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DEVELOPMENT OF MICROENCAPSULATION-BASED

TECHNOLOGIES FOR MICRONUTRIENT

FORTIFICATION IN STAPLE FOODS

FOR DEVELOPING COUNTRIES

By

Yao Olive Li

A dissertation submitted in conformity with the requirements

for the degree of DOCTOR OF PHILOSOPHY

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

Copyright by Yao Olive Li 2009


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Development of Microencapsulation-based Technologies for Micronutrient

Fortification in Staple Foods for Developing Countries

Yao Olive Li

Doctor of Philosophy, 2009

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

ABSTRACT

A microencapsulation-based technology platform for effective delivery of multiplemicronutrients for food fortification has been developed. The technology, consisting of

extrusion agglomeration followed by encapsulation through surface coating, has been

successfully tested on three size scales in typical staple foods: as a surface treatment on salt

and sugar, on 20-100m scale; in salt on a 300-1200 m scale; and on reconstituted rice on

the 5-10 mm scale. The process results in effective delivery systems for one or more active

ingredients with organoleptic properties that are unnoticeable to the average consumer.

Particularly, salt double fortified with iodine and iron using the microencapsulated ferrous

fumarate premix made by the extrusion-based agglomeration process had acceptable sensory

properties and stability when stored at 40oC and 60% relative humidity (RH) for up to a year.

In these tests >85% of iodine and >90% of ferrous iron were retained.

Reconstituted Ultra Rice grains made by extrusion stabilized by internal gelation has

resulted in improved grain integrity and a much simplified process, compared to the original,

patented surface crosslinking technique. The most effective internal gelation system is

composed of alginate, calcium sulphate (CaSO4), and sodium tripolyphosphate (STPP) at a

best ratio of 3%:3%:0.6% (w/w).

It is feasible to incorporate folic acid into the existing fortification programs using the


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technology platform developed in this study. The results indicate that the potential

interactions of folic acid with other added micronutrients or with the food vehicles could be

prevented by incorporating folic acid as a premix made by the extrusion-based technology.

Virtually no folic acid was lost after 9 months storage at 40oC and 60% RH when the folic

acid premix was added into salt or sugar samples.

The technical feasibility of the microencapsulation-based technology platform has been

successfully demonstrated for micronutrient delivery in food vehicles of different size

ranges, resulting in fortified staple foods with desired physical, chemical, nutritional, and

organoleptic properties. The technology should be adaptable to formulating customized

delivery systems of active ingredients for broader applications, and promises to bring

immediate benefits in combatting micronutrient deficiencies, that will have far reaching

effects in health and social development.


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DEDICATIONS

I dedicate the culmination of my formal studies to my parents:

Drs. Wentong Li & Shukun Chen

I dedicate this thesis

to my husband, Shujun,and my daughter, Grace

for their love, patience, support, accommodation

to myself

as a great fortieth birthday gift

and as a new start of my academic career in Canada


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ACKNOWLEDGEMENTS

The over six years that I have spent here at University of Toronto in pursuit my Masters and

doctorate degrees will always be a remarkable period of time in my life. There are so many

people that I would like to express my appreciation for their helps in making my learning

journey here such a great experience.

First of all, I would like to express my greatest gratitude to my supervisor, Dr. L. L. Diosady,

for his continuous guidance and support. His faith in my capability and the research freedom

he gave to me enabled me to develop my professional skills over the course. The appreciable

influence from him, a widely respected professor, has inspired me to re-start my dreamed

career in academia in Canada. I am honoured to be your student!

I am very grateful to my reading committee members, Dr. E. Acosta and Dr. B. Saville, who

have imparted their knowledge, visions, and critical thinking onto me throughout my

dissertation process. Your generous support and invaluable advices are well appreciated!

I am also thankful to my oral committee members, Dr. Y. L. Cheng and Dr. E. Edwards, who

provided me invaluable suggestions from my thesis structure to the technical contents; Dr. V.

Rao and Dr. T. Oshinowo as my chairs of the two oral defenses, who initiated insightful

discussions between me and the exam committees; Dr. D. Rousseau from Ryerson

University as my external appraiser, who reviewed my thesis carefully and provided me

constructive criticisms in helping me improve my thesis. I surely learned a lot from all of

you during the two oral exams, which made me truly understand the meaning of the title of a


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doctorate.

Parts of the research work were financially sponsored by the Micronutrient Initiative (MI)

and the Program for Appropriate Technology in Health (PATH). I would then acknowledge

the financial and technical support from the organizations and the staffs, particularly with Mr.

Venkatesh Mannar and Dr. Annie Wesley from MI, and Dr. Ted Greiner and Ms. Shirley

Jankowski from PATH.

During my six years research many people from U of T or other academic institutions have

kindly offered me the technical assistances in conducting certain analyses and measurements.

I have to say thank you to: Mr. Dan Mathers and Ms. Ying Lei Wania from the ANALEST in

the Department of Chemistry, Mr. Sal Boccia from the Microanalysis Centre of the

Department of Material Science and Engineering, Dr. Rana Sodhi from the Surface Interface

Ontario in the Department of Chemical Engineering and Applied Chemistry, Mr. Dave Sohn

from Professor Yu-ling Chengs research group, Dr. Supratim Ghosh and Dr. Misael

Miranda from Professor Derick Rousseaus research group at the University of Ryerson.

Over the course many undergraduate students and visiting scholars have involved in my

research projects, and it was great experience to work together with them. Special thanks go

to Adrew Barquin, Pauline Rabier, and Haeyeon Lee for their contributions to my

experimental work.

I am especially thankful to the members of the Food Engineering Group, in particular


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Professor Toks Oshinowo and Mr. Bih King Chen, for their great support and suggestions. I

value the friendship of Judy Ue, Jessica Yuan, Katarina Rutkowski, Narongechai

Prapakorenwerzya, Crystal Lo, and Divya Yadava. Thank you for the insightful discussions

between us, and the joy and sadness that we experienced together over the past years.

Finally, I would dedicate all I have achieved to my family my parents, my husband and my

daughter. Without your unconditional love, support, and patience, I could not have come to

thus far.


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TABLE OF CONTENTS

ABSTRACT.........ii

ACKNOWLEDGEMENTS......v

TABLE OF CONTENTS....viii

LIST OF FIGURES......xii

LIST OF TABLES.....xv

LIST OF APPENDICES xvii

1 INTRODUCTION1

2 RESEARCH OBJECTIVES, SCOPE & APPROACHES...1

2.1 Research Objectives7

2.2 Research Scope8

2.3 Research Approaches & Anticipated Outcomes..9

3 RESEARCH BACKGROUND..13

3.1 Micronutrient Deficiencies13

3.2 Food Fortification..14

3.2.1 Food Vehicle Selection15

3.2.2 Fortification Techniques Used in Existing Programs..16

3.2.3 Current Challenges in Food Fortification Programs17

3.3 Microencapsulation18

3.3.1 Microencapsulation Techniques..20

3.3.2 Microencapsulation in the Food Industry27

3.3.3 Coating Materials Used For Microencapsulation in the Food Industry..30

3.3.4 Microencapsulation of Vitamins and Minerals32

3.3.5 Microencapsulation Techniques Used in This Research Group..33

4 RESEARCH APPROACH 1 - SALT GRAIN-SIZED IRON PREMIX MADE BYEXTRUSION & POLYMER COATING FOR SALT DOUBLE FORTIFICATION36

4.1 Research Incentive and Hypothesis...36

4.2 Experimental Materials & Methods..39

4.2.1 Materials..39

4.2.2 Equipment40


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4.2.3 Experimental Procedures.43

4.2.4 Analytical Methods..45

4.3 Results & Discussion.47

4.3.1 Investigation on Extrusion...47

4.3.1.1 Binder selection.48

4.3.1.2 Optimization of the extrusion operation52

4.3.2 Investigation on Colour-Masking Process...54

4.3.2.1 Different techniques for applying TiO2.56

4.3.2.2 Alternative colour-masking agents58

4.3.3 Investigation on Encapsulation Using Glassy Polymer Coatings59

4.3.3.1 Comparison between MethocelTM

and Kollicoat

61

4.3.3.2 Comparison between pan coating and fluidized bed coating61

4.3.4 Formulation Optimization...62

4.3.4.1 Iron content in optimized formulations.64

4.3.4.2 In vitro iron bioavailability in optimized formulations .65

4.3.4.3 Iron premix integrity in pH 4 HCl solution...67

4.3.4.4 Effect of formulation ingredients & processing techniques on density.70

4.3.4.5 Physical characteristics of the microencapsulated iron premixes..71

4.3.5 Stability Test in DFS Samples.72

4.3.5.1 Iodine stability...72

4.3.5.2 Ferrous iron stability..76

4.3.6 Iodine and Iron Interaction in DFS..78

4.3.7 Iodine Degradation Kinetics in DFS80

4.4 Summary of Research Approach 182

5 RESEARCH APPROACH 2 - ULTRA RICEAS MICRONUTRIENT DELIVERY

VEHICLE USING LARGE PARTICLES..83

5.1 Project Background & Research Incentive83

5.2 Literature Review on Alginate Chemistry & Internal Gelation.85

5.3 Experimental Strategy Towards Formulation Design88

5.4 Experimental Materials & Methods ..89

5.4.1 Materials..89


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5.4.2 Experimental Methods.90

