Processing and quality of glutinous rice
Malik Adil Nawaz
BSc (Hons) Agriculture (Food Technology)
MSc (Hons) Agriculture (Food Technology)
Master in Food Studies
A thesis submitted for the degree of Doctor of Philosophy at
The University of Queensland in 2018
School of Agriculture and Food Sciences
Abstract
The loss of stickiness of aged glutinous rice is one of the quality issues. This thesis examined the
various factors that could affect the loss of stickiness of glutinous rice and potential pre-treatments
and storage conditions that could be helpful to maintain the stickiness during storage. In this work,
the effect of modified atmospheric packaging and various pre-processing treatments such as alkali
washing, starch modification, and parboiling were assessed.
Initially, the effect of different rehydration temperatures (30 to 50ºC) and cooking times (2.7 to10.7
min) at 95ºC on the pasting properties of flour of three glutinous varieties (TDK11, TDK8, and
Hom Mali Niaw) was investigated using the RVA. Increased soaking temperature and time
resulted in reduced pasting temperature for all varieties. Extended holding at a cooking
temperature (95ºC) had a more significant (P<0.05) effect on final gel viscosity.
Rice is usually consumed in the form of whole grains. Therefore, it is important to study the
cooking kinetics of whole rice grain. To study the water uptake rate and cooking kinetics of TDK8
(fresh and aged) and TDK11, a novel in situ method using Thermal Mechanical Compression Test
(TMCT) attached to a texture analyzer was developed. The TMCT cooking method was found
valid for in situ analysis of rice cooking by using sample sizes as small as 0.50 g.
A new method (X-ray photoelectron spectroscopy) employed to quantify the surface composition
of raw and cooked rice grains showed that the surface of uncooked grains of glutinous (TDK11)
rice had a higher content of protein and lipids (49.7 and 36.2 %, respectively) to starch (13.4 %)
compared the bulk composition (protein ~ 6.6 %, lipids ~ 0.8 %, and starch ~ 92.6 %). Protein was
hypothesized to contribute to the loss of stickiness. An alkali washing with different concentrations
of NaOH (0 to 0.2 %) of milled grains of TDK8 and Doongara resulted in a significant (P<0.05)
increase in the stickiness and hardness of cooked rice grains, and final viscosity of rice flour, and
a decrease in the amount of retrograded starch. In another study, the acetylation of starch in the
whole grain of TDK8 and Doongara was achieved using various acetic anhydride concentrations
(1-7 g per 100 g of milled grains in 225 mL of water). Results showed that acetylation reduced the
crystallinity of starch with a significant (P<0.05) reduction in peak and final viscosities, and
reduced thermal transition temperatures and enthalpy. Furthermore, the texture of cooked grains
was softer and more adhesive. A significant reduction in the glycemic index (GI) of acetylated
samples was also observed using the in vitro digestion method.
ii
Parboiling of TDK8 and TDK11 was undertaken using various soaking mediums water (control),
3 % NaCl solution and 0.2 % acetic acid solution. These saline and acetic acid soakings improved
the milling efficiency (39 to 41 % HRY for TDK8 and 48 to 53 % HRY for TDK11) when
compared to the control (26 % HRY for TDK8 and 29 % HRY TDK11). Saline and acetic acid
soaking resulted in reduced crystallinity and thermal endotherms. When compared to the control,
saline soaking improved water absorption, resulting in a higher peak (~10 % increase) and final
viscosity (~5 % increase), whereas, the acetic acid soaking restricted swelling, resulting in a
reduced peak (~10 % decrease) and final viscosity (~15 % decrease). Furthermore, parboiling
increased hardness (2.6 to 5 N for TDK8 and 2.3 to 3.5 N for TDK11) and adhesiveness (-0.2 to 0.5 N.s for TDK8 and -0.5 to -0.7 N.s for TDK11) of glutinous rice in saline and acetic acid soaking
as compared to the control (-0.3 to -0.5 N.s for TDK8 and -0.6 to -0.7 N.s for TDK11). Also,
parboiling improved the nutritional quality of glutinous rice by reducing the GI from 116.5 to
100.4 for TDK8 and 94.8 to 72.2 for TDK11. Overall, pre-process treatments of glutinous rice
with alkali washing, acetylation of intact starch and parboiling showed improvement in the grain
quality and which can have commercial potential.
The effect of modified atmospheric packaging (MAP) (control, vacuum, CO₂ and N₂) on the aginginduced changes in the physicochemical properties of TDK8 and TDK11 was also assessed. N2
and CO₂ induced an increase in pasting temperature (72.6ºC ~ control, 73.7ºC ~ N₂ and 74.2ºC ~
CO₂) and a significant (P<0.05) reduction in final viscosity (2276 mPa-s ~ control, 2157 mPa-s ~
N2 and 2216 mPa-s ~ CO2) after 12 months of storage. The in situ TMCT cooking and texture
analysis revealed that MAP slightly slowed the aging-induced changes in the cooking quality and
stickiness of glutinous rice. Overall, among all the storage conditions used, the vacuum was
considered the best to maintain the quality of the glutinous rice.
iii
Declaration by author
This thesis is composed of my original work, and contains no material previously published or
written by another person except where due reference has been made in the text. I have clearly
stated the contribution by others to jointly-authored works that I have included in my thesis.
I have clearly stated the contribution of others to my thesis as a whole, including statistical
assistance, survey design, data analysis, significant technical procedures, professional editorial
advice, financial support and any other original research work used or reported in my thesis. The
content of my thesis is the result of work I have carried out since the commencement of my higher
degree by research candidature and does not include a substantial part of work that has been
submitted to qualify for the award of any other degree or diploma in any university or other tertiary
institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify
for another award.
I acknowledge that an electronic copy of my thesis must be lodged with the University Library
and, subject to the policy and procedures of The University of Queensland, the thesis be made
available for research and study in accordance with the Copyright Act 1968 unless a period of
embargo has been approved by the Dean of the Graduate School.
I acknowledge that copyright of all material contained in my thesis resides with the copyright
holder(s) of that material. Where appropriate I have obtained copyright permission from the
copyright holder to reproduce material in this thesis and have sought permission from co-authors
for any jointly authored works included in the thesis.
iv
Publications during candidature
Peer-reviewed papers
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effect of soaking medium on the
physicochemical properties of parboiled Laotian glutinous rice’, International Journal of Food
Properties, vol. 21, pp. 1896-1910.
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effect of starch modification in the whole
white rice grains on physicochemical properties of two contrasting rice varieties’, Journal of
Cereal Science, vol. 80, pp. 143-149.
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effects of three types of modified
atmospheric packaging on the physicochemical properties of selected glutinous rice’, Journal
of Stored Products Research, vol. 76, pp. 85-95.
Nawaz, MA, Fukai, S & Bhandari, B 2017, ‘In situ analysis of cooking properties of rice by
Thermal Mechanical Compression Test (TMCT) method’, International Journal of Food
Properties, vol. 20, pp. 1174-1185.
Nawaz, MA, Fukai, S & Bhandari, B 2016, ‘Effect of different cooking conditions on the pasting
properties of flours of glutinous rice varieties from Lao People’s Democratic Republic’,
International Journal of Food Properties, vol. 19, pp. 2026-2040.
Nawaz, MA, Fukai, S & Bhandari, B 2016, ‘Effect of alkali treatment on the milled grain surface
protein and physicochemical properties of two contrasting rice varieties’, Journal of Cereal
Science, vol. 72, pp. 16-23.
Nawaz, MA, Gaiani, C, Fukai, S & Bhandari, B 2016, ‘X-ray photoelectron spectroscopic analysis
of rice kernels and flours: Measurement of surface chemical composition’, Food Chemistry,
vol. 212, pp. 349-357.
v
Conference abstracts and presentations
Nawaz, MA, Fukai, S & Bhandari, B 2015, ‘Effect of different cooking conditions on the pasting
properties of flours of glutinous rice varieties from Lao People’s Democratic Republic’, in:
65th Australasian Grain Science Conference, Sydney, NSW, Australia, 16-18 September 2015
(poster presentation).
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2017, ‘Modification of physicochemical
properties of milled rice through alkali treatment: Effect on surface proteins and starch’, in:
Cereals 17, San Diego, CA, USA, 8-11 October 2017 (poster presentation).
Nawaz, MA, Gaiani, C, Fukai, S & Bhandari, B 2017, ‘X-ray photoelectron spectroscopic study
on the chemical composition of rice kernel and flour’, in: 67th Australasian Grain Science
Conference, Christchurch, New Zealand, 20 - 22 September 2017 (abstract).
vi
Publications included in this thesis
Nawaz, MA, Fukai, S & Bhandari, B, ‘2016. Effect of different cooking conditions on the pasting
properties of flours of glutinous rice varieties from Lao People’s Democratic Republic’,
International Journal of Food Properties, vol. 19, pp. 2026-2040. – incorporated as Chapter 3.
Contributor
Statement of contribution
Malik Adil Nawaz (Candidate)
Conception and design (85 %)
Analysis and interpretation (75 %)
Drafting and production (50 %)
Shu Fukai
Conception and design (5 %)
Analysis and interpretation (5 %)
Drafting and production (25 %)
Bhesh Bhandari
Conception and design (10 %)
Analysis and interpretation (20 %)
Drafting and production (25 %)
vii
Nawaz, MA, Fukai, S & Bhandari, B 2017, ‘In situ analysis of cooking properties of rice by
Thermal Mechanical Compression Test (TMCT) method’, International Journal of Food
Properties, vol. 20, pp. 1174-1185. – incorporated as Chapter 4.
Contributor
Statement of contribution
Malik Adil Nawaz (Candidate)
Conception and design (85 %)
Analysis and interpretation (75 %)
Drafting and production (50 %)
Shu Fukai
Conception and design (5 %)
Analysis and interpretation (5 %)
Drafting and production (25 %)
Bhesh Bhandari
Conception and design (10 %)
Analysis and interpretation (20 %)
Drafting and production (25 %)
viii
Nawaz, MA, Gaiani, C, Fukai, S & Bhandari, B 2016, ‘X-ray photoelectron spectroscopic analysis
of rice kernels and flours: Measurement of surface chemical composition’, Food Chemistry, vol.
212, pp. 349-357. – incorporated as Chapter 5.
Contributor
Statement of contribution
Malik Adil Nawaz (Candidate)
Conception and design (80 %)
Analysis and interpretation (70 %)
Drafting and production (40 %)
Claire Gaiani
Conception and design (10 %)
Analysis and interpretation (10 %)
Drafting and production (20 %)
Shu Fukai
Conception and design (5 %)
Analysis and interpretation (5 %)
Drafting and production (20 %)
Bhesh Bhandari
Conception and design (5 %)
Analysis and interpretation (15 %)
Drafting and production (20 %)
ix
Nawaz, MA, Fukai, S & Bhandari, B 2016, ‘Effect of alkali treatment on the milled grain surface
protein and physicochemical properties of two contrasting rice varieties’, Journal of Cereal
Science, vol. 72, pp. 16-23. – incorporated as Chapter 6.
Contributor
Statement of contribution
Malik Adil Nawaz (Candidate)
Conception and design (85 %)
Analysis and interpretation (75 %)
Drafting and production (50 %)
Shu Fukai
Conception and design (5 %)
Analysis and interpretation (5 %)
Drafting and production (25 %)
Bhesh Bhandari
Conception and design (10 %)
Analysis and interpretation (20 %)
Drafting and production (25 %)
x
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effect of starch modification in the whole
white rice grains on physicochemical properties of two contrasting rice varieties’, Journal of
Cereal Science, vol. 80, pp. 143-149. – incorporated as Chapter 7.
Contributor
Statement of contribution
Malik Adil Nawaz (Candidate)
Conception and design (80 %)
Analysis and interpretation (70 %)
Drafting and production (40 %)
Shu Fukai
Conception and design (10 %)
Analysis and interpretation (10 %)
Drafting and production (20 %)
Sangeeta Prakash
Conception and design (5 %)
Analysis and interpretation (5 %)
Drafting and production (20 %)
Bhesh Bhandari
Conception and design (5 %)
Analysis and interpretation (15 %)
Drafting and production (20 %)
xi
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effect of soaking medium on the
physicochemical properties of parboiled Laotian glutinous rice’, International Journal of Food
Properties, vol. 21, pp. 1896-1910. – incorporated as Chapter 8.
Contributor
Statement of contribution
Malik Adil Nawaz (Candidate)
Conception and design (80 %)
Analysis and interpretation (70 %)
Drafting and production (40 %)
Shu Fukai
Conception and design (10 %)
Analysis and interpretation (10 %)
Drafting and production (20 %)
Sangeeta Prakash
Conception and design (5 %)
Analysis and interpretation (5 %)
Drafting and production (20 %)
Bhesh Bhandari
Conception and design (5 %)
Analysis and interpretation (15 %)
Drafting and production (20 %)
xii
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effects of three types of modified
atmospheric packaging on the physicochemical properties of selected glutinous rice’, Journal of
Stored Products Research, vol. 76, pp. 85-95. – incorporated as Chapter 9.
Contributor
Statement of contribution
Malik Adil Nawaz (Candidate)
Conception and design (80 %)
Analysis and interpretation (70 %)
Drafting and production (40 %)
Shu Fukai
Conception and design (10 %)
Analysis and interpretation (10 %)
Drafting and production (20 %)
Sangeeta Prakash
Conception and design (5 %)
Analysis and interpretation (5 %)
Drafting and production (20 %)
Bhesh Bhandari
Conception and design (5 %)
Analysis and interpretation (15 %)
Drafting and production (20 %)
xiii
Manuscripts included in this thesis
No manuscripts submitted for publication.
xiv
Contributions by others to the thesis
Funding source supported by the Australian Center for International Agricultural Research
(ACIAR), Department of Education and Training, Australian Government and the University of
Queensland, Australia.
Prof Bhesh Bhandari, Prof Shu Fukai, Dr Sangeeta Prakash, and Prof Claire Gaiani contributed to
the conception and design of the project as a whole; provided advice; critically reviewed the
conclusion and thesis content. Dr Jaquie Mitchell and Peter Snell provided paddy and milled rice
samples. Dr Barry J Wood, Ying Yu, and Anya Yago supported in XPS, SEM and XRD analysis,
respectively.
Statement of parts of the thesis submitted to qualify for the award of another
degree
“None”.
Research involving human and animal subjects
No animal or human subjects were involved in this research.
xv
Acknowledgements
This project was supported by the Australian Center for International Agricultural Research
(ACIAR). I sincerely thank the Department of Education and Training, the Australian Government
for financial support through the Endeavour Postgraduate Research Scholarship that covered the
cost of my tuition fees and living expenses. I want to thank the University of Queensland for
providing University of Queensland Research Training Tuition Fee Offset. I wish to acknowledge
the National Agriculture and Forestry Research Institute (NAFRI), Lao PDR and Department of
Primary Industries (DPI), NSW, Australia for providing paddy and milled rice samples.
I wish to thank my principal advisor: Prof Bhesh Bhandari for his valuable contribution to my PhD
research, his guidance, and invaluable advice. I also want to thank my associate advisors: Prof Shu
Fukai and Dr Sangeeta Prakash for their support and assistance as well as Dr Jaquie Mitchell
(Research Fellow, School of Agriculture and Food Sciences, University of Queensland) and Peter
Snell (Rice Breeder, NSW DPI) for their useful advice. I am also thankful to Prof Claire Gaiani
(University of Lorraine, Nancy, France), Dr Barry Wood (Scientific Manager, Center for
Microscopy and Microanalysis, University of Queensland) for helping me in surface analysis. I
also acknowledge Ms Ying Yu (AMMRF, CMM, The University of Queensland) for her guidance
in SEM analysis.
A very big “thank you” and appreciation to my friends (PhD candidates) Mr. Pramesh Dhungana,
Ms. Dian Widya Ningtyas, Ms. Bal Kumari Sharma Khanal, Mr. Bhavash Panchal and Mr. Jaspal
Singh for their motivation and helping hand in some of the experiments. Finally, I am very indebted
to my dearest wife, little son, and my parents and relatives for their love, understanding, help,
encouragement and unlimited support during the period of this study.
xvi
Financial support
This research was supported by;
1. The Australian Center for International Agricultural Research (ACIAR).
2. The Department of Education and Training, the Australian Government through
Endeavour Postgraduate Research Scholarship.
3. The University of Queensland through UQRITUIT (University of Queensland Research
Training Tuition Fee Offset) and UQITUITION (University of Queensland International
Tuition).
xvii
Keywords
pasting and thermal properties, grain softening, rice cooking, surface analysis, rice storage, surface
proteins, alkali treatment, acetylation, glycemic index, parboiling.
Australian and New Zealand Standard Research Classifications (ANZSRC)
ANZSRC code: 090899, Food Sciences, 50 %
ANZSRC code: 090802, Food Engineering, 30 %
ANZSRC code: 069999, Biological Sciences, 20 %
Fields of Research (FoR) Classification
FoR code: 0908, Food Sciences, 80 %
FoR code: 0699, Other Biological Sciences, 20 %
xviii
Table of Contents
Abstract ............................................................................................................................ii
Declaration by author ......................................................................................................iv
Publications during candidature ...................................................................................... v
Publications included in this thesis ................................................................................. vii
Manuscripts included in this thesis ................................................................................ xiv
Contributions by others to the thesis ..............................................................................xv
Statement of parts of the thesis submitted to qualify for the award of another degree ...xv
Research involving human and animal subjects ............................................................xv
Acknowledgements ....................................................................................................... xvi
Financial support .......................................................................................................... xvii
Keywords .................................................................................................................... xviii
Australian and New Zealand Standard Research Classifications (ANZSRC) .............. xviii
Fields of Research (FoR) Classification ...................................................................... xviii
List of Figures .............................................................................................................. xxix
List of Tables ............................................................................................................. xxxiv
Rice varieties used in the present study ................................................................... xxxvii
List of abbreviations used in the thesis.....................................................................xxxviii
Chapter 1 General Introduction.................................................................................... 1
1.1. Background ........................................................................................................... 2
xix
1.2. Objectives ............................................................................................................. 4
1.3. Hypothesis ............................................................................................................ 4
1.4. Expected outcomes and significance .................................................................... 5
1.5. Outline of the dissertation ..................................................................................... 5
Chapter 2 Literature review .......................................................................................... 7
2.1. Introduction ........................................................................................................... 8
2.2. Grain Structure...................................................................................................... 9
2.3. The general composition of rice grain ................................................................. 10
2.3.1. Starch ........................................................................................................... 12
2.3.2. Protein .......................................................................................................... 13
2.3.3. Lipids ............................................................................................................ 16
2.3.4. Non-starch polysaccharides.......................................................................... 17
2.3.5. Volatiles ........................................................................................................ 17
2.4. Classification of rice ............................................................................................ 18
2.4.1. Classification by color caryopsis ................................................................... 19
2.4.2. Classification by kernel length and length/width ratio ................................... 19
2.4.3. Classification by type of starch ..................................................................... 20
2.5. Glutinous, waxy or rice variety ............................................................................ 21
2.5.1. Global cultivation and consumption of glutinous rice .................................... 22
2.5.2. Quality characteristics of traditional glutinous rice ........................................ 23
2.5.3. Cooking process of rice ................................................................................ 24
2.6. Pre-treatment processes ..................................................................................... 29
xx
2.6.1. Alkali treatment ............................................................................................. 30
2.6.2. Starch modification ....................................................................................... 30
2.6.3. Parboiling of rice ........................................................................................... 31
2.6.4. Effect of parboiling on the quality attributes of waxy rice .............................. 35
2.7. Aging-induced changes....................................................................................... 36
2.7.1. Aging ............................................................................................................ 36
2.7.2. Mechanism of aging ...................................................................................... 37
2.7.3. Cooked rice texture of the aged rice ............................................................. 37
2.7.4. Effect of storage conditions on physicochemical properties.......................... 38
2.8. Conclusions and perspectives ............................................................................ 38
Chapter 3 Effect of different cooking conditions on the pasting properties of flours
of glutinous rice varieties from Lao People’s Democratic Republic ...................... 40
3.1. Abstract ............................................................................................................... 41
3.2. Introduction ......................................................................................................... 41
3.3. Materials and methods ........................................................................................ 44
3.3.1. Materials ....................................................................................................... 44
3.3.2. Grinding of rice kernels ................................................................................. 44
3.3.3. Apparent amylose content ............................................................................ 44
3.3.4. Pasting properties ......................................................................................... 45
3.3.5. Effect of rehydration time and temperature ................................................... 45
3.3.6. Effect of holding time at 95oC ....................................................................... 46
3.3.7. Statistical analysis ........................................................................................ 46
3.4. Results and discussion ....................................................................................... 46
xxi
3.4.1. Apparent amylose content ............................................................................ 46
3.4.2. Effect of rehydration time and temperature on pasting properties ................ 47
3.4.3. Effect of an extension of holding time at 95ºC on the viscosity ..................... 54
3.5. Conclusions ........................................................................................................ 56
Chapter 4 In situ analysis of cooking properties of rice by Thermal Mechanical
Compression Test (TMCT) method ............................................................................ 58
4.1. Abstract ............................................................................................................... 59
4.2. Introduction ......................................................................................................... 59
4.3. Materials and methods ........................................................................................ 61
4.3.1. The moisture content of rice grains ............................................................... 61
4.3.2. Moisture uptake by rice grains ...................................................................... 61
4.3.3. Measurement of grain softening during hydration and cooking by TMCT device
................................................................................................................................ 61
4.3.4. Pasting properties ......................................................................................... 64
4.3.5. Light microscopy of rice kernels during cooking ........................................... 64
4.3.6. Statistical analysis ........................................................................................ 65
4.4. Results and discussion ....................................................................................... 65
4.4.1. Comparison of grain softening and moisture uptake during hydration .......... 65
4.4.2. The in situ TMCT analysis of cooking properties .......................................... 67
4.4.3. Estimation of the rate of cooking by using the in situ TMCT cooking method69
4.4.4. Pasting properties ......................................................................................... 69
4.4.5. Estimation of cooking time by using light microscopy ................................... 70
4.4.6. Comparison of in situ TMCT cooking, RVA and light microscopy ................. 72
4.5. Conclusions ........................................................................................................ 72
xxii
Chapter 5 X-ray photoelectron spectroscopic analysis of rice kernels and flours:
Measurement of surface chemical composition ...................................................... 74
5.1. Abstract ............................................................................................................... 75
5.2. Introduction ......................................................................................................... 75
5.3. Materials and methods ........................................................................................ 77
5.3.1. Milling of paddy ............................................................................................. 78
5.3.2. Grinding of rice kernels ................................................................................. 78
5.3.3. Chemical analysis of milled white rice .......................................................... 78
5.3.4. Sample preparation of defatted rice kernels and flours ................................. 78
5.3.5. Sample preparation of cooked rice kernels ................................................... 79
5.3.6. Surface chemical analysis ............................................................................ 79
5.3.7. Matrix formula used in the research .............................................................. 79
5.3.8. Confocal analysis of rice kernels and flours .................................................. 80
5.3.9. Scanning electron microscopy of surface and cross-section of uncooked rice
kernels .................................................................................................................... 80
5.3.10. Statistical analysis ...................................................................................... 80
5.4. Results and discussion ....................................................................................... 81
5.4.1. Chemical composition ................................................................................... 81
5.4.2. Pure rice components analysed by XPS to construct the matrix ................... 81
5.4.3. Effect of degree of milling (DOM) on the surface composition of waxy rice
TDK11 .................................................................................................................... 83
5.4.4. Surface composition and microstructure of rice kernels ............................... 84
5.4.5. Surface composition and microstructure of rice flours .................................. 89
5.5. Conclusions ........................................................................................................ 91
xxiii
Chapter 6 Effect of alkali treatment on the milled grain surface protein and
physicochemical properties of two contrasting rice varieties ................................ 93
6.1. Abstract ............................................................................................................... 94
6.2. Introduction ......................................................................................................... 94
6.3. Materials and methods ........................................................................................ 96
6.3.1. Alkali treatment ............................................................................................. 96
6.3.2. Color estimation of alkali treated rice grains ................................................. 96
6.3.3. Confocal laser scanning microscopy (CLSM) ............................................... 97
6.3.4. Crude protein analysis .................................................................................. 97
6.3.5. Textural profile analysis ................................................................................ 97
6.3.6. Pasting properties ......................................................................................... 98
6.3.7. Gelatinization and retrogradation properties ................................................. 98
6.3.8. Statistical analysis ........................................................................................ 98
6.4. Results and discussion ....................................................................................... 99
6.4.1. Color estimation of alkali treated rice grains ................................................. 99
6.4.2. Confocal laser scanning microscopy (CLSM) ............................................. 100
6.4.3. Mass loss during alkali treatment................................................................ 101
6.4.4. Crude protein of control and alkali treated rice samples ............................. 102
6.4.5. Textural profile analysis .............................................................................. 102
6.4.6. Pasting properties ....................................................................................... 104
6.4.7. Gelatinization and retrogradation properties by DSC .................................. 105
6.5. Conclusions ...................................................................................................... 106
Chapter 7 Effect of starch modification in the whole white rice grains on
physicochemical properties of two contrasting rice varieties .............................. 109
xxiv
7.1. Abstract ............................................................................................................. 110
7.2. Introduction ....................................................................................................... 110
7.3. Materials and methods ...................................................................................... 112
7.3.1. Paddy milling .............................................................................................. 112
7.3.2. Grinding of rice kernels ............................................................................... 112
7.3.3. Acetylation of milled rice ............................................................................. 112
7.3.4. Acetyl (%) and degree of substitution ......................................................... 113
7.3.5. X-ray diffraction........................................................................................... 113
7.3.6. Pasting properties ....................................................................................... 114
7.3.7. Gel strength ................................................................................................ 114
7.3.8. Gelatinization and retrogradation properties ............................................... 115
7.3.9. Textural profile analysis .............................................................................. 115
7.3.10. Starch hydrolysis and GI estimation ......................................................... 115
7.3.11. Statistical analysis .................................................................................... 116
7.4. Results and discussion ..................................................................................... 116
7.4.1. Effect of acetic anhydride concentration on acetyl (%) and degree of
substitution (DS) of intact starch in the rice grains ................................................ 116
7.4.2. Mass loss during acetic anhydride treatment .............................................. 117
7.4.3. X-ray diffraction........................................................................................... 118
7.4.4. Pasting properties and gel strength ............................................................ 119
7.4.5. Gelatinization and retrogradation properties ............................................... 120
7.4.6. Textural profile analysis .............................................................................. 120
7.4.7. GI estimation............................................................................................... 121
7.5. Conclusions ...................................................................................................... 123
xxv
Chapter 8 Effect of soaking medium on the physicochemical properties of
parboiled Laotian glutinous rice .............................................................................. 124
8.1. Abstract ............................................................................................................. 125
8.2. Introduction ....................................................................................................... 125
8.3. Materials and methods ...................................................................................... 127
8.3.1. Parboiling .................................................................................................... 127
8.3.2. Head rice yield (HRY) ................................................................................. 128
8.3.3. Mechanical strength .................................................................................... 128
8.3.4. Color estimation .......................................................................................... 129
8.3.5. X-ray diffraction........................................................................................... 129
8.3.6. Pasting properties ....................................................................................... 130
8.3.7. Thermal properties ...................................................................................... 130
8.3.8. Texture profile analysis ............................................................................... 130
8.3.9. Starch hydrolysis kinetics and GI prediction ............................................... 131
8.3.10. Statistical analysis .................................................................................... 132
8.4. Results and discussion ..................................................................................... 132
8.4.1. The hardness of parboiled and milled kernels ............................................ 132
8.4.2. Head rice yield (HRY) ................................................................................. 133
8.4.3. Color change in fresh and parboiled rice .................................................... 133
8.4.4. Pasting properties ....................................................................................... 135
8.4.5. Crystallinity change in starch ...................................................................... 137
8.4.6. Thermal properties ...................................................................................... 138
8.4.7. Textural profile analysis .............................................................................. 139
8.4.8. GI prediction ............................................................................................... 139
xxvi
8.5. Conclusions ...................................................................................................... 142
Chapter 9 Effects of three types of modified atmospheric packaging on the
physicochemical properties of selected glutinous rice ......................................... 143
9.1. Abstract ............................................................................................................. 144
9.2. Introduction ....................................................................................................... 144
9.3. Material and methods........................................................................................ 147
9.3.1. Paddy milling .............................................................................................. 147
9.3.2. Modified atmospheric packaging ................................................................ 147
9.3.3. Scanning electron microscopy of a cross-section of rice kernels ................ 148
9.3.4. Surface chemical analysis .......................................................................... 148
9.3.5. X-ray diffraction........................................................................................... 149
9.3.6. Pasting properties ....................................................................................... 149
9.3.7. Gelatinization and retrogradation properties ............................................... 150
9.3.8. Textural profile analysis .............................................................................. 150
9.3.9. In situ TMCT cooking analysis .................................................................... 151
9.3.10. Statistical analysis .................................................................................... 151
9.4. Results and discussion ..................................................................................... 152
9.4.1 Results. ........................................................................................................ 152
9.4.1.1. Microstructures of fresh and aged rice kernels ........................................ 152
9.4.1.2. Surface composition of uncooked and cooked grains of fresh and aged rice
.............................................................................................................................. 153
9.4.1.3. X-ray diffraction pattern of fresh and aged rice flour ................................ 156
9.4.1.7. In situ TMCT cooking analysis of fresh and aged rice kernels ................. 162
9.4.2. Discussion .................................................................................................. 162
xxvii
9.5. Conclusions ...................................................................................................... 165
Chapter 10 General conclusions and recommendations for future research ...... 166
10.1. General conclusions........................................................................................ 167
10.2. Future direction ............................................................................................... 169
References .................................................................................................................. 171
Appendices ................................................................................................................. 212
xxviii
List of Figures
Figure 2.1 Rice production worldwide in the year 2015-16 (Food and Agriculture
Organization 2018) .......................................................................................................... 8
Figure 2.2 Cross section of a rice kernel (Hoogenkamp et al. 2017) ............................ 10
Figure 2.3 Average protein fractions in milled rice samples (Basak et al. 2002) .......... 14
Figure 2.4 Model for the internal structure of the mature protein body PB-I in rice starchy
endosperm (Saito et al. 2012) ....................................................................................... 15
Figure 2.5 Different ways of rice classification .............................................................. 18
Figure 2.6 Different type of starch found in rice endosperm: (a) Linear structure of
amylose, (b) Branched structure of amylopectin (Belitz et al. 2009) ............................. 20
Figure 2.7 Rice-producing nations are denoted in green. The yellow box highlights the
centre of waxy rice production. Countries in white did not produce rice (Nguyen & Tran
2002) ............................................................................................................................. 22
Figure 2.8 Main stages of the cooking process of glutinous rice .................................. 24
Figure 2.9 State diagram describing the glassy and rubber regions of rice (Cnossen et
al. 2001) ........................................................................................................................ 26
Figure 2.10 Process of starch gelatinization (Singh et al. 2007; Ahmed et al. 2008) .... 27
Figure 2.11 Schematic summarization of the classical chemical methods for starch
modification (Masina et al. 2017) .................................................................................. 31
Figure 2.12 A schematic flow diagram of various parboiling techniques (Dutta & Mahanta
2014) ............................................................................................................................. 33
Figure 2.13 A schematic diagram of the superheated steam dryer (Rordrapat et al. 2005)
...................................................................................................................................... 34
xxix
Figure 2.14 Rapid Visco Analysis curves for rice flour following grain storage for up to 8
months (Tulyathan & Leeharatanaluk 2001) ................................................................. 38
Figure 3.1 Change in pasting temperature (onset of gelatinization) with increasing
apparent amylose content (AAC) .................................................................................. 47
Figure 3.2 Representative RVA curves of selected glutinous varieties (TDK11, TDK8,
and HMN) at standard RVA analysis ............................................................................. 48
Figure 3.3 Effect of soaking time and temperature on the pasting temperature (P temp) of
three different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two nonglutinous rice varieties; (d) IR64, and (e) DG ................................................................ 49
Figure 3.4 Effect of soaking time and temperature on the peak viscosity (Vp) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG................................................................................ 51
Figure 3.5 Effect of soaking time and temperature on the breakdown viscosity (BD) of
three different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two nonglutinous rice varieties; (d) IR64, and (e) DG ................................................................ 52
Figure 3.6 Effect of soaking time and temperature on the trough viscosity (V t) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG................................................................................ 52
Figure 3.7 Effect of soaking time and temperature on the setback viscosity (SB) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG................................................................................ 53
Figure 3.8 Effect of soaking time and temperature on the final viscosity (V f) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG................................................................................ 54
xxx
Figure 3.9 Effect of extended holding time at 95ºC on the pasting properties of glutinous
and non-glutinous rice varieties; (a) Peak viscosity (Vp), (b) Trough viscosity (Vt), (c)
Breakdown (BD), (d) Final viscosity (Vf) and (e) Setback (SB)...................................... 56
Figure 4.1 Illustration of grain softening during soaking and cooking, (a) Overall
experiment assembly, (b) Pictorial representation of the measurement process .......... 62
Figure 4.2 Probe displacement during in situ soaking of rice in a TMCT device at two
compression forces, (a) 0.10 N, and (b) 0.15 N ............................................................ 63
Figure 4.3 The in situ TMCT cooking of rice at 95±1°C with two different rice to water
ratios (1:2 and 1:3) ........................................................................................................ 63
Figure 4.4 Comparison of (a) grain softening measured as probe displacement by using
TMCT and (b) moisture uptake during hydration of fresh TDK8 at different soaking
temperatures (22, 40 and 50ºC) .................................................................................... 65
Figure 4.5 Comparison of (a) grain softening measured by the displacement of the probe
by using TMCT and (b) moisture uptake during hydration of aged TDK8 at different
soaking temperatures (22, 40 and 50ºC) ....................................................................... 66
Figure 4.6 Grain softening measured by probe displacement using TMCT for Doongara
(a) and TDK11 (b) rice varieties during hydration at different soaking temperatures (22,
40 and 50oC) ................................................................................................................. 66
Figure 4.7 The in situ TMCT cooking curves for various rice varieties (Fresh TDK8,
Doongara, TDK11 and aged TDK8) cooked at 95±1ºC using rice to water ratios of 1:3.
Black arrows and numbers correspond to the cooking time .......................................... 68
Figure 4.8 The in situ TMCT cooking of rice at 95±1ºC with two different rice to water
ratios (1:10 and 1:3) ...................................................................................................... 68
Figure 4.9 The RVA viscographs of various rice varieties, (a) Fresh TDK8, (b) Aged
TDK8, (c) TDK11, and (d) Doongara ............................................................................. 70
xxxi
Figure 4.10 The light microscopy of fresh and aged TDK8, TDK11 and Doongara rice
kernels during cooking at 95±1ºC (Black arrows indicate the non-gelatinized white belly
areas) ............................................................................................................................ 71
Figure 5.1 Confocal laser scanning micrographs (CLSM) of control and defatted
uncooked rice kernels of TDK11 (a) and Doongara (DG) (b), cooked rice kernels of TDK11
(c) and Doongara (DG) (d), rice flours of control TDK11 (e), control Doongara (DG) (f),
defatted TDK11 (g), and defatted Doongara (DG) (h). Lipids and proteins are labelled in
red and green respectively ............................................................................................ 86
Figure 5.2 Scanning electron micrographs of uncooked rice kernels of TDK11 and
Doongara (DG); (a) surface of milled TDK11 kernel, (b) cross-section of TDk11 kernel,
(c) surface of milled Doongara (DG) kernel, and (d) cross section of Doongara (DG) kernel
...................................................................................................................................... 87
Figure 6.1 Confocal laser scanning micrographs of control and alkali treated rice grains
of Thadokkham-8 (TDK8) and Doongara (DG). Lipids and proteins are labelled in red and
green, respectively ...................................................................................................... 101
Figure 6.2 Crude protein content of control and alkali treated rice grains; (a)
Thadokkham-8 (TDK8), and (b) Doongara (DG)* ........................................................ 102
Figure 6.3 Textural profile analysis of control and alkali treated rice grains (a)
Thadokkham-8 (TDK8), and (b) Doongara (DG).* Correlation (r) between hardness and
adhesiveness of control and alkali treated rice grains; (c) Thadokkham-8 (TDK8), and (d)
Doongara (DG)+ .......................................................................................................... 103
Figure 7.1 X-ray diffraction and calculated crystallinity (%) of control and acetic anhydride
(1-7 g acetic anhydride per 100 g of milled rice in 225 mL of water) treated rice flour of
Thadokkham-8 (TDK8) and Doongara (DG) as shown on the right side of the figures*
.................................................................................................................................... 118
xxxii
Figure 7.2 Textural profile analysis of control and acetic (1-7 g acetic anhydride per 100
g of milled rice in 225 mL of water) treated rice grains of Thadokkham-8 (TDK8) and
Doongara (DG)* .......................................................................................................... 121
Figure 8.1 Head rice yield (%) and grain hardness (N) of fresh and parboiled rice grains;
(a) Thadokkham-8 (TDK8), (c) Thadokkham-11 (TDK11), and (d) Doongara (DG).*
Correlation (r) between head rice yield (%) and grain hardness (N) in; (b) TDK8, (d),
TDK11, and (f) DG ...................................................................................................... 134
Figure 8.2 Pasting profiles of fresh and parboiled rice grains of Thadokkham-8 (TDK8),
Thadokkham-11 (TDK11), and Doongara (DG) .......................................................... 136
Figure 8.3 X-ray diffraction and calculated crystallinity (%) of fresh and parboiled rice
grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11), and Doongara (DG) as
shown on the right side of the figures* ........................................................................ 137
Figure 8.4 Textural profile analysis of fresh and parboiled rice grains of Thadokkham-8
(TDK8) and Doongara (DG)* ....................................................................................... 140
Figure 9.1 Scanning electron micrographs (SEM) of cross sections of fresh and aged (6
and 12 months under various MAP conditions) milled rice grains of Thadokkham-8
(TDK8), Thadokkham-11 (TDK11) and Doongara (DG). Scale bar on each image = 5 µm
.................................................................................................................................... 152
Figure 9.2 X-ray diffraction pattern of flour of fresh and aged (6 and 12 months under
various MAP conditions) milled rice grains of Thadokkham-8 (TDK8), Thadokkham-11
(TDK11) and Doongara (DG) ...................................................................................... 157
xxxiii
List of Tables
Table 2.1 Range of mean proximate analysis and content (%) of organic fractions of rough
rice and its milling fractions at 14 % moisture (Champagne et al. 2004) ....................... 11
Table 2.2 Vitamin and mineral content of rough rice and its milling fractions at 14 %
moisture (Champagne et al. 2004) ................................................................................ 12
Table 2.3 Properties of starch fractions in rice endosperm (Zobel 1988) ...................... 13
Table 2.4 Composition and energy balance data of selected whole-grain cereals
(Nadathur et al. 2017) ................................................................................................... 13
Table 2.5 Proximate composition and nutrition facts of brown and white glutinous rice
(Amornsin 2003) ............................................................................................................ 21
Table 2.6 Properties of glassy and rubbery states of starch (Truong 2008) .................. 26
Table 3.1 Apparent amylose content (AAC) of selected rice varieties .......................... 47
Table 4.1 The rate of cooking indicated by the rate of probe displacement during in situ
cooking of selected rice varieties (fresh and aged TDK8, TDK11, and Doongara). Higher
rate signifies faster cooking rate* .................................................................................. 69
Table 5.1 Bulk chemical composition of rice varieties TDK11 and Doongara (DG) at 9 %
degree of milling* .......................................................................................................... 81
Table 5.2 Relative elemental compositions of pure rice components measured by XPS*
...................................................................................................................................... 82
Table 5.3 Relative elemental and calculated surface composition (%) of TDK11 with
different DOM (%), control and defatted rice kernels, and control and defatted rice flours
of TDK11 and Doongara (DG)* ..................................................................................... 88
Table 5.4 Relative elemental and calculated surface composition (%) of cooked rice
kernels of TDK11 and Doongara (DG)* ......................................................................... 90
xxxiv
Table 6.1 CIEL*a*b* color space of control and alkali treated rice grains of Thadokkham8 (TDK8) and Doongara (DG)* .................................................................................... 100
Table 6.2 Pasting properties of control and alkali treated flour of Thadokkham-8 (TDK8)
and Doongara (DG)* ................................................................................................... 107
Table 6.3 Gelatinization and retrogradation properties of control and alkali treated flour
of Thadokkham-8 (TDK8) and Doongara (DG)* .......................................................... 108
Table 7.1 Acetylation and degree of substitution of control and acetic anhydride (1-7 g
acetic anhydride per 100 g of milled rice in 225 mL of water) treated flour of Thadokkham8 (TDK8) and Doongara (DG)* .................................................................................... 117
Table 7.2 Pasting properties and gel strength of control and acetic anhydride (1-7 g acetic
anhydride per 100 g of milled rice in 225 mL of water) treated flour of Thadokkham-8
(TDK8) and Doongara (DG)* ....................................................................................... 119
Table 7.3 Experimental results of estimated GI, and gelatinization and retrogradation
properties of control and acetic anhydride (1-7 g acetic anhydride per 100 g of milled rice
in 225 mL of water) treated flour of Thadokkham-8 (TDK8) and Doongara (DG)* ...... 122
Table 8.1 CIEL*a*b* color space of fresh and parboiled rice grains of Thadokkham-8
(TDK8), Thadokkham-11 (TDK11), and Doongara (DG)* ............................................ 135
Table 8.2 Experimental results of estimated GI, and gelatinization and retrogradation
properties of fresh and parboiled rice grains of Thadokkham-8 (TDK8), Thadokkham-11
(TDK11), and Doongara (DG)* .................................................................................... 141
Table 9.1 Relative elemental and calculated surface composition (%) of uncooked rice
kernels of fresh and aged (6 and 12 months under various MAP conditions) milled rice
grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11) and Doongara (DG)* ..... 154
Table 9.2 Relative elemental and calculated surface composition (%) of cooked rice
kernels of fresh and aged (6 and 12 months under various MAP conditions) milled rice
grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11) and Doongara (DG)* ..... 155
xxxv
Table 9.3 Pasting properties of rice flour of fresh and aged (6 and 12 months under
various MAP conditions) milled rice grains of Thadokkham-8 (TDK8), Thadokkham-11
(TDK11) and Doongara (DG)* ..................................................................................... 158
Table 9.4 Gelatinization and retrogradation properties of rice flour of fresh and aged (6
and 12 months under various MAP conditions) milled rice grains of Thadokkham-8
(TDK8), Thadokkham-11 (TDK11) and Doongara (DG)*............................................. 160
Table 9.5 The textural profile and in situ TMCT cooking analysis of milled rice kernels of
fresh and aged (6 and 12 months under various MAP conditions) milled rice grains of
Thadokkham-8 (TDK8), Thadokkham-11 (TDK11) and Doongara (DG)* .................... 161
xxxvi
Rice varieties used in the present study
Doongara (DG)
Hom Mali Niaw (HMN)
IR64
Thadokkham-8 (TDK8)
Thadokkham-11 (TDK11)
xxxvii
List of abbreviations used in the thesis
AACC
American Association for Cereal Chemists
AAC
Apparent amylose content
AC
Amylose content
approx.
Approximately
ACIAR
Australian Centre for International Agricultural Research
BD
Breakdown viscosity
CLSM
Confocal Laser Scanning Microscopy
df
Degree of freedom
DG
Doongara
DMSO
Dimethyl sulfoxide
DOM
Degree of milling
DSC
Differential Scanning Calorimetry
F´ 30s
1st derivative for every 30 sec
g
Gram/Grams
GI
Glycemic index
hr/hrs
hour/hours
Hg
Mercury
HMN
Hom Mali Niaw
Jg-1
Joules per gram
kPa
Kilo Pascal
kV
Kilovolts
MAP
Modified Atmospheric Packaging
mg
Milligram/Milligrams
min
Minute/Minutes
mL
Milliliters
mm
Millimeters
mPa-s
Millipascal-second
xxxviii
N
Newton
n
Number of independent replicates
NAFRI
National Agriculture and Forestry Research Institute
Na2CO3
Sodium carbonate
NaOH
Sodium hydroxide
nm
Nanometers
N.s
Newton second
NSW DPI
New South Wales Department of Primary Industries
PB
Protein body
PBs
Protein bodies
pi
Point of inflection
PT
Peak time
Ptemp
Pasting temperature
r
Correlation
R%
Percentage of retrogradation
RDS
Rapidly digestible starch
RH
Relative humidity
RRAPL
Rice Research Australia Pty Ltd
RVA
Rapid Visco Analyzer
SB
Setback viscosity
SD
Standard deviation
SE
Standard error
sec
Second/Seconds
SEM
Scanning Electron Microscopy
TDK8
Thadokkham-8
TDK11
Thadokkham-11
TMCT
Thermal Mechanical Compression Test
TN
Total nitrogen
xxxix
TPA
Texture Profile Analysis
Tₒ
Onset temperature of gelatinization
Tp
Peak temperature of gelatinization
Tc
Conclusion temperature of gelatinization
Tₒ(r)
Onset temperature of retrogradation
Tp(r)
Peak temperature of retrogradation
Tc(r)
Conclusion temperature of retrogradation
Vf
Final viscosity
Vp
Peak viscosity
Vt
Trough viscosity
v/v
volume by volume
WBPR
weight of brown parboiled rice
WBR
weight of brown rice
WMPR
weight of milled parboiled rice
w/v
weight by volume
w/w
weight by weight
WWR
weight of white rice
XPS
X-ray Photoelectron Spectroscopy
ΔH
Enthalpy of starch gelatinization
ΔH(r)
Enthalpy of retrograded starch
µL
Microliter
µm
Micrometer
xl
Chapter 1 General Introduction
1
1.1. Background
The humans have consumed rice for ages. There is evidence that the rice has been cultivated in
China and Thailand dating from about 6000 BC (Zhang & Hung 2013). Rice (Oryza sativa L.) is
one of the primary food crops in the world and the staple food for almost half of the world’s
population (Childs 2004). In general, rice is classified into two different types, namely glutinous
and non-glutinous based on the native starch present in the endosperm. Glutinous rice primarily
contains amylopectin, and non-glutinous rice contains amylose as well as amylopectin. Glutinous,
waxy or sweet rice is characterized by its opaque appearance and very low amylose content (Wang
& Wang 2002). The pasting and cooking properties of glutinous and non-glutinous rice are quite
different.
