α amylase from aspergillus oryzae

Whey protein isolate (WPI) was purchased from Fonterra Co-operative Group Ltd., New Zealand. The fine powder consisted of 90.31% protein, 0.93% fat, 4.83% moisture and 3.3% ash (w/w). Native wheat starch (WS) was supplied from National Starch (National Starch and Chemical Co., Thailand), with the white powder containing 94.1% starch, 0.3% ash, 0.1% fat and 5.5% moisture (w/w). Low molecular weight chitosan (LCT), medium molecular weight chitosan (MCT) and α-amylase from Aspergillus oryzae was supplied from Sigma-Aldrich, Co. (Castle Hill, Australia). As specified by the manufacturer, the degree of deacetylation for both low and medium molecular weight chitosan, supplied in the form of a pale yellow powder, was about 80% and the viscosity of 1.0% (w/w) solution at ambient temperature was about 150 and 800 cP, respectively. According to the supplier, the molecular weight of LCT and MCT was about 70 kDa and 250 kDa, respectively. All other chemicals utilised were of analytical grade.

Roughly 25 mg of air‐dried stem sample was weighed in a 2 ml tube and extracted twice at 85°C with a total of 2 ml of 80% ethanol. The supernatant was collected in a new 2 ml tube and was re‐extracted with 50 mg activated charcoal (Sigma) to eliminate pigments that interfere with sugar analysis. A 1 ml aliquot of the pigment free extract was incubated overnight in a heating block maintained at 50°C, and the resulting pellet was dissolved in 120 μl of water. A 10 μl aliquot was used for estimation of sucrose and glucose using assay kits (Sigma). Starch from the pellet was digested using 1 U of α‐amylase (from Aspergillus oryzae, Sigma) and amyloglucosidase (from Aspergillus niger, Sigma). After starch removal, the pellet was dried overnight at 95 °C and used for estimating structural sugars. Roughly, 5 mg of sample was weighed in a 2 ml tube and digested with 50 μl of 75% v/v H₂SO₄ for 60 min. The reaction was diluted by adding 1.4 ml of water, and tubes were sealed using lid‐locks and autoclaved for 60 min in a liquid cycle. After cooling, the sample was neutralized with CaCO₃ and sugar composition was estimated using high performance liquid chromatography (HPLC, LaChrom Elite® system, Hitachi High Technologies America, Inc.) as described previously (Fu et al., 2011 (link); Yee et al., 2012 (link)).

RG concentrates (15 brix) were supplied by the Korean Ginseng Corporation (KGC Co., Daejeon, Republic of Korea). Carbohydrate-digesting enzymes, such as α-amylase from Aspergillus oryzae, amyloglucosidase from Aspergillus niger, and pectinesterase from orange peel were all obtained from Sigma (St. Louis, MO, USA), whereas polygalacturonase from Aspergillus aculeatus was purchased from Megazyme (Bray, Ireland). Sephadex G-100 was obtained from GE Healthcare (Uppsala, Sweden). Sheep red blood cells were obtained from Innovative Research, Inc. (Novi, MI, USA). Dulbecco's Modified Eagle's Medium, fetal bovine serum, and other cell culture supplements were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other analytical reagents were purchased from Sigma (USA).
Female Balb/c mice (6 weeks old) were purchased from SaeronBio Inc. (Uiwang, Gyeonggi, the Republic of Korea). They were housed in a specific pathogen–free room maintained at a constant temperature (20 ± 1°C) and humidity (55%) in a clean rack with 12 h of light and dark cycles. Water and pellet diet were supplied ad libitum. All animal experiments were carried out according to the instructions by the Ethics Committee for Use of Experimental Animals at Kyonggi University (admission number: 2017-007).