5.5 Results & Discussion.93

5.5.1 Preliminary Investigations...93

5.5.1.1 Effect of different alginate sources on the internal gelation..94

5.5.1.2 Effect of different calcium salts on the internal gelation...95

5.5.1.3 Effect of different sequestrants on the internal gelation97

5.5.1.4 Effect of different calcium-sequestrant ratios on the internal gelation..98

5.5.1.5 Investigation of other ingredients & procedures for dough mixing..98

5.5.2 Formulation Optimization.100

5.5.2.1 Orthogonal experiment100

5.5.2.2 Verification of the optimal ratios.103

5.5.3 Test of the Optimized Gelation Systems in Nutrient Fortified Formulations110

5.5.4 Texture Measurements on the Best Formulations..112

5.5.5 Mechanism of Alginate-Calcium Interaction in the Ultra RiceSystem..115

5.6 Summary of Research Approach 2..117

6 RESEARCH APPROACH 3 - FOLIC ACID TESTS...120

6.1 Research Incentive Folate Deficiency..120

6.2 Literature Review on Folic Acid Chemistry122

6.2.1 Physiochemical Properties.122

6.2.2 Biological Functions..123

6.2.3 Food Fortification with Folic Acid123

6.2.4 Concerns Related to Folic Acid Fortification125

6.2.5 Issues of Folate Bioavailability.125

6.2.6 Stability of Folic Acid127

6.3 Project Goals & Anticipated Challenges .130

6.4 Experimental Materials & Methods 131

6.4.1 Materials131

6.4.2 Experimental Design.132

6.4.3 Analytical Methods136

6.5 Results & Discussion...138

6.5.1 Folic Acid Fortification in Guatemalan Salt and Sugar.138


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6.5.1.1. Folic acid stability...139

6.5.1.2. Iodine stability in Guatemalan iodized salt....140

6.5.1.3. Vitamin A stability in Guatemalan vitamin A fortified sugar..141

6.5.1.4. Colour Stability...142

6.5.2 Folic Acid Fortification in Multiple Fortified Ultra Rice....143

6.5.2.1 Ultra Riceappearance and colour stability....143

6.5.2.2 Folic acid stability ...147

6.5.3 Interactions of Folic Acid with Other Micronutrients...148

6.6 Summary of Research Approach 3 .150

7 CONCLUSIONS...152

8 RECOMMENDATIONS .154

9 REFERENCES.157

10 NOMENCLATURE168

11 APPENDICES.172


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LIST OF FIGURES

Figure 2.1 Overview of the project scope 9

Figure 3.1 Schematic relationship between the core material, the wall material,and the required technique used in microencapsulation systems

19

Figure 3.2 Microcapsules and microspheres 21

Figure 3.3 Molecular structure and microstructure of beta-cyclodextrin 26

Figure 4.0 Surface defects on encapsulated ferrous fumarate using fluidized-bedagglomeration and soy stearine as the coating material

36

Figure 4.1 Schematic process flow for making microencapsulated FeFum 37

Figure 4.2 Ultra Power Series KitchenAidTMStand Mixer 40

Figure 4.3 La Monferrina P12 Pasta Extruder and other components: die and

cutter

40

Figure 4.4 Schematic diagram for Uni-Glatt top spray fluidized bed assembly 42

Figure 4.5 Laboratory pan coating assembly 42

Figure 4.6 Effect of binder materials on iron digestibility of the extrudedparticles

65

Figure 4.7 Effect of different coating materials on iron digestibility of themicroencapsulated premixes

66

Figure 4.8 Effect of each step of the encapsulation process on iron digestibility 67

Figure 4.9 Effect of binders on particle integrity when dissolved in pH 4 HClsolution

68

Figure 4.10 Effect of each step of the encapsulation process on iron premixintegrity in pH 4 HCl solution

69

Figure 4.11 Iodine stability in DFS samples containing various FeFum forms powder, extruded and colour-masked particles, as well asencapsulated premixes, after one-year storage at 40oC and 60% RH

74

Figure 4.12 Relative iodine retention in the DFS samples containing different

sources of FePP during 6 months storage at 40

o

C and 60% RH

75

Figure 4.13 Ferrous iron stability in various FeFum forms and in DFS samples,after 10 months storage at the ambient condition and one-year storageunder 40oC and 60%RH, respectively

77

Figure 4.14 Iodine-iron interaction in DFS correlation analysis between iodineand ferrous iron losses in the DFS samples containing various ironparticles after one year storage under 40oC and 60% RH

79


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Figure 4.15 Correlation of iodine degradation in the DFS samples with afirst-order degradation pattern

80

Figure 4.16 Apparent first order degradation kinetics of iodine in the DFSsamples made with various iron particles during one-year storageunder 40

oC and 60% RH

81

Figure 5.1 Schematic process flow of Ultra Rice 83

Figure 5.2 Structural characteristics of alginate and the egg-box structure formedby alginate-Ca gelation

86

Figure 5.3 Factors affecting alginate-Ca reaction 88

Figure 5.4 Statistical analyses of the best level for each factor 103

Figure 5.5 Comparison of grain appearance made with glutinous flour in placeof regular rice flour

107

Figure 5.6 Comparison between glutinous flour and regular rice flour using thebest ratio and addition of HPMC

107

Figure 5.7 Colour measurements of the four new formulations made with theoptimized internal gelation systems

111

Figure 5.8 Micronutrient fortified Ultra Riceformulations made with the

optimized internal gelation systems111

Figure 5.9 Two proposed models for alginate-calcium gelation in Ultra Rice

:fish-net model for external/diffusion setting (left) and inter-lockmodel for internal setting (right)

116

Figure 5.10 Alginate-calcium gel structure made with external (left) and internal(right) gelation processes

116

Figure 6.1 Chemical structure of folic acid 122

Figure 6.2 Chemical structure of folate 122

Figure 6.3 Oxidative cleavage products of folates 128

Figure 6.4 Cleavage products of folic acid under acidic and alkaline conditions 128

Figure 6.5 Folic acid retentions in the fortified Guatemalan salt samples during 9months storage at 40oC and 60% RH

139

Figure 6.6 Folic acid retentions in the fortified Guatemalan sugar samples during9 months storage at 40

oC and 60% RH

140

Figure 6.7 Iodine retentions in the fortified Guatemalan salt samples during 9

months storage at 40oC and 60% RH

141

Figure 6.8 Vitamin A retentions in the fortified Guatemalan sugar samplesduring 9 months storage at 40

oC and 60% RH

142

Figure 6.9 Colour stability of the Ultra Ricegrains made with various FePPsources

144


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Figure 6.10 Colour stability in the Ultra Ricegrains made with Dr LohmannFePP at different addition levels

144

Figure 6.11 Colour stability of Ultra Ricegrains with addition of TiO2as the

colour-masking agent at different levels145

Figure 6.12 Colour stability of Ultra Ricegrains made with higher levels of folic

acid and FePP

145

Figure 8.1 Model premix system for salt fortification made by the extrusionagglomeration followed by polymer coatings

155

Figure 8.2 Model Ultra Rice

premix made by extrusion using internal gelationand followed by polymer coatings for delivering multiplemicronutrients

155

Figure 8.3 Model Ultra Ricepremix made by extrusion using internal gelation,containing sub-capsules of microencapsulated premixes of iron andvitamin A made by extrusion-based technology platform

156


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LIST OF TABLES

Table 3.1 Applications of microencapsulation technology 19

Table 3.2 Microencapsulated food ingredients 29

Table 3.3 Commonly used coating materials for microencapsulation of foodingredients 31

Table 4.1 List of chemicals used in the research approach 1 39

Table 4.2 Effect of water content on extrusion operability 52

Table 4.3 Surface defects on the final products caused by the loss of TiO2during coating

56

Table 4.4 Comparison of particle colour when TiO2was incorporated in thedough formulation before extrusion

