The kefir grains used in this study were collected from Tibet,
China and were preserved by the Food Chemistry Laboratory,
Shanxi University. Frozen kefir grains were activated by placing 18 g
of the grains in 500 mL of sterile cow milk, followed by incubation
at 25 C. After 24 h, the grains were sieved out and washed with
cooled sterile distilled water. This step was repeated for three
consecutive days (Witthuhn et al., 2005). The inoculum was
prepared by transferring kefir grains (small grains of approximately
1e2mmdiameter) to a 250 mL Erlenmeyer flask containing 100 mL
of sterile cow milk. Kefir grains cultivationwas carried out statically
in an incubator set at 25 C for 24 h, renewed daily, for a duration of
7 days. Kefir grains as inoculum were stored at 4 C prior to use.