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Application of Thermostable Α-Amylase From APPLICATION OF THERMOSTABLE α-AMYLASE FROM Thermomyces lanuginosus ATCC 58157 TO NUTRITIONALLY ENHANCE STARCH BASED FOOD By THIRILOSHANI PADAYACHEE Submitted in partial fulfillment for the Degree of Doctorate of Technology (Biotechnology) Department of Biotechnology Faculty of Engineering, Science and the Built Environment Durban University of Technology Supervisors: Prof. S. Singh (Ph.D) Dr. K. Permaul (Ph.D) DECLARATION I hereby declare that this dissertation is my own, unaided work. It is being submitted for the degree, Doctorate of Technology, to the Durban University of Technology, Durban, South Africa. It has not been submitted before for any degree or dissertation to any institution. ________________________________ Thiriloshani Padayachee Durban March 2006 DEDICATION This dissertation is dedicated to my husband Kamalasen and children Kamesh, Levard and Sayuri, I thank you for your love, compassion, patience, fortitude, understanding, forbearance and unwavering loyalty and support. You surpass all expectations of husband and children. Of all my blessings you are the most valued and the greatest. Great works are performed, not by strength, but by perseverance. ~Samuel Johnson~ TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS……………………………………………………………...i LIST OF FIGURES…………………………………………………………….……….iii LIST OF TABLES……………………………………………………………………….x CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW…………...1 1.1 Introduction............................................................................................................1 1.1.1 An African perspective of food and nutrition……………………………………..3 1.1.2 Starch based foods………………………………………………………………...6 1.1.3 History of enzymes and food processing………………………………………….7 1.2 Amylases……………………………………………………………….................9 1.2.1 α-Amylase………………………………………………………………………..10 1.2.2 Glucoamylase…………………………………………………………………….16 1.2.3 Pullulanase…………………………………………………………………...…..19 1.3 Production of amylases by solid state fermentation and submerged fermentation…………………………………………………………………….22 1.3.1 Solid state fermentation …………………………………………………………23 1.3.2 Submerged fermentation ………………………………………………………...25 1.3.3 Purification and characterization of amylase…………………………………….28 1.4 Thermostable enzymes ………………………………………………………...32 1.4.1 The evolution of thermostability ………………………………………………...32 1.4.2 Stability of thermozymes………………………………………………………...34 1.5 Industrial enzyme applications ………………………………………………..36 1.5.1 The detergent industry…………………………………………………………...38 1.5.2 Starch liquefaction and saccharification…………………………………………39 1.5.3 Textile applications………………………………………………………………41 1.5.4 Paper industry……………………………………………………………………42 1.5.5 Enzymes for the food industry…………………………………………………...43 1.5.5.1 Baking industry…………………………………………………………………..43 1.5.6 Novel food applications………………………………………………………….45 1.5.7 Analysis in medical and clinical chemistry……………………….......................46 1.6 Animal studies ………………………………………………………………….47 1.6.1 Use of animal models to study dietary effects on human health……………..….47 1.7 Scope of this study……………………………………………………………….50 1.8 Objectives of this study…………………………………………………………..51 1.9 References ……………………………………………………………………….52 CHAPTER TWO: SCREENING OF Thermomyces lanuginosus STRAINS FOR α-AMYLASE ACTIVITY…………...89 2.1 Introduction……………………………………………………………………..89 2.2 Materials and methods………………………………………………………....92 2.2.1 Thermomyces lanuginosus strains…………………………………………….….92 2.2.2 Maintenance of stock cultures………………………………………………...…93 2.2.3 Primary screening of T. lanuginosus strains for amylase activity……………….93 2.2.4 Secondary screening of T. lanuginosus strains for amylase activity…………….93 2.2.4.1 Submerged fermentation…………………………………………………………94 2.2.4.2 Solid state fermentation………………………………………………………….94 2.2.5 Enzyme assay…………………………………………………………………….95 2.3 Results…………………………..……………………………………………….97 2.3.1 Primary screening for α-amylase activity………………………………………..97 2.3.2 Secondary screening……………………………………………………………102 2.4 Discussion……………………………………………………………………...104 2.5 References……………………………………………………………………...107 CHAPTER THREE: PRODUCTION OF AMYLASE BY Thermomyces lanuginosusATCC 58157 USING SOLID STATE FERMENTATION……………….