Suk-Ho Choi

Survival and stability of bifidobacteria loaded in alginate poly-l-lysine microparticles

Bifidobacteria-loaded alginate microparticles were prepared by spraying a mixture of alginate and bifidobacteria culture using an air atomization method. Survival and stability of bifidobacteria loaded in microparticles were then evaluated. Survival of bifidobacteria from alginate poly-l-lysine microparticles was significantly increased when MRS broth or yeast extract was added in simulated intestinal fluid (pH 6.8). The number of bifidobacteria gradually increased for 8 h (10(8) cfu/g) and then reached about 10(9)-10(10) cfu/g when incubated over 12 h in intestinal fluid containing 0.5% yeast extract and 0.05% L-cysteine. The survival of bifidobacteria was highly dependent on the pH of the exposing media. When the bifidobacteria was immobilized with alginate or even poly-l-lysine treatment, the survival of bifidobacteria was highly enhanced in the low pH conditions (ca. > 10(8) vs. < 10(3) cfu/g). The stability of free flowing bifidobacteria-loaded alginate poly-l-lysine microparticles was significantly improved during storage at 4 degrees C in a refrigerator when compared to bifidobacteria cultures. The bifidobacteria-loaded alginate poly-l-lysine microparticles could be applied to various dairy products.

Purification of Peptide Components including Melittin from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis

Objectives : This study was conducted to carry out Purification of Melittin and other peptide components from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis Methods : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. Results : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. The fractions obtained from gel filtration chromatography was analyzed by using SDS-PAGE and propionic acid/urea polyacrylamide gel electrophoresis. The melittin obtained from the gel filtration contained residual amount of phospholipase and a protein with molecular weight of 6,000. The contaminating proteins were removed by the second gel filtration chromatography. Conclusion : Gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis are useful to separate peptide components including melittin from bee venom.

Component Analysis of Sweet BV and Clinical Trial on Antibody Titer and Allergic Reactions

Objectives : The aim of this study was to observe prevention of allergic reactions of Sweet Bee Venom (removing enzyme components from Bee Venom). Methods: Content analysis of Sweet Bee Venom and Bee Venom was rendered using HPLC method and characterization of Anti-Sweet Bee Venom in Rabbit Serum. Clinical observation was conducted for inducement of allergic responses to Sweet BV. Results : 1. Analyzing melittin content using HPLC, Sweet BV contained 34.9% more melittin than Bee venom pharmacopuncture at same concentration. 2. Observing chromatogram of HPLC, removal of the enzyme was successfully rendered on Sweet BV. 3. The anti-serum of Sweet BV showed high titers against melittin and bee venom and relatively low titer against phospholipase A2. 4. After conducting approximately 3,000 cases of Sweet BV administration, not a single case of generalized anaphylatic reaction occurred in clinical observation. 5. Mild compared to the bee venom pharmacopuncture, Sweet BV showed some acute hypersensitive reactions of edema, itchiness, and aching locally. 6. Sweet BV was administered on six patients with previous history of suffering from generalized acute hypersensitive reactions with the bee venom. None of the patients showed allergic reactions with Sweet BV, suggesting it can effectively prevent anaphylatic shock which may occur after the bee venom pharmacopuncture procedure. Conclusion : Summarizing above results, Sweet Bee Venom appears to be an effective measurement against allergic reactions from the bee venom pharmacopuncture especially against anaphylatic shock.

Component analysis of cultivated ginseng, cultivated wild ginseng, and wild ginseng and the change of ginsenoside components in the process of red ginseng

