MOLECULAR DETECTION AND IDENTIFICATION OF POTENTIAL PROBIOTIC LACTIC ACID BACTERIA ISOLATED FROM FERMENTED OLIVES Saxami Georgia1, Panagou Efstathios2, Tassou Chrysoula3, Kourkoutas Yiannis1, Galanis Alex1 1Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece 2Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Human Nutrition, Agricultural University of Athens, Greece 3Institute of Technology of Agricultural Products, Hellenic Agricultural Organization – DEMETER, Lycovrissi, Attiki, Greece INTRODUCTION RESULTS Recent studies showed that several Lactic Acid Bacteria (LAB) strains isolated from naturally fermented table olives display probiotic potential (Argyri et al. 2013). Amongst them, two strains, namely Lactobacillus pentosus B281 and Lactobacillus plantarum B282 display significant potential for the production of novel probiotic food products (Argyri et al. 2014). The aim of this study was the development of a rapid and effective method for the molecular detection and identification of the above strains. RAPD fingerprinting was firstly applied for the development of strain specific primers as both strains were recently isolated and no sequence data was available. A multiplex PCR assay was designed for each strain, based on specific primers derived from RAPD SCAR analysis. A B MATERIALS AND METHODS M 1 2 3 4 5 6 7 8 9 10 Μ M 1 2 3 4 5 6 7 8 9 10 11 Μ Figure 1A:Agarose gel electrophoresis generated with RAPD primer p19, of L. pentosus B281 (lane 1), and 9 L. pentosus wild type strains isolated from table olives (lanes 2-10). Lanes: 2, L. pentosus 141; 3, L. pentosus E95; 4, L. pentosus E106B; 5, L. pentosus E128; 6, L. pentosus E89; 7, L. pentosus E119; 8, L. pentosus E182; 9, L. pentosus E105; 10, L. pentosus 632. M: 1 kb DNA ladder. The PCR product of 872 bp that was isolated from the agarose gel for further analysis is indicated with a circle. B):Agarose gel electrophoresis generated with RAPD primer p69, of L. plantarum B282 (lane 1), and 10 L. plantarum wild type strains isolated from table olives (lanes 2-11). Lanes: 2, L. plantarum E4; 3, L. plantarum E1; 4, L. plantarum E45; 5, L. plantarum E50; 6, L. plantarum E66; 7, L. plantarum E68; 8, L. plantarum E71; 9, L. plantarum E73; 10, L. plantarum E77; 11, L. plantarum E79 (line 11). M: 1 kb DNA ladder. The PCR product of 391 bp that was isolated from the agarose gel for further analysis is indicated with a circle. DNA extraction from pure cultures Genomic DNA from a pure culture was extracted using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The amount of extracted DNA was determined by absorbance at 260 nm using a UV spectrophotometer (Eppendorf). RAPD PCR PCR reactions were carried out in a total volume of 20 μl, containing 2 units Taq DNA polymerase (Kapa Biosystems), 2 μl of 10 x PCR buffer (without MgCl2), 200 μM each dNTPs (Kapa Biosystems), 2.5 mM MgCl2 (Kapa Biosystems), 100 ng template DNA and 10 pmol of selected primer. Amplifications were carried out under the following conditions: initial denaturation at 950C for 5 min, followed by 30 cycles of denaturation at 950C for 30 s, primer annealing for 1 min at 360C and primer extension for 1.5 min at 72°C with a final extension period at 72°C for 5 min. Τhe amplified products were subjected to electrophoresis in 1.5% w/v agarose gels, stained with 0.5 μg ml-1 ethidium bromide, visualized under UV illumination and photographed with a digital camera (Gel Doc EQ System, Biorad). A B M 1 2 3 4 5 6 M 1 2 3 4 5 6 Cloning and sequencing Τhe selected bands were excised from the gel, and DNA was isolated using a NucleoSpin Extract II kit (MACHEREY-NAGEL) according to the manufacturer's instructions. The potential strain-specific RAPD markers were cloned into the pBlueScript SK+ vector following the TA cloning protocol described by Zhou and Gomez-Sanchez, 