Methods
2.2.1. Probiotic activated probiotic culture preparation
The method described by Ding and Shah (2008), with the
modifications proposed by Pimentel et al. (2011)was used to
activate the lyophilised probiotic cultures. A culture aliquot was
inoculated into 5 mL of Man, Rogosa and Sharpe (MRS) media
(Himedia
®
) and incubated at 37
C for 15 h. After this time, 0.05 mL
of the culture was again inoculated into 10 mL of MRS media and
incubated at 37
C for 15 h (pre-inoculum). To obtain the biomass,
0.1 mL of the pre-inoculum was transferred into 300 mL of MRS
media and re-incubated under the same conditions. The biomass
was separated by centrifugation in a refrigerated centrifuge
(Eppendorf
®
, model 5804R) at 14,000 gfor 10 min at 4
C and
washed three times in 0.85 g/100 mL sterile saline solution (NaCl
Dinamica
®
) to remove the residual MRS media. The biomass was
then resuspended in 50 mL of 0.85 g/100 mL sterile saline solution
to obtain the activated probiotic culture.