배양 분리법을 통한 젓갈 내 원핵 세균 군집 분석 및 신규 미생물의 분리

Analysis of Prokaryote Communities in Korean Traditional Fermented Food, Jeotgal, Using Culture-Dependent Method and Isolation of a Novel Strain

  • 발행 : 2009.03.31

초록

우리나라의 전통 발효 식품인 젓갈로부터 배양 분리법과 분자생물학적 분석법을 이용하여 원핵 세균 군집을 분석하고, 신규 미생물 분리를 목표로 하였다. 젓갈은 생산 지역과 주재료를 고려하여 17 종을 선정하였으며, 이들 젓갈 시료를 적정 희석배수로 희석하여 12종류의 미생물 선택배지에 도말, 배양한 후 나타난 집락(colony)을 형태학적 특성에 따라 무작위로 308개를 선정하여 분리하였다. 순수 분리된 미생물은 PCR 방법을 이용하여 16S rRNA 유전자의 염기서열을 분석한 후, 기존에 보고된 미생물 database와 비교함으로서 17종의 젓갈 내 미생물 군집을 확인하였다. 젓갈의 발효 및 숙성 과정에 관여하는 lactic acid bacteria (Leuconostoc 속, Weisella 속, Lactococcus 속, Lactobacillus 속, Carnobacterium 속, Marinilactibacillus 속, Tetragenococcus 속)와 Bacillus 속, Pseudomonas 속, Micrococcus 속, Brevibacterium 속, Microbacterium 속과 Kocuria 속이 17가지 젓갈에서 광범위하게 분리되었으며, Salinicoccus 속, Halomonas 속, Cobetia 속, Lentibacillus 속, Paracoccus 속, Psychrobacter 속이 소수 분리되었다. 또한 분리된 미생물의 계통학적 분석을 통하여 기존에 보고된 적이 없는 신규 미생물 14종을 분리하였다.

This study was aimed at the analysis of prokaryote communities in Korean traditional fermented food, jeotgal, and isolation of a novel strain from jeotgal by using culture-dependent and molecular biological approaches. Seventeen kinds of jeotgal were selected on the basis of its origins and sources. The samples were inoculated on 12 kinds of media. 308 isolates were selected randomly by morphological features, and its 16S rRNA gene sequences was amplified by PCR technique with bacteria and archaea specific primers (8F, 21F, and 1492R). The 16S rRNA gene sequences were compared with those in EzTaxon and GenBank databases. DNA-DNA hybridization was performed to identify a novel strain. As a result, the majority of the isolates were lactic acid bacteria (Leuconostoc, Weisella, Lactococcus, Lactobacillus, Carnobacterium, Marinilactibacillus), Bacillus, Pseudomonas, Micrococcus, Brevibacterium, Microbacterium and Kocuria in 17 kinds of jeotgal. The strains belonging to Salinicoccus, Halomonas, Cobetia, Lentibacillus, Paracoccus, and Psychrobacter were isolated as minor ones. Fourteen novel species were identified based on phylogenetic analysis.

키워드

참고문헌

  1. 도순석, 이영미, 장학길. 1993. 지역별 젓갈의 종류와 이용도에 관한 연구. 한국조리과학회 9, 222-229
  2. 윤지혜, 이명숙. 1993. 저염 창란젓갈 제조 과정중 미생물상의 변화. 한국국제경제학회. PB-25, 140-141
  3. 이철호, 이옹호, 임무현. 1987. 한국의 수산발효식품, 유림문화사
  4. 한국식품개발원. 1990. 한국의 젓갈. 기술신서 제4집
  5. 한국식품개발연구원 전통식품연구실. 1992. 전통식품의 현대화 전략
  6. Baker, G.C., J.J. Smith, and D.A. Cowan. 2003. Review and reanalysis of domain-specific 16S primers. J. Microbiol. Methods 55, 541-555 https://doi.org/10.1016/j.mimet.2003.08.009
  7. Cha, S.K., J.S. Ahn, and B.H. Ahn. 2001. Searching and preservation of mircrobial resources from traditional fermented foods. Food Ind. Nutr. 6, 60-66
  8. Cha, Y.J., S.Y. Chung, J.H. Ha, I.C. Jeong, and E.H. Lee. 1983. Studies on the processiong of low salt fermented sea foods. Bull. Korean Fish Soc. 16, 211-215
  9. Chun, J., J.H. Lee, Y. Jung, M. Kim, S. Kim, B.K. Kim, and Y.W. Lim. 2007. Eztaxon: A web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol. 57, 2259-2261 https://doi.org/10.1099/ijs.0.64915-0
  10. Ezaki, T., H. Hashimoto, and E. Yabuuchi. 1989. Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39, 224-229 https://doi.org/10.1099/00207713-39-3-224
  11. Hirayama, H., J. Tamaoka, and K. Horikoshi. 1996. Improved immobilization of DNA to microwell plates for DNA-DNA hybridization. Nucleic Acids Res. 24, 4098-4099 https://doi.org/10.1093/nar/24.20.4098
  12. Hur, S.H. 1996. Critical review on the microbiological standardization of salt-fermented fish product. J. Food Sci. Nutr. 25, 885-891
  13. Kim, S.J., S.J. Ma, and H.L. Kim. 2005. Probiotic properties of lactic acid bacteria and yeasts isolated from Korean traditional food, Jeot-gal. Kor. J. Food Preserv. 12, 184-189
  14. Lee, J.G. and W.K. Choi. 1974. Studies on the variation of microflora during the fermentation of anchovy, Engraulis japonica. Bull Korean Fish Soc. 7, 105-114
  15. Roh, S.W., Y.D. Nam, H.W. Chang, Y. Sung, K.H. Kim, H.J. Lee, H.M. Oh, and J.W. Bae. 2007. Natronococcus jeotgali sp. nov., a halophilic archaeon isolated from shrimp jeotgal, a traditional fermented seafood from Korea. Int. J. Syst. Evol. Microbiol. 57, 2129-2131 https://doi.org/10.1099/ijs.0.65120-0
  16. Saitou, N. and M. Nei. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425
  17. Stackebrandt, E. and B.M. Goebel. 1994. Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44, 846-849 https://doi.org/10.1099/00207713-44-4-846
  18. Stackebrandt, E., C. Koch, O. Gvozdiak, and P. Schumann. 1995. Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus Cohn 1872 gen. emend. Int. J. Syst. Bacteriol. 45, 682-692 https://doi.org/10.1099/00207713-45-4-682
  19. Suh, H.K. and S.S. Yoon. 1987. The study on the regional characteristics of Korean chotkal. Korean J. Dietary Culture 2, 45-54
  20. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. Mega4: Molecular evolutionary genetics analysis (mega) software version 4.0. Mol. Biol. Evol. 24, 1596-1599 https://doi.org/10.1093/molbev/msm092
  21. Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and D.G. Higgins. 1997. The Clustal_X windows interface: Flexible strategies for multiple sequence alignmentaided by quality analysis tools. Nucleic Acids Res. 25, 4876-4882 https://doi.org/10.1093/nar/25.24.4876
  22. Wayne, L.G., D.J. Brenner, and R.R. Colwell. 1987. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol. 37, 463-464 https://doi.org/10.1099/00207713-37-4-463