processed into a probiotic beverage. Therefore, the aim of this study was to determine the effect of fermentation temperature, fermen- tation time, inoculum size and sucrose concentration on walnut milk fermentation, as well as, to optimize the fermentation conditions of the walnut milk beverage, and investigate the chemical, microbial and sensory characteristics of the walnut milk beverage.
2. Materials and methods
2.1. Kefir grains and inoculum preparation
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
1e2 mm diameter) to a 250 mL Erlenmeyer flask containing 100 mL of sterile cow milk. Kefir grains cultivation was 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.