Media were inoculated with cell suspensions in sterile saline solution (about 6 log CFU ml-1). Tests were performed on three urease positive St. thermphilus strains, namely 309, 82A and 247, and LbGG. Assessment of HA and Hy effect on LAB strains The effect of HA and HA in combination with Hy was evaluated on three St. thermophilus urease positive strains (309, 247, and 82A). The assay was performed in 96-well microplates (Corning Inc., NY, USA). Firstly, 200 μl

of HA + MRS [4, 2, 1, 0,5 and 0.25 mg ml-1] were added in triplicate in each plate. Then 10 μl of LAB cell suspensions (working concentrations of about 1 × 106 CFU ml-1) in sterile saline solution see more were added. Uninoculated MRS was used as control. Plates were incubated at 37°C in an incubator (Ekort 1500, Angelantoni industrie, Milano, Italy). The O.D. values were measured at a wavelength

of 595 nm at 0, 2, 4, 6, 8, 20, 24 and 48 hours by means of a microplate reader (Tecan, Austria). For the evaluation of HA-Hy effect, the procedure above described was repeated by adding to each well 100 μl of Hy [1,8 mg ml-1 in a saline solution] and 10 μl of each strain (about 1 × 106 CFU ml-1). O.D. values were measured at 0, 2, 4, 6, 8, 20, 24, 48 and 72 h of incubation at 37°C. Data analysis Data obtained from the O.D. readings were used to draw charts where O.D. was expressed as a function of time. Each point of the curves is the average value of three replicates (subtracted

of the blank) performed in the same experimental conditions. Statistical Cilengitide concentration analyses were performed at 2 h intervals. At each time, analysis of variance (ANOVA) and Bonferroni post hoc test were carried out to assess overall differences in O.D. readings obtained from different strains in relation to the control. References 1. Maharjan AS, Pilling D, Gomer RH: High and low molecular weight hyaluronic acid differentially regulate human fibrocyte differentiation. PLoS One 2011,6(10):1–10.CrossRef 2. Murai T, Kawashima H: A simple assay for hyaluronidase activity using fluorescence polarization. Biochem Biophys Res Commun 2008, 376:620–624.Vactosertib in vitro PubMedCrossRef of 3. Toole BP: Hyaluronan and its binding proteins the hyaladherins. Curr Opin Cell Biol 1990, 2:839–844.PubMedCrossRef 4. Murai T, Sougawa N, Kawashima H, Yamaguchi K, Miyasaka M: CD44- chondroitin sulfate interactions mediate leukocyte rolling under physiological flow conditions. Immunol Lett 2004, 93:163–170.PubMedCrossRef 5. Kawashima H: Roles of sulfated glycans in lymphocyte homing. Biol Pharm Bull 2006, 29:2343–2349.PubMedCrossRef 6. Masuko K, Murata M, Yudoh K, Kato T, Nakamura H: Anti-inflammatory effects of hyaluronan in arthritis therapy: Not just for viscosity. Int J Gen Med 2009, 2:77–81.PubMedCrossRef 7.