Folate Fortification of Rice Through Parboiling

FOLATE FORTIFICATION OF RICE THROUGH PARBOILING: OPTIMISATION, RICE QUALITY, CONSUMER ACCEPTANCE AND IN-VITRO RELATIVE BIOACCESSIBILITY & ABSORPTION USING CACO-2 CELLS

Ka Yu Karrie KAM

Supervisor: A/Prof. Jayashree ARCOT Co-Supervisors: Prof. Adesoji ADESINA Dr. Rachelle WARD

FOR FULFILMENT OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN FOOD SCIENCE AND TECHNOLOGY

School of Chemical Engineering The University of New South Wales Australia

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Kam

First name: Karrie Other name/s:-

Abbreviation for degree as given in the University calendar:

School: Chemical Engineering Faculty: Engineering

Title: Folate fortification of rice through parboiling: optimisation, rice quality, consumer acceptance and in-vitro relative bioaccessibility & absorption using Caco-2 cells

Abstract 350 words maximum:

Parboiling has been examined as a technique for fortification of rice with folic acid. Multifactorial models were developed to describe folate retention. The optimal parboiling condition was soaking brown rice at 70 ◦C for 2 h. Quality of rice produced at 70 ◦C was further investigated. No significant change in Head Rice Yield and grain dimension was observed between raw and fortified rice, suggesting that the economic value of rice will not be compromised and rice uniformity may likely be achieved after mixing with the untreated rice. Despite the inherent yellow colour of folic acid, significant difference in yellowness was exhibited only in rice fortified at 1.2gfolic acid /300gbrownrice, but not among that fortified at lower concentration. Visual and organoleptic acceptance of the fortified rice by consumers was examined. The appearance of uncooked rice (fortified at 1.2gfolic acid /300gbrownrice ) prepared at 70 ◦C and soaked for 1h resembled the commercial parboiled rice. An informed health claim on the fortified rice increased the consumers’ purchase intent which suggested that they liked the fortified product. Familiarity with parboiled rice improved consumer purchase intent suggesting a higher chance of acceptance in the parboiled-rice- consuming countries. The sensory acceptability of three cooked rice samples (rice soaked in 0.15gfolic acid /300gbrownrice at 70 ◦C for 1, 2 and 3h and then mixed with commercial white rice) was compared to the commercial white rice (ControlWhite). No significant difference was noted in liking attributes between the samples and the ControlWhite. More than 50% of the consumers were willing to purchase the samples. Lastly, the bioaccessibility and absorption of folic acid in the fortified rice was assessed using in-vitro Caco-2 cell model. Fortified rice was more bioaccessible (91%) than the aqueous folic acid control (81%). As for the extent of absorption, no significant difference was observed between the transport results of fortified rice and control, suggesting that the added folic acid in the fortified rice could be absorbed as well as the control.

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ACKNOWLEDGEMENTS

I would like to express my utmost sincere gratitude to my supervisors, A/Prof. Jayashree Arcot, Prof. Adesoji Adesina and Dr. Rachelle Ward for their guidance and support in my PhD study. I would like to thank my principal mentor, A/Prof. Arcot, for supporting me not only academically but also emotionally through this long journey. She has always been there when I needed her advice. I sincerely thank Prof. Adesina for his invaluable advice on data analysis, time spent on providing me with important feedbacks on my work and his encouragements throughout the course of my PhD. I appreciate the continuous input advised by Dr. Ward especially on the preparation of journal manuscripts that form a core part of this thesis. Special thanks to Dr. Jane Murray for her constant support and expert advice on the consumer acceptance studies.

I would also like to thank Mr. Camillo Taraborrelli, Dr. Robert Chan and Dr. Victor Wong for their patience and professional technical assistance that allow the smooth running of my experiments. To the scientists in CISRO (Queensland Bioscience Precinct) Dr. Simone Osborne and Ms. Rama Addepalli, I am thankful for their sharing the knowledge on Caco-2 cells and providing trainings on tissue culture experimentations.

I am grateful to my dear friends for providing me with many joys and laughter in the labs, especially to Yang, Xin, Veronica, Nisha, Amy, Suzi, Lydia, Cheryl, Wilson and Hamish, who shared the many stressful moments in my PhD life. Special thanks to Denny Hioe for his company and encouragement for the entire time.

Last but not least, I want to express my deepest gratitude to my family for their understanding and care over the years. I would never be able to complete my PhD without their supports!

Thank you everyone who walked with me on this long journey. You all have made my life colourful! I would like to dedicate this thesis to you all.

ABSTRACT

Parboiling has been examined in this thesis as the means to fortify rice with folic acid. The development of the fortification method was governed by four major aspects, namely, optimisation based on nutrient retention, rice quality, consumer acceptance and nutrient bioaccessibility and absorption.

Multifactorial models were developed to describe folate retention. The optimal parboiling condition for the fortification purpose was soaking brown rice at 70 ◦C for 2 h. Fortified rice produced at 70 ◦C was further investigated for the rice quality examination, focussing on Head Rice Yield, kernel dimensions and colour. No significant change in Head Rice Yield and grain dimension was observed between raw and fortified rice, suggesting that the economic value of rice will not be compromised with the fortification process and rice uniformity may likely be achieved after mixing with the untreated rice. Despite the inherent yellow colour of folic acid, compared to parboiled rice, significant difference in yellowness was exhibited only in rice fortified at 1.2 gfolic acid/ 300 gbrown rice, but not among that fortified at lower concentrations. Visual and organoleptic acceptance of the fortified rice by consumers was examined. The appearance of uncooked rice (fortified at 1.2 gfolic ◦ acid/ 300 gbrown rice) prepared at 70 C and soaked for 1 h resembled the commercial parboiled rice. An informed health claim on the fortified rice increased the consumers’ purchase intent which suggested that they welcomed the fortified product. Food familiarity (i.e. familiarity with parboiled rice) improved consumers’ purchase intent suggesting a higher chance of acceptance in the parboiled rice-consuming countries. The sensory acceptability of three cooked rice samples (rice soaked in 0.15 gfolic acid/ 300 gbrown ◦ rice at 70 C for 1, 2 and 3 h and then mixed in with commercial white rice) was compared to the commercial white rice (ControlWhite). No significant difference was noted in liking attributes between the samples and ControlWhite. More than 50 % of the consumers were willing to purchase the samples. Their purchase intent increased after they were notified of the additional health benefits from the rice, they especially inclined to purchase the rice soaked for 2 and 3 h. Lastly, the bioaccessibility and absorption of folic acid in the fortified rice was assessed using in-vitro Caco-2 cell model. Fortified rice was more bioaccessible (91 %) than the aqueous folic acid control (81 %). As for the extent of absorption, no significant difference was observed between the transport results of fortified rice and folic acid control, suggesting that the added folic acid in the fortified rice could be absorbed as well as the control.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... i ABSTRACT ...... ii TABLE OF CONTENTS ...... iii LIST OF FIGURES ...... vii LIST OF TABLES ...... ix LIST OF PUBLICATIONS ...... xi CHAPTER 1 ...... 1 INTRODUCTION ...... 1 1.1 Background ...... 1 1.2 Aims ...... 4 Chapter 2 ...... 5 LITERATURE REVIEW ...... 5 2.1 Rice production and consumption statistics ...... 5 2.2 Composition of rice ...... 7 2.3 General rice processing from harvest to packaging ...... 9 2.3.1 Cleaning ...... 9 2.3.2 Dehulling and milling ...... 10 2.3.3 Washing and cooking ...... 13 2.4 Folates and folic acid ...... 14 2.4.1 History of folates ...... 14 2.4.2 Structure of folic acid and folates ...... 15 2.4.3 Role of folates in human health ...... 17 2.4.4 Prevalence of folate deficiency ...... 18 2.4.5 Food source of natural folates ...... 20 2.4.6 Stability of folates ...... 22 2.5 Methods of rice fortification ...... 25 2.5.1 Dusting ...... 26 2.5.2 Surface coating ...... 26 2.5.3 Hot/Cold extrusion ...... 27 2.5.4 Biofortification ...... 28 2.5.5 Parboiling ...... 30 2.6 Parboiling methods and conditions ...... 31 2.6.1 History of parboiling ...... 31 2.6.2 Processes of parboiling ...... 31 2.6.3 Parboiling devices ...... 37 2.7 Quality change of parboiled rice ...... 42 2.7.1 Appearance ...... 42 2.7.2 Milling quality ...... 43 2.7.3 Textural change ...... 44 2.7.4 Nutrients ...... 45

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2.8 Consumer preferences to rice ...... 46 2.9 Folate bioavailability ...... 47 2.9.1 Intestinal absorption and transport of folates ...... 48 2.9.2 Methods in measuring folate bioavailability ...... 51 2.10 Conclusions ...... 57 Chapter 3 ...... 58 THE FEASIBILITY OF FORTIFYING RICE WITH FOLIC ACID THROUGH PARBOILING ...... 58 3.1 Study 1- Preliminary study using water-soluble dye ...... 59 3.1.1 Materials and methods ...... 59 3.1.2 Results and discussions ...... 60 3.1.3 Study 1 Conclusions ...... 62 3.2 Study 2- Feasibility study with folic acid ...... 63 3.2.1 Materials ...... 63 3.2.2 Methods ...... 65 3.2.3 Results and discussions ...... 70 3.2.4 Conclusions...... 75 Chapter 4 ...... 76 OPTIMISATION OF PARBOILING CONDITIONS: FOLIC ACID UPTAKE AND RETENTION ...... 76 4.1 Materials and methods ...... 76 4.1.1 Preparation of brown rice ...... 76 4.1.2 Selection of fortification conditions ...... 76 4.1.3 Folic acid analysis ...... 83 4.1.4 Degree of gelatinisation ...... 83 4.2 Results and discussions ...... 85 4.2.1 Study 1- Multifactorial analysis: Investigation of fortificant concentrations, soaking temperatures and milling durations on folic acid uptake profile in fortified rice ...... 85 4.2.2 Study 2- Multifactorial Analysis: Investigation of fortificant concentrations, soaking durations and milling durations on folic acid uptake profile in fortified rice ...... 98 4.3 Conclusions ...... 108 Chapter 5 ...... 110 RICE QUALITY STUDIES ON FORTIFIED PARBOILED RICE ...... 110 5.1 Materials and methods ...... 111 5.1.1 Preparation of fortified parboiled rice ...... 111 5.1.2 Cooking method...... 112 5.1.3 Folic acid analysis ...... 112 5.1.4 Kernel dimension ...... 113 5.1.5 Instrumental colour evaluation ...... 113 5.1.6 Statistical analysis ...... 114 5.2 Results and discussions ...... 114 5.2.1 Folic acid uptake in uncooked rice and retention in cooked rice ...... 114 5.2.2 Head Rice Yield (HRY) ...... 121

5.2.3 Kernel dimension ...... 122 5.2.4 Colour evaluation ...... 124 5.3 Conclusions ...... 128 Chapter 6 ...... 130 CONSUMER ACCEPTANCE OF FORTIFIED PARBOILED RICE ...... 130 6.1 Materials and methods ...... 131 6.1.1 Preparation of fortified parboiled rice premix ...... 131 6.1.2 Folic acid analysis ...... 131 6.1.3 Instrumental colour evaluation ...... 132 6.1.4 STUDY 1: Visual consumer acceptance of uncooked fortified parboiled rice samples ...... 132 6.1.5 STUDY 2: Consumer acceptance of cooked fortified rice after mixing ..... 137 6.1.6 Data analysis ...... 141 6.2 Results and discussions ...... 142 6.2.1 STUDY 1: Visual consumer acceptance of uncooked fortified parboiled rice ...... 142 6.2.2 STUDY 2: Consumer acceptance of cooked fortified rice after mixing ..... 150 6.3 Conclusions ...... 155 Chapter 7 ...... 157 SHORT TERM RELATIVE BIOACCESSIBILITY AND ABSORPTION OF FOLIC ACID IN FORTIFIED PARBOILED RICE: CACO-2 CELL STUDY .. 157 7.1 Materials and methods ...... 158 7.1.1 Reagents ...... 158 7.1.2 Cell culture...... 158 7.1.3 Sample preparation ...... 159 7.1.4 Caco-2 cell transport model ...... 161 7.1.5 Folic acid analysis ...... 163 7.1.6 Calculations ...... 163 7.1.7 Cell quality controls ...... 164 7.1.8 Statistical analysis ...... 167 7.2 Results and discussion ...... 167 7.2.1 In-vitro bioaccessibility ...... 167 7.2.2 Transport study ...... 169 7.3 Conclusions ...... 173 Chapter 8 ...... 174 CONCLUSIONS AND FUTURE WORK ...... 174 8.1 Conclusions ...... 174 8.1.1 Is parboiling a feasible method for folic acid fortification in rice? ...... 174 8.1.2 What are the effects of soaking temperature, soaking duration, milling duration and fortificant concentration on the retention of added folic acid? ...... 175 8.1.3 How does parboiling change the qualities of fortified rice? ...... 176 8.1.4 How well do the consumers accept the fortified rice visually and organoleptically? ...... 176 8.1.5 Is the folic acid in fortified rice bioaccessible and easily absorbed? ...... 177

8.2 Implication of results from current work ...... 178 8.3 Future work ...... 179 Chapter 9 ...... 183 REFERENCES ...... 183

LIST OF FIGURES

Figure 1-1 Global rice production and consumption, 1961-2007 (Source: International Rice Research Institute (2009b))...... 1 Figure 2-1 Longitudinal section of rice grain (Source: Bhattacharya, 2004) ...... 8 Figure 2-2 Stages of rice post-harvest process at subsistence level...... 10 Figure 2-3 Structure of folic acid and folates (5-methylenetetrahydrofolate, tetrahydrofolate, 5- formyltetrahydrofolate and 10-formyltetrahydrofolate) ...... 16 Figure 2-4 Intracellular folate metabolism (1 = methylenetetrahydrofolate reductase, 2 = methionine synthase, 3 = thymidylat synthase, 4 = dihydrofolate reductase, 5 = serine hydroxymethyl transferase, THF = tetrahydrofolate, HCY = homocysteine, MET = methionine, DH = dihydrofolate (Source: Stanger (2002)) ...... 18 Figure 2-5 Vessel process of parboiling using (a) a single vessel; (b) double vessel (Source: Roy et al. (2006)) ...... 39 Figure 2-6 Small-boiler process of parboiling: (a) single-barrel boiler, (b) double barrel-boiler (Source: Roy et al. (2006)) ...... 40 Figure 2-7 Medium boiler process of parboiling (Source: Roy et al.(2006)) ...... 41 Figure 2-8 Industrial parboiling plant (Source: Schule (2012))...... 41 Figure 2-9 Physiological properties of the gastrointestinal tract (Avdeef, 2003) ...... 50 Figure 2-10 Transport mechanisms of small intestine. a) Transcellular diffusion; B) Paracellular diffusion; C) Transcellular carrier-mediated transport; D) Transcellular endocytose; E) Efflux transport with apical or basolateral located efflux pumps (Source: Verwei (2004)) ...... 50 Figure 2-11 The transwell system consists of the apical compartment, filter (permeable cell insert) and basolateral compartment (Source: Verwei et al. (2005)) ...... 55 Figure 3-1 Soaking conditions for brown and paddy rice with methylene blue ...... 59 Figure 3-2 Paddy rice granules soaked in methylene blue dye for A) 1 h, B) 4 h and C) 20 h at 60 ˚C. (Each column illustrated the paddy grain, intact brown rice and cross-section of the dehulled brown rice) ...... 61 Figure 3-3 Brown rice granules soaked in methylene blue dye for A) 1 h, B) 4 h and C) 20 h at 60 ˚C (Each column illustrated the intact brown rice and cross- section of brown rice) ...... 62 Figure 3-4- Illustration of rubber roll dehuller (left) and rice indented cylinder (right) ...... 65 Figure 3-5 Illustration of the laboratory rice mill ...... 66 Figure 4-1 Parboiled rice after milling for A) 30 s; B) 60 s; C) 120 s and D) commerical parboiled rice ...... 81 Figure 4-2 pH of soaking solution at different soaking temperatures, (i.e. A: 30˚C; B: 50˚C; C: 70˚C; D: 80˚C) with various fortificant concentrations with respect to soaking times (i.e. 1, 2 and 3h) ...... 90 Figure 4-3 Folic acid uptake coefficients (b) of rice soaked at 30, 50, 70 and 80 ◦C for the corresponding soaking durations ...... 91 Figure 4-4 pH change over different soaking temperatures and durations in folic -1 acid-free soaking solution (0 gfolic acid g rice) ...... 93 Figure 4-5 Amount of hydrogen ions at incipient soaking at different soaking temperatures (30, 50, 70 and 80 ˚C) ...... 94

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3 -1 Figure 4-6 The change of residual folic acid concentration (10 gfolic acid g rice) in fortified rice in relation to the moisture content (%) of rice at the end of the soaking stage ...... 96 Figure 4-7 A) Log-scale of residual folic acid concentration in fortified rice versus moisture content of rice; B) Change of ao at different fortificant concentrations; C) Change of b at different fortificant concentrations ...... 97 Figure 4-8 Linear relationship between the residual folic acid concentration in fortified rice and the doped fortificant concentrations at 0 s milling time ...... 99 Figure 4-9 Parity plot of observed and predicted residual folic acid concentration of parboiled rice of the studied conditions (i.e. soaking time: 1h, 2h, 3h; milling time: 0s, 60s, 120s; fortificant concentration: 0.5x10-3, 1x10-3, -3 -3 -1 2x10 and 4x10 gfolic acid g rice)...... 105 Figure 4-10 Example of transient folic acid concentration retention profile at doped -3 -1 fortificant concentration of 2x10 gfolic acid g rice with brown rice soaked for 2h, where slope and intercept are -2 × 10-4 and 0.112 respectively and R2=0.966 ...... 106 Figure 6-1 Flow chart of fortified parboiled rice preparation for Consumer Acceptance Study - STUDY 1 and STUDY 2 ...... 134 Figure 6-2 The distribution of purchase intent responses (%) between the general rice consumers and parboiled rice-experienced groups in Consumer Acceptance Study - STUDY 1. A) Before the notification of health claim; B) After the notification of health claim ...... 148 Figure 6-3 Distribution of Just-About-Right (JAR) score of attributes intensities of rice samples (in percentage) and penalty analysis of Just-about-Right attributes on mean drops for overall liking of rice samples (for the Consumer Acceptance Study- STUDY 2) ...... 152 Figure 6-4 Purchase intent of cooked fortified mixed rice and ControlWhite tested by consumers before and after the notification of additional health claim of rice evaluated in the Consumer Acceptance Study- STUDY 2 ...... 154 Figure 7-1 Confocal microscopy diagram of Caco-2 cells on the filter insert on the harvest day ...... 167 Figure 7-2 Mean folic acid concentration in the extract and digesta of folic acid standard and fortified rice ...... 169

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LIST OF TABLES

Table 2-1 Paddy rice production (million tonnes) in the world's top 10 producing countries, 2003-2007 (Source: USDA (2010)) ...... 6 Table 2-2 Milled rice consumption (million tonnes) in the world's top 10 countries, 2003-2007 (Source: USDA (2010)) ...... 6 Table 2-3 Per cent nutrient losses during washing and cooking in excess water (Source: Juliano and Bechtel (1985)) ...... 14 Table 2-4 Good sources of natural folates in foods (Source: NHMRC (1995) ...... 21 Table 3-1 Linearity range of the method ...... 70 Table 3-2 Recovery of method for folic acid determination ...... 71 Table 3-3 Uptake of folic acid (µg/ g) after 1 h soaking at 60 ◦C ...... 72 Table 3-4 The folic acid concentration (µg/ g) of 3 batches of fortified parboiled rice soaked at 60 and 70 ◦C ...... 74 Table 4-1 Parboiling conditions studied by previous researchers ...... 79 Table 4-2 Concentration of folic acid (µg/g) after soaking rice at 70 ◦C for 1, 2 and 3 h, steamed for 15 and 60 mins and 0 s milling ...... 80 Table 4-3 Summary of parboiling conditions used in Study 1 ...... 82 Table 4-4 Summary of parboiling conditions used in Study 2 ...... 83 Table 4-5 Residual folic acid retention concentration in raw (0 s) and milled (120 s) fortified grains for three doped fortificant concentrations at the respective soaking time and durations ...... 86 Table 4-6 Two-way ANOVA results for A) fortificant concentration by soaking temperature (top); B) soaking temperature by milling time (middle), and C) fortificant concentration by milling time (bottom) at 1 h soaking ...... 88 Table 4-7 Regression coefficients of multifactorial model ...... 89 Table 4-8 Degree of gelatinisation of parboiled rice after different parboiling treatments (without folic acid added to soaking water) ...... 92 Table 4-9 Residual folic acid concentration in milled fortified raw grains at 0, 60 and 120 s for four doped fortificant concentrations at the respective soaking times...... 100 Table 4-10 Moisture content (% dry basis) of rice after soaking stage ...... 101 Table 4-11 Two-way ANOVA results for A) soaking time by milling time (top); B) doped fortificant concentration by soaking time (middle), and C) doped fortificant concentration by milling time (bottom) ...... 103 Table 4-12 Regression coefficients of multifactorial model ...... 105 7 -1 -1 Table 4-13 Folic acid retention rate (10 gfolic acid g rice s ) at various doped fortificant concentrations and soaking durations as a function of milling time ...... 107 Table 5-1 Uptake of folic acid as concentration and % in fortified and unfortified (ControlParboil) rice kernels milled at 0, 60 and 120 s...... 116 Table 5-2 Folic acid concentration of cooked and uncooked form of fortified rice and the corresponding cooking retention percentage ...... 117 Table 5-3 Cost involved in producing 1 kg of mixed (diluted) fortified rice and the relative price increment in India and Bangladesh...... 120 Table 5-4 Head Rice Yield at different fortificant concentrations, soaking and milling durations ...... 121 Table 5-5 Kernel dimensions at different fortificant concentrations, soaking and milling durations ...... 123

Table 5-6 Colour Parameters L* (Lightness), a* (Redness), and b* (Yellowness) of fortified rice milled for 120s and controls (Commercial parboiled and raw rice) ...... 125 Table 5-7 Colour difference (∆L*, ∆a*and ∆b*) between fortified rice and raw (ControlRaw) and parboiled rice (ControlParboil) ...... 126 Table 6-1 Colour Parameters L* (Lightness), a* (Redness), b* (Yellowness) and C* (Chroma) of uncooked fortified rice milled for 120 s and controls (Commercial parboiled and Commercial white rice)...... 135 Table 6-2 The demographics and consumers’ acceptance questions used in STUDY 1 ...... 136 Table 6-3 Scales for liking, intensity, Just-about-Right (JAR) and purchase intent questions ...... 136 Table 6-4 Amount of folic acid (µg) in uncooked and cooked rice and the mixing ratio required to achieve 200 µg of folic acid in 400 g of cooked rice ...... 138 Table 6-5 The demographics and consumers’ acceptance questions used in STUDY 2 ...... 140 Table 6-6 Socio-demographic characteristics of respondents in the Consumer Acceptance Study- STUDY 1 and STUDY 2 ...... 143 Table 6-7 Rice consumption pattern of consumers recruited for the Consumer Acceptance Study- STUDY 1 and STUDY 2 ...... 144 Table 6-8 Mean perceptions (colour intensity, degree of liking of colour, uniformity of colour, overall appearance) of uncooked fortified samples (UF 1: 1 h soaking; UF 2: 2 h soaking; UF 3: 3 h soaking) and ControlCom tested by consumers (A. general consumers (n=87) and B. those who were familiar with parboiled rice (n=28)) in the Consumer Acceptance Study- STUDY 1 ...... 146 Table 6-9 Mean degree of liking of attributes (appearance, colour, odour, texture, taste, aftertaste and overall liking) of rice samples (CFM 1: 1 h soaking; CFM 2: 2 h soaking; CFM 3: 3 h soaking) and ControlWhite tested by consumers in Consumer Acceptance Study - STUDY 2 ...... 151 Table 7-1 The absorbance values of the prepared food digesta and controls in the cell proliferation assay ...... 161 Table 7-2 The mean amount of folic acid (µg/ monolayer) present in the apical and basolateral media before and after the transport study ...... 172 Table 8-1 Concentration of folic acid (µg/g) in uncooked rice, rinsing water, cooking water and cooked rice...... 180

LIST OF PUBLICATIONS

Journal articles

Chapter 3

 Kam K, Arcot J and Adesina A, (2012), “Folic acid fortification of parboiled rice: Multifactorial analysis and kinetic investigation”, Journal of Food Engineering, 108(1): 238-243.  Kam K, Arcot J and Adesina A, “Folic Acid Fortification of Parboiled rice: Investigation of soaking temperature on retention and kinetic profile”, manuscript in progress.

Chapter 4

 Kam K, Arcot J and Ward R, (2012), “Fortification of rice with folic acid using the parboiling technique: effect of parboiling conditions on nutrient uptake & physical characteristics of milled rice”, Journal of Cereal Science, accepted and in press. .

Chapter 5

 Kam K, Murray JM, Arcot J and Ward R, (2012), “Fortification of parboiled rice with folic acid: Consumer acceptance and sensory evaluation”, Food Research International, 49: 354-363.

Conference presentations and abstracts

 2nd International Vitamin Conference – organized by the Technical University of Denmark (DTU) Poster title “Folic acid fortification of rice: a study on parboiling conditions & consumer acceptance of fortified parboiled rice”  XI Asian Congress of Nutrition 2011 – organized by Singapore Nutrition and Dietetics Association Poster title “Micronutrient fortification through parboiling of rice”

 Food Science Summer School Meeting 2011 – organized by Australian Institute of Food Science and Technology Oral presentation title “Consumer acceptance of folic acid-fortified parboiled rice”  3rd International Rice Congress – organized by International Rice Research Institute, Asia Congress and Agroviet in Vietnam Poster title “Predictive modelling of traditional parboiling technique for value-addition in rice with folic acid”  60th Australian Cereal Chemistry Conference organized by Royal Australian Chemistry Institute in Melbourne Poster title “Impact of folic acid- fortification of rice using parboiling on colour of rice”  The 1st International Vitamin Conference – organized by the Technical University of Denmark (DTU) in cooperation with Øresund Food in Copenhagen Poster title “Fortification of folic acid in rice under parboiling”

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Chapter 1 Introduction

CHAPTER 1

INTRODUCTION

1.1 Background

Rice (Oryza sativa L.) is one of the dominant staple crops in the world, particularly for the humid tropics across the globe (Childs, 2004). Global production of rice was estimated at 650 million tonnes in 2007 ( International Rice Research Institute, 2009b).

Figure 1-1 shows the world rice production and consumption from 1960 to 2007. Over the past five decades, except for the decline in rice production between 2000 and 2003, there has been a marked steady increase in rice production. Since 1960, Asia produced over 90 % of rice (International Rice Research Institute, 2009b). World rice consumption correlates closely with rice production trend. In the densely populated countries of Asia, especially China (128 million tonnes), India (92 million tonnes), Indonesia (36 million tonnes), Bangladesh (30 million tonnes), Vietnam (20 million tonnes), the Philippines (12 million tonnes), Myanmar (10 million tonnes), Thailand (10 million tonnes) and Korea (7 million tonnes), the amount of rice consumption in 2007 ranged between ~10 and 128 million tonnes of paddy. These Asian countries contribute from 2.4 % to as much as 30 % of milled rice consumption in the world (423 million tonnes) (c.f. Section 2.1).

Figure 1-1 Global rice production and consumption, 1961-2007 (Source: International Rice Research Institute (2009b)).

Chapter 1 Introduction

According to FAO (2009), rice provides as much as 20 % of the dietary energy supply worldwide. In Asian countries, more than 2 billion people acquire as much as 75 % of the daily caloric intake from rice (Fresco, 2005, FAO, 2009, Golden Rice Humanitarian Board, 2009). Rice is rich in carbohydrates, it also contains a moderate amount of protein, lipids and various vitamins such as thiamin, niacin and riboflavin (Fresco, 2005). Yet, it does not provide the full spectrum of micronutrients required for well-being of humans. The concentrations of the micronutrients are even less after milling. Little or no vitamin C, D or beta-carotene is found in polished rice. The concentration of iron provides 7 % of the RDI for pregnant women (Fresco, 2005, Storozhenko et al., 2007, Golden Rice Humanitarian Board, 2009). According to FAO (2008), 848 million people are undernourished. Of these people, 27 % live in India, 25 % in sub-Saharan Africa, 22 % in Asia and Pacific (excluding China and India), 14 % in China, and the rest scattered over Latin America and the Caribbean, East and North Africa and the developed countries. In addition to the three most common forms of micronutrient malnutrition, i.e. iron, iodine and Vitamin A (Allen et al., 2006), folate is a vitamin gaining more attention globally (Jacques et al., 1999, Honein et al., 2001). The significant roles of this vitamin include its potential ability in preventing abnormalities in early embryonic brain development and malformations of the embryonic brain or spinal cord, namely; Neural Tube Diseases (NTDs) (Czeizel and Dudas, 1992). Its involvement in nucleic acid synthesis and protein metabolism (Choi and Manson, 2002) and low folate status is associated with plasma homocysteine elevation, which is a risk factor for cardiovascular diseases (Wald et al., 2002). Micronutrient deficiencies underlay some of the most prevalent medical maladies across cultures, ethnic and socio-economic groups worldwide (Marshall and Wadsworth, 1994a). Yi et al. (2011) reviewed 14 cost of illness reports and 10 economic evaluations of folic acid based on 143 abstracts selected. Direct (e.g. drugs, hospitalizations and managing comorbidities etc.), indirect (e.g. loss of work time and costs due to premature loss of life) and caregiver time costs were the broad categories of economic burden highlighted as a result of NTDs. A reduction in the prevalence and incidence of folate

Chapter 1 Introduction deficiency, e.g. folic acid fortification, would therefore be economically beneficial in alleviating the burden mentioned.

The majority of rice consumed globally is milled white rice, both regularly milled and parboiled (Childs, 2004). Parboiled rice accounts for about 15 % of the world’s milled rice (Bhattacharya, 2004, International Rice Research Institute, 2009b). Parboiling is a hydro-thermal treatment which is aimed at inducing milling, nutritional and organoleptic improvement in rice (Bhattacharya, 2004, Patindol et al., 2008). The leading countries producing parboiled rice in the World are Asian countries, namely India, Bangladesh, Sri Lanka, Thailand, West Asia and countries of West Africa. They contribute to approximately 92 % of the total parboiled rice production. Central and South America and the Caribbean are the second leading parboiled rice producing countries, followed by a minority of production by the developed countries such as America, Italy and Spain (Bhattacharya, 2004). A large portion of undernourished people are from these major rice consuming countries. Similarly, the dominant countries producing the highest proportion of parboiled rice are those countries where undernourishment is severe. It is therefore essential to enhance the nutrient content of rice as it is their staple food.

Recently, Tulyathan et al. (2007) and Prom-u-thai et al. (2008) presented novel fortification approaches to fortify rice with minerals (iodine and iron, respectively) through parboiling. The authors added the minerals in the soaking water. Both studies reported that the parboiling process achieved a significant penetration of minerals from the fortificant solution through the outer layers into the endosperm of the parboiled rice grains.

Due to the existing parboiling infrastructure and marketing networks, no extra capital instalment of equipment for the fortification process and/or alteration of consumer consumption behaviour are needed if fortification or enrichment of rice can be done through parboiling (Prom-u-thai et al., 2008). It is therefore a cost-effective approach in alleviating the nutritional problems for these countries and populations. Furthermore, for

Chapter 1 Introduction the countries producing parboiled rice without undernourishment issues, successful development of the fortification technique can add value to the existing technology and may be an advantage for their export businesses.

The recent studies examined mineral fortification using a limited range of parboiling conditions. Other fortificants, such as vitamins, may behave differently from minerals and that has not yet been studied. No standardised parboiling condition has been agreed upon and the parboiling process is considered completed based on both the subjective determination of the quality of grains and consumer preference for the final product. Parboiling conditions may vary depending on the target populations’ preferences on the parboiled rice. The variations in the treatment conditions may likely influence the potential uptake and retention of fortificants in the rice grains. Parboiled rice quality may also be altered accordingly. Moreover, evaluation of the dietary folate bioavailability is a prerequisite for understanding the ultimate outcome of the fortified rice to human well- being. It is, therefore, noteworthy to investigate a wider range of parboiling condition in relation to the effect on the fortified parboiled rice in a systematic manner.

1.2 Aims

In order to fill the information gap and explore the potential of fortifying the vitamin into rice through parboiling, the specific objectives of the present study were as follows:

1. To study the feasibility of fortification through parboiling by measuring the retention of folic acid at different stages, i.e. after parboiling and milling - Chapter 3; 2. To optimise the fortification process by maximizing the retention of the micronutrient in rice by means of examining the different combination of processing conditions, namely fortificant concentration, soaking temperature, soaking duration and degree of milling - Chapter 4; 3. To study the change in physical properties of fortified parboiled rice in relation to the treatment conditions - Chapter 5;

Chapter 1 Introduction

4. To study the visual and organoleptic acceptance of fortified parboiled rice by the rice-eating consumers - Chapter 6; and 5. To investigate the short-term bioaccessibility and absorption of folic acid in fortified parboiled rice using in-vitro method, i.e. Caco-2 cells model - Chapter 7.

Chapter 2 Literature review

CHAPTER 2

LITERATURE REVIEW

Rice is one of the most widely consumed staple crops in the world (Bhattacharya, 2004). However, rice is a poor source of naturally occurring micronutrients. Micronutrient deficiencies are prevalent among individuals in the developing countries where the variation in the diet is limited (Kennedy et al., 2003, Garcia-Casal et al., 2005). The following review will give an overview of global rice production and pattern of consumption. It will also describe the impact of rice processing procedures on the nutrient loads and existing rice fortification methods to address the global micronutrient deficiency issues. Folates, in particular, will be discussed in terms of its chemistry, occurrence and need for human well-being. The methods for assessing bioavailability of folates will also be reviewed.

2.1 Rice production and consumption statistics

Global rice production and consumption from 1960 to 2007 is shown in Figure 1-1. Between 1960 and 1970, average growth in rice production was 3.6 % per annum and slowed down to 1.4 % in the last decade. Nonetheless, the absolute increase in world rice production should not be underestimated. World rice production has increased almost three-fold from ~220 million tonnes in 1960 to as much as ~650 million tonnes in 2007. The relative contribution of rice production and consumption from different countries are presented in Table 2-1 and Table 2-2. The top ten rice-producing countries coincide with those which consume the most rice globally. These ten countries alone, contribute to approximately 90 % of total rice production (and rice consumption) in the world. Therefore, any change in rice production in these countries could cause significant impact on the world’s rice economy (FAO, 2002). Between 2003 and 2007, growth in rice production was most rapid in China, Bangladesh and Vietnam. The growth rate was more than 10 % over the observed period.

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Table 2-1 Paddy rice production (million tonnes) in the world's top 10 producing countries, 2003-2007 (Source: USDA (2010)) 2003 2004 2005 2006 2007 China 161 (27.5)+ 179 (29.9) 181 (29.1) 182 (29.0) 186 (28.7) India 133 (22.7) 125 (20.9) 138 (22.2) 140 (22.3) 145 (22.4) Indonesia 54 (9.2) 54 (9.0) 54 (8.7) 55 (8.8) 57 (8.8) Bangladesh 39 (6.7) 38 (6.4) 43 (6.9) 44 (7.0) 43 (6.6) Vietnam 33 (5.6) 34 (5.7) 35 (5.6) 35 (5.6) 37 (5.7) Thailand 27 (4.6) 26 (4.3) 28 (4.5) 28 (4.5) 29 (4.5) South America 23 (3.9) 24 (4.0) 22 (3.5) 21 (3.3) 23 (3.6) Africa 18 (3.1) 19 (3.2) 20 (3.2) 22 (3.5) 22 (3.4) Myanmar 19 (3.2) 17 (2.8) 18 (2.9) 18 (2.9) 19 (2.9) Philippines 14 (2.4) 14 (2.3) 15 (2.4) 16 (2.6) 17 (2.6) Asia 529 538 564 569 587 World 585 598 623 627 647 + Value in parentheses indicates the percentage of contribution in relation to world production.

Table 2-2 Milled rice consumption (million tonnes) in the world's top 10 countries, 2003- 2007 (Source: USDA (2010)) 2003 2004 2005 2006 2007 China 134 (32.6) + 132 (32.5) 129 (31.4) 129 (30.9) 128 (30.3) India 86 (20.9) 81 (20.0) 85 (20.7) 87 (20.9) 92 (21.7) Indonesia 36 (8.8) 36 (8.9) 36 (8.8) 36 (8.6) 36 (8.5) Bangladesh 27 (6.6) 27 (6.7) 29 (7.1) 30 (7.2) 30 (7.1) Africa 19 (4.6) 19 (4.7) 20 (4.9) 20 (4.8) 20 (4.7) Vietnam 18 (4.4) 18 (4.4) 18 (4.4) 19 (4.6) 20 (4.7) South America 14 (3.4) 14 ( 3.4) 14 (3.4) 13 (3.1) 14 (3.3) Philippines 10 (2.4) 10 (2.5) 11 (2.7) 12 (2.9) 12 (2.8) Myanmar 10 (2.4) 10 (2.5) 10 (2.4) 11 (2.6) 10 (2.4) Thailand 9 (2.2) 9 (2.2) 10 (2.4) 10 (2.4) 10 (2.4) Asia 367 360 367 371 377 World 411 406 411 417 423 + Value in parentheses indicates the percentage of contribution in relation to the world consumption.

