A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

Lactobacillus allii sp. nov. isolated from scallion kimchi.

A novel strain of lactic acid bacteria, WiKim39T, was isolated from a scallion kimchi sample consisting of fermented chili peppers and vegetables. The isolate was a Gram-positive, rod-shaped, non-motile, catalase-negative and facultatively anaerobic lactic acid bacterium. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain WiKim39T belonged to the genus Lactobacillus, and shared 97.1-98.2 % pair-wise sequence similarities with related type strains, Lactobacillus nodensis, Lactobacillus insicii, Lactobacillus versmoldensis, Lactobacillus tucceti and Lactobacillus furfuricola. The G+C content of the strain based on its genome sequence was 35.3 mol%. The ANI values between WiKim39T and the closest relatives were lower than 80 %. Based on the phenotypic, biochemical, and phylogenetic analyses, strain WiKim39T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus allii sp. nov. is proposed. The type strain is WiKim39T (=KCTC 21077T=JCM 31938T).

Large-scale targeted metagenomics analysis of bacterial ecological changes in 88 kimchi samples during fermentation.

The microbial communities in kimchi vary widely, but the precise effects of differences in region of origin, ingredients, and preparation method on the microbiota are unclear. We analyzed the bacterial community composition of household (n = 69) and commercial (n = 19) kimchi samples obtained from six Korean provinces between April and August 2015. Samples were analyzed by barcoded pyrosequencing targeting the V1-V3 region of the 16S ribosomal RNA gene. The initial pH of the kimchi samples was 5.00-6.39, and the salt concentration was 1.72-4.42%. Except for sampling locality, all categorical variables, i.e., salt concentration, major ingredient, fermentation period, sampling time, and manufacturing process, influenced the bacterial community composition. Particularly, samples were highly clustered by sampling time and salt concentration in non-metric multidimensional scaling plots and an analysis of similarity. These results indicated that the microbial community differed according to fermentation conditions such as salt concentration, major ingredient, fermentation period, and sampling time. Furthermore, fermentation properties, including pH, acidity, salt concentration, and microbial abundance differed between kimchi samples from household and commercial sources. Analyses of changes in bacterial ecology during fermentation will improve our understanding of the biological properties of kimchi, as well as the relationships between these properties and the microbiota of kimchi.

Metabolites of Kimchi Lactic Acid Bacteria, Indole-3-Lactic Acid, Phenyllactic Acid, and Leucic Acid, Inhibit Obesity-Related Inflammation in Human Mesenchymal Stem Cells.

Given the diversity of vegetables utilized in food fermentation and various lactic acid bacteria (LAB) populations in these materials, comprehensive studies on LAB from vegetable foods, including kimchi, are imperative. Therefore, this study aimed to investigate the obesity-related inflammation response of three metabolites-phenyllactic acid (PLA), indole-3-lactic acid (ILA), and leucic acid (LA)-produced by LAB (Companilactobacillus allii WiKim39 and Lactococcus lactis WiKim0124) isolated from kimchi. Their effects on tumor necrosis factor-α-induced changes in adipokines and inflammatory response in adipose-derived human mesenchymal stem cells were examined. The study results showed that PLA, ILA, and LA, particularly PLA, effectively reduced lipid accumulation and triglyceride, glycerol, free fatty acid, and adiponectin levels. Furthermore, the identified metabolites were found to modulate the expression of signaling proteins involved in adipogenesis and inflammation. Specifically, these metabolites were associated with enriched expression in the chemokine signaling pathway and cytokine-cytokine receptor interaction, which are critical pathways involved in regulating immune responses and inflammation. PLA, ILA, and LA also suppressed the secretion of pro-inflammatory cytokines and several inflammatory markers, with the PLA-treated group exhibiting the lowest levels. These results suggest that PLA, ILA, and LA are potential therapeutic agents for treating obesity and inflammation by regulating adipokine secretion and suppressing pro-inflammatory cytokine production.

