PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF ENZYME MODIFIED TAPIOCA STARCHES

HONG LEE FEN

UNIVERSITI SAINS MALAYSIA 2007

i

PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF ENZYME MODIFIED TAPIOCA STARCHES

by

HONG LEE FEN

Thesis submitted in fulfillment of the requirements for the degree of

Master of Science

January 2007

ii

ACKNOWLEDGEMENTS

First of all, I would like to send my sincere gratitude and appreciation to

both my supervisors, Associate Prof. Dr. Norziah Mohd. Hani and Associate Prof.

Dr. Abdul Karim Alias for their priceless support and guidance throughout the

duration of this research project. Many thanks to all the laboratory assistants in

Food Technology division especially Mr. Joseph for their assistance whenever I

needed them.

I am forever grateful to my collegues and friends, Soo Lee, Yi Shin, Siew

Wai, Yin Hui, Zhang Qian, Bhupinder, Budi, Lai Hoong, Guan Seng, Sabiha,

Zaleha, Ruri, Hayati, Maizura, Dihar, Sapina, Syarifah and Wahidah. Discipline,

determination, focus, and patience are the words that come to my mind when I

think of them. Their advice and attentions were invaluable.

A special word of gratitude is dedicated to Su Yee for his endless support,

encouragement and care which help me a lot in successfully completing my

research project!

Any acknowledgement I make would not be complete without thanking my

parents, sisters and brother for their moral support, care and motivation during

my research project. Thank you once again to all of you!

HONG LEE FEN

January, 2007

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TABLE OF CONTENTS Page

ACKNOLEDGEMENTS ii

TABLE OF CONTENTS iii

LIST OF TABLES vii

LIST OF FIGURES ix

LIST OF ABBREVIATIONS xiii

LIST OF APPENDICES

xiv

ABSTRAK xvi

ABSTRACT xix

CHAPTER ONE: INTRODUCTION

1

1.1 Background 1

1.2 Objectives 5

CHAPTER TWO: LITERATURE REVIEW 6

2.1 Starch 6

2.1.1 Overview of tapioca plant (Manihot esculenta) 6

2.1.2 Tapioca starch in Malaysia and Asia countries 9

2.2 Molecular structure of starch granules 10

2.2.1 Amylose 14

2.2.2 Amylopectin 17

2.3 Granular structure of starch 21

2.4 Gelatinization of starch 29

2.5 Pasting of starch 36

iv

2.6 Swelling and α-glucan leaching 40

2.7 Retrogradation of starch 42

2.8 Starch gelation 45

2.9 Starch modifications 51

2.9.1 Enzyme modifications via enzymatic debranching process

59

2.10 Fat replacers 63

2.11 Food emulsion: mayonnaise 67

2.12 Rheological studies on food emulsion 70

CHAPTER THREE: MATERIALS AND METHODS 73

3.1 Materials 73

3.2 Starch isolation 73

3.3 Proximate analysis 74

3.3.1 Determination of moisture 74

3.3.2 Determination of crude protein 74

3.3.3 Determination of crude fat 75

3.3.4 Determination of ash 76

3.3.5 Determination of crude fiber 77

3.4 Starch content 77

3.5 Debranching process 79

3.6 Reducing value 82

3.7 Fractionation of amylose 83

3.8 Average degree of polymerization 84

v

3.9 Total carbohydrate 85

3.10 Physicochemical characterization of starch samples 85

3.10.1 Molecular weight distribution 86

3.10.2 Scanning electron microscopy (SEM) 87

3.10.3 X-ray diffraction pattern 87

3.10.4 NMR determination 89

3.10.5 Swelling and solubility properties 90

3.10.6 Thermal properties 91

3.10.7 Pasting properties 92

3.10.8 Particle size 93

3.10.9 Lubricating properties 93

3.11 Addition of EMS into several mayonnaise formulations 93

3.11.1 Mayonnaise preparation 94

3.11.2 Rheological properties 96

3.11.3 Textural properties 96

3.11.4 Sensory evaluation 97

3.11.5 Calorie values 97

3.12 Statistical analysis 98

CHAPTER FOUR: RESULTS AND DISCUSSION 99

4.1 Proximate analysis 99

4.2 Percent hydrolysis 101

4.3 Average degree of polymerization of amylose fractions 106

vi

4.4 Physicochemical properties 109

4.4.1 Determination of molecular weight of debranched tapioca starch

109

4.4.2 Morphology studies 117

4.4.3 X-ray diffraction pattern 123

4.4.4 NMR determination 128

4.4.5 Swelling power and solubility 130

4.4.6 Gelatinization properties as determined by DSC 134

4.4.7 Pasting properties 143

4.4.8 Particle size of starch granules 154

4.4.9 Lubricating properties 155

4.5 Fat mimetic properties of EMS fractions 156

4.5.1 Rheological measurements on mayonnaise

157

4.5.2 Texture of mayonnaise 167

4.5.3 Sensory evaluation of mayonnaise 169

4.5.4 Calorie values of mayonnaise 173

CHAPTER FIVE: CONCLUSIONS 174

CHAPTER SIX: RECOMMENDATIONS FOR FUTURE

RESEARCH 176

REFERENCES 177

APPENDICES 199

vii

LIST OF TABLES Table Title

Page

2.1 Characteristics of starch granules of diverse botanical origin.

12

2.2 Characteristics of amylose and amylopectin.

13

3.1 Sample codes of enzyme modified starch fractions and control starch fractions at various incubation times.

86

3.2 Formulations of prepared mayonnaise samples.

95

4.1 Composition (% dry basis, except moisture) of native CT (commercial tapioca) and SK1 (Sri Kanji One).

100

4.2 Percent hydrolysis of native and EMS fractions from CT (commercial tapioca) and SK1 (Sri Kanji One) samples debranched with 30% w/v enzyme concentration.

104

4.3 Average DP of amyloses fractionated from native and EMS from CT (commercial tapioca) and SK1 (Sri Kanji One) samples debranched with 30% w/v enzyme concentration.

109

4.4 Molecular weight distribution of native and EMS fractions from CT (commercial tapioca) starch.

115

4.5 Molecular weight distribution of native and EMS fractions from SK1 (Sri Kanji One) starch.

116

4.6 X-ray diffraction angle (2θ) and d spacing values of the major peaks from the X-ray pattern of native and EMS fractions from CT (commercial tapioca) and SK1 (Sri Kanji One) starches.

127

4.7 Degree of branching of native and EMS fractions from CT (commercial tapioca) starch.

130

4.8 Swelling power and solubility of native and EMS fractions from CT (commercial tapioca) starch.

133

4.9 Swelling power and solubility of native and EMS fractions from SK1 (Sri Kanji One) starch.

134

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4.10 Thermal characteristics of native and EMS fractions from CT (commercial tapioca) starch.

