Bacillus amyloliquefaciens CJW15와 SSD8이 만드는 항균물질들의 특성 Properties of Antimicrobial Substances Produced by Bacillus amyloliquefaciens CJW15 and Bacillus amyloliquefaciens SSD8원문보기
청국장에서 강력한 항균력을 지닌 2개의 Bacillus 균주들인 CJW15와 SSD8이 분리되었다. 16S rRNA와 recA 유전자들 염기서열 결정에 의해 두가지 균주 모두 B. amyloliquefaciens로 동정되었다. CJW15는 B. cereus ATCC14579, Listeria monocytogenes ATCC19111, Lactococcus lactis ATCC11454 들의 증식을 강력히 억제하며 SSD8은 B. cereus ATCC14579와 Enterococcus faecium ATCC19953 증식을 억제하였다. 두 균주들의 항균력은 $100^{\circ}C$, 15분 처리에도 감소하지 않았고 산성인 pH 3과 알칼리인 pH 12에서도 안정하였다. 트립신, 펩신, 프로테아제 K, 프로테아제 효소처리에 의해 CJW15 항균력은 변화가 없었지만 SSD8 항균력은 절반으로 감소하였다. 두 균주 공히 surfactin, fengycin, iturin, iturinA와 같은 lipopeptide 생합성 유전자들을 지니고 있고 subtilin과 같은 박테리오신 유전자들도 지니고 있다. 또 두 균주들은 혈전용해능을 지니고 있다.
청국장에서 강력한 항균력을 지닌 2개의 Bacillus 균주들인 CJW15와 SSD8이 분리되었다. 16S rRNA와 recA 유전자들 염기서열 결정에 의해 두가지 균주 모두 B. amyloliquefaciens로 동정되었다. CJW15는 B. cereus ATCC14579, Listeria monocytogenes ATCC19111, Lactococcus lactis ATCC11454 들의 증식을 강력히 억제하며 SSD8은 B. cereus ATCC14579와 Enterococcus faecium ATCC19953 증식을 억제하였다. 두 균주들의 항균력은 $100^{\circ}C$, 15분 처리에도 감소하지 않았고 산성인 pH 3과 알칼리인 pH 12에서도 안정하였다. 트립신, 펩신, 프로테아제 K, 프로테아제 효소처리에 의해 CJW15 항균력은 변화가 없었지만 SSD8 항균력은 절반으로 감소하였다. 두 균주 공히 surfactin, fengycin, iturin, iturinA와 같은 lipopeptide 생합성 유전자들을 지니고 있고 subtilin과 같은 박테리오신 유전자들도 지니고 있다. 또 두 균주들은 혈전용해능을 지니고 있다.
Two Bacillus strains, CJW15 and SSD8, with strong antibacterial activities were isolated from cheonggukjang. Both were identified as B. amyloliquefaciens strains after gene sequencing of rRNA and recA. CJW15 strongly inhibited the growth of B. cereus (ATCC14579), Listeria monocytogenes (ATCC19111), ...
Two Bacillus strains, CJW15 and SSD8, with strong antibacterial activities were isolated from cheonggukjang. Both were identified as B. amyloliquefaciens strains after gene sequencing of rRNA and recA. CJW15 strongly inhibited the growth of B. cereus (ATCC14579), Listeria monocytogenes (ATCC19111), and Lactococcus lactis (ATCC11454). In comparison, SSD8 inhibited the growth of B. cereus (ATCC14579) and Enterococcus faecium (ATCC19953). The antibacterial activities of the two strains were not affected when exposed to a temperature of $100^{\circ}C$ for 15 min and were quite stable in acidic (pH 3) and alkaline (pH 12) pH conditions. Enzymatic treatments (trypsin, pepsin, proteinase K, and protease) had no effect on the activity of CJW15, but reduced the activity of SSD8 by half. Both isolates possess genes involved in the synthesis of lipopeptides (e.g. surfactin, fengycin, iturin, and iturin A), and genes encoding subtilin, a bacteriocin. Moreover, both isolates have fibrinolytic activities as well.