……………….......112 3.1 Introduction………………………………………………………………….. .112 3.2 Materials and methods………………………………………………………..116 3.2.1 Maintenance and growth of microorganism……………………………………116 3.2.2 Selection of solid substrate……………………………………………………..116 3.2.3 Optimisation of process parameters…………………………………………….117 3.2.4 Production of amylase under optimum conditions……………………………..118 3.2.5 Enzyme extraction and analytical methods……………………………………..118 3.3 Results………………………………………………….....................................120 3.3.1 Optimisation of process parameters…………………………………………….120 3.3.2 Production of amylase under optimized conditions…………………………….125 3.4 Discussion……………………………………………………………………...127 3.5 References...........................................................................................................133 CHAPTER FOUR: PURIFICATIONAND CHARACTERISATION OF α-AMYLASE FROM T. lanuginosus ATCC 58157…………………………………………...140 4.1 Introduction……………………………………………………………………140 4.2. Materials and methods………………………………………………………..143 4.2.1 α-Amylase production…………………………………………………………..143 4.2.2 α-Amylase activity……………………………………………………………..143 4.2.3 Purification of α-amylase……………………………………………………….143 4.2.4 SDS-PAGE……………………………………………………………………..144 4.2.5 Effect of pH and temperature on purified α-amylase activity …………………145 4.2.6 Effect of metal ions and chemical compounds on purified α-amylase activity…………………………………………………………………………..145 4.2.7 Hydrolysis of soluble starch with purified α-amylase………………………….145 4.2.8 Iodine method for residual starch estimation…………………………………...146 4.2.9 Effect of pH and temperature on purified α-amylase stability………………….146 4.3 Results…………………………………………………………………………….147 4.3.1 Purification of α-amylase………………………………………………………..147 4.3.2 Molecular weight of α-amylase from T. lanuginosus ATCC 58157………........147 4.3.3 Effect of pH and temperature on enzyme activities…………………………….151 4.3.4 Effect of metal ions and chemical compounds on α-amylase activity………….152 4.3.5 Effect of pH and temperature on purified α-amylase stability………………….152 4.3.6 Hydrolysis of soluble starch with purified α-amylase from T. lanuginosus ATCC 58157……………………………………………………………………………155 4.4 Discussion……………………………………………………………………...157 4.5 References……………………………………………………………………...162 CHAPTER FIVE: THE APPLICATION OF α-AMYLASE FROM T. lanuginosus ATCC 58157 FOR THE HYDROLYSIS OF MAIZE STARCH…………………………………167 5.1 Introduction……………………………………………………………………167 . 5.2 Materials and methods………………………..………………………………169 5.2.1 Application of purified α-amylase from T. lanuginosus ATCC 58157 in the hydrolysis of maize meal………………………….............169 5.2.2 Optimisation of hydrolysis of maize meal mash with purified α-amylase from T. lanuginosus ATCC 58157……………………………………………………...169 5.2.3 Hydrolysis of maize meal under optimised conditions with α-amylase from T. lanuginosus ATCC 58157……………………………………………………...172 5.2.4 Scanning electron microscopy………………………….………………………172 5.2.5 Nutritional analysis of hydrolysed maize meal with α-amylase from T. lanuginosus ATCC 58157…………………………...........................................173 5.2.5.1 Protein determination……………………………………………………….......173 5.2.5.2 Carbohydrate (CHO) analysis using thin layer chromatography (TLC)…………………………………………..……………………………….173 5.2.5.3 Quantification of carbohydrates in digested and undigested maize meal using high performance liquid chromatography (HPLC)…………………………………..174 5.2.5.3.1 Reagents and standards…………………………………………………………………...…...174 5.2.5.3.2 Sample preparation……………………………………………………...……174 5.2.5.3.3 HPLC conditions……………………………………………………………...175 5.2.5.4 Identification and quantitative analysis of water soluble vitamins using HPLC…………………………………………………………………………...176 5.2.5.4.1 Reagents and Standards………………………………………………………176 5.2.5.4.2 Sample preparation…………………………………………………………...176 5.2.5.4.3 HPLC conditions……………………………………………………………...177 5.2.5.5 Fat soluble vitamin A reagents and standard…………………………………...177 5.2.5.5.1 Sample preparation…………………………………………………………...177 5.2.5.5.2 HPLC conditions……………………………………………………………...178 5.3 Results………………………………………………………………………….179 5.3.1 Optimisation of hydrolysis of maize meal mash………………………………..179 5.3.1.1 Maize mashing using optimised conditions…………………………………….180 5.3.2 Estimation of protein content…………………………………………………...182 5.3.3 Ultrastructure of raw maize meal, undigested maize meal and maize meal digested with α-amylase from T. lanuginosus ATCC 58157……………..182 5.3.4 Carbohydrate analysis using thin layer chromatography (TLC) to identify sugars…………………………………………………………………………...186 5.3.5 Quantification of carbohydrates in digested and undigested maize meal using HPLC…………………………………………………………...........................187 5.3.6 Identification and quantification of water soluble vitamins using HPLC………189 5.3.7 Identification and quantification of fat soluble vitamin A using HPLC………..192 5.4 Discussion……………………………………………………………………...195 5.5 References……………………………………………………………………...200 CHAPTER SIX: EVALUATION OF THE NUTRITIONAL BENEFITS OF HYDROLYSED MAIZE MEAL USING RATS IN FEED TRIALS………………………………………...204 6.1 Introduction……………………………………………………………………204 6.2 Materials and methods………………………………………………………..206 6.2.1 Preparation of the feed………………………………………………………….206 6.2.2 The feed trial……………………………………………………………………206 6.2.3 Statistical Methodology………………………………………………………...208 6.3 Results………………………………………………………………………….210
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