Cucumber Fermentation Research Articles

Purification and Characterization of Plantaricin PQ12: A Novel Bacteriocin from Lactiplantibacillus plantarum Isolated from Fermented Cucumbers, with Inhibitory Activity against Foodborne Pathogens

Purification and Characterization of Plantaricin PQ12: A Novel Bacteriocin from <i>Lactiplantibacillus plantarum</i> Isolated from Fermented Cucumbers, with Inhibitory Activity against Foodborne Pathogens

Complete genome sequence of Escherichia coli O157:H7 phage Φ241.

We report the genome sequence of phage Φ241 infecting Escherichia coli O157:H7. Phage Φ241 was isolated from an industrial cucumber fermentation at high acidity (pH 3.7) and high salinity (5% NaCl). The phage genome consists of a 157,291 bp circular double-stranded DNA with 203 coding regions and 44.96% GC content.

Determination of Post-Fermentation Waste from Fermented Vegetables as Potential Substitutes for Preservatives in o/w Emulsion.

Post-fermentation wastes are rich sources of various biologically active compounds with antimicrobial activity, whose potential is not being fully exploited. One of the possible applications of post-fermentation waste may be its use as a natural preservative that effectively combats pathogens found in formulations. The study aims included the following: (1) compare the antimicrobial and antioxidant activity of fermented vegetable extracts (FVEs), (2) examine the inhibition of cosmetic-borne pathogens by FVEs, and (3) estimate the preservative effectiveness of FVEs in o/w emulsions. It was found that fermented white cabbage, cucumber, celery, and the mixture of fermented white cabbage, cucumber, and celery (1:1:1) showed antibacterial and antifungal activity against all the tested reference microbial strains. The addition of fermented cucumber, celery, and the mixture of fermented white cabbage, cucumber, and celery (1:1:1) to the o/w emulsion fulfilled criterion A of the preservative effectiveness test for S. aureus, E. coli, and A. brasiliensis, but did not fulfill the criterion for P. aeruginosa and C. albicans. The tested FVEs have comparable activity to inhibit pathogens in o/w emulsion as sodium benzoate. The results of our study prove that FVEs can be valuable raw materials supporting the preservative system, which, in turn, can significantly reduce the concentration of preservatives used in o/w emulsion.

Analysis of Pickled Cucumber Products, Based on Microbial Diversity and Flavor Substance Detection.

Changes to the microbial community during pickled cucumber fermentation were studied using the 16S rDNA technique. The changes of volatile organic compounds (VOCs) during pickled cucumber fermentation were studied by gas chromatograph-ion mobility spectrometry. At the phylum level, Cyanophyta and Proteobacteria were the dominant flora in the natural fermentation group, and Firmicutes were the dominant flora in the added-bacteria fermentation group. At the generic level, the addition of Lactobacillus led to changes in the community of the bacteria in the added-bacterial fermentation group and decreased the species abundance of other bacteria. In total, 75 volatile organic compounds were identified from naturally fermented pickled cucumber, and 60 volatile organic compounds were identified from fermented pickled cucumber with bacterial addition. The main metabolites were esters, aldehydes, acids, alcohols, ketones, alkanes, nitriles, and alkenes. These metabolites will bring their unique aroma components to the pickled cucumber. Metabolomic analysis of the O2PLS model showed that Weissella and Lactobacillus were closely and positively correlated with nine alcohols, six esters, five aldehydes, four acids, three ketones, and one pyrazine. Pseudomonas and norank_f_Mitochondria show a close positive correlation with four kinds of alcohols, two kinds of esters, one kind of aldehyde, and one kind of nitrile.

Health Benefits and Antibacterial Properties of Probiotics Isolated From Fermented Food Products

As a result of the continuously expanding scientific evidence probiotic bacteria have become increasingly popular due to their beneficial effects on human health. The main objective of this study was to isolate, identify, and characterize some lactic acid bacterial strains from fermented food products as potential probiotics with antimicrobial activity against some test strains. Total four samples of fermented food products were taken (Sauerkraut, Fermented Cucumber, Idli batter and Dosa batter). The Lactic acid bacteria were isolated from these samples and the probiotics were further tested for their ability to tolerate pH, bile salt at different concentrations. The probiotic strains were found to grow at different pH and also at different conc. of bile and NaCl. These isolates exhibited high antibacterial activity when tested against the test organism (Pseudomonas, Streptococcus, Escherichia coli and Klebsiella) in well diffusion method. The present study concludes that the isolated probiotic strain from the fermented food qualifies pH, bile and NaCl tolerance tests that make an isolate to be an ideal probiotic. Probiotic strains were found to carry antimicrobial property and can be used to control gut microbiota. The result of the present study showed that the fermented food products can be used as a good source of probiotic culture.