Glutinous varieties are grown in many countries, including Lao PDR, Thailand, China, Myanmar,
Vietnam, Cambodia, Japan, Bangladesh, and India (Calingacion et al. 2014; Mar et al. 2015). It is
a staple food of Laotian people. It is usually consumed as a desert, as a breakfast cereal or as
steamed rice in banana leaves in Thailand, Myanmar, Cambodia, India, China and Vietnam
(Schiller et al. 2006). Glutinous rice possesses unique functional and processing properties mainly
due to its distinct starch composition (Kim et al. 2013). The glutinous rice is consumed as milled
raw rice (brown or polished). Milling efficiency and grain quality of rice can be improved by
various preprocessing treatments such as post-harvest tempering and parboiling to reduce the
internal fissures and increase head rice yield (Iguaz et al. 2006).
Usually, rice is harvested at 16-20 % (wet basis) moisture content, dried to 12-14 % moisture,
milled and stored for one year or longer (Truong 2008). However, glutinous rice cannot be stored
for a longer period, as longer storage deteriorates cooking time and the texture by making it harder
and fluffier (Cheaupun et al. 2005). This is a key factor limiting the production efficiency of rice
products such as rice tamale, rice pudding, and rice snacks from glutinous rice (Sung et al. 2008).
Therefore, it is a challenge for rice processing industry to achieve the best quality aged glutinous
rice having smooth and sticky texture after cooking.
Considerable studies have been conducted to investigate the mechanisms of aging (Perdon et al.
1997; Zhou et al. 2002a), storage temperature (Chung & Lim 2003), roles of protein and starch
during aging (Teo et al. 2000). Most of these works have focused on non-waxy rice with very few
studies on waxy rice (Sodhi et al. 2003). Majority of the reported studies on waxy rice elaborate
2
the physicochemical properties (Nicholas et al. 2013; Chun et al. 2015) of waxy rice, influence of
amylopectin chain length (Mar et al. 2015), protein bodies composition (Kim et al. 2013) and,
starch-protein interaction (Zhou et al. 2003a) on the textural attributes of fresh cooked rice. Very
few researchers (Chrastil 1990b; Likitwattanasade & Hongsprabhas 2010; Huang & Lai 2014)
have reported the aging-induced structural changes of macromolecules (starch, protein, and lipid)
in waxy rice cultivars. During rice aging, the enzymatic activity of α- and β-amylase decrease;
however, protease, lipase, and lipoxygenase become more active, increasing the amount of free
fatty acids (FAs) and free amino acids (Dhaliwal et al. 1991). Because of this reason, the surface
of polished aged rice was decreased in pH level (Rehman 2006). In another study, Martin &
Fitzgerald (2002) hypothesised that the polymerisation of rice proteins caused by the disulphide
bond formation during storage, resulting in the decreased protein solubility, increased slurry
viscosity, and reduced cooked aged rice stickiness. Starch is usually considered as an inert
macromolecule; thus the changes of starch properties are considered to be insignificant over the
time during rice aging (Rehman 2006). However, Patindol et al. (2005) proposed enzymatic
degradation of starch might occur during aging, which could cause an increase in the percentage
of short chains (DP 6-12), resulting in harder texture while cooking.
The mechanism of harder texture formation during due to aging reduced water uptake and swelling
ability of aged waxy rice and reduction in the stickiness of cooked waxy rice is still not very clear.
The surface of the grains usually perceives the stickiness of cooked rice grains. Surface
composition of rice can influence the textural attributes especially stickiness of the cooked
glutinous rice. However, the surface composition of cooked rice has not been studied earlier. The
effects of various storage conditions, potential pre-treatments to maintain the stickiness of waxy
rice have not been sufficiently studied. Thus, the present study is mainly focused on the effect of
various factors on the quality of glutinous rice. Effect of various pre-processing techniques such
as surface protein washing by alkali treatment, acetylation of intact starch in the whole grain, and
parboiling on the quality of glutinous rice have been investigated in this study. In addition to this,
the effect of modified atmospheric packaging to slow down the aging-induced quality deterioration
of aged glutinous rice is also included in the study.
3
1.2. Objectives
This project was aimed to investigate the mechanism of textural development and stickiness of the
glutinous rice during cooking and examine the effect of pre-processing treatments and storage on
the cooking quality of glutinous rice.
Following specific objectives were set to target above mentioned broad aim:
I.
To investigate the effect of rehydration time and temperature and extended holding during
cooking on the pasting properties of common Laotian glutinous rice cultivars.
II.
To study the rehydration and cooking kinetics of glutinous rice by in situ Thermal
Mechanical Compression Test (TMCT) cooking.
III.
To study the surface composition of the raw and cooked glutinous rice grains as against
the bulk composition.
IV.
To study the effect of modified atmospheric packaging during aging on the
physicochemical properties of glutinous rice.
V.
To investigate the effect of the removal of protein bodies from the surface of milled
glutinous rice by alkali washing on the stickiness of the cooked grains.
VI.
To study the effect of starch modification by acetylation in the whole rice grains on the
physicochemical properties of glutinous rice.
VII.
To study the efficacy of soaking medium on the physicochemical properties of parboiled
glutinous rice.
1.3. Hypothesis
Following specific hypothesis were tested in this project:
I.
Different rehydration conditions (time and temperature) and cooking times have a distinct
effect on the pasting properties of glutinous rice as a longer rehydration time at higher
temperature, and extended cooking can result in more water absorption, breakdown, and
reduced final viscosity.
II.
The rate of cooking of glutinous and non-glutinous rice can be estimated by grain softening
using the novel in situ TMCT device attached to a texture analyzer.
III.
The aging can affect the rate of water uptake and cooking because of aging-induced
physicochemical changes.
4
IV.
The surface composition, in particular, protein of the grain may influence the stickiness of
cooked glutinous rice. By washing the surface proteins and fat with dilute alkali can expose
more starch on the surface may lead to making sample stickier during cooking.
V.
Acetylation of starches in the grain can slow down the recrystallisation of gelatinised
starch. Therefore, reduced retrogradation due to acetylation may help in maintaining the
stickiness of glutinous rice.
VI.
Thermal treatment such as wet parboiling of fresh glutinous paddy may help in maintaining
to improve quality of parboiled glutinous rice by increasing head rice yield and cooking
quality.
1.4. Expected outcomes and significance
The main expected outcome of this research project is to improve the knowledge of processing
and to optimise the pre-process and storage treatments and influential factors on the stickiness
property of cooked glutinous rice. Also, these findings could assist in understanding the role of
various macromolecules (starch, protein, and fat) on the functional properties of glutinous rice.
The overall outcome of the study is to help rice-producing countries in Asia to improve the quality
of glutinous rice and also to enhance its export.
1.5. Outline of the dissertation
This thesis consists of 10 chapters, starting with a general introduction (Chapter 1), followed by a
literature review (Chapter 2). The research undertaken in the project is described in 7 consecutive
chapters (Chapter 3 to 9) in a format of journal manuscripts. Chapters 3 to 5 are based on the
methodology development and effect of rehydration time and temperature and extended cooking
on the pasting properties of glutinous rice flour. Chapters 6 to 8 are based on the study of the
effects of preprocessing treatments on the physicochemical properties of glutinous rice. Chapter 9
is based on the study of the effects of modified atmospheric packaging on the physicochemical
properties of glutinous rice. Chapter 10 provides the general conclusions and recommendations
from the research.
Chapter 1 describes the background understanding relating to the aging-induced changes in the
physicochemical properties of glutinous rice.
5
Chapter 2 presents a review of literature relating to the general classification of rice based on the
starch type, physicochemical properties of glutinous rice flour and grains. Current literature on the
aging-induced changes of glutinous rice is also reported. Moreover, the literature on the storage
techniques and preprocess treatments of glutinous rice to slow down the aging-induced changes
are also reviewed.
Chapter 3 presents the role of rehydration time and temperature in the gelatinization properties of
glutinous rice flour. This chapter also deals with the effects of extended cooking time on the
pasting profiles of glutinous rice flour.
Chapter 4 illustrates a new procedure of in situ TMCT cooking to study the rate of grain softening
during soaking and cooking. This chapter also deals with the changes in the cooking kinetics due
to the aging of glutinous rice.
Chapter 5 describes a new method of surface analysis of rice grains and flour of glutinous and nonglutinous varieties by using x-ray photoelectron spectroscopy (XPS).
Chapter 6 deals with preprocessing of alkali washing of milled glutinous rice to remove the alkali
soluble protein from the surface glutinous rice to maintain the stickiness of cooked glutinous rice.
Chapter 7 presents the effect of acetylation pre-processing for starch modification in the whole
polished grain of glutinous rice to maintain the stickiness of cooked glutinous rice. This chapter
also deals with in vitro digestion of acetylated glutinous rice.
Chapter 8 presents the preprocessing of parboiling of selected glutinous rice cultivars using the
various soaking medium. This chapter deals also with in vitro digestion of parboiled glutinous rice.
Chapter 9 deals with the efficacy of various modified atmospheric packaging techniques on the
physiochemical properties of glutinous and non-glutinous rice. In this chapter, the novel methods
of in situ TMCT cooking and surface analysis using XPS described in Chapters 4 and 5,
respectively are also used on fresh and aged samples to study the changes in cooking kinetics and
surface composition of fresh and aged samples and how the modified atmospheric packaging
(MAP) can affect the said attributes.
Chapter 10 provides overall conclusions and recommendations for further studies in the future.
6
Chapter 2 Literature review
7
2.1. Introduction
Rice production is geographically concentrated in Eastern and Western Asia with more than 90
percent of world output (Prasad et al. 2017) but the recent increased interest and consumption of
rice in the West, has prompted further research to discover the basis of the distinctive properties
of different cultivars and associated technology (Flor et al. 2016). China and India, which account
for more than one-third of the global population, supply over half of the world's rice (as shown in
Fig. 2.1). Brazil is the biggest non-Asian producer, followed by the United States of America. Italy
is the leading producer in Europe. World production has shown a significant and very stable
growth, mainly due to increased production in Western and Eastern Asia (Muthayya et al. 2014).
Figure 2.1 Rice production worldwide in the year 2015-16 (Food and Agriculture
Organization 2018)
8
Rice is considered as a semiaquatic, annual grass plant and member of the grass family, Poaceae.
This family is divided into some genera or subfamilies, one of which is Oryzoideae. This genus is
further divided into some sections, one of which is Sativae. There is a further subdivision into a
species, the most relevant of which are: Oryza sativa and Oryza glaberrima (Soreng et al. 2015).
O. sativa is the world’s most widespread species because it is used for human consumption. O.
glaberrima, although used as human food, is grown only in Africa on a very small scale. The three
most common subspecies of O. sativa are japonica, javanica, and indica (Wang et al. 2014).
Japonica varieties are mainly found in Japan and Korea. Javanica varieties are commonly found in
Indonesia and the Philippines. Indica varieties are the majority of rice grown all over Asia
including India. Some indica varieties were also brought to America for large scale production
(Awan et al. 2017).
2.2. Grain Structure
The anatomy of mature rough rice (complete grains with husks intact) consists of a brown kernel
enclosed by the husk. The most visible part of a rough rice grain is the husk also known as the
hull. The hull is the outer layer covering the caryopsis and, although inedible, it makes up about
20-25 % of the total grain weight (Ogawa et al. 2002). The hull serves as a protective barrier
against infestation and environmental fluctuations as shown in Fig. 2.2. The hull is comprised of
sterile lemmas, rachilla, palea, and lemma. The lemma covers two-thirds of the seed, with the
edges of the palea fitting inside so that the two close tightly around the seed. The caryopsis contains
the embryo and starchy endosperm, surrounded by the seed coat (tegumen) and the pericarp
(Moldenhauer et al. 1998; Hoogenkamp et al. 2017).
The caryopsis consists of three fibrous bran tissues: pericarp, tegumen, and aleurone. Endosperm
and embryo are also parts of the caryopsis. The bran portion accounts for one-tenth of the weight
of the rough grain and has a high nutritional value because it contains proteins, lipids, and dietary
fibres. The pericarp is made of thin bran layers of proteins. The tegumen consists of arrays of fatty
materials. The aleurone surrounds the endosperm and the embryo. Its tissues are rich in protein
and cellulose. The embryo is the reproductive organ of the grain and is very rich in protein and fat.
9
The endosperm, the largest component of the grain, is mainly composed of starch granules, with
minute amounts of proteins, lipids, and water (Champagne et al. 2004).
Figure 2.2 Cross section of a rice kernel (Hoogenkamp et al. 2017)
2.3. The general composition of rice grain
The chemical composition and properties of rice and its milling fractions are subject to the varietal,
environmental, and processing variability (Bhattacharya 2017). A wide range of values is evident
for all milling fractions (Bond 2004). As shown in Table 2.1, among the milling fractions of rice,
the bran fraction has the highest energy and protein content and the hull fraction has the lowest
(Champagne et al. 2004). Only the brown rice fraction is eatable. Abrasive or friction milling is
10
usually done to remove the pericarp, seed-coat, testa, aleurone layer and embryo to yield milled
rice results in loss of fat, protein, crude and neutral dietary fibre, ash, thiamine, riboflavin, niacin
and α-tocopherol (as shown in Table 2.2). Available carbohydrates, exclusively starch, are higher
in milled rice than in brown rice (Table 2.1) (Belitz et al. 2009). The gradients for the various
nutrients are not identical as evidenced by analysis of successive milling fractions of brown rice
and milled rice. Dietary fibre is highest in the bran layer (and the hull) and lowest in milled rice.
Density and bulk density are lowest in the hull, followed by the bran, and highest in milled rice
because of the low oil content (Champagne et al. 2004).
Table 2.1 Range of mean proximate analysis and content (%) of organic fractions of rough
rice and its milling fractions at 14 % moisture (Champagne et al. 2004)
Nutrient
Rough
Brown
Milled
Hull
Bran
Embryo
Polish
Rice
Rice
Rice
Protein (N x 5.95)
5.8-7.7
4.3-18.2
4.5-10.5
2.0-2.8
11.3-14.9
14.1-20.6
11.2-12.4
Crude Fat
1.5-2.3
1.6-2.8
0.3-0.5
0.3-0.8
15.0-19.7
16.6-20.5
10.1-12.4
Crude Fibre
7.2-10.4
0.6-1.0
0.2-0.5
34.5-45.9
7.0-11.4
2.4-3.5
2.3-3.2
Crude Ash
2.9-5.2
1.0-1.5
0.3-0.8
13.2-21.0
6.6-9.9
4.8-8.7
5.2-7.3
Available
64-73
73-87
77-89
22-34
34-62
34-41
51-55
53.4
66.4
77.6
1.5
13.8
2.1
41.5-47.6
16.4-19.2
2.9-3.9
0.7-2.3
65.5-74.0
23.7-28.6
13.1
-
3.7-5.3
1.2-2.1
0.5-1.4
17.7-18.4
7.0-8.3
7.9-6.4
3.6-4.7
Hemicelluloses
-
-
0.1
2.9-11.8
9.5-16.9
9.7
-
Cellulose
-
-
-
31.4-36.3
5.9-9.0
2.7
-
1,3:1,4 β-glucans
-
0.11
0.11
-
-
-
-
Polyuronic Acid
0.6
-
-
-
1.2
0.4
-
0.5-1.2
0.7-1.3
0.22-0.45
0.6
5.5-6.9
8.0-12
-
Lignin
3.4
-
0.1
9.5-18.4
2.8-3.9
0.7-4.1
2.8
Energy (kJ/g)
15.8
15.2-16.1
14.6-15.6
11.1-13.9
16.7-19.9
-
17.9
Carbohydrates
Starch
Neutral Detergent
Fibre
Pentosans
Free Sugars
11
Table 2.2 Vitamin and mineral content of rough rice and its milling fractions at 14 %
moisture (Champagne et al. 2004)
Thiamine
Riboflavin
Niacin
α - Tocopherol
Calcium
Phosphorus
Phytin P
Iron
Zinc
(mg)
(mg)
(mg)
(mg)
(mg)
(g)
(g)
(mg)
(mg)
Rough rice
0.26-0.33
0.06-0.11
2.9-5.6
0.90-2.00
10-80
0.17-0.39
0.18-0.21
1.4-6.0
1.7-3.1
Brown rice
0.29-0.61
0.04-0.14
3.5-5.3
0.90-2.50
10-50
0.17-0.43
0.13-0.27
0.2-5.2
0.6-2.8
Milled rice
0.02-0.11
0.02-0.06
1.3-2.4
75-0.30
10-30
0.08-0.15
0.02-0.07
0.2-2.8
0.6-2.3
Rice bran
1.20-2.40
0.18-0.43
26.7-49.9
2.60-13.3
30-120
1.1-2.5
0.9-2.2
8.6-43.0
4.3-25.8
Rice hull
0.09-0.21
0.05-0.07
1.6-4.2
0
60-130
0.03-0.07
0
3.9-9.5
0.9-4.0
Rice Fraction
2.3.1. Starch
Starch is the primary constituent of milled rice at about 90 % of the dry matter. Starch is a polymer
of D-glucose linked α-(1-4) and usually consists of an essentially linear fraction, amylose, and a
branched fraction, amylopectin. Various physicochemical properties of starch fractions are shown
in Table 2.3. Branch points are α-(1-6) linkages. Innovative techniques have now shown rice
amylose to have two to four chains with a number-average degree of polymerisation (DPn) of 900
to glucose units and a ß-amylolysis limit of 73-87 % (Belitz et al. 2009). It is a combination of
branched and linear molecules with DPn of 1100 to 1700 and 700 to 900, respectively. The
branched fraction constitutes 25-50 % by number and 30-60 % by weight of amylose. The iodine
affinity of rice amyloses is 20-21 % by weight.
Rice amylopectin has ß-amylolysis limits of 56-59 %, chain lengths of 19 to 22 glucose units, DPn
of 5,000 to 15,000 glucose units and 220 to 700 chains per molecule (Belitz et al. 2009). The iodine
affinity of rice amylopectin is 0.4-0.9 % in low- and intermediate-amylose rice but 2-3 % in highamylose rice. Isoamylase-debranched amylopectin showed longest chain fractions (DPn > 100) (914 %) in high-amylose samples with higher iodine affinity than in low and intermediate-amylose
samples (2-5 %) and waxy rice amylopectin (0 %), (Champagne et al. 2004).
Based on colorimetric starch-iodine color absorption standards at 590 to 620 nm, milled rice is
classified as waxy (1-2 %), very low amylose (2-12 %), low amylose (12-20 %), intermediate (2025 %) and high (25-33 %), (Juliano & Bechtel 1985; Gayin et al. 2015). Recent studies showed
that the maximum true amylose content is 20 % and that additional iodine binding is due to the
long linear chains in amylopectin (Champagne et al. 2004). Hence colorimetric amylose values are
now termed "apparent amylose content" (Delwiche et al. 1995).
12
Table 2.3 Properties of starch fractions in rice endosperm (Zobel 1988)
Property
Amylose
Amylopectin
Linear (α-1-4)
Branched (α-1-4; α-1-6)
Unstable
Stable
Gels
Stiff, irreversible
Soft, reversible
Films
Coherent
-
Favourable
Unfavourable
Blue
Red-Purple
Digestibility, β-Amylase
100 %
60 %
Degree of Polymerization
1500-6000
3 X 105 – 3 X 106
Molecular Structure
Dilute Solutions
Complex Formation
Iodine Color
The waxy endosperm is opaque and shows air spaces between the starch granules, which result in
lower density than the non-waxy endosperm. The structure of the starch granule is still not well
understood, but crystallinity and staling are attributed to the amylopectin fraction (Champagne et
al. 2004). It is broadly accepted that amylopectin molecules are composed of short-amylose-chains
consisting of 6~100 glucosyl residues, and it is very difficult to elucidate the fine structure
assembled these chains because of high molecular weight (Hisamatsu et al. 1996).
2.3.2. Protein
Protein plays an important role in determining the physicochemical properties of rice (Cornejo &
Rosell 2015). As compared to the majority of cereals, the protein content of rice is typically in the
lowest range. It also has low fibre and lipid contents (Table 2.4). However, the net protein
utilisation and digestible energy in rice are the highest among the other cereal grains (Hoogenkamp
et al. 2017).
Table 2.4 Composition and energy balance data of selected whole-grain cereals
(Nadathur et al. 2017)
Property
Brown
Wheat
Corn
Barley
Millet
Sorghum
Rye
Oat
rice
Protein (N x 6.25) (%)
7.3
10.6
9.8
11.0
11.5
8.3
8.7
9.3
Fibre (%)
0.8
1.0
2.0
3.7
1.5
4.1
2.2
5.6
Net protein utilization (%)
73.8
53.0
58.0
62.0
56.0
50.0
59.0
59.1
Digestible energy (kJ (100 g)-1)
1550
1360
1450
1320
1440
1290
1330
1160
13
Protein contents range from 6.6 to 7.3 % for brown rice (Basak et al. 2002; Hoogenkamp et al.
2017), 6.2 to 6.9 % for milled rice (Singh et al. 1998), and 8.2 to 8.4 % for milled basmati rice
(Deka et al. 2000). Some wild varieties of China and North America have high protein contents.
Zhai et al. (2001) figured out that protein content in such varieties could be as high as 12.0 to 15.0
%. The protein (and fat) contents reduced linearly with increase in the degree of polish, as these
constituents were mainly concentrated in the peripheral layers of the kernel (Pal et al. 1999).
Endosperm (milled rice) protein comprises of several fractions comprising of 15.0 % albumin
(water soluble) plus globulin (salt soluble), 5.0 to 8.0 % prolamin (alcohol soluble) and the rest
glutelin (alkali soluble), (Juliano & Bechtel 1985). On the other hand, Basak et al. (2002) found
that the solubility fractions of rice proteins comprised of 9.7 to 14.2 % albumin, 13.5 to 18.9 %
globulin, 3.0 to 5.4 % prolamin and 63.8 to 73.4 % glutelin for non-basmati aromatic, basmati
aromatic and non-aromatic rice samples, respectively. Average protein fractions in milled rice are
shown in Fig. 2.3. Several studies have been conducted by using sequential protein extraction
(Chrastil 1990a; Kato et al. 2000; Shigemitsu et al. 2013; Hoogenkamp et al. 2017). Huebner et al.
(1990) reported that the mean ratio for 33 samples was found to be 9 % prolamin, 7 % albumin
plus globulin and 84 % glutelin.
Figure 2.3 Average protein fractions in milled rice samples (Basak et al. 2002)
14
Protein is most ample in the subaleurone layers and is also present in aleurone cells
(Azhakanandam et al. 2000). Rice bran proteins are richer in albumin than endosperm proteins and
are found as distinct protein bodies containing globoids in the aleurone layer and the germ. These
structures are not similar to endosperm protein bodies (Hoogenkamp et al. 2017).
2.3.2.1. Protein bodies
The endosperm protein is localised mainly in large spherical protein bodies (PB). The estimated
size of proteins is usually around 0.5 to 4 µm. The crystalline (PB-II) protein bodies are rich in
glutelin, and the large spherical protein bodies (PB-I) are rich in prolamin (Masumura et al. 2015).
Ogawa et al. (1989) reported that both PB-I and PB-II were distributed throughout the rice
endosperm. Moreover, storage proteins in endosperm were composed of 60-65 % PB-II proteins,
20-25 % PB-I proteins and 10-15 % albumin and globulin in the cytoplasm (Ogawa et al. 1987).
Saito et al. (2012) proposed the model for the internal structure of mature PB-I in rice starchy
endosperm by using immunofluorescence microscopy. PB-I in rice endosperm consists of a core
region containing 10 kDa prolamin, 13 kDa prolamin-rich inner layer, 13 and 16 kDa prolaminrich middle layers, and a 13 kDa prolamin-rich outermost layer. The 13 kDa prolamins were a
major group of rice prolamins and were grouped into distinct sub-classes (13a and 13b) (Fig. 2.4).
Figure 2.4 Model for the internal structure of the mature protein body PB-I in rice starchy
endosperm (Saito et al. 2012)
15
2.3.2.2. Waxy gene protein
Rice starch granule amylose binds up to 0.7 % protein that is mainly the waxy gene protein or
granule-bound starch synthase, with a molecular mass of about 60 kDa, (Villareal & Juliano 1989).
Rice glutelin consists of three acidic or α-subunits of 30 to 39 kDa and two basic or ß-subunits of
19 to 25 kDa (Belitz et al. 2009). The two kinds of subunits are formed by cleavage of a 57 kDa
polypeptide precursor. Prolamin consists mostly (90 %) of the 13 kDa subunit plus two minor
subunits of 10 and 16 kDa (Belitz et al. 2009).
The waxy gene protein has a huge number of disulphide linkages and mostly presents in greater
proportion in high-amylose compared with low-amylose rice varieties (Zhou et al. 2002b).
Hamaker at al. (1991) reported the correlation of waxy gene protein with amylose contents (r =
0.95) and cooked rice stickiness (r = -0.85). Protein with intact disulphide bonds makes the swollen
granules less prone to break down. When protein disulphide bonds were uninterrupted, rice starch
granules swelled to a huge size, thereby increasing the degree of gelatinization and gel strength
(Hamaker & Griffin 1993). So, the low concentration of waxy gene protein makes the waxy rice
soft, smooth and develops low viscosity while cooking. Traore et al. (2011) figured out that the
presence of a rice waxy gene single nucleotide polymorphism (SNP) marker is associated with
elevated Rapid Visco Analyzer (RVA) properties in specific high amylose rice cultivars.
2.3.3. Lipids
Fat contents or lipids in cereal grains are diverse. Cereal lipids can be divided into neutral lipids,
glycolipids, and phospholipids (Mano et al. 1999). Both japonica and indica rice contain different
lipid classes in almost same ratios (Kang et al. 2011), but they are not uniformly distributed within
the grain. Moreover, the endosperm lipids contained a higher proportion of polar lipids (Kang et
al. 2009). The lipid or fat content of rice is mostly in the bran fraction (20 %, dry basis), specifically
as lipid bodies or spherosomes (0.1-1 µm) in the aleurone layer and bran; however, about 1.5-1.7
% is present in milled rice, mostly as non-starch lipids extracted by ether, chloroform-methanol
and cold water-saturated butanol (Belitz et al. 2009; Zhou et al. 2002b).
Resurreccion et al. (1979) estimated the crude oil in rice at various stages of processing. It was
found that 2.9 % crude oil in brown rice, of which 51 % was found in the germ, 32 % in the polish,
and only 17 % in the endosperm. Lipids are not evenly distributed in the endosperm, with the
16
highest amount in the outer layer and decreasing progressively towards the core of the kernel
(Normand et al. 1966; Houston 1967 and Hogan et al. 1968). Moreover, in protein bodies,
especially the core, are rich in lipids. The key fatty acids of these lipids are linoleic, oleic and
palmitic acids. Essential fatty acids in rice oil are about 29-42 % linoleic acid and 0.8-1.0 %
linolenic acid. The content of essential fatty acids is likely to be increased with temperature during
grain development but at the expense of a reduction in total oil content (Champagne et al. 2004).
Starch lipids (lipids complexed with amylose) are primarily monoacyl lipids (fatty acids and
lysophosphatides). The starch-lipid content is lowest for waxy starch granules (<0.2 %). It is
highest for inter-mediate amylose rice (1.0 %) and may be slightly lower in high-amylose rice.
Waxy milled rice has more non-starch lipids than non-waxy rice. However, starch lipids contribute
little to the energy content of the rice grain. The key fatty acids of starch lipids are palmitic and
linoleic acids, with lesser amounts of oleic acid (Belitz et al. 2009).
2.3.4. Non-starch polysaccharides
Non-starch polysaccharides comprise of water-soluble polysaccharides and insoluble dietary fibre.
They can make a complex with starch and may have a hypocholesterolemic effect. The endosperm
has a lower content of dietary fibre than the rest of brown rice. Reported values for neutral
detergent fibre are 0.7-2.3 %. Also, the endosperm or milled rice cell wall has low lignin content
but a high content of pectic substances or pectin. Endosperm pectin has a higher uronic acid content
but a lower arabinose-to-xylose ratio than the other grain tissues. The hemicellulose of endosperm
also has lower arabinose to xylose ratio than the three other grain tissues (Belitz et al. 2009).
2.3.5. Volatiles
The volatiles characteristic of cooked rice is ammonia, hydrogen sulphide and acetaldehyde. Upon
cooking, all aromatic rice contain 2-acetyl-1-pyrroline as the major aromatic principle. Volatiles
characteristics of fat rancidity are aldehydes, predominantly hexanal, and ketones (Belitz et al.
2009). A wide range of volatiles such as alcoholic, aldehydes, and ketone can be detected in
different rice varieties (Tsugita 1986). Lin et al. (2010) studied the volatile compounds in different
indica and japonica varieties of rice. The amplest volatile alcohols in indica rice were n-hexyl
alcohol, n-octanol, and 2-hexyl-1-octanol, while in Japonica they were n-octanol, 2-hexyl-1octanol and 3,7,11-trimethyl-1-12 alcohol.
17
Both indica and japonica contained pentanal, hexanal, heptanal, 2-heptene aldehyde, octanal,
nonanal, decyl aldehyde and benzene formaldehyde. The most abundant aldehyde was hexanal,
which on average accounted for 13.31 % of the aldehydes (averaging 14.69 % for indica and 1.93
% for japonica), followed by nonanal which accounted for an average of 7.93 %. Pentanal, hexanal,
heptanal, octanal, nonanal, decyl aldehyde and benzene formaldehyde were present at relatively
high levels. A total of twenty-three different volatile ketones were identified of which there were
nineteen in indica and thirteen in japonica rice. The ketones content was much lower than the
aldehyde content (Lin et al. 2010).
2.4. Classification of rice
Several varieties of rice have been developed in the past few decades, which are widely grown in
the world, with immense diversity in physicochemical properties. Therefore, it is very necessary
to classify different varieties. There are different ways of rice classification (Kambo & Yerpude
2014). Rice can be classified either by the color of caryopsis or kernel length or type of starch (Fig.
2.5).
Figure 2.5 Different ways of rice classification
18
2.4.1. Classification by color caryopsis
Rice can be classified on the color of the caryopsis; it can be brown, red or black. White rice is
usually obtained from brown rice by removing the bran layers through a process known as milling.
The red and black varieties are less common and essentially only available in Thailand and the
Philippines (Parrinello 2008).
2.4.2. Classification by kernel length and length/width ratio
According to Codex Alimentarius Commission (1995), rice is categorised as long grain, medium
grain or short grain by one of the following specification:
Option 1: Kernel length/width ratio.
Long/slender grain rice
Husked rice or parboiled husked rice with a length/width ratio of 3.1 or more.
Milled rice or parboiled milled rice with a length/width ratio of 3.0 or more.
Medium grain rice
Husked rice or parboiled husked rice with a length/width ratio of 2.1-3.0.
Milled rice or parboiled milled rice with a length/width ratio of 2.0-2.9.
Short/bold grain rice
Husked rice or parboiled rice with a length/width ratio of 2.0 or less.
Milled rice or parboiled milled rice with a length/width ratio of 1.9 or less.
Option 2: Kernel length.
Long grain rice
Kernel length of 6.6 mm or more.
Medium grain rice
Kernel length of 6.2 mm or more but less than 6.6 mm.
Short grain rice
Kernel length of less than 6.2 mm.
Option 3: A combination of kernel length and length/width ratio.
Long grain rice has either;
19
Kernel length of more than 6.0 mm and with a length/width ratio of more than 2 but less
than 3.
Kernel length of more than 6.0 mm and with a length/width ratio of 3 or more.
Medium grain rice
Kernel length of more than 5.2 mm but not more than 6.0 mm and a length/width ratio of
less than 3.
Short grain rice
Kernel length of 5.2 mm or less and a length/width ratio of less than 2.
2.4.3. Classification by type of starch
Rice can also be classified by starch type found in the endosperm. There are two types of starch,
namely, amylose and amylopectin. As mentioned in Fig. 2.6, amylose consists predominantly of
linear chains of carbohydrates, whereas amylopectin has a more branching tree-like structure. The
proportion of amylose and amylopectin strongly affects the appearance as well as the cooking
characteristics of the grain (Ahromrit et al. 2006).
Figure 2.6 Different type of starch found in rice endosperm: (a) Linear structure of
amylose, (b) Branched structure of amylopectin (Belitz et al. 2009)
20
2.4.3.1. Glutinous rice
Glutinous rice, also known as sticky or waxy rice, has a white and opaque endosperm because of
the air spaces between the starch granules (Zhou et al. 2002a). Its starch consists almost entirely
of amylopectin. When cooked, the grain usually loses its original shape and becomes very sticky
(Noosuk et al. 2003).
2.4.3.2. Non-glutinous rice
Non-glutinous or non-waxy rice has a translucent appearance and contains amylose as well as
amylopectin. The cooked grain tends to retain its shape and is less sticky (Kang et al. 2009).
2.5. Glutinous, waxy or rice variety
The conventional method for determining amylose content has shown that waxy rice could have
up to 5 % amylose. However, including a 0 % amylose standard in the standard curve causes the
amylose content of these varieties to become 0-2 % (Cuevas 2008). The composition of glutinous
rice is depicted in Table 2.5.
Table 2.5 Proximate composition and nutrition facts of brown and white glutinous rice
(Amornsin 2003)
Nutrition Facts Servings: 100 g
Brown Glutinous Rice
White Glutinous Rice
Calories, kcal
362
355
Moisture, g
11.2
11.7
Carbohydrates, g
77.7
81
Protein, g
7.4
6.3
Total Fat, g
2.4
0.6
Dietary Fibre, g
2.8
0
Niacin, g
5.5
1.8
Phosphorus, mg
255
63
Potassium, mg
326
0
Calcium, mg
12
7
Sodium, mg
12
0
Vitamin B1, mg
0.26
0.08
Vitamin B2, mg
0.04
0.03
21
Zeng et al. (2009) investigated the volatile composition of three different varieties of glutinous
rice (Tatsukomochi, Kinunohada, and Miyakadoganemochi) using combined gas chromatographymass spectrometry with modified headspace solid-phase microextraction method. Altogether, 96
different volatile compounds were identified, of which 27 volatile compounds have not been
previously reported in rice. The volatile components detected in the three waxy rice cultivars
during cooking belong to the chemical classes of aldehydes, ketones, alcohols and heterocyclic
compounds, as well as fatty acids and esters, phenolic compounds, hydrocarbons, etc.
2.5.1. Global cultivation and consumption of glutinous rice
Glutinous rice or sticky rice (Oryza sativa L.) is a kind of rice commonly cultivated in Thailand,
Cambodia, Lao PDR, Myanmar, Vietnam, Indonesia, Bangladesh, Northeast India, Japan, Korea,
Taiwan, and China (Fig. 2.7). Waxy rice has regional importance in Lao PDR and Thailand, and a
large majority of the population consumes it as the main component of their diet (Roder et al.
1996). The cultivation of sticky rice has been recorded in the region for at least 1100 years.
Figure 2.7 Rice-producing nations are denoted in green. The yellow box highlights the
centre of waxy rice production. Countries in white did not produce rice (Nguyen & Tran
2002)
22
2.5.2. Quality characteristics of traditional glutinous rice
2.5.2.1. Hydration and gelatinization behavior of glutinous rice
Glutinous rice absorbs very little water during soaking and cooking and therefore have low volume
expansion (Schiller et al. 2006). The grain of most high-amylose rice cultivars shows high volume
expansion (up to 400 %) during cooking (Shinde et al. 2014). Low-amylose rice is moist, sticky,
and glossy when cooked (Dela Cruz & Khush 2000). The texture of the cooked rice is affected by
gelatinization temperature (Peñaflor et al. 2014). Rice that gelatinizes at high temperatures (>74ºC)
takes a longer time to cook and, when finally cooked, is excessively soft; it also collapses when
overcooked (Briffaz et al. 2014). Such rice generally requires more water and time for cooking
than does rice that has low (<70ºC) or intermediate (70-74ºC) gelatinization temperatures. Such
rice is undesirable in all rice markets (Schiller et al. 2006). Low gelatinization temperature is the
most common property of the preferred waxy rice varieties (Amornsin 2003; Horigane et al. 2000).
2.5.2.2. The opaque endosperm of waxy rice varieties
Another characteristic feature of glutinous rice that distinguishes it from the non-glutinous form is
that, if the moisture content of the glutinous form is reduced to about 15 %, the endosperm becomes
opaque and its color changes to milky white or paraffin-like. This white appearance comes as a
result of the way light is differentially refracted in the starch crystals in the absence of amylose. In
non-glutinous rice, the endosperm remains translucent regardless of the moisture content of the
grain (Archer et al. 2008).
2.5.2.3. Nutritional value of waxy rice and consumer preference
There is relatively little specific information available relating to the relative nutritional value of
waxy and non-waxy rice (Schiller et al. 2006). It has been suggested that the higher content of
amylopectin in glutinous rice and the associated larger molecular size and its branched molecular
structure result into a longer stay in the digestive system than non-glutinous rice (Benmoussa et
al. 2007). This, in turn, is reflected in the belief by waxy rice consumers that they only feel “full”
when they consume glutinous rice; they complain that the consumption of non-waxy rice results
in their becoming hungry again within a short time (Schiller et al. 2006).
The low volume expansion of glutinous rice relative to non-glutinous rice on being cooked, and
the resulting higher weight to volume ratio of glutinous vs. non-glutinous rice on consumption,
23
probably explains the effects of perceived differences in “fullness and hunger” between the two
types of rice (Ngaosyvanthn & Ngaosyvanthn 1994).
2.5.3. Cooking process of rice
Rice can be cooked by boiling in excess water or using a fixed rice: water ratio in a rice cooker.
Depending on the cooking conditions (temperature, time, water-to-rice ratio, etc.); the rice grain
undergoes specific structural and physicochemical transformations (Briffaz et al. 2014). During
cooking, several stages occur to transform a raw rice grain into a cooked grain of pleasing textural
attributes. These include glass transition, gelatinization, swelling, pasting and leaching of amylose,
and retrogradation. It is now quite accepted concept that the cooking attributes, texture, water
absorption ability, stickiness, volume expansion, hardness and even the shine and whiteness of the
cooked milled rice are greatly affected by the composition of starch (amylose and amylopectin
contents) (Jie et al. 2010) and protein contents (Teo et al. 2000). Glutinous rice varieties having
high amylopectin contents in endosperm are non-gelling because of the lack of amylose (Jane at
al. 1999). The main stages of the cooking process of glutinous rice are shown in Fig. 2.8.
Figure 2.8 Main stages of the cooking process of glutinous rice
24
2.5.3.1. Glass transition
The concept of ‘glass transition’ has been applied to food science and technology for the last five
decades (Truong 2008). The glass transition or glass-liquid transition is a reversible transition in
amorphous materials (or in amorphous regions within semicrystalline materials) from a hard and
relatively inelastic state into a molten or rubber-like state. It is a branch of material science which
considers food components like starch and protein to be biopolymers (Truong 2008). Before
gelatinization of starch can occur, the amorphous (glassy) regions of the starch must become soft
and rubbery as they go through the glass transition temperature (Tg). The physical and thermal
properties changes in the amorphous regions of the starch during glass transition (which is a second
order transition), while the crystalline regions in starch are disrupted during the first-order
transition, i.e., melting (Roos & Karel 1991).
As shown in Table 2.6, below the glass transition temperature (Tg), the amorphous regions of the
starch are in a glassy state, but they become flexible, viscous and rubbery above the glass transition
temperature (Tg). In the glassy state, molecular movement is quite limited, making it quite viscous,
having low specific volume and low thermal expansion coefficient. During the glass transition
from glassy to rubbery states, many changes in physical properties are observed; like a
discontinuous change in heat capacity increases in thermal expansion coefficient, diffusivity, and
specific volume, and a decrease in viscoelasticity. Some factors including free volume, water
content, average molecular weight, the degree of crystallinity, and degree polymerisation can
affect these properties (Bhandari & Howes 1999; Roos & Karel 1991).
Water is considered as a strong plasticiser, thereby depressing Tg of the amorphous regions at low
water content (<30 %) (i.e., Tg increases with decreased water content) (Biliaderis et al. 1986;
Huang et al. 1994). The relationship between the glass transition temperature and moisture content
hypothesised by Cnossen et al. (2001) and Perdon et al. (2000) is shown in Fig. 2.9. Water reduces
the glass transition temperature, and so at higher moisture contents, glass transition occurs at lower
temperatures (Roos & Karel 1991). The milled rice at 12 % moisture content has a glass transition
temperature (Tg) of about 50ºC (Cnossen et al. 2001). In another study, Thuc et al. (2010) reported
the glass-rubber transition temperature (Tg-r) of rice flour (12-16 % moisture content) ranging from
41.6 to 56.7ºC. During the early hydration stage of cooking, there will be a transition from
amorphous to rubber state facilitating the gelatinization of crystalline starch.
25
Table 2.6 Properties of glassy and rubbery states of starch (Truong 2008)
Figure 2.9 State diagram describing the glassy and rubber regions of rice (Cnossen et
al. 2001)
2.5.3.2. Gelatinization
Gelatinization is the non-equilibrium melting of the crystalline regions, and prerequisite is the presoftening of the amorphous regions (glass transition). Starch gelatinization is an endothermic
26
process that corresponds to the loss of starch crystallinity in the starch granules under particular
heat and moisture conditions. Not all granules in any rice sample gelatinise at the same
temperature; rather gelatinization occurs over a temperature range of about 8-15ºC (Shih et al.
2007). Pictorial representation of starch gelatinization is shown in Fig. 2.10.
Figure 2.10 Process of starch gelatinization (Singh et al. 2007; Ahmed et al. 2008)
27
2.5.3.3. Gelatinization temperature
Gelatinization temperature is a key property of rice because it correlates strongly with the cooking
time and the texture of the cooked product. McGuiness et al. (2000) worked on the relationship
between moisture content and gelatinization temperature. They found that if the moisture content
is too low, then gelatinization cannot take place. Also, the higher the moisture content, the lower
the temperature at which the reaction can occur.
This can be clarified by the plasticizing effect of water on the melting of starch; rice starch
gelatinization endpoint temperature is thus over 100ºC when the water content is less than 50 %
(Briffaz et al. 2012). Moreover, gelatinization behavior also directly affects the perceived texture
of cooked rice; the higher the gelatinization temperature of the grain, the firmer the core of cooked
rice will be (Mestres et al. 2011).
2.5.3.3.1. Rice starches and gelatinization temperature
The composition of rice starches has a great effect on the gelatinization. Rice starches having short
average amylopectin branch chain lengths displayed low gelatinization temperatures (Kalichevsky
et al. 1990).
2.5.3.4. Swelling and pasting
After starch gelatinization, the starch granules begin to swell and, in the absence of shear, can
swell and increase in volume to many folds while maintaining their integrity (Parker & Ring 2001).
Leaching of amylose molecules accompanies the swelling of the starch granules into the liquid
phase only in non-waxy rice. In waxy starches, after swelling granules disrupt, resulting in a
smooth paste.
2.5.3.5. Retrogradation
Gelatinized starch contains no crystalline regions, but under certain conditions of storage and
temperature, the molecules in a starch gel can re-associate into an ordered structure. Retrogradation
describes the rapid recrystallisation of amylose and the slow recrystallisation of amylopectin. The
degree of retrogradation and the nature of newly formed crystals can depend on the time and
temperature of storage (Li et al. 2014), the source of starch (Lian et al. 2015), and the presence of
other molecules (Likitwattanasade & Hongsprabhas 2010) in the system.
28
2.5.3.5.1. Recrystallization of amylose
Recrystallization of amylose is essentially the rapid formation of double helices in parts of the
amylose chains followed by aggregation of these helices. The first stage of retrogradation depends
on the amylose that is free, rather than complex with lipids (Yao et al. 2002). Further, hot-watersoluble components of rice starch with high molecular weight promote retrogradation more than
lower-molecular-weight polymers. Tsai & Lii (2000) suggested that the molecular weight
distribution of the amylose contributes significantly to the first phase of retrogradation.
Retrogradation due to amylose is not reversible at temperatures less than 100ºC because amylose
crystals melt only above 100ºC.
2.5.3.5.2. Recrystallization of amylopectin
Recrystallization of short-chain amylopectin chains constitutes the second process of
retrogradation (Baik et al. 1997). Several studies have been conducted to study the retrogradation
behavior of amylopectin. It is revealed that the fine structure or the chain-length distribution of
amylopectin contributes to differences in the degree of retrogradation by amylopectin, in
particular, the proportion of short A chains in amylopectin (Yao et al. 2002; Tsai & Lii 2000 and
Silverio et al. 2000). The interaction of amylose with amylopectin increases the rate of amylopectin
retrogradation. Retrogradation due to amylopectin is reversible if the retrograded gel is exposed to
a temperature greater than the gelatinization temperature of crystalline amylopectin (Yao et al.
2002).
2.6. Pre-treatment processes
The cooking process of rice involves wetting of the kernels up to a moisture content of 65-70 %.
This wetting of grains results in swelling. Consequently, the melting of amylopectin crystals takes
place, the release of amylose from the starch granules (only in non-waxy rice starches), and
gelatinization takes place. Cooking process leads to the softness of rice and makes it easily
masticateable product, ready for consumption. The food industry has engineered several pretreatment processes for consumers’ convenience, for example, shorter cooking or preparation
times (Mohoric et al. 2009). These processes include wet processing and puffing. During wet
processing, polished rice is cooked and dried afterward, resulting in reduced cooking time with
poor texture product (Lee et al. 2000). However, puffing raw rice results in a product which still
29
requires cooking, but cooking time is reduced with the good texture final product (Mohoric et al.
