• 한국생물공학회:학술대회논문집
• /
• 2003.04a
• /
• pp.40-44
• /
• 2003

Soil-borne infectious disease including Pythium aphanidermatum and Rhizoctonia solani causes severe damage to plants, such as cucumber. This soil-borne infectious disease was not controlled effectively by chemical pesticide. Since these diseases spread through the soil, chemical agents are usually ineffective. Instead, biological control, including antagonistic microbe can be used as a preferred control method. An efficient method was developed to select an antagonistic strain to be used as a biological control agent strain. In this new method, surface tension reduction potential of an isolate was included in the ‘decision factor’ in addition to the other factors, such as growth rate, and pathogen inhibition rate. Considering these 3 decision factors by a statistical method, an isolate from soil was selected and was identified as Bacillus sp. GB16. In the pot test, this strain showed the best performance among the isolated strains. The lowest disease incidence rate and fastest seed growth was observed when Bacillus sp. GB16 was used. Therefore this strain was considered as plant growth promoting rhizobacteria (PGPR). The action of surface tension reducing component was deduced as the enhancement of wetting, spreading, and residing of antagonistic strain in the rhizosphere. This result showed that new selection method was significantly effective in selecting the best antagonistic strain for biological control of soil-borne infectious plant pathogen. The antifungal substances against P. aphanidermatum and R. solani were partially purified from the culture filtrates of Bacillus sp. GB16. In this study, lipopeptide possessing antifungal activity was isolated from Bacillus sp. GB16 cultures by various purification procedures and was identified as a surfactin-like lipopeptide based on the Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), high performance liquid chromatography mass spectroscopy (HPLC-MS), and quadrupole time-of-flight (Q-TOF) ESI-MS/MS data. The lipopeptide, named GB16-BS, completely inhibited the growth of Pythium aphanidermatum, Rhizoctonia solani, Penicillium sp., and Botrytis cineria at concentrations of 10 and 50 mg/L, respectively. A novel method to prevent the foaming and to provide oxygen was developed. During the production of surface active agent, such as lipopeptide (surfactin), large amount of foam was produced by aeration. This resulted in the carryover of cells to the outside of the fermentor, which leads to the significant loss of cells. Instead of using cell-toxic antifoaming agents, low amount of hydrogen peroxide was added. Catalase produced by cells converted hydrogen peroxide into oxygen and water. Also addition of corn oil as an oxygen vector as well as antifoaming agent was attempted. In addition, Ca-stearate, a metal soap, was added to enhance the antifoam activity of com oil. These methods could prevent the foaming significantly and maintained high dissolved oxygen in spite of lower aeration and agitation. Using these methods, high cell density, could be achieved with increased lipopeptide productivity. In conclusion to produce an effective biological control agent for soil-borne infectious disease, following strategies were attempted i) effective screening of antagonist by including surface tension as an important decision factor ii) identification of antifungal compound produced from the isolated strain iii) novel oxygenation by $H_2O_2-catalase$ with vegetable oil for antifungal lipopeptide production.

• Journal of Life Science
• /
• v.32 no.3
• /
• pp.202-209
• /
• 2022

The green alga Chlamydomonas reinhardtii, a unicellular haploid eukaryote, has long been used by researchers and industries as a cell factory to produce high value-added microalgae substances using genetic modification. Microalga K01, presumed to be Chlamydomonas, was isolated from 12 freshwater samples from the Chungcheong and Jeolla regions to replace C. reinhardtii, an introduced species currently used in most basic and industrial research. The isolated K01 strain was identified as C. reinhardtii through morphological and phylogenetic studies of the 18S rDNA gene sequence (NCBI accession number KC166137). The growth and lipid content of the isolated C. reinhardtii K01 were compared with three wild and four mutant strains in TAP medium, and it was found that the K01 strain could produce 1.74×10⁷ cells/ml by the third day of culture. The growth rate of C. reinhardtii K01 was 1.5 times faster than UTEX2244, which showed the highest number of cells (1.20×10⁷ cells/ml) among the compared strains. The lipid content of the isolated C. reinhardtii K01 (20.67%) was similar to those of the wild strains, although the fatty acid oleate C18:1 was not detected in the isolated strain but was identified in the seven others. The cell density of the isolated strain increased to 0.87 g/l during a six-day culture in BG11 medium, where nitrate (NaNO₃) was introduced as a nitrogen source, while the seven acquired strains showed almost no cell proliferation.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.32 no.3
• /
• pp.199-206
• /
• 2010

