Gopod

In vitro amylose digestibility was measured as described previously [37 (link)]. Briefly, samples (~100 mg) were mixed with 10 mL of enzyme solution that was pre-warmed to 37 °C (0.5 mg of pancreatin and 100 μL of amyloglucosidase with 9.9 mL of sodium acetate buffer (0.2 M, pH 6.0)) for digestion. A 100 μL aliquot was collected and mixed with 0.9 mL of absolute ethanol after 720 min of incubation. The glucose concentrations were determined by a UV-1700 Pharma Spectrophotometer at 510 nm after incubation with a glucose oxidase/peroxidase reagent (GOPOD, Megazyme). The data, comprising the concentration of released glucose as a function of time, were fitted using the logarithm of slope (LoS) method and the non-linear least-squares fitting method (NLLS) [44 (link)]. The LoS method shows if one has a single or multiple first-order rate steps during digestion. NLLS fitting then gives accurate coefficients of the digestion rate coefficient, k, and the long-time fraction of undigested starch, C_res, in each of the single-step regions suggested by the LoS method, as described previously [37 (link)].

A modified Association of Official Agricultural Chemists (AOAC) method 2002.02 was applied for RS determination. Each sample (100 mg) was digested by addition of 4 ml of enzyme reagent (30 U/ml of pancreatic α-amylase and 3U/ml of amyloglucosidase) and incubated at 37°C for 16 h. The reaction was terminated by adding 4 mL of ethanol (99%), mixed and centrifuged. The supernatant was discarded and the remaining starch (pellet) was re-suspended and centrifuged two times in 50% ethanol (v/v). The RS fraction was solubilized by addition of 2 mL of 2 M KOH, hydrolyzed with amyloglucosidase. The liberated glucose was quantified using glucose oxidase/peroxidase reagent (GOPOD was purchased from Megazyme Co. Ltd Wicklow, Ireland) and converted to the RS content.

The concentration of reducing sugars released from the substrates was quantified using a scaled dinitrosalicylic acid (DNS) method [23 (link)]. Glucose concentrations were quantified using a glucose-specific kit (GOPOD, Megazyme International Ireland, Bray, Ireland). Substrate and enzyme controls were included wherever necessary.
Terminated fermentations, after centrifugation and filtration through a 0.2 μm filter, were loaded directly onto a Series 200 LC instrument (Perkin Elmer, Seer Green, UK) equipped with a refractive index detector. The analyses were carried out using an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) with matching guard columns operating at 65 °C with ultrapure water as mobile phase at a flow rate of 0.6 mL/min as described [22 (link)].

For the enzymatic hydrolysis, Accellerase 1500 (78.1 mg BCA protein/ml; lot 1662334068; DuPont, Palo Alto, CA, USA) and Multifect xylanase (45.4 mg BCA protein/ml; lot 301-04296-205; DuPont, Palo Alto, CA, USA) were used. The loadings were 80 mg and 20 mg/g biomass for the Accellerase and Multifect enzymes, respectively. Hydrolysis was conducted at 50 °C for 70 h, with 0.02% sodium azide in 140 mM pH 5.0 citrate buffer. Glucose and xylose release values were then measured using GOPOD and XDH enzymatic assays according to the manufacturer’s instructions (Megazyme, Bray, Ireland).

The β-glucan concentration of the supernatant was analyzed by a competitive enzyme-linked immuno-sorbent assay (ELISA) based on Streptococcus (S.) pneumoniae serotype 37 antibodies for the quantification of the bacterial β-glucan [86 (link)]. The assay was performed as previously described [36 (link)]. The D-glucose concentrations of the supernatant samples were determined using a glucose oxidase/peroxidase assay (GOPOD, Megazyme Ltd., Bray, Ireland) according to the manufacturer’s protocol. The assay was adapted to a microtiter plate volume with a 50 μL sample volume and 150 μL of the GOPOD reagent. A standard curve was used for D-glucose (Megazyme Ltd., Bray, Ireland) calculations. All experiments were carried out using four biological replicates.

Liver tissue was homogenized in PBS to prepare 10% liver homogenate, then centrifugated (4 °C, 3000 rpm, 10 min), and the supernatant was collected. The content of MDA, SOD, GPx, CAT, AST, ALT, and ALP in the supernatant of liver homogenate was determined according to the requirements of the instructions provided in ELISA kit methods (Ela science, Boston, MA, USA).
Liver glycogen content was measured using sodium acetate buffer (100 μL; pH 6), 5 μL of amyloglucosidase, and 20 μL of the homogenate to a final volume of 0.5 mL with double distilled water and incubated at 50 °C for 30 min. A control with everything except amyloglucosidase was also analyzed. A 300 μL aliquot of each sample was then added to 1 mL of glucose oxidase/peroxidase reagent (GOPOD, Megazyme) and incubated at 50 °C for 30 min. The glycogen content was calculated at 510 nm based on a calibration curve constructed by reacting _D-glucose of various concentrations with the same GOPOD reagent [38 (link)].
MDA level was expressed as µmol/mg of protein, GSH-px level was expressed as U mol/g of protein, and SOD activity was expressed as U/mg of protein. CAT activity was expressed as U/mg of protein, and glycogen was expressed as mg/g of tissue.