CHEMICAL AND DOUGH FORMING PROPERTIES OF KAFIRIN EXTRACTED FROM CONVENTIONALLY BRED AND GENETICALLY MODIFIED SORGHUMS WITH ALTERED KAFIRIN SYNTHESIS

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INTRODUCTION

This literature review largely deals with the biochemistry, functional quality and nutritional quality of the storage proteins and starch of conventionally bred and genetically modified sorghums. Also, the chemical composition and functional properties of other major cereals such as wheat and maize will be reviewed. The main goal of this study is to improve the quality of sorghum flour as an alternative source for bread making. As maize is known to have similar properties to sorghum flour especially in terms of protein, it will also be reviewed. The major components of sorghum i.e. starch and sorghum prolamin protein (kafirin) will be the focus because of their importance in flour functional properties. Since the research requires different advanced techniques, the review will discuss the principles and application of these techniques.

SORGHUM STARCH

The starch of most sorghum types have the normal ratio of amylose to amylopectin. There are, however, so-called waxy mutants which have a higher proportion of amylopectin. The ratio of amylose/amylopectin in sorghum starch differs depending on genetic background and environment (Beta and Corke, 2001). Sorghum starches have been classified as normal (non-waxy) and waxy based on amylose/amylopectin ratio. Normal sorghum starch has 20–35% amylose. Waxy sorghum starch essentially does not contain amylose (Sang et al., 2008), while the heterowaxy and normal sorghum starches contain about 14% and 23.7% amylose by weight, respectively. These authors found that the side chain lengths of amylopectin within the three types of the sorghum starch (waxy, heterowaxy and normal) do not have significant differences in terms of their distribution by area % of the amylopectin. Within the three types of sorghum starch, about 43-45% is of chain length 6 to 15 degree of polymerization (DP). The chain length of 16 to 36 DP exists in the range from 49-50%. The longest chain length of DP ≥ 37 is in the range from 5-6%.

Water absorption and water solubility of flour

Starch, protein and fibre absorb water in flour. Water absorption and swelling power of starch are closely related together and it can be said that they are two sides of the same coin (Jenkins and Donald, 1998). Flour water absorption index (WAI) is the weight (g) of gel per gram of dry flour. The water solubility of flour can be defined as the maximum quantity of flour which will be dissolved in a unit volume of water. The type of crystallinity of starch granules has an influence on the water solubility of starch, the major flour component (Crochet et al., 2005). The A-type crystal has less solubility than the B-type. Both water absorption and water solubility of starch are affected by the strength and the structure of the micellar network within the starch granule (Qian, Rayas-Duarte and Grant, 1989; Udachan et al., 2012). Water solubility and swelling of starch increase with increasing temperature (Yusuf et al., 2007; Udachan et al., 2012).
This is attributed to induction of strong vibration of starch granules which results in breaking or release of intermolecular linkages. Therefore more sites inside the starch granule become available to take more water through linking via hydrogen bonds (Udachan et al., 2012). Water absorption of starch rises with increasing extent of starch granule damage (Dexter et al., 1994). Noticeable and rapid increase in water absorption of sorghum starch is in the range of its gelatinization temperature approx. 60-70 °C (Udachan et al., 2012). Determination of WSI and WAI at high temperature enables estimation of the textural character of products based on starch. Starches of a white (non-pigmented) high amylose sorghum and red (pigmented) low amylose content sorghum showed different swelling power (SP) and WSI at various temperatures in the range 55-95 °C (Boudries et al., 2009). Normally swelling power and WSI of sorghum starch increase with temperature. The sorghum starches with different amylose content exhibited the same swelling power at temperatures lower than 65 °C (Boudries et al., 2009). Nevertheless, at temperatures above 65 °C sorghum starch of low amylose content displayed higher swelling power than sorghum starch of higher amylose content.
This may confirm what has been stated by Tester and Morrison (1990), that swelling power of cereal starches is related to their amylopectin content, while amylose works as a retarder, especially when amylose forms amylose-lipid complexes in the presence of lipids. The WSI of sorghum starches almost stops increasing at about 85-95 °C (Boudries et al., 2009). Furthermore, WSI of high amylose starch from non-pigmented sorghum is higher than that of lower amylose starch from red sorghum. Alkali treatment of sorghum starch resulted in a decrease in swelling temperature, perhaps due to pre-gelatinization of starch (Beta et al., 2000). It can be said that the WAI and WSI of sorghum increase with temperature (Carcea et al., 1992). However, the swelling powers of some sorghum starches and flours are similar to wheat at a temperature of about 55 °C (Chanapamokkhot and Thongngam, 2007; Phattanakulkaewmorie et al., 2011). However, at temperatures above 75 °C, sorghum starch and flours have a higher swelling power than wheat (Chanapamokkhot and Thongngam, 2007)

