Effects of Thymol, Eugenol and Malate on In vitro Rumen Microbial Fermentation

  • Received : 2009.09.30
  • Accepted : 2009.12.10
  • Published : 2009.12.01


The purpose of this study was to investigate effects of increased levels of eugenol, thymol and malate on pH and the concentrations of VFA, lactate and ammonia-N during in vitro ruminal incubation. One Hanwoo beef steer (741 kg) fitted with a rumen cannula was used and fed 0.5 kg/day rice straw and 10 kg/day corn-based concentrate (ratio of concentrate to rice straw = 95 : 5 on DM basis). Three different doses of thymol, eugenol and malate were used. Treatments of the experiment were as follows: Treatments of thymol were control (1g D-glucose/40ml), T1 (1g D-glucose + 40 mg thymol/40 ml), T2 (1g D-glucose + 50 mg thymol/40 ml) and T3 (1g D-glucose + 60 mg thymol/40 ml). Treatments of eugenol were control (1g D-glucose/40 ml), E1 (1g D-glucose + 55 mg eugenol/40 ml), E2 (1g D-glucose + 65 mg eugenol/40 ml) and E3 (1g D-glucose + 75 mg eugenol/40 ml). Treatments of malate were control (1g D-glucose/40ml), M1 (1g D-glucose + 25 mg malate/40ml), M2 (1g D-glucose + 50 mg malate/40 ml) and M3 (1g D-glucose + 100 mg malate/40 ml). The results of this study showed that eugenol and thymol have improved stability of the ruminal fermentation by decreasing lactic acid concentration and increasing ruminal pH. However, it inhibited the production of total VFA, acetate and propionate. Malate also improved stability of the ruminal fermentation by decreasing lactic acid concentration and increasing ruminal pH, but it had a very little effect on ruminal lactate concentrations and pH. On the other hand, malate did not decrease the concentrations of total VFA, acetate and propionate. Therefore, at the low ruminal pH expected in high-concentrate diets, thymol, eugenol, and malate are potentially useful in Hanwoo finishing diets. Further studies are necessary for determining the effectiveness of these additives on in vivo rumen fermentation and animal performance in Hanwoo finishing steers.


