Please read the abstract in the section 00front of this documentThesis (PhD (Food Science))--University of Pretoria, 2006.Food Scienceunrestricte

Duodu, Kwaku Gyebi

Thesis (PhD (Food Science))--University of Pretoria, 2006.Please read the abstract in the section 00front of this documentFood Scienceunrestricte

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ROLE OF GRAIN ORGANISATIONAL STRlICTURE IN SORGHUM . PROTEIN DIGESTIBILITY BY KWAKU GYEBI DUODU SUBMJTTED IN PARTIAL FULFIL:MENT OF THE REQUlRE:MENTS FOR THE DEGREE ' PhD (FOOD SCIENCE) IN THE DEPARTMENT OF FOOD SCIENCE FACULTY OF NATURAL AND AGRICULTURAL SCIENCES UNIVERSITY OF PRETORIA PRETORIA SOUTH AFRICA OCTOBER 2000                                                   © University of Pretoria I declare that the dissertation herewith submitted for the degree of PhD (Food Science) at the University of Pretoria, has not previously been submitted by me for a degree at any other university or institution of higher education.                                                   © University of Pretoria ABSTRACT ROLE OF GRAIN ORGANISATIONAL STRUCTURE IN SORGHUM PROTEIN DIGESTIBILITY by Kwaku Gyebi Duodu Promoter: Prof JRN Taylor Co-promoter: ProfPS Belton Department: Food Science Degree: PhD (Food Science) Sorghum (Sorghum bie%r (L.) Moench) is a drought-tolerant basic food cereal in many parts of Africa and Asia. Wet cooking decreases the digestibility of sorghum proteins significantly and this is a limitation to the use of sorghum as food . Uncooked sorghum protein digestibility is also considered lower than other cereals. In vitro protein digestibility of uncooked and cooked condensed ..tannin-free sorghum varieties and a white maize variety was examined at four levels of grain organisational structure: whole grain, endosperm, protein body preparations and isolated proteins. The possibility of polyphenols and gelatinised starch having an effect on in vitro protein digestibility of sorghum and maize was also examined. Fourier transform infrared (FTIR) and solid-state l3C NMR spectroscopic methods were used to investigate changes in protein secondary structure on thermal processing. Electrophoresis (SDS-PAGE) was used to investigate the possibility of protein crosslinking on thermal processing by examining molecular weight differences. Uncooked and cooked sorghum protein digestibility improved from whole grain, through endosperm to protein body preparations. However, uncooked and cooked maize protein digestibility was essentially the same at these levels. Uncooked sorghum protein digestibility was lower than uncooked maize at the whole grain level, same as maize at the endosperm level and higher than maize at the protein body-enriched level. Isolated kafirins and zeins from sorghum and maize had similar uncooked protein digestibility. Total polyphenol content                                                   © University of Pretoria of sorghum decreased from whole gram to endosperm and increased from endosperm to protein body preparations. As expected, cooking reduced protein digestibility of sorghum whole grain, endosperm, protein body preparations and extracted kafirins. Cooked sorghum whole grain and endosperm had similar protein digestibilities but cooked protein body preparations were more digestible. There was overall improvement in sorghum protein digestibility with change in organisational level. Protein digestibilities of uncooked and cooked maize were essentially the same at all the organisational levels. Treating cooked whole grain and endosperm samples of sorghum and maize with alpha­amy lase before pepsin digestion improved protein digestibility . In the protein body preparations where the proportion of starch was lower, such treatment had no effect on protein digestibility. SDS-PAGE under non-reducing and reducing conditions of uncooked and cooked protein body preparations from normal sorghum, maize and sorghum mutants (of known high protein digestibility) showed oligomers ofMr 45, 66 and >66 kDa and monomeric kafirins and zeins . Sorghum had more 45-50 kDa oligomers than maize. In comparison with maize, more of these oligomers were