• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.368-377
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• 2020

Enterotoxigenic Bacteroides fragilis (ETBF) is the main pathogen causing severe inflammatory diseases and colorectal cancer. Its biofilm plays a key role in the development of colorectal cancer. The objective of this study was to determine the antagonistic effects of cell-free supernatants (CFS) derived from Clostridium butyricum against the growth and biofilm of ETBF. Our data showed that C. butyricum CFS inhibited the growth of B. fragilis in planktonic culture. In addition, C. butyricum CFS exhibited an antibiofilm effect by inhibiting biofilm development, disassembling preformed biofilms and reducing the metabolic activity of cells in biofilms. Using confocal laser scanning microscopy, we found that C. butyricum CFS significantly suppressed the proteins and extracellular nucleic acids among the basic biofilm components. Furthermore, C. butyricum CFS significantly downregulated the expression of virulence- and efflux pump-related genes including ompA and bmeB3 in B. fragilis. Our findings suggest that C. butyricum can be used as biotherapeutic agent by inhibiting the growth and biofilm of ETBF.

• 한국미생물·생명공학회지
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• 제27권1호
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• pp.46-53
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• 1999

The purpose of this study was to optimize the conditions of continuous mixed culture of C.butyricum and R. spaeroides K-7, which were able to produce hydrogen using biomass-dreived substrate. To investigate the possibility of continuous culture, semi-continuous culture was carried out for 20 days. In semi-continuous culture using the reactor system, the replacement rate of fresh medium was 30% of total medium volume for the highest hydrogen evolution. In continuous culture, the optimum dilution rate was determined to be 0.05$h^{-1}$. The continuous culture produced 3.1 times as compared with the hydrogen on batch culture. On the other hand, the continuous mixed culture produced 1.3~2.1 times as much as hydrogen of the continuous monoculture of C. butyricum. When 10g of glucose in the media (1l) was supplied as a carbon source on continuous culture, mixed culture of C. butyricum and R. sphaeroides K-7 increased hydrogen evolution rate. Because considerable amount of glutamate was contained in waste water of glutamate fermentation, utilization of glutamate was examined in mixed culture. As a result of examination, production of hydorgen was slightly inhibited by high concentration of glutamate, more than 20mM, on continuous monoculture of R. sphaeroides K-7. On the other hand, both on continuous monoculture of C. butyricum and on mixed culture of C. butyricum and R. sphaeroides K-7, production of hydrogen was not inhibited by high concentration of glutamate such as 100mM. Hence this suggests that high concentration of waste water can be used as good substrate for hydrogen production on monoculture of C. butyricum and mixed culture of C. butyricum and R. sphaeroides K-7.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1210-1215
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• 2006

The properties related to the temperature and oxygen stability of the cytoplasmic hydrogenases from the fermentative strict anaerobic bacterium, Clostridium butyricum NCIB 9576 (Cl. butyricum), and purple sulfur phototrophic bacterium, Thiocapsa roseopersicina NCIB 8347 (T. roseopersicina), were compared. The optimum temperatures for the growth of Cl. butyricum and T. roseopersicina were 37$^{\circ}C$ and 25$^{\circ}C$, respectively, whereas those for the H$_2$ evolution of the cytoplasmic hydrogenases prepared from Cl. butyricum (C-H$_2$ase) and T. roseopersicina (T-H$_2$ase) were 45$^{\circ}C$ and 65$^{\circ}C$, respectively. The T-H$_2$ase was more thermostable than the C-H$_2$ase and retained its full activity for 5 h at 50$^{\circ}C$ under anaerobic conditions and 90% of its activity at 60$^{\circ}C$, whereas the C-H$_2$ase lost its activity drastically at 50$^{\circ}C$. The optimum pHs for H$_2$ oxidation of the C-H$_2$ase and T-H$_2$ase were 9.0 and 7.5, respectively. Both enzymes showed a maximum H$_2$ evolution activity at pH 7.0. Under aerobic conditions, 80% of the T-H$_2$ase activity was retained for 10 h at 30$^{\circ}C$, and 50% of the activity remained after 6 days under the same experimental conditions. However, the C-H$_2$ase was labile to oxygen and lost its activity immediately on exposure to air. Therefore, these properties of the T-H$_2$ase are expected to be advantageous for application in in vitro biological H$_2$ production systems.