137

4.11 Thermal characteristics of native and EMS fractions from SK1 (Sri Kanji One) starch.

137

4.12 Pasting properties of native and EMS fractions from CT (commercial tapioca) starch.

146

4.13 Pasting properties of native and EMS fractions from SK1 (Sri Kanji One) starch.

147

4.14 Particle size of starch granules of native and EMS fractions from CT (commercial tapioca) and SK1 (Sri Kanji One) starches.

155

4.15 Lubricating properties of native and EMS fractions.

156

4.16 Physical properties of mayonnaise.

168

4.17 Sensory scores of mayonnaise samples.

172

4.18 Calorie values for mayonnaise samples.

173

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LIST OF FIGURES Figure Title

Page

2.1 Tapioca tuber (Alden, 2005).

9

2.2 Amylose and amylopectin structure.

13

2.3 Helical conformation of amylose, left handed helix containing six glucose units per turn (Cornell, 2004).

16

2.4 Evolution of amylopectin structure proposed by various authors: (A) laminated structure by Haworth, 1937; (B) comb-like model by Staudinger, 1937; (C) bush structure by Meyer, 1940; (D) revision of bush structure, 1971; (E) cluster model by French, 1972; (F) cluster model by Hizukuri, 1986.

19

2.5 Proposed structure for potato amylopectin by Robin et al., (1974). 1 = compact area; 2 = less compact area; arrows indicate branching points; 0 = reducing unit.

20

2.6 (A) Internal structure of crosslinked potato starch after enzyme attack (Hollinger and Marchessault, 1975); (B) Schematic representation of arrangement of amylopectin molecules within a growth ring. The individual molecule may be interwined (French, 1984).

22

2.7 (A) Model of starch crystallite showing the possible positioning and interactions of various components (Blanshard, 1987); (B) Cluster arrangement into concentric crystalline layers of the starch granule (Eliasson et al., 1987).

23

2.8 Schematic model for the arrangement of amylopectin in potato starch showing the ‘super-helical’ structure and the amorphous areas inside the crystalline organization (Oostergetel and van Bruggen, 1993).

24

2.9 Diagramatic representation of the lamellar structure of a starch granule. (A) Stacks of microcrystalline lamellae separated by amorphous growth rings. (B) Magnified view of the amorphous and crystalline regions. (C) Double helical structures formed by adjacent chains of amylopectin give rise to crystalline lamellae. Branching points constitute the amorphous regions (Donald et al., 1997).

25

x

2.10 Structure of a starch granule at different levels of magnification, showing the arrangement of blocklets (with slight modification from Gallant et al., 1997).

26

2.11 Structure of a starch granule (Tang et al., 2006).

27

2.12 X-ray diffraction patterns of different starches (Zobel, 1988b).

28

2.13 The crystal structure for A-type and B-type starches of amylose. The black circles represent water molecules within the structure; glucose molecules are represented by open circles (Wu and Sarko, 1978).

29

2.14 Differential scanning calorimetry profiles of potato starch at various solvent fractions (a volume of 0.6 corresponds to about 50%). The heating rate was 10 ºC/min (Donovan, 1979).

31

2.15 The two stage process involved in the gelatinization of starch in excess water (Waigh et al., 2000).

32

2.16 The two stage process involved in the gelatinization of starch in intermediate water for B-(i) and A-type (ii) starch (Waigh et al., 2000).

33

2.17 The single-stage process involved in the gelatnization of starch at low water contents (Waigh et al., 2000).

33

2.18 A schematic representation of granular changes in relationship to viscosity (Thomas and Atwell, 1997).

37

2.19 Influence of hydrothermic processing on physical starch characteristics (Bornet, 1993).

47

2.20 Minimum substrate requirements for direct debranching enzymes (Allen and Dawson, 1975).

63

2.21 Schematic processes of instability in oil-in-water emulsion (Dalgleish, 1997).

69

3.1 A summary of research protocol.

81

3.2 X-ray diffractogram of maize starch showing crystalline portion (A) and amorphous portion (B) (Köksel et al., 1993).

88

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3.3 1H-NMR spectrum of native potato starch showing proton signals of H-1(1→4), H-1(t) and H-1(1→6) (Nilsson et al., 1996).

90

4.1 Hydrolysis of CT (commercial tapioca) starch debranched with different concentrations of pullulanase upon incubation times. (Vertical bar represents standard deviation).

102

4.2 Hydrolysis of SK1 (Sri Kanji One) starch debranched with different concentrations of pullulanase upon incubation times. (Vertical bar represents standard deviation).

102

4.3 Average DP of amylose fractionated from EMS fractions from CT (commercial tapioca) starch at various incubation times. (Vertical bar represents standard deviation).

107

4.4 Average DP of amylose fractionated from EMS fractions from SK1 (Sri Kanji One) starch at various incubation times. (Vertical bar represents standard deviation).

107

4.5 Size exclusion chromatography elution profile of native CT (commercial tapioca) starch, EMS 4h and 48h.

111

4.6 a) Micrographs of native CT at 3,000 x magnification b) Micrographs of EMS 8h from CT at 3,000 x magnification.

118

4.6 c) Micrographs of EMS 48h from CT at 3,000 x magnification.

119

4.7 a) Micrographs of native SK1 at 3,000 x magnification b) Micrographs of EMS 8h from SK1 at 3,000 x magnification.

120

4.7 c) Micrographs of EMS 48h from SK1 at 3,000 x magnification.

121

4.8 Granular morphology of jacatupé starch (De A. Mélo et al., 1994).

121

4.9 X-ray diffraction patterns of native CT (commercial tapioca), EMS 8h and EMS 48h.

124

4.10 X-ray diffraction patterns of native SK1 (Sri Kanji One), EMS 8h and EMS 48h.

125

4.11 Relative crystallinity of native and EMS fractions from CT (commercial tapioca) and SK1 (Sri Kanji One).

128

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4.12 Thermal profile of 2:1 water/starch system of native and EMS fractions from CT (commercial tapioca) starch.

141

4.13 Thermal profile of 2:1 water/ starch system of native and EMS fractions from SK1 (Sri Kanji One) starch.

142

4.14 Pasting profile of native and EMS fractions from CT (commercial tapioca) starch.

153

4.15 Pasting profile of native and EMS fractions from SK1 (Sri Kanji One) starch.

154

4.16 Dynamic oscillatory response of mayonnaise samples; storage modulus (G’) and loss modulus (G’’): CM (●), CFF (■), CRF (♦), ECT 25 (▼), ECT 40 (▲) and ECT 55 (●). G’ in filled symbols and G’’ in open symbols.

161

4.17 Dynamic oscillatory response of mayonnaise samples; storage modulus (G’) and loss modulus (G’’): CM (●), CFF (■), CRF (♦), ESK1 25 (▼), ESK1 40 (▲) and ESK1 55 (●). G’ in filled symbols and G’’ in open symbols.

