Triple Fortification of Rice: Feasibility, Consumer Acceptanceand In-Vitro (Caco 2-Cell) Absorption Studies
Author:Thiruselvam, Nishaanthini
Publication Date:2015
DOI:https://doi.org/10.26190/unsworks/2727
License:https://creativecommons.org/licenses/by-nc-nd/3.0/au/Link to license to see what you are allowed to do with this resource.
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TRIPLE FORTIFICATION OF RICE: FEASIBILITY,
CONSUMER ACCEPTANCE AND IN-VITRO (CACO 2-CELL)
ABSORPTION STUDIES
By
Nishaanthini Thiruselvam
Supervisor: Associate Professor Jayashree Arcot
Co-supervisor: Dr. Janet Paterson
FOR FULFILLMENT OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN FOOD SCIENCE AND TECHNOLOGY
School of Chemical Engineering
The University of New South Wales
Australia
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1.
PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES
The--si$.10iuertation Sheet
Fi!::!l nmnc NISHA/\NTH NI
Ab'ore•;iation for ~gre'e ;;s grven in th~;; Un ._,~·siiy ::;:.ler.dar Oot:l:.u of Philcsphy
Scho~l: School )f Chem~ :.ng re~r ~.~
Titla: TRIPLE FORTIFICA- ION OF ~ICE =.:ASISIUTY. CONSUMER ACCEPTANCE A'ID IN· viTRO (C:ACO /·CEL- ) ASSORPn0.\1 STUDIES
=acohy FacJI!y of :.ngrr,eering
AbJ~;If::let 350 '"'Ord" m3xlmum: PLEASE TYPE,
R.ioe fcrti"l(::n:on '"'i th !l1tllt't:ole ,.,..;,;·o•' ,llt..ents (folic acd. trQil and P ca'otene) t y utilizing: two diffefent ,:)fOt::eSSes i>a•OOili'!g~ <:u' ilr' c em P•oce~s anc High Pt!!!l!isure 01ocessrnQ ~liPF) a nc·<'t:t pu;ce~.s l"ra~ t:een exanine~ h this l i!:!Sis. The fort ffica: on crocess was ad:fressed tfuough tou-t "' 1;:~1 ph:'lSe~r optlmls;.!t.:>r, n• fl."''hOiltrg r. ·~'~t:~ ~t, ~n(! use or I IPP to:r tort!fi~i'lhon •.-.tth m~ll1ifil!'! mler(lt'tul rl(!nlt; !)h)l~le!) .CJl(t'l'"l~l prepertJOt. ot tl"oC fondled flee, consume• nece;:::l"!:e !::Udf :'lrd I::!O•acc:essit:ll~· :me transpt>rl studieS.
Pa;b:,itn·g ))rocess wus optlnu:!-cd f~,· io·t.fy1119 n.;;c with lhrt:e rn..:·c·• uhu:n l:i us1ng ~he fo lo\.\'lllQ COII:ittrc·l: scaksr09 !he brown rice at ro<-c for 2 hours w~h the fo1t l'i<:;,sr1 se vt-;ou ;r ,\ 150 ·q:~ : DC· 62.5 rng: fe· 2:. ~i fol :>w::d by ste<~rnins; at 10o•c 'or 1 hour i:ml the:'! <1ir d;;•ing lttc noo at rocm te·npera:uu:: until ~·1 -: IIIIJ ':!'.•.I e w ·•t.:-ut d~>Jt,;r;>::U to 10 12%.Fol ic vcid shcrNt:C h glu~~t vpt<.~ke d Uflr¥.,1 p;,n boilin9. Nt~r cooking folic acid was
reu.·tne>::J tile lll!)nes: ~S<a'X:I 1::>11.-:·,#i ~'; l((:<"t •;SO':<): ar"(( tM 1' c .:;~rotene (35~1 Fer HPP ""~ ee$t ~fl(lr,ion tor rortllbtton was s~&Cted: treating milled •Nhite rioe 'Ai!h the ftjltifi:;:;nt : ·:>lu:it:l' a! 203 MPa fer 1 l"ou1 (noMherrnali an.j tl"en sir-dtj'ing :he pr0<:1essec rbe 10·12V, final mosture conrem). Fo11e a:::o !!.Mw<'l:1 , ,;:tt'<!M .J:ll:'lke nf.P.r t-IPP (41~~ f~ttnwec o:,· fi·C.'H<>iene :'ln<l rnr t2C% ea cl'! . ne1ent10"1 in cocked ti PP rloe follt>Wed tile :sa'Tic t.-cn::l as p:ar:>oil c: :: l it::.:- Fcl~e ;;c.::l (!;6%! <- Iron :~%:·~ '$ ·Garc:cnc ( ldYI: Rice fortifiCd Ot the :'N"C :cd"niques was assesMtd 'or pt,ysicod'iemicat pro.:)ertle3 to ~·ne€rslf. l r. (N ln!;e& n $tar<t'l cun"9 Iona ca;ion The proceM ot a<1e1ng nutrient$ Cl o 'lOt r~.a .. -e t~n impact 0t1 S.lard'l prcpertles in ttre foniHed t 'ij- I'Mo:::1 ~j r1ce v1as ~omp.etely gel~t•nisaCI v.'"'lle HPP rice showed OMIJI geta\in zaMn ·,.n.ct atre:te<J hydrolysis of search b'f " -am:.. lose .,.,. (.!' 1r tvr 1niluMced the releo5e o• ..,,t~mll\t;. ;rom tne r~U m;'! ~'il( eunrg eldr;aet•:>n ~HI ·~ '" \'ltrO cllget.:SO'"I. F'ortiflod p arboiled rioo ;•,•as diluted with whi:e r c.e- and ssse~d fo• eo b ur •,at al;on instrum&nt:dly in ccmpari~cr lo •,.tHte rice. which show'ed no sigr i(IC·ent diffaren<E be::.\·een t'lem. Tl'l~ dilutec foJtifieo rice '1•·s<s p~enteo t•j oons<Jmers f~r '.'isual M ::l laste aooeptante. Fortife-::1 diluted rioe uncooke~) w.;s ne» a!'> acr.ept.~hl~ AS .lr<.c'IO·.P.c ,·:hi:P. ru:.e (r:nmmf.rr.i:.l) le r.;>Mume·$ d~1e to n:>n·unifonniiy i r, ;;ppearan:;.e lnitiall;+ ;he pu'dta5e tntent was lo•.v (28%:+ fer lhe di!ul .?::l t::rtd.c:l rico H:h\-a .. ·er a"tar infcrm1ng ccns'~Mers about fnrtifce.:on arv.! :ha add bona I ru;riticral value ;lur-ch a se if'ltent for fortified rice ncreased ro !:1% impl:,'ul-;; trill .:.ll<":as.t 'lr.:lf \"'If lhe ccrcMvrrer:~. were w !ling ro buJ u·e rice 16% at C'.OI1SLm~rs prefP.tre!1 cooked forufied ric:.e m1xed wilh while nee p3rt CIJta·l:: w1.:n Tent•cr.erJ aoou; fo"t1f1cU on Tne bY.J·a·:cessib il• c·f fcnified rmcronLttremi us1ng bolh tecnmques w~ro higher for for.Jicd p:roc l:::c nee : FA· ~-6% BC· 12%) ' clbwed by HPP ( FA· 4:3%; BC· 2%) comp;;u;d 10 the fo."tifi: ant solution \FA· 80%; BC· 0.1 %) contain:ng the :::.:r e ccr:~ntr:mon of 11H.:iems excep; fc· iron (l::u bo ed ri~· 3 1% HFP nee - 42% and fcrt ifJCallt· 38%;.. Fon1tca:;on •udn mulliple mr::ronulrie;ns us ng p<.;:bol ing :;rocEi'5S 'Showed t:e!lel uanosp:n< tFA· 33:% BC· 2::1% and F~:titin 26 nglml) af:er n ;•ilrc :19;!stion and w<:uld be eas.e1 f<tf irnp~e··r ~t·e :<~tic ·
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8. Acknowledgement
I would like to begin by thanking my primary Supervisor A/Prof. Jayashree Arcot for her
guidance during my PhD project. She has not only been my academic support but also my
emotional support whenever I needed her. She has always been very patient motivating and
enthusiastic and knowledgeable and also helped me relieve some financial burden by giving
me some teaching duties. I could not have asked for a better advisor and mentor for my PhD
study. I also would like to extend my gratitude to my co-supervisor Dr. Janet Paterson who
has added invaluable input for my work and also taken time to read my manuscripts and
provided feedback. I express my utmost gratitude to my supervisors who have helped shape
my PhD thesis and my professional life.
Besides my primary guides, I also worked in other Institutes and schools within UNSW who
have helped me with technical issues. My external supervisor Dr. Jagan Mohan from IICPT,
Thanjavur India, Dr. Judith Field, School of Biological Earth and Environmental Sciences,
UNSW, Dorothy Yu and Rabeya Akter Analytical Centre, UNSW, Dr. Andre Bongers from
BRIL- Lowy Cancer research centre, UNSW for assisting me in experimentation and
technical support
I also would like to appreciate the Technical staff from my school, Mr. Camillo Taraborrelli
who provided me lab consumables, Dr. Robert Chan for computer support, Dr. Victor Wong
for technical support for my chromatography issuesand Ms. Ik LingLau for administrative
support. They have been very patient that allowed a smooth run of experiments during my
PhD. I also would like to thank Dr. Karrie Kam who was my mentor during the initial stages
of my PhD who guided me and made me comfortable to initialize my work and to Dr. Maria
Veronica Chandra- Hioe who trained me on the cell culture work for my project. I also would
like to thanks Dr. Alice Lee and Dr. Patrick Spicer for helping me giving some teaching
opportunities to reduce my financial burden, Dr. Robert Driscoll, Dr. George Srzednicki, Dr.
Jian Zhao and Dr. Francisco Trujillo who have been on my review panel and guided my
project in the right direction. I also would like to thanks Siaw Wei who helped with my lab
work.
I am very grateful to have a wonderful bunch of friends and colleagues who made my PhD
journey colorful, provided insight and boosted me during my downtimes. My sincere thanks
to Xin Sun who has been a part of this journey since the beginning, Dr. Van Ho, Ghazaleh
Ghodsizad, Kitty Tang, Gib Uraipong, Na Wang and Dat Hyunh, Lydia and Yang Lu for
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sharing some light moments in the lab. Special thanks to Mr.Chellamariappan Manoharan for
his companionship and encouragement throughout my PhD study, Dr. Subathra Muthukumar
and Suganya Jeyaprakash for proof-reading my thesis writing.
This acknowledgement would be incomplete if I did not mention my father who has always
been my pillar of support for all these years. Words cannot express my gratitude towards him
for supporting me mentally and financially and made me achieve my goals. Without him and
my mothers‘ blessings I would never have been able to complete my PhD degree.
I sincerely thank everyone that I have mentioned once again for being a part of this long
journey and without you all this thesis would have been impossible. I would like to dedicate
this thesis to you all.
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9. ABSTRACT
Rice fortification with multiple micronutrients (folic acid, iron and β-carotene) by utilizing
two different processes: parboiling- an ancient process and High Pressure processing (HPP) –
a novel process has been examined in this thesis. The fortification process was addressed
through four vital phases: optimisation of parboiling process and use of HPP for fortification
with multiple micronutrients, physico-chemical properties of the fortified rice, consumer
acceptance study and bio-accessibility and transport studies.
Parboiling process was optimised for fortifying rice with three micronutrients using the
following condition: soaking the brown rice at 70°C for 2 hours with the fortificant
solution(FA- 150 mg; BC- 62.5 mg; Fe- 25 mg) followed by steaming at 100°C for 1 hour
and then air drying the rice at room temperature until the moisture content dropped to 10-
12%.Folic acid showed highest uptake during parboiling. After cooking folic acid was
retained the highest (98%) followed by iron (90%) and then β-carotene (35%).For HPP,
thebest condition for fortification was selected: treating milled white rice with the fortificant
solution at 200 MPa for 1 hour (non-thermal) and then air-drying the processed rice (10-12%
final moisture content). Folic acid showed highest uptake after HPP (41%) followed by β-
carotene and iron (20% each). Retention in cooked HPP rice followed the same trend as
parboiled rice- Folic acid (98%)> Iron (64%)>β-carotene (18%). Rice fortified by the
twotechniques was assessed for physico-chemical properties to understand changes in starch
during fortification. The process of adding nutrients did not have an impact on starch
properties in the fortified rice. Parboiled rice was completely gelatinised while HPP rice
showed partial gelatinization which affected hydrolysis of starch by α-amylase which in turn
influenced the release of vitamins from the rice matrix during extraction and in vitro
digestion.Fortified parboiled rice was diluted with white rice and assessed for colour
variation instrumentally in comparison to white rice, which showed no significant difference
betweenthem. The diluted fortified rice was presented to consumers for visual and taste
acceptance. Fortified diluted rice (uncooked) was not as acceptable as uncooked white rice
(coomercial) to consumers due to non-uniformity in appearance. Initially the purchase intent
was low (28%) for the diluted fortified rice. Howeverafter informing consumers about
fortification and the additional nutritional value, purchase intent for fortified rice increased to
51% implying that at least half of the consumers were willing to buy the rice. 76% of
consumers preferred cooked fortified rice mixed with white rice particularly when mentioned
about fortification. The bio-accessibility of fortified micronutrients using both techniques
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were higher for fortified parboiled rice ( FA- 96% BC- 12%) followed by HPP ( FA- 46%;
BC- 2%) compared to the fortificant solution (FA- 80%; BC- 0.1%) containing the same
concentration of nutrients except for iron (parboiled rice- 31%, HPP rice – 42% and
fortificant- 38%). Fortification with multiple micronutrients using parboiling process showed
better transport (FA- 33%; BC- 23% and Ferritin 26 ng/mL) after in vitro digestionand
would be easier for implementation.
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10. Table of Contents
1. ACKNOWLEDGEMENT .............................................................................................. II
2. ABSTRACT .................................................................................................................. VII
3. TABLE OF CONTENTS .............................................................................................. IX
LIST OF TABLES ............................................................................................................... XV
LIST OF FIGURES ........................................................................................................ XVIII
LIST OF ABBREVIATIONS ........................................................................................ XXIII
LIST OF PUBLICATIONS ............................................................................................ XXIV
CHAPTER 1 ............................................................................................................................. 1
1. INTRODUCTION ............................................................................................................ 1
1.1 BACKGROUND ................................................................................................................... 1
1.1.1 Vitamin A deficiency ................................................................................................ 1
1.1.2 Iron Deficiency ........................................................................................................ 1
1.1.3 Folic Acid Deficiency .............................................................................................. 2
1.1.4 Rice- Medium of fortification .................................................................................. 2
1.2 AIMS ............................................................................................................................ 5
CHAPTER 2 ............................................................................................................................. 6
2. LITERATURE REVIEW ................................................................................................ 6
2.1 RICE AS A MEDIUM FOR FORTIFICATION ....................................................................... 6
2.1.1 Nutritional properties of rice ................................................................................... 8
2.1.2 Properties of modified rice starch ........................................................................... 8
2.2 PARBOILING TECHNIQUE .............................................................................................. 9
2.2.1 History ..................................................................................................................... 9
2.2.2 Overview of the Process .......................................................................................... 9
2.2.2.1 Soaking ............................................................................................................. 9
2.2.2.2 Steaming/Heating ........................................................................................... 10
2.2.2.3 Drying............................................................................................................. 10
2.3 HIGH PRESSURE PROCESSING (HPP) .......................................................................... 11
2.4 CHOICE OF MICRONUTRIENTS FOR FORTIFICATION ..................................................... 13
2.4.1 Importance of Vitamin A........................................................................................ 13
2.4.2 Vitamin A in the diet .............................................................................................. 14
2.4.3 Importance of Folic Acid ....................................................................................... 15
2.4.4 Importance of Iron ................................................................................................. 17
2.5 FACTORS TO BE CONSIDERED FOR FOOD FORTIFICATION ............................................ 18
2.6 COMMERCIALLY AVAILABLE FORTIFIED RICE ............................................................. 19
2.7 MANDATORY FOOD FORTIFICATION PROGRAMS (FOLIC ACID) .................................. 23
2.7.1 Mandatory Fortification in Australia & New Zealand .......................................... 23
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2.8 NOVELTY IN RICE FORTIFICATION ............................................................................. 24
2.8.1 Parboiling Process ................................................................................................ 24
2.8.2 High pressure processing ...................................................................................... 25
2.8.3 Cost Analysis for parboiling and HPP processes ................................................. 26
2.9 STUDY OF MORPHOLOGICAL AND PHYSICO-CHEMICAL PROPERTIES OF RICE ............. 31
2.9.1 Pasting Properties Using RVA .............................................................................. 31
2.9.2 Polarized light microscope .................................................................................... 32
2.9.3 X-Ray Diffraction .................................................................................................. 35
2.9.3.1 Diffraction of X-Rays..................................................................................... 35
2.9.3.2 Bragg‘s Law ................................................................................................... 35
2.9.3.3 X-ray Diffraction in rice ................................................................................. 36
2.9.4 Magnetic Resonance Imaging ............................................................................... 37
2.10 CONSUMER ACCEPTANCE STUDY ............................................................................... 38
2.11 BIOAVAILABILITY STUDIES USING CACO-2 CELLS (IN VITRO) .................................... 39
2.11.1 Introduction to Cell Culture............................................................................... 39
2.11.2 Previous studies using Caco-2 cells .................................................................. 40
2.11.3 Bioavailability of Iron in Parboiled Fortified Rice ........................................... 41
2.11.4 Bioavailability of β-carotene in carrots ............................................................. 43
2.11.5 Bioavailability of Folic Acid in Fortified Milk Products ................................... 43
2.11.6 Micronutrient Interaction in vitro ...................................................................... 44
2.12 CONCLUSION .............................................................................................................. 44
CHAPTER 3 ........................................................................................................................... 46
3. OPTIMIZATION OF FORTIFICATION OF RICE WITH FOLIC ACID, IRON
AND Β-CAROTENE BY THE PARBOILING METHOD ............................................... 46
3.1. INTRODUCTION ........................................................................................................... 46
3.2. MATERIALS AND METHODS ........................................................................................ 47
3.2.1. Brown Rice Samples .......................................................................................... 47
3.2.2. Fortificants used for Parboiling ........................................................................ 47
3.2.3. Parboiling Procedure ........................................................................................ 47
3.2.4. Parboiling procedure for Scale-up Studies........................................................ 49
3.2.5. Milling of Parboiled rice ................................................................................... 49
3.2.6. Moisture content in fortified rice and mass of solids that leached out after
soaking 50
3.2.7. Cooking of parboiled rice .................................................................................. 51
3.2.8. Sample extraction and analysis for the micronutrients ..................................... 51
3.2.8.1. Analysis of Folic acid ..................................................................................... 51
3.2.8.2. Analysis of β-carotene .................................................................................... 52
3.2.8.3. Analysis of Iron .............................................................................................. 53
3.2.9. Calculation of % concentration of micronutrients in the soak water before and
after the 2 hours soaking: % uptake and % retention of micronutrients in rice after
parboiling and cooking respectively................................................................................. 54
3.2.10. Statistical Analysis ............................................................................................. 55
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3.3. RESULTS AND DISCUSSION ......................................................................................... 56
3.3.1. Optimization of the soaking step of parboiling .................................................. 56
3.3.2. Moisture content of parboiled rice .................................................................... 57
3.3.3. Leaching of solids during the soaking step of the parboiling process ............... 59
3.3.4. Concentration of Nutrients during the soaking stage of the parboiling process
61
3.3.4.1. Folic acid ........................................................................................................ 63
3.3.4.2. Vitamin A (β-carotene) .................................................................................. 64
3.3.4.3. Iron ................................................................................................................. 64
3.3.5. Uptake of micronutrients in the fortified rice and effectiveness of Parboiling as
a method of fortification ................................................................................................... 65
3.3.6. Loss of nutrients on milling................................................................................ 69
3.3.7. Retention of micronutrients on cooking the fortified parboiled rice ................. 71
3.3.8. Pilot scale studies of experimental condition in comparison to conventional
parboiling condition ......................................................................................................... 74
3.3.9. Micronutrients interaction affecting uptake of each other during the parboiling
process 76
3.3.10. Nutrient concentration in dried parboiled rice after milling ............................. 76
3.3.10.1. Folic acid ........................................................................................................ 76
3.3.10.2. Vitamin A (β-carotene) .................................................................................. 77
3.3.10.3. Iron ................................................................................................................. 78
3.3.11. Efficacy of parboiling as a means of fortification ............................................. 80
3.4. CONCLUSIONS ............................................................................................................ 82
CHAPTER 4 ........................................................................................................................... 84
4. FEASIBILITY OF FORTIFYING RICE WITH FOLIC ACID, IRON AND Β–
CAROTENE BY HIGH PRESSURE PROCESSING (HPP) METHOD ......................... 84
4.1 INTRODUCTION ........................................................................................................... 84
4.2 MATERIALS AND METHODS ....................................................................................... 85
4.2.1 Preparation of rice and fortificants mixture.......................................................... 85
4.2.2 High Pressure Treatment of rice samples ............................................................. 86
4.2.3 Analysis of rice moisture content and micronutrient concentration post HPP
treatment ........................................................................................................................... 87
4.2.3.1 Analysis of moisture content in HPP processed rice...................................... 87
4.2.3.2 Analysis of concentration of micronutrients post HPP treatment .................. 88
4.2.3.3 Calculation of % uptake and % retention of micronutrients in fortified rice . 88
4.2.3.4 Data Analysis ................................................................................................. 88
4.3 RESULTS AND DISCUSSIONS ....................................................................................... 88
4.3.1 Moisture analysis of HPP rice............................................................................... 88
4.3.2 Concentration of micronutrients in fortified rice using HPP ................................ 90
4.3.3 Statistical Analysis of micronutrient concentration .............................................. 91
4.3.4 Loss of micronutrients due to longer treatment times and cooking ...................... 92
4.3.5 Efficacy of high pressure processing as a means of rice fortification................... 95
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4.4 CONCLUSIONS ............................................................................................................ 96
CHAPTER 5 ........................................................................................................................... 98
5. PHYSICO-CHEMICAL PROPERTIES OF RICE FORTIFIED BY THE
PARBOILING AND HPP TECHNIQUES ......................................................................... 98
5.1. INTRODUCTION ........................................................................................................... 98
5.2. MATERIALS AND METHODS ....................................................................................... 99
5.2.1. Optimization of method for studying gelatinization of starch in rice ................ 99
5.2.2. Instrumental colorimetric evaluation of fortified rice ..................................... 101
5.2.3. Rapid Viscoanalyser (RVA) of fortified rice flour samples ............................. 102
5.2.4. X-ray diffraction (XRD) of rice flour fortified by parboiling and HPP ........... 103
5.2.5. Polarized light microscopy and Differential Interference Contrast (DIC)
Microscopic images of starch granules in the fortified rice ........................................... 104
5.2.6. Real-time Magnetic Resonance Imaging (MRI) of water migration in rice
during parboiling ............................................................................................................ 105
5.3. RESULTS AND DISCUSSIONS: .................................................................................... 106
5.3.1. Degree of Gelatinization of fortified rice samples ........................................... 106
5.3.2. Colorimetric analysis of fortified processed rice............................................. 108
5.3.2.1. Total colour difference for parboiled rice and HPP rice– against uncooked
commercial white rice (ControlRaw) and parboiled rice (ControlParboil)....................... 111
5.3.3. RVA pasting curves .......................................................................................... 113
5.3.4. X-ray Diffraction (XRD) of fortified rice flour samples showing crystalline
patterns 119
5.3.5. Microscopic images of starch granules processed by parboiling and HPP
treatment ......................................................................................................................... 123
5.3.6. Real-time Magnetic Resonance Imaging of rice during parboiling process ... 131
5.4. CONCLUSIONS .......................................................................................................... 135
CHAPTER 6 ......................................................................................................................... 137
6. CONSUMER ACCEPTANCE STUDY OF FORTIFIED PARBOILED RICE .... 137
6.1 INTRODUCTION ......................................................................................................... 137
6.2 MATERIALS AND METHODS ..................................................................................... 138
6.2.1 Preparation of fortified parboiled rice ................................................................ 138
6.2.2 Micronutrient analysis of the fortified rice.......................................................... 138
6.2.2 Sensory evaluation of parboiled fortified rice: An Overview .............................. 138
6.2.3 Study 1: Visual consumer acceptance of uncooked fortified parboiled rice ....... 140
6.2.3.1 Samples preparation ..................................................................................... 140
6.2.3.2 Survey Methodology .................................................................................... 140
6.2.4 Study 2: Sensory evaluation (tasting) of cooked fortified rice after mixing with
unfortified white rice ...................................................................................................... 141
6.2.4.1 Sample Preparation ...................................................................................... 141
6.2.4.2 Cooking method ........................................................................................... 141
6.2.4.3 Survey Methodology .................................................................................... 142
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6.2.5 Data Analysis ....................................................................................................... 142
6.2.5.1 Study 1: Visual Consumer acceptance of uncooked fortified parboiled rice
142
6.2.5.2 Study 2: Consumer acceptance of cooked fortified rice after mixing .......... 143
6.3 RESULTS AND DISCUSSION ....................................................................................... 143
6.3.1 Analysis of micronutrients in the fortified rice before and after dilution ............ 143
6.3.2 Study 1: Visual Consumer Acceptance of uncooked fortified parboiled rice ...... 146
6.3.2.1 Demographics and rice eating pattern of consumers ................................... 146
6.3.2.2 Degree of visual acceptance of fortified diluted uncooked rice ................... 148
6.3.2.3 Evaluation of purchase intent of uncooked diluted fortified rice with and
without nutritional information ................................................................................... 150
6.3.2.4 Evaluation of purchase intent of uncooked fortified rice- consumers who
were familiar with parboiled rice ................................................................................ 152
Study 2: Consumer acceptance of cooked fortified rice after mixing............................. 154
6.3.2.5 Consumer demographics and rice eating preferences .................................. 154
6.3.2.6 Degree of liking of cooked rice attributes .................................................... 154
6.3.2.7 Consumers‘ perception of attributes JAR (Just about right) ........................ 155
6.3.2.8 Evaluation of purchase intent of cooked rice ............................................... 158
6.4 CONCLUSIONS .......................................................................................................... 160
CHAPTER 7 ......................................................................................................................... 162
7. SHORT TERM RELATIVE BIO-ACCESSIBILITY AND ABSORPTION OF
FOLIC ACID, IRON AND Β – CAROTENE IN FORTIFIED PARBOILED AND
HIGH PRESSURE PROCESSED RICE: CACO-2 CELL STUDY ............................... 162
7.1 INTRODUCTION ......................................................................................................... 162
7.2 MATERIALS AND METHODS ..................................................................................... 163
7.2.1 Reagents for Cell Culture Resuscitation and Maintenance ................................. 163
7.2.2 Reagents for Cell Viability Assay ........................................................................ 163
7.2.3 Reagents for in vitro digestion ............................................................................. 164
7.2.4 Transport Study ................................................................................................... 164
7.2.5 Cell Culture & Maintainence Protocol ............................................................... 165
7.2.5.1 Procedure for resuscitation of frozen Caco-2 cells ...................................... 165
7.2.5.2 Sub-culturing of Caco-2 cells ....................................................................... 165
7.2.5.3 Cell Density .................................................................................................. 166
7.2.5.4 Freezing down Cells ..................................................................................... 167
7.2.6 Sample Preparation ............................................................................................. 167
7.2.6.1 In-Vitro Digestion Protocol .......................................................................... 167
7.2.6.2 Cell Viability Assessment ............................................................................ 168
7.2.7 Transport & Permeability Study.......................................................................... 169
7.2.7.1 Sample Preparation for Quantitative Analysis ............................................. 171
7.2.7.2 Folic acid analysis ........................................................................................ 171
7.2.7.3 β – carotene analysis .................................................................................... 172
7.2.7.4 Iron analysis ................................................................................................. 172
xiv
7.2.7.5 Calculations .................................................................................................. 173
7.3 RESULTS AND DISCUSSION ....................................................................................... 173
7.3.1 Cytotoxicity and cell viability .............................................................................. 173
Note: Mean absorbance values ±standard deviation of triplicate analysis ................... 174
7.3.2 In vitro-bio-accessibility of micronutrients from fortified rice ........................... 174
7.3.3 Transport Study ................................................................................................... 177
7.3.3.1 Folic acid ...................................................................................................... 177
7.3.3.2 β-carotene ..................................................................................................... 178
7.3.3.3 Iron ............................................................................................................... 179
7.3.4 Improvement in the nutritional status after consumption of fortified rice .......... 181
7.4 CONCLUSIONS: ......................................................................................................... 183
CHAPTER 8 ......................................................................................................................... 184
8. CONCLUSION AND FUTURE WORK .................................................................... 184
8.1 EFFICIENCY OF PARBOILING AS A TECHNOLOGY TO FORTIFY RICE: ........................... 184
8.1.1 Feasibility of rice fortification with multiple micronutrients using HPP technology
186
8.1.2 Physicochemical properties of the rice starch due to the two different processing
methods ........................................................................................................................... 187
8.1.3 Acceptability of the fortified parboiled rice by consumers .................................. 187
8.1.4 Bio-accessibility of the micronutrients from the fortified rice ............................ 188
8.1.4.1 Limitations of the in vitro bio-accessibility and transport of micronutrients
using caco-2 cells ........................................................................................................ 189
8.2 RECOMMENDATIONS FOR FUTURE WORK .................................................................. 189
CHAPTER 9 ......................................................................................................................... 191
9. REFERENCES ............................................................................................................. 191
10. SUPPLEMENTARY SECTION ............................................................................. 216
xv
List of Tables
Table 1.1 Macro and micronutrients contribution of rice based on % RDI in the developing
countries (Kenny, 2001)............................................................................................................. 4
Table 2.1 Proximate composition of paddy (rough rice) and its milling fractions at
14%moisture (Eggum. Juliano & Maniñgat, 1982; Pedersen & Eggum, 1983; Juliano, 1985b)
.................................................................................................................................................... 8
Table 2.2 Comparison of Programs and Quality of rice- and Micronutrient-Premixes
(USAID, 2008) ......................................................................................................................... 21
Table 2.3 The Levels of folic acid fortification in countries with mandatory fortification
programs .................................................................................................................................. 23
Table 2.4 Energy consumption in parboiling processes (Roy et al., 2005) ............................. 28
Table 2.5 Installation costs of the parboiling process (US$) (Roy et al., 2005) ...................... 28
Table 2.6 Theoretical Fortification Formulation for Comparison Purposes (USAID, 2008) .. 29
Table 2.7 Comparison of the technologies for premixes production by extrusion, coating and
dusting3 (USAID, 2008)........................................................................................................... 30
Table 2.8 Table Capital and production cost of thermal and pressure processes (Sampedro,
2014) ........................................................................................................................................ 31
Table 3.1 Concentration of micronutrient addition into rice for Parboiling (per 300g of rice)
.................................................................................................................................................. 47
Table 3.2 Micronutrient mass dissolved in 600 mL of soaking solution for the parboiling
process (mg/300g of rice) ........................................................................................................ 50
Table 3.3 Conditions of ICP-OES instrument for Iron analysis .............................................. 54
Table 3.4 % of initial concentrations of micronutrient in soak water before and after
parboiling process and % residual concentration of micronutrients after 120 min of soaking 61
Table 3.5 Percentage uptake of micronutrients after the parboiling process in uncooked rice
showing concentration of micronutrients at different milling times at conditions A, B, C and
D. .............................................................................................................................................. 67
Table 3.6 % retention of micronutrients after cooking fortified parboiled rice (milled at 120s)
(concentrations of the fortificant added in mg/ 300g of rice) .................................................. 73
Table 4.1 HPP treatment conditions used for the rice fortification experiment ...................... 87
Table 4.2 Moisture content of HPP treated rice on wet basis .................................................. 89
xvi
Table 4.3 Concentration of folic acid (in d.w.b) in high pressure processed rice (uncooked
and cooked) (200MPa) at varying time intervals µg/g of rice ................................................. 91
Table 4.4 β-carotene concentration (in d.w.b.) in high pressure processed (200 MPa) fortified
rice at varying time intervals in µg/g ....................................................................................... 91
Table 4.5 Concentration of Na-EDTA (in d.w.b.) iron in high pressure processed rice (200
MPa) at varying time intervals in µg/g of rice ......................................................................... 91
Table 4.6 % uptake (in uncooked rice) and % retention (in cooked rice) of micronutrients in
the fortified rice before and after cooking at various soaking times ........................................ 94
Table 4.7 Concentration of micronutrients consumed from the fortified rice based on RDI (&
NIH-ODS, 2014 & NHMRC (Australia), 2006) ...................................................................... 96
Table 5.1 The RVA program for analysing rice sample ........................................................ 103
Table 5.2 Scan parameters for XRD measurement ................................................................ 104
Table 5.3 Activation energies for gelatinization in Rough rice (paddy) and brown rice (Bakshi
& Singh, 1980) ....................................................................................................................... 108
Table 5.4 Color parameters L* (Lightness), a* (Redness) and b* (Yellowness) of fortified
rice milled at different milling times at various concentrations ............................................ 110
Table 5.5 Colour difference between fortified rice and commercial white rice (Controlraw) and
parboiled rice (Controlparboil) .................................................................................................. 111
Table 5.6 Colour parameters L* (Lightness), a* (Redness) and b* (Yellowness) of HPP
fortified rice at different pressure and time treatment ........................................................... 111
Table 5.7 RVA data for fortified rice sample processed by parboiling and HPP techniques 115
Table 5.8 % Crystallinity (extracted from XRD) of rice sample treated by high pressure
processing and parboiling ...................................................................................................... 122
Table 6.1 Dietary Reference Intake for the selected micronutrients (Vitamin A, folic acid and
Iron) (Dietary reference intake: Elements and Minerals, (NIH-ODS, 2014 & NHMRC, 2006))
................................................................................................................................................ 144
Table 6.2 Socio-demographic characteristics of the participants of consumer acceptance
Study 1 and Study 2 ............................................................................................................... 147
Table 6.3 Rice consumption pattern of participants of the consumer acceptance study 1 and
study 2 .................................................................................................................................... 148
Table 6.4 Mean perceptions of appearance for uncooked rice sample- diluted fortified rice
and p-values (p≤0.05) of t-test on comparing the diluted fortified rice sample with Control –
commercial white rice (Tested by consumers in the Consumer Acceptance Study (colour
intensity, degree of liking of colour, uniformity of colour, overall appearance)) ................. 153
xvii
Table 6.5 Mean degree of liking score for sensory attributes (appearance, colour, odour,
texture, taste, aftertaste and overall liking) of rice samples tested by consumers in Phase 2 of
the Consumer Acceptance Study (n=54) ............................................................................... 155
Table 7.1 Absorbance values for the digesta samples and controls in the cell proliferation
assay after 2 hours incubation ................................................................................................ 174
Table 7.2 Concentration of folic acid, iron and β-carotene (µmol) in fortificant solutions
(control), fortified parboiled rice and HPP rice ..................................................................... 175
Table 7.3 Concentration of folic acid, β-carotene and iron present in the apical chamber of the
transwell in µg/monolayer ..................................................................................................... 177
Table 8.1 Parboiling process cost analysis (Roy, Shimizu, Shiina and Kimura, 2005)......... 185
xviii
List of Figures
Figure 1.1 Global rice production and consumption (1960- 2012) (USDA, 2013) .................. 3
Figure 2.1 Longitudinal cross-section of rice kernel (Juliano, 1993) ........................................ 6
Figure 2.2 Schematic diagram of discontinuous equipment for High Pressure Processing of
packaged food products (Knorr, 1993). ................................................................................... 12
Figure 2.3 Structure of folic acid showing the pteridine ring and the para-aminobenzoic acid
(PABA) (Eitenmiller, 2008)..................................................................................................... 16
Figure 2.4 Flow chart of local parboiling process in Bangladesh and West Bengal, India.
(Roy et al., 2005) ..................................................................................................................... 27
Figure 2.5 Model of amylose molecule with 1000 glucose units (a) and Model of amylopectin
molecule with 1000 glucose units in 30 branches (b) (Nikuni, 1957) ..................................... 33
Figure 2.6 Microscopic images of corn starch granules under normal light (A) and polarized
light (B); where I is untreated and II is pressure treated at 500 MPa for 20 min (Hibi et al.,
1993) ........................................................................................................................................ 34
Figure 2.7 Microscopic images of potato starch granules under normal light (A) and under
polarised light (B) where I is untreated and II is pressure-treated at 500 MPa for 20 min (Hibi
et al., 1993) .............................................................................................................................. 35
Figure 2.8 Diffraction of X-rays by crystal (Skoog and Douglas, 1980) ................................ 36
Figure 2.9 Iron retention rate in rice (as % of the un-rinsed) after rinsing (simulating rice
washing) in the Fe-fortified parboiled rice grains milled for 60 and 120 s, respectively, in the
3 rice cultivars tested (Prom-u-thai et al. 2008) ...................................................................... 41
Figure 3.1 Schematic representation of optimized parboiling condition ................................. 48
Figure 3.2 Moisture content of rice with different fortificants during soaking at 30-min time
interval. BC: β-carotene; Fe: iron; FA: folic acid (The error bars represent standard errors of
corresponding means from three replicates) ............................................................................ 58
Figure 3.3 Moisture content of rice after 2 hours soaking and 1 hour of steaming at 100 °C
(The error bars represent standard errors of corresponding means from three replicates) ...... 59
Figure 3.4 Total solids in rice soaking solution before and after parboiling for the different
soaking solutions (The bars represent standard errors of corresponding means from three
replicates) ................................................................................................................................. 60
Figure 3.5 Mean folic acid %loss in concentration in the soak solution after soaking for 120
minutes with different treatments (The bars represent standard errors of corresponding means
from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) .............................................. 63
xix
Figure 3.6 Mean β-carotene % loss in concentration in the soak solution after soaking for
120 minutes with different treatments (The bars represent standard errors of corresponding
mean values from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) ......................... 64
Figure 3.7 Mean iron % loss in concentration in the soak solution after soaking for 120
minutes with different treatments (The bars represent standard errors of corresponding means
from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) .............................................. 65
Figure 3.8 Concentration of micronutrients (d.w.b) in uncooked rice fortified by parboiling at
varying concentrations and milling times (0s, 60s and 120s) (Error bars indicate the standard
deviations between duplicates (A, B, C and D refers to the mass of the micronutrients added))
.................................................................................................................................................. 66
Figure 3.9 Concentration of micronutrients in the fortified rice bran (Condition A) during
milling (Error bars indicate the standard deviations between duplicates) ............................... 70
Figure 3.10 Schematic representation of mass balance (on a d.w.b.) in terms of nutrients
concentration and loss at key stages of parboiling in rice soaked with all the micronutrient at
condition A (FA- 150mg; BC- 62.5 mg; Fe – 25mg/600mL soaking solution used on 300g
rice) .......................................................................................................................................... 70
Figure 3.11 Retention of micronutrients in cooked rice fortified by parboiling at varying
concentrations (Error bars indicate standard deviation between replicate samples; A, B, C and
D refers to the concentration of the micronutrients added to the soaking water) .................... 71
Figure 3.12 Comparison of micronutrient concentration (in d.w.b) in uncooked rice using
experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4 hours) on
pilot scale (Error bars indicate standard deviation of duplicate samples) ............................... 75
Figure 3.13 Comparison of micronutrient retention in (in d.w.b.) cooked rice using
experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4 hours) on
pilot scale (Error bars indicate standard deviation of duplicate samples) ............................... 75
Figure 3.14 Mean folic acid concentration (in d.w.b) in dried parboiled rice with different
fortifications before and after milling at 60 s and 120 s (The bars represent standard errors of
corresponding means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) ........... 77
Figure 3.15 Mean vitamin A (β-carotene) concentration in (d.w.b) dried parboiled rice (11%)
with different treatments before and after milling at 60 s and 120 s (The bars represent
standard errors of corresponding means from replicates. BC: β-carotene; Fe: iron; FA: folic
acid).......................................................................................................................................... 78
xx
Figure 3.16 Mean iron concentration in (d.w.b.) dried parboiled rice with different
fortifications before and after milling at 60 s and 120 s (The bars represent standard errors of
corresponding means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid) ........... 79
Figure 4.1 Flow chart of sample preparation for Hydrostatic High Pressure Processing
treatment .................................................................................................................................. 86
Figure 4.2 High pressure equipment used for the experiment ................................................. 87
Figure 4.3 % Moisture content of rice on a wet basis at different treatment times and
pressures used .......................................................................................................................... 90
Figure 4.7 Activity of α-amylase by testing glucose release from processed (fortified) and
unprocessed rice samples (Error bars represent standard error in replicates) .......................... 92
Figure 5.1 Optimization of KOH molarity to be added to study the degree of rice
gelatinization .......................................................................................................................... 100
Figure 5.2 % Gelatinization comparison between parboiled rice and HPP processed rice.
Untreated brown rice and white rice were used as controls (Error bars represent standard
errors in replicate samples) .................................................................................................... 106
Figure 5.3 RVA graph for rice treated at 200 MPa at different time intervals using high
pressure process ..................................................................................................................... 116
Figure 5.4 RVA graph for rice treated at 400 MPa at different time intervals using high
pressure process ..................................................................................................................... 117
Figure 5.5 RVA graph for rice treated at 600 MPa at different time intervals using high
pressure processing ................................................................................................................ 117
Figure 5.6 RVA graph for untreated rice and parboiled rice ................................................. 118
Figure 5.7 XRD pattern for parboiled and commercial white, brown and parboiled rice ..... 120
Figure 5.8 XRD pattern for HPP treated rice at 200 MPa for 1,2 and 3h .............................. 120
Figure 5.9 XRD pattern for HPP treated rice at 400 MPa for 1,2 and 3h .............................. 121
Figure 5.10 XRD pattern for HPP treated rice at 600 MPa for 1, 2 and 3h .......................... 121
Figure 5.11 a & b: Starch images for rice treated at 200 MPa for 1 hour (a. under differential
interference contrast; b. under cross-polarized light) ............................................................ 124
Figure 5.12 a & b: Starch images for rice treated at 200 MPa for 2 hours (a. under differential
interference contrast; b. under cross-polarized light) ............................................................ 124
Figure 5.13 a & b: Starch images for rice treated at 200 MPa for 3 hours (a. under differential
interference contrast; b. under cross-polarized light) ............................................................ 124
Figure 5.14 a & b: Starch images for rice treated at 400 MPa for 1 hour (a. under differential
interference contrast; b. under cross-polarized light) ............................................................ 125
xxi
Figure 5.15 a & b: Starch images for rice treated at 400 MPa for 2 hours (a. under cross-
polarized light under; b. differential interference contrast) ................................................... 125
Figure 5.16 a & b: Starch images for rice treated at 400 MPa for 3 hours (a. under
differential interference contrast; b. under cross-polarized light) .......................................... 125
Figure 5.17 a & b: Starch images for rice treated at 600 MPa for 1 hour (a. under differential
interference contrast; b. under cross-polarized light) ............................................................ 126
Figure 5.18 a & b: Damaged starch images for rice treated at 600 MPa for 2 hours (a. under
differential interference contrast; b. under cross-polarized light) .......................................... 126
Figure 5.19 a & b: Healthy starch grain images for rice treated at 600 MPa for 2 hours (a.
under differential interference contrast; b. under cross-polarized light) ................................ 126
Figure 5.20 a & b: Partially integrated starch Starch images for rice treated at 600 MPa for 3
hours (a. under cross-polarized light under; b. differential interference contrast) ................. 127
Figure 5.21 Figure 5.17 a & b: Completely damaged starch Starch images for rice treated at
600 MPa for 3 hours (a. under cross-polarized light under; b. differential interference
contrast).................................................................................................................................. 127
Figure 5.22 a & b: Starch images for parboiled rice milled at 0s (a. under differential
interference contrast; b. under cross-polarized light) ............................................................ 127
Figure 5.23 a & b: Starch images for parboiled rice milled at 60s (a. under differential
interference contrast; b. under cross-polarized light) ............................................................ 128
Figure 5.24 a & b: Starch images from in-house parboiled rice milled at 120s a. shows
birefringence in some starch granules under DIC while in b. it can be seen that there has been
a damaged starch granule ....................................................................................................... 128
Figure 5.25 a & b: Starch images from comemrical parboiled rice showing birefriengence (a.
under DIC and b. cross polarized light) ................................................................................. 128
Figure 5.26 a & b: Starch images from untreated rice showing birefriengence under a. DIC
and b. cross polarized light .................................................................................................... 129
Figure 5.27 Coronal section images of rice after parboiling process at different time intervals
at the optimized condition ...................................................................................................... 131
Figure 5.28 Sagittal section of soaked rice at different time intervals during the optimized
parboiling conditions ............................................................................................................. 132
Figure 5.29 a&b: Migration of water with and without micronutrients where A- is with Fe
and B – is without any micronutrients ................................................................................... 132
Figure 6.1 Schematic representation of fortified parboiled rice prepared for Consumer
Acceptance Study 1 and Study- 2 .......................................................................................... 139
xxii
Figure 6.2 Concentration of micronutrients (in d.w.b.) in fortified premix (cooked and
uncooked) and diluted fortified rice (cooked and uncooked) ................................................ 145
Figure 6.3 Preference of visual attributes investigated in Study 1 tested by participants (n=49)
presented on a hedonic scale .................................................................................................. 149
Figure 6.4 The distribution of purchase intent responses (%) in Consumer Acceptance Study -
STUDY 1 tested by participants (n=49). A) Before the notification of health claim; B) After
the notification of health claim .............................................................................................. 152
Figure 6.5 Bar graph showing consumer preference of cooked fortified rice compared with
unfortified rice ....................................................................................................................... 157
Figure 6.6 (a & b) Graph showing purchase intent of fortified rice before and after
mentioning about fortification ............................................................................................... 159
Figure 7.1 Schematic representation of Transwell permeable support with Caco-2 cells grown
on the membrane .................................................................................................................... 165
Figure 7.2 Diagrammatic representation of the chamber of a haemocytometer (Louis &
Siegel, 2011) .......................................................................................................................... 167
Figure 7.3 Confocal microscopy image of caco-2 cells showing tight junctions (Chandra-Hioe
et al., 2013) ............................................................................................................................ 175
xxiii
List of Abbreviations
Acronym Abbreviation
AM Amylose
ANN Annealing
ANOVA Analysis of Variance
AP Amylopectin
BC β-carotene
BR Brown Rice
d.w.b. Dry weight basis
DIC Differential Interference Contrast
DSC Differential Scanning Calorimeter
FA Folic acid
Fe Iron
HMT Heat and Moistutre treatment
HPP High Pressure Processing
IDA Iron Deficiency Anemia
IICPT Indian Institute of Crop Processing
Technology
JAR Just About Right
MPa Mega Pascals
MRI Magnetic Resonance Imaging
NTD Neural Tube Defects
RDI Recommended Dietary Intake
RVA Rapid ViscoAnalyser
TEER Trans Epithelial Electrical Resistance
VAD Vitamin A Deficiency
w.b. Wet basis
XRD X-Ray Diffraction
xxiv
List of Publications
Journal Articles
Chapter 3
Thiruselvam N, Cheong SW, Mohan J, Paterson J, Arcot J (2014) Micronutrients
Fortification of Rice by Parboiling: Lab Scale and Pilot Scale Studies. J Nutr Food Sci
4: 281,accepted and in press doi: 10.4172/2155-9600.1000281
Thiruselvam N, Mohan J, Paterson J, Arcot J Micronutrient fortification of Rice using
Parboiling: Scale-up Studies‖ at the 3rd International Conference of Food Technology
held at IICPT, Thanjavur, India during January 2013 and the paper was published in
the conference journal.
Conference Presentations
Oral presentation on ―Bio-accessibility of micronutrients from fortified rice using
caco-2 cell culture model‖ held at Washington DC, USA during May 12-15th
, 2014.
Oral Presentation on ―Physicochemical properties and sensory evaluation of fortified
rice‖ at the AIFST summer school held at The University of Queensland during 5-7th
Feb, 2014.
Poster Presentation on ―Folate stability in fortified rice in the presence of an
antioxidantduring parboiling process‖at the International Food Data Conference 2013
held at Granada Spain
Oral presentation on ―Multiple Micronutrient Fortification of Rice: What are the
Challenges?‖ at the 46th
AIFST Annual Convention during 14-16th
July, 2013.
Oral presentation on ―Micronutrient fortification of rice using Parboiling Technique‖
at the AIFST summer school held at The University of NSW during 6-8th Feb, 2013.
Oral presentation on ―A Comprehensive Approach to fortification of Rice with
Micronutrient using Parboiling‖ at the 2nd International Vitamin Conference held at
Copenhagen, Denmark during May 2012.
1
CHAPTER 1
1. INTRODUCTION
1.1 Background
Food fortification has been defined as the addition of one or more essential nutrients to a
food, whether or not it is normally contained in the food, for the purpose of preventing or
correcting a demonstrated deficiency of one or more nutrients in the population or specific
population groups (FAO/WHO 1994). In today‘s rapidly changing lifestyles populations have
become dependent on processed foods to attain nutritional adequacy and hence fortifying
foods with the aid (or during) of processing would be justifiable(LaChance and Bauernfiend,
1991; Hoffpauer and Wright, 1994).Micronutrient Malnutrition (MNM) is not spread
worldwide and not just confined to developing countries but also prevalent in industrialized
countries. Globally, the three most common forms of MNM are iron, vitamin A and iodine.
In the light of public health, MNM not only affects a vast population but is also a potential
risk factor for many diseases, and is the cause of high rates of morbidity and mortality
(Micronutrient Initiative and UNICEF, 2011). This chapter will briefly introduce the project
by explaining the importance of the micronutrients chosen for fortification, the medium of
fortification – rice and the aims of the study.
1.1.1 Vitamin A deficiency
Micronutrient deficiencies are common in developing countries such as Asia, Africa and the
Pacific. Vitamin A deficiency causes diseases such as xerophthalmia, Bitot‘s spot,
conjunctival xerosis, corneal lesion, xerosis, ulcer, scars and keratomalacia all of which can
lead to poor vision or total blindness if left untreated (WHO/UNICEF, 1996). For example,
during the year 2000, in India 5,049,139 people were affected by vitamin A deficiency out of
a total population of 841,523,272 (Food and Agricultural Organization of the United States,
1988).
1.1.2 Iron Deficiency
About one third of the world population suffers from iron deficiency anaemia (IDA)for
example India continues to be one of the countries with a high prevalence. National Health
Family Survey (NHFS, in 1998-1999) revealed that about 70-80% of children, 70% of
pregnant women and 24% of adult men were suffering from IDA (National Family Health
Survey, 2006).
2
1.1.3 Folic Acid Deficiency
Folic acid is one of the important B-complex vitamins essential for biosynthesis of DNA, and
it cannot be synthesized by the body and therefore needs to be obtained from dietary sources.
Folic acid deficiency leads to cardiovascular diseases, major depression, schizophrenia,
Alzheimer‘s disease, and some carcinomas such as colorectal, uterine, cervical, lung and
oesophageal (Gregory, 1996). Various studies and randomized trials over the last three
decades have shown that adequate intake of folate during early pregnancy reduces the risk of
abnormalities in early embryonic brain development and specifically the risk of
malformations of the embryonic brain/spinal cord, collectively referred to as NTDs (Czeizel
& Dudas, 1992; Cuskelly et al., 1996; Scholl & Johnson 2000;Kauwell et al., 2002&Öner et
al., 2006). Fortification of wheat flour to overcome folic acid deficiency diseases has proven
to be effective in many developed countries. Fortification of cereal grain products with folic
acid (100 to 150 µg/ day) resulted in a decrease in NTDs in the United States and Canada
(Bailey et al., 2001 and Berry et al., 2010). Pachón.et al. (2013) state that with the
widespread consumption of wheat flour-based products and advances in technology in the
milling industry, folic acid fortification should be adapted as a public health initiative in
Europe (Pachón et al., 2013). In Australia, two per thousand live births are affected by NTDs
attributed to folic acid deficiency. As a result, fortification of bread making flour with folic
acid ismandatory in Australia since September 2006 (Wesley & Dutta, 2009).
1.1.4 Rice- Medium of fortification
Figure 1.1 shows the growth in global rice production and consumption during the year 1960-
2012. Except for the decline in production during the years 2000 to 2003, there has been a
steady increase in rice production. There is also a consistent increase in rice consumption.
3
Figure 1.1 Global rice production and consumption (1960- 2012) (USDA, 2013)
Table 1.1 shows that rice provides a significant contribution of macro and micronutrients in
the diets of people from developing countries where rice is a staple food. Although rice seems
to contribute moderate suppliesof protein, lipids and other vitamin such as thiamin, niacin
and riboflavin and minerals such as iron, zinc and calcium, it is unable to provide for normal
requirements of humans. Yi et al., (2011) in a review showed that due to NTDs the economic
burden in terms of direct (e.g. drugs, hospitalizations and managing co-morbidities) and
indirect (e.g. loss of work time and costs due to premature loss of life) and caretakers‘ time
was significant. In another study conducted by Stein and Qaim in 2007, it was reported that in
India, due to deficiencies caused by the micronutrients vitamin A and iron, there was a loss of
2.5% to the Indian GDP. Therefore micronutrient deficiencies not only affect the
populations‘health but also an entire nation‘s economy. Hence, an economically beneficial
option of rice fortification with folic acid, iron and vitamin A could be a potential method to
address this public health issue.
