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Calcium Bioavailability and Antiosteoporotic Effects of Cheonggukjang Containing High Poly-Gamma Glutamate Contents (고함량 폴리감마글루탐산(PGA) 청국장에 대한 칼슘 생체이용률 및 골다공증에 미치는 영향)

  • Lee, Mu-Jin;Jung, Ho-Kyung;Sim, Mi-Ok;Jang, Ji-Hun;Kim, Tae-Mook;Lee, Hyun-Joo;Lee, Ki-Ho;An, Byeong-Kwan;Cho, Jung-Hee;Jang, Min-Cheol;Yong, Ju-Hyun;Kim, Jong-Choon;Cho, Hyun-Woo
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.45 no.11
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    • pp.1544-1551
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    • 2016
  • Various types of fermented soybean foods are consumed in various Asian countries, including China, Indonesia, Japan, Korea, and Vietnam. Cheonggukjang is a Korean whole soybean paste fermented by Bacillus subtilis and regarded as a healthy food. The objective of this study was to investigate the Ca-bioavailability and anti-osteoporotic effects of cheonggukjang (CGJ) containing high poly-gamma glutamate (PGA) contents in an animal model. Blood samples were collected from the jugular vein at 0, 0.5, 1, 2, 3, 4, and 5 h after a single oral dose in male rats. Ca-bioavailability in CGJ containing high PGA contents was approximately 3~5 times higher than that of natural CGJ. Female rats were either sham-operated (sham; n=5) or surgically ovariectomized (OVX; five animals per group) and then administered to OVX control, raloxifene hydrochloride (RLX) 1 mg/kg/d, or CGJ containing high contents of PGA (20 and 200 mg/kg/d) for 12 weeks. Serum osteocalcin concentration was significantly lower in the CGJ 200 mg/kg/d group compared with the OVX control group. Serum progesterone concentration was significantly higher in the CGJ 200 mg/kg/d group compared with the OVX control group. Reduction grade of the trabecular bone decreased in the RLX 1 and CGJ 200 mg/kg/d group compared with that of the OVX control group. In conclusion, CGJ 200 mg/kg/d may have inhibitory effects on osteoporosis in OVX rats, and Ca-bioavailability was improved in CGJ containing high PGA contents.

Antioxidative and Antibacterial Activities of Artemisia princeps Pampanini Extracts (사자발쑥 추출물의 항산화 및 항균 활성)

  • Yang, Hyun Gab;Kim, Hye Jin;Kim, Hae Soo;Park, Soo Nam
    • Microbiology and Biotechnology Letters
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    • v.40 no.3
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    • pp.250-260
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    • 2012
  • In the present study, the antioxidative and antibacterial activities of Artemisia princeps Pampanini (A. princeps Pamp.) extract were investigated. The ethyl acetate fraction of A. princeps Pamp. showed the most prominent free radical (1,1-diphenyl-2-picrylhydrazyl, DPPH) scavenging activity ($FSC_{50}=12.27{\mu}g/mL$). Reactive oxygen species (ROS) scavenging activities ($OSC_{50}$) of A. princeps Pamp. extract on $Fe^{3+}-EDTA/H_2O_2$ systems were investigated using a luminol-dependent chemiluminescence assay. The ethyl acetate fraction of the extract ($OSC_{50}=0.33{\mu}g/mL$) had a 5 times greater ROS scavenging activity than L-ascorbic acid ($1.50{\mu}g/mL$), known as a water soluble antioxidant. The cellular protective effects of fractions of A. princeps Pamp. on the rose-bengal sensitized photohemolysis of human erythrocytes were examined. The aglycone fraction of extracts suppressed photohemolysis in a concentration dependent manner. The inhibitory effects of A. princeps Pamp. extract on tyrosinase were investigated to assess their whitening efficiency. The ethyl acetate fraction demonstrated a 7 times higher tyrosinase inhibitory effect ($IC_{50}=29.20{\mu}g/mL$) than albutin, known as a whitening agent. The antibacterial activity of ethyl acetate fractions against various normal skin flora were measured. The results showed that the antibacterial activity of the fraction was the highest on Staphylococcus aureus, Bacillus subtilis, and Propionibacterium acnes. Antioxidant substances were isolated and purified from the ethyl acetate fractions. Eupatilin and jaceosidin were identified. These results indicate that the extract/fractions of A. princeps Pamp. can function as antioxidant and/or antibacterial agents for the skin.