2009).
2.6.1. Alkali treatment
Alkali treatment of cereals is widely used in food industry to produce many value-added food
products such as tortillas, waxy rice dumplings, and yellow alkaline noodles (Nadiha et al. 2010;
Lai et al. 2002; Guo et al. 2017). Alkali application to the starch leads to reduced swelling power
and water binding capacity (Karim et al. 2008; Wang & Copeland 2012). Moreover, starch
particles display Donnan-potential in the presence of water due to its weak acidic ion-exchanging
behavior. The starch particles have a negative charge; therefore, penetration of Na+ into the
amorphous regions of starch granules is promoted (Upma et al. 2017). Moreover, under alkaline
conditions, hydroxyl groups of starch might have a greater tendency to ionize and create even more
binding sites for cations. It is hypothesized by Oosten (1990) that anions might tend to destabilize
starch granules by breaking hydrogen bonds. However, such destabilizing effects of anions might
be much weaker than the stabilizing effects of cations. This electrostatic interactions between
hydroxyl groups of starch and Na+ ions result in increased gelatinized temperature (Lai et al. 2002).
Also, alkali treatment induces structural changes in amylopectin possibly due to alkali-induce depolymerization, resulting in reduced retrogradation (Abhari et al. 2017).
2.6.2. Starch modification
Starch can be modified by using various chemical agents. These chemical agents can be classified
as monofunctional or bi-functional reagents based on their chemical properties (Wolf et al. 1999).
A non-ionic, cationic, hydrophobic or covalently reactive substituent group are provided by
monofunctional reagents (Sui & BeMiller 2013). Etherification modification method or
hydroxypropylation is a common example of starch modification by monofunctional reagents
(Clasen et al. 2018). Tri-meta-phosphates and phosphoryl chlorides are the common examples of
bi-functional reagents as they can react with more than one hydroxyl group and can thus reinforce
starch granules (Masina 2016; Włodarczyk-Stasiak et al. 2017). Moreover, these bi-functional
reagents allow crosslinking of the polymers, resulting in increased starch stability and modify its
swellability, solubility, and mobility (Xiao 2013). Such modifications usually alter the
physicochemical (gelatinization and thermal) properties of starch. In the recent years, great focus
has been given to study the chemical modification of starch and a variety of different starch
30
molecules have been synthesized, each with its distinct chemical property and functionality.
However, there are very few methods applicable to starch particles as most reactions require the
sample to be in solution or slurry (Masina et al. 2017). The three main types of chemical starch
modifications and their derivatives are summarized in Fig. 2.11.
Figure 2.11 Schematic summarization of the classical chemical methods for starch
modification (Masina et al. 2017)
The esterification of starch is the most common chemical modification used in the food industry.
It involves the conversion of the available hydroxyl groups to alkyl or aryl derivatives (Morán et
al. 2011; Ačkar et al., 2015). Esterified starch has reduced retrogradation ability and glycemic
index response (Masina et al. 2017). Therefore, starch esterification can be a potential solution to
maintain the stickiness. However, no reported data is available on the intact starch esterification
in the milled rice.
2.6.3. Parboiling of rice
The parboiling of rice is the major processing technique has been used in South Asian developing
countries especially Indian subcontinent for decades (Islam et al. 2001; Bhattacharya 2004).
31
According to an estimate, 20 % of the world’s rice is parboiled, and production seems to be
growing day by day (Buggenhout et al. 2014).
2.6.3.1. Process of parboiling
Conventionally parboiling is a three-step hydrothermal process given to rough rice. The paddy or
rough rice is first soaked in excess water at a temperature below gelatinization temperature to
increase average moisture contents to 25-35 %. Excess water is drained off, and the soaked paddy
is steamed at 100-130oC for 5-30 min to gelatinize the starch. Steaming of paddy is followed by
cooling and drying of paddy with or without tempering to a moisture content below 14 %. After
drying, the broken husk is removed during milling and followed by polishing (Derycke et al.
2005a; Delcour & Hoseney 2010; Buggenhout et al. 2013).
Rice industrial research has engineered various parboiling techniques in recent years to enhance
the quality of head rice and ready to use products. Use of pressurized steam and roasting of paddy
with or without sand are the latest parboiling techniques introduced by the researchers in the rice
industry (Dutta & Mahanta 2014). The schematic flow diagram of conventional and latest
parboiling techniques is shown in Fig. 2.12.
2.6.3.2. Superheated steam drying
Superheated steam dryers are designed to use superheated steam as a heating source for drying
instead of hot of air. This drying technique is environmentally friendly and economical, as there
are no pollution or odor emissions, and the steam can be recovered (Mujumber 1995). The drying
mechanism of the superheated steam dryer is the condensation of steam onto the surface of rice
grain and a rise in grain temperature to saturation point (100oC) during the initial approximately
30 min. In the second phase of the drying mechanism, there is a reduction in moisture content due
to the high heat transfer rate (Taechapairoj et al. 2003).
Taechapairoj and co-workers (2004) found that the reduction in moisture content from 41 to 25 %
(dry basis) using superheated steam at 150-170oC was initially linear with drying time followed
by the exponential decay when the moisture content was below 25 % (dry basis). The SEM results
revealed that the use of high temperature along with condensation steam enabled the development
of the gel layer. This gel layer caused the very low effective diffusivity when compared with the
case of no gel formation in paddy.
32
Taechapairoj et al. (2003) also reported the similar characteristics of paddy dried by superheated
steam and parboiled rice. The paddy is firmer and tougher after drying in superheated steam, as a
result of gelatinization taking place for paddy with high initial moisture content. Consequently,
head rice production is improved, while a lower value of rice whiteness is usually observed as a
result of the high drying temperature. The schematic diagram of the superheated steam dryer is
shown in Fig. 2.13.
Figure 2.12 A schematic flow diagram of various parboiling techniques (Dutta & Mahanta
2014)
33
Figure 2.13 A schematic diagram of the superheated steam dryer (Rordrapat et al. 2005)
2.6.3.3. Effect of parboiling on rice breakage
The main advantage of parboiling is the reduced level of breakage during dehulling and milling
(Delcour & Hoseney 2010), this only cases when the process of parboiling is carried out properly.
Indeed, the Head Rice Yield (HRY) of parboiled rice depends on the parboiling conditions and the
resulting changes in physicochemical and mechanical properties. The starch gelatinization and
kernel fissuring during parboiling have a great impact on hardness. The more compact and
homogeneous ultrastructure obtained as a result of starch gelatinization increases the grain
hardness (Islam et al. 2001; Islam et al. 2002a, Islam et al. 2002b, Islam et al. 2004; Jagtap et al.
2008; Oli et al. 2014), while the presence of fissures has the opposite effect.
The physicochemical changes in paddy can also influence breakage susceptibility during steam
heating; these changes include protein polymerization, amylose-lipid complex formation, and
34
retrogradation of amylopectin (Buggenhout et al. 2013). The process of starch gelatinization and
fissuring during parboiling are discussed below.
2.6.3.4. Starch gelatinization during parboiling
Both hydration and heating conditions impact the extent of swelling of the starch granules and
degree of gelatinization. Water is absorbed by the rice grains during rehydration and starch
granules inside the endosperm swell. When these swelled starch granules are heated over
gelatinization temperature, their structural order is irreversibly destroyed. These irreversible
changes during gelatinization include loss of birefringence and crystalline melting (Dercyke et al.
2005).
Limited water is available, therefore the gelatinization temperature shifts to a higher value. As the
moisture content and the extent of heating increase, the degree of starch gelatinization increases
by 100 % (Patindol et al. 2008). However, during hydration, moisture content gradients exist inside
the rice grain. It is assumed that without equilibration, the moisture contents at the core of
endosperm may be too low for the starch to gelatinize. As a result, parboiled rice grains have
translucent surfaces with opaque cores known as white bellies (Buggenhout 2013).
Therefore, it is suggested that the starch granule swelling and the degree of gelatinization are the
key factors to determine whether air spaces and fissures, present before parboiling or induced
during hydration, remain present in the parboiled rice grain or are sealed upon parboiling. As a
consequence, conditions adopted during parboiling result in either increased or decreased the Head
Rice Yield (HRY) (Marshall et al. 1993; Miah et al. 2002a; Miah et al. 2002b; Patindol et al. 2008).
2.6.3.5. Fissuring during parboiling
Kernel fissuring can increase the extent of the hardness of parboiled rice. During hydration and
drying, fissures develop as a result of moisture absorption and desorption, respectively. There is
no evidence in the literature on the development of fissures during the heating step in the parboiling
process (Buggenhout et al. 2013).
2.6.4. Effect of parboiling on the quality attributes of waxy rice
Waxy rice varieties contain 0 % to very low amylose contents in the endosperm. Amylopectin is
the main component of starch in such rice varieties. The quality attribute of such varieties is to
35
produce smooth paste after cooking. Conventional and pressurized steam parboiling result in the
hardness of the grains and deteriorate the final product quality. Researchers have engineered a new
technique known as dry heat parboiling (Dutta et al. 2016). It involves conduction heating of fully
soaked paddy at high temperature for shorter durations using sand or hot air. Steam parboiling
causes starch gelatinization during steaming followed by retrogradation during extended drying.
Gelatinization and rapid loss of water result in grain during dry heating which does not allow
retrogradation (Mahanta & Bhattacharya 2010).
Dutta et al. (2016) worked on the cooking quality of traditional Indian waxy rice to make a
speciality product called Bhoja chaul. It was observed that by the use of dry heat parboiling the
cooking time in waxy can be reduced. No white bellies were observed in the grains. After
processing the kernel became bolder. RVA and DSC endotherms suggested molecular damage and
amylose-lipid complex formation by the linear β-chains of amylopectin, respectively.
2.7. Aging-induced changes
Rice is mostly consumed as cooked grains while a small amount of the rice crop is used as
ingredients in processed foods. This varying consumption pattern results in the need to store rice
over varying periods (Zhou et al. 2002a). Moreover, some markets (e.g., India, Pakistan, Sri Lanka,
and Nepal) have a preference for aged rice while other (e.g., Japan, China) favor fresh crop.
Japanese people are so keen about the freshness of rice that tests are devised for its measurement
(Matsukura et al. 2000).
2.7.1. Aging
Aging is a complexed terminology mostly used for the number of physicochemical and
physiological changes occur during storage, potentially leading to both desirable and undesirable
effects on functional properties (Chrastil 1992; Howell & Cogburn 2004). These changes mostly
include pasting properties, color, flavor, and composition (Zhou et al. 2002b). Different
researchers have reported the storage induced changes in both waxy (Tulyathan & Leeharatanaluk
2007) and non-waxy rice (Tananuwong & Malila 2011). These storage-induced changes affect the
rice quality (Katekhong & Charoenrein 2012).
36
2.7.2. Mechanism of aging
The actual mechanisms involved in the process of rice aging have yet to be understood fully
(Truong 2008), and are described in a number ways (Chrastil 1992; Sowbhagya & Bhattacharya
2001; Howell & Cogburn 2004);
i.
The polymerization of protein and the oxidation of esters in non-starch polysaccharides
which resulting in cross-linking and extended strength of cell walls.
ii.
The binding starch to the denatured oryzenin (one type of rice protein).
iii.
Changes in non-soluble amylose.
2.7.3. Cooked rice texture of the aged rice
Aged rice when cooked, the texture becomes fluffier and harder (Suzuki et al. 1999). Three months
of storage is considered as the least period for major changes to occur in the hardness of cooked
rice, gel consistency, and amylograph viscosity values (Perez & Juliano 1981). During the last
couple of decades, several researchers published the pasting properties of different non-waxy and
waxy rice cultivars and the subsequent effect of storage on quality attributes. It has been observed
that the peak viscosity and breakdown of fresh rice were higher than the aged rice (Fig. 2.14)
(Noomhorm et al. 1997; Zhou et al. 2002a; Zhou et al. 2003a; Tulyathan & Leeharatanaluk 2007
and Tanauwong & Malila 2011). Moreover, data from Differential Scanning Calorimetry (DSC)
and Rapid Visco Analyzer (RVA) indicated that longer storage (usually over six months) at room
temperature resulted in extended gelatinization and pasting temperature and setback. All these
changes in the pasting attributes predict the harder texture and decreased stickiness especially in
sticky or waxy rice. This is usually not desirable depending on consumer preference (Sowbhagya
& Bhattacharya 2001).
37
Figure 2.14 Rapid Visco Analysis curves for rice flour following grain storage for up to 8
months (Tulyathan & Leeharatanaluk 2001)
2.7.4. Effect of storage conditions on physicochemical properties
Storage conditions affect the hydration and cooking behavior of rice. Among storage conditions,
storage temperature is of great importance. Zhou and co-workers (2007a) investigated the changes
in hydration and cooking attributes of different rice cultivars stored at 4ºC and 37ºC. Researchers
concluded that the positive correlation between storage temperature and water uptake, higher
temperature storage led to greater water uptake, while the negative correlation between storage
temperature and pH and turbidity of residual cooking liquid.
2.8. Conclusions and perspectives
This literature review has given an account of and the reasons for the attention to the cooking
quality of glutinous and non-glutinous rice. For waxy rice, cooking and eating qualities are
dictated, not by amylose, but by amylopectin. Rice being the staple food of half of the world’s
population has given huge importance in food industries, and several researches have been
conducted in the past to streamline the cooking process of rice. However, maximum researches
focused only on non-glutinous cultivars neglecting waxy cultivars.
38
Stickiness and smoother texture of the cooked rice is the quality attribute of glutinous rice.
Unfortunately, stickiness of glutinous is reduced during aging. This deterioration in the cooking
quality has been associated with the starch type and other macromolecules (like protein bodies and
lipid) in the rice endosperm. Controlled storage conditions can slow down the macromolecules
shift in the endosperm, resulting in improved stickiness and smooth texture in cooked aged
glutinous rice.
Pre-process treatments of freshly harvested rice are very common in the Indian subcontinent.
Processers mostly parboiled paddy to partially gelatinize the starch, resulting in a reduced cooking
time of milled rice. However, these techniques mostly employed for non-glutinous rice cultivars
and very little work has been in the past on the pre-processing of sticky rice. Therefore, it is thought
that pre-processing of paddy could help in improving the cooking quality of glutinous rice or can
avail rice with different quality characteristics.
39
Chapter 3 Effect of different cooking conditions on the pasting properties of
flours of glutinous rice varieties from Lao People’s Democratic Republic
This chapter has been published in the International Journal of Food Properties;
Nawaz, MA, Fukai, S & Bhandari, B 2016, ‘Effect of different cooking conditions on the pasting
properties of flours of glutinous rice varieties from Lao People’s Democratic Republic’,
International Journal of Food Properties, vol. 19, pp. 2026-2040.
40
3.1. Abstract
The effect of different rehydration temperatures (30, 40 and 50ºC) and cooking times (2.7, 4.7,
6.7, 8.7 and 10.7 min) at 95ºC on the pasting properties of three glutinous varieties (TDK11,
TDK8, and Hom Mali Niaw) from Lao PDR was investigated using Rapid Visco Analyzer (RVA).
Non-glutinous varieties (IR64 and Doongara) were also analyzed to compare glutinous (amylose
< 4.5 %) and non-glutinous (amylose > 15 %) varieties. All rice flours took up water at
significantly (P<0.05) higher rates in the case of increased temperature and soaking time, resulting
in a decrease in the onset temperature for pasting. Among the glutinous rice, TDK8 showed a
significant (P<0.05) decrease in peak viscosity in response to increased rehydration time and
temperature. For this variety maximum viscosity (2403.3 mPa-s) was observed at 1 min of
rehydration at 30oC and minimum viscosity (1852.0 mPa-s) at 15 min of rehydration at 50oC. The
viscosity values of TDK11 and Hom Mali Niaw varieties increased to their highest values (1608.7
and 1477.7 mPa-s, respectively) with an increase in temperature to 40ºC for 1 min. In general, the
glutinous rice produced weaker gel than non-glutinous rice. Extended holding at a cooking
temperature (95oC) had a more significant (P<0.05) effect on the glutinous varieties TDK8 and
TDK11 than on the non-glutinous varieties (IR64 and Doongara) used in this study.
3.2. Introduction
Rice (Oryza sativa L.) has been a staple food in the majority of countries in the Asian continent,
and its consumption is also growing in other parts of the world following changes in demography
and eating habits of the population. It has been reported that rice has been cultivated in China and
Thailand dating from about 6000 BC (Zhou et al. 2002a; Childs 2004). There are different cultivars
of rice grown throughout the world (Kambo & Yerpude 2014), each cultivar exhibiting distinct
physicochemical properties, and the type of the starch present influencing the cooking quality (Yu
et al. 2009). Consumer preference is normally dependent on the growing location, and this is a
crucial factor for the selection and utilisation of rice cultivars in a given location (Allahgholipour
et al. 2006).
Rice is classified into glutinous and non-glutinous categories by the type of starch found in the
endosperm. There are two types of starch found in rice, namely, amylose and amylopectin.
Amylose consists predominantly of linear chains of α-D-glucose units, whereas amylopectin has
a more branching tree-like structure (Yu et al. 2015). The proportion of amylose and amylopectin
41
strongly affects the appearance, as well as the cooking characteristics of rice grain (Ahromrit
2006). When the amylose content in rice is lower than 5 %, the rice is classified as glutinous
(Prathepha et al. 2005). Glutinous rice, also known as sticky or waxy rice, has a chalky and opaque
endosperm because of the presence of air spaces between the starch granules (Zhou et al. 2002a).
When cooked, the grain usually loses its shape and becomes very sticky (Noosuk et al. 2003). On
the other hand, non-glutinous or non-waxy rice kernels have a translucent appearance and contain
amylose as well as amylopectin. The cooked grain of non-waxy rice tends to retain its shape and
is less sticky (Kang et al. 2009).
Waxy varieties of rice are grown in many countries, including Lao PDR, Thailand, China,
Myanmar, Vietnam, Cambodia, Japan, Bangladesh, and India (Calingacion et al. 2014; Mar et al.
2015). It is consumed as a sweet dish, as a breakfast cereal or as specialty steam rice in banana
leaves in Thailand, Myanmar, Cambodia, India, China, and Vietnam. In Lao PDR, waxy rice is a
staple food which consumed by most of the population on a daily basis. Lao PDR has the highest
per capita consumption of rice in the world (Schiller et al. 2006), and 85 % of the rice produced is
the glutinous type (Food and Agriculture Organization 2011). Among the wide range of improved
glutinous rice varieties grown in lowland farming systems in Lao PDR are Hom Mali Niaw
(HMN), Thadokkham-8 (TDK8) and Thadokkham-11 (TDK11) (Sengxua et al. 2014).
A knowledge of pasting properties is a key indicator of the processing quality of cereals including
rice and rice products. For example, an understanding of the pasting behavior can help a processor
in optimising ingredient concentrations and temperature-pressure-shear limits when producing the
desired product (Dang & Copeland 2004). Pasting properties are often estimated from pasting
curves obtained using a Rapid Visco Analyzer (RVA), a temperature controlled viscometer that
monitors the resistance of a cereal grain sample to a specified shear. In the beginning, the RVA
was introduced in the 1980s as a means of rapidly measuring the extent of sprout damage in wheat
affected by rain before harvesting (Ross et al. 1987). Originally, it was built to operate at a constant
temperature (95°C), but the addition of variable and controlled heating and cooling made the
instrument more versatile and enabled its use in the measurement of the pasting properties of other
cereal starches under variable conditions (Walker et al. 1988). Currently, the RVA is an industrywide instrument which is used extensively for product development, quality and process control
and quality assurance of various cereals and starches (Doutch et al. 2012). Standard methods for
42
measuring starch pasting properties have been developed and have also been approved by the
American Association of Cereal Chemists (AACC) (Doutch et al. 2012). However, the ability of
RVA to differentiate between samples and to predict the quality of products may vary under
various operating conditions (Konik et al. 1992; Batey et al. 1997; Batey et al. 2000). The pasting
properties measured by RVA can indicate the cooking characteristics of rice to a certain extent
(Champagne et al. 1999). Cooking time is normally the time required for 90 % of the kernels to
become completely translucent when cooked by immersion in distilled water at 95±1oC (Ranghino
1966). However, for RVA analysis the grain is ground to mm size. Therefore, it is not the same as
cooking whole grains, but the technique is still found to provide useful information on the cooking
properties of rice (Zhu et al. 2013). Numerous studies have been done aimed at predicting rice
cooking behavior (Mohapatra et al. 2006; Han & Lim 2009). In most of these studies, the focus
has been to develop a link between cooking time and rice physicochemical characteristics (Vidal
et al. 2007). It has been widely reported that kernel size and shape (especially thickness) are the
key factors influencing cooking time, but cooking time is also dependent on the composition of
the rice kernel, as rice with high protein (Martin & Fitzgerald 2002) and amylose contents (Yu et
al. 2009) has been found to have a longer cooking time. Glutinous rice varieties with low amylose
content have been found to have different pasting properties compared to non-glutinous varieties
(Huaisan et al. 2009; Bao et al. 2004).
Before cooking, the soaking (rehydration) makes the grain softer, enabling water uptake by the
starch during gelatinization (Kashaninejad et al. 2007). Rehydration is a slow and diffusion limited
process. Besides the inherent effect of rice kernel composition and internal structure, the diffusivity
of water is a function of time and temperature (Bello et al. 2010). Warm water rehydration is a
common method used to shorten the soaking time because a higher temperature will increase the
hydration and diffusion rates. Rehydration should be done below the starch gelatinization
temperature to reduce the leaching of solids and unintended gelatinization during this process (Han
& Lim 2009). Thus, to improve the cooking properties and quality, rice is sometimes soaked in
water for hours before cooking; however, there is limited data available relating to the effects of
soaking temperature and soaking time for different types of rice (Lee at al. 2001). This also applies
to glutinous varieties of rice, as systematic studies on the effects of soaking time and temperature
on glutinous varieties are very limited in the literature. Moreover, no such information is available
on the glutinous varieties that are widely grown and consumed in Lao PDR. This study was aimed
43
to investigate the effect of rehydration time and temperature and extended holding during cooking,
on the pasting properties of the common Laotian glutinous rice cultivars. Two non-glutinous rice
varieties were used as reference rice samples for comparison purposes. The results of the study
provide a new data in the literature on the pasting properties of common Laotian glutinous rice
varieties.
3.3. Materials and methods
3.3.1. Materials
Three cultivars of glutinous rice (TDK11, TDK8, and Hom Mali Niaw) were used in this study.
The freshly harvested and milled TDK8 was provided by National Agriculture and Forestry
Research Institute (NAFRI), Lao PDR, while about eight-month-old TDK11, Hom Mali Niaw
(HMN) and the reference non-glutinous rice varieties, IR64 and Doongara (DG), were provided
by Rice Research Australia Pty Ltd (RRAPL), Mackay, QLD, Australia.
3.3.2. Grinding of rice kernels
All rice samples were ground to flour using a hammer mill equipped with a plate of 0.75 mm size.
The samples which passed through this plate were used for RVA analysis.
3.3.3. Apparent amylose content
The apparent amylose content (AAC) of rice samples was determined by the iodine colorimetric
method (Hoover & Ratnayake 2005). This method is based on the fact that iodine-amylose
complex formation gives a blue color, but iodine-amylopectin gives a purple color. This means
that the iodine-amylose complex absorbs other light spectrum but reflects the blue. Measuring the
absorbance in the non-blue region (peak absorbance at 600 nm) will provide the amount of amylose
present in a sample (Knuston & Grove 1994). Samples (20±0.1 mg) in a tube (round bottom with
Teflon cap) were dispersed with 8 mL 90 % DMSO (v/v) and thoroughly mixed with vortex for 5
min. To establish a standard curve, a series of potato amylose (Sigma A-0512) and waxy maize
amylopectin (Sigma S-9679); mixtures at different ratios at the same solids concentrations were
dissolved in 90 % DMSO (v/v) in the same way as the test samples. The tubes containing samples
were heated in a water bath at 85ºC for 15 min with intermittent mixing, allowed to cool to room
temperature (~ 45 min) and then diluted to 25 mL with deionised water. Then, 1 mL of the diluted
solution was mixed with 40 mL of deionised water and 5 mL iodine reagent (2.5 x 10-3 M I2/6.5 x
44
10-3 M KI) in a 50 mL volumetric flask. The tubes with these diluted solutions were vortexed for
thorough mixing and left for 15 min at room temperature for color development. The absorbance
of the standards and samples was measured at 600 nm against a blank reagent as the reference.
The apparent amylose content was determined from the amylopectin/amylose standard curve.
3.3.4. Pasting properties
The pasting properties of rice flours were determined according to the AACC International Method
61-02.01(AACC 1999) by using a Rapid Visco Analyzer (RVA-4D model Thermocline Windows
Control and analysis software, Version 1.2 (New Port Scientific, Sydney, Australia)). To calculate
the sample size for RVA, the moisture contents of all the samples were measured according to the
AACC International Method 44-40.01(AACC 1999) by using a vacuum oven. Based on the
moisture contents, the sample and deionised water required for each cultivar were calculated as
follows: TDK11 (3.00 g sample and 25 mL deionised water), TDK8 (3.01 g sample and 25 mL
deionised water), HMN (3.01 g sample and 25 mL deionised water), IR64 (3.02 g sample and 25
mL deionised water), and DG (3.00 g sample and 25 mL deionised water). Deionised water was
dispensed in RVA canisters. Accurately weighed samples were transferred onto a water surface in
the canisters. Subsequently, an RVA paddle was placed in each canister and firmly inserted into
the RVA. Samples were mixed at 960 RPM for 10 sec to make a homogeneous solution. After 10
sec the RPM was reduced to 160, which was maintained until the end of the run. A standard
program of heating and cooling cycles was: holding the sample at 50ºC for 1 min, followed by
heating to 95ºC in 3.45 min; holding at 95ºC for 2.7 min, then cooling to 50ºC in 3.91 min and
holding at 50ºC for 1.24 min.
3.3.5. Effect of rehydration time and temperature
The effect of rehydration time and temperature on the pasting properties of the rice flours was
investigated by altering the standard AACC International method. The rice flours were rehydrated
at different temperatures for different periods of time: control (50ºC for 1 min)), t1 (50ºC for 15
min), t2 (50ºC for 30 min), t3 (40ºC for 1 min), t4 (40ºC for 15 min), t5 (40ºC for 30 min), t6 (30ºC
for 1 min), t7 (30ºC for 15 min) and t8 (30ºC for 30 min). The remainder of the procedure was left
unaltered. These temperature conditions were chosen based on the practical rehydration
temperature of rice before cooking.
45
3.3.6. Effect of holding time at 95oC
The effect of holding time at 95ºC on the pasting properties of rice flours was investigated by
altering the standard AACC International Method. This was intended to determine the effect of
prolong shear on the rice at the cooking temperature. The rice flours were held at 95ºC for different
time periods, control (2.7 min), T1 (4.7 min), T2 (6.7 min), T3 (8.7 min) and T4 (10.7 min). The rest
of the procedure was left unaltered.
3.3.7. Statistical analysis
All treatments were replicated three times to get mean values. The reported data for the Pasting
temperature (Ptemp), Peak viscosity (Vp), Trough viscosity (Vt), Breakdown (BD), Final viscosity
(Vf) and Setback (SB) for all rice flours were analyzed by analysis of variance (Two-Factor
Factorial Design) using Minitab R16 (Minitab for Windows Release 16, Minitab Inc, Chicago)
in order to determine significant differences. The data was then analyzed using Tukey’s pair-wise
comparison, at 5 % level of significance, to compare the results between different treatments.
3.4. Results and discussion
3.4.1. Apparent amylose content
The rice cultivars selected for this study represented a wide variation in AAC, ranging from 3.72
% to 19.71 % (Table 3.1). Previous studies reported that AAC of various waxy and non-waxy rice
genotypes ranged from 0 % to as high as 29.2 % (Dang & Copeland 2004; Kong et al. 2015). As
expected TDK8, TDK11 and HMN, contained very low levels of amylose, and are therefore
classified as glutinous rice varieties, while IR64 and DG (with amylose contents in the range of
14.92 and 19.71 %) are classified as non-glutinous rice varieties. The variation in AAC has been
reported to differ with the botanical source of the starch and is usually affected by the climatic and
soil conditions during grain development (Tashiro & Wardlaw 1991; Singh et al. 2006; Wang et
al. 2010). This study revealed how the glutinous and non-glutinous group would respond to an
extension of rehydration and cooking time (holding at 95oC). The results (Fig. 3.1), show a positive
correlation between the AAC and Ptemp; higher the AAC the highest Ptemp will be. The onset of
gelatinization is indicated by the pasting temperature (Ptemp).The glutinous varieties had
significantly (P<0.05) lower Ptemp than the non-glutinous cultivars (IR64 and DG) used in this
study.
46
Table 3.1 Apparent amylose content (AAC) of selected rice varieties
Variety
AAC (%)
TDK11
3.72±0.07
TDK8
3.77±0.15
HMN
3.82±0.07
IR64
14.92±0.29
DG
19.71±0.59
85
y = 0.80x + 64.67
R² = 0.92
Pasting temperature (ᵒC)
80
75
70
65
60
0
2
4
6
8
10
12
14
16
18
20
Apparent amylose content (%)
Figure 3.1 Change in pasting temperature (onset of gelatinization) with increasing
apparent amylose content (AAC)
3.4.2. Effect of rehydration time and temperature on pasting properties
The viscograph is designed to study the three basic properties of starch. First, when starch particles
are subjected to heat with water, they will swell, forms a paste and provides a body. The second
important property of the starch is the stability of the body or paste or the viscosity. The third
property is the extent of paste congelation while cooling. The representative RVA curves for
selected glutinous rice varieties (TDK11, TDK8, and HMN) under standard analytical conditions
showing all the pasting attributes, are presented in Fig. 3.2.
47
Figure 3.2 Representative RVA curves of selected glutinous varieties (TDK11, TDK8,
and HMN) at standard RVA analysis
The temperature at the onset of the rise in the viscosity, known as pasting temperature (P temp) is
considered as an indication of the minimum temperature required for cooking (Yadav et al. 2014).
The equilibrium point between starch granules is swelling, and polymer leaching is known as peak
viscosity (Vp). This normally happens close to 95ºC in RVA analysis and can represent the cooking
temperature of rice. It is the maximum viscosity attained by gelatinised starch during heating. Vp
indicates the water binding capacity of starch granules (Shimelis et al. 2006) at this temperature.
In fact, this viscosity value also depicts a balance between water holding capacity and breakdown
of starch at the shear rate. During the hold period at this temperature, the samples are subjected to
a period of constant mechanical shear stress. Continuous stirring of the RVA pedal at high
temperature (95ºC) can result in disruption of starch granules and a reduction in viscosity, which
eventually reaches to a minimum value, known as trough viscosity (Vt) (Song & Shin 2007). The
difference between peak and trough viscosity is commonly referred to as breakdown viscosity
(BD). Re-alignment of starch molecules occurs when the mixture is allowed to cool; consequently,
a gel is formed. This leads to enhanced viscosity known as final viscosity (Vf). It is usually
considered as a quality parameter to measure the strength of gel upon cooling (Cornejo-Villegas
48
et al. 2010). The range of the RVA curve between the trough and final viscosity is usually referred
to as the setback region. The difference between the two ends of this region is known as setback
viscosity (SB) (Zhu et al. 2013). The effect of rehydration time and temperature on the pasting
properties of glutinous and non-glutinous rice cultivars with various amylose contents used in this
study is shown in Fig. 3.3, 3.4, 3.5, 3.6, 3.7 and 3.8.
3.4.2.1. Pasting temperature (Ptemp)
A range of factors governs variations in the Ptemp, but the type of starch in the endosperm is the
most significant factor (Thomas et al. 2014). It is now a very well established concept that water
uptake by the starch granules is directly proportional to time and temperature of rehydration
(Buggenhout et al. 2014; Briffaz et al. 2014). Higher water uptake will result in a decrease in the
onset pasting temperature which is also a trend observed in all glutinous rice varieties investigated
in this study (Fig. 3.3). This effect was not found in the non-glutinous variety IR64 but was found
in the variety DG. This indicates that the effect is variety dependent and will certainly be
influenced by the physicochemical characteristics of the grain (Bao et al. 2004).
Figure 3.3 Effect of soaking time and temperature on the pasting temperature (P temp) of
three different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two nonglutinous rice varieties; (d) IR64, and (e) DG
Pasting temperature (Ptemp) for TDK11 after 1 min of soaking was more significantly (P<0.05)
reduced at 50ºC when compared with 30ºC and 40ºC. However, soaking time did not have a
significant (P>0.05) effect. A similar trend was observed for the varieties TDK8 and HMN. The
49
results revealed that 1 min of soaking of flour particles (≤750 µm) at ambient temperature is
optimum for the onset of gelatinization (Song & Shin 2007). Among the waxy rice, TDK8
exhibited the highest Ptemp during all rehydration conditions (Fig. 3.3b). Pasting properties are
related to amylopectin branch chain length distribution. The presence of long chain branches
contributes to increased pasting temperature (Jane et al. 1999). The higher proportion of long
chains (DP≥37) in amylopectin results in a relatively high level of stabilised starch granules,
leading to higher pasting temperature or the gelatinization temperature (Koroteeva et al. 2007).
IR64 and DG (non-glutinous varieties) had higher Ptemp than the glutinous varieties TDK11, TDK8,
and HMN. This means that the absence or low amount of amylose can also facilitate the
gelatinization of amylopectin. Similar observations have also been reported by Tran and coworkers (2001) for other rice varieties.
3.4.2.2. Peak viscosity (Vp)
The temperature of the soaking had a much greater impact on peak viscosity (Vp) than the duration
of soaking. Different glutinous rice cultivars behaved in a diverse manner when treated with
various soaking times and temperatures (Fig. 3.4). The peak viscosity (Vp) of TDK11 (Fig. 3.4a)
showed a significant (P<0.05) increase when soaked at 40ºC, while a further increase in
temperature had no significant (P>0.05) effect. It is also observed that the duration of soaking had
no significant (P>0.05) effect on peak viscosity (Vp), except 30 min of soaking at 40ºC which
resulted in a significant (P<0.05) decrease in peak viscosity (Vp). TDK8 showed a significant
(P<0.05) decrease in the peak viscosity (Vp) with an increase in soaking temperature. Maximum
viscosity (2403.3 mPa-s) was observed within 1 min of rehydration at 30ºC, and minimum
viscosity (1852.0 mPa-s) was observed after 15 min of rehydration at 50ºC (Fig. 3.4b). HMN
showed a significant (P<0.05) increase in peak viscosity (Vp) at 40ºC, with a further increase in
temperature, resulting in a significant (P<0.05) decrease in viscosity (Fig. 3.4c). TDK8 showed a
significant (P<0.05) decrease in the peak viscosity (Vp) with an increase in rehydration time and
temperature. Maximum viscosity (2403.3 mPa-s) was observed after 1 min of rehydration at 30ºC,
and minimum viscosity (1852.0 mPa-s) occurred after 15 min of rehydration at 50ºC (Fig. 3.4b).
The Vp of TDK11 and HMN increased to highest values (1608.7 and 1477.7 mPa-s, respectively)
with an increase in temperature up to 40ºC for 1 min (Fig. 3.4a and 3.4c). A further increase in
either rehydration time or temperature decreased the Vp in both TDK11 and HMN. HMN showed
50
the lowest Vp for all treatments. You et al. (2014) suggested that the amylopectin with more short
chains (DP6-12) would result in a lower pasting temperature and peak viscosity, as the short branch
chains do not provide strong interactions (Chung et al. 2011) to maintain the integrity of the
swollen granules, resulting in lower peak viscosity. Peak viscosity is the balance between starch
swelling and breakdown at the given shear rate. In the non-glutinous rice, the variety DG showed
susceptibility towards changes in rehydration conditions (Fig. 3.4e), with an increase in time and
temperature of soaking increasing the equilibrium point of granules swelling and leaching of
amylose (Hasjim et al. 2012). IR64 was quite stable in all soaking conditions, and no significant
changes were recorded (Fig. 3.4d).
Figure 3.4 Effect of soaking time and temperature on the peak viscosity (Vp) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG
3.4.2.3. Trough viscosity (Vt) and breakdown (BD)
TDK11 and HMN flours showed significantly higher breakdown at 40ºC (Fig. 3.5a and 3.5c).
Among the waxy rice varieties, the higher breakdown was observed for TDK8 (Fig. 3.5b). Mostly
breakdown is correlated with peak viscosity, the higher the peak viscosity, the greater the level of
breakdown (Higley et al. 2003). In general, a longer soaking time was not reflected in significant
differences in the trough viscosity of the rice samples (Fig. 3.6). However, the temperature of
soaking showed a reduction in the trough viscosity in all rice varieties except TDK8 which showed
an increasing trend. The reduction in trough viscosity was much greater for the samples soaked at
50ºC. Thus, the breakdown of starch granules exhibited a strong positive correlation with soaking
51
time and temperature among both the glutinous and non-glutinous rice cultivars evaluated (Fig.
3.5). A high level of breakdown is associated with a high degree of collapse of swollen starch
granules (low trough viscosity). This may indicate a softer texture of the gel or cooked grain.
Figure 3.5 Effect of soaking time and temperature on the breakdown viscosity (BD) of
three different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two nonglutinous rice varieties; (d) IR64, and (e) DG
Figure 3.6 Effect of soaking time and temperature on the trough viscosity (V t) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG
52
3.4.2.4. Final viscosity (Vf) and setback (SB)
The final viscosity was measured by cooling the sample to 50ºC. The increase in viscosity due to
cooling reflects the effects of temperature on viscosity and retrogradation of starch. Retrogradation
ability of straight chain amylose molecules is much higher and faster than for branched
amylopectin (Suzuki et al. 2006). The glutinous varieties containing a very low amount of amylose
retrogrades very slowly (Singh et al. 2012). Similar observations were recorded in this study
through the observation of reduced final viscosity of the gel (Fig. 3.7 and 3.8). Among the
glutinous rice, TDK11 (Fig. 3.7a) and TDK8 (Fig. 3.7b) showed significantly (P<0.05) high
setback (SB) at 40ºC from soaking for one min while HMN (Fig. 3.7c) showed significantly
(P<0.05) high setback at 50ºC after soaking for 1 min. Non-glutinous rice IR64 and DG (Fig. 3.7d
and 3.7e, respectively) showed significant (P<0.05) increase in setback (SB) with an increase in
soaking temperature. For both varieties, maximum setback (SB) was observed at 50ºC. It is
observed that among the soaking conditions only temperature significantly affects the setback (SB)
rather than the time of soaking.
Figure 3.7 Effect of soaking time and temperature on the setback viscosity (SB) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG
With a decrease in soaking temperature to less than 40ºC, strong resistance was recorded by the
RVA paddle, resulting in high final viscosity (Vf). The soaking time did not show a strong effect
on the final viscosity, but all glutinous varieties showed a decline in final viscosity (Vf) at higher
53
soaking temperature. Among the three varieties, TDK8 (Fig. 3.8b) showed the greatest sensitivity
to soaking temperature. Similar effects were observed in the non-glutinous rice, (both IR64 and
DG) but the retrogradation would be higher in non-glutinous rice due to the presence of higher
amounts of amylose.
Figure 3.8 Effect of soaking time and temperature on the final viscosity (V f) of three
different glutinous rice varieties; (a) TDK11, (b) TDK8, (c) HMN, and two non-glutinous
rice varieties; (d) IR64, and (e) DG
3.4.3. Effect of an extension of holding time at 95ºC on the viscosity
Increased shear at cooking temperature is expected to increase the breakdown, and reduce trough
and final viscosities. The effect of extended holding time at 95ºC on the pasting properties of all
rice cultivars used in this study is shown in Fig. 3.9. As expected, there was a significant difference
(P<0.05) in the pasting properties of all five glutinous and non-glutinous rice cultivars used in this
study. Peak viscosity (Vp) for all cultivars with various amylose contents was significantly
(P<0.05) different (Fig. 3.9a). However, there was no significant (P>0.05) difference in holding
time at 95ºC on the peak viscosity (Vp) of TDK8, HMN, DG and IR64 but peak viscosity (Vp) of
TDK11 flour increased significantly (P<0.05) with an increase in holding time from the control
to 4.7 min, but remained constant with any further increase in holding time. In this case, TDK11
was found to be more resistant than the other varieties to the breakdown of the swelled starch
particles during shearing at 95ºC. This indicates that not only the amylose content but also the
starch grain structure will contribute to the breakdown of gelatinised and swelled starch.
54
The disruption of starch granules followed the peak viscosity during holding at the same
temperature. This period is usually accompanied by a reduction in the viscosity, which eventually
reaches a minimum value. This minimum viscosity attained by the starch granules during RVA
run is known as trough viscosity (Vt) (Dang & Copeland 2004). The loss of viscosity is caused by
leaching of amylose and also a breakdown of the swelled granules due to the shear in RVA. A lot
of variation was observed in trough viscosity (Vt) of the glutinous and non-glutinous cultivars used
in the present study (Fig. 3.9b), with extended holding at a higher temperature (95oC) having a
significant (P<0.05) effect on the glutinous cultivars TDK11 and TDK8, and for non-glutinous
cultivar IR64. The variety DG showed resistance towards extended heating up till 6.7 min, after
which there was a significant (P<0.05) disruption of granules and possibly leaching of amylose
from starch granules was observed, resulting in a decrease in Vt. While HMN flour was quite stable
with extended holding at 95ºC, there was no significant (P>0.05) change in trough viscosity (Vt).
Among the glutinous rice, maximum breakdown (BD) was observed in TDK8 followed by
TDK11, while least breakdown was observed in HMN (Fig. 3.9c). Breakdown (BD) of TDK11
and HMN significantly (P<0.05) increased up to 4.7 min, with further cooking having no
significant (P>0.05) effect on breakdown (BD). Significant (P<0.05) breakdown (BD) was
observed in TDK8 up till 6.7 min of cooking, further cooking had no significant (P>0.05) effect.
In the non-glutinous rice, IR64 showed higher breakdown than DG. Both varieties had
significantly (P<0.05) higher breakdown (BD) and leaching of amylose up till 4.7 min of cooking,
with further cooking having no significant (P>0.05) effect on breakdown (BD) in either of these
varieties.
Flour of glutinous rice cultivars produced weaker gels at the final temperature of cooling (50oC)
than the non-glutinous flours (Fig. 3.9d). Among the glutinous rice, TDK8 had significantly
(P<0.05) higher final viscosity (Vf) and setback (SB) than the other glutinous rice (TDK11 and
HMN) (Fig. 3.9d and 3.9e). Significantly (P<0.05) higher final viscosity and setback was observed
in TDK11 up till 4.7 min of cooking, with a further increase in cooking time affecting setback and
final viscosity. TDK8 behaved in an entirely different manner to TDK11. Increased cooking time
resulted in a significant decline in final viscosity (Vf) and greater setback (SB) up to 4.7 min.
Further cooking resulted in low final viscosity (Vf), but the effect was non-significant. There was
no significant (P>0.05) effect of extended cooking on the final viscosity (Vf) and setback (SB) for
the variety HMN. Among the non-glutinous rice, IR64 showed a significant (P<0.05) decline in
55
final viscosity (Vf) and setback (SB) up till 4.7 min, with any further increase not resulting in
further significant differences in either final viscosity and setback. The variety DG showed quite
a stable behavior towards extended cooking, resulting in no significant differences in final
viscosity (Vf) and setback (SB). Thus in general, the effect of holding at 95ºC and prolonged shear
exhibited the same effects on glutinous and non-glutinous varieties and was variety dependent.
Figure 3.9 Effect of extended holding time at 95ºC on the pasting properties of glutinous
and non-glutinous rice varieties; (a) Peak viscosity (Vp), (b) Trough viscosity (Vt), (c)
Breakdown
(d) Final
viscosity
(Vproduced
(SB)at the final temperature of
f) and (e) Setback
Flour(BD),
of glutinous
rice
cultivars
weaker gels
Hasjim and co-workers (2013) also reported that increasing the cooking time at 95ºC longer than
4 min caused only small changes in the trough and the final viscosity of polished long grain rice
flour.
3.5. Conclusions
A knowledge of rehydration and cooking attributes of various glutinous and non-glutinous rice
varieties is very important, as rice is a major ingredient in many processed foods. As expected,
significant differences in the pasting properties of various glutinous varieties from Lao PDR were
observed. Water uptake by the flour of all rice varieties was directly proportional to the
time/temperature of rehydration. Pasting temperature depends on the amylose content, the higher
the amylose, the higher will be the onset temperature of starch gelatinization. Among the glutinous
rice varieties tested, TDK8 showed the greatest response to rehydration conditions, resulting in
highest peak viscosity. The rice cultivars evaluated in the study showed diverse response when
56
treated with extended cooking. For the glutinous variety TDK8 and non-glutinous variety, IR64
starch granules showed a higher breakdown in response to extended cooking, resulting in reduced
trough viscosity and reduced retrogradation. It is therefore recommended that for glutinous rice
especially TDK8, extended cooking will result in a better-cooked product. This work has generated
the pasting data for the most popular glutinous varieties consumed in Lao PDR. Further work is
being undertaken on the cooking, and textural attributes of whole grain of these glutinous varieties
as RVA analysis can only use ground samples.
57
Chapter 4 In situ analysis of cooking properties of rice by Thermal Mechanical
Compression Test (TMCT) method
This chapter has been published in the International Journal of Food Properties;
Nawaz, MA, Fukai, S & Bhandari, B 2017, ‘In situ analysis of cooking properties of rice by
Thermal Mechanical Compression Test (TMCT) method’, International Journal of Food
Properties, vol. 20, pp. 1174-1185.