Objectives: The aim of this experiment is to provide an objective differentiation of cultivated ginseng, cultivated wild ginseng, and wild ginseng through component analysis, and to know the change of ginsenoside components in the process for making red ginseng. Methods: Comparative analysis of ginsenoside , Rc, Rd, Re, Rf, and from the cultivated ginseng 4 and 6 years, cultivated wild ginseng, and wild ginseng were conducted using High Performance Liquid Chromatography(hereafter HPLC). And the same analyses were conducted in the process of red ginseng. Results: 1. For content comparison of ginsenoside , Rc, Rd, Rf, and , wild ginseng showed high content, followed cultivated ginseng 4 and 6 years, cultivated wild ginseng showed low content than any other samples. 2. For content comparison of ginsenoside and Re, cultivated ginseng 4 years showed high content, followed wild ginseng and cultivated ginseng 6 years, cultivated wild ginseng showed low content than any other samples. 3. For content comparison of ginsenoside , wild ginseng and cultivated wild ginseng were only showed low content. 4. For content comparison of ginsenoside , cultivated wild ginseng was only showed low content. 5. In the process of red ginseng, ginsenoside , Rc, Rd, and were increased, and ginsenoside Re and were decreased in cultivated wild ginseng. 6. In the process of red ginseng, ginsenoside and were increased, and ginsenoside , Rc, and Re were decreased in cultivated ginseng 4 years. 7. In the process of red ginseng, ginsenoside , Rf and were increased, and ginsenoside Rc and Rd were decreased in cultivated ginseng 6 years. Conclusions: Distribution of ginsenoside contents to the cultivated ginseng, cultivated wild ginseng, and wild ginseng was similar and was not showed special characteristics between samples. And the change of ginsenoside to the process of red ginseng, cultivated ginseng and cultivated wild ginseng were showed different aspect.

Development of Reverse Transcriptase-polymerase Chain Reaction of fimA Gene to Detect Viable Salmonella in Milk

ABSTRACT Rapid detection of viable Salmonella in pasteurized milk is important to protect public health from foodpoisoning. Reverse transcriptase-polymerase chain reaction(RT-PCR) is recognized as a molecular geneticalmethod to differentiate between live and dead bacteria. The RT-PCR in this study was designed to detect spe-cifically viable Salmonella in milk by using the primers whose nucleotide sequences were determined based on fimA gene which encodes the subunit of type 1 fimbriae. Treatment of RNA preparation with RNase-freeDNase was adequate enough to destroy DNA, which may otherwise be amplified in the RT-PCR. Sevenstrains of Salmonella were detected in the RT-PCR but Escherichia coli , Shigella sonnei , Citrobacter freundii ,and Klebsiella pneumoniae were not. 10 7 /m and 10 6 /m of dead Salmonella which were heat-treated in milkwere detectable by using the RT-PCR but 10 5 ~10/m of the dead bacteria were not. The sensitivity of theRT-PCR in detecting viable Salmonella was 100 cells/m .

Isolation and Characterization of a 32-kDa Fibrinolytic Enzyme (FE-32kDa) from Gloydius blomhoffii siniticus Venom: Fibrinolytic Enzyme from Gloydius blomhoffii siniticus Venom.

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as Fe2+ and Hg2+ inhibited the fibrin hydrolysis completely, but Zn2+ enhanced it. FE-32kDa hydrolyzed α-chain but did not hydrolyze β-chain and γ-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of 2 μg. Conclusions: FE-32kDa is a α-fibrin(ogen)olytic metalloprotease that requires Zn2+ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

Detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic Assay

Korean native honey (KNH) is much more expensive than European honey (EH) in Korea, because KNH is a favored honey which is produced less than EH. Food fraud of KNH has drawn attention of the government office concerned, which is in need of a method to differentiate between KNH and EH which are produced by the Asiatic honeybee, Apis cerana and the European honeybee, Apis mellifera, respectively. A method to discriminate KNH and EH was established by using duplex polymerase chain reaction (PCR) in this study. Immunochromatographic assay (IC) was examined to analyze the duplex PCR product. The DNA sequences of primers for the duplex PCR were determined by comparing cytochrome C oxidase genes of the two honey bee species. Chelex resin method was more efficient in extracting genomic DNA from honey than the other two procedures of commercial kits. The duplex PCR amplifying DNA of 133 bp were more sensitive than that amplifying DNA of 206 bp in detecting EH in the honey mixture of KNH and EH. Agarose gel electrophoresis and IC detected the DNA of 133 bp at the ratios of down to 1% and 5% EH in the honey mixture, respectively and also revealed that several KNH products distributed by internet shopping sites were actually EH. In conclusion, the duplex PCR with subsequent IC could also discriminate between KNH and EH and save time and labor.

