M.-O. Park, et al.
FoodChemistry300(2019)125225
enzymatic reaction was carried out with 400 U/L of NsAS for 120 h. At
different reaction times, aliquots of the reaction mixture were collected
and boiled for 5 min to inactivate the enzyme. The soluble reaction
product was analyzed by HPAEC as described in the previous study
starch (RDS), non-soluble resistant starch (NSRS), and total RS content
2.8. Branch-chain length distribution analysis by HPAEC of NsAS-treated
starches
2.4.3. Effect of sucrose and fructose concentration on turanose synthesis
yield
Turanose synthesis was investigated by using sucrose as substrate at
various concentrations of substrates (1.0, 1.5, and 2.0 M) amendment of
various fructose (0.25, 0.5, 0.75, and 1.0 M) in 50 mM Tris-HCl (pH 8.0)
at 40 °C. The substrate was pre-incubated at three different tempera-
tures for 10 min. The enzymatic reaction was carried out with 400 U/L
of NsAS for 120 h. At different reaction times, samples were collected
and inactivated by boiling for 5 min. The soluble fraction of reaction
product was analyzed by HPAEC.
The branch-chain length distributions of NsAS-treated starch were
measured by high-performance anion-exchange chromatography
(HPAEC, ICS-5000 SP, Dionex, Sunnyvale, CA) coupled with a pulsed
amperometric detector (PAD, ICS-5000 DC, Dionex). Total 50 mg each
of normal starch or enzyme-treated starch was completely wetted with
0.5 mL of distilled water for 10 min and then dispersed in 4.5 mL of
dimethyl sulfoxide (DMSO). The dispersion in a glass vial was boiled for
1 h with constant stirring, which was additionally stirred for another
12 h at room temperature. The resulting clear solution was mixed with
6 volumes of 99% ethanol, then followed by centrifugation (4500g) for
20 min. The precipitates were dissolved in 4 mL of 10 mM sodium
acetate buffer (pH 3.5) and then boiled for 1 h with constant stirring.
After cooling it, the sample solution was debranched with isoamylase
(Megazyme International Ireland Limited) for the hydrolysis of α-(1,6)-
D-glucosidic branch linkages at 40 °C for 48 h. After the isoamylolysis of
the sample solution, the salts of sodium acetate buffer components were
removed for the HPAEC analysis. The reacted samples were mixed with
0.2 g of resin beads (IONAC NM-60H+/OH− form, J. T. Baker,
Phillipsburg, NJ) and were shaken to desalt for 1 min. And then, the
sample was filtered through a 0.2-μm syringe filter. The filtered sample
(25 μL) was injected into a CarboPac™ PA200 analytical column
(3 × 250 mm, Dionex). Hydrolyzed products were separated via a
linear gradient mode from 150 mM NaOH initially to 600 mM sodium
2.5. In vitro digestibility of turanose in a continuous simulated digestion
fluid system
The degree of turanose hydrolysis in the continuous simulated di-
gestion fluids was carried out by the modified Minekus’s method
water bath. The simulated salivary fluid (SSF), simulated gastric fluid
(SGF), and simulated intestinal fluid (SIF) consisted of electrolyte stock
solutions, CaCl2, digestive enzymes, HCl, and water, depending on their
own individual physiological conditions (Supplement 1). Maltose and
sucrose were used as control sugars at 40 mM level for comparing the
digestion degree to turanose. The first digestion step was an enzymatic
hydrolysis process in SSF at pH 7.0 for 2 min. In this oral phase, final
ratio of sugar sample solution to SSF is targeted to 50:50 (v/v). The
composition of 1 mL SSF was as follows: 0.7 mL of SSF electrolyte stock
solution, 5 μL of 0.3 M CaCl2, 0.1 mL of human salivary α-amylase so-
lution (1500 U/mL of α-amylase in SSF stock solution), and 195 μL of
distilled water. The second step was a gastric phase in SGF at pH 3.0 for
2 h. The 1 mL of SSF was mixed with 1 mL of SGF. The component
contents of 1 mL of SGF were as follows: 0.75 mL of SGF electrolyte
stock solution, 0.5 μL of 0.3 M CaCl2, 5.6 μL of 1 M HCl, 0.16 mL of
porcine pepsin stock solution (25,000 U/mL porcine pepsin) in SGF
stock solution, and 83 μL of distilled water. As the final digestion step,
artificial intestinal phase was prepared in SIF at pH 7.0 for 4 h. The
reaction mixture (1 mL) in the gastric phase was mixed with 1 mL of SIF
of which the composition was as follows: 0.25 mL of SIF electrolyte
stock solution, 2 μL of 0.3 M CaCl2, 35 μL of 1 M HCl, 0.125 mL of
160 mM of fresh bile, 0.25 mL of pancreatin (0.1 g) solution/1 mL in SIF
stock solution, 0.30 mL of intestinal fluid containing 16.7 mg of rat
intestinal acetone powder per 1 mL in SIF stock solution, and 38 μL of
distilled water. At different reaction time intervals for each digestion
step, aliquots (100 μL) were collected, and the degree of hydrolysis was
assessed by the amounts of released glucose from the reaction using a
glucose diagnosis kit (GOPOD kit, Megazyme International Ireland
2.9. Thermal analysis of NsAS-treated starches
Thermal properties of enzyme-modified RS products were analyzed
by using
a differential scanning calorimeter (DSC-200, Netzsch,
and sealed. This sample dispersion was equilibrated at ambient tem-
perature for 2 h. The sample was scanned at a heating rate of 5 °C/min
from 20 °C to 150 °C, using an empty pan as a reference.
2.10. Pasting profile of NsAS-treated starches
Pasting properties of enzyme-modified RS products were measured
by a starch pasting cell attached to a controlled-stress rheometer
prepare a suspension (28 g; 10.7%, w/w) whose viscosity was mon-
itored during the programme heating and cooling cycle. Initially, the
starch samples were warmed up to 50 °C and started heating at a rate of
12 °C/min to 95 °C. This temperature was kept for 2.5 min and cooled at
a rate of 12 °C/min to 50 °C. Pasting temperature, peak viscosity, final
viscosity, breakdown, and setback were determined to explain the
pasting properties of NsAS-treated RS products.
2.6. Starch modification by NsAS treatment
Enzymatic modification of gelatinized corn and rice starches (3%,
w/v) was carried out in 50 mM Tris-HCl buffer (pH 8.0) with 0.3 M of
2.7. Determination of RS content of NsAS-treated starches
2.11. Statistical analysis
The freeze-dried non-soluble precipitates were ground with mortar
and pestle, and sieved through a 100-mesh screen. RS content was in-
vestigated using the Megazyme Resistant Starch Assay Kit (Megazyme
International Ireland Limited) following the AACC Method 32-40.01
ANOVA and Tukey's honestly significant difference tests were per-
formed using Sigmaplot software package 13.0 (Systat Software Inc.,
San Jose, CA).
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