2.4. Isolation of Starch

Wheat starch was isolated from wholemeal semolina by using the protocol described by Budney and colleagues [26] with some modifications. Briefly, bran fractions with a particle size greater than 500 µm were discarded. Then, 2 g of wholemeal semolina was mixed with 3 mL of water to form a round piece of dough, which was then hydrated in water for 20 min. Starch was isolated using 80 mL of water. The starch slurry was filtered through a 70 µm sieve to obtain a non-purified liquid fraction with prime starch, while non-starch particles with a size greater than 70 µm remained on the sieve.

The liquid fraction was cooled to 3 °C for 24 h and centrifuged at 2300× g for 10 min at 4 °C. The pellet was then subjected to a fractionation procedure. From the pellet, the upper, loosely-packed slower sedimenting layer, which is characterized by a pale yellow-brownish color, was carefully removed with a spatula. The white main starch fraction was purified twice with 20 mL of water and centrifuged at 2300× g for 10 min at 4 °C. The starch sample was dried at 40 °C for 24 h.