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Development of Functional Inclusion Complexes Using Cycloamylose and Their Physicochemical Characteristics : 환형아밀로스를 이용한 기능성 포접복합체 개발 및 물리화학적 특성 연구

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dc.contributor.advisor김용노-
dc.contributor.author노신정-
dc.date.accessioned2017-07-13T17:37:38Z-
dc.date.available2017-07-13T17:37:38Z-
dc.date.issued2014-02-
dc.identifier.other000000017518-
dc.identifier.urihttps://hdl.handle.net/10371/120978-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 협동과정 농업생물공학전공, 2014. 2. 김용노.-
dc.description.abstractInclusion complex formation with cycloamylose (CA) can exert a profound effect on the solubility and stability of highly insoluble and unstable guest molecules, since CA can take a hydrophobic cavity geometry as in the case of cyclodextrin (CD). Even though advantages of CA over CD have been reported such as high solubility and diverse cavity size, researches on CA in food and pharmaceutical fields are limited, probably because of its novelty. Therefore, in this study, the complex formation capability of CA with various guest molecules was investigated mainly in terms of its physicochemical characteristics to solubilize and stabilize those molecules.
CA with a range of DP from 23 to 45 was produced by treating amylose with Thermus acuaticus 4-α-glucanotransferase (TAαGTase). The inclusion complex formation capability of CA was investigated using model guest materials, such as iodine, sodium dodecyl sulfate (SDS), and lysolecithin (LL). It was revealed that those model guests were inserted in the cavity of CA as measured using isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). Various functional guest molecules, such as quercetin, polyphenol oxidase substrates (PPOS) and fat-soluble vitamins (FSV), were reacted with CA and two other commercially available complexation agents, cyclodextrin (CD) and maltodextrin (MD), respectively, using a freeze-drying (FD) method. The characterization of functional inclusion complexes was measured using various physicochemical analysis techniques. These results revealed that CA formed stable inclusion complexes with quercetin, PPOS, and FSV. The water solubility of quercetin and FSV significantly increased in the presence of CA. Especially, at high concentration, CA improved the solubility more than CD or MD. The oxidative stability of PPOS was significantly improved by complexation, decreasing the oxidative rate and the activity of polyphenol oxidase (PPO) that caused enzymatic browning. Also, the photo-stability of FSV was significantly improved by forming a complex with CA, showing a decrease in degradation constant (Kd) and an increase in half-life (t1/2), compared to those of pure FSV.
In order to investigate inclusion complexation performance of CA molecules depending on their size, twenty-three different degrees of polymerization (DPs) of CA from 23 to 45 were fractionated using a repeated prep-HPLC. The inclusion complex formation capability of each CA molecule with SDS was analyzed using ITC and NMR. The thermodynamic data obtained using ITC revealed that all the binding processes were enthalpy-driven and the binding stoichiometry (n) increased from about 2 to 3 as DP increased from 23 to 45. The strong binding of the SDS molecule to CA26 was observed, which might be attributed to structural stability of CA26 bound with SDS by forming stable double hydrophobic cavities. The ROESY spectrum of the CA26-SDS complexes confirmed that the alkyl chain of the SDS molecule was inserted into the cavity of CA26. The solubility of β-carotene (BC) was also enhanced in the presence of CA26. The characterization of inclusion complexes between CA26 and BC suggested that the stable inclusion complex was formed with the olefinic region of BC interacting with the hydrophobic cavity of CA26. The photo-stability and oxidative stability of BC were improved upon inclusion complexation with CA26, compared to that of pure BC. In conclusion, CA, as an efficient carrier molecule, formed stable inclusion complexes with various functional materials and greatly improved their solubility and stability, proposing a high potential of CA for various food and pharmaceutical industries.		-
dc.description.tableofcontentsABSTRACT i
TABLE OF CONTENTS iv
LIST OF TABLES x
LIST OF FIGURES xiii
NOMENCLATURE xvii
ABBREVIATIONS xviii

1. INTRODUCTION 1

2. BACKGROUND AND LITERATURE REVIEW 5

2.1. Cycloamylose (CA) 5
2.2. Enzymatic production of CA 6
2.3. Physicochemical properties of CA 9
2.4. Molecular structures of CA 12
2.5. Inclusion complex formation of CA with guest molecules 16
2.6. Applications of CA 20

