Ultracel pl 10

A. dichotoma larvae were lyophilized (Lyoph-Pride 100, Ilshinbiobae, Dongducheon-si, Korea), pulverized into a powder using a grinder (VMO159A, Vitamix, Cleveland, OH, USA), and hydrolyzed with enzymes according to the method described by Park et al. [38 (link)]. Table 3 summarizes the optimal pH, temperature, and characterization of different enzymes. Briefly, 100 mL of distilled water were added to 10 mg of the freeze-dried A. dichotoma larvae powder followed by 0.1 μL (or mg) of an enzyme. The enzymatic hydrolysis was performed for 6 h to achieve an optimal hydrolytic level and was followed by direct heating at 100 °C for 10 min to inactivate the enzymes. After the enzyme-inactivated mixture was centrifuged at 2000 rpm (Optima XE100 ultracentrifuge, Beckman Coulter, Miami, FL, USA), the supernatant was vacuum-filtered and subjected to ultrafiltration (UF, Ultracel PL-10, Millipore, Billerica, MA, USA) with Amicon^® Stirred Cells (Millipore, Billerica, MA, USA) membranes (membrane cut-off: 100 kDa, 10 kDa, 5 kDa, 1 kDa) in a nitrogen atmosphere at a pressure of 5 bar. The proteins were fractionated from 100 kDa or higher to 10 kDa, 10 kDa to 5 kDa, 5 kDa to 1 kDa, or 1 kDa or lower molecular weight, filtered, and lyophilized to obtain the hydrolysate, which was stored at −20 °C until further use.

Different molecular fractions, namely U-1 (<1 kDa), U-2 (1–10 kDa), U-3 (10–100 kDa), and U-4 (>100 kDa), of TMTH were collected as described in our previous study [13 (link)]. Briefly, lyophilized T. molitor larvae were ground to obtain a fine powder and then hydrolyzed with trypsin (1% (w/w) for 6 h at 38 °C (pH 7). The enzymes were inactivated by exposure to direct heating (100 °C) for 10 min. The enzyme-inactivated mixture was then centrifuged (Optima XE100 ultracentrifuge, Beckman Coulter, Miami, FL, USA) at 2000 rpm to obtain the supernatant. The supernatant was vacuum-filtered, followed by ultrafiltration (UF, Ultracel PL-10, Millipore, Billerica, MA, USA) with Amicon^®® Stirred Cells (Millipore, Billerica, MA, USA) with membrane cut-offs of 100 kDa, 10 kDa, and 1 kDa in a nitrogen atmosphere at 5 bar pressure. Different molecular fractions of proteins were thus obtained. Lyophilized molecular fractions were stored at −20 °C until further analysis.