162

4.18 Dynamic oscillatory response of mayonnaise samples; complex viscosity (|η*|): CM (●), CFF (■), CRF (♦), ECT 25 (▼), ECT 40 (▲) and ECT 55 (●).

165

4.19 Dynamic oscillatory response of mayonnaise samples; complex viscosity (|η*|): CM (●), CFF (■), CRF (♦), ESK1 25 (▼), ESK1 40 (▲) and ESK1 55 (●).

166

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LIST OF ABBREVIATIONS Abbreviations

Caption

HPSEC High performance size exclusion chromatography

SEM Scanning electron microscopy

NMR Nuclear magnetic resonance

DSC Differential scanning calorimetry

RVA Rapid visco analyzer

RVU Rapid visco unit

DP Degree of polymerization

Mw Weight-average molecular weight

Mn Number-average molecular weight

P Polydispersity index

To Onset temperature

Tp Peak temperature

Tc Conclusion temperature

Tc-To Melting temperature range

∆H Enthalpy

G’ Storage modulus

G’’ Loss modulus

|η*| Complex viscosity

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LIST OF APPENDICES Table

Title (Appendix 1) Page

4.19 Percent hydrolysis of control starch fractions from CT (commercial tapioca) and SK1 (Sri Kanji One) samples.

199

4.20 Average DP of amylose fractionated from control starch fractions from CT (commercial tapioca) and SK1 (Sri Kanji One) samples.

199

4.21 Swelling power and solubility of control starch fractions from CT (commercial tapioca) starch.

200

4.22 Swelling power and solubility of control starch fractions from SK1 (Sri Kanji One) starch.

200

4.23 Thermal characteristics of control starch fractions from CT (commercial tapioca) starch.

201

4.24 Thermal characteristics of control starch fractions from SK1 (Sri Kanji One) starch.

201

4.25 Pasting properties of control starch fractions from CT (commercial tapioca) starch.

202

4.26 Pasting properties of control starch fractions from SK1 (Sri Kanji One) starch.

203

4.27 Correlation between physicochemical properties of CT (commercial tapioca).

204

4.28 Correlation between physicochemical properties of SK1 (Sri Kanji One).

205

Figure

Title (Appendix 2) Page

4.20 1H-NMR spectrum of native CT (commercial tapioca).

206

4.21 1H-NMR spectrum of EMS 8h from CT (commercial tapioca).

207

xv

4.22 1H-NMR spectrum of EMS 48h from CT (commercial tapioca).

208

4.23 Thermal profile of 2:1 water/starch system of native and control starch fractions from CT (commercial tapioca) starch.

209

4.24 Thermal profile of 2:1 water/ starch system of native and control starch fractions from SK1 (Sri Kanji One) starch.

210

4.25 Pasting profile of native and control starch fractions from CT (commercial tapioca) starch.

211

4.26 Pasting profile of native and control starch fractions from SK1 (Sri Kanji One) starch.

212

No. Title (Appendix 3)

1 Standard calibration curve for starch content determination

213

2 Standard calibration curve for reducing value determination

213

3 Standard calibration curve for total carbohydrate determination

214

4 Sensory evaluation form

215

No. Title (Appendix 4)

1 Proceeding presented in 1st USM-Penang International Postgraduate Convention.

218

2 Proceeding presented in National Conference on Food Science and Nutrition 2006.

223

xvi

SIFAT-SIFAT FIZIKOKIMIA DAN BERFUNGSI KANJI UBI KAYU

TERUBAHSUAI SECARA ENZIMATIK.

ABSTRAK

Dalam kajian ini, proses penyahcabangan telah dijalankan ke atas kanji ubi kayu

iaitu ubi kayu komersil (CT) dan variati Sri Kanji Satu (SK1) dengan

menggunakan 10%, 20% dan 30% enzim pullulanase (v/wt kanji,db), pada suhu

58 ˚C selama 48 j. Sampel dikeluarkan pada pelbagai julat masa (0, 4, 8, 20, 24,

48 j) untuk analisis bagi peratusan hidrolisis dan purata darjah pempolimeran

(DP). Daripada kedua-dua kanji, penyahcabangan menggunakan 30%

pullulanase memberikan peratusan hidrolisis yang paling tinggi dan purata DP

yang paling rendah menunjukkan lebih banyak penyahcabangan berlaku

daripada tindakan pullulanase. Oleh sebab itu, fraksi kanji terubahsuai enzim

(EMS) yang disediakan daripada 30% enzim telah dikaji secara lebih mendalam

terhadap sifat-sifat fizikokimia dan berfungsi. Sifat-sifat fizikokimia, termasuk sifat

pempesan dan termal telah dijalankan. Kromatogram yang diperolehi daripada

‘high performance size exclusion chromatography (HPSEC)’ menunjukkan

puncak tambahan dalam fraksi EMS, iaitu kehadiran fraksi berberat molekul

rendah. Daripada mikroskop penskanan electron (SEM), selepas proses

penayhcabangan oleh pullulanase, retakan yang ketara telah diperhatikan pada

granul kanji akibat daripada tindakan enzim. Didapati tiada perubahan pada

corak pembelauan sinar-X untuk sampel fraksi EMS berbanding dengan kanji

natif. Dengan peningkatan masa penyahcabangan, fraksi EMS menunjukkan

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peningkatan ‘trend’ pada kuasa pembengkakan tetapi penurunan ‘trend’ pada

keterlarutan. Pengukuran kalorimetri penskanan pembezaan (DSC)

menunjukkan penyahcabangan enzimatik pada kanji ubi kayu mengakibatkan

suhu puncak gelatinisasi (Tp) yang lebih tinggi berbanding dengan kanji natif.

Sifat pempesan yang perolehi daripada ‘rapid visco analyzer (RVA)’

menunjukkan semua fraksi EMS memberi kelikatan pempesan dan nilai ‘setback’

(retrogradasi) yang lebih rendah berbanding dengan kanji natif. Selepas proses

penyahcabangan, saiz partikel granul EMS 48j daripada SK1 menjadi lebih kecil

secara signifikan. Ujian lubrikasi menunjukkan fraksi EMS mempunyai sifat-sifat

pengganti lemak dengan membentuk pes yang licin, berkrim dan ‘opaque’.