Combined Non-Thermal Microbial Inactivation Techniques to Enhance the Effectiveness of Starter Cultures for Kimchi Fermentation.

For quality standardization, the application of functional lactic acid bacteria (LAB) as starter cultures for food fermentation is a well-known method in the fermented food industry. This study assessed the effect of adding a non-thermally microbial inactivated starter culture to kimchi, a traditional Korean food, in standardizing its quality. In this study, pretreatment based on sterilization processes, namely, slightly acidic electrolyzed water (SAEW) disinfection and ultraviolet C light-emitting diode (UVC-LED) of raw and subsidiary kimchi materials were used to reduce the initial microorganisms in them, thereby increasing the efficiency and value of the kimchi LAB starter during fermentation. Pretreatment sterilization effectively suppressed microorganisms that threatened the sanitary value and quality of kimchi. In addition, pretreatment based on sterilization effectively reduced the number of initial microbial colonies in kimchi, creating an environment in which kimchi LAB starters could settle or dominate, compared to non-sterilized kimchi. These differences in the initial microbial composition following the sterilization process and the addition of kimchi LAB starters led to differences in the metabolites that positively affect the taste and flavor of kimchi. The combined processing technology used in our study, that is, pre-sterilization and LAB addition, may be a powerful approach for kimchi quality standardization.

Anti-Obesity Effect of Kimchi with Starter Cultures in 3T3-L1 Cells.

Lactic acid bacteria (LAB) isolated from kimchi have various functions, including antioxidant, anti-inflammation, and anti-obesity activities, and are therefore widely used in the food, pharmaceutical, and medical fields. To date, the health functionalities of LAB have been widely reported; however, those of kimchi fermented with LAB as a starter have rarely been reported. Therefore, research on the selection of LAB with anti-obesity activity and the health functionality of kimchi fermented with LAB is needed. In the present study, LAB with anti-obesity activity were initially selected by measuring the Oil-Red O intensity. Among the four LAB strains, anti-obesity activity was confirmed by measuring cell viability, lipid levels, and lipid accumulation. Then, starter kimchi (SK) was prepared by inoculating selected LABs, and its pH, total acidity, and salinity were compared with those of naturally fermented kimchi (NK). Lastly, anti-obesity activity was also investigated in 3T3-L1 cells. Selected LAB showed no cytotoxicity up to 107 CFU/ml, with Lactobacillus brevis JC7 and Leuconostoc mesenteroides KCKM0828 having higher inhibitory effects on TG, TC content and lipid accumulation. Most SKs showed fermentation properties similar to those of the NK. SKs showed no cytotoxicity at concentrations of up to 1,000 µg/ml. SKs showed strong inhibitory effects on TG content, lipid accumulation, and obesity-related gene and protein expressions. Taken together, the utilization of LAB as a starter could improve the health benefits of kimchi.

Anti-obesity effect of vegetable juice fermented with lactic acid bacteria isolated from kimchi in C57BL/6J mice and human mesenchymal stem cells.

This study aimed to investigate the effect of fermented vegetable juice (VJ) obtained from a blend of four crops (Brassica oleracea var. capitata, B. oleracea var. italica, Daucus carota L., and Beta vulgaris) on adipogenesis along with the identification of active compounds. Two lactic acid bacteria (LAB) (Companilactobacillus allii WiKim39 and Lactococcus lactis WiKim0124), isolated from kimchi, were used to ferment the VJ and their effectiveness was evaluated in differentiated human mesenchymal stem cells and obese mice. In vitro antibody array analysis was done to understand signaling proteins in adipogenesis. Gene Ontology enrichment analysis showed that differentially expressed proteins are related to biological processes including immunological processes. These were effectively regulated by LAB and fermented VJ. Supplementation of fermented VJ reduced the weight gain, blood biochemical indicators, and liver fat accumulation in mice. Oil Red O staining indicated that the fermentation metabolites of VJ (indole-3-lactic acid, leucic acid, and phenyllactic acid) had an inhibitory effect on lipid accumulation in vitro. Therefore, it can be concluded that LAB-fermented VJ and its metabolites have the potential to counter obesity, and thus can be therapeutically effective.

