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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
iii
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
viii
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
ix
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
xi
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
xii
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
xiii
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
xiv
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
xvii
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.