Antimicrobial and Hemolytic Activity of Oriental Medicinal Herbs (한약재의 항균 활성 및 인간 적혈구 용혈 활성)

  • Ryu, Hee-Young;Ahn, Seon-Mi;Shin, Yong-Kyu;Sohn, Ho-Yong
    • Microbiology and Biotechnology Letters
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    • v.38 no.2
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    • pp.190-197
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    • 2010
  • To develop the safe and natural antimicrobial agents, the 68 ethanol extracts from the 61 different kinds of oriental herbal medicine were prepared and their antimicrobial activities were evaluated. The herbal medicine used were from China (46 kinds), South Korea (14 kinds), North Korea (5 kinds) and Vietnam (3 kinds), respectively, and the root (27 species) was popular part in this study. The average water content and extraction ratio for ethanol were 7.10% and 6.75%, respectively. Determination of antimicrobial activity by disc-diffusion assay at 0.5 mg/disc concentration showed that the extract of Angelica tenuissima Nakai (china), Illicium verum, Junci medulla, Rhus javanica L., Salvia miltiorrhiza Bunge and Syzygium aromaticum has strong antimicrobial activities against different food spoilage and pathogenic bacteria and fungi. Determination of MIC and MBC/MFC further showed that the extract of Syzygium aromaticum has MIC of 1.25 mg/mL and MBC/MFC of 1.25~5.00 mg/mL against Listeria monocytogenes, Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus, Salmonella typhimurium, Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Saccharomyces cerevisiae. And, the extract of Junci medulla, Rhus javanica L. and Salvia miltiorrhiza Bunge showed strong antibacterial activities with MIC of 0.08~0.63 mg/mL and MBC/MFC of 0.08~2.50 mg/mL against the tested bacteria except E. coli and P. aeruginosa. In a while, the results of hemolytic activity of 68 different herbal extracts against human red blood cells showed that the extract of Angelica tenuissima Nakai has hemolytic activity at 0.5 mg/mL concentration. Therefore, Illicium verum, Junci medulla, Rhus javanica L., Salvia miltiorrhiza Bunge and Syzygium aromaticum were finally selected for natural antimicrobial resources. Further research on active substances and the mode of action of the selected herbal medicine is necessary.

Antioxidative, Antimicrobial and Cytotoxic activities of Fagopyrum esculentum $M{\ddot{o}}ench$ Extract in Germinated Seeds (발아 메밀 추출물의 항산화.항균활성 및 세포독성)

  • Hwang, Eun-Ju;Lee, Sook-Young;Kwon, Su-Jung;Park, Min-Hee;Boo, Hee-Ock
    • Korean Journal of Medicinal Crop Science
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    • v.14 no.1
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    • pp.1-7
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    • 2006
  • This research was conducted to investigate the possibilities of usage of germinated-buckwheat (Fagopyrum esculentum $M{\ddot{o}}ench$) by examining antioxidative, antimicrobial and cytotoxic effects of extracts from different germinated root length of buckwheat. Antioxidant activity $(RC_{50})$ was shown higher in extracts of non-germinated seed $(50.41\;{\mu}g/mL)$ and root length 10 mm $(80.57\;{\mu}g/mL)$, 2 mm $(93.77\;{\mu}g/mL)$, 5 mm $(107.09\;{\mu}g/mL)$ than BHT $(163.96\;{\mu}g/mL)$ as a synthetic antioxidant. In antimicrobial activity, non-germinated and germinated seeds were formed inhibitory zone against S. aureus $(4{\sim}10\;mm)$, P. aeruginosa $(2{\sim}9\;mm)$ at the concentrations of $10{\sim}40\;mg/mL$ but B. subtilis, E. coli and S. typhimurium were not apparent antimicrobial activity. Extracts of germinated seed also decreased their antimicrobial activity compared to non-germinated seed extract. In addition, the growth of Calu-6 was inhibited of both 5 mm root length germinated and non-germinated seeds $(800\;{\mu}g/mL)$ as 95.12% and 87.15%, respectively, but these did not show any influence on cytotoxic effect against MCF-7 and Caco-2 cell lines. Extracts of 2 mm and 5 mm germinated seeds were also inhibited against Calu-6 and SNU-601 cell lines.