4
Table 1.1 Macro and micronutrients contribution of rice based on % RDI in the developing countries (Kenny, 2001)
Consumption
(g/day)
Energy Protein Fat Calcium Iron Thiamine Riboflavin Niacin Zinc
(% of Recommended Daily Intake)
Bangladesh 441 76 66 18 3 8 18 14 25 30
China 251 30 20 17 2 4 10 8 17 17
India 208 31 24 4 1 4 8 6 4 15
Indonesia 414 51 43 8 3 7 17 13 8 29
Myanmar 578 74 68 20 4 10 23 17 20 40
Philippines 267 41 30 5 2 5 10 8 5 17
Sri Lanka 255 38 37 3 2 5 10 8 3 17
Thailand 285 43 33 5 2 5 12 9 5 21
Vietnam 465 67 58 14 3 8 19 14 14 34
5
Parboiling has been used as a successful fortification method in recent times where Prom-u-
thai (2008 & 2010) has utilized this method to fortify rice with iron and zinc individually.
Tyythan et al., (2007) and Kam et al., (2012) fortified rice with iodine and folic acid
respectively using the parboiling technique. Based on the results from the above studies it is
evident that through the parboiling process penetration of minerals and vitamin is significant
from the fortificant solution and the nutrients are able to penetrate through the outer layers
into the endosperm of the rice grain.
A comprehensive fortification program to combat all three deficiencies (folic acid, iron and
β-carotene) will be an ideal approach for those populations affected by all three deficiencies
as often encountered in developing countries. This study attempted to fortify rice with folic
acid, iron and β-carotene using two fortification techniques: Parboiling- an ancient technique
andhigh pressure processing (HPP)-a novel technique despite the difference in processing
properties for the first time.
1.2 Aims
1. To study the feasibility of fortifying rice with 3 micronutrients using parboiling;
performing pilot scale studies for the parboiling process; analysing the retention of
micronutrients after milling and cooking the rice; understanding the efficiency of
micronutrients uptake in the grain using the parboiling technique- Chapter 3.
2. To study the feasibility of fortifying rice with 3 micronutrients using HPP technique; loss
during cooking- Chapter 4.
3. To study the physico-chemical properties of fortified rice by using the Rapid
Viscoanalyser (RVA), microscopy of rice starch, changes in rice crystalline properties of rice
using X-ray diffraction (XRD) and migration of micronutrients using magnetic resonance
imaging -(MRI) Chapter 5.
4. To evaluate consumer acceptance of fortified rice using sensory analysis- Chapter 6.
5. Investigate the short-term relative bio-accessibility of the fortified micronutrients in the
parboiled and HPP rice using in vitro cell culture technique (Caco-2 cells)- Chapter 7.
6
CHAPTER 2
2. LITERATURE REVIEW
2.1 Rice as a medium for fortification
Rice (Oryza sativa L.) comes from the family Poaceae and is harvested as paddy rice, or
―spikelets‖ in botanical terms, where each spikelet consists of caryopsis, four bracts, and a
rachilla (Champagne et al., 2004). The major parts of rice grain are hull, pericarp, testa (seed
coat), embryo (germ) and endosperm (Champagne et al., 2004). The outer layer, called hull
consists of lemma and palea, where they differ in sizes, shapes, and numbers of vascular
bundles (Champagne et al., 2004). Brown rice consists of pericarp, testa and nucellus,
embryo (germ) and endosperm (Juliano, 1993). The pericarp runs along the embryo, dorsal
and the two lateral sides of the grain; testa lies beneath the pericarp; the embryo is located on
the ventral side of the grain; the endosperm consists of the aleurone and starchy endosperm,
where the latter constitutes the majority of the rice grain (Champagne et al., 2004).
Figure 2.1 Longitudinal cross-section of rice kernel (Juliano, 1993)
Rice is the staple food in many Asian countries like India, Myanmar, Bangladesh, Nepal,
Indonesia, Philippines, Sri Lanka and Vietnam where micronutrient deficiency diseases due
to vitamin A, iodine and iron is also common (Dexter , 1998). Rice accounts for nearly 22 %
7
of the world‘s energy intake (Bierlen et al. 1997). According to UNICEF (2009), it is noted
that most of the rice-consuming countries suffer from a higher incidence of low birth weight,
infant mortality and mortality under five. Vitamin A deficiency and nutritional anemia from
iron deficiency are also widespread in rice-consuming countries; vitamin A deficiency is
common particularly in South and East Asian countries such as Bangladesh, India, Indonesia,
Myanmar, Nepal, the Philippines, Sri Lanka and Vietnam (Juliano, 1993). Considering the
role of rice in the diets of these populations it is sensible to fortify rice to adapt a food-based
approach to prevent and control micronutrient deficiencies. Other criteria would be that the
nutrients should be stable under normal conditions of storage. It should also be well absorbed
and mixed with the appropriate vehicle and aesthetically acceptable (Rao, 1981). Therefore,
rice would be a good choice to fortify with Vitamin A, Folic acid and Iron as a
comprehensive fortification strategy as rice is consumed by large populations in India.
In the table below it can be seen that carbohydrates form the highest composition of nutrients
in rice. Milled rice has more carbohydrates compared to brown rice (Barber, 1972). Starch is
the major component of carbohydrate in rice- therefore it is an important determinant of
flavour and quality in rice. The two main polymers in rice starch granules are amylose (AM)
and amylopectin (AP). Other minor constituents associated with these are proteins namely
glutelin, prolamin, globulin and albumin in the rice endosperm (Singh et al., 2000). The
morphology of starch granules depends on the biochemistry of the chloroplast or amyloplast
in conjunction with the physiology of the plant (Badenhuizen, 1969). Rice starch granules are
small polygonal and angular-shaped (Singh et al., 2003) ranging between 3-10 µm (Ellis at
al., 1998) and exhibit unimodal distribution (Dang and Copeland 2004).
8
Table 2.1 Proximate composition of paddy (rough rice) and its milling fractions at
14%moisture (Eggum. Juliano & Maniñgat, 1982; Pedersen & Eggum, 1983;Juliano,
1985b)
Rice
fraction
Crude
protein
(g N x 5.
95)
Crude
fat (g)
Crude
fibre
(g)
Crude
ash (g)
Available
carbohydrates
(g)
Neutral
detergent
fibre (g)
Energy
content
Density
(g/ml)
Bulk
density
(g/ml) (kJ) (kcal)
Rough
rice
5.8-7.7 1.5-2.3 7.2-
10.4
2.9-5.2 64-73 16.4-19.2 1580 378 1.17-
1.23
0.56-
0.64
Brown
rice
7.1-8.3 1.6-2.8 0.6-1.0 1.0-1.5 73-87 2.9-3.9 1520-
1 610
363-
385
1.31 0.68
Milled
rice
6.3-7.1 0.3-0.5 0.2-0.5 0.3-0.8 77-89 0.7-2.3 1460-
1 560
349-
373
1.44-
1.46
0.78-
0.85
Rice bran 11.3-
14.9
15.0-
19.7
7.0-
11.4
6.6-9.9 34-62 24-29 670-1
990
399-
476
1.16-
1.29
0.20-
0.40
Rice hull 2.0-2.8 0.3-0.8 34.5-
45.9
13.2-
21.0
22-34 66-74 1110-
1 390
265-
332
0.67-
0.74
0.10-0.1
2.1.1 Nutritional propertiesof rice
Rice is rich in energy and a good source of protein; contains reasonable concentrations of
thiamine, riboflavin, niacin and vitamin E in the bran layer (Juliano, 1993). Normally, the
level of the vitamin is 2-10 times higher in brown rice than in milled rice (Champagne et al.,
2004). In contrast, the rice grain does not contain any vitamin C, D or A (Juliano, 1993). To
produce milled rice, bran layers, polish (sub-aleurone), germ (embryo) and a small part of the
endosperm are removed. After milling, rice results in loss of nutrients, while starch content
(90% dry weight) is higher in milled rice than in brown rice (Champagne et al., 2004).
2.1.2 Properties of modified rice starch
Starch gelatinization is an important aspect in food modification and it is applicable in
cooking, baking and extruding starch-based foods (Zobel, Young and Rocca, 1988).
Gelatinization is a complex phenomenon that obeys first order kinetics (Riva , Piazza &
Schiraldi , 1991) and alters the physical, chemical and nutritional properties of starch. It also
affects heat diffusivity, viscosity, rheological behaviour, swelling and deformation of original
shape of starchy products and susceptibility to enzymatic digestion (Tsai Li & Lii 1997).
Gelatinization is a dynamic process which disrupts the crystalline and molecular order within
the starch granule (Yifang , Yongzhong , Qifa , Mei & Harold , 2001).
9
The swelling property of starch can be attributed to AP (Tester & Morrison, 1990) where AM
tries to restrict this (Park et al., 2007; Patindol et al., 2007). The variation in the unit chain
length of the AP influences swelling and pasting properties. Presence of lipids and other
morphological structures also affect the solubility and swelling power of starch granules
(Gaillard & Bowler 1987; Singh et al., 2003). The changes that occur to rice starch molecules
upon gelatinization can be studied based on pasting properties. It involves further swelling of
granules, leaching of molecular components from the granules and eventual disruption with
the application of shear force.
2.2 Parboiling Technique
2.2.1 History
Parboiled rice is predicted to have originated in ancient India but it is unknown how and why
it was exactly started (Bhattacharya, 2006). However, parboiled rice reduces the breakage of
rice during milling (Bhattacharya, 1969) and is favoured by South Asians due to its‘ hardened
texture after parboiling (Bhattacharya, 2006). In early 20th
century, parboiled rice gained a lot
of importance as it was seen to preventberiberi (Bhattacharya, 2006). At present, parboiled
rice is popular among the low-income people of Bangladesh, India, Nepal, Pakistan, Sri
Lanka, and parts of West Africa and contributes to one-fifth of the world‘s rice consumed
(Bhattacharya, 1985). Parboiling is desirable not only because of improved nutritional value
but because of improved cooking and processing properties which are desirable from an
industrial point of view (Bhattacharya, 2006).
2.2.2 Overview of theProcess
2.2.2.1 Soaking
The first step in parboiling is soaking, where the paddy is hydrated by soaking in water to
allow gelatinization on subsequent heating (Juliano, 1993). At low temperatures, the paddy
does not absorb water quickly and reaches equilibrium of ~30% moisture content (wet basis)
(Bhattacharya, 2006). Unlike at low temperatures, water absorption of the paddy at high
temperatures increase exponentially after an initial lag period due to starch gelatinization
(Bhattacharya, 2006). Consequently, the ‗swollen‘ starch leaks out of the grain and the grain
deforms as a result of exceeding ~30-32% moisture (Bhattacharya, 2006). Therefore, two
important factors that affect soaking are temperature and time. To achieve practical soaking,
rice is soaked at ~70°C for a certain period of time depending on the gelatinization
temperature (GT) of the variety of rice (Bhattacharya, 2006). According to Biswa and Juliano
10
(1988), it is found that low-GT varieties require soaking temperature of less than 70°C to
avoid over-hydration.
2.2.2.2 Steaming/Heating
Steaming aims to gelatinize the starch of the soaked paddy (Juliano, 1993). As described by
Ayamdoo et al. (2013), covering the rice during steaming creates pressure that aids in inward
movement of water molecules into each rice kernel. According to Wimberly (1983),
gelatinization of starch causes certain changes such as creating translucency and makes the
grain hard, as well as resistant to breakage during milling. Therefore, it results in easier
removal of the husk during milling but more difficult bran removal. Other physiochemical
changes include starch retrogradation, pigment transformations and enzyme deactivation,
where these changes improve flavour and cooking characteristics of the rice (Ayamdoo et al.,
2013).
2.2.2.3 Drying
Drying of steamed paddy takes place at an airy place. During the first stage, moisture
reduction occurs quickly, dropping from 36 to 18% followed by tempering before proceeding
with drying to 14% (Wimberly, 1983). Throughout the process, solute concentrates in the rice
kernel and the gelatinous amylose starch are compressed, where the latter results in easier
husk milling (Ayamdoo et al., 2013).
After parboiling, gelatinized starch and protein bodies occupy the empty spaces in the rice
endosperm thereby giving better consistency and hardness to rice kernel. Therefore, the
kernel becomes translucent and offers great resistance to breakage during milling. Parboiled
rice takes longer time to cook than normal rice and also the color is slightly yellowish to
brown. During parboiling, dextrinisation and destruction of lipase enzyme can occur.
Diffusion and heat-sealing of B-vitamin and other water soluble nutrients to the endosperm
also occur thereby improving the nutritive value of rice. However, heat treatment of rice can
cause destruction of some anti-oxidants resulting in rancidity of parboiled rice during long-
term storage (Nawab & Pandya, 1974).
In a study conducted by Chukwu & Oseh in (2009),the effect on nutritional values of rice
(Oryza sativa) at different parboiling temperatures was analysed. Standard laboratory
conditions, instruments and methods of AOAC (Association of Official Analytical Chemists)
nutritional guidelines were used to obtain the proximate composition of non-parboiled rice
11
and rice that was steam-parboiled at 80, 100 and 120ºC. The results showed that parboiling
leads to variation in the nutritional contents of rice as demonstrated for protein content which
shows a decrease from 6.61% to 5.29% after the parboiling. The results obtained for vitamins
A and C also showed a decrease in values after parboiling at different temperatures of 80, 100
and 120ºC. It was concluded that parboiling rice adversely affects some of the nutritional
content of the product. The moisture content of the rice decreased with gradual increase in
temperature (Chukwu & Oseh, 2009). Parboiled rice is mainly consumed in parts of South
Asia especially, India, Bangladesh and some parts of Pakistan. About 60% of total rice
production in India is through parboiling (Pillayar, 1990).
Traditional drying of parboiled rice involves sun drying. Paddy is laid out on a cemented
floor in a 10-30 mm thick layer. It is continuously stirred with a spiked plank or by feet. After
certain duration the rice is heaped and covered by mats and this process is called tempering.
After tempering for 2-3 hours, the paddy is spread out again and dried for 2 days in sunshine
until the desirable moisture content is reached. The parboiling process incorporates 30 % of
moisture in the rice and this has to be reduced to 13% by the drying process which usually
takes 6 hours in Indian summer and 8-10 hours during the winters in India. Mechanical
dryers, bag dryers and batch dryers, continuous flow dryers, rotating drum dryers, and
modern paddy dryers are the commonly used dryers (Juliano, 1993).
2.3 High Pressure Processing(HPP)
High pressure processing (HPP) is a novel technology which involves treating a product
under a pressure of 100 MPa and above. The transmission of pressure is uniform and
instantaneous and this results in the product being homogenous as well. Conventionally it is
used for the production of ceramics, steels, superalloys and synthetic metals. This technology
gained popularity in the early eighties in biological and food systems. Hite (1899 )tested the
high pressure system for improving milk stability. But it was 70 years later that the first
pressurized jam product was marketed in Japan in 1993 (Konrr 1993; Mertens 1993).
The unit of pressure for high pressure processing is Megapascal (MPa). 1 MPa is equal to
9.869 atm which is equal to 10 bar. Pressure can alter the conformation of secondary, tertiary,
quaternary and supramolecular structures of nucleic acids, polysaccharides and proteins
(Cheftel and Cuiliolo, 1997).
12
Figure 2.2 Schematic diagram of discontinuous equipment for High Pressure Processing
of packaged food products (Knorr, 1993).
The principle of high pressure processing is isostatic transmission of pressure throughout the
food. This prevents deformation of the food products under high pressure and at the same
time the pressure is transmitted instantaneously. This allows homogenous processing
independent of the product size and shape. Another added advantage of this method is that it
takes energy to reach the desired pressure but once the pressure is reached, there is no energy
loss. These properties of high pressure give added advantages in contrast to heat processing.
Pressurization can also be done at low temperatures such as room temperature, refrigeration
and freezing temperature and by doing so the nutrient content and sensory properties can be
preserved. High pressures influence weak interaction bonding in foods and can keep the
covalent bond of primary macromolecules intact (Knorr 1993; Mertens 1993; Heremans
1995).
The most common application of high pressure processing is enhancing food safety by the
killing of microorganisms. High pressure processing can eliminate bacteria and bacterial
spores. When the pressure is between 400 to 600 MPa, vegetative cells can be killed with
temperatures ranging from 5 to 90°C for 10-30 min (Barbosa-Canovas et al., 1998).
Therefore products with good microbiological quality and greater safety and longer shelf life
can be produced. Novel products can be generated by pressurizing food ingredients and
modifying their functionality. This has an effect on the physical properties of the food and
13
induces textural changes different from usual ones. High pressure technology is relatively
new in food science and can be explored more in the nutritive, microbiological, physico-
chemical and sensory aspects.
2.4 Choice of micronutrients for fortification
2.4.1 Importance of Vitamin A
Vitamin A includes pre-formed structures (retinol) and oxidized forms of retinols (retinoic
acid), where they are needed to maintain a healthy vision (Thurnham, 2007). Vitamin A
deficiency(VAD) causes xerophthalmia (dry eyes), which eventually leads to night blindness,
increased morbidity and mortality in children (Sommer and West, 1996). India is one of the
developing countries in which VAD is very common and needs most attention (Food and
Agricultural Organisation of the United Nations, 1988). Serum retinol analysis was done for
children between 3-5 years in the year 2000 and it was found that southern Tamil Nadu
accounted for about 42% of VAD when compared with other states in India. In order to
reduce the prevalence of VAD in India, supplementation and fortification programs arenow
being carried out for young children and lactating women. However, no food fortification
programs have been implemented other than some health drinks (UNICEF, Multiple Indicator
Cluster Survey, 2000).
WHO reported that between 1987 and 1995, vitamin A deficiency increased in prevalence
from 39 countries to 60 countries based on the ocular manifestations of xerophathlmia or
deficient serum (plasma) retinol concentrations (<0.35 µmol/l) (WHO, 2009). In addition, it
indicated that night blindness and biochemical vitamin A deficiency (serum retinol
concentration <0.70 µmol/l) have affected a staggering amount of preschool children, as well
as pregnant women. Globally, night blindness is estimated to affect 5.2 million preschool
children and 9.8 million pregnant women; low serum retinol concentration (<0.70 µmol/l)
affected another 190 million preschool children as well as 19.1 million pregnant women
(WHO, 2009b). To sum up, the affected populations correspond to 33.3% of the preschool
population and 15.3% of pregnant women in populations at risk of vitamin A deficiency, i.e
in low-income countries such as Africa and South-East Asia (WHO, 2009b).
14
Vitamin A deficiency usually results from malnutrition, but can also be due to abnormalities
in intestinal absorption of retinol or carotenoids. Some of the more serious manifestations of
vitamin A deficiency include (Bates, 1995):
Blindness due to inability to synthesize adequate quantities of rhodopsin. Moderate
deficiency leads to deficits in vision under conditions of low light ("night blindness"),
while severe deficiency can result in severe dryness and opacity of the cornea
(xeropthalmia).
Increased risk of mortality from infectious diseaseswhich has been best studied in
malnourished children, but is also seen in animals. In such cases, supplementation
with vitamin A has been shown to substantially reduce mortality from diseases such
as measles and gastrointestinal infections.
Abnormal function of many epithelial cells, manifest by such diverse conditions as
dry, scaly skin, inadequate secretion from mucosal surfaces, infertility, decreased
synthesis of thyroid hormonesand elevated cerebrospinal fluid pressure due to
inadequate absorption in the meninges.
Abnormal bone growth in animals which can result in malformations and, when the
skull is affected, disorders of the central nervous system and optic nerve
2.4.2 Vitamin A in the diet
Vitamin A is a fat-soluble vitamin and an isoprenoid compound mostly obtained from animal
sources. Retinoids are a class of compounds that show vitamin A activity. They differ
structurally from all trans-retinol. Dietary vitamin A is found in the form of provitaminA in
plants. Long chain retinyl esters are mainly found in animals. Both forms of vitamin A are fat
soluble, therefore, normal fat digestion and absorption is essential. Provitamin A can be
found in various forms in nature such as α. Β, γ carotene and cryptoxanthin. β-carotene is
predominantly found in nature. About 50% of dietary carotenoids are absorbed. Low fat
intake reduces absorption of vitamin A. β –carotene is absorbed by enterocytes mainly by
passive diffusion. Vitamin E and other anti-oxidants protect β –carotene from oxidation of
double bonds. In a normal diet, about 1/6th
of the ingested β –carotene is converted to retinol.
Preformed vitamin A is present as retinyl esters in the diet. Retinyl esters are hydrolysed by
pancreatic lipase or cholesterol esterase (Rodriguez- Amaya, 1997; Booth et al., 1992 &
Butití Palm, 1975).
15
Vitamin A deficiency (VAD) is the leading cause of preventable blindness in children. It also
increases the risk of disease and death from severe infections. In pregnant women VAD
causes night blindness and may increase the risk of maternal mortality. Vitamin A deficiency
is a public health problem in more than half of all countries, especially in Africa and South-
East Asia, hitting young children the hardest and pregnant women in low-income countries
(World Health Organization, 1998).
2.4.3 Importance of Folic Acid
Folic acid (water soluble vitamin B9) is a pharmaceutical form of folate that is used for food
fortification, which is important for cell division and reduction of neural tube defects in
babies (Truswell, 2007). It cannot be synthesized by the body and therefore it needs to be
obtained from dietary sources. The major sources of folic acid are fruits, green leafy
vegetables, cow‘s liver, egg yolk, and legumes. Folic acid deficiency leads to cardiovascular
diseases, and some carcinomas such as colorectal, uterine, cervical, lung and
oesophagus.Inadequate intake of folic acid early in pregnancy, results in neural tube defects
(Oner et al., 2006). Recent studies indicate that the daily intake of folate in women in rural
areas of India and that of lower socioeconomic strata in Delhi is less than one third of the
400-micrograms of folate per day required to prevent birth defects. The data showed that the
incidence of NTD was among the highest at 6.57-8.21 per 1000 live births. Fortification of
wheat flour to overcome folic acid deficiency diseases has been implementedbut is yet to
reach the rural population. Rice would be another choice forfortification with folic acid
(Wesley & Dutta, 2009).
Folate helps produce and maintain new cells (Kamen, 1997).This is especially important
during periods of rapid cell division and growth such as infancy and pregnancy. Folate is
needed to make DNA and RNA, the building blocks of cells. It also helps prevent changes to
DNA that may lead to cancer (Fenech Aitken , Rinaldi, 1998). Both adults and children need
folate to make normal red blood cells and prevent anemia. Folate is also essential for the
metabolism of homocysteine, and helps maintain normal levels of this amino acid (Zittoun,
1993). In its naturally occurring form folate is unstable in food storage and preparation.
(Eitenmiller &Landen, 1999). On the other hand folic acid is stable and can be used a
supplement and for food fortification (Eitenmiller & Landen, 1999; O‘Broin, Temperley,
Brown , Scott, 1975; Temple and Montgomery, 1984).
16
Figure 2.3 Structure of folic acid showing the pteridine ring and the para-aminobenzoic
acid (PABA) (Eitenmiller, 2008)
In the year 1965 the relationship between folic acid and neural tube defects was hypothesized
(Hibbard, Hibbardand Jeffcoate, 1965). Neural tube defects (NTDs) are an embryonic
underdevelopment of the neural tube which fails to close resulting in damage of the exposed
underlying neural tissue. There is significant morbidity and mortality depending on the
severity and the location of the lesion. Several Randomized Control trials (RCT) were
performed to explain the relationship between folic acid deficiency and NTDs. In the research
conducted by British Medical Research Council it was found that in pregnant women with a
past history of NTD the incidence of NTD recurrence was reduced by 70% by taking 400 μg
of folic acid daily (Medical Research Council (MRC) Vitamin Study Research Group, 1991).
In 1991 the Centre for Disease Control and Prevention recommended that women with a
history of past NTD-affected pregnancy should consume 4000 μg of folic acid per day
starting at the time of planning for pregnancy. However encouraging women to start
consuming supplements has limitations in terms of a public health programs since in the USA
about 50% of pregnancies are unplanned. The neural tube closes 28 days after conception
which is very early (Finer & Henshaw, 2006) and therefore women who are planning to get
pregnant should start an early dose of folic acid supplementation i.e. during the planning
stage for pregnancy. Education campaigns that encourage women to increase their use of
supplements have not been very effective in reaching the highly affected population (Ray,
Singh, & Burrows, 2004) Therefore it has been suggested that well- implemented mandatory
fortification programs might help reduce the discrepancy (Dowd & Aiello, 2008). Mandatory
fortification programs have been implemented in many countries to maximize their effect and
reduce the cost associated with prevention programs such as education campaign and other
interventions that require behavioural changes (Flour Fortification Initiative, 2010).
17
One of the ways to evaluate the effect of folate fortification programs is to measure the blood
folate levels in the target population. Folate deficiency can be defined as a serum folate
concentration of <7nmol/L (~3 ng/mL) or a red blood cell folate concentration <315 nmol/L
(~140 ng/mL) (Institute of Medicine, 1998). Homocysteine concentration can be used as a
biomarker for folate status in conjunction with blood folate concentrations (Selhub, Jacques,
Dallal, 2008). In an Irish cohort study it was found that the greatest reduction in NTDs was
observed only when blood folate concentration was much higher than the level set for folate
deficiency (Daly et al., 1995).
In Australia, it is mandatory to fortify all bread-making flour with folic acid, where 3 slices of
bread (100g) contains an average of 120 µg of folic acid (FSANZ, 2007). According to Allen
et al. (2006), folate deficiency is common in populations that consume a high intake of
refined cereals that are low in folate as well as a low intake of leafy greens and fruits that are
high in folate.
2.4.4 Importance of Iron
Apart from vitamin A deficiency, approximately 1.6 billion people are anemic worldwide
(WHO/ CDC, 2008). It is reported that iron-deficiency anemia is prevalent in Asia, where
60% of women and over 30% of men are anemic (MacPhail, 2007). Iron is important for
oxidative metabolism and cell functions (NHMRC, 2006). In children, iron- deficiency
anemia can cause impaired psychomotor development; pregnant women are likely to have
premature birth, low birth weight babies and increased perinatal mortality (MacPhail, 2007).
Unlike in Asia, the prevalence of iron-deficiency anemia among women and men in Europe
and North America is less than 5% and 2%, respectively (MacPhail, 2007). Compared to poor
populations, wealthier people have greater access to a variety of fruits and vegetables and
micronutrient-rich foods such as meat and eggs.
United Nations (2002, Vienna)estimated by that about 34% of the world‘s population suffers
from iron deficiency and 80% of this population belonged to developing countries. The
paradoxical fact about iron is that although it is one of the most abundant elements in the
planet its deficiency represents the most common nutritional problems. The reason for this is
that most of the ingested iron has low solubility thereby resulting in low bioavailability
(United Nations, 2002).
Iron is essential for the human body as it integrates many bio-molecules with various
biochemical and physiological functions. Iron deficiency affects most pregnant women. Other
18
consequences of iron deficiency are absorption of toxic metals, low resistance to infections,
and irregular maintenance of body temperature and modification of physical activity, fitness
and work capability (FAO/WHO, 1988).
Iron is present in foods in two forms namely, heme iron which is derived from flesh foods
like fish, meat etc. and non-heme iron which is the inorganic form present in plants like
cereals, pulses, legumes, grains, nuts and vegetables. Heme iron has a better absorption rate
than non-heme iron. Iron absorption can be enhanced by intake of fats and oils, vitamin A,
muscle tissue and ascorbic acid in diet and by reducing foods containing high phytic acid and
tannic acid, other polyphenols, calcium and animal proteins content (Hurrel and Egli, 2010)
(FAO/WHO, 1988). Daily requirement for iron varies based on age. For adults in the age
group of 19-51 the iron requirement is 8 mg/day for men and 18 mg/ day for women
(National Institute for Health, 2007).
2.5 Factors to be considered for food fortification
Fortification of foods with micronutrients involves the basic principle of choosing
theappropriate medium for fortification. The medium should be consumed by the target
population on a daily basis and in adequate intake. Therefore, rice was chosen for
micronutrient fortification as it is the staple food in several developing countries.
Fortification of rice with these micronutrients would improve the nutritional status of the
population suffering from micronutrient deficiency.
In order to fortify foods with nutrients with an expanding range of fortificant compounds and
technologies the need to choose the best medium for fortification becomes a practical
problem in establishing fortification programs. The following factors must be considered to
fortify foods with nutrients (FAO/WHO, 1988):
Consumed by a large majority of target population in adequate intakes
Manufactured in large scale at few centers to supervise efficiently
Nutrient must be stable under normal conditions of storage
Relatively low cost
Have a constant consumption pattern with a low risk of excess consumption
Centrally processed with minimal stratification of the fortificant
Minimal interaction between the fortificant and carrier food
Contained in most meals with availability unrelated to socio-economic status
Linked to energy intake
19
The colour of the food may not change much due to the addition of fortificants. In the case of
iron, commonly used compounds are ferrous sulphate and ferrous fumarate. In recent
research it has been suggested that sodium-iron EDTA is a better fortificant than the other
two as it has iron from the EDTA-complex that remains bioavailable even in the presence of
iron absorption inhibitors and also it does not show adverse effects on humans. The Joint
FAO/WHO Expert Committee on Food Additives (JECFA) has given sodium iron-EDTA
tentative approval for use as a fortificant. In the case of vitamin A fortification, if the
moisture content of the food is greater than about 7-8% it is known to adversely affect the
vitamin A stability. Therefore, the choice of medium for fortification should meet these
criteria (Food and Drug Administration, 1996).
2.6 Commercially available fortified rice
Fortified rice products are commercially available namely, Nutririce and Ultra Rice. The
Ultra Rice micronutrient delivery system employs a unique mechanism for incorporating
vitamin and minerals within an extruded ―rice grain‖ made from rice flour to minimize
nutrient losses. These fortified extruded grains (called ―Ultra Rice grains‖) resemble natural
milled rice in size, shape, and density. After drying, Ultra Rice grains are blended with local
rice, typically in a ratio of 1:100. When cooked, the fortified rice has the same taste, colour,
and texture as unfortified rice. Ultra Rice is fortified with iron, thiamin, zinc, and folic acid
(PATH, 2007).
NutriRice process uses broken rice kernels from the rice mill for production of rice flour and
mixing with vitamin and minerals. To resolve the problems of segregation and washing off
vitamin and minerals, the mixture is formed by extrusion into reconstituted rice kernels. The
fortified reconstituted kernels are then added to the natural whole rice kernels. NutriRice is
fortified with vitamin B1, Vitamin B2, Vitamin B6, Iron, Zinc and Folate. The above two
mentioned fortified products use extrusion as a means of fortification (Global Alliance for
Improved Nutrition GAIN, 2009).
Table 6 gives a comprehensive summary of the existing rice fortification techniques
including the above mentioned. It was concluded that the hot extrusion technique was the
best for premixes excluding the fact that it confers the rice with a strong colour. Rice
premixes produced by cold extrusion technique can be utilized by people who are less
concerned about the rice whiteness and therefore, this rice was successful in Costa Rica. The
disadvantage of the coating technique is that the fortificants give colour to the grain making it
20
less acceptable. Moreover, in The Philippines, the adhesion of fortificant layer on to the rice
in the locally developed method was very weak and therefore leading to heavy loss during
washing and cooking (USAID, 2008).
The dusting technology which is claimed to be the easiest method resulted in a strong
vitaminodour because of the high concentration of micronutrients in the premix. However
after diluting it with the retail rice in the ratio of 1:1600, the odour was not noticeable
(USAID, 2008).
21
Table 2.2 Comparison of Programs and Quality of rice- and Micronutrient-Premixes produced by two different existing rice
fortification technologies (USAID, 2008)
Extrusion and Dusting Technology Coating Technology
Company
(Country)
Type of
Program
Description of Premix Company
(Country)
Type of Program Description of Premix
DSM/ Buhler
& COFCO
(China)
Aimed at
high-end
market
consumers.
White and stable.
A product with β-
carotene is yellowish
and unstable after 3
months at 30°C.
Wright
(USA for the
Philippines)
Aimed at social-
programs under
government
administration.
Product with a strong golden dark
color and off-taste, which is very
easily distinguished from unfortified
kernels. Losses have been reported
after rinse-wash preparation for
cooking.
Superlative
Snacks
(Philippines)
Aimed at
high-end
market
consumers.
Premix has an off
white colour that is
distinguishable upon
close inspection.
CLG-Health
Food
Products
(Philippines)
Responding to
national regulation,
but indeed with a
very small
coverage.
The premix has a grayish color with
dust and strong off-taste. It is
distinguishable from non-fortified
rice. Stability has not been measured,
but losses during the rinse-wash
preparation for cooking might be
large.
22
Vigui- with
DSM support
(Costa Rica)
National
program
mandate by
government
Premix is a rice-shaped kernel of
yellowish colour with no
apparent odour that becomes soft
in contact with water. It is
possible to differentiate from the
non-fortified rice based on
colour. Claims that losses during
washing and cooking are
minimal.
Group NTQ
and Kuruba
with
Fortitech
support
(Costa Rica)
National program
mandate by
government
Slightly yellowish in colour with
some vitamin-like odour. It is
possible to differentiate the rice-
premix from the non-fortified rice
based on colour. Stability of the
micronutrient layer over the grain
surface is attained through a 5-layer
coating process. Losses during
washing have been claimed to be less
than 5%.
Wright &
RPC
(USA)
Required if
claim of
enrichment
is done.
Compulsory
in some
states.
White powder with strong
vitaminodour. It has the
common characteristics of
micronutrient premixes for
cereal fortification. It can
segregate from the rice grain.
Rinse-washing for cooking will
remove the micronutrient premix
Wright &
RPC
(USA)
Required if claim
of enrichment is
done. Compulsory
in some states.
It is claimed that there is no product
odour or colour change if the coating
process is done correctly. Claims
shelf life is 2 years, but storage time
is typically 3 months.
23
2.7 Mandatory Food Fortification Programs (Folic Acid)
Mandatory fortification of wheat flour with folic acid is currently in place in 53 countries
although in many cases regulations are not in place (Centre for Disease Control and
prevention, 2010). Maximum fortification level and the Legal Minimum Level of folic acid to
be used to fortify flour have been established (Allen, de Benoist, Dary, Hurrell, 2006),
Table 2.3 The Levels of folic acid fortification in countries with mandatory fortification
programs
Country Fortification Level Year of Implementation
United States (Food and
Drug Administration, 1996) 140 μg/100g 1998
Canada (Canada Gazette
Food and Drug Regulations,
2008)
150μg/100g 1998
Costa Rica (Chen & Rivera,
2004) 180 μg/100g 1998
Chile (Hertrampf & Cortes,
2004) 220 μg/100g 2000
South Africa (Sayed, Bourne,
Pattionson, Nixon,
Henderson, 2008)
150 μg/100g 2003
2.7.1 Mandatory Fortification in Australia & New Zealand
FSANZ (2007) recommendedthat Australia and New Zealand implement mandatory
programs to fortify bread with folic acid. Australia implemented the mandatory fortification
in September 2009. Cereals and cereal products require that all wheat flour for making bread,
with the exception of flour represented as organic, must be fortified with folic acid from 13
September 2009. The level of fortification required is between 2 and 3 milligrams of folic
24
acid per kilogram of wheat flour for making bread. Folic acid is the only permitted form of
folate that can be used to meet these requirements.
2.8 Novelty in Rice Fortification
2.8.1 Parboiling Process
From the results obtained from the study by Prom-u-thai (2008), it was stated that iron
fortified parboiled rice provided a preliminary basis for further investigation of this
innovative technique. This was further continued by their research group (2009) which
inferred that parboiled rice had a great potential for zinc fortification. Iodine, another mineral
was also successfully fortified in rice by the parboiling method (Tulyathan, 2007). This led to
the possibility of fortifying rice using this technique with vitamin.
Extensive research on rice fortification has been conducted in UNSW (de Ambrosis et al.,
2004, Mulia 2010 and Gunawan 2010 and Kam et al.,2012). Parboiling model for
fortification of rice with folic acid using different times and temperatures have been
extensively studied by Kam et al., 2013 and also the physical and chemical characteristics,
consumer acceptance of the fortified rice. Fortification of β-carotene and iron in rice was
studied by Gunawan and Mulia, respectively (2010). From the above studies it has been
found that the optimum condition for the maximum concentration of each nutrient
individually was found to be 70 °C soaking temperature for 2 hours followed by steaming at
100°C for an hour and shade drying until the moisture content dropped to 14% c.a.
Kam, Arcot and Adesina (2012) have reported on folic acid fortification using parboiling at
70°C for 1, 2 and 3 h using four different concentrations of folic acid. The dried parboiled
rice was milled at 3 different durations i.e. 0s, 60s and 120s. A multifactorial model was
developed to describe the residual folate concentration and it was found that soaking and
milling were significant factors for folic acid fortification. The optimum soaking time was
found to be 1.97 hours and folate uptake rate followed 1st order kinetics. The rate of natural
rice hydrolysis and folate uptake by rice was both time-dependent (Kam, Arcot & Adesina,
2011). From the above literature resources, feasibility of fortifying rice with three
micronutrients was therefore explored through the present study.
25
2.8.2 High pressure processing
High pressure processing (HPP) technology is a novel food processing technique. This novel
technique could be a potential method in delivering fortified rice. Being a novel techniqueit
has not been applied to food fortification. HPP is used in food processing. It involves
application of hydrostatic pressure which can cause gelatinization of egg yolk and white by
the application of 1000-7000 atm pressure at 25-30°C (Yaldagard, Mortazavi, Tabatabaie ,
2008). The application of HPP is independent of sample size and geometry. HPP is
commonly used in food industries for(Knorr, 1993):
1. Inactivation of microorganisms and enzymes
2. Modification of biopolymers
3. Quality retention, such as colour and flavour
4. Changes in product functionality
HPP causes minimal changes to fresh foods as the food is not subjected to thermal
degradation. HPP processed foods have a fresher taste, better appearance, texture and
nutrition. It can be used at high as well as low pressures and it is beneficial for heat sensitive
products(Knorr, 1993).
In a typical HPP process, the product is packaged in a flexible container such as a pouch or
plastic bottle and is loaded onto a high pressure chamber filled with a pressure-transmitting
hydraulic fluid. The hydraulic fluid in the chamber is usually water which is pressurized with
a pump, and this pressure is transmitted through the package into the food itself. Pressure is
applied for a specific time, usually 3 to 5 minutes. The processed product is then removed
and stored/distributed in the conventional manner. Because the pressure is transmitted
uniformly, food retains its shape, even at extreme pressures. And because no heat is needed,
the sensory characteristics of the food are retained without compromising microbial safety
(Ramaswamy, 2006).
Being a novel technique, the usefulness is yet to be explored in detail. However, few
researchers in Japan have tried to treat rice with HPP technique and analysed the property of
rice. Huang, Jao, Hsu, in 2009 analysed the combined effect of pressure (200-500 MPa) and
temperature (20, 40 and 50˚C) on the water uptake and gelatinization of japonica rice variety.
When the pressure was greater than 200 MPa, at all temperatures the moisture content and
volume of grains increased. The highest degree of gelatinization was observed to be 73% at
500 MPa at 50˚C for 120 min. Gelatinization did not occur at pressures below 300 MPa and
26
temperature of 20 or 40˚C. Therefore, it can be said that at higher temperature and pressure
gelatinization of rice occurs using HPP equipment (Huang, Jao, Hsu, 2009).
Yamakura et al., (2005)studied theeffect ofsoaking followed by high pressure treatment on
the cooked quality of rice. In this study, milled rice grains which were pre-soaked in water at
25-550C for 30 min was subjected to HPP treatment at 400 MPa for 10 min and then soaked
in water overnight. The physical and chemical properties of the treated rice were then
analysed. The viscosity of milled grains increased with the soaking process. Total sugar
increased and in internal structure of rice grains changed after HPP treatmentin soaked rice.
The structural change of rice seemed to promote water penetration and brought about higher
degree of swelling in rice granules. This might result in high degree of gelatinization and
higher digestibility of HPP-treated cooked rice. HPP treatment of rice resulted in denaturation
of water soluble proteins and increased free amino acids. Therefore, HPP treated rice proved
to be an advantageous method for cooked rice with better palatability (Yamakura et al.,
2005). Based on literature collected so far in terms of treating rice using HPP fortification of
rice would be novel to explore.
2.8.3 Cost Analysis for parboiling and HPP processes
In a study conducted by Roy, Shimizu, Shiina and Kimura (2005), to evaluate the local
parboiling processes, cost and energy analysis was done. In the eastern part of India (West
Bengal) and Bangladesh, the production of parboiled rice is the main income of many people
in the local area. The commonly used parboiling processes are vessel, small-boiler and
medium-boiler. The major source of energy for this process is biomass and this was
contributed by rice husk. The following paragraph will explain about the common local
parboiling processes and their respective cost and energy analysis.
The local parboiling process is presented in the flow chart in Figure 2.4. The vessel process is
a direct heat method of parboiling at a household level to produce parboiled rice for
consumption. This is the major source of income for these families on a daily basis. The
capacity of the vessel varies from 0.5 to 1.2 tonnes/batch (t/batch) and this varies with the
business capital (Roy et al., 2005).
27
On a commercial scale, a small-boiler process is used. For this process, two conical hoppers
made of aluminium sheets are used for pre-steaming and steaming so that the steam
generation would be continuous and simultaneously applied to the paddy. For the drying
process the home yard itself is used as a drying yard and if the place is too small another
place could also be used. The capacity for this batch processing varied from 2-4 t/batch which
depends on the business capital and on the drying yard area (Roy et al., 2005). .
In the case of a medium-boiler process the purpose is completely dedicated to
commercialization. Two conical hoppers are used as a set for pre-steaming and steaming. The
larger set is used for pre-steaming and the smaller one is used for the steaming process itself.
The capacity of this process varies from 5-10 t/batch (Roy et al., 2005).
Figure 2.4 Flow chart of local parboiling process in Bangladesh and West Bengal, India.
(Roy et al., 2005)
The energy consumption for each of the local parboiling processes described above has been
presented in the Table 2.4. It was observed that the medium-boiler required the lowest energy
and the small-boiler had the highest requirement. However, the energy consumption was
found to be lower than the boilers parboiling process which required 4200 MJ/t where hot
28
soaking was done to the paddy and this is considered to be a modern method (Tiwary & Ojha,
1981). On a lab-scale traditional parboiling process the energy requirement varied from 1400-
2442 MJ/t depending upon the treatment times (Roy et al., 2003a). As it can been seen from
Table (2.4), small-boiler process is a more energy intensive process compared to the vessel
and medium boiler. This was attributed to the usage of two-barrel boiler which caused loss of
heat energy through gaps between the barrels during parboiling and also to the boiler
installation and lower quantity of paddy parboiled and limited capacity of the drying yard.
Therefore, it can be suggested that to reduce the cost of energy a large scale boiler could be
used.
Table 2.4 Energy consumption in parboiling processes (Roy et al., 2005)
Process Biomass consumption, kg/t Total Energy consumption,
MJ/t
Pre-steaming Steaming
Vessel 71.9 113.7 2583
Small- boiler 87.2 109.6 2758
Medium- boiler 43.1 75.1 1659
Table 2.5 Installation costs of the parboiling process (US$) (Roy et al., 2005)
Process Vessel or boiler Hopper Soaking
tank
Tube-well Initial
investment
Cost/unit Used
Units
Life
year
Cost/unit Units
used
Life
year
Cost Life
year
Cost Life
year
Vessel 5.32 2 0.5 42.55 10 21.28 10 397.47
Small-
boiler
42.55 1 1.0 42.55 2 10 148.94 10 21.28 10 1281.87
Medium-
boiler
1170.21 1 1.0 63.83 4 10 638.29 10 212.77 10 5101.09
29
In comparison to the vessel process, boiler process needs a higher installation and investment
cost (Table 2.5). Table 2.6 shows the theoretical fortification formulation from which the
choice of micronutrients relevant to the present study has been presented. It is recommended
that the fortificant should be mixed in the ratio of 1:5000 with the unfortified white rice. If
the fortificants were to be added in the given concentrations, it is suggested that the formula
needs an addition of about 110 g of fortificant mix per metric ton of fortified rice and the
major weight comes from iron. The fortificant mix as per this table costs approximately US$
9 which means that each metric tonne of rice would contain an equivalent of US$ 1.8 of
fortificant mix regardless of the delivery method being a premix or micronutrients mix. From
the table, it is evident that iron is the most expensive fortificant ($1.04) followed by vitamin
A ($ 0.53) and folic acid ($ 0.22).
Table 2.6 Theoretical Fortification Formulation for Comparison Purposes (USAID,
2008)
Micronutrients
(Fortificants)
Micronutrient
content added
to rice
(mg/kg)
Fortificants
to be added
in the
fortified
rice 1(g/MT)
Cost of
fortificants
in the
fortified
rice 2(US$/MT)
Fortificants
in 1
kg
fortificant
mix
(g)
Fortificant
cost in
the
fortificant
mix
(US$/kg)
Vitamin A (Dry
form- 250,000
IU/g)
1.0 13.3 0.53 67 2.68
Folic Acid 1.0 1.1 0.22 6 1.10
Iron
(Micronized
Ferric
Pyrophosphate)
24 96 1.04 484 5.21
Total - 110.4 $1.79 557 $8.99
Note: 1Fortificants are the micronutrient source. These values were calculated dividing the
micronutrient level by the proportion of the micronutrient in the fortificant.
2Using the usual prices in the international market in 2007.
Existing fortification models were compared using a theoretical fortification formula to
understand cost effectiveness of these models. These models were obtained from
DSM/Buhler and COFCO in China (hot extrusion), Vigui in Costa Rica (cold extrusion),
Group NTQ in Costa Rica (coating), and Wright or RPC in the USA (dusting). The
production capacities and therefore the capital costs were different but the capital cost of
30
premix production was found to be in decreasing order of investment i.e. hot extrusion, cold
extrusion, coating, and dusting.
Table 2.7 Comparison of the technologies for premixes production by extrusion, coating
and dusting3 (USAID, 2008)
Technology Dose on rice
(kg/MT)
Annual
Production
(MT)
Capital Cost
(US$)
Recurrent
fortification
costs (US$-
thousands
per year)
Premix
cost
(US$/kg)
Hot Extrusion 4
10 1,500 3,880,000 1724 $ 1.15
Cold
Extrusion 5
10 730 770,000 762 $ 1.05
Coating 6 10 430 300,000 389 $ 0.90
Dusting 7 1 2,500 100,000 11509 $ 4.60
3 This formulation assumes that 10 kilograms of rice-premix for the coating or extrusion
technologies, or one kilogram of micronutrient premix for the dusting technology, are added
to one metric ton of retail unfortified rice. The dilution rates are 1:100 and 1:1000,
respectively. In both cases, the final dilution rate of the fortificants in the rice is 1:5000,
because ~200 grams of the combined fortificants are needed to achieve the proposed
micronutrient levels as described in Table 2.6. The rice-premix is a dilution 1:50 of the
fortificant mix (5000/100 = 50); and the micronutrient mix is a dilution of 1:5 of the same
fortificant mix (5000/1000 = 5). Price of the fortificant mix is assumed as US$15/kg. Price of
broken rice is assumed as US$0.30/kg, and whole rice as US$0.50/kg. The coated technology
uses half broken rice and half whole rice.
4 Using the model of DSM/Buhler and COFCO in China.
5 Using the model of Vigui in Costa Rica.
6 Using the model of the Group NTQ in Costa Rica
7 Using the model of Wright or RPC in USA assuming that process costs are equivalent to
US$1/kg micronutrient premix.
31
Table 2.8 Table Capital and production cost of thermal and pressure processes
(Sampedro, 2014)
Capital cost
Process parameters Unit of measure Thermal HPP
Heat exchanger $ 18,000 2,000
HPP equipment $ - 2,495,000
Process pumps $ 12,000 12,000
Total equipment
cost
$ 66,000 2,545,000
Total Capital cost $ 132,000 5,090,000
Production cost
Total electricity cost $ 3,000 70,000
Total energy cost $/year 8,000 70,000
Compared to thermal processing high pressure processesing is a novel technology. Therefore
it is likely that the capital cost would decrease in the future when they are widely used in food
industries. From the above table it can be seen that the major costs are driven by energy
consumption, and capital cost. The overall cost for HPP is higher than parboiling in terms of
capital and production. Since parboiling is more established than HPP it is likely that the
former method is more economically feasible than HPP.
2.9 Study of Morphological and Physico-chemical properties of Rice
2.9.1 Pasting Properties Using RVA
Rice is classified according to the grain dimensions, amylose content and amylograph
viscosities of rice, degree of gelatinization properties of extracted starches and texture of
cooked rice. Based on these properties rice can be classified into waxy and non-waxy types.
Reddy and co-workers in 1993 proposed that textural changes in cooked rice are attributed to
the fine structure of amylopectin. Starch retrogradation occurs at 40C storage (Lu et al.,
1997; Mohamed et al., 2006) which leads to harder texture and is not desirable (Kadan et al.,
2001; Yu et al., 2010). During starch retrogradation the starch polymer realigns in a more
orderly fashion and creates a more crystalline structure thereby making it less susceptible to
enzymatic breakdown (Bjӧrck, 1996; Holm et al., 1988; Ching et al., 2006). During the
32
gelatinization process the inter- and intra-molecular hydrogen bonds between starch granules
are disrupted and hence retrograded starch has lower GI value (Erlingnen et al., 1994;
Fredriksson et al., 2000; Chung et al., 2006).
Starch is characterized often by RVA for pasting properties, DSC for gelatinization, XRD for
changes in crystalline states. Starches that have undergone ANN and HMT exhibit higher
pasting temperature and stability but lower pasting viscosity (Knutson 1990; Gunaratane and
Hoover 2002; Singh et al., 2005; Vermeylen et al., 2006). In nature, A-type crystals are
present in cereals and legumes. B-type crystals occur natively in tubers. The variation in the
crystal type is attributed to the water content and the alignment of the water molecules are
packed in the crystals (Imberty et al., 1991).
2.9.2 Polarized light microscope
A starch is made up of two types of glucose chains: amylose and amylopectin and they
constitute the main energy storage bodies in plants (Torrence and Barton, 2006). In
archaeology starch is mainly identified by cross-polarized light microscopy which has 200-
400 times magnification. This technique allows real time examination of starch in plant
microfossils (Piperno, 2006). Starch is a very fragile organic material and is very soft. Due to
this property when it undergoes several changes during pre and post-harvest and also during
processing such as milling, grinding, cooking, parching and drying they are prone to damage
(McGee, 1984). Very few studies have been conducted to see the physical effects of cooking
on starch grain alteration that are visible under light microscope (Lamb and Loy, 2005;
Samuel, 2000; del Pilar Babot, 2003).
33
Figure 2.5 Model of amylose molecule with 1000 glucose units (a) and Model of
amylopectin molecule with 1000 glucose units in 30 branches (b) (Nikuni, 1957)
Rice starch grains are small, subangular, faceted and compound. The hilum is centric and the
Maltese crosses are radially symmetric but faint. The size of individual grains range from 3-
10 µm. Light microscope was used to see changes in starch microstructure on baking and
staling of wheat bread by Hug- Iten, Handschin, Conde-Petit and Escher(1999). When
observed under polarized light microscope, starch granules show a Maltese cross. When the
filter of the polarized light is adjusted to an additional λ/4, positive birefringence of starch
granules was observed which theoretically indicates a radial orientation of the principal axis
of the crystallites (Gallant, 1974; Gallant et al., 1992). Native starch is about 15-48%
crystalline and therefore it is not the primary sequence of starch polymers. Starch crystals are
made up of semi-crystalline and crystalline shells which are between 120 to 400 nm thick at
the lowest level of structure. According to Kassensbecj, (1975); Oostergetel & van Bruggen,
(1989); Jenkins et al., (1993) the crystalline shells in starch consists of amorphous and
crystalline lamellae that alternates with each other as a sequence. Robin et al (1974) and
French (1984) stated that the side chain clusters represent the crystalline regions whilst the
branched regions represented amorphous regions of amylopectin molecules. Amylopectin
b a
34
forms the side chain clusters (80-90%) and the inter-cluster connections form the remaining
10-20%
Effect of high pressure processing (600 MPa) on starch and the susceptibility to amylase was
studied by various researchers. High pressure was applied to induce gelatinization in cereals
such as wheat and pulse such as smooth pea (Muhr et al., 1982). The study found that upon
applying 400-500 MPa at 45-50°C on wheat and corn starch showed high susceptibility to
amylase treatment, however potato starch was resistant to high pressure treatment and
therefore only showed slight susceptibility (Hayashi and Hayashida, 1989). Changes in
structure of cassava starch after heating with water was observed using scanning and
transmission electron microscopy (Gracia, Colonna, Bouchet, Gallant, 2006). Effect of high
pressure processing on crystalline structure of corn and potato starch was studied by Hibi et
al., (1993). There is limited information on the changes of rice starch crystallinity after HPP.
Therefore in the current study this gap was tried to be addressed.
Figure 2.6 Microscopic images of corn starch granules under normal light (A) and
polarized light (B); where I is untreated and II is pressure treated at 500 MPa for 20
min (Hibi et al., 1993)
A B
35
Figure 2.7 Microscopic images of potato starch granules under normal light (A) and
under polarised light (B) where I is untreated and II is pressure-treated at 500 MPa for
20 min (Hibi et al., 1993)
2.9.3 X-Ray Diffraction
2.9.3.1 Diffraction of X-Rays
When X-Radiation passes through a sample matter, the electric vector of the radiation
interacts with electrons in the atoms of matter to produce scattering. When X-rays are
scattered by the order of crystals in the sample, constructive and destructive interference
occurs among the scattered rays because the distances between the scattering centres are of
the same order of magnitude as the wavelength of irradiation. As a result of this the
phenomenon of diffraction occurs (Skoog et al., 1980).