56

Table 4.5 Comparison of different colour-masking agents 59

Table 4.6 Formulation variables investigated in the optimization study 63

Table 4.7 Formulation design of microencapsulated FeFum premixes made byextrusion and polymer coatings

63

Table 4.8 Total iron and ferrous iron contents in the 12 final formulations 64

Table 4.9 Effect of coating materials on premix integrity in pH 4 HCl solution 68

Table 4.10 Bulk density changes in iron particles after each processing step 70

Table 4.11 Comparison of particle densities of various premixes and rawingredients

71

Table 4.12 Iodine first order degradation rate constant (k) and the storagehalf-life estimated for the DFS samples containing various FeFumparticles when stored at 40

oC and 60% RH

81

Table 5.1 List of materials used in the formulations of Ultra Rice 89

Table 5.2 Effects of different alginate sources on extrusion operability 94

Table 5.3 Grain integrity and sensory properties of the simulated rice madewith different alginate sources

95

Table 5.4 Effects of different Ca sources on extrusion operability and grain

integrity using the internal gelation process in the absence ofsequestrants

96

Table 5.5 Comparison of different Ca sources using the internal gelationprocess in the presence of STPP as a sequestrant

97

Table 5.6 Comparison of different sequestrants in the internal gelation processusing same alginate and Ca sources

97

Table 5.7 Effect of different CaSO4to sequestrant ratios on grain integrity 98


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Table 5.8 A 3x3 orthogonal design for formulation optimization 101

Table 5.9 Detailed experimental trials for orthogonal study 101

Table 5.10 Results of the grain integrity tests in the formulations prepared by theorthogonal design

102

Table 5.11 Statistical analyses of orthogonal study results 102

Table 5.12 Comparison of two best ratios of alginate/CaSO4/STPP usingalternative alginate sources

103

Table 5.13 Verification of the best ratio (alginate/CaSO4/STPP = 3/3/0.6) usingvarious alginate sources

104

Table 5.14 Investigation on increased concentration levels of alginate andCaSO4

105

Table 5.15 Verification of the best ratio with various calcium compounds 105

Table 5.16 Verification of the best ratio with various sequestrant compounds 106

Table 5.17 Effect of using glutinous rice flour on the grain properties 108

Table 5.18 Effect of HPMC on enhanced grain integrity with the best gelationratio used

108

Table 5.19 Comparison of alternative blending processes on grain integrity withthe best ratio used

109

Table 5.20 Micronutrient retentions in the four new formulations made with theoptimized internal gelation systems

112

Table 5.21 Texture measurements on the Ultra Ricegrains made in the

optimization study113

Table 6.1 List of chemicals used in the study of folic acid fortification 131

Table 6.2 Experimental design for incorporating folic acid in Guatemalan saltand sugar

133

Table 6.3 Experimental design for preparing folic acid fortified Ultra Riceformulations

135

Table 6.4 Folic acid concentration in the final formulations of the Guatemalanfortified salt and sugar samples for storage stability test under 40

oC

and 60% RH

138

Table 6.5 Folic acid retention in the Ultra Ricesamples made with various

FePP sources and at different addition levels

147


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LIST OF APPENDICES

Appendix 11.1.1 Analytical methods used in research approach 1 172

Appendix 11.1.2 Preliminary observations on suitability of different binder

materials

174

Appendix 11.1.3 Specifications of the three cereal flours used as binders in thestudy

175

Appendix 11.1.4 Preliminary investigation of dextrin and HPMC as secondarybinders

176

Appendix 11.1.5 Comparison of extrudability and product characteristicsbetween three cereal flours used in the study as binders

177

Appendix 11.1.6 Comparison on the effectiveness of TiO2 adhesion before andafter drying

178

Appendix 11.1.7 Comparison of surface morphology in the premixes made by

different coating materials

178

Appendix 11.1.8 Development of standard protocols for encapsulation operationusing the fluidized bed and the pan coater

179

Appendix 11.1.9 Detailed composition of the 12 final microencapsulated FeFumpremixes

183

Appendix 11.1.10 Iron in vitro bioavailability test results of the optimizedformulations of microencapsulated FeFum premixes

185

Appendix 11.1.11 Particle integrity dissolution test results of the optimizedformulations of microencapsulated FeFum premixes

186

Appendix 11.1.12 Physical characteristics of the final premixes 187

Appendix 11.1.13 SEM images of the final FeFum premixes (at ~5000magnification)

188

Appendix 11.1.14 Relative iodine retention in DFS samples containing variousFeFum particles during one year storage under 40oC and 60%RH

189

Appendix 11.1.15 Ferrous iron retention in formulated FeFum particles and inDFS samples, when stored at the ambient condition and thehigher conditions of 40oC & 60% RH, respectively

190

Appendix 11.1.16 Detailed data processing for analysing iodine-iron interaction inDFS

191

Appendix 11.2.1 Ranking scheme for measurement of grain integrity duringsoaking and cooking

192

Appendix 11.2.2 Texture measurement on cooked Ultra Rice

grains for grainintegrity

196


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Appendix 11.2.3 Detailed compositions of the final 4 formulations used forverifying the optimal internal gelation systems in the actualnutrient-fortified formulations

197

Appendix 11.2.4 XPS and ToF-SIMS measurements on Ultra Rice 199

Appendix 11.3.1 Folic acid determination protocols 203

Appendix 11.3.2 Colour stability of the double or triple fortified Guatemalan saltor sugar samples after 3 months storage under 40

oC and 60%

RH

209

Appendix 11.3.3 Folic acid-containing multiple fortified Ultra Riceappearance 210


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1 INTRODUCTION

Micronutrient deficiencies are widespread health problems, especially in developing

countries. The deficiencies in vitamin A, iron, and iodine have been identified as the greatest

concern, as they affect over one third of the worlds population (WHO, 1995). In addition,

these micronutrients interact with each other, i.e., synergistic effects between iodine

deficiency disorder (IDD) and iron deficiency anemia (IDA), or between vitamin A

deficiency (VAD) and IDA to deepen their negative impacts (Clydesdale & Weimer, 1985;

Lonnerdal, 2004; Lynch, 1997; Zimmermann et al., 2004). Deficiencies typically coexist in

children in developing countries (Zimmermann et al., 2000). Due to the beneficial metabolic

interactions of iron, iodine and vitamin A (IVACG, 1998), it would be beneficial to develop a

multiple nutrient delivery system to attack the problems simultaneously.

Micronutrient deficiencies can be addressed by changes in the diet, supplementation, and the

fortification of food with selected nutrients. While dietary modification is desirable, it is a

long-range solution and may require changes in food preparation practices and social

customs. Supplementation is an effective and rapid approach, but it requires appropriate

medical infrastructure/administration and thus it is costly. Food fortification is a

cost-effective intervention that does not require any conscious action by the consumer, andneeds no changes in the dietary habits of the target populations. Moreover, it is readily

adapted into existing food production and distribution systems.

Food fortification has been extensively used for many years as a cost-effective strategy for

combating micronutrient deficiencies. Many fortification programs have been implemented

worldwide, including universal iodization of salt and enrichment of B vitamins in wheat

flour. Iodized salt now reaches some 70% of the world's population, significantly reducing

the incidence of iodine deficiency disorders (IDD) over the past two decades (United

Nations, 2008). The introduction of folic acid into cereal-grain products in over 40 countries

has resulted in dramatically increased folate status and significant reduction in the risk of

neural tube defects in newborns (Buttriss, 2005). Successful fortification programs may

involve different social, technical, and political challenges, yet they all have some common


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features, i.e., they are effective in reducing the prevalence of specific micronutrient

deficiencies, they are economically viable, and the fortified products enjoy consumer

acceptance.

To meet these criteria, several technical factors need to be considered, including the

selection of appropriate food vehicles and fortificant forms, as well as determination of

fortification levels and appropriate quality assurance and quality control of the fortified food

products. Among these, food vehicle selection is the primary factor that plays a key role in

determining whether food fortification programs could be more beneficial compared to the

other two strategies supplementation and dietary diversification. Obviously, the selection

of an ideal carrier guarantees that the micronutrients reach the largest number of people and

the fortification strategy remains as the best long-term approach and the cheapest way to

initiate and maintain the desired micronutrient levels in the diet.

