DOI QR코드

DOI QR Code

Analysis of Microbial Diversity in Nuruk Using PCR-DGGE

PCR-DGGE를 이용한 누룩에서의 미생물 다양성 분석

  • Kwon, Seung-Jik (Division of Bacterial Respiratory Infections, Korea National Institute of Health) ;
  • Sohn, Jae-Hak (Department of Bio-food material, College of Medical & Life Sciences, Silla University)
  • 권승직 (국립보건연구원 질병관리본부) ;
  • 손재학 (신라대학교 의생명과학대 바이오식품소재학과)
  • Received : 2011.11.25
  • Accepted : 2012.01.25
  • Published : 2012.01.30

Abstract

Nuruk plays a significant role in the flavor and quality of Takju and Yakju, which are produced through saccharification and alcohol fermentation by various microorganisms. In this study, we identified microbial strains isolated from a plate count and PCR-denaturing gradient gel electrophoresis (DGGE) analysis targeting the 16S and 28S rRNA genes, in order to characterize bacterial and fungal diversity in Sansung Nuruk. The numbers of bacteria and fungi in Nuruk were $1.5{\times}10^9$ CFU/g and $2.2{\tims}10^8$ CFU/g, respectively. The 16S rRNA gene sequence indicated that the predominant bacteria in the isolates and PCR-DGGE profile of Nuruk were Kocuria spp., Pantoea spp., Lactobacillus spp., Pediococcus spp., Weissella spp., Staphylococcus spp., endophytic bacterium, uncultured Gamma-proteobacteria, uncultured Cyanobacteria, and Actinobacteria. Dominant bacteria from the PCR-DGGE profile were Pediococcous pentosaceus and uncultured Cyanobacteria. The 28S rRNA gene sequence indicated the predominant fungi in the isolates and PCR-DGGE profile to be Trichomonascus spp. Pichia spp., Torulaspora spp., Wickerhamomyces spp., Sacharomycopsis spp., Lichtheimia spp., Mucor spp., Rhizopus spp. Aspergillus spp., and Cladosporium spp. Dominant fungi from the PCR-DGGE profile were Pichia kudriavzevii and Aspergillus oryzae. The PCR-DGGE technique was used for the first time in this study to assess a microbial community in Nuruk and proved to be an effective protocol for profiling microbial diversity.

누룩은 탁주와 약주의 제조를 위한 당화효소와 알코올발효를 위한 미생물의 공급원으로서 제품의 맛과 품질을 결정하는 중요한 역할을 한다. 본 연구에서는 산성누룩의 세균과 진균의 다양성을 조사하기 위해 순수분리 종과 16S 및 28S rRNA gene를 대상으로 한 PCR-DGGE를 이용한 분석을 수행하였다. 누룩 내 세균의 수는 $2.7{\times}10^9$ CFU/g이었으며 순수분리와 PCR-DGGE 분석에서 우점종은 Kocuria spp., Pantoea spp., Lactobacillus spp., Pediococcous spp., Weissella spp., Staphylococcus spp. 그 외 endophytic bacterium, uncultured gamma-proteobacteria, uncultured Cyanobacteria와 Actinobacteria였다. PCR-DGGE profile에서 주된 우점종은 Pediococcous pentosaceus와 uncultured Cyanobacteria 이었다. 누룩 내 진균의 수는 $3.5{\times}10^8$ CFU/g이었으며 순수분리와 PCR-DGGE 분석에서 우점종은 Trichomonascus spp., Pichia spp., Torulaspora spp., Wickerhamomyces spp., Sacharomycopsis spp., Lichtheimia spp., Mucor spp., Rhizopus spp., Aspergillus spp., Cladosporium spp.였다. PCR-DGGE profile에서 주된 우점종은 Pichia kudriavzevii와 Aspergillus oryzae이었다. PCR-DGGE 기술은 본 연구에서 누룩의 미생물군집을 평가하기 위해 처음으로 사용되었으며 미생물 다양성을 설명하는 데 효과적임을 입증하였다.