  1. Association of Official Analytical Chemists. 1990. Official Methods of Analysis, Association of Official Analytical Chemists, Washington, D. C.
  2. Benchaar, C., Petit, H. V., Berthiaume, R., Ouellet, D. R., Chiquette, J. and Chouinard, P. Y. 2007. Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations, milk production, and milk composition in dairy cows fed alfalfa silage or corn silage. J. Dairy Sci. 90:886-897.
  3. Bevans, D. W., Beauchemin, K. A., Schwartzkopf-Genswein, K. S., McKinnon, J. J. and McAllister, T. A. 2005. Effect of rapid or gradual grain adaptation on subacute acidosis and feed intake by feedlot cattle. J. Anim. Sci. 83:1116-1132.
  4. Borchers, R. 1965. Proteolytic activity of rumen fluid in vitro. J. Anim. Sci. 24:1033-1038.
  5. Burt, S. 2004. Essential oils: Their antibacterial properties and potential applications in foods-A review. Int. J. Food Microbiol. 94:223-253.
  6. Busquet, M., Calsamiglia, S., Ferret, A. and Kamel, C. 2006. Plant extracts affect in vitro rumen microbial fermentation. J. Dairy Sci. 89:761-771.
  7. Callaway, T. R. and Martin, S. A. 1996. Effect of organic acid and monensin treatment on in vitro mixed ruminal microorganism fermentation of cracked corn. J. Anim Sci. 74: 1982-1989.
  8. Castillejos, L., Calsamiglia, S. and Ferret, A. 2006. Effect of essential oils active compounds on rumen microbial fermentation and nutrient flow in in vitro systems. J. Dairy Sci. 89: 2649-2658.
  9. Chaney, A. L. and Marbach, E. P. 1962. Modified reagents for determination of urea and ammonia. Clin. Biochem. 8:130-137.
  10. Chao, S. C. and Young, D. G. 2000. Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses. J. Essent. Oil Res. 12:639-649.
  11. Davidson, P. M. and Naidu, A. S. 2000. Phyto-phenols. Pages 265-293 in Natural Food Antimicrobial Systems. A. S. Naidu, ed. CRC Press, Boca Raton, FL.
  12. Dawson, D. A. and Allison, M. J. 1988. Digestive disorders and nutritional toxicity. Pages 445-459 in The rumen microbial ecosystem. P. N. Hobson, ed. Elsevier Science Publishers Ltd., London, U.K.
  13. Dawson, K. A., Rasmussen, M. A. and Allison, M. J. 1997. Digestive disorders and nutritional toxicity. In: Hobson, P. N., and C. S. Stewart. (eds), The rumen microbial ecosystem, 2nd edn, pp. 633-660. Blackie Academic and Professional, London.
  14. Dorman, H. J. D. and Deans, S. G. 2000. Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88:308-316.
  15. Erwin, E. S., Marco, G. T. and Emery, E. M. 1961. Volatile fatty acid analysis of blood and rumen fluid by gas chromatography. J. Dairy Sci. 44:1768-1771.
  16. Evans, J. D. and Martin, S. A. 2000. Effects of thymol on ruminal microorganisms. Curr. Microbiol. 41:336-340.
  17. Gershenzon, J. and Croteau, R. 1991. Terpenoids. Pages 165-219 in Herbivores: Their Interactions with Secondary Plant Metabolites. Vol. 1. G. A. Rosenthal, and M. R. Berenbaum, ed. Academic Press, San Diego, CA.
  18. Gottschalk, G. 1986. Bacterial Metabolism (2nd Ed.). Springer Verlag, New York.
  19. Greathead, H. 2003. Plants and plant extracts for improving animal productivity. Proceedings of the Nutrition Society. 62: 279-290.
  20. Helander, I. M., Alakomi, H., Latva-Kala, K., Mattila-Sandholm, T., Pol, I., Smid, E. J., Gorris, L. G. M. and Wright, A. 1998. Characteritzation of the action of selected essential oil components on gram-negative bacteria. J. Agric. Food Chem. 46:3590-3595.
  21. Lehninger, A. L. 1975. Biochemistry (2nd Ed.). Worth Publishers, New York.
  22. Martin, S. A. 1998. Manipulation of ruminal fermentation with organic acids: A review. J. Anim. Sci. 76:3123-3132.
  23. Martin, S. A. and Park, C. M. 1996. Effect of extracellular hydrogen on organic acid utilization by the ruminal bacterium Selenomonas ruminantium. Curr. Microbiol. 32:327-331.
  24. Martin, S. A. and Streeter, M. N. 1995. Effect of malate on in vitro mixed ruminal microorganism fermentation. J. Anim. Sci. 73:2141-2145.
  25. Martin, S. A., Streeter, M. N., Nisbet, D. J., Hill, G. M. and Williams, S. E. 1999. Effects of DL-malate on ruminal metabolism and performance of cattle fed a high-concentrate diet. J. Anim. Sci. 77:1008-1015.
  26. MeDougal, E. I. 1948. Studies on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochem. J. 43:99-103.
  27. Montano, M .F., Chai, W., Zinn-Ware, T. E. and Zinn, R. A. 1999. Influence of malic acid supplementation on ruminal pH, lactic acid utilization, and digestive function in steers fed high-concentrate finishing diets. J. Anim. Sci. 77:780-784.
  28. Nisbet, D. J. and Martin, S. A. 1994. Factors affecting Llactate utilization by Selenomonas ruminantium. J. Anim. Sci. 72:1355-1361.
  29. Panizzi, L., Flamini, G., Cioni, P. L. and Moreli, I. 1993. Composition and antimicrobial properties of essential oils of four mediterranean Lamiaceae. J. Ethnopharmacol. 39:167-170.
  30. Rural Development Administration. 2007. The standard farming textbook. Hanwoo. 6th edition.
  31. Russell, J. B. and Hino, T. 1985. Regulation of lactate production in Streptococcus bovis. A spiraling effect that contributes to rumen acidosis. J. Dairy. Sci. 68:1712-1721.
  32. SAS Institute, 2002. SAS${\circledR}$ User's guide: Statistics. Version 9.1 Edition. Statistical Analysis Systems Institute Inc., Cary, NC.
  33. Slyter, L. L. 1976. Influence of acidosis on rumen function. J. Animal Sci.43:910-929.
  34. Smith-Palmer, A., Stewart, J. and Fyfe, L. 1998. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Letters in Applied Microbiology. 26:118-122.
  35. Tilley, J. M. A. and Terry, R. A. 1963. A two-stage technique for the in vitro digestion of forage crops J. Brit. Grassland Soc. 18:104-111.
  36. Van Soest, P. J., Robertson, J. B. and Lewis, B. A. 1991. Methods of dietary fiber, neutral detergent fiber and non-starch poly-saccharides in relation to animal nutrition. J. Dairy Sci. 74:3583-3597.
  37. Walsh, S. E., Maillard, J. Y., Russell, A. D., Catrenith, C. E., Charbonneau, D. L. and Bartolo, R. G. 2003. Activity and mechanisms of action of selected biocidal agents on grampositive and negative bacteria. J. Appl. Microbiol. 94:240-247.