花芸豆淀粉酶抑制剂（α-AI1）的分离纯化及相关性质的研究
发布时间：2020-12-22 13:37
芸豆（Phaseolus vulgaris）淀粉酶抑制剂亚型1（α-AI1）可被广泛用来辅助治疗肥胖症与糖尿病。通过淀粉酶抑制剂合适的结构、物理化学性质、功能性可影响腔内边缘淀粉酶的活力。最终结果是降低餐后血高血糖与胰岛素水平,增加淀粉消化的抗性和肠内微生物的活力。然而,淀粉酶抑制剂提取物的疗效及安全性取决于加工及提取工艺。抑制剂可被添加到食品中,用来增加糖尿病人的碳水化合物耐受量,降低摄取的能量,从而避免出现肥胖症和提高抗性淀粉的含量。一般的粗提工艺包括水提取,pH分离和乙醇（75%）沉淀,它可用来筛选抑制剂提取合适的品种。几种芸豆中,红芸豆中淀粉酶抑制剂的活力最高,黑芸豆的则最小。随后的粗提取物的SDS-PAGE电泳图显示蛋白质组分的数量减少了,只有两条主要带。1.4-2.6克红芸豆粗提物的特异性血凝活性平均为14222.2U/mg。进一步采用DEAE-Sepharose CL-6B和Sephacryl S-200层析柱分离纯化。在0.09%原芸豆粉（w/w）的基础上通过电泳纯化得到的淀粉酶抑制剂检测不到血凝活性,同时,比酶活由0.32增加到80.78 U/mg。Native-PA...
【文章来源】：江南大学江苏省 211工程院校 教育部直属院校
【文章页数】：82 页
【学位级别】：硕士
【文章目录】：
ACKNOWLEDGEMENTS
DEDICATION
ABSTRACT
摘要
TABLE OF CONTENTS
ABBREVIATIONS USED
CHAPTER 1:INTRODUCTION & LITERATURE REVIEW
1.1 Introduction:Kidney Bean Starch Blockers
1.2 Literature Review
1.2.1 Favorable Physico-chemical Properties of the Phaseolus vulgaris α-amylase inhibitor
1.2.1.1 Structural properties of the Phaseolus vulgaris α-amylase Inhibitor
1.2.1.2 Factors that affect the Phaseolus vulgaris α-amylase inhibitor Activity
1.2.2 The Starch Blocking Mechanism of the Phaseolus vulgaris Alpha-amylase Inhibitor
1.2.3 Efficiency of α-AI1 Extracts in Reducing Activity of Amylases in Man
1.2.4 The Beneficial Effects of the Phaseolus vulgaris α-amylase Inhibitor
1.2.4.1 Decreased obesity due to Phaseolus vulgaris α-amylase extracts
1.2.4.2 The anorexigenic effect of Phaseolus vulgaris α-AI1 extracts
1.2.4.3 Reduced postprandial plasma hyperglycemia and insulin due to α-AI1 extracts
1.2.5 Safety and Toxicity of the Phaseolus vulgaris α-amylase Inhibitor Extracts
1.2.5.1 Toxic effects Associated with kidney beans（Phaseolus vulgaris）
1.2.5.2 Acute toxicity studies
1.2.5.3 Chronic toxicity studies
1.2.5.4 Prospective research on beneficial effects:the potential of α-amylase inhibitor isoform 1 extracts against colorectal cancer
1.3 Justification and Objectives of Study
1.3.1 Justification
1.3.2 Objectives of study
1.3.3 Hypothesis of Study
CHAPTER 2:VARIETY SELECTION,PURIFICATION AND PARTIAL CHARACTERISATION OF PHASEOLUS VULGARIS ALPHA-AMYLASE INHIBITOR（α-AI1）
2.1 Introduction
2.2 Materials
2.3 Methods
2.3.1 Determination of Protein Content
2.3.2 Stabilizing α-amylase activity of Porcine Pancreatic α-amylase
2.3.3 Porcine Pancreatic α-amylase and Inhibitor Activity Measurement
2.3.4 Crude Extraction of the Phaseolus vulgaris α-amylase inhibitor（α-AI1） and Variety Selection
2.3.5 Chromatographic Purification Phaseolus vulgaris α-amylase inhibitor（α-AI1） from Speckled Kidney BeanVariety
2.3.6 Purity Assessment
2.3.6.1 SDS page analysis
2.3.6.2 Native page of purified inhibitor
2.3.6.3 Hemagglutinating activity
2.3.7 Partial Characterization of Purified Speckled Kidney bean（Phaseolus vulgaris） α-amylase Inhibitor（α-AI1）
2.3.8 Statistical Analysis
2.4 Results and Discussion
2.4.1 Stabilizing of Porcine Pancreatic α-amylase Catalytic Activity
2.4.2 Variety Selection through Crude/Partial Extraction from Different Varieties
2.4.3 Chromatographic Purification of Phaseolus vulgaris α-amylase inhibitor（α-AI1） from Speckled kidney Beans
2.4.4 Partial Characterization of the Purified Inhibitor from Speckled Kidney Beans