Isolation from Gloydius blomhoffii siniticus Venom of a Fibrin(ogen)olytic Enzyme Consisting of Two Heterogenous Polypeptides

Objective: This study was undertaken to isolate a fibrin(ogen)olytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate the enzymatic characteristics and hemorrhagic activity of the isolated enzyme as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were determined by using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrin(ogen)olytic enzyme with the molecular weight of 27 kDa (FE-27kDa) isolated from G. b. siniticus venom consisted of two heterogenous disulfide bond-linked polypeptides with the molecular weights of 15 kDa and 18 kDa. When more than 20 μg of FE-27kDa was applied on the fibrin plate, fibrinolysis zone was formed as indicating its fibrinolytic activity. The fibrinolytic activity was inhibited completely by phenylmethanesulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) and partially by thiothreitol and cysteine. Metal ions such as Hg2+ and Fe2+ inhibited the fibrinolytic activity completely, but Mn2+ did not. FE-27kDa preferentially hydrolyzed α- chain of fibrinogen and slowly hydrolyzed β- chain, but did not hydrolyze γ- chain. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into polypeptides with molecular weights of more than 45 kDa. A dosage of more than 10 μg of FE- 27kDa per mouse was required to induce hemorrhage beneath the skin. Conclusion: FE-27kDa was a serine proteinase consisting of two heterogeneous polypeptides, hydrolyzed fibrin, fibrinogen, and gelatin, and caused hemorrhage beneath the skin of mouse. This study suggests that the potential of FE-27kDa as pharmacopuncture agent should be limited due to low fibrinolytic activity and a possible side effect of hemorrhage.

Purification and Characterization of a Fibrinolytic Enzyme from Snake Venom of Macrovipera Lebetina Turanica

Objectives: Fibrinolytic enzyme preparations were isolated from the snake venom of Macrovipera lebetica turanica in this study. Methods: The purity of the preparations was determined using SDS-PAGE and the enzymic characteristics of the purified fibrinolytic enzyme were determined. Results: 1. All of the two preparations with fibrinolytic activity obtained from the snake venom of M. l. turanicat contained the major polypeptide with the molecular weight of 27,500. One of the preparation showed purified fibrinolytic enzyme. 2. The purified fibrinolytic enzyme hydrolyzed -chain of fibrinogen faster than -chain but not -chain. 3. The fibrinolytic activity was inhibited completely by EDTA, EGTA, 1,10-phenanthroline, and dithiothreitol. 4. The fibrinolytic activity was inhibited completely by calcium chloride, iron(III) chloride, mercuric chloride, and cobalt (II) chloride. 5. The fibrinolysis zone formed after addition of zinc sulfate was smaller but clearer than the control. Conclusions: These results suggested that the fibrinolytic enzyme purifed from the snake venom of M. l turanica was a metalloprotease containing dithiol group.

Identification of Fel ursi and Cattle and Pig Bile Juices by speciesspecific PCR and PCR-RFLP

Objective : This study developed species-specific PCR and PCR-RFLP to detect the adulteration of Fel ursi products with cattle and pig bile juices. Methods : All the primers for PCR and PCR-RFLP in this study were designed based on nucleotide sequences of cytochrome b genes in the mitochondria. Results : The species-specific PCR amplified a DNA fragment of 214, 214, 295, and 167 bp from Fel ursi product, bear fur, cattle bile juice, and pig bile juice, respectively. The survey using the speciesspecific PCR indicated that some of commercial Fel ursi products were adulterated with cattle and pig bile juices. PCR-RFLP using the restriction endonucleases, HaeIII and HinfI enabled differentiation among Fel ursi product, cattle bile juice, and pig bile juice. Bear furs from two animals showed variations in PCR-RFLP patterns with HaeIII. Discussion : The detection methods of the species-specific PCR and PCR-RFLP could be useful in eliminating adulterated Fel ursi products from the market.