Keun-Sung Kim

Enhanced Rapidity for Qualitative Detection of Listeria monocytogenes Using an Enzyme-Linked Immunosorbent Assay and Immunochromatography Strip Test Combined with Immunomagnetic Bead Separation

An enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICG) strip test, and immunomagnetic bead separation (IMBS) system based on a monoclonal antibody were individually developed for the detection and isolation of Listeria monocytogenes in meat samples. The three methods showed a strong reaction with Listeria species and a weak reaction with Staphylococcus aureus. To increase the rapidity of L. monocytogenes detection, combinations of the ELISA and ICG strip test with the IMBS system (ELISA-IMBS and ICG-IMBS) were investigated. In comparative analyses of artificially inoculated meat and samples of processed meat, the ELISA and ICG strip test required 24 h of enrichment time to detect the inoculated meat samples with > or =1 X 10(2) CFU/10 g, whereas the ELISA-IMBS and ICG-IMBS required only 14 h of enrichment. Analyses of naturally contaminated meat samples (30 pork samples, 20 beef samples, 26 chicken samples, 20 fish samples, and 20 processed meat samples) performed by ELISA-IMBS, ICG-IMBS, and API kit produced similar results. The ELISA-IMBS and ICG-IMBS provide a more rapid assay than the individual ELISA and the ICG strip test and are appropriate for rapid and qualitative detection of L. monocytogenes (or Listeria species) in meat samples. With the ICG-IMBS, L. monocytogenes could be detected in meat samples within 15 h and the method has potential as a rapid, cost-effective on-site screening tool for the detection of L. monocytogenes in food samples and agricultural products at a minimum detection level of approximately 100 CFU/10 g.

Characterization of Slackia exigua Isolated from Human Wound Infections, Including Abscesses of Intestinal Origin

ABSTRACT Eleven clinical strains isolated from infected wound specimens were subjected to polyphasic taxonomic analysis. Sequence analysis of the 16S rRNA gene showed that all 11 strains were phylogenetically related to Slackia exigua. Additionally, conventional and biochemical tests of 6 of the 11 strains were performed as supplementary methods to obtain phenotypic identification by comparison with the phenotypes of the relevant type strains. S. exigua has been considered an oral bacterial species in the family Coriobacteriaceae. This organism is fastidious and grows poorly, so it may easily be overlooked. The 16S rRNA gene sequences and the biochemical characteristics of four of the S. exigua strains isolated for this study from various infections indicative of an intestinal source were almost identical to those of the validated S. exigua type strain from an oral source and two of the S. exigua strains from oral sources evaluated in this study. Thus, we show for the first time that S. exigua species can be isolated from extraoral infections as well as from oral infections. The profiles of susceptibility to selected antimicrobials of this species were also investigated for the first time.

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 103 cells/ml or 50 cells were detected by the SPR biosensor.

Isolation and Identification of γ-Aminobutyric acid (GABA)-producing lactic acid bacteria from Kimchi

Presumptive lactic acid bacteria (LAB) were isolated from 20 kimchi samples (total of 230 isolates) and screened for their capacity to synthesize γ-aminobutyric acid (GABA). Only 68 isolates (ca. 30%) showed this activity and were identified by a polyphasic approach consisting of morphological characteristics, catalase and biochemical tests, and species-specific polymerase chain reaction and 16S rRNA gene sequence analyses. Five species were found, including Lactobacillus plantarum (55 isolates), Lactobacillus brevis (six), Leuconostoc mesenteroides (four), Leuconostoc lactis (one), and Weissella viridescens (two). The 68 GABA-producing LAB isolates were isolated from only 11 among 20 kimchi samples indicating that they were not evenly distributed. This is the first report on the isolation of two species of Leuconostoc (Le. mesenteroides and Le. lactis) and one species of Weissella (Ws. viridescens) from kimchi with the capacity to synthesize GABA under in vitro conditions. Additionally, in previous screening results, Le. lactis and Ws. viridescens with the capacity to synthesize GABA isolated and identified from fermented food source were not observed.

Functional Applications of Lignocellulolytic Enzymes in the Fruit and Vegetable Processing Industries

Cellulose, hemicellulose, pectin (carbohydrate), and lignin (noncarbohydrate) polymers are the main substrates of lignocellulose-degrading enzymes. They are present in large amounts in the primary cell wall and dietary fibers of major fruits and vegetables. During processing of fruits and vegetables to the corresponding final food products, lignocellulosic substrates are hydrolyzed by different lignocellulolytic enzymes. Currently, lignocellulolytic enzymes such as cellulases, xylanases, pectinases, and laccases are extensively used during the processing of fruits and vegetables, in applications like texturizing and flavoring of products in the food industries. The present article provides an updated overview of functional applications of lignocellulolytic enzymes in the juice processing, oil extraction, and alcoholic beverage processing industries. Extensive use of lignocellulolytic enzymes in different food processing industries not only accelerates the production rates but also improves product quality. It is also possible to ensure the efficient use of fruits and vegetables globally by employing lignocellulolytic enzymes in the corresponding processing industries to convert them into food commodities, which will not only raise their economic value in the global market but also increase food availability, which will help mitigate nutritional problems worldwide.

