Analysis of molecular structure of starch

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Analysis of molecular structure of starch
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Physicochemical properties/ Chemical composition
Molecular structure
Genes
Biosynthesis (enzymes)
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Molecular Structure of Amylose
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Molecular Structure of Amylopectin
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Average chain-length and amount (mole ) of the
fractions of amylopectin unit chain
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Molecular characterization
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Molecular characterization
Average (branch) chain length, overall
Average (branch) chain length, of A-chain
Amylopectin
Average (branch) chain length, of B1-chain
Average (branch) chain length, of B2-chain
Unit chain (A, B1, B2, B3,..) fraction (mole)
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Methods of Analysis

• Colorimetric methods
• chemical reaction
• chemical reaction enzyme reaction
• Chromatographic Techniques
• without enzyme reaction
• with enzyme reaction

Low-angle laser-light-scattering photometer
Refractive index detector
Detector
Pulsed amperometric detector
Fluorescence detector
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Colorimetric methods (chemical reaction)
Determine Total sugar/ Reducing
end/Non-reducing end
Average number of chain (NC) DP/CL
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• Total sugar
• Anthrone-H2SO4
• Phenol-H2SO4

Non-reducing end sugar Rapid Smith
Degradation method Ref 1. J.K. Hamilton and F.
Smith, J. Am. Chem. Soc., 78 (1956), 5907-5909.
2. S. Hizukuri and S. Osaki, Carbohydrate
Research, 63 (1978), 261-264.
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Colorimetric methods (chemical reaction enzyme
reaction)
Isoamylase/pullulanase
Hydrolyze ?-1,6 by isoamylase/pullulanase Determi
ne reducing end sugar by Modified Park
Johnsons method
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Experimental Procedure
Chromatographic Techniques with Enzyme Reaction

• Amylopectin structure studied by HPSEC

Starch
Fractionation (selective precipitation)
Amylose
Amylopectin
Molecular analyses (HPSEC)
Debranched
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Chromatographic Techniques with Enzyme Reaction
A
B
B
A
C
A
B
A
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Chromatographic Techniques with Enzyme Reaction
Column
Mobile Phase
S M L
Detectors
Recorder
Solvent Delivery System
Injector
Response
L
M
S
Injection of debranched amylopectin
Retention time
Chart record
Figure 6 Block diagram showing the component of
an HPSEC instrument.
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Experimental Procedure

• Amylopectin structure studied by HPSEC
• Column Zorbax PSM 60S (? 2)
• MW range 5 ? 102 104
• Column dimension 6.2 mm ID ? 250 mm
• Loading size 40 µl
• Eluent 90 DMSO
• Flow rate 0.5 ml/min
• Pressure lt3,000 psi
• Column temperature 50oC
• Standard maltoheptaose, pullulan6000 and
  pullulan12000 (MW 1,170, 5,900, 11,800,
  respectively)

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Results Discussion
MW 1,170, 17.845 min
MW 11,800, 12.639 min
MW 5,900, 13.727 min
Figure 7 High-performance size exclusion
chromatography of maltoheptaose, pullulan6000 and
pullulan12000.
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Results Discussion
Pullulan12000
Pullulan6000
Maltoheptaose
Log MW -0.1867(Retention time min) 6.3882 R2
0.9906
Figure 8 Standard curve for Zorbax PSM60S (? 2).
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Results Discussion
Normal rice
Waxy rice
Waxy corn
Normal corn
Waxy potato
Normal potato
Figure 9 High-performance size exclusion
chromatography of isoamylolyzate of amylopectin
from starches.
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Results Discussion

• Yuan et al. (1993)
• Refractive index response is proportional to the
  mass of the eluted material.
• The relative mole was derived by dividing the
  relative mass (RI response) by the corresponding
  molecular weight.

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Results Discussion
Waxy rice
Normal rice
Normal corn
Waxy corn
Normal potato
Waxy potato
Figure 10 High-performance size exclusion
chromatography of isoamylolyzate of amylopectin
from starches.
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High performance anion-exchange chromatography
with pulsed amperometric detection (HPAEC-PAD)
System Model 4000i Dionex BioLC system Column
Dionex HPIC-AS6 (now called CarboPac PA-1) 250 ?
4 mm (10 µm) with AG6 guard column (50 ? 4
mm) Detector Model 2 PAD system
Individual members of the components can be
obtained
Ref 1. Koizumi K. and Fukuda M., Estimation of
the distributions of chain length of amylopectins
by HPAEC-PAD, J. of Chromatography, 585 (1991),
233-238. 2. Hanashiro, I., Abe, J., Hizukuri,
S. (1996). A periodic distribution of the chain
length of amylopectin as revealed by
high-performance anion-exchange chromatography.
Carbohydrate Research, 283, 151-159.
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Cannot determine the individual glucans directly
by use of their peak areas in the chromatogram,
as the responses of a pulsed amperometric
detector to glucans having different DPs were
different.
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High performance size-exclusion chromatography
(HPSEC) with fluorescence detector
System HPLC Column For amylose TSK gel
G6000PW, G4000PW and G3000PW (7.5?75 mm)
(Tosoh Co., Tokyo, Japan), connected in series
TSK guard column PWH (7.5?75 mm) Temp. 37
?C, Eluent 0.1 M phosphate buffer (pH 6.1)
containing 0.02 sodium azide Detector
Fluorescence Detector Refractive index
detector Fluorescent reagent 2-aminopyridine
(aromatic primary amine) Std. amylose AS-110 (DP
521), AS-320 (2320), AS-1000 (4400)
Ref Hanashiro, I., Takeda, Y. (1998).
Examination of number-average degree of
polymerization and molar-based distribution of
amylose by fluorescent labeling with
2-aminopyridine. Carbohydrate Research, 306,
421-426.
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Chromatograms of Fluorescence-labeled Amyloses
DP
Fluorescence
RI
DPsample (RI/F)sample
x
DPstd.
(RI/F)std.
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Column for amylopectin (unit chain)
Sample Isoamylolyzate Column Shodex OHpak
SB-803HQ and SB-802.5HQ x 2 (8?300
mm) Eluent Aq. Me2SO (50) containing 50 mM
NaCl Column Temperature 50 ?C Std. amylose G6,
AS-10 (52), AS-30 (141), AS-70 (440)
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Beta-Amylolysis of Amylose Molecule
Linear molecule
Branch molecule
Reducing end
Glucose
alpha-1,4
alpha-1,6
-amylolysis
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Swelling of starch granule
Increase viscosity of starch paste
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Action of amylase on starch
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Phosphorus
1
4
2
3
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Sugar Phosphate
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