• Korean Journal of Environmental Biology
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• v.35 no.4
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• pp.694-705
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• 2017

The aim of this study was to isolate and identify marine bacterium with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and to purify the anti-MRSA compound, as well as to determine its activity and synergistic effects. Among the marine bacteria isolated in this study, the YJ-1 isolate had the strongest anti-MRSA activity. The YJ-1 isolate was identified on the basis of its biochemical characteristics and an analysis of 16S rRNA gene sequences. The YJ-1 isolate showed over 99.2% homology with Pseudomonas stutzeri, and was designated as a Pseudomonas sp. YJ-1. The optimal culture conditions were $25^{\circ}C$ and initial pH 7.0. For the purification of the anti-MRSA compounds, the YJ-1 was cultured in Pa PES-II medium, and the culture filtrates were extracted by ethyl acetate, hexane, and 80% MeOH. The 80% MeOH fraction was separated by a $C_{18}$ ODS column, silica gel chromatography and a reverse phase HPLC, to yield three anti-MRSA agents, the MR1, MR2, and MR3 compounds. When the MR1 compound of $250{\mu}g\;mL^{-1}$ concentration was applied to the MRSA cells, over 95% of bacterial cells was killed within 48 hr. Compared with vancomycin and ampicillin, the MR1 compound showed significant anti-MRSA activity. In addition, the anti-MRSA activity was increased by dose and time dependent manners. Furthermore, the combination of an MR1 compound with vancomycin produced a more rapid decrease in the MRSA cells than did the MR1 compound alone. Taken together, our results suggest that the Pseudomonas sp. YJ-1 and its anti-MRSA compounds could be employed as a natural antibacterial agent in MRSA infections.

• Journal of Life Science
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• v.31 no.1
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• pp.90-99
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• 2021

In this study, to reduce the production cost of poly(3-hydroxyalkanoates) (PHA), optimal cell growth and PHA biosynthesis conditions of the isolated strain Pseudomonas sp. EML8 were established using waste frying oil (WFO) as the cheap carbon source. Gas chromatography (GC) and GC mass spectrometry analysis of the medium-chain-length PHA (mcl-PHAWFO) obtained by Pseudomonas sp. EML8 of WFO indicated that it was composed of 7.28 mol% 3-hydrxoyhexanoate, 39.04 mol% 3-hydroxyoctanoate, 37.11 mol% 3-hydroxydecanoate, and 16.58 mol% 3-hydroxvdodecanoate monomers. When Pseudomonas sp. EML8 were culture in flask, the maximum dry cell weight (DCW) and the mcl-PHAWFO yield (g/l) were showed under WFO (20 g/l), (NH4)2SO4 (0.5 g/l), pH 7, and 25℃ culture conditions. Based on this, the highest DCW, mcl-PHAWFO content, and mcl-PHAWFO yield from 3-l-jar fermentation was obtained after 48 hr. Similar results were obtained using 20 g/l of fresh frying oil (FFO) as a control carbon source. In this case, the DCW, the mcl-PHAFFO content, and the mcl-PHAFFO yields were 2.7 g/l, 62 wt%, and 1.6 g/l, respectively. Gel permeation chromatography analysis confirmed the average molecular weight of the mcl-PHAWFO and mcl-PHAFFO to be between 165-175 kDa. Thermogravimetric analysis showed decomposition temperature values of 260℃ and 274.7℃ for mcl-PHAWFO and mcl-PHAFFO, respectively. In conclusion, Pseudomonas sp. EML8 and WFO could be suggested as a new candidate and substrate for the industrial production of PHA.