resistant to reduction in cooked normal sorghum. SDS-PAGE also showed that residues of the protein body preparations remaining after pepsin digestion consisted mainly of a-zein (uncooked and cooked maize) or a-kafirin (uncooked normal sorghum), whilst cooked normal sorghum had in addition, ~- and y-kafirin and reduction-resistant 45-50 kDa oligomers. FTIR and solid-state l3C NMR spectra of normal sorghum, maize and sorghum mutants indicated a change in protein secondary structure from a-helical to antiparallel intermolecular ~-sheet conformation on cooking. The extent of secondary structural change seemed to be greater in sorghum than in maize. Grain organisational structure does influence sorghum protein digestibility. Interfering factors in the grain outer layers, namely pericarp and germ may be responsible. The decrease in                                                   © University of Pretoria sorghum total polyphenol content from whole grain to endosperm accompanied with an increase in uncooked and cooked sorghum protein digestibility suggests that polyphenols may affect sorghum whole grain protein digestibility. In contrast to earlier reports, uncooked sorghum protein digestibility may not always be lower than that of maize. It depends on the nature of the material being assayed. Gelatinised starch, probably by reducing accessibility of pepsin to protein, reduced digestibility of sorghum and maize whole grain and endosperm. It appears that cooking reduces protein digestibility in sorghum by unravelling of prolamin polypeptides in the a-helical conformation which re-associate either through disul phide or non-disulphide crosslinks to form the anti parallel intermolecular ~-sheet conformation. This conformation may be less digestible due to restricted enzyme access to the protein . Such crosslinking may occur to a greater extent in sorghum than in maize perhaps due to subtle differences in prolamin tertiary structure between the two cereals, contributing to the worse digestibility of cooked sorghum proteins.                                                   © University of Pretoria Do not be afraid of walking slowly Be afraid only of standing still - Old Chinese proverb                                                   © University of Pretoria ACKNOWLEDGEMENTS My deepest gratitude and appreciation go to the following for their assistance with this research: Prof. JRN Taylor of the Department ofFood Science, University of Pretoria, South Africa and Prof. PS Belton of the Institute of Food Research, Norwich, UK (my promoters) for their guidance, leadership and encouragement throughout my doctoral studies. Prof. BR Hamaker of the Department of Food Science, Purdue University, USA and Dr. Arthur Tatham of the IACR-Long Ashton Research Station, UK for their constructive and critical review of this thesis. Thanks to Prof. Hamaker for the highly digestible sorghum mutants and to Arthur for assistance with nitrogen determinations. The Commonwealth Scholarships Commission for much valued financial assistance during my year of study in the UK. Janet Taylor of the Department of Food Science, University of Pretoria, South Africa for the polyphenol and enzyme inhibition assays. Mr. Alex Grant, Dr. Hui-Ru Tang, Dr. Klaus Wellner, Dr. Mary Parker and Dr. Clare Mills all of IFR, Norwich, UK for their technical assistance and encouragement. Thanks to my colleagues in Prof. PS Belton's research group for their wonderful support. Thanks to Alex for those trips to Carrow Road to see the Canaries in action. My friends and colleagues in the Department of Food Science, University of Pretoria for their support and encouragement. My family in Ghana and in East London, South Africa for all their prayers and good will. We did it again. Dr. and Mrs. RO Kumi of Maryland, USA for all those e-mails and telephone calls. God Almighty who has made all this possible.                                                   © University of Pretoria -1-TABLE OF CONTENTS LIST OF TABLES -IV-LIST OF FIGURES -VI-CHAPTER 1: INTRODUCTION 1 CHAPTER 2: LITERATURE REVIEW 4 2.1 Sorghum and maize: Origin, physical characteristics and chemical composition 4 2.1 .1 Proteins of sorghum and maize 5 2.1.2 Structural organisation of sorghum and maize grains 8 2.1. 