Transcriptome responses of lactic acid bacteria isolated from kimchi under hydrogen peroxide exposure.

In this study, five species of lactic acid bacteria (LAB) isolated from kimchi were analyzed in terms of their potential antioxidant activity. Latilactobacillus curvatus WiKim38, Companilactobacillus allii WiKim39, and Lactococcus lactis WiKim0124 exhibited higher radical scavenging activity, reducing power, and lipid peroxidation inhibition than the reference strain and tolerated hydrogen peroxide (H2O2) exposure up to a concentration of 2.5 mM. To investigate the antioxidant mechanism of LAB strains, transcriptomic and proteomic signatures were compared between the H2O2-exposed and untreated group using RNA sequencing and two-dimensional protein gel electrophoresis. Across all LAB strains, cell membrane responses and metabolic processes were the most prominent in the main categories of gene ontology classification, indicating that cellular components and interactions play an important role in oxidative stress responses. Thus, LAB strains isolated from kimchi could be considered for potential use in functional food production and in antioxidant starter cultures.

Molecular Mechanism of L-Pyroglutamic Acid Interaction with the Human Sour Receptor.

Taste is classified into five types, each of which has evolved to play its respective role in mammalian survival. Sour taste is one of the important ways to judge whether food has gone bad, and the sour taste receptor (PKD2L1) is the gene behind it. Here, we investigated whether L-pyroglutamic acid interacts with sour taste receptors through electrophysiology and mutation experiments using Xenopus oocytes. R299 of hPKD2L1 was revealed to be involved in L-pyroglutamic acid binding in a concentration-dependent manner. As a result, it is possible to objectify the change in signal intensity according to the concentration of L-pyroglutamic acid, an active ingredient involved in the taste of kimchi, at the molecular level. Since the taste of other ingredients can also be measured with the method used in this experiment, it is expected that an objective database of taste can be created.

Arginine metabolism and the role of arginine deiminase-producing microorganisms in kimchi fermentation.

In this study, we evaluated the correlation between the arginine deiminase (ADI) pathway and nitrogen cycle during cabbage kimchi fermentation. Nitrite used as a food additive can be converted to carcinogenic N-nitroso compounds via reactions with secondary amines under specific conditions; thus, high nitrate- and nitrite-containing foods present a potential risk to human health. We monitored the bacterial community, levels of ADI metabolites, and nitrogen compounds present in kimchi that contained bacteria that showed low ADI activity during fermentation. The dominant growth of microorganisms with weak ADI activity reduced arginine degradation and ornithine production. Furthermore, nitrite production in kimchi samples was affected by ADI activity. The ornithine and nitrite contents in the control kimchi were 1.7- and 2.6-fold higher at week 2 than at week 1. These results suggest that ADI-associated metabolism is correlated with the nitrogen cycle in kimchi and that the addition of bacteria with weak ADI activity may reduce nitrite production in kimchi.

Supercooling Pretreatment Improves the Shelf-Life of Freeze-Dried Leuconostoc mesenteroides WiKim32.

Storage stability of freeze-dried lactic acid bacteria is a critical factor for their cost-effectiveness. Long-term storage of lactic acid bacteria enables microbial industry to reduce distribution costs. Herein, we investigated the effect of cold adaptation under supercooling conditions at -5°C on the viability of Leuconostoc mesenteroides WiKim32 during the freeze-drying process and subsequent storage. Cold adaptation increased the thickness of exopolysaccharides (EPS) and improved the viability of freeze-dried Leu. mesenteroides WiKim32. Compared to non-adapted cells, cold-adapted cells showed a 35.4% increase in EPS thickness under supercooling conditions. The viability of EPS-hydrolyzed cells was lower than that of untreated cells, implying that EPS plays a role in protection during the freeze-drying process. Cold adaptation increased the storage stability of freeze-dried Leu. mesenteroides WiKim32. Fifty-six days after storage, the highest viability (71.3%) was achieved with cold adaptation at -5°C. When EPS-containing broth was added prior to the freeze-drying process, the viability further increased to 82.7%. These results imply that cold adaptation by supercooling pretreatment would be a good strategy for the long-term storage of Leu. mesenteroides WiKim32.

