• Title/Summary/Keyword: Fermentation period

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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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Quality Characteristics of Doenjang by Aging Period (전통 된장의 숙성 기간에 따른 감각·화학적 품질특성)

  • Ku, Kyung-Hyung;Park, Kyungmin;Kim, Hyun Jung;Kim, Yoonsook;Koo, Minseon
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.43 no.5
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    • pp.720-728
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    • 2014
  • In order to characterize the quality of Doenjang, fermented Korean soybean paste, subjected to long-term aging, this study performed physico-chemical analyses and sensory evaluation according to aging period (from 1 to 9 years). Regarding the proximate composition of Doenjang according to aging period, moisture, crude protein, crude lipid, crude ash, and salt contents showed little differences among Doenjang samples. Amino-type nitrogen content was 1,046.7 mg% in the 1 year-aged sample, 990.9~996.9 mg% in the 2~5 year-aged samples, and 1,214.1~1,304.8 mg% in the samples fermented more than 5 years. ${\Delta}$E value, reflecting total color differences between the samples, increased according to aging period. Ratios of linoleic and linolenic acids, which are essential fatty acids in soybeans, constituted 55% of total fatty acids, which was the most abundant among all fatty acids. The major free sugar in Doenjang was fructose at a content of 1.6~2.2% in 1~9 year-aged Doenjang. Glycoside form of isoflavones in Meju constituted 77.1%, and the aglycon form constituted 22.9%. However, the glycoside type of isoflavones in soybeans was converted to aglycon type in Doenjang through fermentation and aging. In the sensory evaluation of Doenjang samples, brown color, salt smell, soy sauce flavor, and viscosity all increased according to aging period, whereas sweet flavor, roast smell, beany flavor, salty taste, and acrid taste showed no significant differences. In cluster analysis of the sensory attributes of Doenjang according to aging period, 1 year-aged Doenjang was significantly different between 2 year- and 3~5 year-aged Doenjang.

Characteristics of Chungkookjang that Enhance the Flavor and GABA Content in a Mixed Culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 (Bacillus subtilis MC31와 Lactobacillus sakei 383의 혼합배양으로 향상된 풍미와 GABA 함량을 지닌 청국장의 품질 특성)

  • Lee, Ga-Young;Kim, Su-In;Jung, Min-Gi;Seong, Jong-Hwan;Lee, Young-Guen;Kim, Han-Soo;Chung, Hun-Sik;Lee, Byoung-Won;Kim, Dong-Seob
    • Journal of Life Science
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    • v.24 no.10
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    • pp.1102-1109
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    • 2014
  • Chungkookjang has several functional properties, such as fibrinolytic activity, anticancer effects, and antioxidant effects. However, children do not like Chungkookjang because of its foul odor. A mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 was used to improve the production of GABA in Chungkookjang and its flavor. Most of the foul odor of Chungkookjang was removed. The slime content and viscosity of Chungkookjang fermented in the mixed culture were similar to those of commercial Chungkookjang when B. subtilis MC31 and Lactobacillus sakei 383 were inoculated in a 1:1 ratio. The maximum GABA content was obtained when Chungkookjang was fermented with B. subtilis MC31 and L. sakei 383, which was fermented at $37^{\circ}C$ for 72 hr. During the period of fermentation, the viable cell number of B. subtilis MC31 reached a peak (log 9.13 CFU/g) at six days, and L. sakei 383 reached a peak (log 6.78 CFU/g) at two days. The moisture, crude ash, crude protein, crude fat, and crude fiber contents were 61.71%, 2.05%, 17.54%, 8.36%, and 1.95%, respectively. The amino-type nitrogen content of Chungkookjang fermented by B. subtilis MC31 and L. sakei 383 was less than Chungkookjang fermented by B. subtilis MC31 alone. The ammonia-type nitrogen and reducing sugar content of the Chungkookjang fermented by B. subtilis MC31 and L. sakei 383 were higher than that of steamed soybean. The glutamic acid and GABA content detected with an amino acid analyzer were 1.40 mg/g and 0.47 mg/g, respectively. These results suggest that fermentation with B. subtilis MC31 and L. sakei 383 in a 1:1 ratio removes more of the foul odor and increases the GABA content compared with single fermentation.