Many foods or food ingredients have been considered for fortification, including cereal and

grain products, milk and dairy products, fats and oils, infant formula and weaning foods,

condiments such as salt, sugar, and monosodium glutamate (MSG), as well as a range of

processed foods (Lofti et al., 1996). It is generally accepted that staple foods, such as salt,

sugar, wheat flour, and rice, are good carriers for fortification, since they are regularly

consumed by all of the target population at a fairly constant rate, and are relatively

inexpensive so that all segments of the target population could afford them. The global salt

iodization is an example of effective programs that improve human nutrition, mainly due to

the attributes of the food vehicle salt. It is universally consumed and is open to a simple

fortification technique, which makes the program affordable.

Another important technical factor is the selection of appropriate forms of fortificants. Some

vitamins and minerals could be simply added into selected food carriers in powder form,

which involves solid-solid blending or solid-liquid mixing. These methods are

straightforward and low in cost, but usually ineffective in protecting the micronutrients

within the fortified foods. Moreover, the incorporation of these minor ingredients often

causes undesirable sensory changes in the fortified foods, such as off-flavours or colours


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caused either by the additives themselves or the interactions between the additives and the

food vehicles. Ignoring sensory effects and physical/chemical properties leads to major

concerns regarding product stability and consumer acceptance that may jeopardize the

success of a fortification program. Therefore, appropriate technology must be used in

delivering fortificants into fortified foods.

A major challenge for food fortification programs is the development of stable forms of

micronutrients that overcome the instability of vitamins and the reactivity of minerals. For

instance, vitamin A is sensitive to almost all environmental factors, including light, heat,

oxygen, and chemical interactions. The difficulty with iron is in finding an appropriate

chemical form which is adequately absorbed and yet does not alter the appearance or taste of

the food vehicle (Mannar & Gallego, 2002). In addition, the presence of reactive iron

compounds significantly affects the stability of other vitamins added in the same food matrix.

This makes it even more difficult to develop a multiple-fortified food, particularly with

vitamin A and iron. Appropriate technologies are thus required to prevent possible

interactions between added micronutrients and the food system, and subsequently ensure the

stability, bioavailability, and desired sensory properties of the fortified food through

production, distribution, retail, and food preparation.

Previous experience in our research group with double-fortified salt and multiple-fortified

rice has revealed that the best approach for delivering two or more micronutrients

simultaneously in a stable and bioavailable form without interaction and degradation, is to

microencapsulate them in an inert, but digestible matrix. Appropriate microencapsulation

technology will maintain the active ingredients in a stable environment, separated from other

food components and other added micronutrients.

Under the direction of Professor L. L. Diosady, the Food Engineering Group at the

University of Toronto developed microencapsulation-based technology for the double

fortification of salt with iron and iodine. Initially, selected iodine compounds were

encapsulated in modified starches, gelatin, and sodium hexametaphosphate (SHMP) by

spray drying and fluidized bed coating. The encapsulation process was later adapted to


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produce a coated iron premix, consisting of encapsulated ferrous fumarate (Canadian Patent

2238925). R. Yusufali (2002) developed a two-step encapsulation process, starting with

particle agglomeration followed by pan coating. Dextrin was used to mix with the selected

iron or iodine compounds and the mixture was agglomerated into fine particles, which were

then encapsulated by a colour-masking agent and a lipid coating. The double-fortified salt

(DFS) prepared by this approach demonstrated good iodine stability (Yusufali, 2002).

The process was tested on different scales in several countries under the sponsorship of the

Micronutrient Initiative (MI). The field tests have shown that the DFS is effective in

reducing the incidence of iron deficiency anemia (IDA) and iodine deficiency disorder

(IDD); also it is acceptable in terms of organoleptic properties (Oshinowo et al., 2004 &

2007).

Another research activity in this research group is related to rice fortification via the

so-called Ultra Rice technology, which involves producing reconstituted rice grains by

extrusion. Specifically, the selected micronutrients, either vitamin A or iron-containing

multiple nutrients are added into rice flour, blended with other components, including

antioxidants, stabilizers, sodium alginate, water and shortening, to form rice dough, which is

then extruded to form rice-shaped kernels. After extrusion, CaCl2solution is sprayed on the

extruded rice grains, and the crosslinking reaction between calcium and alginate form the

hard surface of the simulated rice, which is the core of the Ultra Ricetechnology. The rice

grains thus produced, which have high concentrations of selected micronutrients, are then

blended with normal market rice to achieve desired dietary intake levels.

So far, two successful formulations of Ultra Ricegrains have been developed, one fortified

with vitamin A and the other containing multiple micronutrients including iron, zinc and

several B vitamins. This technology has also been field tested in several countries, and

demonstrated acceptable results in terms of product stability, sensory properties, and clinical

effectiveness (PATH, 2007).

The Ultra Ricegrains made by extrusion are actually encapsulated forms of the selected


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micronutrients with high concentrations, which are similar to those of microencapsulated

iron premix added into iodized salt. These processes, compared to basic fortification

techniques which involve mainly a powder-mixing process, have introduced a new concept,

where the selected micronutrients are added as concentrated, encapsulated premixes with

modified physical and chemical properties, favourable for adding into selected food carriers.

This research experience has revealed that microencapsulation could protect the stability of

micronutrients without greatly reducing their bioavailability when absorbed in the body.

Microencapsulation can also improve the sensory properties of the fortified foods by hiding

the undesirable colours and tastes from the fortificants and by preventing the interactions

between the fortificants and the food carrier. The microencapsulation-based technology

allows the fortificants to be delivered in appropriate forms that resemble the physical

characteristics of the selected food vehicles, in terms of shape, size, colour, and appearance.

We then hypothesize that microencapsulation-based approach is a feasible solution for

multiple micronutrient fortification of a wide variety of staple foods. Staple foods are

typically presented to the consumer as solids with particle sizes ranging from several

hundred microns to several millimeters. To prevent particle segragation, which may result in

potential under- or over-dosing, micronutrients must be added in forms that either stick to

the food particles, or in agglomerated premixes that match the particle size, and if possible,

the particle density of the food. Succesful food fortification processes require that the added

micronutrients are evenly distributed and are unnoticeable to the consumer. Thus the

complete delivery system must match the food in colour and appearance, and must not alter

the food flavour. Therefore, the added micronutrients must be introduced in a form that

achieves these criteria.

Based on the above considerations, a two-step process based on extrusion agglomeration

followed by surface coating was proposed as the basis of a flexible technology platform for

effective delivery of multiple micronutrients. Specifically, the selected micronutrients could

be agglomerated first to form particles with sizes matching that of the selected staple foods

to ensure stable distribution, without segregation. The appropriately sized micronutrient

particles could then be coated with appropriate polymer materials to obtain modified


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physical/chemical properties, including improved organoleptic properties and enhanced

stability, suitable for adding into the selected food matrices. The goal of the program,

therefore, is to develop a technology platform and to demonstrate its feasibility for effective

delivery of multiple micronutrients in the selected staple foods by using premixes on

different size scales to suit a wide variety of food matrices.


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2 RESEARCH OBJECTIVES, SCOPE & APPROACHES

2.1 Research Objectives

As discussed earlier, the technologies of double fortified salt (DFS) and Ultra Rice have

been field-tested in several countries and achieved acceptable results in terms of product

stability, sensory properties for consumer acceptance, as well as clinical efficacy in reducing

the prevalence of certain micronutrient deficiencies. However, there are several problems

remaining in the current systems. In the case of microencapsulated ferrous fumarate for salt

fortification, the iron premix has a porous texture and surface defects, which may result in

unwanted interactions and sensory changes in the double fortified salt (DFS). Also the low

density of the premix causes the iron particles to float on the surface of water, and then may

be unintentionally removed by the cook as impurities during salt washing.

The problems with Ultra Ricegrains are related to the marginally acceptable colour and

somewhat brittle texture that is probably due to the imperfect crosslinking of

alginate-calcium at the grain surface. The distribution of alginate during extrusion and

calcium during the over-spraying process may not be uniform, which then directly affects

their crosslinking at the grain surface and subsequently the grain integrity during washing

and cooking.

As indicated earlier, the overall objective of the research program is to develop a technology

platform for the delivery of multiple micronutrients in a variety of staple foods. To achieve

this goal, I proposed that processes based on extrusion agglomeration followed by polymer

coating could form the basis of a flexible microencapsulation technology platform for

effective delivery of reactive minor components in a variety of applications including not

only food fortification but also in drug delivery, nutraceutical delivery in functional foods,

and active ingredient delivery in agro-chemicals and cosmetic products. This assumption

was based on the fact that extrusion has several advantages over the currently

commercialized technique of fluidized-bed agglomeration, including high throughput and

low operating cost. It was expected that extrusion could produce agglomerates with better

physical properties, including denser texture and smoother surface, which would make the


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ensuing encapsulation process more effective and efficient by better coverage with less

coating material. Extrusion is flexible and can readily form particles with different particle

sizes, ranging from several hundred microns to several millimeters, which ensures the

premix particles can match the size of a wide variety of staple foods.