Lactic Acid Bacteria Strains Used as Starters for Kimchi Fermentation Protect the Disruption of Tight Junctions in the Caco-2 Cell Monolayer Model.

In this study, we investigated the effect of lactic acid bacteria (LAB) strains used as starters for kimchi fermentation, namely Lactococcus lactis WiKim0124, Companilactobacillus allii WiKim39, Leuconostoc mesenteroides WiKim0121 Leuconostoc mesenteroides WiKim33, and Leuconostoc mesenteroides WiKim32, on the intestinal epithelial tight junctions (TJs). These LAB strains were not cytotoxic to Caco-2 cells at 500 µg/ml concentration. In addition, hydrogen peroxide (H2O2) decreased Caco-2 viability, but the LAB strains protected the cells against H2O2-induced cytotoxicity. We also found that lipopolysaccharide (LPS) promoted Caco-2 proliferation; however, no specific changes were observed upon treatment with LAB strains and LPS. Our evaluation of the permeability in the Caco-2 monolayer model confirmed its increase by both LPS and H2O2. The LAB strains inhibited the increase in permeability by protecting TJs, which we evaluated by measuring TJ proteins such as zonula occludens-1 and occludin, and analyzing them by western blotting and immunofluorescence staining. Our findings show that LAB strains used for kimchi fermentation can suppress the increase in intestinal permeability due to LPS and H2O2 by protecting TJs. Therefore, these results suggest the possibility of enhancing the functionality of kimchi through its fermentation using functional LAB strains.

Root-Knot Nematode (Meloidogyne incognita) Control Using a Combination of Lactiplantibacillus plantarum WiKim0090 and Copper Sulfate.

Lactic acid bacteria (LAB) exert antagonistic activity against root-knot nematodes, mainly by producing organic acids via carbohydrate fermentation. However, they have not yet been used for root-knot nematode (Meloidogyne incognita) control owing to a lack of economic feasibility and effectiveness. In this study, we aimed to isolate organic acid-producing LAB from kimchi (Korean traditional fermented cabbage) and evaluated their nematicidal activity. Among the 234 strains isolated, those showing the highest nematicidal activity were selected and identified as Lactiplantibacillus plantarum WiKim0090. Nematicidal activity and egg hatch inhibitory activity of WiKim0090 culture filtrate were dose dependent. Nematode mortality 3 days after treatment with 2.5% of the culture filtrate was 100%, with a 50% lethal concentration of 1.41%. In pot tests, the inhibitory activity of an L. plantarum WiKim0090-copper sulfate mixture on gall formation increased. Compared to abamectin application, which is a commercial nematicide, a higher control value was observed using the WiKim0090-copper sulfate mixture, indicating that this combination can be effective in controlling the root-knot nematode.

UPLC-QTOF-MS/MS and GC-MS Characterization of Phytochemicals in Vegetable Juice Fermented Using Lactic Acid Bacteria from Kimchi and Their Antioxidant Potential.