2.4.4.1 Effect of inhibitor concentration on activity of speckled kidney bean（Phaseolus vulgaris） α-amylase inhibitor（α-AI1） activity:Determination of IC50 value
2.4.4.2 Effect of pH on the activity of the Phaseolus vulgaris α-amylase inhibitor from speckled kidney beans
2.4.4.3 Effect of Incubation time on the activity of the Phaseolus vulgaris α-amylase inhibitor from speckled kidney beans
2.5 Conclusions
CHAPTER 3:HEAT STABILITY OF THE PHASEOLUS VULGARIS ALPHA AMYLASE（α-AI1） INHIBITOR FROM RED SPECKLED KIDNEY BEANS
3.1 Introduction
3.2 Materials
3.3 Methods
3.3.1 Purification the Phaseolus vulgaris α-amylase Inhibitor（αAI1）
3.3.2 Purity Assessment
3.3.2.1 SDS-PAGE analysis
3.3.2.2 Native-PAGE of purified inhibitor
3.3.2.3 Hemagglutinating activity
3.3.3 Determination of Protein Content
3.3.4 PPA and Inhibitor Activity Assessment
3.3.5 DSC Calorimetry Measurements
3.3.6 Heat Stability Treatments
3.3.6.1 Treatments for Response Surface Methodology experiments
3.3.6.2 Heat treatment for effects of hofmeister series salts and polyols intrinsic fluorescence and ultraviolet absorption spectra
3.3.7 Intrinsic Fluorescence and Ultraviolet Absorption Spectra
3.3.8 Surface Hydrophobicity Measurements
3.3.9 Experimental Design and Statistical Analysis
3.3.9.1 Response Surface methodology design
3.3.9.2 Statistical analysis
3.4 Results and Discussions
3.4.1 Model Selection and Residual α-amylase Inhibitory Activity Variation
3.4.2 DSC Analysis:Phaseolus vulgaris α-amylase Inhibitor（α-AI1）
3.4.3 Molecular Changes Involved in Activity Loss
3.4.4 Effect of Selected Hofmeister Series Salts and Polyols
3.5 Conclusion
CHAPTER 4:GENERAL CONCLUSIONS,RECOMMENDATIONS AND REFERENCES
4.1 General Conclusions
4.2 Recommendations
4.3 Published Papers
4.4 Appendix
4.5 List of Figures and Tables
4.6 References
本文编号：2931855
【文章来源】：江南大学江苏省 211工程院校 教育部直属院校
【文章页数】：82 页
【学位级别】：硕士
【文章目录】：
ACKNOWLEDGEMENTS
DEDICATION
ABSTRACT
摘要
TABLE OF CONTENTS
ABBREVIATIONS USED
CHAPTER 1:INTRODUCTION & LITERATURE REVIEW
1.1 Introduction:Kidney Bean Starch Blockers
1.2 Literature Review
1.2.1 Favorable Physico-chemical Properties of the Phaseolus vulgaris α-amylase inhibitor
1.2.1.1 Structural properties of the Phaseolus vulgaris α-amylase Inhibitor
1.2.1.2 Factors that affect the Phaseolus vulgaris α-amylase inhibitor Activity
1.2.2 The Starch Blocking Mechanism of the Phaseolus vulgaris Alpha-amylase Inhibitor
1.2.3 Efficiency of α-AI1 Extracts in Reducing Activity of Amylases in Man
1.2.4 The Beneficial Effects of the Phaseolus vulgaris α-amylase Inhibitor
1.2.4.1 Decreased obesity due to Phaseolus vulgaris α-amylase extracts
1.2.4.2 The anorexigenic effect of Phaseolus vulgaris α-AI1 extracts
1.2.4.3 Reduced postprandial plasma hyperglycemia and insulin due to α-AI1 extracts
1.2.5 Safety and Toxicity of the Phaseolus vulgaris α-amylase Inhibitor Extracts
1.2.5.1 Toxic effects Associated with kidney beans（Phaseolus vulgaris）
1.2.5.2 Acute toxicity studies
1.2.5.3 Chronic toxicity studies
1.2.5.4 Prospective research on beneficial effects:the potential of α-amylase inhibitor isoform 1 extracts against colorectal cancer
1.3 Justification and Objectives of Study