Keupayaan fraksi EMS untuk bertindak sebagai pengganti lemak juga dikaji

dengan applikasinya ke dalam formulasi mayonis kurang lemak. EMS 20j

daripada CT dan SK1 telah disediakan dalam keadaan sluri dan ditambah ke

dalam enam formulasi mayonis pada tahap penggantian 25%, 40% dan 55%

daripada minyak soya untuk menghasilkan mayonis kurang lemak (RF) dan

dikenali sebagai ECT 25, 40 and 55 dan juga ESK1 25, 40 dan 55. Sampel-

sampel ini dibandingkan dengan satu mayonis kawalan (CM) yang tanpa

penggantian EMS dan dua mayonis komersil, iaitu mayonis penuh lemak (CFF)

dan mayonis kurang lemak (CRF). Daripada kajian reologikal, ujian osilatori

menunjukkan sifat-sifat gel lemah dan viskoelastik dengan ‘storage modulus (G’)’

lebih tinggi daripada ‘loss modulus (G’’)’ dalam semua sampel mayonis.

Walaubagaimanapun, mayonis RF dengan penggantian EMS menunjukkan nilai

G’ yang lebih tinggi berbanding dengan mayonis CM, CFF dan CRF. Nilai G’

xviii

didapati paling tinggi pada mayonis ECT 55 dan ESK1 55 berbanding dengan

semua sampel mayonis yang lain. ‘Absolute complex viscosity (│η*│)’ pada

mayonis RF meningkat dengan darjah penggantian EMS. Daripada ‘back

extrusion test’ dalam analisis tekstur, mayonis RF dengan penggantian EMS

menunjukkan nilai kekerasan dan kelekatan yang lebih tinggi berbanding dengan

mayonis CM, CFF dan CRF. Mayonis RF pada darjah penggantian 55%

memberikan nilai kekerasan dan kelekatan yang paling tinggi berbanding dengan

mayonis RF pada darjah penggantian 25 dan 40%. Penilaian sensori pada

sampel mayonis RF menunjukkan penerimaan keseluruhan di kalangan ahli

panel sehingga tahap penggantian 40% dengan tiada perubahan signifikan

berbanding mayonis CFF dan CRF. Amaun kalori pada mayonis RF menurun

secara signifikan (p<0.05) sebanyak 44% berbanding sampel mayonis CM.

Keputusan ini menunjukkan keupayaan potensi EMS untuk bertindak sebagai

pengganti lemak dalam produk makanan seperti mayonis.

xix

PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF ENZYME

MODIFIED TAPIOCA STARCHES.

ABSTRACT

In the present study, debranching process was carried out on tapioca starches

namely commercial tapioca (CT) and Sri Kanji One variety (SK1) using 10%,

20% and 30% of pullulanase enzyme (v/wt starch, db), at 58 ˚C over a 48 hr

period. Samples were withdrawn at various time intervals (0, 4, 8, 20, 24 and 48

hr) for analysis of percent hydrolysis and average degree of polymerization (DP).

In both starches, debranching using 30% pullulanase gave the highest percent

hydrolysis and lowest average DP indicated more debranching occurred through

the action of pullulanase. Thus, enzyme modified starch fractions (EMS)

prepared from 30% pullulanase enzyme was further investigated for their

physicochemical and functional properties. The physicochemical properties,

including pasting and thermal properties were studied. Chromatograms obtained

from high performance size exclusion chromatography (HPSEC) showed

additional peak in EMS fractions which correspond to lower molecular weight

fractions. From scanning electron microscopy (SEM), after debranching process

by pullulanase, pronounced cracks were observed in starch granules indicative of

enzyme attack. X-ray pattern of EMS fractions remained unchanged compared to

native starches. Upon debranching time, EMS fractions displayed an increasing

trend in swelling power but decreasing trend in solubility. Differential scanning

calorimetry (DSC) measurements revealed that enzymatic debranching of

xx

tapioca starches resulted in higher peak temperature of gelatinization (Tp)

compared to their native starches. Pasting properties obtained from rapid visco

analyzer (RVA) showed that all EMS fractions exhibited lower pasting viscosity

and setback values (retrogradation) as compared to their native starches. After

debranching process, granular particle size of EMS 48h of SK1 became

significantly reduced. Lubricating test showed that EMS fractions showed fat

mimetic properties by forming a smooth, creamy and opaque paste. The potential

use of EMS fractions as fat mimetic was also evaluated by applying them in

reduced fat mayonnaise formulations. EMS 20h from CT and SK1 were prepared

in a slurry form and used in the preparation of six formulations of mayonnaise at

25%, 40% and 55% substitution of soybean oil to produce reduced fat (RF)

mayonnaise abbreviated as ECT 25, 40 and 55 and also ESK1 25, 40 and 55.

These samples were then compared to a control (CM) mayonnaise without

substitution of EMS and two other commercially prepared mayonnaise, full fat

(CFF) mayonnaise and reduced fat (CRF) mayonnaise. From the rheological

studies conducted on these samples, oscillatory tests showed weak gels and

viscoelastic properties with storage modulus (G’) greater than loss modulus (G’’)

in all mayonnaise samples. However, RF mayonnaise with EMS substitution

gave higher G’ values compared to CM, CFF and CRF mayonnaise. G’ values

were found to be highest in ECT 55 and ESK1 55 mayonnaise compared to all

other mayonnaise samples. The absolute complex viscosity (│η*│) in RF

mayonnaise increased with degree of substitution of EMS. From back extrusion

test in texture analysis, RF mayonnaise with substitution of EMS gave higher

xxi

firmness and adhesiveness values compared to CM, CFF and CRF mayonnaise.

RF mayonnaise at 55% substitution level gave highest firmness and

adhesiveness values compared to samples at 25% and 40% substitution level.

Sensory evaluation of RF mayonnaise showed overall acceptability by a panel of

up to 40% substitution level with no significant difference compared to CFF and

CRF mayonnaise. The amount of calories was found to decrease significantly

(p<0.05) by 44% in RF mayonnaise compared to CM mayonnaise. These results

indicate the potential ability of the EMS to behave as fat replacers in food

products such as mayonnaise.

1

CHAPTER ONE

INTRODUCTION

1.1 Background

Starch is an essential source of carbohydrate in human diet. In addition to

its nutritive value, starch plays an important role to impart certain functional

properties such as viscosity and texture. It has contributed a great economic

importance and is isolated on an industrial scale from various sources of starch

such as tapioca, corn, potato, rice, wheat and others. Although native starch

does have its industrial applications, most of the time the industry require

improved functionality from modified starch. This research is focused on the

production of enzyme modified starch using pullulanase enzyme. Starch consists

of amylose and amylopectin. Amylose is essentially a linear molecule with

degree of polymerization (dp) of about 1,500 - 6,000 glucose unit and is

connected through α-(1→4) linkages. Amylopectin, the predominant molecule in

most normal starches, is a branched polymer which is much larger than amylose.

DP of amylopectin is about 300,000-3,000,000 glucose unit and is composed of

α-(1→4) linked D-glucose segments connected by α-(1→6) linked branched

points. The enzyme, pullulanase attacks α-(1→6) linked branched points to

produce more linear fractions (amylose). According to Chiu and Mason (1995),

these enzyme modified starch containing more linear fractions have shown to

have unique functional properties such as fat replacers.

