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Muc5ac Gene Expression Induced by Cigarette Smoke is Mediated Via a Pathway Involving ERK1/2 and p38 MAPK (담배 연기에 의한 Muc5ac 유전자 발현에 관여하는 세포 내 신호 전달 경로로서의 ERK1/2와 p38 MAPK)

  • Kim, Yong Hyun;Yoon, Hyoung Kyu;Kim, Chi Hong;Ahn, Joong Hyun;Kwon, Soon Seog;Kim, Young Kyoon;Kim, Kwan Hyoung;Moon, Hwa Sik;Park, Sung Hak;Song, Jeong Sup;Cho, Kyung Sook
    • Tuberculosis and Respiratory Diseases
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    • v.58 no.6
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    • pp.590-599
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    • 2005
  • Object : Cigarette smoking is a major cause of mucus hypersecretion, which is a pathophysiological feature of many inflammatory airway diseases. Mucins, which are an important part of the airway mucus, are synthesized from the Muc gene in airway epithelial cells. However, the signaling pathways for cigarette smoke-induced mucin synthesis are unknown. The aim of this study was to determine the signal pathway for smoking induced Muc5ac gene expression. Methods : A549 cells were cultured and transiently transfected with the Muc5ac promoter fragment. These cells were stimulated with 5% cigarette smoke extract (CSE) alone or with CSE after a pretreatment with various signal transduction pathway inhibitors (AG1478, PD98059 and SB203580). The Muc5ac promoter activity was examined using the luciferase reporter system, and the level of phosphorylated EGFR, ERK1/2, p38 MAPK and JNK were all examined using Western blot analysis. Muc5ac mRNA expression was also examined using reverse transcriptase polymerase chain reactions (RT-PCR). Results : 1. The peak level of luciferase activity of the Muc5ac promoter was observed at 5% concentration and after 3 hours of incubation with the CSE. The level of EGFR phosphorylation and the luciferase activity of the transfected cells caused by the CSE were significantly suppressed by AG1478 or PD98059 (P<0.01). 2. CSE phosphorylated ERK1/2 or p38 MAPK but not JNK. The Muc5ac mRNA expression level was increased by the CSE but that was suppressed by PD98059 or AG1478. 3. The CSE-induced phosphorylation of ERK1/2 was blocked by PD98059 and that of p38 MAPK was blocked by either PD98059 or SB203580. Either PD98059 or SB203580 suppressed the luciferase activity of the transfected cells (P<0.0001). Conclusion : The Muc5ac mRNA expression level was increased by the CSE. The increased CSE-induced transcriptional activity was mediated via EGF receptor activation, which led to ERK1/2 and p38 MAPK phosphorylation.

Changed in Growth and Chemical Properties of Plastic Film House by Earthworm Cast on Gymnocalycium mihanovichii var. 'Ihong' (비모란 선인장(Gymnocalycium mihanovichii var. 'Ihong') 시설재배에서 지렁이분변토시용에 따른 생육특성 및 토양 화학성 변화)

