Effects of genotype and environment on wheat starch properties

The in vitro enzymatic digestibility of native starch was related to amylose content, starch-granule proteins SGP, lamellar crystallinity, granule size and thermal properties.. The in v

EFFECTS OF GENOTYPE AND

ENVIRONMENT ON WHEAT STARCH PROPERTIES

Minh Tri NHAN

A thesis submitted in fulfilment of the requirement for

the degree of Doctor of Philosophy

Faculty of Agriculture and Environment

The University of Sydney

2013

Statement of originality

I, Minh Tri NHAN, declare that this submission is my own work and that, to the best of my knowledge and belief, this work doesn’t contain any material previously published or written by another person without due acknowledgment, nor has it been submitted for an award of any other degree or qualification of any University

or other institute of higher learning

Minh Tri NHAN

Date: 19 March 2013

Acknowledgements

I would like to acknowledge and extend my heartfelt gratitude to Professor Les Copeland for supervision His advice, guidance, assistance and expertise are unaccountably valuable and helpful for me to complete this thesis

I acknowledge scholarships from Vietnam International Education Development, The University of Sydney World Scholars Program, Christian Rowe Thornett Bequest, and Postgraduate Research Support Scheme of The University of Sydney

I am thankful to the Grain Research and Development Corporation Australian, and the National Variety Trials for providing wheat grain samples and agronomic data

I am grateful for assistance from the staff and use of facilities of the Australian Centre for Microscopy and Microanalysis (Scanning electron microscope), and School of Chemistry (Differential Scanning Calorimetry) I extend my appreciation

to Ms Di Miskelly for her support and use of the facilities at Allied Mills

My acknowledgement extends to Dr Elliot P Gilbert and Dr James Doutch for their assistance and to the Bragg Institute, Australian Nuclear Science and Technology Organisation for access to Small Angle X-ray Scattering instrument

I thank the staff in the Faculty of Agriculture and Environment at The University of Sydney for good support I appreciate kind advice and help from Dr Meredith Wilkes, Dr Thomas Roberts, Dr Bob Caldwell, Dr Edith Lees, Dr Shujun Wang,

Ms Iona Gyorgy, Ms Pamela Stern, Mr Michael Turner, Mr Colin Bailey and Assoc Prof Balwant Singh My thanks extend for help from my colleagues: Michael Nelson, Jingwen Cai and Niza Noor

To achieve my PhD degree, I never forget to thank my teachers who educated and trained me from Phu My Primary, Vinh Phuoc Secondary and Sadec High Schools, and Can Tho University and KULeuven

I am grateful to my father Nghia Nhan and my mother Anh Tran who have raised and educated me Thanks to my brother Tin Nhan and my sister Uyen Nhan for encouragement Thanks to my son Vinh Tien Nhan who always brings me happiness and dynamic to motivate me to study

I would like to thank very much my beloved wife Ngoc My Van Le who always shares, understands, supports and encourages me to study

All are very important for me to complete this thesis

Thank you all,

Minh Tri NHAN

ABSTRACT

Variability of starch between wheat varieties grown in different locations and season causes difficulties in predicting functional performance in food processing and human nutrition The objective of this thesis is to increase the understanding of environmental factors that influence variability of starch structural characteristics, and in turn starch properties that affect wheat grain quality

Five commercial Australian wheat varieties grown in five different geographic regions of Australia in two years were used to isolate and examine starch properties Starch structure at the nanometer scale was characterized by techniques of High Performance Anion Exchange Chromatography (HPAEC) for amylopectin chain length distribution, X-ray diffraction (XRD) for intensity of crystallinity, and small –angle X-ray scattering (SAXS) for crystalline organisation of starch granules Physical and chemical properties of starch were determined in terms of starch granule size distribution, starch-granule proteins, and amylose content Starch functionality was studied by a series of test including swelling power, thermal properties by Differential Scanning Calorimetry (DSC), and pasting properties by Rapid Viscosity

analyser (RVA), gel syneresis, gel strength and in vitro enzymatic digestibility

This study showed that variety was the main influence on starch granule size distribution, starch characteristics (amylose content, starch-granule proteins and amylopectin chain length distribution, starch swelling power) Growth location influenced strongly wheat grain characteristics (grain yield, grain density, grain protein content, total starch content and flower swelling power), lamellar crystallinity, DSC thermal properties and gel hardness Interactions between genotype (varieties) and environmental factors (year, location) contributed significantly to crystallinity, flour swelling power and pasting viscosity Growing conditions (soil type, soil nutrients, temperature, rainfall and the number of clear days) had highly significant correlations with wheat grain yield, protein and starch content, relative crystallinity Environmental temperature parameters before flowering and during grain filling had relationships with DSC thermal temperatures

of starch and hardness of starch gels The temperature parameter, Tmax-Tmin, before flowering had positive correlations with pasting viscosities and gel hardness, whereas Tmax-Tmin during grain filling had negative correlations with breakdown viscosity and gel hardness

With regards to in vitro enzymatic digestibility, variety controlled the initial

digestibility of native starch granules, and the rapidly digestible starch (RDS) of cooked and cooled starch Growth location affected strongly the more advanced digestibility of native starch, the slowly digestible starch (SDS) and resistant starch