Fermentative Properties and Immunomodulating Activity of Low-sodium Kimchi Supplemented with Acanthopanax senticosus and Glycyrrhizae uralensis Extracts (가시오가피와 감초 추출물을 첨가한 저 나트륨 김치의 발효특성과 면역 활성능)

  • Yu, Kwang-Won;Suh, Hyung-Joo;Hwang, Jong-Hyun
    • The Korean Journal of Food And Nutrition
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    • v.25 no.4
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    • pp.878-887
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    • 2012
  • This study investigated the fermentative characteristics and immunomodulating activity in Kimchi added with various salts (salt replacement and herb-salt with Acanthopanax senticosus and Glycyrrhizae uralensis) for the reduction of Na concentration in Kimchi. Kimchi using a salt replacement and herb-salt showed a higher level of acidity (0.8~0.84%) than that of the control (0.7%) at 7-day fermentation. Kimchi using a salt replacement and herb-salt showed a lower level of salinity (1.72~1.98%) than that of control (2.3~2.57%) during fermentation. The growth of Lactobacillus spp. and Leuconostoc spp. recorded the highest level ($2.3{\times}10^8$ and $2.8{\times}10^6cfu/g$, respectively) in control at 6 day-fermentation. However, those levels in Kimchi prepared with salt replacement and herb-salt were $3.5{\sim}5.4{\times}10^8$ and $6.1{\times}10^6cfu/g$, respectively. It is assumed that the high level of acidity of Kimchi prepared with salt replacement and herb-salt was caused by the increase in the growth of Lactobacillus spp. and Leuconostoc spp.. When the macrophage stimulating activity of salt replacement kimchi (Salt-R kimchi) supplemented with hot-water extract from Acanthopanax sentisus (AS) or Glycyrrhiza uralensis (GU) was investigated on aging period, Salt-RA kimchi with AS 5% at 6 days (2.78-fold of saline control at $100{\mu}g/m{\ell}$) and Salt-RG kimchi with GU 5% at 9 days (2.02-fold) significantly increased compared to the Salt-RA kimchi without AS or GU. In addition, Salt-RAG kimchi with AS 3% and GU 3% improved the bitter taste of Salt-RA and potently stimulated the macrophage at 6 days (1.28-fold of Salt-R kimchi) even though its activity was lower than Salt-RA (5%, 1.39-fold).

Investigation on the Amount of Water Evaporation from Composting Facilities Operated in Swine Farms (양돈농가에서 퇴비화시설별 수분변화량 분석에 관한 연구)

  • Kwag, J.H.;Choi, D.Y.;Park, C.H.;Jeong, J.H.;Kim, J.H.;Yoo, Y.H.;Jeon, B.S.;Ra, C.S.
    • Journal of Animal Environmental Science
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    • v.14 no.3
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    • pp.193-200
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    • 2008
  • The results of the research on the amount of water evaporation from composting facilities operated in swine farms are below. The number of swine per a farm was 1433 head/farm for a Simple Composting Facility (SCF) and 3500 head/farm for a Escalator composting facility(ECF) system. The capacities of the SCF and the ECF were $0.33m^3/head$ and $0.25m^3/head$, respectively. The ECF had 24.2% less capacity than the SCF. The average water contents in the swine manure for the CP and the ECF of the surveyed farms were 86.8% and 85.7%, respectively, which revealed the ECF had 1.3% less average water content than the SCF. Daily water inputs into the SCF and the ECF were $4.1kg/m^3/day$ and $6.5kg/m^3/day$, respectively. The ECF had approximately 36.9% higher water input than the SCF. Fermentation temperatures during the composting period for the SCF and the ECF were up to $45^{\circ}C$ and $70^{\circ}C$, respectively. The decreases in water contents per each square meter for the SCF and the ECF were 3.7 kg and 5.2 kg, respectively. The ECF lost approximately 28.8% more water content than the ECF, which would be caused by the difference of fermentation temperature between two systems. Fertilizer components after composting were examined. Nitrogen contents of the SCF and the ECF were similar (0.84% and 0.86%, respectively) and ${P_2}{O_5}$ contents were 0.78% and 0.74%, respectively, showing the SCF had slightly higher content than the ECF. However, OM and OM/N did not show the difference between two systems. Hence, efforts to increase composting efficiency with considerations of the water content of swine manure, fermentation temperature, and water evaporation potential should be done when the SCF and the ECF were used in swine farms.