2.9.3.2 Bragg’s Law
When an incident beam of X-ray strikes on a surface of a crystal at an angle say θ a part of
the beam is scattered by the layer of atoms at the surface. The other part that is unscattered
penetrates to the second layer where another fraction is scattered and the rest is passed
through to the third layer and so-on. X-ray diffraction is based on the spacing between layers
of atoms which should be uniform and same as the wavelength of radiation and the scattering
centres must be spatially distributed in a highly regular way (Skoog et al., 1980).
A B
Typical
Maltese
corss pattern
36
Figure 2.8 Diffraction of X-rays by crystal (Skoog and Douglas, 1980)
Bragg in 1912 treated crystals with X-rays as shown in the figure above. A narrow beam of
radiation strikes the crystal surface at an angle θ; scattering occurs as a result of interaction of
radiation located at A and C. Therefore it can be said that nλ= BC+CD where n is an integer.
However, BC=CD= d sin θ where d is the interplanar distance of the crystal. Due to
constructive interference of the beam, at angle θ nλ= 2d sin θ which is the fundamentally
important Bragg equation. X-rays appear to be reflected from the crystal only if the angle of
incidence satisfies the following condition (Skoog et al., 1980).
sin θ = nλ/2d.
At all other angles, destructive interference occurs.
2.9.3.3 X-ray Diffraction in rice
Parboiling process imparts vital changes to the rice components. Starch gelatinization which
takes place during the heating step in parboiling affects the organoleptic properties of cooked
parboiled rice. Innate starch is generally in a semi-crystalline structure. With the aid of wide-
angle X-ray scattering (WAXS) it was deduced by Imberty & Pérez (1988) that rice is
arranged on a monoclinic crystal lattice (A-type). The structure of the A-type crystal is
affected depending on the heat and moisture treatment during the parboiling process.
(Biliaderis et al., 1993 & Priestly, 1976). According to Bhattacharya (1985), parboiled rice
should have limited presence of white ―bellies‖ or residual A-type crystals in the centre of the
rice kernels. During the drying stage of the parboiling process, retrogradation of the starch
occurs resulting in the formation of A-type or more hydrated B-type crystalline units.
The second change that occurs during the heating step of parboiling process is the
complexation between the lipid and the amylose molecules (Priestly, 1976). The firmer
37
texture of the parboiled rice compared to the normal white rice could be attributed to the level
of crystalline amylase-lipids complexes formed during this stage. This complex is observed
to be stable in the cooking process (Biliaderis et al., 1993; Ong and Blanshard, 1995 &
Priestly, 1977).
The interaction and formation of disulphide bonds between rice proteins has also been
reported during the parboiling process which can be stated as the third change that is also
partly responsible for the changes in cooking properties (Derycke et al., 2005). Apart from
the above mentioned, Pillayar & Mohandoss (1981) reported that the cooling and drying
process and the changes in the rice kernels associated with the process have significant
impact on the properties of the cooked parboiled rice.
Therefore, in this study the variation in the rice crystal changes due to the parboiling process
and HPP was analysed using XRD as the two processes have different effects on the rice
starch. Since the micronutrients adherence was confined to the starchy endosperm of the rice
and the outer layers of the rice are milled away, this study was performed to understand the
variation in the concentrationof nutrients and their interaction with the rice starch.
2.9.4 Magnetic Resonance Imaging
Parboiled rice absorbs high water and therefore leads to lower number of broken grains and
increases the milling yield. This is an essential parameter ofquality for rice grain postharvest
(Miah et al., 2002). When the moisture in the grain is low it results in poor quality after
processing such as cooking. Therefore moisture is a vital quality parameter in rice (Yanase
and Ohtsubo, 1986). Starch is the main component of the rice grain and therefore the degree
of starch gelatinisation brought about by hydration and heating is directly related to the
cooked rice texture (Maruyama and Sakamoto, 1992).
Moisture content of whole grains is usually measured gravimetrically which directly
correlates with the quality of milled and cooked rice. However along with moisture content,
moisture distribution is also essential as the rice grain by itself is inhomogeneous therefore
the distribution of water and diffusion into the grain is also inhomogeneous (Horigane et al.,
2006). Horigane et al. in 2001 measured moisture distribution in cooked spaghetti samples
using magnetic resonance imaging (MRI) and high resolution three-dimensional magnetic
resonance (3D-MR).
38
In Japan an essential preliminary step in cooking rice is to soak rice in excess water which is
a factor in determining cooking quality of rice (Koide et al., 2001 Okuno and Adachi, 1992).
According to Seki and Kainuma (1982), insufficient water penetration into rice grain during
the soaking step results in a hard textured cooked grain as the starch does not fully swell and
gelatinise. For biomaterials such as foods, 3D- MRI is generally performed using pulse
sequence of spin echo (SE) or gradient echo (GE) or single-point imaging (SPI). The most
common method used however is SE which can produce high quality image with good signal
to noise ratios and high spatial resolutions (Callaghan, 1991). SPI is a new imaging technique
that can be used for foods with low moisture content and semi-solid samples (Balcolm et al.,
1996). Jenner et al., (1988) was the first to conduct kinetic study of water movement into
wheat grains using MRI during the intermediate stage of development which had a moisture
content of 62-68%. The total duration of soaking was 1.6 h.
MRI has been used for several cereals such as barley endosperm during storage (Gruwel et
al., 2002) and steeping (Molina-cano et al., 2002) and wheat grain was studied by Song et al
(1998). Development of barley and rice grain was studied by Glidewell (2006) and Horigane
et al. (2001) respectively. Since rice has a moisture content of 13-35%, a fast imaging
technique with high sensitivity for water proton signals is vital. With the aid of high-spatial-
resolution 3D-MR images, water penetration can be observed from all directions. With low
moisture foods, MR images did not show moisture distribution successfully and thus it
required longer measurement time. Real time rice imaging during cooking was achieved by
turbo-spin technique by Mohoric et al(2004) which is a fast imaging technique. However this
was not possible again at low moisture content. Samples with low moisture content have very
short spin relaxation times (T2) of water protons and the echo times (Te) should also be
shortened during measurement (Song et al., 1998).
2.10 Consumer Acceptance Study
Consumer acceptance is the ultimate measure of end-quality of a newly developed product in
terms of preferences based on which the product‘s market is driven. This complex
terminology of consumer acceptance encloses several elements such as sensory descriptors,
packages, food appearance, food labels and health claims and consumer‘s experiences and
knowledge towards the product (Heinemann et al., 2006). It is always a challenge when
value-added foods and functional foods are marketed when it comes to consumer acceptance
as they may not be welcomed by customers (Siro et al., 2008). This scenario can be
explained with an example of Danish consumers who were sceptical about functional food as
39
the food was perceived to be unnatural and the consumers were anxious about the taste of the
product (Poulson, 2009). This scenario was an exemption in the United States where several
fortification programs have been successfully implemented and adopted since 1924
(Backstrand, 2002).
Parboiled rice that was a prototype was evaluated for consumer acceptance against three local
samples and one imported sample by Tomlins et al., 2007. The panel was asked to assess the
appearance of the rice. The end results suggested that the consumers were against the local
parboiled rice which was described as brown, black heads. This shows that appearance is an
important factor for consumer acceptance. The colour of cooked rice is a negative factor for
consumer acceptance (Tomlins et al., 2005). Overall, texture (Tomlins et al., 2005), grain
size and uniformity (Heinemann et al., 2006) are vital factors for consumer acceptance of
parboiled rice.
Apart from appearance, taste is also a major influencing factor for consumer acceptance
(Food Insight, 2010). Folic acid fortified parboiled rice was subjected to consumer evaluation
by Kam et al., (2012) and it was concluded that in terms of visual acceptance an informed
health claim of the fortified rice enhanced consumer‘s purchase intent of the fortified rice.
After cooking and tasting the rice, it was reported that more than 50% of the consumers were
willing to purchase the fortified rice that was mixed with unfortified rice and cooked. This
shows evidence for the acceptance of fortified parboiled rice by consumers. Rice in general
does not create a strong sensory stimulus in terms of tasting (Heinemann et al., 2006).
Although it is described as bland, taste still plays an important role in consumer acceptance
(Diako et al., 2010).
2.11 Bioavailability Studies using Caco-2 cells (in vitro)
2.11.1 Introduction to Cell Culture
The cost of performing animal and human trials for micronutrient bioavailability has shown
limited progress over the past few decades (vanCampen & Glahn, 1999). Therefore an
invitro model which utilises intestinal epithelial cells that can mimic gastric and intestinal
digestion would be able to fill the gap in this area (Glahn et al., 1996-2000). This model
which makes use of caco-2 cells has been approved for bio-accessibility studies at a
conference held by the Office of Dietary Supplements, the National Institute of Health and
the American Society of Nutritional Science (Wood and Tamura, 2001).
40
Cell culture has recently been used extensively as an in vitro method to assess human
bioavailability. In vivo studies have the drawback of dealing with complex systems in which
it is difficult to determine the relative importance of different factors. Therefore, in vitro cell
lines can overcome this problem by identifying an appropriate cell line that can suit the
purpose of our study. Caco-2 cells which are human adenocarcinoma cells have the ability to
differentiate into enterocyte of the microvilli with similar morphological and biochemical
characteristics. These cells can differentiate spontaneously and form polarised monolayers
with well-formed brush border and also produce the associated enzymes. Therefore, these cell
lines could be an appropriate model for the study of transport mechanism related to intestinal
barrier and can be used to study the absorption of nutrients (Pinto et al. 1982 & Ismail M.,
1999)
2.11.2 Previous studies using Caco-2 cells
Several studies have been conducted and reported using Caco-2 cells for analysing uptake,
bioavailability and bio-accessibility of nutrients in various foods. In a study conducted by
Fleshman et al in 2011 Caco-2 cells were used to determine β-carotene bio-
accessibility/bioavailability in stored honeydew and musk melon tissues which were grown
under same conditions. Musk melons and honeydew are good sources of β-carotene.
However, its bio-accessibility/bioavailability was unknown and therefore Caco-2 cells were
used to deduce this. It was found that in orange-fleshed honeydew melons the bio-
accessibility of β-carotene was 3.2±0.3%, bioavailability using Caco-2 cells was found to be
11%.
In another study conducted by Alminger et al.(2012) Caco-2 cells were used to determine the
bioavailability of lycopene and β-carotene in differentially processed soups. Since human
studies are labour intensive and due to the limitations in the number of samples,in vitro
models were sought as a replacement for human study. In this paper in vitro data from 2
humans were compared to results from in vitro bio-accessibility studies. The food sample
used was differentially processed soup which contained carrots, tomatoes and broccoli.
Caco-2 cell uptake model was used by Thakkar, Dixon, Dixon & Failla (2007) for
understanding β-carotene micellarization and β-carotene uptake in different cultivars of
cassava. Cassava is a staple food in Africa where the annual consumption exceeds 80 kg per
capita (Aerni, 2006).
41
Several lines of evidence indicate higher bioavailability of added folic acid than naturally
occurring folates in many foods. As stated previously, the study by Cuskelly et al. (1996)
indicates similar bioavailability of folic acid in fortified foods and dietary supplements. In
addition, labeled folate added to various cereal-grain foods also exhibited bioavailability
similar to that of folic acid in aqueous solution (Pfeiffer et al. 1997), and folate in fortified
breakfast cereal has been shown to be effective in raising folate status in humans (Malinow et
al. 1998).
2.11.3 Bioavailability of Iron in Parboiled Fortified Rice
Prom-u-Thai.,Huang ., Fukai. and Rekasem (2008) fortified rice with iron and found that it
increased the iron content in rice by 10 to 50 fold depending on grain properties among
different rice varieties. The retention rate of Fe in white rice ranged from > 50% to nearly
100% despite rinsing rice with water several times before cooking.
Figure 2.9 Iron retention rate in rice (as % of the un-rinsed) after rinsing (simulating
rice washing) in the Fe-fortified parboiled rice grains milled for 60 and 120 s,
respectively, in the 3 rice cultivars tested (Prom-u-thai et al. 2008)
The bioavailability of the fortified rice was also analysed by Prom-u-Thai , Galhn, Cheng ,
Fukai , Rerkasem and Huang in 2008 by in vitro studies using Caco-2 cells. The
bioavailability of Fe-fortified rice increased with the increase in Fe concentration. The
42
uptakes of the fortified Fe in parboiled rice milled for 120 s was well above those of the
unfortified raw or parboiled rice particularly from the high Fe rice line IR68144-2B-3-2-2
and popular Jasmine rice cultivar KDML 105. Fe bioavailability decreased with increase in
milling time and rinsing of Fe-fortified rice in water due to their negative effects on total Fe
concentrations in the parboiled rice grains, but uptakes were still well above that of their
unfortified raw or parboiled rice grains. Rinsing or washing the Fe-fortified and milled rice
grains decreased the bioavailability to 85 ng ferritin mg protein_1
in the YRF cultivar,
compared to about 100 ng ferritin mg protein_1
in its non-rinsed grains. Dilute acid-
extractable (DAE) Fe was linearly and positively correlated with the uptake of Fe assessed by
the in vitro digestion/Caco-2 cell, which can be used as a rapid method for optimising levels
of bioavailable Fe to be fortified in the parboiled rice by parboiled-rice mills if this Fe-
fortification technique should be adopted in south and southeast (Prom-u-Thai et al., 2009).
Another advantage of using caco-2 model system for iron bioavailability is the formation of
ferritin a molecule that stores iron at an intracellular level and this is used a direct measure of
iron uptake (Glahn et al., 1998). Radioimmunoassay can be used to measure ferritin and
therefore this eliminates the need to radio label iron in the food system. Ferritin, a protein is
controls the levels of iron available in the body. It does so by storing and releasing iron from
it in a controlled fashion and thereby able to maintain a buffering system against iron
deficiency. When the levels of iron in the blood is too low ferritin releases more iron and if
the body experiences iron overload ferritin can store the surplus iron (Glanh et al., 2008).
The structure of ferritin is such that it has a hollow sphere where iron can be stored in the Fe
(III) oxidation state. Iron is converted to mineral ferrihydrite [FeO(OH)]8[FeO(H2PO4)] and
incorporated into the inner wall of the sphere. The body requires iron in the form of Fe (II)
and therefore it has to change its form from Fe (III) before being sent for utilisation by the
body. After this conversion iron leaves the spherical structure. This implies that the stability
of ferritin is essential for iron storage and maintenance in the body. Ferritin is made up of 24
peptides assembled as a hollow spherical shell and has a molecular weight of 474,000 g/mol.
The diameter of the shell is about 80 Angstorm and the walls are 10 Angstorm thick. For the
purpose of movement of ions and molecules through ferritin small channels are formed at the
intersections of three or four peptides and these channels contribute to ferritin‘s ability to
release or capture iron. The channels can be classified into two types: four fold channels
which occur at the intersection of four peptide subunits and three fold channels which occur
43
at the three peptide intersection. The two channels have different chemical properties and also
function differently. Solubilised Fe2+ ions exit through the three-fold channel by the
property of polarity. The three fold channel of ferritin is lined with aspartate (Asp) and
glutamate (Glu) which are polar amino acids. This allows a favourable interaction between
Fe2+ and water (which is positively charged) and attracts negative poles of side chains. The
four folded channels are lined with nonpolar amino acids (leucine) and therefore it does not
interact favourably with Fe2+ ions and therefore Fe
2+ ions do not leave these channels. It is
however speculated that the electron transfer occurs from Fe (III) to Fe (II) at the four-fold
channel (Washington University, 2000).
2.11.4 Bioavailability of β-carotene in carrots
The release and absorption of carotenes from processed carrots using in vitro digestion
coupled with Caco-2 cell trans-well culture model was examined by Netzel et al.,(2011). The
absorption of carotenoids can be strongly affected by the processing conditions used to
prepare the food matrix that they are contained in. In order to determine the effect of
processing on carotenoid bioavailability, the carrots were homogenised and then the raw,
blanched and cooked carrots were exposed to an in vitro gastric and intestinal digestion
model. Final digested sample was placed on a Caco-2 cell trans-well monolayer culture to
mimic intestinal absorption. From the results it was found that cooked carrot puree had the
highest release of carotenes as they consisted mainly of single plant cell proteins followed by
blanched which consisted of plant cell clusters and raw carrot puree which consisted of larger
plant cell clusters. Absorption of carotenes through Caco-2 cells was highest from the digesta
of cooked carrot puree followed by the digesta of blanched puree. Therefore, it was
concluded that if thermal processing/mechanical homogenisation was done to disrupt plant
cell wall matrix, the in vitrobioavailability of carotenes from carrots was quite high (Netzel et
al., 2011)
2.11.5 Bioavailability of Folic Acid in Fortified Milk Products
The intestinal absorption of folic acid and 5-methyl-tetrahydro folate (5-CH3-H4folate) in
fortified milk was studied using mono-layers of human colon carcinoma (Caco-2) cells. Only
a small difference in transport rate and underlying transport mechanisms, across Caco-2 cells
was found between folic acid and 5-CH3-H4-folate. With the presence of Folate Binding
44
Protein (FBP), the absorption of folic acid and 5-CH3-H4folate was found to be lower and
dependent on the extent of binding to FBP at the luminal side of the intestinal cells (Verwei,
2004).
2.11.6 Micronutrient Interaction in vitro
In a National Fortification program implemented in Venezuela in the year 1993 to reduce iron
deficiency anaemia, the fortificants were a mixture of ferrous fumarate, vitamin A and other
vitamins. The uptake of ferrous fumarate from the mixture was studied using Caco 2-cells. It
was found that vitamin A did not enhance the absorption of iron, however when β-carotene
(6µmol/L) was used alongside compared to no β-carotene addition it was found that β-
carotene enhanced iron absorption compared to no β-carotene addition. Also β-carotene
overcame the inhibitory effect of phytates and tannic acid (potential inhibitor of iron) and
improved iron uptake. The experiment was validated for the use of Caco-2 cells to study the
in vitro absorption of iron (García-Cassal, Leets & Layrisse, 1993).
2.12 Conclusion
Based on the information gathered so far from literature significant information regarding the
necessity for fortification of vitamin A, iron and folic acid would clearly be a possible
solution to address the micronutrient deficiency issue on a global scale. Rice as a potential
medium of fortification is also convincing as the rice grain has to be processed and also is a
staple in many developing countries. Parboiling has significantly been a potential method for
fortification of nutrients into rice. The papers on high pressure technology provide potential
ideas and possibilities to fortify rice with the above mentioned micronutrients. Sufficient
evidence from literature on other foods states that bio-accessibility study of fortified nutrients
in rice could be successfully studied using Caco-2 cells. The study focussed on fortifying rice
with vitamin A, folic acid and iron using the parboiling technique the high pressure
processing as a potentially novel technique for fortification.
The aims of the current study were to assess:
1. The feasibility of fortifying rice with three micronutrients (folic acid, iron and β –
carotene) comparing two techniques- parboiling (ancient technique) and HPP (novel
technique).
45
2. The efficiency of uptake of micronutrients using the parboiling process and understanding
micronutrients‘ interaction influencing the uptake of each other.
3. The physicochemical properties of the fortified rice, giving special importance to rice
starch.
4. The Acceptability of fortified rice among rice eating consumers.
5. The bio-accessibility and absorption of the micronutrients from the fortified rice (by
parboiling and HPP methods) using in vitro digestion and caco-2 cell culture model.
46
CHAPTER 3
3. OPTIMIZATION OF FORTIFICATION OF RICE WITH FOLIC ACID, IRON AND
β-CAROTENE BY THE PARBOILING METHOD
3.1. Introduction
As mentioned in section 2.2 (page no. 9) during parboiling, a hydrothermal process, the
crystalline form of rice starch in the paddy is transformed to amorphous form as a result of
irreversible starch swelling and fusion. This process essentially modifies the physical,
chemical and organoleptic properties of rice with added economic and nutritional properties
(Gariboldi 1974, Luh and Mickus 1980, Kasasian 1982, Bhattacharya 1985 & Pillayar 1988,
1990). Some major advantages of the parboiling process are (Ali & Ojha, 1976):
1. Increased head rice yield
2. Prevention of nutrient loss during milling
3. Salvaging of wet or damaged paddy
4. Rice preparation based on consumer requirements
Parboiling process involves soaking the rice in hot water for about 10-24 hours during which
time the paddy is saturated with moisture. The soaked paddy is then steamed till the starch is
gelatinized following which the paddy is milled (Joachim, 2011). This ancient process has
been practised in many Asian countries such as India, Bangladesh, Pakistan, Myanmar,
Malaysia, Thailand, and also in developed countries such as Switzerland, USA and France
(Pillayar, 1981).
Parboiling has been an effective means of fortification for minerals such as Iron (Prom-u-thai
et al.,2007), zinc (Prom-u-thaiet al., 2009), Iodine (Tulyathan, 2007) and also for a water
soluble vitamin - folic acid (Kam et al., 2012). Simultaneous fortification of zinc and iron
into rice has also been made feasible by Prom-u-thai et al.,(2011) using the parboiling
technique. Therefore, the feasibility of fortifying rice with a provitamin of fat soluble vitamin
A vitamin (β- carotene), a water soluble vitamin (folic acid) and a mineral (iron) was
explored in this study using the parboiling method. The aim of this study was to understand
(1) if parboiling would be a plausible rice fortification method for multi-micronutrients and
optimise the fortification conditions (2) the possibility to scale-up the optimized method (3)
47
the efficiency and uptake of the micronutrients by the grain using the parboiling method and
evaluating micronutrient interactions affecting uptake efficiency and (4) studying the loss of
micronutrients at various stages of the parboiling process.
3.2. Materials and methods
3.2.1. Brown Rice Samples
Dehusked brown rice (BR) of a known long grain variety (Pusa 1121) was purchased in large
quantities from a local supermarket in Sydney and stored at 11˚C for the entire period of
study. Prior to use, the rice was cleaned from dust and broken grains using the dehuller.
3.2.2. Fortificants used for Parboiling
Food grade folic acid was purchased from DSM (Catalogue No: 5004357004), Australia.
Lucarotin® 10 CWD S/Y (Catalogue No: 50051378) which has β-carotene encapsulated in a
proteinaceous matrix was purchased from BASF Company, Germany. Sodium EDTA (III)
(Ferrazone®-
Catalogue No: 18154-32-0) forms of iron was purchased from Akzo Nobel
Functional, The Netherlands was chosen as fortificant as it imparted least colour to the rice
post-processing and also had higher bioavailability compared to ferrous sulphate, ferrous
fumarate and ferrious bisglycinate (Trinidad et al., 2002)
3.2.3. Parboiling Procedure
Graded BR was soaked in water in the ratio of 1:2 with the micronutrients at 70˚C in a water
bath for 2 hours. The mass of micronutrients added is listed in Table3.1. The detailed process
of optimised parboiling condition is schematically presented in Figure 3.1:
Table 3.1 Concentration of micronutrient addition into rice for Parboiling (per 300g of
rice)
CONDITION FOLIC ACID (mg) -CAROTENE (mg) IRON (mg)
A 150 62.5 25
B 300 125 50
C 600 250 100
D 1200 500 200
48
Figure 3.1 Schematic representation of optimized parboiling condition
Each condition was replicated and a control sample was also prepared by parboiling the rice
without any micronutrients in the soaking water.
Soaking: Rice was soaked in water
or a water solution of nutrients in
the ratio 1:2 at 70˚C for 2 hours.
After 2 hours, the soaking solution
was discarded.
Steaming: Soaked rice was
steamed at 100°C for 1 hour to
induce starch gelatinization
Drying: The steamed rice was air
dried at room temperature until the
moisture content dropped to 10±2%.
Milling: The dried rice was then
milled at 0s, 60 and 120s for bran
removal.
0s 60s 120s
49
3.2.4. Parboiling procedure for Scale-up Studies
Graded BR (2.5 kg) was soaked in the solution of micronutrients in the ratio 1:2 at 70˚C for 2
hours followed by steaming at 100˚C for 1 hour (experimental condition) and it was
compared to soaking at 70˚C for 4 hours. The soaked rice was steamed for 15 minutes at
100˚C, followed by tempering for 45 min in an air-tight container. The samples were spread
on a tray and air dried at room temperature until the moisture content dropped to 10±2% .
Each condition was replicated and a control sample was also prepared by parboiling the rice
without any micronutrients in the soaking water. The masses of nutrients added were 1.2 g
folic acid, 500 mg β-carotene, 200 mg iron in 5L of water.
3.2.5. Milling of Parboiled rice
For each sample, 100g of parboiled brown rice was milled in a laboratory scale grain mill
(Satake Test Grain Mill, Japan) for 60 and 120 seconds to yield white rice. The
corresponding degree of milling was ~6 and 11% respectively.
Condition A (Table 3.1) was chosen to further explore the efficiency of the parboiling
process. The moisture content during soaking and steaming, leaching of solids during the
soaking step, loss of micronutrients in the soaking step and the concentration of
micronutrients in the presence of each other were examined using condition A. The results
will be presented in Section3.3.9 (page no. 76). The combinations of micronutrients added
during soaking process is presented in Table 3.2. Each nutrient was added singly into rice and
in combination of two and three.
50
Table 3.2 Micronutrient mass dissolved in 600 mL of soaking solution for the parboiling
process (mg/300g of rice)
Rice treatments β-carotene Folic acid Iron
1 62.5 - -
2 - 150 -
3 - - 25
4 62.5 150 -
5 62.5 - 25
6 - 150 25
7 62.5 150 25
3.2.6. Moisture content in fortified rice and mass of solids that leached out after
soaking
The moisture content of brown rice was determined by the standard oven method (AOAC,
2012 (Method 925.09)). Throughout soaking, 20g rice in triplicates was removed from the
water bath at regular intervals of time (0, 30, 60, 90, 120 minutes) using tea infusers. ~20g
samples were placed in moisture dishes, accurately weighed and dried in a vacuum oven at
70°C for 24 h. The dishes were removed from the oven and cooled for 3 h in a desiccator.
Moisture content was expressed on a wet basis (w.b.). The same method was once again
employed to assess the mass of solids leached into the soak water before and after soaking.
51
3.2.7. Cooking of parboiled rice
For cooking the rice, 10g of each sample was washed with approximately 30 mL of water and
was cooked with 25 mL of water in a 100 mL beaker covered by a watch glass. The total time
taken for rice to cook was 24 minutes at boiling temperature of water (100°C). The degree of
cooking was qualitatively assessed by mashing a rice kernel between glass slides and
observing the absence of white belly. Cooked rice was kept for ten minutes in the cooked
state to absorb moisture and then cooled to room temperature in the beaker covered with
watch glass and then mashed and homogenized using mortar and pestle for analysis. The rice
that was milled for 120s was used for cooking as the whiter coloured rice is popular with
consumers.
3.2.8. Sample extraction and analysis for the micronutrients
The soak water in which the rice was soaked during parboiling was analysed for
micronutrients concentration before (0 mins) and after 120 mins of soaking. Milled rice,
brown rice and the rice bran were analysed for each micronutrient concentration in duplicates
to determine the concentration of the fortified micronutrients in the grain and also to
understand the loss on milling. Cooked rice was also analysed for retention of micronutrients.
3.2.8.1. Analysis of Folic acid
Folic acid analysis was done according to Pfeiffer et al., (1997) & Tamura (1998) with some
modifications. Single enzyme extraction with α-amylase (from Bacillussubtilis) purchased
from MP Biomedical LLC, Australia, Catalogue No.: 100447 (100g)) by incubation of the
rice extract for 2h at 37°C was performed in a screw capped centrifuge tube (purchased from
Greiner Bio-one; catalogue no.: 82050-346). At the end of the incubation, the enzyme was
deactivated by heating the sample in boiling water bath for 3min. Sample was cooled and
then centrifuged at 4000rpm for 15min and the supernatant was collected for analysis.
Sample purification was carried out by solid-phase extraction (SPE) on a strong anion
exchange cartridge (500 mg, 3 mL, Phenomenex, Australia). The cartridge was conditioned
with methanol (2x 2.5 mL) and water (2x 2.5mL) to activate the sorbent and remove matrix
interfering components (Patring & Jastrebova, 2007). After loading an aliquot of the sample
extract, the cartridge was washed (3x2.5mL) with water and eluted with 0.1M sodium acetate
52
containing 10% sodium chloride (UNIVAR, Australia) and 1% ascorbic acid (Sigma-Aldrich,
Sydney, Australia) under gravity. Extreme care was taken such that the cartridge did not run
dry during the loading and eluting procedure. The purified sample was filtered through
0.45μm regenerated cellulose (RC) syringe filter (Minisart RC 25, Germany) prior to HPLC
analysis.
Analyses were performed using a HPLC system (model LC AD, Shimadzu Prominence,
USA) consisting of an autosampler, a thermostable column compartment (maintaining the
column temperature at 35°C) and a photodiode array detector (monitoring at 280nm). The
HPLC system was controlled by a computer running LC Solution Shimadzu Chromatogram
Data System.
The separation of folic acid was performed by using a reversed-phase Luna C18 column,
5μm, 150x4.6mm i.d. (Phenomenex, Australia) with a C18 security guard column
(Phenomenex, Australia). Folic acid was determined by using a gradient elution program with
acetonitrile and 30mM phosphate (potassium dihydrogen phosphate purchased from
UNIVAR, Australia) buffer (pH 2.2). The flow rate was 0.8mL/min. The injection volume
was 100μl. The gradient program was as follows: 5% (v/v) acetonitrile maintained
isocratically for the first 8 min. The acetonitrile concentration was then raised linearly to 24%
within 23min then returned to 5% after 5min. The run time between injections was 40min.
Peak was identified based on the retention time and absorption spectrum acquired for the
peak at 280 nm corresponding to the external folic acid standard. HPLC calibration was done
on a daily basis to ensure data integrity.
3.2.8.2. Analysis of β-carotene
β-carotene analysis was done according to Lamberts & Delcour (2008) by saponification of
the sample followed by liquid-liquid extraction.
The extract was dried under nitrogen and then made up with 3 mL of methanol and then
passed through 0.45μm Polytetrafluoroethylene (PTFE) syringe filter (Minisart RC 25,
Germany) prior to HPLC analysis. Analyses were performed using a HPLC system (model
LC AD, Shimadzu Prominence, USA) consisting of an autosampler, a thermostable column
compartment (maintaining the column temperature of 40°C) and a photodiode array detector
(monitoring at 450nm). The HPLC system was controlled by a computer running LC Solution
Shimadzu Chromatogram Data System.
53
The detection of β-carotene was performed by reverse phase HPLC using Luna C18 column,
5μm, 150x4.6mm i.d. (Phenomenex, Australia) with a C18 security guard column
(Phenomenex, Australia). β-carotene was determined using an isocratic elution with methanol
and acetonitrile in the ratio 40:60. The run time between injections was 30min. Peak
identification was based on the retention time and absorption spectrum acquired for the peak
at 450 nm corresponding to the external standard. HPLC calibration was done on a daily basis
to ensure data integrity (Lamberts & Delcour, 2008).
3.2.8.3. Analysis of Iron
Approximately 0.5 g of accurately weighed ground rice sample was digested overnight with
10 mL of 70% nitric acid. The digested samples were heated on a heating mantle until they
produced brown fumes. When the brown fumes subsided the samples were cooled and were
treated with 2 mL of 35% hydrogen peroxide. The samples were heated again for about 5
minutes and then cooled and filtered using Whatman® qualitative filter paper (55 mm
diameter; 11 µm pore size (particle retention)) to remove particulate matter. The filtrate was
then made up to 25 mL and analysed using ICP-OES. One ppm Yttrium was added online as
internal standard to correct for the loss of analyte during sample injection (United States
Environmental Protection Act (USEPA), 1996). The ICP-OES system (Perkin Elmer, Model:
Optima DV7300 ICPOES) was run under the following conditions:
54
Table 3.3 Conditions of ICP-OES instrument for Iron analysis
Radio Frequency Power 1300 watts
Plasma Gas Flow 15 L/min
Auxiliary Gas Flow 0.5 L/min
Nebulizer Gas Flow 0.70 L/min
Sample Introduction System Burgener PEEK Mira Mist nebuliser with
cyclonic spray chamber
Pump rate 0.5 mL/min
Viewing height 15mm above load coil
View mode Axial
Number of Replicates 3
Note:The quantification was performed using the calibration curve with iron standards
diluted from certified 1000 mg/L iron standard that was simultaneously run alongside
samples to maintain integrity of the analysis. The LOD was 0.05 ppm.
3.2.9. Calculation of % concentration of micronutrients in the soak water before and
after the 2 hours soaking: % uptake and % retention of micronutrients in rice after
parboiling and cookingrespectively
This study focused on the % initial concentration of micronutrients in the soak water before
the soaking process (0 min: before soaking) and % residual concentration of micronutrients
after the soaking process (120 min: after soaking), %uptake of micronutrients from the soak
water into the rice and % retention of micronutrients in the rice after cooking. The former two
terminologies (% initial concentration and % residual concentration) were performed when
rice was soaked with the micronutrients solution at 70°C for 120 minutes. In order to
familiarize with each terminology and how they are expressed mathematically they are
described in detail below.
% initial concentration of micronutrients in the soak water here refers to the concentration of
micronutrients present in the soaking water before the soaking step at 70°C (at 0 min soaking
55
time). After 2 hours of soaking the remanent concentration of nutrients can be referred to as
residual concentration, expressed as % in the remaining soaking water.
% initial concentration of micronutrients in the soak water = (F fortificant- S before soaking)/F
fortificant x 100 Equation (1)
% residual concentration in the soak water = (S before soaking- S after soaking)/S before soaking x 100
Equation (2)
Where F fortificant is the mass of fortificant added per 100 mL of water; S before soaking is the mass
of micronutrient per 100mL of the soaking solution before soaking (mg); and S after soaking is
the mass of nutrient remainingin the soaking solution (mg/100mL).
% Uptake of the micronutrients into the rice refers to the concentration of folic acid, iron and
β-carotene present in the rice kernel after parboiling, i.e.after soaking, steaming and drying.
The % micronutrient uptake in fortified parboiled rice (uncooked) was calculated as the ratio
between the concentration of the respective micronutrients present in the fortified parboiled
rice kernel and the corresponding folic acid added to the soaking solution as the fortificant.
Hence, the % uptake was calculated as follows:
% Uptake = (M fort-rice / M fort) x 100 Equation (3)
where Mfort-rice is the analysed concentration of nutrient in the uncooked fortified parboiled
rice; and, Mfort is the initial mass of micronutrient added to the rice in the (Table 3.1) soaking
water (600 mL).
The % retention of micronutrients in the following context refers to the concentration of
nutrients present in the cooked fortified parboiled rice in relation to the concentration of
micronutrients present in the uncooked form of fortified parboiled rice. The % retention was
calculated as follows:
% Retention = (M cooked rice/ M fort-rice) x 100 Equation (4)
where Mcooked rice is the analysed micronutrient concentration in the cooked fortified parboiled
rice.
3.2.10. Statistical Analysis
The data collected was analysed using a descriptive statistic package (Statistical Package for
56
Social Scientist or SPSS (version 21.0, 2013) and the results were interpreted and presented
on tables and charts. The least significant difference (LSD) at p < 0.05 was applied to
compare the means for significant differences of the treatments.
3.3. Results and Discussion
3.3.1. Optimization of the soaking step of parboiling
Husk is a significant barrier for the water absorption process in brown rice (Thakur & Gupta
2006 & Kam et al. 2013). Thakur and Gupta further investigated the diffusion coefficient for
water absorption studies of paddy and brown rice and stated that at different temperatures, the
diffusion coefficient follows an Arrhenius-type relation and the activation energy for the
Arrhenius equation was determined using the Ea/R parameter. The activation energy for
brown rice was 1.2 times higher than the activation energy for paddy and 1.9 times that of the
husk concluding that the husk imparted effective resistance to the water percolation process
in paddy. Therefore, dehusked brown rice was found to be an effective medium for rice
fortification rather than the whole paddy itself (Thakur and Gupta, 2006).
The rate of moisture migration into the rice depends on the temperature of soaking: higher the
soaking temperature, higher the rate of moisture migration (Thakur and Gupta, 2006). For the
present study, 70°C was considered optimum. Beyond 70°C, the rice after soaking was
cooked. The maximum time the rice could withstand temperature beyond 70°C was 15
minutes. Considering the efficiency of migration for the process of fortification, 70°C for 2
hours soaking was deduced to be optimum.
Two hours soaking time is optimum for folic acid fortification by the parboiling process
(Kam et al., 2012). In this study, 2 hours soaking time was adopted. The concentration of the
micronutrients after 1 hour soaking (data not presented) was much lower than 2 hours.
Increasing the soaking time further did not significantly increase the retention of the
micronutrients (data presented in the following section). Therefore 2 hours was chosen for the
soaking time.
Steaming, the next step in the parboiling process causes irreversible changes to the structure
of the starch granules in the rice due to the application of moist heat (100˚C). The kinetic
energy of the starch increased and intermolecular hydrogen bonds are ruptured, leading to the
disruption of structural integrity of starch (Lund, 1984) and starch gelatinisation. During
57
starch gelatinization, the swollen starch may fuse with the inner bran and scutellum layers in
the endosperm, preventing the loss of vitamin during further treatments such as milling (Rao
& Bhattacharya, 1966). For steaming temperature and duration, 100°C was chosen as the
optimum and the duration of steaming was 1 hour based on the quality parameter for
parboiled rice that would render parboiled rice glassy in appearance without any white belly
(Bhattacharya, 1985). This appearance was obtained at 100°Ctemperature and therefore based
on starch gelatinization and rice acceptance, this condition was chosen.
In terms of drying, shade drying at room temperature was chosen rather than mechanical or
sun drying to achieve maximum concentration of nutrients in the fortified rice that would
otherwise not be possible as the nutrients chosen to be added to rice particularly folic acid
and β- carotene are sensitive to heat and light. But by air drying there is an expected loss due
to oxidation at room temperature (Kam et al., 2013). Hence, shade drying was adopted and
the drying took longer (4 days) than mechanical or sun- drying, but the loss of vitamin were
likely to have been minimised due to air drying. Shade drying also resulted in better rice
milling quality and head rice yield from parboiled rice (Bhattacharya & Swamy, 1967).The
drying process was continued until the moisture content dropped to 10-12% (d.b.).
3.3.2. Moisture content of parboiled rice
Normally, soaking is done to achieve quick and uniform water absorption (Wimberly, 1983).
Wimberly (1983) also reported that the grain reaches a moisture level of 30-35% in 2 to 4
hours, which was also observed in this study. The effect of soaking time on moisture content
of rice is shown in Figure 3.2. The moisture analysis was done on samples that were soaked
with micronutrients according to condition A in Table 3.1 (page no.47).
58
Figure 3.2 Moisture content of rice with different fortificants during soaking at 30-min
time interval. BC: β-carotene; Fe: iron; FA: folic acid(The error bars represent standard
errors of corresponding means from three replicates)
Figure 3.2 shows moisture uptake during the 2 hours soaking period. Since the soaking is the
first step in the parboiling process and it is the key step in the inward diffusion of
micronutrients from the soaking water into the rice, moisture analysis is an essential step in
parboiling. After 30-min soaking, the moisture content for all samples increased, from ~22-
28% to ~28-30%. On soaking for 60 min, the moisture content was approximately 31%.
There was a further increase at 90 min for samples fortified with iron; folic acid; iron and
folic acid; and folic acid and β-carotene, where the moisture content reached 32%; whereas
others slightly increased. On soaking for 120 min, rice fortified with β-carotene achieved the
highest moisture content of 37%, whereas for folic acid the moisture content increased the
most from 22% at 0 min to 34% at 120 min. Similarly, in rice fortified with β-carotene and
folic acid, the moisture content increased from 24% at 0 min to 33% at 120 min.
0
5
10
15
20
25
30
35
40
45
50
0 30 60 90 120
Mo
istu
re c
on
ten
t (%
wb
)
Time (min)
No nutrients
BC
Fe
FA
Fe+BC
Fe+FA
FA+BC
Fe+BC+FA
59
Figure 3.3 Moisture content of rice after 2 hours soaking and 1 hour of steaming at
100 °C (The error bars represent standard errors of corresponding means from three
replicates)
After steaming, the moisture content of all samples further increased to35-37% (Figure 3.3).
In particular, rice fortified with Fe and folic acid had the lowest moisture content of 35%,
while rice fortified with folic acid had the highest moisture content of 37%. Steaming adds
water through condensation and therefore, it increases the total water absorption (Wimberly,
1983).
3.3.3. Leaching of solids during the soaking step of the parboiling process
The micronutrients were dissolved in the soaking water for fortification by the parboiling
process as per condition A mentioned in Table 3.1. Some changes in the concentration of the
micronutrients before and after soaking were expected. Therefore, the soaking water was
analysed before and after soaking for the initial and residual micronutrients and also for the
diffusion of solids into and out of the rice. Solids loss (measured after parboiling) increased
in the soaking solution for all treatments (Figure 3.4). The migration of nutrients was by
inward diffusion into endosperm only. While oil and protein present natively in rice are
reported to diffuse outward during the parboiling process (based on microscopic
observations) they cannot diffuse as readily as water-soluble vitamins through cell walls.
When the rice is boiled in excess water other solids such as starch and water soluble vitamins
are also leached out (Juliano, 1993). Bello et al., (2004) also studied the leaching of solids
from the rice during the soaking process. The results from the study conducted by Bello et
05
1015202530354045
Mois
ture
con
ten
t (%
wb
)
60
al., (2004) showed that for raw and dehulled rice the mass of solids leaching remained low
and practically constant with time during soaking. However for the white rice the mass of
solids leaching increased dramatically with time, particularly for the soaking temperature of
65°C and thereby leading to the inference that the presence of bran prevents water absorption
and therefore leaching of solids (Bello et al., 2004). Thus, in the current experiment, the mass
of solids that leached out after soaking could be from the starch, proteins and fat in the rice
and also the some endogenous water-soluble vitamin.
Figure 3.4 Total solids in rice soaking solution before and after parboiling for the
differentsoaking solutions (The bars represent standard errors of corresponding means from
three replicates)
0.000.020.040.060.080.100.120.14
Tota
l so
lid
s (g
)
0 min
120 min
61
3.3.4. Concentration of Nutrients during the soaking stage of the parboiling process
Table 3.4 shows theinitial concentration of micronutrients present in the soak water before
soaking (0 min) and theresidual concentration in the soak water after soaking (120 min)
expressed in % of concentration. The mass of micronutrients added per 300 g of rice was
based on condition A (Table 3.1).
Table 3.4 % of initial concentrations of micronutrient in soak water before and after
parboiling process and % residual concentration of micronutrients after 120 min of
soaking
Nutrient
loss Combination**
Concentration
in mg/ 300g
rice
%
micronutrients
in soak water
at 0 min
% residual
micronutrients
at 120 mins
β-carotene
β-carotene only 62.5 74±7.3 3.5±0.07
β-carotene, Folic acid 62.5,150 70±16.4 4±0.6
β-carotene, Iron 62.5,25 93±3.6 15.3±0.5
β-carotene, Folic acid,
Iron 62.5,150,25 76±17.6 5.6±0.6
Iron
Iron Only 25 99±0.2 15±0.2
Iron, Β-Carotene 25,62.5 99±0.1 0±0.1
Iron, Folic Acid 25,150 99±0.3 77±2.9
Iron, β-Carotene, Folic
Acid 25,62.5,150 98±0.3 54±1.8
Folic acid
Folic Acid Only 150 98±0.4 0.7±0.3
Folic Acid, β-Carotene 150,62.5 97±1.7 48±0.1
Folic Acid, Iron 150,25 98±0.3 37±0.2
Folic Acid, β-Carotene,
Iron 150,62.5,25 98±0.6 8±0.2
Notes:
Concentration values expressed as Mean±Standard Deviation, unit expressed in %
Rice was soaked with each of the nutrient combinations at 70°C for 2 hours for the
steeping process.
It is generally expected that the mass of micronutrients in the soaking water before the
process would be higher compared to after soaking. If this hypothesis was observed it can be
explained that it would be because of the diffusion of micronutrients along with water from
the soak water into the rice grain. The soak water is the micronutrient resource for the rice.
Therefore when there is change in the concentration of micronutrients in the soak water, it
62
can be expected that there would be change in the micronutrient concentration of the rice.
Food grade fortificants were added to the soaking water for the fortification process and each
of these fortificants had analytical purity value which was presented in the quality assurance
certificate for that fortificant. Minor discrepancies in the initial concentration of each
micronutrient in the study as presented in the above table were due to the analytical purity
and the results obtained comply with the product quality assurance certificate. However when
concentration decreases after soaking it implies that the micronutrients have penetrated into
the rice from the soak water thereby resulting in the depletion of initial concentration. Hence,
lower the concentration of micronutrients in the soak water after soaking, the higher its
concentration in the rice.
Therefore, key findings from Table 3.4 are as follows:
Initial analysed concentration for folic acid and iron were close to 100% of the
nominal mass added before soaking. Minor difference could be due to the analytical
purity of the fortificant since food grade micronutrients were used. .
For β-carotene the initial concentration was close to 95% when it was soaked along
with the other micronutrients and this concentration could be covered by the standard
deviation.
After 2 hours of soaking, higher residual iron was present in the soak water when it
was soaked with folic acid and in the mixture of fortificant. When soaked with β-
carotene there was close to 0% of iron in the soak water indicating that most of it has
been absorbed by rice. In the presence of β-carotene the uptake of iron was the highest
and hence it can be concluded that β-carotene enhances iron diffusion.
When folic acid was soaked with β-carotene and with iron there was higher residual
concentration compared to the other 2 conditions thereby indicating that in both these
conditions there is lesser penetration of folic acid than when folic acid was soaked
alone and in mixture of all the micronutrients.
Although β-carotene was present in lower concentration than folic acid and iron in the
initial concentration, after soaking much of it has diffused into the rice.
These results collectively show that the soaking step of the parboiling process is efficient in
the delivery of micronutrients into the rice. However, these results which shows difference in
absorption of micronutrients into the rice at different combinations of micronutrients (Table
3.4) were not statistically significant (p > 0.05) when the analysis of variance was carried out.
63
No correlation was found between β-carotene, Fe and folic acid concentration in soak water
before steeping (p = 0.22; 0.15; 0.46, respectively) and soak water after steeping (p = 0.27;
0.62; 0.18, respectively) in the presence of other nutrients. With p > 0.05, it is therefore
concluded that there are no mean differences between treatments in terms of concentration of
micronutrients present in the soak water.
Overall, it can be concluded that the uptake of nutrients by the rice grain during parboiling is
very effective as the residual concentration of micronutrients in the soak water after soaking
is quite low. This lower concentration of micronutrient in the soak water referred to as loss in
concentration (as previously mentioned) is described in detail below for each micronutrient.
3.3.4.1. Folic acid
As seen in Table 3.4 initially folic acid concentration is close to 100% in the soak water. The
decrease from the soak water is an indication of how much of a nutrient has diffused into the
rice. After 2 hours, there was higher decrease from the soak water. Folic acid soaked with
iron had the lowest loss in concentration followed by soaking with β-carotene implying that
much of the folic acid had not diffused into the rice. On comparing the condition of folic acid
loss to iron loss when these 2 micronutrients were soaked together iron loss was lowest as
well (Figure 3.7). This implied that folic acid uptake by the rice was limited in the presence
of another micronutrient and the uptake efficacy in rice was much higher when there was no
interference from other micronutrients.
Figure 3.5 Mean folic acid %loss in concentration in the soak solution after soaking for
120 minutes with different treatments (The bars represent standard errors of corresponding
means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)
0
20
40
60
80
100
120
FA FA+BC FA+Fe Mix
% l
oss
of
foli
c aci
d i
n t
he
soak
solu
tion
64
3.3.4.2. Vitamin A (β-carotene)
The % loss in concentration of β – carotene remaining in the soak water after the soaking step
at 70°C for 2 hours is presented in Figure 3.6. After 120 min, the concentration of
micronutrients remaining in the soak water is much less implying that the loss is due to
uptake by the rice during the soaking process. Soak water with only β-carotene and β-
carotene with folic acid had the highest loss in concentration while that with iron had the
lowest loss in concentration of residual concentration after soaking. From the results of %
loss in concentration of folic acid and β-carotene it can be said that in the presence of iron
both these micronutrients had the lowest loss after soaking indicating iron was a limiting
factor for uptake in rice during these 2 conditions. Hence the variation can be attributed to the
competition between the micronutrients to penetrate into the grain influencing the uptake
efficiency.
Figure 3.6 Mean β-carotene % loss in concentration in the soak solution after soaking
for 120 minutes with different treatments (The bars represent standard errors of
corresponding mean values from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)
.
3.3.4.3. Iron
Fe loss in concentration in the soaking water also increased after soaking, compared to that of
fresh soak water as depicted in Figure 3.7. An interesting finding in this soaking study was
that in the presence of folic acid the loss in concentration of iron in the soaking water is
lowest. This implies that there is lower uptake of iron into the rice when there is folic acid
78
80
82
84
86
88
90
92
94
96
98
BC BC+FA BC+Fe Mix
% l
oss
of
β-c
aro
ten
e in
th
e so
ak
wate
r
65
present in the soak water. However in the absence of folic acid when iron was soaked alone
and when it was soaked with β-carotene there was higher loss in the soak water and therefore
higher uptake by the rice. This could be due to the lowering of the pH of the soak water in the
presence of folic acid which significantly affected the uptake of iron by the rice. The pH of
the soak water with mixture of micronutrients was 4.71 before soaking and 5.8 after soaking.
In the case of folic acid with iron the pH of soak water before soaking was 4.92 and 5.71 after
soaking. Wu et al., (2010), reported that folic acid solubility in water increases with an
increase in temperature. And also greater solubility is observed in basic and high acidic
conditions. Therefore the increased solubility of folic acid could be a potential barrier for iron
uptake in rice.
Figure 3.7 Mean iron % loss in concentration in the soak solution after soaking for 120
minutes with different treatments (The bars represent standard errors of corresponding
means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)
3.3.5. Uptake of micronutrients in the fortified rice and effectiveness of Parboiling as a
method of fortification
The previous section focused on the micronutrients concentration in the soak water. As
mentioned the decrease of micronutrients in the soak water can be counted as gain in the rice.
Hence in this section, the results from concentration of micronutrients in rice soaked as per
conditions A, B, C and D as mentioned in Table 3.1with all three micronutrients combined
0
20
40
60
80
100
120
Fe Fe+BC Fe+FA Mix
% l
oss
of
iron
in
th
e so
ak
wate
r
66
will be presented. There was no detectable folic acid and - carotene found in the unfortified
(control) brown rice as it is not a natural source of these 2 compounds. Pusa 1121 basmati
rice (BR) variety used in this study had an innate concentration of 12±4.2 mg/kg of iron in it.
This concentration of iron in Pusa (1121) rice variety was comparable with the values
obtained by Anuradha et al., (2012) who reported Pusa 1121 contained 14-18 mg/kg of native
iron.
Figure 3.8 Concentration of micronutrients (d.w.b) in uncooked rice fortified by
parboiling at varying concentrations and milling times (0s, 60s and 120s) (Error bars
indicate the standard deviations between duplicates (A, B, C and D refers to the mass of the
micronutrients added))
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
Folic Acid
β- carotene
Iron
67
Table 3.5 Percentage uptake of micronutrients after the parboiling process in uncooked
rice showing concentration of micronutrients at different milling times at conditions A,
B, C and D.
Sample Name/milling time Folic Acid β-carotene Iron
A 0s 12.7 44a 43
b
A 60s 14.2 26ab
41.5
A 120s 4 21 43
B 0s 23.5 30 23.5
B 60s 21.7 18 16.5
B 120s 21 14 23.5
C 0s 25 11
a 19.3
C 60s 22 9ab
12.2
C 120s 21 8 11.1
D 0s 14 17
a 8.8
D 60s 11 12ab
7.5
D 120s 7 3 6.8b
Note:a, b& ab
denotes significant difference between samples
Figure 3.8 shows the concentration of micronutrients in parboiled fortified uncooked rice. At
various conditions of addition of the fortificants to the rice, folic acid showed the highest
concentration followed by β-carotene and iron. Folic acid concentration ranged between 20 to
560 µg/g rice. For β- carotene the concentration was 50 to 277 µg/g of rice and for iron the
concentration was 28 to 64 µg/g of rice. With the increase in the concentration of fortificants
added to the soaking water the concentration of micronutrients in rice increased. The
quantification of concentration was performed at 3 different milling times (0s, 60s and 120s).
Folic acid concentration was highest in the aleuronic layer of the rice which is in agreement
with an earlier study (Kam et al., 2012). In the case of β-carotene a similar trend was
observed. The aleuronic layer had the highest uptake and with the increase in the milling time
the nutrient concentration decreased. The concentration of iron, however, seemed similar in
the aleurone layer and endosperm. Fortification through parboiling increased the
concentration of iron in the outer surface of rice although,unlike folic acid and β-carotene,
there was very little penetration into the grain. This can be proven with the data from the soak
water where there was high residual concentration of iron when the fortificants were present
in the mixture (Table 3.4). The migration of micronutrients by inward diffusion into the
endosperm during the soaking stage (Lund, 1984) occurs due to the difference in the moisture
gradient between the rice kernels and the surrounding environment. During soaking there was
68
a positive uptake of the nutrients into the rice as the soaking was determined to be the main
step of fortification during the parboiling process.
According to Prom –u- Thai et al., (2008) Fe fortified rice contained 45-96% of added Fe
when milled at 60 s and 20-98% when milled at 120s. The concentration also varied
depending on the cultivar of rice that was used for fortification. In the same study solubility
of Fe by using 2 fortificants (FeSO4 and Na2EDTA-Fe) for rice fortification by parboiling
were studied. There was minimal variation to concentration of Fe due to the solubility of the
fortificants (Prom –u Thai, 2008). In the present study, iron concentration in the fortified rice
did not increase vastly after fortification even when 200 mg of iron was added per 300 g of
rice. Thus the uptake of iron during fortification was limited. The reason for this has been
discussed in the following section where high iron was retained at in the rice bran when
soaked at condition A. Soaking rice at the lowest concentration of fortificant for iron the
uptake was over 100%, perhaps because the innate iron (12±4.2 ppm) in brown basmati rice
contributed to the final concentration.