• 한국식품과학회지
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• 제53권6호
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• pp.775-784
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• 2021

Clostidium butyricum을 분리하기 위하여 국내 모유수유 신생아 분변으로부터 혐기성 균주를 선별하였고 버블을 생성하는 100개의 균을 확보하였다. 이중 Clostridium perfringens에 대한 항균력과 butyric acid의 생산이 가장 우수한 DIMO 52 균주를 선발하였고, 형태학적 특성, 생리 생화적 특성 및 16S rRNA 유전자 분석을 통하여 C. butyricum으로 동정되어 C. butyricum DIMO 52로 명명하였다. 성장률, butyric acid 생산 및 pH 변화를 배양 36시간 동안 모니터링하였다. 배양 24시간 후 DIMO 52 균주의 최대 성장에 도달하였고, butyric acid 최대 농도는 대략 34.73±4.27 mM이었으며, pH는 7.2에서 2.5로 변경되었다. DIMO 52 균주는 낮은 pH와 oxgall에 높은 저항성이 있다. pH 2에서 2시간 동안 접종의 약 67.5%의 유의성 있는 생존율을 보였다(p<0.05). 그리고, 0.3% oxgall이 함유된 RCM 액체배지에서 24시간 동안 접종의 약 64.9%의 유의성 있는 생존율을 보였다(p<0.05). 또한, DIMO 52은 Escherichia coli KCTC 2441와 Salmonella Typhimurium KCTC 1925에 대해 억제효과를 나타냈다. 두 균주에 대한 항균효과는 아마도 butyric acid에 의한 낮은 pH 때문인 것으로 보였다. 5×10³ CFU/mL 생균수 까지는 RAW264.7 세포에 세포독성이 없는 것으로 관찰되었고, NO 생성을 억제할 수 있는 최저 균수를 확인한 결과 약 1×10³ CFU/mL 생균수에서 LPS만 처리한 군 대비 약 33%의 NO 생성을 억제하는 것으로 분석되었다(p<0.01). 이 결과는 C. butyricum DIMO 52이 NO radical 소거 및 항염증 활성을 가지고 있음을 시사한다. 결론적으로, 본 연구에서 분리된 C. butyricum DIMO 52의 프로바이오틱스 특성을 확인하였다.

• 한국미생물·생명공학회지
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• 제17권1호
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• pp.69-73
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• 1989

자가분해 효소는 Cl. butyricum의 포자형성 배지에서 배양할 때 체외로 배출되어 배양액에도 존재하였다. 배양액으로부터 약 50배로 부분정제된 자가분해 효소를 사용하여 효소의 성질을 조사하였다. 자가분해 효소의 최적 pH와 온도는 각각 5.0과 37$^{\circ}C$였으며 중성 pH에서는 안정하나, 열에는 비교적 불안정하여 5$0^{\circ}C$에서 5분간 열처리한 후 효소활성의 70%가 소실되었다. 또한 Cu ion$^{++}$에 의해서 효소활성이 저하되었으나 그 밖의 금속이온에 의하여서는 큰 영향을 받지 않았다. 또한 자가분해 효소는 기질로서 영양세포에는 직접 활성을 나타내지 못하나, 세포벽 fraction에는 활성을 가지고 있었다.

• Journal of Animal Science and Technology
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• 제49권6호
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• pp.819-828
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• 2007