Dough rheology

Starch and protein are the major components in the flour that affect dough rheology. During baking of the dough for bread making, protein denatures and releases water for starch to use it for its gelatinization (Therdthai and Zhou., 2003). As with wheat dough, the rheological properties of sorghum dough are affected by the nature of protein (Goodall et al., 2012). These authors found that dough from sorghum of high protein digestibility had higher maximum resistance to extension than normal sorghum. With regard to the effect of starch, it has been found that starch from red sorghum of lower amylose content exhibited higher peak viscosity (around 4731 cP) than that of high amylose starch from white sorghum (around 4093 cP) (Boudries et al., 2009). This difference in pasting behaviour could be attributed to the amount of amylose rather than the colour of the sorghum. When Beta et al. (2000) tested the pasting properties of starches isolated from 10 sorghum varieties grown in Zimbabwe , they found that sorghum starches had higher peak viscosity (PV) ( 300-398 RVU)

than commercial maize starch (239 RVU). The total time for peak viscosity to reach its maximum

level starting from the beginning of viscosity increase was generally less in sorghum starches, approx. 1.73-3.03 min than maize starch about 3.50 min. While sorghum starch showed a higher rate of shear thinning around 24.4-40.4 RVU/min than maize starch, approx. 21.0 RVU/min. Polyphenol content of the sorghum starch had a positive relationship with starch peak viscosity (r =0.75, p <0.05). Moreover, breakdown and rate of shear thinning of sorghum starch had negative relation with hardness of sorghum starch gel. Sorghum starch from high tannin varieties had a higher rate of shear thinning than sorghum starch from low or non-tannin sorghum. It was concluded that sorghum pasting properties are significantly affected by the genetic diversity and the environmental condition during growing (Beta and Corke, 2001). The pasting properties of hard wheat, barley and sorghum starches were compared (Ragaee and Abdel-Aal, 2006). The peak viscosity (PV), breakdown viscosity (BV) and setback viscosity (SV) were different between these cereals. With regard to wheat starch, PV, BV and SV were about 1335 cP, 755 cP and 842 cP, respectively. Barley starch had a PV of approx. 1355 cP, BV of 989 cP and SV of 695 cP. Generally, sorghum starch had a lower average peak viscosity (821 cP) than wheat and barley starch. Regarding breakdown viscosity, sorghum starch had the lowest extent (2 cP) compared to wheat and barley which may relate to higher stability. Fruthermore, sorghum starch had the highest degree of setback average 1307 cP compared to wheat and barley.

Transmission electron microscopy (TEM)

As with SEM, TEM can be considered as analogous to LM. Since TEM is an electron optical instrument, the visualization of the objects does not occur by light illumination, but by using an electron beam (Klang et al., 2012). For this reason, i.e. use of electrons, TEM needs to be operated under vacuum to avoid electron deviation by air molecules. TEM has an electron gun on top of a column used to generate the electron beam. As with SEM, electromagnetic lenses concentrate the electron beam on the specimen. Electron scattering i.e. the interaction of electrons with specimen is responsible for the image contrast in TEM. The resolution of TEM is positively correlated with the acceleration voltage of the electrons. However, continuous acceleration voltage leads to poor image contrast due to a decrease in electron scattering at high velocity (Kuntsche et al., 2011). TEM can be used to visualize the internal structure of food specimens (Mady Kaye et al., 2007). Samples should be sectioned to a thickness of 15-90 nm after being embedded in epoxy resin or platinum-carbon. The magnified image of the specimens can be viewed on a fluorescent screen or photographed digitally. TEM has, for example, been used to investigate sorghum protein bodies (Da Silva et al., 2011a). TEM facilitated understanding of the effect of suppression of γ-kafirin synthesis on protein body structure. It was found that the genetically modified sorghums with high protein digestibility due to reduction in γ-kafirin had invaginated protein bodies.