The immediate objective of my research project was to improve the current systems, for

producing microencapsulated ferrous fumarate premix and Ultra Rice, by developing

alternate processing techniques to achieve better physical/organoleptic properties of the

microencapsulated fortificants and to simplify the current procedures. In addition, these two

systems could be used as models for testing the proposed technology platform for food

fortification.

2.2 Research Scope

As depicted in Figure 2.1, the project scope was to explore the feasibility of using several

staple foods and/or food ingredients as effective micronutrient carriers. This included salt,

sugar, and rice. Salt is commonly consumed by all societies in the world, and it is almost

always processed. Rice is the primary staple food in some 40 countries and nearly half of the

worlds population (Juliano, 1993). Sugar is a commonly used staple in Latin America.

Clearly, these foods are good choices for fortification.

Micronutrients, including vitamin A, thiamine, folic acid, iron, zinc, and iodine, were

selected on the basis of the prevalence of their deficiencies. These micronutrients were

encapsulated individually and in combinations to attain desirable stability, release properties,

and appearance when incorporated into various foods.

To achieve the goal of the project, the potential interactions between the added ingredients

and the delivery systems had to be identified before designing effective food fortification

processes. The program targeted the development of a broadly applicable technology that

would not only be useful in developing countries, but would be also applicable in typical

Canadian processed food products, and thus prove to be of economic value in the

marketplace.


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Figure 2.1 Overview of the project scope

2.3 Research Approaches & Anticipated Outcomes

As indicated in Figure 2.1, many micronutrients and several staple foods were considered in

the research scope. To develop a broadly applicable technology for effective delivery of

these micronutrients by different food carriers, it was believed that the best approach would

be to start with specific designs for individual applications. Therefore, three specific

research projects were proposed in the study:

1. Microencapsulation of ferrous fumarate (FeFum) premix for double fortification of

salt, after agglomeration by extrusion to match the size of salt grains by polymer

coating;

2. Production of Ultra Rice

grains as carriers of micronutrients by controlled

reactions to form a stable alginate-calcium structure incorporating and protecting

the added micronutrients;

APPROACHES

Microencapsulation: converting the selected vitamins andminerals to appropriate forms which fit into the selected foodvehicles;Multiple Fortification: incorporating several micronutrients intheir proper forms into the selected food vehicles.

FOOD CARRIERS

Staple foods or food ingredients:

Salt, sugar, rice

MICRONUTRIENTS

Vitamins: A, B1, niacin, folic acid

Minerals: iodine, iron, zinc

OBJECTIVE

To develop microencapsulation-based technologiesfor multiple nutrient fortification in staple foods


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3. Folic acid fortification by incorporation into existing programs of salt iodization,

vitamin A fortification of sugar, and iron fortification of Ultra Rice.

The detailed experimental design for each approach and the anticipated outcomes are

discussed in the following.

Research approach 1 microencapsulated FeFum premix made by the extrusion-based

technology platform

Previously, microencapsulated FeFum premix was made by fluidized-bed agglomeration

followed by a lipid coating. To overcome the shortcomings of the current process and

premix product, I proposed to investigate alternate techniques and materials. Specifically,

extrusion technology was proposed to replace the fluidized-bed agglomeration, and

hydrophilic glassy polymers were proposed as improved coating materials. Extrusion is

extensively used in the food industry and has several advantages over fluidized-bed

agglomeration, as it could produce denser particles with fewer pores, regular shape, and

better surface morphology. These improved particle properties could collectively make the

following encapsulation step easier by allowing more uniform coverage with less coating

material used. Hydrophilic glassy polymers have been widely used in oral drug delivery

systems. They can provide excellent physical barriers in the dehydrated, glassy state, while

upon water penetration they can achieve controlled release of the core ingredients by a

swelling mechanism. Therefore, it seemed logical to apply this advance in drug delivery

systems into food applications.

Successful development of a microencapsulated FeFum premix using the proposed

techniques could not only result in a stable salt double fortified with iodine and iron, but

could also provide an effective model system for making microencapsulated particles

ranging in size from two hundred microns to several millimeters that can incorporate one or

more active ingredients simultaneously. Since the current procedure involves several steps,

including granulation, colour-masking, and encapsulation, additional micronutrients could

be added at different stages of processing to different particle layers to prevent direct

interactions between incompatible micronutrients.


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Research approach 2 Extending the extrusion-based technology platform for making

reconstituted rice premix, Ultra Rice, using alginate-calcium internal gelation

Ultra Rice is a product which agglomerates rice flour and added micronutrients to form

reconstituted rice grains that will be indistinguishable from market rice by the average

consumers. Currently the grains contain sodium alginate, which, when sprayed with calcium

chloride, form a stable shell around the grain. We realized that if we could convert all of the

alginate into the cross-linked Ca form, we could, in effect, form a large microencapsulating

structure capable of carrying micronutrients, expanding the technology platform to a larger

size scale. If we could control this reaction and combine it with the extrusion, we could

greatly simplify the existing process and achieve better grain integrity and sensory

properties. The objective of this phase of the work was to understand the Ca-alginate

formation, and to develop a formulation and process that would form the desired

cross-linked structure in the Ultra Riceextrusion process.

Research approach 3 Testing of the developed technology platform using folic acid

fortification incorporated into the existing programs

Folic acid has been identified as the cause of harmful deficiency diseases that could be

prevented by food fortification. Many dietary/nutrition surveys have revealed that most

adults had inadequate serum folate levels, and folate deficiency was common particularly in

elderly populations (Buttriss, 2005). Folate deficiency is clearly linked with the development

of birth defects, several chronic diseases, certain cancers, and reduced cognitive functions in

elderly (Cho et al., 2002; Rampersaud et al., 2002; Choi & Manson, 2000; Miller, 2004).

Prior to mandated supplementation/fortification, it was estimated that folate deficiency

would result in up to 5 neural tube defects (NTD) out of every 1000 pregnancies (Caudill,

2004). Since mandated folic acid fortification of flour in North America, there has been a

dramatic decrease in the incidence of this type of birth defects and of some types of cancer

(Grosse et al., 2006). Folic acid fortification would be relatively inexpensive in developing

countries if the fortification could be combined with existing technologies for the ongoing

fortification programs that have universal coverage in affected areas: salt iodization, sugar

fortification with vitamin A, salt double fortification with iron and iodine, and Ultra Rice


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3 RESEARCH BACKGROUND

3.1 Micronutrient Deficiencies

Micronutrients are essential substances required by the body in small amounts to maintain

its normal functions. Deficiencies in micronutrients have serious health consequences, such

as learning disability, impaired work capability, reduced resistance to infection, illness, or

even death. Various micronutrient deficiencies have occurred due to poor nutrition in many

different regions and populations. Three micronutrients, vitamin A, iron, and iodine, have

been identified as major concerns (WHO, 1995), as over one third of the worlds population

is affected by the deficiencies of these three nutrients. Women and young children in

developing countries are most vulnerable.

Vitamin A deficiency (VAD)is a serious problem in developing countries, and can lead to

partial or total blindness. It is also associated with increased risk of infectious morbidity and

mortality (Levin et al., 1993). It is estimated that over one hundred million children in

developing countries (nearly 40% of the developing worlds children) suffer from a sub

clinical deficiency in vitamin A, and about one million such children would die each year

due to complications arising from VAD (MI, 2004).

Iron deficiency is the most common nutritional deficiency in the world (WHO, 2000). It has

profound negative effects on human health and development, and it is the primary cause of

anemia. Nearly two billion people suffer from iron deficiency anemia (IDA); among these,

over 90% live in developing countries. IDA lowers work capacity and impairs immune

response, which results in reduced resistance to infection and increased risk of maternal and

fetal morbidity (Clydesdale & Weimer, 1985).

Iodine deficiency disorder (IDD) is the worlds single most significant cause of

preventable brain damage and mental retardation (WHO, 2000). It is responsible for

impaired physical and mental development, resulting in significant reduction of intellectual

capability in those affected during childhood. Nearly 20% of the developing worlds

population is iodine deficient and more than 830 million people have goiter a visible


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symptom of iodine deficiency due to swollen thyroid glands (Lotfi et al., 1996).

