Cell-free supernatants (CFS) production
The strains of L. fermentum 5.2, L. plantarum 6.2, andL. plantarum 7.1 were individually inoculated in MRS broth and incubated for 48 hours at 37 ºC. After this period, the material was centrifuged (8000 × g) to separate the biomass and the supernatant was collected, filtered through a membrane with 0.22 µm porosity, and lyophilized [13].
CFS in vitro cytotoxicity
The cytotoxicity of CFS in HT-29 cells (human colon cancer) was determined by the reaction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT); in this analysis, living cells convert MTT (no color) to formazan (purple color) through mitochondrial reactions[14].
HT-29 cells in Roswell Park Memorial Institute medium (RPMI; Hyclone Laboratories Inc, Logan, UT) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA) and 1% antibiotics (Pen Strep; Gibco, Thermo Fisher Scientific, Waltham, MA), were inoculated into 96-well plates at a concentration of 1 × 106 cells/mL and incubated at 37 °C and 5% CO2 until reaching 90% confluence. The medium was then removed, and a new sterile medium was added with CFS concentrations ranging from 40 to 0.625 mg/L, and the cells were incubated for another 24 h. After incubation, the medium was removed and 200 µL of sterile medium containing 50 µL of MTT (5 mg/ml) were added per well, followed by further incubation for 4 h, protected against light. The formazan formed was solubilized with 100 µL of 10% sodium dodecyl sulfate (SDS) during 12 h of incubation. To assess the absorbance, reading was performed in a plate reader (Biochrom ASYS Expert Plus Microplate Reader, Biochrom, Cambridge, United Kingdom) and compared to the positive control (untreated cells).
Autoaggregation and coaggregation between LAB and E. coli