Development of Cabbage Juice Medium for Industrial Production of Leuconostoc mesenteroides Starter.

Leuconostoc mesenteroides is used as a starter to produce high-quality kimchi products. In this study, an efficient and economical cabbage juice medium (CJM) was developed by process optimization of cabbage extraction and pasteurization and by compositional supplementation of various lacking nutrients. The pasteurized cabbage juice was determined to be a good medium candidate to cultivate L. mesenteroides, showing maximal cell numbers (9.85 × 108CFU/ml) after 24 h. Addition of sucrose and yeast extract with soy peptone resulted in increment of bacterial cell counts in CJM, showing the supplementing effect of the lacking nutrients. Furthermore, addition of shell powder gave a protective effect on bacterial cells by preventing pH decline and organic acid accumulation in CJM, resulting in a 2-fold increase of bacterial counts. The optimized composition of CJM was 70% cabbage juice diluted with water, 0.5% (w/v) sucrose, 1% (w/v) yeast extract, 1% (w/v) soy peptone, and 1.5% (w/v) ark shell powder. The CJM developed in this study was able to yield a comparable level of bacterial counts with MRS medium and reduced the cost by almost 10-fold.

Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk.

The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 µg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk.

A Synergy Effect of Trisodium Phosphate and Ethanol on Inactivation of Murine Norovirus 1 on Lettuce and Bell Pepper.

The synergy effect of trisodium phosphate (TSP) and ethanol against murine norovirus 1 (MNV-1), as a surrogate for human noroviruses, on fresh produces was evaluated. More than 2% (w/v) of TSP effectively inactivated MNV-1. The single treatment of 1% TSP or 30% ethanol for 30 min was not effective on MNV-1; however, cotreatment showed inactivation of MNV-1 on stainless steel and the produces of lettuce and bell pepper under 15 min. The results suggest that cotreatment of TSP and ethanol at a low concentration and a short time of exposure might be useful for the reduction of norovirus in some produce.

Norovirus contamination levels in ground water treatment systems used for food-catering facilities in South Korea.

This study aimed to inspect norovirus contamination of groundwater treatment systems used in food-catering facilities located in South Korea. A nationwide study was performed in 2010. Water samples were collected and, for the analysis of water quality, the temperature, pH, turbidity, and residual chlorine content were assessed. To detect norovirus genotypes GI and GII, RT-PCR and semi-nested PCR were performed with specific NV-GI and NV-GII primer sets, respectively. The PCR products amplified from the detected strains were then subjected to sequence analyses. Of 1,090 samples collected in 2010, seven (0.64%) were found to be norovirus-positive. Specifically, one norovirus strain was identified to have the GI-6 genotype, and six GII strains had the GII, GII-3, GII-4, and GII-17 genotypes. The very low detection rate of norovirus most likely reflects the preventative measures used. However, this virus can spread rapidly from person to person in crowded, enclosed places such as the schools investigated in this study. To promote better public health and sanitary conditions, it is necessary to periodically monitor noroviruses that frequently cause epidemic food poisoning in South Korea.

Ascorbyl palmitate-loaded chitosan nanoparticles: characteristic and polyphenol oxidase inhibitory activity.

The aim of this study was to produce ascorbyl palmitate (AP)-loaded nanoparticles in order to inhibit polyphenol oxidase (PPO) in bananas. AP-loaded chitosan nanoparticles were prepared using acetic acid and citric acid (denoted as CS/AA and CS/CA nanoparticles, respectively). As the initial AP concentration increases, the particle size significantly decreases, and the zeta potential, entrapment and loading efficiency significantly increases. The PPO inhibitory activity of AP was effectively improved when AP was nano-encapsulated by chitosan compared to no encapsulation. These results suggest that chitosan nano-encapsulation can be used to enhance the PPO inhibitory activity of AP.

Enzymes hydrolyzing structural components and ferrous ion cause rusty-root symptom on ginseng (Panax ginseng).