Folic acid and iron fortification of rice by the parboiling method has been well studied
previously as discussed in the above paragraphs. However, fortification of rice with β-
carotene was novel as the common forms of β-carotene fortificants include retinyl palmitate
or retinyl acetate(Ranum, 2000 and Stringer, 2000). These are fat soluble and hence pose as a
challenge for rice fortitifcation. Thus by using a water-soluble form of β-carotene this
challenge was overcome in this study. Compared to iron and folic acid this form of β-
carotene (Lucarotin®) was not as soluble initially as it can be seen from Table 3.4 where there
was less concentration of it present in the soak water before the soaking step. However after
soaking, there was less residual concentration of β-carotene in the soak water showing that
much of it had been absorbed in the rice (85% in rice condition A and 14% in rice bran).
The higher the concentration of fortificants added to the rice the lower was the % uptake. At
higher concentrations there was higher loss of fortificants and the uptake was poorer (Table
3.5). Therefore the lowest concentration of fortificants addition was better than higher
concentrations although when higher concentrations of the fortificants were added the
concentration for each micronutrient in the rice grain after milling was higher after
fortification. Addition of lower concentrations of fortificants to the soaking water can also
reduce the overall cost of the finished fortified product and could be an economical solution
for implementing fortification program.
69
3.3.6. Loss of nutrients on milling
During parboiling while soaking the rice with the nutrients, penetration of the nutrients into
the rice kernel occurred: some into the bran and some into the endosperm. Fortified rice was
milled at 0s (no milling), 60s and 120s and the concentration analysis was done for folic acid,
β-carotene and iron in the above section. With the increase in milling time there was a
decrease in the concentration of the micronutrients. Because folic acid was more concentrated
on the outer surface of the grain (Kam et al., 2012), longer milling meant more folate loss.
Experimental data obtained from analysing the concentration of fortified micronutrients in
the rice bran is presented in this section. As it can be seen from the figure 3.9 most of the iron
has been retained in the bran rather than in the endosperm of the rice. According to Doesthale
et al., (1979) the degree of milling affected endogenous iron concentration during parboiling
and that the movement of iron into the endosperm was very weak. Therefore based on the
results from present study it can be inferred that iron, although soluble in water, did not
penetrate very easily in to the grain during the parboiling process. The concentration of β-
carotene in the rice bran was very low. This once again shows that there is high uptake of β-
carotene in the fortified rice and parboiling can be an effective method for β-carotene
fortification using Lucarotin®. It is therefore apparent that the nutrients penetrate several
layers of the rice grain and are retained in the starchy endosperm. There is considerable loss
during milling. The rice bran along with the added micronutrients can be salvaged to produce
by- products for making porridge for example to maximize the utilization of the fortified
product. Lipase present in rice bran causes rancidity due to oxidation and this process is rapid
when bran is separated from rice (Saunders, 1990a). Since parboiling process can deactivate
lipase the shelf-life of rice bran can thus be improved (Silva, Sanches, Amante, 2006, Slavin,
Lampe, 1992). Therefore there is higher value-addition to the fortified rice bran due to
parboiling.
70
Figure 3.9 Concentration of micronutrients in the fortified rice bran (Condition A)
during milling(Error bars indicate the standard deviations between duplicates)
Figure 3.10 Schematic representation of mass balance (on a d.w.b.) in terms of nutrients
concentration and loss at key stages of parboiling in rice soaked with all the
micronutrient at condition A (FA- 150mg; BC- 62.5 mg; Fe – 25mg/600mL soaking
solution used on 300g rice)
From the above schematic Figure 3.10 it can be seen that the maximum loss due to the
process was observed for folic acid and there was no loss for iron. As rice has endogenous
iron present in it and the iron content of the rice was higher after the parboiling process.
0
50
100
150
200
250
60s 120s
Con
cen
trati
on
in
µg/g
Milling Time
Folic acid
β-carotene
Iron
71
There was high concentration of residual iron in the soak water after the soaking step. This
shows that the concentration for iron was close to saturation and therefore there was lack of
concentration gradient which can cause lesser uptake of iron into the grains during the
process compared to the other 2 micronutrients. As it can be seen from Figure 3.10 564% of
iron has been retained in unmilled rice showing that there is exceeding concentration of iron
in the rice itself and hence can be explained by the residual iron in the soak water. Iron
uptake was also limited in the presence of folic acid (Table 3.4-page no.61).For β-carotene
the results from the % loss in soak water implied that ~99% had been lost after soaking and
99% of it was taken up by the rice (Table 3.5- page no.67& Figure 3.9 page no. -70). This
implied that ~1% has been lost due to the parboiling process and thus adding low
concentration of β-carotene fortificant in the form of Lucarotin®was highly efficient for rice
fortification using parboiling.
3.3.7. Retention of micronutrients on cooking the fortified parboiled rice
Rice is always consumed cooked. Therefore it is essential to know the loss of micronutrients
after washing and cooking the fortified rice. This would give a clear picture of whether the
micronutrients from the fortified rice would be able to meet the RDI values. Therefore, in this
section results from the cooked fortified rice has been collated and presented.
Figure 3.11 Retention of micronutrients in cooked rice fortified by parboiling at varying
concentrations (Error bars indicate standard deviation between replicate samples; A, B, C
and D refers to the concentration of the micronutrients added to the soaking water)
Absorption method for cooking was followed and the rice milled for 120s was chosen for
cooking as it had 11% bran removal (on an average) and was therefore whiter than the 0s and
0
20
40
60
80
100
120
140
160
180
200
A B C D
Folic Acid
β-carotene
Iron
72
60s milled rice. Figure 3.11 shows the retention of each micronutrient after cooking the
parboiled rice.Nutrients were lost on cooking. Folic acid was well retained followed by iron
and then β-carotene which showed highest loss compared to uncooked rice after cooking.
73
Table 3.6 % retention of micronutrients after cooking fortified parboiled rice (milled at
120s) (concentrations of the fortificant added in mg/ 300g of rice)
Treatments Folic
Acid
(FA)
%
Initial
concentration
of FA added
β-
carotene
(BC) %
Initial
concentration
of BC added
Iron
(Fe)
%
Initial
concentration
of Fe added
A 98 150 35 62.5 90 25
B 46 300 30 125 95 50
C 35 600 27 250 81 100
D 65 1200 39 500 89 200
Table 3-6 shows the % retention of micronutrients in milled (120s) fortified rice after
cooking. For folic acid the highest retention was at the lowest concentration of fortificant mix
for all three micronutrients after cooking the rice. This means that when low mass of
fortificant was added the % retention is higher after cooking. This could minimize the
quantity of fortificant needed to be added when a food is being fortified by the principle of
diffusion as long as the actual concentration was adequate. For β-carotene highest retention
was with treatment D (500 mg of fortificant) followed by treatment A (62.5 mg) and for iron,
the highest retention was with treatment B (50 mg). It is evident that iron is more stable than
the other micronutrients being a mineral and it is least affected by the cooking process in
fortified rice compared to folic acid and β- carotene. Although ~99% of β-carotene was taken
up by the rice during fortification there was heavy loss during cooking.Degradation of β-
carotene was high when subjected to heat (Borchgrevink and Charley, 1966; Lee and
Ammerman, 1974; Teixeira Neto et al., 1981). Apart from cooking in most Asian countries,
the practice of washing the rice before cooking is common.Due to this about 20 to 100% of
the vitamins are lost depending on the volume of water used in rinsing and the length of
cooking time (Hoffpauer, 1992). In ultra-rice (extruded fortified rice) vitamin A (in the form
of retinyl palmitate) loss was measured under normal rice cooking conditions (approximately
5 min boiling followed by 20-25 min under low-heat) and it was reported that there was a
loss of approximately 26% due to cooking (Flores et al., 1994). Food folates are lost on
cooking by 50%, however folic acid is more easily absorbed and less affected than folates
(tetrahydrofolate, 10-formyltetrahydrofolate, 5,10- Methyltetrahydrofolate, 5-
methyltetrahydrofolate) upon cooking (FAO, 2002). On the whole, lower concentrations of
fortificants seemed effective in terms of uptake during parboiling and after cooking the
fortified rice thus proving the robustness of the fortification process because three
micronutrients could be fortified into rice by a single processing method.
74
3.3.8. Pilot scale studies of experimental condition in comparison to conventional
parboiling condition
The lab scale process (300g of rice) was conducted on a pilot scale (2.5 kg of rice) in order to
see if there was variation in uptake of the micronutrients during parboiling and retention after
cooking due to the scale-up of the parboiling process. If this optimized process is to be
commercialized, it would make more sense economically to produce rice in batches of
several tonnes rather than in small quantities. Therefore as a small comparison to the
industrial batch scale the pilot scale study was performed using condition D (FA- 1.2 g; BC-
500 mg and Fe- 200 mg/ 300g of rice). Condition D used highest concentrations of the
nutrients added to the rice which showed higher concentration in parboiled fortified rice
based on the results from the lab-scale studies (Figure 3.8 -page no. 66).
The uptake of the nutrients performed on a lab scale after soaking for 2 hours (UNSW
optimized condition) and 4 hours (conventional parboiling condition, IICPT, Thanjavur) is
presented in Figure 3.12. There was minimal difference between the different soaking times
for folic acid and iron. In the study conducted by Kam, Arcot & Ward (2012) it was found
that progressive increment in the degree of gelatinization coincided with the increasing folic
acid uptake for prolonged soaking durations (up to 4 hours). Therefore it was reported that
starch gelatinization may be a potential contributor to retain or bind the folic acid (fortificant)
that had diffused into the endosperm from the soaking water (Kam et al., 2012). For β-
carotene the concentration was higher during 2 hours soaking than for 4 hours soaking.
Longer soaking time resulted in poorer uptake of β-carotene. Therefore shorter soaking time
can be better for higher uptake of micronutrients during parboiling.
75
Figure 3.12 Comparison of micronutrient concentration(in d.w.b) in uncooked rice
using experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4
hours) on pilot scale (Error bars indicate standard deviation of duplicate samples)
Figure 3.13 Comparison of micronutrient retention in (in d.w.b.) cooked rice using
experimental condition (70˚C for 2 hours) vs conventional condition (70˚C for 4 hours)
on pilot scale(Error bars indicate standard deviation of duplicate samples)
There was no significant difference in uptake of nutrients between the soaking times (2 hours
and 4 hours) and different milling times using single factor ANOVA with p>0.05 (p = 0.7 for
folic acid; p= 0.3 for β – carotene; p= 0.9 for iron). Folic acid was retained more followed by
β-carotene and then iron. Concentration of nutrients at 0s upto 120s milling was 415– 230
mg/kg for folic acid; 247-86 mg/kg β-carotene; 53-39 mg/kg for iron (on d.w.b.) (Figure
3.12)
0
50
100
150
200
250
300
350
400
450
500
2
hours
0s 60s 120 s 4
hours
0s 60s 120 s
Con
cen
trati
on
in
µg/g
Folic Acid
β-Carotene
Iron
0
20
40
60
80
100
120
140
160
180
200
2 hours 4 hours
Con
cen
trati
on
in
μg/g
Folic Acid
β-Carotene
Iron
76
The retention of micronutrients after cooking in the scale-up study was similar to that of the
lab scale study. There was no significant difference between the two methods (folic acid p=
0.3; β- carotene p = 0.7; iron p = 0.7) using single factor ANOVA (p≥0.05). However, slight
variations between lab scale and pilot scale studies can be attributed to individual variation in
the size and shape of rice kernels as the distribution of the nutrients varied with each rice
kernel. There was no significant difference between the two different soaking times even
after cooking as shown in Figure 3.13 (p ≥0.05). The concentration of micronutrients in the
uncooked fortified rice at pilot scale was also similar to lab-scale concentration results (p ≥
0.05). This shows that the parboiling condition is reproducible for large scale production.
3.3.9. Micronutrients interaction affecting uptake of each other during the parboiling
process
In this study the novelty was that rice was fortified by parboiling with not just one
micronutrient but with three. It is possible that these micronutrients might have a synergistic
or antagonistic effect on each other during the soaking step. This was studied further by
analysing the concentration of each of these micronutrients in the presence of each other and
individually as well. This interaction has not been systematically studied and there is limited
information in terms of micronutrient synergistic effects in food, although there is a lot of
information about micronutrient interaction through in vivo and in vitro studies (Sandström,
2001, Gibson et al., 2006, Arigony et al., 2013). Hence Section 3.3.10 focuses on the
concentration of micronutrients in rice at different combinations using treatment A (FA- 150
mg, BC- 62.5 mg and Fe- 25mg/600 mL of soaking water)
3.3.10. Nutrient concentration in dried parboiled rice after milling
3.3.10.1. Folic acid
Folic acid concentration in rice was high when it was either soaked alone or when it was
combined with both iron and β-carotene. This can also be seen from the soak water data
where there is lesser residual folic acid in these 2 conditions compared to the other two
(FA+Fe and FA+ BC).
77
Figure 3.14 Mean folic acid concentration (in d.w.b) in dried parboiled rice with
different fortifications before and after milling at 60 s and 120 s (The bars represent
standard errors of corresponding means from three replicates. BC: β-carotene; Fe: iron; FA:
folic acid)
Observations are consistent across the different fortification conditions of rice studied
(p<0.05), where concentration of folic acid declined in milled rice (Figure 3.14). In unmilled
rice, folic acid concentration ranged from 39 to 43μg per g rice. At 60 s of milling, folic acid
concentration ranged from 25 to 39 μg/g and from 24 to 26 μg/g for 120 s. The uptake of
folic acid by the rice was similar for all the conditions and as mentioned before there was loss
due to milling. During starch gelatinization, water-soluble vitamin may adhere to the
endosperm (Bhattacharya, 2006). Folic acid concentration n in the starchy endosperm (at
120s milling) is almost consistent showing comparable level of diffusion across different
treatments.
3.3.10.2. Vitamin A (β-carotene)
Rice that was soaked only with β-carotene showed the lowest uptake. When it was
soakedalong with folic acid and iron it showed highest uptake which was comparable to
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
0 60 120
Foli
c aci
d c
on
cen
trati
on
(u
g/g
)
Milling Time (s)
FA
FA+Fe
FA+BC
FA+Fe+BC
78
Table 3.5. From the soak water data (Table 3.4- page no.61) residual concentrations of β-
carotene was not highly varied for the following treatments: β-carotene, β-carotene and folic
acid and β-carotene, folic acid and iron. The reason for lower uptake when soaked alone
could be due to lack of competition during the diffusion process which occurs due to the
change in the moisture gradient between soak water and rice during the soaking step of the
parboiling process.
Figure 3.15 Mean vitamin A (β-carotene) concentration in (d.w.b) dried parboiled rice
(11%) with different treatments before and after milling at 60 s and 120 s (The bars
represent standard errors of corresponding means from replicates. BC: β-carotene; Fe: iron;
FA: folic acid)
The β-carotene concentration decreased with increase in milling time (Figure 3.15). Some
fortified parboiled rice samples showed very small carotenoid peaks when analysed using the
HPLC which was hard to be quantified (Method limit of detection: 0.07µg/mL). Studies by
Lamberts and Delcour (2008) suggested that the decrease of β-carotene in brown rice during
parboiling was due to the loss of soluble solids in the soaking water, oxidation of carotenoids
during soaking and destruction of carotenoids during steamingThe concentration results for
β-carotene in fortified rice (Figure 3.15) were comparable to those in Figure 3.4(page no. 60),
where total solids were higher when β-carotene was present in soaking water after parboiling
compared to other treatments.
3.3.10.3. Iron
Iron uptake was the highest when it was soaked along with β-carotene and this can be
compared to Table 3.4 where there is close to 0% residual Fe in the soak water after soaking.
0
20
40
60
80
100
120
140
160
180
0 60 120
β-c
aro
ten
e co
nce
ntr
ati
on
(µ
g/g
)
Milling Time (s)
BC
BC+Fe
BC+FA
BC+Fe+FA
79
When it was soaked with the other 2 micronutrients there was not much loss due to the
milling process (23 -55%) at varying degrees of milling.
Figure 3.16 Mean iron concentration in (d.w.b.) dried parboiled rice with different
fortifications before and after milling at 60 s and 120 s (The bars represent standard errors
of corresponding means from three replicates. BC: β-carotene; Fe: iron; FA: folic acid)
The concentration of Fe in all milled rice declined, except for milled rice fortified with Fe
and β-carotene which increased from 55 µg/g at 0 s to 64 µg/g of Fe at 120 s (p>0.05). In the
presence of β-carotene the diffusion of iron into the rice grain could be enhanced and
therefore the loss during milling could be lesser implying higher uptake. Studies by Layrisse
et al., (1997) indicated that physiologically vitamin A aided in iron absorption in the presence
of inhibitors by mobilizing iron reserved in haemoglobin synthesis. In the study conducted by
Gracía-Casal et al., (1998) it was concluded that β-carotene had a positive effect on iron
absorption in humans. The form of β-carotene (Lucarotin®
) used in this study also contained
other antioxidants – ascorbyl palmitate (2%) and DL- alpha- tocopherol (2%). The effect of
anti-oxidants on iron could be higher when it was soaked along with β-carotene and therefore
showed higher uptake in this case. Protection of iron in the presence of anti-oxidants was well
studied in vitro and in vivo(Pizzaro et al., 2006& Yamamoto and Niki, 1988).However the
mechanism of increased Fe absorption progressively in rice as seen in this study could
potentially be due to the same mechanism as at physiological level. This could be the reason
why Fe concentration increased with milling time only with BC as this trend is opposite to
normal trend where there is high loss of micronutrients with increase in milling time. This
result needs to be confirmed through further detailed study to understand the exact
mechanism.
0
20
40
60
80
100
120
0 s 60 s 120 s
Iro
n C
on
cen
tra
tio
n in
µg
/g
Milling time (s)
Fe
Fe+BC
Fe+FA
Fe+FA+BC
80
3.3.11. Efficacy of parboiling as a means of fortification
Fortification does not require changes in existing food patterns. Thus, in this study,
estimation of nutrient intake based on rice consumption can be made to deduce the
effectiveness of rice fortification. Average per person consumption of rice differs from
country to country. In Bangladesh, where parboiled rice is commonly eaten, the average
annual rice consumption (kilograms) per person is 160 kg (IRRI b, n.d.), therefore it is
assumed that ~440g of rice is consumed per day. The following calculations are the
estimation on nutrient retention in cooked fortified rice as a premix based on average rice
consumption per day.
For folic acid, its bioavailability in fortified foods is about 85% (NHMRC, 2006). Therefore,
it can be assumed that:
Folic acid concentration in 440 g of cooked fortified rice/ day = 20 μg/g x 440 = 8800 μg
= 8.8mg of folic acid/ 440g of rice
Theoretically folic acid bioavailability (85%) in this fortified rice would be = 0.85 x 8.8 mg =
7.48 mg = 7480 μg/ 440g of rice
However, given that the upper level of intake for dietary folate equivalents is less than 800 μg
per day for children, and 1000 μg per day for females, the folic acid concentration in
parboiled rice needs to be modified to avoid overdose of folate intake for long-term
consumption. However an overdose of folic acid does not have any adverse effect as it a
water-soluble vitamin (Butterworth & Tamura, 1989).
Retinol equivalent expressed as vitamin A -1 μg is assumed to equate to 6 μg of β-carotene
when converted in the human body and the RDI of vitamin A for an adult female is 700 μg
(ODS-NIH, 2006) which is equivalent to 4200 μg of β-carotene. From our study, the
parboiled rice had at least 17.9 μg/g of β-carotene. Assuming that the bioavailability (ranges
from 5% to 65%) of the β-carotene is similar to those in vegetables and fruits (Haskell,
2012),
β-carotene concentration in 440 g of cooked fortified rice/per day = 17.9 μg x 440 = 7876 µg/
440 g of rice
81
β-carotene bioavailability in fortified rice (5% bioavailability) = 0.05 x 7.8 mg = 0.39 mg =
390 μg/ 440 g of rice
β-carotene bioavailability in fortified rice (65% bioavailability) = 0.65 x 7.8 mg = 5.07 mg =
5070 μg/ 440 g of rice
Thus, it can be concluded that the premix fortified rice can supply about 50% of the RDI as
Vitamin A equivalent and the bioavailability of β-carotene ranges between 390- 5070 μg.
Hallberg et al (1998) estimated that only 15% of fortified iron was well absorbed due to the
use of low-bioavailability elemental iron powders and the iron inhibitors in the diet. The iron
bioavailability further decreases to 10-12% when the diet consists of little meat with more
fruits and vegetables, and whole grain cereals (Hallberg et al., , 1998). There is evidence
from Meng et al. (2005) that iron bioavailability in rice can be as low as 1% due to many
inhibitory factors in plant foods. Considering a meal in Bangladesh is made up of fortified
rice with meat and vegetables, it is therefore assumed that:
Iron concentration in 440 g of cooked fortified rice/ day = 0.045 mg/g x 440 g = 19.8 mg of
iron/day.
Iron bioavailability in fortified rice = 0.15 x 19.8 mg = 2.97mg of iron absorbed/ 440 g of rice
According to ODS-NIH (2014), RDI of Fe for men and women at different life stages is 8 mg
and 8-18 mg, respectively. Thus, the daily iron intake of an individual would be fulfilled if
the individual relies on fortified rice as the sole source of dietary iron based on RDI.
The mentioned quantity of rice consumption (440 g) is an approximate average value and the
quantity of rice consumed varies between countries and individuals and these calculated
values are based on the consumption of premix i.e. undiluted fortified rice. This premix is
intended to be diluted to be able to supply at least 50% of RDI for long-term consumption as
the rice premix provides more than 100% of RDI for folic acid and β-carotene. The
concentration of micronutrients are likely to reduce by 1/10th
keeping in mind the dilution
ratio (1:10).The data from this after diluting the fortified rice is described in detail and
presented in chapter 6 when sensory analysis was conducted.
82
Based on the above results it can be concluded that parboiling is an efficient rice fortification
processing method. However like any fortification development programme, rice fortification
is also challenging. The added advantage for rice fortification would be that it is a staple crop
that has been industrialized in terms of large scale processing and milling. Hence with the
data obtained from the scale-up process for rice fortification can be easily implemented
unlike other crops. As previously mentioned in the literature review cost for rice fortification
can be complex and involves a complex supply chain. However with the current scenario of
worldwide micronutrient deficiency, implementation of low cost rice fortification in countries
where rice is a staple can be a comprehensive approach to reduce the prevalence of vitamin
and mineral deficiency (Sydney Wealth Area Health Service, 2009). The collective results
from the current study show that parboiling could be an efficient and possibly a low cost
alternative than extrusion technique for rice fortification with multiple micronutrients.
3.4. Conclusions
The present study was systematic using multiple micronutrients for fortification of rice using
the parboiling process. The concentration of micronutrients at every stage of the parboiling
process (soaking, heating, drying and milling) was analysed and a summarized theoretical
mass balance can be provided for the process. The loss in concentration of micronutrients
from the soak water after 2 hours was close to 99% which indicated that most of the
micronutrients were used in the process. During parboiling process a significant
concentration of the micronutrients diffused into the rice and yet there was loss in particular
for the vitaminsdue to the processesnamely milling and cooking. The effectiveness of
enhancing nutrient uptake in rice through the nutrient-fortified parboiling process is far
greater than expected as more than half of the fortificants were retained in grains based on the
low concentration left in the soaking solution. However, nutrients concentration decreased
after steaming, drying and milling. The combined fortification of β-carotene and Fe in rice
has shown the effectiveness of Fe uptake in milled rice, where Fe concentration was higher at
120s than at 0s and 60s of milling. Also, some micronutrients show higher concentration in
the presence of another micronutrient, as seen in the case of FA and Fe, where both nutrient
concentrations appeared to be the highest among the fortification combinations at 120s
milling. Therefore, despite the fact that low concentrations of all three micronutrients were
seen in milled rice relative to what was added originally these studies indicate that parboiling
83
can still assist in the uptake of micronutrients to a large extent seeming it a successful
technique for fortification.
Fortification of rice with three micronutrients simultaneously using parboiling is feasible.
Lowest concentration of fortificants (Condition A) gave highest uptake during parboiled rice
and retention after cooking. The concentration of micronutrients through this study was as
follows: Folic Acid>β-carotene>iron (at different milling times) in parboiled fortified
uncooked rice with evident loss during milling. When lowest masses of fortificants were
added to rice during parboiling, it was found that approximately 4% of folic acid, and 21% of
- carotene and 43% of iron diffused into the uncooked rice during the fortification process at
120s milling. On an average about 60% of the folic acid, 33% of β-carotene and 78% of iron
was retained after cooking the fortified rice based on the concentration of micronutrients in
the uncooked rice which was taken as 100%. Retention of micronutrients after cooking was
as follows: Folic acid>iron>β-carotene. Rice bran contained significant concentration of
micronutrients when milled and therefore can be used for making by-products. The fortified
rice can be diluted with unfortified rice if used as a premix in the ratio of 1:10. The
concentration after scale-up (in uncooked and cooked rice) did not show significant
difference to that of laboratory scale (single factor ANOVA) which means that the optimized
parboiling condition in the lab is reproducible on a commercial scale.The efficiency of
parboiling as a means of processing technique for rice fortification can be seen with the %
uptake of the micronutrients in the uncooked rice and the % retention of micronutrients in the
cooked rice. Consumption of 100 g of the triple micronutrient fortified rice diluted with white
rice after cooking can provide at least half of RDI for all three micronutrients. This could
vary, depending on the country‘s rice eating pattern. Overall the novelty of this study was to
fortify rice with not just one but three micronutrients without deforming the rice kernel and
this was successfully established using the parboiling process. Fortification of β-carotene in
rice has always been a challenge due to its lipophilic nature. This was also overcome in this
study by using a water-soluble form of β-carotene.
84
CHAPTER 4
4. FEASIBILITY OF FORTIFYING RICE WITH FOLIC ACID, IRON AND β–
CAROTENE BY HIGH PRESSURE PROCESSING (HPP) METHOD
4.1 Introduction
High pressure processing is one of the emerging technologies which could be an alternative
to thermal processing technologies in foods. HPP technique offers a safe and effective
method in food processing by modifying protein structures, formation of new chemicals and
inactivating enzymes in foods (Yaldagard, Mortazavi and Tabatabaie, 2008). The major
advantages of using high pressure processing are (Knorr, 1993):
i. Inactivation of microorganisms and enzymes
ii. Modification of biopolymers
iii. Quality retention such as colour and flavour
iv. Changes in product functionality.
Food products such as jams were found to retain freshness, food colour and flavour upon
chemical and spectrophotometric analysis (Watanabe et al., 1991; Kimura et al., 1994;
Dervisi et al., 2001). Preliminary studies conducted by Kato et al., (2000) showed that rice
grains immersed in distilled water released rice allergenic protein which was dissolved in
water when the rice was treated at 100- 400 MPa.
Despite its wide and novel applications in the field of food processing it was found that there
were limited studies on rice, a staple food in many parts of the world and also a high source
of energy and protein. High pressure treatment of rice caused alterations in the cooking
properties and enzymatic digestion (Arai and Watanabe, 1994). Rice that was soaked and
treated by HPP resulted in changes in the internal rice structure, higher sugars and denatured
water soluble proteins. Rice that was cooked after HPP process also had better palatability
(Yamakrua et al., 2005).
Li, et al., (2011) investigated physicochemical properties of rice starch in water suspension
when subjected to high pressure treatment. The physical properties and structural properties
of the rice starch were studied by performing rapid visco analysis (RVA), differential
scanning calorimetry and X-ray diffraction (XRD) and also by observing the starch molecules
under polarised light microscope and electron microscope respectively. They reported that
85
HPP could be a potential non-thermal processing method to produce a new modified starch
(Li et al., 2011).
Although there are recent studies about the effect of HPP on rice, there was no evidence for
the possibility of fortification of rice with micronutrients. Therefore in this chapter the
feasibility of rice fortification with folic acid, iron and β- carotene were studied. Varying
pressure and time were tried and the most favourable fortification condition was deduced by
analysing the concentration of the micronutrients in the rice. The physicochemical properties
of the fortified rice were also studied (presented in detail in Chapter 5).
4.2 Materials and Methods
4.2.1 Preparation of rice and fortificants mixture
Brown basmati rice (Pusa 1121) was purchased commercially at a local grocery in Sydney,
Australia and milled for 120s in a laboratory scale milling equipment (Satake: Model TM05,
Japan) to produce white rice. When brown rice was treated with HPP it resulted in broken
kernels, even after soaking making it a barrier for rice fortification. Hence milled white rice
was used in this study. The milled white rice (25 ±0.5 g) was weighed into a 50 mL
centrifuge tube (purchased from BD Biosciences, Sydney Australia). To prepare the
fortificant solution folic acid (150 mg), iron (25 mg) and β- carotene (62.5 mg) were
dissolved in 600 mL of deionised water and was incubated at 37°C in a shaking water bath
for 15 minutes for increasing the solubility of the micronutrients in the water. Fortificant
solution (40 mL) was added to the rice in the centrifuge tube and the tube was packed airtight
with the fortificant- rice mixture such that there was no air in the tube. The centrifuge tube
containing rice mixed with excess fortificant solution was ultra-sonicated for 5 minutes to
remove minute air-bubbles in the water (the presence of which might cause breakage of the
centrifuge tube during HPP treatment). The tubes were then packed into triple cryo-bags and
were placed in the high pressure processing chamber for treatment. The high pressure
treatment was done in triplicates and the analysis of micronutrient concentration in the rice
post-treatment was also done in triplicates for each micronutrient. Unlike parboiling the soak
water (Section 3.2.8) was not analysed for residual micronutrients concentration as very little
soak solution was present in the tube after treatment.
86
Figure 4.1 Flow chart of sample preparation for Hydrostatic High Pressure Processing
treatment
4.2.2 High Pressure Treatment of rice samples
High pressure equipment - Mini Food Lab (Model No. S-FL-850-9-W / FPG5620TC25)
(Stansted ,United Kingdom) was used for this study. The pressurising fluid used was 80%
propylene glycol. The high pressure equipment had a sample chamber into which the sample
tubes were loaded and the pressure, time and temperature were adjusted and set to the desired
condition. The sample was loaded onto the sample chamber and then the sample chamber
was locked to the head of the HPP equipment. The head of the HPP equipment attached with
the sample chamber was then lowered into the body of the equipment which contained the
pressurising fluid and the safety lock was turned on. The time, temperature and pressure were
adjusted using the electronic display pad on the equipment. The equipment was connected to
a compressor through which compressed air was passed to the HPP equipment to create the
high pressure atmosphere. The up-ramp rate was 200 seconds and down-ramp rate was set at
5 sec. The decompression timeout was set at 400 sec. The HPP treatment conditions for the
Preparation of white rice by
milling brown rice for 120 s
Preparation of fortificant solution
with folic acid (150 mg), iron (25
mg) and β – carotene (62.5 mg)
Airtight packing of rice + fortificant
solution packed in a Cryobag.
Hydrostatic High Pressure Processing
treatment
87
rice samples were set according to Table 4.1. The temperature was maintained at room
temperature. The pressure was reviewed for 360s, 720s and 1080s for 1h, 2h and 3h
respectively. Post treatment the excess water was discarded from the tubes and the rice
samples were dried at room temperature until the moisture content dropped to 10-12%. The
rice samples were then packed into plastic bags and stored for measuring the concentration.
Table 4.1 HPP treatment conditions used for the rice fortification experiment
Pressure condition in MPa Time in hours
200 1h, 2h & 3h
400 1h, 2h & 3h
600 1h, 2h & 3h
Figure 4.2 High pressure equipment used for the experiment
4.2.3 Analysis of rice moisture content and micronutrient concentration post HPP
treatment
4.2.3.1 Analysis of moisture content in HPP processed rice
After the high pressure process treatment at 200, 400 and 600 MPa for 1, 2 and 3h for each
pressure treatment, moisture analysis was performed on the treated rice. The method adopted
to analyse moisture uptake during HPP treatment was according to AOAC method (1990).
About 10 g of the rice sample was accurately weighed in a moisture dish and the samples
were dried at 70°C to a constant weight (usually 24 hours). The moisture content was
calculated on wet basis.
88
4.2.3.2 Analysis of concentration of micronutrients post HPP treatment
Folic acid and β–carotene were analysed according to section (3.2.8.1-3.2.8.2) by HPLC. Iron
analysis was done using ICP-OES according to USEPA method (3050B) as mentioned in
section (3.2.8.3).
The final concentration was presented in µg/g of rice after correcting the moisture content
(dry basis). Since the rice that was treated at 400 and 600 MPa for 1,2 and 3h was not
physically intact and most of the rice was broken and had a white belly, measurement of
concentration was done only on rice that was treated at 200 MPa for 1,2 and 3h. However the
physicochemical analysis (section 5.2) was done for all the rice samples to understand the
change in starch structure during the processing which will be explained in detail in the
following chapter.
4.2.3.3 Calculation of % uptake and % retention of micronutrients in fortified rice
Calculations for % uptake (in the uncooked HPP treated rice) and % retention (in the cooked
HPP treated rice) of micronutrients in the fortified rice was done according to equation 3 and
4 in section 3.2.9 in Chapter 3.
4.2.3.4 Data Analysis
The uptake of micronutrients into the rice from the soak water during HPP and retention of
the three micronutrients fortified rice after cooking were analysed statistically using single-
factor ANOVA.The factor used being was the treatment time, to see whether there was a
significant difference in micronutrient concentrations in different treatments. Each
micronutrient was analysed individually for the three soaking times. The statistical data
analysis was done using XLSTAT version 2010.
4.3 Results and Discussions
4.3.1 Moisture analysis of HPP rice
The primary mode of nutrient penetration into rice kernels is by water penetration and
difference in concentration gradient inside and outside the rice kernel. The process of
diffusion in this case was aided by high pressure unlike parboiling which used heat. Water
89
molecules penetrate effectively into the peripheral portions of each starch granule in the rice
grain on HPP treatment. Water penetrates into the deep parts of rice grain after the HPP
treatment. HPP treatment injures rice grain microstructure and thereby it is able to penetrate
into the inner parts, aiding starch gelatinisation and digestion. (Yamakura et al., 2005).
Therefore, in this section, the results of the rice moisture content post-HPP treatment have
been presented.
With increase in pressure and time, there is an increase in the moisture content (Table 4.2
&Figure 4.3). Rice treated at 600 MPa for 3 hours had the maximum moisture content while
the rice treated at 200 MPa for 1 hour had the lowest moisture content. However, the key aim
of this work was to study the feasibility of fortifying rice with micronutrients using the HPP
technique. Therefore, 200 MPa (1h, 2h & 3h) treatments were considered suitable for this
study in the perspective of fortification, given that less energy will be needed to dry the
grains post HPP treatment and less structural changes to the grain will be seen which would
make the grain remain intact.
Table 4.2 Moisture content of HPP treated rice on wet basis
Treatments % Moisture (WB)
200 MPa(1hr) 30.8±0.3
400 MPa(1hr) 34.9±0.09
600 MPa(1hr) 53.7±0.4
200 MPa(2hr) 34.4±0.9
400 MPa(2hr) 37.2±9
600 MPa(2hr) 63.4±6.9
200 MPa(3hr) 42.2±1.5
400 MPa(3hr) 39.2±0.9
600 MPa(3hr) 66.0±0.5
Note: All values reported are Means ±SD of triplicate determinations
90
Figure 4.3 % Moisture content of rice on a wet basis at different treatment times and
pressures used
4.3.2 Concentration of micronutrients in fortified rice using HPP
The results of the micronutrients concentration in the fortified rice using high pressure
processing is presented in the figures (4.4 to 4.6) and tables (4.3 to 4.5). For all the three
micronutrients, the concentration in the rice decreases with an increase in time which is quite
the opposite that of the parboiling process. The highest concentration for all the three
micronutrients was seen at 1 hour treatment. Therefore, treating milled rice at 200 MPa for 1
hour would be considered the best possible condition for rice fortification.
0
10
20
30
40
50
60
70
80
0 1 2 3 4
Mo
istu
re c
on
ten
t in
% (
wet
ba
sis)
Treatment time in hours
200 Mpa
400 Mpa
600 Mpa
91
Table 4.3 Concentration of folic acid (in d.w.b) in high pressure processed rice
(uncooked and cooked) (200MPa) at varying time intervals µg/g of rice
Folic Acid 1h 2h 3h
Uncooked 165±33.6 91±32.3 112±34.5
Cooked 162±23.4 86±31.3 94±47
Note: All values reported are Means ± Standard deviation of triplicate determinations
Table 4.4 β-carotene concentration (in d.w.b.) in high pressure processed (200 MPa)
fortified rice at varying time intervals in µg/g
β-carotene in µg/g 1h 2h 3h
Uncooked 44±20.5 28±20.0 9±8.05
Cooked 8±2.8 5.6±3.8 1.19±2.1
Note: All values reported are Means ± Standard deviation of triplicate determinations
Table 4.5 Concentration of Na-EDTA (in d.w.b.) iron in high pressure processed rice
(200 MPa) at varying time intervals in µg/g of rice
Iron 1h 2h 3h
Uncooked Rice 17±2.4 10±5.4 10±7.4
Cooked Rice 11±10.8 8±3.2 6±3.4
Note: All values reported are Means ± Standard deviation of triplicate determinations
4.3.3 Statistical Analysis of micronutrient concentration
When the concentration data for folic acid was analysed statistically to see if there is any
difference between the treatment times, it was seen that between the groups, there was
significant difference. ANOVA results for single factor (treatment time) showed that for both
cooked and uncooked rice there was significant difference in terms of the treatment time (p>
0.05). The single factor ANOVA showed similar results once again for β–carotene indicating
there is significant difference in the cooked and uncooked samples between treatment times.
However for iron the results were different showing that there was no significant difference
between the treatment times for iron concentration. Both uncooked and uncooked samples
had p>0.05 and the F<Fcritical.
92
4.3.4 Loss of micronutrients due to longer treatment times and cooking
Folic acid standard solution was tested for degradation due to HPP at 200 MPa (1h, 2h and
3h). Folic acid did not show degradation after 3 hours HPP treatment and was stable and this
was in agreement with Nguyen et al., (2003) who modelled the stability of pure folic acid
standard subjected to HPP and deduced the stability of the compound. Folic acid was stable
for 7 h when treated at 600 MPa (60°C) and did not show degradation (Nguyen et al., 2003).
Thus the reasons for folic acid decrease in concentration with the increase in treatment time
could be attributed to the change in the starch structure due to the high pressure processing
technique. When the starch gelatinisation is incomplete the effect of amylolytic enzymes is
minimal on the starch (Nasehi & Javaheri, 2012). Activity of α-amylase was tested for release
of glucose in HPP rice treated at 200 MPa for 1h, 2h and 3h (Figure 4.4). With increase in
treatment time at constant pressure there was reduced release of glucose indicating that α-
amylase activity is being hindered due to effect of high pressure processing. Therefore it can
be said that with increase in treatment time, there are structural changes in the starch leading
to partial gelatinisation making it resistant to enzyme digestion and ultimately affecting the
concentration of micronutrients as α- amylase is a key enzyme used for starch digestion and
extraction of folic acid from the fortified rice.
Figure 4.4 Activity of α-amylase by testing glucose release from processed (fortified)
and unprocessed rice samples(Error bars represent standard error in replicates)
Longer HPP treatment times causing partial starch gelatinisation may have affected the assay
rather than the final concentration of folic acid in the fortified rice. Parboiled rice starch can
0
5
10
15
20
25
30
35
40
Glu
cose
con
cen
trati
on
in
mg/g
of
rice
Processed (fortified)and Unprocessedd fortified rice
samples
15 min
30 min
1 h
93
be hydrolysed by α-amylase better than HPP and unprocessed rice and thus the high
concentration of vitamin released from parboiled rice (Figure 3.8) compared to HPP rice.
According to Dhital (2015) parboiled rice is easily digestible than unprocessed rice and this
can reinforces the above results obtained from the current study. Changes in the starch
structure has been studied in detail as discussed in the following chapter which could provide
an insight into the variation in concentration of micronutrients due to longer processing time.
The structural changes in rice starch that possibly affect the micronutrient concentration in
HPP fortified rice is addressed in detail in Chapter 5.
In the present study, unlike the case of folic acid, β-carotene was extracted by saponification
and liquid-liquid extraction. There was no enzyme involved. However, the concentration was
much lower than folic acid and further decreased with longer treatment times. The reason for
this could be that the β – carotene fortificant was encapsulated in a soy protein matrix. Due to
high pressure processing, the destruction of the soy protein matrix (Floury et al., 2002) could
result in degradation of the β – carotene concentration. Apart from that pH was also an
important factor that affected the solubility and stability of soy protein. Torrenzan et al.,
(2002) reported that soy protein showed maximum solubility at pH < 3 or pH >6. In the
present study, the initial pH of the nutrient solution was between 4.7 - 5.03 and this further
increased to 5.8 after the treatment. This is still lower than 6 and therefore it is highly likely
that the protein precipitated out. When soy milk was treated using HPP it was reported that
the emulsifying activity and stability increased but the emulsifying capacity reduced
(Kajiyama et al., 1995). In the present study, β-carotene encapsulated in the protein matrix
was chosen over starch based matrix to improve solubility and form a stable emulsion in
water. The lower concentration of β-carotene with longer treatment time could be due to the
instability of the soy protein matrix during HPP treatment which in turn affects the stability of
β-carotene in which it was encapsulated.
Iron in the form of Na- EDTA has not been studied in detail because using HPP has become
popular only in the past couple of decades. From the results there was no significant
difference in the uptake of iron in the rice during 1h, 2h and 3h thus it can be said there is
lower diffusion of iron in general. Iron being a mineral is stable after cooking and the loss is
lesser than β-carotene after cooking (section 4.3.3). The statistical results also show no
significance after cooking. Lower retention after cooking could be due to washing prior to
cooking the fortified rice. According to Rastogi, (2013) high pressure processing increases the
94
bioavailability of iron by 4.63 to 10.93% in food when treated at 500 MPa implying that there
is added advantage of fortifying rice with iron using HPP.
Table 4.6 % uptake (in uncooked rice) and % retention (in cooked rice) of
micronutrients in the fortified rice before and after cooking at various soaking times
Micronutrients 1 h 2 h 3 h
Folic acid Uncooked fortified rice 41.2% 22.8% 28%
Cooked fortified rice 98% 94.5% 83.9%
Iron Uncooked fortified rice 20.4% 12% 12%
Cooked 64% 81% 60.5%
β- carotene Uncooked fortified rice 21% 13.3% 8%
Cooked fortified rice 18.6% 20.2% 7%
On comparing the % uptake of micronutrients from the soaking water into the rice between
parboiling and HPP process the % uptake was low for all three micronutrients during HPP
compared to the parboiling process (Table 4.6). Parboiled fortified rice had higher
concentration of iron (45 µg/g) compared to HPP treated rice as the raw material used was
brown rice (BR) in the case of the former process and innate iron was higher in BR than in
milled rice and due to diffusion parboiled rice was able to uptake higher iron than HPP where
milled rice was used. In the parboiling process diffusion into the rice was low due to the lower
difference in concentration gradient because of innate iron present in brown rice. Milled rice
had an endogenous iron concentration of ~2 mg/kg (Prom-u-Thai et al., 2008). This could
have resulted in lower uptake of NaEDTA-Fe in rice in current study as it can be seen that the
concentration is almost constant over the three treatment times showing limited migration of
iron and this phenomenon is also comparable to the parboiling process.
High pressure processing can cause disruption of the cell wall in biological matter thereby
making them unstable (Rastogi, 2013). In the current study milled rice was soaked in excess
water and subjected to gelatinisation through high pressure processing. As mentioned
previously approximately 10 mL of the excess soaked water (from initial 40 mL) was
discarded after the treatment and this soak water contained leached out starch from the milled
rice. The rate of starch leaching is higher from milled rice than in brown rice as the rice bran
contains less starch than the endosperm (Juliano, 1985). Thus the reason for lower uptake
95
could be correlated to the leaching of starch to which the micronutrients adhered to and were
present in the soak water that was discarded post HPP treatment. According to Bhattacharya
(2004) during structural changes in starch, water soluble vitamins tend to adhere to the starchy
endosperm. Folate uptake rate in rice followed 1st order kinetics; and the rates of natural rice
hydrolysis (starch gelatinization) and folate uptake are both time-dependent (Kam et al.,
2012). This could be the reason for higher folic acid uptake during the HPP treatment and
lower uptake of β-carotene after HPP treatment. β-carotene, a lipophilic compound was not
as likely to adhere to the starchy endosperm as folic acid even though it was encapsulated in a
protein matrix. This theory could explain the lower uptake of β-carotene in the fortified rice.
From the above table it can be seen that folic acid showed the lowest loss due to cooking
followed by iron and then β-carotene. This retention trend was also similar to that of the
parboiling process.
The adherence of each micronutrient or absorption into the endosperm is to be studied in more
depth to understand the reduction in concentration with time. From previous research it can be
understood that the structure of rice starch changes with high pressure treatment (Yamazaki et
al., 1998). The possibility of adsorption of the micronutrients on to the amylose or
amylopectin could result from this variation. The variation in the ratio of amylose and
amylopectin due to starch gelatinisation when subjected to high pressure treatment could be
one possible reasons for the lower concentration with longer processing time. To study more
in depth, fluorescence tagging of the micronutrients could be performed individually to
unravel this observable fact. This study was the first to identify HPP as a technique for
fortification of rice in a non-thermal environment. Since this was a preliminary study and the
main aim was to look at the feasibility of rice fortification, the most favourable fortification
condition was deduced. In the following chapter, the changes in the rice starch structure due
to the HPP treatment will be further discussed.
4.3.5 Efficacy of high pressure processing as a means of rice fortification
Overall, from the most favourable condition of HPP treatment for fortification, the
concentration of each micronutrient in cooked fortified rice is presented in the table below.
As mentioned in the previous chapter, it is estimated that about 440 g of rice is consumed in
countries where rice is the staple food (IRRI, n.d.). Consequently, based on this quantity (440
g), the concentration of folic acid, iron and β-carotene that can be provided by this fortified
premix rice has been calculated and presented in the Table 4.7. Since our target population
96
mainly includes pregnant women and pre-school children, the RDI is calculated based on
these levels prescribed by USFDA(2014).
Table 4.7Concentration of micronutrients consumed from the fortified rice based on
RDI(NIH-ODS, 2014 & NHMRC (Australia), 2006)
Micronutrient Folic acid (µg) Iron (µg) β – carotene (µg)
Concentration/g of rice 162±23.4 11±10.8 23±4.3
Concentration in premix
rice(µg/440g) 71,280 4840 10,120
Concentration in fortified
rice diluted in the ratio 1:10
with unfortified rice (µg/
440g)
712 48 101
% RDI obtained by
consuming fortified rice 178% 2.6% 12%
Note: The above calculations were made assuming ~440g of rice was consumed per day by a
person living in a country where rice was staple food (IRRI, n,d,)
Hence based on the above it can be seen that folic acid was able to provide more than 100%
of the RDI from the fortified rice. Excessive folic acid consumption has not shown any
adverse effect however, chronic effects of long-term consumption are unknown. Based on the
above calculations iron and β-carotene were only able to provide only2.6% and 12% of the
RDI respectively. As it can be seen from Table 4.4 the concentration of β-carotene retained
after cooking was reduced to almost less than half. Therefore there is a reasonable loss in this
case. On the other hand, folic acid and iron seem to be the more stable after cooking. These
results show that HPP was not able to meet the RDI requirement for iron and β-carotene.
These concentrations are only based on RDI and are susceptible to further changes when they
are actually digested by the body and how much would be bioavailable in the end.
4.4 Conclusions
High pressure processing, a novel food processing method could be a useful tool for rice
fortification. Rice treated at pressures above 200 MPa for three hours resulted in broken
kernels and therefore became unsuitable for fortification. The moisture absorption of rice
97
changed at different pressures with 600 MPa absorbing the maximum water. The water
uptake also increased with time. The best suitable time among those tested for processing was
found to be one hour and the pressure was 200 MPa for maximum concentration of all the
three micronutrients. The uptake during fortification and retention after cooking was the
highest for folic acid, followed by iron and then β- carotene in both uncooked and cooked
rice. There was significant difference between the treatment times for folic acid and β-
carotene but not for iron. By consuming this fortified rice (assuming 440 g of rice is
consumed per day in rice eating populations) the target population would be able to receive
more than 100% of the RDI for folic acid and iron but not for β- carotene. The reduction in
the concentration of the micronutrients could be because of the change in the starch structure
which is affected by HPP. This needs to be studied in detail by investigating the adsorption of
micronutrients on the rice during the high pressure processing. Overall it can be said that high
pressure processing would be a feasible novel method to fortify rice. However the
concentrations of micronutrients to be added should be adjusted to meet RDI in target
populations.
High-pressure equipment on a commercial scale costs between $500,000 to $2.5 million
dollars depending upon equipment capacity and extent of automation. Since it is a relatively
new processing technology with a narrow market, products that are pressure-processed may
cause an increase in the cost by 3 to 10 cents per pound than thermally processed products.
With two 215-litre HPP units operating under typical food processing conditions, the output
of approximately 20 million pounds per year is attainable. With increase in demand for HPP
equipment it is likely that the capital cost and operating cost will decrease (Ramaswamy,
2006).
98
CHAPTER 5
5. PHYSICO-CHEMICAL PROPERTIES OF RICE FORTIFIED BY THE
PARBOILING AND HPP TECHNIQUES
5.1. Introduction
In chapter 3 and chapter 4 the optimization of parboiling procedure to fortify rice with
micronutrients and feasibility of fortification through the HPP technology was described in
detail respectively. The current chapter will focus on the changes in the physicochemical
properties of fortified rice as a result of fortification through parboiling and high pressure
processing techniques used for fortification. For both the fortification techniques the
micronutrients were retained in the starchy endosperm of the rice i.e. for parboiled fortified
rice for which the rice was milled after the fortification process and for the HPP rice for
which milled white rice was used as the medium of fortification. Some of the
physicochemical properties of starch in the processed rice that were studied include:
Degree of gelatinization of the rice
Instrumental colorimetric analysis of the fortified rice
Rapid Visco analysis to understand the pasting properties of the rice
X-ray Diffraction to observe changes in the crystalline nature of the starch
Microscopic images of the processed rice starch under polarized light and differential
interference contrast
Magnetic resonance imaging of migration of water during the parboiling process
Both technologies implemented for rice fortification involved the principle of diffusion. In
the case of parboiling, heat and concentration gradient induced diffusion while for HPP there
was no application of heat whilst pressure played a vital role. The main purpose of this
section of the experiments was to understand how the processing had altered the rice starch.
Essentially in both these processes starch was the main component of fortificant adherence in
rice. Therefore changes in starch properties due to the two processing methods might explain
if there is any relationship that can be established between starch structural changes and the
concentration of micronutrients in the fortified rice. By assessing the physico-chemical
99
properties quantitatively, the qualitative properties of rice starch changes can be studied and
also it can give an understanding of how different the processed rice is compared to
unprocessed rice. Therefore, the rice flour from the fortified and unfortified samples were as
such analysed for the above mentioned physicochemical properties without extracting the
starch alone. The harsh process of starch extraction could lead to further alterations to the
already processed starch and therefore this step was avoided. Grinding of the rice flour and
using alkaline solutions can significantly alter the rice starch structure (Fields, 2013
(pers.comm.) & Ragheb, Abd El-Thalouth and Tawfik, (2006).
5.2. Materials and Methods
5.2.1. Optimization of method for studying gelatinization of starch in rice
Rice starch was gelatinized due to application of heat during parboiling and pressure during
HPP treatment. The degree of gelatinization is likely to vary depending on the process. Hence
the degree of gelatinization was determined through a colorimetric assay. Rice flour was
prepared by grinding the rice that were fortified by the parboiling process and by HPP using a
lab-scale Breville coffee grinder. The ground rice was then passed through a 125 µm mesh
and flour that was >125 µm was used for further experiments. Unprocessed brown and white
rice samples were also processed in the same way and used as controls. The degree of
gelatinization of rice starch was analysed according to Birch and Priestly (1973)& Baryam
(2006) based on the amylose/iodine blue value after dispersion in two different
concentrations of alkali. The principle of the assay was based on relation of gelatinization to
hydrogen bonding effect observed in the infrared absorption spectrum of rice. The colour
change was measured as absorbance using SpectraMax (Model: M2, Sunnyvale, CA) at 600
nm and the data was extracted using the software SoftMax ®Pro, Version 4.8, 2004).
The alkali used in this experiment to induce starch gelatinization was potassium hydroxide
obtained from UNIVAR, Australia. To optimize the molarity of potassium hydroxide (KOH)
which causes gelatinization in rice starch different volumes of 10 M KOH was added to
cooked and uncooked rice. In this preliminary experiment the molarity of KOH that gave
maximum absorbance was used for the assay that was conducted with the rice samples.
Commercial parboiled rice was used as a standard for cooked rice and assumed that it was
100
100% gelatinized and brown rice was used as standard for uncooked rice. Using the
optimized molarity of KOH the standard curve was plotted. The molarity that gave maximum
absorbance for uncooked rice and cooked rice was used for BR and parboiled rice
respectively to build the standard curve. The ratio of absorbance between these values was
used for calculation of degree of gelatinization of unknown samples. The optimized
concentration of KOH is presented in the Appendix 1.
