• KSBB Journal
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• v.22 no.5
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• pp.305-312
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• 2007

It is crucial to develop a miniaturized cultivation method for large and rapid screening of high-yielding mutants of monacolin-K, a powerful anti-hypercholesterolemic secondary metabolite biosynthesized by the fungal cells of Monascus ruber. In order to investigate as many strains as possible in a short time, a miniaturized fermentation method especially suitable for the cultivation of the filamentous Monascus mutants was developed using $50m{\ell}$ culture-tube ($7m{\ell}$ of working volume) instead of the traditional $250m{\ell}$ flask ($50m{\ell}$ of working volume). Generally, in filamentous fungal cell fermentations, morphologies in growth and production cultures should be maintained as thick filamentous and compact-pelleted (usually less than 1 mm in diameter) forms, respectively, for enhanced production of secondary metabolites in final production cultures. In this study, we intended to induce the respective optimal morphologies in the miniaturized culture system for the purpose of rapid screening of overproducers. Miniaturized growth culture system was successfully developed due to the mass production of spores in the statistically optimized solid medium. When large amounts of spores were inoculated into the growth cultures, and brown rice flour (20 g/L) was also supplemented to the growth medium, dense filamentous morphologies were successfully induced in the growth cultures performed with the 50 ml culture tubes. It was implied that the amounts of spores inoculated into the growth tube-cultures and the growth medium components should be the key factors for the induction of the filamentous forms in the growth fermentations. Furthermore, in order to statistically optimize production medium, multiple experiments based on Plackett-Burman design and response surface method (RSM) were carried out, resulting in more than 2 fold enhanced production of monacolin-K in the final production cultures with the optimized production medium. Notably, under the production culture conditions with the statistically optimized medium, optimal pellet sizes below 1 mm in diameter were reproducibly induced, in contrast to the thick and viscous filamentous morphologies observed in the previous production cultures.

• KSBB Journal
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• v.22 no.5
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• pp.297-304
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• 2007

For large and rapid screening of high-yielding mutants of lovastatin produced by filamentous fungal cells of Aspergillus terreus, one of the most important stage is to test as large amounts of mutated strains as possible. For this purpose, we intended to develop a miniaturized cultivation method using $7m{\ell}$ culture tube instead of traditional $250m{\ell}$ flask (working volume $50m{\ell}$). For obtaining large amounts of conidiospores to be used as inoculums for miniaturized cultures, 4 components i.e., glucose, sucrose, yeast extract and $KH_2PO_4$ were intensively investigated, which had been observed to show positive effect on enhancement of spore production through Plackett-Burman design experimet. When optimum concentrations of these components that were determined through application of response surface method (RSM) based on central composite design (CCD) were used, maximum spore numbers amounting to $1.9\times10^{10}$ spores/plate were obtained, resulting in approximately 190 fold increase as compared to the commonly used PDA sporulation medium. Using the miniaturized cultures, intensive strain development programs were carried out for screening of lovastatin high-yielding as well as highly reproducible mutants. It was observed that, for maximum production of lovastatin, the producers should be activated through 'PaB' adaptation process during the early solid culture stage. In addition, they should be proliferated in condensed filamentous forms in miniaturized growth cultures, so that optimum amounts of highly active cells could be transferred to the production culture-tube as reproducible inoculums. Under these highly controlled fermentation conditions, compact-pelleted morphology of optimum size (less than 1 mm in diameter) was successfully induced in the miniaturized production cultures, which proved essential for maximal utilization of the producers' physiology leading to significantly enhanced production of lovastatin. As a result of continuous screening in the miniaturized cultures, lovastatin production levels of the 81% of the daughter cells derived from the high-yielding producers turned out to be in the range of 80%$\sim$120% of the lovastatin production level of the parallel flask cultures. These results demonstrate that the miniaturized cultivation method developed in this study is efficient high throughput system for large and rapid screening of highly stable and productive strains.