(RS) of cooked and cooled starch The in vitro enzymatic digestibility of native starch

had some significant correlations with growing conditions The biphasic kinetic

model described adequately in vitro enzymatic digestion of both native starch and processed starch A first order kinetic model only fitted well to the in vitro enzymatic digestion data of native starch The in vitro enzymatic digestibility of native starch

was related to amylose content, starch-granule proteins (SGP), lamellar crystallinity,

granule size and thermal properties The in vitro enzymatic digestibility of cooked

and cooled had correlations with amylose content, the very short and short chain lengths of amylopectin, starch swelling power (SSP), granule size and pasting viscosity

Storage time was the main factor influencing retrogradation (syneresis and

hardness) and in vitro enzymatic digestibility of stored starch gels However, variety also contributed significantly syneresis and in vitro enzymatic digestibility of stored

starch gels Growth location strongly influenced hardness of stored starch gel Amylose content and size of starch granules played an important role in syneresis

and in vitro enzymatic digestibility of stored starch gels Short chains (DP 7 – 14) of amylopectin had a positive correlation on syneresis and in vitro enzymatic

digestibility of stored starch gels after storage 8 to 17 days

In conclusion, this thesis showed that environment mainly influenced grain quality; both genotype and environment affected strongly starch granule characteristics; and all of genotype, environment and processing influenced significantly starch

functional properties, including in vitro enzymatic digestibility

grain quality

30

1.9.2 Role of environment in plant growth and development 34

1.9.4 Effects of environmental factors on grain development and grain

quality

37

Chapter 4 STARCH CHARACTERISTICS 61

4.2.6 Correlations between growing conditions and starch properties 73

5.2.5 Correlations between growing conditions and structural

6.3.1 Contributions between genotype and growth location for

7.3.1.2 Correlations between digestibility of native starch and starch

properties

122

7.3.1.3 Correlations between growing conditions and in vitro enzymatic

digestibility of native starch

7.3.2.2 Correlation between digestibility of the cooked and cooled

starch with starch properties

130

7.3.2.3 Discussion on variability of the in vitro enzymatic digestibility

of cooked and cooled starch

132

7.4 Comparison in vitro enzymatic digestibility of native starch, and

cooked and cooled starch

8.2.2 In vitro enzymatic digestibility parameters of starch gel during

storage

143

8.2.3 Correlations between stored starch gel syneresis, hardness, in

vitro enzymatic digestibility, and starch properties

146

8.2.3.1 Correlations between stored starch gel syneresis, hardness and

in vitro enzymatic digestibility

146

8.2.3.2 Correlations between stored starch gel syneresis and hardness of

with starch properties

147

8.2.3.3 Correlations between starch properties and in vitro enzymatic

digestibility (RDS, RS and RS-pred) of stored starch gel

149

8.2.3.4 Correlations between starch properties and in vitro enzymatic

digestibility parameters (k1 k2 and k) of stored starch gel

List of Tables

Table 1.1: Characteristics of starch granules from different botanical

sources

7

Table 1.3: Properties of amylopectin molecules from different botanical

Table 4.1: Mean square values from analysis of variance for

characteristics of starch from multiple wheat varieties grown

at different locations in the 2008 and 2009 seasons

63

Table 4.2: Characteristics of starch from multiple wheat varieties grown

at different locations in the 2008 and 2009 seasons

65

Table 4.3: Correlations between characteristics of starch from multiple

wheat varieties grown at different locations in the 2008 and

2009 seasons

72

Table 4.4: Correlations between growing conditions and characteristics

of grains and starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

74

Table 4.5: Summary of significant effects of growing conditions on

characteristics of starch from multiple wheat varieties grown

at different locations in the 2008 and 2009 seasons

76

Table 5.1: Mean square values from variance analysis for structure of

starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

81

Table 5.2: Chain length distribution of amylopectin of starch from

multiple wheat varieties grown at different locations in the

2008 and 2009 seasons

83

Table 5.3: Lamellar crystallinity of starch from multiple wheat varieties

grown at different locations in the 2008 and 2009 seasons

86

Table 5.4: Correlations between structure and properties of starch from

multiple wheat varieties grown at different locations in the

2008 and 2009 seasons

89

Table 5.5: Correlations between growing conditions and structure of

starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

91

Table 5.6: Summary of significant effects of growing conditions on

structure of starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

92

Table 6.1: Mean square values from analysis of variance for functional

properties of starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

97

Table 6.2: Thermal properties, paste viscosities and gel hardness of

starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

100

Table 6.3: Correlations between functional properties of starch from

multiple wheat varieties grown at different locations in the

2008 and 2009 seasons

105

Table 6.4: Correlations between functional properties with structural

characteristics of starch from multiple wheat varieties grown

at different locations in the 2008 and 2009 seasons

106

Table 6.5: Correlations between growing conditions and functional

properties of starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

108

Table 6.6: Summary of significant effects of growing conditions on

functional properties of starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

111

Table 7.1: Mean square values for in vitro enzymatic digestibility of

native starch from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

120

Table 7.2: In vitro enzymatic digestibility parameters of native starch

from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

121

Table 7.3 Correlations between starch properties and in vitro enzymatic

digestibility of native starch from multiple wheat varieties grown at different locations in the 2008 season

123

Table 7.4: Correlations between growing conditions and digestibility of

native starch multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

124

Table 7.5: Mean square values and component of variances for in vitro

enzymatic digestibility of cooked and cooled starch from multiple wheat varieties grown at different locations in the

2008 season

129

Table 7.6: In vitro enzymatic digestibility parameters of cooked and

cooled starch from multiple wheat varieties grown at different locations in the 2008 season

