LINKING CEREAL CHEMISTRY TO NUTRITION: STUDIES OF WHEAT BRAN AND RESISTANT STARCH by LAUREN RENEE BREWER B.S., Clemson University, 2007 M.S., Kansas State University, 2009 AN ABSTRACT OF A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Grain Science and Industry College of Agriculture KANSAS STATE UNIVERSITY Manhattan, Kansas 2012 Abstract Wheat bran is high in dietary fiber. Resistant starch (RS) is considered a source of dietary fiber. Wheat bran and RS have different functional properties and may not have the same nutritional properties. This dissertation covers two areas of importance in cereal chemistry and human nutrition: wheat bran and RS. Wheat bran chemical and physical influence of nutritional components Wheat bran has become a hot topic due to positive nutritional correlations, and industrial /humanitarian needs for healthy ingredients. Evolving wheat bran into a demanded product would impact the industry in a positive way. The overall aim of this research was to understand chemical and structural composition, to provide avenues for wheat bran development as a healthy food ingredient. To achieve this goal, antioxidant properties in dry wheat milling fractions were examined, effects of wheat bran particle size on phytochemical extractability and properties were measured, and substrate fermentation was investigated. It was noted that mixed mill streams, such as mill feed, have antioxidant properties (0.78 mg FAE/g; 1.28 mg/g total antioxidant capacity; 75.21% DPPH inhibition; 278.97 µmol FeSO /g) originating from the bran 4 and germ fractions. Additionally, extraction of reduced particle size whole wheat bran increased measured values for several assays (185.96 µg catechin/g; 36.6 µg/g; 425 µM TE), but did not increase volatile fatty acid production during in vitro rumen fermentation over unmilled bran. RS digestion, glycemic response and human fermentation In vitro action of enzymes on digestion of maize starches differing in amylose contents were studied. The objectives of this study were to investigate the exact role of α-amylase and amyloglucosidase in determining the digestibility of starch and to understand the mechanism of enzymatic actions on starch granules. Starch digestibility differed (30-60%) without combination of enzymes during in vitro assay. Further investigations utilized human glycemic response and fermentation with consumption of a type 3 RS without dietary fiber (AOAC method 991.43). Blood glucose response provided lower postprandial curves (glycemic index value of 21) and breath hydrogen curves displayed low incidences fermentation (40%) with consumption of the type 3 RS, due to the structure of starch and digestion by enzymatic action. LINKING CEREAL CHEMISTRY TO NUTRITION: STUDIES OF WHEAT BRAN AND RESISTANT STARCH by LAUREN RENEE BREWER B.S., Clemson University, 2007 M.S., Kansas State University, 2009 A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Grain Science and Industry College of Agriculture KANSAS STATE UNIVERSITY Manhattan, Kansas 2012 Approved by: Major Professor Yong-Cheng Shi Copyright LAUREN RENEE BREWER 2012 Abstract Wheat bran is high in dietary fiber. Resistant starch (RS) is considered a source of dietary fiber. Wheat bran and RS have different functional properties and may not have the same nutritional properties. This dissertation covers two areas of importance in cereal chemistry and human nutrition: wheat bran and RS. Wheat bran chemical and physical influence of nutritional components Wheat bran has become a hot topic due to positive nutritional correlations, and industrial /humanitarian needs for healthy ingredients. Evolving wheat bran into a demanded product would impact the industry in a positive way. The overall aim of this research was to understand chemical and structural composition, to provide avenues for wheat bran development as a healthy food ingredient. To achieve this goal, antioxidant properties in dry wheat milling fractions were examined, effects of wheat bran particle size on phytochemical extractability and properties were measured, and substrate fermentation was investigated. It was noted that mixed mill streams, such as mill feed, have antioxidant properties (0.78 mg FAE/g; 1.28 mg/g total antioxidant capacity; 75.21% DPPH inhibition; 278.97 µmol FeSO /g) originating from the bran 4 and germ fractions. Additionally, extraction of reduced particle size whole wheat bran increased measured values for several assays (185.96 µg catechin/g; 36.6 µg/g; 425 µM TE), but did not increase volatile fatty acid production during in vitro rumen fermentation over unmilled bran. RS digestion, glycemic response and human fermentation In vitro action of enzymes on digestion of maize starches differing in amylose contents were studied. The objectives of this study were to investigate the exact role of α-amylase and amyloglucosidase in determining the digestibility of starch and to understand the mechanism of enzymatic actions on starch granules. Starch digestibility differed (30-60%) without combination of enzymes during in vitro assay. Further investigations utilized human glycemic response and fermentation with consumption of a type 3 RS without dietary fiber (AOAC method 991.43). Blood glucose response provided lower postprandial curves (glycemic index value of 21) and breath hydrogen curves displayed low incidences fermentation (40%) with consumption of the type 3 RS, due to the structure of starch and digestion by enzymatic action. Table of Contents List of Figures ................................................................................................................................ xi List of Tables ............................................................................................................................... xiii Acknowledgements ....................................................................................................................... xv Dedication ................................................................................................................................... xvii Preface........................................................................................................................................ xviii Chapter 1 - Current topics of wheat bran physical and chemical structure .................................... 1 Abstract ....................................................................................................................................... 1 Introduction ................................................................................................................................. 1 Wheat bran production ............................................................................................................ 3 Wheat bran economics ............................................................................................................ 3 Wheat bran structure ................................................................................................................... 4 Wheat bran description by layers ............................................................................................ 4 The cross section of wheat bran .............................................................................................. 6 Recent structural understanding of wheat bran ....................................................................... 7 Chemical components of wheat bran .......................................................................................... 9 Advances in wheat protein investigations ............................................................................... 