Chemical Characterization

2.3.3.1. Composition

The particle sizes of the milled and defatted WB, MB, and DDG were determined in accordance

with American Association of Cereal Chemists International (AACCI) Method 55-60.01: Guideline for

Determination of Particle Size Distribution (AACC International 2011). The moisture content of each of the

starting materials, defatted materials, and AX was determined in duplicate by following the AACCI Method

44-15.02: Moisture Air-Oven Method (AACC International 1999d). The ash content of all materials was

determined in duplicate following the AACCI Method 08-01.01: Ash-Basic Method (AACC International

1999c). To determine the N content of these materials, AACCI Method 46-30.01: Crude Protein-

Combustion Method. Protein content (estimated by N content) was determined in at least duplicate

(AACC International 1999b). The lipid content in each milled material (WB, MB, and DDG) was analyzed

in duplicate according to the American Oil Chemists Society (AOCS) Method Ba 3-38: Oil (AOCS 2009).

Total Starch analysis was carried out in at least quadruplicate using a Megazyme Enzyme Assay

Kit K-TSTA (Megazyme International Ireland, Wicklow, Ireland) (AACC International 1999a). To begin,

100 mg of each sample was added to a clean, empty test tube (16 X 20 mm). Next, 0.2 mL of aqueous

ethanol (800 g kg-1_{) was added to each test tube and stirred using a VWR Analog Vortex Mixer (VM-3000} Mini Vortexer; Serial No. 061220025). After vortexing, 3 mL of α-amalyse solution (1 mL α-amalyse to 29 mL 3-(N-morpholino)propanesulfonic acid buffer) was added to each test tube. The test tubes were then

vortexed again before being incubated at 100 °C in a VWR Type 89032-226 Water Bath for 10 minutes

with vortexing after two and four minutes. Next, 4 mL of sodium acetate buffer (200 mM; 0.2 g kg-1 _sodium

azide; pH 4.5) and 0.1 mL of amyloglucosidase were added to each test tube. All samples were then

incubated at 50 °C for 30 minutes. After incubation, the WB, MB, and DDG samples were transferred to

100 mL volumetric flasks and diluted to volume. All samples were then filtered into test tubes and 0.1 mL

aliquots were transferred (in duplicate) to small glass test tubes (16 X 100 mm). Once in the small test

tubes, 3 mL of glucose oxidase peroxidase was added to each test tube including the blanks and glucose

50

standard solution. All tubes were then incubated at 50 °C for 20 minutes. The absorbance of all solutions

were then read at 510 nm by a Thermo Electron Corporation Multiskan Ascent Type 354 microplate

reader (Ref No. 51118407) that utilized Ascent Software for Multiskan Ascent (ThermoFisher Scientific

2010).

To determine sugar profile and A:X, acid hydrolysis followed by derivatization to alditol acetates

was completed (Blankeney et al. 1983). After derivatization, the samples were analyzed using a gas

chromatograph with a flame ionization detector (GC-FID). To begin this process, a standard curve was

developed for each sugar. To do this, 250 mg of each sugar standard was placed into individual 25 mL

volumetric flasks. Myo-inositol was used as an internal standard, so 250 mg of this material were also

placed into a 25-mL volumetric flask. For each sugar, four different concentrations were used to make the

standard curve: 250 ng mL-1_{, 500 ng mL}-1_{, 750 ng mL}-1_{, and 1000 ng mL}-1_{. Next, 250 µL of 2 M TFA were}

added to each tube. The samples were then dried under nitrogen in a Thermo Scientific Reacti-Therm III

(Model No. TC-18823).

The next step in this method was reduction, which began with the addition of 100 µL 1 M

ammonium hydroxide. The contents of each standard were then swirled and 0.5 mL of 20 mg mL-1 _sodium

borohydride in dimethyl sulfoxide were added to each tube. The standards were then vortexed on a VWR

Analog Vortex Mixer (Serial No. 061220025) and heated for 90 minutes at 40 °C vortexing every 30

minutes. After heating, 6 drops of glacial acetic acid were added to each standard.

