한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제37권2호
  • /
  • Pages.125-132
  • /
  • 2009
  • /
  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

돼지 장관으로부터 분리한 Lactobacillus sp. HN 235 균주가 생산하는 항균물질의 특성

Characterization of Antimicrobial Substance Produced by Lactobacillus sp. HN 235 Isolated from Pig Intestine

  • 신명수 ((주)오비티 한국생명과학연구소) ;
  • 한선경 (충북대학교 수의과대학) ;
  • 최지현 (충북대학교 수의과대학) ;
  • 지애란 ((주)오비티 한국생명과학연구소) ;
  • 김경수 ((주)오비티 한국생명과학연구소) ;
  • 이완규 (충북대학교 수의과대학)
  • Shin, Myeong-Su (Korea Bio Science Research Institute of Organic Bio Tech Co. Ltd.) ;
  • Han, Sun-Kyung (College of Veterinary Medicine, Chungbuk National University) ;
  • Choi, Ji-Hyun (College of Veterinary Medicine, Chungbuk National University) ;
  • Ji, Ae-Ran (Korea Bio Science Research Institute of Organic Bio Tech Co. Ltd.) ;
  • Kim, Kyeong-Su (Korea Bio Science Research Institute of Organic Bio Tech Co. Ltd.) ;
  • Lee, Wan-Kyu (College of Veterinary Medicine, Chungbuk National University)
  • 투고 : 2009.04.14
  • 심사 : 2009.05.18
  • 발행 : 2009.06.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

항생제 대체물질로서 양돈용 생균제를 개발하기 위하여, 돼지의 소장 및 대장으로부터 500균주의 미생물을 분리하였으며, 항균물질인 박테리오신을 생산하는 5균주를 최종적으로 선발하였다. 선발균주들은 모두 Lactobacillus 속으로 동정되었으며, 생균제를 개발할 목적으로 상기 균주 중 Lactobacillus sp. HN 235 균주에 대한 균주 및 박테리오신 특성을 조사하였다. HN 235 균주의 성장시간에 따른 생균수 및 항균활성을 측정한 결과, 18시간째 최대 $3.5{\times}10^9\;cfu/mL$까지 성장하였으며, 항균활성은 유도기에 나타나기 시작하여 정체기에 6,400 AU/mL로 최고의 활성을 보이다가 급격하게 활성을 상실하였다. HN 235 균주가 생산하는 박테리오신은 병원성 균주로 알려진 L. monocytogenes와 Cl. perfringens에 대하여 강한 항균능력을 나타냈으며, 상기 박테리오신의 항균활성은 단백질 분해효소 처리시 활성을 상실하였다. 또한 $95^{\circ}C$에서도 활성을 유지하였으며, pH 2~10 범위 및 유기용매에 대해서도 비교적 안정한 항균활성을 나타내었다. SDS-PAGE를 실시하여 분자량을 측정한 결과, 대략 5 kDa으로 확인되었다.

In order to develop probiotics which may be a viable alternative of antibiotic use in pig industry, five bacterial strains (Lactobacillus sp. HN 52, 92, 98, 235 and AP 116) possessing antimicrobial properties were selected from 500 strains isolates of pig intestines. The bacteriocin produced by Lactobacillus sp. HN 235 displayed a relative broad spectrum of inhibitory activity against all Enterococcus strains, Pseudomonas aeruginosa, Listeria monocytogenes and Clostridium perfringens using the spot-on-lawn method. The production of antimicrobial substance started in the middle of the exponential growth phase, reached maximum levels (6,400 AU/mL) in the stationary phase, and then declined. Bacteriocin activity remained unchanged after 30 min of heat treatment at $95^{\circ}C$ and stable from pH 2.0 to 10 for 1 h, or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis.

