214 journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XIII, Issue 3 – 2014, pag. 214 – 217 STUDY ON THE ADHESION OF LACTOBACILLUS PLANTARUM STRAINS WITH PROBIOTIC PROPERTIES TO MDCK *Rositsa DENKOVA1, Hristina STRINSKA1, *Zapriana DENKOVA2, Georgi DOBREV1, Daniel TODOROV3, Kirilka MLADENOVA4, Stoyan SHISHKOV3 1University of Food Technology, Department of Biochemistry and Molecular biology, rositsa_denkova@mail.bg 2University of Food Technology, Department of Microbiology, zdenkova@abv.bg 3Sofia University “St. KlimentOhridski”, Laboratory of Virology 4Sofia University “St. KlimentOhridski”, Department of Biochemistry *Corresponding author Received August27th 2014, accepted September 15th 2014 Abstract: One of the requirements for probiotic strains is to adhere to epithelial cells or cell lines. The presence of S-layer proteins in three Lactobacillus plantarum strains with probiotic properties was examined as well as their ability to adhere to the epithelial monolayer model non-cancerous cell line MDCK. The three strains lacked S-layer proteins, but Lactobacillus plantarum X2 and Lactobacillus plantarum LBRZ12 adhered to the cells of MDCK, while Lactobacillus plantarum F3 didn’t. Along with their other probiotic properties these make them suitable for inclusion in the composition of probiotics and probiotic foods. Keywords: adhesion, Lactobacillus, MDCK, probiotic, S-layer proteins 1. Introduction Probiotics are live microorganisms that have beneficial effects on the health of the host, when administered in adequate amounts [1, 2]. Due to their proven health beneficial effect the strains most frequently included in the composition of probiotics belong to the genera Lactobacillus, Bifidobacterium and Propionibacterium [3]. Probiotic microorganisms contribute to the restoration and maintenance of intestinal balance, play an important role in maintaining the overall health condition of the organism and improve significantly the quality of the foods they are incorporated into [4]. Not all strains can be included in probiotic formulas but only those that meet certain requirements [5]. One of these requirements for probiotic strains is to be able to adhere to the intestinal mucosa and cell lines, thereby competitively excluding pathogens [3; 6]. Adhesion may be either non-specific, related to specific physico- chemical factors, or based on specific adhesion molecules located on the surface of the microbial cells and receptor molecules on the surface of the epithelial cells. Adhesion is species specific. Lactic acid bacteria possess moderate to good adhesion properties on human cell lines [7]. Adhesion of probiotic strains to the surface of the gut is of milestone importance as it is related to the subsequent colonization in the human intestinal tract which in turn determines the longer retention time of probiotic bacteria in the intestinal tract and the realization of their inherent immunomodulatory action. A number of in vitro studies demonstrating adherence of different strains of lactic acid bacteria to human epithelial cells have been conducted [8]. S-layer proteins are a strain-specific trait. Surface layer (S-layer) proteins with molecular masses between 40 and 60 kDa [9, 10], responsible for their Food and Environment Safety - Journal of Faculty of Food Engineering, ŞtefancelMareUniversity – SuceavaVolume XIII, Issue 3 – 2014 Rositsa DENKOVA, Hristina STRINSKA, Zapriana DENKOVA, Georgi DOBREV, Daniel TODOROV, Kirilka MLADENOVA, Stoyan SHISHKOV, Study of theadhesion of Lactobacillusplantarumstrainswithprobioticpropertiesto MDCK,Food and Environment Safety,Volume XIII, Issue 3 – 2014, pag. 214 - 217 215 adhesion to epithelial cells or cell lines, are described in lactobacilli [11]. The purpose of the present study was to examine the presence of S-layer proteins and the ability of three strains of Lactobacillus plantarum with probiotic properties to adhere to the cells of the epithelial monolayer model non-cancer cell line MDCK. 2. Materials and methods Microorganisms The studies in this work were performed with three strains of Lactobacillus plantarum with probiotic properties designated as Lactobacillus plantarum X2 and Lactobacillus plantarum F3, isolated from spontaneously fermented sourdough, and Lactobacillus plantarum LBRZ12, isolated from spontaneously fermented vegetables. Determination of the adhesion of the Lactobacillus plantarum strain to MDCK The used non-cancer, kidney cell line MDCK (Madin-Darbey Canine Kidney) is cultured as a monolayer in a 24-well plate in DMEM without antibiotics. The single-strain cultures of each Lactobacillus plantarum strain were incubated overnight at 37°C and centrifuged thrice, the biomass sludge was washed with PBS-buffer. 