  • Choi, I-Jin;Cho, Sang-Tae;Kim, Young-Mun;Kim, Mi-Seon;Lee, Sang-Kweon
    • Korean Journal of Organic Agriculture
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    • v.22 no.4
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    • pp.731-742
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    • 2014
  • In the current study, we investigated effects of a combination of earthworm casting, environment-friendly by-product fertilizer, and cultivation soil of Gymnocalycium mihanovichii in a heavy fertilizing culture on diameter, height, numbers of tubercles, and chemical properties of soil thereby elucidating optimal mixture ratio for securing production as well as providing nutrients throughout cultivation period. The Gymnocalycium mihanovichii var 'Ihong', one of grafted cactus for export (Rootstock: 9 cm, Scion: $1.5{\times}1.3cm$ grafted cactus) was cultured in plastic houses of Agricultural Technology Center located in Naegok-dong, Seocho-gu, Seoul from June, 2013 through December, 2013. For the control group, a mixture of sand and fertilizer (50:50) was used as this ratio is widely utilized in farmhouses. In contrast, a variety mixtures of sand and earthworm casting that was produced with food wastes was compared; the mixture ratios were 80:20, 60:40, 40:60, 20:80, and 0:100 and pH for these mixtures were found to be similar each other (ranging between 7.1 and 7.4) which is in an appropriate range (pH 6.5-7.5) for cultivation of G. mihanovichii. The organic content was increasing along with increasing contents of earthworm casting ratio while it was lower than the treatment practice group (32-43 mg/kg vs. 55 mg/kg). The content of exchangeable cation was also increasing as the ratio of earthworm casting was elevated; although levels of $K^+$, $Na^+$, and $Mg^{2+}$ were lower than the treatment practice group, the level of $Ca^{2+}$ was higher ($9.1cmol^+/kg$ and $11.5-33.7cmol^+/kg$ in the treatment practice group and the earthworm casting group, respectively). Three months after grafting, diameters of G. mihanovichii were compared with the control group; consequently, there was a significant difference noted in between the earthworm casting group and the control group (31.39 mm vs. 32.46-37.59 mm). After 5 months, growth characteristics of G. mihanovichii were evaluated. Similarly, the diameter of G. mihanovichii was significantly increasing in the group with higher ratio of earthworm casting treatment (32.63 mm vs. 32.49-37.59 mm). The height of tubercles was 2.63 mm in the control group while it was significantly elevating along with the ratio of earthworm casting mixture. The more numbers of tubercles, the more incomes for farm-houses; as results, higher mixture ration of earthworm casting resulted more numbers of tubercles compared to the control group (2.7 vs. 3.2-8.3 ea). In particular, in the earthworm casting groups with 80% and 100% ratios, the numbers of tubercles were 6.2 and 8.3 ea, respectively, which is 2.5 times more than those of the control group. These results indicate that earthworm casting treatment may be utilized in G. mihanovichii farming houses for short term production of tubercles. In the group with 40% and 60% of earthworm casting mixture, the numbers of tubercles were found to be 4.5 and 4.8 ea, respectively which is higher than the control group as well; in these groups, there were no issues with soil drainage as well as moss formation. Given the analysis results of growth characteristics of G. mihanovichii, it was concluded that 40% and 60% of earthworm casting mixture might be the optimal ratios.

Growth and Survival by the Breeding Method of Early Young Spats of the Hard Clam, Meretrix petechiails (LAMARCK) (말백합, Meretrix petechiails (LAMARCK) 초기치패의 사육방법별 성장 및 생존)

  • Kim, Byeong-Hak;Cho, Kee-Chae;Jee, Young-Ju;Byun, Soon-Gyu;Kim, Min-Chul
    • The Korean Journal of Malacology
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    • v.27 no.2
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    • pp.115-119
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    • 2011
  • To establish technical development for artificial seed production, growth and survival for early young spats of the hard clam, Meretrix petechialis, were investigated by breeding methods. Adult clams were collected at Hasa-ri, Baeksu-eup, Yeonggwang-gun, Jeollanam-do on July 13, 2010, and then transported to the indoor aquarium at the laboratory. Eggs which were taken from mother clams, were inseminated, and after they were fertilized in the aquarium, 60 million bottom-clinging spats ($198{\pm}12{\mu}m$ in shell length) were produced and bred. The breeding experiments were carried out from July 16 to October 4, 2010 for 80 days. The methods of sand box, sand bottom circulation filter, inclosing net, floor were used for the breeding experiments, and the experimental condition of sea water temperature for larvae were at 25, 28, 31, $34^{\circ}C$. Four marine cultured food organisms were used for this study as follows: Isochrysis galbana, Chaetoceros gracilis, Phaeodactylum tricornutum, Tetraselmis tetrathele. According to the experimental conditions, experimental groups of the spats in the early stage were investigated the growth rate and the survival. As the result, the method of the inclosing net section was the fastest (grew up to $2.64{\pm}0.59{\mu}m$ in shell length), followed by sandbox ($2.59{\pm}0.64{\mu}m$, bottom circulating filter ($2.56{\pm}0.52{\mu}m$), and floor ($2.52{\pm}0.56{\mu}m$). The survival was the highest in the experimental condition of sandbox (35.9%), followed by floor (34.6%), bottom circulating filter (29.5%), and inclosing net (9.3%). Eexperimental condition of water temperature of $34^{\circ}C$ showed the fastest growth rate (grew up to $2.70{\pm}0.76{\mu}m$ in shell length), and showed the latest growth rate (grew up to $2.45{\pm}0.41{\mu}m$ in shell length) at $25^{\circ}C$. The survival (%) was the highest under the water temperature conditions at $31^{\circ}C$, and showed the lowest (14.2%) at $34.^{\circ}C$. The growth rate of the experimental group fed the mixture live food was the fastest with shell length $2.52{\pm}0.66{\mu}m$, and that of experimental group fed P. tricornutum showed the latest (grew up to $2.29{\pm}0.43{\mu}m$ in shell length). The survival was the highest (36.9%) under the experiment condition fed mixture live food and experimental group fed T. tetrathele showed the lowest rate (16.2%).

