2.2. Isolation of MWLs and LCCs

Milled wood lignin (MWL) was isolated according to the method described by Björkman [21], and the isolation procedure of lignin and LCC preparations from ginkgo shells is illustrated in Figure 1. The air-dried ginkgo shells were ground in a Wiley mill. Particles between 40 mesh (0.425 mm) and 80 mesh (0.180 mm) were extracted with ethanol/benzene (1:2, v/v) in a Soxhlet extractor for 12 h to obtain extractive-free samples. The extractive-free shell meals were milled in a planetary ball mill (Fritsch GMBH, Pulverisette 7 premium line, Idar-Oberstein, Germany) at a frequency of 10 Hz for 2 h without solvent. Two 80 mL silicon nitride bowls, with 4 g ginkgo shell meals in each bowl, were filled with 25 zirconium dioxide balls (1 cm diameter). The milling was conducted at room temperature, and 15 min intervals were provided between every 3 min of milling to prevent overheating.

Isolation procedure of MWLs and LCCs from ginkgo shells.

The ball-milled samples were suspended in 1,4-dioxane (96%, v/v) with a liquid-to-solid ratio of 15 (mL/g) at room temperature for 24 h. The extraction procedure was conducted in the dark and under a nitrogen atmosphere. The mixture was centrifuged and washed using 96% 1,4-dioxane until the filtrate was clear. Such operations were repeated thrice. The extractive supernatants were combined and the solvent was recycled by vacuum evaporation. The crude lignin was dissolved in acetic acid (90%, w/w), and the soluble fraction was slowly introduced into deionized water. The supernatants were vacuum evaporated and washed to obtain LCCs (LCC_ML and LCC_FZ). The precipitates were washed with deionized water to obtain lignin preparations (MWL_ML and MWL_FZ). No further purification was performed for the preservation of the structural features of the lignin preparations.