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Effect of Preparation method and Fermentation Conditions on Microbiological Characteristics of Sikhae (어류를 이용한 식해의 제조 방법에 따른 미생물의 특성변화)

  • Kim, Young-Sook;Oh, Seung-Hee;Kim, Soon-Dong
    • Food Science and Preservation
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    • v.15 no.6
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    • pp.909-914
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    • 2008
  • This study investigated microbial populations and the sensory quality of sikhae including globefish (GLS), flounder (FLS), gastropods (GAS) and whelks (WHS) during storage at 4C for 16 d following fermentation at 10C for 4 d. General bacterial numbers increased to 102 in GLS and FLS, and to 1045 in WHS and GAS after the 20 d fermentation/storage period. Lactic acid bacteria increased to 108 log cycle in GLS and FLS after 10 d ripening time, and reached this level in GAS and WHS after 15 d and 20 d, respectively. After 20 days the number of lactic acid bacteria in each of the four samples was 108. There were 104105 yeast cells/g in each of the four samples after 20 d. The number of Leuconostoc increased to over 108 log cycle after 10 d in GLS and FLS, and 15 days in GAS for WHS the increase was to 107 log cycle. The pH values of GLS, FLS, GAS and WHS 4.42, 4.56, 4.31 and pH 4.26, respectively. The Sikhae acidity for all four samples ranged from 1.551.85%. From the sensory evaluation the overall acceptability was in the order of FLS > GLS > GAS > WHS.

Characterization of Biological Chemistry from Over Ripened Kimchi (과숙김치의 생물.화학적 특성)

  • 문영자;백경아;성창근
    • The Korean Journal of Food And Nutrition
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    • v.14 no.6
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    • pp.512-520
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    • 2001
  • Kimchi is one of the traditional Korean food and a very popular side dish in Korea. To obtain funda mental data on how to prevent over ripening in kimchi after acidity of 0.4% was reached during the lactate fermentation, the physicochemical characteristics such as pH. acidity. organic acids, enzyme activity were measured and the time dependent ecology of microorganism were observed. In the initial stages of fermentation, the pH of kimchi was markedly changed and slowly decreased in 0.5% acidity The acidity was slowly increased and markedly increased in pH 4 by growth of microorganism. HPLC analysis showed oxalic acid, lactic acid, acetic acid, malic acid and succinic acid and this results reconfirmed by GC-MSD. Lactic acid was changed a lot during fermentation period as the time of storage went on, where as malic was decreased. Kimchi A, having acidity of 0.75%, showed the highest acidic Protease and lipase activity. Also, the amylase activity was high in kimchi C, having 0.95% acidity. The total viable bacteria showed 8.1$\times$10$^{5}$ , 4.7$\times$10$^4$, 1.2$\times$10$^3$, 3.2$\times$10$^4$, 4.9$\times$10$^{5}$ cfu/ml in the kimchi A, B, C, D and E, respectively. The numbers of lactic acid bacteria counted 1.0$\times$10$^{5}$ , 1.3$\times$10s, 1.2$\times$10$^3$, 2.3$\times$ 10$^3$, 2.1$\times$10$^4$c1u/m1 in the kimchi A, B, C, D and E, respectively. The numbers of acetobactor were counted 1.8$\times$10$^{5}$ , 9.3$\times$10$^4$, 7.0$\times$10$^1$, 4.5$\times$10$^4$, 5.3$\times$10$^3$cfu/m1 in the kimchi A, B, C, D and E, respectively.

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Changes in Physicochemical Properties and Microbial Population during Fermenting Process of Organic Fertilizer (혼합발효 유기질비료의 발효과정 중 이화학성 및 미생물밀도 변화)