Global milled rice consumption has been steadily increasing since 1960, from 156 million tonnes to 423 million tonnes in 2007 (Figure 1-1) and it corresponds with world population growth. Rice is highly consumed in China, India, Indonesia and Bangladesh. Food and Agricultural Policy Research Institute (FAPRI) has produced multi-year projection reports annually on world agricultural sectors and commodity markets (including rice) since 1985 (FAPRI, 2011). In 2001, FAPRI projected that, between 2006 and 2007, total rice consumption would reach 423.6 million tonnes which agreed with the

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total rice consumption figure (423.00 million tonnes) reported by USDA (2010). The latest projection by FAPRI suggests that in 2020 world rice consumption will increase to 477.6 million tonnes. Of the major rice consumers China, India, Indonesia and Bangladesh, demand from China is gradually declining which may due to growth in economic status as diet diversifies towards more protein-based commodities. However, rice consumption from Indonesia and Bangladesh will continue to increase, with India being the second major consumer in the world.(Abdullah et al., 2005, FAPRI, 2011). The absolute amount of rice consumption will likely increase globally mainly due to the increasing world population, given the fact that the world population is projected to grow from 6.9 billion in 2012 to 8.9 billion in 2050. The increase in population is close to 30 % and the average annual population growth rate over these four decades will be about 0.77 % (Baik et al., 1997). This statistic highlights the importance of this staple food to these countries discussed. Furthermore, the expanding world population, the foreseeable demand on rice and finite natural resources such as farmland and water, these pointed out the need to improve the rice production through better farm management and postharvest treatment of rice to enhance the yield of rice.

2.2 Composition of rice

Figure 2-1 illustrates the major components of rice grain, which include: hull (husk), pericarp, seed coat, nucellus, embryo, aleurone layer, and endosperm (Marshall and Wadsworth, 1994a). The hull consists of 18-20 % by weight of the rough rice (Juliano and Bechtel, 1985). It physically protects the grain against insect infestation and against rapid changes in moisture content due to large humidity fluctuations in the external environment, it serves as an antioxidative defence system for the mature rice seed to germinate during storage (Champagne et al., 2004).

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Figure 2-1 Longitudinal section of rice grain (Source: Bhattacharya, 2004)

The content beneath the hull is botanically called caryopsis, which means an indehiscent and single-seeded fruit, and is commonly known as brown rice. Four distinctive layers covering the brown rice grains are the pericarp, seed coat, nucellus and aleurone layer (i.e. bran). Four vascular bundles comprise the spongy parenchyma layers of the pericarp, they run along the ventral (embryo), dorsal (back), and the two lateral sides of the grain (Champagne et al., 2004). Hoshikawa (1993) reported that the dorsal vascular bundle is the largest in diameter and is responsible for transporting nutrients to the seed during development. It may therefore allow the inward diffusion of external nutrients into the endosperm through the vascular bundle. Except for the starch content, compared with milled rice, all other constituents, i.e. macronutrients, minerals and vitamins, are higher in brown rice. The concentration of vitamins, minerals and fibre content, and lipid content are 2-10 times, 2-3 times and about 5 times higher than milled rice, respectively

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(Champagne et al., 2004). Despite its high nutritional value, consumption of brown rice worldwide is limited. Apart from the concern of storage, it is organoleptically less accepted by consumers than milled rice, and cultural reason may be another reason (Zeleznak and Hoseney, 1987).

Milled rice is produced by the removal of bran layer, polish (subaleurone layer and a small part of the starchy endosperm) and germ (embryo). Milled white rice, composed entirely of the starchy endosperm, has a starch content of almost 90 % (dry weight). The starch per unit weight in milled white rice is higher than any other milled fractions, namely hulls, bran and polish (Marshall and Wadsworth, 1994a, Champagne et al., 2004). White rice is a good source of protein and carbohydrate. However, the vitamin and mineral content of white rice is less than in brown rice unless fortified.

2.3 General rice processing from harvest to packaging

The post-harvest systems comprise of a series of operations which start from harvest to consumption (Mejia, 2003). Figure 2-2 illustrates the major stages in converting paddy to edible white rice which is the most widely consumed and profitable form.

2.3.1 Cleaning

Cleaning the paddy is to separate undesirable foreign matters or materials, other than grain particles, before the paddy is stored and processed (Step 4, Figure 2-2). Depending on the harvesting methods involved and the type of milling facilities available, different types of foreign matters or contaminants need to be removed from the grain. Examples are bulk straw, weed, chaff, empty kernels, very light and fine impurities such as stones and metals etc (Bond, 2004). The machines generally involved are (Bond, 2004): 1. Drum sieve – removes impurities that are considerably longer than grains; 2. Scalperator and aspirator – removes impurities that are longer and lighter / less dense than grains; 3. Paddy cleaner or stick machine – remove short straws;

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4. Destoner – removes stones, mud balls, and dense glass; 5. Magnetic separator – removes metals.

The main purpose of cleaning is to prevent the impurities from damaging the machines in the latter process. Less than 2 % of rice may be lost due to the mechanical impact involved in the cleaning process.

1. Harvesting 2. Threshing

4. Paddy Cleaning / De-stoning 3. Drying

5. Dehulling the Paddy 6. Paddy Separation

8. Grading or Classification 7. Milling and separation of white rice

9. Blending & Packaging 10. Cooking

Figure 2-2 Stages of rice post-harvest process at subsistence level

2.3.2 Dehulling and milling

“Dehulling” of paddy rice to brown rice involves the separation of the husk (18-28 %) from the rice kernel (72-82 % caryopsis) (Step 5, Figure 2-2). It is the major step in turning inedible paddy into edible brown rice. The hull is not edible to humans so the loss of the nutrient content in the hull should not be taken into account. However, dehulling disrupts the testa layer, aleurone and germ, in which lipases and oil are compartmentalized. The oil diffuses and reacts with lipases, triggering the hydrolysis of triglycerides to free fatty acids (Champagne, 1994). The downside of high free fatty acid accumulation is associated with rancidity from oxidative deterioration of bran oil,

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resulting in the production of secondary oxidation products (e.g. aldehydes, alcohols, ketones, epoxides etc.). These are responsible for off-flavours and off-odours (Champagne, 1994). The quality of the rice will be affected if lipolytic hydrolysis or oxidation occurs in brown rice.

Following the removal of husk, brown rice is subject to the removal of the germ and the bran (i.e. pericarp, seed coat, nucellus and aleurone layer and embryo) layers from the underlying starchy endosperm (Bond, 2004), resulting in white rice. This process refers to as “milling” (Step 7, Figure 2-2). It is one of the crucial stages in determining the nutrition content of the edible rice grain. According to Mejia (2003), a few factors contribute to the extent of losses in the edible portion of the grains, they are:

1. Variety of paddy— Depending upon the variety of the rice, long and slender rice is more susceptible to breakage than short grains during milling. 2. Condition of paddy at the point of milling— The most prominent component in brown rice is endosperm starch. Therefore any changes on the starch, e.g. fissure development before or after harvest, may influence the milling quality and the quality of end product. The desirable moisture content is 12 – 14 % and the moisture evenly distributed throughout the grain. 3. Degree of milling (DOM) required— DOM determines the extent of bran layer to be removed from the surface of brown rice kernels and results in the whiteness of milled grains (Yadav and Jindal, 2001). DOM relates to the duration of milling. Long duration of milling results in higher DOM, and vice versa. The bran layer is an excellent source of protein (12-15 %), lipid (15- 20 %), vitamins and minerals compared to other milling fractions, e.g. hull, embryo and polish (Bond, 2004). Hinton (1948) examined the distribution of thiamin in rice grain and supported that thiamin is more concentrated in scutellum (part of embryo structure) and aluerone, approximately 44 % and 35 % respectively. These structures are regarded as part of the bran. Therefore, the higher the DOM, the less vitamin and other nutrients can be

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retained in the finish. This phenomenon is supported by Padua and Juliano (1974) who reported that, in three successive millings, thiamin content is almost 4 times higher in the first milling fraction than the second fraction, and as much as 43 times greater than that in the third fraction. As DOM is associated with the whiteness of the rice grain, the colour of the product is in turn dependent on consumer preference. Whiter rice is more acceptable by the market, meaning there is controversy between nutritious product and consumer acceptance. 4. Type of rice mill used— Milling actions used to produce white rice from brown rice are abrasive cutting action or to apply pressure and movement between grains to enable frictional force to tear away the bran layers. Abrasive action produces less broken rice but the finish is relatively rough and the size of the germ is reduced rather than removed from the kernel. The abrasive action may physically penetrate into the starch endosperm while removing the innermost bran layer on the starchy endosperm (Bond, 2004). This means some starch may be lost while the bran is being removed. Economically, it is difficult to achieve optimum total milled rice yield with this type of milling. From a nutritional perspective, removal of the starch content will lower the caloric supply of the rice. The frictional milling method, due to the use of higher pressure, resulted in more broken rice. However, it has more precise control on the amount of bran to be removed. The finish is relatively smoother and more lustrous than the abrasive method (Bond, 2004). In this case the degree of milling can be better managed and hence the nutritional value of the white rice can be controlled. Excessive milling pressure can be avoided, therefore this method has higher total milling yield than the other milling action. 5. Sun-drying process— After the rice is freshly harvested or after post-harvest treatment, such as parboiling, the moisture content of the paddy is well above the condition for safe storage. Paddy needs to be dried to 12-14 % to prevent

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storage issues such as microbial contamination. Section 2.6.2.3 will discuss in detail the effect of sun-drying to the milling quality of rice.

2.3.3 Washing and cooking

Different households have different practices in preparing and cooking rice (Step 10, Figure 2-2). In Asian countries, washing of milled rice prior to cooking is a common practice. It is to remove bran, dirt and dust on the grains as rice is often kept in bins and that may be exposed to contamination (Juliano, 1993). Other common practices include cooking the rice without rinsing, soaking the rice prior to cooking (Ebuehi and Oyewole, 2008) or cooking the rice in excess water and then draining (Brooke, 1972). Table 2-3 illustrates the percentage of nutrient loss due to washing and cooking in excess water. It shows that the washing and cooking method adopted will lead to significant difference in nutrient retention in rice at the point of consumption.

Vitamins, such as thiamin, are water-soluble. Chitpan et al. (2005) observed that during cooking of thiamin-fortified rice, a slight loss of thiamin occurred but the main loss was due to the leaching of the vitamin into the cooking liquid. Shrestha (2003) reported that 49 % of folic acid was lost from the fortified rice during 60 s rinsing in thrice volume of water, and up to 86 % of folic acid was lost on 30 mins boiling in 1:20 rice to water ratio.

As reviewed above, quality and nutritional loss occur in the grain through the series of processes before it reaches the point of consumption. Post-harvest loss in edible portion, and therefore the nutrients, is inevitable. Nonetheless, optimising as many stages in the post-harvest system as possible will essentially minimize the losses and maximize the quality of crop when it reaches the final consumer. This leads to the origin of pre-harvest and post-harvest fortification.

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Table 2-3 Per cent nutrient losses during washing and cooking in excess water (Source: Juliano and Bechtel (1985)) Nutrient Washing Washing Cooking without washing and cooking Raw Brown Parboiled Milled Milled Brown Parboiled milled rice milled rice rice rice milled rice rice rice Weight 1-3 0.3-0.4 - 5-9 2-6 1-2 3 Protein 2-7 0-1 - 2 0-7 4-6 0 Crude fat 25-65 - - 50 36-58 2-10 27-51 Crude fibre 30 - - Crude ash 49 - - 16-25 11-19 29-38 Free sugars 60 - - 40 - - - Total 1-2 - - 10 - - - polysaccharides Free amino acids 15 - - 15 - - - Calcium 18-26 4-5 - 1-25 21 Total 20-47 4 - 5 - - - phosphorus Phytin 44 - - - - phosphorus Iron 18-47 1-10 - 23 - - - Zinc 11 1 - - - - Magnesium 7-70 1 1 - - - Potassium 20-41 5 15 - - - Thiamin 22-59 1-21 7-15 11 47-52 - - Riboflavin 11-26 2-8 12-15 10 3543 - - Niacin 20-60 3-13 10-13 13 45-55 - -

2.4 Folates and folic acid

2.4.1 History of folates

In 1931, Wills (1931) was the first to report that yeast extract/ marmite possessed an active agent that was curative for pernicious anaemia of pregnancy and tropical anaemia in regions of India. The author also drew attention to vitamin B complex, indicating vitamin B to be the potential curative factor. Ten years later, Mitchell et al. (1941) suggested the term “folic acid” (Latin, folium-leaf) to describe the active substance that was extracted from four tons of spinach. This active substance showed similar chemical and physiological properties that occurred also in liver, kidney, yeast and many kinds of

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green leaves in which vitamin B complex, especially vitamin B9, was abundant. Folic acid was later synthesized chemically by Angier et al. (1946). Sixty years after the publication by Wills (1931), Wald (1991) conducted a randomized double-blind trial over 33 centres in seven countries to study the supplementation of folic acid in relation to the prevention of Neural Tube Defects (NTDs). The study successfully proved that folic acid can prevent important birth defects and opened an important chapter for the significance of folic acid (Oakley and Tulchinsky, 2010).

2.4.2 Structure of folic acid and folates

Folic acid belongs to essential B group vitamins and is also known as pteroylglutamic acid (Angier et al., 1946, Gregory, 1996). As illustrated in Figure 2-3, folic acid is composed of three major components, i.e. a pteridine ring, p-aminobenzoic acid and glutamate moieties. Its structure is the backbone for various naturally occurring folate forms (Saxby et al., 1983, Gregory, 1996) , e.g. reduction at 5, 6, 7 and 8 positions and substitution on the 5 and/or 10 positions with methyl, formyl, formimino, methylene or methenyl group thatl give rise to folate forms occurring in nature (Figure 2-3). O'Broin et al. (1975) reported the differences in oxidative degradation of folate in relation to their structures. Folate activity could be destroyed via cleavage at the C9-N10 bond. Among the different folate forms, the pteridine ring of folic acid is fully oxidized which exists as a fully double-bonded conjugate system (Gregory, 1996). This difference potentially explains the greater stability observed in folic acid than the folates (Shrestha, 2003). The order of stability of folates is folic acid > 5-formyl-H4folate > 5-methylH4folate > 10- formyl-H4folate > H4folate. Owing to its stability, folic acid is the major form of folate used by the food processors to fortify foods and in dietary supplements (Gujska and Majewska, 2005, Philips et al., 2005).

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Figure 2-3 Structure of folic acid and folates (5-methylenetetrahydrofolate, tetrahydrofolate, 5- formyltetrahydrofolate and 10-formyltetrahydrofolate)

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2.4.3 Role of folates in human health

Folates are vital for cell division and homeostasis because of their roles as a methyl donor in a range of metabolic and nervous system biochemical processes, protein synthesis and DNA synthesis (Shane, 1989, Stanger, 2002, Garcia-Casal et al., 2005, Hao et al., 2008). As summarized by Bailey and Gregory (1999), the first crucial step in the folate metabolism cycle is the conversion of THF into 5,10-methylene-THF using 3-carbon of serine as the carbon source and turns into glycine (Figure 2-4). Part of the 5,10- methylene-THF formed undergoes irreversible enzymatic reduction by methylene tetrahydrofolate reductase (no. 1, Figure 2-4) into 5-methyl-THF, i.e. the same form of folate enters the cycle through trans-membranous transport. The methyl group on 5- methyl-THF is donated to cobalamin (vitamin B12) forming the intermediate methylcobalamin. Methionine synthase (no. 2, Figure 2-4) helps to transfer the methyl group into homocysteine (HCY, Figure 2-4), the amino acid metabolite, and thus forming amino acid methionine (MET, Figure 2-4. ) Besides protein synthesis, with the incorporation of ATP, methionine converts itself into S-adenosylmethionine (SAM), which serves as the primary methylating agent in over 100 methyltransferase reactions in the body (Bailey and Gregory, 1999).

As shown in Figure 2-4, folate also involved in DNA synthesis, 10-formyl-THF can directly provide carbon groups to C2 and C8 position of adenine and guanine for the biosynthesis purine, which is the building block of DNA (Stanger, 2002). 5,10- methylene-THF, being an active compound, converts into deoxythymidinemonophosphate (thymidylate) by losing one carbon unit to deoxyuridylate with the catalyst thymidylate synthase. It leads to the synthesis of pyrimidine (i.e. thymine) and limits DNA synthesis (Stanger, 2002). Purines and pyrimidine are the building blocks of DNA, which are required for mitotic cell division. Any reduction of folate availability therefore leads to impairment of cell proliferation. It highlights the importance of folates in maintaining the well-being of all humans, especially pregnant women where cell proliferation is essential for foetal development.

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Figure 2-4 Intracellular folate metabolism (1 = methylenetetrahydrofolate reductase, 2 = methionine synthase, 3 = thymidylat synthase, 4 = dihydrofolate reductase, 5 = serine hydroxymethyl transferase, THF = tetrahydrofolate, HCY = homocysteine, MET = methionine, DH = dihydrofolate (Source: Stanger (2002))

2.4.4 Prevalence of folate deficiency

As mentioned in Section 2.4.1, Neural Tube Defects (NTDs) are a severe form of birth defect related to folate deficiency. The association between folate deficiency and enhanced risk for NTD has been established and revealed by various studies (Wald, 1991, Krishnaswamy and Nair, 2001, Bailey et al., 2003). There are three major forms of NTDs, namely, spina bifida, anencephaly and encephalocele, which is the consequence of the failure of closing the neural tube during embryonic development and affecting the brain spine and spinal cord (Wald, 1991). Babies with spina bifida may survive, however, depending on the degree of severity of the condition they may suffer from paralysis, losing control in bladder and bowel, or mental retardation. Anencephaly refers to the incomplete development of bones and skull and partial or complete absence of brain.

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Most babies suffering from this disease are usually stillborn, but usually only live for about a week (Mulinare, 1995).

NTDs are important cause of mortality and morbidity, with greater than 300,000 new cases globally as a conservative estimate (Blencowe et al., 2010). Krishnaswamy and Nair (2001) reviewed the frequency of NTDs in India, USA, Canada, UK and China. The prevalence of NTDs in USA and Canada was the lowest, between 0.90 and 1.41 NTD in 1000 births. The NTD cases in China and UK ranged from 0.8 - 6.5 and 6.4–10.9 in 1000 births, respectively. From the 15 cities reviewed, the mean NTDs cases in India was 6.3 in 1000 births and the highest frequency was 18 NTD in 1000 births.

Moreover, different studies have been conducted to measure the prevalence of folate deficiency, apart from using NTD cases as the indicator. For instance, Hao et al. (2003) reported that about 40 % of the Northerners in China have plasma folate concentration lower than the reference cut off (6.8 nmolfolate/ Lplasma) whereas that of the Southerners was 6 % lower. Approximately 30 % Northerners and 4 % of Southerners have red blood cell folate concentration below the reference level (i.e. 363 nmolfolate/ Lplasma). Men had lower plasma folate concentrations than women in both regions studied. Among the pregnant women in India, around 40 – 50 % of them may suffer from some degree of folate deficiency according to the concentration of folates in their blood serum and red cell samples (Seshadri, 2001). In Bangladesh, roughly 8000 reportable births suffered from severe defects, including infantile paralysis, annually due to folate deficiency (Micronutrient Initiative and UNICEF, 2011). Based on the 5658 serum samples collected from infants, children, adolescents and pregnant women in Venezuela, the result showed that the prevalence of folic acid deficiency was more than 30 % for each group studied, and as high as 82 % in adolescents (Garcia-Casal et al., 2005).

From the available research, despite the vast variations in experimental design between studies, e.g. geography, age group, socio-economic background etc., the results highlighted the fact that folate deficiency is prevalent world-wide. Moreover, the

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populations who are affected by folate deficiency coincidently overlap with the major rice-consuming countries indicated in Section 2.1.

2.4.5 Food source of natural folates

Humans cannot synthesize folate (nor folic acid) so it is necessary to obtain this vitamin from the diet. The major sources of natural folates include green leafy vegetables, liver, yeast, legumes, wheat germ and egg yolk (Subar et al., 1989, Suitor and Bailey, 2000). NHMRC (1995) published a collective list of good sources of folates in foods (Table 2-4). Although folates appear to be present in a wide variety of food (Table 2-4 ), studies have shown that folate status did not increase significantly solely through the consumption of food with high folate content (Cuskelly et al., 1996). A Dutch research group reported that by following the Dutch recommended intake level of vegetables (≥ 200 g/ day) and fruit (≥ 2 pieces/ day) for a month, no effect was found on the plasma folate concentrations or homocysteine concentrations of 71 healthy women (Bogers et al., 2007). Possible explanations included the short intervention period, relatively modest increment in folate intake and the baseline plasma folate concentration of the intervention group was relatively high (Bogers et al., 2007). Furthermore, the stability and bioavailability of dietary folates may also explain the difficulty in improving folate status with nutrition (Castenmiller et al., 2000, Suitor and Bailey, 2000, McKillop et al., 2002).

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Table 2-4 Good sources of natural folates in foods (Source: NHMRC (1995)

# depends on cooking method; - approximately; * unfortified

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2.4.6 Stability of folates

In general, folates are recognized as one of the least stable vitamins (Gregory, 1996). The stability of folates is dependent on various processing parameters and conditions including thermal treatment, pH, reducing agent, oxygen concentration dissolved, food matrix effect and cooking method (Hawkes and Villota, 1989).

2.4.6.1 Temperature, pH and reducing agent

Several research groups have reported evidence of thermal degradation of different folate analogues. The temperature range studied was fairly extensive, varying widely between 20 °C and 160 °C (Ruddick et al., 1980, Day and Gregory, 1983, Mnkeni and Beveridge, 1983, Barrett and Lund, 1989, Nguyen et al., 2003, Butz et al., 2004). Folic acid is more heat stable compared to most of the other folate analogues (Day and Gregory, 1983, Nguyen et al., 2003). Its retention after processing at 100 °C for 2 h, 120 °C for 20 mins and 140 °C for 15 mins was 75-92 %, 68-85 % and 40-55 %, respectively (Day and Gregory, 1983). The degradation rate constant of folic acid was shown to be less temperature dependent relative to 5-methyl-THF, which was one of the most common folate analogues being studied (O'Broin et al., 1975, Day and Gregory, 1983, Mnkeni and Beveridge, 1983, Barrett and Lund, 1989, Viberg et al., 1996, Nguyen et al., 2003, Butz et al., 2004, Indrawati et al., 2004).

Another important influencing factor being studied was pH. Indrawati et al. (2004) reported that the thermostability of 5-methyl-THF was maximal at pH 7. The result concurred with the study by O'Broin et al. (1975), which reported that 5-methyl-THF was most stable in alkaline condition. Thermostability started to decrease below pH 4 (Indrawati et al., 2004). A similar result was observed for folic acid, when the stability dramatically decreased below pH 4. It was reported that at pH above 5, over 700 h were needed to achieve 50 % degradation; whereas below pH 4, less than 80 h would be enough to reach 50 % degradation (O'Broin et al., 1975). The situation altered significantly when ascorbic acid, which acts as an antioxidant, was present. Indrawati et

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al. (2004) stated that at pH 4, with the addition of ascorbic acid (0.5 mg/ g), the thermostability of 5-methyl-THF was largely increased and the activation energy value was decreased by 70 %. Ascorbic acid exerts multiple protective effects on folates through its actions as an oxygen scavenger, reducing agent and free radical scavenger (O'Broin et al., 1975, Butz et al., 2004). O'Broin et al. (1975) stated that comparing ascorbic acid to 2-mercaptoethanol, which is also an antioxidant, ascorbic acid was clearly more superior as it afforded better protection at lower concentration. Ascorbic acid could protect folates from heat treatment at a concentration of as low as 0.1 % concentration. Similar results were suggested by Day and Gregory (1983). They processed different model systems (including unfortified and ascorbic acid fortified) at 100 °C . Significant protection by ascorbic acid was noticeable beyond 100 mins. At 180 mins, folic acid retention was 80 % for ascorbate fortified model system whereas that of unfortified system was 60 %. Ascorbic acid also protected 5-methyl-THF, yet the effect was less pronounced for folic acid. The difference in the retention of 5-methyl-THF was around 89 % and 80 % for ascorbate fortified and unfortified model systems.

2.4.6.2 Oxygen concentration

The presence of oxygen is shown to be a contributing factor to folate and folate derivatives degradation (Ruddick et al., 1980, Day and Gregory, 1983, Mnkeni and Beveridge, 1983, Barrett and Lund, 1989, Viberg et al., 1996, Nguyen et al., 2003). Barrett and Lund (1989) studied the effect of oxygen on thermal degradation of 5-methyl- THF in phosphate buffer. In comparison to the buffer system with no dissolved oxygen, the degradation of 5-methyl-THF was more drastic with the presence of oxygen. Both conditions illustrated the same thermal degradation trend as expected, i.e. the lower the heat treatment, the higher the folate retention. In the case with dissolved oxygen, the percentage of 5-methyl-THF retained in the system was less and the folate was lost at a shorter processing time in contrast to the corresponding thermal treatments without the presence of oxygen. At a fixed processing time, degradation could be as much as 50 % more in the oxygen containing system.

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2.4.6.3 Food matrix

A number of studies done were based on buffer systems or model systems. The advantage of using artificial systems is that complete control of all different process parameters is feasible, leading to more accurate data for model identification (Nguyen et al., 2003). However, the matrix of foods is far more complicated than the artificial systems in a physical or chemical sense. It is reasonable to expect that a certain degree of discrepancy will result in response to the different processing parameters applied. Food samples such as orange juice (pH 3.76) and kiwi puree (pH 3.41) exhibited high stability over different thermal treatments, i.e. 70-120 °C for a fixed treatment time of 30 mins (Butz et al., 2004, Indrawati et al., 2004). The concentration of 5-methyl-THF was not or only slightly decreased during the treatments.

2.4.6.4 General food processing methods

McKillop et al. (2002) highlighted that handling, processing and cooking methods can degrade folates, especially naturally-occurring folates, in food and hence influence their availability. Hoppner and Lampi (1993) studied the pre-treatment of legumes, i.e. pre- soaking stage, before cooking. It was reported that the extent of pre-soaking affected the retention of folate in food (i.e. legumes) for a constant cooking time. Higher retention was found for long overnight soaking than a quick soak (2 mins boiling in hot water and then left soaking for 1 h). About 35 % of folates were retained after long soak whereas only 19 % was retained after quick soak. The additional heating was speculated to cause the fracturing of the legume seed and their outer coat that led to leaching. Numerous studies suggested that the leaching of folate into the soaking water tended to be one of the major causes of loss (Dang et al., 2000, Arcot et al., 2002). Apart from soaking, cooking methods such as boiling, steaming, grilling and baking were investigated (Vahteristo et al., 1998, McKillop et al., 2002). Boiling of spinach resulted in 51 % of folate loss whereas no loss resulted due to steaming. Prolonged grilling of beef (for up to 16 mins) did not result in significant drop in folate in comparison to raw beef. The folate retention

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result was similar for potato before and after boiling, with no effect whether or not the skin was on upon boiling (McKillop et al., 2002). On the contrary, Stea et al. (2007) reported that boiling potatoes resulted in 41 % loss of folates. The discrepancy in results may be due to the source of food supply.

2.4.6.5 Effect of cooking rice in relation to folates stability/ retention

As far as the stability of folates in rice is concerned, the process of rice preparation itself introduced multiple factors (described above) which may lead to the loss or retention of folates, and therefore influencing the final concentration of folate in rice. Generally, the cooking of rice involves the addition of water to rice and subjecting it to thermal treatment. As temperature increases, the level of dissolved gases in the system (i.e. rice and water matrix) constantly alters. Some consumers may prepare rice using the absorption method (add the appropriate amount of water and cook the rice until all the water disappears) and others may use the excess-water cooking method (add excess amount of water to cook the rice and drain off the excess water at the end) (Daomukda et al., 2011, Kam et al., 2012b). Folates that may potentially leach out of rice will be discarded should excess-water cooking method is used. In some countries or some dishes, raw rice may cook together with other food ingredients (e.g. a West African dish called Jollof (Tomlins et al., 2007)). The pH of the system is likely changes with cooking time. Folate protecting substances, e.g. antioxidants, may be present in the food system which may enhance the stability of folates, and vice versa.

2.5 Methods of rice fortification

In response to the nutrient deficiency issues in the rice-consuming populations due to rice processing, several rice fortification technologies have been developed. As opposed to other vehicles used for delivering micronutrients, such as salt (Zimmermann et al., 2003), sugar (Li et al., 2011) and fish sauce (Van Thuy et al., 2005, Watanapaisantrakul et al., 2006), rice fortification faces different challenges. Lotfi and Britton (1998) reviewed the difficulties involved in fortification or enrichment of rice. Rice is essentially consumed as

Chapter 2 Literature review

whole kernels and only occasionally consumed as flour. The fortificant is expected to adhere to or penetrate into the rice in order to consider the method successful. Moreover, rice is often washed thoroughly before cooking and some consumers may cook the rice in excess water and drain. The method of fortification is therefore required to produce fortified rice that is non-rinse and rinse-resistant.

2.5.1 Dusting

This technology has been used mainly in the United States. The fortificant presents in powder form and the “dusted” fortificant is expected to be attached onto the rice surface based on electrostatic forces. Alvai et al. (2008) reported that in order to improve the stability of the micronutrients and the flowability of the fortificant powder blend, corn starch and other ingredients are added to the fortificant blend. Since the fortificant is attached on the rice surface by electrostatic forces, rinsing of rice prior to cooking or draining of the cooking water are not advised to the consumers because the fortificant will be easily lost upon these treatments. This technology is therefore not recommended to countries where rice is washed or cooked in excess water.

2.5.2 Surface coating

The principle of the coating method is placing the milled rice in a rotary cylinder and spraying the rice with a liquid solution which is made of fortificant and ingredients such as waxes and gums (Lotfi and Britton, 1998, Alavi et al., 2008). Fortificant solution may repeatedly spray onto the rice until a desirable fortification level is achieved. Fortified rice is subsequently dried to the safe moisture limit. The rice premix is then blended with commercial rice in order to achieve the desirable fortification level. In comparison to the first patented coating method for cereal products developed by Hoffmann-La Roche (Furter and Lauter, 1949), recent studies improved the method by simplifying the process (Bramall, 1986) and the use of alternative coating materials (Tulyathan et al., 2004, Chatiyanont and Wuttijumnong, 2007). The fortification of folic acid in rice using

Chapter 2 Literature review

coating method in particular, has been described by Shrestha et al. (2003). Shrestha (2003) studied extensively the effect of edible coating materials on folic acid retention. The optimal ingredients for coating were reported to be ethylcellulose, pectin, and locust bean gum and its composite mixtures. In general, the functions of coating layer are to provide physical, chemical and microbiological protection to foodstuff which improves the conservation, distribution and marketing of the product (Falguera et al., 2011). Whole cereal fortification using coating results in higher nutrient retention after cooking in contrast to the dusting method (Shrestha et al., 2003, Chatiyanont and Wuttijumnong, 2007). To produce fortified rice premix on an industrial scale, the equipment potentially required includes fortificant suspension tank, rotary drum and rotary screen (Bramall, 1986). Investment cost may therefore be one of the disadvantages of this method (Tulyathan et al., 2004).

2.5.3 Hot/Cold extrusion

The “fortified rice” produced using this method is so-called fake grains or artificial rice because the grains are made by adding the micronutrients to rice flour to form a dough, which is then extruded with equipment equivalent to noodle manufacturing in order to form a rice-shaped kernel. The fortified artificial rice is then mixed with regular milled rice to achieve the target fortification level (Dexter, 1998, Lotfi and Britton, 1998). Two processes emerged from this technology, namely hot and cold extrusion. For the hot extrusion method, the heated barrel jackets produce relatively high temperatures (70 and 110 ˚C) which result in fully or partially pre-cooked artificial rice kernels that have a similar appearance (e.g. sheen and transparency) to regular milled rice kernel (Alavi et al., 2008). For the cold extrusion method, no additional thermal energy is supplied to the system apart from the heat generated during the process itself, relatively low operational temperature, i.e. 70 ˚C, is primarily used. The artificial grains formed are uncooked, opaque and easier to distinguish from regular rice (Alavi et al., 2008). Ultra Rice is the well-known fortified rice produced using cold extrusion method. Initially, Ultra Rice was mainly used as a vehicle for delivering Vitamin A (Lee et al., 2000, PATH, 2007, Li et

Chapter 2 Literature review

al., 2009), iron, zinc, folic acid and thiamin were subsequently investigated to be included as fortificants (PATH, 2007, Li et al., 2008, Beinner et al., 2010). As the fortification process uses the noodle- or pasta-extruding facilities, the production cost and the need for substantial on-site capital investment will remain low should the equipment be already available.

2.5.4 Biofortification

Biofortification has been defined as a process in boosting the bioavailable concentrations of essential elements in edible portions of crop plants through 1) agronomic intervention or 2) genetic selection (White and Broadley, 2005). In terms of agronomic intervention, the most widely studied minerals were iron and zinc (Cakmak, 2008), followed by calcium, magnesium, iodine, selenium and copper (White and Broadley, 2005). Agronomic practice, e.g. using fertilisers containing trace elements to fortify the crop, is relatively more difficult for developing countries due to the cost and enhancement of complex compounds such as provitamin A being a challenge (Sautter et al., 2006). Moreover, fertilisation of these crops may increase leaf mineral concentrations and enhance yields but the boost in minerals may not always concentrate in fruit, seed or grain to the desired level.

In addition to the traditional breeding methods, biofortification through biotechnology and transgenic strategies is an alternative. The most notable example of biofortification in rice is the Golden Rice (Ye et al., 2000, Potrykus, 2001, Hirschi, 2008). Golden rice is genetically engineered rice that combats vitamin A deficiency in rice-eating populations. Regular rice plants synthesize β-carotene in vegetative tissues but not in grains. By the introduction of two genes, phytoene synthase (psy) and carotene desaturase (crtI ), the reconstituted pathway allows the accumulation of β-carotene in the endosperm, i.e. the edible portion of rice (Ye et al., 2000, Paine et al., 2005). HarvestPlus, a part of the Consultative Group for International Agricultural Research (CGIAR) Research Program on Agriculture for Nutrition and Health, is aimed at reducing micronutrient malnutrition

Chapter 2 Literature review

by breeding higher levels of micronutrients in key staple foods by biofortification. The crops targeted were beans (fortified with iron), maize (Vitamin A), wheat (zinc). Most recently in 2013, biofortified rice with elevated zinc level was released to Bangladesh and India (HarvestPlus, 2013). Publication from HarvestPlus also nominated folate fortification is effective in preventing NTDs (Hertrampf and Cortes, 2004).

Apart from the above micronutrient fortification, folate fortification in rice using metabolic engineering has also been investigated (Bekaert et al., 2007, Storozhenko et al., 2007). The biofortification technology involved the overexpression of two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. The maximum concentration of folate in the rice seeds is approximately 1700 µg / 100g fresh weight, which exceeds the classical daily folate dosage (400 µg) by fourfold (Mastroiacovo and Leoncini, 2011). Given that the biofortified rice is produced by improving the efficiency of biochemical pathways or reconstructing the selected pathways, these modifications locate in the individual rice seed which will give rise to a new plant. The cascade effects therefore may mitigate the micronutrient deficiency issues and ensure the sustainability of the micronutrient-rich product. However, controversial debate has arisen in response to the new technology (Stein et al., 2008). Greenpeace (2009) argues that genetic engineering technology is a lengthy process and the changes in the plant chemistry may cause serious health concerns in the long term. There is a possibility that the genetically modified crop may behave as a weed and the unwanted genes may transfer to wild relatives, therefore disturbing the environment and the ecosystem. Consensus on the effect of biofortified food on humans is yet to be achieved. A comprehensive system for evaluating societal, environmental, regulatory, agricultural and health issues is required to assess the impact of biofortified food (King, 2002). The technology development and conflicting opinions are the challenges faced by this fortification approach.

Chapter 2 Literature review

2.5.5 Parboiling

Parboiling, is a post-harvest treatment of rice that has been practised over a hundred years (Braddon, 1907, Fraser and Stanton, 1909). However, only recently researchers started to explore the possibility of adapting the parboiling process as a method for fortification in rice. In 2006, Tulyathan et al. (2007) first reported that iodine was successfully fortified in brown rice (Thai long grain Charinart 1 variety) through parboiling. Two forms of iodine, potassium iodide and potassium iodate, were added to the 70 ◦C soaking water followed by the addition of brown rice. The soaked rice was subsequently steamed, dried and milled. The study showed that about 19 % of the fortificant penetrated into the rice, regardless of the form of fortificant. The author postulated that iodine (iodide or iodate ions) had diffused into the endosperm through internal cracks developed within the rice grain. Closely after the study by Tulyathan et al. (2007), Prom-u-thai et al. reported the use of parboiling to fortify two different minerals, iron (Prom-u-thai et al., 2008) and zinc (Prom-u-thai et al., 2010). Instead of using brown rice as the starting material, paddy grains were used for both studies. The fortificant of varying molar concentration was added to the soaking water. Two soaking temperatures were employed for the two studies, i.e. 25 ◦C (room temperature) for 24 h and 60 ◦C (elevated temperature) for 6 h, for iron and zinc, respectively. The soaked paddy grains were heated under pressure, sun-dried and then milled to different degrees. Vast difference in the uptake retention was observed for the milled fortified rice. Approximately 1 % of iron penetrated into the endosperm whereas that for the zinc fortified rice it was up to 20 %. It was difficult to compare the two results because of the major differences in the parboiling conditions studied. Nonetheless, both studies showed that parboiling was a feasible process for fortification. Prom-u-thai et al. (2008) used Perls Prussian blue solution to dye the fortified parboiled rice in order to visually identify the distribution of iron in the rice. It was reported that the fortificant (iron) was likely transported from the soaking solution through the husk and entered the grain through the dorsal vascular bundle present in the kernel. This fortification method is beneficial and cost-effective to populations where parboiling is already in practice since no additional investment on infrastructure is needed.

