Antifungal Activity of Lactobacillus plantarum Isolated from Kimchi

김치로부터 항진균 활성 Lactobacillus plantarum의 분리 및 특성 규명

  • Published : 2008.12.28

Abstract

A lactic acid bacterium having antifungal activity was isolated from kimchi. It was identified as Lactobacillus plantarum based on its morphological and biochemical properties, and 16S rRNA sequence, and designated as Lb. plantarum AF1. This isolate inhibited the growth of Aspergillus flavus ATCC 22546, A. fumigatus ATCC 96918, A. petrakii PF-1, A. ochraceus PF-2, A. nidulans PF-3, Epicoccum nigrum KF-1, and Cladosporium gossypiicola KF-2 under a dual culture overlay assay. Also, the antimicrobial activity was found to be active against various species of Gram-positive and Gram-negative bacteria. The antifungal activity was found to be stable after heat ($121^{\circ}C$, 15 min) and proteolytic enzyme treatment, but it was unstable over pH 5.0. The antifungal compound(s) was estimated to have a low molecular mass (below 3,000 Da).

숙성된 김치로부터 항진균 활성을 나타내는 균주 1종과 감수성을 나타내는 곰팡이 1종을 분리하였다. 분리된 균주는 형태학적, 생화학적 특성 조사와 165 rRNA 염기서열 결정을 통한 균주 동정 결과 Lactobacillus plantarum AF1으로 명명하였고, 감수성 곰팡이는 ITS-5.8S rRNA 염기서열 분석을 통하여 Epicoccum nigrum KF-1으로 명명하였다. Dual culture overlay assay를 통한 Lb. plantarum AF1의 항진균 활성 실험 결과 A. ochraceus, A fumigatus, C. gossypiicola 등 식품 부패 곰팡이 및 병원성 곰팡이에 강한 생육 저해 활성을 나타내었다. 또한 항균 물질에 의한 항미생물 활성 범위를 측정한 결과 항진균 활성 외에도 식중독균주를 포함한 그람 양성 및 음성 세균들에 강한 저해 활성을 나타내어 Lb. plantarum AF1은 넓은 항미생물 활성 범위를 가지는 것을 알 수 있었다. Lb. plantarum AF1의 생육에 따른 항진균 활성을 측정한 결과 항진균 활성은 배양 20시간부터 최대 활성(3,200 AU/ml)을 나타내어 120시간까지 활성이 감소되지 않고 유지되었다. 항진균 물질의 안정성 실험을 통하여 AF1 항진균 물질은 산성의 pH(pH $3.0{\sim}4.0$)와 열에 안정한 물질이며, 단백분해효소 처리에 영향을 받지 않으므로 비단백질성 물질이거나 단백분해효소의 영향을 받지않는 구조의 물질임을 추정하였다. AF1 항진균 물질의 분자량을 예측하기 위하여 Lb. plantarum AF1의 배양액을 3,000 Da 이상과 이하의 분획으로 나누어 항진균 활성을 측정한 결과 AF1 항진균 물질은 분자량 3,000 Da 미만의 물질임을 확인하였다. 본 실험에서 분리한 김치유산균인 Lb. plantarum AF1은 넓은 범위의 항진균 활성 및 항세균 활성을 나타내므로 강력한 천연 식품보존제 및 사료보존제로서 활용이 기대된다.

