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.

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

AIMS: Differences in the bacterial population of cucumber fermentations brined with no salt, 100 mmol l-1 (1·1%) calcium chloride (CaCl2 ) or 1·03 mol l-1 (6%) sodium chloride (NaCl) were studied. METHODS AND RESULTS: 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 1·03 mol l-1 NaCl. Leuconostoc, Lactococcus and Weissella dominated in fermentations brined with no salt or 100 mmol l-1 CaCl2 on day 1 as compared to Weissella and enterobacteria in fermentations containing 1·03 mol l-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 100 mmol l-1 CaCl2 (n = 18) or 1·03 mol l-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. CONCLUSION: A reduced NaCl content in cucumber fermentation enhances microbial diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study fills a knowledge gap and aids in the design of improved reduced NaCl cucumber fermentations.

Content of xylose, trehalose and l-citrulline in cucumber fermentations and utilization of such compounds by certain lactic acid bacteria.

This research determined the concentration of trehalose, xylose and l-citrulline in fresh and fermented cucumbers and their utilization by Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus buchneri. Targeted compounds were measured by HPLC and the ability of the lactobacilli to utilize them was scrutinized in fermented cucumber juice. Fresh cucumber juice was supplemented with trehalose, xylose and l-citrulline to observed mixed culture fermentations. Changes in the biochemistry, pH and colony counts during fermentations were monitored. Trehalose, xylose and l-citrulline were detected in fermentations to15.51 ± 1.68 mM, a fresh cucumber sample at 36.05 mM and in fresh and fermented cucumber samples at 1.05 ± 0.63 mM, respectively. Most of the LAB tested utilized trehalose and xylose in FCJM at pH 4.7. l-citrulline was utilized by L. buchneri and produced by other LAB. l-citrulline (12.43 ± 2.3 mM) was converted to ammonia (14.54 ± 3.60 mM) and the biogenic amine ornithine (14.19 ± 1.07 mM) by L. buchneri at pH 4.7 in the presence of 0.5 ± 0.2 mM glucose enhancing growth by 0.5 log CFU/mL. The use of a mixed starter culture containing L. buchneri aided in the removal of l-citrulline and enhanced the fermentation stability. The utilization of l-citrulline by L. buchneri may be a cause of concern for the stability of cucumber fermentations at pH 3.7 or above. This study identifies the use of a tripartite starter culture as an enhancer of microbial stability for fermented cucumbers.

Contribution of Leuconostocaceae to CO2-mediated bloater defect in cucumber fermentation.

Fermented cucumber bloater defect, caused by the accumulation of microbiologically produced carbon dioxide (CO2), creates significant economic losses for the pickling industry. The ability of Leuconostocaceae, indigenous to cucumber, to grow and produce CO2 during a fermentation and cause bloater defect was evaluated. Leuconostocaceae grew and produced over 40% CO2 in cucumber juice medium, used as a model for cucumber fermentation. The inoculation of Leuconostocaceae to 5 Log CFU/g in cucumber fermentations brined with 25 mM calcium chloride and 6 mM potassium sorbate resulted in no significant differences in bloater defect, colony counts from MRS and VRBG agar plates or the fermentation biochemistry; suggesting an inability of the inoculated bacterial species to prevail in the bioconversion. Acidified cucumbers were subjected to a fermentation inoculated with a Leuconostoc lactis starter culture after raising the pH to 5.9 ± 0.4. CO2 was produced in the acidified cucumber fermentations to 13.6 ± 3.5% yielding a bloater index of 21.3 ± 6.4; while 8.6 ± 0.8% CO2 and a bloater index of 5.2 ± 5.9 were observed in the non-inoculated control jars. Together the data collected demonstrate that Leuconostocaceae can produce enough CO2 to contribute to bloater defect, if not outcompeted by the leading lactic acid bacteria in a cucumber fermentation.

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 ±â€¯0.36 and 2.78 ±â€¯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 ±â€¯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.

