions into subclades A.I, A.II, and A.III, suggesting substantial intraspecies diversity in the cucumber fermentation habitat. The hypervariable sequences within CRISPR arrays revealed recent evolutionary history, which aligns with the L. pentosus subclades identified in the phylogenetic trees constructed. While L. plantarum autochthonous to cucumber fermentation only encode for Type II-A CRISPR arrays, autochthonous L. pentosus clade B codes for Type I-E and L. pentosus clade A hosts both types of arrays. L. pentosus 7.8.2, for which phylogeny could not be defined using the varied methods employed, was found to uniquely encode for four distinct Type I-E CRISPR arrays and a Type II-A array. Prophage sequences in varied isolates evidence the presence of adaptive immunity in the candidate starter cultures isolated from vegetable fermentation as observed in dairy counterparts. This study provides insight into the genomic features of industrial Lactiplantibacillus species, the level of species differentiation in a vegetable fermentation habitat, and diversity profile of relevance in the selection of functional starter cultures.

Whole-Genome Sequencing and Annotation of Pediococcus ethanolidurans and Pediococcus pentosaceus Isolates from Commercial Cucumber Fermentation

We report the whole-genome sequences, along with annotations, of five Pediococcus ethanolidurans and three Pediococcus pentosaceus isolates from commercial cucumber fermentations performed in North Carolina (n = 3) and Minnesota (n = 5), USA.

Genome Sequences for Levilactobacillus brevis Autochthonous to Commercial Cucumber Fermentations.

ABSTRACTWe report the whole-genome sequences, along with annotations, of 11 Levilactobacillus brevis isolates from commercial cucumber fermentations performed in North Carolina (n = 9) and Minnesota (n = 2), USA.

Whole-Genome Sequencing and Annotation of Selected Lactobacillales Isolated from Commercial Cucumber Fermentation.

ABSTRACTWe report the whole-genome sequences and annotations of 42 Lactobacillales isolated from commercial cucumber fermentations performed in North Carolina (n = 34) and Minnesota (n = 9), USA. The isolates include representatives from 12 acid-producing species.

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.

Bacteriophages Infecting Gram-Negative Bacteria in a Commercial Cucumber Fermentation

Cucumber fermentations are one of the most important vegetable fermentations in the United States. The fermentation is usually driven by lactic acid bacteria (LAB) indigenous to fresh cucumbers. But LAB are greatly outnumbered by many Gram-negative bacteria on fresh cucumbers, which may influence the growth of LAB and the incidence of bloater defect (hollow cavities formed inside fermented cucumbers) leading to serious economic loss to the pickle industry. Rapid elimination of Gram-negative bacteria is crucial to the dominance of LAB and the reduction of bloater defect in the fermentation. Various factors can affect the viability of Gram-negative bacteria in cucumber fermentation. Bacteriophages (phages) may be one of such factors. This study explored the abundance, diversity, and functional role of phages infecting Gram-negative bacteria in a commercial cucumber fermentation. Cover brine samples were taken from a commercial fermentation tank over a 30-day period. On day 1 and day 3 of the fermentation, 39 Gram-negative bacteria and 26 independent phages were isolated. Nearly 67% of Gram-negative bacterial isolates were susceptible to phage infection. Phage hosts include Enterobacter, Citrobacter, Escherichia, Pantoea, Serratia, Leclercia, Providencia, and Pseudomonas species. About 88% of the isolated phages infected the members in the family Enterobacteriaceae and 58% of phages infected Enterobacter species. Eight phages with unique host ranges were characterized. These phages belong to the Myoviridae, Siphoviridae, or Podoviridae family and showed distinct protein profiles and DNA fingerprints. The infectivity of a phage against Enterobacter cancerogenus was evaluated in cucumber juice as a model system. The phage infection at the multiplicity of infection 1 or 100 resulted in a 5-log reduction in cell concentration within 3 h and rapidly eliminated its host. This study revealed the abundance and variety of phages infecting Gram-negative bacteria, particularly Enterobacteriaceae, in the commercial cucumber fermentation, suggesting that phages may play an important role in the elimination of Gram-negative bacteria, thereby facilitating the dominance of LAB and minimizing bloater defect. To our knowledge, this is the first report on the ecology of phages infecting Gram-negative bacteria in commercial cucumber fermentations.

Modulation of the bacterial population in commercial cucumber fermentations by brining salt type.