This study aims to investigate fermentative metabolites in probiotic vegetable juice from four crop varieties (Brassica oleracea var. capitata, B. oleracea var. italica, Daucus carota L., and Beta vulgaris) and their antioxidant properties. Vegetable juice was inoculated with two lactic acid bacteria (LAB) (Companilactobacillus allii WiKim39 and Lactococcus lactis WiKim0124) isolated from kimchi and their properties were evaluated using untargeted UPLC-QTOF-MS/MS and GC-MS. The samples were also evaluated for radical (DPPH• and OH•) scavenging activities, lipid peroxidation, and ferric-reducing antioxidant power. The fermented vegetable juices exhibited high antioxidant activities and increased amounts of total phenolic compounds. Fifteen compounds and thirty-two volatiles were identified using UPLC-QTOF-MS/MS and GC-MS, respectively. LAB fermentation significantly increased the contents of d-leucic acid, indole-3-lactic acid, 3-phenyllactic acid, pyroglutamic acid, γ-aminobutyric acid, and gluconic acid. These six metabolites showed a positive correlation with antioxidant properties. Thus, vegetable juices fermented with WiKim39 and WiKim0124 can be considered as novel bioactive health-promoting sources.

Production, Characterization, and Antioxidant Activities of an Exopolysaccharide Extracted from Spent Media Wastewater after Leuconostoc mesenteroides WiKim32 Fermentation.

Bacterial exopolysaccharides (EPSs) are important alternatives to plant polysaccharides in fermented products and exhibit antioxidant activity, which is particularly desirable for functional foods. This study evaluated the use of spent media wastewater (SMW) derived from kimchi fermentation for the production of an EPS and analyzed the characterization and antioxidant activity of the resulting EPS. The EPS concentration and conversion yields of sequential purification were 7.7-9.0 g/L and 38.6-45.1%, respectively. Fourier transform infrared spectra and NMR spectra indicated that the EPS was a linear glucan with α-(1 → 6) linkages. The EPS also exhibited thermal tolerance to high temperatures. In vitro antioxidant activity analyses indicated the scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, thiobarbituric acid reactance (TBAR), and ferric ion reducing antioxidant power (FRAP) values of 71.6-79.1, 28.2-33.0%, and 0.04-0.05 mM FeCl3, respectively. These results reveal that the EPS extracted from SMW has potential as a thermally tolerant, nontoxic, and natural antioxidant for industrial applications.

DNAzyme-based quantitative loop-mediated isothermal amplification for strain-specific detection of starter kimchi fermented with Leuconostoc mesenteroides WiKim32.

Leuconostoc spp. are generally utilized as kimchi starters because of their beneficial effects on kimchi fermentation and sensory characteristics. We developed a DNAzyme-based colorimetric method for measuring the abundance of the kimchi starter Leuconostoc mesenteroides WiKim32. A primer set for loop-mediated isothermal amplification and target-specific DNAzyme was designed based on the WiKim32 nucleotide sequence. In the presence of the target amplicon, DNAzyme bound to it, resulting in negligible amounts of green product. In contrast, with the addition of hemin and in the absence of the target amplicon, DNAzyme fragments not bound to the target amplicon formed G-quadruplex-hemin conjugates, generating a visible green product by oxidizing 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt. There was no cross-reaction with other strains. The method had high detection sensitivity and quantitative capacity in kimchi samples without a requirement for DNA isolation. This strategy provides a rapid, sensitive, and simple detection method with possible industrial applications.

A colorimetric Loop-mediated isothermal amplification (LAMP) assay based on HRP-mimicking molecular beacon for the rapid detection of Vibrio parahaemolyticus.

In the world wide, food poisoning accidents related to Vibrio spp. are on the rise, even numbers of food poisoning by other foodborne pathogens are decreasing. Therefore, the requirement of the rapid, sensitive and convenient detection method for V. parahaemolyticus has been grown. The objective of this study is to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay using a molecular beacon (HRPzyme connected with complementary oligonucleotides at the 5' and 3' ends) for the rapid, sensitive, and convenient detection of V. parahaemolyticus. The colorimetric LAMP assay optimized at 58.8°C showed a detection limit of 1 × 100 CFU/mL and was confirmed to be specific to V. parahaemolyticus. The colorimetric LAMP assay can be finished within 1 h including DNA extraction step. The method was successfully applied to flatfish samples artificially inoculated with known amount of V. parahaemolyticus, and its cut-off value for the flatfish samples was 1 × 101 CFU/g. In addition, the colorimetric LAMP assay developed in the study was found to be able to correct false-positive results, which are known to be a disadvantage of conventional LAMP assays. Therefore, these results indicated that the colorimetric LAMP method is a useful tool for the rapid, sensitive and convenient detection of V. parahaemolyticus in fishes and can also be used as a point-of-care molecular diagnostic technique since it does not require any expensive equipment such as a thermocycler and detectors.