Microbial induction of rusty-root was proved in this study. The enzymes hydrolyzing plant structural materials, including pectinase, pectolyase, ligninase, and cellulase, caused the rusty-root in ginseng. Pectinase and pectolyase produced the highest rusty-color formation. Ferrous ion (Fe+++) caused the synergistic effect on rusty-root formation in ginseng when it was used with pectinase. The effect of ferric ion (Fe++) on rusty-root formation was slow, compared with Fe+++, probably due to gradual oxidation to Fe+++. Other metal ions including the ferric ion (Fe++) did not affect rusty-root formation. The endophytic bacteria Agrobacterium tumefaciens, Lysobacter gummosus, Pseudomonas veronii, Pseudomonas marginalis, Rhodococcus erythropolis, and Rhodococcus globerulus, and the rotten-root forming phytophathogenic fungus Cylindrocarpon destructans, caused rusty-root. The polyphenol formation (rusty color) was not significantly different between microorganisms. The rotten-root-forming C. destructans produced large quantities of external cellulase activity (about 2.3 U[micronM/min/mg protein]), which indicated the pathogenecity of the fungus, whereas the bacteria produced 0.1-0.7 U. The fungal external pectinase activities (0.05 U) and rusty-root formation activity were similar to those of the bacteria. In this report, we proved that microbial hydrolyzing enzymes caused rusty-root (Hue value 15 degrees) of ginseng, and ferrous ion worsened the symptom.

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.

Folate contents in human milk and casein-based and soya-based formulas, and folate status in Korean infants.

We assessed folate nutritional status from birth to 12 months in fifty-one infants who were fed human milk (HM; n 20), casein-based formula (CBF; n 12) or soya-based formula (SBF; n 19). Folate contents in ninety-five HM samples obtained from twenty mothers for the first 6-month period and twelve CBF and nineteen SBF samples were measured by bioassay after trienzyme extraction. Folate intake was estimated by weighing infants before and after feeding in the HM group and by collecting formula intake records in the formula-fed groups. After solid foods were introduced, all foods consumed were included to estimate folate intake. Serum folate and total homocysteine (tHcy) concentrations were determined at 5 and 12 months of age, and infant growth was monitored for the first 12 months. Mean HM folate contents ranged from 201 to 365 nmol/l with an overall mean of 291 nmol/l, and the contents peaked at 2 months postpartum. HM folate contents were higher than those reported in North America. Folate contents in CBF and SBF were markedly higher than those in HM and those claimed on the product labels. The overall folate intakes in formula-fed infants were significantly higher than those in HM-fed infants, and this was associated with significantly higher folate and lower tHcy in formula-fed infants than HM-fed infants at 5 months. At 12 months, serum folate was significantly higher in the SBF group than the other groups, whereas serum tHcy and overall growth were similar among all groups.

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.

Development of predictive mathematical model for the growth kinetics of Staphylococcus aureus by response surface model.

A response surface model was developed for predicting the growth rates of Staphylococcus aureus in tryptic soy broth (TSB) medium as a function of combined effects of temperature, pH, and NaCl. The TSB containing six different concentrations of NaCl (0, 2, 4, 6, 8, and 10%) was adjusted to an initial of six different pH levels (pH 4, 5, 6, 7, 8, 9, and 10) and incubated at 10, 20, 30, and 40 degrees C. In all experimental variables, the primary growth curves were well (r2=0.9000 to 0.9975) fitted to a Gompertz equation to obtain growth rates. The secondary response surface model for natural logarithm transformations of growth rates as a function of combined effects of temperature, pH, and NaCl was obtained by SAS's general linear analysis. The predicted growth rates of the S. aureus were generally decreased by basic (pH 9-10) or acidic (pH 5-6) conditions and higher NaCl concentrations. The response surface model was identified as an appropriate secondary model for growth rates on the basis of correlation coefficient (r=0.9703), determination coefficient (r2=0.9415), mean square error (MSE=0.0185), bias factor (B(f)=1.0216), and accuracy factor (A(f)=1.2583). Therefore, the developed secondary model proved reliable for predictions of the combined effect of temperature, NaCl, and pH on growth rates for S. aureus in TSB medium.

Production and characterization of monoclonal and recombinant antibodies against antimicrobial sulfamethazine.