Differences in the bacterial population of cucumber fermentations brined with no salt, 100mmoll-1 (1·1%) calcium chloride (CaCl2 ) or 1·03moll-1 (6%) sodium chloride (NaCl) were studied. Changes in the microbiology and chemistry of commercial and laboratory scale cucumber fermentations occurring as a function of time were monitored using colony counts and metagenetic analysis, and a pH probe and high-performance liquid chromatography analysis respectively. Dissolved oxygen and carbon dioxide content were monitored in commercial fermentations. Fermentations brined with calcium chloride (CaCl2 ) or no salt sustained faster microbial growth and reduction in pH than those brined with 1·03moll-1 NaCl. Leuconostoc, Lactococcus and Weissella dominated in fermentations brined with no salt or 100mmoll-1 CaCl2 on day 1 as compared to Weissella and enterobacteria in fermentations containing 1·03moll-1 NaCl. Lactobacilli dominated all fermentations by the third day, regardless of salt type, and was followed, in relative abundance by Pediococcus, Leuconostoc, Lactococcus and Weissella. From 84 to 96% of the population was composed of Lactobacillus by day 7 of the fermentations, except in the no salt fermentations in which a mixed population of LAB remained. The population of LAB found in commercial cucumber fermentations brined with 100mmoll-1 CaCl2 (n=18) or 1·03moll-1 NaCl (n=9) mimicked that of laboratory fermentations. A declining population of aerobes was detected in commercial fermentations brined with CaCl2 on day 1. A reduced NaCl content in cucumber fermentation enhances microbial diversity. This study fills a knowledge gap and aids in the design of improved reduced NaCl cucumber fermentations.

Assessment of the non-lactic acid bacteria microbiota in fresh cucumbers and commercially fermented cucumber pickles brined with 6% NaCl

Limited documentation of the cucumber fermentation microbiome has impeded the understanding of the role of microbes on the quality of finished products. We characterized the microbiome of fresh and fermented cucumber samples using culture dependent and independent techniques, with an emphasis on the non-lactic acid bacteria (non-LAB) population. Insubstantial microbiome variations were observed among fresh cucumber types with Rhizobium (31.04%), Pseudomonas (14.08%), Pantoea (9.25%), Stenotrophomonas (6.83%), and Acinetobacter (6.5%) prevailing. The relative abundance of LAB remained below 0.4% and 4.0% on fresh cucumbers and day 3 of the fermentations brined with 6% sodium chloride, respectively. Fermentation cover brine samples collected on day 1 harbored Pseudomonas, Pantoea, Stenotrophomonas, Acinetobacter, Comamonas, Wautersiella, Microbacterium, Flavobacterium, Ochrobactrum and the Enterobacteriaceae, Citrobacter, Enterobacter and Kluyvera. Plate counts for presumptive Klebsiella and Pseudomonas from fermentation cover brine samples reached 2.80 ± 0.36 and 2.78 ± 0.83 log of CFU/mL, respectively, in 30% and 60% of the nine tanks scrutinized with selective media. Both genera were found in cover brine samples with pH values at 4.04 ± 0.15. We aim at elucidating whether the low relative abundance of non-LAB in commercial cucumber fermentations, in particular Pseudomonas and Enterobacteriaceae, impacts the quality of fermented cucumbers.

Quality of Cucumbers Commercially Fermented in Calcium Chloride Brine without Sodium Salts

Commercial cucumber fermentation produces large volumes of salty wastewater. This study evaluated the quality of fermented cucumbers produced commercially using an alternative calcium chloride (CaCl2) brining process. Fermentation conducted in calcium brines (0.1 M CaCl2, 6 mM potassium sorbate, equilibrated) with a starter culture was compared to standard industrial fermentation. Production variables included commercial processor(n=6), seasonal variation (June–September, 2 years), vessel size (10,000–40,000 L), cucumber size (2.7–5.1 cm diameter), and bulk storage time (55–280 days). Cucumber mesocarp firmness, color, bloater defects, pH, and organic acids were measured. Complete lactic acid fermentation was achieved, resulting in terminal fermentation pH values of 3.23 ± 0.09 and 3.30 ± 0.12 for CaCl2and NaCl processes, respectively. On average, CaCl2brined, fermented cucumbers were 1.8 N less firm, which remained significant in the finished product(P<0.0001). Color differences evidenced by higher hue and lower chroma values(P<0.0269)were consistent with increased photooxidation in CaCl2brined cucumbers. Commercial implementation of CaCl2brines for cucumber fermentation in open tanks variably resulted in texture and color defects that can impact product quality. Additional research is needed to understand the atypical softening observed at the commercial scale and identify process controls for quality improvements.

Evaluation of the use of malic acid decarboxylase-deficient starter culture in NaCl-free cucumber fermentations to reduce bloater incidence.

