MATERIALS AND METHODS Microencapsulation
The method of Sheu and Marshall (27) was adopted for encapsulating the bifidobacterial cells. Microencapsulation was performed using κ-carrageenan (Sigma Chemical Co., St. Louis, MO). Cells were harvested from 80 ml of a 24-h culture (late log phase) by centrifugation at 5000 g. The cells were then washed twice, under the same centrifugation conditions, with 20 ml of sterile normal saline and resuspended in 10 ml of sterile saline.
The final cell concentration was adjusted to 1.0 to 1.2 109 cfu/ml by diluting with sterile saline and checking the optical density at 600 nm. A 20 ml cell suspension was added to 60 ml of 2% κ-carrageenan, containing 0.9% NaCl to improve the dispersability of κ-carrageenan (21), and tempered in a water bath at 47 to 48C. The κ-carrageenan was boiled but not sterilized, since sterilization prevents encapsulation. Vegetable oil (100 ml, Crisco) at 40C was stirred for 2 to 3 min with 0.1% (vol/vol) Tween 80 (Fisher Scientific, Pittsburgh, PA) added as an emulsifier. The cell/κ-carrageenan mix was then quickly added to the oil and stirred for about 10 min to allow for emulsification and encapsulation to occur. Then, 150 ml of 0.3 M KCl was added quickly down the side of the beaker to break the emulsion. The oil phase was removed from the top with an aspirator and the capsules were harvested from the KCl solution by gentle centrifugation at 350 g for 10 min. The capsules were then washed twice in 0.3 M KCl, using the same centrifugation conditions, and stored at 4C until used. The capsules were used within 1 to 2 d.