Microbial community analysis of an eco-friendly recirculating aquaculture system for olive flounder (Paralichthys olivaceus) using complex microbial probiotics (복합미생물 프로바이오틱을 이용한 환경친화적 넙치 순환여과양식시스템에서의 미생물군집 분석)

  • Rhee, Chaeyoung;Kim, Haham;Emmanuel, S. Aalfin;Kim, Hong-Gi;Won, Seonghun;Bae, Jinho;Bai, Sungchul C.;Koh, Sung-Cheol
    • Korean Journal of Microbiology
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    • v.54 no.4
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    • pp.369-378
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    • 2018
  • This study was conducted to evaluate effects of dietary microbial probiotics on the growth and disease resistance of olive flounder (Paralichthys olivaceus) in a recirculating aquaculture system (RAS), and the effects of the probiotic bioaugmentation on the microbial community structure and water quality. For the analysis, 80 juvenile fish (average weight, $25.7{\pm}7.6g$; average length, $15.2{\pm}1.7cm$) were fed a basal diet containing a commercial microbial product CES-AQ1 (CES; $1{\times}10^9\;CFU/kg$ diet) in an RAS for 8 weeks. Weight gain, the specific growth rate, feed efficiency, and protein efficiency ratio of the fish fed the CES diet in the RAS were 1.5~2.5 times higher than those of fish fed the basal diet alone, or the basal diet containing oxytetracycline (OTC), yeast plus bacterium, or Bacillus subtilis in a still water system. There was no significant difference in the pathogen challenge test between fish fed the OTC diet and fish fed the CES diet in the RAS, suggesting the CES-AQ1 probiotic used in the RAS as a potential replacement for antibiotics. The RAS biofilter maintained the highest microbial diversity and appeared to harbor microbial communities with ammonium oxidation, denitrification, and fish pathogen suppression functions. Ammonia, which is hazardous to fish, was significantly decreased to < 0.5 mg/L in 19 days, indicating the effectiveness of probiotic supplementation to maintain good water quality in RAS. These results suggest that the intestinal microbial communities of fish are stabilized by a probiotic-containing diet (CES) and that bioaugmentation with probiotics may be an eco-friendly and economical supplement for aquaculture of olive flounder, promoting both good water quality and fish health in an RAS.

Efficacy and Safety Evaluation of an Air Sterilizer Equipped With an Electrolytic Salt Catalyst for the Removal of Indoor Microbial Pollutants (염촉매 전기분해 공기살균기의 효능 평가)

  • Sun Nyoung Yu;Ho-Yeon Jeon;Bu Kyung Kim;Ae-Li Kim;Kyung Il Jung;Gye Rok Jeon;Soon Cheol Ahn
    • Journal of Life Science
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    • v.34 no.7
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    • pp.500-508
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    • 2024
  • Recently, there has been increasing interest in enhancing the indoor air quality, particularly in response to the growing utilization of public facilities. The focus of this study was on assessing the efficacy and safety of an air sterilizer equipped with electrolytic salt catalysts. To that end, we evaluated the antimicrobial activity of the vapor spraying from the air sterilizer and its cytotoxicity in condensed form on human cell lines (HaCaT, BEAS-2B, and THP-1). Against the test organisms, which comprised five bacterial strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium) and one fungal strain (Candida albicans), the air sterilizer exhibited relatively high antimicrobial activities ranging from 10.89 to 73.98% following 1 and 3 hr of vapor spraying, which were notably time-dependent. Importantly, cytotoxicity assessments on human cells indicated no significant harmful effect even at a 1.0% concentration. Comprehensive safety evaluations included morphological observations, gene expression (Bcl-2, Bax) tests, and FACS analysis of intracellular ROS levels. Consistent with previous cytotoxicity findings, these estimates demonstrated no significant changes, highlighting the air sterilizer's safety and antimicrobial activities. In a simulated 20-hr operation within an indoor environment, the air sterilizer not only showed an 89.4% removal of total bacteria but also a 100.0% removal of Escherichia sp. and fungi. This research outlines the potential of the developed electrolytic salt catalyst air sterilizer to effectively remove indoor microbial pollutants without compromising human safety, underscoring the solution that it offers for improving indoor air quality.