9 Advances in knowledge of vitamins, minerals, and antioxidants matrices in wheat bran .... 10 Progress in the knowledge of non-starch polysaccharides ................................................... 16 Conclusions ............................................................................................................................... 19 Chapter 2 - Antioxidant contribution within fractions and mill streams produced from the same wheat kernels ......................................................................................................................... 50 Abstract ..................................................................................................................................... 50 Introduction ............................................................................................................................... 50 Materials and methods .............................................................................................................. 53 Wheat and milled wheat fractions ......................................................................................... 53 Soluble and bound phenolic acids ......................................................................................... 53 Phytochemical extraction ...................................................................................................... 54 Total flavonoid content ......................................................................................................... 55 vi Total anthocyanin content ..................................................................................................... 55 Total antioxidant capacity ..................................................................................................... 56 Diphenylpicrylhydrazyl (DPPH) assay ................................................................................. 56 Ferric ion reducing antioxidant power (FRAP) assay ........................................................... 57 Statistical analysis ................................................................................................................. 57 Results ....................................................................................................................................... 57 TPC of wheat fractions ......................................................................................................... 57 Flavonoid concentration in wheat fractions .......................................................................... 58 Anthocyanin concentration in wheat fractions...................................................................... 58 Total antioxidant capacity of wheat fractions ....................................................................... 58 DPPH radical scavenging activity of wheat fractions ........................................................... 59 FRAP of wheat fractions ....................................................................................................... 59 Discussion ................................................................................................................................. 60 Conclusions ............................................................................................................................... 65 Chapter 3 - Wheat bran particle size influence on phytochemical extractability and antioxidant properties ............................................................................................................................... 77 Abstract ..................................................................................................................................... 77 Introduction ............................................................................................................................... 78 Materials and methods .............................................................................................................. 80 Wheat bran samples .............................................................................................................. 80 Chemicals .............................................................................................................................. 81 Particle size determination .................................................................................................... 81 Soluble and bound phenolic acids ......................................................................................... 82 Phytochemical extraction ...................................................................................................... 83 Total flavonoid content ......................................................................................................... 83 Total anthocyanin content ..................................................................................................... 84 Diphenylpicrylhydrazyl (DPPH) assay ................................................................................. 84 Ferric ion reducing antioxidant power (FRAP) assay ........................................................... 85 Oxygen radical absorbance capacity (ORAC) ...................................................................... 85 Total antioxidant capacity ..................................................................................................... 86 Carotenoid Analysis .............................................................................................................. 86 vii Statistical analysis ................................................................................................................. 87 Results ....................................................................................................................................... 87 Particle size distributions ...................................................................................................... 87 TPC extraction ...................................................................................................................... 87 Flavonoid concentrations ...................................................................................................... 88 Anthocyanin concentrations .................................................................................................. 88 DPPH radical- scavenging activity ....................................................................................... 88 FRAP assay ........................................................................................................................... 89 ORAC assay .......................................................................................................................... 89 Total antioxidant capacities .................................................................................................. 89 Carotenoid concentration ...................................................................................................... 90 Discussion ................................................................................................................................. 90 Conclusions ............................................................................................................................... 97 Chapter 4 - Particle size effect on fermentation of wheat bran in rumen fluid ........................... 112 Abstract ................................................................................................................................... 112 Introduction ............................................................................................................................. 