58
4.1. Abstract
A procedure for in situ analysis of rice cooking was developed in this study. Grain softening during
soaking and cooking of selected rice varieties (fresh and aged TDK8, TDK11, and Doongara) were
subjected to in situ analysis by using a thermally controlled sample block (TMCT) attached to a
texture analyser. This technique measures the changes in the mechanical properties of intact rice
grain during cooking continuously. The results obtained from the TMCT technique were validated
against two standard and conventional procedures viz. analysing the pasting properties of rice
flours by Rapid Visco Analyzer (RVA) and microscopic observations during the cooking of rice
grains. The technique developed in this study was found valid for in situ analysis of rice cooking.
This technique can be used for a sample size as small as 0.50 g.
4.2. Introduction
Rice is one of the most popular staple cereals consumed around the globe. There are diverse
varieties of rice, and their aromatic and textural properties can be very distinct. Consumer
preferences for cooked rice vary from region to region, Laotian people like sticky glutinous rice
as staple and the Japanese like it on special occasions (Mohapatra & Bal 2006; Boualaphanh et al.
2011), while Italians consume short grain varieties Baldo and Arborio rices with high amylopectincontent, which release starch during cooking making a creamy and smooth Risotto (Puri et al.
2013). Therefore, it is very difficult to find out a standard procedure for rice cooking. For example,
traditionally, cooked waxy glutinous rice is produced by soaking overnight before steaming the
rice. Prolonged soaking is required to soften the grains and increase the water content to accelerate
the starch gelatinization to reduce the steaming (cooking) time. In some population, freshly
harvested rice grain is preferred while other population may prefer the aged rice due to the
difference in the texture of the cooked rice with aging (Tananuwong & Malila 2011).
Many studies have reported the effect of various cooking conditions such as temperature and
pressure (Leelayuthsoontorn & Thipayarat 2006; Tian et al. 2014), water to rice ratio (Srisawas &
Jindal 2007), steam cooking and stir-frying (Reed et al. 2013) on the texture of cooked rice. A
significant number of researches describing rate and effect of water infusion into non-glutinous
rice grains on their cooking quality have been carried out in the past (Bakshi & Singh 1980;
Hendricks et al. 1987; Miah et al. 2002b; Tian et al. 2014; Rafiq et al. 2015). The cooking quality
of these rice is analysed after the final cooking. This is mainly done by measuring the hardness of
59
the cooked rice using a texture analyser (Bello et al. 2006). Similarly, although the information
about glutinous rice hydration and cooking are very limited, its soaking and softening properties
are characterised by some researchers (Singh et al. 2000; Ahromrit et al. 2006; Peerapattana et al.
2010). Moreover, considerable research has been conducted to find out the mechanism of water
movement and final cooking qualities of cereal grains to develop kinetics model (Bello et al. 2010).
Gelatinization initiated major changes in the physical and chemical properties of starch. Therefore,
it is important to get information on the evolution of cooking of rice during the cooking period.
RVA analysis of the rice has been reported to interpret the pasting properties to the cooking
qualities of rice (Nawaz et al. 2016a). However, during RVA analysis the grain needs to be ground
to flour. The rate of moisture diffusion and gelatinization of starch during cooking can be different
between flour and whole rice kernel. The cooking behavior and grain characteristic changes with
time have also been analysed by microscopic observation such as the presence of un-gelatinised
white belly at the interior of the cereal grains undergoing cooking (Lund & Lorenz 1984; Srikaeo
et al. 2006). This analysis requires periodical sampling, slicing and microscopic observation during
the cooking. The disappearance of the white belly is the indication of the cooking time required.
The cooking quality of the rice is analysed by determining the texture (mainly hardness) of the
cooked grain by the texture analyser. There are no methods which analyse the rate of cooking and
the texture of the whole grain rice as a one-step analysis.
In this study, a new in situ method is introduced to study the textural properties of rice using the
Thermal Mechanical Compression Test (TMCT) device attached to a texture analyser. The TMCT
has been used previously for analysing the stickiness and glass-rubber transition temperature of
various food materials (Liu et al. 2010) including spray dried orange juice powder, milk powders,
spaghetti and rice (Shrestha et al. 2007b; Boonyai et al. 2007; Rahman et al. 2011; Thuc et al.
2010). This method is based on the measurement of the displacement of a probe compressing the
rice grain under a constant force while cooking in a temperature controlled TMCT device. During
hydration and gelatinization, the softening of the grain will cause probe movement to maintain a
set constant force. This study was aimed to establish a standard procedure for in situ cooking
analysis by using TMCT. This novel method will also allow the development of kinetic models of
cooking and also determine the final cooking time of different rice varieties.
60
4.3. Materials and methods
Two Oryza sativa indica cultivars of glutinous rice from Lao PDR viz. TDK8 and TDK11 having
3.77 % and 3.72 % apparent amylose contents (AAC), respectively and one O. sativa japonica
non-glutinous rice from Australia (Doongara, 19.71 % (AAC)) were used in this study. The milled
TDK8 was provided by National Agriculture and Forestry Research Institute (NAFRI), Lao PDR,
while TDK11 and Doongara were provided by Rice Research Australia Pty Ltd (RRAPL),
Mackay, QLD, Australia.
4.3.1. The moisture content of rice grains
The moisture content of rice grain was measured according to the AACC International Method 4440.01(AACC 1999) by using the modified vacuum-oven method. Two grams of well-mixed
sample was accurately weighed in a covered dish, which was previously dried at 98-100ºC and,
cooled in a desiccator to room temperature. The samples were heated at 98-100ºC to constant
weight (for about 5 hrs) in a partial vacuum having pressure equivalent to 25 mm Hg or less. The
dried dishes with sample were cooled in a desiccator and weighed soon after it reached room
temperature.
4.3.2. Moisture uptake by rice grains
Rice grains (10±2 g, known initial moisture contents) were taken in a sieve (a tea filter). The sieve
with the sample was fully immersed in water maintained at 22±1ºC, 40±1ºC, and 50±1ºC. After
every 5 min of soaking, the sample was taken out from the sieve and wiped carefully with blotting
paper to remove the surface moisture. The sample was weighed carefully, and weight gained by
the sample was recorded. The sample was again soaked in water for 5 min. The weight gained by
the sample for 45 min was recorded.
4.3.3. Measurement of grain softening during hydration and cooking by TMCT
device
The TMCT device used in this work is shown in Fig. 4.1. This device is made-up of the temperature
controlled aluminum block (50x50x25 mm) and attached to a Texture Analyser. A single layer of
1 g of rice kernels was soaked in 1 mL of deionised water in thermally controlled (TMCT)
aluminum block. The soaked samples were compressed by a 35 mm Teflon probe at the steady
force of 0.10 and 0.15 N. The hydration and gelatinization of the starch will cause the softening of
61
the grain resulting into the displacement of the probe to maintain the constant force. At a
compression force of 0.10 N, the force was unstable, so the probe displacement was distorted (Fig.
4.2a). In the preliminary experiment, a load of 0.15 N was found appropriate as the compression
of the sample was low (10 % of the grain height at the highest hydration level) and the probe
movement signal was found stable (Fig. 4.2b). Thus, the sample deformation at 0.15 N during the
measurement was not high. The change in distance of probe during soaking was recorded by the
Texture Analyzer TA-XTplus (Stable Microsystems, UK) for 1.5 h.
Figure 4.1 Illustration of grain softening during soaking and cooking, (a) Overall
experiment assembly, (b) Pictorial representation of the measurement process
62
Figure 4.2 Probe displacement during in situ soaking of rice in a TMCT device at two
compression forces, (a) 0.10 N, and (b) 0.15 N
Figure 4.3 The in situ TMCT cooking of rice at 95±1°C with two different rice to water
ratios (1:2 and 1:3)
A similar experiment was conducted during cooking of rice. The sample block was heated to a rice
cooking temperature of 95±1ºC and held it at this temperature. In the preliminary trials various
63
rice to water ratios such as 1:2 and 1:3 were used to streamline the in situ TMCT cooking process.
It was observed that 1:2 rice to water ratio was not stable and probe distortion was started within
the initial 14 min due to evaporation of water from the sample container. However, it was found
that 1:3 provided stable results (Fig. 4.3) with the compression of the sample less than 20 %. The
amount of water required to cook the rice is higher due to loss of water from the annulus space
between the probe and the sample block, although the gap was very small (<1 mm). Therefore, a
single layer of 0.5 g of rice kernels and 1.5 mL of deionised water (rice: water ratio = 1:3) was put
on the sample block. The change in distance of probe was recorded by the texture analyser until
rice sample has taken up all available water and fully cooked (indicated by the vibration in probe
due to the evaporation of water from the cooked grain). The cooking rate of the grains was
estimated by measuring the slope of the initial linear part of the in situ TMCT cooking curves.
4.3.4. Pasting properties
Pasting properties of rice flour (particle size ~ 750 µm) were determined according to the AACC
International Method 61-02.01 (AACC 1999) using a Rapid Visco Analyzer (RVA-4D model
Thermocline Windows Control and analysis software, Version 1.2 (New Port Scientific, Sydney,
Australia)). Rice flour (3.01 g, 12.4 % moisture basis) was mixed with 25.0 g MilliQ water in the
RVA canister. A programmed heating and cooling cycle were used, the samples were held at 50ºC
for 1 min, heated to 95ºC in 3.45 min, held at 95ºC for 2.7 min before cooling to 50ºC in 3.91 min
and holding at 50ºC for 1.24 min. The peak time (PT), peak temperature (Ptemp) and peak viscosity
(Vp) were recorded.
4.3.5. Light microscopy of rice kernels during cooking
The recorded time required for cooking in the grain softening experiment was cross verified by
light microscopy using a Zeiss Axio microscope (Oberkochen, Germany). Five grams rice sample
was added in 15 mL of deionised water (rice: water ratio = 1:3) in a 50 mL glass beaker. The
beaker was placed in a water bath at 95±1ºC. After every 5 min, about half gram of sample was
taken out. Cross sections of rice kernel were studied under a light microscope to see the extent of
gelatinization by observing the non-gelatinized white belly. Cooking was continued until there was
no white belly observed in rice kernel cross-section.
64
4.3.6. Statistical analysis
The reported data was analyzed by analysis of variance (Completely Randomized Design) using
Minitab R17 (Minitab® for Windows Release 17, Minitab Inc., Chicago) to determine significant
differences. The data was then analyzed using Tukey’s pair-wise comparison of different
treatments, at 5 % level of significance.
4.4. Results and discussion
4.4.1. Comparison of grain softening and moisture uptake during hydration
The grain softening and moisture uptake curves of fresh TDK8 soaked at various temperatures
(22±1ºC, 40±1ºC, and 50±1ºC) are shown in Fig. 4.4a and 4.4b, respectively. As expected the
uptake of moisture caused the softening of the grain which was measured by the displacement of
the compression probe in the TMCT device attached to a texture analyser (Fig. 4.1). Results
revealed that grains absorbed moisture quickly and became softer with an increase in soaking
temperature. The rate of water absorption and grain softening was higher in the initial 20 min of
soaking. The probe displacement due to grain softening and moisture uptake results corresponded
very well (Fig. 4.4b).
Figure 4.4 Comparison of (a) grain softening measured as probe displacement by using
TMCT and (b) moisture uptake during hydration of fresh TDK8 at different soaking
temperatures (22, 40 and 50ºC)
65
Figure 4.5 Comparison of (a) grain softening measured by the displacement of the probe
by using TMCT and (b) moisture uptake during hydration of aged TDK8 at different
soaking temperatures (22, 40 and 50ºC)
Figure 4.6 Grain softening measured by probe displacement using TMCT for Doongara
(a) and TDK11 (b) rice varieties during hydration at different soaking temperatures (22,
40 and 50oC)
The results showed that most of the water was taken up in 45 min and showed a slow rate of
moisture adsorption and also probe displacement beyond this time (Fig. 4.4a) might be due to
moisture distribution change within a grain. A similar experiment was undertaken for the aged (6
months) of the same variety (TDK 8) and other varieties of rice. The results also showed the similar
trend for aged (Fig. 4.5) and other rice varieties; Doongara and TDK11 (Fig. 4.6a and 4.6b,
respectively). The rate of infusion of water into the kernels was increased by increasing the soaking
66
temperature, resulting in faster softening of grains. The higher water diffusivity at higher soaking
temperature is well understood (Miah et al. 2002b; Kashaninejad et al. 2009), and this can also be
reflected by the softening of the grain using in situ measurement by TMCT device. To note that
the variability of the results was evident in both methods. Therefore, the statistical significance
(P<0.05) only existed between 22 and 50ºC temperature of rehydration. However, the average
trend of probe displacement or moisture adsorption as a function of temperature and time was very
consistent at all three hydration temperatures. The large error seen in the results are probably
originated by the loss of solids during rehydration and variation while blotting the moisture before
weighing. This type of variability has been reported in the previous studies with rice (Bello et al.
2010). It should also be noted that the water adsorption rate of aged rice samples was more affected
by the temperature of rehydration than the fresh sample. The slow rate of water adsorption by aged
rice is well known (Butt et al. 2008).
4.4.2. The in situ TMCT analysis of cooking properties
The cooking curves of various rice varieties (fresh and aged TDK8, TDK11, and Doongara) are
shown in Fig. 4.7. Results revealed that the fresh and aged TDK8, TDK11 and Doongara grains
took 31.5, 53.8, 26.2 and 19.8 min, respectively to absorb all added moisture and completely
gelatinize (cooked). It was observed that the rice grain was fully gelatinized and the later part of
probe displacement (Fig. 4.7) curve was distorted possibly due to evaporation of moisture from
cooked grains as no free water was visible on the sample holder aluminum block when this
distortion was observed.
In aged rice, the rate of water uptake and gelatinization were slower than the fresh grains possibly
due to age-induced physicochemical changes. Therefore, probe displaced at a much slower rate
after 40 min and distortion started at 53.8 min. This assumption was validated by using excess
water (rice: water ratio = 1:10) by cooking half a gram of grains in 5 mL of deionised water. Grains
were covered with water even after the gelatinization was complete. An indication of completion
was done during preliminary trials; samples were taken out of sample block at different time
intervals to check the ungelatinized white belly. It was observed that when the samples were
completely gelatinised and there was no free water available, “probe distortion” started. So, the
start of probe distortion was the cooking time of respective samples. No probe distortion was
observed in excess water as depicted in Fig. 4.8.
67
Figure 4.7 The in situ TMCT cooking curves for various rice varieties (Fresh TDK8,
Doongara, TDK11 and aged TDK8) cooked at 95±1ºC using rice to water ratios of 1:3.
Black arrows and numbers correspond to the cooking time
Figure 4.8 The in situ TMCT cooking of rice at 95±1ºC with two different rice to water
ratios (1:10 and 1:3)
68
4.4.3. Estimation of the rate of cooking by using the in situ TMCT cooking method
The cooking proceeds from outside the grain to the centre. This process is time dependent. In
general, the initial 20 min of in situ TMCT cooking curves were linear in all rice samples used in
the study. Therefore, only initial cooking curves were used to establish the rate of cooking. The
rate of cooking will thus signify the rate of softening of the grain. In general, it was observed that
higher the cooking rate lower would be the cooking time except for TDK11 (Table 4.1).
Table 4.1 The rate of cooking indicated by the rate of probe displacement during in situ
cooking of selected rice varieties (fresh and aged TDK8, TDK11, and Doongara). Higher
rate signifies faster cooking rate*
Rice varieties
The rate of cooking20 min
Cooking time (min)
(mm/sec)
Fresh TDK8
-0.018±0.002a
31.5±1.1b
Aged TDK8
-0.012±0.003a
53.8±2.5c
TDK11
-0.013±0.003a
26.2±2.02b
Doongara
-0.029±0.006b
19.8±2.47a
* Means ± SD. Within a column, means with different superscripts are significantly
different at 5 % probability level.
4.4.4. Pasting properties
The peak time (PT) estimated by RVA can indicate the cooking characteristics of rice (Champagne
et al. 1999; Zambrano et al. 2016). The RVA viscographs of all rice varieties (fresh and aged
TDK8, TDK11, and Doongara) are shown in Fig. 4.9a, 4.9b, 4.9c, and 4.9d, respectively. The time
required by rice flours to reach the peak viscosity (Vp) is known as peak time (PT). It is the
indication of complete gelatinization of starch. Results showed that the fresh and aged TDK8 rice
flours took 4.6 and 3.9 min to reach Vp (Fig. 4.9a and 4.9b), while TDK11 and Doongara took 3.5
and 6.0 min, respectively (Fig. 4.6c and 4.6d). The PTs of all rice flours used in this study were
significantly (P<0.05) less than the cooking time estimated by the in situ TMCT cooking method.
The grains are ground to mm size before RVA analysis. The reduction in particle size enhances
the gelatinization due to increased heat and mass transfer rate (Zhu et al. 2013). Therefore, PT of
ground rice flour is not the same as the cooking time of whole grains.
69
Figure 4.9 The RVA viscographs of various rice varieties, (a) Fresh TDK8, (b) Aged
TDK8, (c) TDK11, and (d) Doongara
4.4.5. Estimation of cooking time by using light microscopy
The extent of gelatinization of starch while cooking of fresh and aged TDK8, TDK11 and
Doongara rice samples were observed over the time by using light microscopy. The nongelatinized starch was observed by the presence of white belly in the cooking grain (Fig. 4.10).
Fresh TDK8 took 30 min at 95±1ºC of cooking temperature to gelatinize the starch to the core of
the kernel completely. Moreover, aged TDK8, TDK11, and Doongara took 50, 25 and 20 min
respectively to be completely cooked. This method gives actual cooking time (Faruq et al. 2015),
but it is tedious to draw the sample periodically and does not provide the quantitative value.
70
Figure 4.10 The light microscopy of fresh and aged TDK8, TDK11 and Doongara rice
kernels during cooking at 95±1ºC (Black arrows indicate the non-gelatinized white belly
areas)
71
4.4.6. Comparison of in situ TMCT cooking, RVA and light microscopy
It was interesting to note that the in situ TMCT cooking has a possible correspondence to peak
viscosity (Vp) of RVA findings. The in situ TMCT cooking time decreased with decreasing Vp in
all rice varieties used except TDK11. Such as Vp of aged TDK8 is significantly (P<0.05) higher
than fresh TDK8 (Fig. 4.9a and 4.9b). A similar trend was found in in situ TMCT analysis. The
cooking time of aged grains was significantly (P<0.05) higher than fresh grains (Fig. 4.7).
Doongara had the lowest in situ TMCT cooking time and Vp among all the rice varieties used in
the study. So, the RVA results indicated the swelling and integrity of swollen particles but did not
indicate the actual cooking time due to the limitation of flour usage instead of whole grains. The
findings of in situ TMCT cooking analysis correlated well with microscopic observation. The in
situ TMCT cooking analysis was well correlated with the conventional microscopic analysis of
the cooking process with the minor variation of 4 to 7 % in all selected rice samples. The in situ
TMCT results showed that aged TDK8 gelatinised at a very slow rate after 30 min (Fig. 4.7). A
similar trend was observed in the microscopic observation; around 70 % area of the kernel was
gelatinised in first 30 min while the remaining 30 % took 20 min (Fig. 4.10). Microscopic
observations were found to be very tedious, time-consuming, requiring large sample size (at least
3 to 5 g) and providing only subjective observations. The in situ TMCT cooking is very feasible
and easy method and provides detailed information about the rate of water absorption during
soaking and gelatinization or cooking. This technique can be used for sample size as small as 0.50
g. The only limitation of this method is the large standard deviation possibly due to the variation
in multiple kernel dimensions of the sample taken for the analysis. Further work will be undertaken
to relate the in situ TMCT cooking data with the sensory properties of the rice.
4.5. Conclusions
Knowledge of water uptake and cooking behavior of rice is a key indicator in predicting the quality
of rice. In the present study, three different cooking methods (in situ TMCT cooking, RVA and
light microscopy) were used to analyse the cooking behavior of selected rice (fresh and aged
TDK8, TDK11, and Doongara) varieties. Results showed that the new in situ TMCT cooking
method could provide information on the softening of the grain due to water uptake during soaking
at various rehydration temperatures. The rate of cooking can also be calculated from the slope of
in situ TMCT cooking curve. This work represents the starting point of a new and rapid approach
72
for evaluating rice-cooking behavior. Current work focused on representative glutinous and nonglutinous rice varieties and the set-up of the test conditions. A wide range of rice verities showing
different cooking behavior needs to be further tested to validate the method. This provides a new
opportunity for analysis of rice cooking quality.
73
Chapter 5 X-ray photoelectron spectroscopic analysis of rice kernels and flours:
Measurement of surface chemical composition
This chapter has been published in Food Chemistry;
Nawaz, MA, Gaiani, C, Fukai, S & Bhandari, B 2016, ‘X-ray photoelectron spectroscopic analysis
of rice kernels and flours: Measurement of surface chemical composition. Food Chemistry,
vol. 212, pp. 349-357.
74
5.1. Abstract
The objectives of this study were to evaluate the ability of x-ray photoelectron spectroscopy (XPS)
to differentiate rice macromolecules and to calculate the surface composition of rice kernels and
flours. The uncooked kernels and flours surface composition of the two selected rice varieties,
Thadokkham-11 (TDK11) and Doongara (DG) demonstrated an over-expression of lipids and
proteins and an under-expression of starch compared to the bulk composition. The results of the
study showed that XPS was able to differentiate between rice polysaccharides (mainly starch),
proteins and lipids in uncooked rice kernels and flours. Nevertheless, it was unable to distinguish
components in cooked rice samples possibly due to complex interactions between gelatinised
starch, denatured proteins, and lipids. High-resolution imaging methods (Scanning Electron
Microscopy and Confocal Laser Scanning Microscopy) were employed to obtain complementary
information about the properties and location of starch, proteins, and lipids in rice kernels and
flours.
5.2. Introduction
Rice kernels and flour are used to produce a large variety of cereal-based foods, including
semolina, gluten-free bread, noodles, and biscuits. The functional properties (e.g., water
absorption, pasting properties, etc.) and biochemical composition of rice affect the overall quality
of the processed foods (Matos & Rosell 2013). Rice can be classified into waxy and non-waxy
varieties based on the native starch type present in the endosperm. Waxy rice contains branched
amylopectin and becomes very sticky after cooking. However, non-waxy rice contains straightchain amylose and is less sticky (Nawaz et al. 2016a).
A scientific understanding has been established that the functional properties of complex
biological materials are greatly dependent on the surface characteristics (Rouxhet et al. 2008).
Therefore, the spatial distribution of components is a key to understanding complex biological
systems (Saad et al. 2009). Recent studies carried out on biological powders have shown that the
surface chemical composition of particles is significantly different from their bulk composition
(Shrestha et al. 2007a; Baer & Engelhard 2010; Zhao et al. 2015).
X-ray photoelectron spectroscopy (XPS) has become a well-established technique to study the
nature of many different types of surfaces (Gaiani et al. 2011). XPS has been extensively used to
75
investigate the surface composition of biological powders (mainly milk powders) obtained by
spray or freeze drying of complex biological solutions (Kim et al. 2002; Zhao et al. 2011). On the
other hand, there has been very limited research focused on investigating the application of XPS
for natural biological powders obtained by the milling of agricultural produce (Russel et al. 1987;
Saad et al. 2009; Saad et al. 2011; Zhao et al. 2015). The surface composition of rice flour and/or
kernels has received relatively little research attention, despite the importance of these factors in
providing a better understanding of some of the functional properties of cooked rice. The interparticulate interactions (such as stickiness in the case of rice) and exposure to an external
environment that may cause chemical changes (such as oxidation of fat/oil) are depended on the
surface composition and properties of these surface materials.
The determination of the surface composition of a material by XPS is first considered at an
elemental level. XPS provides the relative atomic elemental composition of approximately 5-10
nm of the surface layer (Rensmo & Siegbahin 2015). The elemental composition of the biological
material is defined by considering only the three main elements, carbon, oxygen, and nitrogen.
Usually, minor elements (such as phosphorus, sulphur, silicon, boron, manganese or other
minerals) are ignored, they account for as little as 1 % the bulk composition (Gaiani et al. 2006;
Nijdam & Langrish 2006; Rouxhet et al. 2008; Rensmo & Siegbahin 2015). The relative elemental
composition (carbon, oxygen, and nitrogen) is used to identify components such as proteins, lipids,
and polysaccharides. For milk powders, the XPS apparent atomic stoichiometry has been found to
have reasonable agreement with theoretical stoichiometry based calculations (Nikolova et al.
2015). In addition, the C1s, N1s, and O1s peaks obtained from the XPS survey scans can be
decomposed at specific binding energies into various sub-peaks and assigned to well-identified
chemical functions (e.g., C-C(H), C-O, C-N, C=O, O-C=O, etc.) that are typical for specific
components, such as lipids, sugar derivatives, glucose polymers, and poly-amino acids (Rouxhet
& Genet 2011).
The surface composition of biological materials can be estimated by using the relative elemental
composition of isolated components. Fäldt and co-workers (1993) developed a method by
quantifying the relative atomic concentrations of carbon, oxygen, and nitrogen, and by using a
matrix formula related to the surface content of the different compounds (i.e., polysaccharides,
proteins, and lipids) that make up the sample. These calculations have been found to be reliable
76
only when significant differences between C, O, and N are present in the various components
(Saad et al. 2011). XPS has its limitations when it comes to differentiation of the multiple
functional groups that have similar percentages of atoms. Therefore, isolated components should
be significantly different in elemental composition to be able to be differentiated by XPS. As an
example, it appears not to be possible to differentiate whey proteins and caseins in milk from the
C, O, and N signatures, as these two protein categories are present in the same atomic percentages
(Gaiani et al. 2011).
The application of XPS to biopolymers appears to be very reliable, and versatile with a wide range
of applicability (Kelly et al. 2015). However, natural surface contamination during the experiment
can complicate XPS analysis by overexpression of carbon, because most abundant surface
contaminants on air-exposed samples consist of carbonated particles (McArthur et al. 2014).
Moreover, some biological specimens (such as wood pulps) have shown instability during XPSscanning due to X-ray induced irradiation damage and adsorption or desorption of volatiles in
ultrahigh vacuum conditions during the experimentation. This damage can distort the data and
further complicate the data interpretation (Zhou et al. 2006).
As noted above, over the past ten years XPS has been extensively used to evaluate the surface
composition of dairy powders. However, only a limited number of studies have been undertaken
to investigate natural agricultural products, with most research to date focused on wheat flours
(Rouxhet et al. 2008; Saad et al. 2009; Saad et al. 2011). The objective of the present study was to
evaluate the ability of XPS to identify the surface composition of rice kernels and the flour of waxy
and non-waxy rice varieties. First, XPS survey scans of pure rice components (starch, proteins,
and lipids) were obtained. Then an assessment was made of the surface composition of rice kernels
and rice flours. To quantify the surface composition, only macro-nutrients (starch, protein, and
lipid) were taken into account.
5.3. Materials and methods
Two rice varieties, Thadokkham-11 (TDK11) (glutinous) and Doongara (DG) (non-glutinous)
were used in this study. The rice grains were provided by Rice Research Australia Pty Ltd.
(RRAPL). Powdered rice starch (Sigma S7260, Castle Hill, NSW, Australia), commercially
available rice protein (Bulk Nutrients, TAS, Australia) and pure rice bran oil (Coles, QLD,
77
Australia) were used to estimate the relative elemental composition of pure rice components. The
flow chart of the experimental design is presented in Appendix 1.
5.3.1. Milling of paddy
The effect of the degree of milling (DOM) on the surface composition of rice kernels was analysed
for TDK11 only. Paddy rice was milled to brown rice by using rice husker (Satake, Japan). The
brown rice was milled to white rice using an abrasive polisher (Satake, Japan). Three different
DOMs, 0 % or brown rice, 9 %, and 16 %, were used in the study. DOM was calculated using the
following equation (5.1) as described by Marshall (1992);
𝐷𝑂𝑀 (%) = [1 − (𝑊𝑊𝑅/𝑊𝐵𝑅)] × 100
Eq. 5.1
WWR and WBR are the weight of white rice and brown rice in grams, respectively.
5.3.2. Grinding of rice kernels
The milled white TDK11 and DG rice grains were ground to flour using a hammer mill (Good
Friends of the Guangzhou Machinery Co. Ltd., Guangzhou, China) equipped with a plate of 500
µm size.
5.3.3. Chemical analysis of milled white rice
The starch content of the milled rice flours was determined according to the AACC 76-13.01
method (AACC 1999). Total nitrogen content (TN) was determined by the Kjeldahl method, and
crude protein content was calculated as TN X 5.95. Lipid content was determined by using the
Soxhlet extraction method according to AACC 30-25.01 (AACC 1999). The apparent amylose
content (AAC) was determined by the iodine colorimetric method (Hoover & Ratnayake 2005).
5.3.4. Sample preparation of defatted rice kernels and flours
The milled rice kernels and flour of TDK11 and DG were defatted using solvent extraction.
Kernels/flour (10±1 g) samples were taken in cellulose thimbles and treated with petroleum spirit
at 70ºC for 2 hrs. After 2 hrs of reflux, the petroleum spirit was separated from the sample using
rotary evaporator (RV 10, IKA® Werke GmbH & Co. KG, Germany). The defatted samples were
left in a fume hood overnight to evaporate the petroleum spirit fully.
78
5.3.5. Sample preparation of cooked rice kernels
To five gram samples of milled rice kernels, 15 mL of deionised water (rice to water ratio 1:3) was
added in a glass beaker. The samples were then cooked at 95±1ºC in a water bath, after which they
were held overnight in a freezer. The frozen cooked rice samples were then freeze-dried using an
Alpha 1-4 LSC Freeze Dryer (John Morris Scientific, Australia). The moisture content of the
samples was reduced to 10 % to ensure sample stability in ultrahigh vacuum conditions (base
pressure as low as 1.33 x 10-7 to 1.33 x 10-6) during XPS imaging.
5.3.6. Surface chemical analysis
The surface chemical analysis of pure rice components (starch, proteins, and lipids), rice kernels
with three different DOM (0 %, 9 %, and 16 %) (TDK11 only), uncooked rice kernels (control and
defatted), freeze-dried, cooked rice kernels, and rice flour (control and defatted) were analyzed by
using a Kratos AXIS Ultra Kratos Analytical (Manchester, UK) spectrometer with a
monochromatic Al X-ray source at 150 W. Prior to analysis, the samples were outgassed under
vacuum for 24 hrs. The analysis started with a survey scan from 0 to 1200 eV with a dwell time of
100 ms, pass energy of 160 eV at steps of 1 eV, with a single sweep. For the high-resolution
analysis, the number of sweeps was increased, the pass energy was lowered to 20 eV, at steps of
50 meV, and the dwell time was increased to 250 ms. Data was acquired using a Kratos Axis
ULTRA X-ray photoelectron spectrometer, incorporating a 165 m hemispherical electron energy
analyser. The incident radiation was Monochromatic Al X-rays (1486.6 eV) at 225 W (15 kV, 15
ma). Survey (wide) scans were taken at analyser pass energy of 160 eV, and multiplex (narrow)
higher resolution scans at 80 eV. Base pressure in the analysis chamber was 1.33 x 10 -7 Pa and,
during sample analysis, 1.33 x 10-6 Pa. XPS was applied to measure the relative atomic
concentrations of carbon, nitrogen, and oxygen in the layer of the samples to a maximum thickness
of 10 nm. Typical XPS survey and high-resolution spectra of rice sample are shown in Appendix
2.
5.3.7. Matrix formula used in the research
The relative elemental composition of pure rice components was used in a set of linear relations
in a matrix formula according to a method proposed by Fäldt and co-workers (1993). Adjustments
were made in the matrix formula in accordance with the components/macromolecules to be
79
analyzed (Fäldt 1995). In the present study, the calculations were made by using a matrix, as
presented in equations 5.2, 5.3, and 5.4 for the three rice components.
𝐼 𝐶 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐼 𝐶 𝑠𝑡𝑎𝑟𝑐ℎ 𝛾𝑠𝑡𝑎𝑟𝑐ℎ + 𝐼 𝐶 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 𝛾𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 + 𝐼 𝐶 𝑙𝑖𝑝𝑖𝑑𝑠 𝛾𝑙𝑖𝑝𝑖𝑑𝑠
Eq. 5.2
𝐼 𝑂 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐼 𝑂 𝑠𝑡𝑎𝑟𝑐ℎ 𝛾𝑠𝑡𝑎𝑟𝑐ℎ + 𝐼 𝑂 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 𝛾𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 + 𝐼 𝑂 𝑙𝑖𝑝𝑖𝑑𝑠 𝛾𝑙𝑖𝑝𝑖𝑑𝑠
Eq. 5.4
𝐼 𝑁 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐼 𝑁 𝑠𝑡𝑎𝑟𝑐ℎ 𝛾𝑠𝑡𝑎𝑟𝑐ℎ + 𝐼 𝑁 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 𝛾𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 + 𝐼 𝑁 𝑙𝑖𝑝𝑖𝑑𝑠 𝛾𝑙𝑖𝑝𝑖𝑑𝑠
Eq. 5.3
Where ICstrach, INstrach, IOstarch, ICproteins, INproteins, IOproteins, IClipids, INlipids, and IOlipids are the relative
contents of atomic elements (C, N, and O) measured on the surface of the pure rice components
(Table 5.2). ICsample, INsample, and IOsample are the relative contents of elements found by XPS for the
sample. The parameters γstarch, γproteins, and γlipids are unknown values corresponding to
approximately 100 % component/macromolecules surface contents (starch, proteins, and lipids).
5.3.8. Confocal analysis of rice kernels and flours
Rice kernels and flours were dyed using a mixture (1:1) of 0.01 % (w/v in water) Rhodamine B
(Sigma R6626, Castle Hill, NSW, Australia) and 0.02 % (w/v in poly (ethylene glycol) 200 (Fluka
81150, Castle Hill, NSW, Australia)) Nile Red (Sigma 72485, Castle Hill, NSW, Australia) for
labelling proteins and lipids, respectively. The samples were treated with dyes in the dark with
intermittent shaking. After 10 min dye-labelled samples were washed with deionised water until
the supernatant became clear. The microstructure of rice kernels and flours was observed by using
an LSM 700 confocal laser scanning microscope (CLSM, Zeiss, Germany).
5.3.9. Scanning electron microscopy of surface and cross-section of uncooked rice
kernels
Whole grains and cross sections of milled rice kernels of TDK11 and DG were mounted onto SEM
stubs by placing them on a double-sided carbon adhesive tape. Biological materials suffer from
extensive charge build-up under the electron beam; hence they need to be coated with conductive
material. Samples were iridium-coated for 3 min (~ 15 nm thick). The samples were examined
using a Philips XL30 Scanning Electron Microscope operating at 10 kV accelerated voltage.
5.3.10. Statistical analysis
All measurements presented in this paper are based on two independent samples. The data were
analysed by analysis of variance (Completely Randomized Design) using KyPlot software version
80
2.0 to determine significant differences. The data was then analysed using the Tukey’s pair-wise
comparison of different treatments, at 5 % level of significance.
5.4. Results and discussion
5.4.1. Chemical composition
The bulk chemical composition of the two rice varieties at 9 % DOM is presented in Table 5.1.
Starch (main rice polysaccharide) constituted over 90 % (w/w) of the milled rice in both varieties,
followed by crude protein (6 – 8 %, w/w). Lipid contents were only 0.8 – 1 % (w/w) of the bulk
composition of milled white rice samples. TDK11 had a significantly (P<0.05) higher starch and
lower protein content than DG. Apparent amylose content (AAC) showed that TDK11 has mainly
branched starch (amylopectin) and is classified as a glutinous rice variety. DG has intermediate
amylose content and is classified as a non-glutinous rice variety (Nawaz et al. 2016a).
Table 5.1 Bulk chemical composition of rice varieties TDK11 and Doongara (DG) at 9 %
degree of milling*
Bulk chemical composition
TDK11
DG
Starch
92.63±0.22a
90.44±0.14b
Apparent amylose content (AAC)
3.72±0.05a
19.71±0.42b
Protein
6.55±0.15a
8.52±0.11b
Lipid
0.82±0.07a
1.05±0.03a
content (g/100 g dry matter)
*Means ± SE. Within a row, means with different superscripts are significantly different at
5 % probability level.
5.4.2. Pure rice components analysed by XPS to construct the matrix
To test the usefulness of XPS matrix, pure rice components (starch, proteins, and lipids) were first
individually analysed by XPS. Rice endosperm is comprised of two closely related
polysaccharides, primarily starch and traces of arabinoxylans. XPS analysis has limitations in
distinguishing starch and arabinoxylans, as their atomic percentages are similar (Saad et al. 2011).
Therefore, only starch was considered to be the only polysaccharide in the rice endosperm to
overcome the complexity of analysis. Three atoms elements (C, O, and N) were detected in pure
rice starch and proteins (Table 5.2), and only two (C and O) for lipids. The survey scans obtained
for pure starch (Table 5.2) gave major signals at 286 and 533 binding energy (eV) corresponding
81
to C (59.47±0.07 %) and O (40.22±0.49 %), respectively and a very low signal at 400 eV
corresponding to N (0.63±0.10 %). The XPS survey of pure rice proteins (Table 5.2) gave a major
signal corresponding to C (77.60±0.01 %), a relatively lower signal corresponding to O
(16.10±0.01 %), and a significant signal corresponding to N (6.39±0.01 %). However, the XPS
survey spectra for rice bran oil gave the main signal corresponding to C (91.81±0.01 %), and a
lower signal is corresponding to O (8.20±0.01 %). No N1s peak was detected (Table 5.2).
Table 5.2 Relative elemental compositions of pure rice components measured by XPS*
Binding energy (eV)
Functions
Atomic abundance (%) of elements at the
surface of pure rice components
Starch
Protein
Lipid
286
C
59.47±0.07a
77.60±0.01b
91.81±0.01c
400
N
0.63±0.10a
6.39±0.01b
-
533
O
40.22±0.49a
16.10±0.01b
8.20±0.01c
Starch
Protein
Lipid
C/O
1.47
4.76
11.11
C/N
94.39
12.50
-
Stoichiometry
*Means ± SE. Within a row, means with different superscripts are significantly different at
5 % probability level.
The C/O stoichiometry for pure starch was found to be 1.47 (Table 5.2), which is relatively higher
than the theoretical value for anhydroglucose (1.20) when the theoretical chemical composition
(C6H10O5)n for starch was taken into consideration. Similar findings have been reported for pure
wheat starch by Saad and co-workers (2011). There is a likelihood that the surface components of
the granules other than glucose polymers may be present at a lower level. Moreover, traces of
nitrogen were also found on the starch granules at C/N stoichiometry of only 94.39, suggesting the
possible presence of proteins (mostly protein-based enzymes) on the starch granule surface.
Almost all known cereal proteins have the basic molecular formula C3.3H5.9O1.06N1S0.033 (C/O =
3.12 and C/N = 3.33) (Torabizadeh 2011; Saad et al. 2011). A lower content of both O and N were
found in pure rice protein samples during XPS analysis with calculated values of C/O and C/N
stoichiometry as 4.76 and 12.5, respectively (Table 5.2). These results indicate the presence of
lipid traces within the surface layers of protein particles (Rouxhet et al. 2008). Similar findings
have been reported by Saad and co-workers (2011) for wheat proteins. Also, no traces of sulphur
82
were detected, which suggests the absence of sulphur containing amino acids (cysteine, cysteine,
and methionine) on the surface of the rice protein samples.
The rice lipids C/O stoichiometry (11.11) (Table 5.2) was found to be very similar to that of soya
oil (10) reported by Jones et al. (2013). However, rice lipids differed from wheat lipids where
amide-linked N was detected due to the presence of protein residues (Saad et al. 2011).
5.4.3. Effect of degree of milling (DOM) on the surface composition of waxy rice
TDK11
The effect of the degree of milling on surface properties was investigated for TDK11. The relative
elemental and estimated surface composition of TDK11 kernels with different DOM (0 % or
brown rice, 9 %, and 16 %) are shown in Table 5.3. Four peaks, corresponding to Si, C, N, and O,
were detected during XPS survey scanning. The presence of Si on the surface of grains may be
due to contamination of the husk (Park et al. 2004). The main peak at a binding energy of 286 eV,
corresponding to C, was detected in all three DOMs. The relative C composition was 85.31±0.82
%, 86.54±0.69 % and 82.14±0.86 % for 0 %, 9 %, and 16 % DOM, respectively. Relatively weaker
signals at binding energy 533 eV were attributed to relative O composition. A significant (P<0.05)
increase in O composition was found in 16 % DOM when compared with 0 % and 9 % DOMs.
Very weak signals corresponding to N were also detected at 400 eV binding energy. The relative
N composition in selected DOMs ranged between 2.27±0.02 % to 3.30±0.72 %.
Only C, N, and O relative composition were considered and used in matrix formula for the
calculation of the surface composition of selected DOMs, while Si composition was ignored (Table
5.3). The results show that the calculated surface composition of the upper 10 nm layer of brown
TDK11 was primarily composed of lipids (52.57±9.79 %) and proteins (47.44±9.79 %). Brown
rice endosperm is covered by two distinct layers, the cuticle, and aleurone (Bagchi et al. 2016).
These layers have high contents of proteins and lipids. The average thickness of these layers
usually ranges from 30 to 50 µm (Wu et al. 2016a). The 9 % DOM TDK11 surface was composed
of lipids (63.84±1.51 %), proteins (35.17±0.51 %) with a low content of starch (0.79±0.79 %). A
further increase in the DOM resulted in increased surface starch and proteins and reduced lipids.
Similar results were reported by Gangidi and co-workers (2002), who found a reduction in surface
lipids in response to an increase in DOM (0-40 %) in long and medium-grain rice varieties,
estimated by Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS). The 16 %
83
DOM TDK11 surface was composed of proteins (51±11.41 %), lipids (42.66±9.12 %), and starch
(5.72±2.26 %). It should be noted that the surface composition of milled kernels, even with 16 %
DOM, had a higher content of lipids and proteins than the bulk composition of rice kernels. This
result showed that a thin layer of protein particles and bran oil covering the rice kernels, despite
their overall very low content in the kernel.
5.4.4. Surface composition and microstructure of rice kernels
5.4.4.1. Uncooked rice kernels
The relative elemental composition and estimated surface composition of rice kernels of TDK11
and DG are shown in Table 5.3. XPS spectra showed that the surface composition of rice kernels
of both rice varieties in this study mainly consisted of C, N, and O, with traces of Si. Again, the
traces of Si may reflect husk or bran particles contamination on the surface. The surface
composition of milled TDK11 and DG was found significantly different from bulk chemical
composition (Table 5.1). The surface of TDK11 had high a protein concentration (49.69±8.26 %)
followed by lipids (36.21±6.78 %). However, the surface of variety DG had more lipids
(58.94±0.64 %) than proteins (29.32±0.79 %); possibly due to a higher level of intact bran residues
on the surface of DG, which resulted in more fat being found in bulk composition analysis. It
should be noted that an extremely thin layer of bran oil spread on the surface of the kernel or
particle will increase the surface lipids composition. XPS measures the composition of a 5-10 nm
layer. The surface starch content ranged between 11.77±0.08 % and 13.42±1.51 %. To further
validate the XPS matrix and estimated surface composition, the kernels were defatted using
petroleum spirit and analysed again. A significant increase in surface proteins (75.20±2.09 and
87.14±3.45 %) and starch (24.81±2.09 and 12.87±3.45 %) was found in the defatted TDK11 and
DG rice kernels, respectively. Lipids were absent in the defatted kernel.
The XPS surface analysis was well correlated with the microstructure of rice kernels estimated by
confocal laser scanning microscopy (CLSM). The micrographs of control and defatted rice labelled
with Rhodamine B and Nile Red showed that the surface of both rice kernels (TDK11 and DG)
was covered by a very thin layer of lipids (labelled in red) followed by proteins (labelled in green)
(Fig. 5.1a and 5.1b). However, in defatted rice kernels, only proteins were found on the surface.
In these images, only the surface was visible, as the dye was not able to penetrate the interior of
the kernel.
84
To get greater clarification, scanning electron microscopy (SEM) was performed for whole grains
and cross sections of TDK11 and DG (Fig. 5.2). The surface of both rice samples was found to be
covered with particles (which may be bran residue) as shown in Fig. 5.2a and 5.2c, resulting in
more proteins and lipids than starch. Also, more lipid layers were observed in DG than TDK11, as
was also found in the XPS analysis. Cross sections micrographs (Fig. 5.2b and 5.2d) showed that
the outer surface layer of over 10 µm thickness had no polygonal structure of starch. Possibly, the
outer surface layer was composed of only round protein bodies, lipid droplets, and cell walls. As
the XPS method is unable to differentiate between starch and cell walls (arabinoxylans), more
proteins and lipids were observed on the upper 10 nm surface layer. Around 10-14 % starch
calculated using the matrix formula, may be contributed by cellulose (Saad et al. 2011).
5.4.4.2. Cooked rice kernels
The relative elemental composition of cooked rice kernels of TDK11 and DG is shown in Table
5.4. Four peaks corresponding to C (50.03±1.46 %), N (6.82±1.22 %), O (38.68±1.78 %) and Mn
(4.48±0.54 %) were detected in TDK11. However, the XPS survey of cooked DG was significantly
different and five peaks, corresponding to Si (1.09±0.15 %), B (0.29±0.03 %), C (74.94±0.38 %),
N (2.81±0.44 %), and O (20.89±0.70 %), were detected. Cooked DG had a significantly (P<0.05)
higher percentage of C and lower O than cooked TDK11. It was not possible to calculate the
surface composition of cooked rice kernels using the matrix formula developed for dry samples,
as the relative elemental composition of C (50.03±1.46 %) in cooked TDK11 was found to be
lower than the carbon in pure components, while the N level (6.82±1.22 %) was higher than the
N found in pure components. Therefore, high-resolution XPS scanning was conducted for the C1s,
N1s, and O1s peaks of cooked TDK11 and DG. The deconvolution of C1s showed six distinct subpeaks (C-C, C-COOH, C-N, C-O, C=O, and O-C=O) in both TDK11 and DG, corresponding to
polysaccharides, protein or lipid side chains, etc. Cooking of rice kernels possibly resulted in
complex interactions of gelatinised starch with denatured proteins and lipids. Although there were
no significant (P>0.05) differences found between cooked TDK11 and DG, the deconvolution of
the C1s peak provided good information about the surface composition of cooked rice.