130

Table 7.7 Correlations between starch properties and in vitro enzymatic

digestibility of cooked and cooled starch from multiple wheat varieties grown at different locations in the 2008 season

131

Table 8.1: Mean square values from variance analysis for syneresis,

hardness and in vitro enzymatic digestibility of starch gels after storage at 4 oC

139

Table 8.2: Syneresis, hardness and in vitro enzyme digestibility of

starch gels after storages at 4 oC

141

Table 8.3: Correlations between digestibility, syneresis and hardness of

starch gels after storage at 4 oC

146

Table 8.4: Correlation between starch properties with syneresis and

hardness of starch gels after storage at 4 oC

148

Table 8.5: Correlation between starch properties with RDS, RS and

RS-pred of starch gels after storage at 4 oC

150

Table 8.6: Correlation between starch properties with k, k 1 , and k 2 of

starch gels for storage at 4 oC

152

Table 8.7: Summary of significant correlations between starch

properties with syneresis, hardness and in vitro enzymatic digestibility of starch gels after storage at 4 oC

153

List of Figures

Figure 1.5: Typical X-ray diffraction patterns of A-, B-, C and V-type

Figure 1.10: Molecular modelling representation of amylose–fatty acid

complexes

25

Figure 1.12: Schematic representation of the pathway of starch biosynthesis 32 Figure 1.13: Schematic representation of a wheat plant with photosynthesis 34

Figure 3.1: Map 1 shows the location of the field sites in the Australian

wheat growing districts

54

Figure 3.2: Whole grains of five varieties harvested from the five locations

in the 2008 season

56

Figure 3.3: Whole wheat grains of the five varieties harvested from four

locations in the 2009 season

57

Figure 4.1: Components of variance of characteristics of starch from

multiple wheat varieties grown at different locations in the

2008 and 2009 seasons

64

Figure 4.2: Volume distribution of starch granules from wheat variety

Catalina the 2008 and 2009 seasons

67

Catalina the 2008 and 2009 seasons

69

variety Catalina

79

Figure 5.2: Variance component for structure of starch from multiple

wheat varieties grown at different locations in years 2008 and

2009 seasons

82

Figure 5.3: Small Angle X-ray Scattering of starch granules from wheat

variety Catalina the 2008 and 2009 seasons

85

Figure 5.4: X-ray diffraction patterns of starch granule from wheat variety

Catalina the 2008 and 2009 seasons

87

Figure 6.1: Components of variance for functional properties of starch

from multiple wheat varieties grown at different locations in the 2008 and 2009 seasons

98

Figure 6.2: DSC thermogram of starch from wheat variety Catalina grown

at different locations Beckom (Bec), Delungra (Del), Lockhart (Loc), Merrinee (Mer), and Minyip (Min) in the 2008 and

2009 seasons

99

Figure 6.3: RVA profiles of starch (9%) of wheat variety Catalina grown

at different locations Beckom (Bec), Delungra (Del), Lockhart (Loc), Merrinee (Mer), and Minyip (Min) in the 2008 and

2009 seasons

101

Figure 6.4: Gel texture profiles of starch (9%) from the wheat variety

Catalina grown at different locations Beckom (Bec), Delungra (Del), Lockhart (Loc), Merrinee (Mer), and Minyip (Min) in the 2008 and 2009 seasons

103

Figure 7.1: In vitro enzymatic digestion of native starch from wheat

variety the 2008 and 2009 seasons

Figure 8.1: The two models were used to fit data of in vitro digestibility

data of the 8 day stored gel of starch

137

Figure 8.2: Texture profiles of starch gels (9 %) after storage for the times

shown (2, 4, 8 and 17 days)

138

Figure 8.3: Components of variance for in vitro enzymatic digestibility,

syneresis and hardness of starch gels after storage at 4oC

140

Figure 8.5: In vitro enzyme digestibility parameters of stored starch gels 144

Abbreviations

ANOVA Analysis of variance

D 24h Digestibility after 24 hour

D 2h Digestibility after 2 hour

D 6.5h Digestibility after 6.5 hour

D90 Starch granule size with 90% of particles smaller than itself

DSC Differential scanning calorimetry

k 1 Time to reach half of the maximum digestibility

Mean D Mean diameter of starch granules

RC Relative crystallinity

RC/AP Ratio of crystallinity to amylopectin

RCv Relative crystallinity of V-pattern

SANS Small-angle neutron scattering

SCFA Short chain fatty acid (s)

Tmin Average of minimum temperature in growing locations

Tc-To Thermal transition temperature range

V/S Ratio of total volume to total surface area of starch granules

1 Chapter 1 LITERATURE REVIEW

1.1 Introduction

Globally, wheat (Triticum aestivum) is the second most produced food crop among

the cereals, after maize Wheat is a food crop while maize is grown more for feed The average total production of wheat in worldwide was approximately 650 million tonnes in 2010 – 2011 and is predicted to be 700 million tonnes in 2011 – 2012 (Abbassian and Racionzer, 2012) The largest area for wheat production is the region of Central West Asia and North Africa with 52 million hectares, followed by North America with 40 million, South Asia with 37 million, Eastern Europe and Russia with 36 million, East Asia with 29 million, the European Union with 17 million and Australia with 12 million hectares (Rajaram and Braun 2008)

