• 한국균학회소식:학술대회논문집
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• 2016.05a
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• pp.19-20
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• 2016

Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

• Korean Journal of Agricultural Science
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• v.30 no.1
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• pp.31-40
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• 2003

A research has been done for growing characteristics of Korean ginseng in Geumsan of Chungnam Province. It had been made to determine the transitional element concentrations of the rocks, divided by biotitic granite(GR) and phyllite(PH). The physical and chemical properties of their weathering soils and ginseng nursery soils were analyzed. The texture in the GR weathering and ginseng nursery soils were sandy clay, and the texture of the PH weathering and ginseng nursery soils were heavy or silty clay. The bulk densities of the GR and PH weathering soils were $1.21{\sim}1.32g/cm^3$ and $1.26{\sim}1.38g/cm^3$, respectively. Also, the bulk densities of the GR and PH ginseng nursery soils were $1.02{\sim}1.10g/cm^3$, respectively. The pH (4.80) of the GR weathering soil were lower than the pH of the PH(5.34) weathering soil. The pH in the 2 year and 4 year-ginseng nursery soil of the GR were 4.39 and 4.40. In addition, those of the PH were 5.24 and 5.34, respectively. The difference in pH of the two nursery soils could be from the pH difference between the two parent materials. The organic matter contents of the GR weathering soils(0.24%) were higher than those of the PH(1.02%) weathering soils. The organic matter of the 2 and 4 year-ginseng GR nursery soils were 0.87% and 1.52%, and of the PH nursery soils were 2.06% and 2.96%, respectively. The total nitrogen contents of the GR weathering soils were 259.43ppm and of the PH weathering soils were 657.22ppm. Those of 2 and 4 year-ginseng GR nursery soils were 588.04ppm and 657.22ppm and those of the PH nursery soils were 1037.72ppm and 1227.96ppm, respectively. The nitrate and ammonium contents of the GR weathering soils were the extremely small, and those of the PH weathering soils were 6.7ppm and 9.94ppm. Those of 2 year-ginseng GR nursery soils(223.09ppm and 26.96ppm) were higher than those of PH(19.46ppm and 8.23ppm) nursery soils. And those of 2 year-ginseng PH nursery soils(14.22ppm and 16.84ppm) were lower than those of PH(306.93ppm, 34.21ppm) nursery soils. The difference was due to fertilizer types and more deposits of nitrate after oxidation of ammonium. The phosphate contents of the GR and PH weathering soils were 14.41ppm and 38.60ppm. Those of GR 2 and 4 year-ginseng nursery soils were 46.89ppm and 102.44ppm and those of the PH nursery soils were 147.04ppm and 38.60ppm. The cation exchange capacities of the GR weathering soils were 12.34me/100g and those of the PH weathering soils were 15.40me/100g. Those of 2 and 4 year-ginseng GR nursery soils were 15.80me/100g and 7.70me/100g and those of PH nursery soils were 12.14me/100g and 12.83me/100g. All of exchangeable cation($K^+$, $Ca^{2+}$, $Mg^{2+}$, $Na^+$) contents in the nursery soils were higher than those in the weathering soils. The $SO_4{^2-}$ contents of the weathering soils in both of the GR(5.98ppm) and PH(9.94ppm) were higher than those of the GR and PH ginseng nursery soils. The $Cl^-$) contents of the GR and PH weathering soils were a very small and those of the nursery soils(2-yr GR: 39.06ppm, 4-yr GR: 273.43ppm, 2-yr PH: 66.41ppm, 4-yr PH: 406.24ppm) were high because of fertilizer inputs.

• Applied Biological Chemistry
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• v.9
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• pp.9-27
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• 1968

