2.6. Saccharification of PT-RS

PT-RS #1 was used for the substrates for the saccharification experiments. The wet sample (equivalent to 5 g DM) was mixed with distilled water (68 mL), and the mixture was neutralized with 1 M acetic acid until the pH reached 5.1. The fibrillation of PT-RS #1 was performed as follows: the neutralized PT-RS #1 suspension was dehydrated with a plastic mesh sheet (Mesh Sheet (28 mesh), Advantec Co., Ltd., Tokyo, Japan) set on a Buchner funnel, and the trapped fibers were manually divided to five parts (each equivalent to 1 g dry weight). One of the parts was mixed with the filtrate, and the suspension was set on a blender (Foodmill TML17, Tescom Denki Co., Ltd., Tokyo, Japan) for fibrillation for 30 s. The fibrillated suspension was passed again through the plastic mesh sheet on the Buchner funnel to trap fibrillated fibers and prepare a new filtrate. The next part of the divided fibers was added to the new filtrate for fibrillation. After repeating this procedure four more times, all the divided fibers were obtained in fibrillated form, and the four fractions of the fibrillated fibers were mixed with the fifth suspension after fibrillation. Then, samples of the neutralized PT-RS#1 suspension with and without the fibrillation procedure were mixed with the enzyme mixture solution as described in Section 2.3 for the enzymatic saccharification reaction at a substrate concentration of 5% (w/v). Aliquots (0.5 mL) were taken from the suspension for analysis of solubilized sugars as described in Section 2.3.