After reducing, the standards were acetylated. The first step was to add 100 µL of 1-

methylimidazol and then each sample was swirled. Next, 0.5 mL acetic anhydride were added to each

standard before vortexing on a VWR Analog Vortex Mixer (Serial No. 061220025). The standards were

then allowed to sit for 10 minutes at 23 °C before 4 mL of deionized water were added. The standards

were vortexed once again and 1 mL methylene chloride was added. Each standard was vortexed and the

bottom layer was removed using a long pasture pipette and transferred to a clean test tube. The addition

of 1 mL methylene chloride and removal of the bottom layer was repeated once again. This bottom layer

of each standard was dried under nitrogen at 45 °C in a Thermo Scientific Reacti-Therm III (Model No.

TS-18823). Once dried, 1 mL acetone was added to each test tube and the dissolved contents were then

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After the standard curve was created, 2 to 4 mg of each sample was analyzed. The hydrolysis of

this process began with adding 250 µL 2M TFA to each test tube with sample and sealing tightly with a

cap. Next, the tubes were heated at 121 °C for one hour. After heating, 75 µL of inositol were added to

each test tube and they were dried under nitrogen at 55 °C in a Thermo Scientific Reacti-Therm III (Model

No. TS-18823). After hydrolyzing the samples, they were reduced and acetylated using the same method

described for the control samples.

After sample preparation, the analysis of the sugar composition was performed using GC-FID.

The gas chromatograph used was a Hewlett Packard 5890 Series II GC system with a flame ionization

detector (Agilent Technologies, Incorporated Santa Clara, CA). The column used was a SupelcoSP-2380

fused silica capillary column (30 m x 0.25 mm x 2 µm) (Supelco Bellefonte, PA). The parameters under

which testing was completed were as follows: 827372 Pa flow pressure, 100 °C oven temperature, 0.8

mL min-1 _{flow rate, detector temperature of 250 °C, and injector temperature of 230 °C. Helium was used}

as the carrier gas.

2.3.3.2. High Performance Size Exclusion Chromatography-Multi Angle Light Scattering-Refractive Index

To determine the Mw of each AX extract, a HPSEC-MALS-RI system was used. The Mw and

polydispersity index (PI), which is defined as the ratio Mw Mn-1_{, where Mw is the weight average}

molecular weight and Mn is the number average molecular mass, of AX hydrolyzates was determined

using a modified method of Menids and Simsek (2015). A small amount of sample (4 mg) was dissolved

in deionized water that had been filtered using a 0.1 µm filter by heating at 40 °C with continuous stirring

for 60 minutes. Each sample solution was then filtered through a 5 µm filter and analyzed using an

HPSEC-MALS-RI system. The high performance liquid chromatograph (HPLC) used was an Agilent 1200

with a Wyatt Dawn Helios-II multi-angle light scattering detector (MALS) and a refractive index detector

(RI). The columns used were a Shodex OHpak guard column and a SB 806-HQ column. To normalize the

MALS detector, 300 kDa pullulan was used. The mobile phase used was water with a 0.5 mL min-1 _flow

rate. To calculate the Mw and PI, Astra 6.0.5 software (Wyatt Technology 2116) based on a 3rd _order

Debye plot with second-order polynomial fit was used. The values of dn dc-1_{, which is defined as the}

proportional change in the refractive index as the polymer concentration changes were assumed to be

52 2.3.3.3. 1_{H Nuclear Magnetic Resonance Spectroscopy}

The NMR spectra for each type of AX (WB, MB, and DDG) was determined using duplicate

samples (Mendis and Simsek 2015). To begin the process, 5 mg of each sample were dissolved in 600

µL of deuterium oxide by heating at 40 °C using continuous stirring with a Reacti-therm III No. 18823

(Thermo Scientific, USA). After dissolving the samples, they were freeze-dried on a Labconco Freeze

Zone 4.5 Freeze Dryer (Labconco Corporation Kansas City, MO; Cat No. 7751070; Serial No. 070975444

Rev. S.). The samples were redissolved and freeze-dried two more times. For the NMR analysis, the

samples were dissolved in 650 µL deuterium oxide and analyzed at 80 °C on a 400 MHz spectrometer

(Bruker AV3 HD 400 MHZ NMR that had a 5 mm PABBO BB/19F-!H/D Z-GRD Z probe). Data was

analyzed using TopSpin 3.2 software (Bruker BioSpin Corporation 2015).