[논문]국내 모유수유 유아의 분변에서 분리한 낙산균 Clostridium butyricum DIMO 52의 특징

모상준

doi:10.9721/kjfst.2021.53.6.775

[국내논문] 국내 모유수유 유아의 분변에서 분리한 낙산균 Clostridium butyricum DIMO 52의 특징
Characteristics of butyric acid bacterium, Clostridium butyricum DIMO 52, isolated from feces of Korean breastfeeding infants 원문보기

한국식품과학회지 = Korean journal of food science and technology, v.53 no.6, 2021년, pp.775 - 784

모상준 (단국대학교 의학레이저연구센터)

초록
AI-Helper

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.

Abstract ▼ AI-Helper

After isolating the DIMO 52 strain with a large inhibition zone diameter for Clostridium perfringens and maximum butyric acid production from the fecal sample of a breastfeeding infant, it was identified as Clostidium butyricum. The maximum growth of the DIMO 52 strain was reached 24 h after inoculation, and the maximum butyric acid concentration was approximately 34.73±4.27 mM. The DIMO 52 strain survived approximately 67.5% of the initial inoculum at pH 2.0, and approximately 64.9% survived in RCM broth supplemented with 0.3% (w/v) oxgall. In addition, DIMO 52 showed antibacterial activity against Escherichia coli KCTC 2441 and Salmonella Typhimurium KCTC 1925. In LPS-stimulated RAW264.7 cells, 1×103 CFU/mL viable cells of the DIMO 52 strain also exhibited significant NO (nitric oxide) production inhibitory activity (33%, p<0.01). This result suggests that C. butyricum DIMO 52 has anti-inflammatory activity related to NO radical-scavenging activity. In conclusion, C. butyricum DIMO 52 isolated in this study has the potential to be used as a probiotic.

주제어

표/그림 (11)

그림 Fig. 1. Inhibition zone formed by the isolated DIMO 52 strain on the lawn of C. perfringens and HPLC chromatogram of DIMO 52 strain. (A) About 100 strains were inoculated into the RCM broth at 37℃ for 24 h in an anaerobic chamber, and then the final optimal density of the strains was titrated to 1.0 at A_{660 nm} to measure antibacterial activity. The strain titrated to 1.0 at A_{660 nm} was inoculated into a sterile 8mm diameter paper disk with 30 μL each, and the paper disk was placed on the RCM solid medium smeared with C. perfringens at 1×10⁷ CFU/mL. After being placed on a solid medium and incubated at 37℃ for 18 h, the formation and size of inhibitory zone of strains were compared. (B) The culture solution of DIMO 52 strain with excellent inhibition zone against C. perfringens was subjected to HPLC analysis using a C18 column at UV wavelength of 220 nm. As the mobile phase, a solution in which 10 mM phosphate buffer (pH 3.0) and methanol were mixed in a ratio of 98:2 was used, respectively. The upper is the HPLC chromatogram of the standard solution of acetic acid (top) and butyric acid (middle), and the lower is the HPLC chromatogram of the culture solution of the DIMO 52 strain (bottom).
그림 Fig. 1. Continued
표 Table 1. Inhibition zone formed by the isolated strains from infant feces on the lawn of C. perfringens KCTC 3269 and production of organic acid by them
그림 Fig. 2. Endospores and morphological characteristics of C. butyricum DIMO 52. (A) To stain bacterial endospores, fix the smeared bacteria on a slide glass, cover with absorbent paper, add 5% Malachite Green, and steam in a steam bath for several minutes. After removing the absorbent paper, cool the slide glass and wash the slide glass with running tap water. After counterstaining with 0.5% safranin and washing with tap water, the presence of endospores (arrow) was observed under a light microscope (×1000). (B) Micrograph of a thin section of vegetative cell of C. butyricum DIMO 52 taken by transmission electron microscopy (TEM). Bar represent 0.5 μm. W: Wall (cell); M: Membrane (cytoplasmic); P: periplasmic space; OSC: outer spore coat; ISC: inner spore coat, CT: cortex; CW: core wall; CM: core membrane, C: core.
표 Table 2. Morphological, physiological and biochemical characteristics of the isolate C. butyricum DIMO 52
그림 Fig. 3. Changes in pH and butyric acid concentration by growth of C. butyricum DIMO 52. (A) Changes in pH were measured by using pH paper after removing cells by collecting 1 mL every 6 h. (B) Growth curves of C. butyricum DIMO 52 were measured as viable counts and butyric acid production was performed by HPLC analysis. For CFU counting, cells were serially diluted in RCM medium and 100 uL of the dilution was spread evenly over the agar surface using a sterile glass spreader. The butyric acid produced by the isolated bacteria was analyzed by the analytical method mentioned in materials and methods using HPLC. Each experiment was conducted in triplicate. Data are expressed as mean±standard deviation (n=3). Statistical significance was explored via one-way analysis of variance (ANOVA) followed by post hoc Tukey HDS test. ****p<0.0001 indicates a statistically significant difference in the number of cells per hour compared to time 0. ^####p<0.001 indicates a statistically significant difference in butyric acid concentration per hour compared to time 0.
표 Table 3. Survival of C. butyricum DIMO 52 at pH 2.0 and 3.0 as determined by counts of viable bacteria
그림 Fig. 4. Antibacterial activity of C. butyricum DIMO52. Antibacterial activities were measured by well diffusion method. 20 mL of the appropriate agar (1.5%, w/v) medium were pipetted in 90 mm petri dishes and inoculated (0.5%, v/v) with a standardized indicator suspension (Left, S. Typhimurium KCTC 1925; Right, E. coli KCTC 2441). After 50 μL inoculating of each of cell lysate and supernatant of DIMO 52 strain, each agar plate was incubated at 37℃ for 24 h, and inhibition zones were observed. C: cell lysate; S: supernatant
표 Table 4. Survival of C. butyricum DIMO 52 in RCM broth containing 0.1 and 0.3 % oxgall as determined by counts of viable bacteria
표 Table 5. Antibacterial activity of C. butyricum DIMO 52 against selected bacteria
그림 Fig. 5. Cell viability and NO production by C. butyricum DIMO 52 in RAW264.7 cell. (A) Cells were treated with C. butyricum DIMO 52 at the indicated number of viable cells for 12 h at 37℃. Cell viability was measured by MTT assay. ****p<0.0001 indicates a statistically significant difference in the cell viability compared to control. (B) Inhibition of LPS-induced NO production by C. butyricum DIMO 52. RAW 264.7 cells were treated with LPS (1 μg/mL) for 1 h and then incubated with C. butyricum DIMO 52 at the indicated number of viable cells for 12 h at 37℃. Nitrite produced was measured by Griess reagent as described in material and methods. ****p<0.0001 and **p<0.01 indicate a statistically significant difference in the NO production compared to only LPS treated group. Data are expressed as mean±standard deviation (n=3). Statistical significance was explored via one-way analysis of variance (ANOVA) followed by post hoc Tukey HDS test. ns: no significance.

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표제어: PCR

동의어: Packet Collision Rate

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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