TABLE OF CONTENTS :

• DECLARTION
• ABSTRACT
• DEDICATION
• ACKNOWLEDGEMENTS
• LIST OF TABLES
• LIST OF FIGURES
• 1 INTRODUCTION
• 2 LITERATURE REVIEW
  • 2.1 INTRODUCTION
  • 2.2 SORGHUM STARCH
  • 2.2.1 Sorghum starch granules
  • 2.2.2 Sorghum starch gelatinization
  • 2.3 SORGHUM PROTEINS
  • 2.3.1 Sorghum grain protein distribution
  • 2.3.2 Non-kafirin proteins
  • 2.3.3 Kafirin
    • 2.3.3.1 Kafirin physicochemical characteristics
    • 2.3.3.2 Sub-classes of kafirin
    • 2.3.3.2.1 Alpha-kafirin
    • 2.3.3.2.2 Beta kafirin
    • 2.3.3.2.3 Gamma kafirin
    • 2.3.3.2.4 Delta-kafirin
  • 2.3.3.3 Kafirin digestibility
  • 2.4 WHEAT STARCH AND ITS ROLE IN FLOUR QUALITY
  • 2.5 WHEAT PROTEINS AND THEIR ROLE IN FLOUR QUALITY
  • 2.6 FACTORS RELATED TO FLOUR FUNCTIONALITY
  • 2.6.1 Water absorption and water solubility of flour
  • 2.6.2 Dough rheology
  • 2.6.3 Kafirin modification and its effect on flour functional properties
  • 2.7 ANALYTICAL TECHNIQUES FOR FLOUR COMPOSITION AND QUALITY
    • 2.7.1 Microscopy
    • 2.7.1.1 Scanning Electron Microscopy (SEM)
    • 2.7.1.2 Transmission electron microscopy (TEM)
    • 2.7.1.3 Confocal laser scanning microscopy (CLSM)
    • 2.7.1.4 Fourier transform infrared (FTIR) spectroscopy
  • 2.7.2 Rheometry
  • 2.7.3 Electrophoresis
  • 2.8 CONCLUSIONS
• 3 HYPOTHESES AND OBJECTIVES
  • 3.1 HYPOTHESES
  • 3.2 OBJECTIVES
  • 4 RESEARCH
• 4.1 NOVEL BIOFORTIFIED SORGHUM LINES WITH COMBINED WAXY (HIGH AMYLOPECTIN) STARCH AND HIGH PROTEIN DIGESTIBILITY TRAITS: EFFECTS ON ENDOSPERM AND FLOUR PROPERTIES
  • 4.1.1 Abstract
  • 4.1.2 Introduction
  • 4.1.3 Materials and Methods
    • 4.1.3.1 Sorghum samples
    • 4.1.3.2 Grain endosperm and protein body structure
    • 4.1.3.3 Flour preparation
    • 4.1.3.4 Flour moisture content
    • 4.1.3.5 Protein content
    • 4.1.3.6 Starch amylose content
    • 4.1.3.7 In vitro protein digestibility
    • 4.1.3.8 Flour thermal properties
    • 4.1.3.9 Flour pasting properties
    • 4.1.3.10 Gel texture properties of flour
    • 4.1.3.11 Flour WAI and WSF
    • 4.1.3.12 Statistical analysis
    • 4.1.4 Results and discussion
    • 4.1.4.1 Waxy and high protein digestibility traits
    • 4.1.4.2 Grain endosperm texture and structure
    • 4.1.4.3 Flour thermal properties
    • 4.1.4.4 Flour pasting and gel properties
    • 4.1.4.5 Flour water absorption and solubility
    • 4.1.5 Conclusions
    • 4.1.6 References
  • 4.2 EFFECTS OF GENETICALLY MODIFIED SORGHUMS WITH SUPPRESSED GAMMA-KAFIRIN SYNTHESIS ON THEIR FLOUR AND DOUGH RHEOLOGICAL CHARACTERISTICS
  • 4.2.1 Abstract