Starches with high linear fractions (amylose content) are able to bind

strongly and orient water in such a way as to provide a sensation similar to the

2

rheology of fat in the oral cavity. In food product with formulations high in fat or oil

content, high linear fractions starches can act as an alternative to replace oil to

produce lower calorie food product. Low fat meat patties, frozen desserts, dairy

products, sauces and gravies were successfully formulated by using modified

food starch together with other hydrocolloids (Giese, 1992; Yackel and Cox,

1992; Zoulias et al., 2002). High linear fractions starches are believed to possess

the ability to form good edible films which have good barrier against oxygen and

fats (Anonymous, 1997; Krochta and Mulder-Johnston, 1997). Fried foods coated

with high linear fractions starches absorb less oil and make the fried food

become less greasy. Amylose, the linear fraction of starch, is known to form

coherent, relatively strong, free-standing films in contrast to amylopectin films

which are brittle and non-continuous (Gennadios et al., 1997). Edible films and

coatings prepared from high linear fractions starches are used in dried fruits,

candied fruits and deep fried potato products (Krochta and Mulder-Johnston,

1997).

Fat replacer can be a promising additive in the future. This is because

society today is very much concern with healthy food. As we know, obesity is the

side effect of excessive intake of fat. Obesity can link to several chronic disease

including cardiovascular disease and Type 2 diabetes. Production of high linear

fractions tapioca starch can serve as an alternative choice to food developers.

Furthermore, high linear fractions tapioca starch produced can boost our

country's economy by exporting it to overseas and reduce the import of other fat

replacers.

3

Using tapioca starch to produce fat replacer has several advantages.

Food developer is always aiming for improved functionality ingredient but at the

same time, the cost of an ingredient still remains as an important factor due to

cost constraint. Starch being the cheapest form of polysaccharides has become

the choice for food product innovation by the food industry. Even though modified

starch is more expensive than native starch but its price is still considered

reasonable compared to other hydrocolloids, according to a survey done by

Williams and Phillips (2000).

Under European Union labeling regulation, most of the chemically

modified food starches that have been approved as a food additive have to be

assigned with E-number classification. Modified starches which are categorized

as additives are defined as ”substances obtained by one or more chemical

treatments of edible starch which may have undergone a physical or enzymatic

treatment and may be acid- or alkali-thinned or bleached”. Thermally treated

starches, native starches, dextrins, starch modified by acid or alkali treatment,

bleached starch and starch treated by enzymes are categorized as food

ingredients and have no E-number classification (Murphy, 2000). Thus, enzyme

modified starches may appear more natural and appealing to consumers.

Apart from that, tapioca starch has the potential to serve as a fat replacer

in food formulation because it forms a bland and clearer gel and is non allergen

(gluten free), easy to digest and has ideal viscosity (Anonymous, 2006b). With

these advantages, it is believed that high linear fractions tapioca starch has the

4

potential to compete with other fat replacers in the market not only as a food

ingredient but also to serve as alternative yet economic fat replacers.

In addition, through this research, knowledge of the effect of pullulanase

enzyme towards the properties of starch can be gained. Most researchers carried

out debranching process on solubilised starch either solubilised with DMSO,

NaOH or autoclave (Kimura and Robyt, 1996; Wang et al., 1996; Kujawski et al.,

2003). At present, to our knowledge, there are very few articles reported on

debranching raw starch granule. Many previous studies investigate hydrolysis

treatments with amyloglucosidase, α- and β-amylase towards raw starch granule

to examine how these enzymes affect patterns of hydrolysis (surface erosion,

endoerosion, and the erosion at the equatorial groove plane), x-ray diffraction

pattern, gelatinization, granular size and degree of hydrolysis (Franco, 1988;

Planchot et al., 1995; Wang et al., 1996; Sarikaya, 2000; Li et al., 2004). This

study is expected to provide an insight of the action of pullulanase on raw starch

granule and may become useful information to other scientific studies in the

future.

There is a high demand for tapioca starch in Malaysia (Tengku Ahmad,

1998) as the total imports of tapioca had risen from RM 2.8 million to almost RM

47 million in 1995. Thailand is the world’s largest exporter of tapioca starch and

its derivatives (Anonymous, 2001). They produced various type of modified

tapioca starch including hydrolyzed starch by enzyme, acid thinned starch, pre-

gelatinized starch, oxidized starch, dextrinized starch, starch ether, starch ester

and cross-linked starch. The Thai government had put a lot of effort on the

5

research and also policies in order to improve tapioca production, modification,

application, and also local and international trading (Ratanawaraha et al., 2000).

It is hoped that this research on the preparation of modified starch from tapioca

starch could increased utilization of local tapioca starch as well as provide value-

added food ingredients based on tapioca starch.

1.2 Objectives

The objectives of this project were to produce modified tapioca starch

containing higher linear fractions (or amylose content) by using an enzymatic

debranching process. The debranching was carried out on two varieties of

tapioca starch. Studies on the properties of modified starch would be carried out

using instrumental methods and chemical analysis. The debranched tapioca

starch was applied as a fat replacer in a food model system such as in

mayonnaise formulation.

The specific objectives of the research are:

i) To produce enzyme modified starch with high linear fractions from two

tapioca starch varieties using pullulanase enzyme

ii) To study the effects of debranching enzyme on starch granule in terms

of physical and chemical properties

iii) To investigate the functional properties of these modified starch

fractions in mayonnaise formulations as fat mimetic through

rheological, textural and sensorial studies.

6

CHAPTER TWO

LITERATURE REVIEW

2.1 Starch

Most of the starch utilized world-wide comes from a relatively small

number of crops, the most important being maize, potato, wheat and tapioca with

smaller amounts from rice, sorghum, sweet potato, arrowroot, sago and mung

beans. Within Europe, the main sources of starch are maize, wheat and potato

(Batchelor et al., 1996). Starch is the primary source of stored energy in cereal

grains, tubers and other parts of plant. Starch provides 70-80% of the calories

consumed by humans worldwide. In addition to being a major component of the

human diet and their nutritive value, starches and modified starches are an

excellent raw material for modifying the physical properties of many foods for

example as gelling, thickening, adhesion, moisture retention, stabilizing,

texturizing, and antistaling applications. Starch and products derived from starch

are also important in numerous nonfood applications, such as pharmaceuticals,

paper, textile industries, alcohol-based fuels and adhesives (Thomas and Atwell,

1997; Biliaderis, 1998; Tester and Karkalas, 2002).

2.1.1 Overview of tapioca plant (Manihot esculenta)

The plant known as tapioca in the East is also known as cassava in Africa

and manioc or yucca in South America (Watson, 1979; Tester and Karkalas,

2002). Originally it is native to South America and southern and western Mexico.

7

From Mid and South America, tapioca spread to other parts of the world. It was

widely grown in Africa and Southeast Asia by 1850s (Okigbo, 2006).