3. ENZYMATIC PRODUCTION OF CA AND THEIR COMPLEX FORMATION CAPABILITY 22

3.1. INTRODUCTION 22
3.2. MATERIALS AND METHODS 25
3.2.1. Materials 25
3.2.2. Enzymatic production of CA 27
3.2.2.1. Purification of TAαGTase 27
3.2.2.2. Production of CA 28
3.2.3. Identification of CA 30
3.2.3.1. High-performance size exclusion chromatography (HPSEC) 30
3.2.3.2. High-performance anion exchange chromatography (HPAEC) 30
3.2.3.3. MALDI-TOF MS 31
3.2.4. Confirmation of Inclusion complex formation 32
3.2.4.1. Complex formation of CA with iodine 32
3.2.4.2. Complex formation of CA with SDS and lysolecithin 33
3.2.4.2.1. Isothermal titration calorimetry (ITC) 33
3.2.4.2.2. 1H NMR 35
3.2.4.2.3. 2D ROESY 36
3.2.5. Complex formation capability of CA with functional materials 37
3.2.5.1. Complex formation of CA with quercetin 37
3.2.5.1.1. Phase solubility studies 37
3.2.5.1.2. Preparation of inclusion complexes 39
3.2.5.1.3. Characterization of inclusion complexes 40
3.2.5.1.3.1. Powder X-ray diffractometry (XRD) 40
3.2.5.1.3.2. Differential scanning calorimetry (DSC) 41
3.2.5.2. Complex formation of CA with polyphenol oxidase substrate (PPOS) 41
3.2.5.2.1. Preparation of inclusion complexes 41
3.2.5.2.2. Characterization of inclusion complexes 41
3.2.5.2.2.1. Fluorescence measurements 42
3.2.5.2.2.2. ITC and 1H NMR 47
3.2.5.2.3. Stability of inclusion complexes 48
3.2.5.2.3.1. Oxidative stability during storage 49
3.2.5.2.3.2. Stability of PPOS against enzyme oxidation 51
3.2.5.2.3.3. Color-stability of apple juice 53
3.2.5.3. Complex formation of CA with fat-soluble vitamins (FSV) 55
3.2.5.3.1. Phase solubility studies 55
3.2.5.3.2. Preparation of inclusion complexes 57
3.2.5.3.3. Characterization of inclusion complexes 58
3.2.5.3.3.1. Fluorescence measurements 58
3.2.5.3.3.2. Fourier transform infrared (FT-IR) spectroscopy 58
3.2.5.3.3.3. 1H NMR and 2D ROESY 60
3.2.5.3.4. Photo-stability (irradiation tests) 61
3.2.6. Statistical analysis 63
3.3. RESULTS AND DISCUSSION 64
3.3.1. Enzymatic production of CA 64
3.3.1.1. Purification of TAαGTase 64
3.3.1.2. Production of CA 64
3.3.2. Identification of CA 65
3.3.2.1. HPSEC 65
3.3.2.2. HPAEC 66
3.3.2.3. MALDI-TOF MS 67
3.3.3. Confirmation of Inclusion complex formation 70
3.3.3.1. Complex formation of CA with iodine 70
3.3.3.2. Complex formation of CA with SDS 72
3.3.3.2.1. ITC 72
3.3.3.2.2. 1H NMR 78
3.3.3.2.3. 2D ROESY 84
3.3.3.3. Complex formation of CA with lysolecithin 89
3.3.3.3.1. ITC 89
3.3.3.3.2. 1H NMR 91
3.3.3.3.3. 2D ROESY 94
3.3.4. Complex formation capability of CA with functional materials 96
3.3.4.1. Complex formation of CA with quercetin 97
3.3.4.1.1. Phase solubility studies 97
3.3.4.1.2. Formation of inclusion complexes 100
3.3.4.1.3. Characterization of inclusion complexes 100
3.3.4.1.3.1. XRD 100
3.3.4.1.3.2. DSC 102
3.3.4.2. Complex formation of CA with polyphenol oxidase substrate (PPOS) 106
3.3.4.2.1. Characterization of inclusion complexes 106
3.3.4.2.1.1. Fluorescence measurements 106
3.3.4.2.1.2. ITC 114
3.3.4.2.1.3. 1H NMR 118
3.3.4.2.1.4. 2D ROESY 123
3.3.4.2.2. Stability of inclusion complexes 125
3.3.4.2.2.1. Oxidative stability during storage 125
3.3.4.2.2.2. Stability of PPOS against enzyme oxidation 134
3.3.4.2.2.3. Color-stability of apple juice 141
3.3.4.3. Complex formation of CA with fat-soluble vitamins (FSV) 147
3.3.4.3.1. Phase solubility studies 147
3.3.4.3.2. Formation of inclusion complexes 152
3.3.4.3.3. Characterization of inclusion complexes 153
3.3.4.3.3.1. Fluorescence measurements 153
3.3.4.3.3.2. XRD 157
3.3.4.3.3.3. DSC 158
3.3.4.3.3.4. FT-IR 165
3.3.4.3.3.5. 1H NMR 173
3.3.4.3.3.6. 2D ROESY 179
3.3.4.3.4. Photo-stability 183
3.4. CONCLUSIONS 199

