Sucrose:sorbitol (S:S), trehalose, and inulin HD at 2, 4, 6, and 8 % (w/v) were prepared to determine their effect on freezing point depression . Each sample (1 mg) was accurately weighed into 50-μL aluminum pans using a high-precision balance (Model S1500, Stuart, UK) and immediately sealed with a Universal press (PerkinElmer).