Studies were carried out in order to elucidate chemical components and microflora in three types of soy-sauce, 12-year aged soy-sauce prepared by improved method. 7-year aged and 20-year aged soy-sauce prepared by ordinary method. They results are summarized as follows: 1. The followings are found to be the important factors affecting the quality of soy sauce. a. Organic acids, reducing sugars and free amino acids were increased in the course of storage. b. In the aged soy-sauces under study non-volatile organic acid increased while volatile organic acid decreased and the total acidity was dependent only upon tie latter. c. It was found that suit concentration decreased during the storage. 2. The results of investigation of microflora in the stored soy-sauce are shown as follows. Soy-sauce Improved Ordinary Microbe 12-Y. 20-Y. 7-Y. Aerobic bacteria colony/1ml. 6 123 2 Halophilic lactic acid bacteria colony/1ml. 4 6 10 Osmophilic yeast colony/1ml. $828{\times}10^4$ 248 - b. In the stored soy-sauces, aerobic bacteria are incapable of growing due to drop in pH value and the influence of salt concentration. c. Halophilic lactic acid bacteria are incapable of growing due to drop in pH value, even the salt concentrations decreased during the storage. d. Osmophilic yeast are still growing in low pH value and in the decreasing salt concentration during the strage. 3. The results of amino acid analysis by paper partition chromatographic and calorimetric methods are shown as follows. a. Fourteen kinds of amino acid and thirteen amino acids were detected in the soy-sauce of 12-year aged improved soy-sauce and 7-year aged and 20-year aged ordinary one, respectively. b. The contents of aspartic acid, glutamic acid, serine, valine, leucine, lysine, histidine and methionine increased in the 20-year aged ordinary soy-sauce compared to the 7-year aged one. On the other hand those of alanine, tyrosine, phenylalanine and cystine decreased. 4. The results of sugar analysis by paper chromatography are as follows. a. In the 12-year aged improved soy-sauce, galactose, glucose, arabinose, xylose, rhamnose, maltose and an unknown were detected, and their amounts were in the above order except maltose and an unknown. b. Both in the 7 and 12-year aged ordinary soy-sauces, galactose, arabinose, xylose, glucose and rhamnose were detected and the amounts of the sugars were in the above order. c. In the non-aged ordinary soy-sauce, glucose was not detected but detected from 7-year and 20-year aged ordinary soy-sauce. 5. The results of organic acid analysis by paper chromatography were as follows. a. As volatile acids, acetic, propionic and butyric acids were detected in the 7-year aged ordinary soy-sauce. On the other hand in both the 20-year aged ordinary soy-sauce and the 12-year aged improved ones, only acetic acid was abundant while propionic and butyric acids were round in trace. It was found that propionic and butyric acids, as the unpleasant flavor components, decreased during the storage. b. In the ordinary soy-sauce, citric acid were produced during the storage and lack, malic and tartaric acids increased in the course of aging while succinic, glycolic, fumaric and malonic acids were shown to decrease. Glutaric and oxalic acids disappeared. Citric acid was produced also in the improved soy-sauce, but lactic, tartaric, succinic, malic, and glycolic acids decreased, while both malonic and glutaric acids disappeared. From the above results the citric acid production was considered to be a favorable factor for the taste. c. In the aged soy-sauces, pyruvic, α-ketoglutaric and probably acetoacetic and oxaloacetic acids (both in trace) were present and their amounts were in the above order. All of the α-keto acid abruptly decreased during the storage.

• Journal of Food Hygiene and Safety
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• v.26 no.4
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• pp.417-423
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• 2011

Salted Cabbage products purchased from different companies at 4 different districts in South Korea were detected in this study. Cabbage and salt are the main materials for kimchi manufacture. The results of general bacteria contaminated in the samples were $1.4{\times}10^5$, $6.4{\times}10^5$, $1.7{\times}10^7$, $3.6{\times}10^7$ CFU/g in cabbage and $2.7{\times}10^3$ CFU/g in salt, respectively. The results of coliforms were detected as $2.4{\times}10^4$ CFU/g, and there was no Escherichia coli in any sample. Staphylococcus aureus was detected in cabbage as $9.9{\times}10^2$, $8.0{\times}10^1$, and $3.0{\times}10^3$ CFU/g, Bacillus cereus was also found in cabbage as $4.1{\times}10^3$ and $1.0{\times}10^1$ CFU/g. The results of Campylobacter jejuni and Vibrio paraheamolyticus were $2.4{\times}10^6$ and $1.0{\times}10^4$ CFU/g in cabbage, respectively. $1.0{\times}10^3$ CFU/g for Yersinia enterocolitica was determined in salt. In case of Listeria monocytogenes, the results were $1.5{\times}10^1$, $1.1{\times}10^2$, and $4.5{\times}10^1$ CFU/g in cabbage. Total batcteria ranged from $1.4{\times}10^1$ to $4.4{\times}10^5$ CFU/g were detected in salting solution, from $1.5{\times}10^4$ to $1.2{\times}10^8$ CFU/g in dehydrated salted-cabbage, from $9.4{\times}10^4{\sim}1.3{\times}10^8$ CFU/g in minced salted-cabbage. The results of E. coli in samples from different companies were different from one to anther. The results of the contamination of S. aureus and B. cereus showed positive in salting solution and dehydrated salted-cabbage at a portion of companies. V. paraheamolyticus was detected in salting solution. The contamination of Y. enterocolitica ranged from $9.5{\times}10^2$ to $1.8{\times}10^3$ CFU/g in salting solution, from $1.7{\times}10^1$ to $2.7{\times}10^2$ CFU/g in dehydrated salted-cabbage, from $1.2{\times}10^2$ to $1.3{\times}10^8$ CFU/g in minced salted-cabbage. The contamination of L. monocytogenes ranged from $8.0{\times}10^2$ to $1.7{\times}10^4$ CFU/g in salting solution, from $2.8{\times}10^2$ to $1.2{\times}10^4$ CFU/g in dehydrated salted-cabbage. During the manufacture processing of Kim chi, microorganisms were detected in cabbages salted in different concentrations of salt solution at 8%, 10%, 12% and 15% for 5-20 hours. As the results, $3.5{\times}10^5-1.7{\times}10^6$, $3.4{\times}10^5-2.5{\times}10^6$, $5.4{\times}10^5-2.3{\times}10^6$, $4.0{\times}10^5-2.3{\times}10^6$ CFU/g were detected for E. coli in samples at different treatment conditions. $1.9{\times}10^4-4.1{\times}10^4$, $4.1{\times}10^3-2.8{\times}10^4$, $1.5{\times}10^3-7.8{\times}10^3$, $2.2{\times}10^4-6.6{\times}10^4$ CFU/g were detected for S. aureus in samples at different treatment conditions. Salmonella typhimurium was detected in salted cabbage with various salt concentration after salting for 5 hrs, the result ranged from $2.5{\times}10^5$ to $3.8{\times}10^6$ CFU/g, and change of microorganism was the smallest in salted cabbage under the concentration of salting solution at 10% for 15 hours. The cabbage salted in 10% salting solution for 15 hours were washed with water for 2 and 3 times, with chlorine for 3 times, and with acetic acid for 3 times. E. coli was detected in the samples washed with water for 2 and 3 times, washed with chlorine for 3 times. The contamination of S. aureus was $3.0{\times}10^5$ CFU/g in the samples washed with water for 2 times, $5.6{\times}10^3$ CFU/g in the samples washed with acetic acid for 3 times, $3.6{\times}10^5$ CFU/g in the samples washed with water for 3 times and same amount in the samples washed with chlorine for 3 times. According to the results, the contamination of S. aureus was $5.6{\times}10^3$ CFU/g lower in samples washed with chlorine and acetic acid than that in samples washed with water. In case of S. typhimurium, it has been detected in samples washed with water and chlorine, $3.0{\times}10^1$ CFU/g as the lowest concentration among all the samples was measured in the samples washed with acetic acid for 3 times.

