S201 53090

The thermal properties of purified starch were analyzed by DSC (LabSolutions TA software version 1.01, DSC 60 Plus; Shimadzu, Kyoto, Japan). Briefly, 3 mg of purified starch was suspended in 9 μL of distilled water and encapsulated in an aluminum seal pan (S201-53090; Shimadzu, Kyoto, Japan). Then, 45 mg of activated alumina was encapsulated in the aluminum seal pan and used as a reference sample. The thermal properties of starch were analyzed using the temperature program described previously [60 (link),61 (link)].
The pasting properties of purified starch were analyzed by RVA (Thermoclin software version 2.1, RVA-4; Newport Scientific, Inc., Jessup, MD, USA) using 3.5 g of purified starch suspended in 25 mL of distilled water, as described previously [60 (link)].

Perimysium extraction and denaturation temperature assessment were conducted according to methods described by Vierck et al. (2018) (link) with modifications. Pulverized muscle tissue was homogenized in 0.05 M CaCl₂ using a bead homogenizer (Bead Blaster 24; Benchmark Scientific, Sayreville, NJ). The homogenate was filtered through a 1 mm standard testing sieve (VWR International, Radnor, PA). The perimysial fraction collected on the screen was transferred to a microcentrifuge containing 1X PBS and hydrated for at least 1 h at 4°C prior to peak transitional temperature measurement. Ten milligram of hydrated samples was placed in a hermetic aluminum crucible (S201-53090; Shimadzu, Kyoto, Japan), and the aluminum crucible was sealed using a sealing press (SSC-30; Shimadzu). The sample crucible and a sealed empty crucible, for a reference crucible, were placed at the stage of the differential scanning calorimeter (DSC-60; Shimadzu). The temperature program was set from room temperature to 100°C with a 5°C/min temperature increase. To analyze the data the TA-60WS software (Shimadzu) was used to determine the peak transitional temperature (°C).