Changes in quality characteristics of makjang depending on fermentation location and complex starters (발효 장소와 복합 종균에 따른 막장의 품질 특성 변화)

  • Jieon Park;Myeong-Hui Han;Woosoo Jeong;Soo-Hwan Yeo;So-Young Kim
    • Food Science and Preservation
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    • v.30 no.6
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    • pp.1056-1071
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    • 2023
  • This study aimed to investigate the quality and microbial population changes for 90 days under two fermentation conditions, outdoors and indoors (35℃), with starters (single or mixed) in soybean paste. Bacillus velezensis NY12-2 (S1), Debaryomyces hansenii D5-P5 (S2), Enterococcus faecium N78-11 (S3), and their mixtures (M) were used for the makjang fermentation. The content of amino-type nitrogen among the makjang samples was highly shown in the indoors, followed by M, S3, and S2. The glutamic and aspartic acid contents in the M sample fermented in the indoors showed the highest values of 867.42±77.27 and 243.20±15.79 mg/g, respectively. By the electronic tongue analysis, the M sample fermented in the indoors exhibited lower saltiness and higher umami than the others. Consequently, we expect that using mixed strains, such as Bacillus, Debaryomyces, and Enterococcus, under constant conditions showed potential to the quality improvement of soy products.

Microbiological Studies of Korean Native Soy-sauce Fermentation -A Study on the Microflora Changes during Korean Native Soy-sauce Fermentation- (한국재래식(韓國在來式) 간장의 발효미생물(醱酵微生物)에 관(關)한 연구(硏究)(제2보(第二報)) -한국재래식(韓國在來式) 간장의 담금중(中)에 있어서의 발효미생물군(醱酵微生物群)의 소장(消長)에 관(關)한 연구(硏究)-)

  • Lee, Woo-Jin;Cho, Duck-Hiyon
    • Applied Biological Chemistry
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    • v.14 no.2
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    • pp.137-148
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    • 1971
  • Studies were carried out to investigate the main fermentation microorganisms and their flora changes during Korean native soy-sauce fermentation. Korean native Maeju loaves collected from 5 Do's were separated into surface and inner parts. Four different soy-sauces-the surface part Maeju fermented soy-sauce, the inner part, the surface and inner part combined Maeju fermented soy-sauce, and the semi-Japanese type soy-sauce were fermented and the changes of fermentation microorganism flora and the various chemical components during the period of their fermentations were studied. Besides, 14 home-made soy-sauces collected from 14 different places all over Korea were examined in comparison with the laboratory soy-sauces and to determine the characteristics of Korean native soy-sauce. The results were as follows: 1. The main microorganisms in Korean native soy-sauce fermentation were determined as; Aerobic bacteria: Bacillus subtilis, Bacillus pumilus Lactic acid bacteria: Pediococcus halophilus, Leuconostoc mesenteroides Yeasts: Torulopsis datila, Saccharomyces rouxii 2. Microflora changes during Korean native soy-sauce fermentation were as follows; Aerobic bacteria increased until the 2nd week of fermentation and then gradually decreased. The lactic acid bacteria increased until the 3rd week, after which decreased. When the lactic acid fermentation lowered the pH value to below the 5.4, yeasts were able to grow and participate the fermentation. As the production of organic acids amounted, to a certain height, the growth of all microorganisms lead to the period of decline or death at about the 2nd month of fermentation. After boiling of soy-sauce most microorganisms except a few of Bacillus sp. disappeared. Occosionally yeasts and lactic acid bacteria survived depending upon the composition of soy-sauce. 3. Changes of general chemical components influencing the microflora were investigated for the period of Korean native soy-sauce fermentation. Tetal acidity, salt concentration and total nitrogen were increasing steadily over the entire period of fermentation. pH values were dropping to a certain degree of about 4.5. Salt concentration and pH value seemed to be the important factors influencing the microflora. 4. The microflora were influenced by chemical components of soy-sauce. Aerobic bacteria were able to survive in all soy-sauce as they made spores. Growth of lactic acid bacteria was inhited at 23-26% of salt concentration and pH 4.8. Soy-sauce yeasts started to grow only at pH below 5.4 and seemed to be inhibited at around 26% of salt concentration under pH 4.5-4.7. 5. The open kettle boiling of soy-sauce, the characteristic process of Korean native soy-sauce manufacturing, was effective to sterilize microorganisms, increase the salt concentration, and coagulate proteins. 6. The average viable cell counts of microorganism found in collected samples of home-made Korean native soy-sauces were; Aerobic bacteria: $53{\times}10^2\;cell/ml$ Lactic acid bacteria: 34 cell/ml Yeasts: 14 cell/ml The average values of chemical compositions of samples of home-made Korean native soy-sauce were; Salt concentration: 28.9% pH value: 4.79 Total acidity(lactic acid): 0.91g/100ml Total nitrogen: 1.09g/100ml