There is evidence showing that the three deficiencies interact with each other. Clydesdale &

Weimer (1985) reported that IDD and IDA have synergistic effects which lead to severe

retardation of physical, mental, and social development. IDA interferes with thyroidal

metabolism of iodine and may reduce the efficacy of iodine treatment for IDD by

supplementation or fortification (Lonnerdal, 2004). Lynch (1997) reported there was a direct

correlation between serum retinol and hemoglobin levels in several surveys. VAD may

impair iron metabolism and aggravate iron deficiency anemia (IDA) (Zimmermann et al.,

2004). In addition, Zimmermann et al. (2000) indicated that the three deficiencies often

coexist in children in many developing countries. Due to the detrimental interactions of iron,

iodine, and vitamin A deficiencies, a simultaneous attack by an effective multiple-nutrient

delivery system will be required.

In addition to the three key micronutrient deficiencies, significant populations are deficient

in other micronutrients, including vitamin B1 (thiamine), B2 (riboflavin), B12

(cyanocobalamin), folate, and zinc. These problems are more prevalent in rice-consuming

populations (OMNI/USAID, 1998). Recently folate deficiency was recognized as a serious

but readily treatable problem. Abundant evidence has linked this vitamin deficiency to

severe birth defects and several chronic diseases. It is reported that folate deficiency was

responsible for approximately 250,000 severe birth defects neural tube defects (NTD) -

each year prior to mandated supplementation and/or fortification programs (Berry et al.,

1999).

3.2 Food Fortification

Several interventions can be used to alleviate micronutrient deficiencies, including

supplementation, dietary education/diversification, and food fortification. The uptake of

essential micronutrients through a healthy, balanced diet is a desirable and sustainable

approach for prevention of micronutrient deficiencies, but may not be sufficient to cure

severe deficiency syndromes. It requires intensive cooperation and individual compliance,

and depends on adequate supply of nutrient-rich foodstuffs and sufficient income to obtain


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them. Direct supplementation is an effective, rapid strategy to combat micronutrient

deficiencies, but it requires consistent, well-educated participation from individuals and

well-established medical infrastructure from the governments. Due to its relatively high cost,

it is usually used only as a short-term medical intervention. Food fortification is recognized

as the most cost-effective measure to overcome micronutrient deficiencies. It is relatively

simple and does not require active cooperation from the consumers.

Food fortification has been used for over a century (Bonner et al., 1999). Many programs

have been successfully implemented worldwide. It is well known that wheat flour has been

fortified with B vitamins and minerals in North America, and salt has been used worldwide

to provide iodine in the normal diet for more than two decades (Salt Institute Website).

Despite these examples, the development and implementation of a successful food

fortification program is generally constrained by technical, socio-economic, infrastructural,

and political factors (Lofti et al., 1996). The major technical issues are 1) selection of

appropriate food vehicles and micronutrient forms to be added, 2) determination of the

appropriate addition levels and the methods/techniques to be used, and 3) ensuring product

stability and bioavailability (Wirakartakusumah & Hariyadi, 1998). A successful

fortification program should integrate all these technical considerations and ensure the final

product meets the following three criteria:

Clinical effectiveness: which requires that the fortified foods maintain the desirable

micronutrient stability and bioavailability,

Consumer acceptance: which requires that the final products be acceptable to the

consumers, through education about its benefits, or more desirably through the foods

attractive appearance and taste,

Technical feasibility: which requires that the fortification technology can be

implemented and maintained economically.

3.2.1 Food Vehicle Selection

Among the technical factors, the selection of the food vehicle is the key factor in developing

an effective fortification program. An ideal food vehicle is regularly consumed by all of the

target population at a relatively constant rate. Appropriate food vehicles will deliver the


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selected micronutrients in such way that the added micronutrients retain their activity and

bioavailability without adversely affecting the sensory properties of the final products during

food production, distribution, retail, food preparation and consumption. Staple foods are

most likely to meet these criteria. Salt is the ideal carrier as it is universally consumed and

industrially processed. In urban settings rice is a good carrier, as it forms the basis of the diet

of the urban poor. Other population-specific staple foods have been identified earlier,

including noodles, sugar, oil, and monosodium glutamate (MSG).

Numerous foods have been tested for fortification by a single nutrient and achieved

desirable results, including iodine fortification of salt; vitamin A fortification of oils/fats,

sugar, milk and dairy products; and iron fortification for wheat flour, breakfast cereals, bread,

and weaning foods. Unfortunately, vehicles for multiple micronutrient fortification are more

limited. A few acceptable examples include soy or fish sauce fortified with iron and iodine

in the Philippines and Thailand, and commercially produced breakfast cereals fortified with

iron and multi-B vitamins (Lofti et al., 1996; Wirakartakusumah & Hariyadi, 1998). Due to

the complex nature of food systems and the many variables involved in the fortification

processes, the selection of effective food vehicles for delivering multiple micronutrients

needs to be carefully examined, with consideration of chemical and technical factors to

prevent nutritional degradation.

3.2.2 Fortification Techniques Used in Existing Programs

Typically the techniques used in food fortification are rather simple, involving mainly

mixing processes (Lofti et al., 1996). For example, solid-solid mixing is used for fortifying

dry foods with small quantities of micronutrients in powder form, such as in the process of

fortifying B vitamins and iron powder into wheat flour. For delivering fortificant to a liquid

food matrix, such as milk and oil, solid-liquid mixing is used through dissolution or

dispersion. Other techniques include liquid-liquid mixing, solid-solid adhesion, and coating

by spraying the liquid fortificant on the dry food vehicle. Water-soluble fortificant may be

carried by aqueous solutions, while fat-soluble ingredients by organic solvents. For example,

salt iodization is achieved by spraying an aqueous solution containing iodine compounds,

either potassium iodate or iodide, onto salt grains (Salt Institute website).


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These basic methods work well for single fortification; however, when multiple

micronutrients consist of fortificants with greatly different physical characteristics from the

selected food carrier, the fortificants or food vehicle must be treated to ensure an acceptable

product. For example, the selected micronutrients in powder forms may need to be

granulated first to match the particle size of a granular food matrix to avoid particle

segregation (Diosady et al., 2002). Processes may be required to hide the undesirable colour

or taste of some vitamins and minerals. Moreover, there are potential interactions between

the added fortificants and the food vehicle, which subsequently results in loss of the

micronutrients and altered sensory properties of the end products (Hurrell, 2002; Rutkowski,

2003; Li, 2005). Technical solutions are thus required to overcome or prevent these

problems.

3.2.3 Current Challenges in Food Fortification Programs

As discussed earlier, the scope of the current program involves development of technology

for delivering several vitamins and minerals in at least three typical staple foods salt, sugar,

and rice. The selected food vehicles are all solids, where directly added powder ingredients

would cause segregation during production and distribution, resulting in uneven distribution

of the added micronutrients (Johnson et al., 2004). This may lead to potential under- or

over-dosing when consumed. Therefore, proper modifications to the fortificants particle

size distribution are required.

The studied micronutrients present chemical challenges in the form of instability of vitamins

and reactivity of minerals. Most vitamins, particularly vitamin A, are unstable under a

number of chemical and physical conditions, such as presence of oxygen, acids, and

reducing agents as well as humidity, heat, and light. The problem with minerals, particularly

iron, is the difficulty in selecting an appropriate form which is adequately absorbed and yet

does not alter theappearance or taste of the food vehicle (Manner & Gallego, 2002).

Water-soluble iron compounds, mostly in the ferrous form, are more bioavailable, but they

are also more reactive, often causing unacceptable colour and flavour changes in the food

matrices. In contrast, insoluble iron compounds may not cause sensory changes but are often


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so poorly absorbed as to be of little or no nutritional benefit (Hurrell, 2002). Therefore, iron

bioavailability and organoleptic properties must be balanced in designing/developing iron

fortified foods.

Previous research revealed that reactive iron compounds significantly affect the stability of

other micronutrients present in the same food matrix (Hurrell, 1999; Diosady et al., 2002;

Rutkowski, 2003). This makes it even harder to develop iron-containing multiple

fortification systems.

3.3 Microencapsulation

A promising approach to multiple fortification is to microencapsulate the selected

micronutrients, especially iron, in an inert but digestible coating system to ensure that the

micronutrients are effectively delivered in a stable and bioavailable manner without altering

the organoleptic properties of the food vehicle. Proper microencapsulation techniques could

also integrate colour/flavour masking and size enlargement to convert the selected powder

materials into proper granular forms with desirable appearance and size.

Generally, microencapsulation is defined as a technology of enveloping small solid particles,

liquid droplets or gases in a coating matrix (Benita, 1996). The coated or entrapped material,

also know as the core, fill, internal phase or payload, is usually the active ingredient which

needs to be protected from the environment and/or released at a controlled rate. The coating

material is called the capsule, wall, shell, membrane, carrier, encapsulant, or matrix (Benita,

1996).