Anatomical Studies on Root Formation in Hypocotyl and Epicotyl Cuttings of Woody Plants (임목(林木)의 배축(胚軸) 및 유경삽수발근(幼茎揷穗発根)의 해부학적(解剖学的) 연구(研究))

  • Choi, Man Bong
    • Journal of Korean Society of Forest Science
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    • v.52 no.1
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    • pp.1-30
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    • 1981
  • The origin and development of adventitious roots was studied using hypocotyl and epicotyl cuttings of 34 species, 24 genus of woody plants. These cuttings obtained from young seedlings cultured in vials containing distilled water only. The several characteristics of cuttings materials studied are shown in Table 1. The results are summerized as follows: 1. The circumference shapes of cross-sections of hypocotyl and epicotyl cuttings can be divided into six categories, namely, round, irregular round, ellipse, irregular ellipse, square, and triangle. Species differences within a genus did not show any difference of hypocotyl and epicotyl cross-sections shape, however, a noticeable variation among genus or higher taxa. 2. The arrangements of vascular bundles in the cross-sections of hypocotyls or epicotyls were almost all collateral types and generally showed generic characteristics differing one to the other. However, there were some variations between species within the genus. Six models of vascular bundle arrangement were proposed for all the above speices. 3. The rooting portions of hypocotyl and epicotyl cuttings in this experimental materials can be grouped as follows: (1) Interfascicular parenchyma; (Thuja orientalis. T. orientalis for. sieboldii, Acer microsieboldianum, A. palmatum, A. saccharinum, Cercis chinensis, Lespedeza bicolor, Magnolia obovata, M. sieboldii, Mallotus japonicus, Staphylea bumalda) (2) Cambial and phloem parenchyma: (Chamaecyparis obtusa, C. pisifera, Albizzia julibrissin, Buxus microphylla var. Koreana, Cereis chinensis, Euonymus japonica, Firmiana platanifolia, Lagerstroemia indica, Ligustrum salicinum, L. obtusifolium, Magnolia kobus, M. obovata, Mallotus japonicus, Morus alba, Poncirus trifoliata, Quercus myrsinaefolia, Rosa polyantha, Styrax japonica, Styrax obassia) (3) Primary ray tissues; (Euonymus japonica, Styrax japonica) (4) Leaf traces; (Quercus acutissima, Q. aliena) (5) Cortex parenchyma; (Ailanthus altissima) (6) Callus tissues; (Castanea crenata, Quercus aliena, Q. myrsinaefolia, Q. serrata) 4. As a general tendency throughout the species studied, in hypocotyl cuttings, the adventitious root primordia were originated from the interfascicular parenchyma tissue, however, leaf traces and callus tissues were contributed to the root primordia formation in epicotyl cuttings. The hypocotyl cuttings of Ailanthus altissima exhibited a special performance in the root primordia formation, this means that cortex parenchyma was participated to the origin tissue. And in Firmiana platanifolia, differening from the other most species, the root primordia were formed at the phloem parenchyma adjacent outwardly to xylem tissue of vascular bundle system as shown photo. 48. 5. All the easy-to, or difficult-to root species developed adventitious roots in vials filled with distilled water. In the difficult-to-root species, however, root formations seemed to be delayed because they almost all had selerenchyma or phloem fiber which gave some mechanical hindrance to protrusion of root primordia. On the other hand, in the easy-to-root species they seemed to form them more easily because they did not have the said tissues. The rooting portions between easy-to-root and difficult-to-root species have not clearly been distinguished, and they have multitudinous variations. 6. The species structured with the more vascular bundles in number compared with the less vascular bundles exhibited delayed rooting. In the cuttings preparation, the proximal end of cuttings was closer to root-to-stem transition region, the adventitious root formation showed easier. 7. A different case occured however with the mature stem cuttings, in both the needle-leaved and the broad-leaved species. In the hypocotyl cuttings, parenchymatous tissues sited near the vascular bundles become the most frequent root forming portions in general and relevant distinctions between both species were hardly recognizable. 8. In the epicotyl cuttings, root primordia originated mainly in leaf traces in connection with cambial and phloems or callus tissues itself. In the hypocotyl cuttings, interfascicular parenchyma was the most frequent portion of the root primordia formation. The portions of root primordia had more connection with vascular cambium system, as the tissues were continuing to be developed.

