• Korean Journal of Microbiology
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• v.41 no.3
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• pp.216-224
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• 2005

Chromatography has now been used successfully to provide the requisite purity for human plasma-derived biop-harmaceuticals such as coagulation factors and immunoglobulins. Recently, increasing attention has been focused on establishing efficient cleaning procedures to prevent potential contamination by microorganisms as well as carry-over contamination from batch to batch. The purpose of present study was to develop a cleaning validation system for the assurance of virus removal and/or inactivation during chromatography process. In order to establish an assay system for the validation of virus clearance during chromatography cleaning process, a quantitative real-time PCR method for porcine parvovirus(PPV) was developed, since PPV, a model virus for human parvovirus B19, has a high resistance to a range of physico-chemical treatment. Specific primers for amplification of PPV DNA was selected, and PPV DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1.5 $TCID_{50}/ml$. The established real-time PCR assay was successfully applied to the validation of PPV removal and cleaning during SP-Sepharose cation chromatography for thrombin purification and Q-Sepharose anion chromatography for factor VIII purification. The comparative results obtained by real-time PCR assay and infectivity titrations suggested that the real-time PCR assay could be a useful method for chromatography cleaning validation and that it could have an additive effect on the interpretation and evaluation of virus clearance during the virus removal process.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Korean Journal of Microbiology
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• v.49 no.4
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• pp.424-428
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• 2013

To identify risk factors for Legionella contamination, water quality variables routinely measured in examination of natural and city waters were meta-analyzed for significance of correlation to Legionella incidences. For evaluation of abundance of Escherichia coli as a risk factor, which is currently used as an indicator of Legionella contamination in an official guideline in Korea, odds ratio (OR) of above-cutoff total coliform counts for Legionella presence/absence was used as the effect size in the meta-analysis. The OR was estimated as 1.05 (0.36-3.12, 95% CI), and the probability of having identical odds reached 0.92. Also, ORs from individual studies showed significant heterogeneity (P=0.008), which contributed to 63% of total variance of the ORs. In the case of heterotrophic plate count (HPC), the OR for Legionella presence/absence was 2.72 (2.04-3.63) with highly significant deviation from identical odds (P<0.0001). ORs from different studies were seemingly homogeneous ($Q_{df=8}$=12.7, P=0.12). Turbidity and concentrations of chlorine, iron ion and cupper ion were other routine variables that could be considered as risk factors. However, statistical measures from different studies were not uniform enough to develop an appropriate effect size while the number of studies reporting the variables was also small (3-5). In conclusion, HPC appeared to be appropriate as indicator of Legionella contamination, rather than fecal bacteria contamination. HPC may imply abundance of habitats (amoebas and biofilms) of Legionella in water. This result warrants further studies for standardizing protocols and cutoff values to infer Legionella risks from HPC.

• Korean Journal of Microbiology
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• v.49 no.4
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• pp.360-368
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• 2013

Recently interactive water fountains are gaining popularity in making public facilities in South Korea. The total number of interactive fountains is rapidly growing at the rate of >50% annually. In this study, we performed quantitative microbial risk assessment to estimate infection risks in children by Legionella spp. while playing in interactive fountains. The exposure dose for a given concentration of Legionella in water was calculated using water-aerosol partition rate of Legionella, exposure duration, inhalation rate, and deposit rate of aerosols in the lungs following inhalation. The dose was converted to infection risk by using the dose-response function developed for L. pneumophila. High weight and/or old children, i.e., 12-year children, running around in fountains were the highest risk group by showing >0.05 infection probability for fountain waters containing ${\geq}10^4$ CFU/L Legionella. The result supported the current guideline by Korea Centers for Disease Control and Prevention, which permits use of water with < $10^3$ CFU/L Legionella cells for all purposes. However, the results still warrant further evaluation of the guideline to accommodate risks for children because the dose-response relationship in the model was developed for healthy adults. Further risk assessment studies need to be conducted by employing dose-response model for children who generally carries weaker immune system than adults.