• Journal of Applied Biological Chemistry
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• v.65 no.4
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• pp.421-427
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• 2022

In this study, Jeju Tatary buckwheat tea's chemical composition and physiological activities were compared according to the leaching temperature (60, 80, 100 ℃). As the leaching temperature is increased, the degree of browning is induced. However, there was no significant change in pH. The total polyphenol content was higher at 80 ℃ than at 60 ℃ leaching temperature, but significantly decreased at 100 ℃ leaching temperature (60 ℃: 17.06 mg GA/g, 80 ℃: 20.09 mg GA/g, 100 ℃ :18.45 mg GA/g). There were high content of flavonoid and rutin as the leaching temperature increased. Consistently, 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging activity and tyrosinase inhibitory activity were significantly higher with increasing temperature (DPPH % inhibition: 60 ℃: 41.88%, 80 ℃: 46.01%, 100 ℃: 46.80%/tyrosinase inhibitory activity: 60 ℃: 9.38%, 80 ℃: 22.94%, 100 ℃: 28.17%). However, there was no significant difference in DPPH radical scavenging activity between 80 and 100 ℃. A cytotoxicity test was performed by treating with Jeju Tatary buckwheat extract into mouse macrophage cells (Raw264.7). 100 and 200 ㎍/mL treatment (100 ℃ extract) were significantly upregulated the survival rate, but there was no significant difference in other concentrations. Collectively, most of the bioactive components, antioxidant activity, and tyrosinase inhibitory activity were induced as the leaching temperature increased. However, the content of polyphenols which are known to have antioxidant activity, was significantly reduced at 100 ℃ leaching temperature. Several reports have demonstrated that leaching at too high temperature lowered the overall acceptability, so the optimal leaching condition of Tatary Buckwheat is 80 ℃, 5 min in this study.

• Journal of Food Hygiene and Safety
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• v.38 no.6
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• pp.449-456
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• 2023

In this study, we developed Rapid Enrichment Broth for Vibrio parahaemolyticus (REB-V), a broth capable enriching V. parahaemolyticus from 10⁰ CFU/mL to 10⁶ CFU/mL within 6 hours, which greatly facilitates the rapid detection of V. parahaemolyticus. Using a modified Gompertz model and response surface methodology, we optimized supplement sources to rapidly enrich V. parahaemolyticus. The addition of 0.003 g/10 mL of D-(+)-mannose, 0.002 g/10 mL of L-valine, and 0.002 g/10 mL of magnesium sulfate to 2% (w/v) NaCl BPW was the most effective combination of V. parahaemolyticus enrichment. Optimal V. parahaemolyticus culture conditions using REB-V were at pH 7.84 and 37℃. To confirm REB-V culture efficiency compared to 2% (w/v) NaCl BPW, we assessed the amount of enrichment achieved in 7 hours in each medium and extracted DNA samples from each culture every hour. Real-time PCR was performed using the extracted DNA to verify the applicability of this REB-V culture method to molecular diagnosis. V. parahaemolyticus was enriched to 5.452±0.151 Log CFU/mL in 2% (w/v) NaCl BPW in 7 hours, while in REB-V, it reached 7.831±0.323 Log CFU/mL. This confirmed that REB-V enriched V. parahaemolyticus to more than 10⁶ CFU/mL within 6 hours. The enrichment rate of REB-V was faster than that of 2% (w/v) NaCl BPW, and the amount of enrichment within the same time was greater than that of 2% (w/v) NaCl BPW, indicating that REB-V exhibits excellent enrichment efficiency.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.13 no.2
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• pp.121-128
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• 2008