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THE FOOD AND GROWTH OF THE LARVAE OF THE ARK SHELL ANADARA BROUGHTONI SCHRENCK (피조개의 먹이와 성장)

  • Yoo Sung Kyoo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.2 no.2
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    • pp.147-154
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    • 1969
  • The larvae of the ark shell Anadare broughtoni(Schrenck) were grown at room temporature (approximately $20.4^{\circ}C$), and fed laboratory-cultured Cyclotella nana. The egg of the ark shell produced in the laboratory measured about $54.9\mu$ in diameter. The embryos gradually developed into larvae up to $110.8\mu$ shell length, $83.9\mu$ shell height and with shell breadth of $58.2\mu$ even in the absence of the algal food. Beyond this sire, however, the growth of the larvae was considerably retarded. The larvae showed better growth rate when they were fed the algal food two days after spawning, i. e., early straight-hinge stage. Daily rate of food consumption varies according to the larval sizes. But the rate increases considerably when the larvae begin to form umbos. In general the rate Is indicated by the following formula: $Y=0.0025161\;X^{2.76459}$. The growth experiments of the larvae indicate that the efficiency of food conversion was higher when fed centrifuged food. Regarding to the difference in the slopes of growth curve, centrifuged food showed better growth rate as compared to those grown with the non-centrifuged food. The smaller the larval size, the greater will be the difference in growth. The larvae began settling when they reathed 261.7 to $289.6\;{\mu}$ in shell length, 199.2 to $221.7\mu$ in shell height and 147.6 to $170.8\mu$ in shell breadth. The time which elapsed from spawning to the larval settlement was about 28 days. The mean growth of the larvae is indicated with regression line and exponential curve equations as follows. Regression line shell length. 94.3 to $133.9\mu$ : Y==85.22857+3.35000X 141.6 to $269.3\mu$: Y=10.83036X-36.05357 296.8 to $373.2\mu$ : Y=19.10000X-279.30000 shell height: 72.7 to $89.7\mu$ : Y=67.11429+2.15714X 108.4 to $206.4\mu$ : Y=8.31607X-27.45357 228.6 to $282.1\mu$: Y=173.46700+13.37500X shell breadth: 45.3 to $77.8\mu$ : Y=38.08510X+2.73570X 87.4 to $157.7\mu$: Y=5.77320X-5.99640 175.4 to $214.0\mu$: Y=19.65000X-114.13300 Exponential curve shell length. 94.3 to $373.2\mu$: Y=72.45 $e^{0.04697x}$ shell height: 72.7 to $282.1\mu$: Y=54,96 $e^{0.04720x}$ shell breadth: 45.3 to $214.0\mu$ : Y=39.82 $e^{0.04927x}$ The relationships between the shell length and shell height and between the shell length and shell breadth are indicated as follows- shell height: 72.7 to $98.7\mu$ : Y=12.87780+0.63817X 108.4 to $206.4\mu$ : Y=0.90220+0.76456X 228.6 to $282.1\mu$ : Y=25.02630+0.69156X shell breadth: 45.3 to $77.8\mu$:Y=0.81373Xx-31.18914 87.4 to $157.7\mu$ : Y=13.37549+0.53230X 175.4 to $214.0\mu$: Y=30.24328+0.49545X

