Gas pack system

Cell-free supernatants (CFS) of L. reuteri LR47 were prepared according to the method described by Lüthi-Peng et al. (31 (link)). A single colony with typical morphology (small pinpointed and creamy white colony) from the MRS agar plates was picked and inoculated into MRS broth and incubated anaerobically (gas pack system, HiMedia) at 37 °C for 20 h. After incubation, the culture was centrifuged (Precision Biotech Instruments Pvt. Ltd., Delhi, India) at 4000×g and 4 °C for 10 min. The supernatant was filter-sterilised through a 0.22-μm syringe filter (Millipore, New Delhi, India) to remove any remaining cells. The cell pellet was used for secondary fermentation to prepare a suspension of MRS supplemented with glycerol (360 mM). The supernatant obtained from secondary fermentation of MRS with glycerol was designated MRS-Gly, while the supernatant derived from MRS broth was designated MRSB. Finally, the pH values of the above supernatants were determined using a portable pH meter (Orion Star™ A111; Thermo Fisher Scientific, Waltham, MA, USA). The pH of the supernatants was adjusted from pH=4.6 to 6.5 (in order to eliminate the cause of microbial death due to acidic pH) using 0.1 M sodium hydroxide at room temperature prior to testing their antimicrobial activity.