Alpha amylase

Trolox, DPPH, trichloroacetic acid, ABTS, potassium persulpahte, 2,4,6-tripyridyl-s-triazine (TPTZ), soluble starch, Folin–Ciocalteu reagent, alpha glucosidase from Saccharomyces cerevisiae, gallic acid, acarbose, quercetin, aluminum chloride, p-nitrophenyl a-d-glucopyranoside, bovine serum albumin (BSA), and alpha-amylase were purchased from Sigma Aldrich (Allentown, PA, USA). The chemicals and reagents used for all experiments were of analytical grade.

Alpha-amylase (porcine pancreas), 2-chloro-4-nitrophenyl-D-maltotrioside (CNPG3) and tetramethylsilane were from Sigma-Aldrich (St. Louis, MO, USA) while the remaining chemicals were from UFC Biotechnology. Spectrophotometric readings were performed using the microplate reader (Anthos Zenyth 200RT, Cambridge, UK). ¹H-NMR (400 MHz) analysis was carried out using Bruker NMR Spectrometer (Bruker, Yokohama, Japan). Spectra were recorded in CD₃OD or DMSO-d₆ with tetramethylsilane being employed as an internal standard, and chemical shift values were expressed in ppm.

Alpha-amylase, CaCl₂ ∙ 2H₂O, 4′,6′-diamidino-2-phenylindole (DAPI), 2′,7′-dichlorofluorescin-diacetate (DCFH-DA), MgCl₂ ∙ 6H₂O, mucin, ox bile, pancreatin, paraformaldehyde, pepsin, trypsin and ZnO nanopowder (#677450 and #544906) were purchased from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany. Dulbecco’s Modified Eagle Medium (DMEM), foetal bovine serum (FBS), non-essential amino acids, penicillin/streptomycin, and trypsin/EDTA were obtained from PAN-Biotech GmbH, Aidenbach, Germany. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide, fluorescein isothiocyanate (FITC)-dextran, hydrogen peroxide (30%), nitric acid (Suprapur), and triton X-100 were acquired from Merck KGaA, Darmstadt, Germany. 2-((3-Chlorophenyl) hydrazinylidene) propanedinitrile (CCCP) and ZnCl₂ were procured from Thermo Fisher Scientific Inc., Waltham, MA, USA. Carbamide, ethylene glycol tetraacetic acid (EGTA), KCl, KH₂PO₄, NaCl, NaHCO₃, and Na₂HPO₄ ∙ 2H₂O were purchased from Carl Roth GmbH & Co. KG, Karlsruhe, Germany. Cacodylic acid and glutaraldehyde were bought from Serva Electrophoresis GmbH, Heidelberg, Germany. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was obtained from Enzo Life Sciences GmbH, Lörrach, Germany. Phalloidin-iFlour 488 reagent was acquired from Abcam plc., Cambridge, UK.

Pretreatment and enzymatic hydrolysis of biomass was carried out as described 196 previously with some modifications [35 (link)]. Before the 197 analyses, biomass was treated with alpha-amylase (0.47 U per mg biomass, Sigma 198 Cat # A6255) in 100 mM ammonium formate (pH 5.0) buffer at 25 °C for 48 h to 199 remove starch, followed by three water and two acetone washes. Then biomass 200 samples were kept under the hood for 72 h for drying. This was followed by 201 ethanol Soxhlet extraction for an additional 24 h to remove extractives. After drying 202 overnight, pretreatment was carried out with 5 mg of dry biomass at 180 °C for 17.5 203 min. About 40 μl of buffer-enzyme stock, 8% CTec2 (Novozymes) in 1.0 M sodium 204 citrate buffer, was added to the pretreated biomass. The samples were incubated at 205 50 °C for 70 h. After 70 h of incubation, the hydrolysate was analyzed using 206 Megazyme’s GOPOD and XDH assays as described earlier.

Alpha-amylase and 3,5-dinitro salicylic acid (DNS) were purchased from Sigma-Aldrich, Saint Louis, MO, USA, while starch, sodium dihydrogen phosphate, and di-sodium hydrogen phosphate were purchased from Hi-Media, PA, USA.

The following chemicals were obtained commercially from the sources given in parentheses: rat serum (Biozol, Eching, Germany), chicken pancreas (Difco, Sparks, MD, USA), acetic acid, acetonitrile, sodium phosphate dibasic dihydrate, formic acid, hexane, methanol, potassium phosphate monobasic, sodium hydroxide, sodium chloride (Merck, Darmstadt, Germany), alpha-amylase, ammonium formate, ascorbic acid, pteroylmonoglutamic acid, 4-morpholineethanesulfonic acid (MES), 2-mercaptoethanol, protease type XIV, sodium acetate (Sigma, Deisenhofen, Germany), (6S)-tetrahydrofolic acid, calcium (6S)-5-methyltetrahydrofolate, 10-formylfolic acid, and (6S)-5-formyltetrahydrofolic acid (Schircks, Jona, Switzerland). All chemicals were at least of analytical-reagent grade. [²H₄]-5-methyltetrahydrofolic acid, [²H₄]-5-formyltetrahydrofolic acid, [²H₄]-tetrahydrofolic acid, [²H₄]-10-formylfolic acid, and [²H₄]-pteroylmonoglutamic acid were synthesized as previously reported (14 (link)).

Proximate analysis consisted of determining moisture, protein, lipid and ash contents of the diet using standard methods (AOAC, 1995 ).Crude protein (N×6.25) was determined by the Kjeldahl method after an acid digestion using an auto-Kjeldahl System (Hanon, Jinan, China). Crude lipid was determined by the ether-extraction method. Moisture was determined by oven drying at 105°C for 24 h. Ash was determined using a muffle furnace at 550°C for 24 h. Starch content of the diet was determined by spectrophotometric determination of glucose after hydrolysis by heat-stable alpha-amylase and amylo-glucosidase (Sigma, St Louis, USA) (Hall, 2000 ).