Wheat consumption has been increasing by about 5.6 million tonnes/season due to population growth and people migrating from rural to urban areas (Carter 2002) For example, wheat-based foods such as Asian noodles or flat breads are now commonly consumed in Western countries, while pan breads, hamburger buns, pizza, and pasta are now common in Asia and the Middle East Consequently, the wheat processing industry demands wheat grains with specific quality attributes to satisfy the processing requirements of diverse traditional and non-traditional wheat-based foods Therefore, countries need to develop new wheat varieties that combine high yield to satisfy the needs of farmers and high-quality to satisfy the demands of local consumers and the export trade (Carter 2002)

Wheat grains contain mostly starch, then proteins and lipids Starch is the major carbohydrate stored in wheat grains Protein and lipid are also important for quality of wheat and food products However, as this research focuses on starch, the role of proteins and lipids in grain quality will not be considered Starch presents as a macro–constituent of many foods, contributing 50 – 70% of the energy in the human diet and providing a direct source of glucose for generation of metabolic energy (Copeland,

Blazek et al 2009) Starch is also an important industrial material Annually, about 60 million tonnes of starch are isolated worldwide from cereal crops (maize, wheat and rice), tuber and root crops (cassava, potatoes and sweet potatoes) Sixty percent of starch is used for foods (bakery products, confectionery, sugar syrups and snack foods), and forty percent for pharmaceutical and non-edible uses (seed coating, paper, bioplastics and textiles) (Burrell 2003) To ensure fitness-for-purpose for such a diverse range of end uses, it is necessary to understand the wide range of structure and functional properties of starch (Copeland, Blazek et al 2009)

Starch is a polysaccharide that contains two glucan polymers: amylose with very few branches, and amylopectin with multiple branches The glucan polymers are compacted in starch granules Starches vary in the size and shape of the granules,

in the content of amylose and amylopectin, and in the branching architectures of amylopectin (Hoover, Hughes et al 2010) These characteristics determine starch functional and nutritional properties A property that is of particular current nutritional interest is the susceptibility of starch to digestion Starch that is not degraded rapidly by human digestive enzymes in the upper gut has been associated with health benefits, such as reduction of risks for colon cancer, obesity and diabetes, due to lowering of the glycaemic effect (Perera, Meda et al 2010) The increased resistance of starch to digestion may be due to its intrinsic properties or the result of changes during processing, or due to interactions with other food constituents, especially lipids

Multiple interacting factors influence the structure and properties of starch, including the genetic variability and environment for crop growth, crop management, and processing technology Many studies have reported effects of genotypes (varieties of wheat), the crop management (irrigation, soil types and fertilizers) and growing environment (temperature and rainfall) during grain filling

on yield and protein content of wheat (Bahrani, Abad et al 2009; Bogard, Allard et

al 2010; Bogard, Jourdan et al 2011) In respect of processing technology, many studies have focused on effects of additives (salt, sugar, lipid, and surfactants) and cooking temperatures on starch properties (Kurakake, Hagiwara et al 2004;

Gunaratne, Ranaweera et al 2007; Mira, Persson et al 2007) However, we still lack a good understanding of how the interplay between genetic and environmental factors throughout the growing period influences starch quality

This project will examine the effect of the growing environment (temperature, rainfall, soil types, and soil nutrients) during the crop growth period on the properties of starch in wheat grains The main aim of the project is to investigate genotype and environmental effects on starch structural characteristic properties,

functional properties, in vitro enzyme digestibility and gel retro gradation The

review of the literature will focus on topics relevant to this aim

1.2 Wheat structure and morphology

Wheat composition may vary due to several factors, such as varieties and growing conditions including rainfall, temperature, irrigation and soil properties (Panozzo and Eagles 1998; Mikhaylenko, Czuchajowska et al 2000; Hoover, Hughes et al 2010) The wheat grain has a complex structure composed of different tissues, which have distinct functions and biochemical composition (Fig 1.1)

Figure 1.1: Structure of a wheat grain, from Saulnier, Sado et al (2007)

The starchy endosperm (80–85% of the grain) is mostly composed of starch and proteins, while most of the fibre, vitamins, minerals and antioxidants are concentrated in the outer layers (12–17% of the grain) and the wheat germ (3% of

the grain) (McKevith 2004)

The bran fraction is produced after milling and includes the outer layers surrounding wheat endosperm (including pericarp, testa, and aleurone layer) In wheat, the bran material comprises 14 – 16% of the kernel by weight, and it is rich

in fibre, vitamins and minerals The aleurone layer is usually considered as a part of the endosperm, but it is biologically active due to the presence of lipases and lipoxidases These enzymes can cause rancidity in wheat flour Consequently, the aleurone layer is generally removed as a part of the bran during most flour milling operations (Atwell 2001)

The germ is the embryonic plant It is a rich source of several essential nutrients including vitamins (E, B1), proteins, minerals (Zn, Mg) and dietary fibre It comprises only about 2 – 3% of the kernel The germ contains a large amount of unsaturated fats and oxidative as well as hydrolytic enzymes, rendering the product highly susceptible to rancidity and thus posing major limitations in its utilization (Alok, Srivastava et al 2007) Therefore most commercial milling industries aim at the maximum extraction of the endosperm with the minimum possible contamination

by germ, which is used in other food applications (Sun, Zhu et al 2008)

The endosperm makes up about 80% of the wheat grain and is the primary constituent of flour The endosperm cells are packed with starch granules embedded

in a protein matrix (panel a, Fig.1.2) The cell walls are mostly ruptured in mature grains Starch accounts for 65 – 75% of wheat grain weight, but it can exceed 80%

of the endosperm weight (Hurkman, McCue et al 2003)