Two Bacillus strains, CJW15 and SSD8, with strong antibacterial activities were isolated from cheonggukjang. Both were identified as B. amyloliquefaciens strains after gene sequencing of rRNA and recA. CJW15 strongly inhibited the growth of B. cereus (ATCC14579), Listeria monocytogenes (ATCC19111), and Lactococcus lactis (ATCC11454). In comparison, SSD8 inhibited the growth of B. cereus (ATCC14579) and Enterococcus faecium (ATCC19953). The antibacterial activities of the two strains were not affected when exposed to a temperature of $100^{\circ}C$ for 15 min and were quite stable in acidic (pH 3) and alkaline (pH 12) pH conditions. Enzymatic treatments (trypsin, pepsin, proteinase K, and protease) had no effect on the activity of CJW15, but reduced the activity of SSD8 by half. Both isolates possess genes involved in the synthesis of lipopeptides (e.g. surfactin, fengycin, iturin, and iturin A), and genes encoding subtilin, a bacteriocin. Moreover, both isolates have fibrinolytic activities as well.
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제안 방법
B. amyloliquefaciens CJW15 and SSD8 were grown in TSB for 60 h and the resulting FS was used as the sample for the stability test (Table 2). The pH of FS (3 ml) was adjusted to 3−11 using 1 N HCl or 1 N NaOH.
대상 데이터
Colonies showing typical Bacillus morphologies were saved and examined for the antimicrobial activity by agar spot method [5]. Bacillus cereus ATCC14579 was used as an indicator. Bacilli were grown in LB for 48 h at 37°C and 1 μl culture was spotted on a LB plate.
이론/모형
After 2 h incubation at 25°C, the pH was readjusted to 7 and the remaining activity was measured by agar well diffusion method using B. cereus ATCC14579 as the indicator.
At every 6 h, 4 ml of each culture was taken and the OD 600 value and antibacterial activity were measured. The antimicrobial activity of FS was assayed by agar well diffusion method using B. cereus ATCC14579 as the indicator. All measurements were done in triplicates and the means were shown.
The plate was incubated for overnight before inhibition zons were examined [8]. The strength of antimicrobial activity was determined by two-fold serial dilution method as described previously [11]. The activity was defined as the reciprocal of the highest dilution that still produced a definite zone of inhibition and expressed as activity units (AU) per milliliter after multiplied by a conversion factor.
성능/효과
B. amyloliquefaciens CJW15 and B. amyloliquefaciens SSD8 were grown in different media (LB, NB, TSB, and BHI) and the growth (OD600) and the antibacterial activities were measured during growth.
B. amyloliquefaciens CJW15, B. amyloliquefaciens SSD8, and B. subtilis EMD4 (control, a bacteriocin producer) inhibited the growth of B. cereus ATCC14579, B. amyloliquefaciens CH86-1 and B. subtilis 168 (Table 1).
B. subtilis EMD4 showed broader inhibition spectra than two B. amyloliquefaciens isolates, inhibiting the growth of B. licheniformis ATCC4527, Streptococcus thermophilus KFRI193, Pediococcus pentosaceus NRRLB-14009, and Enterococcus faecalis ATCC29212.
amyloliquefaciens strains and 99% with other species. From these results, both CJW15 and SSD8 were identified as B. amyloliquefaciens. The Genbank accession numbers for the partial 16S rRNA and recA genes are KR095447 and KR095446, respectively, for B.
Inhibiting activities were also stable at high temperature, 100% of the activities were remained after 15 min exposure to 50−100 oC. From these results, it was concluded that the dominant antibacterial substance in the FS from B. amyloliquefaciens CJW15 and SSD8 was not proteinaceous. High stability of an antimicrobial substance is a desirable property when the inhibitory substance or the producing strain is included into foods as a biopreservative to inhibit the growth of pathogens like B.
BHI was the most unfavorable medium for the antibacterial activity and the highest activity was just 80 AU/ml. In conclusion, the best medium for the antibacterial activity of B. amyloliquefaciens CJW15 and SSD8 was TSB and LB, respectively.
amyloliquefaciens. The Genbank accession numbers for the partial 16S rRNA and recA genes are KR095447 and KR095446, respectively, for B. amyloliquefaciens CJW15, and KR095443 and KR095442, respectively, for B. amyloliquefaciens SSD8.
08 log CFU/ml at 12 h. The results indicated that the inhibitory substance(s) had bacteriocidal effect although the degree of reduction in the viable counts was not great when compared with BacEMD4 from B. subtilis EMD4. When BacEMD4 (12,800 AU) was added into B.
후속연구
More studies are required to find out the nature of the antimicrobial substance(s) produced by B. amyloliquefaciens CJW15 and SSD8 strains. Other properties of these 2 isolates such as sensory properties of soy products fermented with these isolates should be investigated before they are employed as starters for fermented soy foods or biopreservatives for foods.
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