CFS inhibitory activity
Lyophilized CFS of L. fermentum 5.2, L. plantarum 6.2, andL. plantarum 7.1 had their antimicrobial activity againstE. coli F4 evaluated using the agar diffusion technique[13]. To this end, 100 µL of inoculum withE. coli F4 at a concentration of 108 CFU/mL were inoculated in Mueller-Hinton (MH) agar containing wells with 100 µL of the CFS at a concentration of 40 mg/mL. Each CFS was evaluated individually.
The material was incubated for 24 h at 37 ºC, and the formation of an inhibition halo of bacterial growth was determined as effective CFS antimicrobial activity. As a negative control, MRS broth without previous growth of bioprotective microorganisms was used.
Inhibition of E. coli F4 biofilm production
To evaluate the biofilm production of E. coli F4, the strain was cultivated in BHI broth for 24 h at 37 ºC, and 100 µL were inoculated in 96-well plates at a concentration of 105 CFU/mL, followed by incubation at 37 ºC for 24~48 h. Biofilm formation was verified by visual reading and confirmed by removal of the culture medium and reading in a plate reader at 492 nm, with the comparison of the OD obtained in the negative control (which received 100 µL of sterile culture medium). After confirmation, the bacteria were inoculated again in 96-well plates in the presence of CFS at concentrations of 105 CFU/mL and 40 mg/mL, respectively.
To assess the inhibition of biofilm formation, removal of the culture medium and reading in a plate reader at a wavelength of 492 nm was performed. The biofilm formation index (BFI) was calculated with the following formula, where OD refers to the OD492nmobtained from the plate reading and 0.07 to the OD492nmof the reference strain (Streptococcus pyogenes ):
\begin{equation} BFI=\frac{\text{OD}}{0.07}\nonumber \\ \end{equation}