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.

CO2-mediated bloater defect can be induced by the uncontrolled growth of Enterobacteriaceae in cucumber fermentation.

Enterobacteriaceae are known to proliferate in cucumber juice, deriving energy from the fermentation of sugars to organic acids and ethanol, and theoretically generating carbon dioxide (CO2). We hypothesized that the CO2 produced by the indigenous Enterobacteriaceae in the early stage of cucumber fermentation accumulates in the fermenting fruits causing bloater defect. The ability of seven Enterobacteriaceae, indigenous to cucumber, to grow and produce CO2 in cucumber juice medium (CJM), a sterile model system for cucumber fermentation, was characterized. The induction of bloater defect in cucumber fermentation conducted with pasteurized and acidified fruits was also evaluated. The generation times of the seven Enterobacteriaceae in CJM ranged between 0.25 and 8.20 h and resulted in carbon dioxide (CO2) production to estimated amounts of 7.22-171.5 mM. Enterobacter cancerogenus and Enterobacter nimipressuralis were among the bacteria that produced the most and the least CO2 in CJM, respectively, at estimated mM concentrations of 171.58 ± 42.96 and 16.85 ± 6.53. Inoculation of E. cancerogenus and E. nimipressuralis in acidified and pasteurized cucumbers resulted in the production of 138 and 27 mM CO2, respectively. Such Enterobacteriaceae produced 2% hydrogen in the model cucumber fermentations. A bloater index of 25.4 and 17.4 was calculated from the cucumbers fermented by E. cancerogenus and E. nimipressuralis, respectively, whereas no defect was observed in the fruits collected from uninoculated control fermentation jars. It is concluded that the metabolic activity of the Enterobacteriaceae indigenous to cucumber can produce sufficient CO2 in cucumber fermentations to induce bloater defect.

Levilactobacillus brevis, autochthonous to cucumber fermentation, is unable to utilize citric acid and encodes for a putative 1,2-propanediol utilization microcompartment.

The metabolic versatility of Levilactobacillus brevis, a heterofermentative lactic acid bacterium, could benefit environmentally compatible and low salt cucumber fermentation. The biodiversity of Lvb. brevis autochthonous to cucumber fermentation was studied using genotypic and phenotypic analyses to identify unique adjunct cultures. A group of 131 isolates autochthonous to industrial fermentations was screened using rep-PCR-(GTG)5 and a fermentation ability assay under varied combinations of salt (0 or 6%), initial pH (4.0 or 5.2), and temperature (15 or 30°C). No apparent similarities were observed among the seven and nine clusters in the genotypic and phenotypic dendrograms, respectively. A total of 14 isolates representing the observed biodiversity were subjected to comparative genome analysis. The autochthonous Lvb. brevis clustered apart from allochthonous isolates, as their genomes lack templates for citrate lyase, several putative hypothetical proteins, and some plasmid- and phage-associated proteins. Four and two representative autochthonous and allochthonous Lvb. brevis, respectively, were subjected to phenotype microarray analysis using an Omnilog. Growth of all Lvb. brevis strains was supported to various levels by glucose, fructose, gentiobiose, 1,2-propanediol, and propionic acid, whereas the allochthonous isolate ATCC14890 was unique in utilizing citric acid. All the Lvb. brevis genomes encode for 1,2-propanediol utilization microcompartments. This study identified a unique Lvb. brevis strain, autochthonous to cucumber, as a potential functional adjunct culture for commercial fermentation that is distinct in metabolic activities from allochthonous isolates of the same species.

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.

Changes in the free amino acid profile of pickling cucumber during lactic acid fermentation.