지난 20여년 동안 항생제나 화학 요법의 문제점을 줄이고 외부 환경의 변화에 민감하게 반응하는 장내 미생물들의 균총을 유지시켜주기 위해 살아있는 미생물제제(direct-fed microbial agents, DFM)를 반추가축의 사료에 직접 첨가하고 있다. 반추동물 사료에도 생산성을 개선하기 위하여 여러 종류의 DFM이 널리 이용이 되고 있는데, DFM은 건물섭취량(dry matter intake, DMI)의 증가 및 반추위내에서의 영양소 분해율 향상 그리고 유생산량의 증가와 체온감소 등에 효과적이다(Piva 등, 1993; Higginb- otham 등, 1994; MacGilliard와 Stallings, 1998; Nocek와 Kautz, 2006). 대표적인 DFM으로는 효모균, 곰팡이, 박테리아 및 세포 구성물질 등이 이에 속한다(Beharka와 Nagaraja, 1993; Sullivan과 Martin 1999). 효모 및 효모배양물은 대표적인 DFM의 한 종류인데, 반추가축에게 효모(yeast) 및 효모 배양물(yeast culture)을 급여하였을 때 반추위 내 발효 특성을 변화시키고(Harrison 등, 1988; Martin과 Nisbet, 1990) 반추위 내에서 조단백질과 조섬유의 반추위내 분해율을 개선시키며(Gomez-Alarcon 등, 1990; Williams 등, 1991), 체내 전장소화율(Gomez-Alarcon 등, 1990; Wiede- mier 등, 1987)을 향상시키는 효과가 있다고 보고된 바 있다. 한편, 일반적으로 대부분의 Clostridium 균들은 독소를 생산하거나 인체에 유해한 물질들을 생산하여 사람과 가축에게 좋지 않은 영향을 주고 있으나, Clostridium butyricum은 장내 유익한 균으로서 유산균과의 공생이 가능하고 반추위 내에서 주요 VFA 생산에 관여하기도 하며(Araki 등, 2002a, 2002b) 많은 종류의 비타민 B군을 생산하여 유산균이 이용할 수 있게 한다. 또한 생산되어진 acetic acid와 butyric acid는 장관 상피세포를 흡수되어 상피세포에서 에너지원으로 사용되어진다고 하였다(김 등, 2001). 낙산균은 주로 단위동물에서 사용되어 왔는데, 일부 복합생균제의 형태로 사용하였을 경우 유해세균이 장 점막을 침입하는 것을 방지하고(Fuller와 Brooker, 1974), butyric acid를 생산하여 장내 pH를 저하시키며 항균작용(Under-dahl 등, 1982)을 하는 것으로 알려져 있다. S. cerevisiae는 가장 일반적으로 사용되는 균주로써 이의 효과와 효능에 관한 연구는 비육우와 착유우를 대상으로 많이 이루어져 왔지만, 낙산균은 일부 국내 젖소 사육농가에서 사용되고 있음에도 불구하고 이에 대한 연구는 단위동물 및 어린송아지의 경우(최 등, 1991)를 제외하고는 거의 이루어지지 않았다. 따라서 본 연구는 S. cerevisiae 및 C. butyricum을 단독으로 또는 혼합하여 급여할 때 면양에서의 반추위내 발효성상과 소화율, 그리고 젖소를 이용한 사양시험을 통해 산유량과 유조성에 미치는 효과를 비교하기 위하여 실시되었다.

• 한국수소및신에너지학회논문집
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• 제16권3호
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• pp.219-228
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• 2005

Fermentative strict anaerobic bacterium, Clostricium butyricum NCIB 9576 (Cl. butyricum) and purple sulfur phototrophic bacterium, Thiocapsa roseopersicina NCIB 8347 (T. roseopersicina) were compared on their temperature and oxygen stabilities of cytoplasmic hydrogenases. Cell growth phase and the specific activities of evolution $H_2ase$ were related for both strains, exhibiting the highest cytoplasmic $H_2ase$ activities during the logarithmic growth phases which were 4 and 18 hrs after the incubation for Cl. butyricum and T. roseopersicina, respectively. The optimum temperatures for the growth of Cl. butyricum and T. roseopersicina were 37$^{\circ}C$ and 27$^{\circ}C$, respectively, while those for $H_2$ evolution of cytoplsmic hydrogenases prepared from Cl. butyricum ($C-H_2ase$) and T. roseopersicina ($T-H_2ase$) were 45$^{\circ}C$ and 65$^{\circ}C$, respectively. $T-H_2ase$ was more thermo-stable than $C-H_2ase$. $T-H_2ase$ retained its full activity for 5 hrs at 50$^{\circ}C$ and retained 90% of its original activity for 5 hrs at 60$^{\circ}C$, however, $C-H_2ase$ lost its activity drastically at 50$^{\circ}C$. The optimum pHs for $H_2$ oxidation of $C-H_2ase$ and $T-H_2ase$ were 9.0 and 7.5 respectively. The both enzymes showed maximum $H_2$ evolution activity at pH 7.0. Under the aerobic condition, 80% of $T-H_2ase$ activity was retained for 10 hrs at 30$^{\circ}C$, and 50% of activity was still remained after 6 days at the same experimental conditions. But the $C-H_2ase$ was labile to oxygen and lost its activity immediately after the exposure to air.