1.3.1 Justification
1.3.2 Objectives of study
1.3.3 Hypothesis of Study
CHAPTER 2:VARIETY SELECTION,PURIFICATION AND PARTIAL CHARACTERISATION OF PHASEOLUS VULGARIS ALPHA-AMYLASE INHIBITOR（α-AI1）
2.1 Introduction
2.2 Materials
2.3 Methods
2.3.1 Determination of Protein Content
2.3.2 Stabilizing α-amylase activity of Porcine Pancreatic α-amylase
2.3.3 Porcine Pancreatic α-amylase and Inhibitor Activity Measurement
2.3.4 Crude Extraction of the Phaseolus vulgaris α-amylase inhibitor（α-AI1） and Variety Selection
2.3.5 Chromatographic Purification Phaseolus vulgaris α-amylase inhibitor（α-AI1） from Speckled Kidney BeanVariety
2.3.6 Purity Assessment
2.3.6.1 SDS page analysis
2.3.6.2 Native page of purified inhibitor
2.3.6.3 Hemagglutinating activity
2.3.7 Partial Characterization of Purified Speckled Kidney bean（Phaseolus vulgaris） α-amylase Inhibitor（α-AI1）
2.3.8 Statistical Analysis
2.4 Results and Discussion
2.4.1 Stabilizing of Porcine Pancreatic α-amylase Catalytic Activity
2.4.2 Variety Selection through Crude/Partial Extraction from Different Varieties
2.4.3 Chromatographic Purification of Phaseolus vulgaris α-amylase inhibitor（α-AI1） from Speckled kidney Beans
2.4.4 Partial Characterization of the Purified Inhibitor from Speckled Kidney Beans
2.4.4.1 Effect of inhibitor concentration on activity of speckled kidney bean（Phaseolus vulgaris） α-amylase inhibitor（α-AI1） activity:Determination of IC50 value
2.4.4.2 Effect of pH on the activity of the Phaseolus vulgaris α-amylase inhibitor from speckled kidney beans
2.4.4.3 Effect of Incubation time on the activity of the Phaseolus vulgaris α-amylase inhibitor from speckled kidney beans
2.5 Conclusions
CHAPTER 3:HEAT STABILITY OF THE PHASEOLUS VULGARIS ALPHA AMYLASE（α-AI1） INHIBITOR FROM RED SPECKLED KIDNEY BEANS
3.1 Introduction
3.2 Materials
3.3 Methods
3.3.1 Purification the Phaseolus vulgaris α-amylase Inhibitor（αAI1）
3.3.2 Purity Assessment
3.3.2.1 SDS-PAGE analysis
3.3.2.2 Native-PAGE of purified inhibitor
3.3.2.3 Hemagglutinating activity
3.3.3 Determination of Protein Content
3.3.4 PPA and Inhibitor Activity Assessment
3.3.5 DSC Calorimetry Measurements
3.3.6 Heat Stability Treatments
3.3.6.1 Treatments for Response Surface Methodology experiments
3.3.6.2 Heat treatment for effects of hofmeister series salts and polyols intrinsic fluorescence and ultraviolet absorption spectra
3.3.7 Intrinsic Fluorescence and Ultraviolet Absorption Spectra
3.3.8 Surface Hydrophobicity Measurements
3.3.9 Experimental Design and Statistical Analysis
3.3.9.1 Response Surface methodology design
3.3.9.2 Statistical analysis
3.4 Results and Discussions
3.4.1 Model Selection and Residual α-amylase Inhibitory Activity Variation
3.4.2 DSC Analysis:Phaseolus vulgaris α-amylase Inhibitor（α-AI1）
3.4.3 Molecular Changes Involved in Activity Loss
3.4.4 Effect of Selected Hofmeister Series Salts and Polyols
3.5 Conclusion
CHAPTER 4:GENERAL CONCLUSIONS,RECOMMENDATIONS AND REFERENCES
4.1 General Conclusions
4.2 Recommendations
4.3 Published Papers
4.4 Appendix
4.5 List of Figures and Tables
4.6 References
本文编号：2931855
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