Antimicrobial activity Reduction of Enterococcus faecium in the TIM-1 dynamic gastrointestinal model by Lactobacillus sakei subsp. sakei 2a, and evaluation of the sakacin activity KRUGER, Monika F. 1,2,4; Van RIJN, Sanne 2; VENEMA, Koen 2; MARTINEZ, Rafael C.R. 2,3,4; De MARTINIS, Elaine C.P. 3; SAAD, Susana M.I. 1; FRANCO, Bernadette D.G.M. 1; SMIDT, Hauke 4; ZOETENDAL, Erwin G.4 1 Universidade de São Paulo, Food Microbiology - São Paulo, Brazil; 2 TNO Nutrition and Food Research, Zeist, The Netherlands; 3 Universidade de São Paulo, Bioscience – Ribeirão Preto, Brazil; 4 Wageningen University and Research Centre - Molecular Ecology , Wageningen, NL; Introduction Lactic acid bacteria (LAB) are present naturally in the gut and have been intensively investigated as a dietary adjunct for gastrointestinal disorders in animals and humans [1]. A major concern in studies of LAB as probiotic bacteria is the assessment of their persistence in the gastrointestinal tract (GIT) and also their ability to inhibit the pathogenic bacteria inside the intestinal environment [2]. Probiotic cultures must be adapted to different stresses procedures during food production to provide adequate amounts of active, living cells to withstand several conditions in the stomach and duodenum before reaching the colon in large quantities [3]. Considering that the viability of these cultures in the GIT is affected mainly by acid conditions in the stomach and bile salts in the duodenum, we evaluated the survival of Lactobacillus sakei subsp. sakei 2a [4], and its impact on the survival of Enterococcus faecium in the TNO in vitro model simulating the human small intestine track (TIM-1).  As L. sakei 2a is a bacteriocin producer strain, we also analyzed the sakacin activity during the transit in the model. Materials and Methods Cultures: L. sakei 2a and E. faecium were cultivated overnight in MRS broth at 30°C and BHI at 37°C, respectively. The cultures were washed twice and resuspended in PFZ (Peptone Physiological Salt Solution) or in commercial cow’s milk, getting 6-7 log cfu/ml when introduced in the TIM-1 Vancomicyn (5mg-1) and Neomycin (5mg-1) were used in MRS and BHI agar respectively, for the differentiation of the bacteria when the strain were tested in co-culture. TIM-1 model and sampling: The dynamic, computer-controlled TNO Intestinal Model (TIM-1) was used. The viability of the microorganisms was assessed by pour plate method, counting the numbers of colony-forming units (CFU/ml) on MRS and BHI agar. Samples were analyzed from time 0 till 6 hours of the intestinal transit. Bacteriocin assay: The sakacin activity was measured by the spot-on-lawn test [5], using E. faecium as indicator strain (Figure 2). Results The results showed that L. sakei 2a is able to persist in the small intestine until the sixth hour of analysis, but their number was drastically reduced (5 log cycle). The opposite was observed for E. faecium, that presented high ability to colonize the TGI, establishing a number of 108 cfu/ml from the second till the sixth hour of analysis. However, when the strains were co-inoculated, L. sakei 2a was able to persist in an order of 104 cfu/ml, causing a substantive decrease in the number of E. faecium (presenting 101 cfu/ml). No antimicrobial activity by sakacin was founded on the spot-on-lawn assay. Figure 3: Survival rate of L. sakei 2a and E. faecium during the transit in the TIM-1 system, when cultivated alone in PFZ or in milk. Figure 4: Survival rate of L. sakei 2a and E. faecium during the transit in the TIM-1 system, when co-cultivated in PFZ or in milk. Figure 5: Survival rate of L. sakei 2a and E. faecium during the transit in the TIM-1 system, when cultivated alone (A) or co-cultivated (B) in PFZ or in milk. 1 4 7 2 6 5 3 9 8 10 11 12 13 A B 1 2 3 4 5 6 7 8 9 10 11 12 13 TIM-1 samples 12.000 x g / 10m, 4°C Antimicrobial activity supernatant Conclusion Due to the survival rate and the ability to inhibit E. faecium in the simulated small intestinal conditions, L. sakei 2a becomes an interesting strain for future studies as a potential probiotic culture. We are currently investigating the mechanisms underlying the inhibitory effect by L. sakei 2a. Figure 1: TIM-1 (TNO gastrointestinal model) used to simulate human intestines, consisted of four chambers to simulate in vivo conditions in the stomach and small intestine (Jejunum, Duodenum and Ileum), such as the kinetics of pH, bile salt concentrations, and transit of the chyme. Figure 2: Schematic representation of the spot-on-lawn test. Acknowledgements This work has been carried out as part of a collaborative project between CAPES-Wageningen 17/07 and Wageningen University, and with additional financial support of the European Community, FP6-FOOD-506144 (Feed for pig health). Wageningen UR: erwin.zoetendal@wur.nl Phone: NL (31)0317 483111 Universidade de São Paulo: bfranco@usp.br Phone: BR (55)1130912191 Kruger, M.F.: monikakruger@ig.com.br Phone: BR (55)1130912191 References: [1] Blay et al., (2007) L Appl Microbiol 187:6750–6761; [2] Maragkoudakis et al., (2006) Int. Dairy Journal 16: 189–199 ; [3] Konstantinov et al., (2006) Int J Syst Evol Microbiol 56: 29-32; [4] De Martinis & Franco, (1998) Int J Food Microbiol 42: 119–126; [5] Lewus & Montville, (1991) J Microbiol Methods 13: 145–150;