1. Analysis of molecular structure of starch

2. Physicochemical properties/ Chemical composition Molecular structure Biosynthesis (enzymes) Genes

3. Molecular Structure of Amylose

4. Molecular Structure of Amylopectin

5. Average chain-length and amount (mole %) of the fractions of amylopectin unit chain

6. Molecular characterization Amylose Number average degree of polymerization (molecular size, DP) Average chain length (CL) Average number of chain (NC) Linear amylose fraction (mole%) Branched amylose fraction (mole%)

7. Molecular characterization Amylopectin Average (branch) chain length, overall Unit chain (A, B1, B2, B3,..) fraction (mole%) Average (branch) chain length, of A-chain Average (branch) chain length, of B1-chain Average (branch) chain length, of B2-chain

8. Methods of Analysis  Colorimetric methods  chemical reaction  chemical reaction + enzyme reaction  Chromatographic Techniques  without enzyme reaction  with enzyme reaction Detector Low-angle laser-light-scattering photometer Refractive index detector Pulsed amperometric detector Fluorescence detector

9. Colorimetric methods (chemical reaction) Determine: Total sugar/ Reducing end/Non-reducing end Average degree of polymerization = total sugar (molecular size, DP)reducing end sugar Average chain length (CL) = total sugar non-reducing end sugar Average number of chain (NC) = DP/CL

10. Total sugar: Anthrone-H 2 SO 4 Phenol-H 2 SO 4 Reducing end sugar Modified Park-Johnson’s method Ref; 1. J. Park and M.J. Johnson, J. Biol. Chem., 181 (1949), 149-151. 2. S. Hizukuri, Y.Takeda, M. Yasuda, Carbohydrate Research, 94 (1981), 205-213. 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.

11. Colorimetric methods (chemical reaction + enzyme reaction) Branch chain length (CL) = total sugar non-reducing end sugar Isoamylase/pullulanase Hydrolyze  -1,6 by isoamylase/pullulanase Determine reducing end sugar by Modified Park & Johnson’s method

12. Experimental Procedure Amylopectin structure studied by HPSEC Fractionation (selective precipitation) Starch Amylose Debranched Amylopectin Molecular analyses (HPSEC) Chromatographic Techniques with Enzyme Reaction

13. C B A B A A B Isoamylase or pullulanase. A

14. Chromatographic Techniques with Enzyme Reaction Figure 6Block diagram showing the component of an HPSEC instrument. Mobile Phase Solvent Delivery System Injector |S| |M| |L| Detectors Recorder Injection of debranched amylopectin Column Chart record Retention time Response L MS

15. Experimental Procedure Amylopectin structure studied by HPSEC  Column:Zorbax PSM 60S (  2)  MW range:5  10 2 – 10 4  Column dimension:6.2 mm ID  250 mm  Loading size:40 μ l  Eluent:90% DMSO  Flow rate:0.5 ml/min  Pressure: <3,000 psi  Column temperature:50 o C  Standard: maltoheptaose, pullulan6000 and pullulan12000 (MW 1,170, 5,900, 11,800, respectively)

16. Results & Discussion Figure 7High-performance size exclusion chromatography of maltoheptaose, pullulan6000 and pullulan12000. MW 1,170, 17.845 min MW 11,800, 12.639 min MW 5,900, 13.727 min

17. Results & Discussion Figure 8Standard curve for Zorbax PSM60S (  2). Log MW = -0.1867(Retention time; min) + 6.3882 R 2 = 0.9906 Maltoheptaose Pullulan12000 Pullulan6000

18. Results & Discussion Figure 9High-performance size exclusion chromatography of isoamylolyzate of amylopectin from starches. Normal riceWaxy rice Waxy potatoNormal potato Waxy corn Normal corn

19. 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.

20. Results & Discussion Figure 10High-performance size exclusion chromatography of isoamylolyzate of amylopectin from starches. Normal rice Normal corn Waxy rice Waxy potato Normal potato Waxy corn

21. 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. 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

23. 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.

24. High performance size-exclusion chromatography (HPSEC) with fluorescence detector 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. 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)

27. DPn = RI response (RI) fluorescence response (F)

28. Chromatograms of Fluorescence-labeled Amyloses Fluorescence RI DP DP sample = (RI/F)sample (RI/F)std. x DP std.

29. Column for amylopectin (unit chain) Sample: Isoamylolyzate Column: Shodex OHpak SB-803HQ and SB-802.5HQ x 2 (8  300 mm) Eluent: Aq. Me 2 SO (50%) containing 50 mM NaCl Column Temperature: 50  C Std. amylose: G6, AS-10 (52), AS-30 (141), AS-70 (440)

33. Beta-Amylolysis of Amylose Molecule Linear molecule Branch molecule Reducing end Glucose alpha-1,4 alpha-1,6 -amylolysis

34. Swelling of starch granule Increase viscosity of starch paste

35. Action of amylase on starch

36. Phosphorus 1 2 3 4 5 6

37. Sugar Phosphate