Chapter 2 Literature review

2.6 Parboiling methods and conditions

2.6.1 History of parboiling

Parboiling of rice is thought to have originated in ancient India (Ramiah, 1937, Hargrove, 1994). Bhattacharya (2004) thoroughly reviewed the nutritional benefit of parboiled rice. The author reported a number of ancient incidences related to machine-milled rice. In the 19th century, Chinese immigrants in Malaya consumed milled raw rice while the Tamil immigrants preferred parboiled rice (Braddon, 1907). Surprisingly those who consumed parboiled rice did not suffer from beriberi, i.e. thiamin deficiency. The rice was called “cured rice” by Braddon (1907). Two years later Fraser and Stanton (1909) revisited anti- beriberi property of parboiled rice. They recruited 300 Javanese labourers and divided them into two groups, i.e. parboiled rice-eating and white rice-eating. Beriberi occurred within six-months in the white rice-eating group, and once the rice was interchanged, ceased within the former white rice-eating group but appeared in the second group. They concluded that no evidence was obtained to show that any food other than rice was the possible source of a causative agent of beriberi. Similarly, Sharp and Sharp (1994) highlighted the high incidence of beriberi in areas of the Orient where diet was composed largely of milled rice. Beriberi was absent in specific areas where parboiled rice was consumed instead. Although this may not be the fundamental reason how rice parboiling began, the dramatic discovery of the health benefit of parboiled rice created worldwide attention and the practice has spread throughout the general area of India, Bangladesh and Sri Lanka (Hargrove, 1994).

2.6.2 Processes of parboiling

Parboiling, as the name suggests, means “par”-tially “boil” the rice (Bhattacharya, 2004). It is a treatment of rice after harvest and before rice milling that mainly consists of three major steps, which are soaking, heating and drying (Bhattacharya and Ali, 1985, Biswas and Juliano, 1988, Guha and Ali, 1998, Elbert et al., 2001b, Miah et al., 2002,

Chapter 2 Literature review

Bhattacharya, 2004). The three major elements involved in this process are water, heat and time.

2.6.2.1 Soaking

The first step in parboiling is to sufficiently hydrate the rice grains by soaking in water for a period of time to allow gelatinisation of starch upon subsequent heating (Bhattacharya, 2004). Different hydration temperatures have been tested in previous studies (Padua and O. Juliano, 1974, Damir, 1985, Kar et al., 1999, Bhattacharya, 2004, Manful et al., 2007, Himmelsbach et al., 2008, Sareepuang et al., 2008). The soaking temperature employed consists of two broad groups, i.e. soaking temperature below gelatinisation temperature of rice and soaking temperature above gelatinisation temperature of rice.

Soaking temperature below the gelatinisation temperature is often referred to as cold soaking (Bhattacharya, 2004). Temperature employed ranged from ambient to about 60- 65 °C . The soaking duration varies from 12 h to 1 or 2 days (Guha and Ali, 1998, Roy et al., 2003, Igathinathane et al., 2005). The advantage of low temperature soaking is that less control of soaking time is required since the equilibrium moisture content achieved under these conditions does not exceed 30-32 % wet weight basis. The chance of over- soaking or splitting of the husk is minimized and hence less solid-loss results (Luh, 1991, Soponronnarit et al., 2006). The downsides were that long soaking time is not economical from a production point of view. Moreover, low temperature soaking, especially at room temperature, is a slow process that favours microbial contamination or fermentation which may lead to the development of off-odour (Bhattacharya, 2004, Igathinathane et al., 2005).

The alternative is to implement the soaking temperature of above 70 °C which is above the gelatinisation temperature (Kar et al., 1999, Larsen et al., 2000, Manful et al., 2007, Himmelsbach et al., 2008). The soaking temperature may reach as high as 90 °C (Manful et al., 2007, Himmelsbach et al., 2008). The elevation of soaking temperature is an

Chapter 2 Literature review

advance of traditional parboiling and is often used in the commercial parboiling process because it reduces the soaking duration from over 24 h (Larsen et al., 2000, Prom-u-thai et al., 2008) to less than 6 h (Larsen et al., 2000). A long soaking time is a limitation within the industry (Chitpan et al., 2005). The significant reduction in soaking time is economically favourable due to the increased product throughput (Pillaiyar et al., 1996). Apart from reducing the soaking time due to the increased rate of water diffusion, the application of the elevated soaking temperature likely results in the change in the rice structure, i.e. the softening of the bran layer, due to the higher soaking temperature (Cheevitsopon and Noomhorm, 2011). Control in maintaining the high water temperature and soaking time is necessary. Over-soaking may cause splitting of the husk, leaching of rice content and deformation of grains. The high moisture gradient from the soaking water and the grain would cause the gelatinised shell to continue absorbing water, achieving the total grain moisture content to exceed 30-32 % before the core of the grain fully hydrated (Bhattacharya, 2004). Soaking of the paddy until saturation is a usual practice (Miah et al., 2002, Bhattacharya, 2004). However, hydration of the paddy with lower moisture content is still enough to reach gelatinisation if the grains are subsequently steamed under pressure (Bhattacharya, 2004).

2.6.2.2 Heating

An example of the way of heating is steaming. The purpose of steaming is to further complete the gelatinisation of starch after soaking the rice grains (Bhattacharya, 2004). Steaming (heating with saturated steam) at atmospheric pressure is the most widely used condition after the soaking of rice grains (Roy et al., 2003, Ahiduzzaman and Sadrul Islam, 2009). Mild heating at between 80 ◦C and less than 100 ◦C (Islam et al., 2002a, Bello et al., 2006, Lamberts et al., 2008) and severe heating conditions of above 100 ◦C under elevated pressure (Damir, 1985, Miah et al., 2002, Himmelsbach et al., 2008, Lamberts et al., 2008, Prom-u-thai et al., 2008, Sareepuang et al., 2008) have also been investigated on a laboratory and industrial scale.

Chapter 2 Literature review

Apart from using steaming as the means of heating in parboiling, other heating systems were explored to shorten the processing time and/or to improve the quality of the end- product. Examples include electrical resistance heating and thermic fluid heating. Electrical resistance heating, also known as ohmic heating, is done by passing electricity through the food material which serves as an electrical resistor. The electrical energy is transformed into heat energy and results in rapid heating (Ramaswamy et al., Vasan and Ganesan, 1981, Sivashanmugam and Arivazhagan, 2008). In addition to the enhanced milling performance of the parboiled rice compared to using conventional parboiling conditions, Sivashanmugam and Arivazhagan (2008) suggested that electrical resistance heating would not impose any chemical toxicity to rice quality, although the risk was deemed low compared to other heating methods. Pillaiyar et al. (1996) studied the use of pre-heated thermic fluid, Servotherm oil with a flash point of 208 ◦C and viscosity index of 90 minimum, as the heating medium of the soaked rice. The major advantage of this heating method is that the moisture content of the parboiled paddy was lower than that of heating using open steaming (atmospheric pressure). Lower initial moisture content of the parboiled paddy could reduce the drying time of rice and hence energy consumption could be lowered.

Generally, the indicative moisture content at the end of steaming varies between 30 and 38 % wet weight basis, depending on the heating method chosen (Bhattacharya, 2004, Bello et al., 2006). In terms of the degree of gelatinisation (DOG), no specific cutoff has been suggested. DOG of as low was 40 % was suggested to be sufficient for maximum head rice yield for the two rice varieties (Lemont and Tebonnet) (Marshall et al., 1993) although the commercial parboiled rice usually shows the DOG of above 90 % (Himmelsbach et al., 2008). The desirable DOG appears to depend on the rice variety used and the visual appearance of any white bellies, which will be discussed further in Section 2.7.1.

Chapter 2 Literature review

2.6.2.3 Drying

Drying is the final step in the parboiling process. Usually the moisture content for the regular parboiled paddy can reach up to 38 % (Bhattacharya, 2004). The moisture content needs to be reduced to around 12-14 % for safe storage and milling. Sun-drying is a common practice in local traditional parboiling (Roy et al., 2003, Roy et al., 2006, Ahiduzzaman and Sadrul Islam, 2009, FAO, 2012). Rapid drying in the sun was found to give high rice breakage during milling (Ali and Pandya, 1974) but damage (such as cracks) occurs only when the critical moisture content of 16 – 18 % was reached and increased markedly upon further drying (Bhattacharya and Swamy, 1967, Bhattacharya and Ali, 1985). Slow drying, such as shade drying, is an alternative to sun-drying and gives excellent milling quality, only the drying time is prolonged due to the less severe moisture gradients (Velupillai and Verma, 1982, Islam et al., 2002a, Lamberts et al., 2008). Zoerb (1958) suggested that moisture content was the greatest influence on the mechanical properties of grains. Starch is brittle a t a moisture content of < 15 % and behaves like plastic at a higher moisture content (Rhind, 1962). This may explain the rapid increase in rice breakage at a moisture content of < 15 %. Perdon et al. (2000) and Cnossen and Siebenmorgen (2000) also examined rice drying in relation to the concept of glass transition temperature of rice. The existence of a temperature or moisture gradient within a kernel may generate regions with different mechanical properties (e.g. expansion coefficients, specific volumes). Upon drying, the region close to the surface has lower moisture content compared to the core of the grain. The intra-kernel moisture gradient creates uneven glass transition zone within the kernel. The differences in properties between regions result in expansion/contraction in the different glass transition zone and the stresses created may be sufficient to lead to fissure/crack of kernels, and hence compromise the milling quality of rice ( Yang et al., 2003).

In order to solve the problem, the concept of rice tempering is introduced. Rice can be dried as quickly as possible to ~16 % (i.e. when the rice is at the rubbery region), and the rice is allowed to temper for 4 to 24 h for the re-distribution of moisture within the grains

Chapter 2 Literature review

before further drying (Cnossen and Siebenmorgen, 2000, Elbert et al., 2001a, Yang et al., 2003, Bhattacharya, 2004), this minimises the chance of non-uniform glass transition at different parts of the rice kernel. The moisture from the core of the grain diffuses to the surface of the grain and therefore the drying rate of the second-stage drying is facilitated and rice quality can be improved compared to continuous drying. In Southeast Asia, a few popular types of hot air dryers are used as they reduce drying cost, benefit grain quality and reduce weather exposure. They are flat bed dryer, box dryer and re-circulating batch dryer (International Rice Research Institute, 2009a).

Apart from drying rice with hot air, other drying means have been studied. Soponronnarit et al. (2006) studied the use of superheated steam fluidization as a technique for drying. Fluidization technique is a drying method that allows air and solid particles to be rigorously mixed. The advantages of using superheated steam drying are that, by recycling the exhaust steam, energy supplied to the dryer can be reduced. Also, the exhaust steam due to the evaporation of moisture inside the solid can be recovered so energy can be further conserved. Microwave heating is also gaining popularity as it saves energy and conserves the nutritional and sensory attributes of food (Sumnu, 2001, Khan et al., 2011). Marshall et al. (1993) studied the effect of parboiling rice using conventional and microwave energy. The processing variables were found to be more precisely monitored using microwave energy than the conventional method. Far-infrared radiation is another drying method being studied because of its fast transient response and simplicity in equipment installation (Ratti and Mujumdar, 1995, Meeso et al., 2008).

It is worth noting that apart from starch gelatinisation and retrogradation which may happen to starch as described above, starch may also undergo glass transition and recrystallization (Zeleznak and Hoseney, 1987 , Baik et al., 1997). As soaking proceeded, the uneven distribution of moisture from the core to the surface of the rice kernel and the subsequent heating to rice may result in localised starch gelatinisation and glass transition (Cnossen and Siebenmorgen, 2000). Aside from the mechanism behind parboiling, it is

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clear that migration of water was present during soaking and the moisture was retained in the endosperm.

2.6.3 Parboiling devices

Parboiling of rice has practised for a century. Nowadays, traditional parboiling processes are commonly practised in local villages (Roy et al., 2003). The parboiling device consists of pottery to boiler, used for direct or indirect heating. The eastern part of India (West Bengal) and Bangladesh employ the same parboiling processes. Roy et al. (2006) conducted a survey investigating the local parboiling process at Gazole in the Malda district of West Bengal, India. Figure 2-5 illustrates the equipment of the local vessel for parboiling. The vessel process is used at household level and is capable of producing 0.5 to 1.2 tonnes per batch of parboiled rice. A clay-pot (pottery), which is known as a char or masonry tank locally, is used for soaking. The reservoir has a drain cock to remove the excess water at the end of soaking before the steaming stage. The soaked paddy rice is poured into another vessel (i.e. steaming vessel) and exposed to direct heating where fires are lit underneath. The vessel is made of aluminium sheet and the oven is made of clay or bricks. In Benin, West Africa, a similar parboiling setup is used. A cooking pot made of clay is used for soaking, which is placed on the stove to achieve a desirable soaking temperature. Water is drained and the paddy is transferred to a parboiler, i.e. equivalent to the vessel except it is perforated and is to a shape that can fit on top of the cooking pot. The cooking pot is filled with water (without touching the bottom of the parboiler) and placed on the stove to generate steam which steams the soaked paddy ( FAO, 2012).

In addition to the domestic scale production of parboiled rice described above, Roy et al. (2006) reported that small-boiler and medium boiler processes are used for commercial purposes only (Figure 2-6 and Figure 2-7). The batch capacity of the process varies from 2 to 4 tonnes and 5 to 10 tonnes, respectively. Again, a masonry reservoir is used for soaking the paddy. A shallow tube-well is the source for water to pump into the reservoir from a nearby pond. Indirect heating is applied to these parboiling processes, i.e. not

Chapter 2 Literature review

directly exposed to the stove. Steam is generated in a boiler made from a used barrel or mild-steel sheets and conducted to the paddy in the hopper through connecting pipes for pre-steaming and steaming. The small boiler process is regarded as relatively more dangerous than the medium boiler process because steam flow is controlled by valves and there is no pressure gauge, water meter or chimney used in the former process. The steamed paddy is then unloaded to dry on the drying yard. Natural sun drying is usually chosen for the traditional parboiling process. A concrete floor or any clean surface is preferred to dry the parboiled paddy on instead of clay, earth and pots because the latter materials tend to dry too fast under the sun and cause cracking of the parboiled rice (Ahiduzzaman and Sadrul Islam, 2009, FAO, 2012).

In addition to the home scale parboiling systems, parboiling has also been practised on an industrial scale in countries such as the United States, Europe, Thailand and Central and South America (Bhattacharya, 2004). The fundamental principle of parboiling remains the same, although the commercial processes details are not publicly revealed and some variations are expected. An example of a rice parboiling plant is illustrated in Figure 2-8. The throughput of parboiled rice varies between 12 and 500 tonnes per day. Most of the industrial parboiling devices allow flexible choice of parboiling parameters and the process is highly automated ( Schule, 2012).

Chapter 2 Literature review

Figure 2-5 Vessel process of parboiling using (a) a single vessel; (b) double vessel (Source: Roy et al. (2006))

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Figure 2-6 Small-boiler process of parboiling: (a) single-barrel boiler, (b) double barrel- boiler (Source: Roy et al. (2006))

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Figure 2-7 Medium boiler process of parboiling (Source: Roy et al.(2006))

Figure 2-8 Industrial parboiling plant (Source: Schule (2012))

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2.7 Quality change of parboiled rice

As described in Section 2.6, parboiling is a post-harvest treatment prior to rice milling. Considering rice as a biological sample, the physicochemical properties of rice are expected to change with hydrothermal treatment.

2.7.1 Appearance

One of the most noticeable features of parboiled rice is its yellow or amber colour compared to normal unparboiled rice. It is an important quality to be considered from a market and consumer perspective because the first impression of a food is generally visual (Kratochvil et al., 1994). The colour change, or discolouration, of rice due to parboiling has been studied and widely reviewed (Bhattacharya, 1995, Bhattacharya, 2004, Lamberts et al., 2008, Lv et al., 2009). Bhattacharya (1995) reported that most of the colour development in rice followed zero-order kinetics. High heating processes, i.e. steaming pressure and duration, resulted in darkening and marked increase in the yellowness of rice, although the change in redness appeared insensitive to heating duration but was enhanced by high pressure. Thus, low steaming pressure and short steaming duration are favoured in order to restrict colour development in parboiled rice. This observation was supported by Lamberts et al. (2006b) who reported that the effect of parboiling on yellowness was more pronounced than that on the redness of milled parboiled rice for all soaking and steaming conditions studied. Colour changes of parboiled rice have mostly been explained by pigment diffusion and Maillard reactions. The pigments in rice are not uniformly distributed. The yellow and red pigments are more concentrated in the bran layer than in the endosperm (Lamberts et al., 2007). The soaking process during parboiling facilitates the migration of bran pigments through the soaking water into the endosperm (Lamberts et al., 2006a). Upon soaking, noticeable enzymatic conversion of sucrose into reducing sugars and de novo production of sugars and amino acid was observed (Ali and Bhattacharya, 1980). Due to the presence of reducing sugars and free α-amino nitrogen containing proteins and peptides in rice, when the rice is

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subjected to heat (e.g. heating stage in the parboiling process), they serve as the precursors for Maillard reactions and result in the prominent yellow/ brown colour of rice (Lamberts et al., 2006b, Parnsakhorn and Noomhorm, 2008, Lv et al., 2009).

Unlike ordinary rice grains, parboiled rice grains are more translucent or glassy rather than opaque. Bhattacharya (2004) and Raghavendra Rao and Juliano (1970) proposed that the starch granules in parboiled rice were gelatinised, the disrupted protein bodies adhere to each other to form a compact mass. This led to reduced light scattering at the boundaries of granules. Researchers have reported that inadequate parboiling, i.e. insufficient starch gelatinisation, resulted in the formation of an opaque core called “white bellies” (Raghavendra Rao and Juliano, 1970, Bello et al., 2006, Lamberts et al., 2008). The grains with the opaque core chalk, or white belly, generally showed higher milling breakage because they were more susceptible to cracking (Srinivas and Bhashyam, 1985). Apart from the transition from opaque to translucent core, parboiled rice was observed to be shorter and broader than ordinary milled rice (Bhattacharya and Ali, 1985, Oludare et al., 2012). It was believed that starch gelatinisation and some realignment were the contributors.

2.7.2 Milling quality

The gelatinisation of starch during parboiling does not only affect the appearance of parboiled rice. Numerous reports have mentioned that the remarkable improvement of milling quality in parboiled rice was the result of starch gelatinisation (Bhattacharya and Swamy, 1967, Miah et al., 2002). An early study investigated this observation through deliberately induced cracking of the rice. The milling quality of rice was dramatically restored after parboiling. The examination of the rice under transmitted light confirmed the absence of cracks in the parboiled rice, suggesting the realignment and cementing of starch granules after parboiling can eliminate kernel defects (Bhattacharya, 1969). The result was confirmed by Miah et al. (2002) who reported that soaking period in

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conjunction with steaming cemented internal cracks of rice and therefore improved the milling quality and therefore, the head rice yield. The change in starch structure also led to a harder rice kernel than unparboiled rice (Ali and Pandya, 1974, Pillaiyar and Mohandas, 1981, Bello et al., 2006). Higher heating temperature and duration enhanced the hardness of uncooked parboiled rice grains (Ali and Pandya, 1974). Bhattacharya (2004) summarized that due to the increase in kernel hardness, it was found that greater milling time or force and therefore higher energy, was required for milling the rice.

2.7.3 Textural change

The changes in cooking and eating properties of parboiled rice are important and relevant to consumers (Bhattacharya, 2004). Studies have shown that parboiled rice required longer cooking time than ordinary rice (Bello et al., 2006). The texture of cooked parboiled rice was harder, less sticky, fluffier and retained better shape (Priestley, 1976, Bello et al., 2006). The textural change in cooked rice was thought to be related to starch reassociation or complexation (Raghavendra Rao and Juliano, 1970, Priestley, 1976). In addition to the previous studies, Ramesh et al. (1999) investigated the quality change in relation to starch breakdown. There was a fair correlation between the starch degradation and firmness (hardness) of parboiled rice, suggesting that the thermal degradation of starch that took place during heat treatment in parboiling may be another possible contributor to the textural change in rice. Recent studies improved the understanding of textural change by incorporating the concept of starch polymorphism (Derycke et al., 2005). Several terminologies were introduced: 1) retrograded starch existed in two forms, i.e. retrograded amylose (R-Am) showed a higher melting point (140-150 ˚C) and retrograded amylopectin (R-Ap) had a lower melting point (50-55 ˚C); 2) there are two types of lipid-amylose complexes , i.e. (L-Am) in parboiled rice, namely L-Am I and L- Am II. The former one is formed at relatively low temperatures and melted at about 100 ˚C whereas the latter one formed at higher temperatures and melted at 110-120 ˚C. Bhattacharya (2004) summarised that severe parboiling conditions, such as high steaming

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pressures would likely produce annealed starch and L-Am II complex, and possibly R-Ap. L-Am II was unlikely to melt at boiling temperature, i.e. the temperature of rice cooking. It therefore explains the longer cooking time for the parboiled rice and its harder texture.

2.7.4 Nutrients

As discussed earlier in Section 2.6.1, rice parboiling has been practised for over 100 years because of its advantages in storage and rectifying micronutrient deficient disease, especially thiamin deficiency. In 1974, Padua and Juliano studied the effect of parboiling on thiamin distribution in rice. They reported that thiamin content in rice was reduced after parboiling compared to unparboiled brown rice. Nonetheless, at the same degree of milling, milled parboiled was found to have higher amount of thiamin than milled raw (unparboiled) rice. By milling the rice to three different degrees, Padua and Juliano suggested that parboiling resulted in dramatic redistribution of thiamin. For unparboiled rice, thiamin was concentrated only in the first milling (i.e. outer layer of rice) and significantly dropped in subsequent millings. However, after parboiling thiamin appeared to be distributed fairly evenly throughout the rice kernel, indicating the extensive diffusion of thiamin from the bran layer into the endosperm. The observation was supported by other recent researches (Otegbayo et al., 2001, Manful et al., 2007). Apart from thiamin, Manful et al. (2007) reported that the level of riboflavin was initially increased with parboiling but dropped eventually as the parboiling intensity increased further. Unlike vitamins, minerals did not appear to draw as much attention. Early studies revealed that parboiling caused negligible composition change in brown rice. Anyhow, parboiling appeared to affect the distribution of some minerals in rice but not zinc, magnesium and copper. In general, parboiled rice resulted in less milling loss in comparison to unparboiled rice (Doesthale et al., 1979).

Apart from micronutrients, rice is a biological sample which also contains enzymes. Enzymatic activities are expected to be altered when the rice is subjected to soaking (hydration) and heating (thermal treatment). Xavier and Raj (1999) focussed on the

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enzymatic changes in paddy and soaked water during parboiling. Amylase activity increased almost five-fold after 12 h soaking and steaming of the soaked grains deactivated about 70 % of the enzymatic activity. The study clearly showed the relationship between amylase activity and starch breakdown during soaking. The amount of total sugars did not change significantly but the amount of reducing sugars markedly increased for the soaked paddy. Similarly, protease activity in paddy grains was increased more than ten-fold after soaking. The increased protease activity coincided with the higher level of amino acids in the soaked paddy. The results agreed with that observed by Lamberts et al. (2006b), and also supported the presence of reducing sugars and free α- amino nitrogen in the parboiled rice, thereby serving as the starting materials for non- enzymatic discolouration in parboiled rice (Lamberts et al., 2006b).

2.8 Consumer preferences to rice

To some extent, consumer preference is an end-use quality measure of rice as their preferences drive the market. Consumer acceptance is referred to as a key success factor for functional food, which includes fortified food (Siro et al., 2008). Value-added food, with enhanced nutritional benefits, may not always be well accepted by consumers. For instance, Danish consumers were sceptical about functional foods (Poulsen, 1999). The foods were perceived as unnatural and the consumers were apprehensive about the changes in taste of food. Unlike in the United States, where the first food fortification program was implemented and adopted in 1924 and different fortification practices have been carrying out until present time (Backstrand, 2002). Consumer acceptance of food, therefore, cannot be taken for granted.

Consumer acceptance is a complex terminology which embraces a range of elements such as objective sensory descriptors, packages, food appearance, food labels and health claims, and consumers’ experiences and knowledge towards the product (Heinemann et al., 2006). Tomlins et al. (2007) evaluated consumer acceptance of a prototype parboiled rice in comparison to three local samples and one imported parboiled rice. The panel

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recruited were asked to assess only the appearance of the uncooked form of rice. The results suggested that some consumers were negatively associated with local parboiled rice which was described as brown, black heads. These consumers were associated with clean, uniform colour and bright parboiled rice. The experimental design itself highlighted that visual attributes of product, i.e. appearance, was an important factor related to consumer acceptance. The study also revealed that consumers do show particular preferences to rice. In general, brown colour of cooked rice poses negative impact on consumer acceptance (Tomlins et al., 2005). Texture (Tomlins et al., 2005), grain uniformity and grain size (Heinemann et al., 2006) were other visual factors previously studied in relation to consumer acceptance of parboiled rice.

Taste is often one of the major influencing factors in the decision to purchase food (Food Insight, 2010). Some consumers are not willing to compromise on the taste of food for health (Verbeke, 2006) although the provision of nutrition information of the product may improve consumer acceptance (Chowdhury et al., 2011). In terms of rice, although it does not normally generate a strong sensory stimulus (Heinemann et al., 2006) and is normally described as bland for general consumers, taste of cooked rice is still a critical attribute that consumers are seeking (Diako et al., 2010).

2.9 Folate bioavailability

The intestinal epithelium is the gatekeeper which controls the entry of nutrients from the ingested food (Le Ferrec et al., 2001). There are different terminologies used to describe the specific concepts in relation to the fate of nutrients after being ingested into the body.

 Bioaccessibility: it refers to the fraction of nutrients (i.e. folates in this context) released from the food product (after the digestion through gastrointestinal tract) into the intestinal lumen and become available for absorption by cells (Verwei, 2004).

 Bioconversion: it refers to the fraction of the bioavailable nutrient being converted into the active form (Castenmiller and West, 1998)

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 Absorption: it refers to the uptake and transport across the intestinal epithelial cells (Olinger et al., 1973, Öhrvik et al., 2010). Different transport mechanisms may be involved for different nutrients (will be discussed in Section 2.9.1).

 Bioavailability: it comprises of bioaccessibility and absorption, i.e. the target nutrients must be absorbed and delivered to the target tissue for utilisation or storage (Failla et al., 2008).

2.9.1 Intestinal absorption and transport of folates

After the ingestion of folates, both dietary folates and folic acid (pteroylmonoglutamic acid), folates are mainly absorbed in the upper half of small intestine, i.e. in jejunum (Olinger et al., 1973, Yarbaeva, 2009) (Figure 2-9). Prior to the transport of folates across the intestinal epithelia, the glutamyl residues on the natural folates are cleaved by ɣ- glutamylcarboxy conjugase at the brush border membrane of the duodenum and jejunum, resulting in monoglutamate folate (resembling folic acid) (Sirotnak and Tolner, 1999, Verwei et al., 2005, Subramanian et al., 2009).

The transport mechanism of folates in small intestines is summarized in Figure 2-10. As folates are highly hydrophilic divalent anions, they do not diffuse across biological membranes and passive transcellular absorption is not expected (Figure 2-10, route A) (Bridges et al., 2001). Route B refers to paracellular diffusion which may contribute to intestinal transport of folate at particularly high folate concentration (Figure 2-10, route B). Vincent et al. (1985) reported that a non-saturable transport (diffusion) became predominant only when the folic acid concentrations were greater than 10 µM. At physiological folate concentrations, transport via passive diffusion is unlikely. Specific transport mechanisms, therefore, are required for folates to enter the cells. Transcellular carrier-mediated (Figure 2-10, Route C) and endocytose (Figure 2-10, Route D) are the two important specific transport mechanisms for folates (Antony, 1992, Sirotnak and Tolner, 1999, Suh et al., 2001). Reduced folate carrier (RFC) and membrane-associated

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folate binding protein (mFBP) (or folate receptor: FR) are the proteins associated with the respective mechanisms. RFC is an integral membrane protein with a molecular weight of 80-120 kDa in human cells (Westerhof et al., 1995). RFC-mediated folate transport functions as anion-exchange mechanism (Verwei, 2004). mFBP is anchored to the plasma membrane by glycosylphosphatidylinositol tail (Luhrs and Slomiany, 1989). This membrane-bound receptor mediates unidirectional transport of folate into the cell. The uptake mechanism involving mFBP has been described as the classic receptor-mediated endocytosis pathway (Antony, 1992). Low levels of mFBP receptors are present on the gut mucosal cells, therefore, the absorption and transport of folate via route D in intestinal lumen is low (Verwei, 2004). Verwei (2004) studied the contribution of some efflux transporters, i.e. multi-drug resistance proteins (MRP), to the transport of folates, by using an inhibitor of MRPMK571. The result showed that the transport of folic acid and 5-CH3-THF dropped to 48-44 % and 69-72 %, respectively. It indicated that MRP- efflux was a significant pathway for folate transport (Figure 2-10, Route E).

Approximately 40-50 % of the total folate polyglutamates in the red blood cells are made up of 5-methyl-THF. 5-methyl-THF exhibits as monoglutamate in serum and polyglutamate intracellularly (conjugation done by ɣ-glutamate synthase in order to form polyglutamate for storage and metabolism) (Perry and Chanarin, 1970, Shane, 1989).

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Figure 2-9 Physiological properties of the gastrointestinal tract (Avdeef, 2003)

Figure 2-10 Transport mechanisms of small intestine. a) Transcellular diffusion; B) Paracellular diffusion; C) Transcellular carrier-mediated transport; D) Transcellular endocytose; E) Efflux transport with apical or basolateral located efflux pumps (Source: Verwei (2004))

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2.9.2 Methods in measuring folate bioavailability

Due to the important health implications associated with folates, different experimental approaches have been developed to determine the bioavailability of folates. Each method comprises both advantages and limitations (McNulty and Pentieva, 2004).

2.9.2.1 Animal bioassays

Chicks and rats were the animal models used to assess the folate bioavailability in the early studies (Ristow et al., 1982, Abad and Gregory, 1987). Graham et al. (1980) examined the bioavailability of natural folates and folic acid added to frozen convenience foods, TV dinners and meat pies using a chick bioassay. Unfortified and fortified diets (prepared by adding varying concentrations of folic acid into the basal diet) were fed to White Leghorn chicks in groups of 10 each for 21 days. TV dinners and pies with the addition of folic acid demonstrated elevation of plasma and RBC folacin, but did not cause consistent elevation in fortified vegetables. The authors noted that should the animal model resemble that of human subjects, chick model could be used for screening of food for folate bioavailability in place of the difficult and expensive human trials. Abad and Gregory (1987) reminded researchers to consider the usefulness and limitations of using rat as the animal models for assessing folate bioavailability. Several differences in conceptual and practical characteristics in animal models restrained its application to human nutrition, which included: 1) the mechanism of intestinal folate deconjugation in animals which were different from humans (Gregory, 1995); 2) blending of the dry powdered test doses into basal diets, which may not represent the form of food that is consumed by human subjects; 3) the application of oral antibiotics to suppress the endogenous folate production from intestinal microflora (Gregory, 2001).

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2.9.2.2 Intervention studies with humans

Acute /short term studies

The determination of folate bioavailability using human subjects can broadly be divided into acute and chronic studies by measuring their change in plasma or serum folate and/or urinary folate excretion (McNulty and Pentieva, 2004). Human trials often involve blood sample collections at multiple times after the ingestion of test food, in order to include the maximal plasma or serum folate concentration and the time required to attain the maximal plasma or serum folate response. This allows the establishment of area under the curve (AUC). This bioavailability study approach is regarded as acute studies as the blood collection duration typically ranged between hours (Perry and Chanarin, 1970, Fenech et al., 1999, Pentieva et al., 2004). Early studies often compared the bioavailability of unlabelled reduced folates against that of folic acid, which is the synthetic form which is being used widely for supplementation and food fortification. Gregory (2001) and McNulty and Pentieva (2004) summarized the special considerations needed when acute or short-term protocols are used: 1) relatively high folate content (at least ~300 µg/ dose) is required in order to elicit measurable folate response in blood or urine; 2) subjects with high folate status (without suffering from folate deficiency) or folate presaturation protocol are needed in order to minimise the chance that the test folate is preferentially directed to the depleted folate pools, and subjects with similar state of folate nutriture is preferred as the folate response in blood or urine depends on the subjects’ folate status; 3) sufficient number of subjects in order to ensure the statistical power and to reduce the variability of folate response; 4) folate intake during the treatment period need to be strictly controlled and the inclusion of a placebo group is essential to draw meaningful conclusion for any changes in folate measurement due to the test conditions. Recent studies introduced the use of stable-isotopes which largely improved the specificity of the study, because the measured labelled folates in blood or urine can only come from the ingested dose (Gregory, 2001, de Ambrosis, 2006, Vishnumohan, 2008). The stable-isotope folates are considered safe for human use which

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increases its popularity in bioavailability studies. The stable-isotopes are, in general, expensive in nature and some of the folate forms may not be commercially available. Some investigators synthesized and purified the stable-isotopes for their studies (Maunder et al., 1999). The detection of the labelled folates and its metabolism often requires the use of sophisticated liquid chromatography-MS (Finglas et al., 2002, Vishnumohan, 2008), which increases the cost of the study.

Chronic / long-term studies

The advantage of chronic or long-term studies is that the folate response in blood is based on the overall utilisation of folate in total diets consumed, but not due to a single meal. Serum folate change is more responsive than that of erythrocyte folate because less than 1 % of circulating erythrocytes is replaced daily; whereas the biomarker plasma homocysteine requires several weeks of intake to reflect the differences in folate status (Gregory, 2001). The typical length of the study is between 4 and 24 weeks (Venn et al., 2003, Hao et al., 2008) and so subject compliance during the intervention is fundamentally important to the quality and validity of the results obtained. Frequent contacts with the subjects and on-going records are needed during the intervention (McNulty and Pentieva, 2004). Ashfield-Watt et al. (2003) conducted a 4-month intervention study investigating the change in plasma folate and homocysteine due to the effect of natural folates and folic acid from fortified food sources. Subjects were divided into three groups, placebo group (subjects consumed their normal diet through the study), fortified food test group (subjects were required to take an extra 100 µg/ day folic acid from fortified food products without exceeding that limit), and natural folate test group (subjects were advised to consume an extra 100 µg/ day folate from natural products, such as vegetables and fruit, without exceeding that limit). The results showed that fortified food increased plasma folate more easily than by natural folate-rich foods, although both food groups increased plasma folate by a similar extent. McNulty and Pentieva (2004) commented that poor compliance of subjects with the protocol may result in less food folate intake; hence no difference was revealed between the two test

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groups. McNulty and Pentieva (2004) noted that contrary result reported by Cuskelly et al. (1996) showed that only folic acid supplement or fortified food significantly improved the plasma folate concentration against the baseline level. Intervention through the intake of natural folate did elevate plasma folate level but the change was not significant. It may be because the subjects in the supplement group were required to simply ingest a tablet daily along with their usual diet, this protocol may be easier to comply with than other interventions (McNulty and Pentieva, 2004).

2.9.2.3 In-vitro studies-Caco-2 cell model

Due to the fact that the intestine is an organ with considerable cellular heterogeneity and complex geometric structure, it is challenging to study the nutrient transport through the intestine. In-vitro model of intestinal cells may overcome the issue (Grasset et al., 1984). Moreover, the use of in-vitro models reduced the need for experimental animals or human subjects which lowers the associated cost and bypasses the potential ethical limitations.

Caco-2 cell culture model has been widely used in the academic and industrial laboratories as a tool to predict the intestinal permeability and absorption of drugs (Faassen et al., 2003, Hubatsch et al., 2007, Volpe, 2008). The model was first developed at Borchardt’s laboratories in the Department of Pharmaceutical Chemistry at University of Kansas and its uptake and barrier properties between the cell model and small intestinal epithelial layer had been confirmed (Hidalgo et al., 1989). The Food and Drug Administration (FDA) has recognized this model as a useful system to perform absorption studies for drug classification according to the Biopharmaceutics Classification System (Hu et al., 2004).

The emerging popularity of this cell assay may due to the fact that this cell line was derived from human origin and the cells are a moderately well-differentiated colon adenocarcinoma possessing both enterocytic and colonocytic characteristics (Grasset et al., 1984, Sambuy et al., 2005). Caco-2 cells undergo spontaneous morphological and

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biochemical differentiation when the cells are grown on microporous membrane in culture condition. Unless induced with differentiating reagent (e.g. butyric acid) (Yamashita et al., 2002), Caco-2 cells typically take 21 days for differentiation (Biganzoli et al., 1999, Verwei, 2004, Netzel et al., 2011) in order to form enterocyte-like phenotype features such as tight junctions, microvilli and functional brush-border enzymes which resembles the human intestinal barrier (Vincent et al., 1985, Hu et al., 2004, Yarbaeva, 2009), which is illustrated in Figure 2-11. Apical compartment resembles the vicinity of lumen and basolateral compartment represents the serosal side of intestinal epithelium.

Figure 2-11 The transwell system consists of the apical compartment, filter (permeable cell insert) and basolateral compartment (Source: Verwei et al. (2005))

Apart from using the model to investigate the pharmacokinetics of drugs for decades (Artursson and Karlsson, 1991, Faassen et al., 2003, Crowe and Wright, 2012), Caco-2 model has become more attractive to researchers who study the transport mechanisms and bioavailability of different nutrients. Garcia et al. (1996) assessed the usefulness of Caco-2 cell as a model for studying the bioavailability of dietary iron from different food sources, i.e. beef, soybean, egg. The investigators conducted in total of 40 experiments to evaluate the mean iron uptake by Caco-2 cells. The results supported that Caco-2 cells metabolise iron in a similar manner to the human intestinal tract. Recently, the bioavailability of iron in fortified parboiled rice has been studied (Prom-u-thai et al.,

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2009a). In comparison to the raw and unfortified parboiled rice, the iron uptake of fortified parboiled rice showed about 11 times higher than the former two groups. The presentation of data could be improved by expressing the iron uptake/ iron bioavailability in percentage in order to facilitate comparisons between studies. In terms of folates in particular, Vincent et al. (1985) examined the Caco-2 cell model which was at the beginning stage for describing the characteristics of folic acid uptake. Caco-2 cell model has lately been used to measure the in-vitro folate (labelled [6S]-5-CH3-tetrahydrofolate) uptake of fortified bread (Verwei et al., 2005). Stable isotope was used in the experiment to allow the endogenous folate to be distinguished from the added folates in the bread sample. The result indicated that bread matrix appeared to possess inhibitory effect to folate uptake and transport in comparison to the aqueous folate standard. Other dietary compounds such as catechins, tea extracts (Alemdaroglu et al., 2007), ethanol, phenolic compounds (Lemos et al., 2007) also showed to limit the intestinal uptake of folates.

