Amyloglucosidase

Oat bran was purchased from Tong Yuan Gong He Co., Ltd. (Yantai, China). TermamylSC and amyloglucosidase were obtained from Novozymes Co., Ltd. (Bagsvaerd, Denmark). Pepsin and trypsin were obtained from Amresco Co., Ltd. (Solon, OH, USA). Lichenase was purchased from Megazyme (Wicklow, Ireland). Mannose, glucose, galactose, xylose, arabinose, and fucose standard were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). DEAE52 and SephadexG200 were purchased from GE Healthcare. All other chemicals used were of analytical grade.

Heat-stable α-amylase (Termamyl 120 L, 120 KNU-S/mL), protease (Alcalase 2.4 L, 4.42 AU/mL), and amyloglucosidase (AMG 300 L, 300 AGU/mL) were purchased from Novozymes (Beijing, China) biotechnology Co., Ltd. Folin—Ciocalteu reagent, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tripyridyl-s-triazine (TPTZ), 3′,6′-dihydroxy-spiro[isobenzofuran-1(³H),9′-(⁹H)-xanthene]-3-one disodium salt (FL) and 2,2′-azobis-(2-amidinopropane) dihydrochloride (ABAP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Gallic acid, chlorogenic acid, protocatechuic acid, p-hydroxybenzonic acid, vanillic acid, catechin, epicatechin, caffeic acid, sinapic acid, syringic acid, vanilline, isoquercitrin, p-coumaric acid, ferulic acid, quercetin, caffeic acid methyl, ferulic acid methyl were purchased from Aladdin Reagents (Shanghai, China). Fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS) and Dulbecco’s modified eagle’s medium (DMEM) were purchased from Atlanta Biologicals (Lawrenceville, GA, USA), GibcoLife Technologies (Grand Island, NY, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. HepG2 human liver cancer cells were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). All other chemicals used were of analytical grade or above.

Intracellular glycogen content was calculated as previously described^{69 (link)} with minor modifications. Briefly, Synechococcus cells were collected after centrifugation at 10,000 × g for 15 min. The pellet was washed three times with ddH₂O, resuspended in 30% (w/v) KOH, heated at 95 °C for 2 h, and finally, ice-cold ethanol was added to a final concentration of 70–75% (v/v). The mixture was cooled overnight at −20 °C, then centrifuged at 10,000 × g for 15 min to collect the glycogen pellet. Glycogen pellets were washed twice consecutively with 70% ethanol (v/v) and 98% ethanol (v/v) and then dried by vacuum centrifugation. The resulting glycogen was suspended in 100 mM sodium acetate and digested with amyloglucosidase (Novozymes). To measure the amount of glucose produced in the glycogen solution, a sucrose/D-glucose assay kit (Megazyme, Ireland) was used.

Wheat starch was provided by Yuanye Biotechnology Co., Ltd. (Shanghai, China). Spectral-grade KBr was provided by Aladdin Reagent Co., Ltd. (Shanghai, China). A leached amylose assay kit and 3,5-dinitrosalicylic acid (DNS) were provided by Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China). Amyloglucosidase (300 AGU/g, AMG 300 L) was provided by Novozymes Biotechnology Co., Ltd. (Shanghai, China). Porcine pancreatic α-amylase (40.4 U/mg) was provided by Sigma-Aldrich (St. Louis, MO, USA). Kiwifruit SDF and IDF were isolated from fresh kiwifruit by our previously described research method [23 (link)].

Glycogen contents of Synechococcus strains were measured as previously introduced with modifications (Grundel et al., 2012 (link)). Synechococcus cells cultivated in BG11 broth under normal condition were collected at day 6. The cells were centrifuged and washed twice with distilled water. The cells were finally resuspended with 400 μl 30% KOH and heated at 95°C for 2 h. Chilled ethanol was added to the lysate solution to the final concentration of 70% (V/V). The mixture was cooled on ice for 4 h, and then centrifuged (14000 g, 15 min, and 4°C). The pellet was washed with 70% ethanol (V/V) and 98% ethanol (V/V) successively, and then dried by vacuum centrifugation. The glycogen finally obtained was suspended in 500 μl 100 mM sodium acetate and digested by 2 mg amyloglucosidase (Novozymes). Glucose contents generated in the glycogen solution was determined with an SBA-40c biosensor analyzer (Shandong Academy of Sciences, China) and used for calculation of the glycogen contents.