85
Figure 5.1 Confocal laser scanning micrographs (CLSM) of control and defatted
uncooked rice kernels of TDK11 (a) and Doongara (DG) (b), cooked rice kernels of TDK11
(c) and Doongara (DG) (d), rice flours of control TDK11 (e), control Doongara (DG) (f),
defatted TDK11 (g), and defatted Doongara (DG) (h). Lipids and proteins are labelled in
red and green respectively
86
Figure 5.2 Scanning electron micrographs of uncooked rice kernels of TDK11 and
Doongara (DG); (a) surface of milled TDK11 kernel, (b) cross-section of TDk11 kernel,
(c) surface of milled Doongara (DG) kernel, and (d) cross section of Doongara (DG) kernel
87
Table 5.3 Relative elemental and calculated surface composition (%) of TDK11 with different DOM (%), control and defatted
rice kernels, and control and defatted rice flours of TDK11 and Doongara (DG)*
The degree of milling (DOM)
Rice kernels
Control kernels
0%
9%
a
16 %
a
TDK11
a
DG
ab
Rice flour
Defatted kernels
TDK11
Control flour
DG
a
TDK11
b
Defatted flour
DG
TDK11
DG
Elemental atomic
Si
0.59±0.16
0.45±0.08
0.55±0.04
0.58±0.03
-
0.76±0.15
0.11±0.11
-
-
-
-
percentage (%)
C
85.31±0.82a
86.54±0.69a
82.14±0.86a
79.78±0.76a
83.86±0.02b
65.55±0.07c
71.37±0.07d
74.13±0.47a
79.17±0.18b
66.37±0.02c
68.66±0.31d
N
3.10±0.69a
2.27±0.02a
3.30±0.72a
3.29±0.52a
1.97±0.05a
6.79±0.35b
6.38±0.43b
4.43±0.74ab
2.67±0.52b
7.04±0.47a
6.11±0.20a
O
11.00±0.29a
10.75±0.75a
14.01±0.18b
16.37±0.17a
14.29±0.05b
26.91±0.14c
21.46±0.66d
21.44±0.28a
18.16±0.34b
26.60±0.48c
25.24±0.12c
ab
a
b
ab
a
a
-
24.76±0.87a
100±0.00a
75.25±0.87a
-
-
a
Surface composition
Starch
-
(%) after using matrix
Protein
47.44±9.79a
35.17±0.51a
a
a
formula
Lipid
52.57±9.79
0.79±0.79
63.84±1.51
a
5.72±2.26
51.00±11.41a
42.66±9.12
a
13.42±1.51
11.77±0.08
49.69±8.26a
29.32±0.79b
a
b
36.21±6.78
58.94±0.64
24.81±2.09
75.20±2.09c
-
12.87±3.45
87.14±3.45c
-
24.89±3.83
66.88±11.96a
a
8.12±8.12
21.35±3.15
39.68±8.45b
38.87±5.30
b
*Means ± SE. For DOM, Rice kernels and Rice flour, in each respective section, the means within a row with different
superscripts differ significantly at 5 % probability level.
88
The calculated values for C/O and C/N stoichiometry for cooked TDK11 were found to be 1.29
and 7.14, respectively (Table 5.4). The results reflected that the cooked TDK11 (waxy rice) kernel
surface was mainly covered by gelatinised amylopectin (branched starch) with high levels of
proteins. More starch on the kernel surface may induce the adhesive properties of sticky rice
(Keawpeng & Venkatachalam 2015). However, the cooked DG kernels had C/O and C/N
stoichiometry values of 3.57 and 25, respectively (Table 5.4). Therefore, the kernel surface was
mainly covered by fat with some proteins. Fat and proteins are responsible for the hardness and
cohesiveness of cooked non-waxy rice kernels (Choi et al. 2015). These findings need to be the
subject of further investigation.
The results were validated by using CLSM. The dye labelled microstructures of both cooked
TDK11 and DG are shown in Fig. 5.1c and 5.1d. The results show lipids and protein networks
fluoresced in red and green, respectively in cooked DG (Fig. 5.1d). The rice proteins exist in the
form of round protein bodies in the endosperm (Saito et al. 2012), but form a continuous
honeycomb-like structure during cooking, due to heat denaturation (Likitwattanasade &
Hongsprabhas 2010). However, only gelatinised starch (not in granular shape) was visible in
TDK11 (Fig. 5.1c). This may be due to variations in the functional properties of glutinous (TDK11)
and non-glutinous (DG) rice varieties. The glutinous rice starch consists almost entirely of
amylopectin (Zeng et al. 2016). When cooked, the grain usually loses its original shape and
becomes very sticky (Nawaz et al. 2016a). On the other hand, non-glutinous rice starch contains
amylose as well as amylopectin, and the cooked grain tends to retain its shape and is less sticky
(Nawaz et al. 2016a; Li et al. 2016). It is understood that starch is stickier than protein (Hamaker
et al. 1991). More starch and proteins were found on the surface of cooked TDK11 kernel (sticky
rice variety) and fat on the surface of cooked DG kernel (non-sticky rice variety). This suggests
the role of protein in the stickiness of the cooked rice.
5.4.5. Surface composition and microstructure of rice flours
The relative elemental composition of rice flours (TDK11 and DG) is shown in Table 5.3. In both
rice samples, three peaks were detected during the XPS survey scans. The relative C composition
of TDK11 and DG was found to be 74.13±0.47 % and 79.17±0.18 %, respectively. Relatively
weaker signals corresponding to O were detected in both rice flours. The calculated O composition
of TDK11 and DG was found to be 21.44±0.28 % and 18.16±0.34 %, respectively, while the
89
calculated N composition of TDK11 and DG was estimated to be 4.43±0.74 % and 2.67±0.52 %,
respectively.
The surface composition of both rice flours was also found to be significantly different from the
bulk composition (Table 5.1). The calculated surface composition of TDK11 flour consisted of
proteins (66.88±11.96 %), starch (24.89±3.83 %), and lipids (8.11±8.12 %). The DG flour surface
consisted of proteins (39.68±8.45 %), starch (21.35±3.15 %), and lipids (38.87±5.30 %).
Table 5.4 Relative elemental and calculated surface composition (%) of cooked rice
kernels of TDK11 and Doongara (DG)*
Elements
Functions
TDK11
DG
Si
-
1.09±0.15
B
-
0.29±0.03
C
50.03±1.46a
74.94±0.38b
C-C
52.49±0.10a
56.54±2.09a
C-N
9.78±6.36a
12.69±0.41a
C=O
8.68±1.05a
6.46±0.36a
O-C=O
3.84±0.17a
3.41±0.02a
C-COOH
3.84±0.17a
3.42±0.02a
C-O
21.38±4.88a
17.51±2.11a
N
6.82±1.22a
2.81±0.44a
O
38.68±1.78a
20.89±0.70b
4.48±0.54
-
TDK11
DG
C/O
1.29
3.57
C/N
7.14
25
Mn
Stoichiometry
*Means ± SE. Within a row, means with different superscripts are significantly different at
5 % probability level.
The surface composition of both rice flours had significant amounts of lipids and proteins. To
validate the XPS matrix and estimated surface composition, the flours were defatted with
petroleum spirit and analysed again. Significant changes in the relative elemental (C, N, and O)
composition were recorded (Table 5.3). Significant increases in N and O and a decrease in C
concentrations were found in both defatted rice flours. The surface composition of TDK11 showed
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only proteins, with no lipids and starch. However, DG had 75.25±0.87 % proteins and 24.76±0.87
% starch. Similar results have also been reported by Saad and co-workers (2011) for wheat flours,
in which an over-expression of proteins (54 %) and lipids (44 %) and an under-expression of starch
only 2 %, were found when compared to the bulk composition. It was assumed that this reflected
the heterogeneous distribution of components and molecular masking effects between the
components. Also, the grinding procedures may result in the XPS analysis to give different results
from the actual composition, due to the variations in exposed surfaces of the particles produced by
grinding and spreading of lipids. Zhou et al. (2015) reviewed and illustrated the presence of protein
bodies and nanometric fat droplets (100-500 nm) surrounding the endosperm cells. However, this
work suggests that these free lipid droplets are in fact spread in a thin layer on the endosperm
surface. The rice proteins may have been absorbed into the thin layer of lipids formed on the kernel
surface due to rice proteins’ hydrophobic properties. The over-expression of both protein and fat
in the rice kernel and flour surfaces was an interesting result and needs to be further researched to
investigate its implications for the functionality and chemical stability of these products.
The microstructures of control and defatted rice flours of TDK11 and DG were also analysed using
CLSM (Fig. 5.1e, 5.1f, 5.1g and 5.1h). The results showed that control the flours of both rice
varieties have lipids (labelled in red by Nile Red) and protein bodies (labelled in green by
Rhodamine B) on the surface (Fig. 5.1e and 5.1f). However, only proteins were observed on the
surface in defatted rice samples (Fig. 5.1g and 5.1h). Also, the surface lipids were found to be
significantly (P<0.05) higher in DG flour than TDK11 during XPS scanning (Table 5.3). Similar
findings were observed during CLSM analysis. DG flour (Fig. 5.1f) had bigger and more lipid
bodies than TDK11 (Fig. 5.1e).
5.5. Conclusions
The results of the study demonstrated that XPS was able to be used for the evaluation of the surface
of two different rice varieties. XPS imaging provided detailed information about the elemental
composition of the upper 5-10 nm layer of rice kernels and flours. This relative elemental
composition could be used in the calculation of macromolecules on the surface of rice kernels.
Higher amounts of proteins and lipids were found on the surface of kernels and flours than in the
bulk composition. The findings from XPS imaging were well correlated with CLSM
microstructure analysis. These results had significance in relation the chemical stability and
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functionality of rice grain. Further research needs to be done in future for optimising the XPS
imaging and use of this technique for the analysis of aged rice samples and process related changes
on the kernel surfaces which can potentially lead to a reduction in the functional quality of rice.
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Chapter 6 Effect of alkali treatment on the milled grain surface protein and
physicochemical properties of two contrasting rice varieties
This chapter has been published in the Journal of Cereal Science;
Nawaz, MA, Fukai, S & Bhandari, B 2016, ‘Effect of alkali treatment on the milled grain surface
protein and physicochemical properties of two contrasting rice varieties’, Journal of Cereal
Science, vol. 72, pp. 16-23.
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6.1. Abstract
A systematic study was conducted to explore the effect of grain surface proteins on the
physicochemical properties (pasting, retrogradation, and textural quality) of rice. Milled rice grains
of two selected glutinous (Thadokkham-8 (TDK8)) and non-glutinous (Doongara (DG)) varieties
were treated with different concentrations (0 %, 0.004 %, 0.02 %, 0.04 %, and 0.2 % w/v) of NaOH
solution for 1 h. After surface protein removal, the cooked rice grains showed a significant
(P<0.05) increase in adhesiveness. Similarly, protein removal showed a significant (P<0.05)
decrease in the final viscosity (Vf) of rice flours. Furthermore, NaOH treatment at a concentration
of 0.04 % induced yellow color development in grains. The differential calorimetric study showed
that alkali treatment resulted in increased onset (Tₒ), peak (Tp), conclusion (Tc) temperatures and
enthalpy (ΔH) of both rice varieties. No significant (P>0.05) effect of alkali treatment was
observed on the retrogradation thermal temperatures (To(r), Tp(r), and Tc(r)), but the amount of
retrograded starch (as indicated by reduction in ΔH(r)) was decreased significantly (P<0.05) in both
varieties. These findings suggest a good potential of applying alkali pre-treatments in the
processing of rice to alter the hardness and stickiness properties of rice.
6.2. Introduction
An increasing trend in the consumption pattern of rice has been observed due to rising interest in
gluten-free products. Rice can be broadly divided into two distinct types based on the native starch
type present in the endosperm; glutinous rice cultivars are primarily containing branched
amylopectin and non-glutinous rice cultivars containing linear chain amylose as well as
amylopectin (Yu et al. 2015). The textural attributes of cooked glutinous and non-glutinous rice
are quite different from each other due to this compositional difference. Good quality glutinous
rice should be very sticky and vice versa for non-glutinous rice (Nawaz et al. 2016a). However,
aging induces functional changes in the stored glutinous rice (Nawaz et al. 2017) making it less
sticky. The mechanism of reduction in the cooked rice stickiness is still an area of research interest.
The functional attributes of rice have long been ascribed to starch composition and property. Many
studies to date have focused on the role of amylose content (Lu et al. 2013; Syahariza et al. 2013),
fine structures of amylopectin (Syahariza et al. 2013), solubility of amylose (Fu et al. 2015), the
gelatinization and melting temperatures of amorphous and crystalline regions of amylopectin
(Zeng et al. 2014), and the amount of native structures remaining in starch granules after heating
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(Klaovhanpong et al. 2015). Extensive consideration of investigation on only starch is not
surprising considering that starch accounts for 92-95 % of the dry matter in milled rice grain.
However, it has now been realised that starch may not be the only factor affecting the
cooking/eating attributes of rice grains (Yadav et al. 2013).
Protein is the second most abundant macromolecule in rice endosperm after starch. Rice contains
6-8 % protein and does not fluctuate widely from this level (Yadav et al. 2013). Proteins in a rice
kernel are present in the form of round discrete protein bodies (PBs). The estimated size of PB is
usually around 4-5 µm. There are two types of PBs; Protein body I and protein body II (Han &
Hamaker 2002). PBs in the subaleurone layer are not similar to those present in the endosperm
(Baxter et al. 2004). Subaleurone PBs are rich in glutelin (alkali soluble) and albumin (water
soluble). While endospermic PBs are rich in prolamin (alcohol soluble) (Baxter et al. 2004).
Various studies have been conducted in the past to find out the effect of protein (Yadav et al. 2013;
Xie et al. 2008) and shown a weak correlation between the gross protein content and the texture of
cooked rice, higher protein content rice is harder than low protein content rice (Baxter et al. 2004).
Moreover, in a recent study, the surface analysis of rice kernels using X-ray Photoelectron
Spectroscopy (XPS) and Confocal Laser Scanning Microscopy (CLSM) showed an overexpression of proteins and lipids and an under-expression of starch on the surface of rice
endosperm compared to the bulk composition of endosperm (Nawaz et al. 2016b). Alkali
extraction has been used in recent studies to extract protein from cereal flours, especially in rice
(Souza et al. 2016). Alkaline treatment by agents such as lye or sodium hydroxide is widely used
in the production of many value-added food products from cereals, including tortillas, waxy rice
dumplings (Lai et al. 2002), and various extruded products such as instant noodles and yellow
alkaline noodles (Nadiha et al. 2010). It is assumed that dilute alkali treatment to the whole rice
grains may be a useful technique to remove surface protein residues, resulting in more starch on
the surface. An increase in stickiness/adhesiveness in stored rice may be improved by removing
surface proteins, as starch is stickier than protein (Hamaker et al. 1991). Alkali treatment may also
wash surface lipids by saponification. However, alkali application to food products especially
cereals should be employed carefully as steeping with higher concentration of alkali (such as 0.4
% NaOH) for longer time (7-14 days) can lead to structural changes in rice starches (Cai et al.
2014), resulting in changes in functional properties such as swelling power, water binding
95
capacity, gelatinization and pasting attributes (Karim et al. 2008; Wang & Copeland 2012). Our
study has avoided the inappropriate alkali steeping by using lower NaOH concentration for a
shorter period. The objective of the present study is to investigate if the removal of the protein
bodies from the surface of the grain alters the stickiness of the cooked grain. For this, the milled
rice grains of two contrasting rice varieties (waxy and non-waxy, respectively) were treated with
various concentrations of sodium hydroxide solution to wash surface proteins and lipids. This
washing was expected to lead to increasing in the stickiness of cooked rice grains which is one of
the most important quality attributes of waxy rice.
6.3. Materials and methods
One Oryza sativa indica cultivar of glutinous rice from Lao PDR (Thadokkham-8 (TDK8) having
3.77 % apparent amylose contents (AAC)) and one O. sativa japonica non-glutinous rice from
Australia (Doongara (DG), 19.71 % (AAC)) were used in this study. The milled TDK8 was
provided by the National Agriculture and Forestry Research Institute (NAFRI), Lao PDR, while
Doongara were provided by Rice Research Australia Pty Ltd (RRAPL), Mackay, QLD, Australia.
6.3.1. Alkali treatment
The milled rice grains of selected varieties were soaked in various concentrations of NaOH
solution (Cc0 ~ 0 %, C0.004 ~ 0.004 %, C0.02 ~ 0.02 %, C0.04 ~ 0.04 %, and C0.2 ~ 0.2 %) at 40oC for
1 hr with a rice to solution ratio of 1:8. After 1 hr the treated rice grains were washed with deionised
water until completely neutralised (pH = 7.0 approx.). These concentrations corresponded to 7.0,
11.0, 11.7, 12.0 and 12.7 pH, respectively. The treated samples were spread on blotting paper and
kept in a fume hood at room temperature (22±1oC, RH~50 %) for 72 hrs to reduce the moisture
contents to 14 %. One control (Cc) sample without any treatment was also kept for comparison.
6.3.2. Color estimation of alkali treated rice grains
A Konica Minolta Chroma Meter CR-400 (Tokyo, Japan) was used for all color measurements.
Before color measurements, the color meter was calibrated with a white tile. Color measurements
were made at least in three folds of samples placed in a clear petri dish. The color was measured
in CIEL*a*b* color space. L* is a measurement of brightness from black (0) to white (100).
Parameter a* refers red-green color with positive a*-values indicating redness and negative a*-
96
values indicating greenness. Whereas, parameter b* refers yellow-blue color with positive b*values indicating yellowness and negative b*-values indicating blueness (Good 2002).
6.3.3. Confocal laser scanning microscopy (CLSM)
Alkali-treated rice grains were dyed with a mixture (1:1) of 0.01 % (w/v in water) Rhodamine B
(Sigma R6626) and 0.02 % (w/v in poly (ethylene glycol) 200 (Fluka 81150)) Nile Red (Sigma
72485) for labelling protein and lipid, respectively. The samples were treated with dyes in the dark
with intermittent shaking. After 10 min dye-labelled samples were washed with deionised water
until the supernatant became clear. The microstructure of rice grains was observed by using an
LSM 700 confocal laser scanning microscope (CLSM, Zeiss, Germany).
6.3.4. Crude protein analysis
Rice samples were ground to flour using a disc mill (Good Friends of the Guangzhou Machinery
Co. Ltd., Guangzhou, China). The flour particles were sieved through 500 µm sieve to attain
particle size < 500 µm. Crude protein content in rice flour was determined by the semi-microKjeldahl method using a Kjeltec 2300 Autoanalyser (Foss AB, Sweden). A nitrogen conversion
factor of 5.95 was used to compute the protein value.
6.3.5. Textural profile analysis
Rice grains (5 g) were added in 15 mL of MilliQ water (rice: water ratio =1:3) in 50 mL glass
beaker. The beaker was placed in a water bath at 95±1ºC. Cooking was continued until there was
no ungelatinized white belly observed in rice kernel cross section (data not shown). Analysis of
textural attributes was performed on a TA-XTplus Texture Analyzer (Stable Microsystems, UK)
using 35-mm circular probe. Three cooked grains were placed on the flat stage, and the texture
determined. The texture analyzer settings were as follows: pre-test speed, 2.00 mm/sec; post-test
speed, 2.00 mm/sec; distance, 2.00 mm; time, 10.00 sec; (auto) trigger force, 0.05 N. From the
force-time curve obtained, textural attributes of hardness (height of the force peak on cycle 1, N)
and adhesiveness (negative force area of the first cycle, N.s) were computed using the EXPONENT
Stable Micro Systems software supplied with instrument. The TPA values reported are the
averages of 3 different determinations.
97
6.3.6. Pasting properties
Pasting properties of rice flour (particle size ≤ 500 µm) were determined according to the AACC
International Method 61-02.01 using a Rapid Visco Analyzer (RVA-4 model Thermocline
Windows Control and analysis software, Version 1.2 (New Port Scientific, Sydney, Australia)).
Rice flour (3.01 g, 12.4 % moisture basis) was mixed with 25.0 g MilliQ water in the RVA canister.
A programmed heating and cooling cycle were used, the samples were held at 50ºC for 1 min,
heated to 95ºC in 3.45 min, held at 95ºC for 2.7 min before cooling to 50oC in 3.91 min and holding
at 50oC for 1.24 min. Pasting temperature (Ptemp), Peak viscosity (Vp), Trough viscosity (Vt),
Breakdown (BD), Final viscosity (Vf) and Setback (SB) were recorded.
There was no peak viscosity found in DG viscographs. Therefore, Point of inflection (pi) was
calculated using the 1st derivative for every 30 sec (F´ 30s) as shown in Appendix 3. Viscosity at
the point of inflection (Vpi) was estimated by using pi.
6.3.7. Gelatinization and retrogradation properties
Differential Scanning Calorimeter (DSC) (Mettler Toledo, Schwerzenbach, Switzerland) with
internal coolant and nitrogen/air purge gas was used to determine the gelatinization characteristics
of rice flours. The DSC was calibrated for the heat flow using indium as standard. Rice flour (4
mg, dry weight basis) was accurately weighed into an aluminum pan and six µL MilliQ water was
added. The pan was hermetically sealed and equilibrated at room temperature for 30 min, then
scanned at the heating rate of 15ºC/min from 0 to 100ºC with the empty sealed pan as a reference.
The onset (To), peak (Tp) and conclusion (Tc) temperatures, and enthalpy (ΔH) of gelatinization
was determined by Stare Software Version 9.1 (Mettler Toledo).
After cooling, the scanned samples pans were placed in a refrigerator at 4±1ºC for seven days.
Retrogradation properties were measured by rescanning these samples at the rate of 15ºC/min from
0 to 100ºC. The onset (To(r)), peak (Tp(r)) and conclusion (Tc(r)) temperatures, and enthalpy of
retrograded starch (ΔH(r)) were determined. The percentage of retrogradation (R %) was calculated
as ΔH(r)/ΔH X 100.
6.3.8. Statistical analysis
All treatments were replicated three times to obtain mean values. The reported data for the
CIEL*a*b* color space, crude protein, textural profile analysis, pasting properties, and
98
gelatinization and retrogradation for each variety were analysed separately by analysis of variance
(Completely Randomized Design) using Minitab R17 (Minitab® for Windows Release 17,
Minitab Inc, Chicago) to determine significant differences. The data was then analysed using
Tukey’s pair-wise comparison, at 5 % level of significance, to compare the results between
different treatments.
6.4. Results and discussion
6.4.1. Color estimation of alkali treated rice grains
The color parameters of the raw and cooked grains of control and alkali treated TDK8 and DG are
shown in Table 6.1. Significant (P<0.05) decrease in the brightness (L*) of raw TDK8 kernels was
observed when treated with 0.2 % of NaOH. However, no change in L* was observed in raw DG
rice kernels. From 0.02 % to 0.2 % of NaOH concentration induced a significant (P<0.05) increase
in greenness (-a*) in raw TDK8 rice; whereas, only 0.2 % of NaOH induced a significant (P<0.05)
greenness in raw DG rice grains. For raw TDK8 rice grains, yellowness (b*) increased
significantly with 0.2 % of NaOH. Raw DG rice grains were susceptible to alkali yellowness (b*)
and significant (P<0.05) increase in yellowness (b*) was observed with an increase in alkali
concentration. In cooked TDK8 rice kernels significant (P<0.05) decrease in the brightness (L*)
at 0.004 % of NaOH concentration was observed. However, no significant (P>0.05) decrease in
L* was observed with further increase in NaOH concentration. Moreover, NaOH concentration of
0.04 % induced significant (P<0.05) increase in L* of cooked DG rice kernels. NaOH
concentration of 0.02 % induced a significant (P<0.05) increase in greenness (-a*) in cooked
TDK8 rice. Further increase in NaOH concentration had no significant (P>0.05) effect on a* of
cooked TDK8 rice kernels. Whereas, NaOH concentration of 0.04 % induced a significant
(P<0.05) increase in greenness (-a*) in cooked DG rice kernels. From 0.02 % to 0.2 % of NaOH
concentration induced a significant (P<0.05) increase in yellowness (b*) in both cooked TDK8
and DG rice kernels. Previous studies on the effect of alkali treatment on cereal products also
reported the induced yellowness in sodium hydroxide treated grains and flour (Lai et al. 2004;
Nadiha et al. 2010). The induced yellow color in alkali treated rice grains may be attributable to
the naturally occurring flavonoids (such as apigenin-C-diglycosides) present in cereal aleurone
and sub-aleurone layers. These compounds are colorless at acidic or neutral pH but turn yellow at
99
basic pH (Asenstorfer et al. 2006). The scanned images showing color differences of raw and
cooked grains of control and alkali treated TDK8 and DG are presented in Appendix 4.
6.4.2. Confocal laser scanning microscopy (CLSM)
The CLSM images of control and alkali treated rice kernels are shown in Fig. 6.1. The surface of
Cc and Cc0 in both rice varieties (TDK8 and DG) showed a layer of lipids (labelled as red with Nile
red) and protein (labelled as green with Rhodamine B). Reduction of surface proteins and lipids
was observed with increase in NaOH concentration from 0.004 to 0.2 %, showing washing of
surface proteins (possibly mostly glutelin) and lipids. Also, besides surface proteins, some of the
proteins located internally might have been removed. Although CLSM was unable to detect this,
as the dyes (Rhodamine B and Nile red) were unable to penetrate the interior of the kernel.
Table 6.1 CIEL*a*b* color space of control and alkali treated rice grains of Thadokkham8 (TDK8) and Doongara (DG)*
Rice variety
Raw TDK8 rice
grains
Cooked TDK8 rice
grains
Raw DG rice
grains
Cooked DG rice
grains
Treatment
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
L*
97.4±0.18ab
97.6±0.01a
97.2±0.41ab
97.7±0.04a
97.6±0.52a
96.4±0.09b
96.8±0.35ab
97.7±0.37a
95.6±0.21c
95.4±0.57cd
94.8±0.14cd
94.2±0.05d
94.1±0.23a
94.6±0.71a
93.9±1.38a
93.9±0.60a
94.3±0.55a
94.1±0.18a
93.1±0.26a
94.3±0.35ab
93.4±0.21a
93.8±0.57a
95.2±0.07bc
95.5±0.19c
CIEL*a*b* color space
a*
-0.41±0.04a
-0.38±0.05a
-0.41±0.02a
-0.59±0.04b
-0.86±0.04c
-1.88±0.03d
-0.96±0.02a
-0.68±0.32a
-0.84±0.05a
-1.69±0.02b
-1.79±0.01b
-1.85±0.04b
0.43±0.04a
0.38±0.02a
0.34±0.04a
0.35±0.01a
0.39±0.02a
-0.55±0.10b
1.20±0.28a
0.73±0.04ab
0.65±0.04b
0.39±0.01b
-0.13±0.04c
-0.35±0.07c
b*
7.56±0.09a
7.99±0.12a
7.82±0.28a
9.00±0.29a
9.27±0.95a
13.21±0.20b
0.55±0.01a
0.37±0.01a
0.65±0.04a
2.54±0.06b
3.55±0.10c
4.35±0.32d
8.87±0.08a
9.69±0.03b
9.97±0.18bc
10.34±0.18d
10.30±0.03bc
13.10±0.05e
7.25±0.35ab
6.45±0.35a
7.55±0.21ab
8.55±0.21b
10.34±0.09c
14.10±0.71d
*Means ± SD (n = 3). For a particular rice variety, means with different letters in the same
column denote significant difference at 5 % probability level within each rice variety.
100
Figure 6.1 Confocal laser scanning micrographs of control and alkali treated rice grains
of Thadokkham-8 (TDK8) and Doongara (DG). Lipids and proteins are labelled in red and
green, respectively
6.4.3. Mass loss during alkali treatment
Less than 3 - 4.5 % of mass loss was recorded in the water, and maximum alkali (0.2 % NaOH
w/v) treated samples (data not shown). Soaking of milled rice kernels with alkali solution not only
101
washed surface proteins and saponify surface lipids but also removed intact dust and bran residues.
No doubt there might be a loss of water-soluble components from grains which were not analysed
in the present study and needed further investigation.
6.4.4. Crude protein of control and alkali treated rice samples
The crude protein content of control and alkali treated TDK8 and DG are presented in Fig. 6.2a
and 6.2b, respectively. Results showed that DG might have more alkali-soluble protein (such as
glutelin) content than TDK8. There was no significant (P>0.05) reduction of the total crude protein
content of TDK8 up to 0.04 % of NaOH when compared to Cc (control). However, significant
(P<0.05) reduction of total protein content was found in DG even at 0.004 % of NaOH.
Figure 6.2 Crude protein content of control and alkali treated rice grains; (a)
Thadokkham-8 (TDK8), and (b) Doongara (DG)*
*Means ± SD (n = 3). Within figure, different letters denote significant difference at 5 %
probability level.
6.4.5. Textural profile analysis
Textural profiles of cooked control and alkali treated TDK8, and DG rice grains are shown in Fig.
6.3a and 6.3b, respectively. Cooked TDK8 rice hardness values ranged from 2.12±0.004 N (C c0)
to 7.84±0.011 N (C0.2) and from 8.03±0.058 N (Cc0) to 18.56±0.157 N (C0.2) for cooked DG rice.
The rice samples treated with 0 % water showed the least hardness in both rice varieties. Results
showed a significant (P<0.05) increase in cooked rice hardness with an increase in NaOH
concentration from C0.004 to C0.2 in both rice varieties. Baxter and co-workers (2004) reported that
102
removal of prolamin by 100 % propan-2-ol resulted in significant (P<0.05) increase in hardness
which is similar to our results where we removed the water-soluble proteins by treating with
alkaline conditions.
Figure 6.3 Textural profile analysis of control and alkali treated rice grains (a)
Thadokkham-8 (TDK8), and (b) Doongara (DG).* Correlation (r) between hardness and
adhesiveness of control and alkali treated rice grains; (c) Thadokkham-8 (TDK8), and (d)
Doongara (DG)+
*Means ± SD (n = 3). Within figure, significant differences are denoted by lowercase
letters for hardness and uppercase letters for adhesiveness at 5 % probability level.
+The
negative sign associated with adhesiveness was ignored while calculating the
correlation (r) due to more adhesiveness associated with a greater negative value.
The effect of protein removal through alkali (NaOH) treatment on the adhesiveness/stickiness of
cooked rice grains of selected rice varieties TDK8 and DG is shown in Fig. 6.3a and 6.3b,
respectively. Cooked rice stickiness increased significantly (P<0.05) with a decrease in grain
103
surface protein contents, as indicated in CLSM images of control and alkali treated rice grains of
selected varieties (Fig. 6.1). Similar to the current findings, Chrastil (1990b) also reported that the
adhesiveness/stickiness of rice could be increased with the reduced amount of glutelin it contained.
Also, there was significant (P<0.05) correlation found between hardness and adhesiveness of alkali
treated rice grains TDK8 and DG as shown in Fig. 6.3b and 6.3c, respectively. Cooking is a
complex process due to several physicochemical changes taking place simultaneously. The surface
layers, cells and granules disintegrate, and components leach out from the cells (Tamura et al.
2014). This disintegration allows starch granules to interact with protein bodies, mostly
amylopectin with glutelin (Chrastil 1990b; Baxter et al. 2014). This starch-protein interaction
affects the overall stickiness of the cooked rice, greater the interaction higher the stickiness will
be. Previous studies reported the reduced starch-protein interaction in aged rice resulted in
decreased stickiness (Chrastil 1990b; Derycke et al. 2005b; Baxter et al. 2014), probably due to a
thin layer of non-interacted protein (glutelin) bodies on the rice endosperm surface. This
explanation supports the hypothesis and findings of the present study; washing of surface proteins
led to the increased stickiness of cooked rice.
6.4.6. Pasting properties
The pasting properties of control and alkali treated TDK8, and DG rice flours are shown in Table
6.2. Results showed the diverse behavior of samples due to alkali treatment. Increase in pasting
temperature (Ptemp) with an increase in surface proteins removal was observed for TDK8. However,
a slight decrease in Ptemp was found in DG with an increase in surface proteins removal. Increase
in surface proteins removal restricted the swelling of starch granules in TDK8 flour, resulting in
significant (P<0.05) decrease in peak viscosity (Vp). However, protein removal did not affect
viscosity at the point of inflection (Vpi) in the case of DG. Probably, sodium hydroxide (NaOH)
treatment had masked the effect of protein on Vp. It was also found that an increase in alkali
concentration also significantly (P<0.05) reduced the Vp. Interestingly, these results are not in
agreement with the findings of Lai et al. (2004), who reported significant (P<0.05) increase in Vp
of native cereal starches in sodium carbonate (Na2CO3) and NaOH solutions. However, similar
results of restricted Vp of alkali treated sago starch were reported by Karim et al. (2008). The
possible reason for the variation in the pasting behaviors may be the difference in the sample
preparation. In this study and the study carried out by Karim et al. (2008), cereal samples were
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treated with alkali and subsequently washed with deionised water for 15 min to neutralise the pH
before examining the pasting behavior. However, Lai et al. (2004) carried out pasting studies on
starch samples suspended in alkali solutions such as 1 % Na2CO3 and 1 % NaOH. As the sample
preparation in the present study is similar to that of Karim et al. (2008), the reduced Vp could be
due to the disrupted amorphous regions and weakened granular rigidity of alkali treated starch
samples.
In general, protein removal resulted in decreased Breakdown (BD) and Setback (SB) of TDK8
flour and Final viscosity (Vf) of both TDK8 and DG flours. Although there was no BD and Trough
viscosity (Vt) observed in DG, increase in alkali concentration reduced the rate of increase in
viscosity during holding at 95ºC as shown in Appendix 5. The results are on par with the findings
of Baxter and co-workers (2004), who studied the effect of prolamin on the pasting properties of
rice flour. They reported that extraction of approx. 95 % of total prolamin fractions in rice flours
resulted in significant (P<0.05) reduction in BD and Vf and a slight reduction in SB. However, this
study did not investigate the effect of surface protein removal from the rice flour or kernels.
6.4.7. Gelatinization and retrogradation properties by DSC
The gelatinization and retrogradation properties of control and alkali treated TDK8, and DG rice
flours are shown in Table 6.3. Protein removal via alkali treatment resulted in increased onset (Tₒ),
peak (Tp), conclusion (Tc) temperatures and enthalpy (ΔH) during gelatinization of both rice
varieties. The rise in gelatinization transition temperatures and enthalpy with an increase in NaOH
concentration may be attributed to the starch granule stability, possibly through electrostatic
interactions between hydroxyl groups of starch and Na+ ions. Starch exhibits Donnan-potential in
the presence of water due to its weak acidic ion-exchanging behavior (Oosten 1990). The starch
particles have a negative charge; therefore, penetration of Na+ into the amorphous regions of starch
granules is promoted. Moreover, under alkaline conditions, hydroxyl groups of starch might have
a greater tendency to ionise and create even more binding sites for cations. It is hypothesised by
Oosten (1990) that anions might tend to destabilise starch granules by breaking hydrogen bonds.
However, such destabilising effects of anions might be much weaker than the stabilising effects of
cations. Several researchers have reported similar stabilising effects of sodium salts such as sodium
chloride (Abd Ghani et al. 1999; Evans & Haisman 1982), sodium acetate (Evans & Haisman
1982), sodium sulphate (Evans & Haisman 1982), and sodium carbonate (Lai et al. 2002).
105
On the other hand, no significant (P>0.05) effect of protein removal was observed on the
retrogradation thermal temperatures (To(r), Tp(r), and Tc(r)), but enthalpy of retrograded starch
(ΔH(r)) was decreased significantly (P<0.05) in the flour samples of both varieties. This indicates
that a higher concentration of alkali treatment on starch restricts retrogradation. This was the
highest amount of decrease in retrogradation at the highest concentration (0.2 % w/v) of alkali
treatment. In fact, ΔH(r) provides an overall measure of the energy requirement for melting or
uncoiling of double helices of recrystallised amylopectin (Russell 1987). Significant (P<0.05)
decrease in ΔH(r) with an increase in alkali treatment may be indicative of structural changes in
amylopectin, possibly alkali-induced depolymerisation. This will need further investigation.
6.5. Conclusions
This study showed that the protein surface layer from rice kernel or rice flour could be washed by
using very dilute (as low as 0.004 %) alkali (NaOH) solutions. It was found that NaOH treatment
at a concentration of 0.04 % induced yellow color development in grains. Moreover, it was also
observed that textural, pasting, thermal attributes and retrogradation properties were also affected
by alkali solution washing. The stickiness of cooked glutinous and non-glutinous (TDK8 and DG,
respectively) rice could be significantly increased by washing with dilute NaOH solutions. The
contrasting effects of washing of surface proteins and NaOH concentration mean that it might be
promising to manipulate the textural properties of glutinous and non-glutinous rice kernels to
achieve desirable sensory outcomes by varying the proportions of the surface proteins in milled
rice kernels.
106
Table 6.2 Pasting properties of control and alkali treated flour of Thadokkham-8 (TDK8) and Doongara (DG)*
Rice variety
TDK8 flour
Rice variety
DG flour
Treatment
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
Treatment
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
Ptemp (ºC)
74.7±0.67a
73.3±0.78a
73.9±0.49a
73.8±0.64a
74.1±0.28a
79.7±0.46b
Ptemp (ºC)
83.3±0.60a
82.5±1.73a
82.1±0.78a
80.8±0.49a
80.7±0.18a
79.9±0.35a
Vp (mPa-s)
2417±27.58a
2784±108.19b
2692±25.46b
2357±70.00a
2184±62.23a
1535±14.14c
Vpi (mPa-s)
304±44.55a
297±87.68a
341±50.91a
363±87.68a
316±3.54a
225±31.82a
Pasting properties
Vt (mPa-s)
BD (mPa-s)
2121±46.67a
296±74.25ab
a
2183±61.52
601±169.71a
2063±41.01ab
629±15.59a
1966±7.78b
391±77.78ab
1722±12.73c
462±74.95a
d
1481±16.26
255±2.12b
Vf (mPa-s)
3469±151.32a
3014±177.48b
3037±62.93b
2848±75.66bc
2466±3.54c
2594±11.31c
Vf (mPa-s)
2527±43.13a
2584±45.96a
2473±17.68a
2244±22.63b
2029±52.33c
1751±14.14d
SB (mPa-s)
406±3.54a
401±15.56a
410±23.33a
279±14.85b
307±39.60b
271±2.12b
*Means ± SD (n = 3). For a particular rice variety, means with different letters in the same column denote significant
difference at 5 % probability level within each variety.
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Table 6.3 Gelatinization and retrogradation properties of control and alkali treated flour of Thadokkham-8 (TDK8) and
Doongara (DG)*
Rice
variety
TDK8
flour
DG flour
Treatment
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
Tₒ (ºC)
64.4±0.40a
64.9±0.45a
65.1±0.75a
64.1±0.71a
64.8±0.16a
68.2±1.24b
70.3±0.45a
70.7±0.08ab
70.9±0.16ab
70.9±0.02ab
70.6±0.16ab
71.5±0.47b
Gelatinization
Tp (ºC)
Tc (ºC)
71.6±0.01a
84.6±0.47a
72.5±0.16b
87.8±0.81b
b
72.5±0.22
86.3±0.18ab
b
72.5±0.19
87.8±0.27ab
72.9±0.43b
88.7±0.13b
75.1±0.03c
92.6±1.74c
a
76.1±0.49
83.3±0.73a
76.3±0.43a
83.8±0.34a
76.6±0.23a
84.1±0.17a
a
76.5±0.25
83.7±0.62a
a
76.3±0.22
83.1±0.04a
78.2±0.15b
88.3±1.67b
ΔH (Jg-1)
10.4±0.23ab
9.3±0.78a
9.3±0.19a
10.6±0.21ab
11.0±0.08b
11.1±0.02b
6.9±0.35ab
6.0±0.12a
6.2±0.17a
6.2±0.06a
7.4±0.40b
8.4±0.09c
Tₒ(r) (ºC)
41.1±0.18a
41.4±1.22a
41.7±1.28a
41.5±1.97a
42.0±0.23a
41.8±0.02a
39.2±2.17a
40.2±0.54a
41.3±0.52a
42.0±1.10a
42.2±0.68a
42.3±0.62a
Tp(r) (oC)
51.9±0.33a
52.2±1.01a
52.2±0.34a
52.3±0.86a
52.7±0.34a
52.7±0.33a
53.4±0.01a
53.7±1.00a
53.6±0.54a
54.1±0.66a
54.6±0.01a
54.8±0.52a
Retrogradation
Tc(r) (ºC)
ΔH(r) (Jg-1)
60.0±0.45a
4.4±0.26ab
60.3±0.23a
4.5±0.14a
a
59.8±0.16
3.5±0.06c
a
60.3±0.18
3.8±0.09c
60.2±0.25a
3.9±0.02bc
60.5±0.04a
3.9±0.04bc
a
62.4±0.11
4.1±0.12ab
62.7±0.30a
4.2±0.06b
63.1±0.07a
3.5±0.02bc
62.8±0.37a
3.2±0.05c
a
62.3±0.13
3.3±0.07c
62.8±1.46a
2.4±0.36d
R (%)
42.2±3.44ab
48.2±2.53a
38.0±0.10b
35.9±1.56b
35.5±0.47b
35.0±0.39b
58.6±1.26b
70.2±2.47a
56.6±1.21b
50.9±1.26bc
45.1±3.36c
29.0±3.97d
*Means ± SD (n = 3). For a particular rice variety, means with different letters in the same column denote significant
difference at 5 % probability level within each variety.
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Chapter 7 Effect of starch modification in the whole white rice grains on
physicochemical properties of two contrasting rice varieties
This chapter has been published in the Journal of Cereal Science;
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effect of starch modification in the whole
white rice grains on physicochemical properties of two contrasting rice varieties’, Journal of
Cereal Science, vol. 80, pp. 143-149.
109
7.1. Abstract
The effect of acetylation of milled rice of selected rice varieties viz. TDK8 and DG on their
physicochemical properties were investigated at different acetic anhydride concentrations (1 – 7 g
per 100 g of milled rice samples in 225 mL of water). Results showed that the intact starch of
milled grains of both selected verities could be acetylated (Acetyl % for TDK8 = 2.18 and DG =
0.89) even with 1 g of acetic anhydride. X-ray diffraction patterns showed that acetylation resulted
in reduced crystallinity. Acetylation resulted in reduced peak and final viscosities and gel strength,
particularly in glutinous (TDK 8) and non-glutinous (DG) rice. The thermal study showed
acetylation resulted in reduced thermal transition temperatures and enthalpy of both varieties.
Although the increase in retrogradation thermal temperatures was observed, the amount of
retrograded starch was decreased in both varieties. Furthermore, the texture of cooked acetylated
grains was less hard and more adhesive. In vitro digestion showed a significant decrease in GI
possibly due to structural changes in the native starch during acetylation. These findings suggest a
good potential of applying acetic anhydride pre-treatments in rice processing, especially glutinous
varieties to control the hardness and maintain the stickiness properties of rice.
7.2. Introduction
Rice (Oryza sativa L.) together with wheat and maize provides the major portion of daily food
calories to more than 4.5 billion people in developing countries around the globe (Shiferaw et al.
2011). Rice grain quality is mostly assessed on four main aspects; milling yield, grain appearance,
nutritional value and cooking/eating attributes. Among these quality aspects, the cooking and
eating attributes are the most important traits affecting consumer acceptability of rice. Starch, is
the major component of rice, is considered as the main contributor influencing the
physicochemical properties such as endosperm physical appearance, water absorption and
swelling, gelatinization and retrogradation behavior (Pan et al. 2017).
Among all factors, the amylose-amylopectin ratio is the most important parameter affecting
physicochemical properties of starch pastes/gels and characteristics of cereals (Jiamjariyatam et
al. 2015). Amylose contents (AC) of the rice endosperm are widely used to classify the rice
varieties such as glutinous rice (< 5 % AC), very low amylose rice (5-10 % AC), low amylose rice
(10-19 % AC), intermediate amylose rice (20-25 % AC), and high amylose rice (> 25 % AC)
(Gayin et al. 2015; Nawaz et al. 2016a). Glutinous rice, also known as sticky or waxy rice primarily
110
contains branched amylopectin or very low amylose content, has a white and opaque endosperm
because of the air spaces between the starch granules. When cooked, the grain loses its original
shape and becomes very sticky. Non-glutinous or non-waxy rice has a translucent appearance and
contains amylose as well as amylopectin. The cooked grain tends to retain its shape and is less
sticky (Zhu et al. 2017).
Rice quality is usually assessed by functional properties such as water absorption, pasting
properties and thermal properties (Pang et al. 2016; Nawaz et al. 2016b). Fresh, good quality
glutinous rice usually swells to a greater extent, and cooked grains have high adhesiveness than
their non-glutinous counterparts. However, storage at ambient temperature can lead to decrease in
water absorption, longer cooking time and reduced stickiness of cooked glutinous rice, which is
usually unacceptable attribute by the consumers (Thanathomvarakul et al. 2016). Therefore,
controlled storage is usually recommended for glutinous rice to slow down the age-related changes
in functional properties of glutinous rice. The current controlled storage techniques to maintain the
stickiness of rice are mainly chilling temperature and vacuum storage which increase the retail
price of the milled grains due to high infrastructure and operational cost. Pre-processing of milled
grains such as alkali and acid treatment can be a potential option to slow down the storage
deterioration of glutinous rice (Nawaz et al. 2016b; Hatae et al. 1995).