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Anti-inflammatory Effects of Pentoxifylline and Neutrophil Elastase Inhibitor on Lipopolysaccharide-Induced Acute Lung Injury In Vitro (In Vitro 내독소 유도성 급성 폐손상에서 Pentoxifylline과 Neutrophil Elastase Inhibitor의 항염효과)

  • Kim, Young-Kyoon;Kim, Seung-Joon;Park, Yong-Keun;Kim, Seok-Chan;Kim, Kwan-Hyoung;Moon, Hwa-Sik;Song, Jeong-Sup;Park, Sung-Hak;Kim, Sang-Ho
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.6
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    • pp.691-702
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    • 2000
  • Background : Acute lung injury (ALI) is a commonly encountered respiratory disease and its prognosis is poor when the treatment is not provided promptly and properly. However no specific pharmacologic treatment is currently available for ALI, although recently several supportive drugs have been under scrutiny. We studied anti-inflammatory effects of pentoxifylline (PF), a methylated xanthine, and ONO-5046, a synthetic neutrophil elastase inhibitor on lipopolysaccharide (LPS)-induced ALI in vitro. Methods : To establish an in vitro model of LPS-induced ALI, primary rat alveolar macrophages and peripheral neutrophils in various ratios (1:0, 5:1, 1:1, 1:5, 0:1) were co-cultured with transformed rat alveolar epithelial cells (L2 cell line) or vascular endothelial cells (IP2-E4 cell line) under LPS stimulation. Each experiment was divided into five groups-control, LPS, LPS+PF, LPS+ONO, and LPS+PF+ONO. We compared LPS-induced superoxide anion productions from primary rat alveolar macrophages and peripheral neutrophils in various ratios, and the resultant cytotoxicity on L2 cells or IP2-E4 cells between groups. In addition we also compared the productions of tumor necrosis factor (TNF)-$\alpha$ interleukin (IL)-$1{\beta}$, monocyte chemotactic protein(MCP)-1, IL-6, and IL-10 as well as mRNA expressions of TNF-$\alpha$ inducible nitric oxide synthetase(iNOS), and MCP-1 from LPS-stimulated primary rat alveolar macrophages between groups. Results : (1) PF and ONO-5046 in each or both showed a trend to suppress LPS-induced superoxide anion productions from primary rat alveolar macrophages and peripheral neutrophils regardless of their ratio, except for the LPS+PF+ONO group with the 1:5 ratio, although statistical significance was limited to a few selected experimental conditions. (2) PF and ONO-5046 in each or both showed a trend to prevent IP2-E4 cells from LPS-induced cytotoxicity by primary rat alveolar macrophages and peripheral neutrophils regardless their ratio, although statistical significance was limited to a few selected experimental conditions. the effects of PF and/or ONO-5046 on LPS-induced L2 cell cytotoxicity varied according to experimental conditions. (3) PF showed a trend to inhibit LPS-induced productions of INF-$\alpha$ MCP-1, and IL-10 from primary rat alveolar macrophages. ONO-5046 alone didnot affect the LPS-induced productions of proinflammatory cytokines from primary rat alveolar macrophages but the combination of PF and ONO-5046 showed a trend to suppress LPS-induced productions of INF-$\alpha$ and IL-10 PF and ONO-5046 in each or both showed a trend to increase LPS-induced IL-$\beta$ and IL-6 productions from primary rat alveolar macrophages. (4) PF and ONO-5046 in each or both showed a trend to attenuate LPS-induced mRNA expressions of TNF-$\alpha$ and MCP-1 from primary rat alveolar macrophages but at the same time showed a trend increase iNOS mRNA expression. Conclusion : These results suggest that PF and ONO-5046 may play a role in attenuating inflammation in LPS-induced ALI and that further study is needed to use these drugs as a new supportive therapeutic strategy for ALI.