• Journal of Korean Society of Forest Science
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• v.29 no.1
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• pp.20-53
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• 1976

For the purpose of detecting the capability of the trees to resist air pollution and of determining the tree species best suited for purification of polluted air, particularly with regard to $SO_2$ contamination, six following ornamental tree species were selected as experimental materials: i.e., Hibiscus syriacus L., Ginkgo biloba L., Forsythia koreana Nak., Syringa dilatata Nak., Larix leptolepis Gordon, and Pinus rigida Miller. The susceptiblities of the trees were observed and analyzed on the basis of area ratio of smoke injury spots to the total leaf area. The results of the experiments are as follows: I. The Susceptibilities to Sulfur Dioxide. (1) The decreasing order of tolerance to $SO_2$ by species was as follows: 1. Hibiscus syriacus 2. Ginkgo biloba, 3. Forsythia koreana, 4. Syringa dilatata, 5. Larix leptolepis, and 6. Pinus rigida. In general, Hibiscus syriacus and Ginkgo biloba can be grouped as the most resistant and Larix leptolepis and Pinus rigida as the least resistant and Forsythia koreana and Syringa dilatata as of intermediate resistance. (2) The sulfur content of the leaves treated by $SO_2$ increased in proportion to the increase of the concentration of the fumigation. The content in the coniferous species proved to be less than that of the broad-leaved species, but Ginkgo biloba proved to contain as much sulfur as broad-leaved species. (3) The earlier-stage leaves fumigated in June with the $SO_2$ concentration up-to-l-ppm showed that sulfur content increases in proportion to the increase of the concentration of the fumigation, but the difference between concentration was not so significant. (4) The later-stage leaves fumigated in October showed higher sulfur content than the earlier stage leaves, and a wider range of difference in sulfur content was detected among different concentrations. The limit of fumigation resulting in culmination of sulfur absoption in broad-leaved species, such as Syringa dilatata, Hibiscus syriacus, and Forsythia koreana proved to be around 0.6 ppm. (5) Due to the sprouting ability and the adventitious bud formation, the recovery from $SO_2$ fumigation was prominent in Hibiscus syriacus, Syringa dilatata, and Forsythia koreana. (6) The differences in the smoke spot color were recognized by species: namely, dirt-brown in Syringa dilatata, brilliant yellowish-brown in Pinus rigida and Ginkgo biloba, whitish-yellow in Hibiscus syriacus and reddish-brown in Forsythia koreana. (7) The leaf margins proved to be most susceptible, and the leaf bases of the mid-rib most tolerant. In both Ginkgo biloba and Larix leptolepis, the younger leaves were more resistant to $SO_2$ than the older ones. II. The ulfur Content of the Leaves of the Ornamental Trees Growing in the City of Seoul. (1) The sulfur contents in the leaves of the Seoul City ornamental trees showed a remarkably higher value than those of the leaves in the non-polluted areas. The sulfur content of the leaves in the non-polluted area proved to be in the following descending order: Salix pseudo-lasiogyne Leveille, Ginkgo biloba L., Alianthus altissima swingle, Platanus orientalis L., and Populus deltoides Marsh. (2) In respect to the sulfur contents in the leaves of the ornamental trees in the city of Seoul, the air pollution proved to be the worst in the areas of Seoul Railroad Station, the Ahyun Pass, and the Entrance to Ewha Womans University. The areas of Deogsu Palace, Gyeongbog Palace, Changdeog Palace, Changgyeong Park and the Hyehwa Intersection were least polluted, and the areas of the East Gate, the Ulchi Intersection and the Seodaemun Intersection are in the intermediate state.