  • 4.2.2 Introduction
  • 4.2.3 Materials and Methods
    • 4.2.3.1 Sorghum samples
    • 4.2.3.2 Sorghum milling
    • 4.2.3.3 Protein content
    • 4.2.3.4 Starch amylose content
    • 4.2.3.5 In vitro pepsin protein digestibility
    • 4.2.3.6 Differential scanning calorimetry (DSC) of flour thermal behaviour
    • 4.2.3.7 Flour pasting profile
    • 4.2.3.8 Gel strength (texture)
    • 4.2.3.9 Flour WAI and WSF
    • 4.2.3.10 Stress relaxation behaviour of doughs
    • 4.2.3.11 Dynamic rheological analysis
    • 4.2.3.12 Confocal laser scanning microscopy (CLSM)
    • 4.2.3.13 Statistical analysis
  • 4.2.4 Results and discussion
    • 4.2.4.1 Starch amylose content
    • 4.2.4.2 Protein digestibility
    • 4.2.4.3 Thermal characteristics
    • 4.2.4.4 Pasting profile and gel properties
    • 4.2.4.5 Water absorption and solubility
    • 4.2.4.6 Amplitude and temperature sweeps
    • 4.2.4.7 Stress relaxation
    • 4.2.4.8 Sorghum dough (thick slurry) microstructure
    • 4.2.5 Conclusions
  • 4.2.6 References
  • 4.3 CHEMICAL AND DOUGH FORMING PROPERTIES OF KAFIRIN EXTRACTED FROM CONVENTIONALLY BRED AND GENETICALLY MODIFIED SORGHUMS WITH ALTERED KAFIRIN SYNTHESIS
  • 4.3.1 Abstract
  • 4.3.2 Introduction
  • 4.3.3 Materials and Methods
    • 4.3.3.1 Sorghum lines
    • 4.3.3.2 Kafirin extraction
    • 4.3.3.3 Kafirin dough formation
    • 4.3.3.4 Electrophoresis
    • 4.3.3.4.1 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
    • 4.3.3.4.2 Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)
    • 4.3.3.5 Fourier Transform Infrared Spectroscopy (FTIR)
    • 4.3.3.6 Microscopy
    • 4.3.3.6.1 Stereomicroscopy
    • 4.3.3.6.2 Confocal Laser Scanning Microscopy (CLSM)
    • 4.3.3.6.3 Scanning Electron Microscopy (SEM)
    • 4.3.3.7 Statistical analysis
  • 4.3.4 Results and discussion
    • 4.3.4.1 Kafirin dough formation
    • 4.3.4.2 Secondary structure by FTIR
    • 4.3.4.3 Electrophoresis
  • 4.3.5 Conclusions
  • 4.3.6 References
• 5 GENERAL DISCUSSION
  • 5.1 METHODOLOGICAL CONSIDERATIONS
  • 5.2 EFFECT OF THE MODIFIED KAFIRIN EXPRESSION ON ENDOSPERM TEXTURE
• • 5.3 PROPOSED MECHANISM FOR KAFIRIN DOUGH FORMATION BY COACERVATION FROM A SOLUTION OF KAFIRIN IN GLACIAL ACETIC ACID
• • 5.4 PROPOSED MECHANISM OF HOW MODIFIED KAFIRINS IMPROVE SORGHUM FLOUR DOUGH RHEOLOGICAL PROPERTIES
  • 5.5 FUTURE RESEARCH WORK AND DEVELOPMENT OF THE KAFIRIN DOUGH APPROACH
• 6 CONCLUSIONS AND RECOMMENDATIONS
• 7 REFERENCES
• 8 PUBLICATIONS, PRESENTATIONS AND POSTERS BASED ON THIS RESEARCH

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