As an aquatic ecotoxicity test method, a bioassay using the inhibition of sporualtion of the green macroalga, Ulva pertusa, has been developed. Optimal test conditions determined for photon irradiance, pH, salinity and temperature were $100\;{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, $7{\sim}9$, $25{\sim}35\;psu$ and $15{\sim}20^{\circ}C$, respectively. The validity of the test endpoint was evaluated by assessing the toxicity of four metals (Cd, Cu, Pb, Zn) and elutriates of sewage or waste sludge collected from 9 different locations. When the metals were assayed, the $EC_{50}$ values indicated the following toxicity rankings: Cu ($0.062\;mg{\cdot}L^{-1}$) > Cd ($0.208\;mg{\cdot}L^{-1}$) > Pb ($0.718\;mg{\cdot}L^{-1}$) > Zn ($0.776\;mg{\cdot}L^{-1}$). When compared with other commonly used bioassays of metal pollution listed on US ECOTOX database, the sporualtion test proved to be the most sensitive. Ulva sporulation was significantly inhibited in all elutriates with the greatest and least effects observed in elutriates of sludge from industrial waste ($EC_{50}=6.78%$) and filtration bed ($EC_{50}=15.0%$), respectively. The results of the Spearman rank correlation analysis for $EC_{50}$ data versus the concentrations of toxicants in the sludge presented a significant correlation between toxicity and four heavy metals(Cd, Cu, Pb, Zn). The method described here is sensitive to toxicants, simple to use, easy to interpret and economical. It is also easy to procure samples and maintain cultures. The present method would therefore probably make a useful assessment of aquatic toxicity of a wide range of toxicants. In addition, the genus Ulva has a wide geographical distribution and species have similar reproductive processes, so the test method would have a potential application worldwide.

• Journal of Food Hygiene and Safety
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• v.39 no.2
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• pp.72-82
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• 2024

This study aimed to develop a scientifically and systematically standardized xylooligosaccharide analytical method that can be applied to products with various formulations. The analysis method was conducted using HPLC with Cadenza C₁₈ column, involving pre-column derivatization with 1-phenyl-3-methyl-5-pyrazoline (PMP) and UV detection at 254 nm. The xylooligosaccharide content was analyzed by converting xylooligosaccharide into xylose through acid hydrolysis. The pre-treated methods were compared and evaluated by varying sonication time, acid hydrolysis time, and concentration. Optimal equipment conditions were achieved with a mobile phase consisting of 20 mM potassium phosphate buffer (pH 6)-acetonitrile (78:22, v/v) through isocratic elution at a flow rate of 0.5 mL/min (254 nm). Furthermore, we validated the advanced standardized analysis method to support the suitability of the proposed analytical procedure such as specificity, linearity, detection limits (LOD), quantitative limits (LOQ), accuracy, and precision. The standardized analysis method is now in use for monitoring relevant health-functional food products available in the market. Our results have demonstrated that the standardized analysis method is expected to enhance the reliability of quality control for healthy functional foods containing xylooligosaccharide.

• Journal of Microbiology
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• v.44 no.6
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.71-78
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• 1983

The number of aerobic free-living nitrogen fixation bacteria and factors in soil at different stands covered with Pinus rigida, Quercus acutissima and Zoysia japonica in Cheongju area were investigated from Feb. to Sept 1981. 1. The numbers of $N_2-fixation$ bacteria, according to the seasonal changes, increased gradually from winter to spring and summer. But the growth pattern revealed some differences in accordance with plant cover stands : the numbers increased abruptly in May at Pinus, May-June at Quercus and Apr. May at Zoysia stand. The pick of numbers represented in Aug. Sept, at Pinus, Jul-Aug. at Quercus and May-Jun. at Zoysia stand, respectively. 2. The interrelationship between the monthly changes of enviotnmental factors and numbers of $N_2-fixation$ bacteria at different stands, mainly depends upon the soil temperature than other soil factors (r=0.71-0.84). The numbers of $N_2-fixation$ bacteria may increase 5-7 times according to increase $10^{\circ}C$ of soil temperature, and optimal range was $20{\sim}30^{\circ}C$ for growth. Equation of the interrelation between soil temperature and numbers could be stated as follows : log y=ax+b. 3. In the case of high soil temperature, the bacterial numbers presented high level in drought periods. Therefore, the $N_2-fixation$ bacterial species in these soil seem to consist of resistant to desication. 4. The influence of soil organic matter for growth of $N_2-fixation$ bacteria indicated low conrelation. The reason may seen the content of organic matter in these soil existed abundantly above the quantities of limitation for growth. 5. In artifical gradients, the $N_2-fixation$ bacteria were predominated at $20{\sim}30^{\circ}C$ same as natural condition, pH7-8, and 20-30% of soil water contents. 6. The vertical distribution of bacteria marked decreasing trends from surface to lower layers, and the decreasing degree was shown well in Zoysia, Quercus and Pinus stand in order. But in the trees, the numbers increased at 30cm layer estimated the region of root than 20cm layer. 7. Both catalase megative and positive group of $N_2-fixation$ bacteria in soil increased according to the rise of the soil temperature. Catalase positive group was revealed as dominant group in winter, and catalase negative group revealed in summer. The change of dominant pattern was shown during Feb. to Apr.