2.1 Pericarp 8 2.1.2.2 Germ II 2.1. 2. 3 Endosperm I I 2.l.3 Localisation of proteins in the various anatomical parts of sorghum and maize 12 2.2 Food uses of sorghum and maize 16 2.3 Protein nutritional value of sorghum and maize 16 2.3.1 Amino acid composition 16 2.3.2 Protein digestibility 17 2.4 Factors affecting protein digestibility of sorghum and maize 19 2.4.1 Starch and cell walls 19 2.4.2 Polyphenols 20 2.4.3 Phytic acid 23 2.4.4 Protein crosslinking 24 2.4.4.1 Disulphide crosslinking and kafirin solubility 25 2.4.4.2 Racemization and isopeptideformation 29 2.5 Analytical methods for protein digestibility and protein conformation 30 2.5.1 In vitro protein digestibility assays 30 2.5.2 Fourier Transform Infrared spectroscopy 32 2.5.3 Nuclear Magnetic Resonance (NMR) spectroscopy 35                                                   © University of Pretoria -ii­2.6 Gaps in knowledge 40 2.7 Objectives and hypotheses 41 CHAPTER 3: MATERIALS AND METHODS 42 3.1 Grain samples 42 3.2 Sample preparation 42 3.2.1 Whole grain meal 42 3.2.2 Decorticated sorghum and degermed maize 42 3.2.3 Endosperm meal 42 3.2.4 Preparation of protein body-enriched samples 43 3.2.5 Preparation ofunalkylated and alkylated total kafirin and zein 43 3.2.6 Cooked whole grain meal, cooked endosperm meal, cooked protein body-enriched samples and cooked protein fractions (unalkylated and alkylated kafirin and zein) 44 3.2.7 Alpha-amylase-treated samples 44 3.2.8 Popped grain 44 3.2.9 Starch hydrolysis prior to Fourier Transform Infrared (FTIR) spectroscopy 44 3.3 Analytical methods 45 3.3.1 Protein content 45 3.3.2 In vitro protein digestibility (IVPD) 45 3.3 .3 Total polyphenols 46 3.3.4 Enzyme inhibition by whole grain 47 3.3 .5 Transmission electron microscopy 47 3.3 .6 Fourier Transform Infrared (FTIR) spectroscopy 48 3.3.7 13C Nuclear Magnetic Resonance (NMR) spectroscopy 48 3.3 .8 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 49 3.4 Statistical analyses 51                                                   © University of Pretoria -111-CHAPTER 4: RESULTS 52 4.1 Total protein and polyphenol contents of sorghum and maize samples 52 4.2 Ultrastructure of protein body-enriched samples 55 4.3 In vitro protein digestibility of whole grain, endosperm and protein body­enriched samples and enzyme inhibition by whole grain 62 4.4 In vitro protein digestibility of reduced/alkylated and reduced/non-alkylated kafirin and zein 71 4.5 SDS-PAGE of protein body-enriched samples of sorghum and maize under non-reducing and reducing conditions 72 4.6 FTIR and HC NMR spectroscopy of uncooked and cooked protein body­enriched samples of sorghum and maize 81 4.7 In vitro protein digestibility and FTIR spectroscopy of popped sorghum and maize 94 CHAPTER 5: DISCUSSION 99 CHAPTER 6: CONCLUSIONS AND RECOMMENDATIONS 119 CHAPTER 7: REFERENCES 125 LIST OF PUBLICATIONS 149                                                   © University of Pretoria -iv-Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 LIST OF TABLES Proximate composition of sorghum and maize grain in g per 100 g edible portions at 12% moisture. 5 Osborne protein fractions of sorghum and maize. 6 Essential amino acid composition (mglg crude protein) of maize and sorghum whole grain in comparison with suggested amino acid requirements for infants and adults and amino acid composition of egg as a high quality animal protein. 17 In vitro protein digestibility, IVPD (%) of uncooked sorghum and maize reported by different workers. 18 L-M fractions 2 and 3 proteins in sorghum and maize (% of total protein) 26 Characteristic chemical shifts of protein carbons in 13C NMR spectra 38 Total protein contents (gilOO g dry basis) of whole grain, endosperm and protein body-enriched samples of sorghum (NK 283, KAT 369) and maize (pAN 6043) and protein body-enriched samples ofP851171 and P850029 sorghum mutants and total polyphenol contents of whole grain, endosperm and protein body enriched samples of sorghum (NK 283, KAT 369) and maize (pAN 6043). 