Microencapsulation was originally developed by Barrett K. Green of the National Cash

Register Corp. (NCR) in 1950s, with a process called coacervation to create carbonless

paper (Benita, 1996). The process involved a soluble polymer, such as gelatin, induced to

come out of solution and form a shell around dispersed droplets of an oil at the interface

with a water medium. The gelatin shell is hardened by the addition of glutaraldehyde, and

the microscopic beads are collected and dried (Clark, 2002). Since then, many technologies

for preparing microparticles have been developed for applications (Table 3.1) in


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pharmaceutical, food, cosmetic, chemical, and printing industries (Madene et al., 2006).

Table 3.1 Applications of microencapsulation technology

Figure 3.1 Schematic relationship between the core material, the wall material, and therequired technique used in microencapsulation systems

As shown in Figure 3.1, the design of a microencapsulated system generally involves a core

material, a wall material, and an appropriate technique/process required to coat or entrap the

Wall materialCore ingredient

Microparticles with desired properties

Technique/Process

Application Microencapsulated actives References

PharmaceuticalOral, topical or transdermal, parenteral

drugs

Wang, et al., 2003; Lamprecht, et al.,

2000; Carafa, et al., 2004; Kshirsagar,

2000; Chen, et al., 1999; Park e t al., 2005

BiologicalCells, vaccines, hormons, antigens,

plasmid DNAs, enzymes

Sefton, et al., 2000; Cleland, et al., 1997;

Lee, et al., 1997; Tinsley-Bown, et al.,

2000; Hao, et al., 2001; Genta, et al.,

2001

Food

Acidulents, flavours, artificial

sweeteners, colourants, enzymes,

microorganisms, probiotics, leavening

agents, antioxidants, preservatives,vitamins, minerals, amino acids,

essential oils

Kirby, 1991; Gibbs, et al., 1999;

Gouin, 2004; Schrooyen, 2001;

Desai, et al., 2005

Agro-chemical Pesticides, herbicides Tsuji, 2001

Cosmetic and

personal care

Vitamin E, fragrances, perfumes, plant

extracts

Dingler, 1999; Schmitt, et al., 1998;

Benita, 1996


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core by the wall material. The core material is the key factor that needs to be protected or

released at a defined rate, while the wall material and process/technique play an important

role in the physical and chemical properties of the formed microparticles, such as particle

size, permeability, porosity, density (bulk and particle density), flowability, integrity,

reactivity/stability, release properties, and bioavailability. For each active ingredient, the

appropriate choice of process and wall materials depends on the end use of the

microencapsulated particles. For example, microencapsulated flavours for extrusion-based

processed foods should be heat resistant and insoluble while in the barrel so the flavour is

protected against thermal degradation and flash-off at the exit of the die (Bhandari et al.,

2001).

In the current project, various vitamins and minerals were selected as core ingredients. The

goal of the development program for a microencapsulation system is to find an effective

combination of appropriate wall materials and encapsulation techniques, which could

present the selected micronutrients in stable, bioavailable, and organoleptically desirable

forms suitable for food fortification. In the following sections the literature on available

techniques of microencapsulation and coating materials is discussed.

3.3.1 Microencapsulation Techniques

Current microencapsulation techniques can be classified based on the microparticle

formation mechanism. These include physical or mechanical processes (such as spray drying,

spray chilling/cooling, extrusion, and fluidized bed coating), and chemical processes (such

as coacervation, co-crystallization, molecular inclusion, and interfacial or in-situ

polymerization). In some cases, a combination of processes is used, for example in

formation of a single or double emulsion followed by spray drying (Madene et al., 2006).

The formed microparticles are categorized as either microcapsules or microspheres (Figure

3.2), based on the structure, or more precisely, the mutual position of the core and the shell.

In a microcapsule, the active ingredient is a continuous, concentrated phase and enveloped

by a protective layer of coating material. Usually a two-step process will be needed to

produce microcapsules, including the formation of the core particles followed by a coating


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process. In contrast, in a microsphere, the active substance is dispersed in the structure and

entrapped within the matrix material, which sometimes only involves a single-step of

entrapment (Adamiec et al., 2004). Microcapsules or microspheres may have diameters

ranging from a few microns to a few millimeters.

Figure 3.2 Microcapsules and microspheres (adapted from Gibbs et al., 1999)

Physical/mechanical processes

Spray drying is a commonly used method of drying a liquid feed through a hot gas. The

liquid feed is pumped through an atomiser device that produces fine droplets into the main

drying chamber. Typically, the hot gas is air, but when sensitive materials are processed or

oxygen-free drying is required, nitrogen gas is used instead. It is a well-established

technology involving commercially available equipment. It is extensively used to produce

powdery particles ranging in size from 1 m to 150 m, which contain value-added

ingredients, such as fragrances or flavours. The advantages of this process are relatively low

in cost and ease of scale up. The microparticles prepared by the technique can quickly

release the core ingredients without leaving any shell debris, due mainly to the high water

solubility of the shell materials used. However, the suitable shell materials for this process

are limited. Also, the concerns of solvent flammability and toxicity severely restrict the use

of organic solvents for conventional spray drying operations. Other limitations of this

technique include a generally low payload (


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a cooling medium and subsequently solidified into powder form (Madene et al., 2006). This

is probably the least expensive process, which can be used to convert liquid hydrophilic

ingredients into free flowing powders with improved heat stability and delayed release in

wet environments. However, as a matrix encapsulation process, rather a true core/shell

encapsulation, it leads to a significant proportion of unprotected active ingredients on the

particle surface or sticking out of the wall material, which subsequently affects the

effectiveness of the encapsulation.

Freeze drying, also called lyophilization, is one of the most useful processes for drying

thermo-sensitive ingredients in aqueous solutions that are unstable. It involves the

sublimation/removal of water content from dissolved or dispersed solids. The food industry

widely uses the technology to preserve plant or animal products in dehydrated powder forms.

In the case of microencapsulation operation, it can be used to dehydrate and convert food

emulsions into powders. The technique is relatively simple and can provide better particle

properties compared to spray drying and drum/tray drying, such as resistance to oxidation

and intact shape of microcapsules (Madene et al., 2006). Nonetheless, it normally requires a

long processing time for dehydration, ~20 hours depending on the materials and the loads

(Desai & Park, 2005a).

Fluidized bed coatinginvolves suspending a bed or column of solid particles in a moving

gas stream, usually air, and a liquid coating formulation is sprayed onto the individual

particles. The freshly coated particles are cycled into a zone where the coating formulation is

dried either by solvent evaporation or cooling. Three types of fluidized beds are available, as

top-spray, tangential-spray, and bottom-spray. They vary in the nozzles location or

configuration used to apply the coating solution. This technique is generally an efficient way

to apply a uniform layer of shell materials onto solid particles. Basically all kinds of shell

materials can be used in this process, such as polysaccharides, proteins, lipids, and

emulsifiers. In addition, it is highly thermal efficient due to good gas-solid contact in which

optimal heat and mass transfer rates could be reached. On the other hand, its limitations are

also obvious; it can be only used for encapsulating solid particles, and the particle size of the

end products cannot be less than ~10 m (Gouin, 2004).


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Extrusion was first introduced as an encapsulation process by Swisher in 1957 to coat

volatile and unstable flavours (Madene et al., 2006; Gibbs et al., 1999). Essential oils were

dispersed in glassy carbohydrate matrices (such as corn syrup solids and glycerin) at >100oC,

and then extruded into a dehydrating liquid such as isopropyl alcohol. The solidified

material was then separated into small pieces and vacuum-dried. This process was later

modified to encapsulate microorganisms and enzymes at low temperatures (Gouin, 2004).

The active ingredients were mixed with plasticized composite matrices, such as starch/fat or

starch/polyethylene glycol. The dry mixture was converted to a wet paste by incorporating

~20% (w/w) water and then extruded. The exiting rope was cut into pieces between 500 m

to 1000 m and air-dried. This technique can provide virtually full protection to the core

ingredients by the surrounding wall materials. Also, the use of glassy polymers can provide

an essentially impermeable barrier against oxygen, which enables prolonged shelf life of the

end product. However, this process can only produce large particles, typically >500 m,

which greatly limits its applications. In addition, the suitable shell materials or binders are

limited to glassy carbohydrates and carbohydrate derivatives (Gibbs et al., 1999).