Figure 5.1 Optimization of KOH molarity to be added to study the degree of rice
gelatinization
The present method is based on the principle of analysing the amylose content upon
gelatinization of starch using a colorimetric approach based on the iodine-amylose blue
complex. This method facilitates the determination of rice parboiling index and milled white
rice quality. By adopting this method, measurement of absolute starch concentration in
samples need not be considered and the degree of gelatinization measurement can be quick
and efficient. Gelatinization of rice starch was done using alkali potassium hydroxide (KOH)
and the optimum concentration was found to be (0.86) for cooked and 0.6 for uncooked rice
(presented in Appendix 1). For uncooked rice, gelatinization occurred at a lower
concentration of KOH compared to cooked rice and this was indicated by the absorbance
value.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 1 2 3 4
Ab
sorb
an
ce a
t 6
00
nm
Molarity of KOH added
Optimized absorbance for cooked and uncooked rice
Uncooked
Rice
Cooked Rice
101
5.2.2. Instrumental colorimetric evaluation of fortified rice
Rice that was fortified will have different appearance compared to unfortified and
unprocessed rice due to the addition of the fortificants which impart colour to the rice. The
colour change is evident to the human eye and it is subjective and highly varies between
individuals (Bergman et al., 2004, Champagne et al., 2010). However instrumental colour
evaluation is objective and can provide numerical values that can be used to standardize a
products‘ colour. In this study the fortified rice samples had a yellowish colour compared to
unprocessed white rice. Rice that was fortified by parboiling had a different colour compared
to the HPP rice. Thus to get an objective understanding of colour variation among the rice
samples, parboiling (Conditions A, B, C and D- Table 3.1) and HPP fortified rice (200, 400
& 600 MPa for 1h, 2h & 3h) were evaluated instrumentally for colorimetric values.
Instrumental colour evaluation was performed for the rice samples using a colorimeter
(Minolta CR-400 series). The instrument was calibrated using a white calibration tile. The
colour was measured as three parameters namely; L*, a* and b* colour space (McLaren,
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 values representing yellowness
whereas negative values representing blueness (Lamberts et al., 2006b).
Samples for colour evaluation were placed on a plastic petridish and covered by cling wrap.
The colorimeter was placed on the samples and operated to obtain L*, a* and b* values.
Parboiled rice that was optimized at condition A (mentioned in Chapter 3, section 3.2.3) was
diluted with commercial white rice in the ratio 1:10 as it was chosen to be the way to
commercialize. This will be referred to as diluted fortified rice and was used in Chapter 6 for
sensory evaluation of the fortified rice. Colour differences were calculated based on the
differences in L*, a* and b* between samples and reference materials. Brown rice that was
milled for 120s was used as a control for the untreated rice (ControlRaw) and parboiled rice
that was prepared with the same parboiling conditions but without fortificants added to the
soaking solution (ControlParboil) was used as control for the processed rice. The total colour
difference ΔE, was calculated as a single value that takes into account the differences
between L*, a* and b* of the samples and references.
102
ΔE =
𝐿∗𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐿∗𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒
2+ 𝑎∗
𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑎∗𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒
2+ (𝑏∗
𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑏∗𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 )2
The total colour difference (ΔE) between fortified and unfortified commercial white rice is
being denoted as ΔERaw whereas the difference between fortified and unfortified parboiled
rice is being denoted as ΔEParboil.
5.2.3. Rapid Viscoanalyser (RVA) of fortified rice flour samples
Rapid Viscosity analysis can be used to determine the degree of gelatinization of starch by
assessing the pasting properties of the rice starch. Graphs obtained from RVA can serve as a
sensory quality index for starches (Champagne, 1999). Therefore fortified rice samples were
assessed for the variation in the pasting properties due to the two different processing
techniques.
The instrument used was Model RVA 3D+ (Newport scientific). About 24.89±0.01 g of
water was weighed into a canister and 2.89±0.01g of each rice sample was transferred to the
water surface. The sample weight and volume of water added were adjusted for moisture
according to the following formula:
S= 86 × 𝐴 ÷ 100 − 𝑀
Where S= corrected sample mass (g),
A= Sample weight at 10% moisture
M= Actual Moisture content
Water that was added was also corrected based on the sample weight according to the
formula:
𝑊 = 25 − (𝐴 − 𝑆)
Where W is the corrected water mass (g).
The slurry was mixed using a paddle for 10 times to homogenize it. The paddle and the
canister were placed into the instrument and the RVA cycle was initiated by pressing down
103
the tower of the instrument when the software was ready for analysis. The total run time was
12 minutes and 30 seconds.
Table 5.1 The RVA program for analysing rice sample
Time (mm:ss) Type Value
00:00 Temperature (°C) 50
00:00 Speed (rpm) 960
01:00 Temperature (°C) 50
04:45 Temperature (°C) 95
07:15 Temperature (°C) 95
11:06-13:00 Temperature (°C) 50
5.2.4. X-ray diffraction (XRD) of rice flour fortified by parboiling and HPP
Starch (composed of amylose and amylopectin) is defined by amorphous and semi-crystalline
rings. Amorphous region of starch contains amylose and less ordered amylopectin and the
semi-crystalline region comprises of repeated sequences of amorphous and crystalline
lamellae (Morrison, 1995). The crystalline nature of starch is altered due to processes during
which gelatinization occurs. Therefore the processed fortified rice samples were analysed for
crystallinity using XRD. Rice flour samples that were prepared for the gelatinization
experiment was used for XRD work as well. The instrument used for XRD analysis was
X‘Pert PRO Multi-purpose X-ray Diffraction system (MPD system-PANalytical, Australia).
During the experimentation process the voltage used was 45 kV/40 mA. The program for the
continuous scan has been described in Table 5.3
104
Table 5.2 Scan parameters for XRD measurement
Scan parameters Values
Start angle (°) 3.008
End angle (°) 60.008
Step size (°) 0.05
Time per step (sec) 0.5
Scan speed (°/sec) 0.1
No. Of steps 1140
Total scan time 9 min 33 sec
The rice flour samples were evenly distributed onto a sample plate to make a smooth surface
for analysis and this was loaded onto the sample changer which automated the process of
analysis. The XRD scan program was chosen for analysis using HighScore Plus software
(Version 3.0.5, 2012).
After the analysis, the XRD data was extracted using X‘Pert HighScore plus (version 1.3.2.6,
2010) and % crystallinity was calculated using the software Magicplot Student (Version
2.5.1, 2013).
5.2.5. Polarized light microscopy and Differential Interference Contrast (DIC)
Microscopic images of starch granules in the fortified rice
Due to gelatinization of rice starch under the influence of heat and pressure, the starch
molecules are likely to have physical and structural changes. These changes vary depending
upon how harsh the treatment is to the starch (Hibi et al., 1993). Hence fortified rice flour
was observed for physical intactness using polarized light microscope. Ground rice flour was
further ground to make smaller particle size manually using mortar and pestle for 10 minutes.
The rice flour was once again passed through a 125 µm and the flour that was < 125 µm was
stored in screw capped Nunc®
CryoTubes® (purchased from Sigma-Aldrich, Sydney
Australia). For observation under microscope a small quantity of the rice flour was mounted
onto a slide and was spread evenly with water. The excess flour was tipped off. To the
remaining flour 50% glycerol was added and the sample was covered with a cover slip. The
105
starch granules were observed under a polarized light microscope and differential
interference contrast microscopy and the images were captured.
Starch samples mounted on glass slides with 50% Glycerol were scanned using a Zeiss
Axioskop II – a brightfield transmitted light microscope with polarising filters and Nomarski
Optics for viewing under differential interference contrast. Images were captured using a
Zeiss HRc digital camera and archived using Zeiss Axiovision software. Images were
photographed under a 63x oil immersion lens.
5.2.6. Real-time Magnetic Resonance Imaging (MRI) of water migration in rice during
parboiling
MRI can be used to observe moisture distribution in rice grain in three dimensions with time
(Horigane et al., 2013). Moisture migration into the rice grain can be observed using MRI. In
this study, migration of micronutrients into the grain in real-time was observed based on the
assumption that migration of micronutrients will correlate with moisture uptake into the rice.
This is the first time that real-time MRI has been used for this purpose. For loading samples
on the MRI machine for evaluation, rice grain was soaked at the optimum parboiling
condition of 70 0C for 2 hours and rice kernels were removed at particular time intervals
(0,15,30, 45 , 60 and 120 min). One kernel from each interval (all in all 6 kernels) was
prepared on a plastic board (size) to which a layer of Blue-Tack ™ (Bostik) was attached.
Extreme care was taken so that all rice kernels aligned in a uniform plane for imaging. The
plastic board was placed in a water filled Greiner cryovial ™. MR Imaging was performed
using a 9.4T preclinical imaging system.
Bruker biospec 94/20 Avance III which was equipped with a 15mm Quadrature
Receive/Transmit RF-coil and running Paravision 5.1 To image the samples from all time
points in one image the rice kernel assembly was scanned in longitudinal and coronal
directions using a 2D gradient echo, Fast Low Angle Shot (FLASH) method. Field of View
(FoV): 10mmx14mm, Slice Thickness: 200mm Echo time (TE) =4ms, Repetition time
(Tr)was 64.2 ms, and the flip angle (FA) was 40.0 deg. It was taken care to use the shortest
possible TE for the available set-up to collect enough signals from the hard and still relatively
dry rice kernels. This enabled to effectively detect free water in cracks. The total scan time
was 26 min 17 s.
106
5.3. Results and Discussions:
5.3.1. Degree of Gelatinization of fortified rice samples
Processing methods in foods can significantly alter the starch structure and this can in turn
have impact on the postprandial response when consumed after cooking the rice (Björck,
1996). When rice is subjected to heat or pressure treatment the starch granules are disrupted
and therefore they are more susceptible to enzymatic digestion (Holm et al., 1988). During
starch retrogradation after gelatinization rice starch is converted to a crystalline form from an
amorphous form (Bhattacharya, 1985). Therefore in this section the rice that was subjected to
gelatinization due to heat and pressure was retrograded by cooling to room temperature.
Following this degree of gelatinization was assessed.
The result for the gelatinization study has been presented below:
Figure 5.2 % Gelatinization comparison between parboiled rice and HPP processed
rice. Untreated brown rice and white rice were used as controls(Error bars represent
standard errors in replicate samples)
In the present study, milled white basmati rice has 34% amylose and therefore classified as
very high amylose content rice according to the classification by Juliano (1992).
Gelatinization in the current study occurs due to application of heat and pressure on the rice
kernels. Depending upon the starch type, the pressure and heat required for gelatinization
varies. Wheat starch for instance begins to gelatinize below 300 MPa and complete
gelatinization occurs at 600 MPa (Douzals, Marechal, Coquilee & Gervais, 1996). Potato
starch on the other hand begins to gelatinize only at 600 MPa (Bauer & Knorr, 2005).
Douzals, Cornet, Gervais and Coquille (1988) found out that the release of amylose in
0.00
20.00
40.00
60.00
80.00
100.00
120.00
% Gelatinization
107
pressure induced gelatinization was lower than with the heat-induced treatment. Experimental
results from the current study showed that gelatinization induced my HPP resulted in the
lower swelling and release of amylose compared to heat treatment. This can be attributed to
the lowering of % amylose during HPP treatment at 200 MPa for 3 hours. By increasing the
pressure (400 and 600 MPa) and time (3 h) more amylose is released and this can be seen in
the results (Figure 5.2).Hence it can be said that pressure influences the level of amylose
released and thereby impacting the degree of gelatinization. This could explain the variation
in degree of gelatinization in pressure treated rice and heat treated rice as the swelling of
starch did not completely solubilize the amylose therefore leading to lower release due to
pressure treatment (Hermansson & Svegmark, 1996). The disintegration of the crystalline
region of starch was not completed by pressure due to the side-by-side dissociation and helix
unwinding of amylopectin units being suppressed by van der Waals‘ forces and stabilization
of hydrogen bonds in normal rice starch (Buckow et al., 2007). Hence, the gelatinization of
rice starch induced by pressure depends on the intensity of pressure, duration of the
treatment, size of the starch granules and ratio of amylose/amylopectin.
Parboiled rice showed maximum absorbance and the degree of gelatinization was close to
100% showing complete starch gelatinization due this process. The degree of gelatinization is
responsible for various attributes of parboiled rice (Marshall et al., 1994). Gelatinization
causes amylose molecules to leach out of the micellar network and diffuse into the
surrounding aqueous media (Hermansson & Segmark, 1996). The degree of gelatinization
has also shown to have effect on the colour of parboiled rice. Different soaking time affects
the relative darkness of the milled parboiled rice. However, translucency is also another
factor affected by parboiling and with longer soaking treatments rice quality improved in
terms of translucency (Miah et al., 2002). The rate of starch gelatinization is highly
temperature dependent (Priestly 1976 & Bakshi and Singh, 1980). For complete starch
gelatinization in paddy it took about 60 minutes at 1100
C and 40 minutes at 1200C. For
brown rice to achieve about 80% of gelatinization it took about 50 minutes at 700C and 20
minutes at 800C but it took 240 min at 70
0C and 80 min at 80
0C for paddy to attain same
degree of gelatinization. Thus it can be seen that paddy requires more time than brown rice. It
was also deduced that brown rice requires less resistance to water movement than paddy
(Bakshi and Singh 1980). These properties make BR a better medium for fortification than
paddy.
108
Table 5.3 Activation energies for gelatinization in Rough rice (paddy) and brown rice
(Bakshi & Singh, 1980)
Temperature range °C Ea Cal/g-mole
Rough rice (paddy) Brown rice
50-85 18534 24672
85-120 10474 9586
From the above table it can be seen that the activation energy for paddy (rough rice) is lower
than brown rice for temperatures below 850C. On the other hand, at temperatures above 85
0C
the activation energy for brown rice is lower than paddy. This type of reaction is common in
the case of catalytic reactions (Bakshi and Singh 1980). Sttaterfield (1970) observed that the
activation energy for diffusion-limited reaction is about one-half of the activation energy
observed in the case of just the reaction only. In parboiling for temperatures below 850C, the
proportion of rice components when soaked is a limiting factor while for temperatures above
850C diffusion of water in rice is a limiting factor. This explains the results obtained in the
present study when soaked at 700C using the parboiling method the added nutrients to brown
rice may have contributed to be a limiting factor for uptake of micronutrients. This can be
further explained by comparing the iron concentration in the fortified rice after parboiling
process. The uptake of iron is almost constant at different conditions (A, B, C and D (Table
3.1)) during parboiling showing that brown rice could have restricted the uptake of iron
during the soaking step.
Generally, both pressure and heat treatment have significant effects on the degree of
gelatinization. However, the magnitude of difference for gelatinization varied between the
treatments. Heat gelatinized rice produced more translucent and acceptable rice compared to
the pressure treatment. Thus variation in degree of gelatinization can be attributed as a major
factor for concentration of micronutrients in the rice as it varied between the two methods for
each micronutrient.
5.3.2. Colorimetric analysis of fortified processed rice
Evaluation of colour is an important aspect of food products as it influences the consumer‘s
behaviour towards purchasing and consuming a particular food (Brewer and McKeith, 1999,
VanHurley, 2007). According to Champange et al., (2010) white rice consumers expect the
cooked and uncooked rice to be white in colour with minimal discolouration. However
109
parboiled rice consumers can accept some colour change in the rice consumed such as a tinge
of yellow or brown (Tomlins et al., 2007). Instrumental colour evaluation of fortified rice in
comparison to commercial white rice is presented in the table below.
110
Table 5.4 Color parameters L* (Lightness), a* (Redness) and b* (Yellowness) of
fortified rice milled at different milling times at various concentrations
Sample L* a* b*
A 0s 61.9 a,b,
2.7c,d
13.3 e,f
A 60s 64.6 a,b,
0.9c,d
16.2 e,f,n
A120s 66.4 a,b,g,i
-0.7c,d,k
16.7 e,f
B 0s 65.0 a,b
2.5c,d
17.4 e,f
B 60s 66.7 a,b
2.3c,d
18.6 e,f
B120s 69.5 a,b,h,i
0.9c,d,h,k
17.4 e,f,o
C 0s 60.0 a,b,
2.9c,d
18.4 e,f
C 60s 65.3 a,b,
1.0c,d
18.2 e,f
C 120s 69.5 a,b,h,i
0.97c,d,h,k
17.7 e,f,n
D 0s 57.3 a,b,h
3.2c,d
19.0 e,f
D 60s 65.5 a,b,
1.9c,d
20.2 e,f
D 120s 64.4 a,b,
1.5 c,d,h,k
22.0 e,f,n
Control parboiled
rice (unfortified) 65.9
a,b,h,j 0.25
c 15.2
Commercial
Parboiled rice 64.5 0.19
15.9
Commercial white
rice 71.6 -0.6
9.9
Diluted Fortified
rice 70.6
l -0.54
,l 10.2
m
Note:Values followed by different letters in the columns indicate significantly different
means at p < 0.05.
Based on the L*, a* and b* values obtained from the colorimeter the colour difference was
calculated based on the formula mentioned in section 5.2.2 (page no.101) to see the
difference in mean colour between parboiled rice and raw rice. Commercial white rice and
commercial parboiled rice were used as reference for respective samples.
111
Table 5.5 Colour difference between fortified rice and commercial white rice
(Controlraw) and parboiled rice (Controlparboil)
Sample ΔE parboil ΔE raw
Diluted Fortified rice 8.4 1.0
In house parboiled unfortified
rice milled at 0s 1.6 7.8
Premix fortified rice -0s (A) 3.8 12.3
Premix fortified rice 60s (A) 3.3 9.5
Premix fortified rice 120s (A) 0.8 6.2
Premix fortified rice 0s (B) 5.3 15.9
Premix fortified rice 60s (B) 4.1 10.4
Premix fortified rice 120s (B) 2.9 8.0
Premix fortified rice 0s © 5.9 14.8
Premix fortified rice 60s © 5.4 10.5
Premix fortified rice 120s © 2.6 8.3
Premix fortified rice 0s (D) 8.4 17.4
Premix fortified rice 60s (D) 6.3 14.2
Premix fortified rice 120s (D) 4.8 12.3
Table 5.6 Colour parameters L* (Lightness), a* (Redness) and b* (Yellowness) of HPP
fortified rice at different pressure and time treatment
Pressure L a B ∆E raw
200 1h 69.1 a,b
-1.1 12.8 c,d
3.8
200 2h 64.4 a,b
-0.8 12.7 c,d
7.7
200 3h 70.9 a,b
-1.4 12.6 c,d
2.8
400 1h 67.9 a,b
-1.0 13.0 c,d
4.9
400 2h 51.6 a,b
-1.3 10.9 c,d
20.0
400 3h 67.4 a,b
-1.6 11.2 c,d
4.5
600 1h 58.3 a,b
-1.2 10.1 c,d
13.4
600 2h 52.8 a,b
-1.1 9.0 c,d
18.9
600 3h 59.0 a,b
-1.1 10.6 c,d
12.6
Note: Values with different alphabets indicate significantly different means at p <
0.05.
5.3.2.1. Total colour difference for parboiled rice and HPP rice– against uncooked
commercial white rice (ControlRaw) and parboiled rice (ControlParboil)
Rice eating populations commonly consume milled rice (Bhattacharya, 2004) and therefore it
can be set as a standard for fortified rice. Instrumental colour evaluation can be an objective
way to see the colour difference between the fortified rice and white rice for consumer
acceptance.
112
For the parboiled rice, the total colour difference ΔE increased with concentration of the
fortificant added and also with the milling time. ΔE was comparable statistically at different
fortificant concentration levels to the commercial fortified rice. ΔEparboil value was the lowest
for the in-house parboiled fortified rice- A120s (0.8) and ΔERaw was lowest for the diluted
fortified rice (1.0) making them comparable in colour to commercial parboiled rice and
commercial white rice respectively. Based on the fact that the commercial parboiled rice has
already been widely accepted by consumers it can be said that the product can be marketed
and acceptable locally and internationally. Also the variation between white rice and fortified
rice was much higher compared to parboiled rice making it distinctly different in terms of
colour. The major contributors for colour change was deduced to be L* and b* when highest
mass of fortificant was added and this has been reflected in the ΔE that was calculated in the
present study (Kam et al., 2013).
For HPP treated rice, commercial white rice was used as a benchmark. The results show that
the L*, a* and b* values were comparable to commercial white rice. Similar to results from
parboiled rice, the major contributors for variation in colours were L* and b* while a* was
more or less the same and also there was no significant difference statistically as well
between treatments for a*.The lowest ΔERaw value was obtained for 200 MPa 3 hours
treatment and highest was for 600 MPa 2 hours treatment. Fortified premix rice (condition A)
milled at 120s and the diluted fortified rice (milled at 120s) had the lowest ΔEparboil and ΔERaw
value respectively. This shows that the optimized parboiled fortified rice had the lowest
difference in colour compared to its corresponding reference.
Overall, ΔEparboil was lower than ΔEraw indicating that the colour difference between fortified
rice and parboiled rice was lesser than when compared with uncooked commercial white rice.
When the fortified parboiled rice was diluted with white rice the ΔEraw was the lowest
implyingthat there is minimal variation from the commercial white rice and therefore it is
suitable for commercialization at this dilution (1:10). The likelihood of product acceptance
would increase if there is no foreign pigmentation in rice (Hurrell, 1997). From the ΔEparboil
value it can be seen that fortification during parboiling does not create a huge magnitude of
colour change. However, colour change is evident due to fortification using both processing
methods. ΔEraw for HPP rice treated at 200 MPa (1h, 2h and 3h) was relatively lower than
parboiled rice as parboiling introduces enzymatic and non-enzymatic reactions and also
113
diffusion of pigments from the rice bran into the rice endosperm (Bhattacharya, 2004;
Lamberts et al., 2006a; Lamberts et al., 2006b). However on higher (400 & 600 MPa) and
longer (2h & 3h) pressure treatments HPP rice ΔEraw was high due to low redness value(a*)
making it less acceptable.
5.3.3. RVA pasting curves
Treating starch in excess water or applying heat and moisture treatment is known to cause
variation in the pasting properties and lower the viscosity of the pasting starch (Knutson
1990; Gunaratne and Hoover 2002; Singh et al 2005; Vermeylen et al 2006). In this study
rice has been treated by heat and moisture treatment and also subjected to high pressure.
Therefore, the two processes are expected to have an impact on the starch pasting properties.
It was tried to deduce if fortification had any impact on the rice pasting properties by
comparing the fortified rice to commercial white rice and parboiled rice which were used as
control.
The pasting curve data for the processed rice starch has been presented in Table 5.9 below. It
can be seen that with an increase in the pressure treatment, there is a decrease in the viscosity
(peak viscosity and final viscosity). However, more or less all the peak times were the same
for all the samples. The highest peak and final viscosity was observed for unprocessed white
rice. This shows that high pressure treatment and parboiling modifies the starch structure and
in agreement with previous studies from literature which show that similar results were
obtained for modified starches and high pressure processed starches (Stolt et al., 2001 & Pei-
Ling, 2011). The process of fortification did not have any impact on the pasting properties
unlike the processing. The reasons for lower peak and trough viscosities were explained by
Pei-ling et al., (2011) by three steps. The first reason was that HPP treatment weakened the
resistance to shear and therefore withholding the starch granules from breakdown prior to
arrival at peak viscosity. The second reason was that continued shear lead to sustained
fragmentation of the ruptured granules. Finally, HPP affects molecular weight polymers
including crystallinity melting, amorphous hydration and gelatinization of starch granules
(Blaszcak et al., 2005).
The peak time value for unprocessed and processed starch was almost the same i.e. at 7
minutes. The pasting temperature on the other hand increased slightly between treatments. It
was more or less similar for 200 MPa treatment and the control rice samples. However there
was a slight increase for the treatment at 400 and 600 MPa indicating that pasting
114
temperature was higher for HPP modified starches than native starches. The breakdown value
indicates the end point of gelatinization due to the melting of starch crystals or complex
viscosity increase. It was due to the disintegration of gelatinized and swollen starch granules
(Hermanson & Sevgmark, 1996). In the present study, most of the breakdown values were
negative except for 600 MPa treated rice and parboiled rice indicating that these 2 types of
rice showed higher degree of pre-gelatinization and this can be correlated with the degree of
gelatinization data as well. Setback value extracted from RVA implies the starch thaw
stability. Lower setback values mean lower tendency to retrograde which is commonly
observed in pre-gelatinized and modified starches (Deffenbaugh & Walker, 1989). In this
study lowest setback value was observed for the commercial parboiled rice and the highest
for the unprocessed white rice.
115
Table 5.7 RVA data for fortified rice sample processed by parboiling and HPP
techniques
Note: RVU stands for rapid viscosity unit. Each viscosity values are Mean±SD of
replicateanalysis.
RVA graph for parboiled rice (Figure 5.6) showed dead peak implying that it has been
completely gelatinized. Commercial parboiled rice also showed RVA pattern similar to in-
house parboiled rice. Therefore the RVA results are comparable to the degree of
gelatinization in the present study which was close to 100% for in-house parboiled rice. In
this current study, glucose released from parboiled rice was found to be high after digesting
Sample
Peak
viscosity
(RVU)
Trough
viscosity
(RVU)
Breakdown
(min:sec)
Final
Viscosity
(RVU)
Setback
Viscosity
(RVU)
Peak
Time
(mins:
sec)
Pasting
Temperature
(°C)
200 MPa 1 hour 1188±2.8 1190.5±7.7 0.00 2618±8.4 1427.5±9.1 6.93 92.65
200 MPa 2
hours 1035±14.1 1028.5±0.7 -4.00 2416.5±0.7 1388.5±3.5 7 93.3
200 MPa 3
hours 1195.5± 1199±1.4 3.00 2611±7.0 1412±4.2 7 92.6
400 MPa 1 hour 771±24.0 788.5±3.5 -3.00 1791±2.8 1002±4.2 7.00 94.85
400 MPa 2
hours 899±14.1 910±14.1 -11.00 1913.5±7.7 1001±2.8 7.00 95.00
400 MPa 3
hours 860±23.0 871.5±9.1 -1.00 1627±4.2 756±12.7 7.00 93.4
600 MPa 1 hour 912.5±6.3 911±9.8 4.00 1960.5±40.3 1064±24.0 7.00 94.25
600 MPa 2
hours 538±36.7 505±8.4 1.00 934±19.7 429±9.8 7.00 94.95
600 MPa 3
hours 442±57.9 486±8.8 3.00 1152±8.4 658.5±23.3 7.00 94.95
In-house
parboiled Rice 215.5±0.7 210.5±10.6 -3.00 428±2.8 206±7.0 7.00 Nil
Unprocessed
White Rice
1297.5±12.
0 1304.5±6.3 -3.00 3252±31.1 1972±5.6 7.00 92.45
Unprocessed
brown Rice
1458.5±43.
1 1418.5±24.7 -8.00 2613.5±6.3 1166.5±33.2 6.87 93.35
Cooked Rice 250±9.8 251.5±12.0 -3.00 456±5.6 200.5±3.5 7.00 Nil
Commercial
Parboiled Rice 155.5±19.0 170±2.8 1.00 226±8.4 60.5±3.5 6.93 Nil
116
with α-amylase compared to HPP rice and therefore completely gelatinized starch is more
susceptible to hydrolysis of starch than partially gelatinized starch. By comparing the
concentration of folic acid and β-carotene in the parboiled fortified rice and HPP rice (for
both the treatments condition A from Table 3.1 (page no.47) was the initial mass of added
micronutrients) it was found that parboiled rice had higher concentration of the 2 vitamin
(folic acid: 367 µg/g & β-carotene: 235 µg/g) than HPP rice (folic acid: 164 µg/g & β-
carotene: 43 µg/g). This could be due to the higher degree of gelatinization due to which folic
acid and β-carotene were able to be released better from the rice matrix in parboiled rice than
HPP rice during extraction. This can also be correlated with HPP results where increase in
treatment time resulted in lower concentration of folic acid and β-carotene in the rice (Table
4.4 (page no.91), 4.7 (page no.91)) due partial gelatinization and formation of resistant starch.
Hence the degree of gelatinization of rice starch during the processing can be correlated with
the variation in concentration of the micronutrients in the fortified rice.
Figure 5.3 RVA graph for rice treated at 200 MPa at different time intervals using high
pressure process
117
Figure 5.4 RVA graph for rice treated at 400 MPa at different time intervals using high
pressure process
Figure 5.5 RVA graph for rice treated at 600 MPa at different time intervals using high
pressure processing
119
5.3.4. X-ray Diffraction (XRD) of fortified rice flour samples showing crystalline
patterns
The results from previous section illustrate that crystalline nature of starch is changed due to
processing affecting the birefringence of starch. In this section the crystalline nature and
degree of crystallinity of starch samples that were processed and unprocessed were examined
using XRD. Although parboiled rice was well studied for crystalline properties using XRD
not many studies have looked at the processing of the whole rice kernels under high pressure
and then studying its physiochemical properties. In this study the influence of heat and
pressure on the native crystal pattern of rice starch was explored.
Crystals of native starch show A-type crystals (Katz, 1928). This can be seen in Figure 5.7for
the unprocessed control rice. There is not much change in the 2Θ value for unprocessed and
parboiled rice. The crystal structure also seems not altered. However there was a decrease in
the intensity of counts for the same 2Θ value for parboiled rice. In this study, it can be
observed that for commercial parboiled rice, there is minor change in type of crystal but it is
incomplete. This type of crystal is alike with V-type of crystal formed by the amylose-lipid
interaction under severe parboiling conditions (Dercyke et al., 2005). Change in native starch
structure can be correlated with the degree of gelatinization as discussed in the previous
sections.
120
Figure 5.7 XRD pattern for parboiled and commercial white, brown and parboiled rice
Figure 5.8 XRD pattern for HPP treated rice at 200 MPa for 1,2 and 3h
NPB- Non-parboiled
PB- Parboiled
121
Figure 5.9 XRD pattern for HPP treated rice at 400 MPa for 1,2 and 3h
Figure 5.10 XRD pattern for HPP treated rice at 600 MPa for 1, 2 and 3h
122
Li et al., (2011) studied physicochemical properties of purified rice starch suspended in
excess water and subjected to high pressure processing. The results obtained in the present
study were in agreement with the study by Li et al., (2011). In the present study when the rice
flour was treated at 600 MPa for 2 hours it was seen that there was evident change in the
predominant crystal type, i.e. from A-type to B-type. These findings were also comparable to
the study conducted by Li et al., (2011). This shows that not only purified rice starch but also
rice flour exhibits same XRD pattern after processing and also the addition of micronutrients
did not impact the crystalline pattern of rice starch. It is also in agreement with the data
obtained from microscopic images and pasting properties. Pressure treatment at 600 MPa
resulted in complete disintegration of starches and partial gelatinization. The threshold
pressure for conversion of crystal type from A-type to B-type existed in the range between
480- 600 MPa (Li et al., 2011). For B- type starches water fills up the channel in the cell unit
of the crystallite and stabilizes the structure(Katopo et al. 2002). But for A-type starches
amylopectin branches are more scattered and therefore more flexible allowing rearrangement
of double helices and thereby generating a channel that includes water molecules under
pressure (Jane et al., 1997). As a result crystal structure changes from A-type to B-type and
this requires a threshold pressure treatment of 600 MPa and 2 hours approximately.
Table 5.8 % Crystallinity (extracted from XRD) of rice sample treated by high pressure
processing and parboiling
Sample % Crystallinity
Brown rice 46.6±2.1
White rice 37.8±2.6
200 MPa (1h) 38.5±2.1
200 MPa (2h) 35.3±1.3
200 MPa (3h) 36.5±1.7
400 MPa (1h) 30.8±3.2
400 MPa (2h) 30.5±1.2
400 MPa (3h) 24.2±2.5
600 MPa (1h) 23.8±3.8
600 MPa (2h) 22.6±2.2
600 MPa (3h) 17.1±2.5
Parboiled (0s) 23±2.1
Parboiled (60s) 24.9±1.6
Parboiled (120s) 20.8±1.4
Notes: Crystallinity values are Mean±SD of duplicate analysis.
Bracketed alphanumerical values represent treatment time in hours for HPP and milling time
in seconds for parboiled rice.
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From the above table, native starch had a % crystallinity of 38.5%. Typical native A-type
crystals from cereals exhibit strong peaks at 15.04°, 16.84°, 17.96° and 23.03° 2Θ and
weaker peaks at 20.04°, 26.3° and 30.26° 2Θ. This pattern can be observed from the XRD
graphs for all the rice flour samples. With an increase in the pressure treatment, there was a
decrease in the % crystallinity. Parboiling treatment also resulted in lower crystallinity
compared to native starches although there was no change in the type of the rice crystal.
Basmati rice (Pusa 1121) has not been previously studied for XRD properties after being
subjected to HPP or parboiling treatment. The results from XRD can be correlated with the
degree of gelatinization and RVA results. Degree of gelatinization is inversely proportional to
the % crystallinity whereas RVA results (peak, final and setback viscosity) are directly
proportional to the % crystallinity. The degree of milling of parboiled rice did not
significantly affect the % crystallinity and also the parboiling treatment was not severe to
impose change in crystal structure.
Collectively the data from degree of gelatinization, RVA, microscopy and XRD denote that
physico-chemical properties are affected by the swelling property of amylose in the rice. The
rice variety used in this study had high amylose (34%) and hence the variation in the starch
properties can be linked to the micronutrients concentration in the rice. Although the addition
of micronutrients (folic acid in particular) did not have any influence on the starch alteration,
its uptake to the fortification medium (rice) can be associated with the amylose modification
due to processing.
5.3.5. Microscopic images of starch granules processed by parboiling and HPP
treatment
In the above sections physicochemical properties of starch granules were explored by
quantitative techniques. In this section, the change in the rice starch was examined visually
using polarized light microscopy and differential interference contrast microscopy (DIC).
Parboiled rice has been previously well studied under polarized light microscopy (Juliano,
1985; Pillaiyar, 1988) however there are limited studies which used DIC to observe starch
changes under microscope. DIC has the advantage of capturing images in striking colour
(optical contrast) with a 3-dimensional shadowed-like appearance and at outstanding
resolution (John Innes Centre, n.d.). Starch granules from the fortified processed rice
observed under polarized light microscopy and DIC are presented in the current section.
124
a . b.
Figure 5.11a & b: Starch images for rice treated at 200 MPa for 1 hour (a. under
differential interference contrast; b. under cross-polarized light)
a. b.
Figure 5.12a & b: Starch images for rice treated at 200 MPa for 2 hours (a. under
differential interference contrast; b. under cross-polarized light)
a. b.
Figure 5.13a & b: Starch images for rice treated at 200 MPa for 3 hours (a. under
differential interference contrast; b. under cross-polarized light)
125
a. b .
Figure 5.14a & b: Starch images for rice treated at 400 MPa for 1 hour (a. under
differential interference contrast; b. under cross-polarized light)
a. b.
Figure 5.15a & b: Starch images for rice treated at 400 MPa for 2 hours (a. under cross-
polarized light under; b. differential interference contrast)
a. b.
Figure 5.16a & b: Starch images for rice treated at 400 MPa for 3 hours (a. under
differential interference contrast; b. under cross-polarized light)
126
a. b.
Figure 5.17a & b: Starch images for rice treated at 600 MPa for 1 hour (a. under
differential interference contrast; b. under cross-polarized light)
a. b.
Figure 5.18a & b: Damaged starch images for rice treated at 600 MPa for 2 hours (a.
under differential interference contrast; b. under cross-polarized light)
a. b.
Figure 5.19a & b: Healthy starch grain images for rice treated at 600 MPa for 2 hours
(a. under differential interference contrast; b. under cross-polarized light)
127
a. b.
Figure 5.20a & b: Partially integrated starch Starch images for rice treated at 600 MPa
for 3 hours (a. under cross-polarized light under; b. differential interference contrast)
a. b.
Figure 5.21 Figure 5.17 a & b: Completely damaged starch Starch images for rice
treated at 600 MPa for 3 hours (a. under cross-polarized light under; b. differential
interference contrast)
a. b.
Figure 5.22a & b: Starch images for parboiled rice milled at 0s (a. under differential
interference contrast; b. under cross-polarized light)
128
a. b.
Figure 5.23a & b: Starch images for parboiled rice milled at 60s (a. under differential
interference contrast; b. under cross-polarized light)
a. b.
Figure 5.24 a & b: Starch images from in-house parboiled rice milled at 120s a. shows
birefringence in some starch granules under DIC while in b. it can be seen that there
has been a damaged starch granule
a. b.
Figure 5.25a & b: Starch images from comemrical parboiled rice showing
birefriengence (a. under DIC and b. cross polarized light)
129
a. b.
Figure 5.26 a & b: Starch images from untreated rice showing birefriengence under a.
DIC and b. cross polarized light
Birefringence (Maltese-cross –pattern) is caused by the radial orientation of crystallites in
native starch granules under polarized light microscope (Yuryev, Wasserman, Andreev, &
Tolstoguzov, 2002). From the images it can be seen that there is some visible birefringence
and Maltese cross in most samples. For samples treated at 200 MPa at 1hour, 2 hours and 3
hours Maltese cross and birefringence is visible for all the samples with minor damage with
longer treatment times. For rice samples treated at 400 MPa losses in birefringence can be
observed with increase in treatment time. Rice samples treated at 600 MPa there was evident
loss of birefringence with increase in treatment time. The damage was higher when the rice
was treated at 600 MPa for 2 hours and 3 hours. Birefringence was completely lost for some
intact starch granules while the granular structure and the crystalline region are deformed.
Method of grinding can also play a role in damaging starch granules (Li et al., 2014).
Damage to starch due to grinding does not follow any linear relationship. When isolated
starch granules from different botanical sources were milled cryogenically, it showed non-
linear relationships in damage level, disruption of double helices and crystalline structure and
degradation of starch (Dhital et al., 2011 & Stark& Yin, 1986). When parboiled, the starch
granules stop exhibiting birefringence under polarized light, and the orderly polyhedral
structure of the compound granules is converted into a consistent mass (Juliano, 1985;
Pillaiyar, 1988). Loss of birefringence in in-house parboiled samples shows that in house
parboiling treatment caused more damage compared to rice starch than commercial parboiled
sample which retained partial birefringence. This shows that heat and moisture treatment can
cause higher degree of distortion to the crystalline region of the starch thereby causing loss in
birefringence.
130
HPP modification of non-crystalline granule tapioca starch slurry in water occurred at 600
MPa for 30 min treatment. Swelling and disintegration of waxy corn starch was observed at
600 MPa treatments. Variation in swelling was also observed at different concentrations of
the starch-water slurry (Pei-Ling et al., 2012). Similar results were observed in the present
study as well. High pressure was a much milder treatment than heating in terms of
gelatinization (Muhr & Blanshard, 1982). Cooking results in swelling of starch grains and
making them lose definitions in polarization cross and other signs of gelatinization. In
general, cooking affects microscopic features of each starch species in a unique way. Watson
& Dikeman (1977) observed different layers of rice – hull, aleurone layer and starchy
endosperm before and after parboiling using scanning electron microscope (SEM). It was
found that parboiling showed complete gelatinization of endosperm and it was hard to
distinguish between the parenchymal cell of the hull and aleurone layer after parboiling.
Birefringence is associated with the semi-crystalline nature of starch which is made up of
orderly sequences of amylose and amylopectin. Swelling of amylose followed by
amylopectin followed by change in sequence of starch can thus be accounted for
birefringence loss. Hydrolysisof starch amylases can be aided by gelatinization (Blish et al.,
1938). For in-house parboiled rice in the present study most of the starch granules lost the
crystalline structure. Also the starch was almost completely gelatinized in contrast to HPP
processed rice which showed partial gelatinization (5.3.1). This implies that α-amylase that
was used to breakdown starch to release folic acid from the fortified rice would be able to
hydrolyse parboiled rice starch better than HPP rice. This confirms the results where there
was decrease in concentration of folic acid with increase in pressure treatment time (section
4.3.2) as being due to the formation of resistant starch during HPP treatment which was not
susceptible to hydrolysis.
Combining the results from sections 5.3.2, 5.3.3 and 5.3.5 it can be suggested that the starchy
endosperm where the fortified micronutrients are adhered to the rice is altered due to the
processes. Correlating the microscopic images to XRD, the experimental data shows that
HPP caused variation to native starch crystal and parboiling retained native starch crystal
pattern although it was a harsher treatment. Therefore it can be said that the two processes
alter the arrangement of amylose and amylopectin uniquely and this variation in the sequence
during processing can be attributed to the variation in micronutrient concentration in fortified
rice.
131
5.3.6. Real-time Magnetic Resonance Imaging of rice during parboiling process
Migration of water during the soaking stage of parboiling was investigated using MRI. Water
migration into the kernel would likely indicate how much of the nutrients are likely to be
absorbed into the grain as they are dissolved in the solution for the fortification process. Real-
time MRI was used for the first time in this study to see if water migration can provide
insight into absorption of the nutrients into the grain.
Figure 5.27 Coronal section images of rice after parboiling process at different time
intervals at the optimized condition
0 mins
30 mins
45 mins
60 mins
120 mins
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Figure 5.28 Sagittal section of soaked rice at different time intervals during the
optimized parboiling conditions
a. b.
Figure 5.29 a&b: Migration of water with and without micronutrients where A- is with
Fe and B – is without any micronutrients
Figure 5.27 shows the variation in the water molecules measured as resonance among rice
kernels soaked at different times. The top image of the rice kernel shows the reference kernel
which was dry rice, not parboiled prior to imaging. It can be seen that – due to the low proton
0 min
A 60 mins
B 60 mins
A 90 mins
B 90 mins
A 120 mins
B 120 mins
0 mins
A 15 mins
B 15 mins
A 30 mins
B 30 mins
A 45 min
B 45 mins
0 mins
30 mins
45 mins
60 mins
120 mins
133
abundance- this reference shows essentially very low signal which could be due to the
minimal content of moisture in dry rice. With increase in soaking time, more water starts to
diffuse into rice kernels, which can be measured as additional signal from MRI. From the
MRI images the cracks in the rice kernel are visible. With the progress in time the centre of
the rice kernel can be seen as brighter which is presumably due to increased uptake of water
and softening of the kernel core. The sagittal section shows a similar trend (Figure 5.28). It
can also be observed that the innermost core of the rice looks brightest compared to other
layers. This contrast is evident especially during the last 30 minutes which means that there
are some modifications and changes occurring during this step altering the starchy
endosperm. The previous sectionsdetermined the qualitative changes in rice starch structure
and this alteration could be narrowed down to occur during this period of the soaking time.
This essentially reinforces that this method measures a combination of water content and
structural features of starch. However, pure diffusion of water during the later stages could
not be obtained as a result of changes in starch degradation. But adopting a lower soaking
temperature it would be possible to unravel water diffusion more effectively where starch
decomposition does not initialize sooner or by concentrating more on the earlier stages of
diffusion. However, in both cases it is not really pure variation in water but will contain the
structural features as well.
Figure 5.29 shows that there is not much signal variation with and without iron (25 mg) in the
soaking water. Very likely this is due the low level of iron concentration in the soaking water
which is below the limit where significant image alterations can be detected in the MRI. Also
the diffusion of water molecules into the rice kernel is independent of the micronutrients
present in the soaking water so that iron content might not be increased by the same quantity
as water. In other words, irrespective of the concentration of the of micronutrients the
moisture distribution remains the same in rice after the soaking step of parboiling. This can
be substantiated with the moisture data presented in Chapter 3 section 3.3.2 (Figure 3.2-page
no. 58) Signal intensity of the rice increases with time as seen in the MRI by about 35% on an
average after 120 minutes soaking and the trend is similar for rice in the presence and
absence of micronutrients. However, for methodological reasons the signal intensity of a
gradient echo method does not depend solely on water distribution in the sample but is also
influenced by local sample structures. This is why MRI imaging signal cannot be directly
translated to moisture content and it is not possible to actually quantify the water content
from MRI methods.
134
From the experimental data for uncooked parboiled rice (section 3.5.5) it can be said that the
maximum concentrations of the micronutrients were on the outer surface of the rice kernel
rather than in the centre. Inference from the MRI data showed that there is a nominal degree
of structural changes observed in the rice core indicating that the core was softened due to the
process. This can be seen by the signal which was higher on the inside of the rice kernel
rather than the outside. It can be speculated that there is evident movement of water during
the parboiling process but the intensity of movement of the micronutrients were much less
compared to the water. This could be the reason for higher concentration of micronutrients on
the outer layer rather than in the rice core. Similar results were obtained by Horigane et al.,
(2006) where moisture absorption was slower on the periphery of the white rice grain
especially on the dorsal side from the apex of the grain despite the closest contact with water
when rice grain was soaked at room temperature. Cracks observed on the transverse section
of the rice kernel were formed on the ventral side and can be seen in the 30 min soaking
image (Figure 5.27). The central line has the highest intensity of signal and is proposed to be
the main route for water infiltration which then passes on to the cracks on grain surface. Rice
aleurone layer thickness varies depending on the rice variety however it is thicker on the
dorsal side than on the ventral side (del Rosario et al., 1968).After 30 minutes soaking, the
cracks become obscure. Generally, water migrates from the pericarp vascular bundle into the
embryo (Horigane et al., 2001). Water distribution was highest in the cracks and regions
along the central line than other parts of endosperm (Horigane et al., 2006).
In brown grains of Koshikari and Yamadanishiki varieties, water penetration was inhibited by
the embryo, pericarp and seed coat due to which the rate of uptake was much lower than
milled rice. In case of BR, infiltration started from the embryo and the surface of the rice
grain. However it was observed that the final moisture distribution was much more
homogenous in milled rice although water tended to be more retained around the central line
of the softer tissue than other parts of the endosperm. The core endosperm of rice contains
amyloplasts with thin walled starch granules (Horigane et al., 2006). The outermost cell
layers i.e. the sub-alourone layer contained high protein and lipids and lesser amyloplasts and
compound starch granules than inner endosperm (Marshall & Wadswoth, 1994). This could
be a reason for uptake of water molecules around the central core which is more hydrophilic
than other parts which contain relatively more hydrophobic compounds.
135
5.4. Conclusions
Qualitative changes in rice starch structure due to parboiling and high pressure processing
was collectively studied in this chapter by analysing the degree of gelatinization, evaluating
pasting properties of starch using RVA and crystalline alterations using XRD. Microscopic
examination of starch due to processing was also investigated and migration of water during
parboiling was also explored using MRI.
Parboiled rice showed close to 100% gelatinization of starch while for the HPP
treatment it was proportional to the increase in pressure.
Pasting properties of HPP treated rice exhibited partial gelatinization of starch but not
complete gelatinization as in the case of parboiled rice where there were dead peaks
present.
Parboiling treatment did not result in change in starch crystal structure however for
600 MPa treatments after 1 hour, starch crystallinity changed from A-type to B-type.
Treatment of rice starch at 600 MPa and parboiled rice starch showed complete
distortion of starch granules compared to other pressure treatments (200 and 400
MPa). Loss in birefringence and Maltese cross was evident from polarized light
microscope and differential interference contrast microscope.
Moisture distribution for parboiled rice kernels examined by MRI was more
concentrated on the rice core- endosperm than in other parts. Gradual uptake of water
during the parboiling process could be seen using this imaging method. The
movement of water into rice was independent of the micronutrients in the soaking
water.
To summarize, starch properties are significantly altered due to the processing methods
and they vary between the two processing methods. The degree of alteration varied
between different HPP treatment and parboiling process. HPP resulted in partial
modification of rice starch while parboiling caused complete gelatinization. Different
parameters studied to gain an insight into starch structural changes showed that the
variation in the amylose swelling and leaching is speculated to be associated with the
dissimilarity in vitamin uptake during the fortification by both the processes. There is
evident matrix effect that influences the release of vitamin from the starch matrix and
therefore there is variation in concentration of vitamin in rice after parboiling and HPP.
Although 400 MPa and 600 MPa treatments were not suitable for implementing rice
fortification, the qualities of rice starch make them suitable for other products where they
136
can replace pre-gelatinized starch. Whilst the findings in this chapter relate to the
modification in rice starch in terms of fortification another perspective that could affect
the starch properties due to processing would be the digestibility. Alterations in the rice
starch due to processing can result in the formation of resistant starch and thereby
influence the taste and digestion of the rice in vivo after consumption. The acceptability
of the fortified rice among consumers and the bio-accessibility of the fortified rice would
therefore be addressed in the following chapters (6 & 7).
137
CHAPTER 6
6. CONSUMER ACCEPTANCE STUDY OF FORTIFIED PARBOILED RICE
6.1 Introduction
In the previous chapters optimisation of fortification process through two different techniques
(parboiling and HPP) were discussed and the changes that occurred to rice starch due to
processing was also addressed. The success of a fortified rice product would depend on
consumer acceptance of the product, when cooked. Acceptance of cooked rice is a vital
attribute especially to consumers for whom rice is a staple food. Rice preparation methods
and degree of cooking is very subjective and varies highly depending on the region of the
world. For instance, the Japanese prefer cooked rice that is soft and sticky and of short-grain
variety such as Japonica. Consumers from the U.S. like medium and long grain rice that is
firm and not sticky. In the Asian countries such as Thailand, Pakistan and India fragrant rices
are favoured (Lyon, 1999).
With quality attributes on one hand, nutritional value of rice also plays an important role in
rice consumption worldwide. Brown rice is considered healthy in the U.S. (Marshall and
Wadsworth, 1994) despite better palatability and digestibility of milled rice compared to
brown rice (Misaki and Yasumatsu, 1985). Hence nutritional value of rice is also a significant
factor in consumer purchase and acceptance.
Acceptability of fortified rice can be a challenge as it is highly likely that the addition of
extraneous nutrients could alter the native rice taste. Several studies have been performed by
researchers all over the world to understand acceptance of fortified rice. Lee et al., (1995)
evaluated the sensory qualities of calcium fortified rice. The data from their study proved that
there is significant decrease in tenderness and adhesiveness when the rice had additional
calcium. Iron-fortified extruded rice was evaluated for acceptance using a triangle test and it
was deduced that when ferric pyrophosphate was used as a fortificant, the tested rice closely
resembled that of the control rice which was natural unfortified white rice (non –extruded) in
both cooked and uncooked forms (Moritteo et al., 2005). Folic acid fortified rice using the
parboiling method was also investigated for consumer acceptance by Kam et al., (2012).
The aim of this study was to analyse consumer acceptance of parboiled fortified rice. Rice
fortified using the HPP technique could not be used for sensory studies as the pressurising
138
fluid used was 80% propylene glycol which seeped into the rice during the HPP treatment in
small quantities. Due to safety reasons this study could not be conducted. The sensory study
for parboiled fortified rice was conducted in two phases i.e.by investigating if people would
buy the rice from the supermarket and if they liked the taste of the fortified rice. This was
conducted by performing descriptive sensory tests and the data was analysed statistically for
interpretation of results.
6.2 Materials and Methods
6.2.1 Preparation of fortified parboiled rice
Brown rice was prepared as described in section 3.2.1. For the two phases of the sensory
evaluation study, folic acid (150 mg), β- carotene (62.5 mg) and iron (25 mg) were dissolved
in the soaking water. To this, 300g of brown basmati rice that was purchased commercially
was added to the fortificant solution in rice-water ratio of 1:2. The rice was soaked at 70°C
for 2 hours and then it was steamed at 100°C for 1 hour. Following this the rice was dried at
room temperature until the moisture content dropped to 12% (wet weight basis). The
parboiled brown rice was milled in a food-grade lab scale mill (Satake Test grain Mill, Japan)
for 120s such that the bran removal was about 10-11% (Section 3.2.5). The parboiled rice
was then diluted in the ratio 1:10 with unfortified commercial white rice since the parboiled
rice was prepared as a premix and intended to be mixed with white rice for better acceptance
among consumers.
6.2.2 Micronutrient analysis of the fortified rice
The fortified parboiled rice (cooked and uncooked) was analysed for concentration of folic
acid, iron and β – carotene as discussed in sections 3.2.8.1 – 3.2.8.3. Both concentrated
premix and the diluted rice were tested for concentration of micronutrients in uncooked rice
and retention after cooking. Folic acid and β – carotene were analysed using HPLC while
iron analysis was performed using ICP-OES.
6.2.2 Sensory evaluation of parboiled fortified rice: An Overview
A schematic representation of the sensory evaluation performed for the parboiled fortified
rice is presented in Figure 6.1. Phase 1 of the sensory study was performed on uncooked rice
to understand if consumers were willing to purchase the fortified diluted rice if made
commercially available. In Phase 2 of the sensory study the parboiled fortified rice that was
diluted with the unfortified white rice was cooked and evaluated by consumers for
acceptability. Methodology for the two studies that were conducted is explained in detail in
the following sections.
139
Figure 6.1 Schematic representation of fortified parboiled rice prepared for Consumer
Acceptance Study 1 and Study- 2
Brown rice
(300 g)
Soaked in micronutrients
solution in the ratio 1:2 at
700C for 2 hours
Excess water drained off and
steamed at 1000C for 1 hour.
Dried in the shade until
moisture content <12%
Milled for 120s
Study 1: Visual Consumer
Acceptance Study of uncooked
fortified parboiled rice
Fortificant (mg/300g rice) added to
soaking water: FA- 150mg; BC- 62.5
mg; Fe- 25 mg.
Samples:
1. Commercial parboiled rice
2. Commercial White rice
3. Fortified diluted rice
4. Unfortified parboiled rice
Study 2: Consumer Acceptance Study
of cooked fortified rice after diluting.
Fortificant (mg/300g rice) added to
soaking water: FA- 150mg; BC- 62.5
mg; Fe- 25 mg.
