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