53' Effect of cooking and addition of alpha-amylase after cooking on percentage in vitro protein digestibility of whole grain, endosperm and protein body­enriched samples ofNK. 283 red sorghum. 62 Effect of cooking and addition of alpha-amylase after cooking on percentage in vitro protein digestibility of whole grain, endosperm and protein body­enriched samples ofKAT 369 white sorghum, 64 Effect of cooking and addition of alpha-amylase after cooking on percentage in vitro protein digestibility of whole grain, endosperm and protein body­enriched samples ofPAN 6043 maize. 66 Effect of cooking and addition of alpha-amylase after cooking on percentage in vitro protein digestibility of protein body- enriched samples ofP851171 and P850029 sorghum mutants in comparison with red sorghum NK 283, white sorghum KAT 369 and maize PAN 6043 . 68                                                   © University of Pretoria -v-Table 12 Table 13 Table 14 Table 15 Percentage enzyme (amylase) inhibition caused in whole grain ofNK 283 sorghum, KAT 369 sorghum and PAN 6043 maize in comparison with DC 75 sorghum (a high-tannin hybrid). 70 Effect of cooking and alkylation on in vitro protein digestibility (PD) of total kafirin (kafirin 1 and kafirin 2) and total zein (zein 1 and zein 2). 71 In vitro protein digestibility of popped NK 283 sorghum and PAN 6043 maize in comparison with uncooked and wet cooked whole grain. 94 Proposed factors affecting protein digestibility of uncooked and cooked sorghum and maize and their levels of importance at the whole gram, endosperm, protein body and extracted protein levels. 123                                                   © University of Pretoria Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 -vi-LIST OF FIGURES A sorghum kernel. 9 Longitudinal and cross sections of a maize kernel. 10 Development of protein bodies in maize endosperm as proposed by Lending and Larkins (1989). 15 Basic structures of phenolic acids, flavonoids and proanthocyanidin 21 Structure of phytic acid . 24 Changes a protein undergoes during heat treatment. 25 Representation of precessing nuclei and the energy transition between the aligned and opposed conditions. 36 Transmission electron micrographs of uncooked protein body-enriched samples of sorghum (NK 283 and KAT 369), maize (PAN 6043) and sorghum mutants (P851171 and P850029). 55 SDS-PAGE of uncooked and cooked protein body-enriched samples of sorghum (NK 283 and KAT 369) and sorghum mutant (P850029). 72 SDS-PAGE of pepsin-indigestible residues of sorghum (NK 283 and KAT 369), maize (PAN 6043) and sorghum mutant (p850029) protein body­enriched samples under non-reducing and reducing conditions (200 mM DTT). 78 FTIR spectra of uncooked and cooked protein body-enriched samples of sorghum (NK 283 and KAT 369), maize (PAN 6043) and sorghum mutants (p851171 and P850029). 81 Fourier deconvoluted FTIR spectra of uncooked and cooked protein body­enriched samples of sorghum and maize varieties. 85 I3C CPMAS N1vfR. spectra of uncooked and wet cooked protein body­enriched samples of sorghum and maize. 88 Fourier deconvoluted FTIR spectra of uncooked, wet cooked and popped whole grain of sorghum and maize. 95 Proposed model for maize bran cell walls. 104 Structures of proposed covalent crosslinks between polysaccharides and proteins in cell walls. 105                                                   © University of Pretoria 106 Figure 17 Figure 18 Figure 19 -vii-Structure of the hydroxyproline-rich region of plant cell wall glycoproteins. Proposed structures of oxidatively coupled products of tyrosine which may lead to formation of non-disulphide crosslinks in proteins and possible structure of a tyrosyl-tyrosyl crosslink between proteins. I I I Representation of a-helix to ~-sheet conformational change on cooking followed by crosslink formation between polypeptide chains. 117                                                   © University of Pretoria 

Role of grain organisational structure in sorghum protein digestibility

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Role of grain organisational structure in sorghum protein digestibility

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