2.2. Methods
2.2.1. Preparation of alginate microparticles
The emulsification/internal gelation technique to form alginate
microparticles has been described previously by Poncelet et al.
(1995). The procedure was modified as follows to encapsulate
L. fermentum. Glassware and reagents used in the experimentswere
sterilized before used. A (5 g/100 mL) sodium alginate mixture was
prepared by mixing with 1 g of lyophilized culture and 0.04 mol/l
CaCO3 and after homogenization, was added to 2 parts of soya oil
(v/v) (continuous phase) containing 2.5% (w/v) Span 80. The
mixturewas stirred at 700 rpmfor 10minwith amechanical stirrer
to form a uniformwater-in-oil-emulsion.With continuous stirring,
0.450 mL glacial acetic were added to the emulsion to initiate in-
ternal gelation. After 10 min under stirring, microparticles were
separated fromthe oil dispersion bymixingwith 0.45mol/l calcium
chloride solution. The phases were allowed to separate overnight.
The supernatant was discarded and the microparticles were
centrifuged, collected and washed using 100 mL sterile 20 g/l
peptone water by vacuum filtration and stored at 4 C.