This study investigated the effects of strontium on osteoblastic phenotypes of cultured human periostealderived cells. Periosteal tissues were harvested from mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the periostealderived cells were further cultured for 28 days in an osteogenic induction DMEM medium supplemented with fetal bovine serum, ascorbic acid 2-phosphate, dexamethasone and at a density of $3{\times}10^4$ cells/well in a 6-well plate. In this culture medium, strontium at different concentrations (1, 5, 10, and 100 ${\mu}g$/mL) was added. The medium was changed every 3 days during the incubation period. We examined the cellular proliferation, histochemical detection and biochemical measurements of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and von Kossa staining and calcium contents in the periostealderived cells. Cell proliferation was not associated with the addition of strontium in periosteal-derived cells. The ALP activity in the periosteal-derived cells was higher in 5, 10, and 100 ${\mu}g$/ml strontium-treated cells than in untreated cells at day 14 of culture. Among the strontium-treated cells, the ALP activity was appreciably higher in 100 ${\mu}g$/ml strontium-treated cells than in 5 and 10 ${\mu}g$/ml strontium-treated cells. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in strontium-treated cells than in untreated cells at day 14 of culture. Their levels were increased in a dose-dependent manner. Von Kossa-positive mineralization nodules were strongly observed in the 1 ${\mu}g$/ml strontium-treated cells at day 21 and 28 of culture. The calcium content in the periosteal-derived cells was also higher in 1 ${\mu}g$/ml strontium-treated cells at day 28 of culture. These results suggest that low concentration of strontium stimulates the osteoblastic phenotypes of more differentiated periosteal-derived cells, whereas high concentration of strontium stimulates the osteoblastic phenotypes of less differentiated periosteal-derived cells. The effects of strontium on osteoblastic phenotypes of periosteal-derived cells appear to be associated with differentiation-extent.

• International Journal of Oral Biology
• /
• v.33 no.3
• /
• pp.77-82
• /
• 2008

Innate immune response is initiated by the recognition of unique microbial molecular patterns through pattern recognition receptors (PRRs). The purpose of this study is to dissect the expression of various PRRs in gingival epithelial cells of differentiated versus undifferentiated states. Differentiation of immortalized human gingival epithelial HOK-16B cells was induced by culture in the presence of high $Ca^{2+}$ at increased cell density. The expression levels of various PRRs in HOK-16B cells were examined by realtime reverse transcription polymerase chain reaction (RTPCR) and flow cytometry. In addition, the expression of human beta defensins (HBDs) was examined by real time RT-PCR and the amounts of secreted cytokines were measured by enzyme linked immunosorbent assay. In undifferentiated HOK-16B cells, NACHT-LRR-PYDcontaining protein (NALP) 2 was expressed most abundantly, and toll like receptor (TLR) 2, TLR4, nucleotide-binding oligomerization domain (NOD) 1, and NOD2 were expressed in substantial levels. However, TLR3, TLR7, TLR8, TLR9, ICE protease-activating factor (IPAF), and NALP6 were hardly expressed. In differentiated cells, the levels of NOD2, NALP2, and TLR4 were different from those in undifferentiated cells at RNA but not at protein levels. Interestingly, differentiated cells expressed the increased levels of HBD-1 and -3 but secreted reduced amount of IL-8. In conclusion, the repertoire of PRRs expressed by gingival epithelial cells is limited, and undifferentiated and differentiated cells express similar levels of PRRs.

• Korean Journal of Agricultural Science
• /
• v.34 no.1
• /
• pp.1-11
• /
• 2007

This study is aimed at finding an optimum density for 6-Dimethylaminopurine (6-DMAP) and cycloheximide which have an effect on the revitalization of porcine oocytes. The results were summarized as follows: 1. When 6-DMAP was treated with 2 mM for 2 hours, It showed a significantly (P<0.05) different high result in activation rate, cleavage rate and blastocyst growth rate of 51.2%, 52.7% and 25.2% respectively. 2. When Cycloheximide was treated with 5 ug/ml for 6 hours, It showed a significantly (P<0.05) different high result in activation rate, cleavage rate and blastocyst growth rate of 47.7%, 46.8%, and 27.3% respectively. 3. When it was cultured in the culture medium, NCSU, for 7 days after inducing activation with 6-DMAP and cycloheximide, it showed no differences in the number of inner cell mass (ICM) and total cell of blastocysts. To conclude, it has been examined for porcine oocytes to be suitable when 6-DMAP was treated with 2 mM for 2 hours, Cycloheximide with 5 ug/ml for 6 hours.