It has been reported that partial starch modification can be a useful processing technique to
improve the glossiness, stickiness, and softness of cooked starches (Bao et al. 2003). This can be
achieved by acetic anhydride reaction with starch by esterification to produce acetylated starch. In
this reaction, the hydroxyl groups on anhydrous-glucose units are substituted with acetyl groups
of acetic acid (Ačkar et al., 2015). Shon & Yoo (2006) performed the rheological analysis of
acetylated rice starch pastes with different acetyl substitution degrees and reported that the
acetylation could increase the swelling power and solubility of starch pastes. They also reported
that the increase in the degree of acetylation resulted in high shear-thinning fluids with high
magnitudes of yield stresses.
The nutritional quality of food is also assessed by its glycemic index (GI). Glycemic response of
dietary starch is directly related to the rate of digestion. Starchy foods such as rice and rice flour
products have a high GI. With the new advances in human nutrition and dietetics, products with a
low GI are preferable (Han & BeMiller 2007). It has been reported that modification of isolated
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starches resulted in reduced production of postprandial hyperglycemic and hyperinsulinemic
spikes associated with rapidly digestible starch (RDS) fractions (Hung et al. 2016). To our
understanding, the effect of acetylation of intact starch in rice grain endosperm to improve
physiochemical properties and nutritional quality of whole rice grains is least studied.
Therefore, the objective of current study is to treat uncooked milled rice grains of varieties with
varying amylose contents with dilute acetic anhydride solution (1 – 7 g per 100 g of milled rice
grains in 225 mL of water) to partially acetylate the starch prior to cooking and investigate the
changes in the physicochemical properties of rice. Acetic anhydride may induce structural changes
in proteins and fat, but these changes are negligible as protein and fat contents are less than 6 and
1 %, respectively of the bulk composition. This partial or limited acetylation was expected to
increase the stickiness of cooked rice grains which is one of the most important quality attributes
of low amylose-containing glutinous rice.
7.3. Materials and methods
One Oryza sativa indica cultivar of glutinous rice Thadokkham-8 (TDK8) having 3.77 % apparent
amylose contents (AAC) and one O. sativa japonica non-glutinous rice Doongara (DG), 19.71 %
(AAC) were used in this study. Both rice paddies were provided by the Department of Primary
Industries (DPI), Yanco, NSW, Australia.
7.3.1. Paddy milling
Paddy was milled to brown rice using rice husker (Satake, Japan). The brown rice was milled to
white rice using an abrasive polisher (Satake, Japan). To avoid the effect of the degree of milling
(DOM), brown rice of all selected rice varieties were kept at 9 % DOM.
7.3.2. Grinding of rice kernels
For the analysis of pasting and thermal properties, the milled white rice grains were ground to flour
using a disc mill (Good Friends of the Guangzhou Machinery Co. Ltd., Guangzhou, China)
equipped with a plate of 500 µm size.
7.3.3. Acetylation of milled rice
Acetylation of starch in intact rice grain followed the method mentioned by Sodhi & Singh (2005)
for rice flour with some modifications. Milled rice grains (100 g) were dispersed in 225 mL of
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MilliQ water and kept in shaking water bath at 25ºC for 60 min. pH of the mixture was adjusted
at 8.0 using 3.0 % (w/v) NaOH solution. Acetic anhydride (AA1 ~ 1 g, AA3 ~ 3 g, AA5 ~ 5 g, and
AA7 ~ 7 g per 100 g of milled rice samples in above mixture) was added dropwise to the mixture
while maintaining pH within 8.0 – 8.4 using 3.0 % (w/v) NaOH solution. Acetylation reaction was
allowed to proceed for 10 min after the addition of acetic anhydride. The mixture was then adjusted
to pH 4.5 with 0.5 N HCl. Grains were then washed free of acid twice with deionised water and
once with 95 % ethanol and then dried at 40ºC. One set of the sample (AA0 ~ 0 g acetic anhydride)
was also prepared to see the effect of soaking. The treated samples were reweighed to estimate the
mass loss of water-soluble components during acetic anhydride treatment. Moreover, one set of
control (C) sample without any treatment was also kept for comparison.
7.3.4. Acetyl (%) and degree of substitution
The degree of substitution is defined as the average number of hydroxyl groups per glucose unit
substituted by acetyl group (Whistler & Daniel 1995). Acetyl (%) and the degree of substitution
(DS) were determined by the titration method. Flour of acetylated grains (1.0 g) was placed in a
250 mL conical flask, and 50 mL of 75 % (v/v) of ethanol in water was added. The flasks were
loosely stoppered and put in shaking water bath at 50oC for 30 min, cooled to room temperature
(22±1ºC), and 40 mL of 0.5 N KOH was added (Colussi et al. 2014). The excess alkali was then
back-titrated with 0.5 N HCl using phenolphthalein as an indicator. The solution was kept for 2
hrs in a fume hood, and any additional alkali, which might have leached out from the sample was
titrated. A blank, using the flour of control (C) sample was also used. Acetyl (%) and degree of
substitution (DS) were calculated using equation 7.1 and 7.2 (Garg & Jana 2011).
𝐴𝑐𝑒𝑡𝑦𝑙 (%) =
[(𝑏𝑙𝑎𝑛𝑘−𝑠𝑎𝑚𝑝𝑙𝑒) ×𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝐻𝐶𝑙+𝑚 ×100]
𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡
𝐷𝑆 = (162 × 𝑎𝑐𝑒𝑡𝑦𝑙 %)/((𝑀 × 100) − [(𝑀 − 1) × 𝑎𝑐𝑒𝑡𝑦𝑙 %])
Eq. 7.1
Eq. 7.2
Where m is the decimal molecular weight of substituent and M is molecular weight of substituent.
7.3.5. X-ray diffraction
X-ray diffraction pattern measurement of rice flour of control and acetylated samples (particle size
≤ 125 µm) was analysed by using Bruker Advance D8 X-Ray diffractometer equipped with a
LynxEye detector and Cu-kα (1.54 Å) radiation. The accelerating voltage and current of 30 kV
113
and 30 mA, respectively, in combination with scan rate 2o/min, were used. The diffractograms
were recorded in a 2θ ranged from 4o to 35o with sampling width of 0.02o. Traces were analysed
using the Diffractplus Evaluation Package Release V3.1, PDF-2 Release 2014.
The crystallinity percentage was calculated with normalised values of the intensities at each
diffraction angle, using the method of Htoon & coworkers (2009). The ratio of the upper diffraction
peak area taken as the crystalline portion, to total diffraction area (two-phase model), represented
the percentage of crystallinity. The diffractograms were smoothed by 13 points using Traces
version 3.01 software (Diffraction Technology Pty LTD, Mitchell, ACT, Australia) before
calculating the percentage of crystallinity.
7.3.6. Pasting properties
Pasting properties of rice flour of control and acetylated samples (particle size < 500 µm) were
determined according to the AACC International Method 61-02.01 using a Rapid Visco Analyser
(RVA-4 model Thermocline Windows Control and analysis software, Version 1.2 (New Port
Scientific, Sydney, Australia). Rice flour (3.01 g, 12.4 % moisture basis) was mixed with 25.0 g
MilliQ water in the RVA canister. A programmed heating and cooling cycle were used, the
samples were held at 50ºC for 1 min, heated to 95ºC in 3.45 min, held at 95ºC for 2.7 min before
cooling to 50ºC in 3.91 min and holding at 50ºC for 1.24 min. Pasting temperature (Ptemp), Peak
viscosity (Vp), Trough viscosity (Vt), Breakdown (BD), Final viscosity (Vf) and Setback (SB) were
recorded.
7.3.7. Gel strength
The RVA canisters after the estimation of pasting properties of acetylated and untreated samples
were sealed with parafilm tape and stored at refrigeration temperature (4±1ºC) for 24 h. A similar
method was used by Saartrat & co-workers (2005). The refrigerated RVA canisters were left at
room temperature (23±1ºC) for 10 min before analysis. The gel was compressed at a speed of pretest 2.0 mm/sec, test 0.2 mm/sec, and post-test 0.2 mm/sec, to a distance of 5.0 mm with 2.5-mm
cylindrical probe under the texture profile analysis (TPA) test mode. The peak force of the first
penetration was termed gel strength. The gel strength values reported are the averages of 3 different
determinations.
114
7.3.8. Gelatinization and retrogradation properties
Differential Scanning Calorimeter (DSC) (Mettler Toledo, Schwerzenbach, Switzerland) with
internal coolant and nitrogen/air purge gas was used to determine the gelatinization characteristics
of rice flours. The DSC was calibrated for the heat flow using indium as standard. Rice flour
(approximately 4 mg, dry weight basis) was accurately weighed into a pan and 6 µL MilliQ water
was added. The pan was hermetically sealed and equilibrated at room temperature for 30 min, then
scanned at the heating rate of 15oC/min from 0 to 100ºC with the empty sealed pan as a reference.
The onset (To), peak (Tp) and conclusion (Tc) temperatures, and enthalpy (ΔH) of gelatinization
were determined by Stare Software Version 9.1 (Mettler Toledo).
After cooling, the scanned samples pans were placed in a refrigerator at 4±1ºC for 7 days.
Retrogradation properties were measured by rescanning these samples at the rate of 15ºC/min from
0 to 100ºC. The onset (To(r)), peak (Tp(r)) and conclusion (Tc(r)) temperatures, and enthalpy of
retrograded starch (ΔH(r)) were determined (Nawaz et al. 2016c). The percentage of retrogradation
(R %) was calculated as ΔH(r)/ΔH X 100.
7.3.9. Textural profile analysis
Rice grains (5 g) were added in 15 mL of Milli-Q water (rice: water ratio =1:3) in 50 mL glass
beaker. The beaker was placed in a water bath at 95±1ºC. Cooking was continued until there was
no ungelatinized white belly observed in rice kernel cross section (data not shown). Analysis of
textural attributes was performed on a TA-XTplus Texture Analyzer (Stable Microsystems, UK)
using 35-mm circular probe. Three cooked grains were on the flat stage, and the texture
determined. The texture analyzer settings were as follows: pre-test speed, 2.00 mm/sec; post-test
speed, 2.00 mm/sec; distance, 2.00 mm; time, 10.00 sec; (auto) trigger force, 0.05 N. From the
force-time curve obtained, textural attributes of hardness (height of the force peak on cycle 1, N)
and adhesiveness (negative force area of the first cycle, N.s) were computed using the EXPONENT
Stable Micro Systems software supplied with instrument. The TPA values reported are the
averages of 3 different determinations.
7.3.10. Starch hydrolysis and GI estimation
Starch hydrolysis was done by using the in vitro digestion method proposed by Goñi et al. (1997).
Starch hydrolysis rate was estimated at 30, 90 and 180 min. Glucose concentration was estimated
115
with a glucose oxidase-peroxidase kit (Sigma 510-A), and GI values for each treatment were
estimated using the non-linear first-order kinetic model.
7.3.11. Statistical analysis
All treatments were replicated three times to obtain mean values. The reported data of acetylation
and degree of substitution, x-ray diffraction patterns, pasting properties, gel strength, gelatinization
and retrogradation properties, textural profile analysis and starch hydrolysis for each variety was
analyzed separately by analysis of variance (Completely Randomized Design) using Minitab R17
(Minitab® for Windows Release 17, Minitab Inc, Chicago) in order to determine significant
differences. The data was then analysed using Tukey’s pair-wise comparison, at 5 % level of
significance, to compare the means between different treatments.
7.4. Results and discussion
7.4.1. Effect of acetic anhydride concentration on acetyl (%) and degree of
substitution (DS) of intact starch in the rice grains
The acetyl (%) and degree of substitution were increased by increasing the quantity of acetic
anhydride as shown in Table 7.1. In general, TDK8 acetylated starch showed high acetyl (%) and
degree of substitution than DG. This might be attributed to the difference in amylose contents and
the difference in the intra-granule packing of rice starch in the endosperm. Moreover, the chemical
substitution reaction in the glucose units of starch macromolecules may be affected by the pattern
in which the amylose chains are packed in amorphous regions and the arrangement of amylopectin
and amylose chains (Sodhi & Singh 2005). The addition-elimination mechanism takes place during
starch acetylation and all the free hydroxyl groups of starch viz. C6OH, C2, and C3 have different
reactivity (Garg & Jana 2011). It has been reported by Colussi et al. (2014) that C 6OH (primary
hydroxyl group) is more reactive than C2 and C3 (secondary hydroxyl groups) due to steric
hindrance and can be readily acetylated. These findings can justify the higher degree of substitution
in TDK8 (glutinous) than that of DG (non-glutinous), and further work will be conducted to
analyze the structural changes due to acetylation in the starch fine structures of glutinous and nonglutinous rice varieties. Food products with acetyl (%) more than 2.5 % are not generally
recognized as safe (GRAS) by Food and Drug Administration of USA (FDA 2012). Results
showed that only AA1 for TDK8 and AA1 as well as AA3 for DG have acetyl (%) lower than 2.5
% and can be considered as GRAS. Further study should be conducted by using lower
116
concentrations (< 1 g acetic anhydride per 100 gram of rice in 225 mL of water) of acetic anhydride
to achieve intact starch esterification in the whole grain less than the FDA standard.
The outer surface of grain might have higher acetylation than the grain core due to more exposure
to the acetic anhydride. However, this assumption could not be verified in the present study due to
technical difficulties. Soaking and subsequent drying resulted in increased internal fissures making
it impossible to separate the outer layers without damaging (and therefore mixing) the grain core
while milling. Further studies will be conducted in the future to overcome this challenge.
Table 7.1 Acetylation and degree of substitution of control and acetic anhydride (1-7 g
acetic anhydride per 100 g of milled rice in 225 mL of water) treated flour of Thadokkham8 (TDK8) and Doongara (DG)*
Rice variety
Treatment
TDK8
DG
Acetyl (%)
Degree of Substitution
C
-
-
AA0
-
-
AA1
2.18±0.13a
0.08±0.01a
AA3
3.01±0.12b
0.12±0.00b
AA5
3.81±0.21c
0.15±0.01c
AA7
4.86±0.02d
0.19±0.00d
C
-
-
AA0
-
-
AA1
0.89±0.11a
0.03±0.00a
AA3
2.18±0.15b
0.08±0.01b
AA5
3.69±0.26c
0.14±0.01c
AA7
5.88±0.06d
0.24±0.00d
*Means ± SD (n =3). Within each variety, means with different letters in the same column
denote significant difference at 5 % probability level.
7.4.2. Mass loss during acetic anhydride treatment
In general, less than 3-4.5 % of solid mass loss was recorded in water, and acetic acid treated
samples of all rice varieties (data not shown). There might be a loss of water-soluble components
from grains which were not analysed in the present study and needed further investigation. We
assume that such a loss will be negligible affecting the results.
117
7.4.3. X-ray diffraction
X-ray diffraction analysis was performed to check if acetylation altered the diffraction and the
crystallinity of rice flour. The x-ray diffraction spectra of control and acetylated rice flours of all
varieties are presented in Fig. 7.1. As expected, the x-ray diffraction pattern of control samples of
selected rice varieties exhibited A-type diffraction (XRD) pattern detected with main peaks at
14.9º, 16.9º, and 22.8o. The crystallinity for TDK8 and DG was recorded as 25.87±0.57 % and
18.73±0.36 %, respectively. It is now a well-established concept that the crystallinity depends upon
the amylose/amylopectin ratio. The results confirm the earlier finding that the lower the ratio of
amylose/amylopectin, the higher the degree of crystallinity (Park et al. 2007).
Figure 7.1 X-ray diffraction and calculated crystallinity (%) of control and acetic anhydride
(1-7 g acetic anhydride per 100 g of milled rice in 225 mL of water) treated rice flour of
Thadokkham-8 (TDK8) and Doongara (DG) as shown on the right side of the figures*
*Means ± SD (n = 3). Within each variety, means with different letters in the same figure
denote significant difference at 5% probability level.
Native starch modification of rice showed that the samples treated with a higher acetic acid
concentration depicted significant (P<0.05) decrease in crystallinity, possibly because of more
starch acetylation and subsequently reduced reordering. Highly ordered crystalline structures in
starch are due to the intra- and intermolecular hydrogen bonds. Acetylation damaged the ordered
crystalline structure by reducing the formation of intermolecular hydrogen bonds (Luo & Shi
2012).
118
7.4.4. Pasting properties and gel strength
The pasting properties of control and acetic anhydride treated rice flours are shown in Table 7.2.
Results showed the diverse pasting behavior of samples due to acetylation. The decrease in pasting
temperature (Ptemp) with an increase in acetylation was observed. Increase in starch acetylation
restricted the swelling of starch granules especially in glutinous (TDK 8) rice, resulting in
significant (P<0.05) decrease in peak viscosity (Vp). Starch swelling may be predominantly
controlled by the amylose and/or amylopectin amorphous regions close to the flour particle surface
(Wu et al. 2010).
Table 7.2 Pasting properties and gel strength of control and acetic anhydride (1-7 g acetic
anhydride per 100 g of milled rice in 225 mL of water) treated flour of Thadokkham-8
(TDK8) and Doongara (DG)*
Rice
Treatment
Ptemp (ºC)
Vp (mPa-s)
Vt (mPa-s)
BD (mPa-s)
Vf (mPa-s)
C
71.5±0.53a
3370.5±17.7b
SB (mPa-s)
Gel strength (N)
2218.5±21.9c
1152.0±39.6c
2757.5±14.8b
a
1557.5±26.2
a
a
539.0±7.1ab
0.48±0.006a
513.5±55.9
604.5±6.4a
variety
TDK8
DG
a
a
AA0
71.3±0.33
4191.0±2.8
AA1
70.1±0.04b
4164.5±17.7a
2515.5±16.3b
1649.0±1.4a
3120.0±22.6a
AA3
69.2±0.11
bc
c
2163.5±13.4
c
c
c
AA5
68.6±0.21cd
2776.0±26.9d
2015.5±9.2d
AA7
67.4±0.26
d
e
C
78.2±0.10a
AA0
AA1
76.3±0.14
AA3
75.7±0.19c
AA5
74.6±0.18
d
1830.0±21.2
AA7
73.8±0.13e
1824.0±8.5c
3096.0±24.0
2633.5±29.0
932.5±37.5
2546.0±11.3c
d
d
676.5±36.1
1771.5±21.9c
1473.5±17.7a
77.1±0.14b
2086.5±13.4a
1470.0±8.5a
c
b
1949.5±14.8
1914.0±15.6
a
2634.5±33.2
760.5±17.7d
e
2590.5±20.5
3147.0±26.9
0.41±0.011c
b
0.36±0.005d
530.5±20.5ab
0.32±0.004e
471.0±19.8
b
0.29±0.004f
473.0±7.1
298.0±4.2d
4877.5±14.8a
3404.0±2.8a
0.62±0.004a
616.5±21.9a
3522.5±16.3b
2052.5±7.8b
0.58±0.004ab
b
b
0.58±0.003ab
c
481.5±0.7
1928.5±12.0b
1427.0±8.5ab
501.5±20.5bc
3326.0±18.4c
c
b
c
d
1255.0±17.0c
0.44±0.004b
2387.0±22.6
1468.0±14.1
1376.0±14.1
ab
454.0±35.4
569.0±8.5ab
3473.0±11.3
2006.0±2.8
1899.0±26.9c
d
3169.0±11.3
1793.0±2.8
2925.5±12.0e
1670.5±29.0e
0.58±0.004ab
0.55±0.040bc
0.49±0.002c
*Means ± SD (n = 3). Within each variety, means with different letters in the same column
denote significant difference at 5% probability level.
Increase in starch acetylation may degrade these amorphous regions, resulting in reduced water
absorption and starch swelling in glutinous and low amylose varieties. In general, increased starch
acetylation resulted in decreased breakdown (BD), setback (SB) and final (Vf) viscosity values of
rice. The starch substitution with acetyl group of acetic anhydride may restrict the recrystallisation
of amylose and/or amylopectin, resulting in reduced Vf and weak gelation, therefore, significant
(P<0.05) reduction in the gel strength was recorded.
119
7.4.5. Gelatinization and retrogradation properties
The thermal (gelatinization and retrogradation) properties of control and acetic anhydride treated
rice flours are presented in Table 7.3. Results showed reduced thermal transition temperatures viz.
onset (To), peak (Tp), and conclusion (Tc), and enthalpy (ΔH) of gelatinization with an increase in
acetic acid pre-treatment. This shift to lower thermal transition temperatures and enthalpy of
gelatinization indicated that the greater extent of starch acetylation might accelerate the starch
gelatinization at a lower temperature with less energy requirement (Ohishi et al. 2007). The DSC
findings are in agreement with pasting properties results, where a slight reduction in pasting
temperature (Ptemp) was observed with increase in acetic anhydride concentration during pretreatment.
On the other hand, increase in retrogradation temperatures viz. onset (To(r)), peak (Tp(r)), and
conclusion (Tc(r)) was observed, but the amount of retrograded starch (as indicated by reduction in
enthalpy (ΔH(r)) of retrograded starch) was decreased with increase in acetic anhydride
concentration during pre-treatment. It was hypothesised that greater extent of starch acetylation
might restrict the retrogradation possibly due to structural changes in the amylose and/or
amylopectin fine structures. Modified starch molecular structures may be unable to recrystallise
during storage. Interestingly, rice varieties with a higher amount of amylopectin showed a more
significant decrease in retrogradation (R %) after acetic anhydride pre-treatment than the one with
the higher amount of amylose. These findings revealed that branched amylopectin might be
acetylated to a greater degree than the straight chain amylose. This will need further investigation.
7.4.6. Textural profile analysis
The textural profiles of cooked control and acetic anhydride treated grains of different varieties
are presented in Fig. 7.2. Results showed that acetic anhydride pre-treated cooked rice grain
samples of all varieties were significantly (P<0.05) softer and showed significantly (P<0.05)
higher stickiness than the control cooked samples. During cooking several physicochemical
changes take place simultaneously such as the disintegration of surface layers, cells and granules,
and leaching out of components from the cells (Tamura et al. 2014). This cellular disintegration
and leaching of cell components result in softness and stickiness of cooked rice grains. On the
other hand, the leached starch mainly amylose and amylopectin with smaller molecular weight and
fewer branches can rearrange the double helical fine structures during storage after cooking. This
120
rearrangement or recrystallisation of fine structures is known as retrogradation, result in increased
hardness and reduced adhesiveness of the cooked grains (Nawaz et al. 2016b). The starch
modification caused by the addition of acetyl group during esterification of starch can retard the
recoiling and rearrangement of starch fine structures during storage, resulting in softer and stickier
cooked rice grains.
Figure 7.2 Textural profile analysis of control and acetic (1-7 g acetic anhydride per 100
g of milled rice in 225 mL of water) treated rice grains of Thadokkham-8 (TDK8) and
Doongara (DG)*
*Means ± SD (n = 3). Within each variety, significant differences are denoted by
lowercase letters for hardness and uppercase letters for adhesiveness at 5% probability
level.
7.4.7. GI estimation
The results of estimated GI using in vitro digestion of 180 min are presented in Table 7.3. The GI
value of TDK8 (glutinous rice) was relatively higher than that of DG (non-glutinous rice). The
lower GI of DG might be due to the formation of complexes between amylose and lipids upon
heating, resulting in reduced enzyme susceptibility (Frei et al. 2003). It was observed that the
increase in acetyl (%) and DS of both selected verities resulted in significant (P<0.05) decrease in
GI. These results demonstrated that the esterification of starch might have changed the structure
of starch resulted in a reduced glycemic response. Reported observations are in agreement with
the previous research conducted by Zhou et al. (2014).
121
Table 7.3 Experimental results of estimated GI, and gelatinization and retrogradation properties of control and acetic
anhydride (1-7 g acetic anhydride per 100 g of milled rice in 225 mL of water) treated flour of Thadokkham-8 (TDK8) and
Doongara (DG)*
Rice
Treatment
Estimated GI
Gelatinization
variety
Retrogradation
Tₒ ( C)
Tp ( C)
Tc ( C)
ΔH (Jg )
Tₒ(r) ( C)
Tp(r) ( C)
Tc(r) (oC)
ΔH(r) (Jg-1)
R (%)
o
o
o
-1
o
o
TDK8
C
97.05±0.18a
61.95±0.62a
69.77±0.53a
85.97±0.55a
9.85±0.54a
38.98±0.55d
52.61±0.73ab
61.29±1.27a
1.59±0.03a
16.17±0.61a
flour
AA0
93.77±0.24b
61.24±0.18a
68.45±0.01b
80.85±0.20b
8.50±0.40b
41.09±0.44cd
52.27±0.19b
60.76±0.78a
1.14±0.07b
13.45±1.47a
AA1
90.95±0.77c
60.89±0.28ab
67.94±0.08bc
77.85±0.35c
8.34±0.23b
43.00±1.20bc
53.07±0.15ab
59.83±0.27a
0.74±0.10c
8.87±0.94b
AA3
85.24±0.18
d
a
bc
c
b
ab
ab
a
d
3.77±0.56c
AA5
73.11±0.41e
60.94±0.01ab
67.16±0.07cd
76.99±0.05c
7.97±0.04b
47.91±0.04a
53.53±0.20ab
61.89±0.15a
0.23±0.01d
2.89±0.19c
AA7
68.69±0.61f
59.82±0.09b
66.89±0.03d
76.33±0.15c
7.63±0.05b
48.18±0.09a
53.78±0.15a
62.04±0.22a
0.20±0.00d
2.62±0.04c
a
a
a
a
d
c
a
a
39.46±2.70a
DG flour
61.11±0.01
61.09±0.16
68.04±0.21
77.64±0.43a
60.65±0.18ab
66.75±0.04b
77.27±0.13b
AA1
73.11±0.41
b
bc
c
c
AA3
68.69±0.61c
60.21±0.04bc
66.31±0.03cd
75.04±0.16d
8.31±0.17d
39.69±0.06b
50.57±0.09ab
AA5
64.07±0.61
d
cd
d
e
de
ab
a
AA7
62.92±1.42d
60.01±0.12
59.70±0.11d
66.08±0.06
65.78±0.10e
74.37±0.11
10.37±0.17b
9.42±0.09
7.91±0.11
73.90±0.17e
c
7.48±0.06e
35.70±0.38
53.64±0.38
AA0
76.33±0.07
11.31±0.14
a
46.20±1.67
78.24±0.02
66.39±0.04
79.15±0.15
8.23±0.09
C
60.27±0.06
67.48±0.09
77.93±0.82
48.67±0.50
61.28±1.61
58.81±0.11
0.31±0.04
4.47±0.36
37.50±0.40c
49.89±0.18b
58.31±0.21b
2.69±0.25b
25.91±2.81b
c
b
b
c
20.06±1.17bc
58.02±0.10bc
1.23±0.06cd
14.81±0.98c
c
d
13.72±0.26c
1.02±0.01d
13.65±0.07c
38.17±0.13
40.09±0.13
40.82±0.09a
49.89±0.17
50.90±0.05
51.12±0.06a
58.32±0.04
57.68±0.09
57.18±0.08d
1.89±0.13
1.09±0.04
*Means ± SD (n = 3). For a particular rice variety, means with different letters in the same column denote significant
difference at 5 % probability level within each variety.
122
7.5. Conclusions
This study showed that the rice kernel could be acetylated by using very dilute acetic anhydride.
It was observed that the textural, pasting, thermal attributes, retrogradation properties and glycemic
index were affected by acetic anhydride treatment of native starch. The stickiness of cooked rice
with varying apparent amylose contents (AAC) could be significantly increased by washing with
acetic anhydride. The contrasting effects of limited acetylation mean that it might be promising to
manipulate the textural properties of glutinous and non-glutinous rice kernels to achieve desirable
sensory outcomes by modifying the native starch with varying extent of acetylation in milled rice
kernels. The level of esterification and the effect of esterification of milled rice on the changes
during storage will be another potential further study. Further study is needed on the level of
acetylation across the grain.
123
Chapter 8 Effect of soaking medium on the physicochemical properties of
parboiled Laotian glutinous rice
This chapter has been published in the International Journal of Food Properties;
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effect of soaking medium on the
physicochemical properties of parboiled Laotian glutinous rice’, International Journal of Food
Properties, vol. 21, pp. 1896-1910.
124
8.1. Abstract
The effect of various soaking mediums viz. water (control), 3 % NaCl and 0.2 % acetic acid and
without soaking on the physicochemical properties of parboiled selected glutinous (TDK8 and
TDK11) and non-glutinous (Doongara) was investigated in the present study. Results showed that
the chemistry of soaking had a significant effect on the head rice yield (HRY), grain hardness,
crystallinity, color, pasting and thermal properties, textural attributes and glycemic index of these
rice varieties. Soaking with NaCl and acetic acid significantly (P<0.05) increased the grain
hardness and HRY than that of control and without soaking treatments. Acetic acid and NaCl
soaking significantly (P<0.05) affected crystalline regions of starch, resulting in reduced
crystallinity in X-ray diffraction analysis and thermal endotherms in DSC analysis. NaCl soaking
induced swelling of starch granules, resulting in high peak and final viscosities. However, acetic
acid restricted swelling, resulting in reduced peak and final viscosities. NaCl and acetic acid
soakings also resulted in increased hardness and adhesiveness of cooked grains than normal water
soaked and un-soaked parboiled rice samples. Interestingly, change in textural attributes was
prominent in parboiled glutinous rice. The color difference values for fresh parboiled samples was
significantly (P<0.05) lower for acetic acid soaked samples than NaCl soaked and un-soaked
samples probably due to the bleaching effect of acetic acid. Moreover, parboiling also resulted in
significant (P<0.05) reduction in glycemic index which shows nutritional benefits of parboiling of
glutinous rice. These findings revealed the potential application of parboiling with modified
soaking techniques to improve the grain quality.
8.2. Introduction
Parboiling is hydrothermal processing of paddy and/or brown rice to improve the head rice yield,
nutritional and organoleptic properties by using a three-stage process viz. soaking, steaming and
drying (Kar et al. 1999; Ballogou et al. 2015; Kwofie & Ngadi 2017). According to the recent
statistics, 130 million tonnes of paddy is parboiled annually around the globe with about 3-4
million tonnes of high-value parboiled milled rice being moved in world trade (Bhattacharya
2013). Parboiling was initially originated and is largely practiced in India (Dutta & Mahanta 2014).
It is also processed and consumed in other South Asian countries including Bangladesh (Hotz et
al. 2015) and Sri Lanka (Gunaratne et al. 2013) as well as some Sub-Saharan African countries
125
including Ghana (Amissah et al. 2003), Benin (Demont et al. 2012), Senegal (Demont 2013), and
Nigeria (Usman et al. 2014).
Parboiling results in some profound changes in the physicochemical and functional properties of
rice (Haspari et al. 2016). Starch granules are swelled due to gelatinization, protein bodies are
disrupted due to denaturation and disulfide cross-linking, and lipid-amylose complex formation
takes place (Oli et al. 2014). Moreover, parboiling also results in the inward diffusion of watersoluble vitamins and minerals from bran to endosperm improving the nutritional quality (Patindol
et al. 2017; Paiva et al. 2016). These changes in physicochemical properties of rice due to
parboiling increase grain hardness and improve the milling yield (Buggenhout et al. 2013).
Furthermore, physicochemical changes create more translucency and amber coloration in head rice
(Lamberts et al. 2006). These changes also influence cooking properties such as the harder texture
of cooked grains. Moreover, parboiling also results in increased resistant starch fraction and a low
glycemic index (GI) which are health-promoting (Kale et al. 2017).
Traditionally to make parboiled rice, paddy is soaked in water for 48 hrs followed by steaming and
drying in the rural rice producing communities (Kwofie & Ngadi 2017). As the global food index
is taking a step forward from food security to food safety, innovations and improvements in the
parboiling process are becoming a subject of research interest. Therefore, several types of research
have been conducted in the past to improve the parboiling operation by varying the soaking
temperature (Sareepuang et al. 2008), reduction in soaking process of paddy by using a tumbler
(Hapsari et al. 2016), modifications in steaming process (Soponronnarit et al. 2006) and
modifications in drying process (Swasdisevi et al. 2010). In recent years, dry-heat parboiling
operations have also been introduced to improve parboiling efficiency (Dutta et al. 2015; Dutta et
al. 2016).
In the past, scientists have considered on the soaking time and temperature on parboiled rice
quality, but to our understanding, the effect of the modified soaking medium by the addition of
organic acids and/or salt on the parboiled glutinous rice is still not well defined. Previous studies
have established a weak correlation between water uptake by paddy during soaking and chemistry
of soaking medium and reported that the rate of soaking could be altered by the addition of organic
acids, alkalis, and salts (Bello et al. 2004). They reported that the incorporation of acids
(hydrochloric acid, acetic acid, and phosphoric acid) into the soaking water reduced the water
126
absorption than control soaking. However, the addition of alkali (sodium hydroxide and sodium
bicarbonate) significantly increased the water absorption than control soaking. Thammapat et al.
(2015) reported that saline soaking of Thai glutinous rice (RD6) before parboiling significantly
increased the amounts of total phenolic contents, phenolic acid, γ-oryzanol, saturated fatty acids
and monosaturated fatty acids and decreased α-tocopherol and polyunsaturated fatty acids
compared to controlled samples, resulting in improvement of bioactive compounds and cooking
quality.
The researches reported on the parboiling of glutinous rice in the past focused only on head rice
yield and nutrient diffusion, and less focus was given on the cooking quality and glycemic index
(GI) of parboiled glutinous rice. Moreover, there is no data available on the parboiling of Laotian
glutinous varieties such as TDK8 and TDK11. Therefore, in this study, the focus is given on the
effect of soaking medium on the physicochemical properties of parboiled selected glutinous rice
mainly focusing on cooking attributes and GI using in vitro digestion method.
8.3. Materials and methods
Fresh paddies (Feb. 2017) of two Oryza sativa indica cultivar of glutinous rice viz. Thadokhham8 (TDK8) and Thadokhham-11 (TDK11) having 3.77 % and 3.72 % (w/w) apparent amylose
contents (AAC), respectively were used in this study. Moreover, fresh paddy of one O. sativa
japonica cultivar of non-glutinous rice viz. Doongara (DG) having 19.71 % (w/w) AAC was also
studied for comparison.
8.3.1. Parboiling
8.3.1.1. Soaking
Paddies (250 grams each) were soaked in three different soaking mediums viz. Water, 0.2 % acetic
acid and 3 % NaCl for 24 hrs at room temperature (22±1°C) with paddy to solvent ratio of 1:8.
The concentrations of acetic acid and NaCl viz. 0.2 and 3 % (w/w), respectively were selected by
doing preliminary soaking experiments and above-mentioned concentrations (>3 % NaCl and >0.2
% acetic acid) of soaking medium, no difference was observed on physicochemical properties of
parboiled rice (data not shown). Fresh paddy without soaking was also analyzed for comparison.
127
8.3.1.2. Steaming
The overnight soaked paddies in various soaking mediums were drained and pressure cooked using
super-heated steam at 50 kPa (gauge pressure) for 10 min in a pressure cooker (Breville, VIC,
Australia). The actual time of steaming was 30 min as shown in time-pressure profile Appendix 6.
Sample without soaking was also parboiled using same steaming conditions.
8.3.1.3. Drying
The parboiled paddy samples were spread on blotting paper and kept in a fume hood at room
temperature (22±1oC, RH~50 %) to dry for 72 hrs to the moisture content of 14 % (w/w).
8.3.2. Head rice yield (HRY)
The fresh and parboiled paddies (without soaking, water (control) soaking, 3 % NaCl soaking, and
0.2 % acetic acid soaking) were milled to brown rice using husker (Satake, Japan). The brown rice
was then milled to white rice using an abrasive polisher (Satake, Japan). The degree of milling
(DOM) was 9 % for all treatments, which was calculated by using equation 8.1.
𝐷𝑂𝑀 (%) = [1 − (𝑊𝑀𝑃𝑅/𝑊𝐵𝑃𝑅)] × 100
Eq. 8.1
WMPR and WBPR are the weight of milled parboiled rice and brown parboiled rice in grams,
respectively.
The head rice yield (HRY %) was calculated as a percentage of whole milled grains with respect
to the paddy rice.
8.3.3. Mechanical strength
The mechanical strength of individual sound milled grain (9 % DOM) of fresh and parboiled
(without soaking, water (control) soaking, 3 % NaCl soaking, and 0.2 % acetic acid soaking)
samples of three varieties were measured by using three-point bending test according to the method
of Truong (2008). A special attachment including a cutting probe and a sample holder plate with
grooves of five different sizes was used. The cutting probe was attached to the TA-XTplus Texture
Analyzer (Stable Microsystems, UK) and grains were placed in the grooves of the sample holder.
The bending test was performed in a compression test mode using pre-test, test, and post-test
128
speeds of 1 mm/sec, 2 mm/sec, and 10 mm/sec, respectively. The hardness (N), the maximum
force required to break the grains was calculated.
8.3.4. Color estimation
The color measurements of samples for all treatments were done by using Konica Minolta Chroma
Meter CR-400 (Tokyo, Japan). The color meter was calibrated with a white tile. Samples were
packed in a clean petri dish. The color was measured in CIEL*a*b* color space. The color
∗
difference (∆𝐸𝑎𝑏
) between fresh and parboiled rice (without soaking, water (control) soaking, 3 %
NaCl soaking, and 0.2 % acetic acid soaking) was also calculated using equation 8.2.
∗
∆𝐸𝑎𝑏
= √(𝐿∗2 − 𝐿∗1 )2 + (𝑎2∗ − 𝑎1∗ )2 + (𝑏2∗ − 𝑏1∗ )2
Eq. 8.2
Where, 𝐿∗2 is the brightness of parboiled rice, 𝐿∗1 is the brightness of fresh rice, 𝑎2∗ refers to the red
-green color of parboiled rice, 𝑎1∗ refers to the red-green color of fresh rice, 𝑏2∗ refers to the yellow-
blue color of parboiled rice, and 𝑏1∗ refers to the yellow-blue color of the fresh rice.
8.3.5. X-ray diffraction
Samples were ground to a flour (particle size ≤ 125 µm) using disc mill (Good Friends of the
Guangzhou Machinery Co. Ltd., Guangzhou, China) equipped with a plate of 125 µm size. X-ray
diffraction pattern measurement of rice flour was analysed by using Bruker Advance D8 X-Ray
diffractometer equipped with a LynxEye detector and Cu-kα (1.54 Å) radiation. The accelerating
voltage and current of 30 kV and 30 mA, respectively, in combination with scan rate 2o/min, were
used. The diffractograms were recorded in a 2θ ranged from 4o to 35o with sampling width of
0.02o. Traces were analysed using the Diffractplus Evaluation Package Release V3.1, PDF-2
Release 2014.
The crystallinity percentage was calculated with normalised values of the intensities at each
diffraction angle, using the method of Htoon et al. (2009). The ratio of the upper diffraction peak
area taken as the crystalline portion, to total diffraction area (two-phase model), represented the
percentage of crystallinity. The diffractograms were smoothed by 13 points using Traces version
3.01 software (Diffraction Technology Pty LTD, Mitchell, ACT, Australia) before calculating the
percentage of crystallinity.
129
8.3.6. Pasting properties
Samples were ground to a flour (particle size ≤ 500 µm) using disc mill (Good Friends of the
Guangzhou Machinery Co. Ltd., Guangzhou, China). Pasting properties of rice flour samples were
determined according to the AACC International Method 61-02.01 using a Rapid Visco Analyser
(RVA-4 model Thermocline Windows Control and analysis software, Version 1.2 (New Port
Scientific, Sydney, Australia)) (AACC, 1999). Rice flour (3.01 g, 12.4 % moisture basis) was
mixed with 25.0 g MilliQ water in the RVA canister. A programmed heating and cooling cycle
were used, the samples were held at 50oC for 1 min, heated to 95oC in 3.45 min, held at 95oC for
2.7 min before cooling to 50oC in 3.91 min and holding at 50oC for 1.24 min. Pasting temperature
(Ptemp), Peak viscosity (Vp), Trough viscosity (Vt), Breakdown (BD), Final viscosity (Vf) and
Setback (SB) were recorded.
8.3.7. Thermal properties
Differential Scanning Calorimeter (DSC) (Mettler Toledo, Schwerzenbach, Switzerland) with
internal coolant and nitrogen/air purge gas was used to determine the gelatinization characteristics
of rice flours. The DSC was calibrated for the heat flow using indium as standard. Rice flour
(approximately 4 mg, dry weight basis) was accurately weighed into pan and 6 µL MilliQ water
was added. The pan was hermetically sealed and equilibrated at room temperature for 30 min, then
scanned at the heating rate of 15oC/min from 0 to 100oC with the empty sealed pan as a reference.
The onset (To), peak (Tp) and conclusion (Tc) temperatures, and enthalpy (ΔH) of gelatinization
was determined by Stare Software Version 9.1 (Mettler Toledo).
After cooling, the scanned samples pans were placed in a refrigerator at 4±1oC for 7 days.
Retrogradation properties were measured by rescanning these samples at the rate of 15oC/min from
0 to 100oC. The onset (To(r)), peak (Tp(r)) and conclusion (Tc(r)) temperatures, and enthalpy of
retrograded starch (ΔH(r)) were determined. The percentage of retrogradation (R %) was calculated
as ΔH(r)/ΔH X 100.
8.3.8. Texture profile analysis
Rice grains (5 g) were added in 15 mL of MilliQ water (rice: water ratio =1:3) in 50 mL glass
beaker. The beaker was placed in a water bath at 95±1oC and samples were cooked. Analysis of
textural attributes was performed on a TA-XTplus Texture Analyzer (Stable Microsystems, UK)
130
using 35-mm circular probe. Three cooked grains were on the flat stage, and the texture
determined. The texture analyzer settings were as follows: pre-test speed, 2.00 mm/sec; post-test
speed, 2.00 mm/sec; distance, 2.00 mm; time, 10.00 sec; (auto) trigger force, 0.05 N. From the
force-time curve obtained, textural attributes of hardness (height of the force peak on cycle 1, N)
and adhesiveness (negative force area of the first cycle, N.s) were computed using the EXPONENT
Stable Micro Systems software supplied with instrument.
8.3.9. Starch hydrolysis kinetics and GI prediction
8.3.9.1. Starch hydrolysis
The starch hydrolysis was conducted by adapting the method of Goñi et al. (1997). Rice samples
(200 mg) were cooked in 4 mL of MilliQ water at 80oC for 30 min. Then, 10 mL of HCl-KCl
buffer (pH 1.5) was added to the cooked samples for protein digestion, and the mixture was
smashed to make a paste. Solution (0.2 mL) containing 1.0 mg of pepsin in 10 mL of HCl-KCl
buffer (pH 1.5) was added to samples. Samples were transferred to 50 mL beaker and incubated at
40oC for 60 min in a shaking water bath (100 RPM). Then, 15 mL of Tris-maleate buffer (pH 6.9)
to adjust the volume to 25 mL. For starch hydrolysis, 5 mL of Tris-maleate buffer containing 2.6
IU of porcine pancreatic α-amylase (A-3176, Sigma, Missouri, USA) was added and samples were
incubated at 37oC in a shaking water bath. Aliquots of 0.1 mL were taken in a test tube at 30, 90
and 180 min and boiled in a water bath (100oC) for 5 min to inactivate the α-amylase. One mL of
0.4 M sodium acetate (pH 4.75) and 30 µL of amyloglucosidase from Aspergillus niger (A-1602,
Sigma, Missouri, USA) was added to each tube followed by incubation at 60oC for 40 min in a
water bath to allow amyloglucosidase to decompose starch into glucose. The glucose released by
in vitro digestion of rice was estimated by using glucose oxidase-peroxidase kit (GAGO-20,
Sigma, Missouri, USA).
8.3.9.2. GI prediction
The kinetics of starch hydrolysis and Glycemic index (GI) were calculated by using the non-linear
first order kinetic model as described by Rattanamechaiskul et al. (2014). Following equations
(8.3, 8.4, 8.5, and 8.6) were used;
𝐶 = 𝐶∞ (1 − 𝑒 −𝑘𝑡 )
Eq. 8.3
131
𝐴𝑈𝐶 = 𝐶∞ (𝑡𝑓 − 𝑡0 ) − (
𝐻𝐼 = (
𝐶∞⁄
𝑘 ) (1 − 𝑒𝑥𝑝 (−𝑘(𝑡𝑓 − 𝑡0 )))
𝐴𝑈𝐶𝑠𝑎𝑚𝑝𝑙𝑒
⁄𝐴𝑈𝐶
) × 100
𝑤ℎ𝑖𝑡𝑒 𝑏𝑟𝑒𝑎𝑑
Eq. 8.4
Eq. 8.5
𝐺𝐼 = 39.71 + 0.549(𝐻𝐼)
Eq. 8.6
Where, C is the percentage of starch hydrolyzed at time t, C∞ is the equilibrium concentration of
hydrolyzed starch, k is the kinetic constant (min-1), t is the time (min), AUC is the area under the
hydrolysis curve, tf is the final time (180 min), t0 is the initial time (0 min), and HI is the hydrolysis
index, which is the percentage of area under hydrolysis curve of sample divided by the area under
the hydrolysis curve white bread as a reference.
8.3.10. Statistical analysis
All treatments were replicated three times to obtain mean values. The reported data of head rice
yield, CIEL*a*b* color space, pasting properties, thermal properties, texture profile analysis and
GI prediction for each variety was analyzed separately by analysis of variance (Completely
Randomized Design) using Minitab R17 (Minitab® for Windows Release 17, Minitab Inc,
Chicago) in order to determine significant differences. The data was then analyzed using Tukey’s
pair-wise comparison, at 5 % level of significance, to compare the means between different
treatments.
8.4. Results and discussion
8.4.1. The hardness of parboiled and milled kernels
The mechanical strength of milled grains of fresh and parboiled grain samples was expressed as
grain hardness, which is the force (N) required to break the grains. Results showed that the
parboiling and subsequent drying of paddy resulted in significant (P<0.05) increase in grain
hardness as compared to the fresh non-parboiled grains (Fig. 8.1). These results are in agreement
with the findings of previous researches, who reported that the increased grain hardness is due to
less internal fissures in parboiled rice grain because of the gelatinization and fusion of starch
granules during parboiling process (Buggenhout et al. 2013; Oli et al. 2014). Among various
parboiling treatments, acetic acid treated grains were significantly (P<0.05) harder than NaCl
treated followed by water (control), without soaking treatment and non-parboiled rice for all
132
varieties. It could be that the acetic acid and saline soakings might reduce internal fissures than in
control soaking, resulting in harder grains.
