In vitro dose–response of carvacrol, thymol, eugenol and trans-cinnamaldehyde and interaction of combinations for the antimicrobial activity against the pig gut flora ☆ J. Michiels a,b, ⁎ , J. Missotten b , D. Fremaut a , S. De Smet b , N. Dierick b a Departement Biowetenschappen en Landschapsarchitectuur, Hogeschool Gent, Voskenslaan 270, 9000 Ghent, Belgium b Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium Abstract In vitro simulations of the gastric and small intestinal fermentation with varying doses of carvacrol, thymol, eugenol and trans- cinnamaldehyde and binary combinations thereof were carried out to calculate dose–response equations for the antimicrobial effect against the main pig gut flora components. Interaction effects were evaluated following the isobole method. In gastric simulations eugenol and trans-cinnamaldehyde showed clearly less inhibitory activity towards lactobacilli than carvacrol and thymol, which could also be observed in the small intestinal simulations. The minimum concentration for carvacrol, thymol, eugenol and trans- cinnamaldehyde in small intestinal simulations to reduce the number of total anaerobic bacteria compared to control with a probability of 99.7% was 255, 258, 223 and 56 mg/l respectively. This strong activity of trans-cinnamaldehyde was due to its effect against coliform bacteria; a dose of 104 mg/l gave a reduction of 1 log CFU/ml vs. 371, 400 and 565 mg/l for carvacrol, thymol and eugenol respectively. Few combinations demonstrated synergism; most mixtures showed zero interaction or antagonism. Only carvacrol + thymol was slightly synergistic. © 2007 Elsevier B.V. All rights reserved. Keywords: Antimicrobial activity; Carvacrol; Thymol; Cinnamaldehyde; Eugenol; Gut flora; In vitro; Pigs 1. Introduction The antimicrobial properties of carvacrol, thymol, eugenol and cinnamaldehyde have been described extensively, mostly based on observations from semi- quantitative diffusion methods or microtiter plates assays. However, problems associated with the assess- ment of the antimicrobial activity by diffusion methods are that the method in itself is highly dependent on water solubility and the ability of test components to diffuse Livestock Science 109 (2007) 157 – 160 www.elsevier.com/locate/livsci ☆ This paper is part of the special issue entitled “Digestive Physiology in Pigs” guest edited by José Adalberto Fernández, Mette Skou Hedemann, Bent Borg Jensen, Henry Jørgensen, Knud Erik Bach Knudsen and Helle Nygaard Lærke. ⁎ Corresponding author. Departement Biowetenschappen en Land- schapsarchitectuur, Hogeschool Gent, Voskenslaan 270, 9000 Ghent, Belgium. Tel.: +32 9 2488863; fax: +32 9 2424279. E-mail address: joris.michiels@hogent.be (J. Michiels). 1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.132