Accumulation of carbon dioxide (CO2 ) in cucumber fermentations is known to cause hollow cavities inside whole fruits or bloaters, conducive to economic losses for the pickling industry. This study focused on evaluating the use of a malic acid decarboxylase (MDC)-deficient starter culture to minimize CO2 production and the resulting bloater index in sodium chloride-free cucumber fermentations brined with CaCl2 . Attempts to isolate autochthonous MDC-deficient starter cultures from commercial fermentations, using the MD medium for screening, were unsuccessful. The utilization of allochthonous MDC-deficient starter cultures resulted in incomplete utilization of sugars and delayed fermentations. Acidified fermentations were considered, to suppress the indigenous microbiota and favour proliferation of the allochthonous MDC-deficient Lactobacillus plantarum starter cultures. Inoculation of acidified fermentations with L. plantarum alone or in combination with Lactobacillus brevis minimally improved the conversion of sugars. However, inoculation of the pure allochthonous MDC-deficient starter culture to 107 CFU per ml in acidified fermentations resulted in a reduced bloater index as compared to wild fermentations and those inoculated with the mixed starter culture. Although use of an allochthonous MDC-deficient starter culture reduces bloater index in acidified cucumber fermentations brined with CaCl2 , an incomplete conversion of sugars is observed. Economical losses due to the incidence of bloaters in commercial cucumber fermentations brined with CaCl2 may be reduced utilizing a starter culture to high cell density.

Reassessment of the succession of lactic acid bacteria in commercial cucumber fermentations and physiological and genomic features associated with their dominance

A compositional re-assessment of the microbiota present in commercial cucumber fermentation using culture independent and dependent methods was conducted, with emphasis on lactic acid bacteria (LAB). Two commercial cucumber fermentation tanks were monitored by measuring pH, dissolved oxygen and temperature, and used as sources of samples for microbial plating, genomic DNA extraction and measurement of organic acids and carbohydrates by HPLC. Six additional commercial tanks were included to identify the dominant microorganisms using molecular methods. A comparative analysis of the publically available genome sequences corresponding to the LAB found in cucumber fermentations was completed to gain an understanding of genomic features possibly enabling dominance. Analyses of the microbiota suggest Lactobacillales prevail in cucumber fermentations, including in order of prevalence Lactobacillus pentosus, Lb. plantarum, Lb. brevis, Weissella spp., Pediococcus ethanolidurans, Leuconostoc spp. and Lactococcus spp. It was observed that Lb. pentosus and Lb. plantarum have comparatively larger genomes, higher gene counts, uniquely distribute the ribosomal clusters across the genome as opposed to close to the origin of replication, and possess more predicted amino acids prototrophies and selected biosynthesis related genes. It is theorized that Lb. pentosus and Lb. plantarum dominance in cucumber fermentations is the result of their genetic make-up.

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

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.

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

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. An understanding of the microorganisms that cause commercial cucumber fermentation spoilage may aid in developing methods to prevent the spoilage from occurring.

Bacteriophage Ecology in a Commercial Cucumber Fermentation

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.

Development of a Model System for the Study of Spoilage Associated Secondary Cucumber Fermentation during Long‐Term Storage

Calcium chloride fermentations represent an alternative to reduce chloride concentrations in the wastewaters generated from commercial cucumber fermentations, currently performed in cover brine solutions containing 6% to 12% sodium chloride. However, preliminary attempts to commercially ferment the cucumbers in the presence of oxygen led to the development of a secondary cucumber fermentation or spoilage. The development of cucumber secondary fermentation has also been occasionally reported by processors using cover brine solutions containing sodium chloride. This study focused on the development of a model system to characterize CaCl(2) and NaCl secondary cucumber fermentations under conditions similar to those present on the commercial scale. Cucumber fruits mixed with cover brine solutions, containing 100 mM CaCl(2) or 1.03 M NaCl, and 25 mM acetic acid, were fermented in 2 L fermentation vessels subjected to air-purging at a rate of 5 mL/min. Microorganisms and selected biochemical changes detected in the experimental cucumber fermentations had been previously observed in commercial spoilage samples, suggesting the successful reproduction of the secondary fermentation in the laboratory. Experimental secondary fermentations were characterized by the rapid oxidation of the lactic acid produced during the primary fermentation, which, in turn, increased pH. Lactic acid disappearance seemed to be the result of yeast metabolism that also led to the chemical reduction of the environment to levels at which other bacteria could become established and produce butyric, propionic, and acetic acids. This model system will be applied for the identification of strategies to prevent the initiation of the cucumber secondary fermentation and reduce economic losses in the pickling industry. The study of secondary cucumber fermentation has represented a challenge for many years. The successful development of a model system for the study of this phenomenon in the laboratory is instrumental in furthering the study of the event and in optimizing the sodium-chloride-free fermentation at the commercial scale.

Isolation and characterization of a Lactobacillus plantarum bacteriophage, ΦJL-1, from a cucumber fermentation

A virulent Lactobacillus plantarum bacteriophage, ΦJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of ΦJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that ΦJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl 2) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of ΦJL-1 at pH 6.5 were estimated to be 2.7 min at 70 °C and 0.2 min at 80 °C by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage ΦJL-1 may be important for the development of controlled vegetable fermentations.

Characteristics of Lactic Acid Bacteria from Commercial Cucumber Fermentations

Characteristics of Lactic Acid Bacteria from Commercial Cucumber Fermentations