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

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

  • 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)
  • 신명수 ((주)오비티 한국생명과학연구소) ;
  • 한선경 (충북대학교 수의과대학) ;
  • 최지현 (충북대학교 수의과대학) ;
  • 지애란 ((주)오비티 한국생명과학연구소) ;
  • 김경수 ((주)오비티 한국생명과학연구소) ;
  • 이완규 (충북대학교 수의과대학)
  • Received : 2009.04.14
  • Accepted : 2009.05.18
  • Published : 2009.06.28

Abstract

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.

Keywords

Antimicrobial substance;bacteriocin;lactic acid bacteria;probiotics;pig

Acknowledgement

Supported by : 충북대학교

References

  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. 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
  3. 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
  4. 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
  5. Klaenhammer, T. R. 1988. Bacteriocin of lactic acid bacteria. Biochimie. 70: 337-349 https://doi.org/10.1016/0300-9084(88)90206-4
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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
  12. Farber, J. M. and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55: 476-511
  13. 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
  14. 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
  15. 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
  16. 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
  17. Diez-Gonzalez, F. 2007. Applications of bacteriocins in livestock. Curr. Issues Intestinal Microbiol. 8: 15-24
  18. 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
  19. 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
  20. Zimmerman, D. R. 1986. Role of subtherapeutic antimicrobials in pig production. J. Anim. Sci. 62: 6-17
  21. 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
  22. 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
  23. 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
  24. 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
  25. 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
  26. 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
  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. 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
  29. Fuller, R. 1992. History and development of probiotics, pp. 1-8. In R. Fuller (ed.)., Probiotics. The scientific basis, Chapman and Hall, London
  30. 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
  31. 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
  32. 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
  33. 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
  34. 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
  35. 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
  36. Delves-Broughton, J. 1990. Nisin and its uses as a food preservative. Food Technol. 44: 100-117