This phenomenon could be related to ability of tested strains to metabolize N-acetyl-D glucosamine, one of the precursor of hyaluronic acid. Methods Media and reagents MRS (Oxoid LTD, Basingstoke, Hampshire, Selleckchem AZD8931 U.K.) was employed for bacterial strains growth, strain maintenance and viable count assessment. Sterile saline solutions of High Molecular Weight HA (1837 kDa, 8 mg ml-1) where kindly provided by IBSA (Institute selleck screening library Biochemique SA, Lugano, CH). Hyaluronidase solution (Jaluronidasi 100 I.U., 3.2 mg ml-1) was purchased from Farmacia Testi snc, Milan, Italy. Evaluation of minimal inhibitory concentration for HA Dilutions for HA MIC determination were performed

in sterile deionized water with concentrations ranging from 0.0625 up to 4 mg ml-1

for a total of 7 levels of exposure. 50 μl of each dilution were loaded into wells in MRS agar plates seeded with tested strains. pH values of HA solutions were evaluated by means of pH-meter (Beckman PHI43). LAB tested are reported in Table 1. Tolerance to HA of strain Lb. rhamnosus LbGG (ATCC) was also evaluated. Briefly, strain was subcultured twice in MRS (incubation at 30°C). Cells in early stationary phase (7.91 ± 0.29 Log CFU ml-1) were collected by centrifugation (6.500 rpm, 10 min), washed once with sterile Ringer solution (Oxoid) and resuspended in the same saline. 200 μl of sterile water solutions of HA (0.0625, 0.125, click here 0.25, 0.5, 1, 2, 4 and 8 mg ml-1) were added to 200 μl of cell suspensions. Positive control was realized by adding 200 μl of sterile saline instead of HA. After 30 min of incubation at 37°C, living cells were enumerated by drop counting method (Collins et al., 1989) on MRS agar plates, followed by incubation for 72 h at 37°C. Effect of HA on Lb.GG tolerance to simulated gastric juice The effect of HA on LbGG tolerance to simulated gastric juice was determined according to the procedure reported by Michida et al. (2006) [22]. Briefly, cells were harvested from cultures in exponential phase isothipendyl of growth

by centrifugation (6.500 rpm, 10 min), washed twice with sterile saline (0.5%, w/v), and resuspended in the same sterile saline. Simulated gastric juice was prepared daily by suspending pepsin (1:10 000, ICN) in sterile saline (0.5%, w/v) to a final concentration of 3 g l-1 and adjusting the pH to 2.00 with concentrated HCl using a pH meter. Aliquots (0.2 ml) of the cell suspensions were transferred to a 2.0 ml capacity Eppendorf tube, mixed with 0.3 ml of sterile water solutions of HA (0.125, 0.25, 0.5, 1, 2, 4, and 8 mg ml-1) and finally mixed with 1.0 ml of simulated gastric. After incubation at 37°C for 90 min, cells viability was assayed by drop counting method [23] on MRS agar plates (incubation for 72 h at 30°C).