Grapevine and Horseradish Leaves as Natural, Sustainable Additives for Improvement of the Microbial, Sensory, and Antioxidant Properties of Traditionally Fermented Low-Salt Cucumbers

The agro-food industry produces large amounts of secondary by-products, which can act as a source of bio-active ingredients. These ingredients can be used as valuable additives to support the sustainable circular economy concept. This study aimed to analyze the potential application of horseradish and grapevine leaves in the fermentation process of low-salt pickled cucumbers to improve their sensory and functional properties. The pour plate technique, RT-qPCR, HPLC, and a nine-point hedonic scale test with penalty analysis were used to analyze the traditionally fermented product. The research showed that the addition of both horseradish and grapevine leaves did not negatively affect the kinetics of fermentation and had a positive effect on the overall desirability. Moreover, they contributed to an increase in the concentration of antioxidant compounds, namely gallic acid (grape leaves) and ellagic acid (grapevine and horseradish leaves). Bacterial metabiome analysis showed the positive effect of all analyzed additives on an increase in the relative expression of genes responsible for the synthesis of selected bacteriocins (plantaricin and acidocin). Research results indicated a high potential for sustainable use of by-products (horseradish and grapevine leaves) in the production of traditional low-salt fermented cucumbers with high health-promoting potential.

Traditional Bulgarian Fermented Foods as a Source of Beneficial Lactic Acid Bacteria.

Traditional Bulgarian fermented foods are prominent for their uniqueness of local ingredients, production methods, and endemic microbial species. The present research investigated the diversity and beneficial biological potential of lactic acid bacteria (LAB) isolated from various types of unique Bulgarian fermented foods. Species identification was performed via 16S rDNA sequencing. Biological activity was evaluated by determining antibacterial activity (via agar well diffusion assay), H2O2 production, spectrophotometrically determined auto- and co-aggregation, microbial adhesion to hydrocarbon, and biofilm formation. The biosafety of the isolated lactic acid bacteria was established based on hemolytic activity and phenotypic and genotypic antibiotic susceptibility. Forty-five strains were isolated from fermented foods (sauerkraut, fermented green tomatoes, fermented cucumbers, kefir, white cheese, and Izvara (curdled milk)). Five species were detected: Lactiplantibacillus plantarum, Levilactobacillus koreensis, Levilactobacillus brevis, Lactobacillus helveticus, and Levilactobacillus yonginensis. The most prominent species was L. plantarum, at 47%. For the first time, L. koreensis and L. yonginensis, isolated from unique Bulgarian fermented foods, are reported in this study. The antibacterial effect of the cell-free supernatants was evaluated. An antagonistic effect was observed against Escherichia coli (57%) and Salmonella enterica subsp. enterica serotype Enteritidis (19%) for several L. plantarum strains. Only one L. brevis (Sauerkraut, S15) strain showed activity against E. coli. The best autoaggregation ability at hour 4 was observed for L. koreensis (fermented cucumbers, FC4) (48%) and L. brevis S2 (44%). The highest percentage of co-aggregation with Candida albicans, at hou 4 in the experiments, was observed for strains L. koreensis (fermented green tomato, FGT1) (70%), L. plantarum strains S2 (54%), S13 (51%), and S6 (50%), while at hour 24 for strains L. koreensis FGT1 (95%), L. brevis (Kefir, K7) (89%), L. plantarum S2 (72%), and L. koreensis FC2 (70%). Seven of the isolated LAB strains showed hydrophobicity above 40%. Our results showed that the ability of biofilm formation is strain-dependent. No hemolytic activity was detected. The antibiotic resistance to 10 antibiotics from different groups was tested phenotypically and genotypically. No amplification products were observed in any strains, confirming that the isolates did not carry antibiotic-resistance genes. Traditional fermented Bulgarian foods can be considered functional foods and beneficial LAB sources.

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.