  • Lee, Jong-Tae;Lee, Chan-Jung;Kim, Hee-Dae
    • Korean Journal of Soil Science and Fertilizer
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    • v.37 no.2
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    • pp.116-123
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    • 2004
  • This study was conducted to investigate the changes in physicochemical and microbiological properties during fermenting process of organic fertilizer which was made from the mixture of organic materials such as sesame oil cake, fish meal, blood meal, rice bran, ground bone meal, and natural minerals such as illite, crusted oyster shell and loess. They were mixed and fermented for 70 days. The sesame oil cake and rice bran, major ingredients for organic fertilizers, consisted of 7.6 and 2.6% total nitrogen, 3.6 and 4.6% $P_2O_5$, 1.4 and 2.2% $K_2O$, respectively. The ground bone meal included 29.2% $P_2O_5$ and illite included 3.8% $K_2O$. Temperature of organic fertilizer during the fermentation rapidly increased over $50^{\circ}C$ within 2 days after mixing and stabilized similar to outdoor temperature after 40 days. Moisture content decreased from 36.3 to 16.0% after 1 month. C/N ratio of organic fertilizer slightly increased until 30 days and thereafter, it slowly decreased, It resulted from the faster decrease of total nitrogen concentration compared with organic matter. Concentration of $NH_4-N$ in organic fertilizer rapidly increased from 1,504 to $5,530mg\;kg^{-1}$, the highest concentration after 10 days. Meantime, $NO_3-N$ concentration was low and constant about $150mg\;kg^{-1}$ over the whole fermenting period. This result seemed to be due to the high pH. The organic ferfilizer fermented for 70 days was composed of 2.7% N, 2.8% $P_2O_5$, 1.8% $K_2O$, and 35.9% organic matter. Total populations of aerobic bacteria, Bacillus sp. and actinomycetes, after fermenting process, were $12.5{\times}10^{10}$, $45.5{\times}10^{5}$ and $13.6{\times}10^{5}cfu\;g^{-1}$ respectively. Pseudomonas sp. was $71.9{\times}10^{7}cfu\;g^{-1}$ at first, but it rapidly decreased according to the rise of temperature. Yeasts played an important role in the early stage of fermentation and molds did in the late stage.

The Development of Korean Traditional Wine Using the Fruits of Opuntia ficus-indica var. saboten - I. Characteristics of Mashes and Sojues - (손바닥 선인장 열매를 이용한 전통주 개발 - I. 전통주 제조기법을 이용한 발효주 및 증류주의 특성 -)

  • Bae, In-Young;Yoon, Eun-Ju;Woo, Jeong-Min;Kim, Joo-Shin;Yang, Cha-Bum;Lee, Hyeon-Gyu
    • Applied Biological Chemistry
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    • v.45 no.1
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    • pp.11-17
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    • 2002
  • Fermentation characteristics with/without nitrogen source and quality of the fruit distillate of Opuntia ficus-indica var. saboten were investigated during the manufacturing process of a Korean traditional liquor. As the fermentation period increased, acidity, brix degree, and alcohol concentration increased, whereas pH and contents of reducing sugar decreased. Acidity, pH, and brix degree were higher, whereas the content of reducing sugar lower, in the nitrogen source-added distillate than in the distillate without nitrogen source. The growth of yeast increased, while that of bacteria decreased; this trend was more prominent with the addition of a nitrogen source. Sojues, distilled from two types of mashes and diluted with $H_2O$ and tails of distillate into 22% alcohol concentration, showed pH $3.7{\sim}4.0$, acidity $0.02{\sim}0.10$, and $5.4{\sim}6.1$ $^{\circ}Brix$. Analysis through GC using direct injection methods revealed common volatile flavor compounds in sojues, including acetaldehyde, acetyl acetone, acetic acid ethyl ester, ethyl alcohol, 2-propyl alcohol, acetone, n-propyl alcohol, butanoic acid methyl ester, 2-phenyl ethanol, thymol, acetic acid phenyl ester, and vanillic aldehyde. As revealed through the sensory evaluation, no significant difference (p>0.05) in overall acceptability was shown among four experimental groups, while color and flavor showed significant differences(p<0.05).

Studies on the Bacteriophages of Brevibacterium lactofermentum (L-글루타민산 생산균 Brevibacterium lactofermentum의 Bacteriophag에 관한 연구)

