Effects of Protease-resistant Antimicrobial Substances Produced by Lactic Acid Bacteria on Rumen Methanogenesis

  • Reina, Asa (Graduate School of Animal Science, Obihiro University of Agriculture and Veterinary Medicine Obihiro) ;
  • Tanaka, A. (Graduate School of Animal Science, Obihiro University of Agriculture and Veterinary Medicine Obihiro) ;
  • Uehara, A. (Fermentation & Biotechnology Laboratory, Amino Acids Company, Ajinomoto Co., Inc.) ;
  • Shinzato, I. (Fermentation & Biotechnology Laboratory, Amino Acids Company, Ajinomoto Co., Inc.) ;
  • Toride, Y. (Fermentation & Biotechnology Laboratory, Amino Acids Company, Ajinomoto Co., Inc.) ;
  • Usui, N. (Fermentation & Biotechnology Laboratory, Amino Acids Company, Ajinomoto Co., Inc.) ;
  • Hirakawa, K. (Fermentation & Biotechnology Laboratory, Amino Acids Company, Ajinomoto Co., Inc.) ;
  • Takahashi, J. (Graduate School of Animal Science, Obihiro University of Agriculture and Veterinary Medicine Obihiro)
  • Received : 2009.08.27
  • Accepted : 2009.11.06
  • Published : 2010.06.01


Effects of protease-resistant antimicrobial substances (PRA) produced by Lactobacillus plantarum and Leuconostoc citreum on rumen methanogenesis were examined using the in vitro continuous methane quantification system. Four different strains of lactic acid bacteria, i) Lactococcus lactis ATCC19435 (Control, non-antibacterial substances), ii) Lactococcus lactis NCIMB702054 (Nisin-Z), iii) Lactobacillus plantarum TUA1490L (PRA-1), and iv) Leuconostoc citreum JCM9698 (PRA-2) were individually cultured in GYEKP medium. An 80 ml aliquot of each supernatant was inoculated into phosphate-buffered rumen fluid. PRA-1 remarkably decreased cumulative methane production, though propionate, butyrate and ammonia N decreased. For PRA-2, there were no effects on $CH_4$ and $CO_2$ production and fermentation characteristics in mixed rumen cultures. The results suggested that PRA-1 reduced the number of methanogens or inhibited utilization of hydrogen in rumen fermentation.