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45 ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml, compared with 1.6 ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

Production of monoclonal antibody against Listeria monocytogenes and its application to immunochromatography strip test.

An immunochromatography (ICG) strip test based on a monoclonal antibody for the rapid detection of L. monocytogenes in meat and processed-meat samples was developed in this study. A monoclonal antibody (MAb) specific to L. monocytogenes was produced from cloned hybridoma cells (FKLM-3B12-37) and used to develop an ICG strip test. The antibody showed a stronger binding to L. monocytogenes than other Listeria species, and a weak cross-reaction to S. aureus based on an ELISA. The detection limit of the ICG strip test was 10(5) cell/ml. In total, 116 meat and processed-meat samples were collected and analyzed using both the ICG strip test and a PCR. The ICG strip test and PCR indicated L. monocytogenes contamination in 34 and 27 meat samples, respectively. The 7 meat samples not identified as L. monocytogenes positive by the PCR were also tested using an API kit and found to be contaminated by Listeria species. In conclusion, the ICG strip test results agreed well with those obtained using the PCR and API kit. Thus, the developed ICG has potential use as a primary screening tool for L. monocytogenes in various foods and agricultural products, generating results within 20 min without complicated steps.

Morphology and adhesion of Campylobacter jejuni to chicken skin under varying conditions.

The adhesion of Campylobacter jejuni to chicken skin, along with the associated morphological changes under aerobic conditions at 4, 25, and 37 degrees C and microaerobic (O2 5%, CO2 10%, N2 85%) conditions, were investigated using confocal laser scanning microscopy (CLSM), flow cytometry, and plate counting. The morphological change of C. jejuni from a spiral shape to a coccoid form or VBNC form (viable but nonculturable form) progressed rapidly under aerobic conditions at 25, 37, and 4 degrees C. As regards adhesion, the C. jejuni cells were mostly located in the crevices and feather follicles of the chicken skin, where the cells in the feather follicles floated freely in the entrapped water, even after the skin was rinsed quite thoroughly. CLSM also revealed the penetration of some spiral-shaped C. jejuni cells into the chicken skin. Even after changing their shape at various temperatures, coccoid-form C. jejuni cells were still found in the crevices and feather follicles of the chicken skin.

Molecular cloning and characterization of a large subunit of Salmonella typhimurium glutamate synthase (GOGAT) gene in Escherichia coli.

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

Marinimicrobium koreense gen. nov., sp. nov. and Marinimicrobium agarilyticum sp. nov., novel moderately halotolerant bacteria isolated from tidal flat sediment in Korea.

Two moderately halotolerant Gram-negative bacteria were isolated from tidal flat sediment of the South Sea in Korea (the Korea Strait). The strains, designated M9T and M18T, were strictly aerobic, rod-shaped and non-spore-forming and motile with a flagellum and their major fatty acids were C(16:0) and C(19:0) cyclo omega8c. Strains M9T and M18T could grow in the presence of up to 13-15% (w/v) NaCl, but their optimum salt concentrations were relatively low (0-3%, w/v). The major predominant isoprenoid quinone was Q-8 and the G + C content of the genomic DNA was 57-58 mol%. Phylogenetic analyses and comparative 16S rRNA gene sequence studies revealed that strains M9T and M18T formed a phylogenetic lineage distinct from the genus Teredinibacter within the class Gammaproteobacteria and were most closely related to the genera Microbulbifer, Saccharophagus and Teredinibacter, with less than 92.5% 16S rRNA gene sequence similarity. The level of 16S rRNA gene sequence similarity between the two strains was 96.7%. On the basis of physiological and phylogenetic properties, strains M9T and M18T represent separate species within a novel genus of the class Gammaproteobacteria, for which the names Marinimicrobium koreense gen. nov., sp. nov. (type species) and Marinimicrobium agarilyticum sp. nov. are proposed. The type strains of Marinimicrobium koreense and Marinimicrobium agarilyticum are M9T (= KCTC 12356T = DSM 16974T) and M18T (= KCTC 12357T = DSM 16975T), respectively.

PCR method based on the ogdH gene for the detection of Salmonella spp. from chicken meat samples.

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.