Samples:
1. Fortified diluted rice
2. Commercial White rice
140
6.2.3 Study 1: Visual consumer acceptance of uncooked fortified parboiled rice
6.2.3.1 Samples preparation
For the first phase of the sensory evaluation four rice samples were presented to 49 untrained
participants/consumers. The samples used were as follows: commercial parboiled unfortified
rice, unfortified commercial white rice, diluted parboiled fortified rice with unfortified white
rice and unfortified parboiled rice. Commercial parboiled rice and commercial white rice
were used as controls. Parboiled rice has a characteristic yellow colour and colour is a major
influential factor for rice purchase from a consumer‘s perspective. The study was performed
with uncooked rice to determine if the rice would be purchased from the supermarket if it
were available commercially (Tomlins et al., 2007). Therefore, as a preliminary step to
understand consumers‘ choice about purchasing fortified parboiled rice from the market,
phase 1 of the sensory evaluation study was performed.
6.2.3.2 Survey Methodology
The four uncooked rice samples as mentioned in the above section were evaluated by 49
untrained consumers who were students and staff of the University of New South Wales,
Sydney, Australia. About 20g of each rice sample packed in transparent Ziploc® pouches
were presented to the consumers with a randomized 3-digit code. A survey questionnaire was
also provided along with the samples for the consumers to fill up. The questionnaire was
divided into 2 parts: the first part included questions about the demography and rice
consumption pattern of the consumers; while Part II evaluated their opinion, likes and
purchase intent of each rice type. Table illustrating the items that were presented in the
consumer questionnaire along with the likert scale is in Appendix (2&3).
For the study to evaluate the purchase intent of fortified rice, a five point purchase intent
scale was used based on Mcdaniel and Gates, (1998) & Kam et al., (2013) i.e. ―Definitely
would not buy‖, ―Probably would not buy‖, ―Undecided Probably would buy‖ and
―Definitely would buy‖ For simpler calculations and convenience of the study ‗Definitely
would not buy‘ and ‗probably would not buy‘ were categorised under ―Not buy‖ (1 and 2).
On the other hand ―Probably would buy‖ and: definitely would buy‖ were categorised into
―Would buy‖ (4 and 5) (Moskowitz, 2004 & Kam et al., 2013).
141
6.2.4 Study 2: Sensory evaluation (tasting) of cooked fortified rice after mixing with
unfortified white rice
6.2.4.1 Sample Preparation
For the second phase of the study fortified rice was prepared by adding folic acid: 150 mg; β-
carotene: 62.5 mg; iron: 25 mg/ 300g of brown rice. The parboiling conditions followed are
described in section 3.2.3. Two samples were provided to the consumers for the sensory
study which included commercial unfortified white rice and fortified rice mixed with
unfortified white rice in the ratio1:10. The two rice samples were cooked and presented to the
consumers for sensory evaluation labelled with a random 3-digit number code.
Preparation of uncooked fortified rice with unfortified white rice was essential to monitor and
ensure that the added nutrient intake from the rice were within the safe upper limits of daily
intake. It has been estimated that in rice eating countries such as India and Bangladesh
approximately 440g of rice is usually consumed on a daily basis (IRRI, n.d.). In Table
6.1(page no. 144) the concentration of folic acid, β-carotene and iron that was retained in the
cooked rice has been presented in µg/440 g of rice. The fortified premix rice and unfortified
white rice were mixed in the ratio of 1:10 in a laboratory food roller mixer (Thermoline
Scientific, ten roller mixer, Model 210 RM) on a lab scale. The mixing ratio was based on the
daily requirement for target populations. The mixing ratio (1:10) was chosen such that it
would be able to provide at least half of RDI for each micronutrient i.e. 400 µg of folic acid,
18 mg of iron and 700 µg of vitamin A i.e. 44 g of fortified rice in 396 g of unfortified rice.
The mixing was done to derive an appropriate concentration of the micronutrients added as
well as to dilute the colour intensity of the fortified rice to increase consumer acceptance. It
also masked any unique aroma of the parboiled rice. Mixing with white rice also ensured that
the consumers will choose the fortified diluted rice over normal white rice.
6.2.4.2 Cooking method
The mixed rice (250 g) was washed thrice in approximately 500 mL of water and drained.
The rice was cooked in an electric cooker (Breville, BRC200, Australia) in a rice: water ratio
of 1: 1.75 for 19 minutes at boiling temperature. It worth noting that there is variation in the
cooking time of the rice where fortified parboiled rice took 24 minutes to cook at room
temperature while when mixed with white rice the cooking time reduced 19 minutes (Section
142
3.2.7). The rice was kept for about 10 minutes in the cooked state to absorb moisture and then
it was kept in the rice cooker at a temperature of 60±1 °C until served.
6.2.4.3 Survey Methodology
The cooked rice was served in a plastic container (approximately 20g) to the consumers and
the samples were labelled with a randomised 3-digit code. Untrained consumers (No: 54)
were recruited and most of them were staff and students from the University of New South
Wales. The sensory evaluation was performed in a partitioned booth under uniform light
conditions and the subjects were not informed about the nature of the study.
The survey questionnaire for the second phase of the study was also similar to the first phase
of the study (Appendix 4). The former part of the questionnaire included demographic
questions about the consumers and about the rice consumption pattern. The latter part of the
questionnaire evaluated their opinion about the cooked rice based on 5 different attributes.
The intensity of each attribute was evaluated on a 5-point just-about-right (JAR) scale. For
convenience sake the 5-point scale was categorized into ―Not enough‖ (1 and 2), ―Just-about
right‖ (3) and ―Too much‖ (4 and 5) (Popper and Gibes, 2004 & Kam et al., 2013).
6.2.5 Data Analysis
6.2.5.1 Study 1: Visual Consumer acceptance of uncooked fortified parboiled rice
To understand and interpret the data obtained from the results of the sensory evaluation
study, statistical tests were performed. For interpreting the results obtained regarding the
perception and liking of attributes in Appendix 2 (question 2-5), one way analysis of variance
ANOVA was used to compare the difference between the samples (including the control).
The average of the means was compared using Tukey‘s (HSD) means comparison test (p<
0.05). To compare the variability among different attributes between general consumers and
consumers who were familiar with parboiled rice, independent samples t-test was performed.
Chi-squared (χ2) statistical test was performed for the purchase intent questions in Appendix
2 (questions 6-7) to compare the response distribution between ―Not buy‖, ―Undecided‖ and
―Would buy‖ between the samples and control and also for the response between general
consumers and consumers who were familiar with parboiled rice. To analyse the effect of
143
additional health benefits associated with the consumers on rice purchase intent it was further
analysed using paired t- test.
6.2.5.2 Study 2: Consumer acceptance of cooked fortified rice after mixing
One way ANOVA was performed for questions regarding the liking of attributes in Appendix
4 (Questions 1, 3,6,8,11,14 and 15) to study the mean differences between samples including
control. Tukey‘s test was performed to compare the means (p< 0.05). Chi square (χ2)
statistical test was performed to compare purchase intent between samples and control
(Question 16-17 (Appendix 4) in Table 6.5). To understand the consumers overall liking (9-
point hedonic scale) in Study 2, penalty analysis was performed. One - way ANOVA and
Tukey‘s test was performed to evaluate significant difference between groups.
Homogeneity of variance within the data was measured on a 10- point intensity scale, 9-
point hedonic scale and 5- point likert scale. Levene‘s test was performed. The data which
were all above p <0.05 showed equal variance across samples. All statistical analysis was
performed using XLSTAT (version 2010).
6.3 Results and Discussion
6.3.1 Analysis of micronutrients in the fortified rice before and after dilution
The parboiled fortified rice with 3 micronutrients is intended to be a premix. Consuming the
fortified rice without dilution would result in overdose of each micronutrient on a daily basis
based on RDI. Therefore it is essential to dilute the concentration of the micronutrients for
consumption. The ratio for dilution was chosen to be 1:10 in order to meet at least half of the
RDI (according to NIH-ODS, 2014 & NHMRC, 2006) for each micronutrient and also to
account for the loss due to milling (120s) and cooking. Concentration of the micronutrients
in the fortified rice (premix and diluted rice – cooked and uncooked) are presented in Figure
6.2. Assuming that approximately 440g of cooked rice is consumed on a daily basis by rice
eating populations, folic acid in the fortified diluted rice was able to meet more than 100 % of
RDI- close to the upper limit and for β-carotene about half of RDI and just about the RDI for
iron (Table 6.1). Therefore, this dilution ratio was considered reasonable based on the
concentration of the added nutrients of the cooked fortified rice diluted with white rice. In the
following sections, the results from the sensory evaluation have been presented and discussed
to understand if this dilution factor of the premix influences the acceptance of the fortified
rice in terms of purchase intent and taste.
144
Table 6.1 Dietary Reference Intake for the selected micronutrients (Vitamin A, folic
acid and Iron) (Dietary reference intake: Elements and Minerals, (NIH-ODS, 2014 &
NHMRC, 2006))
Micronutrients
Concentration in
440g of cooked
diluted fortified
rice
Adequate Intake Upper limit
Vitamin A (retinol
equivalents, precursors
of carotenoids)
334.4 µg
(β-carotene)
Infants (1-3 years old–
pre-schoolers) 300 µg/day 600 µg/day
Male (19-50 years old) 900 µg/day 3000 µg/day
Female (19 – 50 years
old) 700 µg/day 3000 µg/day
Folic acid 1100 µg
Infants (1-3 years old–
pre-schoolers) 150 µg/day 300 µg/day
Male (19-50 years old) 400 µg/day 1000 µg/day
Female (19 – 50 years
old) 400 µg/day 1000 µg/day
Iron 19.8 mg
Infants (1-3 years old–
pre-schoolers) 7 mg/day 40 mg/day
Male (19-50 years old) 8 mg/day 45 mg/day
Female (19 – 50 years
old) 18 mg/day 45 mg/day
145
Figure 6.2 Concentration of micronutrients (in d.w.b.) in fortified premix (cooked and uncooked) and diluted fortified rice (cooked and
uncooked)
0100020003000400050006000700080009000
Diluted
Uncooked
Diluted
Cooked
Premix
Uncooked
Premix
CookedCon
cen
trati
on
in
µg/1
00g
Folic acid concentration in fortified rice
012345678
Con
cen
trati
on
in
mg/1
00g
Iron concentration in fortified rice
154
76
211194
0
50
100
150
200
250
Diluted
Uncooked
Diluted
Cooked
Premix
Uncooked
Premix
Cooked Con
cen
trati
on
in
µg/1
00g
β-carotene concentration in fortified rice
146
6.3.2 Study 1: Visual Consumer Acceptance of uncooked fortified parboiled rice
6.3.2.1 Demographics and rice eating pattern of consumers
For part 1 of the consumer acceptance study a total of 49 people were recruited comprising
comprised of 39% men and 61% women. Majority of the participants were Asian (71.4%)
and the rest were from Australia or other countries mentioned in Table 6.2. Majority of the
people belonged to the age group of 20-30 years (75.5%) and 72% of this cohort consumed
rice on a daily basis or at least 2-3 times a week. Hence, this population seemed suitable for
this study as they were representative of the rice-eating populations. Among various types of
rice, white rice was consumed by 94% of the selected subjects which indicated that these
consumers were also representative of white-rice eating populations. It is also to be noted that
none of the recruited consumers ate parboiled rice though about half of the cohort (52%)
were aware of the availability of parboiled rice.
147
Table 6.2 Socio-demographic characteristics of the participants of consumer acceptance
Study 1 and Study 2
Demographic variable Percentage (%)
(Study 1 n=49) (Study 2 n= 54)
Gender
Men 39 26
Women 61 74
Age
<19 2 1.8
20-30 75.5 81.4
31-40 18.3 12.9
41-50 0 1.8
51-60 2 1.8
Nationality
Australian 20.8 24
Chinese 22.9 20.3
Indonesian 27 16.6
Indian 2.08 7.4
Malaysian 4.16 12.9
Others (Iranian, Bangladesh, British, Singapore,
Thailand, Samoan, New Zealand, Filipino) 22.9 14.6
Employment Status
Casual 20.4 42.5
Full time 30.6 35.1
Part time 10.2 5.5
Unemployed 36.7 16.6
148
Table 6.3 Rice consumption pattern of participants of the consumer acceptance study 1
and study 2
Item Percentage (%)
Study 1 (n= 49) Study 2 (n=54)
Type of rice consumed usually
White rice 93.7 72.2
Brown rice 6.2 18.5
Red/Purple/Black rice
7.4
Parboiled rice
1.8
Consumption frequency
Daily 72.9 72.2
At least once a week 20.3 18.5
At least once a month 2 3.7
Rarely 4.1 5.5
Methods to cook rice
Rice cooker 100 88.8
Rapid cooking (Cooking in excess water)
3.7
Absorption cooking ( cook in a pot)
7.4
Heard about parboiled rice?
Yes 52 75.9
No 47.9 24
6.3.2.2 Degree of visual acceptance of fortified diluted uncooked rice
Figure 6.3 shows the hedonic preferences of rice samples for visual acceptance attributes
(commercial parboiled rice, commercial white rice, fortified diluted rice and unfortified
parboiled rice). The sample of interest was fortified diluted rice and the aim of phase 1 of
consumer acceptance study was to investigate if the participants of the study showed any
preference based on colour and appearance of the fortified diluted rice and their purchase
intent.
149
Figure 6.3 Preference of visual attributes investigated in Study 1 tested by participants (n=49) presented on a hedonic scale
Note: Uniformity represents the appearance based on the colour of the rice samples.
02468
10
Commercial
parboiled rice
Commercial
white rice
Fortified
diluted rice
Unfortified
Parboiled
Uniformity in appearance
02468
10
Commercial
parboiled
rice
Commercial
white rice
Fortified
Diluted rice
Unfortified
Parboiled
Pre
fere
nce
on
a h
ed
on
ic
scal
e
Colour preference
0
2
4
6
8
10
Commercial
parboiled
rice
Commercial
white rice
Fortified
Diluted Rice
Unfortified
Parboiled
Pre
fere
nce
in h
ed
on
ic s
cale
Colour Intensity
0
2
4
6
8
10
Commercial
parboiled rice
Commercial
white rice
Fortified
diluted rice
Unfortified
Parboiled
Overall acceptance of
appearance
150
As it can be seen from the Figure 6.3, uniformity of the samples based on colour for fortified
diluted rice had the lowest score indicating that there is evident heterogeneity in the sample.
The interesting observation regarding uniformity was that commercial parboiled rice gained
the highest score compared to commercial white rice which was the most common type of
rice consumed by the participants of the study. Parboiled rice from this study was also
accepted to be uniform compared to the fortified diluted rice. This shows that consumers
generally prefer uniformity in colour and they are willing to purchase despite the colouration
as long as they are uniform.
In terms of colour intensity, unfortified parboiled rice was considered to have the highest
intensity compared to the other samples. Colour intensity of unfortified white rice was similar
to the fortified diluted rice. Hence it can be interpreted that unfortified white rice was able to
lessen the intensity of colour of the parboiled rice and could be chosen as an appropriate
diluent for the premix. When it came to colour preference clearly most consumers preferred
commercial white rice over the other samples and the least preference was observed for the
fortified diluted rice. Commercial parboiled rice was preferred second to commercial white
rice indicating that despite many of the participants not being familiar with parboiled rice; it
was preferred and is possible for consumers to accept it even if not mixed with white rice. For
the overall acceptance fortified dilute rice was least preferred among the 4 samples. Therefore
it can be said that appearance is an important factor for consumer acceptance and since the
fortified rice was diluted with white rice there was a major impact on the uniformity of the
sample. Although the concentration of micronutrients in the fortified rice diluted with white
rice met the RDI requirements (Table 6.1) the dilution factor influenced the consumer
acceptance behaviour. The fortified premix diluted with commercial parboiled rice is likely to
be preferred over dilution with white rice as consumer acceptance study indicates that
commercial parboiled rice was the second preference after white rice for consumers.
6.3.2.3 Evaluation of purchase intent of uncooked diluted fortified rice with and without
nutritional information
As mentioned in the questionnaire (Appendix 2) two consecutive purchase intent questions
were asked of the fortified rice to understand if people would buy the fortified parboiled rice
diluted with unfortified white rice when sold commercially in supermarkets. Results from the
present study showed that 67% of people were willing to buy commercial white rice if it were
fortified with micronutrients. However, only 24.5% were willing to buy the fortified diluted
151
rice. Moreover t-tests revealed that the purchase intent for fortified parboiled rice was
significantly different from the commercial white rice. When subsequently asked about
purchase intent of the rice that might offer better nutritional value the likelihood of purchase
of the fortified rice increased. About 67% of the recruited population said that they would
probably buy the diluted fortified rice. But, 88% of the people still preferred to buy
commercial white rice if it were fortified. This result was unfavourable in terms of success of
the fortification study. One of the reasons for this was that consumers could detect fortified
rice in the diluted mixture as a speck or discolouration indicating a quality defect that may be
a factor in rejecting the lot. This was the reason for low purchase intent compared to
commercial white rice as reported by the prospective consumers who participated in the
study.
When Chi square (χ2) test was performed for the response distribution of purchase intent
before and after mentioning the health benefits of the rice it was found that there were
significant differences before mentioning the health benefit between the control white rice
and the diluted fortified rice (p= 0.002; p≤0.05). However, after mentioning about the
fortification there was no significant difference between the control sample and the diluted
fortified rice (p =0.9; p≤0.05). Moreover the results from the Chi square test (χ2) when
analysing the response distribution for purchase intent for each rice sample before and after
indicating that they were fortified, did not show any significant difference for commercial
parboiled and white rice but there was significant difference for diluted fortified rice and
unfortified parboiled rice. Hence it can be interpreted that based on the response distribution
after notification of ―health claim‖ there was an increase in the number of people who
preferred to buy the diluted fortified rice. Overall the results indicate that consumers were
willing to buy the diluted fortified rice if they got prior information about the health claim.
152
A) B)
Figure 6.4 The distribution of purchase intent responses (%) in Consumer Acceptance
Study - STUDY 1 tested by participants (n=49). A) Before the notification of health
claim; B) After the notification of health claim
6.3.2.4 Evaluation of purchase intent of uncooked fortified rice- consumers who were
familiar with parboiled rice
The perception of people who consumed parboiled rice varies compared to those who do not
consume parboiled rice as they are more familiar with the distinct colour and flavour of
parboiled rice (Prom-u-Thai et al., 2009b). The specific target population in this study would
be people who consume parboiled rice as there is a specific market for it. To gain a deeper
perspective on purchase intent among people who had previous knowledge of parboiled rice a
further study was conducted (n=26).
0102030405060708090
Per
cen
tag
e o
f re
spo
nse
Purchase Intent before notification of
health claim
Commercial
parboiled rice
Commercial
white rice
Fortified
diluted rice
Unfortified
Parboiled
0102030405060708090
100
Per
cen
tag
e o
f re
spo
nse
Purchase Intent after notification of
health claim
Commercial
parboiled rice
Commercial
white rice
Fortified
diluted rice
Unfortified
Parboiled
153
Table 6.4 Mean perceptions of appearance for uncooked rice sample- diluted fortified
rice and p-values (p≤0.05) of t-test on comparing the diluted fortified rice sample with
Control – commercial white rice (Tested by consumers in the Consumer Acceptance
Study (colour intensity, degree of liking of colour, uniformity of colour, overall
appearance))
Colour
intensity
p -value
for t-test
Colour
preference
p-value
for t-test Uniformity
p-value
for t-
test
Overall
acceptance
p-value
for t-
test
General
consumers 5.1±1.0
0.5
4.6±1.6
0.7
3.6±1.6
0.19
4.6±1.1
0.5 Consumers
familiar
with
parboiled
rice
4.9±1.6 4.7±1.1 4.4±2.1 4.3±1.6
Note: General consumers (n=22) and those who were familiar with parboiled rice (n=27))
The mean liking scores for the appearance attributes (colour intensity, degree of liking of
colour, colour uniformity and degree of overall liking) are presented for the diluted fortified
rice in Table 6.4. The overall mean for the two groups – general consumers and consumers
familiar with parboiled rice was not very different. However on performing t-test for
individual parameters, except for colour preference there was no significant difference for the
other three attributes between the two groups. This shows that there is not much difference in
the way of perception of the fortified rice when people had prior experience or knowledge
about parboiled rice for individual parameters.
Moreover, when the response distribution was analysed statistically using Chi square test (χ2)
there was no significant difference between the general consumers and consumers who were
familiar with parboiled rice in terms of their purchase intent before and after the health claim
for the diluted fortified rice (p=0.9). This was further investigated by performing paired t-test
for investigating the additional health claim between the 2 groups. When the sample of
interest – diluted fortified rice was compared with the control – commercial white rice and
paired t-test was performed to understand additional health claim benefits for consumers who
were familiar with parboiled rice and general consumers there was significant difference for
both groups (p= 0.03- general consumers; p=0.04-people familiar with parboiled rice) before
154
and after mentioning the health claim indicating that both these groups of consumers are
more likely to purchase the fortified diluted rice compared to commercial white rice. For
other samples there was no significant difference between the two groups and also within and
between samples. In this study therefore it can be said that the willingness to buy was
independent of people‘s prior experience with parboiled rice. However the willingness to buy
diluted fortified rice increased after mentioning the health claim.
Study 2: Consumer acceptance of cooked fortified rice after mixing
6.3.2.5 Consumer demographics and rice eating preferences
For Study 2, a total of 54 participants were recruited and this comprised of 26% men and
74% women. The demographics of consumers recruited for Study 1 and 2 were similar.
Majority of the participants belonged to the age group of 20-30 years (81%). From the
demographic data 68.5% were of Asian origin and the others identified themselves as
Australian, British, Samoan etc. Over 70% of these people consumed rice on a daily basis or
at least 2-3 times a week. Hence, this cohort was also considered to be representative of rice -
consuming populations.
6.3.2.6 Degree of liking of cooked rice attributes
Table 6.5 presents average liking of the rice sensory attributes (appearance, colour, odour,
texture, taste and aftertaste) after cooking. Overall there was no significant difference
between the control white rice sample and the test sample (diluted fortified parboiled) for any
attribute. Unlike for the uncooked rice, the cooked rice appeared to be uniform in colour.
Also it has to be noted that for each attribute the average for the test sample i.e. the diluted
fortified rice was slightly higher than the control white rice indicating that the fortified rice
after cooking was better liked than the control white rice by the taste panel. In terms of
tasting, none of the panel members commented on any specific difference or adverse taste
such as being ―metallic‖ which is highly likely in iron fortified foods. Hence, it can be said
that in terms of key sensory attributes, both the fortified rice and control white rice appeared
and tasted similar and therefore, it is highly likely to be accepted by the wider community
after cooking.
155
Table 6.5 Mean degree of liking score for sensory attributes (appearance, colour, odour,
texture, taste, aftertaste and overall liking) of rice samples tested by consumers in Phase
2 of the Consumer Acceptance Study (n=54)
Attributes Diluted fortified rice Control White rice
Appearance 6.5±1.1 6.4±1.1
Colour 6.2±1.2 6.4±1.3
Odour 6.2±1.3 6±1.2
Texture 6±1.2a,b
5.6±1.2a,b
Taste 6.2±1.2a,b
5.7±1.3a,b
Aftertaste 5.7±1 5.4±1
Overall 6.2±1.2 6±1.2
Note: Values indicate Mean ± standard deviation for the sensory attributes
Values followed by (a,b) indicate significant difference of means at p ≤0.05.
6.3.2.7 Consumers’ perception of attributes JAR (Just about right)
For an attribute to be acceptable and nominal it is generally expected that at least 70% of the
response from the consumers lie in the JAR region (Meullenet et al., 2007). From figure 6.5 it
can be said that about 70% of the attributes evaluated in the sensory study is prevalent in the
JAR area. Colour change that was evident in uncooked rice when fortified rice was diluted
with white rice was not evident when the rice was cooked and hence not considered as a
foreign particle. The acceptability of the product thus increased. According to Hurrell (1997),
iron-fortified rice was disliked by many consumers since the rice was black in colour and it
was considered as specks from the bulk of rice. This scenario was not apparent in the given
study. Also there was no significant difference in colour between the control white rice and
the fortified rice in this study which clearly showed that after cooking, colour was no longer a
concern for consumer acceptability.
When the texture of the control and fortified rice was compared, there was no significant
difference once again between the two samples (p ≤ 0.05). Hardness and stickiness are the
two parameters that define texture of rice. For hardness there was no significant difference
between the two groups however for stickiness there was significant difference indicating that
the fortified rice did affect the overall texture of the diluted fortified rice. Normally parboiled
rice is firmer and harder in texture compared to white rice (Bhattacharya, 2004). This
156
phenomenon was not observed when the fortified parboiled rice was mixed with white rice in
the ratio 1:10. Thus the texture of the fortified rice was comparable and quite similar to
commercial white rice.
For taste intensity and after-taste, there was no significant difference statistically between the
two samples. However for taste preference there was significant difference. This indicated
that due to the presence of parboiled rice there is alteration in the normal rice taste which can
be perceived by consumers. Similarly for odour the overall preference did not show
significant difference, however there was significant difference for odour intensity indicating
that parboiled rice imparts particular flavour even after dilution with white rice which
seemingly influenced consumer perception. Since the attributes fell in the JAR region and
also overall fortified rice was preferred over white rice it can be said that fortified rice is
acceptable by consumers although there were minor differences when compared with
commercial white rice.
157
Figure 6.5 Bar graph showing consumer preference of cooked fortified rice compared
with unfortified rice
0
20
40
60
80
Not Enough JAR Too Much
% F
req
uen
cyUniformity
Fortified
Rice
Unfortified
Rice0
20406080
% F
req
uen
cy Taste
Fortified Rice
Unfortified
Rice
020406080
% F
req
uen
cy Aroma
Fortified Rice
Unfortified
Rice
0204060
% F
req
uen
cy
Stickiness
Fortified
Rice
Unfortified
Rice
020406080
% F
req
uen
cy
Hardness
Fortified rice
Unfortified
rice
0
50
100
% F
req
uen
cy
Colour Intensity
Fortified Rice
Unfortified
Rice
5.95
6
6.05
6.1
6.15
6.2
6.25
Fortified Rice Unfortified rice
Pre
fere
nce
on
a H
edon
ic
scale
of
10
Overall Liking
158
6.3.2.8 Evaluation of purchase intent of cooked rice
The purchase intent of fortified rice was classified into ―Not buy‖, ―Undecided‖ and ―Would
buy. From Figure 6.6 it can be seen that the distribution of the response skewed towards
‗would buy‘ for both fortified and unfortified rice. ―Willingness to buy‖ showed no
significant difference when fortified rice was compared with white rice when t-test was
performed. Similar results were obtained for the chi square (χ2) test for response distribution
as well and there was no significant difference. Experimental data from Figure 6.6 shows that
consumers preferred to buy fortified rice compared to unfortified rice and this was higher
after mentioning the health claim. Fortified rice was equally accepted as unfortified rice in
terms of purchase intent. It has to be noted that fortified rice was mixed with white rice
instead of parboiled rice and hence there is a possibility that it can be less preferred because
of the white rice background. Despite this, the data from the sensory study shows that it is
apparent that people would prefer to buy the fortified rice. From the demographic survey in
this study about 75% of the people were aware of parboiled rice. So if the fortified rice was
mixed with unfortified parboiled rice, this population would have accepted the rice anyway.
As parboiling of rice is quite common in many rice eating countries, such populations will
not have difficulty in accepting fortified parboiled rice and frequent consumption will
guarantee adequate intake of the added nutrients.
159
a.
b.
Figure 6.6 (a & b) Graph showing purchase intent of fortified rice before and after
mentioning about fortification
0
10
20
30
40
50
60
70
80
Purchase Intent (before
mentioning fortification
Purchase Intent (after
mentioning fortification)
% F
req
uen
cy
Purchase Intent for Unfortified rice
Would not Buy
Undecided
Would buy
0
20
40
60
80
100
Purchase Intent (before
mentioning fortification
Purchase Intent (after
mentioning fortification)
% F
req
uen
cy
Purchase Intent for Fortified rice
Would not Buy
Undecided
Would buy
160
6.4 Conclusions
Retention analysis of micronutrients in fortified cooked rice showed that the dilution ratio of
1:10 was optimum and was able to meet half of the RDI for β-carotene (700 µg), just about the
RDI for iron (18 mg) and more than 100% of RDI for folic acid (400 µg) (NIH-ODS (USA),
2014, NHMRC (Australia), 2006).
Visual acceptance of uncooked fortified rice was performed in Study 1. People who were
familiar and unfamiliar with parboiled rice both accepted diluted fortified rice. However,
commercial white rice was the most acceptable among all. The colour intensity and uniformity in
appearance were the two factors where the diluted fortified rice was scored down due to
variation in rice kernel caused by the fortification process. On the other hand, commercial and
unfortified parboiled rice was also widely accepted by the consumers indicating that people are
willing to purchase parboiled rice and hence the process can be successful as a method of
fortification. By declaring the health claim of the fortified rice, the willingness to buy increased.
However this aspect did not influence the decisions of people who were familiar with parboiled
rice. The primary reason for this would be the non-uniformity in the parboiled rice. To overcome
this issue innovative packaging that would be able to mask this variation could be implemented
as this could possibly overcome the issue of non-uniformity in rice samples and influence the
purchase intent decision in a positive way between general consumers and people who were
familiar with parboiled rice to a higher extent.
In Study 2, the cooked rice was assessed for sensory acceptance of taste and it was deduced that
after cooking there was no variation between commercial white rice and fortified diluted rice.
However, there was some slight differences observed with odour intensity and stickiness
compared to white rice. But these were covered by the JAR responses, which indicated that the
fortified rice had better or comparable taste attributes to the white rice. Also another notable
finding was that the fortified diluted rice had overall better acceptance compared to the
commercial white rice indicating that parboiling itself caused favourable taste on cooking
thereby increasing consumer acceptance. The ultimate success of any fortified new product
would be acceptability by consumers followed by bioavailability after consumption. Therefore,
from the data shown in chapter 3 and the sensory study, it is reasonable to study the
bioavailability of fortified rice at the optimised condition (70°C and 2 hours soaking). By
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performing bioavailability studies (discussed in the following chapter-7), essential information
on how much of the fortified micronutrient would actually be bio-available can be estimated.
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CHAPTER 7
7. SHORT TERM RELATIVE BIO-ACCESSIBILITY AND ABSORPTION OF FOLIC
ACID, IRON AND β – CAROTENE IN FORTIFIED PARBOILED AND HIGH
PRESSURE PROCESSED RICE: CACO-2 CELL STUDY
7.1 Introduction
Understanding the bioavailability of the micronutrients fortified in the rice by the two different
processing methods will be the ultimate mark of the effectiveness of the fortification and thereby
giving an estimate of the absorption by humans. Bioavailability studies are expensive although
they can provide relative estimates of actual absorption in humans. This study focussed on
obtaining bio-accessibility data using Caco-2 cell lines. Caco-2 cells are adenocarcinoma cells
from the human colon. Well isolated and differentiated primary cell lines were utilized for this
experiment. These cells when grown in an appropriate medium can proliferate and differentiate
into enterocytes (epithelial cells of the small intestine) (Said, 2011; Simon- Assmann, Truck,
Sidhoum- Jenny, Gradwohl & Kedinger, 2007). Cell lines such as T-84 and HT-29 based on
literature have been found to be suitable for in vitro studies. However, caco-2 cells were the most
commonly used for drug interaction and absorption studies (D‘Souza, Shertzer, Menon &
Paultetti, 2003).
When the Caco-2 cell lines become confluent they form a polarized monolayer resembling the
brush border segment of the intestine with tight junctions and therefore can be convenient to
study drug metabolism in vitro (Travelin, 2002; Simon-Assmann, Truck, Sidhoum- Jenny,
Gradwohl & Kedinger, 2007). Netzel et al., (2011) studied release and absorption of carotenes
from processed carrots; O‘Callaghan & O‘Brien, (2010) studied bio-accessibility, cellular uptake
and trans-epithelial transport of α-tocopherol and retinol from a range of supplemented foods;
Ohrvik, Talkvist & Witthoft, (2010) investigated folate bio-accessibility at an in vitro level in
breads; Rodriguez-Amaya, (2010) reviewed in vitro assessment of bioavailability and
antioxidant activity of food carotenoids; Sy, Gleize, Dangles, Landrier,Veyrat & Borel, (2012);
Verwei, Van Den Berg, Havenaar & Groten, (2005) analysed the effect of folate-binding protein
on intestinal transport of folic acid and 5-methyltetrahydrofolate across Caco-2 cells. A validated
system for in vitro absorption to predict in vitro permeability and absorption using caco-2 cells
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has been well designed for experimental purposes by Delie & Rubas, (1997); Fernandez-Gracia,
Carvajal- Lerida & Perez- Galvez, (2009) and Simon-Assmann, Truck, Sidhoum-Jenny, Grawohl
& Kedinger, (2007). This chapter has been divided into the following sub-sections: reagents
required for cell culture maintenance, procedure for resuscitation of the cell line, sub-culturing of
the cells, in vitro digestion model, optimization of the in vitro digestion and absorption model for
folic acid, iron and β – carotene from the fortified rice from parboiling and HPP processes. The
aim of this chapter was thus was to study the bio-accessibility of micronutrients (folic acid, iron
and β-carotene) from the fortified rice processed by parboiling and HPP technology using in
vitro techniques. The results from the transport study will also be presented which may provide
future insight into bioavailability studies of nutrients from foods.
7.2 Materials and Methods
7.2.1 Reagents for Cell Culture Resuscitation and Maintenance
Human colon carcinoma cell lines namely, Caco-2 cells were a kind gift from the Lowy Cancer
Centre (The University of New South Wales). The viable cells were used between passage (sub-
culturing of viable cell population) numbers 30 to 50. The growth medium for the cells was
composed of Dulbeccos modified eagle medium (DMEM), foetal bovine serum (FBS), penicillin
(5000 u/mL), streptomycin (5000 µg/mL), non-essential amino acids and GlutaMAX™ and
these were purchased from Invitrogen (Sydney, NSW, Australia). The growth media composition
was as follows: 435 mL of DMEM, 10 % (v/v) FBS, 5 % (v/v) non-essential amino acids, 5 %
(v/v) Penicillin/Streptomycin and 5 % (v/v) Glutamax, and stored in the fridge for usage.
7.2.2 Reagents for Cell Viability Assay
Lucifer yellow (LY) which was non-toxic to the cells were obtained from Sigma-Aldrich
(Sydney, Australia). The cyto-toxicity assay was performed in 96-well microtitre plates
(Nunc™) obtained from Thermo Fischer (Sydney, Australia) and the integrity of the caco-2
monolayer assessed using LY was analysed using a black titre plate supplied by BD Biosciences
(Sydney, Australia). Cell viability and toxicity assays were performed using CellTiter 96 ®
a non-
radioactive cell proliferation assay (MTT) purchased from Promega (cat no. G5421). The assay
was performed using MTS ([3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4sulfophenyl) -2H-tetrazolium) and PMS (phenazine methosulfate (PMS), respectively). The
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assay solutions – MTS and PMS were thawed and warmed in a 37 0C water bath and the PMS
solution was then added to the MTS solution and 1200 µL of this solution was used for the cyto-
toxicity assay.
7.2.3 Reagents for in vitrodigestion
Fortified rice samples were digested using enzymes that would be secreted in the human system
to mimic in vivo digestion. The enzymes used were α-amylase, hog bile extract, porcine
pancreatin purchased from MP Biomedicals (Solon, OH). Proteolytic enzyme – pepsin (15.2
mg/mL) was prepared by dissolving in 0.1 M Hydrochloric acid (Univar, Ajax-Finechem,
Sydney, Australia). Post-gastric enzymes pancreatin (2 mg/mL) combined with hog bile extracts
(11.7 mg/mL) were dissolved in 0.1 M NaHCO3 (Univar, Ajax-Finechem, Sydney, Australia)
for in vitro digestion. The enzyme mixture was vortexed and placed in a 37 °C water bath for 15
minutes. Enzyme α-amylase (20 mg/mL) was prepared by vortexing in Milli-Q water and
centrifuged at 10,000 g at room temperature for 10 minutes (Chandra- Hioe et al., 2013).
7.2.4 Transport Study
Transport study was performed using 24-well transwell plates which consisted of 12 permeable
wells (12 mm insert diameter) with polycarbonate membranes which were purchased from
Corning (Sydney, Australia). Trans-epithelial electrical resistance (TEER) which was used to
measure integrity of the monolayer of caco-2 cells was purchased from Millipore (Billerica,
MA). Hank‘s Balanced Salt Solution (purchased from Life Technologies, Sydney Australia) was
added to the outer well of the transwell plates to maintain humidity for the cell growth during
incubation period.
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Figure 7.1 Schematic representation of Transwell permeable support with Caco-2 cells
grown on the membrane
7.2.5 Cell Culture & Maintainence Protocol
7.2.5.1 Procedure for resuscitation of frozen Caco-2 cells
Frozen Caco-2 cells presented in an ampoule (1mL) was removed from liquid nitrogen and
rapidly thawed in a 37°C water bath as this avoids osmotic damage to cells and improves cell
viability (Gull et al., 2009). The premixed growth media (~ 10 mL) was warmed in 37°C water
bath and added to the thawed cells and the cells were centrifuged to remove the dimethyl
sulfoxide (DMSO, Sigma-Aldarich, Australia). Cells were grown in 75 cm2
flasks (EasyFlasks™
,NUNC, Thermoscientific, Denmark) until it was 70% confluent. After the cells have reached the
desired confluence level (based on microscopic observation) they were split for them to be viable
until the assay was performed.
7.2.5.2 Sub-culturing of Caco-2 cells
Sub-culturing of the Caco-2 cells is essential for maintaining the cells in the optimal growing
phase and it was performed twice a week. This was performed by removing the old growth
media followed by washing of the cells with about 10 mL of Dulbecco‘s Phosphate-buffered
saline (DPBS) with no Calcium and Magnesium. This was done to remove serum which contains
Trypsin inhibitors and also other cell debris. Following this about 3 mL of trypsin (0.25%)-
EDTA was added to detach the cells from the 75 cm2
flasksurface and the cells were incubated at
37°C with 5% CO2 for about 3 minutes. Then about 10 mL of the growth media was added and
the cells were centrifuged (Boeco U-320, Hamburg, Germany) at 2000 rpm for 5 minutes. After
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centrifugation the supernatant was discarded and the cells were re-suspended in 10 mL growth
medium. The cells were split in the ratio of 1:10 in a T75 flask and incubated at 37°C with 5%
CO2 thereby allowing sub-culturing approximately in 4 days.
7.2.5.3 Cell Density
Cell density was calculated per mL of the growth media using a haemocytometer by visualizing
and counting the cells under a microscope. Trypan blue was the dye used to distinguish the cells
in an exclusion assay. The principle of the assay is that Trypan blue has the property to dye non-
viable cells when their cell walls are not intact. On the other hand viable cells are excluded from
the dye and thereby distinguished from the non-viable cells (Stoddart, 2011). It is essential to
keep a count of the cell density as it is vital for cell sub-culturing, freezing down, and seeding on
the transwell membrane for the transport study. During cell passage process 1 mL of the
dispersed cells was retained separately for calculating cell density. The cells were then diluted
with Trypan blue in the ratio of 1:5 or 1:6 depending on the number of cells that could be
observed under the microscope. The dyed cells were then mounted on the haemocytometer with
the cover slip on it. The cells were fixed between the cover slip and the haemocytometer
inscription by capillary effect and were then counted immediately after loading them as the dye
is toxic to the cells and could lead to death if delayed. Figure 7.2 is a diagrammatic
representation of the chambers in a haemocytometer (Louis & Siegel, 2011). The cell density
was henceforth calculated depending on the number of viable cells according to the following
formula:
Cell density/ mL = Total no. of cells from the 4 quadrants x dilution factor x 104
4
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Figure 7.2 Diagrammatic representation of the chamber of a haemocytometer (Louis &
Siegel, 2011)
7.2.5.4 Freezing down Cells
For long-term storage of the viable cells and maintaining the inventory of the caco-2 cells they
were frozen down in liquid nitrogen. To do this the re-suspended cells from an early passage
number (24- 30) were centrifuged to remove the growth medium and the pellet was re-suspended
in cold FBS (90%) supplemented with DMSO (10%) and mixed gently. The cells (containing 3 x
106 / mL) were then aliquoted into a cryo-tube (Thermo Fisher, Sydney Australia). The cells
were frozen gradually by freezing them first in a -20ºC freezer and then freezing them overnight
at -85ºC and then the tubes were submerged in liquid nitrogen stored in a cryogenic vessel.
Under this condition the cells are ever-lasting as long as the liquid nitrogen is replenished until
the cells are frozen.
7.2.6 Sample Preparation
7.2.6.1 In-Vitro Digestion Protocol
The protocol for performing in vitro digestion was followed according to Netzel et al., (2011)
with some modifications. Fortificant mix (150 mg of folic acid, 62.5 mg of β-carotene, 25 mg of
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iron / 600 mL of distilled water) and cooked rice in duplicates as mentioned previously in section
(3.2.7) for analysis of folic acid, iron and β – carotene was used. To the cooked rice about 5 ±
0.01 g 2.5 mL of lukewarm de-ionized water was added along with 5 mL of α- amylase (20
mg/mL, pH 6.56) into 50 mL centrifuge tubes. The tubes were vortexed and placed in a 37˚C
shaking water bath for 5 minutes. The action of α- amylase was to mimic the hydrolysis of starch
in the mouth. Following this the pH of the samples was adjusted to 2.1-2.2 with 0.1 M HCl. The
gastric digestion was initiated with pepsin (250 µL) in the shaking water bath at 370C for 1h.
Following this the pepsin was inactivated by increasing the pH to 5.7 with 0.1 M NaHCO3 (pH
8.43) and 12 mM calcium chloride (pH 6.42). The mixture was incubated in the water bath at
37˚C for 30 minutes. The mixture was then treated with pancreatin and bile extract (1 mL) for 2h
in the 37˚C water bath after adjusting the pH to 6.8- 7 (gastric pH) with a mixture of 0.1 M
NaHCO3 and 12 mM CaCl2 (pH 8.07). The resulting mixture will be referred to as intestinal
digesta from this point of the document. The digesta was centrifuged at 10000 g for 20 min (at
4˚C) and the supernatant was collected and stored at -80˚C until further analysis.
As a negative control nil digesta was prepared and as a positive control the fortificants in
solution were also prepared and digested using the above protocol. In addition, for analysis of β –
carotene 0.5 mL of soy bean oil, vegetable oil and canola oil were added individually to the
digesta in order to improve the efficiency of the digesta to extract β – carotene. The efficiency of
extraction of β – carotene was determined between the three oils.
7.2.6.2 Cell Viability Assessment
It is important to understand the cell viability because non-viable or lysed cells could detach
from the filter at the washing stage which is mentioned below resulting in an increased filter
exposure during the transport study thereby affecting the movement of the substance from the
apical compartment to the basolateral compartment leading to a false positive result. In order to
ensure that the cells utilized in the transport absorption model are healthy it is vital to analyse the
cell viability (Stoddart, 2011). A 96 well titre plate was used and the caco-2 cells were seeded on
the wells of the plate with a cell density of 1 x 105
cells/well. The plates were incubated at 37°C
for 1 week to allow the cells to attach and differentiate. After the mentioned incubation time the
cell culture medium was removed and 100 µL of the digesta (prepared as mentioned in section
7.3.3.1) was added to the well. As a positive control Hanks Balanced Salt Solution (HBSS) was
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added to the wells with confluent cells and as a negative control H202 was added to the wells
without any cells in them.
The rice digesta, nil digesta and the fortificant digesta were all assessed for toxicity. The positive
control was HBSS added to the cells and the negative control was HBSS without the cells and
H202 added to the cells. To assess the cytotoxicity caco-2 cells were cultured in a 96 well plate
with a cell density of 1 x 105 cells per well. The outermost border wells contained HBSS in order
to maintain moisture for the cells (Addepalli & Osborne, 2011). The plate with the seeded cells
was incubated at 37˚C in a humidified atmosphere with 5% CO2 for a period of one week.
After the incubation period Lucifer yellow (LY) prepared on the day of analysis was used to test
cell toxicity. A standard LY with a concentration of 1 mM was prepared by dissolving 4.6 mg in
10 mL of HBSS. The stock solution was stored at -20˚C for future use. The stock solution was
diluted further at x 6 dilution (0.025-1 mM). The working solution was stored in the dark while
the 96- well plates were prepared. For the assay the growth media from the wells were removed
and the cells were washed with 100 µL of HBSS. Following this LY (100 µL) was added to the
wells in increasing concentrations in triplicates along with the sample namely rice digesta, nil
digesta and fortificant digesta. The plates were incubated at 370C and 5% CO2 for 1-2 hours. As
a positive and negative control, HBSS (100 µL) was added into the wells with cells and without
cells. Post incubation cell viability was tested using a commercial CellTiter 96® non-radioactive
cell proliferation assay (MTT) which was was used to assess the cell viability in response to the
in vitro digesta using a colorimetric method. The protocol was followed as provided by the
supplier. This colorimetric assay consisted of MTS and PMS. The aliquot of MTS/PMS was
thawed and 20 µL of this was added into each well and mixed thoroughly by pipetting. The
plates were again incubated at 37°C for 2 hours for colour development. The absorbance was
measured at 485 nm and the data was recorded using an ELISA plate reader (Spectra Max M2,
Molecular device, Australia).
7.2.7 Transport & Permeability Study
According to Addepalli & Osborne (2011) the cells have to be split at least thrice before being
seeded on to the 24-well Transwell membrane plate. The cell density was then determined using
the haemocytometer as mentioned in section 7.2.2.3 and then the cells were diluted such that the
final cell density of 4 x 105 cells/mL was achieved. The Transwells were then seeded with 200
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µL of the cells which contained 8 x 104 cells and cultured in a 5% CO2 atmosphere at 37ºC for
21 days. For the cell viability and toxicity test LY transport study was conducted in parallel and
for this purpose 4 x 104 cell density was utilized. The basolateral chamber of the Transwell
contained 600 µl of the growth media (Figure 7.1). The first and the last row (which did not
contain the cell or the inserts) of the Transwell contained 1 mL of sterile DPBS for maintaining
humidity within the plate (Addepalli & Osborne, 2011). The growth media in the apical and
basolateral chamber was replaced systematically twice a week to restock the nutrients and
maintain optimum pH for cell growth.
To begin, the transport study was conducted on the 21st day after seeding the Transwell plate.
Firstly the (Trans Epithelial Electrical Resistance) TEER was measured and recorded using a
Millicell voltohm-meter to assess the integrity of the cell monolayer (Verwei, Arkbåge, Groten,
Witthöft, VandenBerg, & Havenaar, 2005). The TEER measurement was done using an
electrode probe which had two ends. The longer end was immersed in the basolateral side of the
Transwell and the shorter electrode was immersed in the apical side with precaution such that the
probe does not damage the integrity of the monolayer. The baseline for the TEER was based
upon the reading obtained from the HBSS without any cells. Wells which gave TEER above 500
Ω was used for the transport study.
For the transport study the growth media was removed from both the apical and the basolateral
chambers. Following this the chambers were washed with sterile HBSS twice and then 200 µL
and 600 µL of HBSS was added to the apical and basolateral chambers respectively and the
plates were incubated at 370C with 5%CO2 for 4 hours prior to starving the cells and rinsed off
any remaining growth media from the chambers. After the incubation time the HBSS was
removed from the chambers and to the apical chamber 200 uL of LY, nil digesta, rice digesta and
the fortificant digesta were added and to the basolateral chamber 600 µL of HBSS were added.
The plates were then incubated at 37°C with 5% CO2 in a humidified incubator for 1 hour for
folic acid uptake, 16 hours for b- carotene and 22 h for iron uptake. The application of LY to the
Tranwell was to confirm the integrity of the cells monolayer.
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The LY standards (0.1 mM ) was prepared fresh on the day of the analysis and 200 µL of the
LY was applied to the apical chamber in triplicates and the plates were incubated for 2 hours.
After the incubation period the TEER was measured and the samples were collected from the
apical and the basolateral chambers and analysed using a spectrophotometer on the same day. A
standard curve of LY was constructed with solutions prepared in the concentration range of 0-
250 µM. The standards and the samples were added to a 96 well microtitre plate. The sample
obtained from the apical chamber was diluted in the ratio 1: 20 with HBSS in order to fall within
the calibration range and the basolateral chamber was not diluted because if the membrane
retained its integrity the LY should not be able to pass through the Transwell membrane. The
absorption was then measured using fluorescence spectrometer with an excitation wavelength of
430 nm and emission wavelength of 535 nm.
After the transport study the samples were collected from the apical and the basolateral chambers
and stored in a -80ºC until further analysis. To the apical and the basolateral chambers 200 µL
and 600 µL of growth media was added respectively and the TEER was measured right away.
Then the cells were removed from the Transwell insert by applying warm Trypsin and incubating
at 37ºC and 5% CO2 for 20 minutes. The cells were then collected from the apical chamber and
the trypsin was removed by centrifugation. The cells were then washed and suspended in HBSS
and stored at -80ºC until further analysis. For the transport studies, caco-2 cell lines with passage
number of 25-40 were used throughout the experimental period.
7.2.7.1 Sample Preparation for Quantitative Analysis
For quantification of the micronutrients from the transport study 6 wells from the apical and the
basolateral chambers were pooled to obtain a single sample for folic acid. For β- carotene and
iron 12 wells of samples from the apical and the basolateral chambers were pooled to obtain a
single sample. The cells were lysed using 1 M KOH with 2% sodium ascorbate (w/v) (Ohrvik et
al., 2010).
7.2.7.2 Folic acid analysis
The samples (output from the transport studies obtained from the apical and basolateral
chambers and the cell lysate) were purified using SAX SPE cartridge. The purified extract was
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analysed using HPLC and the method was followed according to section (3.2.8.1). The digesta
which was prepared according to the in vitro model was also analysed for folic acid using the
above procedure.
7.2.7.3 β – carotene analysis
The samples and the cell lysates were prepared according to Netzel et al., (2011) with some
modifications. The samples from the transport study and the digesta were spiked with β- apo
carotenoid (purchased from Sigma-Aldrich –Catalogue No: 10810-1G) and then extracted with
equal volumes of hexane: acetone (1:1) with 0.5 mL of 10% sodium chloride to remove the
emulsion. The hexane layer was collected and washed with water until it ran clear without any
visible solid particles and emulsion. The combined extracts were dried under N2 and dissolved in
methanol for analysis using HPLC as mentioned in section (3.2.8.2- page no. 52).
7.2.7.4 Iron analysis
Iron analysis for the sample from the apical chamber and digesta was analysed using ICP-OES
and the extraction procedure was the same as in section (3.2.8.3 page no.53). For the sample
collected from the basolateral chamber of the cell culture assay , iron analysis was performed
using a Ferritin assay kit (obtained from MP Biomedicals, Diagnostics Division, Orangeburg
NY) as the absorbed iron is converted to a protein (ferritin) by the human body and the same was
observed in the caco-2 cells as well. The protocol was strictly followed according to the kit
manual provided by the manufacturer.
The principle of the assay was based on solid-phase enzyme linked immunosorbent assay. The
microwell plates are coated with rabbit anti-ferritin for solid phase-immobilization and antibody-
enzyme (horseradish peroxidase) was monoclonal anti-ferritin obtained from mouse. When the
samples containing ferritin are added to the wells, the analyte is sandwiched between the solid-
phase and the enzyme-linked antibodies. After 45 minutes incubation the wells are washed with
water to remove unbound labelled antibodies. A solution of 3,3‘,5,5‘- Tetramethylbenzidine
(TMB) was added to the wells and incubated for 20 minutes for colour development. A stop
solution of 1M HCl was added to stop the reaction and the resulting colour was measured with a
spectrophotometer at 450 nm. The concentration of ferritin in the sample directly corresponds to
the color intensity of the sample. To quantify the ferritin present by the current analysis standards
with concentrations between 0- 1000 ng/mL (provided in the kit) was used. The samples and
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standards were analysed in triplicates. The sample from the basolateral chamber and the cells
were also analysed for ionisable iron using ICP-OES as mentioned above.
7.2.7.5 Calculations
The bio-accessibility study indicated the % uptake of three micronutrients in the fortified rice
and the transport was measured based on the nutrients passing through the apical chamber into
the basolateral chamber- across the human intestinal wall. Bio-accessibility refers to the portion
of micronutrients released from the food matrix and made available for the cell uptake after
digestion and it is applicable only for in vitro procedures (Hendren et al., 2002). It can be
calculated according to the following equation:
Bio-accessibility % = M digesta/ M cooked rice x 100% (Equation 5)
where M digesta and M cooked rice are the concentrations of the micronutrients (folic acid, iron and β
– carotene) present in the digesta and cooked rice respectively.
The transport of each micronutrient is referred to as the concentration of each micronutrient
detected in the basolateral chamber of the Transwell plate at the end of the designated transport
incubation time in the context of the digesta. The transport % can be calculated according to the
following equation:
Transport % = M basolateral/ M digesta x 100% (Equation 6)
where M basolateral is the concentration of micronutrients present in the basolateral compartment.
7.3 Results and Discussion
7.3.1 Cytotoxicity and cell viability
In order to perform in vitro transport study the cells that are to be seeded onto the insert of the
Transwell must be assessed for viability and also the cells should be non-toxic to the digesta that
will be used for the study. If the digesta is toxic it will lead to cell death and thereby invalidate
the transport study. Stoddart (2011) stated that cell viability implies the number of living cells in
a sample. Caco-2 cell viability was assessed according to section 7.2.3.2. The cells are
considered viable after application of digesta when there is intense color change and the
absorbance value increases progressively with time indicating that the digesta is not toxic to the
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cells. The principle behind assessing the viability of cells is that the living cells synthesise an
enzyme that can convert MTS into an aqueous and soluble formazan product. Due to this there is
a transformation in the colour from pale yellow of MTS to dark yellow or brown colour in the
presence of viable cells. The number of viable cells is directly proportional to the intensity of
colour change caused by the formation of formazon (Stoddart, 2011).