  • 이태우
    • Korean Journal of Microbiology
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    • v.17 no.3
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    • pp.97-130
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    • 1979
  • Many industrial processes those employ bacteria are subjected to phage infestations. In L-glutamic acid fermentions using acetic acid, the phage infestations of the organisms have been recently recognized. In efforts to elucidate the sources of phage contamination involved in the abnormal fermentation, a series of study was conducted to isolate the phages both from the contents of abnormally fermented tanks and the soil or sewage samples from the surroundings of a fermentation factory, to define major charateristics of the phage isolates, and finally to determine the correlation between the phage isolates and temperate phages originating from the miscellaneous bacterial species isolated from the soil or sewage samples. The results are summarized as follows; 1) All phages were isolated from the irregular fermentation tanks and soil or sewage samples, and they were designated as phage PR-1, PR-2, PR-3, PR-4, PR-5, PR-6, and PR-7, in the order of isolation. These PR-series phages were proved to be highly specific for the variant strains of Br. lactofermentum only, namely, phage PR-1 and PR-2 for Br. lactofermentum No. 468-5 and phage PR-3~PR-7 for Br. lactofemrentum No. 2256. By cross-neutralization test, the 7 phagescould be subdivided into 3 groups, i. e., phage PR-I and PR-2 the first, phage PR-3, PR-4, PR-5, PR-6 the second, and the phage PR-7 the third. 2) The 7 phages were virulent under the experimental conditions. They produced plaques with clear and relatively sharp margins without distinct halo. The mean sizes of plaques were 1.5mm in diameter for phage PR-1 and PR-2, and 1. Omm for phages PR-3~PR-7. Double layer technique modified by Hongo and described by Adams, was applied to assay of the PR-series phages. The factors influencing the plaques were as follows;young age cells of host bacteria cultured for 3-6 hours represented the largest number and size, optimum was pH 7.0, incubation temperature was $30^{\circ}C$, and agar concentration and amount of overlayer medium were 0.6% and 0.2ml, respectively. 3) PR-series phages were stable in 0.05M tris buffer and 0.1M ammonium acetate buffer solution. The addition of $5{\times}10^{-3}M$ magnesium ion effectively increased the stability. Thermostability experiments indicated that PR-series phages were stable at the teinperture between $50^{\circ}{\sim}55^{\circ}C$ in nutrient medium, $45^{\circ}{\sim}50^{\circ}C$ in buffer solution. However, the phages mere completely inactivated at 603C and 65$^{\circ}$C within 10 minutes. The phages were stable at the range of pH6~9 in nutrient medium and of pH 8-9 in buffer solution, respectively. Exposure of the phages to UV for 25, 60 and 100 seconds resulted in the complete loss of infectivily, respectively. 4) Electron microscopy showed that PR-series phage particles exhibited rather similar morphology, differing in the size All of PR-series phages had a multilateral head and had a simple long tiil about three to five times long as compared with head. By the size, phage PR-1 and PR-2, PR-3, PR-4, PR-5, and PR-6 and PR-7 were classified into same groups, respectively. The head and tail size of phage PR-1, PR-5, PR-5(T) and PR-7 were 85nm, 74nm and 235nm and 350mm, and 72nm and 210nm, respectively. 5) Nucleic acids of PR-series phages were double stranded DNA. The G+C contents of phage PR-1, PR-5 and PR-7 were 56.1, 52.9 and 53.7, respectively. The values of G+C contents derived from the $T_m$ were in agreement with the chemically determined values. 6) PR-series phages effectively adsorbed on their host bacteria at the rate of more than 90% during 5 min. K value for phage PR-1, PR-5 and PR-7 were calculated to be $6{\times}10^9 ml$ per minute, respectiveky. The pH of the medium did effect adsorption rate, but both temperature and age of host cells did not. Generally, optimum adsorption condition of phages seemed to be almost same as optimum growth conditions of host bacteria. 7) In one-step growth experiments, the latent periods at $30^{\circ}C$ for PR-1, and PR-7 were about 70, 50 and 55 min, respectively. The corresponding average burst size was 200, 70 and 90, respectively. Lpsis period according to the multiplicity of infection and a phage series. In case of m. o. i. 100, strain No. 2256 (PR-5) and No. 468-5(PR-1) failed to grow and turbidity decreased after 50 and 70min, respectively. 8) In the lysate of a plaque purified phage PR-5 infected bacteria, there observed 2 types ofphage particles, i. e., phage PR-5 and PR-5 (T) of similar morphology but differing at the length of phage tail, and phage tail like particles. The phage taillike particles could be divided into 4 types by the length. Induction experiments of Br. lactofermentum with UV irradiation, mitomycin C or bacitracin treatment produced neither phage PR-5 (T) or phage tail-like particles. 9) No lysis occured when the growth of 7 strains of miscellaneous bacteria, isolated from soil and sewage samples, were inoculated with either phage PR-5 (T) or phage tail-like particles the inoculation of phage PR-5 pellet resulted in the growth inhibition of the orgainsms in the spot test. The lysates obtained from 3 miscellaneous soil derived bacteria following mitomycin C treatment the growth of Br. lactofermentum, but did not lyze the bacterium.

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