Similar to any other bioavailability approach, there are limitations associated with in- vitro models: 1) Caco-2 cells exhibit as single monolayer on the porous membrane which may be an over-simplified model for the complex human intestines, which possess villi and microvilli to greatly increase the effective absorption area than that of Caco-2 cells (Sun et al., 2008, Avdeef and Tam, 2010); 2) The absence of mucus covering the Caco-2 cell monolayer and the expression of some enzymes and transporters may not be fully comparable to human intestine (Le Ferrec et al., 2001, Hu et al., 2004); 3) Caco-2 cells originated from tumours and the experimental condition may not reflect the in-vivo physiological environment (Le Ferrec et al., 2001). Regardless, Caco-2 cell model still remains as a viable method to assess nutrient absorption and as an alternative to human/animal bioavailability studies.

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2.10 Conclusions

Rice processing and preparation are the inevitable steps in causing a loss of nutrients. Given the fact that micronutrient deficiencies occurred widely in rice-consuming populations and that rice itself is not a perfect source for micronutrients, this clearly demonstrates that there is a need to replenish or fortify the micronutrients in rice for those rice-consuming populations. Earlier researchers illustrated the pros and cons for different rice fortification methods. Among all, parboiling appears to be a recent fortification method with growing potential, due to its cost-effectiveness and least possible disturbance to the populations. To the best of our knowledge, the recent studies have been focussing on using this parboiling method to fortify rice with minerals. No study has yet reported to fortify rice with vitamins, e.g. folic acid.

Hence, the following gaps were identified for the current study:

1. To explore the potential of fortifying rice with vitamin (i.e. folic acid) using parboiling technique based on feasibility study; 2. To systematically study the relationship between parboiling conditions and the uptake and retention of folic acid; 3. To investigate the parboiling conditions based on: rice quality, i.e. head rice yield, grain dimension and colour; 4. To evaluate consumer acceptance of the fortified parboiled rice based on visual appearance and sensory assessment; 5. To examine the bioaccessibility and absorption of fortified rice using Caco-2 cell model.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

CHAPTER 3

THE FEASIBILITY OF FORTIFYING RICE WITH FOLIC ACID THROUGH PARBOILING

As mentioned in Section 2.7, parboiling primarily involves soaking, heating and drying of the grains before any other routine processes of rice handling (Bhattacharya, 2004). Moisture content of the rice inevitably increases during soaking as water migrates inwards from the surrounding soaking water (Elbert et al., 2001b, Miah et al., 2002, Sareepuang et al., 2008). Due to the moisture gradient between the soaking water and rice grains, it is expected that the dissolved fortificant in the solution will penetrate into the endosperm and therefore the soaking stage was chosen as the point of fortification in this study.

Firstly, paddy is used as the starting material in traditional parboiling (Bhattacharya and Ali, 1985, Bhattacharya, 2004, Roy et al., 2006). The natural structure of paddy consists of an outermost siliceous husk which neither wets easily nor facilitates water movement (Kar et al., 1999). Disregarding the extra energy input required for parboiling paddy rice, the presence of the husk acts as an effective barrier to hinder the access of water to the kernel (Kar et al., 1999). So, folic acid may also be prevented from penetrating into the endosperm. Secondly, beneath the siliceous husk is the oil-rich bran layer (Luh, 1991). As folic acid is a water-soluble vitamin, it is doubtful whether the vitamin can penetrate across the lipid-rice layer. If no vitamin can migrate into the endosperm across the hull, bran and aleurone layer, the purpose for using parboiling as an approach for rice fortification will be defeated.

Therefore, the aims of this chapter were to address the two uncertainties indicated above through two studies: Study 1 was a preliminary study using a water-soluble dye, methylene blue, to visually evaluate whether a water-soluble compound could migrate from the soaking water into the rice endosperm through the husk and the bran layer;

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

Study 2 was a feasibility study to investigate the retention of folic acid in the parboiled rice.

3.1 Study 1- Preliminary study using water-soluble dye

The most straight-forward approach to address the above issues is to observe the migration of the fortificant during parboiling. Unfortunately, folic acid is not a strong dye and does not provide an apparent visual illustration of penetration. Due to this, methylene blue was chosen for the preliminary study because, it is a water-soluble dye which may resemble the water-soluble vitamin, i.e. folic acid; and, methylene blue which has an intense blue colour which readily shows any dye penetration into the grain during soaking.

3.1.1 Materials and methods

Methyl blue dye (0.25 g / 100 g water. Merck, Australia) was prepared and the solution added to the rice under the following conditions:

Paddy rice (Langi) Brown rice (Langi)

60°C 60°C 60°C Soaking for Soaking for Soaking for 1 h 4 h 20 h

Figure 3-1 Soaking conditions for brown and paddy rice with methylene blue

After the rice grains were soaked for the designated durations (Figure 3-1), soaking solution was drained and dried in the shade until the moisture content reached between 12 and 14 %. Rice grains were investigated under the stereo microscope (WildM3C, Leica)

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling to study the surface and horizontal cross-section of the rice. The horizontal section was prepared by scrapping the surface of the grains gently against an abrasive surface until the endosperm was exposed.

3.1.2 Results and discussions

Figure 3-2 and Figure 3-3 illustrate the paddy and brown rice grain which were soaked in the methylene blue dye solution at 60 ˚C for 1, 4 and 20 h, respectively. After 1 h soaking, the husk of the paddy grain was only slightly stained, and the intensity of the colour had obviously increased over the prolonged soaking duration (A-1, B-1 and C-1, Figure 3-2). However, a negligible amount of blue dye was present on the kernel when the husk was removed (A-2, B-2 and C-2, Figure 3-2), and increasing soaking duration did not significantly enhance the penetration of the dye. For the paddy rice which was soaked for 20 h, some of the husks split open during soaking, the appearance of the rice is shown in the C-2 of Figure 3-2. The rice grain located on the top is the rice grain soaked for 20 h with the intact husk, whereas the one at the bottom indicated the paddy grain with the split husk during soaking. The obvious contrast in the two grains suggests that the outermost husk provided a significant barrier to the transfer of dye into the kernel.

Considering the soaking of brown rice, the entire bran layer was intensely stained by the blue dye for all three soaking durations studied (A-1, B-1 and C-1, Figure 3-3). The cross-sections of the grains illustrate the benefits of prolonged soaking. Only a thin layer of outermost bran layer was dyed blue for 1 h soaking, leaving the major portion of the endosperm white and unstained. The relatively thicker outer layer of rice was stained after 4 h soaking, and the endosperm of the rice appeared blue. The result extended to the 20 h soaking of brown rice in the dye solution.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

Figure 3-2 Paddy rice granules soaked in methylene blue dye for A) 1 h, B) 4 h and C) 20 h at 60 ˚C. (Each column illustrated the paddy grain, intact brown rice and cross-section of the dehulled brown rice)

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

Figure 3-3 Brown rice granules soaked in methylene blue dye for A) 1 h, B) 4 h and C) 20 h at 60 ˚C (Each column illustrated the intact brown rice and cross-section of brown rice)

3.1.3 Study 1 Conclusions

Based on the experiment, the results showed that:

1. The husk of rice should be removed for effective transport of fortificant into the inner rice kernel;

2. Methylene blue, and therefore folic acid, is likely able to penetrate through the bran layer into the rice endosperm, and; 3. Brown rice is an ideal starting material for fortification using parboiling and soaking is the point of fortification.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

3.2 Study 2- Feasibility study with folic acid

With the preliminary speculation that a water-soluble substance can migrate into the rice kernel, the immediate research question was “Can the actual fortificant, i.e. folic acid, also penetrate through the bran layer into the endosperm?” In this context, the method was considered feasible if folic acid penetrated and was retained in the rice kernel after the parboiling treatments.

3.2.1 Materials

The following reagents stated were prepared with analytical grade chemicals MilliQ water was used for preparing the solutions, unless stated otherwise.

3.2.1.1 Parboiling of brown rice

1. Paddy grains: Long grain variety, Langi, was supplied by NSW Department of Primary Industries, Yanco Agriculture Institute, Australia. The samples were stored in 4±1 ◦C cool room until use. 2. Food grade folic acid (pteroylmonoglutamate): Purity > 95 % purchased from DSM, Australia. 3. Water source: Tap water was chosen to prepare the rice throughout the parboiling study because no detectable trace of folic acid was present. Also tap water better mimics the conventional parboiling method in India and Bangladesh (Roy et al., 2006).

3.2.1.2 Sample extraction

1. Extraction buffer (0.1 M phosphate, 1.0 % ascorbic acid, pH 6.1): Extraction buffer was prepared according to Patring and Jastrebova (2007). 13.61 g

potassium dihydrogen-orthophosphate (KH2PO4) and 17.42 g dipotassium

hydrogen-orthophosphate (K2HPO4) and 10 g ascorbic acid were dissolved in 1 L water.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

2. α-amylase (20 mg/ mL): 1.0 g of α-amylase (EC 3.2.1.1, type IV-B from porcine pancreas, Sigma Chemical) was suspended in 50 mL of water and stirred for 5 min with a magnetic stirrer until completely dissolved. The enzyme solution was always prepared fresh on the day of use.

3.2.1.3 Sample purification

1. Elution buffer for Solid Phase Extraction (SPE): 0.1 M sodium acetate containing 10 % (w/v) sodium chloride, 1 % (w/v) ascorbic acid and 0.1 % (v/v) of 2-mercaptoethanol prepared with water (Nilsson et al., 2004). Elution buffer was prepared fresh on the day of use.

3.2.1.4 Folic acid analysis

1. Mobile phase: a) Phosphate buffer - 3.14 g of anhydrous potassium dihydrogen-orthophosphate was dissolved in 1 L water. pH was adjusted to 2.2 using concentrated phosphoric acid. Phosphate buffer was then filtered through a 0.45 µm nylon membrane filter. b) 100 % Acetonitrile (HPLC grade). Both of the mobile phases were sonicated for 30 mins to sufficiently remove dissolved gas. 2. Standard buffer: 1.36 g dipotassium hydrogen-orthophosphate, 3.06 g potassium dihydrogen-orthophosphate and 2 g of sodium ascorbate were dissolved in 100 mL water. pH was adjusted to 6.1 using concentrated phosphoric acid. 3. Folic acid standard (100 µg/ mL): 0.025 g of folic acid was dissolved in 250 mL standard buffer. The volumetric flask was placed in a sonicator for 15 min to ensure complete dissolution of the standard. The standard was prepared under UV-free light to minimize the chance of degradation.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

3.2.2 Methods

3.2.2.1 Preparation of brown rice

Paddy was dehulled by a rubber roll dehuller (model THU-35A, Satake Engineering Co Ltd., Japan) with the roll gap of 0.5 mm. Broken grains were separated from the dehulled brown rice using the rice indent cylinder (model TRG, Satake Engineering Co Ltd., Japan). The angle used was 30◦ and the rotation time was set to 3 min. Broken rice was collected in the middle of the container and the intact brown rice was in the cylinder. Only whole brown grains were used in the study (Figure 3-4).

Figure 3-4- Illustration of rubber roll dehuller (left) and rice indented cylinder (right)

3.2.2.2 Parboiling of brown rice

Food grade folic acid (1.2 g) was first dissolved in tap water (600 mL) and 300 g of graded brown rice was added to the folic acid solution. The temperature of the soaking water was adjusted so that when the brown rice was added the initial starting temperature was 60 or 70 °C. Brown rice was soaked for 1 h. After the designated soaking duration,

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling fortificant soaking solution was discarded and the sample was subsequently heated in a steam box at atmospheric pressure for 15 mins and 60 mins at 100 °C. Steamed samples were spread to dry at room temperature under shade until moisture content dropped to 12 - 14 % wet weight basis.

3.2.2.3 Degree of Milling

For each sample, 150 g of parboiled brown rice was milled in a food-grade laboratory scale grain mill (model Satake TM05, Satake Engineering Co Ltd., Japan) for 30 and 60 s to yield white rice (Figure 3-5). The degree of milling was obtained based on the weight of milled rice divided by the weight of brown rice (Kawamura et al., 1999).

The Head Rice Yield (HRY) was expressed as a percentage calculated as:

 WHRY Wt)/'( x100% (Equation 3-1) where W’ is considered as the mass of rice kernels which are three-fourth the size of intact milled rice and Wt is the total mass of the sample paddy (Parnsakhorn and Noomhorm, 2008).

Figure 3-5 Illustration of the laboratory rice mill

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

3.2.2.4 Sample preparation for extraction

Dried rice samples (i.e. moisture content less than 14 % wet weight basis) were ground into rice flour with a domestic coffee grinder (Breville, CG2B, Australia) and passed through a 700 µm sieve. Aliquots of rice flour was packed in polyethylene bags and stored in a 4 ◦C cool room until analysis.

To identify the optimal cooking protocol for the cooked rice, rice was cooked in three different rice-to-water ratios, i.e. 1: 1.6, 1:1.8 and 1:2, using a rice cooker (BRC200, Breville, Australia). During the cooking of rice, about 3 grains were withdrawn at one minute intervals after 10 mins and the kernels were pressed against two glass slides. The absence of opaque centres and complete translucence of the pressed rice kernels represented sufficient cooking and therefore the optimal cooking time (Kar et al., 1999). At the end of the cooking cycle (i.e. 19 mins), opaque cores were shown in rice sample cooked with rice-to-water ratio of 1:1.6 when the kernels were pressed against the glass slides, whereas the pressed kernels were translucent for rice samples cooked with 1:1.8 and 1:2 rice-to-water ratios. Based on the visual observation of rice cooked at 1:2 rice-to- water ratio, the cooked rice was more sticky than that cooked at 1:1.8 rice-to-water ratio due to the content of rice kernels was exposed. The optimal cooking protocol was identified to be rice-to-water ratio of 1:1.8 and cooking time of 19 mins.

Cooked rice samples were ground into rice slurry using mortar and pestle until no intact rice kernel was present. Cooked rice samples were prepared and extracted on the same day. Moisture content of both uncooked and cooked rice samples were measured in order to correct for the moisture for data processing and sample comparison.

3.2.2.5 Heat extraction and single-enzyme extraction

For the uncooked rice, 3±0.01 g of ground and sieved rice flour was added to 20 mL extraction buffer into plastic centrifuge tubes and vortexed thoroughly (Patring and Jastrebova, 2007). For the cooked rice, 5±0.01 g of the ground rice slurry was mixed with 20 mL extraction buffer and vortexed thoroughly. The mixture was subjected to heating

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling in 100 ◦C boiling water for 10 min and immediately cooled under running water until the temperature dropped to ambient. The enzyme, α-amylase (3.2 mL), was added to the heat-extracted sample and vortexed to ensure the sample was homogeneous. The tube was placed in a 37 ◦C waterbath for 2 h and was centrifuged at 4500 rpm for 15 mins. The supernatant was aliquoted into amber bottles and flushed with nitrogen gas before capping. This is because folates are sensitive to both UV light and oxygen which may lead to degradation of analyte (Tannenbaum et al., 1985). Samples were kept frozen at - 20 ◦C and were subjected to further sample purification within no more than 3 days.

3.2.2.6 Sample purification

Extracts were purified by solid-phase extraction (SPE) on strong anion exchange (SAX) cartridges (500 mg, 3 mL, Phenomenex, Australia). The cartridge was preconditioned with methanol (2x 2.5 mL) and water (2x2.5 mL) to activate the sorbent and remove matrix interfering components (Patring and Jastrebova, 2007, Jastrebova et al., 2011). After loading an aliquot of sample extract, the cartridge was washed with 3 volumes of water and then eluted with 0.1 M elution buffer under gravity. The purified sample was filtered through 0.45 µm regenerated cellulose (RC) syringe filter (Minisart RC 25, Germany) prior to HPLC analysis. Analysis was always performed directly after sample purification.

3.2.2.7 Folic acid analysis

Instrumentation

The HPLC method by HarvestPlus (2013) was used for the current study. Analyses were performed using HPLC system (model LC AD, Shimadzu Prominence, USA) consisting of an autosampler, a thermostable column compartment (maintaining the column temperature of 35 °C) and a photodiode array detector (monitoring at 280 nm). The HPLC system was controlled by a computer running LCSolution Shimadzu Chromatogram Data System.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

The separation of folic acid was performed using a reverse-phase Luna C18 column, 5 µm,

150 x 4.6 mm i.d. (Phenomenex, Australia) with a C18 security guard column (Phenomenex, Australia). Gradient elution was performed with phosphate buffer (pH 2.2) and acetonitrile. The gradient started with 5 % (v/v) acetonitrile which was maintained isocractically for the first 8 mins then raised linearly to 24 % within 23 mins then returned to 5 % after 5 mins. The flow rate was 0.8 mL/ min and the run time between injections was 40 mins. Injection volume was 100 µL. The peak was identified based on retention time and absorption spectrum acquired for the peak corresponding to the external folic acid standard. HPLC calibration was done on a daily basis to ensure data integrity (Gregory et al., 1984).

3.2.2.8 Quality Control

In-house reference

In-house reference material was prepared by grinding 250 g of commercial white rice into rice powder. Exactly 0.022 g of folic acid was added to the rice powder which was contained in a polyethylene bag covered with aluminium foil. The bag was then attached onto the rotary wheel and allowed to rotate for 3 h for sufficient mixing. The folic acid concentration of the in-house standard was 88 µg/ g.

3.2.2.9 Recovery study

In order to study the recovery of sample extraction and purification, folic acid standard (i.e. 1 and 10 µg) was spiked into cooked fortified rice. The fortified rice sample was cooked in a rice-to-water ratio of 1:1.8. The cooked rice samples were extracted according to Section 3.2.2.5, followed by purification (Section 3.2.2.6) and HPLC analysis (Section 3.2.2.7). In addition, the extraction buffer was spiked with known amounts of folic acid (1, 5 and 10 µg) and the same extraction and purification procedures were followed. Recovery of folic acid was calculated as: (amount of folic acid measured in spiked sample – amount of folic acid measured in unspiked sample) / (amount of folic acid added in spiked sample) x 100 %.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

3.2.3 Results and discussions

3.2.3.1 Evaluation of HPLC method

A reverse-phase gradient HPLC method was adapted for the current study (HarvestPlus, 2013). The following parameters, namely linearity, repeatability, recovery and accuracy, were studied to ensure the applicability and reliability of method performance.

3.2.3.2 Linearity and repeatability

A series of folic acid concentrations was prepared, 0.5, 1, 5, 10, 20, 30, 50, 80 and 100 µg/ mL (Table 3-1). The correlation coefficient (R2) was 0.999 for standard curve of 0.5 - 20 µg/ mL, and decreased to 0.996 when the upper concentration increased to 30 µg/ mL. R2 was further reduced to 0.982 when the upper concentration increased to 50 µg/ mL. In this study, 0.5 – 20 µg/ mL was chosen because of the high linearity shown (R2= 0.999). Furthermore, most of the samples analysed were fortified with folic acid. The wide linear range ensured the folic acid concentration in the fortified samples fall within this range even after dilution due to extraction and purification processes. The samples could be treated the same way without further dilution or concentration. Standard solutions of low (0.5 µg/ mL), medium (5 µg/ mL) and high (20 µg/ mL) concentrations were injected 5 times and the coefficients of variation were 7.1, 6.2 and 3.0 %.

Table 3-1 Linearity range of the method Linearity range (µg/ mL) Correlation coefficient (R2) 0.5 – 20 0.9992 0.5 – 30 0.9958 0.5 – 50 0.9816 0.5 – 80 0.9914 0.5 – 100 0.9945

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

In order to detect samples with low folic acid concentration, such as samples in the apical and basolateral compartment (cf. Chapter 7), a calibration curve covering the lower end, i.e. 0.01, 0.025, 0.05, 0.01, 0.02, 0.05, 1 and 5 µg/ mL was tested, and R2 was 0.9993.

Three lowest standard concentrations were injected 5 times, i.e. 0.01, 0.025 and 0.05 µg/ mL. The coefficients of variation obtained were 11.9, 6.5 and 6.7 %, respectively. As the coefficient of variation % at 0.01 µg/ mL was relatively higher than the other two concentrations, the linearity range of 0.025 - 5 µg/ mL was used for analysis and R2 was 0.9992.

3.2.3.3 Recovery and accuracy

The accuracy of the HPLC method was determined by a recovery study (Jastrebova et al., 2003). In Table 3-2, the recovery percentage was above 90, which showed that the method used was accurate for analysing folic acid (Jastrebova et al., 2003).

Table 3-2 Recovery of method for folic acid determination Sample Amount of folic Amount of folic Total folic acid Recovery (%) acid in unspiked acid added recovered sample (µg) (µg) (µg) Fortified rice 3.2±0.2 1 4.2±0.1 99.8 3.2±0.2 10 12.3±0.1 90.6 Extraction buffer 0 1 1.2±0.0 116.5 0 5 4.9±0.1 98.0 0 10 9.6±0.2 95.5

Moreover, accuracy of sample extraction and purification method was also determined by measuring the in-house reference material 10 times. The reference material was individually extracted (and purified). The average folic acid concentration found was 86.2 µg/ g with coefficient of variation % of 3.4. Given the folic acid concentration was 88 µg/ g, the recovery % was therefore as high as 98.0.

3.2.3.4 Feasibility of folic acid fortification through parboiling

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

Based on the methylene blue dye experiment (Study 1), it was shown that 1 h soaking at 60 ◦C resulted in a minute amount of penetration, and the penetration progressed as soaking duration increased. Soaking for 1 h at 60 ◦C was therefore chosen as the starting point to find if folic acid could penetrate into the endosperm the same way as methylene blue dye. It was speculated that if uptake was occurred at this soaking point, prolonged soaking would likely increase the extent of folic acid uptake.

As folic acid does not produce a visually distinct colour as methylene blue, the penetration of folic acid in rice was assessed by milling the fortified grains to different degrees by different milling durations. The longer the milling time, the higher the degree of milling and more outer layers were removed (Padua and O. Juliano, 1974, Prom-u-thai et al., 2008). For this preliminary study, two milling durations (30 s and 60 s) were chosen. The milled rice was then subjected to extraction, purification and finally analysed using HPLC method.

From Table 3-3, the uptake of folic acid at this parboiling condition agreed with the visual observation from the methylene blue dye, i.e. fortificant was concentrated on the outermost layer and decreased towards the inner endosperm. The result shows that parboiling is a feasible method to fortify folic acid in rice through the soaking stage of parboiling. Given the folate content in raw untreated rice is less than 0.2 µgfolic acid/ grice, the significant amount of folic acid found in the rice suggested that the folic acid originated externally, i.e. from the soaking solution.

Table 3-3 Uptake of folic acid (µg/ g) after 1 h soaking at 60 ◦C Steaming time (mins) Milling time (s) 15 60 0 206.8±7.4 (5.2)* 212.7±20.8 (5.3) 30 145.0±8.4 (3.6) 154.1±13.5 (3.9) 60 99.9±10.4 (2.5) 140.8±13.8 (3.5)

Mean value ± standard deviation. * Values in parenthesis indicate the percentage of folic acid uptake.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

3.2.3.5 Reproducibility of fortification through parboiling

Inter-batch variation of folic acid concentration in the fortified parboiled rice is an important factor because the laboratory-scale production is usually the preliminary phase for commercial scale-up. The method shows higher potential for future scale-up if the inter-batch variation is low in the initial laboratory-scale production.

As well as from soaking the rice at 60 ◦C which was previously chosen in Study 1, an exploratory soaking temperature, 70 ◦C, was also studied to 1) investigate the reproducibility of producing a consistent amount of folic acid in the fortified rice, and 2) to determine the potential of fortifying rice under a different soaking conditions. Three individual batches of fortified parboiled brown rice were prepared on three different days. Brown rice was soaked at the designated temperature and then subjected to heating and drying. Dried parboiled rice was milled for 60 s for this study and the mean folic acid concentration and coefficient of variation was shown in Table 3-4. The inter-batch coefficient of variation % for the mean folic acid at 60 ◦C and 70 ◦C were 7.8 and 3.0, respectively. The variation obtained by this method was lower than the polymer coating method reported by de Ambrosis (2006), where the coefficient of variation % was found to be between 14.5 and 18.7. de Ambrosis (2006) suggested that the large inter-batch variation was primarily due to the manual and pilot-scale nature of the rice fortification process during rice coating. Moreover, significant differences occurred because of the changes in the pectin solution concentration, drying time, rice grain agitation, spray parameters, folic acid losses to the air and adheres onto the rotating pan.

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

Table 3-4 The folic acid concentration (µg/ g) of 3 batches of fortified parboiled rice soaked at 60 and 70 ◦C Fortified parboiled rice 60 ◦C* 70 ◦C* Sample no. 1 135.7±0.9 169.8±2.4 2 140.8±6.5 174.2±0.2 3 148.7±18.3 180.5±1.5 Mean ± SD 140.3±10.9 174.3±5.2 Coefficient of variation (%) 7.8 3.0

* After soaking the rice at the designated soaking temperature for 1 h, rice was steamed at 100 ◦C for 60 mins and dried below 14 % moisture content. Dried rice was milled for 60 s.

The low inter-batch variation in the current study may be due to the two precautions made during the soaking stage (as soaking was identified to be the point of fortification), they were 1) the control of soaking temperature and 2) the transfer of folic acid into the soaking water.

In the initial phase of this study, brown rice was added directly to water that was heated to the designated soaking temperature, i.e. 60 or 70 ◦C. However, it was found that the temperature of brown rice was about 10 ◦C lower than the desirable temperature after added to the soaking water. The observation was similar to that suggested by Islam et al. (2002b) and Miah et al. (2002). Based on the result reported by Islam et al. (2002b) and from the repeated trials, in this study the soaking water was heated to 15 ◦C above the desirable soaking water temperature and then added to the brown rice (which was removed from the coolroom and brought to room temperature overnight) in order to successfully achieve the designated soaking temperature.

With regard to the transfer of folic acid into the soaking water, a weighing paper was previously used as the container to weigh out the exact amount of fortificant to be added to the soaking water. It was found that residual folic acid remained on the weighing paper after adding the fortificant to the soaking water. Two individual batches of fortified rice (with identical parboiling conditions) were prepared using weighing paper to transfer the fortificant. The mean folic acid found in the rice was 218.6±5.6 and 160.6±2.0 µgfolic acid/

Chapter 3 The feasibility of fortifying rice with folic acid through parboiling

grice. The distinctive difference (~30 %) in mean folic acid concentration suggested that weighing paper was not appropriate to transfer the fortificant. A plastic centrifuge tube was then chosen to replace the weighing paper because the fortificant could be quantitatively transferred into the soaking water using aliquots of water. All of the fortificant could be thoroughly washed into the soaking water.

3.2.4 Conclusions

The foremost research challenge regarding the possibility that the husk and oil-rich bran layer of rice may prevent the penetration of water-soluble materials was addressed with the visual illustration of methylene blue dye. Sufficient soaking allowed the penetration of dye into the endosperm, and the effect was more noticeable when brown rice was used as the starting material rather than paddy rice. The experiment was repeated with the actual fortificant, i.e. folic acid. The evidence of folic acid penetration into the grain was proved by quantifying the folic acid content in the successively milled fortified parboiled rice. The significant amount of folic acid present in the fortified rice milled for 60 s suggesting that parboiling was feasible for fortifying rice. Also, the inter-batch variation of fortified rice was less than 8 %, confirming that the proposed method was reliable and robust.

Chapter 4-Optimisation of parboiling conditions: Folic acid uptake and retention

CHAPTER 4

OPTIMISATION OF PARBOILING CONDITIONS: FOLIC ACID UPTAKE AND RETENTION

Subsequent to the confirmation of the feasibility of brown rice parboiling as a means of fortification, the choice and optimisation of parboiling conditions is the next milestone of the project. The experiment by Prom-u-thai et al. (2008) indicated that, among others, fortificant level, soaking time and milling time were key control variables for the retention of nutrient in the parboiled rice ( Tulyathan et al., 2007, Prom-u-thai et al., 2008, Prom-u-thai et al., 2010). Nonetheless, there is a paucity of information on the multifactorial nutrient fortification process. The objective of this chapter was to seek a unifying framework that may be used to delineate regions for optimal processing conditions during folic acid fortification of rice. Specifically, two consecutive studies were performed with specific orthogonal main effect plan in order to obtain a regression polynomial model useful for constrained optimisation. This investigation provides, to the best of our knowledge, the first attempt to procure insights into fortificant retention kinetics during the soaking and milling operations. This information is useful in the design of improved soaking and milling process vessels.

4.1 Materials and methods

4.1.1 Preparation of brown rice

Intact brown rice was prepared according to Section 3.2.2.1.

4.1.2 Selection of fortification conditions

4.1.2.1 Soaking temperature and duration

Chapter 4-Optimisation of parboiling conditions: Folic acid uptake and retention

As shown in Table 4-1, a wide variance of soaking conditions was previously studied for rice parboiling, the soaking conditions ranged from room temperature to 100 ˚C and soaked for minutes up to hours (Table 4-1). The variation of conditions may be due to the fact that different conditions may impose a different impact on rice depending on their varieties (Parnsakhorn and Noomhorm, 2008). To the best of our knowledge, the rice variety of the current study, Langi, has not been previously studied for parboiling. The suitable soaking temperatures and durations were therefore determined experimentally, ranging from 30 ◦C (which represented the traditional room temperature soaking in India or Bangladesh) to 80 ◦C, because a significant portion of rice kernel split open and the content of the endosperm is exposed when the brown rice is soaked above 85 ◦C for less than 30 mins. Sridhar and Manohar (2003) also reported that at approximately 80 ◦C, splitting of the husk occurred. The effective soaking temperature was restricted to between 30 and 80 ◦C. Based on Table 4-1, the soaking duration of brown rice was normally less than 4 h at elevated temperature (i.e. > 50 ◦C), preliminary experimentation suggested that 4 h soaking at 70 ◦C softened the brown rice kernel and it was difficult to handle. Therefore, the soaking duration was chosen to be up to 3 h for the soaking temperatures studied.

4.1.2.2 Heating temperature and duration

According to Section 2.6.2.2, heating of soaked rice was to promote starch gelatinisation further and aimed to produce translucent rice without the white opaque core (“white belly”) (Bello et al., 2006). The use of steam under pressure has gained popularity as it normally shortened the heating duration. However, the installation cost of the high- pressure parboiling equipment is general higher than using the regular steaming unit (Pillaiyar and Mohandas, 1981, Parnsakhorn, 2009). Moreover, the traditional parboiling procedure (i.e. soaking, steaming and drying) is still used by the majority of parboiling countries, partly because of their tradition, existing machinery and familiarity (Igathinathane et al., 2005). Therefore, steaming (100 ◦C) at atmospheric pressure was chosen as the heating method in the current study. In a preliminary selection process, 1.2

Chapter 4-Optimisation of parboiling conditions: Folic acid uptake and retention

gfolic acid/ 300 gbrown rice was added to the soaking water (600 mL). The rice was soaked at 70 ◦C for 1, 2 and 3 h and subsequently steamed at 100 ◦C for two durations, i.e. 15 mins and 60 mins (Table 4-2 ). No significant difference in folic acid concentration resulted between 15 and 60 mins steaming at the respective soaking duration. Nonetheless, based on visual assessment, white belly was observed in the core of rice after 15 mins steaming and a translucent rice kernel resulted after 60 mins steaming. As reviewed in Section 2.7.1, the kernel of parboiled rice is expected to be translucent. Therefore, 60 mins steaming was deemed to be the appropriate heating duration.

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

Table 4-1 Parboiling conditions studied by previous researchers Soaking Heating Drying Type of rice Soaking Soaking Temp: Temp: Heating Drying Drying References temp. time steaming pressure time method time (◦C) (h) (◦C) (psi) (mins) (h) Paddy 60 6-7 100 - 14 Ambient - (Raghavendra Rao and Juliano, 1970) Paddy 65 6 80, 90, 100 120 10-60 Shade drying 1-2 (Islam et al., 2002b)

Paddy 25, 80 0.25, 2 - 121 - Thin-layer dryer - (Miah et al., 2002) (31-37 ◦C) Paddy 35, 45, 55, 5-6 100 - 10 Shade drying 2 (Sridhar and Manohar, 60, 65, 75 2003) Paddy 85,95 5-10 85, 95 - 45-180 - - (Bello et al., 2006) Paddy 30, 50, 70, 16 100 - 4-12 Tray dryer 4 (Himmelsbach et al., 90 (40 ◦C) 2008) Paddy 40, 50, 60 3 - 121 15 Cabinet dryer - (Sareepuang et al., (60 ◦C) 2008) Brown 70, 80, 90, 1, 2, 4 100 - 5-20 Shade drying - (Kar et al., 1999) 100 Brown 65, 68 0.2, 2.5 - 110, 135, 4-44 Ambient 48 (Lamberts et al., 140 2006b) Brown 50 0.5 80, 100 105, 115, 2-17 Ambient 48 (Lamberts et al., 2008) 125 Brown 70, 80 1,2,3,4 100 - 10-20 Shade drying - (Parnsakhorn and Noomhorm, 2008)

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

Table 4-2 Concentration of folic acid (µg/g) after soaking rice at 70 ◦C for 1, 2 and 3 h, steamed for 15 and 60 mins and 0 s milling Steaming time (mins) Soaking time (h) 15 60 1 247.3±15.3a+ 230.7±23.1a 2 256.3±15.6a 262.1±9.6a 3 333.1±19.6a 296.4±19.7a

+ Values followed by different letters in the same row indicate significantly different means at p < 0.05.

4.1.2.3 Drying conditions

Shade drying was reported to be a traditional and preferable drying method which resulted in excellent milling quality and head yield from parboiled rice (Bhattacharya and Swamy, 1967, Ali and Pandya, 1974). As discussed in Section 2.6.2.3, the desirable end- point of drying is approximately 12-14 % moisture content (wet weight basis) in order to ensure safe storage. Shade drying was therefore chosen as the subsequent step after the steaming parboiled rice. Experimental results indicated that approximately 4 days were required to reduce the moisture content of parboiled rice to ~12 % after the steaming process. Parboiling process was considered to be completed.

4.1.2.4 Milling duration

At the end of parboiling, brown rice is generally milled to produce white rice (according to Section 3.2.2.3), which is the preferred form of consumable rice because brown rice often possesses distinctive cooking properties and sensory attributes (Bhattacharya, 2004). White rice is obtained by removing the bran layer of brown rice which is approximately 5-8 % of brown rice weight (Unnikrishnan and Bhattacharya, 1987, Lamberts et al., 2006b). Figure 4-1 illustrates the appearance of parboiled rice milled between 30 and 120 s was compared to commercial parboiled rice. The degree of milling was ~3, ~6 and ~9 % at the respective milling durations. A significant amount of bran remained after 30 s

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

milling which resembled under-milling. Milling for 60 s improved the appearance of the grains and produced rice with only a small portion of residual bran layer remaining on the endosperm whereas 120 s milling resulted in rice with essentially no bran layer. Therefore, 120 s milling was chosen as the optimal milling duration to yield white rice. Anyhow, for the purpose of studying the behaviour of folic acid retention in parboiled rice, milling durations of 0, 60 and 120 s milling were considered in the following studies.

Figure 4-1 Parboiled rice after milling for A) 30 s; B) 60 s; C) 120 s and D) commerical parboiled rice

4.1.2.5 Fortificant concentration

The addition of folic acid in the soaking water of brown rice during parboiling was a new fortification approach. Therefore at this exploratory stage, an arbitrary folic acid -3 -1 concentration, i.e. 1.2 gfolic acid/ 300 gbrown rice (4 x10 gfolic acid g brown rice) was studied -3 - (Chapter 3). Moreover, 3 additional fortificant concentrations, 0.6 (2 x10 gfolic acid g 1 -3 -1 -3 brown rice), 0.3 (1 x10 gfolic acid g brown rice) and 0.15 gfolic acid/ 300 gbrown rice (0.5 x10 gfolic -1 acid g brown rice), were studied in order to investigate the potential contribution of fortificant concentration to the folic acid retention upon parboiling.

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

4.1.2.6 Parboiling conditions summary

According to the fortification conditions discussed in Section 4.1.2.1 to 4.1.2.5, two studies were conducted (Study 1 and Study 2) and the corresponding conditions studied are summarised in Table 4-3 and Table 4-4, respectively.

Table 4-3 Summary of parboiling conditions used in Study 1 Soaking temp. Fortificant conc. Soaking time Milling time ◦ 3 -1 ( C) (x10 gfolic acid g brown rice) (h) (s) 30 4 1 0,120 2 0,120 3 0,120 2 1 0,120 2 0,120 3 0,120 0.5 1 0,120 2 0,120 3 0,120 50 4 1 0,120 2 0,120 3 0,120 2 1 0,120 2 0,120 3 0,120 0.5 1 0,120 2 0,120 3 0,120 70 4 1 0,120 2 0,120 3 0,120 2 1 0,120 2 0,120 3 0,120 0.5 1 0,120 2 0,120 3 0,120 80 4 0.25 0,120 0.5 0,120 1 0,120 2 0.25 0,120 0.5 0,120 1 0,120 0.5 0.25 0,120 0.5 0,120 1 0,120

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

Table 4-4 Summary of parboiling conditions used in Study 2 Soaking temp. Fortificant conc. Soaking time Milling time ◦ 3 -1 ( C) (x10 gfolic acid g brown rice) (h) (s) 70 4 1 0,60,120 2 0,60,120 3 0,60,120 2 1 0,60,120 2 0,60,120 3 0,60,120 1 1 0,60,120 2 0,60,120 3 0,60,120 0.5 1 0,60,120 2 0,60,120 3 0,60,120

4.1.2.7 Parboiling procedures

The fundamental parboiling procedure was followed as described in Section 3.2.2.2. Folic acid was dissolved in the soaking water and 300 g of graded brown rice was added at water-to-rice ratio of 2:1. Brown rice was soaked at the desirable temperature and duration, and subsequently subjected to steaming in a 100 ◦C steaming bath for 60 min. Steamed samples were spread out to dry at room temperature under shade until moisture content dropped to 12 - 14 % (wet weight basis). The dried parboiled rice was milled to different degrees between 0 and 120 s milling duration as described in Section 3.2.2.3.