• KSBB Journal
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• v.24 no.1
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• pp.30-40
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• 2009

Statistical optimization of the production medium was carried out in order to find an optimal medium composition in itaconic acid fermentation process. Itaconic acid utilized in the manufacture of various synthetic resins is a dicarboxylic acid biosynthesized by fungal cells of Aspergillus terreus in a branch of the TCA cycle via decarboxylation of cis-aconitate. Through OFAT (one factor at a time) experiments, six components (glucose, fructose, sucrose, soluble starch, soybean meal and cottonseed flour) were found to have significant effects on itaconic production among various carbon- and nitrogen-sources. Hence, using these six factors, interactive effects were investigated via fractional factorial design, showing that the initial concentrations of sucrose and cottonseed flour should be high for enhanced production of itaconic acid. Furthermore, through full factorial design (FFD) experiments, negative effects of $KH_2PO_4$ and $MgSO_4$ on itaconic acid biosynthesis were demonstrated, when excess amounts of the each component were initially added. Based on the FFD analysis, further statistical experiments were conducted along the steepest ascent path, followed by response surface method (RSM) in order to obtain optimal concentrations of the constituent nutrients. As a result, optimized concentrations of sucrose and cottonseed flour were found to be 90.4g/L and 53.8g/L respectively, with the corresponding production level of itaconic acid to be 4.36 g/L (about 7 fold higher productivity as compared to the previous production medium). From these experimental results, it was assumed that optimum ratio of the constituent carbon (sucrose) and nitrogen (cottonseed flour) sources was one of the most important factors for the enhanced production of itaconic acid.

• Journal of Life Science
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• v.27 no.1
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• pp.57-66
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• 2017

Traditional Korean fermented herbal plants are potential sources of new food that promote health, but they are still produced by yeast, fungi or bacteria fermentation. In the present work, mushroom (Paecilomyces tenuipes and Cordyceps militaris) fungal dongchunghacho were used to fermented Glycyrrhiza uralensis Fischer (licorice) or mixed with pupa. The pupa were tested as solid substrates for the production of corcycepin, liquiritin, and liquiritigenin. The fermented substrates were analyzed the content of cordycepin, liquiritin, liquiritigenin, and glycirrhizin productivity and inhibitory activity of NO. The cordycepin content of 70% EtOH extract from the fermented mixture of licorice and 50% pupa with C. militaris increased maximum at 33 times. Pupa was very excellent for the production of cordycepin. The liquiritin content was decreased in all the assays inoculated with P. tenuipes and C. militaris dongchunghachos. The liquiritigenin content was higher when fermented with P. tenuipes than C. militaris. The addition of pupa significantly reduced the liquiritin content and glycyrrhizin production. As a result, the liquiritigenin content increased in fermented P. tenuipes and C. militaris, and liquiritin and glycyrrhizin decreased. The inhibition of NO production in the different ethanolic extracts fermented with licorice and pupa was also significantly increased and higher than that of a nonfermented extract in higher polar solvent extracts. The contents of cordycepin and biological active compounds were altered in accordance with the concentration of pupa and fungi. This study provides basic data for use in developing dongchunghacho fungi as a functional food resource.

• Journal of Food Hygiene and Safety
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• v.35 no.5
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• pp.438-446
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• 2020

Meju and nuruk (respectively soybean and malt) are traditional Korean fermentation starters that are vulnerable to contamination by harmful microorganisms such as aflatoxigenic fungi and their associated aflatoxins (AFs). In this study, Aspergillus spp. were isolated and identified from a total of 57 meju and 18 nuruk samples collected from Korean markets. Their potential aflatoxigenicity was investigated by examining the presence of three aflatoxin biosynthetic genes (aflO, aflP, and aflR) using multiplex polymerase chain reaction (mPCR) assays. Thereafter, aflatoxin production of isolates and the natural occurrence of AFs in meju and nuruk samples were analyzed by high-performance liquid chromatography (HPLC). A total of 177 Aspergillus isolates were identified and 130 isolates were obtained from meju samples. Of these, 25 isolates (19.2%) contained all three aflatoxin biosynthetic genes, and five (20%) of these isolates produced aflatoxins. Forty-seven of the Aspergillus isolates were obtained from nuruk samples, five of which (10.6%) expressed all three AF biosynthetic genes; however, none of these strains produced AFs. HPLC analysis showed that 88% (51/58) of the meju samples and 39% (7/18) of nuruk samples were not contaminated with AFs (below limit of detection). Among the isolates isolated from meju and nuruk, there were aflatoxigenic strains containing all three aflatoxin biosynthetic genes or producing aflatoxin in medium, but the frequency of aflatoxin contamination was low in the meju and nuruk samples.

