Amyloglucosidase solution

Muscle glycogen concentrations were determined as described by Dreiling et al [15 (link)]. Briefly, 200 mg of muscle sample was homogenized with 2 mL cold 8% (v/v) perchloric acid using a polytron (Ultra Turrax T25 disperser, IkA-Labortechnik, Staufen, Germany) and then centrifuged at 15,000×g for 10 min. The supernatant was neutralized with saturated sodium bicarbonate solution and 0.2 M sodium acetate buffer (pH 4.8). A glycogen standard curve was established by dissolving 0 to 90 μg of bovine liver glycogen (Cas no. 9005-79-2, Sigma-Aldrich, Darmstadt, Germany) in neutralized perchloric acid. Each standard and neutralized supernatant were incubated in amyloglucosidase solution (A7255, Sigma-Aldrich, Germany) for 30 min at 55°C to convert glycogen to glucose. The concentration of glucose was determined using Enzymatic Glucose Reagent (Infinity Glucose Oxidase Reagent, Thermo Scientific, Lidcomb, NSW, Australia), by measuring the absorbance at 550 nm on a 96-well microplate reader (Infinite F50, Tecan, Switzerland).

The total dietary fibre (TDF) of winemaking by-products was measured following the standard enzymatic-gravimetric method [40 ]. First, 1 g of dry sample was mixed into 50 mL of distilled water. Then, samples were digested with 200 µL of α-amylase (Sigma-Aldrich, St. Louis, MO, USA) at 80 °C for 1 h with constant agitation. After digestion, 100 µL of amyloglucosidase solution (50 mg/mL) (Sigma-Aldrich) was added and the mixture was kept at 60 °C for 3 h. Next, the pH was adjusted to 7.0 with NaOH 10% (w/v), followed by incubation with 200 µL of protease (Sigma-Aldrich) at 80 °C for 1 h. Finally, digested samples were vacuum filtered with cellulose-free filters (Whatman glass microfiber filters, 934-AHTM). The solid fraction contained in the filter represented insoluble dietary fibre (IDF). To precipitate the soluble dietary fibre (SDF), 4 volumes of 96% ethanol were added to the filtrate at 60 °C. Both fractions of fibre were dried overnight at 45 °C in an oven and were then weighed. The TDF was calculated as the sum of IDF and SDF. The results were expressed as g/100 g dry sample. All experiments were conducted in triplicate.

TAG and glycogen measurements were essentially as in [40 (link)]. We seed 60 1st instar larvae per vial to grow under defined density conditions. Hatching adults were collected within a 12-hour window, and allowed to age 3 or 4 days as indicated. Three times 8 animals were crushed in 500μl 1xPBS with 0.05% Tween-20 (Applichem, A1389) and heat-inactivated for 5 minutes on 70°C. 100μl lysate were cleared of debris and used for Protein concentration measurement using Bradford reagent (BIORAD protein assay, 500–0006). 200μl lysate were used for TAG measurement by adding 2μl of Lipase (Calbiochem, cat 437707) to the lysate, incubating at 37°C ON, spinning down debris and analyzing by using the free glycerol reagent from Sigma (F6428). For glycogen measurement, another 200μl of the original lysate were split into two times 30μl–a control lysate and one lysate to which 1 μl of amyloglucosidase solution (14 U per μl, Sigma 10115) was added. After 1h incubation at 50°C, both lysates were analyzed using the Glucose reagent from Sigma (G3293) and subtracting the glucose content of the control lysate from the amyglucosidase lysate to yield relative glycogen levels of each sample.