Keywords

References

  1. Bowman, J. P., S. A. McCammon, J. A. Gibson, L. Robertson, and P. D. Nichols. 2003. Prokaryotic metabolic activity and community structure in Antarctic continental shelf sediments. Appl. Environ. Microbiol. 69, 2448-2462. https://doi.org/10.1128/AEM.69.5.2448-2462.2003
  2. Chang, H. W., K. H. Kim, Y. D. Nam, S. W. Roh, M. S. Kim, C. O. Jeon, H. M. Oh, and J. W. Bae. 2008. Analysis of yeast and archaeal population dynamics in kimchi using denaturing gradient gel electrophoresis. Int. J. Food Microbiol. 126, 159-166. https://doi.org/10.1016/j.ijfoodmicro.2008.05.013
  3. Cocolin, L., L. F. Bisson, and D. A. Mills. 2000. Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiol. Lett. 189, 81-87. https://doi.org/10.1111/j.1574-6968.2000.tb09210.x
  4. Ha, D. M., D. C. Kim, S. M. Hong, and C. W. Lee. 1989. Identification and properties of starch utilizing yeasts isolated from Nuruk. J. Korean Agric. Chem. Soc. 32, 408-415.
  5. Jo, K. Y. and D. M. Ha. 1995. Isolation and identification of the Lactic acid bacteria from Nuruk. Agri. Chem. Biotechnol. 38, 95-99.
  6. Jung, H. K., C. H. Park, G. D. Lee, S. C. Park, H. H. Park, and J. H. Hong. 2007. Characteristics of Pichia anomala K15 producing killer toxin isolated from traditional nuruk. J. Korean Soc. Food Sci. Nutr. 36, 1077-1082. https://doi.org/10.3746/jkfn.2007.36.8.1077
  7. Lane, D. J. 1991. 16S/23S rRNA sequencing, pp. 115-175, In Stackebrandt, E. and M. Goodfellow (eds.), Nucleic acid techniques in bacterial systematic, John Wiley & Sons. New York.
  8. Lee, S. H., H. J. Jung, S. H. Yeo, H. S. Kim, and T. S. Yu. 2004. Characteristics of a-amylase of a new species Aspergillus coreanus NR 15-1. Korean J. Biotechnol. Bioeng. 19, 301-307.
  9. Kim, H. S., J. S. Hyun, J. Kim, H. P. Ha, and T. S. Yu. 1998. Enzymological characteristics and identification of useful fungi isolated from traditional Korean nuruk. Korean J. Appl. Microbiol. Biotechnol. 26, 456-464.
  10. Kim, J. Y. and J. S. Kho. 2004. Screening of brewing yeasts and saccharifying molds for foxtail millet-wine making. J. Korean Soc. Appl. Biol. Chem. 47, 78-84.
  11. Marmur, J. 1961. A procedure for the isolation of DNA from microorganisms. J. Mol. Biol. 3, 208-218. https://doi.org/10.1016/S0022-2836(61)80047-8
  12. Masoud, W., L. B. Cesar, L. Jespersen, and M. Jakobsen. 2004. Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis. Yeast 21, 549-556. https://doi.org/10.1002/yea.1124
  13. Meroth, C. B., W. P. Hammes, and C. Hertel. 2003. Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 69, 7453-7461. https://doi.org/10.1128/AEM.69.12.7453-7461.2003
  14. Mills, D. A., E. A. Johannsen, and L. Cocolin. 2002. Yeast diversity and persistence in botrytis-affected wine fermentations. Appl. Environ. Microbiol. 68, 4884-4893. https://doi.org/10.1128/AEM.68.10.4884-4893.2002
  15. Muyzer, G., E. D. Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction- amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695-700.
  16. Rantsiou, K., R. Urso, L. Iacumin, C. Cantoni, P. Cattaneo, G. Comi, and L. Cocolin, 2005. Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages. Appl. Environ. Microbiol. 71, 1977-1986. https://doi.org/10.1128/AEM.71.4.1977-1986.2005
  17. Reeson, A. F., T. Jankovic, M. L. Kasper, S. Rogers, and A. D. Austin. 2003. Application of 16S rDNA-DGGE to examine the microbial ecology associated with a socialwasp Vespula germanica. Insect Mol. Biol. 12, 85-91. https://doi.org/10.1046/j.1365-2583.2003.00390.x
  18. Sharma, S., Z. Szele, R. Schilling, J. C. Munch, and M. Schloter. 2006. Influence of freeze-thaw stress on the structure and function of microbial communities and denitrifying populations in soil. Appl. Environ. Microbiol. 72, 2148-2154. https://doi.org/10.1128/AEM.72.3.2148-2154.2006
  19. Shin, Y. D. and D. H. Cho. 1970. A study on the microflora changes during Takju brewing. Korean J. Microbiol. 8, 53-64.
  20. So, M. H. and Y. S. Lee. 2009. Effects of culture conditions of Rhizopus sp. ZB9 on the production of saccharifying amylase during the preparation of rice koji. Korean J. Food Nutr. 22, 644-649.
  21. Suh, M. J. and K. H. Nho. 1987. Studies on the screening and properties of raw starch saccharifying microorganism. Korean J. Mycol. 15, 169-174.
  22. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL_$\chi$ windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876-4882. https://doi.org/10.1093/nar/25.24.4876
  23. Xia, S., Y. Shi, Y. Fu, and X. Ma. 2005. DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical-biological flocculation and chemical coagulation systems. Appl. Environ. Microbiol. 69, 99-105.
  24. Yang, J. Y. and K. H. Lee. 1996. Shelf-life and microbiological study of Sansung Takju. Korean J. Food Sci. Technol. 28, 779-785.
  25. Yeates, C., M. R. Gillings, A. D. Davison, N. Altavilla, and D. A. Veal. 1998. Methods for microbial DNA extraction from soil for PCR amplification. Biol. Proced. Online 1, 40-47. https://doi.org/10.1251/bpo6
  26. Yu, T. S., H. S. Kim, H. P. Ha, T. Y. Kim, and I. W. Yoon. 1996. Bibliographical study on microorganisms of Nuruk (Until 1945). J. Korean Soc. Food Nutr. 25, 170-179.

Cited by

  1. Change in the composition and enzyme activity of culturable lactic acid bacteria in Nuruk during fermentation at different temperatures vol.22, pp.6, 2015, https://doi.org/10.11002/kjfp.2015.22.6.920
  2. Pyrosequencing reveals bacterial diversity in Korean traditional wheat-based nuruk vol.53, pp.12, 2015, https://doi.org/10.1007/s12275-015-5516-3
  3. Comprehensive analysis of fungal diversity and enzyme activity in nuruk, a Korean fermenting starter, for acquiring useful fungi vol.55, pp.5, 2017, https://doi.org/10.1007/s12275-017-7114-z