Tasting of traditional Polish fermented cucumbers: Microbiology, morpho-textural features, and volatilome

In the present study, naturally fermented and unpasteurized cucumbers (Cucumis sativus L.) collected from 4 producers located in different regions of Poland were studied. The fermented cucumbers were characterized by significant nutritional features in terms of polyphenols content and antioxidant activity. Microbiological analyses revealed active bacterial populations of lactococci, thermophilic cocci, lactobacilli, and coagulase-negative cocci. The microbiological characterization of cucumber and brine samples through metataxonomic analysis allowed the dominant species to be detected, being Lactococcus and Streptococcus in cucumbers, and Lactiplantibacillus, Leuconostoc, Pediococcus, Secundilactobacillus, and Lentilactobacillus in brine. The isolation activity offered a clear picture of the main active lactic acid bacteria at the end of fermentation, being Pediococcus parvulus and Lactiplantibacillus plantarum group. All the studied isolates showed a good attitude in fermenting a cucumber-based broth, thus suggesting their potential application as starter or adjunct cultures for guided cucumber fermentation. Moreover, for the same isolates, strong aminopeptidase activity (due to leucine arylamidase and valine arylamidase) was observed, with potential effect on the definition of the final sensory traits of the product. Only a few isolates showed the ability to produce exopolysaccharides in synthetic medium. Of note, the presence of the hdcA gene in some Pediococcus ethanolidurans isolates also confirmed the need for a thorough characterization of starter candidates to avoid undesired adverse effects on consumer’s health. No isolate showed the production of bacteriocins against Listeria innocua used as surrogate for Listeria monocytogenes. Based on the results of Headspace Solid-Phase Microextraction-Gas Chromatography/Mass Spectrometry analysis, a rich and complex volatilome, composed by more than 80 VOCs, was recognized and characterized. In more detail, the detected compounds belonged to 9 main classes, being oxygenated terpenes, alcohols, terpenes, ketones, acids, aldehydes, esters, sulfur, and sesquiterpenes.

Screening of Wild Lactobacillus Plantarum Found in Brine Solution of Naturally Fermented Cucumbers

Fermentation has been used as a simple technique for preserving vegetables since ancient days. This research work aimed to isolate, characterize, and identify wild Lactobacillus plantarum found in a brine solution (8% w/v NaCl) of naturally fermented cucumber. Cucumber samples were naturally fermented at ambient temperature for 14 days in a brine solution, in which lactic acid bacteria (LAB) were then cultured (37℃, 48 h) within 24 h for 3, 5, 7, 10, and 14 days. Seventeen different LAB strains were isolated and identified within the different time intervals of fermentation using morphological, biochemical API 50 CHL, PCR verification using Lactobacillus subspecies-specific genes primer, and 16S rRNA gene sequencing. The API confirmed strains were further verified by PCR to be L. plantarum subsp. plantarum M23 based on the amplified gene fragment separation by gel electrophoresis and gene sequencing. After 24 h of fermentation, the most dominant LAB-identified strains were Leuconostoc mesenteroides, Tetragenococcus halophilus, and L. plantarum M23, respectively. Starting from the fifth day of fermentation, L. plantarum M23 controlled the fermentation process as the dominant LAB. The drop in the pH value was significant (from 6.8 to 3.18) due to the variations in the LAB strains throughout the 14 days of fermentation. Further investigation will be carried out to study the production of plantaricin, which has great importance in food biopreservation.

Characterization of the Bacterial Composition of 47 Fermented Foods in Sweden.

Fermentation has long been utilized to preserve and enhance the flavor and nutritional value of foods. Recently, fermented foods have gained popularity, reaching new consumer groups due to perceived health benefits. However, the microbial composition of many fermented foods re-mains unknown. Here, we characterized the bacterial composition, diversity, and richness of 47 fermented foods available in Sweden, including kombucha, water kefir, milk kefir, yogurt, plant-based yogurt alternatives, kimchi, sauerkraut, and fermented vegetables. Via 16S rRNA gene sequencing, we identified 2497 bacteria (amplicon sequence variants). The bacterial composition was strongly associated with the type of fermented food, and lactic acid bacteria and/or acetic acid bacteria dominated most samples. However, each fermented food had a unique composition, with kombucha and water kefir having the highest diversity across and within samples. Few bacteria were abundant in multiple foods and food groups. These were Streptococcus thermophilus in yogurts and plant-based yoghurts; Lactococcus lactis in milk kefirs and one water kefir; and Lactiplantibacillus plantarum in kimchi, sauerkraut, and fermented cucumber. The broad range of fermented foods included in this study and their diverse bacterial communities warrant further investigation into the implications of microbial compositions for product traits and potential impact on human health.

Culture condition optimization of naturally lacto-fermented cucumbers based on changes in detrimental and functional ingredients.