Since it was a colorimetric assay, HBSS was chosen as a positive control and wells without cells
and those with cells and had added H202 in it were chosen as negative control for colour change.
The positive control (HBSS) showed color change over time while there was no substantial
colour change in the negative control (H202). This is because H202 was toxic to the cells and
hence did not produce the enzyme that utilizes the substrate to create colour change. The other
samples such as LY, fortified rice digesta and nil digesta showed dark colour change but did not
reach OD 1 after 6 hours incubation. However, after overnight incubation there was significant
colour change where the OD was measured to be over 1 indicating that the cells are viable in the
presence of the digesta and hence the digesta is non-toxic to the cells. Therefore the digesta
produced according to the above mentioned protocol (section 7.2.3.1) was used for transport
study.
Table 7.1 Absorbance values for the digesta samples and controls in the cell proliferation
assay after 2 hours incubation
Sample/Control Absorbance at 485 nm
Unfortified Rice 0.61 ± 0.164
Fortified Parboiled Rice 0.56±0.058
Fortified HPP processed rice 0.49±0.013
Nil digesta 0.44±0.090
Fortificant 0.30±0.187
Positive Control: HBSS 0.75±0.044
Negative Control: H2O2 0.13±0.018
Note: Mean absorbance values ±standard deviation of triplicate analysis
7.3.2 In vitro-bio-accessibility of micronutrients from fortified rice
The integrity of the caco-2 monolayer after 21 days of growing it in vitro was assessed by adding
0.1 mM of LY to the apical chamber. After the designated inbcubation time (2 hours) there was
no LY present in the basolateral chamber of the transwell indicating that the junctions between
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the caco-2 cells were intact and therefore it can be used for performing the transport study. The
TEER values were recorded and for further experiments wells with TEER ≥ 1000Ω was used for
transport study. In the following sections, bio-accessibility of the micronutrients from the
fortified rice and transport data will be presented and discussed.
Figure 7.3 Confocal microscopy image of caco-2 cells showing tight junctions (Chandra-
Hioe et al., 2013)
Table 7.2 Concentration of folic acid, iron and β-carotene (µmol) in fortificant solutions
(control), fortified parboiled rice and HPP rice
Digesta Folic acid β-carotene Iron
Fortificant 0.9±0.053 0.0009±0.0004 0.034±0.01
Parboiled rice 0.32±0.28 0.00042±0.00005 0.25±0.19
HPP rice 0.16±0.003 0.0005±0.0004 0.103±0.19
Note: Value represent Mean±Standard deviation of replicates.
After performing in-vitro digestion the folic acid concentration in the digesta was measured in
the fortificant standard solutions, fortified parboiled rice and fortified high pressure processed
rice. The fortificant standards with 150 mg of folic acid/ 600mL of water was used as a control
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and had a measured concentration of 400 µg/ mL of folic acid; parboiled fortified rice had a
measured concentration of 20 µg/ g of cooked rice on wet basis and HPP processed rice had 160
µg/g of folic acid. The concentrations of folic acid in the 3 samples after in vitro digestion are
presented in the above Table 7.2 and there was a significant difference observed in the
concentration of folic acid in the 2 different samples after in vitro digestion. This shows that the
release of folic acid after in vitro digestion varied with the food matrix.
From calculating the % bio-accessibility from the control folic acid standard solutions and the
rice it was found that the fortified rice (96%) was more bio-accessible compared to the fortificant
solution (80%). Similar observations were made by Kam et al., (2013) where folic acid fortified
rice had higher bio-accessibility compared to standard folic acid solution. However in the case of
high pressure processed rice the % bio-accessibility was much lower (46%) compared to fortified
parboiled rice. The reason for this could be attributed to the formation of resistant starch during
HPP making the starch less susceptible to in vitro enzymatic digestion leading to lower folic acid
bio-accessibility from HPP rice. This was likely due to the addition of 0.1 M HCl and 0.1 M
NaHC03 at gastric and intestinal stage of digestion which was favourable in the case of rice as the
folic acid was entrapped in a solid matrix compared to the standard solution. The rice matrix
would have provided a buffering environment for the folic acid making it more stable and less
susceptible to degradation. This also suggests that folic acid is stable in the gastric environment
when released from the rice and it is also easily released from the matrix. Folate bio-accessiblity
was better in wholemeal breads (above 75%) and breakfast foods (about 94%) compared to
fortified milk (60%) (Öhrvik et al.,2010 and Verwei et al., 2003).
Similar results were also observed for β-carotene (12% from parboiled rice 2% from HPP rice
and 0.1% from control) as well. From the digesta concentration it was calculated that iron bio-
accessibility was 38% from the fortificant alone; 31% from fortified parboiled rice and 42% from
high pressure processed rice. Fortified rice had higher concentration of iron in the digesta (Table
7.2). Vitamin A can affect iron metabolism at several stages and these include erythropoiesis and
release of iron from ferritin stores. Vitamin A and β-carotene enhanced iron absorption from
fortified maize and wheat and rice meals (Garcia –Casal et al., 1996 & Garcia –Casal, 2006).
The same group also reported that non- pro-vitaminA carotenoids such as lycopene, lutein and
zeaxanthin also increase iron absorption 2-3 folds when added to maize and wheat – bread meals.
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Davidson et al., (2003) added vitamin A to iron-fortified maize gruels and fed to vitamin A
deficient Ivorian children and reported that vitamin A significantly decreased iron absorption.
This shows that the relationship between β-carotene and iron is complex and is subject to the
methodology adopted. However, this area of micronutrient interaction needs to be highlighted as
many fruits and vegetable are good sources of both iron and β-carotene.
Table 7.3 Concentration of folic acid, β-carotene and iron present in the apical chamber of
the transwell in µg/monolayer
Apical Basolateral
Folic acid
Fortificant (control) 0.15±0.01a 2.8±0.2
a,b
Parboiled rice 4.3±0.98 a,b
0.42±0.85a,b
HPP rice 0.17±0.01a,b
0.42±0.8a
β – carotene
Fortificant (control) 0.053±0.002a 0.005± 0.002
a
Parboiled rice 0.026±0.003a,b
0.0065± 0.001a
HPP rice 0.001±0.003 a,b
0.0009±0.0002a
Iron
Fortificant (control) 0.015±a 19±1.2
a*
Parboiled rice 0.03±0.01a 26±0.75
a*
HPP rice 0.02±0.005a 24±1.5
a*
*Note: iron was quantified as ferritin in the basolateral chamber. Different superscripts within
the column indicate significant differences (p≤0.05)
7.3.3 Transport Study
7.3.3.1 Folic acid
For the transport study the digesta was added onto the apical chamber of the transwell plate and
the initial concentration of the apical chamber was the same as that of the digesta before the
transport study. After1 hour transport for folic acid, the concentration of folic acid in the apical
chamber was lower in the fortificant (control) than in the rice digesta and in the basolateral
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chamber, folic acid concentration from the fortificant (control) was higher than from that of the
parboiled rice digesta. For HPP rice after 1 hour transport, there was less concentration of folic
acid remaining in the apical chamber and much of it had been transported to the basolateral
chamber. This indicated that there was significant transportation of folic acid from the apical
chamber to the basolateral chamber. Overall from Table 7.3, it can be said that from the
fortificant control 12% of folic acid was transported and 33% and 9.4% for parboiled and HPP
rice respectively. The molecular structure of folic acid transported from the apical chamber to the
basolateral chamber was unchanged (Verwei, 2004). However if folate polyglutamates were
present they would be retained in the cells until they are converted into folate polygulatamtes in
the cytoplasm or mitochondria (Verwei, 2005) indicating that folic acid is more readily
bioavailable then folates. The concentration of folic acid detected in the basolateral chamber can
be considered as the concentration of folic acid that would be absorbed and it can also be
reported that the % transport is higher for the rice than the control folic acid solution.
7.3.3.2 β-carotene
In the case of β-carotene there was higher concentration remaining in the apical chamber rather
than in the basolateral chamber which had lower concentration after 16 hours of transport study.
This implies that there is less β-carotene transported across compared to folic acid. However, an
interesting observation was that when the cells were analysed for β-carotene concentration in
HPP processed rice and the fortificant 0.007 µg/ monolayer of β-carotene was present and ready
to be converted into chylomicrons and transported. This scenario was not observed in the case of
folic acid indicating that most of the transport had already occurred during the incubation time.
For transportation study of β-carotene (in vitro) 16 hours was deduced optimum incubation time
as after the designated incubation time the digesta became toxic to the cells hence the results
after this time could not be taken into account. Overall it was found that 4% of β-carotene was
transported from the fortificant and 23% and 13% was transported from the parboiled rice and
HPP rice respectively.
Very low concentrations of β-carotene were present in the digesta of the fortificant (control) and
in the rice. β-carotene bioavailability from fortified rice is very novel and it has not been
explored very well. However in other foods such as cassava, maize etc this has been well studied
using the caco-2 model. Micellization of β-carotene and other carotenoids into chylomicrons in
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cassava was studied by Thakkar et al., (2007) who reported that β-carotene in vitro digestion and
uptake by caco-2 cells was directly proportional to the content of β-carotene in different Cassava
cultivars. According to Failla et al., (2011) bio-accessibility of β-carotene from fortified cassava
is greater than in wild type roots. Information from literature thus suggests that assessing β-
carotene using caco-2 models is well established but not explored for fortified rice and this is the
first time it has been explored. Experimental data from the present study implies that there is
significant potential of having high bio-accessibilityof β-carotene compared to unfortified foods.
Courraud et al., (2013) studied bio-accessibility of β-carotene in various foods with and without
matrix effects. It was found that pure β-carotene stability decreased over time and in vitro
digestion stages and showed loss of approximately 60% at the end of the intestinal phase. Carrot
juice on the other hand showed greater than 100% of β-carotene at the end of the intestinal phase
of digestion. Compared to cooked spinach and raw spinach, pure form of β-carotene showed
lowest uptake post in vitro digestion. This shows that there is significant effect of matrix in the
bio-accessibility of foods. In this study although there is very low transport of β-carotene from
the fortificant there is higher concentration that has migrated from the parboiled rice compared to
high pressure processed rice. This could be because of the lower retention of β-carotene in the
cooked HPP rice than in the parboiled rice. The change in the structure of the soy protein in
which β-carotene is encapsulated could be a barrier for micellization. This could also be a
possible reason for lower transport of β-carotene compared to the other 2 micronutrients. Overall
it can be said that β-carotene from parboiled rice showed better bio-accessibility and transport
compared to HPP rice.
7.3.3.3 Iron
When iron transportation across the caco-2 monolayer was studied, it was encapsulated in the
form of ferritin protein and was quantified as such in the basolateral chamber and in the cells.
This phenomenon was definitive from the fortified rice digesta as when nil digesta and
unfortified rice was assessed by the transport study there was negligible concentration of ferritin
that was not detectable by the ELISA method. Hence ferritin was positively from the fortified
rice digesta. From Table 7.3 it can be seen that highest concentration of ferritin was from the
parboiled rice followed by HPP rice and the level were not very different. However from the
fortificant there were lower concentrations of ferritin present in the basolateral chamber. This
shows that there is once again effect of matrix as in the case of folic acid which enhances the
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concentration of iron transported from the rice rather than from the pure fortificant. As
mentioned previously in the study by Gracia et al., (2002) that β-carotene enhancing iron
absorption in the fortified rice could also be another reason for better iron bioavailability from
the rice rather than the fortificant. However it is worth noting that here was no significant
difference in the concentration of iron in the basolateral chamber for fortificant, parboiled and
HPP fortified rice.
Based on the normal range for serum ferritin (15-200 ng/mL, Iron Disorders Institute (2009)) and
the calculation of ferritin in the basolateral chamber, it can be said that with the given fortified
rice, there is high likelihood of showing improvement in the serum ferritin level of people who
consume it. A comparisson with the bioavailability study performed by Prom-u-thai (2009) (82
ng of ferritin/mg protein) using caco-2 cells shows that, the levels of ferritin was much lower in
this study. This can be attributed to the higher concentrations of ferrous sulphate and Na2EDTA-
Fe that was used as a fortificant (150mL of Fe solution with concentration range between 5.5 –
23.3 g per was added per 150 g of rice) in their study. In the present study however, 3
micronutrients were used for fortification and there was higher loss expected due to the
competition between the nutrients during the uptake process. It is also important to note that
prior to cooking the rice was washed for analysis. This could also add to the loss of the iron. In
another study conducted by Prom-u-thai et al., (2006), Fe bioavailability in the presence of anti-
nutrients such as phytates and phenolic acids which are potential iron absorption inhibitors was
assessed. In this study, it was concluded that in polished grains due to bran removal there was
high loss of Fe from raw rice and also substantial proportion of anti-nutrients such as phytates
and extractable phenols which enhanced the bio-availability of Fe in spite of the low
concentration. Despite all these factors significant concentrations of ferritin can be absorbed by
the body from the fortified rice and this indicates the success of the product in terms of bio-
accessibility.
Iron deficiency anaemia occurs in three stages namely: Iron depletion, iron deficiency and iron
deficiency anaemia. In the iron depletion stage levels of iron in the haemoglobin is normal but
the iron reserve would deplete soon with no obvious symptoms occurring at this stage. When the
body experiences iron deficiency, the stored iron and blood-borne iron in the body is low and
haemoglobin levels are lower than normal and the patient may experience symptoms such as
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tiredness. In the ultimate state of iron deficiency the patient becomes anaemic with very low
levels of haemoglobin in the blood where there is not enough oxygen to be delivered to the cells
and the patient looks pale, experiences breathlessness, dizziness and fatigue. This also causes
reduced immune functions, impaired growth and cognitive functions as symptoms (de Souza
Querioz & Torres, 2000). By consuming the fortified rice by parboiling or high pressure
processing, there is a high possibility that the iron stores would be accumulated in the body and
hence prevent the causes and symptoms of anaemia when the rice is eaten for a prolonged time.
This can also address symptoms of iron deficiency and there by prevent anaemia. Incidence of
iron deficiency anaemia can be reduced by consuming about 440 g of diluted fortified rice per
day on an average. The given concentration of ferritin in the basolateral chamber is only for 5 g
of rice which will increase when 440 g of rice is consumed on an average and therefore be able
to counter balance symptoms of IDA.
7.3.4 Improvement in the nutritional status after consumption of fortified rice
As mentioned in the previous chapter, the mixing ratio of fortified rice to commercial white rice
was 1:10 assuming that consumers would consume 440 g of cooked rice per day. For the in vitro
bio-accessibility study premix rice was used. From the above results it can be said that parboiling
process is overall much more promising than HPP treatment in terms of efficacy of nutrients
retention after cooking and also bio-accessibility. In the end for a consumer it matters as to how
much of each micronutrient is absorbed from the portion of the rice that would be consumed. In
this study, 5 g of cooked rice containing 68 µg of folic acid; 10 µg of β-carotene and 0.3 mg of
iron were used to prepare the digesta (final volume 50mL). From this 0.2 mL was added onto the
apical chamber of the transwell. This represents 1/250 times of the total digesta (50 mL). Hence
in the following paragraphs theoretical calculations of micronutrient absorption from the fortified
parboiled rice for each micronutrient is to be discussed.
In the case of the rice that was diluted in the ratio 1:10 and assuming 440g of cooked rice
would be consumed, 44g of fortified premix rice is present in it. The cooked rice would
therefore have a concentration of 13.6 µg of folic acid/g of cooked rice or 600 µg per
440g of cooked rice. Based on the current experiment the % transported from the digesta
to the basolateral chamber was 33%. Thus it can be said that theoretically 197 µg of folic
acid would be absorbed by the body from the given fortified rice. This value is very close
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to the upper limit of expected fortification level proposed by the current study. The
normal serum folate range is 2.7- 17 ng/mL (National Institute of Health, 2014). With the
concentration of folic acid absorbed from the fortified rice, it is highly likely that serum
levels after consumption may increase favourably.
For β-carotene it can be said that in the diluted fortified rice (in the ratio 1:10) there
would be 88 µg/ 440 g of rice. With the given 23% of transport from the digesta to the
basolateral chamber, 20 µg (3.3 Vitamin A equivalent) would be absorbed by the body
and metabolised. The normal serum range for Vitamin A is 50-200 µg/dL (National
Institute of Health, 2014). Therapeutic doses of vitamin A (1000-9000 IU/kg) have
shown to induce oxidative stress in sub-mitochondrial particles isolated from cerebral
cortex and cerebellum of adult rats (deOliveria and Moreira, 2007). Long-term
therapeutic dose of vitamin A can thus be harmful. Hence by administering lower levels
of vitamin A in the form ofβ-carotene through fortified foods after a high dose of
supplementation of vitamin A can help to maintain retinol balance in the body in
deficient populations.
In the case of iron the diluted fortified rice (cooked) would contain 2.64 mg/ 440g of rice
and if 31% of this transported 0.8 mg would be absorbed by the body. This can be
extrapolated to a ferritin level of 69 ng/mL was present in the basolateral chamber.
Normal serum ferritin range for men is 12-300 ng/mL and 12-150 ng/mL for women
(National Institute of Health, 2014). With the given concentration of ferritin from the
fortified rice it is possible to improve serum ferritin status with prolonged consumption of
the fortified rice.
In this study, caco-2 cells were used as an alternative to human bioavailability trials. The results
obtained from this study were an estimation of the in vitro uptake and transport in the short term.
However to understand the real picture, human trials are to be performed that could serve as a
comparison to the in vitro studies. Nevertheless, from the results it can be seen that bio-
accessibility of the micronutrients in the fortified rice through the parboiling methods and HPP
method was feasible and overall the micronutrients from the parboiled rice was better absorbed.
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7.4 Conclusions:
Overall, for the three micronutrients, the study suggests that the fortified rice had high potential
for improving the nutritional status of folic acid, iron and β-carotene. As mentioned previously in
chapter 3, rice does not have measurable concentrations of the three nutrients present naturally.
However there are minor concentrations of native non-haeme iron in the rice. From the fortified
rice there were higher concentrations of these micronutrients bio-accessible compared to the
unfortified rice. Therefore the results imply that fortification by parboiling and HPP is both
feasible but parboiled fortified rice is better option in terms of acceptability and bioavailability.
Rice eating population can highly benefit from this fortified rice and their nutritional status is
likely to improve even better if they are regularly consumed parboiled rice. These populations
can experience an improvement in their Folic acid, iron and β-carotene status and also be
benefitted if they have limited access to other food rich in these micronutrients.
The digesta from the fortified rice was shown to be non-toxic to the caco-2 cells based on the
cytotoxicity results. Overall the % transport and bio-accessibility was better for fortified rice
compared to the fortificant control solution except for iron transport. Parboiled rice which has
shown better bio-accessibility can deliver expected nutritional benefits in target populations. The
process of adding β-carotene along with iron in a fortified food itself has the possibility of
increasing blood haemoglobin concentration due to the synergistic relationship between β-
carotene and iron. This study however was performed in vitro using adenocarcinoma cell model.
Caco-2 in vitro model is not a replica of a human study although was able to give an insight on
absorption of the micronutrients from the triple-fortified rice.
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CHAPTER 8
8. Conclusion and Future work
Micronutrient deficiencies due to vitamin A, folic acid and iron are remarkably high and
prevalent worldwide. In developing countries more than 40% of women are anaemic and about
25% of children suffer from subclinical vitamin A deficiency (Micronutrient Initiative, 2001).
There are many programmes in place by governments and non-governmental organisations
(NGO) to address this issue. In terms of a long term solution reduction in poverty would be a
possible solution. However this is unlikely to happen in the near future. Food fortification could
provide an alternative in the short to medium term. Deficiency diseases are prevalent in rice
eating populations. Several technologies such as dusting, coating, extrusion and biofortification
have been employed to overcome this problem in the past. Parboiling: although an old technique
has recently been used for micronutrient fortification. High pressure processing on the other hand
is a novel technique for food preservation and has been explored for rice fortification in this
study. Both are post-harvest technologies and have their pros and cons. In many developing
countries parboiled rice is commonly consumed by rice-eating populations and thus there is no
need for capital investment. However in the case of high pressure processing there is necessity
for capital investment but this technology can save energy compared to parboiling process. HPP
process involves only 1 hour treatment and the energy is expended as pressure whereas
parboiling involves heating for 2 hours followed by steaming for an hour and the expenditure of
energy is through heat. Thus in this project the process of developing a method to fortify rice
with multiple micronutrients using 2 different technologies was explored in order to address the
public health concerns in the rice eating populations. Based on the results and discussions
presented in the earlier chapters some conclusions can be drawn.
8.1 Efficiency of parboiling as a technology to fortify rice:
Based on the results from Chapter 3 it can be concluded that parboiling is a feasible method for
fortification of rice with three micronutrients (folic acid, iron and β-carotene). The optimized
soaking (70°C for 2 hours) condition clearly would be able to maximize the uptake of the
micronutrients rather than shorter or longer soaking times. Based on the steaming time, the best
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favourable condition was 100°C for 1 hour for starch gelatinization and absence of white belly in
the rice which is an important quality parameter for purchase. By adapting to air drying at room
temperature, loss of micronutrients can be minimized compared to heat drying methods. The
retention of micronutrients (as measured after cooking) in the rice was according to the following
trend: folic acid> β-carotene>iron in the uncooked rice and folic acid>iron>β-carotene in the
cooked rice. Scale up of the conditions from 300 g to 2.5 kg showed concentrations similar to lab
scale for all the micronutrients in both uncooked and cooked parboiled rice. Also the
concentration of micronutrients in rice treated by the conventional parboiling condition was
similar to the lab scale condition implying that 2 hours is sufficient for maximum uptake of
micronutrients and by utilizing this optimized condition energy could be saved.
Table 8.1 shows the cost analysis performed by Roy et al., (2005) which showed that all the
processes were economical but the small-boiler was a clear choice as it had the shortest payback
period (the time period required to recoup the initial investment) compared to the others although
it consumes higher energy compared to the other methods. Keeping in mind the uncertainty of
investment and limitation of business capital as well, small-boilers were found to be the most
cost and energy effective process according to this study.
Table 8.1 Parboiling process cost analysis (Roy, Shimizu, Shiina and Kimura, 2005)
Process Vessel or
boiler Hopper
Soaking
tank Tube-well
Initial
Investment
Equipment $USD $USD $USD $USD $USD
Vessel (0.5- 1
t/batch) 5.32 - 42.55 21.28 397.47
Small- boiler
(2-4 t/batch) 42.55 42.55 148.94 21.28 1281.87
Medium-
boiler (5-10
t/Batch)
1170.21 63.83 638.29 212.77 5101.09
Parboiling is a well established process and the cost analysis has been performed in the above
table to get a glimpse of the financial aspect of the process. From the above table it can be said
that processing rice in bigger batches would be more economical than small scale processing
although it had a shorter payback period. The cost can be further reduced by adding more than
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one micronutrient. Also compared to hot extrusion technique, parboiling seems much more cost
efficient. With added cost of nutrients in the process the overall cost is likely to add up slightly
higher (USAID, 2008).
An approximate estimate of the cost of the rice-premix is around $1-2/kg, and the total cost for
rice fortification is from US$10/MT to US$20/MT.It can be deduced that the annual investment
is US$0.36-0.73/year for an individual consuming 100 g/day of fortified rice and $1.09-2.20/year
for an individual consuming 300 g/day. The process can be made even more effective if the rice
processing and fortification were centralized and the mills involved had production capacities
larger than 5 MT/hour (i.e., around 15,000 MT/year). Small mills have the disadvantage of
creating logistic issues and also increase the overall cost of the program (USAID, 2008).
8.1.1 Feasibility of rice fortification with multiple micronutrients using HPP technology
High pressure processing has been used for extending shelf-life of various food products and is a
popular processing method for over a decade. However the application of the technology in the
light of food fortification has never been explored. In this study, the feasibility of using this
technique as a potential method for rice fortification was explored. It can be concluded that rice
can be fortified using the HPP technology and the best condition for fortification was 200 MPa
treatment for 1 hour. Prolonged pressure treatment resulted in broken kernels and as opposed to
parboiling technique brown rice could not be used in this process as milling of the rice after the
process resulted in minimal salvaging of the kernels. After HPP treatment and cooking the rice
the retention of the micronutrients were as follows: folic acid>iron>β-carotene.
In comparison to thermal processing methods such as pasteurization and heat sterilization high
pressure processing is much more cost effective (Wang, 2009). Therefore, in terms of energy
efficiency HPP could be a possible technology that can be used for rice fortification. However it
involves a huge capital investment which is not needed in the case of parboiling.
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8.1.2 Physicochemical properties of the rice starch due to the two different processing
methods
Parboiled rice and high pressure processed rice have been previously been extensively studied
for alterations in starch physiochemical properties due to processing. However in the present
study, the starch in which the micronutrients were embedded in the fortified rice was studied to
understand how the 2 processing techniques altered native starch which was the primary novelty
of this section of work. Essentially, the physicochemical properties of the rice starch processed
by parboiling and high pressure processing was assessed for degree of gelatinization, pasting
properties, microscopic imaging of starch, crystallinity using XRD. Apart from these analytical
parameters, diffusion of micronutrients during the parboiling process was imaged in real-time
using MRI.
Collectively the results from this section of work demonstrate that both the processing methods
altered the rice starch properties significantly with the modifications pertaining to be unique to
the respective processing method. Addition of micronutrients did not significantly alter the rice
properties making the variations in rice starch dependent on the processing method. There is
evident correlation between release of micronutrients from fortified rice and the modified starch
matrix. Formation of partially gelatinized starch during HPP could hinder release of vitamins
from the matrix while parboiled rice which was fully gelatinized released more vitamins from the
embedded matrix. The variations in colour due to the processing could be of vital importance in
consumer preference and the changes in the rice starch could affect the starch digesting property.
Rice treatment at 400 and 600 MPa were not suitable for fortification as it resulted in broken
kernels, although rice starch processed at these pressures could be of use in other products as a
functional ingredient.
8.1.3 Acceptability of the fortified parboiled rice by consumers
A successful fortification does not stop with achieving higher concentrations of the
micronutrients in the grain. It has to be accepted and preferred as a choice for consumption by
consumers. In this case, rice a staple crop in Asian countries was the fortification vehicle.
Consumer acceptance study was performed to investigate if people are willing to buy the
uncooked fortified rice when commercialized and also if any changes to the taste of the fortified
rice was acceptable to the consumers. Sensory studies were performed in 2 stages: Study 1-
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Visual consumer acceptance; Study 2- Sensory evaluation of the cooked fortified rice. Due to
technical issues as mentioned in Chapter 6, HPP rice could not be tested for sensory evaluation.
Fortified parboiled rice diluted with white rice in the ratio 1:10 was pre presented to consumers
for consumer acceptance study. The dilution ratio was adjusted such that the fortified product
would be able to deliver 50% of the recommended dietary intakes of the nutrients added.
The results from Phase 1 of the sensory study showed that diluted fortified rice was not well
accepted in the uncooked form and the key factor that affected the acceptance was the non-
uniformity in rice samples. Familiarity with parboiled rice did not influence the acceptability of
the rice. Nevertheless after cooking, the fortified rice was well accepted and the non-uniformity
was neither recognized by the panel nor did it affect the acceptability of the product. In both the
studies, the purchase intent of the rice increased after mentioning about the health claim which
denotes that appropriate labelling would increase the purchase intent and consumer preference of
the fortified rice. There was slight variation in the flavour and texture when fortified rice was
diluted with white rice; nonetheless using the hedonic scale, fortified rice was preferred over
commercial white rice by consumers.
8.1.4 Bio-accessibility of the micronutrients from the fortified rice
The next step after consumer acceptance would be to understand if the fortification has
significant impact on the target population by trying to address the insufficiency of the
micronutrients and thereby improving the nutritional status of the affected populations. In this
study, caco-2 cells in vitro model was used to estimate the bio-accessibility of the micronutrients
from the fortified rice. As mentioned in Chapter 6, folic acid was able to deliver more than100%
of RDI requirement through the fortified rice, close to 50% of RDI for β-carotene and 100% of
RDI for iron. The results from the in vitro studies show that there is possibility that blood level
of folic acid, vitamin A and iron are likely to fall in the acceptable range of sufficiency if target
population consumes the triple-fortified rice over a long period of time. In vitro digestion, bio-
accessibility and transport studies were done using the premix rice and not the diluted fortified
rice. Further studies using the diluted fortified rice should be tested on humans to get a good
indication of absorption. The fortified rice diluted with white rice was not used in the caco-2 cell
study as a limitation of the analytical methodology used namely HPLC-PDA which could not
detect concentrations at ng levels especially for β-carotene. An LC-MS method for carotenoids
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should be developed for analysis in the diluted fortified rice digesta to address the low level
quantification issue of the compound.
8.1.4.1 Limitations of the in vitro bio-accessibility and transport of micronutrients using
caco-2 cells
There are several limitations due to the in vitro approach of analysing micronutrients from the
fortified rice and also due to the usage of caco-2 cells per se that was used in this study. They are
listed below:
In this study 3 micronutrients were added in the fortified rice. Therefore the micronutrient
interaction between the micronutrients could be explored which could provide answers
for increased or decreased transportation due to the effect of the micronutrients.
The in vitro static digestion performed in this study cannot exactly resemble or replicate
the functions of human (in vivo) digestive system, as the process of digestion in vivo
involves shear, mixing, hydration, changes in conditions over time and peristalsis
(Fernandez-Garcia, Carvajal-Lerida, & Perez-Galvez, 2009). These processes can alter
the absorption of micronutrients at intestinal level in vivo and the results of which can be
different from the in vitro studies.
It has to be noted that the in vitro approach is time consuming. It requires 21 days for the
cells to differentiate fully until the stage where it can be used for transport study (Tavelin,
2002).
The cells are also highly susceptible to contamination if not well taken care of. They can
get contaminated with fungal colonies and it is also expensive to purchase the materials
and maintain the lab at suitable conditions that are favourable for cell growth.
Caco-2 cells do not have protective mucus layers; therefore the pH at the cell surface
rapidly balances the pH of the applied samples (Galkin, Pakkanen, & Vuorela, 2008).
This condition could significantly impact the transport of micronutrients across the
membrane.
8.2 Recommendations for future work
Between the two fortification methods, it is likely that parboiling could be more feasible for
implementation compared to HPP due to the fact that it is relatively novel and further studies are
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still required to produce fortified rice on a larger scale using HPP. Therefore based on the results
from the current study, there are certain recommendations for future work.
When cooking the fortified rice, washing step can be reduced or avoided if the rice is
processed in a hygienic facility. This could reduce the loss of the micronutrients
especially for β-carotene as its loss was highest after cooking in the fortified rice after
both the processing methods. One possible solution could be to mention ―washing not
required prior to cooking‖ on the label of the packaged rice.
A suitable nutrition education program for the implementation of the fortification
program such that the target population would profit from the consumption of the rice. In
order to do this, the government along with non-governmental organisations such as
GAIN or Micronutrients Initiative could assist in putting together an effective approach
to educate people.
Based on the results from the scale-up study the optimized fortification process using
parboiling is reproducible. Therefore, it can be produced at an industrial scale by which
there would be lesser cost involved in processing and also the overall cost of the finished
rice product would not be very high. It is important that the fortified rice is available
without a substantial increase in the price. As mentioned in Chapter 7, in vitro studies
that were conducted using caco-2 cell was only an estimate of likely absorption in
humans and therefore human trials should be performed to understand the effectiveness
of the fortified rice in reality.
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CHAPTER 9
9. REFERENCES
Addepalli R., and Osborne S., (2011). ―Standard Operating Procedure for Caco-2 cell culture ans
in vitro transport study‖. Brisbane: The Commonwealth Scientific and Industrial Research
Organisation (CSIRO).
Aerni P. (2006); Mobilizing science and technology for development: the case of the cassava
biotechnology network (CBN).AgBioForum. 9:1–14
Ali, N. and Ojha, T.P. (1976) Parboiling. In: Araullo, E.V., de Padua, D.B. and Graham, M.,
Eds., Rice Postharvest Technology, International Development Research Center, Ottawa, 163-
204.
Allen, L., de Benoist, B., Dary, O. &Hurrell, R. (Ed.). (2006).‖Guidelines on food fortification
with micronutrients‖, France: World Health Organization and quality. Food Technology. 47(6),
156-161
Alminger, M. (2012). "Applicability of in vitro Models in Predicting the in vivo Bioavailability
of Lycopene and &β;-Carotene from Differently Processed Soups." Food and Nutrition Sciences
3(4): 477-489.
Anuradha K et al. (2012). "Evaluating rice germplasm for iron and zinc concentration in brown
rice and seed‖, Journal of Phytology 4(1): 19-25.
AOAC INTERNATIONAL (2012). Official Methods of Analysis, 19th ed., Association of
Official Analytical Chemists, Gaithersburg, MD, USA, Official Method 925.09.
Arigony, A. L., Vargas, I. M. de Oliveira, M. Machado, D. L. Bordin, L. Bergter, Pr, D. J. gas
Henriques and o. Antonio (2013). "The Influence of Micronutrients in Cell Culture: A Reflection
on Viability and Genomic Stability." BioMed Research International 2013: 22.
Ashognon,A.O. & Akintayo E.T. (2012) Morphological, functional and pasting properties of
starches separated from rice cultivars grown in Nigeria, International Food Research Journal
19(2): 665-671.
Ayamdoo J.A., Demuyakor, B., Dogbe, W., &Owusu, R. (2013). Parboiling Of Paddy Rice, The
Science And Perceptions Of It As Practiced In Northern Ghana. International Journal of
Scientific & Technology Research 2(4): 13-18.
Backstrand, J. R. (2002). "The history and future of food fortification in the United States: A
public health perspective." Nutrition Reviews. 60(1): 15-26.
Badenhuizen, N.P. (1969), The Biogenesis of Starch Granules in Higher Plants, Starch 22(3):
102-103
192
Bailey L.B., Moyers, S., Gregory, J.F. (2001) ―Folate‖. In: Bowman B.A., Russell, R.M., ed.
Present Knowledge in Nutrition. 8th Edition ed. Washington, DC: International Life Sciences
Institute Press; 214-229.
Bakshi, A. and Singh, R. (1980)."Kinetics of water diffusion and starch gelatinization during rice
parboiling."Journal of Food Science.45: 1387-1392.
Balcom, B.J., MacGregor, R.P., Beyea, S.D., Green, D.P., Armstrong, R.L., Bremner, T.W.,
(1996), Single-point ramped imaging with T1 enhanced (SPRITE), Journal of Magnetic
Resonance A, 123:131–134.
Barber, S., (1972),.―Milled rice and changes during aging‖. In D.F. Houston, ed. Rice chemistry
and technology,St Paul, MN, USA, American. Association of Cereal Chemistry. 215-263
Barbosa-Canovas, G.V., Pothakamury, U.R., Palou, E., and Swanson, B.G., (1998), ―Nonthermal
processing of Foods and Emerging Technologies‖, Food Engineering vol.3.
Bates CJ (1995), Vitamin A (Retinol), Lancet, Vol. 345(31).
Bauer, B. A., & Knorr, D. (2005), ―The impact of pressure, temperature and treatment time on
starches: Pressure-induced starch gelatinisation as pressure time temperature indicator for high
hydrostatic high pressure‖, Journal of Food Engineering 68(3): 329-334.
Bayram, M. (2006)."Determination of the cooking degree for bulgur production using
amylose/iodine, centre cutting and light scattering methods."Food Control17(5): 331-335.
Bello, M., et al. (2004). ―Factors affecting water uptake of rice grain during soaking." LWT -
Food Science and Technology 37(8): 811-816.
Bergman, C., Bhattacharya, K. and Ohtsubo, K. (2004).Rice end-use quality analysis. T. Elaine
(ed). In: Rice Chemistry and Technology. St. Paul, Minnesota, American Association of Cereal
Chemists, Inc. pp: 415-472.
Berry RJ, Bailey L, Mulinare J, Bower, Dary O (2010). ―Fortification of flour with folic acid‖,
Food and Nutrition Board 31(1): S22- S35.
Bhattacharya, K. and Swamy, Y. M. I. (1967)."Conditions of Drying Parboiled Paddy for
Optimum Milling Quality."Cereal Chemistry.44(6): 592.
Bhattacharya, K. R. (2004). ―Parboiling of rice‖, In: Rice Chemistry and Technology (. St. Paul,
Minnesota: American Association of Cereal Chemists, Inc, pp: 329-394)
Bhattacharya, K.R. (1985). ―Parboiling of rice:. In: ―Rice Chemistry and technology‖ ed. By
Juliano. B.O American. Association of Cereal Chemists, Inc., St. Paul, Minnesota, 289–348.
Bierlen R, Wailes E, Cramer, E (1997) The Mercosur Rice Economy, Arkansas Experiment
Station Bulletin. No. 954, University of Arkansas.
193
Biliaderis, C.G., Tonogai, J.R., Perez, C.M., Juliano, B.O., (1993), ―Thermo-physical properties
of milled rice starch as influenced by variety and parboiling method‖, Cereal Chemistry 70, 512–
516
Birch, G. G. and R. J. Priestley (1973)."Degree of Gelatinisation of Cooked Rice."Starch -
Stärke25(3): 98-100.
Biswa, S.K., & Juliano, B.O. (1988), ―Laboratory parboiling procedures and properties of
parboiled rice from varieties differing in starch properties‖, Cereal Chemistry 65:417-423.
Björck, I. 1996. Starch: Nutritional Aspects. Page 505 in: Carbohydrates in Food. A.-C. Eliasson,
ed. Marcel Dekker: New York.
Błaszczak, W., J. Fornal, S. Valverde and L. Garrido (2005). "Pressure-induced changes in the
structure of corn starches with different amylose content." Carbohydrate Polymers61(2): 132-
140.
Blaszczak, W., Valverde, S., & Fornal, J. (2005).―Effect of high pressure on the structure of
potato starch‖.Carbohydrate Polymers, 59:377-383.
Blish, M.J., Sandstedt, R M., and Kneen, E. (1938)― The cereal amylases with reference to flour
and malt behaviour‖, Cereal Chemistry, 15: 629:657.
Boccio J R & Iyengar V (2003), ―Iron deficiency causes, consequences and strategies to
overcome this nutritional problem‖, Biological Trace Element Research, 94:1-32.
Booth, S.L., Johns, T. & Kuhnlein, H.V. (1992). Natural food sources of vitamin A and pro-
vitamin A. UNU Food and Nutrition Bulletin, 14: 6-19.
Borchgrevink, N. C.; Charley, H. (1966) ―Color of cooked carrots related to carotene content‖
Journal of American Diet Association, 49, 116-121.
Brewer, M. S. and McKeith, F. K. (1999)."Consumer-rated quality characteristics as related to
purchase intent of fresh pork."Journal of Food Science.64(1): 171-174.
Buckow, R., V. Heinz and D. Knorr (2007). "High pressure phase transition kinetics of maize
starch." Journal of Food Engineering81(2): 469-475.
Burití Palm. (1975). In: Report, Ad Hoc Panel of the Advisory Committee on Technology
Innovations, Board on Science and Technology for International Development, Commission on
International Relations. Underexploited tropical plants with promising economic value. P. 133-
137. Washington, DC, National Academy Sciences.
Canada Gazette Food and Drugs Regulations, authors.SOR/96-527. [(accessed on October 7,
2012)]. Available online:<http://canadagazette.gc.ca/partII/1998/19981125/html/sor550-e.html>
Champagne, E. T., Bett-Garber, K. L., Fitzgerald, M. A., Grimm, C. C., Lea, J., Ohtsubo, K.,
Jongdee, S., Xie, L., Bassinello, P. Z., Resurreccion, A., Ahmad, R., Habibi, F. and Reinke, R.
(2010). "Important sensory properties differentiating premium rice varieties." Rice.3: 270-28
194
Champagne, E.T. (Eds.) (2004). ―The rice grain and its gross composition‖. T. Elaine (ed). 3rd,
In: Rice Chemistry and Technology St. Paul, Minnesota: American Associatiation of Cereal
Chemists, Inc. (pp.77-107).
Champagne, E.T., Bett, K.L., Vinyard, B.T., McClung, A.M., Barton, F.E. II,Moldenhauer, K.,
Linscombe, S., McKenzie, K., (1999). ―Correlation between cooked rice texture and rapid visco
analyser measurements‖.Cereal Chemistry 76(5), 764-771.
Chandra-Hioe, M. V., R. Addepalli, S. A. Osborne, I. Slapetova, R. Whan, M. P. Bucknall and J.
Arcot (2013). "Transport of folic acid across Caco-2 cells is more effective than 5-
methyltetrahydrofolate following the in vitro digestion of fortified bread." Food Research
International53(1): 104-109.
Cheftel, J.C. and Culioli, J., (1997), ―Effect of High Pressure on Meat: Review‖, Meat Science
46(3):211-235.
Chen L.T., Rivera M.A. (2004). The Costa Rican experience: reduction of neural tube defects
following food fortification programs. NutritionReviews ;62:S40–S43.
Chukwu &, Oseh (2009), ―Response of nutritional contents of rice (Oryzasativa) to parboiling
temperatures‖, American-Eurasian Journal of Sustainable Agriculture 3(3):381-387.
Chung, H.-J., H. S. Lim and S.-T.Lim (2006). "Effect of partial gelatinization and retrogradation
on the enzymatic digestion of waxy rice starch", Journal of Cereal Science 43(3): 353-359.
Courraud, J., J. Berger, J.-P.Cristol and S. Avallone (2013)."Stability and fibility of different
forms of carotenoids and vitamin A during in vitro digestion."Food Chemistry 136(2): 871-877.
Cuskelly, G. J., McNulty, H. & Scott, J. M. (1996), ―Effect of increasing dietary folate on red-
cell folate: implications for prevention of neural tube defects‖, Lancet, 347:657-659.
Czeizel AE & Dudas I, (1992), ―Prevention of the first occurrence of neural tube defects by
periconceptual vitamin supplementation‖, The New England Journal of Medicine 327(26):1832-
5.
D‘Souza, Shetzer, Menon & Pauletti (2003). ―High glucose concentration in isotonic media
alters Caco-2 permeability‖. Aaps Pharmsci, 5(3).
Daly, L.E.; Kirke, P.N.; Molloy, A.; Weir, D.G.; Scott, J.M. Folate levels and neural
tubedefects.Implications for prevention (1995).JAMA, 274, 1698–1702.
Dang, J.M.C. and Copeland, L. (2004) ―Studies of the fracture surface of rice grains using
environmental scanning electron microscopy‖, Journal of the Science of Food and Agriculture,
84(7):707-713.
Davidsson L, Adou P, Zeder C, Walczyk T, Hurrell R., (2003). ―The effect of retinyl palmitate
added to iron-fortified maize porridge on erythrocyte incorporation of iron in African children
with vitamin A deficiency‖,. British Journal of Nutrition 90:337–43.
de Ambrosis, A. Arcot, J., Haber, P. Paterson, JL., Smythe, G and Guilhaus, M. (2004)
Bioavailability of folate fortified rice in humans using stable isotope techniques. In Proceedings
of EUROFOODFOLATE - First International Conference on Folates, Analysis, bioavailability
195
and health. 11-14 February Warsaw, Poland. P113. Edited by A. Brzozowska, P. M. Finglas, D.
Wright and M. Araucz. Warsaw Agricultural Press, Warsaw. (ISBN 83-7244-474-9)
de Oliveira, M. R. and J. C. Moreira (2007). "Acute and chronic vitamin A supplementation at
therapeutic doses induces oxidative stress in submitochondrial particles isolated from cerebral
cortex and cerebellum of adult rats." Toxicology Letters 173(3): 145-150.
de Souza Queiroz, de. A. Torres. M. A. (2000). "Iron deficiency anemia in children." Journal of
Pediatrics76(Suppl. 3): S298-S-304.
Deffenbaugh L.B. & Walker C.E. (1999), ―Comparisson of starch pasting properties in the
Barbender Viscoamylograph and the Rapid Visco-Analyser‖, American Association of Cereal
Chemists, 66 (6): 493-499.
del Pilar Babot, M., (2003). Starch grain damage as an indicator of food processing, In: Hart,
D.M., Wallis, L.A. (Eds.), Phytolith and Starch Research in the Australian–Pacific–Asian
regions: the state of the art. Pandanus Press, 69–81.
Delcour, J. A. (2005),.―Starch gelatinization and amylose-lipid interactions during rice
parboiling investigated by tem-perature resolved wide angle X-ray scattering and differential
scanningcalorimetry‖, Journal of Cereal Sciences. 42:334-343.
Delie & Rubas (1997). ―A human colonic cell line sharing similarities with enterocytes as a
model to examine oral absorption: Advantage and limitations of the Caco-2 model‖. Critical
Reviews in Therapeutic Drug Carrier Systems, 14(3), 221-286.
Dervisi P, Lamb J, Zabetakis I (2001). ―High pressure processing in jam manufacture: effect on
textural and colour properties‖ Food Chemistry 73: 85-91.
Derycke, V., Veraverbeke, W.S, Vandeputte, G.E. De Man W., Delcour J.A.,(2005), ― Role of
proteins in the pasting and cooking properties of non-parboiled and parboiled rice‖, Cereal
Chemistry 82, 468–474.
Dexter. P.B., (1998), Rice Fortification for developing countries, OMNI/USAID, Available at <
http://www.mostproject.org/PDF/rice4.pdf>, Accessed on 15th March, 2011.
Dhital, S., Shrestha, A. K., Flanagan, B. M., Hasjim, J., Gidley, M. J., (2011), ―Cryo-milling of
starch granules leads to differential effects on molecular size and conformation‖ Carbohydrate.
Polymers., 84, 1133–1140.
Dhital, S., et al. (2015). "In vitro digestibility and physicochemical properties of milled rice."
Food Chemistry 172(0): 757-765.
Diako, C., Sakyi-Dawson, E., Bediako-Amoa, B., Saalia, F. and Manful, J., (2010), "Consumer
perceptions knowledge and preferences for aromatic rice types in ghana", Nature and Science.
8(12): 12-19.
196
Doesthale, Y. G., S. Devara, S. Rao and B. Belavady (1979). "Effect of milling on mineral and
trace element composition of raw and parboiled rice."Journal of the Science of Food and
Agriculture30(1): 40-46.
Douzals, J. P., Cornet, J. M. P., Gervais, P., & Coquille, J. C. (1998) ―High-pressure
gelatinization of wheat starch and properties of pressure-induced gels‖, Journal of Agricultural
and Food Chemistry, 46(12), 4824–4829.
Douzals, J. P., Marechal, P. A., Coquille, J. C., & Gervais, P. (1996).―Microscopic study of
starch gelatinization under high hydrostatic pressure‖, Journal of Agricultural and Food
Chemistry, 44(6), 1403–1408.
Dowd, J.B.; Aiello, A.E. Did national folic acid fortification reduce socioeconomic and racial
disparities in folate status in the US? (2008). International Journal of Epidemiology., 37, 1059–
1066.
Eerlingen, R.C., Jacobs, H., Delcour, J.A., (1994). ―Enzyme-resistant starch: V. Effect of
retrogradation of waxy maize starch on enzyme susceptibility‖. Cereal Chemistry 71, 351–355.
Eggum, B.O., Juliano, B.O. & Maniñgat,, C.C. (1982). Protein and energy utilization of rice
milling fractions by rats. Qualitas Plantarum. Plant Foods Human Nutrition., 31: 371 -376.
Eitenmiller, R.; Landen, W. Folate. (1999). In Vitamin Analysis for the Health and Food
Science; CRC Press: Baco Raton, FL, USA,; pp. 411–465.
Eitenmiller.(2008). Folate Vitamin Analysis for the Health and Food Sciences, Taylor & Francis
Group.
Ellis, R.P., Cochrane, M.P., Dale, M.F.B., Duffus, C.M., Lynn, A., Morrison, I.M., Prentice,
R.O.M., Swanston, J.S. and Tiller, S.A. (1998).Starch production and industrial use.Journal of
Food Science and Agriculture 77: 182-187
Failla M.L.,ChitchumroonchokchaiC., SiritungaD., de MouraF.F., Fregene M. and Sayre R.,
(2011). ―Retention and bioaccessibility of B-carotene (BC) in biofortified cassava‖, The Journal
of the Federation of America Societies for Experimental Biology, 25:976.3.
Fenech,. (1996), ―Folate and cancer initiation; Will folate fortification help prevent genetic
events that could initaiate cancer?‖, Australian Journal of Nutrition and Dietetics 53:S13-S17.
Fernandez-Garcia, Carvajal-Lerida, & Perez-Galvez.(2009). In vitro bioaccessibility assessment
as a prediction tool of nutritional efficiency.Nutrition Research, 29(11), 751-760.
Finer, L.B.; Henshaw, S.K. Disparities in rates of unintended pregnancy in the United States,
1994 and 2001. (2006) Perspectives on Sexual and Reproductive Health, 38, 90–96
Fleshman,M.K., Lester,G.E., Riedl, K.M., Kopec,R.E., Narayanasamy, S., Curley,R.W.Jr.,
Schwartz,S.J., and Harrison,E.H.(2011), ―Carotene and novel apocarotenoid concentrations in
orange-fleshed Cucumismelo melons: determinations of β-carotene bioaccessibility and
bioavailability‖,Journal of Agriculture and .Food Chemistry. 59: 4448–4454.
197
Flores H, Guerra NB, Cavalcanti ACA, Campos FACS, Azevedo MCNA, Silva MBM. (1994),
―Bioavailability of vitamin A in a synthetic rice premix‖, Journal of Food Science 59(2):371-
377.
Flour Fortification Initiative. Report of the Workshop of Wheat Flour Fortification: Cuernavaca,
Mexico. Available online: http://www.sph.emory.edu/wheatflour/CKPAFF/index.htm (accessed
on August 8, 2011).
Floury, D. L. (2002)."Effect of Ultra-high-pressure Homogenization Structure and on
Rheological Properties of Soy Protein-stabilized."Journal of Food Science, 67(9): 3388-3395.
Food and agricultural Organization of the United States (1988), ―Requirement of Vitamin A,
iron, folate and vitamin B12: report of a joint FAO/WHO expert consultation‖, Rome.
Food and Agricultural Organization of the United States, (2002), Chapter 2.Human Vitamin and
Mineral requirement,: Report of a joint FAO/WHO expert consultation Bangkok, Thailand,
Food-based approaches to meeting vitamin and mineral needs.
Food and Agriculture Organization /World Health Organisation (1994). Food Fortification:
Technology and Quality Control. Rome.
Food and and Agricultural Organization/World Health Organization, (1994), Codex
Alimentarius, Volume 4, 2nd edition.
Food and Drug Administration (1996) Food standards: amendment of standards of identity for
enriched grain products to require addition of folic acid. Final Rule.21 CFR Parts 136, 137, and
139. Fed. Regist;61:8781–8789.
Food and Nutrition Board, Institute of Medicine, National Academies 2010, Dietary Reference
Intakes (DRIs):
Food Insight.(2010). "Food fortification in today's world." Retrieved 6/8/2014, from:
http://www.foodinsight.org/Newsletter/Detail.aspx?topic=Food_Fortification_in_Today_s_Worl
d
Food Standard Australia and New Zealand (2007) ―Mandatory Folic acid Foritification guide‖
Reterived October 8 2013 from <
http://www.foodstandards.gov.au/code/userguide/Documents/Mandatory%20Folic%20Acid%20
Fortification%20User%20Guide.pdf >
Food Standards Australia and New Zealand (FSANZ) (n.d.).Folic acid/folate and pregnancy.
Retrieved October 12 2013, from
<http://www.foodstandards.gov.au/consumer/generalissues/pregnancy/folic/Pages/default.asp>x
Fredriksson, H., Björck, I., Andersson, R., Liljeberg, H., Silverio, J., Eliasson, A.-C.,Åman, P.,
(2000). ―Studies on a-amylase degradation of retrograded starch gelsfrom waxy maize and high-
amylopectin potato‖.Carbohydrate Polymers 43, 81–87
198
French, D. (1984), ―Organisation of the starch granules, in Starch: Chemistry and Technology,
ed. R. L. Whistler, J. N. BeMiller and J. F. Paschall. Academic Press, Orlando, pp. 183-247.
GAIN (Global Alliance for Improved Nutrition) BUHLER Nutrition solutions Nutririce Process
2009, NutriRice Process. A breakthrough in rice fortification, Switzerland.
Galkin, Pakkanen, & Vuorela.(2008). Development of an automated 7-day 96-well Caco-2 cell
culture model.Pharmazie, 63(6), 464-469
Gallant, D. J. (1974) Contribution i l‘etude de la structure et de I‘ultrastructure du grain
d‘amidon‖, PhD. Thesis, University of Paris VI, France. N‖ CNRS A0 10823.
Gallant, D. J., Bouchet, B., Bulion, A. and Perez, S. (1992) ―Physical characteristics of starch
granules and susceptibility to enzymatic degradation‖.European Journal of Clinical Nutrition 46,
S3-S 16.
Galliard, T. and Bowler, P. (1987). ―Morphology and composition of starch‖, In: Starch:
Properties and potential. Ed. T. Galliard. John Wiley & Sons, NewYork, 281.
Garcia, V., P. Colonna, B. Bouchet and D. J. Gallant (1997)."Structural Changes of Cassava
Starch Granules after Heating at Intermediate Water Contents."Starch 49(5): 171-179.
García-Casal M.N. (2006),―Carotenoids increase iron absorption from cerealbased food in the
human‖, Nutrition Research; 26:340–4.