There are several advantages regarding tapioca crop. It can adapt to poor

soils and resist drought, making it a good famine crop. It is easily propagated by

stem cuttings. It gives a relatively high yield and an excellent source of calories.

Furthermore, it is relatively inexpensive to produce and (i) requires very little

weeding when planted in optimal plant populations; (ii) has no critical planting

date, provided there is enough moisture at planting; and (iii) its roots can be left

stored in the ground and harvested when required (Okigbo, 2006).

There are bitter and sweet tapioca varieties. Figure 2.1 shows a typical

picture of tapioca tuber. Tapioca tubers contain a cyanohydrin (a β-glucoside

also known as linamarin or phaseolunatin), which liberates hydrogen cyanide

(HCN), benzaldehyde and glucose on hydrolysis in an acid medium. It can be

removed after fermentation and by repeated washing of the crushed tubers.

Nowadays, the tubers (containing ~ 25% starch) are processed in modern plants.

Processing comprises of rasping or hammer milling of the tubers, sieving to

remove fibrous and cellular material, repeated washing in centrifuges or

hydrocyclones, dewatering and drying. A thoroughly purified starch may contain

0.04% protein, <0.1% lipid, and ~0.1% ash (Tester and Karkalas, 2002).

Tapioca products generally can be divided into three groups including

human consumption, livestock feed and industrial products. In Far East, Africa,

India and other developing countries tapioca product become their important

staple food (Hrishi and Balagopal, 1979; Tester and Karkalas, 2002). Since

8

1970s, tapioca product as animal feed has became important industry in Tonga,

New Caledonia and Western Samoa and research on animal feed even being

carried out in Western Samoa (Chandra, 1979; Lambert, 1979).

Industrial products including starch production, substrate for single cell

protein and alcohol production, conversion of tapioca starch into alcohol,

glucose, dextrin, monosodium glutamate and single cell protein (Philips, 1979;

Okigbo, 2006). Tapioca starch is widely used in noodle, bakery products,

confectionery, snacks and tapioca pearl which has commercial value. Tapioca

starch has remarkable characteristic including white colour, odorless, bland

flavour, paste clarity, high viscosity and freeze-thaw stability. Tapioca starch has

been modified to improve its functionality such as pre-gelatinized starch, acid-

thinned starch, starch ethers, starch ester, and cross-linked starch (Anonymous,

2001). In 1979, the feasibility of using tapioca as a substrate in the production of

single cell protein (SCP) has been demonstrated by research carried out at

Universities of Guelph, and the International Center for Tropical Agriculture

(CIAT). SCP production technique combined with improved cassava production

techniques is believed to provide means of increasing both the quality and

quantity of animal feed. Another emerging market for tapioca is as substrate for

production of ethyl alcohol (ethanol). Brazil is promoting ethanol production in an

attempt to become nearly self-sufficient in fuel production. The ethanol can be

added to gasoline (up to 20%) to replace imported fuel (Philips, 1979).

9

Figure 2.1 Tapioca tuber (Alden, 2005).

2.1.2 Tapioca starch in Malaysia and Asia countries

Tapioca is known as ‘ubi kayu’ in Malaysia. Malaysia harvested 400,000

tons of tapioca in 1999 from an area of 39,000 hectares. Yield of tapioca is 10.26

tons per hectares in 1999. Malaysia exported 52 tons of dried cassava, 18 tons

of tapioca starch and tapioca of 1,215 tons in 1998 (FAO database, 2000). In

Peninsular Malaysia, production of tapioca is concentrated in the Perak state

(Onwueme, 2002). Sole cropping of tapioca is commonly practice in Malaysia but

sometimes it is also intercropped with maize or groundnut (Onwueme, 2000).

Tapioca is mainly used to manufacture starch and starch products as well as

animal feeds (Senik, 1995; Tengku Ahmad, 1998; Onwueme, 2002). Small

amount of tapioca tubers are usually processed into snack foods or fermented

into a product called ‘tapai’ (Senik, 1995). This is usually done by the small scale

food processing enterprise. Small scale enterprises played an important role in

Malaysia economy, especially in generating vacancy, better income distribution

and as a training ground for entrepreneurs prior to investment in a larger

enterprise (Senik, 1995). Malaysia also imports some tapioca starch and dried

10

tapioca from Thailand. Tapioca research is done in the Malaysian Agricultural

Research and Development (MARDI) (Onwueme, 2002).

Nearly 37% of starch produced in the world is contributed by Asia in 2002.

While about 70% of world starch production was derived from maize. Maize

accounted for only 45 percent of starch production in Asia. Root and tuber crops

supplied more than half of Asia’s starch needs, especially tapioca (24.7 percent),

sweetpotato (23.5 percent) and potato (6.0 percent) (Fuglie et al., 2005). In Asia

countries, Thailand is the world largest exporter of tapioca, representing about 80

percent of the total world exports. However, Thailand only ranks the third among

the world’s producers of tapioca, after Brazil and Nigeria (Anonymous, 2003).

According to Anonymous (2005b) on the global market analysis by FAO,

Asia countries will become major destination of international trade in tapioca.

China will become leading cassava importer. In 2005, China accounted for 53

percent of the global market and is expected to remain the major destination of

trade in tapioca starch and tapioca feed ingredients in 2006. Demand in tapioca

starch in Japan, as well as in China, Indonesia and Malaysia, will remain

increasing in the coming years.

2.2 Molecular structure of starch granules

Starch is synthesized in many parts of plants, e.g., pollen, leaves, stems,

roots, tubers, bulbs, rhizomes, fruits, flowers, and seeds. The biosynthesis of

starch occurs within three types of membrane-bound organelles: chloroplasts,

chloroamyloplasts, and amyloplasts (Shannon and Garwood, 1984; Buléon et al.,

11

1998a; Tester and Karkalas, 2002). Starch is unique among carbohydrate as it

occurs naturally as discrete particles, called granules. Starch granules are

relatively dense, are insoluble, and hydrate slightly in room temperature water

(Whistler and Bemiller, 1997; Biliaderis, 1998). Characteristics of starch granules

are different among the plants botanical origin (Table 2.1) in terms of the size,

distribution, shape, and morphology (Banks and Greenwood, 1975; Whistler and

Bemiller, 1997; Tester and Karkalas, 2002). From Table 2.1, most of the starches

show single size distribution (unimodal), however, barley, rye and wheat exhibit

bimodal distributions (spherical B-granules, 2-10 µm, and lenticular A-granules,

10-40 µm). Rice starch and oat starch granules tend to exist in clusters of

individual granules which are known as compound granules (Juliano, 1984;

Hoover et al., 2003; Bao and Bergman, 2004).