0,5 cm3 of each cell suspension, suspended in PBS-buffer, with concentration of 109cfu/cm3 was resuspended in 1 cm3 DMEM and 0,5 cm3 of the mixture were pipetted into the wells with the developed monolayer cell line MDCK. After 3 hours of incubation at 37°C the MDCK monolayer was washed with 250 μl PBS-buffer. 250 μl of fixating agent (96% ethanol : and acetic acid = 3: 1) were pipetted in each well and the plate was incubated for 20 minutes at room temperature. It was washed once with saline solution. A few drops of Gimza stain (diluted in a ratio of 1: 7) as to cover the bottom of the wells were pipetted in each well. After 5 minutes, the dye was removed and the cells were washed twice with 300 μl of saline solution. A light microscope (Dundee), at a magnification of 100x, was used for each well of the plates Characterization of surface layer proteins by SDS-PAGE [9]. Untreated bacterial cells were resuspended in 1% SDS, incubated for 30 min at 37ºC for the isolation of surface layer proteins, centrifugedfor 5 min at 9000 x g and the supernatant was analyzed by SDS- polyacrylamide gel electrophoresis using 10% polyacrylamide gel. The used marker for the electrophoresis was Precision Plus ProteinTM Standards (BioRad, Cat. # 161-0373). Protein bands were visualized by staining with Coomassie Blue R-250. 3. Results and discussion The presence of surface layer (S-layer) proteins with molecular masses between 40 and 60 kDa is described in lactobacilli. The examination of the presence of S-layer proteins showed that the strains Lactobacillus plantarum X2, Lactobacillus plantarum F3 and Lactobacillus plantarum LBRZ12 did not possess S-layer proteins (Fig. 1). The ability of Lactobacillus plantarum X2, Lactobacillus plantarum F3 and Lactobacillus plantarum LBRZ12 to adhere to the cells of the monolayer model non-cancerous epithelial cell line MDCK was examined. In the control the cells were arranged in a dense monolayer, their shape and intercellular contacts being clear. The strains Lactobacillus plantarum X2 and Lactobacillus plantarum LBRZ12 adhered to MDCK cells, while Lactobacillus plantarum F3 didn’t (Fig. 2). Food and Environment Safety - Journal of Faculty of Food Engineering, ŞtefancelMareUniversity – SuceavaVolume XIII, Issue 3 – 2014 Rositsa DENKOVA, Hristina STRINSKA, Zapriana DENKOVA, Georgi DOBREV, Daniel TODOROV, Kirilka MLADENOVA, Stoyan SHISHKOV, Study of theadhesion of Lactobacillusplantarumstrainswithprobioticpropertiesto MDCK,Food and Environment Safety,Volume XIII, Issue 3 – 2014, pag. 214 - 217 216 Marker 1. Marker 2. L. plantarum F3 3. L. plantarum LBRZ12 4. L. plantarum X2 Fig. 1. Presence of S-layer proteins in Lactobacillus plantarum X2, Lactobacillus plantarum F3 and Lactobacillus plantarum LBRZ12 When examining the adhesion of Lactobacillus plantarum LBRZ12 and Lactobacillus plantarum X2 to the cells of MDCK there was visible mounting of the cells of the Lactobacillus plantarum strains to the cells of the monolayer. Furthermore disturbances in the structure, as well as the appearance of individual cell line cells were observed. 4. Conclusion The ability of Lactobacillus plantarum strains of different origin with probiotic properties to adhere to the epithelial monolayer model non-cancerous cell line MDCK was studied. Control Lactobacillus plantarum X2 Lactobacillus plantarum F3 Lactobacillus plantarum LBRZ12 Fig. 2. Adhesion of L. plantarum strains to MDCK cell line Food and Environment Safety - Journal of Faculty of Food Engineering, ŞtefancelMareUniversity – SuceavaVolume XIII, Issue 3 – 2014 Rositsa DENKOVA, Hristina STRINSKA, Zapriana DENKOVA, Georgi DOBREV, Daniel TODOROV, Kirilka MLADENOVA, Stoyan SHISHKOV, Study of theadhesion of Lactobacillusplantarumstrainswithprobioticpropertiesto MDCK,Food and Environment Safety,Volume XIII, Issue 3 – 2014, pag. 214 - 217 217 The three studied strains did not possess S- layer proteins, but Lactobacillus plantarum X2 and Lactobacillus plantarum LBRZ12 adhered to the monolayer model non- cancerous cell line MDCK, which is a prerequisite for their inclusion in the composition of probiotics and probiotic foods. 5.References [1]. CHARALAMPOPOULOS D, PANDIELLA S, WEBB C, Evaluation of the effect ofmalt, wheat and barley extracts on the viability of potentially probiotic actic acidbacteria under acidic conditions. Int. 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