키워드

참고문헌

  1. Aarnisalo, K., E. Vihavainen, L. Rantala, R. Maijala, M-L. Suihko, S. Hielm, P. Tuominen, J. Ranta, and L. Raaska. 2008. Use of results of microbiological analyses for riskbased control of Listeria monocytogenes in marinated broiler egs. Int. J. Food Microbiol. 121: 275-284 https://doi.org/10.1016/j.ijfoodmicro.2007.11.037
  2. Aasen, I. M., T. Moretro, T. Katla, L. Axelsson, and I. Storro. 2000. Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42678. Appl. Microbiol. Biotechnol. 53: 159-166 https://doi.org/10.1007/s002530050003
  3. Barefoot, S. F. and T. R. Klaenhammer. 1984. Purification and characterization of the Lactobacillus acidophilus bacteriocin lactacin B. Antimicrob. Agents Chemother. 26: 328-334 https://doi.org/10.1128/AAC.26.3.328
  4. Byun, J. W., G. T. Kim, H. S. Bae, Y. J. Baek, and W. K. Lee. 2000. In vitro selection of lactic acid bacteria for probiotic use in pigs. Korea J. Vet. Res. 40: 701-706
  5. Carolissen-Mackay, V., G. Arendse, and J. W. Hastings. 1997. Purification of bacteriocins of lactic acid bacteria: problems and pointers. Int. J. Food Microbiol. 34: 1-16 https://doi.org/10.1016/S0168-1605(96)01167-1
  6. Cleveland, J., T. J. Montville, I. F. Nes, and M. L. Chikindas. 2001. Bacteriocins: safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71: 1-20 https://doi.org/10.1016/S0168-1605(01)00560-8
  7. Czanderlova, L., P. Hlozek, D. Chmelar, and P. Lany. 2006. Clostridium perfringens in suckling piglets with diarrhoea and its PCR typing and prevalence in the Czech Republic in 2001-2003. Veterinarni Medicina. 51: 461-467
  8. Daba, H., S. Panadian, J. F. Gosselin, R. Simard, J. Huang, and C. Lacroix. 1991. Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides. Appl. Environ. Microbiol. 57: 3450-3455
  9. Dahiya, J. P., D. C. Wilkie, A. G. Van Kessel, and M. D. Drew. 2006. Potential strategies for controlling necrotic enteritis in broiler chickens in post-antibiotic era. Anim. Feed Sci. Technol. 129: 60-88 https://doi.org/10.1016/j.anifeedsci.2005.12.003
  10. Delves-Broughton, J. 1990. Nisin and its uses as a food preservative. Food Technol. 44: 100-117
  11. De Vuyst, L., R. Callewaert, and K. Crabbe. 1996. Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin under unfavourable growth conditions. Microbiology. 142: 817-827 https://doi.org/10.1099/00221287-142-4-817
  12. Diez-Gonzalez, F. 2007. Applications of bacteriocins in livestock. Curr. Issues Intestinal Microbiol. 8: 15-24
  13. Elegado, F. B., W. J. Kim, and D. Y. Kwon. 1997. Rapid purification, partial characterization, and antimicrobial spectrum of the bacteriocin, pediocin AcM, from Pediococcus acidilactici M. Int. J. Food Microbiol. 37: 1-11 https://doi.org/10.1016/S0168-1605(97)00037-8
  14. Ennahar, S., T. Sashihara, K. Sonomoto, and A. Ishizaki. 2000. Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol. Rev. 24: 85-106 https://doi.org/10.1111/j.1574-6976.2000.tb00534.x
  15. Farber, J. M. and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55: 476-511
  16. Fuller, R. 1992. History and development of probiotics, pp. 1-8. In R. Fuller (ed.)., Probiotics. The scientific basis, Chapman and Hall, London
  17. Gillor, O., B. C. Kirkup, and M. A. Riley. 2004. Colicins and microcins: the next generation antimicrobials. Adv. Appl. Microbiol. 54: 129-146 https://doi.org/10.1016/S0065-2164(04)54005-4
  18. Guyonnet, D., C. Fremaux, Y. Cenatiempo, and J. M. Berjeaud. 2000. Method for rapid purification of class IIa bacteriocins and comparison of their activities. Appl. Environ. Microbiol. 66: 1744-1748 https://doi.org/10.1128/AEM.66.4.1744-1748.2000