4.1.3 Folic acid analysis

Folic acid in the fortified rice samples was extracted and purified according to Section 3.2.2.4 to 3.2.2.6, respectively. The purified extract was analysed using HPLC with the instrumental conditions detailed in Section 3.2.2.7.

4.1.4 Degree of gelatinisation

Amylose/iodine method was used to determine the degree of gelatinisation of parboiled rice. The method is based on the release of amylose during gelatinisation, and reaction

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

with iodine to form a blue colour complex which was measured at 600 nm, before and after complete dispersion of the starch at two different concentrations of alkali (Birch and Priestley, 1973).

Standard uncooked (ungelatinised) rice flour was prepared by grinding rice to pass through a 200 µm mesh screen. It represented 0 % gelatinised rice. Standard gelatinised rice was prepared by soaking rice (Langi) at 60˚C for 3 h, the excess water was drained and the rice autoclaved at 121 ˚C for 1 h. The heated rice was dried in a forced air oven at 50 ˚C and the flour was passed through a 200 µm mesh screen. This sample represented 100 % gelatinised rice. A standard calibration curve was prepared by mixing the appropriate amount of ungelatinised and gelatinised rice flour (made up to a total of 0.1 g) to achieve a 5-point standard curve between 0 and 100 % gelatinisation.

Degree of gelatinisation was determined for fortified rice soaked at 1, 2 and 3 h. For the amylose/iodine blue value, 0.1 g of ground rice sample (passed through a 200 µm mesh screen) was mixed with 48.5 mL water and 1.5 mL of 10 M KOH (Merck, Germany) and gently agitated for 5 mins. The slurry was then centrifuged for 10 mins at 4500 rpm. The supernatant (1 mL) was removed and neutralised with 0.6 mL of 0.5M HCl. It was then diluted to 10 mL with water. Subsequently, 0.1 mL of iodine reagent (0.5 g iodine and 2 g potassium iodide per 50 mL water) was then added. Three minutes after adding the iodine reagent, absorbance was read at 600 nm using a Spectrophotometer (Spectra Max M2, Molecular Device, Australia). The above procedure was repeated on a duplicate sample except an addition of 3 mL instead of 1.5 mL of 10 M KOH was made to the rice flour/water mixture and 1.2 mL instead of 0.6 mL of 0.5 M HCl for final neutralization. The standard flour was similarly treated. The degree of gelatinisation was calculated as the ratio of the two absorbances obtained, relative to the established standard curve.

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

4.2 Results and discussions

4.2.1 Study 1- Multifactorial analysis: Investigation of fortificant concentrations, soaking temperatures and milling durations on folic acid uptake profile in fortified rice

4.2.1.1 Variance analysis

In the following context, the amount of fortificant (i.e. folic acid) was defined as doped fortificant and folic acid that was present in the fortified parboiled rice is referred to as residual folic acid.

As mentioned in Section 4.1.2.1, the maximum soaking temperature was restricted to 80 ◦C because brown rice underwent physical deformation when soaked at 85 ◦C for even less than 30 mins. To maintain the consistency of the number of variables and yet to conserve the validity of experimental quality, three soaking durations were investigated for 80 ◦C soaking but at reduced soaking times, i.e. 0.25, 0.5 and 1 h. As shown in Table 4-5, prolonged soaking times enhanced folic acid retention although the doped fortificant concentration showed more noticeable impact. Longer milling resulted in the reduction of residual folic acid concentration. Moreover, higher soaking temperature improved the residual folic acid concentration at the respective parboiling conditions. These qualitative observations were further analysed in order to study the impact of different parboiling variables on folic acid retention in parboiled rice. Due to the deformation of rice kernel at the elevated soaking temperature, 1 h soaking was the common soaking duration across all the soaking temperatures studied and hence it was chosen for the following analyses.

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

Table 4-5 Residual folic acid retention concentration in raw (0 s) and milled (120 s) fortified grains for three doped fortificant concentrations at the respective soaking time and durations 3 1 Residual folic acid concentration in rice (10 gfolic acid grice ), in replicate Milling time, s (XMT) Soaking temp Fortificant conc. Soaking time 0 120 ◦ 3 -1 ( C) (x10 gfolic acid g rice) (h) (0) (1) (XSTemp) (XFC) (XST) 30 (0)* 4 (1) 1 0.153, 0.172 0.061, 0.058 2 0.159, 0.179 0.085, 0.079 3 0.183, 0.199 0.087, 0.080 2 (0.429) 1 0.082,0.106 0.038, 0.037 2 0.084, 0.117 0.050, 0.048 3 0.091, 0.126 0.057, 0.054 0.5 (0) 1 0.021, 0.027 0.009, 0.011 2 0.023, 0.031 0.016, 0.017 3 0.026, 0.037 0.018, 0.019 50 (0.4) 4 (1) 1 0.200, 0.210 0.095, 0.081 2 0.201, 0.219 0.122, 0.083 3 0.239, 0.233 0.133, 0.096 2 (0.429) 1 0.108, 0.119 0.053, 0.054 2 0.118, 0.129 0.065, 0.060 3 0.130, 0.143 0.074, 0.071 0.5 (0) 1 0.025, 0.029 0.012, 0.012 2 0.031, 0.033 0.020, 0.014 3 0.034, 0.042 0.023, 0.032 70 (0.8) 4 (1) 1 0.204, 0.244 0.167, 0.179 2 0.265, 0.273 0.200, 0.186 3 0.292, 0.307 0.247, 0.248 2 (0.429) 1 0.116, 0.107 0.083, 0.080 2 0.115, 0.112 0.089, 0.087 3 0.125, 0.116 0.101, 0.098 0.5 (0) 1 0.027, 0.031 0.020, 0.020 2 0.032, 0.037 0.025, 0.024 3 0.045, 0.039 0.037, 0.033 80 (1) 4 (1) 0.25 0.221, 0.190 0.091, 0.057 0.5 0.223, 0.237 0.090, 0.090 1 0.522, 0.489 0.260, 0.189 2 (0.429) 0.25 0.099, 0.100 0.042, 0.042 0.5 0.115, 0.115 0.059, 0.050 1 0.260, 0.182 0.147, 0.099 0.5 (0) 0.25 0.025, 0.024 0.012, 0.010 0.5 0.028, 0.027 0.016, 0.013 1 0.069, 0.037 0.048, 0.047 * Values in parentheses represent the dimensionless conversion of variables.

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

Two-way analysis of variance (ANOVA) was performed on the replicated data for the residual folic acid concentration of raw rice fortified at three levels of doped fortificant concentrations, four levels of soaking temperatures and two levels of milling time (Table 4-3). For consistency and to minimise numerical errors, all the variables, i.e. doped fortificant concentration (XFC), soaking temperature (XSTemp) and milling time (XMT), were expressed in dimensionless form as:

VVii ,min X i  (Equation 4-1) VVii,max,min

th where Xi is the i dimensionless variable, min and max representing the lower and upper

-3 1 values of original variable, Vi respectively. Specifically, VFC,max = 410 gfolic acid grice ; -3 ◦ ◦ VFC,min = 0.510 gfolic acid ; VSTemp,max = 80 C; VSTemp,min = 30 C. VMT,max = 120 s; VMT,min = 0 s.

The F-values of the doped fortificant concentration, soaking temperature and milling time were higher than the corresponding critical F-values, indicating that these factors were statistically significant to residual folic acid retention (Table 4-6). Furthermore, strong interactions were found between fortificant concentration-soaking temperature (XFCStemp), fortificant concentration-milling time (XFCMT) and soaking temperature-milling time

(XSTempMT), suggesting that with the incorporation of fortificant concentration, soaking temperature, and milling time affect folic acid retention dependently. Consequently, the relevant regression model is given by the multilinear expression:

(Equation 4-2)

where is the residual folic acid concentration; a0 is the concentration of natural folate present in rice ( ) at XFC = XSTemp = XMT = 0; aFC is the doped fortificant concentration coefficient; XFC is doped fortificant concentration; aSTemp is the soaking

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

temperature coefficient; XSTemp is the soaking temperature; aMT is the milling time coefficient; XMT is the milling time; aFCSTemp is the doped fortificant concentration- soaking temperature interaction coefficient; XFCXSTemp is the doped fortificant concentration-soaking temperature interaction; aFCMT is the doped fortificant concentration-milling time interaction coefficient; XFCXMT is the doped fortificant concentration-milling time interaction; aSTempMT is the soaking temperature-milling time interaction coefficient; XSTempXMT is the soaking temperature-milling time interaction.

Table 4-6 Two-way ANOVA results for A) fortificant concentration by soaking temperature (top); B) soaking temperature by milling time (middle), and C) fortificant concentration by milling time (bottom) at 1 h soaking A Fortificant conca Soaking tempb Interaction Dimensionless (XFC) (XSTemp) (Fortificant conc x Milling time Soaking temp)c (XFCXSTemp) 0 74.8 253.5 23.1 1 28.2 76.9 6.2 B Soaking tempd Milling timee Interaction (Soaking Dimensionless (XSTemp) (XMT) temp x Milling Fortificant conc time)d (XSTempXMT) 0 6.3 4.3 0.2 0.429 26.5 15.5 1.4 1 142.1 94.4 18.4 C Fortificant concf Milling timeg Interaction Dimensionless (XFC) (XMT) (Fortificant conc x Soaking temp Milling time)g (XFCXMT) 0 107.4 121.7 24.3 0.4 462.9 351.2 75.8 0.8 206.5 21.8 4.2 1.0 76.7 38.8 13.0 a Critical value of F(2,12) = 3.9 b Critical value of F(3,12)=3.5 c Critical value of F(6,12)=3.0 d Critical value of F(3,8)=5.3 e Critical value of F(1,8)=4.1 f Critical value of F(2,6)=5.1 g Critical value of F(1,6)=6.0

The coefficients of the multifactorial model are displayed in Table 4-7. Among the variables studied, doped fortificant concentration has the most impact on the retention of folic acid in rice followed by soaking temperature and milling time. It is reasonable as the

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

intrinsic natural folate in rice is minimal compared to the doped fortificant in the soaking water.

Table 4-7 Regression coefficients of multifactorial model Regression Coefficient Degree of significance (106) a0 12.01 0.71 aFC 130.56 0.01 aSTemp 37.47 0.42 aMT -12.19 0.76 aFCSTemp 201.09 0.00 aMTFC -124.16 0.01 aMTSTemp -44.03 0.38

4.2.1.2 pH study

Folic acid uptake coefficients at different soaking temperatures

o Folic acid (C19H19N7O6) is a triprotic acid soluble in water (160 mg/100g H2O at 25 C) with three pKa values of 4.65, 6.75 and 9.00 (Dawson, 1989). As a result, within the concentration range used in this study, folic acid would exist as folate anions in the soaking water. (Tyagi and Penzkofer, 2010). The dynamic movement of folic acid (folate anions) between the solid (brown rice) and liquid (water and folic acid solution) phases of the system may affect the pH value of the soaking solution at the end of the soaking stage. The pH of the soaking water was therefore measured at the end of the designated soaking duration for all the parboiling conditions studied (Figure 4-2). Equation 4-3 is used to describe the pH of the soaking water at different fortificant levels at the respective soaking temperature:

(Equation 4-3)

where pH0 is the pH value in the soaking water used for the undoped rice (folic acid-free rice), b is the folic acid uptake coefficient and CFC is the doped fortificant concentration.

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

Figure 4-2 pH of soaking solution at different soaking temperatures, (i.e. A: 30˚C; B: 50˚C; C: 70˚C; D: 80˚C) with various fortificant concentrations with respect to soaking times (i.e. 1, 2 and 3h)

From a plot of 1/pH against CFC based on Equation 4-3, the slope (b/pH0) and intercept

(1/pH0) may be used to estimate b (folic acid uptake coefficient) in Equation 4-4, which may be captured by:

(Equation 4-4)

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

where  and n are both empirical constants and t is the soaking time.

With the current parboiling conditions investigated, γ values were 71.7, 101.2, 91.7 and 89.1 for 30, 50, 70 and 80 ˚C, respectively whereas n values were -0.3, -0.2, -0.4 and -0.3 in which the n value was essentially temperature-invariant at about -0.3 (Figure 4-3).

Figure 4-3 Folic acid uptake coefficients (b) of rice soaked at 30, 50, 70 and 80 ◦C for the corresponding soaking durations

The decreasing folic acid uptake rate in relation to soaking time (as suggested by the negative value of n) was consistent over the four soaking temperatures. This suggests that the folic acid retention mechanism was governed by the same mechanism at the range of soaking temperature studied. The decreasing folic acid uptake rate may be due to the diminishing moisture gradient between the soaking water and rice kernel with soaking time (Bakshi and Singh, 1980, Kashaninejad et al., 2007).

Given that 1 h soaking was the soaking duration in common over the studied soaking temperatures, by substituting tsoak = 1 into Equation 4-4, the highest folic acid uptake

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

coefficient, b, was between 50 and 70 ˚C, followed by 80 ˚C and dropped at 30 ˚C soaking. Elevated soaking temperatures therefore appeared beneficial to folic acid uptake in fortified rice. Based on this result, 50 ˚C appears to be the best soaking temperature for the enhancement of folic acid in parboiled rice. However, given that the gelatinisation temperature of the rice is about 73 ˚C, soaking at 50 ˚C for 1 h is not sufficient to produce parboiled rice free from white belly, i.e. opaque core present in the middle of the rice kernel despite the subsequent steaming procedure (Igathinathane et al., 2005, Bello et al., 2006) (Table 4-8). A soaking temperature of 70 ˚C between 1 and 3 h is therefore recommended as a minimum amount of white belly resulted and the folic acid uptake coefficient was the second highest among the temperatures investigated.

Table 4-8 Degree of gelatinisation of parboiled rice after different parboiling treatments (without folic acid added to soaking water) Soaking temp Soaking time DOG White belly (◦C) (h) (%) 30 1 10.0 + 2 22.1 + 3 37.9 + 50 1 48.1 + 2 72.8 + 3 83.0 - 70 1 78.9 - 2 83.0 - 3 99.1 - 80 0.25 61.7 + 0.5 78.9 - 1 100.0 - a ControlCom - 100.0 - + White bellies were observed. - White bellies were not observed. a ControlCom refers to commercial parboiled rice.

Activation energy of the fortification system

Based on Figure 4-4, the change of pH upon soaking at different temperatures may be described by the linear model:

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

pHo = kXST+pHIS (Equation 4-5) where is the pH value in the soaking water used for the undoped rice (folic acid free rice); the slope ( ) representing the rate of change of pH; is the soaking time; is the intercept which indicates the pH for incipient soaking ( = 0).

Figure 4-4 pH change over different soaking temperatures and durations in folic acid-free -1 soaking solution (0 gfolic acid g rice)

Considering where is the concentration of hydrogen ions, the relationship between pH and soaking temperature may be described by the usual Arrhenius equation:

(Equation 4-6) where is the concentration of hydrogen ions; is the activation energy for the change of pH; R universal gas constant; T is the soaking time.

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

Given that the incipient soaking values were found to be 7.02, 6.93, 6.61 and 7.39 for 30, 50, 70 and 80 ˚C, respectively. By plotting ln (of the incipient soaking pH values) against 1/T, the slope represents and therefore the activation energy of the process can be estimated (Figure 4-5).

Figure 4-5 Amount of hydrogen ions at incipient soaking at different soaking temperatures (30, 50, 70 and 80 ˚C)

Figure 4-5 illustrates that between 30 and 70 ˚C the change in ln was relatively linear but a break point occurred beyond 70 ˚C. Given that the slope represents , the activation energy of the process between 30 and 70 ˚C was found to be 20.1 kJmol-1. Previous studies suggested that the activation energy values estimated for diffusion of water in rice kernel ranged between 14.2 and 32.9 kJmol-1 for soaking temperature from 30 ◦C to 85 ◦C (Bakshi and Singh, 1980, Lin, 1993, Bello et al., 2007). The variations between the activation energy found by different research groups may be due to 1) the range of soaking temperature studied; 2) the type of rice used, i.e. rough rice, brown rice and white rice; and, 3) the varieties of rice chosen (Suzuki et al., 1976, Bakshi and Singh, 1980, Bello et al., 2007). Bakshi and Singh (1980) reported that the activation energy of

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

diffusion required by rough rice (32.9 kJmol-1) was higher than brown rice (16.1 kJmol-1) for the same temperature range, 50-85 ◦C. The elevated activation energy is likely due to the presence of the husk which acts as an effective moisture-barrier to the rice kernels (Kar et al., 1999). Given the measure of pH gives a collective overview of activities undergone by the rice kernel during soaking, by comparing the activation energy of the current process between 30 and 70 ˚C to previous studies, the activation energy (Ea30-70◦C) of the current experiment was 20.1 kJmol-1, which aligned with the previously reported activation energy values for diffusion. It therefore likely indicates that between 30 and 70 ˚C, the system for folic acid fortification is a symptomatic diffusional transport phenomenon.

Beyond 70 ˚C, the direction of the slope was reversed as opposed to that between 30 and 50 ˚C (Figure 4-5). Previous studies had also observed the break point, a marked change in the slope of activation energy, occurred both in water diffusivity and starch reactivity, at temperatures between 60 and 85 ˚C for paddy and 110 ˚C for cooked rice (Suzuki et al., 1976, Bakshi and Singh, 1980, Bello et al., 2007). Although no explicit explanation was given to the occurrence of the break point, it was believed that it relates to a significant change in the structural properties of the grain at the break point temperature (Bello et al., 2007). Sayar et al. (2001) reported that the break point temperature was in close agreement with the experimentally determined gelatinisation temperature of the food sample studied, i.e. chickpeas. Note that pH is an indicator of the summation factors present in the current system. Further analysis is required to specifically isolate the contributing factors. Based on the consistency between the gelatinisation temperature and the remarkable change of activation energy at 70 ˚C and beyond, 70 ˚C appeared to be a critical temperature for the fortification of folic acid in rice.

4.2.1.3 Moisture content study

Based on the earlier discussion, the underlying mechanism of folic acid fortification in rice (i.e. liquid-solid phase system) may be based on the diffusional transport of folic acid

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

from the soaking solution into rice. Therefore moisture content of rice is an important aspect to be investigated. Figure 4-6 illustrates the change in residual folic acid concentration with the corresponding moisture content.

3 -1 Figure 4-6 The change of residual folic acid concentration (10 gfolic acid g rice) in fortified rice in relation to the moisture content (%) of rice at the end of the soaking stage

The relationship between residual folic acid concentration and moisture content may be described by an Arrhenius-type equation:

(Equation 4-7a)

where is the residual folic acid concentration; is the asymptotic folic acid concentration for high moisture content; b is the moisture content coefficient and MC is the moisture content (wet weight basis %) of fortified rice. Equation 4-7a which is plotted into Figure 4-7, which is described by Equation 4-7b:

(Equation 4-7b)

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

The plot of vs was shown in Figure 4-7A. The intercepts ( ) (i.e. asymptotic folic acid concentration for high moisture content) at each fortificant concentration was further plotted into Figure 4-7B whereas the slopes (b), which represented the activation moisture content for folic acid uptake, was illustrated in Figure 4-7C.

Figure 4-7 A) Log-scale of residual folic acid concentration in fortified rice versus moisture content of rice; B) Change of ao at different fortificant concentrations; C) Change of b at different fortificant concentrations

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

2 Figure 4-7 B and Figure 4-7C showed the linear change in ao and b (R = 0.999 and 0.974, respectively) as a function of fortificant concentration and the equations for describing the trend were:

(Equation 4-7c)

(Equation 4-7d) where is the doped fortificant concentration. By substituting in Equation 4-7c, -6 -1 was found to be 3.8x10 gfolic acidg rice. This is the minimum amount of fortificant required to allow the transfer of folic acid in the liquid phase into the solid -6 -1 phase. Subsequently, by computing 3.8x10 gfolic acidg rice into Equation 4-7d, was found to be 30.0 %, which implies 30 % (wet weight basis) is the activation moisture content required for the uptake of folic acid from the soaking water into the brown rice. Igathinathane et al. (2005) and Miah et al. (2002) also reported that the end-point moisture content of the soaked rice was between 30 and 37 % (wet weight basis). The result suggested that the critical moisture content required for the fortification purpose in this system matches with the conventional parboiling requirements, therefore again indicating that fortification may likely occur during parboiling. Proper hydration of soaked rice is required to prevent the occurrence of white bellies which would affect the milling quality of rice resulting in the higher chance of rice breakage (Roy et al., 2003).

4.2.2 Study 2- Multifactorial Analysis: Investigation of fortificant concentrations, soaking durations and milling durations on folic acid uptake profile in fortified rice

According to Study 1, the optimal soaking temperature was identified to be 70 ◦C based on the overall achievement of the computed folic acid uptake coefficients and the quality of parboiled rice (i.e. disappearance of white bellies). Study 2 was therefore subsequently conducted focussing on this soaking temperature. The parboiling conditions were extended to four levels of doped fortificant concentrations (4 x10-3, 2 x10-3, 1 x10-3 and

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

-3 -1 0.5 x10 gfolic acid g rice), three levels of soaking time (1, 2 and 3 h) and three levels of milling time (0, 60 and 120 s) ( Table 4-4).

4.2.2.1 Variance analysis

From the residual folic acid concentration in parboiled rice presented in Table 4-9, a significant increase in the residual folic acid concentration was observed in the parboiled rice. Negligible amount of folic acid was found in the unfortified parboiled rice (control), indicating that the detected residual folic acid in the fortified grains was due to the addition of the fortificant in the soaking solution. In addition, a linear increase (R2 > 0.98) in residual folic acid concentration with fortificant content in the soaking solution was seen in the parboiled rice on a dry weight basis (Figure 4-8).

Figure 4-8 Linear relationship between the residual folic acid concentration in fortified rice and the doped fortificant concentrations at 0 s milling time

This again suggests that fortificant uptake likely proceeded via diffusional transport of the folic acid anions from the bulk liquid solution to the outer surface of the rice grain, referred to as the aleurone layer where adsorption takes place. Intuitively, as the folic acid soaking solution content increased, the retained folic acid in the rice grain would continue

Chapter 4 Optimisation of parboiling conditions: Folic acid uptake and retention

to increase until a critical (saturation) folic acid concentration in the liquid phase where equilibrium is established with solid (rice) phase concentration and beyond which mass transport would cease. It would therefore seem that the linear increase in residual folic acid concentration with increasing liquid phase folic acid content is indicative of the fact that the saturation folic acid concentration in the rice was higher than the highest value observed in the study and hence, the data were suitable for kinetic analysis.

Table 4-9 Residual folic acid concentration in milled fortified raw grains at 0, 60 and 120 s for four doped fortificant concentrations at the respective soaking times 3 1 Residual folic acid concentration in rice (10 gfolic acid grice ), in duplicate Milling time, s (XMT) Fortificant conc., Soaking time, 0 60 120 3 -1 10 gfolic acid g rice h (0) (0.5) (1) (XFC) (XST) 1 0.204 0.170 0.166 (0) 0.244 0.184 0.179 4 2 0.265 0.211 0.200 (1.000)* (0.5) 0.273 0.203 0.186 3 0.292 0.253 0.247 (1) 0.306 0.247 0.248 1 0.116 0.083 0.083 (0) 0.107 0.084 0.080 2 2 0.115 0.099 0.089 (0.429) (0.5) 0.112 0.095 0.087 3 0.125 0.104 0.101 (1) 0.116 0.132 0.098 1 0.055 0.047 0.044 (0) 0.067 0.055 0.053 1 2 0.058 0.058 0.043 (0.143) (0.5) 0.067 0.061 0.050 3 0.090 0.073 0.071 (1) 0.090 0.072 0.072 1 0.027 0.024 0.020 (0) 0.031 0.021 0.020 0.5 2 0.033 0.024 0.025 (0) (0.5) 0.037 0.025 0.024 3 0.045 0.041 0.037 (1) 0.039 0.033 0.033 * Values in parentheses represent the dimensionless conversion of variables

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Table 4-10 displays the moisture content of rice measured after the soaking stage. It is evident that moisture content generally increased with the soaking duration at all fortificant levels, which is likely due to the difference in moisture content gradient between the rice kernels and the surrounding environment, i.e. soaking water with the added folic acid (fortificant). Bakshi and Singh (1980) have also indicated that moisture content of rice increases with increased soaking period. The result also agreed with the previous finding in Study 1 that significant folic acid retention occurred as the moisture content (in wet weight basis) was higher than 30 % (Section 4.2.1.3).

Table 4-10 Moisture content (% dry basis) of rice after soaking stage Fortificant Soaking time (h) 3 -1 conc. (10 gfolic acid g rice) 1 2 3 4 55.1 (35.6)* 57.5 (36.5) 58.1 (36.8) 2 55.2 (35.6) 57.9 (36.7) 56.9 (36.3) 1 56.9 (36.3) 60.8 (37.8) 64.6 (39.3) 0.5 57.2 (36.4) 58.7 (37.0) 59.3 (37.2) * Values in parentheses represent the moisture content in wet basis.

Steaming was the subsequent step in the parboiling procedure after soaking. The increased moisture content in rice kernels after soaking and the supply of heat (steaming at 100 ◦C) caused irreversible changes to the starch granules. The kinetic energy of the starch increased and intermolecular hydrogen bonds were ruptured, leading to the disruption of structural integrity of starch (Lund and Lorenz, 1984, Stoddard, 2004) and gelatinised starch. In the present study, the degree of gelatinisation of 1, 2 and 3 h soaking (at 70 ◦C) was found to be 78.9, 83.0 and 99.1 %, respectively (Table 4-8). The progressive increment in the degree of gelatinisation coincided with the increasing folic acid uptake trend for prolonged soaking durations. It indicated that starch gelatinisation may also be a potential contributor to retain or bind to the folic acid (fortificant) that had diffused into the endosperm from the soaking water. During starch gelatinisation, the swollen starch may fuse with the inner bran and scutellum layers in the endosperm,

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preventing the loss of the vitamin by further treatments such as milling (Rao and Bhattacharya, 1966).

The successive decrement of folic acid concentration with milling time evident from Table 4-9 also supports the proposition of fortificant migration from the soaking water, through the aleurone layer, into the inner layer of the endosperm. Between 0 s and 60 s milling) the loss of residual folic acid concentration varied from 2 % to 41 %, with the average loss of 20 %. The loss of residual folic acid concentration between 60 s and 120 s milling ranged from 1 % to 21 %, while the average loss was 6 %. It is clear that the rate of folic acid loss (upon milling) was higher in the first 60 s milling. It therefore implies that the fortificant was concentrated mostly on the outer aleurone layer consistent with a higher folic acid concentration on the rice grain surface (due to adsorption). Subsequent milling for another 60 s resulted in 6 % loss of residual folic acid concentration, indicating that once the fortificant migrated beyond the outer aleurone layer, the folic acid was most likely more strongly bonded to the hydrolysed carbohydrate molecule of the rice.

Based on Table 4-9, increasing fortificant concentration improved residual folic acid concentration in milled rice. The residual folic acid concentration was enhanced by prolonged soaking time but was reduced by increased milling time. These qualitative observations were further analysed in order to study the impact of different parboiling variables on folic acid uptake in parboiled rice. Two-way analysis of variance (ANOVA) was performed on the replicated data for the residual folic acid concentration of raw rice th fortified based on Equation 4-1, where Xi is the i dimensionless variable, min and max representing the lower and upper values of original variable, Vi respectively. Specifically,

-3 1 -3 VFC,max = 410 gfolic acid grice ; VFC,min = 0.510 gfolic acid ; VMT,max = 120 s; VMT,min = 0 s;

VST,max= 3 h; VST,max = 1 h.

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Calculated F-values for both soaking and milling times were greater than the critical F- values (i.e. 4.26 at 95 % confidence interval) but not for the interaction between soaking and milling times (cf. Table 4-11A).

Table 4-11 Two-way ANOVA results for A) soaking time by milling time (top); B) doped fortificant concentration by soaking time (middle), and C) doped fortificant concentration by milling time (bottom) A Soaking timea Milling timea Interaction (XST) (XMT) (Soaking time x Milling time)b (XSTXMT) Dimensionless fortificant concentration 1 58.148 44.822 0.747 0.429 11.791 18.094 1.621 0.143 46.875 16.598 1.491 0 31.011 13.219 0.524

B Fortificant Soaking timed Interaction c conc. (XST) (Fortificant (XFC) conc x Soaking time)e (XFCXST) Dimensionless milling time 0 617.501 20.259 5.223 0.5 729.269 50.360 6.478 1 1631.955 103.862 19.623

C Fortificant Milling timed Interaction c conc. (XMT) (Fortificant (XFC) conc x Milling time)e (XFCXMT) Dimensionless soaking time 0 320.860 15.862 2.423 0.5 2145.473 106.065 26.842 1 1127.955 24.929 5.727 a Critical value of F (2,9) = 4.26 b Critical value of F (4,9) = 3.63 c Critical value of F (3,12) = 3.49 d Critical value of F (2,12) = 3.98 e Critical value of F (6,12) = 3.00

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This suggests that folic acid retention during parboiling is a function of both soaking time and milling time and they affect the retention of folic acid independently. Soaking and milling times are statistically significant at each fortificant level, indicating that the folic acid uptake mechanism was the same regardless of the doped fortificant concentration employed. This was further examined by two-way ANOVA between doped fortificant concentrations by soaking time (Table 4-11B), and doped fortificant concentrations by milling time (Table 4-11 C).

Based on Table 4-11, the associated soaking time-milling time interaction (XSTXMT) appeared to have a non-contributory effect on the residual folic acid concentration, Cfolic acid, in rice. However, interactions between doped fortificant concentration-soaking time and doped fortificant concentration-milling time were both statistically significant.

Multifactorial models were generated both with and without XSTXMT. No significant difference in the regression coefficients was observed between the two models confirming that the interaction between soaking time and milling time has minimal contribution to folic acid uptake. Therefore, the 2-factor interaction term, XSTXMT, was excluded from the model.

Consequently, the relevant regression model is given by the multilinear expression:

(Equation 4-8)

1 where a0 is the concentration of folic acid present in rice (gfolic acid grice ) at XST = XMT = XFC

= 0; aST is the soaking time coefficient; XST is the soaking time; aMT is the milling time coefficient; XMT is the milling time; aFC is the doped fortificant concentration coefficient;

XFC is doped fortificant concentration; aFCST is the doped fortificant concentration- soaking time interaction coefficient; XFCXST is the doped fortificant concentration-soaking time interaction; aFCMT is the doped fortificant concentration-milling time interaction coefficient; XFCXMT is the doped fortificant concentration-milling time interaction.

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Table 4-12 Regression coefficients of multifactorial model Regression Coefficient Degree of significance (106) a0 27.73 0.00 aST 9.71 0.29 aMT -7.95 0.35 aFC 190.01 0.00 aFCST 58.31 0.00 aFCMT -50.68 0.93

Table 4-12 displays the coefficients of the multifactorial model. The sign of the coefficient for soaking time (positive), milling time (negative) and doped fortificant concentration (positive) was in agreement with earlier discussion. The adequacy of the multivariable linear model is demonstrated in the parity plot shown in Figure 4-9 which is characterised by a diagonal angle,  = 45o and an R-squared coefficient of 0.983.

Figure 4-9 Parity plot of observed and predicted residual folic acid concentration of parboiled rice of the studied conditions (i.e. soaking time: 1h, 2h, 3h; milling time: 0s, -3 -3 -3 -3 -1 60s, 120s; fortificant concentration: 0.5x10 , 1x10 , 2x10 and 4x10 gfolic acid g rice)

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4.1.2.2 Milling kinetics

In general, the transient residual folic acid concentration profile in fortified rice was linear as seen from Figure 4-10 and consequently, the slope of the plot may be regarded as the folic acid retention rate, ( ), for a particular run. Table 4-13 summarises the retention rate data at different doped fortificant concentrations and soaking times. Although the linear relation between folic acid concentration and time may at first suggest that the milling kinetics has a zero-order behaviour, the data in Table 4-13 clearly shows that the folic acid retention rate during milling varied with initial fortificant concentration implicating a non-zero-order rate.

Figure 4-10 Example of transient folic acid concentration retention profile at doped -3 -1 fortificant concentration of 2x10 gfolic acid g rice with brown rice soaked for 2h, where slope and intercept are -2 × 10-4 and 0.112 respectively and R2=0.966

To be sure, the general equation describing the transient folic acid concentration, CFC vs t, may be obtained from the integration of;

dCFC m kCint FC (Equation 4-9a) dt

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which rewrites as;

dCFC mm1  k"  k ' CkFC kwhere C ' or k int " kFC C  int FC (Equation 4-9b) dt where dCFC/dt (= k”) is the slope of the CFC vs t profile, m is the reaction order, kint is intrinsic rate constant and k’ is a pseudo-rate constant. It is evident that if m is zero, then the slope, dCFC/dt, is equal to kint regardless of the value of CFC. However, if the experimentally obtained slope depends on CFC (as is the case here), then the reaction is not an intrinsically 1st order reaction.

7 -1 -1 Table 4-13 Folic acid retention rate (10 gfolic acid g rice s ) at various doped fortificant concentrations and soaking durations as a function of milling time Fortificant Soaking time (h) 3 -1 conc. (10 gfolic acid g rice) 1 2 3 4 4.250 6.364 4.296 2 2.512 2.088 1.748 1 1.035 1.324 1.586 0.5 0.722 0.897 0.568

4 -1 kFA (× 10 s ) 1.10 1.48 1.06

Indeed, it is evident from the present data (Table 4-13) that the folic acid retention rate has a linear dependency on the doped fortificant concentration for each soaking period and hence, a 1st order kinetic expression (cf., Equation 4-10) was used to describe the rate data.

(Equation 4-10)

where is the folic acid retention rate during milling, CFC is the initial doped fortificant concentration and kFA,milling is the specific (or intrinsic) folic acid retention -1 constant (s ). It is readily seen that kFA,milling is identical to kint in Equation 4-9b since m = 1. In fact, Equation 4-10 was suggested after initial assessment of the slope values (k”) based on a plot of ln k” against ln CFC where m, the slope of the resulting straight line,

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was obtained as 0.987 (essentially 1). The estimates of kFA,milling shown in Table 4-13 suggests an optimum at a soaking period of about 2 h (a fit of the data in the last row to a quadratic model showed a maximum at tsoak = 1.97 h).

4.3 Conclusions

This chapter showed the process of kinetics investigation for folic acid fortification in rice under parboiling using brown rice as the starting material.

In Study 1, it was suggested that fortificant concentration (XFC) showed a positive effect on the retention of folic acid in rice whereas milling time (XMT) was negative to folic acid retention. Interactions between doped fortificant concentrations, soaking temperatures and milling durations were significant. The rate of folic acid uptake during soaking is time-dependent and it is also a function of temperature. It showed that at 1 h soaking, the folic acid uptake coefficient changed from 71.7, 101.2, 91.7 to 89.1 over 30 – 80 ◦C of soaking. The soaking temperature at 70 ◦C was considered optimal due to the high folic acid uptake coefficient and relatively better visual quality of rice compared to other soaking temperatures.

The activation energy of changes in the system between 30 and 70 ◦C was 20.1 kJmol-1 which was in line with the activation energy for diffusional mechanism reported earlier (Bakshi and Singh, 1980, Lin, 1993, Bello et al., 2007), suggesting folic acid penetration into rice was likely based on diffusion. Break-point temperature occurred at 70 ◦C which coincided with the gelatinisation temperature of rice, again highlighting the critical soaking temperature at 70 ◦C. The retention of folic acid in rice was a function of moisture content. The critical (minimum amount) fortificant concentration required for -6 -1 this fortification system was 3.8x10 gfolic acidg rice and the activation moisture content for the system was 30 % (wet weight basis). This moisture content agreed with the desirable end-point for the soaking stage of parboiling, suggesting the fortification of folic acid in rice is feasible with no major alteration of the traditional parboiling procedure.

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In Study 2, the parboiling conditions (i.e. fortificant concentration, soaking durations and milling durations) were focussed on 70 ◦C soaking temperature as it was indicated to be the optimal soaking condition in Study 1. Analysis of variance revealed that doped fortificant concentration as well as milling and soaking times were all statistically significant although the effect of factor-interactions appeared negligible. The transient folic acid concentration profiles during milling were used to procure the folic acid retention kinetics. The associated 1st order rate constant exhibited an optimum at a soaking time of about 1.97 h, i.e. essentially 2 h.

In this chapter, it only showed the practical parboiling conditions which would maximize the uptake of folic acid after parboiling. Anyhow, the quality of fortified parboiled rice was another important aspect to be examined, because the practicality of the fortification method is dependent on the end-quality of rice. The value of the method would be reduced should it degrade the quality of end product, and vice versa.

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CHAPTER 5

RICE QUALITY STUDIES ON FORTIFIED PARBOILED RICE

After identifying that 70 ◦C as the recommended soaking temperature for nutrient retention in the fortified rice, the rice quality associated with such parboiling conditions was investigated. This is because rice grains undergo physical, chemical and functional changes during this process (cf. Chapter 2). For instance, water migrates into the rice kernel during soaking and subsequent heating leads to irreversible swelling and fusion of starch granules. The starch changes from orderly crystalline to an amorphous form resulting in gelatinisation (Lund and Lorenz, 1984, Himmelsbach et al., 2008). Parboiled rice is harder and takes longer to cook than unparboiled rice (Bhattacharya, 2004). The cooked parboiled rice is fluffier, less sticky and harder. The physical dimension of the grain can change after parboiling (Bhattacharya, 2004). The nutritional composition of parboiled rice changes as the concentrations of water-soluble constituents in parboiled rice is higher than in milled white rice (Bhattacharya, 2004). Enzymes and some antioxidants have been reported to be inactivated by the supplied heat (Belitz et al., 2009).