Another uniqueness of starch is that most starch granules are composed

of a mixture of two polymers: amylose and amylopectin (Banks and Greenwood,

1975; Whistler and Bemiller, 1997; Biliaderis, 1998; Taggart, 2004). These two

polymers are built up of the six-carbon sugar D-glucose, often referred to as the

“building block” of starch (Fig. 2.2). Despite both polymers composed of D-

glucose, the structural differences between these two polymers contribute to

large differences in starch properties and functionality. Some important

characteristics of amylose and amylopectin are listed in Table 2.2.

12

Table 2.1 Characteristics of starch granules of diverse botanical origin.

Starch Type Character Shape Diameter (µm) Barley Cereal Bimodal Lenticular (A-type)

Spherical (B-type) 15-25 2-5

Rye Cereal Bimodal Lenticular (A-type) Sperical (B-type)

10-40 5-10

Wheat Cereal Bimodal Lenticular (A-type) Spherical (B-type)

15-35 2-10

Oat Cereal Unimodal Polyhedral 3-10 (single) 80 (compound)

Rice Cereal Unimodal Polyhedral 3-8 (single) 150 (compound)

Sorghum Cereal Unimodal Spherical 5-20 Triticale Cereal Unimodal Spherical 1-30 Millet Cereal Unimodal Polyhedral 4-12 Amylomaize Cereal Unimodal Irregular 2-30 Maize (waxy and normal) Cereal Unimodal Spherical/ polyhedral 2-30 Pea Legume Unimodal Reniform (single) 5-10 Potato Tuber Unimodal Lenticular 5-100 Tapioca Tuber Unimodal Spherical/ Lenticular 5-35 (Tester and Karkalas, 2002).

13

Figure 2.2 Amylose and amylopectin structure.

Table 2.2 Characteristics of amylose and amylopectin.

Characteristic Amylose Amylopectin

Shape Essentially linear Branched

Linkage α-(1→4); [some α-(1→6)-] α-(1→4)and α-(1→6)

Molecular Weight Typically <0.5 million g/mol 50-500 million g/mol

Films Strong Weak

Gel Formation Firm Non gelling to soft

Colour with iodine Blue Reddish brown

(Thomas and Atwell, 1997).

14

The ratio of the two components varies according to the botanical origin

with “waxy” starches containing less than about 15% amylose, normal starches

20-35% and high amylose starches greater than about 40% (Thomas and Atwell,

1997; Tester and Karkalas, 2002). Besides from that, starches also may contain

some minor constituents of proteins, lipids, moisture and ash (Galliard and

Bowler, 1987; Thomas and Atwell, 1997; Tester et al., 2004).

2.2.1 Amylose

Amylose is essentially linear polymer composed almost entirely of α-

(1→4)-linked D-glucose (Fig 2.2). However there is evidence suggested that

some branches are present on the amylose polymer (Banks and Greenwood,

1975; Hizukuri et al., 1981; Curá et al., 1995). The molecular weight (Mw) is

approximately 1 x 105 to 1 x 106 g/mol (Biliaderis, 1998; Buléon et al., 1998a).

According to Hizukuri et al. (1981) there are 9-20 branch points per molecule and

the side chains range in chain length (CL) from 4 to over 100 D-glucose units. It

has been calculated that there are about 1.8x109 amylose molecules per average

starch granule (Buléon et al., 1998a). Degree of polymerization (DP) of amylose

range from 1,500-6,000 (Zobel, 1988a). The size and structure of amylose and

its polydispersity, however, varies according to the plant source, the method of

polymer isolation (usually a solvent precipitation method is used), and the

method of Mw determination (Thomas and Atwell, 1997; Tester and Karkalas,

2002). Amylose chain contain reducing end which is the highly reactive aldehyde

group (Thomas and Atwell, 1997).

15

There are two typical properties of amylose, namely its behaviour to form

complex with complexing agents and also its tendency to aggregate in neutral

aqueous solution. In neutral solutions, amylose exists as a random coil, but in the

presence of complexing agents, including iodine, fatty acids, fatty acid

components of glycerides and some alcohols, it will form a regular helical

structure with about six D-glucose per helical turn with the complexing agent

occupying a position at the centre of the helix (Banks and Greenwood, 1975;

Rappenecker and Zugenmaier, 1981; French, 1984; Fennema, 1985; Tester and

Karkalas, 2002). The interior of the helix is hydrophobic with a diameter of 0.6 nm

whereas outer diameter of helix is 1.4 nm and a pitch per helical turn is 0.8 nm

(Fig. 2.3) (Tester and Karkalas, 2002; Cornell, 2004). Iodine complexation has

become an important diagnostic tool for the characterization of starch. Amylose

molecule binds approximately 20% (w/w) iodine at 20 ºC under conditions in

which amylopectin does not absorb iodine to any appreciable amount (Banks and

Greenwood, 1975). The complex formation with fats and food emulsifiers such as

mono- and diglycerides can shift starch gelatinization temperatures, alter textural

and viscosity profiles of the resultant paste, and limit retrogradation (Thomas and

Atwell, 1997). Another interesting attribute of amylose is its ability to form a gel

after the starch granule is cooked, i.e., gelatinized and pasted (Table 2.2).

Amylose in aqueous solution is unstable in nature. During cooling, amylose

chains which are linear have the tendency to reassociate by hydrogen bonding to

precipitate (retrograde) and set up to form a gel. High amylose starches

16

particularly are quick to set into a gel form after cooling (Zobel, 1988a; Thomas

and Atwell, 1997; Biliaderis, 1998; Taggart, 2004).

Figure 2.3 Helical conformation of amylose, left handed helix containing six glucose units per turn (Cornell, 2004).

17

2.2.2 Amylopectin

Amylopectin, the predominant molecule in most normal starches, is a

branched polymer which is much larger than amylose. Amylopectin with a Mw of

the order of 107-108 g/mol (Banks and Greenwood, 1975; Morrison and Karkalas,

1990; Biliaderis, 1998) is one of the largest known naturally occurring polymers.

It is much more heavily branched, with about 95% α-(1→4) linkages (Fig 2.2). DP

of amylopectin varies from 3x105-3x106 (Zobel, 1988a). The size of this polymer

and its polydispersity varies as a function of botanical origin (Thomas and Atwell,

1997; Tester and Karkalas, 2002). It has been estimated that about 4-6% of the

linkages within an average amylopectin molecule are α-(1→6) linkages. This may

appear to be a small percentage, but it results in more than 20,000 branches in

an average molecule, although the branches themselves are not large (Banks

and Greenwood, 1975; Thomas and Atwell, 1997). Amylopectin is unable to form

stable complexes with iodine because of the short side chains, so only small

amounts of iodine are bound (<0.6%); a red brown complex is formed with iodine

(λmax ≈ 530-540 nm) (Banks and Greenwood, 1975).