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Microbiological and Enzymological Studies on Takju Brewing (탁주(濁酒) 양조(釀造)에 관(關)한 미생물학적(微生物學的) 및 효소학적(酵素學的) 연구(硏究))

  • Kim, Chan-Jo
    • Applied Biological Chemistry
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    • v.10
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    • pp.69-100
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    • 1968
  • 1. In order to investigate on the microflora and enzyme activity of mold wheat 'Nuruk' , the major source of microorganisms for the brewing of Takju (a Korean Sake), two samples of Nuruk, one prepared at the College of Agriculture, Chung Nam University (S) and the other perchased at a market (T), were taken for the study. The molds, aerobic bacteria, lactic acid bacteria, and yeasts were examined and counted. The yeasts were classified by the treatment with TTC (2, 3, 5 triphenyltetrazolium chloride) agar that yields a varied shade of color. The amylase and protease activities of Nuruk were measured. The results were as the followings. a) In the Nuruk S found were: Aspergillus oryzae group, $204{\times}10^5$; Black Aspergilli, $163{\times}10^5$; Rhizogus, $20{\times}10^5$; Penicillia, $134{\times}10^5$; Areobic bacteria, $9{\times}10^6-2{\times}10^7$; Lactic acid bacteria, $3{\times}10^4$ In the Nuruk T found were: Aspergillus oryzae group, $836{\times}10^5$; Black Aspergilli, $286{\times}10^5$; Rhizopus, $623{\times}10^5$; Penicillia, $264{\times}10^5$; Aerobic bacteria, $5{\times}10^6-9{\times}10^6$; Lactic acid bacteria, $3{\times}10^4$ b) Eighty to ninety percent of the aerobic bacteria in Nuruk S appeared to belong to Bacillus subtilis while about 70% of those in Nuruk T seemed to be spherical bacteria. In both Nuruks about 80% of lactic acid bacteria were observed as spherical ones. c) The population of yeasts in 1g. of Nuruk S was about $6{\times}10^5$, 56.5% of which were TTC pink yeasts, 16% of which were TTC red pink yeasts, 8% of which were TTC red yeasts, 19.5% of which were TTC white yeasts. In Nuruk T(1g) the number of yeasts accounted for $14{\times}10^4$ and constituted of 42% TTC pink. 21% TTC red pink 28% TTC red and 9% TTC white. d) The enzyme activity of 1g Nuruk S was: Liquefying type Amylase, $D^{40}/_{30},=256$ W.V. Saccharifying type Amylase, 43.32 A.U. Acid protease, 181 C.F.U. Alkaline protease, 240C.F.U. The enzyme activity of 1g Nuruk T was: Liquefying type Amylase $D^{40}/_{30},=32$ W.V. Saccharifying type amylase $^{30}34.92$ A.U. Acid protease, 138 C.F.U. Alkaline protease 31 C.F.U. 2. During the fermentation of 'Takju' employing the Nuruks S and T the microflora and enzyme activity throughout the brewing were observed in 12 hour intervals. TTC pink and red yeasts considered to be the major yeasts were isolated and cultured. The strains ($1{\times}10^6/ml$) were added to the mashes S and T in which pH was adjusted to 4.2 and the change of microflora was examined during the fermentation. The results were: a) The molds disappeared from each sample plot since 2 to 3 days after mashing while the population of aerobic bacteria was found to be $10{\times}10^7-35{\times}10^7/ml$ inS plots and $8.2{\times}10^7-12{\times}10^7$ in plots. Among them the coccus propagated substantially until some 30 hours elasped in the S and T plots treated with lactic acid but decreased abruptly thereafter. In the plots of SP. SR. TP. and TR the coccus had not appeared from the beginning while the bacillus showed up and down changes in number and diminished by 1/5-1/10 the original at the end stage. b) The lactic acid bacteria observed in the S plot were about $7.4{\times}10^7$ in number per ml of the mash in 24 hours and increased up to around $2{\times}10^8$ until 3-4 days since. After this period the population decreased rapidly and reached about $4{\times}10^5$ at the end, In the plot T the lactic acid becteria found were about $3{\times}10^8$ at the period of 24 fours, about $3{\times}10$ in 3 days and about $2{\times}10^5$ at the end in number. In the plots SP. SR. TP, and TR the lactic acid bacteria observed were as less as $4{\times}10^5$ at the stage of 24 hours and after this period the organisms either remained unchanged in population or ceased to exist. c) The maiority of lactic acid bacteria found in each mash were spherical and the change in number displayed a tendency in accordance with the amount of lactic acid and alcohol produced in the mash. d) The yeasts had showed a marked propagation since the period of 24 hours when the number was about $2{\times}10^8$ ㎖ mash in the plot S. $4{\times}10^8$ in 48 hours and $5-7{\times}10^8$ in the end period were observed. In the plot T the number was $4{\times}10^8$ in 24 hours and thereafter changed up and down maintaining $2-5{\times}10^8$ in the