Free amino acid (FAA) profiles of fresh, acidified, naturally fermented, and starter culture fermented cucumbers were analyzed by liquid chromatography triple quadrupole mass spectrometry. Fermented cucumbers contained more total FAA than acidified cucumbers (1,302 ± 102 mg/kg and 635 ± 35 mg/kg, respectively). Total FAA content of fermented cucumber was similar regardless of brine salt levels (2-6% NaCl) and starter culture addition. Glutamine (1491.4 ± 69.3 mg/kg), γ-aminobutyric acid (GABA, 269.6 ± 21.4 mg/kg), asparagine (113.0 ± 6.4 mg/kg), and citrulline (110.3 ± 8.5 mg/kg) were the most abundant FAA in fresh pickling cucumber, whereas GABA (181.3 ± 21.5 mg/kg), isoleucine (165.2 ± 11.2 mg/kg), leucine (129.8 ± 10.9 mg/kg), and lysine (110.9 ± 5.0 mg/kg) were the most abundant in fermented cucumber. GABA and ornithine were produced during fermentation, indicating glutamate decarboxylase and arginine deiminase activities. Notably, ornithine was significantly higher in natural (63.3 ± 31.5 mg/kg) versus starter culture fermented cucumbers (3.0 ± 0.7 mg/kg). This new information on FAA composition of fresh and fermented pickling cucumbers shows the impact of fermentation conditions on cucumber amino acid profiles while providing insight for manipulating fermentations for health promotion and consumer acceptance. PRACTICAL APPLICATION: This study reports changes in the free amino acid profiles of raw, fermented and acidified cucumbers, which may be valuable for understanding the impact of these foods on human health and nutrition. This information is useful for food microbiologists studying the metabolism of lactic acid bacteria during fermentation and/or designing starter cultures and could contribute to the development of novel fermented cucumber pickle products with enhanced nutritional value.

Identification of potential causative agents of the CO2-mediated bloater defect in low salt cucumber fermentation.

Development of bloater defect in cucumber fermentations is the result of carbon dioxide (CO2) production by the indigenous microbiota. The amounts of CO2 needed to cause bloater defect in cucumber fermentations brined with low salt and potential microbial contributors of the gas were identified. The carbonation of acidified cucumbers showed that 28.68 ± 6.04 mM (12%) or higher dissolved CO2 induces bloater defect. The microbiome and biochemistry of cucumber fermentations (n = 9) brined with 25 mM calcium chloride (CaCl2) and 345 mM sodium chloride (NaCl) or 1.06 M NaCl were monitored on day 0, 2, 3, 5, 8, 15 and 21 using culture dependent and independent microbiological techniques and High-Performance Liquid Chromatography. Changes in pH, CO2 concentrations and the incidence of bloater defect were also followed. The enumeration of Enterobacteriaceae on Violet Red Bile Glucose agar plates detected a cell density of 5.2 ± 0.7 log CFU/g on day 2, which declined to undetectable levels by day 8. A metagenomic analysis identified Leuconostocaceae in all fermentations at 10 to 62%. The presence of both bacterial families in fermentations brined with CaCl2 and NaCl coincided with a bloater index of 24.0 ± 10.3 to 58.8 ± 23.9. The prevalence of Lactobacillaceae in a cucumber fermentation brined with NaCl with a bloater index of 41.7 on day 5 suggests a contribution to bloater defect. This study identifies the utilization of sugars and malic acid by the cucumber indigenous Lactobacillaceae, Leuconostocaceae and Enterobacteriaceae as potential contributors to CO2 production during cucumber fermentation and the consequent bloater defect.

Gentiobiose and cellobiose content in fresh and fermenting cucumbers and utilization of such disaccharides by lactic acid bacteria in fermented cucumber juice medium.