• 한국미생물·생명공학회지
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• 제17권1호
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• pp.63-68
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• 1989

낙산균 Cl. butyricum ID의 자가분해 최적 조건과 특징을 조사하였다. 영양세포의 자가분해 최적 pH는 0.05M 완충용액에서 7.0 이었으며, 최적 온도는 37$^{\circ}C$였다. 자가분해 속도는 대수기의 영양세포가 가장 높았으며, 대수기 이후의 영양세포에서는 급격히 감소하였다. 대수기의 영양세포는 0.3M 이상의 NaCl, sucrose, glucose를 포함하는 배양액에서는 자가분해가 일어나 정상적인 증식을 할 수 없었다. 자가분해 효소는 균체내에서 세포벽에 결합된 상태로 존재하였고 배양중 대수 증식기에 체외로 배출되어 배양액에도 존재하였다.

• 한국미생물·생명공학회지
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• 제27권1호
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• pp.62-69
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• 1999

Clostridium butyricum NCIB 9576 evolved hydrogen gas and produced various organic acids from glucose, lactose, starch, and glycerol. Total amount of hydrogen gas produced from 1 and 2% glucose were 630 and 950ml $H_2$/l-broth, respectively, for the first 24 hrs of incubation and the maximum hydrogen production rates were 42 and 94ml $H_2$/hr/1-broth, respectively. Teh initial pH 6.8 decreased to 4.2~4.5 during the first 12~16 hrs of fermentation when the pH was not controlled, resulting in ceasing the cell growth and hydrogen evolution and in degradation of 82 and 40% glucose after 24hrs of incubation from 1 and 2% glucose, respectively. When pH was controlled to 5.5, glucose was consumed completely and resulted in increasing hydrogen production approximately 38~50% compared to the experiments without the pH control. C. butyricum NCIB 9576 produced hydrogen gas approximately 644, 1,700 and 3,080 ml $H_2$/l-broth with 0.5, 1 and 2% lactose, respectively and the maximum hydrogen production rates were 41, 141 and 179ml $H_2$/hr/l-broth, respectively. All of the lactose added was degraded completely during fermentation even though pH was not controlled. C. butyricum NCIB 9576 produced 183 and 709ml $H_2$/l-broth with 0.1 and 0.5% starch for 48 hrs, respectively, when pH was not controlled. The maximum rates of hydrogen gas production were 43 and 186ml $H_2$/l-broth, respectively and 80~100% of starch added was fermented. Approximately 107ml $H_2$/l-broth was produced using 1% glycerol by C. butyricum NCIB 9576 and the pH was maintained higher than 6.1 during fermentation without pH control. The degradation of glucose, lactose, starch and glycerol by C. butyricum NCIB 9576 were affected by the pH of fermentation broth and the organic acids released during fermentation. The pH of feremtntation broth dropped to 4.2~4.6 after 12~14 hrs incubation when glucose was used as a substrate while pHs were maintained above pH 5 under the same experimental conditions when lactose, starch and glycerol were used. The organic solvents and acids produced during glucose fermentation were mainly ethanol, butyrate, acetate and a little of propionate, while butyrate was the main organic acids during the lactose, starch, and glycerol fermentation by C. butyricum NCIB 9576.

• Journal of Microbiology and Biotechnology
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• 제33권10호
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• pp.1337-1350
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• 2023

Caproic acid is a precursor substance for the synthesis of ethyl caproate, the main flavor substance of nongxiangxing baijiu liquor. In this study, Clostridium butyricum GD1-1, a strain with high caproic acid concentration (3.86 g/l), was isolated from the storage pit mud of nongxiangxing baijiu for sequencing and analysis. The strain's genome was 3,840,048 bp in length with 4,050 open reading frames. In addition, virulence factor annotation analysis showed C. butyricum GD1-1 to be safe at the genetic level. However, the annotation results using the Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server predicted a deficiency in the strain's synthesis of alanine, methionine, and biotin. These results were confirmed by essential nutrient factor validation experiments. Furthermore, the optimized medium conditions for caproic acid concentration by strain GD1-1 were (g/l): glucose 30, NaCl 5, yeast extract 10, peptone 10, beef paste 10, sodium acetate 11, L-cysteine 0.6, biotin 0.004, starch 2, and 2.0% ethanol. The optimized fermentation conditions for caproic acid production by C. butyricum GD1-1 on a single-factor basis were: 5% inoculum volume, 35℃, pH 7, and 90% loading volume. Under optimal conditions, the caproic acid concentration of strain GD1-1 reached 5.42 g/l, which was 1.40 times higher than the initial concentration. C. butyricum GD1-1 could be further used in caproic acid production, NXXB pit mud strengthening and maintenance, and artificial pit mud preparation.