2000. The clones were sent for sequencing to VBC-Biotech, Austria. The forward and reverse primers were designed including the decamer primer sequence extending few nucleotides (8–10 bases). NCBI Primer-BLAST was used to check the designed primers for specificity. Figure 2A,B: Agarose gel electrophoresis of PCR products from multiplex PCR assay. A) Lanes: 1, L. pentosus B281; 2, L. pentosus 141; 3, L. pentosus E95; 4, L. pentosus E106B; 5, L. pentosus E128; 6, L. pentosus E89. M: 1 kb DNA ladder. The numbers on the left of the figure indicate the DNA size markers in base pairs (bp). The PCR product generated from the primer set p19FF/p19R of 872 bp is unique to L. pentosus B281, whereas the PCR product of the primer set P1/P2 of 89 bp is universal for lactobacilli. B) Lanes: 1, L. plantarum B282; 2, L. plantarum E4; 3, L. plantarum E1; 4, L. plantarum E45; 5, L. plantarum E50; 6, L. plantarum E66. M: 1 kb DNA ladder. The PCR product generated from the primer set p69F/p69R of 391 bp is unique to L. plantarum B282, whereas the PCR product of the primer set P1/P2 of 89 bp is universal for lactobacilli. Both products are indicated with the corresponding circles. CONCLUSIONS A multiplex PCR assay was designed for each strain based on specific primers derived from RAPD SCAR analysis. The method designed offers rapid and efficient identification of potential probiotic lactic acid bacteria isolated from fermented olives. Two unique primer sets were developed (Fig. 1). Each strain-specific primer set was used in a multiplex PCR assay along with a set of universal primers. For L. pentosus B281 a distinct product of 872 bp was generated, along with the 89 bp a positive control product (Fig. 2A). Accordingly, for L. plantarum B282 two PCR products were only produced (391 and 89 bp) (Figure 2B). The specificity of the assay was successfully tested with a total of 32 and 27 different probiotic LAB strains for L. pentosus B281 and L. plantarum B282, respectively. In conclusion, the multiplex PCR assay represents an efficient tool for rapid and accurate identification of L. pentosus B281 and L. plantarum B282. It could be used to monitor the presence of these strains in food products and subsequently evaluate their probiotic character. Multiplex PCR Multiplex PCR reactions were carried out in a total volume of 20 μl, containing 2 units of Taq DNA polymerase (Kapa Biosystems), 2 μl of 10x PCR buffer, 200 μM each dNTPs (Kapa Biosystems), 2.5 mM MgCl2 (Kapa Biosystems), and 100 ng template DNA and the strain specific designed primers. For L. pentosus B281: p19F: 5’-GGTGAAGCTGATATTTATG-3’ (10pmol) p19R:5’-GGTGAAGCTGGTGGTGGTATC-3’ (10pmol) For L. plantarum B282: p69F: 5’-CCACAGCAGTAGGGCGCGAG-3’ (10pmol) p69R: 5’-CCACAGCAGTCTGCCCAACC-3’ (10pmol). Universal Primers: P1: 5’-AGCAGTAGGGAATCTTCCA-3’ (10pmol), P2: 5’-ATTYCACCGCTACACATG-3’ (10pmol) (used as positive control). Amplification was carried out in a under the following conditions: for L. pentosus B281, 95°C (2 min), followed by 26 cycles of 950C (30 s), 630C (30 s), 720C (45 s), followed by a final extension step at 720C (2 min); for L. plantarum B282, 950C (2 min), followed by 22 cycles of 950C (30 s), 680C (30 s), 720C (45 s), followed by a final extension step at 720C (2 min). The PCR products were separated on 1 % (wt/vol) agarose gels, visualized under UV illumination, and photographed. REFERENCES Argyri, A., Zoumpopoulou, G., Karatzas, K.A., Tsakalidou, E., Nychas, G.J., Panagou, E., and Tassou, C. (2013) Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiol 33, 282-291. Argyri, A., Nisiotou, A., Malouchos, A., Panagou, E. and Tassou, C. (2014) Performance of two potential probiotic Lactobacillus strains from the olive microbiota as starters in the fermentation of heat shocked green olives. Int J Food Microbiol 171, 68–76.