Keywords

References

  1. Atanossova, M., Y. Choiset, M. Dalgalarrondo, J. M. Chobert, X., Dousset, I. Ivanova, and T. Haertle. 2003. Isolation and partial biochemical characterization of proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3. Int. J. Food Microbiol. 87: 63-73 https://doi.org/10.1016/S0168-1605(03)00054-0
  2. Aziz, N. H. and L. A. A. Moussa. 2002. Influence of gamma-radiation on mycotoxin producing moulds and mycotoxins in fruits. Food Control 13: 281-288 https://doi.org/10.1016/S0956-7135(02)00028-2
  3. Bello, F. D., C. I. Clarke, L. A. M. Ryan, H. Ulmer, T. J. Schober, K. Strom, J. Sjogren, D. van Sinderen, J. Schnurer, and E. K. Arendt. 2007. Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7. J. Cereal Sci. 45: 309-318 https://doi.org/10.1016/j.jcs.2006.09.004
  4. Brul, S. and P. Coote. 1999. Preservative agents in foods. Mode of action and microbial resistance mechanisms. Int. J. Food Microbiol. 50: 1-17 https://doi.org/10.1016/S0168-1605(99)00072-0
  5. Galvano, F., A. Piva, A. Ritieni, and G. Galvano. 2001. Dietary strategies to counteract the effects of mycotoxins: A review. J. Food Prot. 64: 120-131 https://doi.org/10.4315/0362-028X-64.1.120
  6. Gould, G. W. 2001. New processing technologies: an overview. Proc. Nutr. Soc. 60: 463-474
  7. Hoover, D. G. and S. K. Harlander. 1993. Screening methods for detecting bacteriocin activity, pp. 23-39. In Hoover, D. G. and L. R. Steenson. (eds.), Bacteriocins of Lactic Acid Bacteria. Academic Press, Inc., San Diego, U.S.A
  8. Hussein, H. S. and J. M. Brasel. 2001. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicol. 167: 101-134 https://doi.org/10.1016/S0300-483X(01)00471-1
  9. Kim, J.-D. 2005. Antifungal activity of lactic acid bacteria isolated from Kimchi against Aspergillus fumigatus. Mycobiol. 33: 210-214 https://doi.org/10.4489/MYCO.2005.33.4.210
  10. Kim, S. I., I. C. Kim, and H. C. Chang. 1999. Isolation and identification of antimicrobial agent producing microorganisms and sensitive strain from soil. J. Kor. Soc. Food Sci. Nutr. 28: 526-533
  11. Klaenhammer, T. R. 1988. Bacteriocin of lactic acid bacteria. Biochim. 70: 337-349 https://doi.org/10.1016/0300-9084(88)90206-4
  12. Lavermicocca, P., F. Valerio, A. Evidente, S. Lazzaroni, A. Corsetti, and M. Gobetti. 2000. Purification and chracterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B. Appl. Environ. Microbiol. 66: 4084-4090 https://doi.org/10.1128/AEM.66.9.4084-4090.2000
  13. Lee, H.-J., J.-H. Baek, M. Yang, H.-U. Han, Y.-D. Ko, and H.-J. Kim. 1993. Characterizations of lactic acid bacterial community during kimchi fermentation by temperature downshift. Kor. J. Microbiol. 31: 346-353
  14. Magnusson, J. and J. Schnürer. 2001. Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broadspectrum proteinaceous antifungal compound. Appl. Environ. Microbiol. 67: 1-5 https://doi.org/10.1128/AEM.67.1.1-5.2001
  15. Magnusson, J., J. Strögren, and J. Schnürer. 2003. Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiol. Lett. 219: 129-135 https://doi.org/10.1016/S0378-1097(02)01207-7
  16. Niku-Paavola, M. L., A. Laitila, T. Mattila-Sandholm, and A. Haikara. 1999. New types of antimicrobial compounds produced by Lactobacillus plantarum. J. Appl. Microbiol. 86: 29-35 https://doi.org/10.1046/j.1365-2672.1999.00632.x
  17. Okkers, D. J., L. M. T. Dicks, M. Silvester, J. J. Joubert, and H. J. Odendaal. 1999. Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans. J. Appl. Microbiol. 87: 726-734 https://doi.org/10.1046/j.1365-2672.1999.00918.x
  18. Sanglard, D. 2002. Resistance of human fungal pathogens to antifungal drugs. Curr. Opin. Microbiol. 5: 379-385 https://doi.org/10.1016/S1369-5274(02)00344-2
  19. Schnurer, J. and J. Magnusson. 2005. Antifungal lactic acid bacteria as biopreservatives. Trends Food Sci. Technol. 16: 70-78 https://doi.org/10.1016/j.tifs.2004.02.014
  20. Sjogren, J., J. Magnusson, A. Broberg, J. Schnurer, and L. Kenne. 2003. Antifungal 3-hydroxy fatty acids from Lactobacillus plantarum MiLAB 14. Appl. Environ. Microbiol. 69: 7554-7557 https://doi.org/10.1128/AEM.69.12.7554-7557.2003
  21. Stiles, M. E. 1996. Biopreservation by lactic acid bacteria. Antonie van Leewenhoek 70: 331-345 https://doi.org/10.1007/BF00395940
  22. Strom, K., J. Sjogren, A. Broberg, and J. Schnurer. 2002. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and phenyl lactic acid. Appl. Environ. Microbiol. 68: 4322-4327 https://doi.org/10.1128/AEM.68.9.4322-4327.2002