• Journal of The Korean Society of Grassland and Forage Science
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• v.40 no.2
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• pp.80-86
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• 2020

This study was conducted to research on the efficacy of chemical treatment as an effective method for reducing mycotoxin in rice straw silage. As a chemical treatment method, ammonia and sodium hydroxid were treated at 4% level of rice straws contaminated with mycotoxin, and the effects of silage storage on fungal toxin reduction, fermentation quality, and fiber digestion were evaluated. Aflatoxin B₁, B₂, G₁, G₂ and fumonisin B₁, B₂ as well as deoxynivalenol were not detected in all experimental groups, and ochratoxin A and zearalenone were detected. Ochratoxin A was detected lower in the chemical treatment than control (41.23 g / kg) (p<0.05). Zearalenone showed lower results in sodium hydroxide treatment (297.44 ㎍ / kg) than control (600.33 ㎍ / kg) and ammonia treatment (376.00 ㎍ / kg) (p<0.05). The pH of rice straw silage was the lowest in ammonia treatment and the highest in sodium hydroxide treatment (p<0.05). The lactic acid contents of control and ammonia treatments were similar, but sodium hydroxide treatment was the lowest (p<0.05). Propionic acid was higher in the control than in the chemical treatments (p<0.05), and showed similar contents in the ammonia and sodium hydroxide treatment. Both the rumen microbial degradation rate of NDF and ADF showed the highest in sodium hydroxide treatment, followed by ammonia treatment, and the control showed the lowest level (p<0.05). Therefore, the results of this study are demonstrated to have a good effect on the treatment of ammonia and sodium hydroxide to reduce the mycotoxins and increase the rumen microbial degradation rate in the rice straw silage. Sodium hydroxide treatment was more effective in reducing mycotoxins and improving fiber degradation rate than ammonia treatment, but it is thought to have an inefficient effect on silage fermentation in rice straw silage.

• Journal of Mushroom
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• v.12 no.3
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• pp.201-208
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• 2014

The separation of the bacteria inhibiting Trichoderma sp. mold, the strain causing blue mold disease that occurs frequently when cultivating mushroom while carrying out the efficient fermentation of mushroom medium, from the growth was done. In about 200 strains isolated primarily from fungus garden samples, 6 strains were secondly isolated, which had fast growth rates and a clear zone on the plate medium of SM, AM, and CM. Among the 6 strains isolated, the C-1 strain showed high enzymatic activity of cellulase, amylase, and protease, and strong antibacterial activity for the T. virens and T. harzianum, selected finally. The selected C-1 strain was identified as Paenibacillus polymyxaby the result of the identification by Bergey's Manual of Systematic Bacteriology and the analysis of the nucleotide sequence of 16S rRNA, and named as P. polymyxa CK-1. In reviewing the growth conditions of the P. polymyxa CK-1 strain, the optimum cultivation temperature was $45^{\circ}C$, and the optimum pH for growth was in the range of 6.0~7.0. Appropriate incubation time of P. polymyxa CK-1 for the growth inhibition of the fungus T. virens and T. harzianum was 22 to 36 hours. And the fungal growth was not observed, even when leaving two molds inoculated on each petri dishes, which were treated with 24 hour culture solution of P. polymyxa CK-1 strain for 10 days. As a result of studying the thermal stability of the antagonists produced by the P. polymyxa CK-1 strain, no mycelial growth of the two fungi was observed in the test group treated for 20 minutes at $60^{\circ}C$ and $100^{\circ}C$, but mycelial growth was slightly observed in the test group treated for 20 minutes at $121^{\circ}C$. As aresult of reviewing the impact of the P. polymyxa CK-1 culture medium on mushroom mycelial growth, it showed no effect on a variety of mushroom mycelial growth including enoki mushroom and shiitake mushroom.