Coffee residue as a valorization bio-agent for shelf-life extension of lactic acid bacteria under cryopreservation.

The present work describes the feasibility of coffee residue extracts as cryoprotective agents in the storage stability of freeze-dried lactic acid bacteria. Coffee residue extracts were extracted from coffee residue, produced after coffee extraction for coffee powder and instant coffee preparation, using an autoclave. Leuconostoc mesenteroides WiKim32 was selected to evaluate the ability of coffee residue extracts to protect bacteria during freeze-dried storage. The storage stability of freeze-dried Leu. mesenteroides WiKim32 with coffee residue extracts was comparable to those with commercial cryoprotective agents. Coffee residue extracts contributed to storage stability immediately after freeze-drying (61.2%) and subsequent storage (48.7%). Our data indicate that the protective effect of the coffee residue extracts is associated with ions, carbohydrates, and phenolic compounds. Coffee residue extracts are feasible materials, which can reduce the storage and distribution costs compared to commercial agents currently available.

Identification of a replicon from pCC3, a cryptic plasmid from Leuconostoc citreum C4 derived from kimchi, and development of a new host-vector system.

Analysis of the structural properties of pCC3, a cryptic plasmid from Leuconostoc citreum C4 isolated from kimchi, determined its length as 3,338 bp and revealed three open reading frames (ORFs): ORF1-ORF3. ORF3 showed high homology with a replication initiation protein of the theta-type plasmid pTXL1. The fragment encompassing ORF3 and its upstream sequences (nt 1,299-1,634) was found to contain a functional plasmid replicon. A new shuttle vector, pUCC3E1, was constructed based on pCC3. Using Southern hybridization analysis, no single-stranded DNA intermediate was detected from Leu. citreum harboring pUCC3E1, which indicates that pCC3 replicated via the theta mechanism. The pUCC3E1 could be replicated in E. coli TG1 (5.8 x 10(4) CFU/microg DNA) and the developed cloning hosts, Leu. citreum C16 (2.1 x 10(2) CFU/microg DNA) and Leu. citreum GJ7 (8.0 x 10(1) CFU/microg DNA). pUCC3E1 was stably maintained in Leu. citreum C16 (for 100 generations, ca. 94.2%) in the absence of erythromycin (5 microg/ml).

Bacterial diversity in Korean temple kimchi fermentation.

Kimchi is manufactured using salted vegetables and various seasonings, including garlic, scallion, and jeotgal (fermented seafood). However, similar to vegan diets, Korean temple food does not contain animal products (meat- and seafood-free) and is restricted to five pungent herbs: garlic, scallion, leek, onions, and chives. In this study, we investigated the fermentation characteristics of 25 kimchi samples from traditional Korean temples or commercial sources using Illumina MiSeq sequencing. The initial pH of the kimchi samples ranged from 5.05 to 5.95 and the bacterial diversity-index showed a significantly high value in temple-style kimchi. Moreover, differences in microbial community were significantly reflected in kimchi types using non-metric multidimensional scaling plots and analysis of similarity. Additionally, the distribution patterns of the core bacterial genera differed according to kimchi type, especially during early phases of fermentation. These findings offer novel insights into the microbial ecology and quality characteristics of kimchi lacking vital ingredients, which are generally reported based on the origin of the microorganisms.