Hcp 2

The extracellular matrix composition was evaluated by electron microscopic examination. For scanning electron microscopy (SEM), fresh and decellularized tissues were fixed with 2% glutaraldehyde in 0.1 M PBS at 37°C for 1 h, washed with PBS twice, and dehydrated through a graded series of ethanol. All specimens were critical point dried in an HCP-2 (Hitachi, Tokyo, Japan) and coated with platinum using an HCP-2 (Hitachi). Images were observed and captured using an S-4300 (Hitachi). For transmission electron microscopy (TEM), specimens were fixed, dehydrated, and embedded in Epon 812 (Sigma-Aldrich). Ultrathin sections (70 nm) were generated by using an EM UC7 (Leica, Wetzlar, Germany) and stained with 2% uranyl acetate and 1% phosphotungstic acid (Sigma-Aldrich), pH 3.2. TEM images were captured at 100 kV on a HT7700 Bio-TEM (Hitachi).

To further compare the cytological differences between the calli and different structures, transmission and scanning electron microscopy were performed. The calli of different structures were selected and divided into sections of 3 mm × 3 mm. The samples were fixed with 2.5% glutaraldehyde for longer than 4 h in PBS (0.1 M, pH 7.0), washed with the same buffer, post-fixed with 1% OsO₄ in phosphate buffer (0.1 M, pH 7.0) for 1–2 h, dehydrated for 15–20 min for each step in a graded series of ethanol (30, 50, 70, 80, 90, 95, and 100%), and transferred to a 1:1 mixture of alcohol and iso-amyl acetate for 0.5 h, before being transferred to pure iso-amyl acetate for 1 h. The samples were also dehydrated in an HCP-2 critical point dryer (HCP-2, Hitachi, Japan) with liquid carbon dioxide (CO₂). Further, gold–palladium was utilized to coat the dehydrated samples in an E-1010 Ion Sputter (E-1010, Hitachi, Japan) for 4–5 min, which we observed using a Model SU-8010 SEM (SU-8010, Hitachi, Japan).

After fixation with 2.5% glutaraldehyde for 2 days, the samples from different treatments were immediately dehydrated in 10 mL of ice-cold 200 proof ethanol (Sigma-Aldrich), point-dried in a critical point dryer (HCP-2, Hitachi High-Technologies Corporation, Japan) and coated with 60% Au/Pd in a sputter coater (Sputter Coater Baltec SCD500, Bal-Tel) [43 (link)]. The surface morphologies of different samples were observed by using an S-4800 II field emission scanning electron microscope (Hitachi, Japan). The respiratory rate of NJAU4742 strain during the solid-state fermentation process was evaluated by tracking the release of CO₂. Briefly, the triangular flasks of solid-state fermentations were sealed with a parafilm 2 h before sampling. When sampling, 20 mL of gas in the triangular flask was extracted with a syringe and then injected into the gas sampling bag (E-SWITCH, China), and the contents of CO₂ was determined by a gas chromatography (Agilent 7890A) equipped with Porapak Q column and a flame ionization detector (FID) according to Zhang et al. [44 (link)] with some modification: 500 µL of gas were injected through a septum with the temperature of 80 °C, and the carrier gas (N₂) flow-rate was 30 mL min⁻¹; meanwhile, the temperature of the methanizer and detector were set as 450 °C and 250 °C, respectively.