α amylase

Considering the possible effect of starch to the structure and purity, amylase was adopted. The dried crude PPC (5 g) was put in distilled water with α-amylase at a suitable pH and temperature (activity of enzyme ≥ 2 × 104 U/g, pH 5.5−7.0, 90 °C, Beijing Solar Bio Science & Technology Co., Ltd. Beijing, China) for 20 min. The mixed suspension was transferred to a 100 °C water bath and maintained for 10 min to inactivate the enzyme. The mixture was precipitated by the addition of ethanol in 75% (v/v) at room temperature and the precipitate was dried.
Then non-starch polysaccharide was purified by Sephadex G-100 filtration chromatography according to the reported method with little modifications. In brief, the non-starch polysaccharide solution (3 mL, 10 mg/mL) was applied to a column (2.6 cm × 70 cm) of Sephadex G-100 dextran. Then, the column was eluted with ultrapure water at a flow rate of 0.4 mL/min. The obtained elute was collected automatically (4 mL/tube) and the polysaccharides were detected by the phenol-sulfuric acid method. As a result, one PPC fraction was obtained. The fraction was collected, concentrated, dialyzed and dried for further research.

RNCF and natural raw corn flour (RNCOF, purchased from a farmer’s market in Nanning, China) were used as substrates for enzymatic hydrolysis. For raw starch hydrolysis by RSDEs from P. oxalicum A2-13 and OXPoxGA15A, hydrolysis reactions with 10% (w/v) solid substrate and an enzyme loading of 50 U/g substrate were conducted in citrate–phosphate buffer (pH 4.5) at 40 °C for 72 h. The reducing sugars produced were determined using the DNS method [38 (link)].
A combination of RSDEs from the P. oxalicum strains with commercial α-amylase (Solarbio, Beijing, China) at a ratio of 1:1 was also tested, as described above. RSDEs were added at several concentrations ([U/g substrate], 50, 100, 150, and 200).

The broken rice used to produce the hydrolysate in this study was the by-product of rice processing provided by Zhongkou Fu Rice Industry Co Ltd, Dongguan, China. The microalgae strain used in this study, C. vulgaris FACHB-32, was obtained from the Institute of Hydrobiology, Chinese Academy of Science, P. R. China. The soybeans used in this study were purchased from the market in Nanchang, China.
The preparation of hydrolysate from broken rice was based on the method former reported [18 (link)]. Briefly, broken rice was gelatinized in boiling water for 30 min, and then hydrolyzed with 0.24% α-amylase (3,700 U/g, Solarbio Science and Technology Co., Ltd.) and 0.59% glucoamylase (10⁵ U/g, Solarbio Science and Technology Co., Ltd.). After that, the sample was boiled to inactivate the enzyme, and filtered with 0.45 µm filter membrane and stored at 4 °C for later use. The concentration of TOC, reducing sugar and TN content in BRH were 39.04, 87.12 and 0.81 g/L, respectively.