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}