Garcıa-Casal, M.N., Layrisse, M., Solano, L., Barón, M.A., Arguello, F., Llovera, D., Ramırez,
J., Leets, I., &Tropper, E. (1998), ― Vitamin A and β-Carotene Can Improve Nonheme Iron
Absorption from Rice, Wheat and Corn by Humans‖,. The Journal of Nutrition, 128(3):646-650.
Gariboldi, F. (1985).―Parboiled Rice‖. In: Rice Chemistry and Technology. ed. By B.O. Juliano,
American Association of Cereal Chemists, Inc., St. Paul, Minnesota, USA.
Gibson, R. S., L. Perlas and C. Hotz (2007)."Improving the bioavailability of nutrients in plant
foods at the household level."Proceedings of the Nutrition Society65(02): 160-168.
Glahn R. P.; Lai, C.; Hsu, J.; Thompson, J. F.; Van Campen, D. R. (1998), ―Decreased Citrate
Improves Iron Availability from Infant Formula: Application of an In Vitro Digestion/Caco-2
Cell Culture Model‖, Journal of .Nutrition., 128: 257-264.
Glahn, R. P., M. Rassier, M. I. Goldman, O. A. Lee and J. Cha (2000)."A comparison of iron
availability from commercial iron preparations using an in vitro digestion/caco-2 cell culture
model."The Journal of Nutritional Biochemistry11(2): 62-68.
Glahn, R. P.; Lee, O. A.; Miller, D. D. (1999) ―In Vitro Digestion/ Caco-2 Cell Culture Model to
Determine Optimal Ascorbic Acid to Fe Ratio in Rice Cereal‖, Journal of Food Science 64, 925-
928.
199
Glahn, R. P.; Lee, O. A.; Yeung, A.; Goldman, M. I.; Miller, D. D., (1998) ―Caco-2 Cell Ferritin
Formation Predicts Nonradiolabeled Food Iron Availability in an In Vitro Digestion/Caco-2 Cell
CultureModel‖ The Journal of Nutrition., 128: 1555-1561.
Glahn, R. P.; Wien, E. M.; Van Campen, D. R.; Miller, D. D. Caco-2 cell iron uptake from meat
and casein digests parallels in vivo studies: Use of a novel in vitro method for rapid estimation of
iron bioavailability. The Journal of Nutrition. 1996, 126, 332-339.
Glidewell, S.M., (2006). NMR imaging of developing barley grains, Journal of Cereal Science
43: 70–78.
Gracĺa.-Casal et al., (1998), "Vitamin A and beta-carotene can improve non-heme iron
absorption from rice, wheat and corn by humans‖ The Journal of Nutrition ,128(3): 646-650.
Granick (1949), ―General Review of Iron Metabolism‖.Acta Paediatricia, 42:S93: 9-17.
Gregory, J. (1996). Vitamin. O. Fennema (ed). In: Food Chemistry. New York, Dekker Inc. pp:
590-601.
Gruwel, M.L.H., Yin, X.S., Edney, M.J., Schroeder, S.W., MacGregor, A.W., Abrams, S.,
(2002), ―Barley viability during storage: use of magnetic resonance as a potential tool to study
viability loss‖, Journal of Agricultural and Food Chemistry 50: 667–676.
Guidelines on food fortification with micronutrients 2006, World Health Organisation, in Allen
L., Benoist B., Dary O, Hurrell R. (eds), pp.- 97-123.
Gunaratne, A., and Hoover, R. (2002).―Effect of heat- moisturetreatment on the structure and
physicochemical properties of tuber and root starches‖.Carbohydrate Polymers.49:425.437.
Guwanan S. and Arcot J (2010), ―Fortification of rice with β-carotene using parboiling‖,
Honours thesis- Food Science and Technology, UNSW, Australia.
Hallberg L, Hulthén. L. and Garby L. (1998). "Iron stores in man in relation to diet and iron
requirements." European Journal of Clinical Nutrition 52: 623-632.
Haskell, M.J. (2012), ―The challenge to reach nutritional adequacy for vitamin A: b-
carotene bioavailability and conversion—evidence in humans‖, American Journal of Clinical
Nutrition 96(suppl):1193S–203S.
Hedren, E., Diaz, V., Svanberg, U., ―Estimation of carotenoid accessibility from carrots
determined by an in vitro digestion method‖ European Journal of Clinical Nutrition, 56 (5)
(2002), pp. 425–430
Heinemann, R., Behrens, J. and Lanfer-Marquez, U. (2006). "A study on the acceptability and
consumer attitude towards parboiled rice." International Journal of Food Science and
Technology. 41: 627-634.
Heremans K., (1995), ―High pressure effects on biomolecules. In High Pressure Processing of
Foods: (Ledward DA, Johnston DE, Earnshaw RG and. Hasting A.P.M, eds) Loughborough:
Nottingham University economic value. pp. 81-98.
200
Hermansson, A. M., & Svegmark, K. (1996).―Developments in the understanding of high
hydrostatic pressure processing‖.Journal of Food Engineering, 68(3), 329–334
Hertrampf E., Cortes F. (2004). Folic acid fortification of wheat flour: Chile. Nutrition
Reviews;62:S44–S48. doi: 10.1111/j.1753-4887.2004.tb00074.x. discussion S49.
Hibbard, B.M.; Hibbard, E.D.; Jeffcoate, T.N. (1965).Folic acid and
reproduction.Acta Obstetricia et Gynecologica Scandinavica 44, 375–400.
Hibi Y., Matsumoto T., and Hagiwara S., (1993). ―Effect of High Pressure on the Crystalline
Structure of Various Starch Granules‖, American Association of Cereal Chemists, 70(6): 671-
676.
Hoffpauer, D.W. 7 Wright S.L., (1996) Enrichment of rice.In Rice Science and Technology. ed.,
W. E. Marshall and J. I. Wadsworth.Marcel Rekker, New York, NY.
Holm, J., Lundquist, I., Björck, I., Eliasson, A.C., & Asp, N.G. (1988), ―Degree of gelatinisation,
digestion rate of starch in vitro, and metabolic response in rats‖, American Journal of Clinical
Nutrition. , 47:1010–1016.
Horigane, A. K., H. Takahashi, S. Maruyama, K. i. Ohtsubo and M. Yoshida (2006). "Water
penetration into rice grains during soaking observed by gradient echo magnetic resonance
imaging." Journal of Cereal Science 44(3): 307-316.
Horigane, A. K., K. Suzuki and M. Yoshida (2013). "Moisture distribution of soaked rice grains
observed by magnetic resonance imaging and physicochemical properties of cooked rice grains."
Journal of Cereal Science 57(1): 47-55.
Horigane, A.K., Engelaar, W.M.H.G., Toyoshima, H., Maruyama, S., Yoshida, M., Okubo, A.,
Nagata, T., (2001), ―Visualization of moisture distribution during development of rice caryopses
(Oryza sativa L.)by nuclear magnetic resonance microimaging‖, Journal of Cereal Science
33:105–114.
Horigane, A.K., Takahashi, H., Maruyama, S., Ohtsubo, K., Yoshida, M., (2006), ―Water
penetration into rice grains during soaking observed by gradient echo magnetic resonance
imaging‖, Journal of Cereal Science, 44:307-316.
Huang, S. L., C. L. Jao and K. C. Hsu (2009). "Effects of hydrostatic pressure/heat combinations
on water uptake and gelatinization characteristics of japonica rice grains: a kinetic study."
Journal of Food Science 74(8): E442-448.
Hug- Iten, Handschin, Conde-Petit and Escher., (1999), ―Changes in starch microstructure on
baking and staling of wheat bread‖, LWT- Food Science and Technology., 32:255-260
Hurrell, R. (1997). "Preventing iron deficiency through food fortificaion."Nutrition Reviews.
55(6): 210-222.
Hurrell, R. and I. Egli (2010). "Iron bioavailability and dietary reference values." American
Journal of Clinical Nutrition 91(5): 1461S-1467S.
201
Imberty, A. and S. Perez (1988)."A revisit to the three-dimensional structure of B-type
starch."Biopolymers27(8): 1205-1221.
Imberty, A., Buléon, A., Tran. V., and Perez, S. 1991. ―Recent advances in knowledge of starch
structure‖, Starch ,43:375-384.
Institute of Medicine.Folate. In Dietary Reference Intakes for Thiamin, Riboflavin,
Niacin,Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline (1998). National
Academy Press: Washington, DC, USA,; pp. 196–305.
International Rice Research Institute (IRRI a) (n.d.).Rice Basics. Retrieved on October 22nd
2013, from
<http://www.irri.org/index.php?option=com_k2&view=item&layout=item&id=9081>.
International Rice Research Institute (IRRI). Which countries consume the most rice? Retrieved
October 17 2013, from
<http://irri.org/index.php?option=com_k2&view=item&id=12109&lang=en>
Iron (2006), Nutritive Reference Value for Australia and New Zealand, NHMRC, Australia.
Iron Disorders Institute (2009), Serum Ferritin. Retrieved on September 12th
2014, from
<http://www.irondisorders.org/iron-tests/>
Ismail M., 1999, ―The use of Caco-2 cells as an in vitro method to study bioavailability of iron‖,
Malaysian Journal of Nutrition, 5: 31-45
Jane, J., Wong, K. S., & McPherson, A. E., (1997), ― Branch-structure difference in starches of
A- and B-type X-ray patterns revealed by their Naegeli dextrins‖,. Carbohydrate Research, 300:
219–227.
Jenkins, P. J., Cameron, R. E. and Donald, A. M., (1993), ―A universal feature in the structure of
starch granules from different botanical sources‖, Starch, 45:417- 420.
Jenner, C.F., Xiar, Y., Eccles, C.D., Callaghan, P.T., 1988. ―Circulation of water within wheat
grain revealed by nuclear magnetic resonance micro-imaging‖, Nature 336: 399–402
John Innes Centre (n.d.). Nomarski (DIC) microscopy accessed on September 12th
2014
<https://www.jic.ac.uk/microscopy/more/T5_5.htm>
Juliano, B. and Bechtel, D. (1985).The rice grain and its gross composition. B. Juliano (ed). 2nd,
In: Rice Chemistry and Technology. St. Paul, Minnesota, American Association of Cereal
Chemists pp: 17-58.
Juliano, B.O. (1992). Structure and function of the rice grain and its fractions.Cereal Foods
World 37: 772-774.
Juliano, B.O., (1993), ―Rice in human nutrition‖, Rome: Food and Agriculture Organization of
the United Nations.
202
Kadan, R. S., M. G. Robinson, D. P. Thibodeaux and A. B. Pepperman (2001)."Texture and
other Physicochemical Properties of Whole Rice Bread."Journal of Food Science 66(7): 940-944.
Kajiyama et al., (1995), "Changes of soy protein under ultra-high hydraulic pressure",
International Journal of Food Science and Technology 30: 147-158.
Kam K., Adesina A., and Arcot J ., (2012), ―Folic acid fortification of parboiled rice:
Multifactorial analysis and kinetic investigation‖, Journal of Food Engineering 108(1): 238-243.
Kam, K., Arcot, J., & Ward, R., (2012), ―Fortification of rice with folic acid using parboiling
technique: Effect of parboiling conditions on nutrient uptake and physical characteristics of
milled rice‖, Journal of Cereal Science. 56:587-594.
Kam, K., & Arcot J., (2013), ―Folate fortification of rice through parboiling: optimisation, rice
quality, consumer acceptance and in-vitro relative bioaccessibility & absorption using caco-2
cells‖, PhD, The University of New South Wales, Sydney.
Kassenbeck P., (1975), ―Eleckronenmikroskopisher Beitrag zur Kenntnis der Feinstruktur der
Weizenstarke‖, Starch 27: 217-227.
Katopo, H., Song, Y., & Jane, J. L. (2002), ―Effect and mechanism of ultrahigh hydrostatic
pressure on the structure and properties of starches‖, Carbohydrate Polymers, 47(3): 233–244.
Katz, J.R., (1928). ―The X-ray spectrography of starch‖, in: Walton, R.P. (Ed.),A
Comprehensive Survey of Starch Chemistry, vol. I. Reinhold, New York, NY, 68–76
Kauwell GP, Wilsky, CE, Cerda, JJ, Herrlinger-Garcia, K, Hutson, AD, Theriaque, DW, Boddie
A, Rampersaud, G.C., Bailey, L.B., (2000) ,―Methylenetetrahydrofolate reductase mutation
(677C-->T) negatively influences plasma homocysteine response to marginal folate intake in
elderly women‖, Metabolism, 49(11):1440-1443.
Kenny, G. (2001). ―Nutrient impact assessment of rice in major rice consuming
countries‖. FAO-ESNA Consultancy Report.
Key findings: National Family Health Survey (NHFS-3) (2005-2006), Ministry of Health and
Family Welfare Government of India, International Institute for Population Sciences, Mumbai,
India. Accessed on 12th June 2012; available at
http://www.measuredhs.com/pubs/pdf/SR128/SR128.pdf
Kimura K, Ida M, Yoshida Y, Ohki K, Fukumoto T, Sakui N (1994), ―Comparison of keeping
quality between pressure-processed and heat-processed jam: changes in flavor components, hue
and nutritional elements during storage‖ Bioscience Biotechnology. Biochemistry 58:1386-1391.
Knorr D., (1993), ―Effect of high-hydrostatic pressure process on food safety and quality‖ Food
Technology 47(6): 156-161.
Knutson, C. A., (1990). ―Annealing of maize starches at elevated temperatures‖, Cereal
Chemistry 67(4): 376-384.
203
Kyritsi, A., Tzia, C. &Karathanos, V.T., (2010), ―Vitamin fortified rice grain using spraying and
soaking methods‖, LWT - Food Science and Technology, 44:312-320.
Lachance, P. A. and Bauernfeind, J.C. (1991).―Concepts and practices of nitrifying foods‖.In
Nutrient Additions to Food, ed. J. C. Bauernfeind and P. A. Lachance.Food and Nutrition Press,
Connecticut.
Lamb, J., Loy, T.H., (2005), ―Seeing red: the use of Congo Red dye to identify cooked and
damaged starch grains in archaeological residues‖, Journal of Archaeological. Science. 32:1433–
1440.
Lamberts, L. &Delcour, J. A.,(2008), ―Carotenoids in Raw and Parboiled Brown and Milled
Rice‖, Journal of Agriculture and. Food Chemistry., 56 (24):11914-11919
Lamberts, L., Brijs, K., Mohamed, R., Verhelst, N. and Delcour, J. (2006a)."Impact of browning
reactions and bran pigments on colour of parboiled rice."Journal of Agricultural and Food
Chemistry.54: 9924-9929.
Lamberts, L., De Bie, E., Derycke, V., Veraverbeke, W., De Man, W. and Delcour, J. (2006b).
"Effect of processing conditions on colour change of brown and milled parboiled rice."Cereal
Chemistry.83(1): 80-85.
Lamberts, L., Rombouts, I., Brijs, K., Gebruers, K. and Delcour, J. A. (2008)."Impact of
parboiling conditions on Maillard precursors and indicators in long-grain rice cultivars."Food
Chemistry.110(4): 916-922.
Layrisse M. (1976), ―Nutritional anaemia: Nutrition in Preventive Medicine- The Major
Deficiency Syndromes, Epidemiology, and Approaches to Control‖ in Bengoa J. M.&. Beaton G.
H, Geneva World Health Organisation 55-82.
Layrisse, M., Garcia-Casal, M.N., Solano, L., Baron, M.A., Arguello, F., Llovera, D., Ramirez,
J., Leets, I., & Tropper, E. (1997), ―The role of vitamin A on the inhibitors of nonheme iron
absorption: preliminary results‖, The Journal of Nutritional Biochemistry, 8(2):61–7.
Lee M.H., Hettiarachchy N.S., McNew R.W., Gnanasambandam R., (1995), Physicochemical
Properties of Calcium-Fortified rice, Cereal Chemistry, 72(4): 352-355.
Lee, W. G.; Ammerman, G. A. (1974) ―Carotene stereoisomerization in sweet potatoes as
affected by rotating and still retort canning processes‖ Journal of Food Science,39, 1188-1190.
Li, W., Y. Bai, S. A. S. Mousaa, Q. Zhang and Q. Shen (2011), "Effect of High Hydrostatic
Pressure on Physicochemical and Structural Properties of Rice Starch", Food and Bioprocess
Technology, 5(6): 2233-2241.
Li, Y. O., L. L. Diosady and S. Jankowski (2011). "Folic acid stability in the presence of various
formulation components including iron compounds in fortified extruded Ultra Rice® over
prolonged storage at 40 C and 60% relative humidity (RH)." International Journal of Food
Science & Technology, 46(2): 379-385.
204
Louis,& Siegel. (2011). ―Cell viability analysis using trypan blue: manual and automated
methods‖ Methods in molecular biology(Clifton, N.J.), 740, 7-12.
Lu, S., Chen, L., and Lii, E., (1997). ―Correlations between the fine structure, physicochemical
properties, and retrogradation of amylopectins from Taiwan rice varieties‖. Cereal Chemistry.
74: 34-39.
Lund, D. (1984). "Influence of time, temperature, moisture, ingredients, and processing
conditions on starch gelatinization." Critical Reviews in Food Science and Nutrition .,20(4): 249-
273.
Lyon B.G., C. E. T., Vinyard B.T., Windham W.R., Barton F.E., Webb B.D., McClung A.M.,
Moldenhauer K.A., Linscombe S., McKenzie K.S. and Kohlwey D.E. (1999). "Drying
Condition, and Final Moisture Content on Sensory Texture of Cooked Rice." Cereal Chemistry
76(1): 56-62.
MacPhail, P. (2007). Iron.In Mann, J. &Truswell, A. S. (Eds.), Essentials of human nutrition (pp.
125-137). United States: Oxford University Press.
Malinow, M. R., Duell, P. B., Hess, D. L., Anderson, P. H., Kruger, W. D., Phillipson, B. E.,
Gluckman, R. A., Block, P. C. & Upson, B. M. (1998) ―Reduction of plasma homocysteine
levels by breakfast cereal fortified with folic acid in patients with coronary heart disease‖,The
New England. Journal of. Medicine, 38:1009-1015.
Maruyama, E., Sakamoto, K.,(1992). ―A basic study on cooking of rice (Part 1): the influence of
rice soaking by hot water‖, Journal of Home Economics of Japan 43:97–103 (in Japanese with
English abstract)
Mason J.B., Mahshid L., Dalmiya N., Sethuraman K., and Deitcheler M., (2001), ―The
Micronutrient Report Current Progress and Trends in the Control of Vitamin A, Iodine, and Iron
Deficiencies‖, Micronutrient Initiative & International Development Research Centre, Ottawa,
Canada.
McGee, H., (1984), ―On Food and Cooking: The Science and Lore of the Kitchen‖, Charles
Scribner‘s Sons, New York.
McLaren, K. (1976). "XIII—The Development of the CIE 1976 (L* a* b*) Uniform Colour
Space and Colour-difference Formula."Journal of the Society of Dyers and Colourists92(9): 338-
341.
Meng, F., Wei, Y., & Yang, X. (2005), ―Iron content and bioavailability in rice‖, Journal of
Trace Elements in Medicine and Biology, 18:333–338.
Mertens, B. A., (1993), ―Developments in high pressure food processing‖, I. Z. Lebensm,
44:100-104
Meullenet, J., Xiong, R. and Findlay, C., Eds. (2007). Multivariate and probabilistic analyses of
sensory science problems.Analysis of Just About Right data U.S.A, Blackwell Publishing.
205
Miah, M.A.K., Haque, A., Douglass, M.P., Clarke, B., (2002), ―Parboiling of rice, Part I: effect
of hot soaking time on quality of milled rice‖, International Journal of Food Science and
Technology, 37:527–537
Micronutrient Initiative and UNICEF.(2011). "Vitamin&Mineral deficiency- A damage
assessment report for Bangladesh."Retrieved 22/02/2012, from:
http://www.micronutrient.org/vmd/CountryFiles/BangladeshDAR.pdf.
Misaki, M., and Yasumatsu, K.(1985), ―Rice enrichment and fortification. In: Rice: Chemistry
and Technology‖, 2nd ed. Am. Assoc.Cereal Chem.: St. Paul, MN.
Mohamed, A., S. C. Peterson, L. A. Grant and P. Rayas-Duarte (2006), "Effect of jet-cooked
wheat gluten/lecithin blends on maize and rice starch retrogradation.", Journal of Cereal Science,
43(3): 293-300.
Mohoricˇ , A., Vergeldt, F., Gerkema, E., de Jagar, A., van Duynhoven, J.,van Dalen, G., van As,
H., (2004), ―Magnetic resonance imaging of singlerice kernels during cooking‖, Journal of
Magnetic Resonance, 171:157–162
Molina-Cano, J.-L., Sopena, A., Polo, J.P., Bergareche, C., Moralejo, M.A., Swanston, J.S.,
Glidewell, S.M., (2002), ―Relationships between barley hordeins and malting quality in a mutant
of cv. Triumph, II: genetic and environmental effects on water uptake‖, Journal of Cereal
Science, 36:39–50.
Moritteo, D., T.-C.Lee, M. B. Zimmermann, J. Nuessli and R. F. Hurrell (2005)."Development
and Evaluation of Iron-fortified Extruded Rice Grains." Journal of Food Science 70(5): S330-
S336.
Morrison W.R, Tester R.F., Snape C.E., Law R., Gidley M.J. (1993) “Swelling and
gelatinisation of cereal starches IV. Some effects of lipid-complexed amylose and free amylose
in waxy and normal barley starches‖ Cereal Chemistry, 70, pp. 385–391.
Moskowitz, H. (2004). Hedonics, Just-about-Right, purchase and other scales in consumer tests.
H. Moskowitz, A. Lunoz and M. Gacula (ed). In: Viewpoints and controversies in sensory
science and consumer product testing John Wiley & Sons pp: 145-172.
MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical
Research Council Vitamin Study (1991). Lancet, 338, 131–137
Muhr, A. H., Wetton, R. E., & Blanshard, J. M. V. (1982) . ―Effect of .hydrostatic pressure on
starch gelatinisation, as determined by DTA‖ Carbohydrate Polymers 2: 91-102.
Mulia C.V., and Arcot J., (2010) ―Iron fortification of rice through parboiling‖ Honours thesis-
Food Science and Technolgoy, UNSW, Australia.
Nasehi and Javaheri, (2012) ―Application of high hydrostatic pressure in modifying functional
properties of starches: a Review‖ Middle-East Journal of Scientific. Research 11: 856–861
206
National Health and Medical Research Council (NHMRC) (2006). Nutrient Reference Values for
Australia and New Zealand Including Recommended Dietary Intake, Australia: Commonwealth
of Australia.
Nawab Ali and Pandya A. C, (1974),―Basic concept of parboiling of paddy‖, Journal of
Agricultural Engineering research, 19:111-115.
Netzel M., Netzel G., et al. (2011). "Release and absorption of carotenes from processed carrots
(Daucus carota) using in vitro digestion coupled with a Caco-2 cell trans-well culture model."
Food Research International, vol. 44, pp-868-874.
NFHS (2000).―National Family Health Survey-2 (NFHS-2).Mortality, Morbidity and
Immunization-India 1998-1999‖, International Institute for Population Sciences, Mumbai.
Nguyen M.T., Indrawati & Hendrickx M., (2003). "Model Studies on the Stability of Folic Acid
and 5-Methyltetrahydrofolic acid degradation during Thermal in Combination with High
Hydrostatic Pressure." Journal of Agricultural and Food Chemistry 51: 3352-3357.
Nikuni.Z., (1957), ―Proposed model of a starch granule or a starch molecule‖, Chori Kaguku
(Japan).
Nutrient reference values for Australia and New Zealand including recommended dietary
intakes, 2006, National Health and Medical Research Council
<http://www.nhmrc.gov.au/_files_nhmrc/publications/attachments/n35.pdf>, accessed on 16th
April, 2014.
O‘Broin, J.D.; Temperley, I.J.; Brown, J.P.; Scott, J.M. (1975).Nutritional stability of various
naturallyoccurring monoglutamate derivatives of folic acid. American Journal of Clinical
Nutrition 28, 438–444
O‘Callaghan, Y. and N. O‘Brien (2010). "Bioaccessibility, cellular uptake and transepithelial
transport of α-tocopherol and retinol from a range of supplemented foodstuffs assessed using the
caco-2 cell model." International Journal of Food Science & Technology45(7): 1436-1442.
Oh, H. E., Y. Hemar, S. G. Anema, M. Wong and D. Neil Pinder (2008). "Effect of high-
pressure treatment on normal rice and waxy rice starch-in-water suspensions."Carbohydrate
Polymers 73(2): 332-343.
Öhrvik, V., H. Öhrvik, J. Tallkvist and C. Witthöft (2010). "Folates in bread: retention during
bread-making and in vitro bioaccessibility." European Journal of Nutrition49(6): 365-372.
Öner, N (2006) ―The prevalence of folic acid deficiency among adolescent girls living in Edirne,
Turkey‖, Journal of Adolescent Health 38(5): 599-606.
Ong, D. E.,(1994), Absorption of Vitamin A, Vitamin A in Health and Disease R. Blomhoff.
New York Marcel Dekker, Inc.,37-72.
207
Ong, M.H., Blanshard, J.M.V., (1995), ―Texture determinants of cooked, parboiled rice. II.
Physicochemical properties and leaching behaviour of rice‖, Journal of Cereal Science 21:261–
269.
Oostergetel, G. T. and van Bruggen, E. F. J., (1989) ―On the origin of a low angle spacing in
starch‖, Starch, 41:331-33s.
Osseyi, E.S., Wehling, R.L. &Albrecht, J.A. (1998). ―Liquid chromatographic method for
determining added folic acid in fortified cereal products‖, Journal of Chromatography A,
826:235–240.
Pachón, H., (2013). "Folic acid fortification of wheat flour: A cost-effective public health
intervention to prevent birth defects in Europe." Nutrition Bulletin 38(2): 201-209
Park, T.M., Ibanez, A.M., Zhong, F. and Shoemaker, C.F. (2007). ―Gelatinization and pasting
properties of waxy and non-waxy rice starches‖, Starch 59:388-396
Parnsakhorn S. & Noomhorm A., (2008), ―Changes in Physicochemical Properties of Parboiled
Brown Rice during Heat Treatment‖.Agricultural Engineering International: the CIGR.,10:1-20.
PATH (2013).Ultra Rice ® Technology. Retrieved October 22 2013, from
http://www.path.org/publications/files/TS_update_ultra_rice.pdf
PATH: A catalyst for global health 2008, PATH‘s ultra-rice project, brochure, PATH,
Washington, USA.
Patindol, J., Gonzalez, B., Wang, Y.-J., and McClung, A., (2007), ―Starch fine structure and
physicochemical‖ properties of specialty rice for canning, Journal of Cereal Science, 45:209-218.
Pedersen, B. & Eggum, B.O. 1983.The influence of milling on the nutritive value of flour from
cereal grains. IV. Rice. Qualitas Plantarum Plant Foods Human Nutrition., 33: 267-278.
Pei-Ling, L., (2012). "Effect of high hydrostatic pressure on modified non-crystalline granular
starch of starches with different granular type and amylose content." LWT - Food Science and
Technology, 47(2): 450-458.
Pfeiffer C, Rogers L & Gregory J (1997), ―Determination of folate in cereal-grain food products
using trienzyme extraction and combined affinity and reversed-phase liquid chromatography‖,
Journal of Agricultural and Food Chemistry, 45: 407-413
Pfeiffer, C. M., Rogers, L. M., Bailey, L. B. & Gregory, J. F. (1997), ―Absorption of folate from
fortified cereal-grain products and of supplemental folate consumed with or without food
determined using a dual-label stable-isotope protocol‖, American. Journal of.Clinical.
Nutrition.,66:1388-1397.
Pillaiyar, P. and Mohandas, R. (1981). "Hardness and color in parboiled rice produced at low and
high temperature." Journal of Food Science and Technology.18: 7-9.
Pillaiyar, P., (1990), ―Rice Parboiling Research in India‖. Cereal Foods World, 35: 225-57.
Pillaiyar, P., Mohandoss, R., (1981b). ―Cooking qualities of parboiled rices produced at low and
high temperatures‖, Journal of the Science of Food and Agriculture, 32: 475–480.
208
Pillaiyar, P., Sabarathinam, P. L., Subramaniyan, V. and Sulochana, S. (1996)."Parboiling of
paddy using thermic fluid."Journal of Food Engineering.27(3): 267-278.
Pinto M., Appay M.D., Simon-assmann P., Chevalier G., Dracopoli N., Fogh J. and Zweibaum
A., (1982), ―Enterocytic differentiation of cultured human colon cancer cells by replacement of
glucose by galactose in the medium‖, Biology of the. Cell., 44:193-196.
Piperno, D.R., (2006), ―Phytoliths: A Comprehensive Guide for Archaeologists and
Paleoecologists‖, in AltaMira Press, Lanham, MD.
Pizarro F., Olivares M., Hertrampf E., Nunez S., Tapia M., Cori H. And de Romana D.L.,
(1988). ―Ascorbyl palmitate enhances bioavailability in iron fortified bread‖, American Journal
of Clinical Nutrition 84:830-834.
Planning Department: Govt. Of Uttar Pradesh, 2008, Modern project on Improved Rice Mill,
available at <http://planning.up.nic.in/innovations/inno3/ae/rice.htm> accessed on 15th March,
2011. Press, Loughborough
Popper, R. and Gibes, K. (2004). "Workshop summary: Data analysis workshop: getting the
most out of just-about-right data - Abstracts." Food Quality and Preference.15(7-8): 891-899.
Poulsen, J. (1999). "Danish consumers‘ attitudes towards functional foods." MAPP Working
paper 62. The Aarhus School of Business, Aarhus.
Priestley, R.J., (1977). Studies on parboiled rice.Part 3.Characteristics of parboiled rice on
recooking. Food Chemistry 2, 43–50
Prom-u-thai C., P. Glahn R., Cheng Z., Fukai S, Rerkasem B, Huang L., (2009), ―The
bioavailability of iron fortified in whole grain parboiled rice‖, Journal of Food Chemistry,
112:982-986.
Prom-u-thai C., and Benjavan R., İsmail Ç., and Longbin H., (2009), ―Zinc fortification of whole
rice grain through parboiling process, Food Chemistry‖ 120 (3):858-863.
Prom-u-thai C., Huang L.,Cakmak I,. Rerkasem B. ,(2011), ―Simultaneous fortification of iron
and zinc in parboiled rice kernel‖, Science Asia 37 296-302
Prom-u-thai CP Glahn R, Cheng Z, Fukai S, Rerkasem B, Huang L, (2008),―The bioavailability
of iron fortified in whole grain parboiled rice‖. Journal of Food Chemistry, 112: 982-986.
Promuthai, C., et al. (2008). "Iron-fortified parboiled rice – A novel solution to high iron density
in rice-based diets." Food Chemistry 110(2): 390-398.
Prom-u-thai, C., et al. (2010). "Zinc fortification of whole rice grain through parboiling
process.",Food Chemistry 120(3): 858-863.
Prom-u-thai, C., Fukai, S. and Godwin, I., (2008). ―Iron-fortified parboiled rice-a novel solution
to high iron density in rice-based diets‖, Food Chemistry.110:390-398.
Prom-u-thai, C., Glahn, R., Cheng, Z. and Fukai, S. (2009a). ―The bioavailability of iron fortified
in whole grain parboiled rice‖. Food Chemistry. 112: 982-986.
209
Prom-u-thai, C., Rerkasem, B., Fukai, S. and Huang, L. (2009b). ―Iron fortification and parboiled
rice quality: appearance, cooking quality and sensory attributes‖. Journal of Science and Food
Agriculture. 89: 2565-2571.
Prom-u-thai, C., Rerkasem, B., Fukai, S. and Huang, L. (2010).―Key factors affecting Fe density
in Fe-fortified-parboiled rice: Parboiling conditions, storage duration, external Fe-loading rate
and genotypic differences‖ Food Chemistry. 123:628–634
Ragheb, A. A., I. Abd El-Thalouth and S. Tawfik (1996). "Gelatinization of Thermally Treated
Starch Mixed with Sodium Hydroxide in the Solid State." Starch - Stärke48(2): 57-64.
Ramaswamy (2006), High Pressure Processing Fact Sheet for Food Processors, <
http://ohioline.osu.edu/fse-fact/pdf/0001.pdf> accessed on March 25th 2011.
Ranum P., (2000), ―Fortification of high extraction wheat flour”Cereal Foods World, 45 (6) pp.
267–268
Rao B.S.N.,(1981), ―Physiology of iron absorption and supplementation‖, British Medical
Bulletin, 37(1): 25-30.
Rao, B.S.N. and Juliano, B.O., (1970a) ―Effect of parboiling on some physicochemical
properties of rice‖. Journal of Agricultural and Food Chemistry, 18: 289-294.
Rao, P. B. S. and Bhattacharya, K. (1966)."Effect of parboiling on thiamine content of
rice."Journal of Agricultural and food chemistry.14(5): 479-482.
Rastogi (2013), ecent Developments in High pressure processing of Foods In: Springer Chapter 2
High-Pressure Processing of Plant Products pp (1-50).
Ray, J.G.; Singh, G.; Burrows, R.F. Evidence for suboptimal use of periconceptional folic
acidsupplements globally. (2004) BJOG, 111, 399–408.
Recommended Dietary Allowances and Adequate Intakes, Vitamins &Minerals, The National
Acacdemies Press
<http://iom.edu/Activities/Nutrition/SummaryDRIs/~/media/Files/Activity%20Files/Nutrition/D
RIs/RDA%20and%20AIs_Vitamin%20and%20Elements.pdf> last accessed on 4/4/2012.
Reddy, R.K., Ali, Z.A., & Bhattacharya, K.R. (1993).―The fine structure of rice-starch
amylopectin and its relation to the texture of cooked rice‖.Carbohydrate Polymers 22:267–276.
Report of a Joint FAO/WHO Expert Consultation, 1988, Requirements of Vitamin A, Iron,
Folate and Vitamin B12 Rome Food and Agricultural Organization of the United States
Rodriguez-Amaya, D. B. (2010)."Quantitative analysis, in vitro assessment of bioavailability and
antioxidant activity of food carotenoids—A review."Journal of Food Composition and
Analysis23(7): 726-740.
Roy, P., N. Shimizu, T. Shiina and T. Kimura (2006). "Energy consumption and cost analysis of
local parboiling processes." Journal of Food Engineering76(4): 646-655.
210
Said (2011).Intestinal absorption of water-soluble vitamins in health and disease.Biochemical
Journal, 437 (3): 357-372.
Sampedro, F., A. McAloon, W. Yee, X. Fan and D. J. Geveke (2014). "Cost Analysis and
Environmental Impact of Pulsed Electric Fields and High Pressure Processing in Comparison
with Thermal Pasteurization." Food and Bioprocess Technology7(7): 1928-1937.
Samuel, D., (2000). ―Brewing and baking‖. In: Nicholson, P.T., Shaw, I. (Eds.), Ancient
Egyptian Materials and Technology. Cambridge University Press, Cambridge 537–576.
Sandström, B. (2001). "Micronutrient interactions: effects on absorption and bioavailability."
British Journal of Nutrition 85(Suppl. 2): S181-S185.
Satterfield, C.N. 1970. ―Diffusion and reaction in porous catalysis, Mass Transfer in
Heterogeneous Catalysis‖.MIT Press. Cambridge
Saunders, R. M. (1990a) ―Stabilized rice bran: a new world food resource‖. Newsletter /
International Rice Commission, 39(1):179-183,
Sayed A.R., Bourne D., Pattinson R., Nixon J., Henderson B. (2008).Decline in the prevalence of
neural tube defects following folic acid fortification and its cost-benefit in South Africa. Birth
Defects Res. A Clin. Mol. Teratol. ;82:211–216.
Scholl T.O.,Johnson W. G., (2000) ―Folic acid: influence on the outcome of pregnancy‖
American Journal of Clinical Nutrition 71(5 Suppl):1295S-303S.
Seki, C., Kainuma, Y., (1982). ―A study of rice cooking (Part 2): Soaking time as a factor
controlling rice cooking‖. Journal of Home Economics of Japan 33:228–234.
Selhub, J.; Jacques, P.F.; Dallal, G.; Choumenkovitch, S.; Rogers, G. (2008) ―The use of
bloodconcentrations of vitamins and their respective functional indicators to define folate and
vitamin B12 status‖ Food and Nutrition Bulletin., 29, S67–S73.
Silva, M. A.; Sanches, C.; Amante, E. R. (2006) ―Prevention of hydrolytic rancidity in rice
bran‖ Journal of Food Engineering, 75(4): 487-491.
Simon-Assmann, P., Turck, N., Sidhoum-Jenny, M., Gradwohl, G., & Kedinger, M. (2007).―In
vitro models of intestinal epithelial cell differentiation‖, Cell Biology and Toxicology, 23(4),
241–256.
Singh P (2007),―Micronutrient deficiency in India‖, Journal of Indian Society of Agricultural
Statistics 6 (2):128-131.
Singh, N., Singh, J., Kaur, L., Sodhi, N.S. and Gill, B.S.( 2003). ―Morphological, thermal and
rheological properties of starches from different botanical sources‖, Food Chemistry 81: 219-231
Singh, S., Raina, C. S., Bawa, A. S., and Saxena, D. C. (2005).―Effect of heat-moisture treatment
and acid modification on rheological, textural, anddifferential scanning calorimetry
characteristics of sweetpotato starch‖ .Journal of.Food Science. 70:E373-E378.
211
Singh, V., Okadome, H., Toyoshima, H., Isobe, S. and Ohtsubo, K. (2000). Thermal and
physicochemical
Siro, I., Kapolna, E., Kapolna, B. and Lugasi, A. (2008)."Functional food.Product development,
marketing and consumer acceptance-A review."Appetite. 51(3): 456-467.
Skoog, D.A., and West, D.M., (1980) ―Principles of Instrumental analysis‖, 2nd Edition,
Saunders college, Philadelphia, PA, 427-457.
Slavin, J. L. &Lampe, J. W. (1992) ―Health benefits of rice bran in human nutrition‖ Cereal
Foods World, 37 (10):760-763.
Song, H.P., Delwiche, S.R., Line, M.J., (1998). Moisture distribution in a mature soft wheat
grain by three-dimensional magnetic resonance imaging.Journal of Cereal Science 27:191–197.
Song, H.P., J.B. Litchfield and H.D. Morris.(1992). ―Three dimensional microscopic MRI of
maize kernels during drying‖.Journal of Agricultural Engineering Research 53(1):51-69.
Stark, J. R., Yin, X. S., (1986), ―The effect of physical damage on large and small barley starch
granules‖, Starch, 38:369–374.
Stein, A. J., Sachdev, H. P. S. and Qaim, M. (2008). "Genetic engineering for the poor: Golden
Rice and public health in India." World Development.36(1): 144-158.
Stoddart (2011). ―Methods in molecular biology Cell viability assays: introduction‖. Methods in
molecular biology.(Clifton, N.J.).
Stolt, M., Oinonen, S., & Autio, K. (2001). ―Effect of high pressure on the physical properties of
barley starch‖, Innovative Food Science and Emerging Technology 1(3):167-175.
Stringer, H. (2000).―The surprising history of cake‖. The peerless cake bakers.
Stute, R., Heilbronn, R. W. Klingler, S. Boguslawski, M. N. Eshtiaghi and D. Knorr
(1996)."Effects of High Pressures Treatment on Starches."Starch - Stärke48(11-12): 399-408.
Subba Rao, P. B. and Bhattacharya K. R., (1966)."Effect of Parboiling on Thiamine Content of
Rice."Journal of Agricultural and Food Chemistry14(5): 479-482.
Sy, C., B. Gleize, O. Dangles, J.-F. Landrier, C. C. Veyrat and P. Borel (2012). "Effects of
physicochemical properties of carotenoids on their bioaccessibility, intestinal cell uptake, and
blood and tissue concentrations." Molecular Nutrition & Food Research56(9): 1385-1397.
Synthesis of Heme 2008, Rensselaer Polytechnic Institute, available at
<http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/heme.htm> accessed on 25th
July 2011.
Tamura T. (1998), ―Determination of food folate‖, The Journal of Nutritional Biochemistry
9(5):285-293.
Tavelin.(2002). Applications of Epithelial Cell Culture in Studies of Drug Transport. C. Wise,
Epithelial Cell Culture Protocols. 10.1385/159259185X.
212
Tester R. & Morrsion (1990), ―Swelling and Gelatinization of Cereal Starcher II, Waxy Rice
starches‖, American Association of Cereal Chemists 67(6): 558-563.
Thakkar, S. K., B. Maziya-Dixon, A. G. Dixon and M. L. Failla (2007). "Beta-carotene
micellarization during in vitro digestion and uptake by Caco-2 cells is directly proportional to
beta-carotene content in different genotypes of cassava." Journal of Nutrition 137(10): 2229-
2233.
Thakur, A. K. and A. K. Gupta (2006)."Water absorption characteristics of paddy, brown rice
and husk during soaking."Journal of Food Engineering 75(2): 252-257.
Tomlins, K. I., Manful, J. T., Larwer, P. and Hammond, L. (2005). "Urban consumer perferences
and sensory evaluation of locally produced and imported rice in West Africa." Food Quality and
Preference. 16: 79-89
Tomlins, K., Manful, J., Gayin, J., Kudjawu, B. and Tamakloe, I. (2007). "Study of sensory
evaluation, consumer acceptability, affordability and market price of rice." Journal of the Science
Food and Agriculture, 87: 1567-1575.
Torrence, R., Barton, H., eds. (2006). Ancient Starch Research.Left Coast Press, Walnut Creek,
CA.
Torrezan, R., Thamb, W. P., Bell, A. E., Frazier, R. A., & Cristianini, M. (2007). ―Effects of
high pressure on functional properties of soy protein‖ Food Chemistry, 104:140-147.
Truswell, S. (2007).The B vitamins.In Mann, J. &Truswell, A. S. (Eds.), Essentials of human
nutrition (pp. 184-200). United States: Oxford University Press.
Tsai M.L., Li C.F. and Lii C.Y. (1997) ―Effect of Granular Structure on the Pasting Behaviour of
Starches‖, Cereal Chemistry 74(6): 750-757.
Tulyathan, V., Laokuldilok, T. and Jongkaewwattana, S. (2007). "Retention of iodine in fortified
parboiled rice and its pasting characteristics during storage." Journal of Food Biochemistry.31:
217-229.
U.S Environmental Protection Agency (1996).Acid Digestion of Sediments, Sludges, and Soils,
retrieved October 15 2013, from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3050b.pdfU.S. Food and Drug
Administration.( June 20, 2014). Guidance for Industry: A Food Labeling Guide (14. Appendix
F: Calculate the Percent Daily Value for the Appropriate Nutrients) retived from
http://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/labelingn
utrition/ucm064928.htm on> 13th August 2014.
UNICEF (2009).The state of the world‘s children. Retrieved October 13 2013, from
http://www.unicef.org/rightsite/sowc/pdfs/statistics/SOWC_Spec_Ed_CRC_Statistical_Tables_E
N_111809.pdf
United Nations International Children's Emergency Fund (UNICEF) (2000). Multiple Indicator
Cluster Survey Estimated prevalence of VAD, UNICEF, India.
213
United States Environmental Protection Act (1996) ACID DIGESTION OF SEDIMENTS,
SLUDGES, AND SOILS Method 3050B
USAID (2008). Rice Fortification in Developing Countries: A Critical Review of the Technical
and Economic Feasibility accessed on February 24 2014< http://www.spring-
nutrition.org/sites/default/files/a2z_materials/508-food-rice-fortification-report-with-annexes-
final.pdf >
USDA (2013). Global rice production and consumption (1960-2012) accessed on February 4
2014 <http://www.oryza.com/content/rice-yield-must-grow-2-annually-meet-rising-global-
demand-says-irri-chief>
van Stuijvenberg M. E., Dhansay M.A., Smuts, C.M., Lombard, C.J., Jogessar, B. &Benade,
A.J.S. (2001). ―Long-term evaluation of a micronutrient-fortified biscuit used for addressing
micronutrient deficiencies in primary school children‖, Public Health Nutrition, 4:1201–1209.
vanCampen,D.R.,andGlahn,R.P. (1999). ―Micronutrientbioavailabil-
itytechniques:accuracy,problems and limitations‖, FieldCropsRes. 60, 93–113.
VanHurley, V. L. (2007). The influence of packaing color on consumer purchase intent: The
influence of color at the point of purchase, Michigan State University.
Vasconcelos M et al. (2003). ―Enhanced iron and zinc accumulation in transgenic rice with the
ferritin gene‖, Plant Sci. 64 (3): 371-378.
Vermeylen, R., Goderis, B., and Delcour, J. A. (2006) ― X-ray study of hydrothermally treated
potato starch‖. Carbohydr.Polym. 64:364-375.
Verwei, M., H. van den Berg, R. Havenaar and J. P. Groten (2005). "Effect of folate–binding
protein on intestinal transport of folic acid and 5–methyltetrahydrofolate across Caco–2
cells."European Journal of Nutrition44(4): 242-249.
Wang, X.-S., C.-H.Tang, B.-S.Li, X.-Q.Yang, L. Li and C.-Y.Ma (2008)."Effects of high-
pressure treatment on some physicochemical and functional properties of soy protein
isolates."Food Hydrocolloids22(4): 560-567.
Washington University (2000), Iron Use and Storage in the Body:
Ferritin and Molecular Representations in Iron in Biology: Study of the Iron Content in Ferritin,
The Iron-Storage Protein
<http://www.chemistry.wustl.edu/~edudev/LabTutorials/Ferritin/Ferritin.html> accessed on 28th
October 2013.
Watanabe M, Aria E, Kumeno K, Homma K (1991).‖A new method for producing non-heated
jam sample: the use of freeze concentration and high pressure sterilization‖, The Journal of
Biological Chemistry 55: 2175-2176
Watson & Sikeman (1977) , ―Structure of the rice grain by scanning electron microscope‖,
American Association of Cereal Chemists 54 (I): 120-130.
214
Wesley A & Dutta S 2009, ―Update on Wheat Flour Fortification in India‖ Flour Fortification
Initiative Country Information: India, United States.
West, KP (2002). ―Extent of Vitamin D Deficiency among preschool children and women of
reproductive age.‖Journal of Nutrition 132: 2857S-2866S
WHO (2009).Weekly iron-folic acid supplementation (WIFS) in women of reproductive age: its
role in promoting optimal maternal and child health, retrieved October 5 2013, from
<http://www.who.int/nutrition/publications/micronutrients/weekly_iron_folicacid.pdf>
WHO/UNICEF (1996).Indicators of VAD and their use in monitoring intervention
programmes.WHO/NUT/96 (10), 66. World Health Organization, Geneva.
Wimberly, J.E. (1983). ―Parboiling‖ In: Technical Hand Book for the Paddy Rice Post Harvest
Industry in Developing Countries, 101-116.
Wood, R. J.; Tamura, T. (2001) Methodological issues in assessing bioavialability of nutrients
and other bioactive substances in dietary supplements: summary of workshop discussion. J.
Nutr., 131:1396S-1398S.
World Health Organization & Centers for Disease Control and Prevention (WHO/CDC)
(2008).Worldwide prevalence of anaemia 1993- 2005.WHO Global Database on Anaemia.
Geneva: World Health Organization.
World Health Organization (WHO) (2009a). Global health risks: mortality and burden of disease
attributable to selected major risks. Geneva: World Health Organization.
World Health Organization (WHO) (2002). The World Health Report 2002: reducing risks,
promoting healthy life: overview. Geneva, World Health Organization.
World Health Organization (WHO) (2004).Focusing on Anaemia: Towards an integrated
approach for effective anaemia control, retrieved October 5 2013, from
<http://www.who.int/nutrition/publications/micronutrients/WHOandUNICEF_statement_anaemi
a_en.pdf>
World Health Organization (WHO) (2007). Preventing and controlling micronutrient
deficiencies in populations affected by an emergency: Multiple vitamin and mineral supplements
for pregnant and lactating women, and for children aged 6 to 59 months, retrieved October 5
2013, from
<http://www.who.int/nutrition/publications/micronutrients/WHO_WFP_UNICEFstatement.pdf>
World Health Organization (WHO) (2009).Global prevalence of vitamin A deficiency in
populations at risk 1995–2005. WHO Global Database on Vitamin A Deficiency. Geneva:
World Health Organization, 2001 (WHO/NHD/01.3) Iron deficiency anaemia: assessment,
prevention, and control. A guide for programme managers. Geneva.
Yaldagard M, Mortazavi S A, Tabatabaie F (2008), ―The principles of ultra-high pressure
technology and its application in food processing/preservation: A review of microbiological and
quality aspects‖, African Journal of Biotechnology, 7 (16): 2739-2767.
215
Yamakura M., et al. (2005). "Effects of Soaking and High-Pressure Treatment on the Qualities
of Cooked Rice."Journal of Applied Glycosciences 52: 85-93.
Yamamoto, K. and E. Niki (1988). "Interaction of α-tocopherol with iron: antioxidant and
prooxidant effects of α-tocopherol in the oxidation of lipids in aqueous dispersions in the
presence of iron." Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism958(1):
19-23.
Yamazaki A., & Sasagawa A., (1998) ―Development of rice food products processed by high
pressure treatment‖ ,Nippon Nogeikagaku Kaishi-Joural of the Japan Society for Bioscience
Biotechnology and Agrochemistry 74(5): 619-623.Agrochemistry 74(5): 619-623.
Yanase, H., Ohtsubo, K., (1986). Relation between rice milling methods and palatability of
cooked rice (Part 3): milling and cooking qualities of brown rice with a low moisture content.
Report of National Food Research Institute 49:1–5 (In Japanese with English abstract).
Yi, Y., Lindemann, M., Colligs, A. and Snowball, C. (2011). "Economic burden of neural tube
defects and impact of prevention with folic acid: a literature review." European Journal of
Pediatrics.170(11): 1391-1400.
Yifan T, Yongzhong X, Qifa Z, Mei S & Harold C, (2001). "Quantitative Genetic basis of
Gelatinization Temperautre of Rice."Cereal Chemistry 78(6): 666-674.
Yu, S., Y. Ma and D.-W.Sun (2010). "Effects of freezing rates on starch retrogradation and
textural properties of cooked rice during storage." LWT - Food Science and Technology 43(7):
1138-1143.
Yuryev, V. P., Wasserman, L. A., Andreev, N. R., & Tolstoguzov, V. B.(2002). Structural and
thermodynamic features of low- and high amylose starches. A review.In V. P. Yuryev, A.
Cesaro, & W. J. Bergthaller (Eds.), Starch and starch containing origins: Structure, properties
and new technologies (pp. 23–55). New York: Nova Science Publishers, Inc.
Zittoun J (1993), ―Anemias due to disorder of folate, vitamin B12 and transcobalamin
metabolism‖, Rev Prat. 43:1358-63.
Zobel H.F., Young. S. N. and. Rocca. L. A. (1988). "Starch Gelatinization: An X-ray Diffraction
Study." American Journal of Cereal Chemistry 65(6): 443-446.
216
10. SUPPLEMENTARY SECTION
Appendix 1 Optimization of the volume of KOH added to rice for gelatinization study
Volume of KOH added (10 M) Absorbance for Uncooked
Rice
Absorbance for Cooked
Rice
0 0.063 0.063
0.25 0.309 0.403
0.5 0.532 0.836
0.75 0.433 0.714
1 0.398 0.759
1.25* 0.514 0.867*
1.5 0.51 0.73
1.75 0.584 0.699
2 0.507 0.744
2.25 0.559 0.703
2.5 0.57 0.718
2.75* 0.6* 0.716
3 0.576 0.739
3.25 0.521 0.668
Note: *Values highlighted were the optimized concentration of KOH used for optimization of
degree of gelatinization experiment.
217
Appendix 2 Questionnaire used in Study I of the consumer acceptance study
Part I- Demographic and rice consumption pattern questions
A Gender
B Age
C Nationality
D Employment Status
E How often do you consumer rice?
F What type of rice do you usually consume?
H Have you heard of parboiled rice previously?
Part II- Consumer acceptance study- Study 1 questionnaire
1. How would you describe the COLOUR? (In
TWO or THREE WORDS)
Open question
2. How INTENSE is the COLOUR of the
rice? (Circle one number only)
10- point scale
3. How much do you like the COLOUR of the
rice? (Circle one number only)
9- point scale
4. How UNIFORM is the colour of the rice
sample? (Circle one number only)
10- point scale
3. How much do you like the OVERALL
APPEARANCE of the rice? (Circle one
number only)
9- point scale
6. How WILLING are you to buy this rice?
(Circle one number only)
5- point likert
7. If you are told that the rice is fortified with
vitamins and minerals which gives higher
nutritional value, how WILLING are you to
buy this rice? (Circle one number only)
5- point likert
218
Appendix 3 Sensory analysis liking scale for intensity, Just about right (JAR) and purchase intent
questions
Liking (Studies 1 &2) Intensity (Study 1) JAR (Study 2) Purchase intent
(Study 1 &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
dislike
5 5 Much too intense 5 Definitely
would buy
6 Like moderately 6
7 Like 7
8 Like very much 8
9 Like extremely 9
10 Extremely 10 Too intense
219
Appendix 4 Questionnaire used in Study 2 (Demographics and consumers acceptance)
Part I – Demographic and consumers rice preference questions
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 Preference 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 this rice? Just- about right (JAR)
3. How much do you like the COLOUR of this rice? 9- point hedonic
4. How would you describe the COLOUR? (In TWO or THREE
WORDS eg: white, yellow, off-white etc.)
Open question
5. What do you think about the COLOUR INTENSITY of this
rice?
JAR
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 which
gives higher nutritional value, how WILLING are you to buy
this rice?
5-point likert
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