• Journal of the Korean Recycled Construction Resources Institute
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• v.8 no.1
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• pp.97-104
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• 2020

The present study conducted mock-up tests under the simulated sewage environments to examine the practical significance and limitation of coating materials that were previously developed on the basis of the bacterial glycocalix as a protection of concrete structures exposed to microbiological and sulphate attacks. The variations of the compressive strength and mass of the concrete due to the sulphate attack were measured using cylinder specimens. The bacteria growth and glycocalix formulation were calculated from the samples extracted from the sewage pipes. The next generation sequencing analysis was also conducted for environmental damage assessment due to the use of Rhodobacter capsulatus in the simulated sewage environments. The mock-up tests revealed that the developed coating materials have a good potential in resisting the sulphate attack, indicating no reduction on compressive strength and mass of the coated concrete under the sewage environment. At the age of 91 days, the concentrations of viable bacteria and glycocalix measured from the hardened coating materials were 1.4×10⁴cell/mL and 67.5mg/㎤, respectively. Moreover, harmful strains were not observed in the sewage water including glycocalix-coated concrete pipes. This implies that Rhodobacter capsulatus used in the coating materials does not influence negatively the microorganism cluster in the sewage environments.

• Journal of Applied Biological Chemistry
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• v.58 no.1
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• pp.55-60
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• 2015

To secure the microbial safety of frozen strawberries, they were treated with the combined solution of aqueous chlorine dioxide and acetic acid prior to freezing and the effects of different freezing methods (at $-20^{\circ}C$ in a freezer, at $-70^{\circ}C$ in a gas nitrogen convection chamber, and at $-196^{\circ}C$ in liquid nitrogen) on the quality changes of strawberries were examined. Regarding the color of frozen strawberries, there were negligible changes among freezing treatments. In contrast, vitamin C content and sensory evaluation scores of strawberries frozen at $-70^{\circ}C$ were the highest among the samples. Drip loss of strawberries frozen at $-70^{\circ}C$ was the lowest as 14.39%, compared with strawberries frozen at -20 and $-196^{\circ}C$. In addition, the effects of combined treatment of 50 ppm chlorine dioxide and 1% acetic acid on the microbial growth in frozen strawberries were investigated, and the populations of preexisting microorganisms in the frozen strawberries were not detected by the combined pre-treatment. These results suggest that rapid freezing at $-70^{\circ}C$ using a gas nitrogen convection chamber is an appropriate freezing method for preserving quality of strawberries, and as a pre-freezing treatment, the combined treatment of aqueous chlorine dioxide and acetic acid can be effective for improving microbiological safety of frozen strawberries.

• Journal of dental hygiene science
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• v.18 no.5
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• pp.296-304
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• 2018

The purpose of this study was to propose a method for the effective management of toothbrush contamination. Toothbrush microbial contamination was analyzed according to the duration of toothbrush use, frequency of toothbrush use per day, and toothbrush storage location. We also analyzed the microbial reduction effect of vinegar, antimicrobial mouth rinse, bamboo salt, and baking soda, which are sterilization materials that can be easily used every day. We collected 45 toothbrushes from university dormitories from May to June 2018. To determine the degree of microbiological contamination with general bacteria, coliform bacteria, and Staphylococcus aureus, bristle samples were cultured at $36^{\circ}C$ for 24 hours using 3M$^{TM}$ Petrifilm plates and then measured based on Petrifilm evaluation criteria. Toothbrush microorganisms were analyzed according to the duration of use, frequency of use per day, storage location, and effect of each sterilization material. General bacteria, coliforms, and S. aureus contamination increased with frequency and duration of use (p<0.05). In particular, S. aureus showed a statistically significant increase to 36.15 CFU/ml after 1 month, 504.23 CFU/ml after 2 months, and 2,386.67 CFU/ml after 3 months (p<0.05). We found that 1% vinegar was the most effective substance for reducing general bacteria, coliforms, and S. aureus. In addition, 1% antimicrobial mouth rinse solution applied for 5 minutes was the most effective in reducing S. aureus. It is crucial to recognize the importance of toothbrush care and store toothbrushes in a dry place and replace them periodically. We recommend use of vinegar and antimicrobial mouth rinse solution to disinfect toothbrushes. These should be applied as a 1% solution for at least 1 minute. Proper care of toothbrushes is important in maintaining oral health as well as overall health. Instructions on toothbrush care should be given when teaching children or adults how to brush teeth.