Coextrusion is a relatively new technique for encapsulation. It creates fibers consisting of

active ingredients within fluid, high-viscosity, glassy shell materials. These fibers can be

chopped to form microcylinders, or when the viscosity is low and the surface tension of the

fluid is high these extrudates would thermodynamically break up into tiny droplets, forming

microcapsules. The typical coextrusion systems include stationary nozzle coextrusion,

centrifugal coextrusion, or slightly altered spinning disk coextrusion. In the former two

processes, concentric nozzles are used to pump the core material through the inner nozzle

and the shell formulation through the annulus, allowing true core-shell morphologies.

Spinning disk coextrusion involves a suspension of the core material dispersed in the carrier

material. The mixture is then extruded through the rotating disc in such a way that the excess

coating fluid is atomized and separated from the coated particles (SwRI website).

This technique can be treated as true encapsulation, which gives the microcapsules unique

properties allowing release of the core ingredients at a defined rate (Gouin, 2004). The high


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operating cost and specific requirements of the equipment greatly limit the application of

this technique. In addition, the core and shell materials must be mutually immiscible liquids,

(i.e., polar liquids like aqueous solutions in the core require hot melt shell materials like

waxes; whereas with water-immiscible oils as the core, an aqueous polymer solution that can

gel rapidly is required for the shell) (SwRI website).

Chemical processes

Coacervation is a separation process involving two liquid phases in a colloidal system

(IUPAC, 1997). It starts with an aqueous colloid solution in an appropriate solvent.

According to the nature of the colloid, when the environmental conditions changes, such as

pH change, the solubility of the colloid is reduced and a large part of the colloid can be

separated out into a new phase. The original one phase system becomes two phases, one is

rich and the other is poor in colloid concentration. The colloid-rich phase in a dispersed state

appears as amorphous liquid droplets called coacervate droplets, which form the wall

material of the resultant capsules. This concept initiated the development of

microencapsulation technology by B. K. Green in 1957.

The first coacervative capsules developed by B.K. Green for carbonless paper were made

using gelatin as a wall in an "oil-in-water" system. Later developments could produce

"water-in-oil" systems for highly polar and water soluble cores. The process involves three

steps: particle or droplet formation, coacervative wall formation, and capsule isolation. It is

considered as a true microencapsulation technique, as the core material is completely

entrapped by the matrix.

Although this technique has a very high payload, >99% (Gouin, 2004), and the formed

particles are able to achieve sustained or controlled release of the core ingredients, its high

operating cost and complexity restrict its commercial utilization.

Gelation involves the formation of gelled microcapsules or microspheres using techniques

such as cooling, crosslinking, or a chemical reaction. A well-known system is

alginate-calcium crosslinked beads, which were initially developed for immobilization of


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live cells and enzymes. It has been rapidly adapted to many other applications due mainly to

its extreme ease of preparation on a lab-scale and mild processing conditions. Unfortunately,

the process is difficult to scale up and the operation is costly. In addition, the obtained

microcapsules are very porous and allow fast and easy diffusion of water and other fluids in

and out of the matrix. Such good permeability is desired for carrying live cells or enzymes

but is not suitable for protecting most active ingredients.

Liposome entrapment was originally used in the pharmaceutical industry, and in recent

years it has been used in many other applications, such as food-based delivery systems. The

processcan be achieved by dispersing a bilayer-forming polar lipid, such as lecithin, in an

aqueous medium containing dissolved active ingredients. The formed particles are typically

spherical in shape with a relatively narrow size range from several nanometers to several

hundreds nanometers. Liposomes may contain a single or multiple layers of amphiphilic

polymolecular membranes, which closely resemble the natural structure of cell membranes.

Generally, liposomes are kinetically stable, i.e., they are only stable for a short period of

time, similarly to emulsions. Because of this, many principles and techniques of emulsion

formation can be also applied to the development of liposomes (Taylor & Davidson, 2005).

Other technical issues with this technique include that the process is hard to scale up.

Moreover, liposomes are usually in aqueous forms, which impart great stability of

water-soluble materials in high water activity applications, but limit their usefulness when

the coated ingredients need to be in a dry state.

Molecular inclusionis an advanced technique that is highly specific. It generally refers to

the supra-molecular association of a ligand (the core material) into a cavity-bearing substrate

(the shell material) (Gouin, 2004). Particularly, beta-cyclodextrin, an enzymatically

modified starch molecule, is used in this molecular-level technique. Cyclodextrins are

hollow truncated cone-shaped molecules with an inner diameter of approximately 5-8 A

(Figure 3.3), sufficient for inclusion of one or more volatile flavour molecules or essential

oil compounds (Desai & Park, 2005a). The guest molecules, which are apolar, can be


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entrapped within the hydrophobic internal cavity by hydrogen bonding, van der Waals forces,

or entropy-driven hydrophobic effect. In contrast, its hydrophilic external part requires water

as the suspension medium. When the water molecules in the center of the cyclodextrin are

replaced by less polar molecules, the complex is precipitated (Gibbs et al., 1999).

Figure 3.3 Molecular structure and microstructure of beta-cyclodextrin (adapted from

Yuliani et al., 2004)

The microparticles obtained from this technique have unique, sustained release properties

and thermal/chemical stability of the active ingredients entrapped. However, the relatively

high cost of the shell material, cyclodextrin, and the low yield of the end product greatly

restrict its current commercial applications (Gouin, 2004).

Other chemical techniques include co-crystallization, solvent evaporation/extraction from

emulsions, and interfacial or in-situ polymerization. These methods have been reported in a

small number of studies with limited applications. Co-crystallization has been used to

incorporate aroma compounds into supersaturated sucrose syrup at the time of spontaneous

crystallization under the condition of high temperature (>120oC) and low moisture (95-97 o

Brix). Due to its relative simplicity, co-crystallization may evolve into an economical and

flexible process in the future (Madene et al., 2006). Solvent evaporation/extraction is

applied to convert droplets formed by emulsification to solid particles. The mechanism of

solvent elimination from the emulsion droplets is not well known, but considered to havegreat influence on the particle morphology and release behaviour (Rosca et al., 2004). These

techniques are primarily used in the development of drug delivery systems, but attract some

interest from food scientists and technologists recently. Interfacial or in-situ

polymerization involves the formation of capsule shell on the emulsion droplet/particle

surface by indirect or direct polymerization. Interfacial polymerization occurs based on the


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classical Schotten-Baumann reaction between an acid chloride and a compound containing

an active hydrogen atom, such as an amine or alcohol, esters, urea, and urethane. In-situ

polymerization, without the presence of reactants in the core material happens when

monomers are directly added to the system. Both polymerization methods are used for the

encapsulation of herbicides and pesticides with rare applications in food (Gibbs et al., 1999;

Benita, 1996).

As discussed above, all techniques have pros and cons. Generally, physical techniques are

less expensive and easy to scale-up, but have the drawbacks of relatively low payloads and

imperfect particle properties. In contrast, chemical processes are costly and involve

complicated concepts, but typically can provide well-defined particle structures and desired

controlled release properties. Chemical processes are often reported in formulating drug

delivery systems or for making value-added products. For most applications in the food

industry, physical processes are used.

As our program is focused on developing countries, technical/economic feasibility becomes

the main consideration, i.e., low cost of the process, availability of equipment, and ease of

implementation into existing production lines. It would be preferable therefore to

concentrate on physical/mechanical processes. In some cases chemical methods will be

required, as in making Ultra Ricegrains using internal gelation.

3.3.2 Microencapsulation in the Food Industry

Many well developed microencapsulation techniques and numerous wall materials have

been used in food since the 1980s, which results in many food ingredients being

microencapsulated, as shown in Table 3.2. Different techniques and shell materials will be

reviewed in the following sections.

There are a number of reasons for the food industrys use of microencapsulation

technologies, including:

Encapsulation or entrapment can protect the active ingredients from degradation due to

environmental conditions, such as heat, moisture, air, and light;


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The physical properties of original core materials can be modified to make it easy for

handling, e.g., liquid ingredients can be converted to free-flowing powders;

Evaporation or leakage of the core material to the outside environment can be reduced

or controlled, with components such as the volatile flavouring agents;

The unpleasant taste or appearance of some food ingredients can be masked;

Several active ingredients can be segregated within a food matrix in separate forms,

which prevents undesirable interactions between them;

The food ingredients can be tailored to either release slowly or at a certain point within

the process of the digestive system.

Many of these approaches are applicable to our program.


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Table 3.2 Microencapsulated food ingredients (Kirby, 1991; Gibbs et al. 1999, Gouin, 2004; Schrooyen e

Food ingredients Examples Functions or properties Purposes for microencapsulation

Acidulants Adipic, fumaric,citric, lactic and

ascor

fortification in stample foodsdf

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