Figure 1.2: Scanning electron micrographs of wheat starch granules shown in a central endosperm region: (a) starch packed in protein matrix, (b) starch granules with indentations From Rahman et al (2000)

Most modern milling processes for wheat grains focus mainly on production of white flour without bran and germ When the bran is not separated, or all the flour streams are reunited, the flour is known as whole grain flour, but otherwise the completeness of separation determines the resulting different grades of flour In general, only about 72 – 78% of the endosperm is obtained in white flour, the remainder constituting bran and shorts (Owens 2001)

1.3 The form of the starch granules

In the endosperm, starch is deposited as granules inside amyloplasts, which are specialised starch biosynthetic organelles derived from the same proplastids as chloroplasts, but containing no photosynthetic apparatus

Starch granules vary in size (1 to 100 µm in diameter), shape (round, lenticular, polygonal), size distribution (uni- or bi-modal), as individuals (simple) or associated

in granule clusters (compound), and composition (α-glucan, lipid, moisture, protein and mineral content) reflecting the botanical origin (Zobel and Stephen 1996; Buléon, Colonna et al 1998; Rahman, Li et al 2000; Tester and Karkalas 2002; Tester, Karkalas et al 2004; Copeland, Blazek et al 2009) Wheat, barley, rye and triticale have two types of granules: A-type starch granules with large diameter (10 – 35 μm)

type granules with diameter less than 10 μm (Dai, Yin et al 2009)

Protein

b

In wheat, granule initiation occurs in two phases In the period 3 – 7 days after anthesis, large A-granules are initiated and continue to form until mid-endosperm development In the second phase, which is much more prolific, a period of initiation occurs to develop the small B-granules population Figure 1.2 shows a scanning electron micrograph of a wheat grain in the late stage of development (Rahman, Li et

al 2000) The A and B granules have been packed tightly into the cell (panel a, Fig.1.2), and their surfaces have characteristic indentations (panel b, in Fig 1.2)

Formation of starch granules in wheat endosperm follows a characteristic development pathway The first structures that can be defined as starch are spherical granules of about 0.5 – 1 µm diameter, and these granules continue to grow radially until 2 – 4 µm in diameter Plates in A-granules are developed circularly around the granules The equatorial plate then expands further at its rim to a diameter approaching the maximum granule diameter A period of active deposition on the faces of the equatorial plate then occurs, producing the characteristic lenticular shape of the mature wheat A-granules (Rahman, Li et al 2000; Yin, Qi et al 2012)

On the other hand, deposition of small granules (B-type) is initiated between 16 and

22 days post-anthesis The B granules are located inside the amyloplast membrane These granules remain spherical and do not proceed through the equatorial plate formation stage like the A granules (Rahman, Li et al 2000; Yin, Qi et al 2012)

There are marked differences between botanical sources in the patterns of starch deposition, starch granule size distributions and morphologies The size and properties of various cereal starch granules are summarised in Table 1.1

Although there is considerable natural variation in starch granule dimensions and size distributions, plant breeding technologies can select plant mutants to provide a broader compositional variation under more controlled conditions (Davis J P., Supatcharee N et al 2003) This diversity in the morphology and molecular constituents of starch granules influences starch functionality (Copeland, Blazek et al 2009)

Table 1.1: Characteristics of starch granules from different botanical sources (from Tester and Karkalas, 2002) (2002)

Barley Lenticular (A-type),

Spherical (B-type)

Bimodal 15–25

2–5 Maize (waxy and

normal)

Spherical/polyhedral Unimodal 2–30

80 (compound) Pea Rentiform (single) Unimodal 5–10

150 (compound)

Tapioca Spherical/lenticular Unimodal l 5–45

Wheat Lenticular (A-type)

capacity than the corresponding lipid-extracted material The starch lipids have the potential to moderate starch functionality (Blazek and Copeland 2009)

1.4.2 Structures of amylose and amylopectin

Amylose and amylopectin have different structures and properties (Abdel-Aal, Hucl et al 2002; Blazek and Copeland 2008; Copeland, Blazek et al 2009) Amylose is a relatively long, linear α-glucan containing around 99% α-(14) and less than 1% α-(16) linkages and it differs in size and structure depending on botanical origin (Fig 1.3)

Amylose has a molecular weight of approximately 105 – 106 and a degree of polymerisation by number (DPn) of 300 – 5000 with around 9 – 20 branch points equivalent to 3 – 11 chains per molecule Each chain contains approximately 200 –

700 glucose residues equivalent to a molecular weight of 3 x 104 – 1 x 105 (Tester, Karkalas et al 2004)

Figure 1.3: Structure of amylose and amylopectin Adapted from Tester and Karkalas (2004)

Compared to amylose, amylopectin is a much larger molecule with molecular weight of 107 – 109 and a heavily branched structure containing 95 % α-(1 4) linkages at positions and 5 % α- (16), as shown in Fig 1.3 The DPn is typically the range 9600 – 15,900, but comprises three major species with DPn 13,400 – 26,500, 4400 – 8400 and 700 – 2100 (Tester, Karkalas et al 2004)

Table 1.2: Properties of amylose from different sources (Shibanuma, Takeda et al 1994; Tester, Karkalas et al 2004)

Figure 1.4: Schematic representation of amylopectin Adapted from Hizukuri (1986)