8.4.2. Head rice yield (HRY)
The primary parameter used in the industry to quantify rice milling efficiency is the head rice yield
(HRY) (Cnossen et al. 2003). The head rice yield (HRY) of fresh and parboiled samples of selected
varieties using different soaking conditions are presented in Fig. 8.1. HRY was expressed as a
percentage of whole milled grains (70 % of the full length) produced in the milling of paddy at 9
% DOM. Results showed that parboiling significantly (P<0.05) increased the HRY in all varieties,
glutinous TDK8 (Fig. 8.1a), TDK11 (Fig. 8.1c) and non-glutinous DG (Fig. 8.1d). Moreover, it
was observed that the chemistry of soaking medium also significantly (P<0.05) affected the HRY.
Maximum yield was gained in the acetic acid soaked samples followed by NaCl soaked, water
(control) and without soaking treatments in all selected varieties. As expected, there was a positive
correlation between grain hardness and HRY (Fig. 8.1b, 8.1d, and 8.1f).
8.4.3. Color change in fresh and parboiled rice
∗
CIEL*a*b* color space of fresh and parboiled milled rice and color difference (∆𝐸𝑎𝑏
) of parboiled
grains (without, water (control), 3 % NaCl, and 0.2 % acetic acid soaking) with fresh samples of
all selected varieties are presented in Table 8.1. Color represented in terms of L*, a*, and b* values
of parboiled samples are significantly (P<0.05) different from the fresh samples in all varieties.
There was a significant (P<0.05) decrease in L* value and significant (P<0.05) increase in b* value
due to parboiling in all varieties. However, a* value significantly (P<0.05) increased in glutinous
(TDK8 and TDK11) varieties and significantly (P<0.05) decreased in non-glutinous (DG) variety
due to parboiling.
This shade conversion from milky white (fresh) to an amber color (parboiled) in glutinous rice is
possibly due to the diffusion of husk color into the endosperm (Lamberts et al. 2006). The
chemistry of soaking medium had a significant effect on the color of the parboiled rice. Without
∗
soaking, control soaking and NaCl soaking had significantly (P<0.05) higher ∆𝐸𝑎𝑏
values than
acetic acid soaking in glutinous (TDK8 and TDK11) varieties possibly due to the bleaching effect
of acetic acid. Acetic acid acts as a weak inhibitor of browning due to its metal-chelating
characteristics by lowering the pH and can also interact with phenols in rice (Son et al. 2001).
133
Figure 8.1 Head rice yield (%) and grain hardness (N) of fresh and parboiled rice grains;
(a) Thadokkham-8 (TDK8), (c) Thadokkham-11 (TDK11), and (d) Doongara (DG).*
Correlation (r) between head rice yield (%) and grain hardness (N) in; (b) TDK8, (d),
TDK11, and (f) DG
*Means ± SD (n = 3). Within figure, significant differences are denoted by lowercase
letters for head rice yield and uppercase letters for grain hardness at 5 % probability level.
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Table 8.1 CIEL*a*b* color space of fresh and parboiled rice grains of Thadokkham-8
(TDK8), Thadokkham-11 (TDK11), and Doongara (DG)*
Rice
Treatment
L*
a*
b*
Fresh
96.75±0.25a
-0.96±0.02a
0.55±0.01a
∆𝑬∗𝒂𝒃
Without soaking
89.00±1.00bc
-0.03±0.02b
11.23±0.33c
13.25±1.01bc
Water (control) soaking
86.50±0.50d
0.47±0.03c
11.85±0.45c
15.32±0.84a
3 % NaCl soaking
87.50±0.35cd
0.82±0.03d
11.62±0.59c
14.33±0.07ab
0.2 % acetic acid soaking
89.50±0.50b
1.58±0.13e
9.61±0.11b
11.88±0.52c
96.40±0.40a
-0.97±0.02a
0.56±0.02a
-
Without soaking
93.60±0.40b
0.15±0.03b
8.40±0.60b
8.44±0.27c
Water (control) soaking
87.84±0.41d
1.41±0.10d
12.81±0.31d
15.14±0.74a
3 % NaCl soaking
90.52±0.32c
1.03±0.04c
11.57±0.32c
12.64±0.34b
0.2 % acetic acid soaking
90.75±0.30c
0.89±0.09c
10.65±0.33c
11.71±0.36b
Fresh
93.19±0.19a
1.20±0.20a
7.25±0.25a
-
Without soaking
89.55±0.55c
0.94±0.09ab
7.90±0.30a
3.73±0.44b
Water (control) soaking
91.50±0.50b
0.16±0.02c
10.19±0.39b
3.56±0.62b
3 % NaCl soaking
88.50±0.27d
0.85±0.14b
11.94±0.40c
6.68±0.12a
0.2 % acetic acid soaking
92.33±0.33ab
0.20±0.02c
13.12±0.32d
6.02±0.08a
variety
TDK8
TDK11 Fresh
DG
CIEL*a*b* color space
-
*Means ± SD (n = 3). For a particular rice variety, means with different letters in the same
column denote significant difference at 5 % probability level within each rice variety.
8.4.4. Pasting properties
The pasting profiles of fresh and parboiled rice flour of selected varieties are presented in Fig. 8.2.
Results showed the diverse behavior of glutinous (TDK8 and TDK11) and non-glutinous (DG)
samples due to parboiling. Parboiling significantly (P<0.05) reduced the pasting temperature in
both glutinous flours except without soaking parboiling, where pasting temperature significantly
(P<0.05) increased as compared to the fresh flour. However, pasting temperature of parboiled nonglutinous (DG) was increased as compared to fresh flour.
135
Figure 8.2 Pasting profiles of fresh and parboiled rice grains of Thadokkham-8 (TDK8),
Thadokkham-11 (TDK11), and Doongara (DG)
Moreover, significant (P<0.05) increase in peak and final viscosities was recorded in parboiled
glutinous flours which were significantly (P<0.05) decreased in non-glutinous flour when
compared to fresh flours. This variation in the pasting profiles of glutinous and non-glutinous rice
after parboiling may be due to increased starch damage owing to parboiling in DG than TDK8 and
TDK11, resulting in resistance of starch granules to absorb water and swell during RVA analysis.
Dutta and Mahanta (2012) reported that the parboiled rice varieties (Ranjit and Kola Chowkua)
with higher amylose content showed reduced pasting profiles than glutinous rice varieties (Aghoni
bora and Bhogali bora) possibly due to an extensive breakdown of straight chain amylose during
parboiling (Mir & Bosco 2013). Among various parboiling treatments, saline soaking induced
significant swelling of starch, resulting in high peak and final viscosities than water (control) in all
samples. This might be due to the inter- or intra-molecular cross-linking of Na+ ions with the
amylopectin during parboiling (Samutsri and Suphantharika 2012), resulting in increased water
absorption and viscous gels during RVA analysis. However, acetic acid soaking restricted the
136
swelling leading to reduced peak and final viscosities. Acetic acid soaking before parboiling may
have degraded the amorphous regions (Ohishi et al. 2007), resulting in reduced water absorption
and starch swelling.
8.4.5. Crystallinity change in starch
The X-ray diffraction spectra of fresh and parboiled rice flour of rice samples are shown in Fig.
8.3. The fresh flour samples of both glutinous (TDK8 and TDK11) and non-glutinous (DG)
varieties displayed a typical A-type pattern with main crystalline peaks at 14.9°, 16.9°, 18° and
22.8° and crystallinity was recorded as 25.95, 22.62 and 18.79 %, respectively.
Figure 8.3 X-ray diffraction and calculated crystallinity (%) of fresh and parboiled rice
grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11), and Doongara (DG) as
shown on the right side of the figures*
*Means ± SD (n = 3). Within each variety, means with different letters in the same figure
denote significant difference at 5 % probability level.
137
Parboiled TDK8 in all soaking medium was almost completely amorphous, and no A-type pattern
from residual starch was detected. However, a weak A-type pattern was noted in parboiled TDK11
and DG with significant (P<0.05) decrease in crystallinity as compared to untreated samples. The
variation in the crystallinity of parboiled TDK8 and TDK11 is possibly due to the difference in the
amylopectin chain lengths. It has been reported that external and inter-block chain lengths
correlated with the retrogradation of amylopectin (Bertoft et al. 2016). Very short external chains
(DP 6-8) prevent retrogradation (Vamadevan & Bertoft 2018), resulting in no crystalline region
during XRD analysis. However, further studies on the amylopectin chain lengths of TDK8 and
TDK11 should be done in the future to get a clearer picture.
The gelatinization temperature of starch of untreated rice flour samples was recorded as ~65°C for
glutinous (TDK8 and TDK11) and ~75°C for non-glutinous (DG) (Fig. 8.2), therefore, almost
complete gelatinization has occurred during steaming process of parboiling, resulting in complete
disappearance of crystalline peaks in TDK8 and weak peaks in TDK11 and DG. The appearance
of weak peaks could be attributed to the retrogradation of amylopectin in TDK11 and amylose in
DG. Among various soaking medium before parboiling, acetic acid and saline soaking affected the
residual starch more than water (control) and without soaking, resulting in significant (P<0.05)
decrease in crystallinity.
8.4.6. Thermal properties
The thermal (gelatinization and retrogradation) properties of fresh and parboiled rice flour of rice
samples are presented in Table 8.2. Parboiling resulted in significant (P<0.05) decrease in thermal
transition temperatures (To, Tp, and Tc) and enthalpy (ΔH) of gelatinization than fresh flour in
glutinous (TDK8 and TDK11) and non-glutinous (DG) flours. Moreover, the retrogradation
thermal temperatures (To(r), Tp(r), and Tc(r)) and enthalpy (ΔH(r)) was also significantly (P<0.05)
reduced in parboiled DG and without soaking parboiled TDK8 and TDK11. Interestingly, no
retrogradation peaks were detected in water (control), saline and acetic acid soaking parboiled
TDK8 and TDK11. However, significant (P<0.05) reduction in ΔH(r) was recorded in without
soaking parboiled samples led to significant (P<0.05) reduction in the percentage of retrogradation
(R %). Results showed that the parboiled glutinous rice samples made without soaking might
contain the residues of ungelatinized starch, resulting in the melting enthalpy of gelatinization and
138
retrogradation (Sittipod & Shi 2016). The gelatinization endotherms are in agreements with the
XRD spectra (Fig. 8.3), where a significant reduction in the crystallinity was recorded.
8.4.7. Textural profile analysis
Textural profiles of cooked fresh and parboiled grains of glutinous (TDK8 and TDK11) and nonglutinous (DG) are shown in Fig. 8.4. Parboiling resulted in significant (P<0.05) increase in
hardness (N) and adhesiveness (N.s) when compared to the freshly cooked samples for all
varieties. Parboiling results in several physicochemical changes such as disruption of surface
layers and leaching of components from cells. This cells disruption and leaching of cellar
components possibly facilitate more damaged starch exposure on the surface, resulting in the
increased stickiness of cooked parboiled glutinous rice (Ong & Blanshard 1995).
Among various parboiling treatments, saline soaking exhibited the hardest texture followed by
acetic acid, water (control) and without soaking. Interesting, parboiling also resulted in significant
(P<0.05) increase in adhesiveness (N.s) especially in glutinous (TDK8 and TDK11) varieties.
Chemistry of soaking medium significantly (P<0.05) affected the adhesiveness in TDK8 as shown
in Fig. 8.4, where adhesiveness of acetic acid parboiled rice was significantly (P<0.05) higher than
saline, water (control) and without soaking. However, soaking medium had no significant (P>0.05)
effect on the adhesiveness of TDK11 and DG. The TPA results are in agreement XRD analysis
(Fig. 8.3), where no crystalline region was found in parboiled TDK8, and a weak A-type crystalline
pattern was found in parboiled TDK11 and DG.
8.4.8. GI prediction
The results of estimated GI using in vitro digestion of 180 min is presented in Table 2. As expected,
the predicted GI of glutinous rice varieties viz. TDK8 and TDK11 was higher than non-glutinous
(DG) rice variety. Moreover, parboiling resulted in significant (P<0.05) decrease in GI in both
TDK8/TDK11 and DG. Gelatinization and recrystallization are the major physicochemical
changes that occur during parboiling (Zavareze et al. 2010). These changes may lead to higher
levels of resistant starch, resulting in low glucose response and GI (Boers et al. 2015). The proteinstarch interaction may have restricted the starch digestibility, resulting in reduced GI (Kaur et al.
2016). However, saline soaked parboiled glutinous rice showed significantly (P<0.05) higher GI
than fresh. NaCl might have increased the postprandial plasma glucose by accelerating the
139
digestion of starch especially amylopectin by stimulating α-amylase activity (Thorburn et al.
1986).
Figure 8.4 Textural profile analysis of fresh and parboiled rice grains of Thadokkham-8
(TDK8) and Doongara (DG)*
*Means ± SD (n = 3). Within each variety, significant differences are denoted by
lowercase letters for hardness and uppercase letters for adhesiveness at 5% probability
level.
140
Table 8.2 Experimental results of estimated GI, and gelatinization and retrogradation properties of fresh and parboiled rice
grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11), and Doongara (DG)*
Rice
Treatment
Estimated
Gelatinization
Retrogradation
variety
GI
Tₒ (oC)
Tp (oC)
Tc (oC)
ΔH (Jg-1)
Tₒ(r) (oC)
Tp(r) (oC)
Tc(r) (oC)
ΔH(r) (Jg-1)
R (%)
TDK8
Fresh
116.2±0.2b
66.4±0.0a
73.7±0.4a
89.9±1.6a
10.4±0.5a
51.7±0.0a
58.5±0.5a
65.2±1.3a
0.2±0.0a
2.1±0.1a
flour
Without soaking
111.8±0.3c
50.6±0.4b
58.1±0.2b
62.6±0.2b
5.1±0.1b
40.6±0.4b
52.4±0.4b
60.3±0.2b
0.1±0.0b
2.0±0.1a
Water (control) soaking
108.0±1.0d
48.2±0.1c
56.1±0.4c
61.1±0.2b
3.5±0.2c
NDˆ
NDˆ
NDˆ
NDˆ
NDˆ
3 % NaCl soaking
119.2±1.0a
47.3±0.2d
54.4±0.4d
58.0±0.0c
3.5±0.0c
NDˆ
NDˆ
NDˆ
NDˆ
NDˆ
0.2 % acetic acid soaking
100.4±0.2e
45.8±0.2e
54.0±0.0d
57.4±0.3c
3.0±0.1c
NDˆ
NDˆ
NDˆ
NDˆ
NDˆ
TDK11
Fresh
105.9±0.2b
64.8±0.1a
71.0±0.2a
81.1±0.2a
9.1±0.2a
50.6±0.4a
57.5±0.4a
63.82±0.17a
0.20±0.01a
2.14±0.02a
flour
Without soaking
101.5±0.3c
49.5±0.5b
57.1±0.1b
61.1±0.2b
4.4±0.2b
40.1±0.1b
50.6±0.4b
59.06±0.17b
0.10±0.00b
2.15±0.00b
Water (control) soaking
97.7±1.0d
47.3±0.2c
54.4±0.4c
58.2±0.2c
3.6±0.0c
NDˆ
NDˆ
NDˆ
NDˆ
NDˆ
3 % NaCl soaking
108.9±1.0a
45.8±0.2d
53.7±0.3c
57.3±0.3d
3.1±0.1d
NDˆ
NDˆ
NDˆ
NDˆ
NDˆ
0.2 % acetic acid soaking
90.1±0.2e
43.4±0.4e
52.6±0.4d
56.4±0.3e
2.0±0.1e
NDˆ
NDˆ
NDˆ
NDˆ
NDˆ
DG
Fresh
94.8±1.4a
72.3±0.7a
77.1±0.5ab
83.0±1.5a
3.4±0.0a
43.7±0.2a
56.4±0.0a
62.9±0.2a
2.0±0.0a
59.2±0.0a
flour
Without soaking
84.3±0.5b
70.9±0.2b
77.6±0.3a
82.6±0.2a
3.1±0.1b
43.1±0.1b
55.9±0.1b
61.8±0.1b
1.6±0.1b
51.8±1.3b
Water (control) soaking
78.4±0.8c
70.1±0.1bc
76.8±0.1bc
81.5±0.1a
3.0±0.0c
42.9±0.1b
55.5±0.1c
60.8±0.0c
1.4±0.0c
48.9±1.5c
3 % NaCl soaking
85.6±1.4b
70.1±0.1c
76.1±0.1cd
78.7±0.2b
2.4±0.1d
41.0±0.1c
54.9±0.1d
59.9±0.0d
1.1±0.0d
45.7±0.3d
0.2 % acetic acid soaking
72.2±0.8d
69.9±0.1c
75.5±0.1d
77.6±0.2b
1.9±0.1e
39.9±0.1d
54.0±0.1e
59.4±0.2e
0.9±0.0e
44.9±1.2d
ˆNot detected.
*Means ± SD (n = 3). For a particular rice variety, means with different letters in the same column denote significant
difference at 5 % probability level within each rice variety.
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8.5. Conclusions
This study showed that the soaking medium affected the physicochemical properties of glutinous
(TDK8 and TDK11) rice varieties. Milling efficiency of glutinous rice was improved by adding
NaCl and acetic acid to the soaking water of paddy before parboiling. Induced coloration from the
husk during steaming can be minimized by the bleaching effect of acetic acid used during soaking.
Saline soaking can also improve water absorption of parboiled rice. NaCl and acetic acid affected
the residual starch, resulting in reduced crystalline regions which may be responsible for more
adhesiveness during textural profile analysis. The parboiling of rice significantly reduced the
glycemic index which may be due to more resistant starch contents. This should be further
investigated. Current findings showed the potential of parboiling with modified soaking techniques
to improve grain quality of glutinous rice.
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Chapter 9 Effects of three types of modified atmospheric packaging on the
physicochemical properties of selected glutinous rice
This chapter has been published in the Journal of Stored Products Research;
Nawaz, MA, Fukai, S, Prakash, S & Bhandari, B 2018, ‘Effects of three types of modified
atmospheric packaging on the physicochemical properties of selected glutinous rice’, Journal
of Stored Products Research, vol. 76, pp. 85-95.
143
9.1. Abstract
The effect of modified atmospheric packaging (MAP) on the physicochemical properties of
selected glutinous (Thadokkham-8 and Thadokkham-11) rice was studied and compared with nonglutinous rice (Doongara). The freshly harvested/milled grains were packed in four different MAP
conditions viz. control, vacuum, CO2, and N2 for 12 months at room temperature (23±1ºC). Gas
(N2 or CO2) was flushed in aluminum bags at the pressure of 300 kPa for 3 sec and subsequently
hermetically sealed. Vacuum packaging was done at -100 kPa. Results showed that aging-induced
changes in the starch granules were less prominent in a vacuum and/or MAP samples using CO2
or N2. Surface analysis showed that control storage significantly reduced the percentage of lipids
and increased the percentage of proteins on the surface in all selected varieties. N2 and CO2 storage
of TDK8 and DG slowed down the shift of properties of macromolecules and maintained the
surface starch/proteins/lipids ratios during 6 months of storage. Moreover, the grains stored in a
vacuum maintained the lipids with a lower proportion of proteins exposed to the surface after
cooking. N2 and CO2 induced increase in pasting temperature but significant reduction in final
viscosity when compared to control. The findings correlated well with thermal analysis. The in
situ Thermal Mechanical Compression Test (TMCT) device cooking and texture analysis revealed
that modified storage slightly slowed the aging-induced changes in the cooking quality and
stickiness of glutinous rice. Among all storage conditions used vacuum was relatively the best to
maintain the quality of the grain.
9.2. Introduction
The significance of food storage especially for cereals such as rice and wheat has become
important because of frequent natural disasters such as floods, drought, and earthquakes to ensure
food security (Tulyathan & Leeharatanaluk 2007). Rice being the primary staple food of more than
half of the world’s population requires adequate storage with preservation of its eating quality to
ensure uninterrupted supply to end users all year round (Thongrattana 2012). Storage of rough and
milled rice undergoes a wide range of physicochemical changes on the characteristics of rice such
as pasting properties (Ziegler et al. 2017), thermal properties (Faruq et al. 2015; Ziegler et al.
2017), and textural changes in the cooked rice over a time period (Nawaz et al. 2017; Zhou et al.
2007b; Keawpeng & Venkatachalam 2015; Jungtheerapanich et al. 2017).
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Age-related effect on the cooking quality of rice has been obvious to the consumers. Aging is very
complex process and attributed to several changes (Zhou et al. 2015) in cell walls and proteins
(Zhou et al. 2002a; Sodhi et al. 2003), starch/protein interaction (Tananuwong & Malila 2011),
and oxidation of lipids, resulting in breakdown of products (Park et al. 2012). Loss of stickiness
is one of the most noticeable changes occurring in glutinous rice (Chen et al. 2015). Several factors
are responsible for this physicochemical deterioration. Previous studies have demonstrated the
strong association of aging and endogenous enzymatic reactions on rice starch (Zhou et al. 2003a;
Huang & Lai 2014), proteins (Zhou et al. 2003a) and lipids (Zhou et al. 2003b). Huang and Lai
(2014) reported the effects of endogenous amylase on the isolated starch fine structures of aged
waxy rice varieties (TNW1 and TCSW1) and reported that the structural changes in the starch fine
structures with a decreasing percentage of longer chains and an increasing percentage of shorter
chains of amylopectin when rice was stored for a longer period (15 months) of time. Although, the
studies on rice protein suggested that there was no significant difference in the gross protein
contents of fresh and aged rice (Zhou et al. 2002a), storage in ambient conditions could induce
oxidation reaction of proteins, resulting in the formation of intermolecular disulphide bonds (S-S)
(Teo et al. 2000). The polymerization of rice proteins due to intermolecular disulfide bond
formation can increase the average molecular weight of rice storage proteins especially oryzenin
(Zhou et al. 2007a). The formation of bigger protein bodies in stored rice can lead to increased
final viscosity during rapid visco analysis and reduced stickiness of cooked waxy rice (Ohno &
Ohisa 2005; Zhou et al. 2007b). In addition to this, previous studies (Shin et al. 1986; Park et al.
2012) have reported that there was no significant change found in the fatty acids (both unsaturated
and saturated) in the propanol-water extractable lipid (PWE-L) fraction between fresh and stored
rice grains. However, unsaturated fatty acids fractions in the petroleum ether extractable lipid
(PEE-L) of aged rice grains was significantly (P<0.05) lower than that of fresh rice grains (Zhou
et al. 2015). This suggests that the unsaturated fatty acids in the PEE-L fraction are more unstable
and could be more responsible for the oxidation than those in PWE-L fraction during storage
(Tsuzuki et al. 2014). It is now quite well defined that the lipid peroxidation during storage can
cause quality deterioration of rice grains, especially, lipoxygenase-3 (LOX-3), which is one of the
key enzymes catalyzing the reaction (Shin et al. 1985; Hildebrand 1989; Suzuki & Matsukura
1997). The above studies suggest that an efficient direction for improving the storage property of
the rice grains can be achieved through proper storage environment.
145
To slow down aging-induced physicochemical changes in stored rice, storage at chilling
temperatures (at or below 8oC, targeting 5oC) is usually reported as the best method (Pearce et al.
2001). However, cold storage is not cost effective due to initial capital investment for cooling
system installation, high energy consumption during its operation, and also expensive equipment
maintenance (Evans et al. 2014). Also, the suggested method cannot be effectively applied in the
developing nations facing an energy shortage. Thus, modified atmospheric packaging of rice can
be the best option to reduce aging-induced physicochemical changes in ambient temperature
conditions and maintain the cooking quality.
In modified atmospheric packaging (MAP), extra nitrogen (N2) or carbon dioxide (CO2) is added
to alter the ratio of oxygen (O2), N2, and CO2 (Caleb et al. 2012). Altered ratio of O2, N2, and CO2
in the micro-environment of the food product can maintain the physical and cooking quality of
aged food product by slowing down the physicochemical changes such as the speed of oxidation
reactions (Caleb et al. 2013) and other physicochemical changes. The MAP of fresh fruits and
vegetables is quite well-established technique mainly focusing on the prolonging shelf life by
reducing the respiration rate and microbial growth (Oliveira et al. 2015; Shaarawi & Nagy 2017).
However, to the best of our knowledge MAP of rice has not been widely reported by researchers
due to its least susceptibility towards microbial spoilage, having a relatively long shelf life and less
importance given to the cooking quality of rice.
Good quality glutinous rice grains usually lose their shape during cooking and become very sticky.
However, storage at ambient temperature induces physicochemical changes in glutinous rice,
resulting in significant reduction in the stickiness during cooking. As mentioned earlier, although
aging-induced changes can be minimized by storing the rice in the refrigerated condition, this
method is not feasible due to the cost factor and large volume of the grains. Therefore, the objective
of the present study is to investigate the effect of altered atmospheric (vacuum, N 2, and CO2)
storage on the functional properties of selected glutinous rice varieties. For this, the milled rice
grains of two glutinous rice varieties viz. Thadokkham-8 (TDK8) and Thadokkham-11 (TDK11)
were selected and stored in four different modified atmospheric conditions for one year. For
comparison purpose, one non-glutinous rice variety (Doongara (DG)) was also milled and stored
in the same condition. This modified storage is expected to maintain the functional quality of aged
146
glutinous rice grains stored at ambient temperature by reducing the rate of physicochemical
changes in rice, in particular, the loss of stickiness.
9.3. Material and methods
Two Oryza sativa indica cultivars of glutinous rice from Lao PDR viz. Thadokkham-8 (TDK8)
and Thadokkham-11 (TDK11) having 3.77 % and 3.72 % apparent amylose contents (AAC),
respectively and one O. sativa japonica non-glutinous rice from Australia (Doongara (DG), 19.71
% (AAC)) were used in this study. The milled TDK8 with 9 % degree of milling (DOM) (harvested
March/April 2015) was provided by the National Agriculture and Forestry Research Institute
(NAFRI), Lao PDR, while TDK11 and DG (harvested March/April 2015) were provided by Rice
Research Australia Pty Ltd (RRAPL), Mackay, QLD, Australia.
9.3.1. Paddy milling
TDK11 and DG paddies were milled to brown rice by using a rice husker (Satake, Japan). The
brown rice of both cultivars was milled to white rice using an abrasive polisher (Satake, Japan).
The DOM of both TDK11 and DG was maintained at 9 % to be consistent with milled TDK8
provided by NAFRI, Lao PDR.
9.3.2. Modified atmospheric packaging
The milled rice kernels (250 g per bag) of TDK8, TDK11, and DG were packed in sealed
aluminum bags (West’s Packaging Services, Melbourne, VIC, Australia) using various modified
atmospheric conditions viz. vacuum, N2, CO2 by using a tabletop vacuum chamber machine
equipped with gas flushing system; Vacumatic 282 (Vacumatic Australia Pty Ltd, Pakenham, VIC,
Australia). N2 and CO2 were flushed in aluminum bags at a pressure of 300 kPa for 3 sec and
subsequently hermetically sealed. Similarly, vacuum packaging was done at -100 kPa. Moreover,
one control (MAPctrl) without any treatment was also kept in sealed aluminum bags for
comparison. The control and MAP packets of rice grains of selected rice varieties were stored at
room temperature (23±1ºC). The experimental design of the present study is presented in Appendix
7.
147
9.3.3. Scanning electron microscopy of a cross-section of rice kernels
The cross sections of milled rice kernels of fresh, 6 and 12 months aged TDK8, TDK11, and DG
stored in the various MAP were mounted onto SEM stubs by placing them on a double-sided
carbon adhesive tape. Biological materials suffer from extensive charge build-up under the
electron beam; hence they need to be coated with conductive material. Thus, samples were iridiumcoated for 3 min (~ 15 nm thick). The samples were examined using a Philips XL30 Field Emission
Scanning Electron Microscope operating at 8 kV accelerated voltage.
9.3.4. Surface chemical analysis
The surface chemical analysis of pure rice components (starch, proteins, and lipids), milled
uncooked rice kernels, and freeze-dried cooked rice kernels of fresh, 6 and 12 months aged TDK8,
TDK11, and DG stored in various MAP were analyzed by using a Kratos AXIS Ultra Kratos
Analytical (Manchester, UK) spectrometer with a monochromatic Al X-ray source at 150 W. This
method follows the procedure previously used by our research group (Nawaz et al. 2016b). Before
analysis, the samples were outgassed under vacuum for 24 hrs. The analysis started with a survey
scan from 0 to 1200 eV with a dwell time of 100 ms, pass energy of 160 eV at steps of 1 eV, with
a single sweep. For the high-resolution analysis, the number of sweeps was increased, the pass
energy was lowered to 20 eV, at steps of 50 meV, and the dwell time was increased to 250 ms.
Data was acquired using a Kratos Axis ULTRA X-ray photoelectron spectrometer, incorporating
a 165 m hemispherical electron energy analyzer. The incident radiation was Monochromatic Al
X-rays (1486.6 eV) at 225 W (15 kV, 15 ma). Survey (wide) scans were taken at analyzer pass
energy of 160 eV, and multiplex (narrow) higher resolution scans at 80 eV. Base pressure in the
analysis chamber was 1.33 x 10-7 Pa and, during sample analysis, 1.33 x 10-6 Pa. XPS was applied
to measure the relative atomic concentrations of carbon, nitrogen, and oxygen in the layer of the
samples to a maximum thickness of 10 nm.
The surface composition of samples was calculated by using the relative elemental compositions
of samples and pure rice components in a set of linear relations in a matrix formula according to
the method developed by Fäldt et al. (1993). The matrix formula was adjusted by
components/macromolecules to be analyzed. In the current study, the calculations were made by
using a matrix, as presented in equations 9.1, 9.2, and 9.3 for the three rice components.
148
𝐼 𝐶 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐼 𝐶 𝑠𝑡𝑎𝑟𝑐ℎ 𝛾𝑠𝑡𝑎𝑟𝑐ℎ + 𝐼 𝐶 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 𝛾𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 + 𝐼 𝐶 𝑙𝑖𝑝𝑖𝑑𝑠 𝛾𝑙𝑖𝑝𝑖𝑑𝑠
Eq. 9.1
𝐼 𝑂 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐼 𝑂 𝑠𝑡𝑎𝑟𝑐ℎ 𝛾𝑠𝑡𝑎𝑟𝑐ℎ + 𝐼 𝑂 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 𝛾𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 + 𝐼 𝑂 𝑙𝑖𝑝𝑖𝑑𝑠 𝛾𝑙𝑖𝑝𝑖𝑑𝑠
Eq. 9.3
𝐼 𝑁 𝑠𝑎𝑚𝑝𝑙𝑒 = 𝐼 𝑁 𝑠𝑡𝑎𝑟𝑐ℎ 𝛾𝑠𝑡𝑎𝑟𝑐ℎ + 𝐼 𝑁 𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 𝛾𝑝𝑟𝑜𝑡𝑒𝑖𝑛𝑠 + 𝐼 𝑁 𝑙𝑖𝑝𝑖𝑑𝑠 𝛾𝑙𝑖𝑝𝑖𝑑𝑠
Eq. 9.2
Where ICstrach, INstrach, IOstarch, ICproteins, INproteins, IOproteins, IClipids, INlipids, and IOlipids are the relative
contents of atomic elements (C, N, and O) measured on the surface of the pure rice components,
presented in Table 5.2 in Chapter 5. ICsample, INsample, and IOsample are the relative contents of elements
found by XPS for the sample. The parameters γstarch, γproteins, and γlipids are unknown values
corresponding to approximately 100 % component/macromolecules surface contents (starch,
proteins, and lipids).
9.3.5. X-ray diffraction
The milled kernels of fresh, 6 and 12 months aged TDK8, TDK11, and DG stored in the various
MAP were ground to flour using a disc mill (Good Friends of the Guangzhou Machinery Co. Ltd.,
Guangzhou, China) according to the method previously reported by Nawaz et al. (2016b). The
flour particles were sieved through 500 µm sieve to attain particle size ≤ 500 µm. X-ray diffraction
pattern measurement of rice flour was analyzed by using Bruker Advance D8 X-Ray
diffractometer equipped with a LynxEye detector and Cu-kα (1.54 Å) radiation. The accelerating
voltage and current of 30 kV and 30 mA, respectively, in combination with scan rate 2o/min, were
used. The diffractograms were recorded in a 2θ ranged from 10o to 35o with sampling width of
0.02o. Traces were analyzed using the Diffractplus Evaluation Package Release V3.1, PDF-2
Release 2014.
The percentage of crystallinity was calculated with normalized values of the intensities at each
diffraction angle, using the method of Htoon et al. (2009). The ratio of the upper diffraction peak
area taken as the crystalline portion, to total diffraction area (two-phase model), represented the
percentage of crystallinity. The diffractograms were smoothed by 13 points using Traces version
3.01 software (Diffraction Technology Pty LTD, Mitchell, ACT, Australia) before calculating the
percentage of crystallinity.
9.3.6. Pasting properties
Pasting properties of fresh and aged rice (6 and 12 months under various MAP) flour (particle size
≤ 500 µm) of all three rice varieties were determined according to the AACC International Method
149
61-02.01 using a Rapid Visco Analyzer (RVA-4 model Thermocline Windows Control and
analysis software, Version 1.2 (New Port Scientific, Sydney, Australia)) (AACC 1999). To
calculate the sample size for RVA, the moisture contents of all the flour samples were measured
according to the AACC International Method 44-40.01(AACC 1999) by using a vacuum oven.
Rice flour (3.01 g, 12.4 % moisture basis) was mixed with 25.0 g MilliQ water in the RVA canister.
A programmed heating and cooling cycle were used, the samples were held at 50oC for 1 min,
heated to 95oC in 3.45 min, held at 95oC for 2.7 min before cooling to 50oC in 3.91 min and holding
at 50oC for 1.24 min. Pasting temperature (Ptemp), Peak viscosity (Vp), Trough viscosity (Vt),
Breakdown (BD), Final viscosity (Vf) and Setback (SB) were recorded.
9.3.7. Gelatinization and retrogradation properties
Differential Scanning Calorimeter (DSC) (Mettler Toledo, Schwerzenbach, Switzerland) with
internal coolant and nitrogen/air purge gas was used to determine the gelatinization characteristics
of rice flours (particle size ≤ 500 µm) of fresh and aged rice (6 and 12 months under the various
MAP). The DSC was calibrated for the heat flow using indium as standard. Rice flour (approx. 4
mg, dry weight basis) was weighed into an aluminum pan and 6 µL MilliQ water was added. The
pan was hermetically sealed and equilibrated at room temperature for 30 min, then scanned at the
heating rate of 15ºC/min from 0 to 100ºC with the empty sealed pan as a reference. The onset (To),
peak (Tp) and conclusion (Tc) temperatures, and enthalpy (ΔH) of gelatinization were determined
by Stare Software Version 9.1 (Mettler Toledo).
After cooling, the scanned samples pans were placed in a refrigerator at 4±1ºC for 7 days.
Retrogradation properties were measured by rescanning these samples at the rate of 15ºC/min from
0 to 100ºC. The onset (To(r)), peak (Tp(r)) and conclusion (Tc(r)) temperatures, and enthalpy of
retrograded starch (ΔH(r)) were determined. The percentage of retrogradation (R %) was calculated
according to equation 9.4.
𝑅% = [
𝛥𝐻(𝑟)
⁄ ] × 100
𝛥𝐻
Eq. 9.4
9.3.8. Textural profile analysis
Fresh and aged (6 and 12 months under various MAP conditions) milled rice grains (5 g) were
added in 15 mL of MilliQ water (rice to water ratio =1:3) in 50 mL glass beaker. The beaker with
150
the rice and water was placed in a water bath at 95±1ºC. Cooking was continued until there was
no ungelatinized white belly observed in rice kernel cross section (data not shown). Analysis of
textural attributes was performed on a TA-XTplus Texture Analyzer (Stable Microsystems, UK)
using 35-mm circular probe as previously described (Nawaz et al. 2016c). Three cooked grains
were placed on the flat stage, and the textural property was determined. The texture analyzer
settings were as follows: pre-test speed, 2.00 mm/sec; post-test speed, 2.00 mm/sec; distance, 2.00
mm; time, 10.00 sec; (auto) trigger force, 0.05 N. From the force-time curve obtained, textural
attributes of hardness (height of the force peak on cycle 1, N) and adhesiveness (negative force
area of the first cycle, N.s) were computed using the EXPONENT Stable Micro Systems software
supplied with instrument. The textural values reported are the averages of three different
determinations.
9.3.9. In situ TMCT cooking analysis
The in situ rate of water absorption, grain softening and gelatinization time during cooking of fresh
and aged milled rice kernels was analyzed by using a temperature controlled aluminum block
(50x50x25 mm) attached to a texture analyzer; TA-XTplus Texture Analyzer (Stable
Microsystems, UK) according to the optimized method reported by Nawaz et al. (2017). The
experiment assembly is shown in Fig. 4.1 in Chapter 4. The sample block was heated to a rice
cooking temperature of 95±1ºC and held it at this temperature. A single layer of 0.5 g of rice
kernels and 1.5 mL of deionized water (rice to water ratio = 1:3) was put on the sample block. The
soaked samples were compressed by a 35 mm Teflon probe at a steady force of 0.15 N attached to
the texture analyzer. The texture analyzer recorded the change in distance of probe until rice
sample has taken up all available water and fully cooked (indicated by the vibration in probe due
to the evaporation of water from the cooked grain). The cooking rate of the grains was estimated
by measuring the slope of the initial linear part of the in situ Thermal Mechanical Compression
Test (TMCT) cooking curves.
9.3.10. Statistical analysis
All treatments were replicated three times and the data presented are their mean values. The
reported data for the surface chemical composition of raw and cooked rice kernels, X-ray
diffraction, pasting properties, gelatinization and retrogradation, textural profile analysis, and in
situ TMCT cooking analysis for each variety was analyzed separately by analysis of variance using
151
Minitab R17 (Minitab® for Windows Release 17, Minitab Inc, Chicago, USA) in order to
determine significant differences. The data was then analyzed using Tukey’s pair-wise
comparison, at 5 % level of significance, to compare the different treatments.
9.4. Results and discussion
9.4.1. Results
9.4.1.1. Microstructures of fresh and aged rice kernels
The microstructures of fresh and aged (6 and 12 months under various MAP conditions) rice
kernels of selected glutinous (TDK8 and TDK11) and non-glutinous (DG) rice varieties are
presented in Fig. 9.1. Scanning electron micrographs of fresh rice kernels have shown that the
endosperm was mainly packed with polyhedral starch granules with an average size of 6-7 µm in
TDK8 and TDK11 and 8-10 µm in DG.
Figure 9.1 Scanning electron micrographs (SEM) of cross sections of fresh and aged (6
and 12 months under various MAP conditions) milled rice grains of Thadokkham-8
(TDK8), Thadokkham-11 (TDK11) and Doongara (DG). Scale bar on each image = 5 µm
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9.4.1.2. Surface composition of uncooked and cooked grains of fresh and aged rice
The relative elemental and calculated macromolecules (starch, proteins, and lipids) of the upper
10 nm surface layer of uncooked and cooked fresh and aged (6 and 12 months under various MAP
conditions) rice grains of selected glutinous (TDK8 and TDK11) and non-glutinous (DG) rice
varieties are presented in Table 9.1 and Table 9.2. XPS spectra showed that the surface
composition of rice kernels of selected rice varieties mainly consisted of C, N, and O. The surface
of uncooked grains of fresh glutinous rice varieties (TDK8 and TDK11) had more lipids (52.5 %
and 57.7 %, respectively) followed by proteins (34.2 % and 41.1 %, respectively) and less
concentration of starch (13.3 % and 1.2 %, respectively). The surface of non-glutinous uncooked
fresh DG rice grains showed more lipids (51.7 %) followed by starch (25.2 %) and proteins (23.1
%) as shown in Table 9.1. The control storage conditions induced significantly (P<0.05) higher
concentration of surface proteins and reduced surface lipids in all selected rice varieties (Table
9.1).
The XPS spectra of cooked rice grains showed diversity among all rice varieties. Three peaks
corresponding to C, N, and O were detected in TDK8. However, four peaks corresponding to C,
N, O, and Mn were detected in TDK11 as shown in Table 9.2. In addition to this, five peaks
corresponding to C, N, O, B, and Si were detected in DG. The relative elemental composition for
C, N, and O of cooked rice samples did not fit in the matrix formula developed for dry samples
due to the complex nature of gelatinized starch, denatured proteins, and starch/proteins/lipids
interaction. Therefore, high-resolution XPS scans for C1s, N1s, and O1s peaks of cooked fresh and
aged rice grains of selected rice varieties were conducted. The deconvolution of C1s showed six
distinct sub-peaks (C-C, C-COOH, C-N, C-O, C=O, and O-C=O) in all samples, corresponding to
various macromolecules such as polysaccharides, proteins or lipids side chains. The XPS survey
and high-resolution scans of fresh cooked TDK8, TDK11, and DG are presented in Appendix 8, 9
and 10, respectively. Moreover, the calculated values for C/O and C/N stoichiometry for cooked
rice samples were also calculated and compared with C/O and C/N stoichiometric values of pure
rice starch, rice protein and rice lipid (presented in Table 5.2 in Chapter 5).
In the present study, the calculated C/O and C/N stoichiometry of fresh cooked glutinous rice were
2.7 and 14.9, respectively for TDK8 and 3.6 and 12.4, respectively for TDK11 (Table 9.2).
However, the calculated C/O and C/N stoichiometry of fresh cooked non-glutinous rice (DG) were
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3.6 and 26.6, respectively (Table 9.2) indicating that lipids mainly covered the grain surface with
some proteins. The deconvolution of C1s of fresh and aged cooked TDK8 stored under various
MAP conditions for 6 and 12 months showed that the modifications in the storage environment
might lead to significant (P<0.05) variations in the surface of the cooked rice.
Table 9.1 Relative elemental and calculated surface composition (%) of uncooked rice
kernels of fresh and aged (6 and 12 months under various MAP conditions) milled rice
grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11) and Doongara (DG)*
Rice
Aging
MAP
Elemental atomic percentage (%)
Surface composition (%) after using
variety
matrix formula
C
TDK8
Fresh
-
6 months
12 months
12 months
12 months
2.8±0.1
ab
14.8±0.1
13.3±0.4
52.5±2.8a
ab
41.0±2.3
43.2±3.4b
34.2±3.2
16.7±0.4c
16.7±0.8b
42.9±1.7ab
40.4±2.5b
N2
81.6±0.3b
2.6±0.4a
15.9±0.1b
15.5±2.0ab
38.4±6.5a
46.2±4.5ab
c
a
cd
d
20.5±0.5
a
34.0±2.1
45.5±2.6ab
CO2
80.5±0.5
control
78.3±0.5d
4.0±0.4d
17.7±0.1ce
15.3±1.2ab
60.5±5.8d
24.2±4.7c
vacuum
de
78.0±0.1
bc
f
d
21.0±0.3
50.1±1.2
bc
28.9±0.8c
N2
78.3±0.2de
3.9±0.2cd
17.8±0.1e
15.8±0.3ab
59.5±2.3cd
24.7±2.0c
e
cd
f
19.7±0.7
cd
cd
23.3±0.2c
3.4±0.0
3.8±0.1
17.2±0.3
17.1±1.2
bc
2.9±0.1ab
2.3±0.1
16.8±0.2
c
Lipids
a
80.5±0.5c
18.7±0.0
CO2
77.4±0.1
95.0
30.9
118.1
21.8
28.8
43.6
-
85.9±1.5a
2.6±0.6a
11.5±0.9a
1.2±0.3a
41.1±9.4a
57.7±9.7a
control
81.4±0.1abc
4.7±0.0bc
14.0±0.1ab
0.4±0.4a
73.8±0.1bc
25.9±0.3bcd
vacuum
81.6±0.5abc
5.0±0.2bc
13.4±0.7ab
0.1±0.1a
78.6±3.1c
24.0±0.3bcd
ab
bc
ab
a
bc
25.5±3.9bcd
70.3±6.8c
29.6±7.7bc
18.2±0.2
N2
81.7±0.2
4.9±0.4
CO2
82.0±1.3ab
4.5±0.4bc
13.6±0.9ab
0.3±0.5a
cde
c
cd
ab
5.4±1.0
13.5±0.1
9.9±12.4cd
80.4±3.5bcd
3.2±0.9ab
16.4±2.6bc
14.7±4.2b
47.8±14.2ab
37.5±18.4ab
N2
75.6±0.2de
5.6±0.5c
18.9±0.7cd
12.6±4.1b
85.4±7.8c
2.6±2.9d
e
c
d
b
78.2±13.5
c
7.0±9.4cd
CO2
74.6±2.3
11.6
7.4
16.4
13.0
7.4
10.3
-
80.6±0.3c
1.7±0.2c
17.8±0.1c
25.2±0.6c
23.1±3.1b
51.7±2.5ab
control
84.0±0.6a
2.7±0.1ab
13.4±0.6a
7.1±1.7a
40.7±0.6cd
52.2±2.2ab
vacuum
84.3±0.8a
2.5±0.3b
13.3±0.6a
7.7±0.7a
37.7±3.8c
54.7±4.5ab
a
ab
a
a
cd
53.3±4.5a
46.8±1.7d
47.3±1.0ab
ab
39.5±2.6c
N2
84.5±0.7
2.7±0.3
CO2
83.5±0.1a
3.0±0.1a
13.5±0.1a
f
cd
e
5.9±0.6a
vacuum
77.2±0.5e
1.0±0.0d
21.8±0.5e
40.9±1.5e
10.9±0.3a
48.2±1.7ab
d
c
d
d
b
45.5±1.2bc
78.7±0.0
1.8±0.1
CO2
81.9±0.3b
155.9
42.0±1.4
e
41.6±4.6
75.7±0.1
N2
22.8±0.1
5.1±0.1
control
F value
1.5±0.2
12.8±0.4
15.2±5.0
83.2±16.0
c
vacuum
20.2±1.4
6.7±5.9
76.4±5.7
76.4±3.1
5.1±0.9
18.2±2.0
0.0±0.0
57.1±1.0
control
Fresh
6 months
2.3±0.2
Protein
a
vacuum
F value
DG
c
Starch
a
80.5±0.0
Fresh
6 months
82.9±0.3
O
a
control
F value
TDK11
N
a
18.5±4.0
19.6±0.2
30.7±1.1
23.8±2.3
2.4±0.0b
15.8±0.3b
16.1±1.1b
34.7±0.5c
49.3±0.6ab
42.9
348.1
578.4
58.5
9.2
*Means ± SD (n = 3, df = 8). For a particular variety, means with different letters in the
same column denote significant difference at 5 % probability level within each rice variety.