Methane Production;Lactic Acid Bacteria;In vitro Fermentation


  1. Callaway, T. R., M. S. Alexandra, Carneiro De Melo and J. B. Russell. 1997. The effect of nisin and monensin on ruminal fermentations in vitro. Curr Microbiol. 35:90-96
  2. Daeschel, M. A., M. C. Mckenny and L. C. McDonald. 1990. Bacteriocidal activity of Lactobacillus plantarum C11. Food Microbiol. 7:91-99
  3. Delves-Broughton, J., P. Blackburn, R. Evans and J. hugenholtz. 1996. Applications of the bacteriocin, nisin. Antonie Van Leeuwenhoek 69:193-202
  4. Farkas-Himsley, H. 1980. Bacteriocins-are they broad-spectrum antibiotics? J. Antimicrob. Chemother. 6:424-426
  5. Gonz$\'{a}$lez, B., P. Arca, B. Mayo and J. E. Su$\'{a}$rez. 1994. Detection, purification and partial characterization of plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin. Appl. Environ. Microbiol. 6:2158-2163
  6. Jim$\'{e}$nez-D$\'{i}$az, R., R. M. Rios-S$\'{a}$nchez, M. Desmazeaud, J. L. Ruiz-Barba and J. C. Piard. 1993. Plantaricin S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation. Appl. Environ. Microbiol. 59:1416-1424
  7. Sauer, F. D., V. Fellner, R. Kinsman, J. K. Kramer, H. A. Jackson, A. J. Lee and S. Chen. 1998. Methane output and lactation response in Holstein cattle with monensin or unsaturated fat added to the diet. J. Anim. Sci. 76:906-914
  8. Sang, S. L., Jih-Tay Hsu, Hilario C. Mantovani and James B. Russell. 2002. The effect of bovicin HC5, A bacteriocin from Streptococcus bovis HC5, on ruminal methane production in vitro. FEMS Microbiol. Lett. 217:51-55
  9. Sang, S. L., Hilário C. Mantovani and James B. Russell. 2002. The binding and degradation of nisin by mixed ruminal bacteria. FEMS Microbiol. Ecol. 42:339-345
  10. Todorov, S., B. Onno, O. Sorokine, J. M. Chobert, I. Ivanova and X. Dousset. 1999. Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough. Int. J. Food Microbiol. 48:167-177
  11. Yoshida, N., N. Takahashi and A. Hiraishi. 2005. Phylogenetic characterization of a polychlorinated-dioxin-dechlorinating microbial community by use of microcosm studies. Appl. Environ. Microbiol. 71:4325-4334
  12. Yuan, J., Z.-Z. Zang, X.-Z. Chen and W. Yang. 2004. Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants. Appl. Microbiol. Biotechnol. 64:806-815
  13. Leal, M. V., M. Baras, J. L. Ruiz-Barba, B. Floriano and R. Jimenez-Diaz. 1998. Bacteriocin production and competitiveness of Lactobacillus plantarum LPCO10 in olive juice broth, a culture medium obtained from olives. Int. J. Food. Microbiol. 43:129-134
  14. Aymerich, M. T., M. Garriga, J. M. Monfort, I. Nes and M. Hugas. 2000. Bacteriocin-producing lactobacilli in Spanish-style fermented sausages: haracterization of bacteriocins. Food Microbiol. 17:33-45
  15. Enan, G., A. A. El-Essawy, M. Uyttendaele and J. Debevere. 1996. Antibacterial activity of Lactobacillus plantarum UG1 isolated from dry sausage:characterization, production and bactericidal action of plantarcin UG1. Int. J. Food. Microbiol. 30:189-215
  16. Muyzer, G., E. C. De waal and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700
  17. SAS. 1996. SAS/STAT$^{{\circledR}}$ Software: Changes and Enhancements through release 6.11. SAS Institute Inc., Cary, NC, USA
  18. Brijesh, K. T., P. V. Vasilis, P. O. D. Colm, M. Kasiviswanathan, B. Paula and P. J. Cullen. 2009. Applivation of natural antimicrobials for food preservation. J. Agric. Food Chem. 57: 5987-6000
  19. Nakatsu, C. H., V. Torsvik and L. ${\O}$vreas. 2000. Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Sci. Soc. Am. J. 64:1382-1388
  20. Santoso, B., B. Mwenya, C. Sar, Y. Gamo, T. Kobayashi, R. Morikawa, K. Kimura, H. Mizukoshi and J. Takahashi. 2004. Effects of supplementing galacto- oligosaccharides, Yucca schidigera or nisin on rumen methanogenesis, nitrogen and energy metabolism in sheep. Livest. Prod. Sci. 91:209-217
  21. Ennahar, S., K. Sonomoto and A. Ishizaki. 1999. Class IIa bacteriocins from lactic acid bacteria: Antibacterial activity and food preservation. J. Biosci. Bioeng. 87:705-716
  22. Sar, C., B. Mwenya, B. Santoso, K. Takaura, R. Morikawa, N. Isogai, Y. Asakura, Y. Toride and J. Takahasi. 2005a. Effect of Escherichia coli W3110 on ruminal methanogenesis and nitrate/nitrite reduction in vitro. Anim. Feed Sci. Technol. 118:295-306
  23. Sar, C., B. Mwenya, B. Pen, R. Morikawa, K. Takaura, T. Kobayashi and J. Takahashi. 2005b. Effect of nisin on ruminal methane production and nitrate/nitrite reduction in vitro. Aust. J. Agric. Res. 56:803-810
  24. Aslim, B., Z. N. Yuksekdag, E. Sarikaya and Y. Beyatli. 2005. Determination of the bacteriocin-like substances produced by some lactic acid bacteria isolated from Turkish dairy products. LWT 38:691-694
  25. Guan, H., K. M. Wittenberg, K. H. Ominski and D. O. Krause. 2006. Efficacy of ionophores in cattle diets for mitigation of enteric methane. J. Anim. Sci. 84:1896-1906
  26. McAuliffe, O., R. P. Ross and C. Hill. 2001. Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol. Rev. 25:285-308
  27. Kalmokoff, M. L., F. Bartlett and R. M. Teather. 1996. Are ruminal bacteria armed with bacteriocins? J. Dairy Sci. 79:2297-2306
  28. Green, S. J. and D. Minz. 2005. Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates. Appl. Environ. Microbiol. 71:4721-4727
  29. Mwenya, B., C. Sar, B. Pen, R. Morikawa, K. Takaura, S. Kogawa, K. Kimura, K. Umetsu and J. Takahashi. 2006. Effects of feed additives on ruminal methanogenesis and anaerobicfermentation of manure in cows and steers. In:Greenhouse Gases and Animal Agriculture (Ed. C. R. Soliva, J. Takahashi and M. Kreuzer). Amsterdam. ELSEVIER B.V. pp. 209-212
  30. WHO Expert Committee on Food Additives. 1969. Specifications for the identify and purity of food additives and their toxicological evaluation: some antibiotics. World Health Organ. Tech. Rep. Ser. No. 430
  31. Kelly, W. J., R. V. Asmundson and C. M. Huang. 1996. Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum. J. Appl. Bacteriol. 81:657-662
  32. Chen, H. and D. G. Hoocver. 2003. Bacteriocins and their food applications. CRFSFS 12:82-99
  33. ${\O}$rskov, E. R. and I. McDonald. 1979. The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J. Agric. Sci. Camb. 92:499-503
  34. Garriga, M., M. Hugas, T. Aymerich and J. M. monfort. 1993. Bacteriocinocinogenic activity of lactobacilli from fermented sausages. J. Appl. Bacteriol. 75:142-148
  35. Johnson, K. A. and D. E. Johnson. 1995. Methane emission from cattle. J. Anim. Sci. 73:2483-2492
  36. Conway, E. J. and E. O'Malley. 1942. Microdiffusion methods:ammonia and urea using buffered absorbents (revised methods for ranges greater than 10 $\mu$g N). Biochem. J. 36:655-661
  37. Mantovani, H. C. and J. B. Russell. 2002. The ability of a bacteriocin of streptococcus bovis HC5 (bovicin HC5) to inhibit clostridium aminophilum, an obligate amino acid fermenting bacterium from the rumen. Anaerobe 8:247-252
  38. MacDougall, E. I. 1948. Studies on ruminal saliva 1. The composition and output of sheep's saliva. Biochem. J. 43:99-109
  39. Rekhif, N., A. Atrih and G. Lefebvre. 1995. Activity of plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6 isolated from fermented sausage. J. Appl. Bacteriol. 78:349-358

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