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Studies on the Amylase Production by Bacteria (세균(細菌)에 의(依)한 Amylase생산(生産)에 관한 연구(硏究))

  • Park, Yoon-Joong
    • Applied Biological Chemistry
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    • v.13 no.2
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    • pp.153-170
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    • 1970
  • 1. Isolation and identification of amylase-producing bacteria. The powerful strain A-12 and S-8 were respectively isolated from air and soil after screening a large number of amylase-producing bacteria. Their bacterial characteristics have been investigated and it has been found that all characteristics of strain A-12 and S-8 are similar to Bac. subtilis of Bergey's manual except for the acid formation from a few carbohydrates and the citrate utilization, i.e., the strain A-12 shows negative in the citrate utilization, and the acid formation from arabinose and xylose, S-8 shows negative in the acid formation from xylose. 2. Amylase production by Liquid cultures with solid materials. Several conditions for amylase production by strain A-12 in stationary cultures have been studied. The results obtained are as follows. (1) The optimum conditions are:temperature $35^{\circ}C$, initial pH 6.5 to 7.0 and incubation time 3 to 4 days. (2) The amylase production is not affected by the preservation period of the stock cultures. (3) Among the various solid material, the defatted soy bean is found to be the best for t1e amylase production. However, the alkali treatment of the defatted soy bean gives no effect contrary to the cage of defatted rape seed. The addition of soluble starch to the alkali extract of defatted soy bean shows the increased amylase production. (4) Up to 1% addition of ethanol to carbon dificient media gives the improved amylase production, whereas the above effect is not found in the case of carbon rich media. (5) The amylase production can be increased 2.5 times when 10% of defatted soy bean is admixed to cheaply available wheat bran. (6) The excellent effect is found for amylase production when 20% of wheat bran is admixed to defatted dry milk which is a poor medium. The activity is found to be $D^{40^{\circ}}_{30'}$ 7,000(L.S.V. 1,800) in 10% medium. (7) No significant effect is observed due to the addition of various inorganic salts. 3. Amylase production by solid cultures. Several conditions for amylase production by strain A-12 in wheat bran cultures have been studied and the results obtained are as follows. (1) The optimum conditions: are temperature $33^{\circ}C$, incubation lime 2 days, water content added 150 to 175% and the thickness of the medium 1.5cm, The activity is found to be $D^{40^{\circ}}_{30'}$ 36,000(L.S.V. 15,000) (2) No significant effect is found in the case of the additions of various organic and inorganic substances.

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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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