In the structure of amylopectin, the A-chains are defined as unsubstituted, whereas B-chains are substituted by other chains The macromolecule also contains a single C-chain that carries the sole reducing end group The B-chains are also referred to

as B1- B4 (one to four clusters) according to their positions in the cluster structure model proposed by Hizukuri (1986) in Fig 1.4 Thus, B1-chains are short chains, being components of the units of clusters, whereas B2 and B3 are long chains that span over two, three, or more clusters, thereby interconnecting them Typical chain lengths for A, B1, B2, B3 and B4 chains from different starches (after debranching with isoamylase) are 12 – 16, 20 – 24, 42 – 48, 69 –75 and 101 – 115, respectively The ratio of A- to B-chains depends on the starch source and is typically of the order of < 1:1 to > 2:1 on a molar basis or < 0.5:1 to > 1:1 on a weight basis (Tester, Karkalas et al 2004; Kim and Huber 2008)

Table 1.3 shows that the average chain length of starch varies depending on botanical origin, crystallinity and genotype of starch granules

Table 1.3: Properties of amylopectin molecules from different botanical sources

(Tester, Karkalas et al 2004)

1.4.3 Interactions within starch granules

Both amylose chains and exterior chains of amylopectin can form double helices which may in turn associate to form crystalline domains Crystalline double helices (A- and B-type polymorphs) have single helix pitch lengths of 2.1 nm (equivalent to double helical pitch of 1.05 nm) with six glucose residues per single helix pitch The minimum CL of amylopectin required to form double helices is 10 glucose residues, although shorter chains may form helical structures in the presence of longer chains (Gidley and Bulpin 1987; Gidley, Cooke et al 1995) The exterior chains with various lengths have different capacities to form double helices The relatively short exterior chains of cereal starch amylopectin molecules (CL 14 – 20) favours the formation of A-type crystalline polymorphs, while the longer exterior chains of tuber starches (CL 16 – 22, or > 22) favours the formation of B-type polymorphs (Gidley and Bulpin 1987; Hizukuri 1993)

Amylopectin is the predominant crystalline component in starch granules and amylose may be considered as a diluent to amylopectin Nuclear magnetic resonance (NMR) studies provide evidence that amylose forms double helices (and potentially crystalline arrays) in high amylose starches (Tester, Debon et al 2000)

Table 1.4 shows variation in the proportions of double helices and that only some of the amylopectin fraction in starches forms double helices

Table 1.4: Proportion of double helical material in starches of different botanical origins

helical amylopectin (%)

References

Maize, amylose 38 Tester, Debon et al (2000)

Maize, normal 38 – 43 Cooke and Gidley (1992)

Maize, waxy 36 – 50 Bogracheva, Wang et al.(2001) and Paris,

Bizot et al (1999) Potato 48 – 64 Bogracheva, Wang et al (2001); Paris,

Bizot et al (1999); Tester R, Stephane (1999) and Yusuph, Tester et al (2003) Potato, waxy 52 Bogracheva, Wang et al (2001)

Rice 49 – 63 Gidley and Bociek (1985) and Qi, Tester et

al (2003) Wheat 32 – 46 Bogracheva, Wang et al (2001) and Cooke

and Gidley(1992); Morrison, Tester et al (1994)

The tendency for amylose molecules to aggregate, especially around DP 100, is limited in native starches because of the presence of amylopectin and lipid However, the lipid fraction within starch granules is usually insufficient to saturate the entire amylose fraction and hence form fully saturated amylose–lipid complexes Therefore, amylose is referred to as free-amylose (F-AM) and lipid complexed amylose (L-AM) (Famá, Goyanes et al.) These forms may have specific locations within starch granules (Morrison, Tester et al 1993; Morrison and Tester 1994; Morrison, Tester et al 1994; Tester, Debon et al 2000) The amount of lipid-complexed amylose ranges from 15 – 20 % of total amylose for maize and wheat, and 37 – 59% for waxy barley However, Kiseleva, Tester et al (2003) suggests that not all of the lipid in starch granules is complexed

1.4.4 Starch crystallinity

Native starch granules exhibit two main types of X-ray diffraction patterns, the A type for cereal starches and the B type for tuber and amylose-rich starches (Fig 1.5) Another C-type diffraction diagram, which is considered to be a mixture of the A- and B-type, is characteristic of most legume, root and some fruit and stem

starches, and also from cereals grown in specific conditions of temperature and water availability (Gidley and Bulpin 1987; Buléon, Colonna et al 1998; Sevenou, Hill et al 2002) The X-ray patterns of these kinds of starch give stronger diffraction peaks at around 15o, 17o, 18o, and 23o 2θ (Zobel 1988a; Cheetham and Tao 1998; Wang, Yu et al 2007) The crystalline V-form, which is characteristic of amylose complexed with fatty acids and monoglycerides, appears in gelatinized starch and is rarely detected in native starches (Buléon, Colonna et al 1998)

Figure 1.5: Typical X-ray diffraction patterns of A-, B-, C and V-type starch (from Buleon, Colonna et al 1998) (1998)

As represented schematically in Fig 1.6, the packing of the double helices in A- and B-type polymorphic structures are different (Wu and Sarko 1978; Gidley and Bulpin 1987) The packing of these double helices within the A-type polymorphic (crystalline) structure is relatively compacted with low water content, whereas the B-type polymorph has a more open structure containing a hydrated helical core (Fig 1.6) Therefore, it is thought that temperature and hydration conditions during plant growth may induce some important changes in the A: B composition The A type is produced preferentially in dry and warm conditions as opposed to the B type

in wet and cold conditions (Buleon, Duprat et al 1984)

Reflection angle 2θ

Figure 1.6: Schematic presentation of A- and B-type polymorphs of starch Adapted from Wu, H and Sako, A (1978)