The above are only some of the examples of physicochemical changes that can occur to parboiled rice. With the spectrum of changes involved with parboiling, the quality of parboiled rice is an important aspect to examine to ensure that the end-quality of the product fulfils the specific preferences of specific users. The two main users in the rice industry can be broadly categorized into 1) those at the beginning of the supply chain, e.g. rice growers and processors, and 2) those at the end of the chain, i.e. rice consumers. Before the rice is delivered to the market, rice industries throughout the world have established certain criteria to grade the commodity. The common assessments include physical properties such as grain length, degree of milling, percentage of broken and damaged grains, moisture content and level of impurity (Kaosa-ard and Juliano, 1990). The ultimate goal of grain quality assessment is to assure economic rewards to the industry, i.e. both growers and processors, by satisfying consumers’ needs. During rice

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production, milling is an early and significant process but it imposes mechanical stresses on the grains which may lead to rice breakage. The value of broken rice is much lower than the whole kernel rice (Marshall and Wadsworth, 1994b, Bhattacharya, 2004). Head Rice Yield (HRY), the portion of whole white rice resulting from processing the paddy rice, is therefore an important quality control factor to maintain the economic return from rice marketing. After milling and polishing, the bran layer and embryo are removed which give the rice its final appearance before launching into the market. The colour of the rice and kernel dimensions are the two important visual characteristics perceived by the consumers. Consumers have different preferences for rice colour and kernel dimensions (Bergman et al., 2004, Champagne et al., 2010).

Based on the results from Chapter 4, parboiling conditions of 70 ◦C and soaking for up to 3 h for fortification was optimal based on folic acid retention and the preliminary visual assessment (i.e. white bellies). Therefore, the aims of this chapter were to focus on evaluating the quality of the parboiled rice produced using this set of parboiling conditions based on: 1) study of the concentration of folic acid present in the uncooked and cooked fortified rice that is available to the consumers, 2) impact on Head Rice Yield, and 3) observation of the visual appearance of the rice, i.e. kernel dimension and colour.

5.1 Materials and methods

5.1.1 Preparation of fortified parboiled rice

Brown rice was prepared according to Section 3.2.2.1 in order to produce graded intact brown rice prior to the following parboiling treatments.

Five soaking solutions were prepared to soak the brown wholegrain rice in rice-to-water ratio of 1:2. They were the control (with no folic acid added) and folic acid concentrations of 0.15, 0.3, 0.6 and 1.2 gfolic acid/ 300 gbrown rice. The soaking temperature for the brown rice was 70 °C and the soaking durations evaluated were 1, 2 and 3 h. After the designated soaking duration, the soaking solution was discarded and the sample was

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subsequently heated in a steam box at atmospheric pressure for 60 mins at 100 °C. Steamed samples were dried in shade at room temperature until the moisture content dropped to 12-14 % (wet weight basis). Parboiled brown rice was then milled to 60 and 120 s according to Section 3.2.2.3 and Head Rice Yield (HRY) was calculated based on the ratio between the weight of the intact milled rice and that of the initial paddy sample (Equation 3-1).

5.1.2 Cooking method

Based on a cooking method survey (result presented in Chapter 6 (Table 6-7)) over 90 % of the subjects (n = 115) indicated that either a rice cooker or absorption method was used by them to cook rice. As using a rice cooker essentially mimicked the absorption cooking method, it was chosen as the way to cook the rice.

Rice-to-water ratio and cooking time used to prepare the cooked rice were 1:1.8 and 19 mins, respectively, in reference to Section 3.2.2.4.

5.1.3 Folic acid analysis

The uncooked and cooked rice samples were prepared according to Section 3.2.2.4. Samples were subsequently extracted, purified according to Section 3.2.2.5 to 3.2.2.6. The purified extract was analysed using HPLC with the instrumental conditions as detailed in Section 3.2.2.7.

In the following pages, the amount of folic acid present in the rice kernel after parboiling, i.e. soaked, steamed and dried was referred to as folic acid uptake. Folic acid uptake in uncooked fortified parboiled rice (Uptake Percentage) was calculated as the ratio between the amount of folic acid present in the fortified parboiled rice kernel and the corresponding folic acid added to the soaking solution as the fortificant. Hence, the uptake percentage was described as follow:

Uptake %  ricefort folicacid)/( xFF 100% (Equation 5-1 )

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where F fort-rice is the analysed folic acid amount in the uncooked fortified parboiled rice; and, Ffolicacid is the initial amount of folic acid (i.e. 0.15, 0.3, 0.6 and 1.2 g folic acid/ 300 g brown rice) added to the soaking water.

The amount of folic acid present in the cooked fortified parboiled rice in relation to the amount of folic acid present in the uncooked form of fortified parboiled rice is referred to as Cooking Retention. The cooking retention percentage was calculated as follows:

Cooking retention %  )/( xFF 100% (Equation 5-2) cookedrice ricefort where Fcookedrice is the analysed folic acid amount in the cooked fortified parboiled rice whereas Ffort-rice is the folic acid amount in the uncooked fortified parboiled rice.

5.1.4 Kernel dimension

A grain inspector (CervitecTM 1625, FOSS Analytical, Denmark) was used to measure the kernel dimension of fortified and raw untreated rice. CervitecTM 1625 comprises of two cameras for recording two images of each kernel, which are separated and transported by a rotating disk. Through the patented technology, one image is recorded for transmission analysis whereas the other one is for reflectance analysis. Each kernel is analysed by the calibration model according to pre-set kernel specification. An average of 400 milled rice kernels were analysed per sample. Average length and width of rice kernels were measured in millimetres.

5.1.5 Instrumental colour evaluation

A colorimeter (Minolta CR-3000 series) was used for all colour determinations. The instrument was calibrated with a white calibration tile. The colour was measured as L*, a* and b* colour space (CIE 1976), where L* describes lightness from black (0) to white (100); a* describes red-green colour with positive a* values representing redness and negative values referring to greenness; b* describes yellow-blue colour with positive

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values representing yellowness whereas negative values represent blueness (Lamberts et al., 2006b).

Colour parameter differences were calculated based on the difference in L*, a* and b* between sample and reference materials, i.e. raw untreated Langi milled for 120 s resulting in white rice (ControlRaw) and parboiled rice prepared with the same parboiling conditions but without fortificant added to the soaking solution (ControlParboil). The total colour difference, ΔE, was calculated as a single value that takes into account the differences between L*, a* and b* of the sample and references.

( * () *2* () *2* bbaaLL)2* ∆E = sample ref sample ref sample ref (Equation 5-3)

The total colour difference (∆E) between fortified and raw white rice is denoted as ∆ERaw whereas that between fortified and parboiled rice is denoted as ∆EParboil.

5.1.6 Statistical analysis

To analyse the effect of parboiling conditions on folic acid uptake, retention, HRY and colour, differences between means were considered significant when p < 0.05 using Tukey’s HSD. PASW Statistics 18 software (SPSS Inc., Chicago, II, USA) was used for the analysis.

5.2 Results and discussions

5.2.1 Folic acid uptake in uncooked rice and retention in cooked rice

For the unfortified rice sample (ControlParboil), the folic acid concentration was below the lower end of the linear range of folic acid detection, i.e. 0.025 µg/g. Folic acid and 10- formyl-THF was suggested to be present in rice, however they were the product of oxidation due to degradation of natural folates (De Brouwer et al., 2010). Between 5 (De Brouwer et al., 2010) and 13 (Pfeiffer et al., 1997) % of folic acid was present in uncooked rice, folic acid concentration was found to be less than 2 µg/100 grice.

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Considering the total folate content is 20 µg/100 grice or less (De Brouwer et al., 2010,

U.S. Department of Agriculture Agricultural Research Service, 2012), i.e. 0.2 µg/100 grice, the folic acid concentration found in the fortified rice was therefore at least 95 higher than the unfortified rice (Table 5-1). In order to investigate further the extent of folic acid uptake from the soaking solution into the rice kernel, uptake percentages of fortified rice samples at the corresponding parboiling conditions were also considered (Table 5-1). The uptake of folic acid in the fortified rice sample was between 3.8 and 9.0 %. Tulyathan et al. (2007) reported about 19 % of iodine penetration in the rice kernel from the soaking solution, whereas the study conducted by Prom-u-thai et al. (2008) showed approximately 1 % of iron penetration into the milled rice kernel. Despite the fact that direct comparison of the fortificant penetration percentage obtained by the different research groups may be difficult due to the difference in the experiment conditions examined (as mentioned in Section 2.5.5), these earlier studies still reflected a potential range of penetration (or uptake) percentage of fortificant subjected to parboiling. The extent of fortificant (i.e. folic acid) uptake into the rice kernel appeared to be within this range of estimation.

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Table 5-1 Uptake of folic acid as concentration and % in fortified and unfortified (ControlParboil) rice kernels milled at 0, 60 and 120 s.

Uptake Concentration Uptake Percentage (µg folic acid/ g fortified rice) (%) Fortificant Soaking 0 60 120 0 60 120 conc.1 time (s) (s) (s) (s) (s) (s) (h) 1.2 1 224.0±28.4d2 177.2±10.3g 173.0±9.1e 5.6±0.7a 4.4±0.3a,b,c 4.3±0.2a 2 269.0±5.0e 206.5±5.6h 192.6±10.0f 6.7±0.1a,b 5.2±0.1a,b,c 4.8±0.3a 3 299.0±10.6e 249.8±4.4i 247.5±1.1g 7.5±0.3a,b,c 6.2±0.1c,d 6.2±0.0b 0.6 1 111.8±6.3c 83.6±0.9d,e 81.7±2.3c,d 5.6±0.3a 4.2±0.0a,b 4.1±0.1a 2 113.3±2.0c 96.7±2.6e,f 88.2±1.1c,d 5.7±0.1a 4.8±0.1a,b,c 4.4±0.1a 3 120.4±6.5c 118.3±19.5f 99.5±2.7d 6.0±0.3a 5.9±1.0b,c,d 5.0±0.1a 0.3 1 61.1±8.4a,b 50.9±5.3b,c 48.6±6.3b 6.1±0.9a 5.1±0.5a,b,c 4.9±0.6a 2 62.6±6.5a,b 59.4±1.8b,c,d 46.7±4.7b 6.3±0.7a 5.9±0.2b,c,d 4.7±0.5a 3 90.4±0.7b,c 72.6±0.5c,d,e 71.4±0.8c 9.0±0.1c 7.3±0.1d 7.1±0.1b 0.15 1 29.0±2.8a 22.5±2.1a 20.0±0.0a 5.8±0.6a 4.5±0.4a,b,c 4.0±0.0a 2 35.0±2.8a 24.5±0.7a 24.5±0.7a 7.0±0.6a,b 4.9±0.1a 4.9±0.1a 3 42.0±4.2a 37.0±5.7a,b 35.0±2.8a,b 8.4±0.8b,c 7.4±1.1d 7.0±0.6b 0 1 n.d. n.d. n.d. - - - (ControlParboil) 2 n.d. n.d. n.d. - - - 3 n.d. n.d. n.d. - - - 1 Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. 2 Values followed by different letters in the columns indicate significantly different means at p < 0.05. n.d. Below the detection limit of the method. .

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Chapter 5 Rice quality studies on fortified parboiled rice

Given that nutrients will inevitably be lost during washing and cooking (Rubin et al., 1977, Juliano and Bechtel, 1985), the amount of folic acid remaining in cooked rice needs to be examined. This is because the retention percentage of folic acid in cooked rice represents the initial amount of folic acid that is available to the rice consumers.

Table 5-2 shows the folic acid concentration in the fortified rice (after 120 s milling) before and after cooking. The folic acid concentration of both the cooked and uncooked rice is expressed on a dry weight basis. The mean folic acid retention after cooking was approximately 90 %. Given the high retention of folic acid after cooking, the folate status of the target population may be improved through the consumption of the fortified rice, therefore increasing its chance to be a successful fortification strategy (Allen et al., 2006).

Table 5-2 Folic acid concentration of cooked and uncooked form of fortified rice and the corresponding cooking retention percentage Fortificant Soaking Uncooked rice Cooked rice* Cooking conc.# time (µg/g) (µg/g) retention (h) (%) 1.2 1 182.7±13.5 147.4±7.4 80.7 2 191.3±0.6 159.9±5.2 83.6 3 240.0±0.5 175.9±23.0 73.3 0.6 1 79.1±1.1 74.0±8.6 93.5 2 84.1±0.1 87.6±3.7 104.2 3 103.2±0.5 94.6±8.3 91.8 0.3 1 48.5±1.2 46.0±0.6 94.9 2 43.1±0.0 31.6±2.2 73.3 3 61.4±0.2 56.4±0.6 91.8 0.15 1 21.1±0.2 20.5±3.4 97.2 2 27.8±0.6 26.1±0.6 94.0 3 29.5±1.3 25.2±1.1 85.4 0 1 n.d. n.d. - (ControlParboil) 2 n.d. n.d. - 3 n.d. n.d. - # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. * Folic acid concentration expressed on dry weight basis.

Referring to Table 5-1, the maximum uptake percentage of folic acid was about 10, suggesting that more than 90 % of the added fortificant was remained in the soaking solution after parboiling. From the cost effective point of view, it is beneficial to re-use

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the soaking solution as significant amount of folic acid remained after parboiling. Nonetheless, one of the potential issues associated with the re-use of soaking solution is that the soaking solution is nutrient-rich, together with the rice content released during parboiling, such as sugars, these create an environment which may encourage microbial growth. Future studies may be conducted to investigate the potential of recycling the soaking solution.

In the present study, the fortificant solution was not recycled for any subsequent batch, the cost involved in manufacturing the fortified rice is therefore another aspect in evaluating the feasibility of this fortification process. Assuming the required infrastructure was present for the population who practised rice parboiling, the additional cost involved in using this parboiling technique would predominantly be the purchase of the fortificant. Based on the retail price of the food grade folic acid supplied by DSC (AUD 164.8/ kg), the cost for fortifying rice at the concentration of 1.2, 0.6, 0.3 and 0.15 gfolic acid/ 300 gbrown rice was therefore AU$0.2, 0.1, 0.05 and 0.02, respectively. These were the cost for producing every 300 g of folic acid fortified rice-premix, which was required to be diluted further to achieve the safe folic acid level for human consumption. The desirable folate intake was deemed to be between 350 µg/ day (beneficial to adults in preventing homocysteinemia) (deBree et al., 1997) and 400 µg/ day (to prevent the occurrence of neural tube defects in pregnant women) (Hao et al., 2008, Mastroiacovo and Leoncini, 2011) while the estimated upper limit of folate intake is 1000 µg for general populations (Mastroiacovo and Leoncini, 2011). Considering that approximately 400 g rice/ day in parboiled rice-consuming countries such as India and Bangladesh (Doesthale et al., 1979, Moretti et al., 2006) and the folate intake is around 180 µg/ day for the target population (Krishnaswamy and Nair, 2001), an extra 150-200 µg folate would be needed to match the classical 400 µg of folate/ day dose or at least reach the beneficial 350 µg of folate/ day level (Mastroiacovo and Leoncini, 2011). In order to achieve an additional 200 µg of folic acid in a serving of rice, the appropriate mixing ratios were illustrated in Table 5-3. Column 4 estimated the amount of fortified rice that would present in 1 kg of mixed rice and the associated cost of producing the fortified rice

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was summarised in column 5. Based on FAO Global Information and Early Warning System (GIEWS) food price data and analysis tool, the average retail price for rice in India over 2008-2012 was USD 0.45, 0.44, 0.50, 0.50 and 0.47 per kg, respectively, whereas that in Bangladesh was USD 0.46, 0.33, 0.43. 0.44 and 0.35 per kg, respectively. Over the 5-year period, the retail price changes in India were -2.0, 12.5, 1.3 and -6,8 % between each year, and those in Bangladesh were -28.7, 31.1, 3.3 and -20.7 %.

If considering the retail price of rice on 2012, the relative price increase in producing the fortified rice is between 0.8 and 1.9 %. This percentage increment is at the lower end of the increase in retail price of rice previously experienced in these countries. Moreover, the current cost of the study for producing the fortified rice is based on the retail price of folic acid purchased. The investment for folic acid would likely be reduced if large quantity of folic acid was purchased for pilot or industrial scale study, the % increase in price for producing the fortified rice would be expected to be lower than that estimated in Table 5-3.

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Table 5-3 Cost involved in producing 1 kg of mixed (diluted) fortified rice and the relative price increment in India and Bangladesh. Fortificant Soaking Cooked fortified Mixing ratioa Fortified rice in Price elevation % Increment % Increment conc.# time rice* 1 kg of mixed (USD)b in price in price (h) (µg/serving) rice (g) (India)c (Bangladesh)c 1.2 1 23582.6±1189.7 118 8.5 0.0068 1.4 1.9 2 25584.2±836.8 128 7.8 0.0052 1.1 1.5 3 28145.6±3679.7 141 7.1 0.0047 1.0 1.3 0.6 1 11832.1±1379.7 59 16.9 0.0056 1.2 1.6 2 14023.9±598.1 70 14.3 0.0047 1.0 1.3 3 15143.6±1331.7 76 13.2 0.0044 0.9 1.2 0.3 1 7359.4±93.1 37 27.2 0.0045 1.0 1.3 2 5050.5±350.2 25 39.6 0.0065 1.4 1.9 3 9021.6±100.2 45 22.2 0.0037 0.8 1.0 0.15 1 3279.8±549.4 16 61.0 0.0050 1.1 1.4 2 4181.2±508.4 24 47.9 0.0039 0.8 1.1 3 3789.4±895.9 20 49.6 0.0041 0.9 1.2 # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. * Concentration of folic acid in 400 g of cooked rice based on the results presented in Table 5-2. a 1 g of fortified rice in x g of unfortified rice (i.e. background rice) in order to achieve 200 µg of folic acid in a serving of rice consumed. b The cost involved in producing 1 kg of fortified rice after mixing with background rice c Based on the average retail price of coarse rice in India (Delhi) and Bangladesh (national average) on 2012.

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5.2.2 Head Rice Yield (HRY)

In the rice industry, brown rice is generally milled to produce white rice because it is the preferred form of consumable rice (Bhattacharya, 2004). White rice is obtained by removing the bran layer of brown rice which equates to approximately 5-8 % of brown rice weight (Unnikrishnan and Bhattacharya, 1987, Lamberts et al., 2006b). 60 s and 120 s milling durations were studied, and they resulted in ~6 and 9 % degree of milling, respectively, based on the weight of milling portions, suggesting the durations chosen were sufficient to remove the bran layers. During post-harvest drying and polishing of rice, breakage is one of the major problems in rice processing because the market price of broken grains is approximately half the price of whole grains (Marshall and Wadsworth, 1994b). The HRY of fortified rice under different treatments is shown in Table 5-4.

Table 5-4 Head Rice Yield at different fortificant concentrations, soaking and milling durations Head Rice Yield (%)

Fortificant Soaking conc.# time 60 120 (h) (s) (s) 1.2 1 65.7a+ 64.8a,b 2 66.8a 64.0a 3 66.4a 63.3a 0.6 1 66.0a 64.6a,b 2 65.5a 63.4a 3 66.5a 66.5a,b 0.3 1 66.4a 65.1a,b 2 66.0a 64.6a,b 3 66.4a 64.8a,b 0.15 1 65.7a 64.1a 2 65.7a 63.7a 3 65.7a 64.8a,b Raw a a,b (ControlRaw) 66.4 64.6 # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. + Values followed by different letters in the columns indicate significantly different means at p < 0.05.

The HRY for the raw rice (ControlRaw) was 66.4 % and 64.6 % at 60 s and 120 s milling, respectively. For the fortified rice parboiled at different soaking durations, HRY was

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found to be between 65.5 % and 66.8 % at 60 s milling and 63.3 % and 66.5 % at 120 s milling. Parboiling has been reported to have a positive effect on reducing rice breakage in other studies (Ali and Pandya, 1974, Miah et al., 2002, Bhattacharya, 2004). This study, however, showed no significant difference in HRY before and after parboiling (p > 0.05). Marshall et al. (1993) indicated that the HRY of both Lemont and Tebonnet rice improved to a maximum HRY of ~67 % from ~44 and 53 %, respectively, at about 40 % gelatinisation of rice starch. Here, the HRY was maintained at ~65 % with a degree of gelatinisation more than 78 % (Table 4-8) to show that the HRY was already high with limited margins for improvement. Parnsakhorn & Noomhorm (2008) studied the relationship between HRY and soaking time of brown rice with different rice varieties. They reported that the change in HRY appeared to be variety-dependent in addition to the parboiling conditions used. Although the parboiling conditions did not show significant improvement on the HRY in the present study, these results on the other hand indicate that folic acid fortification of rice under the tested parboiling conditions is unlikely to pose any negative effect on the HRY of parboiled rice. Degree of milling between 6 and 9 % can likely be employed at an industrial scale without compromising on the HRY, and therefore no HRY related loss on the economic return on the grains.

5.2.3 Kernel dimension

In some countries, consumers tend to associate sensory qualities with kernel dimension (Bergman et al., 2004), while other consumers consider grain dimension and uniformity as significant visual characteristics in rice (Heinemann et al., 2006). Grain dimension is therefore an important trait to be considered for the rice industry. Table 5-5 illustrates the average length and width of whole kernels of fortified, parboiled and raw rice in the present study. After parboiling, the length and width of fortified rice did not differ significantly from the unfortified parboiled and raw rice, unlike in a previous study which reported that parboiled rice is shorter and broader than milled raw rice due to the realignment of the cooked rice (Bhattacharya and Ali, 1985). Minimal change in grain dimension of fortified rice is considered positive in the present study because the chance

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of consumers associating any change in sensory characteristics due to the alteration of kernel dimension is reduced. In the present study, the concentration of folic acid in fortified rice produced using the method described was highly concentrated at between 20 and 130 times above the Recommended Dietary Intake of the classical 400 µg/ day (Mastroiacovo and Leoncini, 2011), with the estimation of around 400 g of cooked rice consumption per day (Agrahar-Murugkar and Pal, 2004). Fortified rice, therefore, serves as a nutrient premix to be diluted by mixing with a suitable portion of unfortified parboiled rice or unfortified white rice to achieve the desirable folic acid concentration for human consumption, depending on the origin of consumers and the amount of daily rice consumption (cf. Section 6.1.5.1).

Table 5-5 Kernel dimensions at different fortificant concentrations, soaking and milling durations Kernel Dimension

60 120 (s) (s) Fortificant Soaking conc.1 time Length Width Length Width (h) (mm) (mm) (mm) (mm) 1.2 1 6.86a2 1.93a 6.81a 1.90a 2 6.86a 1.94a 6.77a 1.91a 3 6.85a 1.93a 6.85a 1.92a 0.6 1 6.89a 1.96a 6.89a 1.94a 2 6.84a 1.93a 6.78a 1.91a 3 6.93a 1.94a 6.93a 1.94a 0.3 1 6.88a 1.94a 6.89a 1.92a 2 6.87a 1.96a 6.85a 1.95a 3 6.88a 1.96a 6.88a 1.94a 0.15 1 6.91a 1.96a 6.87a 1.93a 2 6.87a 1.97a 6.83a 1.95a 3 6.92a 1.96a 6.88a 1.95a 0 1 6.91a 1.95a 6.88a 1.92a a a a a (ControlParboil) 2 6.92 1.93 6.86 1.90 3 6.92a 1.93a 6.86a 1.91a Raw a a a a (ControlRaw) 6.97 2.01 6.89 1.98 # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. + Values followed by different letters in the columns indicate significantly different means at p < 0.05.

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For mixing granular products, small differences in either size or density may lead to flow- induced segregation (Ottino and Khakhar, 2000). As the length and width of fortified rice were comparable for both parboiled (ControlParboil) and raw rice (ControlRaw), it suggests that fortified rice can be mixed with both parboiled rice (for parboiled rice-consuming populations) and white rice (for white rice-consuming populations). With proper mixing to achieve grain uniformity, the fortification method proposed may extend the potential of delivering the nutritional benefits of the fortified rice to even white rice-consuming countries.

5.2.4 Colour ev aluation

Colour is an important attribute to consider for food commodities as consumers’ perception of colour can strongly associate with their purchase intent (Brewer and McKeith, 1999, VanHurley, 2007). White rice consumers expect the raw rice and cooked rice to be white in colour (Champagne et al., 2010) while parboiled rice consumers can accept some margins of yellow or brown colour in rice (Tomlins et al., 2007).

5.2.4.1 Overall colour comparison (L*, a*, and b*) of fortified, parboiled and raw rice

Table 5-6 presents the colour parameters L*, a*, and b* of the fortified, parboiled rice milled for 120 s (ControlParboil), commercial parboiled rice (ControlCom) and raw rice

(ControlRaw). For the parboiled rice (ControlParboil), the L-values were 69.7, 65.1 and 61.3 at the respective soaking times and they were all significantly lower than the raw rice

(ControlRaw) (L-value was 75.0). The marked decrease in L-values indicate that the parboiling process alone darkened the product as previously described by Bhattacharya (1995) and Lamberts et al. (2006b). The increase in b-value is likely caused by the migration of yellow pigments from bran to the endosperm upon soaking and steaming (Lamberts et al., 2006a, Lamberts et al., 2006b). Contrary to the significant difference in lightness, parboiled rice (ControlParboil) was not significantly more yellow than the raw rice, i.e. ControlRaw (p > 0.05).

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Table 5-6 Colour Parameters L* (Lightness), a* (Redness), and b* (Yellowness) of fortified rice milled for 120s and controls (Commercial parboiled and raw rice) Fortificant Soaking conc.# time (h) L* a* b* 1.2 1 66.8f,g* -2.2b,c,d,e 18.6d 2 64.2b,c,d,e,f -2.4a,b,c 21.3e 3 62.7a,b,c,d -2.5a,b 23.7f 0.6 1 65.6d,e,f -2.1b,c,d,e 16.4b,c 2 64.1b,c,d,e,f -2.3a,b,c,d 16.6b,c 3 63.4a,b,c,d,e -2.8a 17.3c,d 0.3 1 65.0c,d,e,f -2.0b,c,d,e 15.8a,b,c 2 63.6a,b,c,d,e,f -1.8d,e,f 16.1b,c 3 63.4a,c,d,e -2.4a,b,c 16.3b,c 0.15 1 66.3e,f -2.3a,b,c,d 15.7a,b,c 2 61.5a,b -2.0b,c,d,e 15.7a,b,c 3 60.8a -1.9c,d,e 16.9c,d 0 1 69.7g -1.9c,d,e 15.5a,b c,d,e,f e,f,g a,b,c (ControlParboil) 2 65.1 -1.7 15.8 3 61.3a,b -1.3f,g 15.9a,b,c Commercial 62.2a,b,c -1.7e,f,g 20.5e parboiled (ControlCom) Raw 75.0h -1.2g 14.4a (ControlRaw) # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. * Values followed by different letters in the columns indicate significantly different means at p < 0.05.

5.2.4.2 Total colour difference

To gain some insight into the effect of parboiling conditions and fortificant on colour change, the colour parameters (L*, a* and b*) of fortified rice were evaluated against two references; raw rice (ControlRaw) and parboiled rice (ControlParboil) (Table 5-7).

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Table 5-7 Colour difference (∆L*, ∆a*and ∆b*) between fortified rice and raw (ControlRaw) and parboiled rice (ControlParboil) Raw Parboiled (ControlRaw) (ControlParboil)

Fortificant Soaking conc.# time (h) ∆L* ∆a* ∆b* ∆ERaw ∆L* ∆a* ∆b* ∆EParboil 1.2 1 -8.2f,g* -1.0c,d,e 4.2c 9.3b,c -2.9a,b,c,d -0.3c,d 3.1b 4.3b,c,d 2 -10.7b,c,d,e,f -1.2a,b,c 6.9d 12.8d,e,f,g -0.9c,d,e,f -0.8b,c 5.5c 5.7d,e 3 -12.3a,b,c,d -1.3a,b 9.3d 15.6g 1.3e,f -1.2a,b 7.8d 8.1e 0.6 1 -9.4d,e,f -0.9b,c,d,e 2.0a,b 9.7b,c -4.1a,b -0.3c,d 0.9a 4.3b,c,d 2 -10.9b,c,d,e,f -1.1a,b,c,d 2.3a,b 11.2b,c,d,e -1.0c,d,e -0.7b,c,d 0.8a 1.8a,b 3 -11.6a,b,c,d,e -1.6a 2.9b,c 12.1c,d,e,f 2.1f -1.5a 1.4a 3.1a,b,c,d 0.3 1 -10.0c,d,e,f -0.8b,c,d,e 1.4a,b 10.1b,c,d -4.7a -0.2d 0.3a 4.8c,d 2 -11.3a,b,c,d,e,f -0.6d,e,f 1.7a,b 11.5b,c,d,e,f -1.5b,c,d,e -0.2c,d 0.3a 1.6a,b 3 -11.6a,b,c,d,e -1.2a,b,c,d 1.9a,b 11.8b,c,d,e,f 2.1f -1.1a,b 0.4a 2.4a,b,c 0.15 1 -8.7e,f -1.1a,b,c,d 1.3a,b 8.9b -3.4a,b,c,d -0.4c,d 0.2a 3.5a,b,c,d 2 -13.4a,b -0.8b,c,d,e 1.3a,b 13.5e,f,g -3.6a,b,c -0.4c,d -0.1a 3.6a,b,c,d 3 -14.1a -0.7c,d,e 2.5a,b,c 14.4f,g -0.5d,e,f -0.6b,c,d 0.9a 1.3a 0 1 -5.3g -0.7c,d,e,f 1.1a 5.5a - - - - c,d,e,f e,f a,b b,c,d (ControlParboil) 2 -9.8 -0.5 1.4 10.0 - - - - 3 -13.6a,b -0.1f 1.6a,b 13.7e,f,g - - - - Commercial - - - - parboiled -12.74a,b,c -0.5e,f 6.1d 14.2f,g (ControlCom) # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. * Values followed by different letters in the columns indicate significantly different means at p < 0.05

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A. Total colour difference - against raw rice (ControlR)

Milled rice is widely consumed by the rice-consuming populations (Bhattacharya, 2004) and therefore set as a benchmark for fortified rice. The colour difference can be interpreted as that perceived by the white rice consumers.

Considering the total colour difference (∆E) compared to raw rice, ∆ERaw increased as soaking duration increased at constant fortificant concentration while ∆E was comparable at the constant parboiling conditions across the several fortificant levels (Table 5-7). ∆E values of all the fortified rice were either lower than or statistically comparable with the commercial parboiled rice (ControlCom). The parboiling conditions studied were capable of producing parboiled rice which resembled the commercial parboiled rice, or even showed less colour change in the final product. Note that the parboiling conditions of commercial parboiled rice (ControlCom) are noticeably different from the present laboratory conditions studied. Given that it is a commercial product available in the local and international market, the colour of the rice has likely been accepted by consumers already. Its colour evaluation describes objectively the colour parameters which are considered desirable by consumers or at least acceptable. The magnitude of the difference in colour parameters, ∆L* and ∆b*, appear to be the two main contributors to total colour difference when the highest fortificant concentration was added. Interestingly, the major contribution shifted to solely ∆L* at fortificant levels at 0.6gfolic acid/ 300 gbrown rice or less.

B. Total colour difference-against parboiled rice (ControlParboil)

Assuming that the colour changes due to parboiling, i.e. enzymatic and non-enzymatic reactions and the diffusion of pigments from bran layers (Bhattacharya, 2004, Lamberts et al., 2006a, Lamberts et al., 2006b), were to the same extent in both fortified and parboiled rice (ControlParboil) at constant parboiling conditions, the colour differences between the fortified and parboiled rice likely reflect the contribution due only to the added fortificant. Given that folic acid is yellow in colour, intuitively fortified rice is expected to have progressive yellow intensity corresponding to the increasing fortificant levels. However,

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apart from ∆b* of rice fortified at 1.2 gfolic acid/ 300 gbrown rice (3.1, 5.5 and 7.8) were significantly higher than the rest of the samples with lower fortificant levels, and no significant difference was observed between the samples treated with 0.6 gfolic acid/ 300 gbrown rice or lower (0.153 < p < 0.999). The results suggested that at the fortificant concentration above 0.6 gfolic acid/ 300 gbrown rice, the dominant cause of yellowness in the fortified parboiled rice was likely due to folic acid (fortificant). Considering the magnitude of the colour parameter differences, it is interesting to note that different fortificant levels appeared to have relatively greater impact on the lightness of the fortified parboiled rice rather than on their yellowness. Prolonged soaking in folic acid rich solution surprisingly, had less impact on darkening of the rice, but even improved the lightness of the rice at the constant fortificant concentration.

The resultant low ∆EParboil indicated that the fortification does not introduce dramatic colour change in the end product. The ∆EParboil was lower than ∆ERaw which implies that less colour difference between fortified rice and parboiled rice than in white rice, indicating that parboiled rice is a more suitable diluent for the fortified rice (Table 5-7). Without any foreign colour, the chance of the product being accepted by the consumers will be increased (Hurrell, 1997).

5.3 Conclusions

This study has demonstrated a robust method to produce fortified rice using the parboiling technique. An average of 90 % of folic acid was retained in the fortified rice after cooking. The high retention percentage of folic acid in the cooked rice strengthens the applicability of using parboiling as a fortification approach. The minimum loss of folic acid also suggests that fortified rice is of nutritionally beneficial for the consumers.

Moreover, the HRY and grain dimension of folic acid fortified rice were maintained at all parboiling conditions studied, indicating that the milling and mixing efficiency are maintained for the rice industry. It also suggests that no extra investment is needed on top of the existing infrastructure for parboiling.

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The fortified rice, similar to any parboiled rice, is darker and more yellow than the raw rice. Soaking duration decreases L-value and increases b-value across the fortificant levels studied. Total colour difference (∆E) is a relative measure between the sample and a reference control, which accounts for L* (lightness), a* (redness) and b* (yellowness).

1. Considering the raw rice as the reference control, ∆ERaw of fortified rice increased with increasing soaking duration. The major contributors of ∆E

were ∆L* and ∆b* at 1.2 gfolic acid/ 300 gbrown rice, but the effect of ∆L*

outweighed ∆b* at fortificant levels of 0.6 gfolic acid/ 300 gbrown rice or less. This implies that lightness of the fortified parboiled rice was a more significant factor to colour change in comparison to the raw rice.

2 Considering the parboiled rice as the reference control, ∆EParboil reflected the effect of the folic acid added to the colour change of fortified rice. Only ∆b*

of parboiled rice fortified at 1.2 gfolic acid/ 300 gbrown rice was significantly higher than those fortified at other lower fortificant levels. The ∆b-values of fortified

rice fortified between 0.15 and 0.6 gfolic acid/ 300 gbrown rice exhibited no significant difference. Interestingly, ∆L-values increased with soaking duration which indicates that prolonged soaking in the folic acid solution improved the lightness of fortified rice which was soaked at the constant fortificant level.

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CHAPTER 6

CONSUMER ACCEPTANCE OF FORTIFIED PARBOILED RICE

As discussed in Chapters 2 and 5, rice with low milling quality can be improved through parboiling which reduces the breakage of the kernel during milling, and this provides a more efficient yield and better economic return for the rice (Islam et al., 2002b, Bhattacharya, 2004, Bello et al., 2006). Moreover, the concentration of water-soluble constituents in parboiled rice is higher than in milled white rice (Luh, 1991, Bhattacharya, 2004). In terms of texture, parboiled rice is fluffier and less sticky (Bhattacharya, 2004). Parboiling does, however, impact on the visual and sensory characteristics of the rice which include changes in colour (becomes yellow), texture and flavour (Bhattacharya, 2004, Prom-u-thai et al., 2009b).

To some consumers and cultures, the changes in rice characteristics are unacceptable. Some consumers may believe that changes in colour of rice relate to staleness and thus indicate poor quality (Kratochvil et al., 1994). The first impression of any food is generally visually-based. Appealing colour and other visual attributes are therefore the predominant factors of consumers’ willingness to consume/purchase a particular food (Kratochvil et al., 1994).

The ultimate goal of fortification is to improve consumers’ nutritional status through gaining acceptance thereby encouraging the purchase and consumption of fortified rice. The objective of this work was to ascertain whether the fortified parboiled rice would be accepted by consumers in rice-consuming (especially parboiled rice consuming) populations. The objective was achieved by assessing the acceptability of fortified rice based on visual and sensory comparisons with the appropriate commercial rice control.

Two consumer acceptance studies based on visual and taste attributes were therefore conducted to investigate this issue. In STUDY 1, visual consumer acceptance of

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uncooked fortified parboiled rice (UF 1, UF 2 and UF 3) was examined. The visual acceptability of three uncooked fortified parboiled rice samples was compared to a commercial parboiled rice control. In STUDY 2, uncooked fortified parboiled rice was mixed with unfortified commercial white rice and cooked. Consumer acceptance of cooked fortified rice after mixing (CFM 1, CFM 2 and CFM 3) was studied.

6.1 Materials and methods

6.1.1 Preparation of fortified parboiled rice premix

Brown rice preparation was followed according to Section 3.2.2.1 to produce graded intact brown rice prior to the following parboiling treatments.

The parboiling procedure was followed as described in Section 3.2.2.2. Two fortificant concentrations, 1.2 gfolic acid/ 300 gbrown rice and 0.15 gfolic acid/ 300 gbrown rice, were dissolved in each soaking water, respectively (Figure 6-1). Graded brown rice (300 g) was added to each solution in a rice-to-water ratio of 1 :2. Brown rice was soaked in each folic-acid solution at 70 °C for 1, 2 and 3 h. Soaking solution was removed and the rice was subsequently steamed at 100 °C for 60 mins. Steamed samples were dried in shade at room temperature until the moisture content was dropped to 12 % (wet weight basis). The parboiled brown rice samples were milled in a food-grade laboratory scale grain mill (Satake Test Grain Mill, Japan) for 120 s to yield about 9 % degree of milling (Section 3.2.2.3).