4. COMPLEX FORMATION CAPABILITY OF FRACTIONATED CA OF DIFFERENT DP 202

4.1. INTRODUCTION 202
4.2. MATERIALS AND METHODS 205
4.2.1. Materials 205
4.2.2. Fractionation of CA23-CA45 205
4.2.3. Identification of CA23-CA45 206
4.2.4. Confirmation of inclusion complexes of CA23-CA45 with SDS 208
4.2.5. Complex formation capability of CA26 with β-carotene 208
4.2.5.1. Phase solubility studies 208
4.2.5.2. Preparation of inclusion complexes 208
4.2.5.3. Characterization of inclusion complexes 209
4.2.5.3.1. XRD, DSC, FT-IR, and NMR 209
4.2.5.3.2. Focused ion beam-scanning electron microscopy (FIB-SEM) 209
4.2.5.4. Stability of inclusion complexes 210
4.2.5.4.1. Photo-stability of inclusion complexes 210
4.2.5.4.2. Oxidative stability of inclusion complexes 210
4.2.6. Statistical analysis 211
4.3. RESULTS AND DISCUSSION 212
4.3.1. Fractionation of CA23-CA45 212
4.3.1.1. Optimization of fractionation procedure 212
4.3.1.2. Fractionation of CA23-CA45 214
4.3.2. Identification of CA23-CA45 217
4.3.2.1. HPAEC 217
4.3.2.2. NMR 217
4.3.2.3. MALDI-TOF MS 219
4.3.3. Confirmation of inclusion complexes of CA23-CA45 with SDS 221
4.3.3.1. ITC 221
4.3.3.2. 1H NMR 228
4.3.3.3. 2D ROESY 231
4.3.4. Complex formation capability of CA26 with β-carotene 233
4.3.4.1. Phase solubility studies 233
4.3.4.2. Characterization of inclusion complexes 236
4.3.4.2.1. XRD 236
4.3.4.2.2. DSC 238
4.3.4.2.3. FT-IR 241
4.3.4.2.4. FIB-SEM 245
4.3.4.2.5. 1H NMR 248
4.3.4.2.6. 2D ROESY 251
4.3.5. Stability of inclusion complexes 253
4.3.5.1. Photo-stability of inclusion complexes 253
4.3.5.2. Oxidative stability of inclusion complexes 256
4.4. CONCLUSIONS 261

5. RECOMMENDATIONS FOR FUTURE WORK 263

6. LIST OF REFERENCES 265

국문 초록 282		-
dc.formatapplication/pdf-
dc.format.extent9977585 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectcycloamylose-
dc.subjectinclusion complex-
dc.subject4-α-glucanotransferase-
dc.subjectpolyphenol oxidase substrates-
dc.subjectfat-soluble vitamins-
dc.subject.ddc660-
dc.titleDevelopment of Functional Inclusion Complexes Using Cycloamylose and Their Physicochemical Characteristics-
dc.title.alternative환형아밀로스를 이용한 기능성 포접복합체 개발 및 물리화학적 특성 연구-
dc.typeThesis-
dc.contributor.AlternativeAuthorShin-Joung Rho-
dc.description.degreeDoctor-
dc.citation.pagesxix, 284-
dc.contributor.affiliation농업생명과학대학 협동과정 농업생물공학전공-
dc.date.awarded2014-02-
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