Ri 2031 refractive index detector

The purified EPS were hydrolyzed for 6 h at 100°C with 8N HCl, evaporated in an Eppendorf AG centrifugal concentrator (Eppendorf, Hamburg, Germany) and re-suspended in ultrapure water. Monosaccharide composition of EPS was determined by automated thin-layer chromatography (TLC) (CAMAG, Muttenz, Germany) using the ascending technique with silica gel 60 F254 precoated glass sheets (Merck, Damstadt, Germany). The sugars were eluted with a mixture of 1-butanol/acetic acid/water, 6/1/2 (v/v) and the bands were visualized by spraying with p-aminobenzoic acid (Wall, 2005) . Glucose, galactose, rhamnose, manose, ribose, xylose (Fluka, Sigma-Aldrich, Switzerland), fructose (Merck KGaA, Darmstadt, Germany), arabinose (Veb Berlin Chemie, Germany), glucosamine and galactosamine (both from Calbiochem, Inc. San Diego, Calif., USA) were used as standards. In another option, HPLC was used to determine the sugar composition of the hydrolyzed EPS. A Jasco HPLC system (Jasco Europe, Cremella, Italy), equipped with a Carbo Sep Coregel 87Pcolumn (Teknokroma, Spain), kept at 85 °C, and coupled with a RI-2031 refractive index detector (Jasco) was used for separation. Elution was performed with MilliQ water, at a flowrate of 1 ml/min. Arabinose, fructose, galactose, glucose, maltose, mannose, rhamnose, ribose, sorbose, sucrose, and xylose at a concentration of 0.1 mg/ml were used as standards.

The nine LAB used throughout this study were isolated from kradi cheese. These strains were isolated by plating on MRS agar (De Mann et al. 2013) . Also modified MRS agar with 50 g/l of sucrose (MRS-s) and modified MRS agar with 20 g/l of fructose (MRS-f) were used as isolation media. All the strains were stored at -85°C in their corresponding isolation medium, containing 25% (v/v) of glycerol as a cryoprotectant. LAB strains were propagated twice in fresh liquid medium for obtaining fresh culture from frozen stock, before the experiments.
LAB strains were grown in MRS-s while screening for EPS production. The glucomannans in the growth medium which could interfere with the EPS screening were removed (Vander Meulen et al. 2007 ). The LAB strains were screened for EPS production by Gel Permeation Chromatography (GPC), using a Jasco HPLC System (Jasco Europe, Cremella, Italy), equipped with an Ultra-hydrogel Linear column (Waters Corp., Milford, Mass., USA), kept at 35°C, and coupled to RI-2031 refractive index detector (Jasco). Samples were prepared (Vander Meulen et al. 2007 ) prior to the injection on the GPC column. The EPS were eluted with 0.1M NaNO3 at a flow rate of 0.6ml/l. Dextran standards with molecular masses ranging from 80 kDa to 1.4 MDa (Sigma-Aldrich, Switzerland) were used to calculate the molecular mass of the purified EPS.