6.1.2 Folic acid analysis

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6.1.3 Instrumental colour evaluation

A colorimeter (Minolta CR-3000 series) was the instrument used for colour determination of fortified rice. The protocols were followed according to Section 5.1.5. In addition, Chroma (C*) was calculated for this study. Chroma (C*) is the coordinate which is perpendicular from the lightness and it is mathematically defined in Equation 6-1 (Sahin and Sumnu, 2006):

√ (Equation 6-1)

6.1.4 STUDY 1: Visual consumer acceptance of uncooked fortified parboiled rice samples

6.1.4.1 Samples

In the first phase of the acceptance study, three uncooked samples of fortified rice (which were fortified at 1.2 gfolic acid / 300 gbrown rice and soaked for 1, 2 and 3 h) were denoted as

UF 1, UF 2 and UF 3, respectively (Figure 6-1). Commercial parboiled rice (ControlCom) was chosen as a blind control because it serves as a standard which possesses the characteristics of parboiled rice that has already been accepted by consumers of that commodity.

Samples prepared using the highest folic acid concentration were evaluated in STUDY 1 because the rice showed significantly higher b-value, i.e. yellowness, based on the instrumental colour analysis (Table 6-1). Colour is an influential quality attribute as it is presupposed by some consumers that colour change (deviation from the expected colour) may relate to food deterioration (Kratochvil et al., 1994). The yellowness of rice is the characteristic colour change due to parboiling (Bhattacharya, 2004). It was therefore assumed that if the consumers could accept the fortified rice which potentially has the most noticeable “discolouration”, they would likely accept that with a lesser degree of colour change. All the samples (including the ControlCom) were assessed visually in the

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uncooked state because it is the form of rice presented to consumers in markets or supermarkets where they make a purchase decision (Tomlins et al., 2007). STUDY 1 therefore served as a preliminary exploration of the first level of acceptance of fortified parboiled rice by consumers.

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Figure 6-1 Flow chart of fortified parboiled rice preparation for Consumer Acceptance Study - STUDY 1 and STUDY 2

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Table 6-1 Colour Parameters L* (Lightness), a* (Redness), b* (Yellowness) and C* (Chroma) of uncooked fortified rice milled for 120 s and controls (Commercial parboiled and Commercial white rice) Fortificant Soaking time L* a* b* C* conc.# (h) 1.2 1 66.8d,e+ -2.2a,b,c 18.6c 18.7a,b 2 64.2b,c,d -2.4a 21.3d 21.5b,c 3 62.6b,c -2.5a 23.7e 23.9c 0.15 1 66.3a,d -2.3a,b 15.7a,b 15.9a 2 61.5a,b -2.0a,b,c,d 15.7a,b 15.8a 3 60.8a -1.9b,c,d 16.9b,c 17.0a 0 1 69.7e -1.9b,c,d 15.5a,b 15.6a 2 65.1c,d -1.7c,d,e 15.8a,b 15.9a 3 61.3a,b -1.3e 15.9a,b 16.0a Commercial - 62.2a,b,c -1.7d,e 20.5d 20.6b,c parboiled (ControlCom) Commercial - 75.0f -1.2e 14.4a 14.4d white (ControlWhite) # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. + Values followed by different letters in the same columns indicate significantly different means at p < 0.05.

6.1.4.2 Survey methodology

The 4 samples (3 Uncooked Fortified rice and ControlCom) were evaluated by 115 naive (untrained) consumers who were students and staff recruited from the University of New South Wales, Sydney, Australia. Approximately 5 g of each sample which were individually coded in 3-digit numbers were presented to consumers. The survey was divided into two parts: Part I included demographic questions and consumers’ pattern of consumption pattern of rice; Part II evaluated their opinion, liking and purchase intent. The items presented in the questionnaire are shown in Table 6-2 and the corresponding scales used are illustrated in Table 6-3.

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Table 6-2 The demographics and consumers’ acceptance questions used in STUDY 1 Part I- Demographics and consumers’ rice consuming preference

a. Gender b. Age c. Nationality d. Employment status e. How often do you consume rice? f. What type of rice do you usually consume? g. How do you normally cook rice? h. Have you heard of parboiled rice previously? Part II. Consumer acceptance study- STUDY 1 questionnaire

1. How would you describe the colour of the rice? Open question 2. How intense is the colour of the rice? 10-point scale 3. How much do you like the colour of the rice? 9-point hedonic 4. How uniform is the colour of the rice sample? 10-point scale 5. How much do you like the overall appearance of the 9-point hedonic rice? 6. How willing are you to buy this rice? 5-point likert 7. If you are told that the rice is fortified with folic acid 5-point likert which gives higher nutritional value, how willing are you to buy this rice?

Table 6-3 Scales for liking, intensity, Just-about-Right (JAR) and purchase intent questions Liking Intensity JAR Purchase intent (STUDIES 1 and 2) (STUDY 1) (STUDY 2) (STUDY 1 and 2) 1 Dislike extremely 1 Not at all 1 Not nearly enough 1 Definitely would not buy 2 Dislike very much 2 2 2 Probably would not buy 3 Dislike 3 3 3 Undecided 4 Dislike moderately 4 4 4 Probably would buy 5 Neither like nor 5 5 Much too intense 5 Definitely would buy dislike 6 Like moderately 6 7 Like 7 8 Like very much 8 9 Like extremely 9 10 Extremely

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The purchase intent of the uncooked fortified rice was evaluated on the five-point purchase intent scale (Mcdaniel and Gates, 1998), i.e. “Definitely would not buy”, “Probably would not buy”, “Undecided”, “Probably would buy”, and “Definitely would buy” (Table 6-3 ). For convenience the responses of “Definitely would not buy” and “Probably would not buy” were summarized into “Not buy”; (1 and 2) while “Probably would buy” and “Definitely would buy” were grouped into “Would buy” (4 and 5) (Moskowitz, 2004).

6.1.5 STUDY 2: Consumer acceptance of cooked fortified rice after mixing

6.1.5.1 Samples

In the second phase of the acceptance study, uncooked fortified rice (fortified at 0.15 gfolic acid/ 300 gbrown rice and soaked for 1, 2 and 3 h) was mixed with commercial white rice as the background base. The mixed rice was then cooked and the samples were denoted as

CFM 1, CFM 2 and CFM 3, respectively. Commercial white rice (ControlWhite) was used as the control (Figure 6-1 ).

Mixing of uncooked fortified rice with white rice was required because:

1. The folic acid concentration in the cooked rice had to be monitored to ensure it was within the safe consumption level, considering that approximately 400 g rice/ day in parboiled rice-consuming countries such as India and Bangladesh (Doesthale et al., 1979, Moretti et al., 2006), Table 6-4 illustrates that the amount of folic acid present in a serving of cooked rice (i.e. 400 g) varied between 3500 and 28000 µg per serve.

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Table 6-4 Amount of folic acid (µg) in uncooked and cooked rice and the mixing ratio required to achieve 200 µg of folic acid in 400 g of cooked rice Fortificant Soaking Uncooked rice Cooked rice Mixing ratioa conc.# time (µg/serve) (µg/serve) (h) 1.2 1 29238.3±2167.8 26781.2±1408.0 134 2 30608.7±90.9 23493.9±1720.0 118 3 38400.0±74.0 27881.1±2910.2 140 0.15 1 3375.6±36.1 3458.0±211.1 17 2 4440.3±95.9 4882.5±463.9 24 3 4724.7±200.8 3838.2±838.1 19 # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. a 1 g of fortified rice in x g of diluent or background rice.

As detailed in Section 5.2.1, an additional 150-200 µg folate was required by the target populations to achieve the desirable 400 µg of folate/ day dose or at least reach the beneficial 350 µg of folate/ day level (Mastroiacovo and Leoncini, 2011). The concentration of folic acid in the uncooked fortified rice was therefore, 17 to 140 times higher than the desirable folate intake level. Fortified rice had to be mixed at the appropriate mixing ratio to achieve the recommended folate intake;

2. The mixing ratio (uncooked-fortified-rice-to-white-rice ratio) increased as folic acid concentration in the fortified rice decreased. The mixing ratio of

fortified rice fortified at 1.2 gfolic acid/ 300 gbrown rice was as high as 1 in 140

whereas that fortified at 0.15 gfolic acid/ 300 gbrown rice was up to 1 in 24 (Table 6-4). Homogeneity of fortified rice at low fortificant concentration was achieved successfully at the laboratory scale.

The rationale for using white rice (unparboiled and unfortified rice) as the background rice was:

1. Greater contrast was expected when the fortified rice (being parboiled rice) was mixed (diluted) with commercial white rice than commercial parboiled rice. If no noticeable difference was observed in appearance and sensory attributes between the diluted samples and commercial white rice control,

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minimum differences would likely be expected when the fortified rice was mixed with parboiled rice, which is known to possess unique flavours; and

2. It allowed the investigation of the potential to deliver the fortified rice to white rice-consuming populations.

Fortified rice (20.8 g) was mixed with 479.2 g long grain commercial white rice in a drum mixer with circular motion for 20 mins. The mixed rice was then cooked (Section 6.1.5.2) prior to presentation to consumers.

6.1.5.2 Cooking method

The mixed rice (250 g) was washed twice in approximately 500 mL of water and drained. It was cooked in an electric household electric rice cooker (BRC200, Breville, Australia) with the rice:water ratio of 1:1.8 (w/w) for 19 mins at boiling temperature. The cooked rice was left in the rice cooker for 10 mins to absorb excess moisture. It was kept at 60 ± 1 °C in the rice cooker for a maximum of 1 h until served.

6.1.5.3 Survey methodology

Approximately 20 g of each sample was served warm in a plastic container labelled with a randomized 3-digit code. One hundred naive (untrained) consumers from the student and staff community were recruited from the University of New South Wales. All tests were performed in partitioned booths under uniform white light conditions, and the subjects were not informed about the background of the study. A complete block design was used in the study.

The survey was again divided into two parts: the first part of the questionnaire included the demographic questions and consumers’ consumption pattern of rice; the second part of the questionnaire evaluated their opinions of the cooked rice and was divided into 5 major parts (Table 6-5). The intensity of the attributes was measured using a five-point Just-About-Right (JAR) scale. The five-point scale was collapsed and summarized into

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Chapter 6 Consumer acceptance of fortified parboiled rice three categories, namely, “Not enough” (1 and 2), “Just about right” (3) and “Too much” (4 and 5) according to Popper and Gibes (2004) (Table 6-3).

Table 6-5 The demographics and consumers’ acceptance questions used in STUDY 2 Part I- Demographics and consumers’ rice consuming preference

a. Gender b. Age c. Nationality d. Employment status e. How often do you consume rice? f. What type of rice do you usually consume? g. Which attributes are most important to you when consuming rice? h. Have you heard of parboiled rice previously? Part II. Consumer acceptance study- STUDY 2 questionnaire

A. Visual Appearance 1. How much do you like the appearance of this rice? 9-point hedonic 2. What do you think about the uniformity of appearance Just-About-Right of this rice? (JAR) 3. How much do you like the colour of this rice? 9-point hedonic 4. How would you describe the colour? Open question 5. What do you think about the colour intensity of this JAR rice? B. Smell 6. How much do you like the odour of this rice? 9-point hedonic 7. How intense is the rice odour? JAR C. Texture 8. How much do you like the texture of this rice? 9-point hedonic 9. How hard is this rice? JAR 10. How sticky is this rice? JAR D. Taste 11. How much do you like the taste of this rice? 9-point hedonic 12. How would you describe the taste of this rice? Open question 13. How intense is the taste of this rice? JAR 14. How much do you like the aftertaste of this rice? 9-point hedonic E. Overall liking 15. How much do you like this rice overall? 9-point hedonic 16. Would you buy this rice? 5-point likert 17. If you are told that the rice is fortified with folic acid 5-point likert which gives higher nutritional value, how willing are you to buy this rice?

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6.1.6 Data analysis

6.1.6.1 STUDY 1: Visual consumer acceptance of uncooked fortified parboiled rice

For the questions regarding the perceptions and the liking of attributes (Questions 2-5, Table 6-2), one-way analysis of variance (ANOVA) was used to verify the differences between the samples (including ControlCom), followed by comparison of averages by Tukey’s (HSD) means comparison test (p < 0.05). Independent samples t-test was used to study the differences in attribute scores between general consumers and consumers who were familiar with parboiled rice. For the purchase intent questions (Questions 6-7, Table 6-2), Chi-square statistical test (χ²) was performed to compare the response distribution

(i.e. Not buy, Undecided and Would buy) between the samples and ControlCom, and the response distribution between general consumers and consumers who were familiar with parboiled rice. Paired t-test was applied considering the effect of additional health benefits associated with the rice on consumers’ purchase intent.

6.1.6.2 STUDY 2: Consumer acceptance of cooked fortifie d rice after mixing

For the questions regarding the liking of attributes (Questions 1, 3, 6, 8, 11, 14 and 15, Table 6-5), one-way ANOVA was used to verify the mean differences between the samples (including ControlWhite), followed by Tukey’s (HSD) means comparison test (p < 0.05). χ² statistical test was used to compare the response distribution of purchase intent between the samples and control (Questions 16-17, Table 6-5). Penalty analysis was performed to determine the effect of consumers scoring of the cooked-rice attributes (on JAR scale) over their overall liking (9-point hedonic scale) in STUDY 2 (Meullenet et al., 2007). One-way ANOVA and Tukey’s (HSD) tests were performed to identify the significant mean drop between groups.

Levene’s test was used to assess the homogeneity of variance within the data measured on the 10-point intensity scale, 9-point hedonic scale and 5-point likert scale. The data was all above 0.05 which showed equal variances across samples. All the statistical

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Chapter 6 Consumer acceptance of fortified parboiled rice procedures were computed using IBM SPSS Statistics V.18 and XLSTAT (version 2011.4.02).

6.2 Results and discussions

6.2.1 STUDY 1: Visual consumer acceptance of uncooked fortified parboiled rice

6.2.1.1 Consumer demographics and rice eating preferences

Consumers (n = 115) from both genders, 52 % females and 48 % males, were recruited, with the majority of consumers being Asian (68.7 %) and the rest were Australian or other (Table 6-6). A large portion of recruited consumers was aged between 21 and 38 years (82 %) which may not necessarily represent the age distribution of the populations in countries such as India and Bangladesh. Table 6-7 shows that almost 90 % of the participants consumed rice on a daily basis or at least 2-3 times a week. The recruited consumers with high consumption frequency of rice suggested that they were good candidates resembling the rice-consuming populations. Moreover, of all rice types, white rice was consumed the most (94 %), indicating that the recruited consumers may represent the white rice-consuming population. None of the consumers usually consumed parboiled rice. Only 24 % of the consumers recruited in Study 1 were aware of parboiled rice.

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Table 6-6 Socio-demographic characteristics of respondents in the Consumer Acceptance Study- STUDY 1 and STUDY 2 Demographic variable Percentage (%) STUDY 1 STUDY 2 (n = 115) (n = 100) Gender Male 47.8 39.0 Female 52.2 61.0 Age < 20 7.0 11.0 21-29 66.1 70.0 30-38 15.7 12.0 39-47 2.6 1.0 48-55 3.5 3.0 > 56 5.2 3.0 Nationality Australian 13.0 21.0 Chinese 51.3 26.0 Indonesian 8.7 20.0 Indian 5.2 3.0 Malaysian 3.5 4.0 Others 18.3 26.0 Employment status Employed Full-time 17.4 13.0 Employed Part-time 1.7 5.0 Employed Casual 3.5 3.0 Unemployed 2.6 2.0 Student 74.8 75.0 Others 0.0 2.0

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Table 6-7 Rice consumption pattern of consumers recruited for the Consumer Acceptance Study- STUDY 1 and STUDY 2 Item Percentage (%) STUDY 1 STUDY 2 (n = 115) (n = 100) Type of rice consumed usually White rice 93.9 83.0 Brown rice 3.5 11.0 Parboiled rice 0.0 2.0 Others 2.6 4.0 Consumption Frequency Daily 58.3 73.0 2-3 times per week 28.7 23.0 Monthly 10.4 4.0 Rarely 2.6 0.0 Method to cook rice Rice cooker 83.5 - Rapid cooking (cook in excess water) 6.1 - Absorption method (cook in a pot) 8.7 - Others 1.7 - Heard of parboiled rice? Yes 24.3 36.0 No 75.7 64.0

6.2.1.2 Degree of visual acceptance of fortified uncooked rice

The b-values of samples and ControlCom measured were positive and significantly higher than of commercial white rice, suggesting that the uncooked fortified rice and commercial parboiled rice were more yellow than white rice (Table 6-1). The results agreed with the colour of uncooked fortified rice and ControlCom perceived by the consumers, which they indicated as either brown, yellow or golden (Question 1, Table 6-2).

The mean colour intensity of the uncooked fortified rice and ControlCom evaluated were

4.1, 4.6, 5.0 and 6.0 for UF 1, 2, 3 and ControlCom, respectively (Table 6-8) and the samples were significantly different from the control (p < 0.05). Intelmann et al. (2005) suggested that the psychophysical magnitude of C* (Chroma) may be calculated to facilitate the interpretations in relation to sensory properties. C* is a measure of colour saturation which may resemble the colour intensity of rice perceived by the consumers. The trend of C*-values was only consistent with the colour intensity rated by the

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Chapter 6 Consumer acceptance of fortified parboiled rice consumers among the uncooked fortified rice, i.e. 18.7, 21.5 and 23.9 for UF 1, 2 and 3, respectively, unlike the colour intensities of ControlCom which was perceived the most intense in colour. C* of ControlCom showed that it was less saturated (or intense) than UF 3 and was comparable to UF 2. The measure of colour saturation alone may not be sufficient to correlate to consumers’ perception of colour intensity. Despite the significant difference in the colour intensity perceived by the consumers (Table 6-8), it did not significantly impact on the liking of colour between the uncooked fortified rice and

ControlCom (Calculated-F(3,456) = 1.448; p = 0.228). The consumers liked the colour of

ControlCom best although it appeared to be the darkest to them. It may be that the colour the consumers perceived rather than its intensity that dominated their liking of colour. Luh (1991) reported that the darkening of finished parboiled rice products can be an issue for consumer acceptability. As the degree of colour liking of UF 1, 2 and 3 are comparable to ControlCom, it suggests that the uncooked fortified rice meets the first visual requirement to being acceptable.

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Table 6-8 Mean perceptions (colour intensity, degree of liking of colour, uniformity of colour, overall appearance) of uncooked fortified samples (UF 1: 1 h soaking; UF 2: 2 h soaking; UF 3: 3 h soaking) and ControlCom tested by consumers (A. general consumers (n=87) and B. those who were familiar with parboiled rice (n=28)) in the Consumer Acceptance Study- STUDY 1 UF 1 UF 2 UF 3 Commercial parboiled rice t-test t-test t-test (ControlCom) t-test (p value) (p value) (p value) (p value) * Colour intensity A. General consumers 4.1±1.7a,b+ 4.6±1.7b,c 5.0±1.7c 6.0±1.9d B. Consumers who know 4.0±1.8a (0.782) 4.5±1.6a (0.790) 5.0±1.8a,b (0.977) 6.3±1.8b (0.300) about parboiled rice

Liking of colour A. General consumers 5.2±1.3a 5.0±1.5a 5.0±1.3a 5.3±1.8a B. Consumers who know 5.3±1.0a (0.941) 4.9±1.6a (0.691) 5.2±1.1a (0.257) 5.8±1.4a (0.083) about parboiled rice

Colour uniformity A. General consumers 5.6±2.0a 5.4±2.0a 5.4±1.8a 5.8±2.1a B. Consumers who know 5.6±1.9a (0.980) 5.3±2.2a (0.636) 5.4±2.0a (0.932) 5.3±2.2a (0.145) about parboiled rice

Liking of overall appearance A. General consumers 5.3±1.2a,b 5.1±1.4b 5.0±1.4b 5.6±1.3a B. Consumers who know 5.4±1.2a,b (0.606) 5.1±1.5b (0.873) 5.3±1.3b (0.244) 6.1±1.7a (0.110) about parboiled rice

+ Values followed by different letters in the same rows indicate significantly different means at p < 0.05. * Values in the parentheses indicate the p-value of independent samples t-test performed between group A. general consumers and group B. consumers who know about parboiled rice.

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In terms of the overall appearance liking, ControlCom was the most liked followed by UF 1 which showed no difference (Calculated-F(3,456) = 3.597; p = 0.518), whereas UF 2 and 3 were significantly lower than ControlCom (Calculated-F(3,456) = 3.597; p < 0.041).

Note that ControlCom was a commercial product available in the local and international market, it was a suitable benchmark to assess consumers’ acceptance of rice. Although the mean scores of the liking of colour, colour uniformity and overall appearance of all uncooked fortified rice and ControlCom were approximately 5, which represented “Neither like nor dislike”, the result still highlighted the fact that the fortified rice was comparable to a commercially available product and was as likely to be accepted by consumers as the commercial rice control.

6.2.1.3 Evaluation of purchase intent of uncooked fortified rice- with and without the nutritional information

Two consecutive purchase intent questions were asked in the questionnaire. When the consumers were asked directly about their purchase intent of the rice samples, the response distributions of all the fortified rice were significantly different from the

ControlCom (calculated χ² (2) = 16.9 (UF 1), 13.1 (UF 2) and 18.8 (UF 3); p <0.001). The percentage of “Would buy” was between 32-36 % for all the fortified rice which was far lower than that of ControlCom (51 %) (Figure 6-2).

In the second purchase intent question, when the consumers were subsequently asked to re-evaluate the purchase intent of the same rice with notification that the rice may have been fortified and offered higher nutritional benefits, their purchase intent for all uncooked fortified rice and ControlCom increased (Figure 6-2). Based on the same sample, notification of a nutritional benefit significantly altered the response distribution of purchase intent (paired t-test (95% CI); p < 0.0001), with most of the responses skewed towards “Would buy”. Moreover, the uncooked fortified rice was no longer significantly different from the ControlCom response after the consumers were notified about the associated nutritional benefit from the rice (calculated χ² (2) = 2.007 (UF 1), 2.641 (UF 2)

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Chapter 6 Consumer acceptance of fortified parboiled rice and 2.821 (UF 3); p > 0.224). The result indicated that the general consumers were as likely to purchase the fortified rice as the commercial rice (ControlCom).

Figure 6-2 The distribution of purchase intent responses (%) between the general rice consumers and parboiled rice-experienced groups in Consumer Acceptance Study - STUDY 1. A) Before the notification of health claim; B) After the notification of health claim

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6.2.1.4 Evaluation of purchase intent of uncooked fortified rice- consumers who were familiar with parboiled rice

Note that the parboiled rice can be considered as a specific market for the rice industry and the parboiled rice-consuming population are the primary target consumers for this fortified rice. The perception of parboiled rice consumers may be different from those who do not normally consume parboiled rice (Prom-u-thai et al., 2009b) as they likely have more experience with the distinct appearance and flavour of parboiled rice. Further analysis was therefore conducted focusing on those consumers who were familiar with parboiled rice (n = 28). These consumers would better represent the parboiled rice- consuming markets.

Based on the independent samples t-test results shown in Table 6-8, the responses to the parameters (i.e. colour intensity, degree of liking of colour, colour uniformity and degree of liking of overall appearance) obtained from this particular group was not significantly different from that of the general consumers, only the mean scores were slightly higher in the former group. In contrast, when the purchase intent (before the notification of health claim) between the general and experienced consumers was compared, the response distribution of purchase intent was similar between the two consumer groups for UF 1 and 2 (calculated χ² (2) = 4.789 (UF 1), p = 0.091 and calculated χ² (2) = 1.851 (UF 2), p

= 0.396) but significantly different for UF 3 and ControlCom (calculated χ² (2) = 10.296

(UF 3), p = 0.006 and calculated χ² (2) = 25.447 (ControlCom), p ,< 0.001). The results indicated that the consumers who knew about parboiled rice were more inclined to buy

UF 3 and ControlCom (Figure 6-2A). The notification of the health benefits associated with the rice significantly improved their willingness to buy the rice (Figure 6-2B). The response distribution for the corresponding rice samples (including ControlCom) was significantly different from that of the general consumers accordingly (calculated χ² (2) = 46.127 (UF 1), p < 0.001; calculated χ² (2) = 8.551 (UF 2), p < 0.014; calculated χ² (2) =

8.978 (UF 3), p < 0.011; and calculated χ² (2) = 7.211 (ControlCom), p < 0.027). The target group (i.e. parboiled rice consuming populations) showed increased intention to purchase

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Chapter 6 Consumer acceptance of fortified parboiled rice the rice than general consumers and their purchase intents was further enhanced by the relevant health claim. This result suggested that familiarity with parboiled rice may improve consumers’ preference and, more importantly, the purchase intent for parboiled rice.

6.2.2 STUDY 2: Consumer acceptance of cooked fortified rice after mixing

6.2.2.1 Consumer demographics and rice eating preferences

One-hundred consumers were recruited for STUDY 2 (39 % men and 61 % women). The demographics of consumers recruited in STUDY 2 were similar to those recruited in STUDY 1 (Table 6-6). The recruited participants were mostly between 21 and 38 years old (82 %). The majority of the consumers were Asian (53 %) and 16 % of the consumers who chose “Others” identified themselves as Singaporean, Thai or Vietnamese. Over 95 % of the recruited consumers ate rice daily or at least twice per week (Table 6-7). The results showed that the consumers were representative of the rice-consuming populations.

6.2.2.2 Degree of liking of cooked rice attributes

Table 6-9 illustrates the mean liking of the rice attributes, i.e. appearance, colour, odour, texture, taste and aftertaste. Apart from their liking of the appearance of CFM 1 which was significantly lower than ControlWhite (calculated F(3, 396) = 3.142; p < 0.018), there was no significant difference between all samples from ControlWhite for the rest of the attributes. Despite the significant difference noted in the degree of liking of appearance between CFM 1 and ControlWhite, it did not affect consumers’ overall acceptance for CFM 1. The overall liking for all three fortified diluted samples was not significantly different from ControlWhite (calculated F(3, 396) = 0.656; p > 0.580), indicating that the consumers liked all the diluted samples as much as the commercial white rice control.

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Table 6-9 Mean degree of liking of attributes (appearance, colour, odour, texture, taste, aftertaste and overall liking) of rice samples (CFM 1: 1 h soaking; CFM 2: 2 h soaking; CFM 3: 3 h soaking) and ControlWhite tested by consumers in Consumer Acceptance Study - STUDY 2 Attributes CFM 1+ CFM 2 CFM 3 Commercial white rice (ControlWhite) Appearance 6.2±1.5b 6.3±1.3a 6.4±1.3a 6.7±1.2a Colour 6.3±1.2a 6.2±1.3a 6.3±1.2a 6.6±1.2a Odour 5.7±1.3a 5.8±1.3a 5.9±1.4a 5.8±1.3a Texture 5.6±1.3a 5.9±1.4a 6.1±1.3a 5.9±1.4a Taste 5.8±1.3a 6.0±1.3a 6.1±1.3a 6.0±1.4a Aftertaste 5.5±1.2a 5.6±1.4a 5.6±1.4a 5.7±1.2a Overall 6.0±1.4a 5.9±1.5a 6.1±1.5a 6.2±1.3a + Values followed by different letters in the rows indicate significantly different means at p < 0.05.

6.2.2.3 Consumers’ perceptions of attributes “JAR”

According to Meullenet et al. (2007), at least 70 % of responses are expected to be in the JAR group to consider the attribute to be optimal. Based on Figure 6-3, more than 75 % of the consumers rated the colour intensity of the samples as just right. Since the samples evaluated in STUDY 2 were cooked fortified rice that was mixed with white rice, any foreign colour from the background rice may be seen as off-colour which may jeopardize consumers’ acceptability (Kratochvil et al., 1994). Iron-fortified rice is an example of how consumers disliked fortified rice which appeared black in colour. They considered this colour as foreign particles from the bulk of the rice (Hurrell, 1997). This highlighted the concern of rice colour in rice fortification (Darnton-Hill and Nalubola, 2002, Winger et al., 2008). Fortunately the consumers’ degree of colour liking for the CFM did not differ significantly from ControlWhite (Table 6-9), which confirmed that the colour of this mixed sample was not an issue.

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Number on the bar indicates the mean drop compared with consumers who responded as JAR compared with overall liking. (-) indicates less than 20% of consumers scored in this category. *Mean drop considered significant p < 0.05 from the Tukey’s (HSD) comparison. Figure 6-3 Distribution of Just-About-Right (JAR) score of attributes intensities of rice samples (in percentage) and penalty analysis of Just-about-Right attributes on mean drops for overall liking of rice samples (for the Consumer Acceptance Study- STUDY 2)

In terms of texture, after the uncooked fortified rice was mixed with commercial white rice, the response distributions of hardness and stickiness for CFM 1 and CFM 2 was not different from ControlWhite (p > 0.075) except CFM 3 (p < 0.002), in which the response was more prevalent at “Just about right” (~70 %) for both “hardness” and “stickiness”. The texture of rice deviated from the expectation that parboiled rice would be harder than

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Chapter 6 Consumer acceptance of fortified parboiled rice normal rice after cooking (Bhattacharya, 2004). The reason may be due to the mixing ratio (1:24) chosen was high enough to sufficiently dilute the characteristic texture of parboiled rice, resulting in the CFM having a comparable texture to commercial white rice.

The response distributions of taste intensity of all the diluted samples was not significantly different from ControlWhite (calculated χ² (2) = 1.100 (CFM 1), p = 0.577; calculated χ²(2) = 0.810 (CFM 2), p = 0.667; χ²(2) = 2.480 (CFM 3), p = 0.289). Between 28 and 36 % of the consumers felt the taste of rice was “Not Enough” for all rice samples

(including ControlWhite) and a significant mean drop in the overall liking score resulted (mean drop range of 0.659-1.263) (Figure 6-3). Despite the significant mean drop, the result appeared positive for the mixed rice because normal white rice has a typical bland and subtle flavor (Heinemann et al., 2006), so it is a weak agent to mask any apparent taste and odour. This result highlighted that the fortified parboiled rice did not convey any detectable unpleasant flavours to the mixed rice (end product) and it was acceptable to the consumers.

6.2.2.4 Evaluation of purchase intent of cooked rice

By grouping the responses of purchase intent into “Not buy”, “Undecided” and “Would buy”, the distribution of the CFM and ControlWhite all inclined towards “Would buy” (Figure 6-4). CFM 2 and 3 showed no difference in the response distribution as

ControlWhite, with the majority who rated “Would buy” (61 – 65 %). Only CFM 1 was significantly lower than ControlWhite (χ²(2) = 12.870; p < 0.002). More consumers either chose not to buy or were undecided, although almost 50 % of the consumers would still decide to buy this CFM 1.

After the consumers were informed about the additional health benefits from the rice, their mean purchase intent significantly increased (paired t-test; p < 0.0001). The distribution of the purchase intent responses of all the CFM samples was more negatively skewed, although the shift was mostly from “Not buy” to “Undecided”. CFM 2 and 3,

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again, had a statistically comparable distribution to ControlWhite. This indicated that CFM 2 and 3 were comparable to the commercial white rice and implied that the two fortification conditions, i.e. 2 h and 3 h soaking and mixing the concentrate with white rice were acceptable to consumers.

Figure 6-4 Purchase intent of cooked fortified mixed rice and ControlWhite tested by consumers before and after the notification of additional health claim of rice evaluated in the Consumer Acceptance Study- STUDY 2

Given that the contrast in mixing the fortified rice with the background white rice was expected to be larger than mixing with the background parboiled rice, based on the degree of liking and purchase intent, consumers still expressed their acceptance of the fortified rice. Moreover, because the consumers recruited resembled the general rice- consuming populations, it is postulated that the fortified rice would also be accepted by the parboiled-rice-consuming populations if the rice was mixed with the parboiled rice,

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Chapter 6 Consumer acceptance of fortified parboiled rice which was a better masking agent. This may be an important finding for populations who do not consume parboiled rice and yet are at risk of folic acid deficiency. It also enhanced the universal appeal of the methodology used in this study as an acceptable tool for rice fortification.

6.3 Conclusions

In STUDY 1, visual assessments were performed for uncooked fortified rice. General consumers and those who were familiar with parboiled rice accepted the visual attributes of fortified rice. Based on the attributes evaluated, UF 1 (i.e. 1 h soaking) resembled commercial parboiled rice the best. It implies that the parboiling conditions studied for folic acid fortification were capable of producing parboiled rice quality that is comparable to the commercial standard. Additional health claims improved consumers’ willingness to buy the rice and the purchase intent of those who knew about parboiled rice was significantly higher than the general consumers, suggesting that food familiarity may affect consumers’ food choice and fortified products are welcomed by consumers.

In STUDY 2, consumers liked the attributes of cooked fortified mixed rice samples as much as the commercial white rice control. The attribute intensities were found comparable or even better than the white rice control (based on the JAR responses). No difference was observed in texture, odour and taste of the CFM and ControlWhite, indicating that the present mixing ratio and parboiling conditions chosen to produce the fortified rice (70 ◦C for 1, 2 and 3 h) did not adversely affect the appearance and sensory qualities of the final product (i.e. cooking of the fortified rice after mixing). If higher mixing ratios are to be used, it is possible to postulate that negligible changes would be detected by the consumers. Looking at the purchase intent in particular, consumers were most willing to purchase CFM 2 and 3, which referred to as soaking the rice for 2 and 3 h, respectively. With the additional information from Chapter 4, the optimal soaking condition was 70 ◦C for 2 h, which coincided with the condition most accepted by the consumers. It is therefore a reasonable condition to be examined further for its

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Chapter 6 Consumer acceptance of fortified parboiled rice bioavailability after ingestion. Bioavailability study is an essential information to confirm whether the fortified rice is useful to improve the folate status of the populations which have high prevalence of folic acid deficiency and to prevent the complications associated with low folate status, i.e. the ultimate goal for the current study.

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CHAPTER 7

SHORT TERM RELATIVE BIOACCESSIBILITY AND ABSORPTION OF FOLIC ACID IN FORTIFIED PARBOILED RICE: CACO-2 CELL STUDY

Considering that the purpose of folic acid fortification is to improve the folate status and reduce the incidence of the associated deficiency diseases of the target populations, the fortificant would only be functional after being ingested, digested, absorbed through the intestinal tract, taken up and utilised by tissues and cells. The fortification process therefore indicated the importance of studying the bioavailability of the nutrient in the food vehicle.

For rice in particular, de Ambrosis (2006) studied the bioavailability of pectin-coated folic acid fortified rice using human subjects. de Ambrosis (2006) reported that the extent of absorption, intestinal transit times and gastric motility differed between individual subjects. It was difficult to accurately capture the plasma peaks and elimination rates for designing the area under the curve (AUC) sampling times (cf. Section 2.9.2.2). As discussed in Section 2.9.2.3, in-vitro studies are an emerging tool to assess bioavailability of nutrients in addition to human trials (Verwei et al., 2005, Öhrvik et al., 2010, Netzel et al., 2011), especially Caco-2 cell model owing to its enterocytic and colonocytic characteristics which resembles human intestinal epithelium (Grasset et al., 1984, Vincent et al., 1985). Apart from Prom-u-thai et al. (2009a) who assessed the bioavailability of iron fortified parboiled rice, parboiled rice fortified with folic acid has not been examined, especially using the Caco-2 cell model.

Therefore, the aims of this chapter were to 1) investigate the in-vitro bioaccessibility of added folic acid from fortified rice; and, 2) assess the transport and absorption of the fortificant using Caco-2 model. Notably, fortified rice soaked at 70 ◦C for 2 h (at 0.15

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gfolic acid/ grice) was the most appealing condition from a nutritional and consumer acceptance point of view (cf. Chapter 4 and Chapter 6), so this condition was chosen for the following experiment.

7.1 Materials and methods

7.1.1 Reagents

The human colon carcinoma cell line, Caco-2 cells obtained from the American Type Culture Collection (ATCC) and used between passage number 30 to 40. Dulbeccos modified eagle medium (DMEM), foetal bovine serum (FBS), penicillin (5000 U/ mL), streptomycin (5000 µg/ mL), non-essential amino acids and GlutaMAXTM were purchased from Invitrogen (Sydney, NSW, Australia). These chemicals made up the growth media for the cell culture work. CellTiter 96® non-radioactive cell proliferation assay (MTT) was purchased from Promega (cat# G5421) which was used to determine the cell viability. All other chemicals were purchased from Invitrogen, Sydney, unless stated otherwise.

7.1.2 Cell culture

7.1.2.1 Handling procedure for bringing up frozen Caco-2 cells

The Caco-2 cells were initiated from a frozen ampoule. The frozen ampoule (1 mL) was removed from the liquid nitrogen and rapidly thawed in a 37 °C water bath because rapid thawing helps to avoid osmotic damage to the cells and improve cell viability (Gull et al., 2009). The growth medium (~12 mL) was pre-warmed to 37 °C , added to the thawed cells slowly and centrifuged to remove the dimethyl sulfoxide (DMSO, Sigma-Aldarich, Australia). Cells were grown in 75 cm2 flasks (EasyFlasksTM, NUNC, ThermoScientific, Denmark) until ~70 % confluence and subsequently split (Section 7.1.2.2).

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7.1.2.2 Subculturing of Caco-2 cells

Caco-2 cells were subcultured weekly to maintain the growing phase of the adenocarcinoma cells. Cells were washed with 3 mL of Dulbecco’s Phosphate-buffered saline (DPBS) with no calcium and magnesium to remove serum which contains trypsin inhibitor. Trypsin (0.25%)-EDTA (3 mL) was added to detach the cells from the 75 cm2 ◦ flask surface and the cells were incubated in 5 % CO2 at 37 C for approximately 3 mins, then 12 mL of growth media was added and they were centrifuged. After centrifugation, the washed cell pellet was resuspended with 10 mL growth medium. The split ratio of 1:10 optimally allowed subculturing once in approximately 4 days. The cells were grown ◦ in vented culture flasks incubated in 5 % CO2 at 37 C.