• Journal of Environmental Health Sciences
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• v.34 no.1
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• pp.86-94
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• 2008

Lake Shiwha, an artificial lake located near metropolitan Seoul, offers a unique water environment and has been suspected to have high levels of chemical and microbiological contaminations. Lake Shiwha was originally connected to the sea but currently has four major surface water inputs from agricultural, municipal, industrial areas and in addition an occasional inflow from the sea. The objectives of this study are to investigate the relative contribution of microbial contaminants from each of the inflowing surface waters and to identify appropriate microbial indicator organisms in this unique water environment. We measured the levels of microbial contaminations in the four inflowing surface waters. A number of microbial indicator organisms including total coliform (TC), fecal coliform (FC), E. coli, Enterococci, somatic and male-specific coliphages were analyzed. Bacterial indicator microorganisms were detected and quantified by the $Colilert^{(R)},\;Enterolert^{(R)}$ kit. Surface water (50 l) was sampled by $ViroCap^{TM}\;5"$ cartridge filters and analyzed by the single agar layer method for detecting coliphages. The concentrations of TC, FC, E. coli, and Enterococci were 1543 CFU/100 ml${\sim}1.99{\times}10^6$ CFU/100 ml, 0 CFU/100 ml${\sim}202$ CFU/100ml, 0 CFU/100 ml${\sim}1.80{\sim}10^5$ CFU/100ml, 74 CFU/100 ml${\sim}3408$ CFU/100 ml, respectively. The male-specific and somatic coliphages were detected in three different inflowing surface waters. Isolated E. coli and Enterococci strains were further analyzed by 16s rDNA amplification and subsequent phylogenetic analysis from Jungwang-chun, Ansan-chun, Banwol-chun and penstock of inflowing surface water. Our results indicated that the concentrations of different fecal indicator microorganisms might not be highly correlated with each other. Multiple microbial indicator organisms should be used for monitoring microbial contamination and microbial source tracking methods.

• Korean Journal of Food Science and Technology
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• v.49 no.2
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• pp.117-122
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• 2017

The aim of this study was to evaluate the quality characteristics of fresh red paprika treated by electron-beam irradiation at quarantine doses. The initial microbial loads were low with $10^4$ and $10^2$ colony-forming units/g for total aerobic bacteria and coliform, respectively; however, a dose of 1 kGy resulted in load reduction of 1 log cycle. A dose level of more than 1 kGy caused significant decreases in the hardness and carotenoid content parameters. An applied dose level of less than 2 kGy did not affect vitamin C content; however, a decrease of 87-90% was observed after 40-day storage. Samples treated with 2 kGy showed significantly lower acceptance compared to the control, with lower sensory evaluation scores for color and texture. Therefore, e-beam irradiation at dose range of 0.4 and 1 kGy was found to be appropriate for quarantine applications for microbiological control and quality maintenance of paprika.

• Korean Journal of Food Science and Technology
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• v.24 no.4
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• pp.353-360
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• 1992

This study was carried out to investigate the changes of quality in yogurt during delivery and storage. To do this, microbiological, physico-chemical and sensory properties in stirred yogurt were studied at different temperatures $(10^{\circ}C$ & $20^{\circ}C)$ and shakings (100 rpm & 200 rpm for 30 min). Titratable acidity and TCA soluble nitrogen in yogurt were increased in accordance with increase of temperature and time, while pH, lactose and lactic acid bacteria tended to be decreased. Interestingly enough, shaking conditions almost did not influence these studies. In sensory evaluation it showed that acidity was strong and sweetness was weak as increasing the storage temperature and time, but there were no significant differences until 10 days at $10^{\circ}C$ and 3 days at $20^{\circ}C$. Off-flavor was developed after 12 days at $10^{\circ}C$ with only 200 rpm shaking and after 6 days at $20^{\circ}C$ in both shaking conditions. These yogurts were not suitable for consumer. Viscosity in the yogurt became high as the temperature and time were increased. Viscosity was lower in yogurt shaked at 200 rpm than in non-shaked yogurt. Finally, this study indicates that temperature and shaking during delivery of yogurt may be critical in keeping quality of yogurt.