• Journal of Veterinary Clinics
• /
• v.28 no.5
• /
• pp.486-496
• /
• 2011

A special mesenchymal tissue layer called perichondrium has a chondrogenic capacity and is a candidate tissue for engineering of cartilage. To overcome limited potential for chondrocyte proliferation and re-absorption, we studied a method of cartilage tissue engineering comprising chondrocyte-hydrogel pluronic complex (CPC) and cultured perichondrial cell sheet (cPCs) which entirely cover CPC. For effective cartilage regeneration, cell-sheet engineering technique of high-density culture was used for fabrication of cPCs. Hydrogel pluronic as a biomimetic cell carrier used for stable and maintains the chondrocytes. The human cPCs was cultured as a single layer and entirely covered CPC. The tissue engineered constructs were implanted into the dorsal subcutaneous tissue pocket on nude mice (n = 6). CPC without cPCs were used as a controls (N = 6). Engineered cartilage specimens were harvested at 12 weeks after implantation and evaluated with gross morphology and histological examination. Biological analysis was also performed for glycosaminoglycan (GAG) and type II collagen. Indeed, we performed additional in vivo studies of cartilage regeneration using canine large fullthickness chondrial defect model. The dogs were allocated to the experimental groups as treated chondrocyte sheets with perichondrial cell sheet group (n = 4), and chondrocyte sheets only group (n = 4). The histological and biochemical studies performed 12 weeks later as same manners as nude mouse but additional immunofluorescence study. Grossly, the size of cartilage specimen of cPCs covered group was larger than that of the control. On histological examination, the specimen of cPCs covered group showed typical characteristics of cartilage tissue. The contents of GAG and type II collagen were higher in cPCs covered group than that of the control. These studies demonstrated the potential of such CPC/cPCs constructs to support chondrogenesis in vivo. In conclusion, the method of cartilage tissue engineering using cPCs supposed to be an effective method with higher cartilage tissue gain. We suggest a new method of cartilage tissue engineering using cultured perichondrial cell sheet as a promising strategy for cartilage tissue reconstruction.

• Korean Journal of Korean Medical Institute of Dermatology and Aesthetics
• /
• v.1 no.1
• /
• pp.127-144
• /
• 2005

This study compared the activity of the aerial part of P. japonicum with its root in order to examine the possibility of the medicinal use of the aerial part, which has not been used as medicine, in substitute for the root that has traditionally been used as medicine. For this purpose, the author measured the proliferation of Human $CD4^-$ T cells, which are related to immunity, the differentiation of HL-60 cells, and the contents of IL-6, IgE and $TNF-{\alpha}$ and compared their anti-cancer effect on Hep3B and A549 cells. The results of this study are as follows: 1. As for Human $CD4^-$ T cells, $1.0\;g/{\ell}$ methanol extract from the aerial part promoted the proliferation of the cells 1.8 times higher while $1.0\;g/{\ell}$ methanol extract from the root did by 1.76 times higher compared to the control group. 2. As for HL-60 cells, methanol extract and water extract from the aerial part showed differentiation 1.14 times higher and 1.12 times higher respectively while methanol extract and water extract from the root did 1.14 times higher and 1.07 times higher compared to tile control group. 3. Cell density was highest on Day 4 of culture in all samples, Methanol extracts from the aerial part and the root showed activities of $7.9{\times}10^3\;cells/m{\ell}$ and $7.5{\times}10^3\;cells/m{\ell}$ respectively, and water extracts from the aerial part and the root did activities of $5.3{\times}10^3\;cells/m{\ell}$ and $6.1{\times}10^3\;cells/m{\ell}$. 4. The secretion of IL-6 was highest on Day 4 of culture. Methanol extracts from the aerial part and the root showed secretions of $6.7{\times}10^{-3}\;pg/cells/m{\ell}$ and $7.2{\times}10^{-3}\;pg/cells/m{\ell}$ respectively, and water extracts from the aerial part and the root did secretions of as high as $7.0{\times}10^{-3}\;pg/cells/m{\ell}$ and $6.0{\times}10^{-3}\;pg/cells/m{\ell}$. 5. As for the production of IgE, water extract from the root effectively inhibited the product at $1,000\;{\mu}g/m{\ell}$, methanol extract from the root at $10\;{\mu}g/m{\ell}$ and $100\;{\mu}g/m{\ell}$, water extract from the aerial at $1,000\;{\mu}g/m{\ell}$, and methanol extract from the aerial part at $1,000\;{\mu}g/m{\ell}$. 6. According to the result of measuring the content of $TNF-{\alpha}$, methanol extracts from the root and the aerial part showed inhibition effect at $10\;{\mu}g/m{\ell}$, $100\;{\mu}g/m{\ell}$ and $1,000\;{\mu}g/m{\ell}$. 7. As for liver cancer cell Hep3B, $1.0\;g/{\ell}$ methanol extracts from the root and the aerial part showed inhibition effects of 78% and 70% respectively, and $1.0\;g/{\ell}$ water extracts from the root and the aerial part did inhibition effects of 56% and 59%. 8. As for lung cancer cell A549, $1.0\;g/{\ell}$ methanol extracts from the root and the aerial part showed inhibition effects of 75% and 70% respectively, and $1.0\;g/{\ell}$ water extracts from the root and the aerial part did inhibition effects of 48% and 45%. The results of this study presented above show that the aerial part of P. japonicum has immunizing and anti-cancer effects as high as its root, which has commonly been used as medicine. There should be more in-depth research on the aerial part of P. japonicum in the future.