113 Materials and methods ............................................................................................................ 114 Materials ............................................................................................................................. 114 Milling and preparation of cooked and destarched wheat bran .......................................... 114 General methods ................................................................................................................. 115 Particle size determination .................................................................................................. 116 In vitro rumen fermentation ................................................................................................ 116 VFA ..................................................................................................................................... 117 Digesta analysis .................................................................................................................. 117 Statistical analysis ............................................................................................................... 118 Results ..................................................................................................................................... 118 Composition of wheat bran ................................................................................................. 118 ANKOM fermentation and gas production ......................................................................... 119 VFA ..................................................................................................................................... 120 Discussion ............................................................................................................................... 120 In vitro fermentations .......................................................................................................... 120 viii Effect of WHC .................................................................................................................... 121 Effect of protein content ..................................................................................................... 122 Effect of fiber components .................................................................................................. 123 Effect of total starch content ............................................................................................... 124 Effects of total gas production and contributing VFA ........................................................ 125 Conclusions ............................................................................................................................. 126 Chapter 5 - Mechanism and enzymatic contribution to in vitro test method of digestion for maize starches differing in amylose content .................................................................................. 136 Abstract ................................................................................................................................... 136 Introduction ............................................................................................................................. 137 Materials and Methods ............................................................................................................ 138 Samples ............................................................................................................................... 138 Methods ............................................................................................................................... 139 Digestion method and modifications .............................................................................. 139 High-performance anion-exchange chromatography (HPAEC) ..................................... 140 SEM ................................................................................................................................ 140 Molecular size distribution by GPC ................................................................................ 141 WAXD and SAXS .......................................................................................................... 141 Statistics .......................................................................................................................... 141 Results and discussion ........................................................................................................ 142 RDS, SDS, and RS content of maize starches ................................................................ 142 Starch granular morphology before and after enzyme digestion .................................... 144 Molecular size distribution ............................................................................................. 145 Starch structure by SAXS and WAXD ........................................................................... 146 Chapter 6 - Glycemic response and breath hydrogen testing by human consumption of crystalline short-chain amylose ............................................................................................................. 161 Abstract ................................................................................................................................... 161 Introduction ............................................................................................................................. 163 Materials and Methods ............................................................................................................ 168 Production of CSCA ........................................................................................................... 168 General methods ................................................................................................................. 169 ix Ethics ................................................................................................................................... 169 Subjects ............................................................................................................................... 170 Glycemia ............................................................................................................................. 171 Fermentation ....................................................................................................................... 172 Statistical analysis ............................................................................................................... 173 Results and discussion ............................................................................................................ 173 CSCA .................................................................................................................................. 173 RS and TDF content ............................................................................................................ 174 D-glucose analysis .............................................................................................................. 175 Glycemia ............................................................................................................................. 175 Gas production .................................................................................................................... 180 Conclusions ............................................................................................................................. 184 Chapter 7 - Appendix list ............................................................................................................ 202 x