Various amylopectin structures have been proposed since 1930s. The

classic structures of the ‘laminated’ and the ‘comb-like’ models (Fig. 2.4A and B)

of amylopectin were proposed (Haworth et al., 1937; Staudinger and Huseman,

1937). The amylopectin structure further evolved to ‘bush structure’ (Fig. 2.4C)

(Meyer et al., 1940). However, in 1960s, question arose regarding the Meyer’s

irregular structure of amylopectin. In answer to these questions, Whelan (1971)

18

submmitted a revision of the structure (Fig. 2.4D) proposed by Meyer. Later on,

‘cluster model’ was proposed by other authors as describe below.

French (1972) proposed a ‘cluster model’ for amylopectin molecule. This

model also explained the high viscosity of amylopectin and the possibility of

building the high molecular weight amylopectin (107-108 g/mol) with the short side

chains of DP 20-25. According to the cluster model, it is easy to increase the

molecular weight by simply increasing the number of clusters (Fig. 2.4E). He also

suggested that the exterior chains of amylopectin molecules as double helices

forming crystalline region, which interspersed with amorphous regions.

In 1974, Robin and co-workers used gel chromatography and enzymatic

treatment on native and lintnerized potato starch which had lead to a new model

of amylopectin (Fig. 2.5) containing clusters of highly ordered side chains with

DP of 15. They indicated the crystalline region of amylopectin consisting of

closely packed chains of about 15 glucose units with a dimension of 60 Å. This

crystalline region appeared to be acid resistant. Amylopectin chains were said to

be responsible for the crystallinity of starch whereas there was no evidence of

amylose participation in the crystalline areas. The cluster model was further

developed by Manners and Matheson (1981), who claimed the branch points are

arrange in “tiers” or clusters and not distributed randomly throughout the

macromolecule.

19

Fig 2.4 Evolution of amylopectin structure proposed by various authors: (A)

laminated structure by Haworth, 1937; (B) comb-like model by Staudinger, 1937; (C) bush structure by Meyer, 1940; (D) revision of bush structure, 1971; (E) cluster model by French, 1972; (F) cluster model by Hizukuri, 1986.

20

Figure 2.5 Proposed structure for potato amylopectin by Robin et al., (1974). 1 = compact area; 2 = less compact area; arrows indicate branching

points; 0 = reducing unit.

Hizukuri (1986) used enzymes to debranch amylopectin, followed by high

performance liquid chromatography (HPLC) to determine the branch size

distribution. Using this method it was possible to separate the chains into two

main peaks, one with an average DP of 11-16 and the second with an average

dp of 40-45 (Hizukuri, 1986). This study contributed to the formation of the

generally accepted cluster model describing the three dimensional structure of

amylopectin (Fig. 2.4F). This model proposes that the amylopectin molecules is

21

made up of three broad classes of glucose chains, A, B and C chains. A-chains

are unbranched and are linked to the molecule through their reducing end-group;

B chains (B1-B4) which are joined to the molecule in the same way but carry one

or more A-chains; and one C-chain, which has the reducing end-group of the

molecule. Typical chain lengths (CL) for A-, B1, B2, B3, and B4 chains for starches

are 12-16, 20-24, 42-48, 69-75, and 101-119 respectively (Hizukuri, 1986, 1988;

Wang and White, 1994; BelloPerez et al., 1996). The A and B1 chains (Fig 2.4F)

are the most external (exterior) and form double helices (and crystallites) within

the native granules (Tester and Karkalas, 2002). Their CL is typically ~12-24

depending on genetic origin (Hizukuri, 1985, 1993; Mua and Jackson, 1997;

Jane, et al., 1999; Franco et al., 2002; Li et al., 2001) and starches with ‘A-type’

crystallinity, (most cereals) having shorter chain lengths on average than ‘B-type’

starches (like potato).

2.3 Granular structure of starch

Early microscopic work with plane-polarized light showed that the granules

are positively birefringent which implies that molecules are radially oriented

(Banks and Greenwood, 1975; Banks and Muir, 1980). Using a more

sophisticated microscopy technique, granule internal structure (Fig. 2.6A) was

revealed when lightly crosslinked potato starch was attacked by α-amylase

(Hollinger and Marchessault, 1975). By using microscopic observation,

Yamaguchi et al. (1979) reported that granules from waxy maize starch show

“rippled” fibrous structures which correspond to growth rings. Their observations

22

and results had lead to the proposal of various ultrastructure of granule by

scientists which will be discussed below.

In 1984, French proposed the growth ring of starch structure which

involves possible arrangement of amylopectin clusters (Fig. 2.6B). The growth

ring is thought to consist of 16 clusters per growth ring and growth rings 120-400

nm long. The crystalline lamellae consisting of double helices were 5 nm long,

and interspersed with amorphous branch regions 2 nm long. This model fitted

well either with crystalline polymorphs of A- or B-type.

Figure 2.6 (A) Internal structure of crosslinked potato starch after enzyme

attack (Hollinger and Marchessault, 1975); (B) Schematic representation of arrangement of amylopectin molecules within a growth ring. The individual molecule may be interwined (French, 1984).

A

B

23

Blanshard (1987) further reviewed starch structure, and presented a

model to describe starch structure to accommodate chemical and physical data

(expecially X-ray scattering) to provide spatial location for amylopectin (double

helices) and amylose (free, as V-type helices with lipids and as double helices as

shown in Figure 2.7A. He pointed out that amylose and lipid are arrayed radially

which certainly allows for the existence of V-amylose in the native starch granule.

In 1987, Eliasson and co-workers combined the cluster structure of amylopectin

into the analogy to the quartz structure (Fig. 2.7B).

Figure 2.7 (A) Model of starch crystallite showing the possible positioning and interactions of various components (Blanshard, 1987); (B) Cluster arrangement into concentric crystalline layers of the starch granule (Eliasson et al., 1987).

A

B

24

In 1993, however, a new idea was proposed by Oostergetel and van

Bruggen using electron optical tomography and cryoelectron diffraction. Their

data imply a ‘super helical’ arrangement of both amorphous and crystalline

lamellae. Moreover distinct superhelices are interlocked through their respective

amorphous and crystalline lamellae to yield a tetragonal symmetry (Fig. 2.8). In

this three dimensional arrangement, the double helical glucans are pointing in the

axis of the superhelix towards the surface of the granule.

Figure 2.8 Schematic model for the arrangement of amylopectin in potato starch showing the ‘super-helical’ structure and the amorphous areas inside the crystalline organization (Oostergetel and van Bruggen,1993).

Small angle and wide angle X-ray scattering (Cameron and Donald, 1992;

Jenkins et al., 1993; Jenkins et al., 1994) contribute to the ‘lamellar stack’ model

proposed by Donald et al. (1997). Starch granules contain alternating amorphous

and crystalline lamellae, with a periodicity of ~9nm as shown in Figure 2.9.