• Clean Technology
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• v.22 no.3
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• pp.175-180
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• 2016

Tyrosinases catalyze the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to o-quinone (diphenolase activity), which are mainly involved in the modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (DOPA) and DOPA/DOPAquinone-derived intermolecular cross-linking. Previously, we obtained a slightly acidic and cold-active tyrosinase, tyrosinase-CNK, by our recombinant protein approach. The enzyme showed optimal activity at pH 6.0 and 20 ℃ with an abnormally high monophenolase/diphenolase activity ratio and still had approximately 50% activity compared with the highest activity even in ice water. Here, we investigated reaction stability of the recombinant tyrosinase-CNK as a psychrophilic enzyme. The enzyme showed remarkable thermal stability at 0 ℃ and the activity was well conserved in repeated freeze-thaw cycles. Although water-miscible organic solvent as reaction media caused the activity decrease of tyrosinase-CNK as expected, the enzyme activity was not additionally decreased with increased concentration in organic solvents such as ethanol and acetonitrile. Also, the enzyme showed high salt tolerance in chaotropic salts. It was remarkably considered that 2⁺ metal ions might inhibit the incorporation of Cu²⁺ into the active site. We expect that these results could be used to design tyrosinase-mediated enzymatic reaction at low temperature for the production of catechols through minimizing unwanted self-oxidation and enzyme inactivation.

• Food Science of Animal Resources
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• v.37 no.5
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• pp.735-742
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• 2017

This study was conducted to isolate and characterize Paenibacillus sp. MBT213 possessing ${\beta}$-glucosidase activity from raw milk, and examine the enzymatic capacity on the hydrolysis of a major ginsenoside ($Rb_1$). Strain MBT213 was found to have a high hydrolytic ability on ginsenoside $Rb_1$ by Esculin Iron Agar test. 16S rDNA analysis revealed that MBT213 was Paenibacillu sp. Crude enzyme of MBT213 strain exhibited high conversion capacity on ginsenoside $Rb_1$ into ginsenoside Rd proven by TLC and HPLC analyses. The API ZYM kit confirmed that Paenibacillu sp. MBT213 exerted higher ${\beta}$-glucosidase and ${\beta}$-galactosidase activity than other strains. Optimum pH and temperature for crude enzyme were found at 7.0 and $35^{\circ}C$ in hydrolysis of ginsenoside $Rb_1$. After 10 d of optimal reaction conditions for the crude enzyme, ginsenoside $Rb_1$ fully converted to ginsenoside Rd. Ginseng roots (20%) were fermented for 14 d, and analyzed by HPLC showed that amount of ginsenoside $Rb_1$ significantly decreased, while that of ginsenoside Rd was significantly increased. The study confirmed that the ${\beta}$-glucosidase produced by Paenibacillus sp. MBT213 can hydrolyze the major ginsenoside $Rb_1$ and convert to Rd during fermentation of the ginseng. The ${\beta}$-glucosidase activity of this novel Paenibacillus sp. MBT213 strain may be utilized in development of variety of health foods, dairy foods and pharmaceutical products.