Heraeus contifuge stratus

Surface proteins of CD were isolated using a “washing off” method employing 1 M LiCl [15 (link)]. Shortly, 1 g of bacterial mass was suspended in 7 mL of freshly prepared 1 M LiCl and incubated at room temperature (RT) for 1 h, with shaking. Then, the mixture was centrifuged at 6000× g for 5 min (Heraeus Contifuge Stratus, Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was collected and dialyzed against water for at least 48 h with frequent water change. The protein solution was then concentrated with Amicon Ultra (Thermo Fisher Scientific, Waltham, MA, USA), MWCO = 10,000 Da. Protein concentration was estimated with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to instruction provided by supplier. The protein solution was aliquoted and stored at −20 °C until used.

Bifidobacterium longum ssp. longum CCDM 372 and Bifidobacterium longum ssp. longum CCM 7952 were originally isolated from feces of breast-fed healthy child. Both strains were provided by Culture Collection of Dairy Microorganisms (Milcom, Czech Republic). The isolates were cultivated in MRS medium (Difco) supplemented with 0.05% L-cysteine-hydrochloride (Millipore) at 37°C in anaerobic conditions (GasPak EZ Anaerobe Container System BD, USA) for 48 h. Lactobacillus paracsei LOCK 0912 for sera preparation was isolated form feaces of healthy newborn and cultivated in MRS medium (Difco) at 37°C in microaerophilic conditions for 48 h. Cells were harvested by centrifugation (6000 g for 15 min) (Heraeus Contifuge Stratus, Thermo Scientific, Germany) and washed two times in PBS buffer (phosphate buffered saline pH 7,4). Strain B. longum ssp. longum CCM 7952 is characterized by high amount of slime production, therefore, before protein isolation, the culture was centrifuged at 14,500 g several times to get rid of salts and bacterial polysaccharides.