Introduction

1. Cecum Cecum -ve control +ve control DNA ladder Colon Colon M 1 2 3 4 5 6 Immobilized Free 500 bp 300 bp 100 bp Saxami G, Nikolaou M, Kiourtzidis M, Belik D, Ypsilantis P, Simopoulos C, Kourkoutas Y, Galanis A Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece Email: agalanis@mbg.duth.gr Introduction The development of novel foods containing probiotic microorganisms, such as lactic acid bacteria (LAB), represents a growing field in the food industry1,2. Among LAB, Lactobacillus casei ATCC 393 strain has been extensively incorporated into food products, due to its excellent technological properties3,4. We have recently investigated the survival of L. caseiATCC 393 contained in fermented milk after transit through the gastrointestinal (GI) tract and its role as potential regulator of the intestinal microbial flora of Wistar rats5. Daily oral administration led to reduction of staphylococci, enterobacteria and streptococci counts5. Additionally, probiotic cells were detected in feces at levels ≥ 6 logCFU/g (suggested minimum levels for conferring a probiotic effect). These findings do not, however, provide information on the adhesion ability of L. casei ATCC 393. The aim of our study was to investigate the in vivo adhesion properties of Lactobacillus casei ATCC 393, using the rat as a model system. • Results • At single dose administration, 12 rats were randomly assigned into 2 groups of 6 animals each. They were administered a single dose of fermented milk containing free (group F-1) or immobilized (group I-1) L. caseiATCC 393 on apple pieces. Microbiological and multiplex PCR analysis demonstrated that in both groups, L. casei ATCC 393 was detected at levels of ≥ 5 logCFU/g at the cecumand colon, whereas levels were reduced to 3 logCFU/g at the jejunum and ileum (Table 1, Fig. 1) • At daily administration 12 rats were randomly assigned into 2 groups of 6 animals each. Fermented milk containing either free (group F-2) or immobilized (group I-2) L. caseiATCC 393 was administered orally for 7 days. The results of the microbiological and molecular analyses indicated adhesion of the large intestine by bothfree and immobilized L. caseiATCC 393 at levels of ≥ 6 logCFU/g. The levels of the probiotic strain were lower at the duodenum, jejunum and ileum (Table 2). Notably, the levels of L. caseiATCC 393 were slightly higher in the cecal and colon fluids following single and daily administration of immobilized cells (Tables 1, 2). • Materials and Methods • Animals:The animals used in the present study, were Wistar rats, 3-4 months of age, weighing 250-300g. They were housed in polycarbonate cages, 1 rat per cage, at 20-22oC room temperature, on a 12 hour light: 12 hour dark cycle and were provided with commercial pelleted diet and tap water ad libitum. The facilities were in accordance with Directive 86/609/EEC. • In vivo experiments:The experimental protocol was divided in two parts; in the first part the rats were orally administrated with a single dose of probiotic fermented milk containing either free or immobilized L. casei ATCC 393 on apple pieces (1 g/rat), while in the second part, daily administration of probiotic fermented milk was performed for a 7-day period. In both cases, rats administrated no probiotic fermented milk were used as negative controls. 24h post administration,intestinal content and tissue samples from distinctive parts of the small (duodenum, jejunum, ileum) and the large (cecum and colon) intestine, were collected and subjected to microbiological and molecular analysis, in order to monitor L. casei ATCC 393 levels.The animals were subjected to sevoflurane anesthesia. The small and large intestines were removed aseptically and 3-cm long individual sections were cut longitudinally. Intestinal fluids were collected and then the tissue samples were washed twice with sterile buffered peptone water and vortex mixed to break down bacterial clumps and to remove loosely attached bacteria from the tissue sections. The tissues were subsequently homogenized, to release firmly adherent bacteria. Aliquots of dilutions were plated on MRS agar following microbiological analysis. • PCR reactions: Multiplex PCR reactions were carried out in a total volume of 50 μL, containing 5 units Taq DNA polymerase (HyTest Ltd), 400 μM each dNTPs (Promega), 1.5 mM MgCl2 (HyTest Ltd) and 100 ng template DNA. The specific for L. casei ATCC 393 primers consisted of GGCGACCAAGGCAGCG (10pmol), CTTCGGTTTCATCTTCC (50 pmol) and GGCCAACTTTTTCCATA (50 pmol). Additionally, a set of primers consisted of AGCAGTAGGGAATCTTCCA (10 pmol) and ATTYCACCGCTACACATG (10pmol) was used as positive control. Amplification was carried out in a Thermal Cycler (MastercyclerEppendorf) under the following conditions: 94oC (2min), followed by 25 cycles of 94oC (15sec), 51oC (15sec), 72oC (30sec), followed by a final extension step at 72oC (1min). Figure 1:Agarose gel electrophoresis of PCR products from multiplex PCR assay. The multiplex PCR was assayed for the identification either free or immobilized L.caseiATCC 393 cells at cecum and colon. Conclusions In conclusion, in this study the adhesion to the GI tract of Wistar rats by L. casei ATCC 393 was examined, after oral administration of fermented milk, containing either free or immobilized cells on apple pieces. We demonstrated that L. caseiATCC 393 cells adhered to the rat GI tract. The adhesion at the large intestine (cecum and colon) was recorded at levels ≥ 6 logCFU/g, following daily administration. Their levels were lower at the small intestine (duodenum, jejunum, ileum) (≤3 logCFU/g), indicating that adhesion was a targeted process. Also, the colonization of the GI tract was transient. Daily consumption of probiotic products containing the specific strain appears to be a prerequisite for retaining its levels at an effective concentration, information that could be valuable in food industry. References 4. Kourkoutas Y., et al., Lactobacillus casei immobilization on fruit pieces for probiotic additive, ermented milk and lactic acid production. Process Biochem. 2005;40:411–416. 5. Sidira M, et al., Effect of probiotic-fermented milk administration on gastrointestinal survival of Lactobacillus casei ATCC 393 and modulation of intestinal microbial flora.J Mol Microbio Biotechnol. 2010;19:224-230. 1. Saxelin M., Probioticformulationsandapplications, thecurrent probiotics market, andchanges in themarketplace: a European perspective. Clin Infect Dis 2008; 2:S76–S79. 2. Stanton C., et. al., Market potential for probiotics. Am J Clin Nutr 2001; 73:476S–483S. 3. Kourkoutas Y., et al., Probiotic cheese production using Lactobacillus casei cells immobilized on fruit pieces. J. Dairy Sci. 2006; 89:1431-1451.