The content of cellobiose and gentiobiose, cellulose-derived dissacharides, in fresh and fermented cucumber was evaluated along with the ability of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus buchneri and Lactobacillus brevis to utilize them during and after fermentation. The disaccharide content in fresh and fermenting cucumbers was below the detection level (10 µM) using HPLC for analysis. Utilization of cellobiose and gentiobiose by lactic acid bacteria (LAB) was tested in fermented cucumber juice medium (FCJM), a model system for the bioconversion and postfermentation lacking glucose and fructose. Changes in the fermentation metabolites were followed using HPLC and pH measurements as a function of time. The disaccharides were utilized by L. plantarum, L. pentosus, and L. buchneri in FCJM at pH 4.7 ± 0.1, representative of the active fermentation period, and converted to lactic acid. The disaccharides were not utilized in FCJM at pH 3.7 ± 0.1, representative of the end of fermentation. While L. brevis was unable to utilize cellobiose efficiently in FCJM, they were able to remove gentiobiose at pH 4.7 ± 0.1. Some strain level differences in cellobiose utilization were observed. It is concluded that the disaccharides are absent in the fresh cucumber and the typical fermentation. The LAB prevalent in the bioconversion utilizes cellobiose and gentiobiose, if available, at pH 4.7 ± 0.1. The LAB would not remove the disaccharides, which could become available from cellulose degradation by the acid resistant indigenous microbiota, after the pH is reduced to 3.7 ± 0.1.

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.

Characterization of robust Lactobacillus plantarum and Lactobacillus pentosus starter cultures for environmentally friendly low-salt cucumber fermentations.

Seven candidates for starter cultures for cucumber fermentations belonging to the Lactobacillus pentosus and Lactobacillus plantarum species were characterized based on physiological features desired for pickling. The isolates presented variable carbohydrate utilization profile on API® 50CHL test strips. The L. pentosus strains were unable to utilize d-xylose in MRS broth or the M medium. The lactobacilli were unable to produce histamine, tyramine, putrescine, and cadaverine in biogenic amine broth containing the necessary precursors. Production of d-lactic acid by the lactobacilli, detected enzymatically, was stimulated by growth in MRS broth as compared to cucumber juice medium (CJM). The lactobacilli utilized malic acid in the malate decarboxylase medium. Exopolyssacharide biosynthesis related genes were amplified from the lactobacilli. A sugar type-dependent-ropy phenotype was apparent for all the cultures tested in MRS and CJM. The genes associated with bacteriocin production were detected in the lactobacilli, but not the respective phenotypes. The antibiotic susceptibility profile of the lactobacilli mimics that of other L. plantarum starter cultures. It is concluded that the lactobacilli strains studied here are suitable starter cultures for cucumber fermentation. PRACTICAL APPLICATION: The availability of such starter cultures enables the implementation of low salt cucumber fermentations that can generate products with consistent biochemistry and microbiological profile.

Fermentation Cover Brine Reformulation for Cucumber Processing with Low Salt to Reduce Bloater Defect.

Reformulation of calcium chloride (CaCl2 ) cover brine for cucumber fermentation was explored as a mean to minimize the incidence of bloater defect. This study particularly focused on cover brine supplementation with calcium hydroxide (Ca[OH]2 ), sodium chloride (NaCl), and acids to enhance buffer capacity, inhibit the indigenous carbon dioxide (CO2 )- producing microbiota, and decrease the solubility of the gas. The influence of the cover brine formulations tested, on the cucumber fermentation microbiota, biochemistry, CO2 production, and bloating defect was studied using metagenetics, HPLC analysis, a portable gas analyzer and bloater index, respectively. Cover brine supplementation with Ca(OH)2 and acetic acid resulted in complete fermentations with final pH values 0.5 units higher than the un-supplemented control. Lactic acid production increased by approximately 22%, possibly inducing the observed reduction in the relative abundance of Enterobacteriaceae by 92%. Ca(OH)2 supplementation also resulted in an increased relative abundance of Leuconostocaceae by 7%, which likely contributed to the observed increment in CO2 levels by 25%. A 50% reduction on acetic acid formation was detected when cover brines were supplemented with Ca(OH)2 and 690 mM (4%) NaCl. No significant difference was observed in bloater index as the result of Ca(OH)2 or NaCl supplementation in cover brines, given that the CO2 levels remained at above the 20 mg/100 mL needed to induce the defect. It is concluded that the modified cover brine formulation containing Ca(OH)2 and NaCl enables the complete conversion of sugars, decreases production of CO2 and levels of Enterobacteriaceae, but insignificantly reduces bloater index. PRACTICAL APPLICATION: A cucumber fermentation cover brine containing Ca(OH)2 , 0.26% CaCl2 , 345 mM (2%) NaCl, and acetic acid to pH 4.7 has a functional combination of ingredients enabling a complete conversion of sugars to lactic acid with reduced production of acetic acid and CO2 . It represents a process ready cover brine formulation with the potential to allow the manufacture of cucumber pickles with low salt, enhanced food safety, and reduce environmental impact and water usage. Pilot commercial scale cucumber fermentations brined with such ingredients are to reveal the efficacy of this process ready formulation in the presence of oxygen from air in tanks, as opposed to 3.8 L (1-US gal) closed jars in the laboratory.