A two-step trial was used to optimize the culture condition of naturally lacto-fermented cucumbers. In the first trial, changes in pH values and total biogenic amines were measured to optimize the pickling juice formula. A 15% crystal sugar solution with low-salt brine at 4°C was proved to be the best formula. In the second trial, pH values, organic acids, total phenolics, flavonoids, saponins and free amino acids, as well as biogenic amines and nitrites under the optimal pickling formula were measured. The optimal fermentation day was suggested at around 8days. During the cucumber's fermentation process, the pH value was quickly lowered to <4.6. Meanwhile, the functional ingredients increased significantly. In contrast, total biogenic amines and nitrites did not exceed the risk limit, evidencing the safety and functional characteristics for the naturally lacto-fermented cucumbers. The two-step trial has evidenced the possibility to develop desirable lacto-fermented cucumbers.

Lactiplantibacillus plantarum subsp. plantarum BCRC10069 co-fermented cucumber safety and functional characteristics

Lactiplantibacillus (Lpb.) plantarum subsp. plantarum BCRC10069 was selected to co-ferment immature cucumbers for 8 days. The pH values, total biogenic amines and organic acids were measured. Extracts from 8-days Lpb. plantarum co-fermented cucumbers (LPCFC) were collected to analyze their anti-inflammatory characteristics using RAW 264.7 macrophages with four inflammatory models. Pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines profiles by RAW 264.7 cells were assessed with ELISA. Results showed that pH value was rapidly lowered to 4.33 ± 0.10 (aka high-acid foods). Functional lactic acid quickly increased to 44.4 ± 1.6 μg/ml in the fermented pickling juice. However, detrimental total biogenic amines in solid fermented cucumbers were estimated much lower than the risk limit, suggesting the safety for LPCFC. LPCFC extracts demonstrated strong potential to repair inflammation in lipopolysaccharide-stimulated macrophages. Water extract had a better effect than ethanol extract. Our results evidence the safety and anti-inflammatory effect of Lpb. plantarum co-fermented cucumbers.

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.

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.

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.

Determination of selected biogenic amines in fermented vegetables juices

Fermented vegetables and fruits are a good source of selected vitamins, antioxidants, microelements and revealed immune-boosting properties. The appropriate content of health-promoting compounds and probiotics allows you to qualify juices from fermented vegetables or fruits as better-for-you food. Notwithstanding, biogenic amines in this type of food are also detected due to the fermentation process, which may negatively affect their health-promoting properties. In this study, various biogenic amines were determined in juices obtained during the pressing of fermented vegetables (white and red cabbage, cucumbers, tomatoes, cauliflowers, celery, celery leaves, broccoli, and beetroots). The level of monamines (histamine, tyramine, tryptamine, and phenylethylamine), diamines (putrescine, cadaverine), and polyamines (spermidine and spermine) was discussed. The significant discrepancies between all the tested determined compounds as well as the sums of the BAs in tested samples, were observed. The calculated values of BAs sum varied from 6.36 mg L−1 (fermented tomato juice) to 116.52 mg L−1 (fermented cucumber juice). Among the determined amines, the majority were histamine, tyramine, putrescine, and cadaverine (from 66% to 97–98% of total biogenic amines level). Moreover, depending on the type of tested juice, the amount of histamine and tyramine in the full content of BAs was between 0.44% (for fermented red cabbage juice) and 68% (for fermented broccoli juice). According to our knowledge, this is the first time that the determination of biogenic amines in fermented vegetable juices has been studied.

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.

Sensory and microbial analyses on naturally lacto-fermented cucumbers

A solution of 2.5% salt with 15% crystal sugar was used for developing naturally fermented cucumbers. Fermented food sensory characteristics, color difference, and microbial analyses have not been yet investigated. To clarify this puzzle, changes in naturally fermented cucumber characteristics through the aging period were analyzed in this study. The results show that sensory evaluation and color difference in the fermented product improved significantly through the optimal fermentation process. Both total plate count and lactic acid bacteria count significantly and time-dependently increased. Importantly, Leuconostoc mensenteroides were identified as the dominant lactic acid bacteria. Leuconostoc mensenterosides significantly increased from 33.64% to 80.72% through the 8-days optimal aging process. Detrimental Enterobacter unclassified bacteria markedly decreased from 35.45% to 3.34%. Based on the overall changes in sensory evaluation, color difference and microbiota, naturally lacto-fermented cucumbers were developed and may be further exploited in the food industry.