Species-Specific Detection of Listeria monocytogenes Using Polymerase Chain Reaction Assays Targeting the prfA Virulence Gene Cluster

Sequence comparison of individual genes in the prfA virulence gene cluster (pVGC), a central virulence gene cluster, from different Listeria species indicated that priming sites within the genes appeared to be specific for Listeria monocytogenes exclusively. Therefore, the pVGC was targeted for polymerase chain reaction (PCR) assays to detect and specifically identify L. monocytogenes. Each gene of the pVGC was specifically amplified in L. monocytogenes but not in other Listeria species.

Quantitative microbiological profiles of brown rice and germinated brown rice

Among health conscious people, brown rice (BR) and germinated brown rice (GBR) are increasingly more popular for consumption in Korea because their nutritional values are greater than those of ordinary white rice (WR). The overall microbial counts for BR were higher than those for WR and those of GBR were higher than those of BR. Interestingly, the lactic acid bacteria (LAB) counts in GBR increased markedly and their selected representatives were Weissella confusa, Pediococcus pentosaceus, and Lactobacillus fermentum. To our knowledge, this is the first attempt to enumerate and compare LAB loads on WR, BR, and GBR.

Isolation and characterization of a novel endo-β-1,4-glucanase from a metagenomic library of the black-goat rumen

The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30–50 °C at a pH of 6–7. The thermostability was retained and the pH was stable in the range of 30–50 °C at a pH of 5–7.

Metagenomic Mining and Functional Characterization of a Novel KG51 Bifunctional Cellulase/Hemicellulase from Black Goat Rumen

A novel KG51 gene was isolated from a metagenomic library of Korean black goat rumen and its recombinant protein was characterized as a bifunctional enzyme (cellulase/hemicellulase). In silico sequence and domain analyses revealed that the KG51 gene encodes a novel carbohydrate-active enzyme that possesses a salad-bowl-like shaped glycosyl hydrolase family 5 (GH5) catalytic domain but, at best, 41% sequence identity with other homologous GH5 proteins. Enzymatic profiles (optimum pH values and temperatures, as well as pH and thermal stabilities) of the recombinant KG51 bifunctional enzyme were also determined. On the basis of the substrate specificity data, the KG51 enzyme exhibited relatively strong cellulase (endo-β-1,4-glucanase [EC 3.2.1.4]) and hemicellulase (mannan endo-β-1,4-mannosidase [EC 3.2.1.78] and endo-β-1,4-xylanase [EC 3.2.1.8]) activities, but no exo-β-1,4-glucanase (EC 3.2.1.74), exo-β-1,4-glucan cellobiohydrolase (EC 3.2.1.91), and exo-1,4-β-xylosidase (EC 3.2.1.37) activities. Finally, the potential industrial applicability of the KG51 enzyme was tested in the preparation of prebiotic konjac glucomannan hydrolysates.

Isolation and Characterization of Spore‐Forming Bacilli (SFB) from Shepherd's Purse (Capsella bursa‐pastoris)

Shepherds purse (Capsella bursa-pastoris), native to Europe, is commonly consumed fresh and sometimes inadequately washed before consumption in Korea. The objective of this study was to characterize isolates of spore-forming bacilli (SFB) in samples of fresh Shepherds purse. Three genera were identified: Bacillus (9 species), Paenibacillus (3 species), and Brevibacillus (1 species). None of the genes of the hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE) complexes, or of the emetic toxin, was detected in the 25 SFB isolates, except for 2 Bacillus pseudomycoides isolates, where all 3 genes of the HBL enterotoxin complex were detected. There were significant sequence variations between the 2 species (Bacillus cereus and B. pseudomycoides) in the 3 genes of the HBL enterotoxin complex. These findings may provide insights into the diverse characteristics of the B. pseudomycoides HBL enterotoxin complex. Antibiotic resistance was assessed using 8 antibiotics. Among the 25 SFB isolates, 11 showed resistance to antibiotics, of which 5 were multiresistant. Assessment of the spoilage potential showed that all 25 SFB isolates could produce enzymes that can cause spoilage of foods. In conclusion, our findings may serve as integrative information for food research and industrial sectors.