• Korean Journal of Microbiology
• /
• v.37 no.1
• /
• pp.92-99
• /
• 2001

High-cell-density cultivation of Ralstonia eutopha KHB 8862 by fed-batch fermentation in a 200 l pilot plant was carried out for the mass production of poly(3-hydroxybutyrate) (PHB). After 80 h of cultivation, the dry cell weight (DCW), PHB concentration, and PHB yield from fructose syrup reached 168 g/l, 74%DCW, and 0.27 (w/w), respectively, resulting in a productivity of 1.6 g of PHB/L/h. Based on these results, the PHB production cost from bacterial fermentation was analyzed and economic evaluation was performed. In the case of new investment being implemented or not, the production cost of PHB was US$ 3.15/kg and US$ 2.41/kg, respectively. PHB productivity and PHB yield on a carbon substrate were both important factors to be optimized. The increase of PHB yield on a carbon sources significantly decreased the PHB production cost but the increase in productivity had a relatively slight effect on the decrease in PHB production cost because the cost of carbon sources (37%) for PHB was larger in proportion to total cost than the depreciation cost (17%). These results suggest that the increased PHB yield from carbon sources and the development of new cheaper substrates would be more effective in decreasing PHB production cost than the increase in productivity. It was demonstrated that PHB is not in competition with consumable plastics such as PET in present market. Therefore, it is essential to lower production cost to be used as a bulk product and desirable to develop new application fields for PHB such as biomedical and cosmeceuticals.

• KSBB Journal
• /
• v.25 no.2
• /
• pp.142-154
• /
• 2010

The protein-bound innerpolysaccharides (IPS) produced by suspended mycelial cultures of Inonotus obliquus have promising potentials as an effective antidiabetic as well as an immunostimulating agents. To enhance IPS production, intensive strain improvement process should be carried out using large amount of UV-mutated protoplasts. During the whole strain-screening process, the stage of solid growth-culture was found to be the most time-requiring step, thus preventing rapid screening of high-yielding producers. In order to reduce the cell growth period in the solid growth-stage, therefore, solid growth-medium was optimized using the statistical methods such as (i) Plackett-Burman and fractional factorial designs (FFD) for selecting positive medium components, and (ii) steepest ascent (SAM) and response surface (RSM) methods for determining optimum concentrations of the selected components. By adopting the medium composition recommended by the SAM experiment, significantly higher growth rate was obtained in the solid growth-cultures, as represented by about 41% larger diameter of the cell growth circle and higher mycelial density. Sequential optimization process performed using the RSM experiments finally recommended the medium composition as follows: glucose 25.61g/L, brown rice 12.53 g/L, soytone peptone 12.53 g/L, $MgSO_4$ 5.53 g/L, and agar 20 g/L. It should be noted that this composition was almost similar to the medium combinations determined by the SAM experiment, demonstrating that the SAM was very helpful in finding out the final optimum concentrations. Through the use of this optimized medium, the period for the solid growth-culture could be successfully reduced to about 8 days from the previous 15~20 days, thus enabling large and mass screening of high producers in a relatively short period.

• Microbiology and Biotechnology Letters
• /
• v.44 no.3
• /
• pp.293-302
• /
• 2016

To determine the dominant bacterial species during the Saeu-jeotgal ripening process, the cultivable bacterial population was examined over a 135-day period using six different growth media. The greatest numbers of bacteria were identified when marine agar was used for culture, with maximum cell density identified at day 65 (2.51 × 10⁷ colony forming units/g). Over the course of 135 days, the bacterial diversity was analyzed eight times. A total of 467 isolates, comprising 87 species from 42 genera, as well as 16 isolates belonging to previously unknown species, were identified. The number of species detected decreased from 39 at day 1 to 13 at day 135. The order of dominance at the genus level was as follows: Staphylococcus, Salimicrobium, Kocuria, and Psychrobacter. Staphylococcus and Salimicrobium accounted for 2% of the diversity at day 1, and then increased to 39% and 36%, respectively, at day 135. The dominant species Staphylococcus equorum, Salimicrobium salexigens, and Kocuria palustris accounted for 23.6%, 16.1%, and 10.9% of all isolates, respectively. Importantly, both St. equorum and Sm. salexigens remained viable at a NaCl concentration of 21% (w/v), which indicates their strong involvement in the ripening of Saeu-jeotgal.