However, it is still a major challenge for researchers working on starch biosynthesis and structure to elucidate the mechanism of starch crystallization and granule formation

Certain pea mutants show different proportions of A- and B-type polymorphs within the starch granule and serve as useful models to understand the development of the different polymorphs (Hedley, Bogracheva et al 2002) Indeed, the center of pea starch granules is rich in B-type, while peripheral regions are rich in A-type polymorphs (Wang, Blazek et al 2012)

Table 1.5 shows the variation in amount of crystallinity within starch granules from different botanic origins as measured using X-ray diffraction

Table 1.5: Proportion of crystalline amylopectin in starches of different botanical origins, from Tester, Karkalas et al (2004)

amylopectin (%)

References

Maize, normal 17–42 Cheetham and Tao (1998)

Maize, waxy 37–48 Cooke and Gidley (1992); Paris,

Bizot et al (1999) Potato 23–53 Cooke and Gidley (1992); Paris,

Bizot et al (1999); Yusuph, Tester

et al (2003)

and Tester (1994)

Barley (normal) 20–24 Tang, Watanabe (2002)

1.4.5 Semi-crystalline structure and relationship with growth rings

Some publications (for examples, (Zobel 1988b; Jenkins and Donald 1996; Buléon, Colonna et al 1998; Hoover 2001) have schematically represented the relationship between crystallinity and the molecular composition of the starch granule based on the disposition of amylopectin molecules The diagram in Fig 1.7 is one such representation showing an internal model of starch granules with radial growth rings

of amylopectin in clusters The growth rings are estimated to be 120 – 400 nm thick The double helices forming the subgrowth ring crystalline lamellae represent about 5

nm thick regions (repeats) interspersed with amorphous branch regions about 2 nm long Different crystalline polymorphs (A- and B-type) may be accommodated in this model for the structure of starch granules (Jenkins and Donald 1996)

Figure 1.7: Overview of starch granule structure: (a) the whole grain, (b) the lamellae and (c) the polymer chain Adapted from Jenkins and Donald (1996)

Amylopectin branches are envisaged as radiating from the hilum (the centre of growth) towards the periphery of the granule Amylose does not appear to have any significant effect on crystallinity in normal and waxy starches, both of which display strong birefringence In high amylose starches, the amylose may contribute significantly to the crystallinity (Zobel 1988b; Buléon, Colonna et al 1998; Tester, Debon et al 2000; Hoover 2001; Wang, Blazek et al 2012), although the exact nature of the crystalline polymorphs may be different In waxy starches, the origin

of crystallinity is due to the intertwining of the outer chains of amylopectin (exterior

or external chains, representing A- and B1 type) in the form of double helices These associate together to form ordered regions or ‘crystalline lamellae’ Adjacent double helices give rise to the regular three-dimensional geometrical patterns (Zobel 1988b; Buléon, Colonna et al 1998; Tester, Debon et al 2000; Hoover 2001; Wang, Blazek et al 2012)

Intertwining of adjacent branches of amylopectin to form of helical structures can

be indicated by electromagnetic waves of short wavelength (rays) Wide angle ray scattering (WAXS) is an X-ray diffraction (XRD) technique to obtain useful information about density or intensity of crystallinity of amylopectin in starch

X-granules Small angle X-ray scattering (SAXS) is used to identify the laminar order and crystalline spacing in starch granules (Blazek and Gilbert 2011) WAXS and SAXS are used in parallel to reveal the complex ultrastructure of the granule and to quantify crystallinity and polymorphic forms and crystalline laminates, respectively Small angle neutron scattering (SANS) and SAXS are able to probe structures over

a size range from approximately 1 nm to several hundreds of nm, to understand architectural aspects of the starch granules and complement the X-ray techniques (Donald, Kato et al 2001; Blazek and Gilbert 2011)

Normally, the average diameter of an amylopectin side chain cluster is about 10 nm The small and large blocklets have average diameter of 20 – 50 nm and 50 – 500

nm, respectively The small blocklets contain between two and five amylopectin side chain clusters, while larger blockets contain 5 – 50 amylopectin side chain clusters (Figure 1.8) The presence of the blockets and amorphous channels are considered to play a role in both the resistance of starch to enzymatic attack and the structure of the semicrystalline shells (Gallant, Bouchet et al 1997)

Under light and electron-microscopy, starch granules (for example, pea, potato, wheat) have been observed to have a characteristic layered structure, so-called

‘growth rings’, which are considered to represent periodical, diurnal deposition of starch (Baker, Miles et al 2001; Li, Vasanthan et al 2003; Pilling and Smith 2003; Wang, Blazek et al 2012) This feature is seen as multiple concentric shells (or lamellae) of increasing diameter extending from the hilum towards the surface of granules (like the layers of an onion) Figure 1.9 (Wang, Blazek et al 2012) presents the growth ring in starch granules based on data generated from acid hydrolysis treatment of starch granules

Figure 1.9: Model of pea starch granule organization (from (Wang, Blazek et al 2012)

As discussed by Wang, Blazek et al (2012), Figure 1.9 shows a model of pea starch

granule organization of a pea starch granule hydrolysed by acid in panel (a), a starch granule model showing the growth rings is in (b) and a model for the distribution of amylose and amylopectin is in panel (c) Panels (b) and (c) depict the core as an amorphous region composed mainly of amylose molecules and amylopectin molecules not organised into crystalline arrays The core is surrounded by concentric semicrystalline growth rings composed mainly of crystalline amylopectin interspersed with amylose molecules The amorphous growth rings are composed mainly of extended chains of amylopectin interconnecting the crystalline regions and interspersed amylose molecules

