Fdu 2110

The heavy metal content of raw materials and residues was determined. All samples needed to be dried in a freeze-dryer (FDU-2110, EYELA, Tokyo, Japan) and ground through a 200-mesh screen before analysis. The heavy metal content was measured by the method proposed by the Chinese Ministry of Ecology and Environment [62 ]. The metal concentrations were detected by inductively coupled plasma mass spectrometry (ICP-MS, Perkin Elmer NexION 300X, Waltham, MA, USA).

All digesta samples were freeze-dried at −50 °C (FDU-2110, EYELA, Tokyo, Japan). Before analyses, the diets and dried digesta samples were ground by an analytic mill processing to pass through a 0.18 mm screen. The diets and digesta samples were analyzed for gross energy, crude protein, and amino acids on a dry matter (DM) basis. The DM was determined by drying at 105 °C for 24 h (method 934.01; AOAC, 2005). The gross energy was determined using a bomb calorimeter (Parr Instrument Co., Moline, IL, USA). The concentration of crude protein was measured according to the method of AOAC (method 990.3; AOAC, 2005). The concentrations of amino acids were determined using an amino acid analyzer (L-8900, Hitachi, Tokyo, Japan). In brief, about 0.2 g sample was hydrolyzed by 6 N HCl containing 6 g/L phenol for 24 h at 110 °C in a glass tube sealed under vacuum. For the cysteine and methionine analyses, the samples were oxidized by performic acid for 16 h at 0 °C, and then the reaction was terminated by adding hydrobromic acid before acid hydrolysis. The concentration of chromium was determined by digesting the samples in concentrated nitric acid and perchloric acid, and the absorption was measured using a spectrophotometer at 440 nm (Beckman DU-800; Beckman Coulter, Inc., Brea, CA, USA). All analyses were performed in duplicate.

The sugars were extracted according to a published protocol (Zhou et al., 2022 (link)). Samples were taken at different aging time points; the seed embryos were freeze-dried (FDU-2110, EYELA, Japan), then ground in a clean mortar, and put into an Eppendorf tube. A 200-mg aliquot of the sample was put into a 10-ml graduated centrifuge tube with the addition of 5 ml of 80% ethanol and placed in an 80°C water bath for 10 min, shaken three to five times in the middle of the process. Next, the supernatant was collected after centrifugation at 5,000g for 5 min. The above operation was repeated and the two supernatants were combined. The extracts were filtered through 0.22-μm water phase filters (Jinteng, China). Sugars were measured directly in the extract by HPLC using a Waters binary HPLC system (Waters 1525-2707, Milford, USA) equipped with a refractive index detector (2414, Waters, Milford, USA). Analytical conditions were as follows: column Agilent Hi-Plea Ca (8% cross-linked), 300 × 7.7 mm, 8 µm in diameter (Agilent Technologies, Inc., USA); column temperature, 85 °C; mobile phase, Milli-Q water; and flow rate, 0.6 ml min⁻¹. Data were collected and processed by the Waters chromatography station DataApex. Sugars were identified by comparison with retention times and coelution of authentic standard solutions.