154
Table 9.2 Relative elemental and calculated surface composition (%) of cooked rice kernels of fresh and aged (6 and 12
months under various MAP conditions) milled rice grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11) and Doongara
(DG)*
Rice
variety
Aging
TDK8
Fresh
6 months
TDK11
DG
MAP
control
vacuum
N2
CO2
12 months
control
vacuum
N2
CO2
F value
Fresh
6 months
control
vacuum
N2
CO2
12 months
control
vacuum
N2
CO2
F value
Fresh
6 months
control
vacuum
N2
CO2
12 months
control
vacuum
N2
CO2
F value
C
69.3±0.4a
61.8±1.6cde
60.9±0.7de
62.4±0.7cde
60.1±0.5e
63.4±1.0bcd
66.3±0.0b
64.2±2.1bc
62.6±0.8cde
22.2
73.5±1.8b
68.1±0.3c
81.6±0.5a
81.8±0.1a
82.0±1.3a
67.7±0.2cd
65.5±0.2cde
62.8±0.6e
64.8±2.3de
154.9
75.9±0.4abc
76.4±1.2ab
73.6±0.8abcd
70.7±1.2d
74.0±0.7abcd
76.9±2.0a
74.8±2.1abc
73.1±1.0bcd
72.7±0.4cd
7.6
C-C
35.0±1.0ab
28.1±6.6abc
18.3±3.6c
22.1±0.3bc
36.1±11.5a
25.9±0.3abc
29.5±0.5abc
33.1±0.5ab
34.1±1.1ab
5.3
52.3±0.1a
1.4±1.4f
13.8±0.5b
11.9±0.6cd
12.1±0.3c
10.3±0.3d
7.2±0.3e
13.8±0.3b
0.5±0.5f
2040.7
14.7±1.9a
24.1±0.9d
21.0±1.0bc
23.2±0.4cd
30.6±0.6e
23.7±0.7d
19.5±0.5b
18.9±0.1b
15.2±0.2a
97.8
C-N
34.9±0.7a
20.9±0.4c
17.0±2.4d
29.3±0.3b
22.6±0.7c
22.8±0.5c
26.8±0.8b
10.2±0.8e
5.4±1.4f
216.8
12.6±3.5a
46.8±1.3d
68.3±0.1e
69.3±1.1e
71.0±0.0e
36.1±1.0c
37.6±0.4c
31.6±0.6b
30.7±0.3b
680.0
61.0±2.2a
34.6±0.5c
19.8±0.9e
19.1±0.9e
34.6±0.5d
12.3±0.3f
58.3±1.3a
50.9±0.9b
48.7±0.3b
865.3
Elemental atomic percentage (%)
C
C=O
O-C=O
C-COOH
9.3±1.0a
4.3±1.2b
4.3±1.2b
13.9±0.8bcd
2.6±0.5cd
2.6±0.5cd
12.5±1.4bc
4.1±0.1bc
4.1±0.1bc
11.8±1.4ab
1.2±0.3de
1.1±0.2e
14.3±1.7bcd
0.4±0.4e
0.4±0.4e
14.9±0.2cd
1.5±0.2de
1.5±0.2de
12.2±0.3abc
3.0±0.5bc
3.0±0.4bc
14.2±1.2bcd
6.6±0.6a
6.7±0.5a
16.4±0.6d
7.3±0.4a
7.3±0.4a
11.3
59.2
65.1
8.8±1.2bc
3.3±0.4a
3.3±0.4a
8.5±4.0bc
0.6±0.6c
0.0±0.0d
5.8±0.4c
2.5±0.3ab
2.5±0.3ab
5.2±0.6c
3.2±1.0a
3.3±1.1a
7.0±0.6c
1.0±1.0c
1.0±0.0cd
12.1±0.1b
1.1±0.1c
1.0±0.0cd
11.6±0.6b
1.4±0.1bc
1.4±0.1bc
0.2±0.2d
0.4±0.4c
0.5±0.5cd
52.5±1.4a
0.5±0.5c
0.6±0.6cd
312.9
17.0
19.7
6.1±0.3a
1.5±0.0b
1.5±0.0a
7.5±1.3ab
4.3±0.3c
4.4±0.2b
17.2±0.2e
6.3±0.3d
6.3±0.2c
18.0±0.7e
6.0±0.1d
6.0±0.0c
10.1±0.4cd
4.2±0.3c
4.3±0.3b
11.5±0.5d
0.7±0.7ab
0.8±0.8a
5.9±0.1a
1.2±0.2ab
1.3±0.2a
8.4±0.7b
1.3±0.2ab
1.3±0.2a
8.7±0.3bc
0.5±0.5a
0.6±0.6a
173.9
139.7
122.8
C-O
12.1±0.4a
32.0±4.4bc
44.1±7.2c
34.6±0.4bc
26.1±11.7b
34.0±0.2bc
25.3±0.7ab
29.1±0.9b
29.4±0.9b
9.6
19.8±3.3c
42.7±0.7b
7.1±0.6e
7.0±0.0e
7.9±0.5e
39.1±0.9b
40.6±0.7b
53.4±0.9a
14.8±0.8d
611.8
15.2±0.1ab
22.0±0.0d
29.4±0.6f
27.7±0.3ef
16.2±0.8b
51.0±1.0g
13.8±0.3a
19.3±0.3c
26.4±1.4e
821.5
N
O
4.7±0.2a
3.3±1.5ab
3.4±1.4ab
4.5±0.2a
3.9±2.0ab
5.0±0.1a
2.5±2.5ab
2.4±1.1ab
0.4±0.4b
3.5
5.9±1.1ab
6.5±0.2a
5.0±0.2abc
4.8±0.3bc
4.5±0.4bc
3.8±0.3cd
3.5±0.1cd
2.6±0.1de
1.7±1.0e
24.1
2.9±0.4ab
3.6±0.5a
2.6±0.1abc
2.0±0.5bc
2.4±0.1bc
2.9±0.6ab
3.1±0.0ab
2.3±0.5bc
1.7±0.3c
6.6
26.1±0.3a
35.5±2.5bc
35.5±1.8bc
33.2±0.4bc
36.0±1.5bc
31.2±0.8b
31.3±2.4b
33.4±3.2bc
37.0±1.2c
10.1
20.6±0.7b
25.4±0.5c
13.4±0.6a
13.4±0.2a
13.6±0.9a
28.6±0.2cd
30.9±0.2de
34.6±0.7f
33.5±3.3ef
158.3
21.2±0.8abc
20.0±0.7a
23.8±0.9cde
27.3±1.7f
23.6±0.7bcde
20.2±1.4ab
22.1±2.1abcd
24.9±0.7def
25.6±0.7ef
13.4
Stoichiometry
C/O
C/N
2.7
1.7
1.7
1.9
1.7
2.0
2.1
1.9
1.7
14.9
18.9
17.9
13.9
15.5
12.8
27.0
26.6
148.9
3.6
2.7
6.1
6.1
6.0
2.4
2.1
1.8
1.9
12.4
10.4
16.4
17.0
18.3
18.1
19.0
24.6
37.3
3.6
3.8
3.1
2.6
3.1
3.8
3.4
2.9
2.8
26.6
21.2
28.3
35.2
30.6
26.7
24.2
31.6
42.4
*Means ± SD (n = 3, df = 8). For a particular variety, means with different letters in the same column denote significant
difference at 5 % probability level within each rice variety.
155
9.4.1.3. X-ray diffraction pattern of fresh and aged rice flour
The x-ray diffraction pattern of fresh and aged (6 and 12 months under various MAP conditions)
rice flour of selected glutinous (TDK8 and TDK11) and non-glutinous (DG) rice varieties are
presented in Fig. 9.2. As expected, all the three selected rice varieties exhibited A-type diffraction
(XRD) pattern detected with main peaks at 14.9º, 16.9º, and 22.8o. The crystallinity for TDK8 and
TDK11 was recorded as 50 and 48 %, respectively. However, the crystallinity for DG was recorded
as 45 %. Moreover, no significant (P>0.05) change in crystallinity was recorded during various
MAP conditions in all three selected rice varieties.
9.4.1.4. Pasting properties of fresh and aged rice flour
The pasting properties of fresh and aged (6 and 12 months under various MAP conditions) rice
flour of selected glutinous (TDK8 and TDK11) and non-glutinous (DG) rice varieties are presented
in Table 9.3. The pasting temperature (Ptemp) of flour of fresh rice grains of selected glutinous
(TDK8 and TDK11) and non-glutinous (DG) rice varieties was significantly (P<0.05) lower than
aged the flours. However, vacuum storage of TDK8 exhibited lower physicochemical changes
which resulted in no significant (P>0.05) increase in Ptemp even after 12 months of storage. On the
other hand, N2 and CO2 storage resulted in significant (P<0.05) increase in Ptemp in all selected rice
varieties.
The flour of selected rice varieties showed a diverse behavior of viscosity (peak ~ Vp, breakdown
~ BD, trough ~ Vt, setback ~ SB, and final ~ Vf) during heating-cooling cycles of rapid visco
analysis. TDK8 and DG showed significant (P<0.05) increase in Vp during aging in all MAP
conditions. Vp indicates increased swelling of starch. However, TDK11 showed decreased
swelling during the first 6 months of storage followed by increased Vp only for control storage
after 12 months. Results showed that the MAP conditions especially vacuum and N2 slowed down
the physicochemical changes, which is reflected by significantly (P<0.05) lower Vf of vacuum and
N2 storage than the control in all selected rice varieties. Low Vf indicates softer texture of the final
gel.
156
Figure 9.2 X-ray diffraction pattern of flour of fresh and aged (6 and 12 months under
various MAP conditions) milled rice grains of Thadokkham-8 (TDK8), Thadokkham-11
(TDK11) and Doongara (DG)
157
Table 9.3 Pasting properties of rice flour of fresh and aged (6 and 12 months under
various MAP conditions) milled rice grains of Thadokkham-8 (TDK8), Thadokkham-11
(TDK11) and Doongara (DG)*
Rice
Aging
MAP
Fresh
-
variety
TDK8
6 months
12
months
Vt (mPa-s)
BD (mPa-s)
Vf (mPa-s)
2371.7±47.7a
1443.3±17.0a
928.3±31.8b
1779.7±18.0a
ab
cd
cd
b
cd
2652.5±7.5
vacuum
72.4±0.5ab
2462.0±28.0b
1691.5±5.5b
770.5±33.5a
ab
cd
de
886.5±10.5
b
1744.5±19.5
2107.5±34.5
2026.5±5.5b
363.0±15.0ab
335.0±0.0a
cd
376.5±12.5bc
2658.0±8.0
CO2
73.1±0.5bc
2695.0±5.0cd
1778.0±5.0de
917.0±0.0b
2158.3±12.6d
380.3±11.6bc
control
72.6±0.2ab
2939.0±40.0e
1869.0±4.0f
1070.0±36.0c
2275.5±8.5f
406.5±4.5cd
vacuum
ab
72.5±0.1
c
N2
73.7±0.2cd
2654.0±17.0cd
1763.0±2.5de
CO2
d
74.2±0.2
d
2719.0±31.0
e
17.6
101.5
176.1
32.8
189
21.9
-
68.3±0.2a
2521.5±5.5b
1084.0±18.0a
1437.5±12.5a
1377.0±7.0a
293.0±11.0a
control
69.6±0.3b
2077.5±44.5e
1359.0±52.0c
718.5±7.5c
1679.0±13.0d
320.0±39.0a
vacuum
69.9±0.6b
1805.5±25.5h
1213.5±3.5b
592.0±22.0e
1516.5±4.5b
303.0±1.0a
bc
g
1216.0±31.0
69.9±0.2b
1800.5±18.5h
1784.5±6.0b
d
a
3016.0±34.0
2246.0±25.0
CO2
70.8±0.2
1881.5±25.5
1717.0±30.0
1799.0±9.0
bc
b
2106.0±23.0
c
389.0±7.0bcd
891.0±4.0b
2156.5±6.5cd
393.5±14.5cd
e
416.5±11.5d
906.5±3.5
920.3±22.0
b
665.5±5.5
b
d
657.0±12.5f
f
2148.0±10.0
336.3±8.4a
72.9±0.1
2623.5±26.5
1771.5±2.5
908.0±27.0
SB (mPa-s)
N2
N2
c
370.5±5.5b
1558.5±2.5bc
310.5±8.5a
1586.5±36.5
b
2732.5±12.5
g
486.5±12.5c
control
72.7±0.4
months
vacuum
70.8±0.7bc
1984.5±8.5f
1369.5±0.5c
615.0±8.0e
1677.0±5.0d
307.5±4.5a
e
c
e
d
f
383.0±1.0b
N2
74.6±0.3
CO2
72.0±0.9cd
2177.0±8.5d
1454.5±25.0d
722.5±20.5c
1838.5±37.5e
384.0±22.0b
48.7
708.2
719.4
1302.4
1490.7
43.5
Fresh
6 months
a
2441.5±6.5
a
1784.5±0.5
770.0±9.0
2215.5±20.5
12
F value
DG
Vp (mPa-s)
72.3±0.1a
72.6±0.3
Fresh
6 months
Ptemp (ºC)
control
F value
TDK11
Pasting properties
a
657.0±6.0
a
1226.0±16.0a
393.5±25.5de
4039.0±10.0d
2814.5±14.5d
e
b
2348.0±25.0b
-
75.5±0.4
1325.5±34.5
1180.0±17.0
control
79.5±0.1b
1618.0±21.0c
1224.5±4.5ab
vacuum
80.2±0.5
b
cd
N2
79.9±0.2b
1653.0±37.0cd
1229.5±18.5ab
423.5±18.5e
3675.5±42.5bc
2446.0±24.0c
CO2
80.4±0.2b
1752.0±26.0d
1273.5±24.5bc
478.5±1.5f
3796.5±3.5c
2523.0±21.0c
d
b
bc
1665.0±29.0
1254.0±39.0
404.0±17.0
3609.0±37.0
3058.5±19.5e
84.0±0.4
months
vacuum
82.9±0.3cd
1668.5±64.5cd
1319.5±44.5cd
349.0±20.0cd
4515.0±92.0f
3195.5±47.5f
N2
82.6±0.2
c
1619.0±35.0
c
ab
cde
e
3028.5±27.5e
CO2
81.7±0.9c
1672.0±4.0cd
1342.5±3.5d
329.5±0.5c
4362.0±56.0e
3019.5±52.5e
109.8
33.0
12.9
100.7
529.0
1203.3
373.0±22.0
4312.5±58.5
e
control
1246.0±13.0
222.0±20.0
b
2406.0±33.0
12
F value
1476.0±59.0
1261.0±12.0
bc
145.5±17.5
a
2167.5±1.5
4274.5±40.5
*Means ± SD (n = 3, df = 8). For a particular variety, means with different letters in the
same column denote significant difference at 5 % probability level within each rice variety.
9.4.1.5. Gelatinization and retrogradation properties of fresh and aged rice flour
The gelatinization and retrogradation properties of fresh and aged (6 and 12 months under various
MAP conditions) rice flour of selected glutinous (TDK8 and TDK11) and non-glutinous (DG) rice
varieties are presented in Table 9.4. Aging under modified atmospheric conditions resulted in
slightly decreased onset (To), peak (Tp) and conclusion (Tc) temperatures. In general, the enthalpy
158
(ΔH) of gelatinization for TDK11 and DG flour was increased significantly (P<0.05) during aging
with the more prominent increase during N2 and CO2 storage. Increase in ΔH can be related to the
increase in Ptemp as shown in Table 9.3. However, no significant (P>0.05) change in ΔH for TDK8
was observed.
Similarly, the retrogradation thermal temperatures (To(r), Tp(r), and Tc(r)) were also reduced in TDK8
and DG. The retrogradation thermal temperatures for fresh TDK8 were recorded as To(r) ~ 51.7ºC,
Tp(r) ~ 58.5ºC, and Tc(r) ~ 65.2ºC. However, after 6 and 12 months of storage, the retrogradation
thermal temperatures were reduced. Moreover, the retrogradation thermal temperatures for fresh
DG were recorded as To(r) ~ 43.7ºC, Tp(r) ~ 55.4ºC, and Tc(r) ~ 62.9ºC but during first 6 months of
storage, no significant (P>0.05) reduction in thermal transition temperatures was observed.
Interestingly, TDK11 did not show any retrogradation (as no retrogradation peak was detected)
during the first 6 months in any MAP conditions. The results showed that vacuum condition for
all rice varieties reduced the rate of physicochemical changes whether they were glutinous or nonglutinous as reflected by no significant (P>0.05) increase in the percentage of retrogradation (R
%) (Table 9.4).
9.4.1.6. Textural profile analysis of cooked grains of fresh and aged rice
The textural profiles of cooked fresh and aged (6 and 12 months under various MAP conditions)
are presented in Table 9.5. Results showed that there was a significant increase in hardness and a
significant decrease in adhesiveness of cooked rice during storage under various MAP conditions
in all rice varieties (P = 0.05). Interestingly, the rice grains stored in N2 and CO2 had harder texture
after cooking compared to that of fresh and aged rice grains of control and vacuum storage.
However, the reduction in the stickiness or adhesiveness of cooked rice grains was found relatively
slower in N2 and CO2 than the control and vacuum storage even after 12 months of storage. The
results of pasting properties especially setback (SB) and final (Vf) viscosities (Table 9.3) and
thermal analysis (Table 9.4) of N2 and CO2 storage well correlated with TPA results.
159
Table 9.4 Gelatinization and retrogradation properties of rice flour of fresh and aged (6 and 12 months under various MAP
conditions) milled rice grains of Thadokkham-8 (TDK8), Thadokkham-11 (TDK11) and Doongara (DG)*
Rice
variety
TDK8
Aging
Fresh
6 months
12 months
TDK11
Fresh
6 months
12 months
DG
Fresh
6 months
12 months
MAP
control
vacuum
N2
CO2
control
vacuum
N2
CO2
F value
control
vacuum
N2
CO2
control
vacuum
N2
CO2
F value
control
vacuum
N2
CO2
control
vacuum
N2
CO2
F value
Tₒ (oC)
66.4±0.0ab
65.2±0.1cd
66.3±0.0abc
66.8±1.0a
65.1±0.1d
66.3±0.1abc
65.3±0.4bcd
65.6±0.0bcd
66.0±0.6abcd
6.8
64.3±0.1b
64.8±0.1a
64.2±0.1b
64.3±0.2b
64.3±0.0b
61.6±0.0e
61.8±0.1d
61.1±0.0f
62.2±0.0c
1232.2
72.3±0.7a
70.3±0.3b
70.4±0.2b
71.0±0.1b
70.6±0.1b
68.6±0.3c
68.2±0.3c
67.9±0.0c
68.3±0.0c
88.1
Gelatinization
Tp (oC)
Tc (oC)
73.7±0.4ab
89.9±1.6a
72.6±0.2c
85.6±1.5b
abc
73.5±0.1
85.4±0.8b
a
73.9±1.0
86.9±2.4ab
72.7±0.2bc
87.4±0.7ab
73.4±0.3abc
84.2±0.2b
72.7±0.2bc
84.2±1.4b
c
72.5±0.0
85.1±0.7b
abc
73.3±0.1
87.6±0.2ab
5.1
6.5
70.6±0.1b
77.4±0.1a
71.0±0.2ab
81.1±0.2bc
ab
70.9±0.1
80.4±1.0bc
70.8±0.1ab
79.7±1.0ab
71.3±0.0a
82.8±0.9c
d
68.6±0.1
79.3±1.0ab
de
68.3±0.4
79.2±1.5ab
e
68.0±0.2
79.4±1.2ab
69.1±0.1c
80.2±0.5bc
165.2
8.0
77.1±0.5a
83.0±1.5ab
76.1±0.3b
83.3±0.5a
76.2±0.2b
83.1±0.0ab
76.6±0.1ab
83.8±0.4a
b
76.3±0.2
83.1±0.0a
c
74.2±0.1
81.5±0.2bc
73.9±0.0c
80.9±0.0c
74.0±0.1c
81.0±0.4c
c
74.0±0.1
81.0±0.0c
95.8
13.7
ΔH (Jg-1)
10.4±0.5a
10.8±0.3a
9.7±1.0a
9.4±1.0a
10.6±0.5a
9.7±0.3a
10.8±0.5a
10.3±0.7a
9.8±1.8a
1.0
7.7±0.1a
9.6±0.3bc
8.8±0.4ab
9.3±0.4bc
9.2±0.5bc
10.4±0.7cd
10.1±0.6cd
10.3±0.5cd
11.0±0.3d
14.9
3.4±0.0a
6.9±0.3bcd
6.6±0.1bc
8.4±0.3e
7.4±0.3cd
7.5±0.4d
7.2±0.4cd
7.7±0.4de
6.2±0.3b
72.8
Tₒ(r) (oC)
51.7±0.0a
44.6±0.4de
46.8±0.1c
45.8±0.1cd
41.6±0.1f
47.9±0.6bc
44.0±1.1de
43.1±1.8ef
49.0±1.1b
53.1
ND^
ND^
ND^
ND^
ND^
37.9±0.3a
37.3±0.8a
37.2±0.7a
37.9±0.9a
5195.1
43.7±0.2a
39.1±1.5bc
41.6±0.6ab
42.4±0.5ab
39.1±1.5ab
35.1±0.3d
36.5±0.1cd
37.6±0.2cd
35.1±2.9d
24.4
Tp(r) (oC)
58.5±0.5a
54.4±0.5cd
56.9±0.2ab
56.1±1.4bc
53.9±0.8d
56.5±0.1ab
55.3±0.8bcd
54.4±1.3cd
56.3±0.2bc
12.5
ND^
ND^
ND^
ND^
ND^
48.9±0.2a
48.8±0.1a
48.8±0.1a
49.2±0.3a
22219.3
56.4±0.0a
53.4±0.0c
54.0±0.8bc
54.6±0.0bc
54.8±0.4b
49.2±0.5d
49.2±0.3d
49.9±0.1d
49.2±0.7d
136.9
Retrogradation
Tc(r) (oC)
65.2±1.3a
62.7±0.8ab
63.6±0.2ab
62.9±0.0ab
64.1±1.3ab
64.0±0.3ab
62.5±1.4ab
61.8±1.8b
62.9±0.1ab
3.1
ND^
ND^
ND^
ND^
ND^
56.4±0.2a
56.5±0.3b
57.1±0.0c
57.5±0.0d
187048.1
62.9±0.2a
62.4±0.1a
62.4±0.4a
62.3±0.1a
62.8±1.0a
59.5±0.2b
59.2±0.4b
59.1±0.5b
59.2±0.4b
42.2
ΔH(r) (Jg-1)
0.2±0.0a
0.4±0.0ab
0.3±0.0ab
0.3±0.1ab
0.4±0.0abc
0.4±0.1abc
0.3±0.0ab
0.5±0.1bc
0.7±0.3c
6.1
ND^
ND^
ND^
ND^
ND^
2.4±0.2b
1.7±0.1a
3.0±0.0c
4.5±0.1d
1097.0
2.0±0.0a
4.7±0.1d
3.5±0.0b
3.3±0.1b
2.3±0.1a
5.2±0.2e
4.6±0.2d
5.2±0.4e
4.1±0.1c
172.1
R (%)
2.1±0.1a
3.5±0.1ab
2.8±0.4ab
3.5±0.4abc
3.8±0.1bc
4.0±0.5bc
3.1±0.0ab
5.1±0.1cd
6.5±1.4d
17.2
ND^
ND^
ND^
ND^
ND^
22.7±0.4a
16.9±0.0d
28.7±1.0b
40.8±0.2c
5204.1
59.2±0.0d
68.4±1.3ab
52.9±0.8e
39.7±1.8f
30.6±0.1g
69.6±1.2a
64.6±0.5c
67.2±0.8abc
66.4±1.1bc
560.6
^Not detected.
*Means ± SD (n = 3, df = 8). For a particular variety, means with different letters in the same column denote significant
difference at 5 % probability level within each rice variety.
160
Table 9.5 The textural profile and in situ TMCT cooking analysis of milled rice kernels of
fresh and aged (6 and 12 months under various MAP conditions) milled rice grains of
Thadokkham-8 (TDK8), Thadokkham-11 (TDK11) and Doongara (DG)*
Rice
Aging
MAP
variety
TDK8
Hardness (N)
12 months
12 months
(min)
30.2±1.2a
control
2.3±0.2b
-0.20±0.01cd
-0.013±0.001c
37.2±1.0b
vacuum
3.4±0.1c
-0.32±0.01bc
-0.017±0.004ab
34.7±1.2b
N2
4.4±0.3de
-0.28±0.01bcd
-0.014±0.002bc
35.1±0.3b
CO2
4.5±0.3ef
-0.34±0.02b
-0.014±0.000bc
35.6±0.4b
control
2.8±0.2bc
-0.15±0.00d
-0.012±0.000c
53.0±0.9d
vacuum
3.5±0.0cd
-0.18±0.01cd
-0.014±0.000abc
47.2±1.0c
N2
5.0±0.7ef
-0.20±0.01cd
-0.014±0.001abc
48.8±0.6c
CO2
5.3±0.3f
-0.27±0.01bcd
-0.013±0.000bc
49.0±1.1c
78.2
950.4
5.6
254.1
-
1.6±0.1a
-0.33±0.02a
-0.019±0.001a
25.0±0.3a
control
1.6±0.0a
-0.15±0.01cd
-0.015±0.001cd
28.1±0.6cd
vacuum
4.3±0.2e
-0.33±0.03a
-0.017±0.001a
25.5±0.6a
N2
3.6±0.1d
-0.30±0.02a
-0.017±0.001a
26.8±0.3b
CO2
3.4±0.0d
-0.21±0.03b
-0.016±0.001bc
25.0±0.4a
control
2.7±0.1b
-0.10±0.02e
-0.012±0.000f
32.6±0.6e
vacuum
6.1±0.1f
-0.13±0.01de
-0.015±0.000de
28.4±0.3cd
N2
3.6±0.0d
-0.19±0.02bc
-0.013±0.000e
29.2±0.6d
CO2
3.1±0.0c
-0.20±0.01bc
-0.013±0.000de
27.4±0.3bc
609.8
66.5
57.5
81.3
-
6.2±0.2a
-0.14±0.02a
-0.029±0.002a
19.8±0.5a
control
6.3±0.0a
-0.03±0.01efg
-0.017±0.000de
24.7±0.4c
vacuum
8.5±0.0cd
-0.10±0.01b
-0.022±0.001b
21.8±0.6b
N2
8.4±0.1c
-0.08±0.01bc
-0.019±0.001c
22.6±0.6b
CO2
8.7±0.1d
-0.07±0.01cd
-0.019±0.000cd
22.6±0.4b
control
7.5±0.1b
-0.01±0.01g
-0.013±0.001g
29.7±0.5d
vacuum
8.7±0.1d
-0.02±0.01fg
-0.015±0.001ef
24.8±0.7c
N2
9.5±0.3e
-0.05±0.01def
-0.015±0.000efg
26.0±0.3c
CO2
10.0±0.1f
-0.05±0.01cde
-0.015±0.001fg
25.7±0.6c
351.6
57.5
126.4
95.4
Fresh
6 months
Cooking time
(mm/sec)
-0.019±0.002a
F value
DG
Rate of cooking20 min
(N.s)
-2.79±0.14a
Fresh
12 months
Adhesiveness
0.3±0.0a
F value
6 months
In situ TMCT cooking
-
Fresh
6 months
TDK11
Textural Profile Analysis
F value
*Means ± SD (n = 3, df = 8). For a particular variety, means with different letters in the
same column denote significant difference at 5 % probability level within each rice variety.
161
9.4.1.7. In situ TMCT cooking analysis of fresh and aged rice kernels
The rate of cooking (rate of softening of the grain during cooking) during first 20 min and complete
cooking time of fresh and aged (6 and 12 months under various MAP conditions) rice grains using
the in situ TMCT cooking method are shown in Table 9.5. The cooking process initiates from the
starch gelatinization of the outer layers of the grain and proceeds to the inner layers of the
endosperm. Moreover, this process is time-dependent (Nawaz et al. 2017). The in situ TMCT
records this change by measuring the softness of the grain (indicated by probe movement) with
the time. In general, the initial 20 min of the in situ TMCT cooking curves were linear in all rice
samples used in the study, as shown in Appendix 11. Therefore, only the initial 20 min of cooking
were used to establish the rate of cooking. Results showed that aging of rice grains significantly
(P<0.05) reduced the rate of cooking and significantly (P<0.05) increased the cooking time in
control storage conditions. Moreover, a similar trend was observed during the cooking of rice
grains stored in MAP conditions. Findings are also well correlated with pasting temperature (Ptemp)
as shown in Table 9.3.
9.4.2. Discussion
In the current study, we have analyzed the effect of three different modified atmospheric packaging
viz. vacuum, N2 and CO2 on the aging of selected glutinous (TDK8 and TDK11) and non-glutinous
(DG) milled rice grains. Milled rice samples were packed in aluminum bags instead of plastic bags
as aluminum is more impermeable to gas and water vapor than plastic (Marsh & Bugusu 2007).
As aluminum bags are not cost effective, therefore, future studies on the MAP will focus on other
cost-effective packaging materials.
The current findings for the microstructures of fresh grains are in agreement with previous studies
that have reported larger starch granules in non-waxy rice cultivars than in waxy rice cultivars
(Wani et al. 2012; Vandeputte & Delcour 2004). In the control sample, results showed aginginduced structural changes in the starch granules mainly depicted by the loss of polyhedral shape.
This might be due to macromolecules (starch, proteins, and lipids) interactions, oxidation reactions
and enzymatic activity (Sharp & Timme 1986). However, this structural change was seen less
prominent in a vacuum and/or modified atmospheric packaged samples using an increased
percentage of carbon dioxide or nitrogen. These findings revealed that the MAP conditions
162
(vacuum, CO2, and N2) might slow down the macromolecular interactions, oxidation reactions,
and enzymatic activities and maintain the polyhedral shape (Wu et al. 2016).
The surface analysis of freshly milled grains showed that a nano-metric scale layer of bran oil from
bran might remain on the surface of grain even after 9 % degree of milling of the grain. Similar
observations were also reported in the past researches (Saad et al. 2011; Nawaz et al. 2016b). The
control storage induced higher concentration of surface proteins and reduced surface lipids in all
selected rice varieties. This shows some loss of lipids from the grain surface. This may be due to
the oxidation of lipid or re-adsorption of lipid and oxidized lipid products to the interior of the
grain as the liquid state bran oil is the main lipid of rice. Another possibility is the formation of the
starch-lipid complex (Villwock et al. 1999). Ohno and Ohisa (2005) reported the outer layer of
rice proteins oxidized more than inner layer during aging, resulting in a bigger size of surface
protein bodies. This might have also contributed to the increased level of protein on the surface.
However, N2 and CO2 MAP of TDK8 and DG slightly slowed down the shift of macromolecules
and maintained the surface starch/proteins/lipids ratios during 6 months of storage. Detection of
Mn on the surface of TDK11 might be due to accumulation in the endosperm (Sperotto et al. 2013).
Moreover, the traces of B and Si in DG may be due to the bran particles intactness to the surface
of endosperm (Uraguchi & Fujiwara 2011; Nawaz et al. 2016b).
From our previous research, the C/O stoichiometry values for pure rice starch, proteins and lipids
were found to be 1.47, 4.76 and 11.11, respectively and the C/N stoichiometric values for the same
were found to be 94.39, 12.50 and 0, respectively (Nawaz et al. 2016b). These results reflected
that the surfaces of fresh cooked TDK8 and TDK11 were mainly covered by gelatinized
amylopectin (branched starch) with high levels of proteins, resulting in surface adhesiveness. The
surface analysis of cooked aged glutinous rice (TDK8 and TDK11) under various storage
conditions showed slight decrease in C/O stoichiometric values (except TDK11 during first 6
months in vacuum, N2 and CO2) and slight increase in C/N stoichiometric values suggesting
increase in starch and proteins and decrease in lipid exposure during cooking.
Lipids and proteins are responsible for the hardness and cohesiveness of cooked non-glutinous rice
kernels (Choi et al. 2015). Interestingly, the modified storage conditions and time of storage does
not affect C/O and C/N stoichiometry of DG suggesting least interaction of macro-molecules in
non-glutinous rice during aging making them more resistant to changes in cooking quality during
163
storage as compared to glutinous rice. The rice grains in vacuum conditions slightly maintained
the surface lipids with fewer proteins exposed to the surface, as more O-C=O, C-COOH, and C-O
sub-peaks and less C-N sub-peak were deconvoluted. However, rice grains stored in N2 and CO2
showed more polysaccharides side-chains (starch) on the surface. In addition to this, rice grains in
control storage showed a reduction in surface lipids possibly due to more oxidation or readsorption of lipids by the grains as discussed earlier.
X-ray diffraction technique is mostly used to study the change in the percentage crystallinity of
starch carried out by various physical treatments such as milling and storage (Ye et al. 2016). The
crystallinity depends upon the ratio of amylose and amylopectin. Higher the ratio of amylopectin
higher will be the crystallinity (Park et al. 2007). Interestingly, the calculated crystallinity of the
rice varieties used in this work was much higher than the reported crystallinity of similar glutinous
and non-glutinous varieties in previous studies.
XRD results showed that there was no V-type crystalline peak found at 20o in the aged glutinous
and non-glutinous rice stored in various MAP conditions, showing undetectable starch-lipid
complex formation. Interestingly, XRD findings are not in agreement with the XPS results where
we assumed that one of the causes of reduction in surface lipids was possibly due to starch-lipid
interaction. These findings may also suggest that the starch-lipid interaction may have occurred
only on the exposed surface of the grain that is undetectable during XRD analysis of flour.
In rapid visco analysis, Ptemp is the indication of water binding and initiation of gelatinization. The
influence of modified atmosphere on the restricted water binding capacity of starches have been
reported in the past (Cofie-Agblor et al. 1998). Higher concentrations of atmospheric CO2 and N2
might block the water binding sites, resulting in reduced hydrogen bonding between amylopectin
branches (Noomhorm et al. 2009). This may lead to less water absorption/binding and high Ptemp.
Moreover, increased concentration of atmospheric N2 and CO2 may interact with water binding
sites of amylopectin and restrict the gelatinization, resulting in more energy requirement to
gelatinise the starch. This might be due to retarded enzymatic activity and starch/proteins/lipids
interactions. The cooking process initiates from the starch gelatinization of the outer layers of the
grain and proceeds to the inner layers of the endosperm. Moreover, this process is time-dependent
(Nawaz et al. 2017). The in situ TMCT recorded this change by measuring the softness of the grain
(indicated by probe movement) with the time. The atmospheric CO2 and N2 might retard the
164
recrystallisation of gelatinised starch during cooling, resulting in increased adhesiveness of cooked
rice.
9.5. Conclusions
Aging-induced physicochemical changes can affect the cooking quality and textural attributes of
the glutinous rice. Stickiness or adhesiveness of the cooked rice is one of the textural parameters
most sensitive towards aging of rice. The findings of the present study revealed that aging-induced
physicochemical changes of milled glutinous and non-glutinous rice could be slightly slowed
down using the modified atmospheric packaging (MAP). Vacuum and/or higher concentration of
atmospheric CO2 and N2 during storage can slow down the physicochemical deterioration. Overall,
in relative term vacuum packaging was found more effective than any other storage conditions
investigated in this work.
165
Chapter 10 General conclusions and recommendations for future research
166
10.1. General conclusions
Glutinous varieties are grown in many countries, including Lao PDR, Thailand, China, Myanmar,
Vietnam, Cambodia, Japan, Bangladesh, and India. It is a staple food of Laotian people. It is
usually consumed as a desert, as a breakfast cereal or as steamed rice in banana leaves in Thailand,
Myanmar, Cambodia, India, China, and Vietnam. Glutinous rice contains amylopectin with a very
low level (<5 %) of amylose, which contributes to a typical stickiness of cooked rice. Good quality
fresh or aged glutinous rice should be reasonably sticky in texture after cooking. This research
mainly focused on the underline causes of loss of stickiness of glutinous rice during storage and
different processing interventions to maintain its quality. This study covered a number of aspects
of glutinous rice; (i) Characterization of rehydration and gelatinization behavior of glutinous rice;
(ii) Development of an in situ method to study the cooking kinetics; (iii) Effect of modified
atmospheric storage on the stickiness attribute of glutinous rice; (iv) Characterization and
modification of surface composition of raw and cooked rice grains and its correlation to stickiness
property of cooked rice; (v) Pre-treatments including parboiling of glutinous rice and their effect
on stickiness property. The major outcomes of this project are described below.
i.
Rehydration and gelatinization behavior of rice flour is usually used as an indicator of
cooking properties of rice grains. The glutinous rice flour of selected Laotian varieties viz.
TDK8, TDK11, and Hom Mali Niaw were subjected to different rehydration time and
temperatures and cooking times at 95oC. Water uptake by the flour was directly proportional
to the time/temperatures of rehydration. Starch granules showed a higher breakdown in
response to extended cooking, resulting in reduced trough viscosity and reduced
retrogradation. It is therefore recommended that for glutinous rice especially TDK8,
extended cooking will result in a better-cooked product. This work also generated the pasting
data for the most popular glutinous varieties consumed in Lao PDR.
ii.
Type of rice (glutinous or non-glutinous) and aging temperature can affect the rate of water
uptake and cooking properties. The physicochemical properties of rice flour are different
from whole grain. Therefore, water uptake and cooking behavior of Laotian glutinous rice
varieties (fresh and aged TDK8 and TDK11) was analyzed by a novel in situ method using
Thermal Mechanical Compression Test (TMCT) attached to a texture analyzer. The in situ
TMCT cooking method provided information on the softening of the grain due to water
167
uptake during soaking at various rehydration temperatures. The slope of in situ TMCT
cooking curve can be used to estimate the rate of cooking.
iii.
The textural attributes especially the stickiness of cooked rice are perceived by the surface
of the grain. Therefore, it is important to study the surface of the composition. A new method
was developed to quantify the surface composition of raw and cooked rice grains by using
X-ray photoelectron spectroscopy (XPS). The detailed elemental composition of the upper
5-10 nm layer of rice grains and flour through XPS was used to calculate the percentage of
major components on the grain surface. We found a higher percentage of proteins and lipids
on the surface of grains and flour as compared to their respective percentages in bulk
composition. These results for XPS analysis correlated well with CLSM microstructure
analysis verifying the accuracy of the new technique.
Surface analysis showed that higher contents of protein present on the surface of milled
grains might also contribute to the reduced stickiness of cooked rice. Proteins in rice are
mostly glutelin which is soluble in alkali. Therefore, glutinous rice grains were washed with
different concentrations of NaOH (0 to 0.2 %). We found that surface proteins of rice grains
were removed with as low as 0.004 % NaOH. Protein removal by alkali washing possibly
restricted the realignment of starch during retrogradation, resulting in maintaining stickiness
and freshness of cooked glutinous rice. These results suggested that varying the proportion
of surface proteins of the rice grain through alkali washing can be used to improve the
textural attributes and sensory properties of glutinous rice.
iv.
Starch modification by various pretreatments can improve the glossiness, stickiness, and
softness of gelatinized starches. The intact starch of whole rice grains was esterified by using
dilute acetic anhydride. Acetylation of starch in the glutinous rice grains affected the
crystalline structure of starch granules which was reflected by the reduced peak and final
viscosities during rapid visco analysis and reduced thermal transition temperatures and
enthalpy during thermal analysis. Moreover, the texture of cooked acetylated glutinous rice
grains was softer and more adhesive. In vitro digestion of acetylated glutinous rice showed
reduced glycemic index possibly due to the increased amount of resistant starch. These
results suggested that varying the acetylation of starch in the glutinous rice grain can be used
to achieve the desirable textural property of intact rice grains.
168
v.
Thermal treatment such as wet parboiling of fresh glutinous paddy may help in maintaining
the stickiness and overall quality of glutinous rice. Parboiling of glutinous rice with various
soaking mediums water, 3 % NaCl saline solution and 0.2 % acetic acid solution showed that
saline and acetic acid soakings improved the milling efficiency. Saline and acetic acid
soaking also resulted in reduced crystallinity and thermal endotherms. The amber
discoloration from the husk during steaming was minimized by the bleaching effect of acetic
acid used during soaking. The acetic acid soaking restricted swelling, resulting in a reduced
peak (~10 % decrease) and final viscosity (~15 % decrease), whereas, the saline soaking
improved water absorption, resulting in a higher peak (~10 % increase) and final viscosity
(~5 % increase). Furthermore, parboiling increased hardness (2.6 to 5 N for TDK8 and 2.3
to 3.5 N for TDK11) and adhesiveness (-0.2 to -0.5 N.s for TDK8 and -0.5 to -0.7 N.s for
TDK11) of glutinous rice in saline and acetic acid soaking as compared to water only soaking
(-0.3 to -0.5 N.s for TDK8 and -0.6 to -0.7 N.s for TDK11). Also, the GI value of parboiled
rice was reduced from 116.5 to 100.4 for TDK8 and 94.8 to 72.2 for TDK11 showing some
improvements in the nutritional quality. Overall, parboiling of glutinous showed
improvement in the grain quality which can have commercial potential.
vi.
Cooking quality and textural attributes especially stickiness of the glutinous rice is affected
by aging-induced physiochemical changes. We found that the higher concentration of
atmospheric gases (CO2 or N2) and/or vacuum during storage can slow down aging-induced
changes in milled glutinous rice and can maintain the stickiness attributes of cooked aged
glutinous rice. Overall, among all the storage conditions used, the vacuum was considered
the best to maintain the quality of the glutinous rice.
10.2. Future direction
i.
The in situ TMCT cooking method was found useful to calculate the cooking kinetics of a
range of samples. However, it is recommended that further work should be done to relate the
in situ TMCT cooking data with the sensory evaluation of rice.
ii.
The pre-processing treatment with alkali may induce the structural changes in amylopectin,
possibly due to alkali-induced depolymerization. Therefore, the impact of alkali washing on
starch molecular-structure should be studied by using advanced techniques such as Size
Exclusion Chromatography.
169
iii.
It was challenging to estimate the extent of acetylation in the whole rice grains due to
technical difficulties. Soaking and subsequent drying resulted in increased internal fissure
making it impossible to separate outer layers without damaging the grain core during milling.
Therefore, further studies should be conducted to study the acetylation gradient across the
grain.
iv.
In the present study, only the fresh samples of glutinous rice were analyzed for preprocessing treatments of alkali washing, starch esterification of whole grains and parboiling.
It would be interesting to see the effect of aging on the pre-processed glutinous rice.
v.
In the present study, the textural attributes were assessed using a texture analyzer, and no
sensory evaluation was conducted due to unavailability of the trained sensory panel.
Therefore, it is recommended that all the pre-processing treatments and modified storage
techniques reported in this study should be validated with the trained sensory panel.
vi.
The glycemic index of pre-processed glutinous rice by using starch modification and
parboiling was predicted using in vitro digestion method. Therefore, it is recommended that
the digestibility of pre-processed glutinous rice may be tested in vivo using an animal model.
vii.
The use of digital rice cookers with sticky rice program, pressure cooking and microwave
cooking are gaining popularity around the globe. Use of these modern techniques may
overcome the overnight soaking of aged glutinous rice before cooking. Therefore, it is
important to study the dynamics of rice cooking under these modern cooking conditions.
170
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Appendices
Appendix 1: The flow chart of experimental design for Chapter 5.
212
Appendix 2: Typical XPS survey and high resolution spectra of rice sample with
decomposition of C1s peak into distinct sub-peaks.
213
Appendix 3: Point of inflection (pi) calculation by using first derivative for every 30 sec
(F´ 30 sec).
214
Appendix 4: Scanned images of control and alkali treated Thadokkham-8 (TDK8) and
Doongara (DG); (a) uncooked rice grains, and (b) cooked rice grains.
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
C0.004
C0.02
C0.04
C0.2
TDK8
(a)
Cc
Cc0
DG
Cc
Cc0
C0.004
C0.02
C0.04
Cc
Cc0
C0.004
C0.02
C0.04
C0.2
TDK8
(b)
DG
215
C0.2
Appendix 5: RVA viscographs of control and alkali treated flour of Thadokkham-8 (TDK8)
and Doongara (DG).
216
Appendix 6: Cooking curve during parboiling.
217
Appendix 7: Experimental design for Chapter 9.
218
Appendix 8: XPS survey and high resolution spectra of fresh cooked Thadokkham-8
(TDK8).
219
Appendix 9: XPS survey and high resolution spectra of fresh cooked Thadokkham-11
(TDK11).
220
Appendix 10: XPS survey and high resolution spectra of fresh cooked Doongara (DG).
221
Appendix 11: In situ TMCT cooking curves of fresh and aged (six and twelve months
under various MAP conditions) Thadokkham-8 (TDK8), Thadokkham-11 (TDK11), and
Doongara (DG).
222