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ESTABLISHMENT of HIGH-THROUGHPUT TECHNIQUES for STUDYING STARCH FUNCTIONALITIES by Miguel Angel Alvarez Gonzales

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ESTABLISHMENT of HIGH-THROUGHPUT TECHNIQUES for STUDYING STARCH FUNCTIONALITIES by Miguel Angel Alvarez Gonzales ESTABLISHMENT OF HIGH-THROUGHPUT TECHNIQUES FOR STUDYING STARCH FUNCTIONALITIES by Miguel Angel Alvarez Gonzales A Thesis Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Master of Science Department of Food Science West Lafayette, Indiana August 2019 2 THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Yuan Yao, Chair Department of Food Science Dr. Bruce Hamaker Department of Food Science Dr. Clifford Weil Department of Agronomy Approved by: Dr. Arun K. Bhunia Head of the Graduate Program 3 Dedicated to my family 4 TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................... 8 LIST OF FIGURES ........................................................................................................................ 9 LIST OF ABREVIATIONS ......................................................................................................... 12 ABSTRACT .................................................................................................................................. 14 CHAPTER 1. LITERATURE REVIEW ................................................................................... 16 1.1 Introduction ....................................................................................................................... 16 1.2 Clean label movement ....................................................................................................... 17 1.3 Genetic modification of corn ............................................................................................ 17 1.3.1 Increasing genetic variation of corn .......................................................................... 18 1.3.2 Single corn mutant effects ......................................................................................... 18 1.4 Starch and starch composition .......................................................................................... 19 1.4.1 Starch granule architecture ........................................................................................ 19 1.5 Starch properties in water: GelatiniZation, pasting, and retrogradation. ........................... 20 1.5.1 GelatiniZation ............................................................................................................. 20 1.5.2 Pasting ........................................................................................................................ 21 1.5.3 Shear resistance ......................................................................................................... 21 1.5.4 Retrogradation ........................................................................................................... 21 1.6 Enhancement of starch properties ..................................................................................... 22 1.6.1 Cross-linking of starch ............................................................................................... 23 1.6.2 StabiliZation of starch ................................................................................................ 23 1.6.3 Increasing genetic variability for the modification of starch properties .................... 24 1.7 Conventional methods for studying starch properties. ..................................................... 25 1.7.1 Conventional methods for studying gelatiniZation. ................................................... 25 1.7.2 Conventional methods for studying paste properties and shear resistance. ............... 25 1.7.3 Conventional methods for studying retrogradation. .................................................. 26 1.8 Starch properties studied using high-throughput platform ............................................... 27 1.9 Single kernel sampling ...................................................................................................... 28 1.9.1 Mechanical approach for individual kernel sampling ............................................... 29 1.9.2 New mechanical approaches for single kernel sampling ........................................... 29 5 1.10 Molecular rotors ............................................................................................................. 30 1.10.1 Mode of action ........................................................................................................ 30 1.10.2 Application of molecular rotors in biological samples ........................................... 31 1.10.3 Potential application of molecular rotors in food science ....................................... 31 1.11 Experimental aims .......................................................................................................... 32 CHAPTER 2. MILLIGRAM CRUDE STARCH ISOLATION FROM INDIVUDUAL CORN KERNELS ............................................................................................................................ 34 2.1 Introduction ....................................................................................................................... 34 2.2 Materials and methods ...................................................................................................... 35 2.2.1 Chemicals and Materials ............................................................................................ 35 2.2.2 Single Kernel Sampling ............................................................................................. 36 2.2.2.1 Extraction of endosperm sample by mechanical approach: trephine bur and carbon steel blade ............................................................................................................................ 36 2.2.3 Crude starch isolation from endosperm sample. ........................................................ 37 2.2.3.1 Steeping method ................................................................................................... 37 2.2.3.2 Sonication and protease digestion method ........................................................... 37 2.2.4 Particle siZe ................................................................................................................ 38 2.2.5 Microscope images .................................................................................................... 38 2.2.6 Germination test. ........................................................................................................ 38 2.2.7 Statistical Analysis ..................................................................................................... 38 2.3 Results and discussion ...................................................................................................... 39 2.3.1 Evaluation of mechanical approach as a method for single kernel sampling ............ 39 2.3.2 Vitality of kernel after SKS ....................................................................................... 40 2.3.3 Comparison of crude starch yield by two isolation methods. .................................... 40 2.3.4 Particle siZe distribution ............................................................................................ 42 2.3.5 Microscope images of corn starch granules ............................................................... 43 2.4 Conclusions ....................................................................................................................... 43 CHAPTER 3. STARCH RETROGRADATION AT MILLIGRAM LEVEL OF COMMERCIALLY AVAILABLE CORN STARCH ................................................................. 45 3.1 Introduction ....................................................................................................................... 45 3.2 Materials and methods ...................................................................................................... 47 6 3.2.1 Chemicals and materials ............................................................................................ 47 3.2.2 Preparation of chemically modified normal corn starch and chemically modified waxy corn starch .............................................................................................................................. 47 3.2.3 Analysis of hydroxypropylated starch ....................................................................... 47 3.2.4 Preparation of starch paste using standard methods .................................................. 48 3.2.5 Preparation of starch pastes at milligram level .......................................................... 48 3.2.6 Rheological measurements ........................................................................................ 49 3.2.7 Turbidity measurements ............................................................................................ 49 3.2.8 Fluorescence intensity profiles of low-concentration starch pastes at milligram level by molecular rotor. ................................................................................................................. 50 3.2.8.1 Preparation of CCVJ solutions ............................................................................. 50 3.2.8.2 Fluorescence intensity measurement .................................................................... 50 3.2.9 Accelerated retrogradation by freeZe-thaw cycling, temperature cycling and isothermal treatment. ............................................................................................................. 50 3.2.10 Statistical Analysis .................................................................................................
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