Newly synthesized α-1,3 linkages on starch molecules by 4,3-α-glucanotransferase contribute to enhancing the slowly digestible properties of various banana flours

다국어 초록 (Multilingual Abstract)

Bananas are representative plant-based raw materials containing a large amount of starch, and green banana flour prepared from unripe bananas has attracted attention as a starchy ingredient that can reduce the loss of banana resources that are likely to be discarded during distribution and processing, while also being applicable to various food. In particular, starch in banana flour has a structural characteristic of containing a relatively high proportion of long B3-chains compared with other plant- based starches, and this chain structure may provide suitable substrate properties for efficient chain-transfer reactions by glucanotransferase. Therefore, in this study, the starch structure of banana flours was modified using 4,3-α-glucanotransferase derived from Lactobacillus fermentum NCC 2970, and the effect of α-1,3 linkage introduction on its digestive properties was investigated. 4,3-α-Glucanotransferase treatment effectively rearranged the molecular structure of starch in banana flour and newly synthesized α-1,3 linkages at a level of 4.83-5.50%, which were not detected before enzymatic treatment. This change in linkage structure significantly affected the starch digestibility of banana flour. After treatment with human pancreatic α-amylase, the proportion of α-limit dextrins resistant to α-amylase increased in the enzymatically modified samples, and these structures were considered to contribute to delayed glucose generation at the small intestinal mucosal α-glucosidase level. Indeed, digestion assays using rat intestinal α-glucosidase and recombinant human α-glucosidase showed that the enzymatically treated samples exhibited a more gradual glucose release pattern than the raw samples and rapidly digestible controls. These in vitro results were similarly confirmed in the in vivo postprandial glycemic response, showing properties that attenuated the initial glucose spike while continuously releasing glucose. These findings suggest that 4,3-α- glucanotransferase treatment can be used to develop slowly digestible starch materials capable of attenuating rapid postprandial blood glucose elevation. In addition, the absence of marked differences in structural changes and hydrolysis properties among banana varieties indicates that this enzymatic modification strategy may be broadly applicable as a digestion-modulating approach for banana flours, regardless of variety.

목차 (Table of Contents)

• Ⅰ. Introduction 13
• 1. Structural organization of starch granules 13
• 2. Enzymatic digestion of starch 17
• 3. Enzymatic modification for designing slowly digestible starch 21
• 4. Banana: production, diversity and nutritional significance 26
• 5. Green banana flour as a starch-based food ingredient 28
• 6. 4,3-α-Glucanotransferase 30
• 7. Research hypothesis & Objectives 33
• Ⅱ. Materials and methods 34
• 1. Materials 34
• 2. Methods 35
• 2.1 Production of 4,3-αGT from Lactobacillus fermentum NCC 2970 and measurement of enzyme activity 35
• 2.2 Enzymatic modification of banana flour using 4,3-αGT 36
• 2.3 Analysis of the molecular size distribution of 4,3-αGT treated banana flour using high-performance size exclusion chromatography (HPSEC) 37
• 2.4 Determination of chain length distribution of debranched starch using high performance anion-exchange chromatography (HPAEC) 38
• 2.5 Analysis of glycosidic linkage patterns using gas chromatography-mass spectrometry (GC-MS) 39
• 2.6 Determination of anomeric configuration and glycosidic linkage ratio in 4,3-αGT treated banana flour 40
• 2.7 Analysis of the molecular size distribution and content of human α-amylase (HPA) resistant α-limit dextrins 41
• 2.8 Total glucose content 42
• 2.9 In vitro digestibility of 4,3-αGT treated banana flour 43
• 2.10 Analysis of estimated glycemic index (eGI) using an in vitro digestion model 44
• 2.11 In vitro analysis of glucose generation characteristics using an intestinal mucosal α-glucosidase complex 45
• 2.11.1 In vitro analysis using rat intestinal α-glucosidase 45
• 2.11.2 In vitro analysis using recombinant human α-glucosidase 46
• 2.12 In vivo postprandial glycemic response assay 47
• 2.13 Swelling power and water solubility 48
• 2.14 Thermal stability of banana flour after 4,3-αGT treatment 49
• 2.15 Statistical analysis 49
• Ⅲ. Results and discussion 50
• 1. Changes of molecular size distribution of banana flours after 4,3-αGT treatment 50
• 2. Chain length distribution of α-1,6 debranched banana flours after 4,3-αGT treatment 53
• 3. Changes in FT-IR spectra and short-range molecular order of banana flours after 4,3-αGT treatment 56
• 4. Glycosidic linkage patterns in 4,3-αGT treated banana flours analyzed by GC-MS 60
• 5. 1H NMR analysis of glycosidic linkage changes in banana flours after 4,3-αGT treatment 63
• 6. In vitro starch digestibility of 4,3-αGT treated banana flours determined by the Englyst assay 66
• 7. Estimated glycemic index (eGI) of 4,3-αGT treated banana flour 70
• 8. Hydrolysis patterns of 4,3-αGT treated banana flours by human pancreatic α-amylase 73
• 9. In vitro assay of glucose generation property of 4,3-αGT treated banana flours by mammalian small intestinal mucosal α-glucosidase 77
• 10. In vitro assay of glucose generation property of 4,3-αGT treated banana flours by recombinant human α-glucosidase 81
• 11. In vivo postprandial glycemic response (mouse model) 85
• 12. Swelling power and water solubility of 4,3-αGT treated banana flours 88
• 13. Thermal stability of 4,3-αGT treated banana flours analyzed by TGA 91
• Ⅳ. Conclusion 94
• References 95