Commercial Scale Cucumber Fermentations Brined with Calcium Chloride Instead of Sodium Chloride.

Development of low salt cucumber fermentation processes present opportunities to reduce the amount of sodium chloride (NaCl) that reaches fresh water streams from industrial activities. The objective of this research was to translate cucumber fermentation brined with calcium chloride (CaCl2 ) instead of NaCl to commercial scale production. Although CaCl2 brined cucumber fermentations were stable in laboratory experiments, commercial scale trials using 6440 L open-top tanks rapidly underwent secondary cucumber fermentation. It was understood that a limited air purging routine, use of a starter culture and addition of preservatives to the cover brine aids in achieving the desired complete cucumber fermentation. The modified process was used for subsequent commercial trials using 12490 and 28400 L open-top tanks packed with variable size cucumbers and from multiple lots, and cover brines containing CaCl2 and potassium sorbate to equilibrated concentrations of 100 and 6 mM, respectively. Lactobacillus plantarum LA0045 was inoculated to 10(6) CFU/mL, and air purging was applied for two 2-3 h periods per day for the first 10 d of fermentation and one 2-3 h period per day between days 11 and 14. All fermentations were completed, as evidenced by the full conversion of sugars to lactic acid, decrease in pH to 3.0, and presented microbiological stability for a minimum of 21 d. This CaCl2 process may be used to produce fermented cucumbers intended to be stored short term in a manner that reduces pollution and waste removal costs.

Escherichia coli O157:H7 bacteriophage Φ241 isolated from an industrial cucumber fermentation at high acidity and salinity.

A novel phage, Φ241, specific for Escherichia coli O157:H7 was isolated from an industrial cucumber fermentation where both acidity (pH ≤ 3.7) and salinity (≥5% NaCl) were high. The phage belongs to the Myoviridae family. Its latent period was 15 min and average burst size was 53 phage particles per infected cell. The phage was able to lyse 48 E. coli O157:H7 strains, but none of the 18 non-O157 strains (including E. coli O104:H7) or the 2 O antigen-negative mutants of O157:H7 strain, 43895Δper (also lacking H7 antigen) and F12 (still expressing H7 antigen). However, the phage was able to lyse a per-complemented strain (43895ΔperComp) which expresses O157 antigen. These results indicated that phage Φ241 is specific for O157 antigen, and E. coli strains lacking O157 antigen were resistant to the phage infection, regardless of the presence or absence of H7 antigen. SDS-PAGE profile revealed at least 13 structural proteins of the phage. The phage DNA was resistant to many commonly used restriction endonucleases, suggesting the presence of modified nucleotides in the phage genome. At the multiplicity of infection of 10, 3, or 0.3, the phage caused a rapid cell lysis within 1 or 2 h, resulting in 3.5- or 4.5-log-unit reduction in cell concentration. The high lytic activity, specificity and tolerance to low pH and high salinity make phage Φ241 a potentially ideal biocontrol agent of E. coli O157:H7 in various foods. To our knowledge, this is the first report on E. coli O157:H7 phage isolated from high acidity and salinity environment.