7.1.3 Sample preparation

7.1.3.1 In-vitro digestion model

The in-vitro digestion model used was a modification of that described by Netzel et al. (2011). Duplicate rice samples were cooked according to Section 5.1.2. 5±0.01 g of the cooked rice was sampled for folic acid analysis (Section 7.1.5) and another 5±0.01 g of the cooked rice sample was ground with a mortar and pestle with 5 mL of water and placed in a 50 mL centrifuge tube. In order to simulate gastric digestion, 0.1 M HCl was added drop-wise until the mixture was reduced to pH 2.0. Pepsin mixture (250 µL, 40mg/ mL pepsin from porcine gastric mucosa dissolved in 0.1 M HCl) was added to the mixture and incubated in a shaking water bath at 37 ◦C for 1 h. At the end of the gastric digestion, the pH of the mixture was adjusted to pH 7.0 using 0.1 M NaHCO3 followed by the addition of 1 mL of pancreatin bile solution (2 mg/ mL pancreatin, 12 mg/ mL hog bile extract in 0.1 M NaHCO3), to mimic intestinal digestion. The mixture was incubated in a shaking water bath at 37 ◦C for 2 h. After the completion of in-vitro digestion, the extract was named “digesta”. The total volume of the digesta was approximately 30 mL. The digesta was centrifuged at 3500 rpm for 15 mins to separate the solid component of

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Chapter 7 Short term relative bioaccessibility and absorption of folic acid in fortified parboiled rice: Caco-2 cell study the sample. The supernatant was divided into aliquots and stored frozen at -20 ◦C until further analysis. Two controls were prepared for this experiment, namely, 1) positive control: 5 mL of folic acid solution (10 µg/ mL), and 2) negative control (NIL digesta): 5 mL of MilliQ, was each pipetted into 50 mL centrifuge tube at the start and the above gastric and intestinal digestion simulation was followed accordingly.

7.1.3.2 Cell viability assessment

The viability of cells in response to in-vitro digesta was determined using a colorimetric method, i.e. a commercial CellTiter 96® non-radioactive cell proliferation assay (MTT). The cell viability is important because non-viable or lysed cells could detach from the filter at the washing stage described below. This would result in increased filter exposure and facilitate the movement of substance from the apical compartment to the basolateral compartment, and a false positive conclusion may be drawn on the transport study. The protocol provided by the supplier was strictly followed. On a 96-well plate, 5 approximately 1x10 cells/ well were seeded and subsequently incubated in 5 % CO2 at 37 ◦C for 1 week to allow cell adherence and cell differentiation. Following the incubation, the culture medium was removed and replaced with 100 µL of digesta (based on Section 7.1.3). Positive control was adding Hanks Balanced Salt Solution (HBSS) on wells with confluent cells and negative control was adding HBSS on wells without any cells seeded. Samples and controls were prepared in triplicate to obtain an average value.

The sample digesta and controls (i.e. rice and HBSS) were incubated for 1 h in 5 % CO2 at 37 ◦C which resembled the incubation duration that was to be investigated for the transport study (Section 7.1.4). The solution in the well was meticulously removed without disturbing the attached cells. After washing the cells with HBSS, MTT solution was added and incubated for 2 h for colour development. Absorbance at 490 nm was recorded using the ELISA plate reader (Spectra Max M2, Molecular Device, Australia). The result of the absorbance for samples and controls was illustrated in Table 7-1. As shown in Table 7-1, the absorbance value for the negative and positive controls was 0.075 and 0.779, it suggests that absorbance values above 0.779 indicated the cells were

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Chapter 7 Short term relative bioaccessibility and absorption of folic acid in fortified parboiled rice: Caco-2 cell study viable. All the digesta prepared were all above 0.955, which means that the samples investigated and the incubation period studied did not affect the viability of cells. The result obtained enhanced the validity of the in-vitro transport study as it eliminated the possibility of drawing a false conclusion on the transport result.

Table 7-1 The absorbance values of the prepared food digesta and controls in the cell proliferation assay Sample/Control Absorbance -3 -1 Uncooked fortified rice (4 x10 gfolic acid g rice) 0.957±0.029 -3 -1 Uncooked fortified rice (2 x10 gfolic acid g rice) 1.007±0.047 -3 -1 Uncooked fortified rice (0.5 x10 gfolic acid g rice) 0.957±0.048 -3 -1 Cooked fortified rice (4 x10 gfolic acid g rice) 1.007±0.077 -3 -1 Cooked fortified rice (2 x10 gfolic acid g rice) 0.956±0.077 -3 -1 Cooked fortified rice (0.5 x10 gfolic acid g rice) 0.955±0.033 Positive Control: HBSS (with cells in well) 0.779±0.056 Negative Control: HBSS (without cells in well) 0.075±0.012

7.1.4 Caco-2 cell transport model

The protocol for the in-vitro study was followed based on that reported by Netzel et al. (2011). For preparing the transport study, 200 µL of Caco-2 cells (cell density: 4x105 cells/ mL) was seeded on the polycarbonate filter insert (apical compartment) of a 24- well transwell plate with 0.4 µm mean pore size (Corning-Costar, cat# 3413). Approximately 80000 cells per well was achieved on the transwell membrane (Hubatsch et al., 2007). Six hundred microliters of growth media was added into the well under the cell culture insert (basolateral compartment) (Figure 2-11). The cells were incubated for ◦ 21 days in 5 % CO2 at 37 C to allow cell differentiation and a confluent monolayer formation. Within the 21-day incubation, the growth medium in the apical and basolateral compartment was changed regularly to maintain the viability of the cells.

At the beginning of the transport study, the culture medium was first removed from the basolateral compartment and then the apical compartment. The apical and basolateral compartments were washed twice with HBSS (200 and 600 µL, respectively). When HBSS was added to the compartments the second time, the solution was left in the

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◦ transwell compartments and incubated in 5 % CO2 at 37 C for 1 h prior to the addition of samples. After the 1 h incubation, the transport study was started by carefully removing the HBSS from the apical compartment without disrupting the monolayer of Caco-2 cells on the filter, and replaced with 200 µL of digesta samples (fortified rice, folic acid standard and NIL digesta) and 600 µL of fresh HBSS (transport medium) was placed in the basolateral compartment. The 200 µL digesta in the apical compartment was labelled “apical” and the 600 µL of sample in the basolateral compartment after the transport study was labelled “basolateral” (Öhrvik et al., 2010). Duplicate samples were prepared and each sample was loaded into 6 individual wells. At the end of the transport study, apical and basolateral samples were collected in eppendorf tubes and stored at -20 ◦C prior to folic acid analysis (Section 7.1.5). The cells (from the same sample) on the insert filter were removed using a scalpel and the cells were lysed in 1 M KOH with the addition of 2 % sodium ascorbate (w/v) (Öhrvik et al., 2010). All the samples were analysed within 7 days to prevent possible degradation.

The cells were first incubated in HBSS at the beginning of the transport study in order to ensure that the cells released all the materials that may have been taken up during the 21- day incubation in the growth medium (Hu et al., 2004). In a preliminary study, NIL digesta was applied to the apical compartment in a transport study to observe any possible folic-acid-carry-over from the growth medium that the cells were previously incubated in. No detectable amount of folic acid was found in NIL digesta or in the apical compartment, yet, trace amounts of folic acid (~0.012 µg/ mL) were detected in the basolateral compartment after the transport study. However, folic acid was no longer detectable in the basolateral compartment once the cells were incubated in HBSS for 1 h prior to NIL digesta loading. The result suggested that by incubating the cells in HBSS for 1 h could possibly eliminate the chance of over-estimation of the percentage of transport from the apical compartment.

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7.1.5 Folic acid analysis

Apical and basolateral samples were each pooled from the 6 wells to produce a single sample. The lysed cells were subjected to rat serum deconjugation of polyglutamate chains and incubated at 37 ˚C for 3 h in a shaking water bath. The extracted lysed cells, the pooled apical and basolateral samples, and the digesta samples, were purified using SAX SPE according to Section 3.2.2.6. The purified extract was analysed using HPLC with the instrumental conditions detailed in Section 3.2.2.7. For the purpose of identifying the fate of the added folic acid from the start of the simulated in-vitro digestion, the amount of folic acid in cooked fortified rice was also analysed.

7.1.6 Calculations

Two terms are used in the following context to describe the change in the amount of folic acid at different stages of the bioavailability study, namely the percentage of bioaccessibility and transport.

As defined in Section 2.9, folic acid bioaccessibility refers to the amount of folic acid released from the food portion and made available for cell absorption after digestion. The percentage of bioaccessibility may be described by the following equation:

⁄ (Equation 7-1)

where and are the amounts of folic acid present in the digesta and cooked rice, respectively.

The folic acid transport is referred to as the amount of folic acid detected in the basolateral media at the end of the 1 h transport incubation in relation to the digesta. The transport percentage was calculated as:

⁄ (Equation 7-2)

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where is the amount of folic acid present in the basolateral compartment.

7.1.7 Cell quality controls

As mentioned previously, the usefulness of Caco-2 cells for studying intestinal absorption of nutrients is due to its enterocytic characteristics and these characteristics would only be expressed after full differentiation (Sambuy et al., 2005). Studies had suggested that Caco-2 cells required 21 days for differentiation of the cells (Netzel et al., 2011, Turco et al., 2011). Due to the importance of cell differentiation, three different markers were used to assess the level of Caco-2 cell differentiation 21 days after seeding: transepithelial electrical resistance (TEER) measurement, Lucifer Yellow (LY) permeability and confocal microscopy analysis.

7.1.7.1 Transepithelial electrical resistance (TEER) measurement

When the cells achieved a confluent monolayer on the filter insert, tight junctions (Figure 2-11) became well-defined and detectable (Sun et al., 2008). The resistance between the apical and basolateral compartments is therefore higher for the intact monolayer as opposed to ‘leaky’ cells. Therefore, measuring the TEER between apical and basolateral compartment ensures the integrity of the monolayer and, indirectly, the level of differentiation of the cells.

On the day of harvest (21 days after seeding), TEER between the monolayer was measured using the Millicell ERS-2 Volt-ohm meter (Merck Millipore, Germany) prior to the start of the transport experiment (Faassen et al., 2003, Verwei et al., 2005). TEER readings were corrected by subtracting the intrinsic resistance of the cell-free filter (~50- 100 Ω/ cm2) from the reading (Biganzoli et al., 1999). Although as low as 300 Ω/ cm2 was accepted by some researchers (Öhrvik et al., 2010), in this study only the cell monolayers with a corrected TEER reading greater than 800 Ω/ cm2 were used (De Brouwer et al., 2010) in order to provide greater confidence in obtaining intact cell layer.

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At the end of the transport study, TEER was again measured to determine whether the monolayer remained undisrupted.

7.1.7.2 Lucifer Yellow (LY)

LY was used as the second marker to examine the formation of tight junction as the marker has a low apparent permeability coefficient. As well as the reasons in relating tight junction formation with cell differentiation, its formation is important for the quality of folic acid transport because folates are expected to be transported through the epithelial cells largely by carrier-mediated transport and folate transporter but not through passive diffusion (cf. Section 2.9.1). The confirmation of tight junction formation prevents the possibility of over-estimating folate transport due to paracellular diffusion.

In the preliminary study, two apical concentrations of LY (0.1 and 0.5 mM) were investigated. The protocols described in Section 7.1.4 were followed. TEER readings were measured at the beginning of the study and each concentration of LY (200 µL) was loaded into the respective apical compartments in triplicate. At the end of the transport study, TEER readings were measured and 100 µL of basolateral medium from each well was subsequently collected and transferred into the corresponding well of a 96-well black plate. Apical medium was diluted with HBSS (1:20) and 100 µL aliquots of the diluted medium were similarly loaded into the 96-well black plate. A 10-point standard curve (0- 0.25mM of LY) was constructed to quantify the amount of LY present in the apical and basolateral compartments. An additional 100 µL of HBSS was added to each well as a diluent. The fluorescent reading was measured with a spectrophotometer at excitation 430 nm, 535 nm and manual gain of 40.

The results showed that the TEER readings (~ 800 Ω/ cm2) were maintained after the transport study, and a negligible amount of LY (< 0.5 % of the amount of LY in the apical medium) was present in the basolateral compartment for both concentrations studied, suggesting that the tight junctions were formed and there was negligible paracellular diffusion.

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7.1.7.3 Confocal microscopy

TEER measurement is arguably the most convenient marker in relation to cell integrity although it only is an indicator (U.S. Department of Agriculture Agricultural Research Service, 2012). Microscopic analysis will be the most accurate assessment for the formation of tight junction because it provides visual confirmation of the cell integrity.

After sampling the apical and basolateral media after the transport study, cells were rinsed with 500 µL of HBSS and subsequently fixed using 200 µL of paraformaldehyde (4 %) and stored at room temperature for 15 mins. They were then rinsed three times with Phosphate-buffered saline (PBS, with 5 mins incubation each) and were then permeabilised with 300 µL of 0.1 % Triton X100 in PBS (Sigma-Aldarich, Australia) for 15 min. After removing the Triton solution, the cells were incubated with HOECHST 33342 and then fluorescein Phalloidin FITC for nuclear and F-actin staining, respectively. Bovine serum albumin in PBS (1 %, 300 µL) was used to rinse the cells 3 times and the cells were then removed from the insert using a scalpel. Mounting fluid was then applied. The samples were observed under the confocal microscope. Figure 7-1 illustrates an example of a cross-sectional confocal microscopy diagram of Caco-2 cells on the harvest day. HOECHST 33342 is a nucleic acid stain which emits blue fluorescence when bound to dsDNA whereas fluorescein Phalloidin FITC has high affinity to F-actin which emits green fluorescence when bound. Figure 7-1 clearly shows that distinct cell membranes and nuclei were formed on the polycarbonate filter and no obvious ‘leaks’ were identified, suggesting that tight junctions were formed on the cell layer which were suitable for the transport study.

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Figure 7-1 Confocal microscopy diagram of Caco-2 cells on the filter insert on the harvest day

7.1.8 Statistical analysis

Comparisons between group means were performed by Student’s two-tailed t-test. A significant difference between means was considered when p < 0.05.

7.2 Results and discussion

7.2.1 In-vitro bioaccessibility

Prior to the start of the in-vitro bioaccessibility study, the folic acid concentration of fortified rice digesta was measured and 10.8 µgfolic acid/ gcooked fortified rice, on wet weight basis, was detected. Therefore, an aqueous folic acid standard with a comparable amount of folic acid was prepared as a control. The average amount of folic acid in the folic acid standard and fortified rice sample was 50 and 54 µg per extract (5 g of cooked rice in each extraction). There was no significant difference in folic acid concentration measured

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Chapter 7 Short term relative bioaccessibility and absorption of folic acid in fortified parboiled rice: Caco-2 cell study in the respective extracts (sample or control prior to gastrointestinal digestion simulation) (Figure 7-2). Therefore, equivalent amounts of folic acid were available in the control and fortified rice for in -vitro digestion and subsequent absorption.

The folic acid present in the digesta represented the bioaccessible fraction and the bioaccessibility of folic acid in the control and fortified rice was 81 (40 µg) and 91 (51 µg) %, respectively. The bioaccessibility of folic acid in the aqueous standard control was comparable to that of early reports on yogurt (80 % bioaccessibility) (FAO, 2013). The fortified rice, which was presented as a solid food, was expected to show lower bioaccessibility than liquid food (e.g. aqueous folic acid standard) due to its relatively complicated matrix, but in fact the folic acid was found to be significantly more bioaccessible (91 %) (Figure 7-2). Öhrvik et al. (2010) had also suggested that folate bioaccessibility in solid food such as wholemeal breads was above 75 % and that in the breakfast foods (mixed diet with bread, liver pate, orange juice, sour milk, bran flakes and kiwifruit) was as high as 94 %, compared to the fortified milk (60 %) (Verwei et al., 2003). During the preparation of the fortified rice digesta, it was noted that in response to the addition of acid (0.1 M HCl) and alkali (0.1 M NaHCO3) at the gastric and intestinal digestion stages, the change in pH was relatively more gentle for rice sample compared to preparing the folic acid standard. It appeared that the rice matrix may have provided a certain degree of buffering as opposed to folic acid solution, that may be beneficial in protecting or stabilizing folic acid during the in-vitro digestion. Given the high bioaccessibility of folic acid in fortified rice, it suggests that the fortificant may be released from the food matrix easily and that folic acid is stable during gastrointestinal digestion.

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Figure 7-2 Mean folic acid concentration in the extract and digesta of folic acid standard and fortified rice

7.2.2 Transport study

In order to initiate the transport study, the digesta (FA standard control and fortified rice) prepared was applied to the apical compartment of the transwell. Therefore, the initial concentration of folic acid in the digesta was the same as the apical media at the start of the transport study. The transport mechanisms governing the intestinal transport of folate should be studied at a concentration range that would minimise non-saturable transport. An early study suggested that non-saturable transport component was dominating only at a folic acid concentration greater than 10 µM (Vincent et al., 1985) but not at a concentration less than 10 µM (Mason et al., 1990). Similarly, Verwei (2004) concluded that at the folic acid concentration of 0.02-10 µM, the linear relation between transport and concentration of folic acid was maintained and it indicated that the transport mechanisms involved in folate transport were not saturated up to 10 µM. For this reason, the folic acid concentrations of the digesta of FA standard and fortified rice were adjusted

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Chapter 7 Short term relative bioaccessibility and absorption of folic acid in fortified parboiled rice: Caco-2 cell study such that they were within the linear region for the transport study, i.e. ~ 3 and 4 µM, respectively (Figure 7-2, the final volume of the digesta was 30 mL).

At the beginning of the transport study, the initial amount of folic acid in the standard was significantly lower than that in the fortified rice due to its lower bioaccessibility in in-vitro digestion (Table 7-2). Interestingly, after the 1 h incubation for the transport study, the amount of folic acid in the apical media for the control was marginally lower but insignificantly different from that in the fortified rice. A comparable amount of folic acid (~ 0.02 µg/ monolayer) was transported from the apical to the basolateral compartment for both standard and fortified rice (Table 7-2). The folic acid transport percentage of the standard solution (6.77 %) was slightly higher than that of the fortified rice (5.12 %) although the difference was not significant. The current transport percentages obtained were in agreement with the previous study, which was essentially 6 % (Öhrvik et al., 2010).

In this study, the aqueous folic acid standard was used as the reference dose and it was assumed that the aqueous folic acid standard would be well absorbed by the human body. In this case, in the Caco-2 cell layer, absorption of folic acid in the fortified rice was measured relative to the reference standard. As suggested by the Food and Drug Administration (FDA) guidelines, the formulations whose extent of absorption differs by 20 % or less are generally considered bioequivalent, i.e. the mean absorption of a test product relative to a reference control lies within 80-125 % (FDA, 2005). Relative absorption of folic acid was therefore expressed as the amount of folic acid in the basolateral media of fortified rice against that of the folic acid standard (0.018 µg/ 0.19 µg x 100 %), which was 94.7 %. Folic acid in the fortified rice was therefore considered as bioequivalent to the aqueous folic acid standard, indicating that the absorption of folic acid in the fortified rice was high.

Considering the amount of folic acid present in the apical media was 100 % at the start of the transport study for the aqueous folic acid standard, 6.7 % of the folic acid was present

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Chapter 7 Short term relative bioaccessibility and absorption of folic acid in fortified parboiled rice: Caco-2 cell study in the basolateral media for the control and only 67.1 % of that was detected in the apical media at the end of the transport. Therefore the recovery of folic acid in the Caco-2 cell model was approximately 74 % whereas that of fortified rice was about 87 %, if only these two compartments were considered. The recovery of Caco-2 cell model for folate standard (control) and bread (sample) previously reported was as high as 99 and 88 %, respectively (Öhrvik et al., 2010). The relatively low recovery for the current study was likely because no detectable folic acid was found in the cell layer, for both the control and fortified rice. Verwei (2004) reported that the structure of folic acid transported from the apical to the basolateral compartment was unaltered. However, for the portion of folate that were retained in the Caco-2 cell layer, if folate monoglutamates were the analytes, they would not accumulate in the cell layer unless they are converted to folate polyglutamates in the cytoplasm or mitochondria (Verwei et al., 2005). Öhrvik et al. (2010) also reported that folate monoglutamate was retained in the cells in the form of polyglutamate which was shown by the increased content of the labelled folate monoglutamate in lysed cells after enzymatic deconjugation. The lysed cells were subjected to enzymatic treatment in the present study, and no folic acid peak was identified. The results obtained confirmed that the added folic acid was not retained by the cells in its original form (Verwei et al., 2005). It may be converted into a different folate form and it was out of the scope of the present study to detect those forms. Considering the amount of folic acid transported through the Caco-2 cell monolayer represents the absorbed portion of added folic acid that will be available for cell utilisation and metabolism, the folic acid transport percentage in the basolateral compartment may be a more important indicator for assessing the absorption of folic acid in the aqueous folic acid standard and fortified rice, which addressed the aim of this chapter.

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Table 7-2 The mean amount of folic acid (µg/ monolayer) present in the apical and basolateral media before and after the transport study Folic acid standard Fortified rice Initial condition (before transport study)1 Apical media 0.278±0.011 (100 %)a+ 0.355±0.015 (100 %)b Basolateral media n.d. n.d. Final condition (after1 h transport study)1 Apical media 0.187±0.060 (67.11 %)a 0.290±0.017 (81.79 %)a Basolateral media 0.019±0.002 (6.77 %)a 0.018±0.002 (5.12 %)a * Value in parentheses indicates the percentage of folic acid present in the designated media in respect to the digesta (i.e. apical media at initial condition) 1 The calculated amount of folic acid present in one well (from the average of pooled 6 wells) + Values followed by different letters in the same row indicate significantly different means at p < 0.05

The above discussion focuses on the absorption at the cellular level. From the consumers’ point of view, it may be more practical to understand the amount of folic acid being absorbed in relation to the portion of rice consumed. In this study, 5 g of cooked fortified rice (containing 54 µg of folic acid) was used to prepare the digesta (with the final volume of 30 mL). To initiate the transport study, an aliquot (0.2 mL) of the digesta was then loaded onto each apical compartment. The apical media of each transwell, therefore, represents 1/150 times of the total 30 mL digesta. The amount of folic acid present in the basolateral compartment after the transport study was 0.018 µg per transwell (Table 7-2), which relates to 2.7 µg (i.e. 0.018 µg x 150) out of the 5 g of cooked fortified rice sample. The absorption percentage corresponded to the cooked rice sample was therefore 5 % (i.e. 2.7 µg / 54 µg x 100 %), which was similar to the absorption of folic acid at the cellular level (5.12 %) (Table 7-2).

As suggested in Chapter 6, the mixing ratio chosen for the present study was 1:24, and assuming that the consumers consumed 400 g of cooked rice per day, there would be 16.7 g of cooked fortified rice present in the 400 g portion of mixed and cooked rice. Given that the concentration of folic acid in the cooked fortified rice is 10.8 µg/ gcooked fortified rice, the amount of folic acid present in 16.7 g of cooked fortified rice would therefore be 180 µg, which would lie within the expected fortification level proposed by the current study, i.e. 150-200 µg/ day. By extrapolating the absorption result, 16.7 g of cooked fortified

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Chapter 7 Short term relative bioaccessibility and absorption of folic acid in fortified parboiled rice: Caco-2 cell study rice in the 400 g mixed and cooked rice would expect 9 µg of folic acid (180 µg x 5 %) to be absorbed by the body.

In this study, Caco-2 cell model was chosen as an alternative to human bioavailability study but it did not intend to replace human trial. Note that the extrapolated result was an estimation and that the in-vitro transport and absorption results need to be interpreted with this in mind. Human trials are expected to be performed in the future to confirm and relate the in-vitro results with in-vivo results. Nonetheless, based on the available results, they likely suggest the usefulness of the proposed parboiling method as the approach for folic acid fortification in rice.

7.3 Conclusions

With reference to the aqueous folic acid standard, the study demonstrated that the present in-vitro digestion procedure used was sufficient to release most of the added folic acid in the sample matrix, and that the parboiled rice matrix did not appear to limit the bioaccessibility of folic acid. The high bioaccessibililty of fortified rice (90 %) may, again, highlight the potential of parboiling in contributing to produce fortified rice that would enable a high bioaccessibility of folate.

Moreover, no significant difference was found in the extent of folic acid transport through the Caco-2 cell layer between the aqueous folic acid standard and fortified rice (~ 6 %), which was consistent with the previous study using a bread matrix (Öhrvik et al., 2010). Given that aqueous folic acid standard was the reference dose for the transport study, the comparable transport percentage of fortified rice against the standard may suggest high absorption of folic acid in the fortified rice. The in-vitro transport study, therefore, served as a preliminary indicator that the fortified rice is useful and is a beneficial vehicle to deliver the expected nutritional advantages to its target population.

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CHAPTER 8

CONCLUSIONS AND FUTURE WORK

Folate deficiency is one of the most prevalent nutritional diseases in the world. The high prevalence coincides with many rice-consuming countries. Poverty reduction may be a more sustainable and a long term approach to not only reduce one form of micronutrient deficiency but also other forms of malnutrition. However, this will not happen in the near future (Stein et al., 2008). Folic acid fortification in food is a short term intervention deemed necessary for these populations to fight against folate deficiency. Several rice fortification methods have been developed to-date, namely, dusting, coating, extrusion (“artificial rice”) and biofortification. These methods have both pros and cons. Fortification using parboiling, being a relatively new approach, appears to be a useful method because many countries have been practising this post-harvest treatment procedure locally. Therefore no additional capital investment on infrastructure is needed for these countries. This study was conducted with this general background information on parboiling and the urge to address population health issues in rice-consuming countries. This thesis demonstrated in detail the process of developing and understanding a systematic folic acid fortification method in rice using parboiling. The following 5 research questions were used to form the framework in the development of the fortification method.

8.1 Conclusions

8.1.1 Is parboiling a feasible method for folic acid fortification in rice?

A water-soluble dye, methylene-blue, was added to the soaking water and soaked with both paddy and brown rice. The dye was used to visually assess rice on soaking and it mimicked the result of soaking done to the rice when the fortificant (i.e. folic acid) was applied instead. The preliminary experiment indicated that brown rice was a better

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starting material than paddy rice due to its physical barrier, the husk, which prevented the migration of dye (cf. fortificant). The fortificant was expected to be concentrated on the outer layer of rice and a reasonable amount of fortificant was present in the endosperm of the brown rice.

Trial studies were performed by adding folic acid (1.2 gfolic acid/ 300 gbrown rice) to the soaking water and soaking the rice at 60 and 70 ◦C for 1 h and then milling for 60 s. The inter-batch variation was less than 8 % which indicated that parboiling was a reproducible approach for rice fortification.

8.1.2 What are the effects of soaking temperature, soaking duration, milling duration and fortificant concentration on the retention of added folic acid?

The fundamental parboiling processing parameters, i.e. soaking temperature and duration, heating temperature and duration, drying condition, milling duration and fortificant concentration, were examined in order to identify the possible factors which may be applicable to the parboiling method. Moreover, multifactorial analysis of the chosen parameters was conducted to assess their roles in folic acid retention in the fortified rice. Generally, higher soaking temperature, longer soaking duration and higher fortificant concentrations affected folic acid retention positively, whereas milling duration had a negative effect on folic acid retention. Folic acid in the brown and milled parboiled rice was retained proportionately to the level of fortificant added. This suggested the modelling of the fortification process facilitated the precise control of the final folic acid concentration in the product (Kam et al., 2012a).

The mechanism of fortification in parboiling is likely due to diffusional transport, between the liquid phase (folic acid soaking solution) and solid phase (brown rice), as indicated by the Ea value of ~20.1 kJmol-1. The existing moisture gradient between the rice and fortificant solution allowed inward migration of the fortificant into the endosperm of the rice kernel. The critical moisture content for fortification to occur was

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about 30 %, which was in agreement with the end-point moisture content found by other researchers (Miah et al., 2002, Igathinathane et al., 2005). Further heat treatment followed by soaking caused rice gelatinisation which appeared to be another potential contributor to folic acid retention. Among the soaking temperatures studied, 70 ◦C was the desirable soaking temperature because rice was fully gelatinised (absence of white bellies) and it showed the second highest folic acid uptake coefficient while 2 h was the optimal soaking duration based on the present fortification modelling.

8.1.3 How does parboiling change the qualities of fortified rice?

Parboiling had no effect on the head rice yield or the kernel dimension of fortified rice compared to unfortified parboiled rice (rice parboiled using the same condition but without the addition of folic acid to the soaking water). The economic return based on head rice yield and the necessary mixing process associated with rice dilution was therefore not an issue. Fortified rice appeared darker than white rice after parboiling. Compared to unfortified parboiled rice, the addition of folic acid to the parboiling process appeared to mostly affect the lightness of parboiled rice. The inherent yellow colour of the fortificant was only passed on to the fortified rice when a concentration as high as 1.2 gfolic acid/ 300 gbrown rice was used, and lower fortificant concentration did not show an increase in yellowness intensity.

8.1.4 How well do the consumers accept the fortified rice visually and organoleptically?

After identifying rice fortified at 1.2 gfolic acid/ 300 gbrown rice was the condition resulting in the most distinct colour change compared to those fortified at other fortificant concentrations. The fortified rice was subjected to a visual acceptance study using 115 untrained consumers. Both nutritional claim (i.e. fortified rice contains folic acid which gives higher nutritional value) and familiarity of product affected consumers’ purchase intent in a positive way. Among the three different soaking conditions studied, the rice produced after 1 h soaking resembled the commercial parboiled rice the most, in terms of

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the degree of liking of colour, colour uniformity, overall appearance and purchase intent. This preliminary study suggested that by simply focussing on the appearance of fortified rice there is a potential market in the rice-consuming populations.

Furthermore, the organoleptic acceptance of the cooked fortified rice was assessed

(fortified at 0.15 gfolic acid/ 300 gbrown rice) by 100 untrained consumers. The consumers liked the cooked fortified rice as much as the commercial white rice control, in terms of appearance, colour, odour, texture, taste, aftertaste and overall appearance. Health claim associated with the rice, again, improved consumers’ purchase intent and rice soaked for 2 and 3 h appeared to be most widely accepted by the consumers, as no significant difference was detected compared to the commercial rice control. This experiment indicated that fortified rice was accepted by the consumers, and that soaking for 2 and 3 h were preferred.

8.1.5 Is the folic acid in fortified rice bioaccessible and easily absorbed?

The bioaccessibility and extent of absorption were measured using an in-vitro method, i.e.

Caco-2 cell model. Rice fortified at 0.15 gfolic acid/ 300 gbrown rice and soaked for 2 h was subjected to in-vitro digestion, in reference to an aqueous folic acid standard as control. The high bioaccessibility of fortified rice (91 %) showed that the rice matrix did not entrap the added folic acid and that almost all of the fortificant was released at the end of the digestion. When the digesta was loaded into the apical compartment of the well, it mimicked the moment when the digesta arrives at the intestinal tract and comes in contact with the epithelial surface. The amount of folic acid present in the basolateral compartment therefore represented the extent of fortificant absorption. At the end of the transport study, the transport percentage of the fortified rice was about 6 %. The transport result was in line with the previous study (Öhrvik et al., 2010) with fortified bread and was not significantly different from the aqueous folic acid standard. As the aqueous standard was used as a reference which was anticipated to demonstrate a high absorption level, the comparable transport result between the fortified rice and control suggested that folic acid in the fortified rice was as easily absorbed as the control.

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8.2 Implication of results from current work

The results of the current work have demonstrated the value of fortifying rice with the conventional parboiling technique on a laboratory scale, and showed its potential to be adopted by the industry. The predictive models developed identified the effect of different parameters on folic acid retention and therefore may easily predict the residual folic acid in rice when different parboiling conditions are used. This provides flexibility to users to conveniently adjust the parboiling conditions according to their available resources and preferences. The parboiling parameters studied were within the practicable range which can be adopted by the industry or local users. Given that rice is the staple food of many countries, change in prices of staple food impose more drastic impact on people than changes in price of any other food commodities. For the fortification method studied, no additional infrastructure is deemed required. The additional investment involved for this technique is likely due to the cost in purchasing the fortificant, i.e. folic acid. With the current cost analysis, between 0.8 and 1.9 % of price increment was estimated for producing 1 kg of mixed fortified rice. The increment in price is expected to be further lowered when the fortificant acid is purchased in bulk. Moreover, the uptake of folic acid in fortified rice was up to ~9 %, suggesting that approximately 90 % of the added folic acid was remained in the soaking solution, which was discarded at the end of the soaking stage in the current study. The cost of the fortification technique could be further reduced if the fortificant soaking solution was able to be recycled. The rice quality assessments confirmed that economic return was not affected using this fortification process. Consumers’ preferences are an important factor in the rice supply chain. Although many of the white rice consumers recruited were not familiar with parboiled rice, they did show acceptance and were willing to purchase the fortified rice. As food familiarity is likely to improve food choice, it is therefore expected that the rice would be accepted by the parboiled rice-consuming populations as well. The results also highlighted the opportunity for expanding the market to not only target the parboiled rice- consuming populations but to white rice-consuming countries, especially as folate deficiency is not restricted to parboiled rice-consuming populations. Therefore, more

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people may benefit from this fortified rice. The ultimate purpose of fortifying rice is anticipating the fortificant is consumed and utilised in the human body. The high bioaccessibility and absorption of folic acid further confirms the value of this fortified rice and, therefore, this fortification process. Based on the current laboratory scale study, scaling up to pilot and eventually commercial scale is required in the future in order to bring the expected nutritional benefit (i.e. improve the consumers’ folate status) to the consumers.

8.3 Future work

In light of the current research, the following research ideas and recommendations are suggested in order to advance the existing knowledge of folic acid fortification in rice using the parboiling technique:

1. The present study only focussed on a single fortificant (i.e. folic acid) fortification. Future investigations on multiple fortificants will add value to the fortification process itself (from an economic perspective) and more micronutrient deficiency diseases may be addressed (PATH, 2007). Potential fortificants to be investigated are iron, β-carotene and iodine because these are the associated micronutrient deficiencies which are the most prevalent in the world (Chapter 1) (Allen et al., 2006). More people may benefit from the enhanced nutritional value of the fortified rice. 2. In the preliminary study, a water-soluble dye, methylene-blue was used to predict the consequence of soaking rice in the fortificant solution. Although methylene blue and folic acid are both water soluble, the properties of the two compounds may not be similar. If a dye is not to be used as an indicator, electrical resistance tomography (ERT) may be an alternative to visually identify the migration of the actual fortificant into the rice during soaking. For ERT measurement, multiple electrodes are placed around the periphery of the processing vessel, in this case, the soaking vessel in contact with the soaking solution. The alternating current is applied between the electrode pair

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and there may be a change in resistance in the course of the soaking process with the uptake of folic acid in the rice kernel, and signals are therefore generated and images may be generated reflecting the distribution of folic acid in rice (Blencowe et al., 2010). Tomography may be a better method compared to the use of water-soluble dye to produce visual assessment of folic acid distribution in rice.

3. In the current study, only absorption cooking method, i.e. cooking with a rice cooker, was studied in depth. Other cooking methods, such as cooking in excess water (i.e. rapid cooking), could be investigated to cater consumers who practise rapid cooking method (Daomukda et al., 2011). As a starting point, preliminary experiments were conducted to examine the effect of rapid cooking on the retention of folic acid in fortified rice. Fortified rice (initial

folic acid added: 1.2 g/ 300 gbrown rice) was rinsed twice with 5 volumes of tap water and subsequently boiled at the rice-to-water ratio of 1:20. The samples were simmered for 19 mins. The preliminary results were as follow:

Table 8-1 Concentration of folic acid (µg/g) in uncooked rice, rinsing water, cooking water and cooked rice. Fortificant Soaking Uncooked Rinsing Cooking water Cooked conc.# time rice water (drained) rice (h) 1.2 1 173.0±9.1 20.2±1.2 145.4±7.7 32.3±5.7 (11.7%)* (84.1%) (18.7%) 2 192.6±10.0 18.9±1.1 125.9±5.7 57.6±21.3 (9.8%) (65.4%) (29.9%) 3 247.5±1.1 18.2±0.8 124.9±10.5 47.8±3.1 (7.3%) (50.5%) (19.3%) # Fortificant concentration was measured in gfolic acid/ 300 gbrown rice. * The percentage of folic acid in the respective composite relative to the amount of folic acid in the uncooked rice.

The preliminary results indicated that a major portion of folic acid was leached into the cooking water when excess cooking was used (Table 8-1). Less than 30 % of folic acid was retained in the cooked rice, which was significantly lower than 90 % retention of folic acid in cooked rice when

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absorption method was used (Section 5.2.1, Table 5-2). This experiment suggested that absorption method would be a preferred cooking method than rapid cooking. Future market campaign may need to educate potential consumers to be aware of the reduced nutritional benefits associated with rapid cooking.

4. In order to gauge the acceptance of the fortified rice by the consumers, an untrained panel was used for the studies. In the future, trained panellists may be used to specifically identify different rice attributes which may be useful for product development. Moreover, majority of the consumers recruited was Chinese, who are not regular consumers for parboiled rice. Future studies shall be conducted by recruiting consumers who are familiar with parboiled rice, e.g. West African, Bangladeshis and Indian etc. The results may then confirm with postulation that “fortified rice would also be accepted by the parboiled-rice-consuming populations if the rice was mixed with the parboiled rice” (Section 6.2.2.4).

5. Caco-2 cell model was used for studying the in-vitro absorption of folic acid in fortified rice. Although the standard deviations of the results obtained in the current result was not huge, Rothen-Rutishauser et al. (2000) suggested that the phenotypes of Caco-2 cells may express high variability from well- to-well and day-to-day. The expression of reduced folate carrier and proton- coupled folate transporter differs due to the stage of differentiation and the transport of folic acid differs accordingly (Subramanian et al., 2009). Due to the relationship between transporters present on the Caco-2 cell layer and the transport of folates, it may be worthwhile to identify the number of transporter present on the cell membrane using confocal microscopy and relate it to the extent of nutrient transport. This may also improve the correlation between in-vitro and in-vivo results as Verwei (2004) also indicated that the activity of the transporters in the small intestine may be one of the important determinants for folate absorption from the intestine.

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6. Despite a range of difficulties associated with human subjects, in-vivo especially, human bioavailability study is necessary in the future to verify the result obtained from the in-vitro study because nothing yet can totally replace the human response. This is the way to confirm the usefulness of Caco-2 cell model as a tool to mimic and assess absorption of folic acid in fortified rice.

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CHAPTER 9

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