  19. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams. 1994. Regular, nonsporing gram-positive rods. pp. 565-570. In Bergey's Manual of Determinative Bacteriology. 9th ed. Williams and Wilkins. Baltimore, USA
  20. Hong, J. W., I. H. Kim, Y. K. Han, S. H. Lee, O. S. Kwon, J. H. Kim, and K. H. Kang. 2002. Probiotic properties of Enterococcus durans LP44 isolated from pigs feces. J. Korean Soc. Food Sci. Nutr. 31: 939-944
  21. Johansson, T. 1998. Enhanced detection and enumeration of Listeria monocytogenes from foodstuffs and food-processing environments. Int. J. Food Microbiol. 40: 77-85 https://doi.org/10.1016/S0168-1605(98)00022-1
  22. Jung, B. Y., H. S. Lim, and B. H. Kim. 2003. Prevalence of Listeria spp. in cecal contents of livestock. Kor. J. Vet. Publ. Hlth. 27: 41-46
  23. Kim, N. Y., E. H. Oh, K. P. Houng, G. C. Kang, I. H. Chung, and S. J. Park. 1998. Studies on the Clostridium perfringens isolated from piglets with diarrhea in western area of Chonnam province. Kor. J. Vet. Serv. 21: 141-148
  24. Klaenhammer, T. R. 1988. Bacteriocin of lactic acid bacteria. Biochimie. 70: 337-349 https://doi.org/10.1016/0300-9084(88)90206-4
  25. Mateu, E. and M. Martin. 2001. Why is anti-microbial resistance a veterinary problem as well? J. Vet. Med. 48: 569-581 https://doi.org/10.1046/j.1439-0450.2001.00475.x
  26. Mayr, H. A., A. J. Hedges, and R. C. W. Berkeley. 1972. Methods for studying bacteriocin. pp. 313-342. In Bergen, T. and J. R. Norris (ed.), Methods in Microbiology, Academic Press, New York
  27. Parente, E. and A. Ricciardi. 1994. Influence of pH on the production of enterocin 1146 during batch fermentation. Lett. Appl. Mcirobiol. 19: 12-15 https://doi.org/10.1111/j.1472-765X.1994.tb00891.x
  28. Parente, E., A. Ricciardi, and G. Addario. 1994. Influence of pH on growth and bacteriocin production by Lactococcus lactis subsp. lactis 140 NWC during batch fermentation. Appl. Microbiol. Biotechnol. 41: 388-394
  29. Petersen, L. and M. Madsen. 2000. Listeria spp. in broiler flocks: recovery rates and species distribution investigated by conventional culture and the EiaFoss method. Int. J. Food Microbiol. 58: 113-116 https://doi.org/10.1016/S0168-1605(00)00258-0
  30. Rodríguez, E., J. L. Arqués, R. Rodríguez, M. Nuñez, and M. Medina. 2003. Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Lett. Appl. Microbiol. 37: 259-263 https://doi.org/10.1046/j.1472-765X.2003.01390.x
  31. Ryan, M. P., W. J. Meaney, R. P. Ross, and C. Hill. 1998. Evaluation of lacticin 3147 and a teat seal containing this bacteriocin for inhibition of mastitis pathogens. Appl. Environ. Microbiol. 64: 2287-2290
  32. Sch$\"{a}$gger, H. and G. Von Jagow. 1987. Tricine-sodium dodecyl sulfate -polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368-379 https://doi.org/10.1016/0003-2697(87)90587-2
  33. Shin, M. S., S. K. Han, J. S. Ryu, K. S. Kim, and W. K. Lee. 2008. Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi. J. Appl. Microbiol. 105: 331-339 https://doi.org/10.1111/j.1365-2672.2008.03770.x
  34. Th$\'{e}$venot, D., A. Dernburg, and C. Vernozy-Rozand. 2006. An updated review of Listeria monocytogenes in the pork meat industry and its products. J. Appl. Microbiol. 101: 7-17 https://doi.org/10.1111/j.1365-2672.2006.02962.x
  35. Yang, R., M. C. Johnson, and B. Ray. 1992. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl. Environ. Microbiol. 58: 3355-3359
  36. Zimmerman, D. R. 1986. Role of subtherapeutic antimicrobials in pig production. J. Anim. Sci. 62: 6-17