1.5 Starch functionality

When starch is heated in water with shear forces, several processes can occur including swelling, gelatinization, pasting, amylose release, and interactions between amylose and other constituents such as lipids In the cooling phase, a connection between starch chains occurs to form a gel that retrogrades gradually into semicrystalline aggregates with different structure from native granules Cooking and cooling of starch-rich foods can lead to the formation of substantial amounts of retrograded starch It is important to understand the processes of starch gelatinization and retrogradation to predict the functional properties of processed starch from knowledge of the structure of native granules (Copeland, Blazek et al 2009) Differential scanning calorimetry (DSC) is used to determine thermal properties (melting temperature and enthalpy change) of starch by measuring the amount of energy absorbed or released by starch when it is heated or cooled Instruments such as Rapid Visco Analyser (RVA) are used to measure viscosity changes in starch pastes during heating and cooling

1.5.1 Starch gelatinisation, melting and dissolution

According to the theory of Jenkins and Donald (1998), starch gelatinisation occurs when water firstly enters the amorphous growth rings, starch swells to a certain degree, and disruptive stress is transmitted through connecting molecules from the

study starch gelatinization, other researchers (Ratnayake, Jackson et al 2008) suggested that there are three distinct stages in the granule disruption process: (i) water diffusion into the starch granule, (ii) loss of birefringence or ‘‘hydration facilitated melting,’’ and (iii) granular swelling predominantly after loss of birefringence occur during gelatinization when starch is heated in water The gelatinization temperature has been defined as the temperature at which 98 % of the granules lose birefringence using of polarized light hot stage microscopy (Ratnayake, Jackson et al 2008)

During gelatinization, hydrogen bonds between chains are disrupted and the starch granules absorb water and swell to many times their original size At the same time amylose (if present) is preferentially solubilised Amylose molecules begin to leach from the granules as they are disrupted under shear to enhance the paste viscosity to

a maximum The maximum is followed by a decrease in paste viscosity, as the granules rupture and starch molecules are dispersed in the aqueous phase (Parker and Ring 2005) Without mechanical shearing, the swollen granules with high amylopectin content maintain their integrity at temperatures up to 100 oC When cereal starch concentrations are greater than about 6 %, the gelatinized granules fill the available volume producing a viscoelastic material Hence, amylopectin concentration and deformability of the swollen starch granule influences properties

of the paste The swelling of the starch granules on gelatinization clearly has a major impact on the rheology of starch pastes (Parker and Ring 2005)

The rate and extent of swelling and breakdown are dependent on the type and amount of starch, the temperature gradient and shear force (Debet and Gidley 2007) Extent of swelling and the endothermic transition measured by DSC also depend on amount of water in the mixtures (Wang and Copeland 2012) The gelatinization temperature of most starches ranges between 60 and 80 oC depending on factors such as the origin of the starch, size of starch granules, amylose content, lipid content and rate of heating (Buléon, 1998; Lelievre, 1984;

Salman, 2009; Vermeylen, 2005; Wang, 2012)

Table 1.6: Properties of starch from various botanical origins compiled from various authors (Jane, Tunyawat K et al 1994; Singh, Singh et al 2003; Knill and Kennedy 2005)

Pasting temp ( o C)

Cooked properties

of gelatinization, a starch paste consists of a continuous phase of solubilised starch (amylose and/or amylopectin) and a discontinuous phase of granules remnants (unswollen granules, partially swollen granules, fragments of swollen granules, swollen starch aggregates) and retrograded starch precipitates (Berski, Ptaszek et

al 2011) The pasting temperature, which initiates formation of a paste, is usually higher than the gelatinization temperature, as measured by the loss in birefringence (Biliaderis 2009)

Complete molecular dispersion can only be accomplished under conditions of high temperature, high shear, and excess water, which are seldom, encountered

in most food processing applications However, in many instances of processing, what is required is the breakdown of the granule structure via gelatinization (Jenkins and Donald 1998) The starch paste viscosity changes over a broad range upon applied shearing (Mason 2009)

1.5.3 Physicochemical aspects of pastes and gels

The starch gelation process has been described by two possible mechanisms: (i) a thermodynamic process of phase separation followed by development of crystallinity and (ii) non–covalent interactions, e.g., hydrogen bonding, which initiates gel formation followed by aggregation (Case, Capitani et al 1998)

Starch pastes with gel properties are considered useful as thickeners or as gelling agents These properties of viscosity, texture, the paste transparency and retrogradation play very important roles in commercial applications of starch (Mason 2009) Above 6% starch concentration in water, most cooked starches can form elastic gels and there is an approximately linear relationship between the rigidity modulus and concentration

The development of elastic properties in gels is dependent on the concentration

of starch and amylose (Miles, Morris et al 1984; Miles, Morris et al 1985) Amylose concentrations between 0.3 – 2.0% can develop an infinite network structure for gel formation Amylose molecules influence the structural order and levels of macromolecular organization of the starch gels (Tang and Copeland 2007) The aged amylose networks would thus consist of double helices and aggregates of double helices that form junction zones Amylose gel networks can develop in different ways, depending on molecular size, polymer concentration and gelation conditions (Mason 2009)

Gelation for amylopectin is a slower process than that for amylose under appropriate conditions (low temperature and high polymer concentration)