Sb 12l

SBC was determined following a method modified from Dubey et al. [20 (link)]. First, 0.1 g of SCF was dispersed in 2.5 mL deionized water. Subsequently, 5 mL 0.01 M glucose was added, and the mixture was subjected to shaking under incubated conditions at 200 rpm and 37 °C for 6 h (SB-12l, Benchmark Scientific, Sayreville, NJ, USA). The mixture was centrifuged at 2000× g for 15 min, and the supernatant was filtered through a 0.45 µm filter. Then, 2 mL of filtrate was collected and subsequently analyzed using the phenol-sulfuric assay. Absorbance of the mixtures was recorded at 490 nm (Cary 60 UV-Vis Spectrophotometer, Agilent Technologies, Santa Clara, CA, USA) [20 (link)]. To quantify the glucose content of SCF, absorbance values of the samples were compared against a glucose standard curve.

The release of reducing sugars during the digestion process was quantified using the dinitrosalicylic acid (DNS) colorimetric method [27 (link),28 (link)]. Inactivated aliquots were centrifuged at 1000× g at 25 °C for 10 min. Then, 500 µL of the supernatant was transferred to 250 µL of enzyme solution containing 1% (v/v) amyloglucosidase (and 0.05% (w/v) invertase dissolved in 0.1 M sodium acetate buffer (pH 5.2). The mixture was incubated at 37 °C for 15 min (SB-12l, Benchmark Scientific, Sayreville, NJ, USA). Subsequently, 750 µL of DNS mixture (comprising of a 1:1:5 mixture of 0.5 mg/mL of glucose solution, 4 M NaOH, and DNS reagent respectively) was added, and the contents were heated in a water bath at 95 °C for 15 min. Samples were cooled in ice water and 4 mL of deionized water was added and thoroughly mixed. Absorbance of the mixtures was recorded at 530 nm (Cary 60, Agilent Technologies, Santa Clara, CA, USA). To quantify the glucose content of SCF, absorbance values of the samples were compared against a glucose standard curve.