range. e) Over 90% of the yeasts found in the mashes of S and T plots were TTC pink type while both TTC red pink and TTC red types held range of $2{\times}10-3{\times}10^7$ throughout the entire fermentation. f) The population of TTC pink yeasts in the plot SP was as $5{\times}10^8$ much as that is, twice of that of S plot at the period of 24 hours. The predominance in number continued until the middle and later stages but the order of number became about the same at the end. g) Total number of the yeasts observed in the plot SR showed little difference from that of the plot SP. The TTC red yeasts added appeared considerably in the early stage but days after the change in number was about the same as that of the plot S. In the plot TR the population of TTC red yeasts was predominant over the T plot in the early stage which there was no difference between two plots there after. For this reason even in the plot w hers TTC red yeasts were added TTC pink yeasts were predominant. TTC red yeasts observed in the present experiment showed continuing growth until the later stage but the rate was low. h) In the plot TP TTC pink yeasts were found to be about $5{\times}10^8$ in number at the period of 2 days and inclined to decrease thereafter. Compared with the plot T the number of TTC pink yeasts in the plot TP was predominant until the middle stage but became at the later stage. i) The productivity of alcohol in the mash was measured. The plot where TTC pink yeasts were added showed somewhat better yield in the earely stage but at and after the middle stage the difference between the yeast-added and the intact mashes was not recognizable. And the production of alcohol was not proportional to the total number of yeasts present. j) Activity of the liquefying amylase was the highest until 12 hours after mashing, somewhat lowered once after that, and again increased around 36-48 hours after mashing. Then the activity had decreased continuously. Activity of saccharifying amylase also decreased at the period of 24 hours and then increased until 48 hours when it reached the maximum. Since, the activity had gradually decreased until 72 hours and rapidly so did thereafter. k) Activity of alkaline protease during the fermentation of mash showed a tendency to decrease continusously although somewhat irregular. Activity of acid protease increased until hours at the maximum, then decreased rapidly, and again increased, the vigor of acid protease showed better shape than that of alkaline protease throughout. 3. TTC pink yeasts that were predominant in number, two strains of TTC red pink yeasts that appeared throughout the brewing, and TTC red yeasts were identified and the physiological characters examined. The results were as described below. a) TTC pinkyeasts (B-50P) and two strains of TTC red pink yeasts (B-54 RP & B-60 RP) w ere identified as the type of Saccharomyces cerevisiae and TTC pink red yeasts CB-53 R) were as the type of Hansenula subpelliculosa. b) The fermentability of four strains above mentioned were measured as follows. Two strains of TTC red pink yeasts were the highest, TTC pink yeasts were the lowest in the fermantability. The former three strains were active in the early stage of fermentation and found to be suitable for manufacturing 'Takju' TTC red yeasts were found to play an important role in Takju brewing due to its strong ability to produce esters although its fermentability was low. c) The tolerance against nitrous acid of strains of yeast was marked. That against lactic acid was only 3% in Koji extract, and TTC red yeasts showed somewhat stronger resistance. The tolerance against alcohol of TTC pink and red pink yeasts in the Hayduck solution was 7% while that in the malt extract was 13%. However, that of TTC red yeasts was much weaker than others. Liguefying activity of gelatin by those four strains of yeast was not recognized even in 40 days. 4. Fermentability during Takju brewing was shown in the first two days as much as 70-80% of total fermentation and around 90% of fermentation proceeded in 3-4 days. The main fermentation appeared to be completed during :his period. Productivity of alcohol during Takju brewing was found to be apporximately 65% of the total amount of starch put in mashing. 5. The reason that Saccharomyces coreanuss found be Saito in the mash of Takju was not detected in the present experiment is considered due to the facts that Aspergillus oryzae has been inoculated in the mold wheat (Nuruk) since around 1930 and also that Koji has been used in Takju brewing, consequently causing they complete change in microflora in the Takju brewing. This consideration will be supported by the fact that the original flavor and taste have now been remarkably changed.

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