Beruflich Dokumente
Kultur Dokumente
By
Yao Olive Li
ABSTRACT
A microencapsulation-based technology platform for effective delivery of multiple
micronutrients 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
ii
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.
iii
DEDICATIONS
I dedicate the culmination of my formal studies to my parents:
Drs. Wentong Li & Shukun Chen
to myself
as a great fortieth birthday gift
and as a new start of my academic career in Canada
iv
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
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.
vi
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.
vii
TABLE OF CONTENTS
ABSTRACT.........ii
ACKNOWLEDGEMENTS......v
TABLE OF CONTENTS....viii
LIST OF FIGURES......xii
LIST OF TABLES.....xv
LIST OF APPENDICES xvii
INTRODUCTION1
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
ix
CONCLUSIONS...152
RECOMMENDATIONS .154
REFERENCES.157
10
NOMENCLATURE168
11
APPENDICES.172
xi
LIST OF FIGURES
Figure 2.1
Figure 3.1
19
Figure 3.2
21
Figure 3.3
26
Figure 4.0
36
Figure 4.1
37
Figure 4.2
40
Figure 4.3
40
Figure 4.4
42
Figure 4.5
42
Figure 4.6
65
Figure 4.7
66
Figure 4.8
67
Figure 4.9
Figure 4.11
Figure 4.12 Relative iodine retention in the DFS samples containing different
sources of FePP during 6 months storage at 40oC and 60% RH
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 storage
under 40oC and 60%RH, respectively
Figure 4.14 Iodine-iron interaction in DFS correlation analysis between iodine
and ferrous iron losses in the DFS samples containing various iron
particles after one year storage under 40oC and 60% RH
xii
68
69
74
75
77
79
80
Figure 4.16 Apparent first order degradation kinetics of iodine in the DFS
samples made with various iron particles during one-year storage
under 40oC and 60% RH
81
Figure 5.1
83
Figure 5.2
86
Figure 5.3
Figure 5.4
103
Figure 5.5
107
Figure 5.6
Comparison between glutinous flour and regular rice flour using the
best ratio and addition of HPMC
Colour measurements of the four new formulations made with the
optimized internal gelation systems
Micronutrient fortified Ultra Rice formulations made with the
optimized internal gelation systems
Two proposed models for alginate-calcium gelation in Ultra Rice:
fish-net model for external/diffusion setting (left) and inter-lock
model for internal setting (right)
107
Figure 5.7
Figure 5.8
Figure 5.9
88
111
111
116
Figure 5.10 Alginate-calcium gel structure made with external (left) and internal 116
(right) gelation processes
Figure 6.1
122
Figure 6.2
122
Figure 6.3
Figure 6.4
128
128
Figure 6.5
139
Figure 6.6
140
Figure 6.7
Figure 6.8
Figure 6.9
Colour stability of the Ultra Rice grains made with various FePP
sources
xiii
141
142
144
Figure 6.10 Colour stability in the Ultra Rice grains made with Dr Lohmann
FePP at different addition levels
Figure 6.11 Colour stability of Ultra Rice grains with addition of TiO2 as the
colour-masking agent at different levels
Figure 6.12 Colour stability of Ultra Rice grains made with higher levels of folic
acid and FePP
Figure 8.1 Model premix system for salt fortification made by the extrusion
agglomeration followed by polymer coatings
144
145
145
155
Figure 8.2
Figure 8.3
Model Ultra Rice premix made by extrusion using internal gelation, 156
containing sub-capsules of microencapsulated premixes of iron and
vitamin A made by extrusion-based technology platform
xiv
155
LIST OF TABLES
Table 3.1
19
Table 3.2
29
Table 3.3
31
Table 4.1
39
Table 4.2
52
Table 4.3
56
Table 4.5
Table 4.6
63
Table 4.7
63
Table 4.8
Table 4.9
68
Table 4.10
70
Table 4.11
71
Table 4.12
81
Table 5.1
89
Table 5.2
94
Table 5.3
95
97
Table 4.4
Table 5.4
Table 5.5
Table 5.6
Table 5.7
xv
56
59
64
96
97
98
Table 5.8
101
Table 5.9
101
Table 5.10
102
103
Table 5.15
105
Table 5.16
106
Table 5.17
108
Table 5.18
108
Table 5.11
Table 5.12
Table 5.13
Table 5.14
Table 5.19
Table 5.20
Table 5.21
Table 6.1
Table 6.2
Table 6.3
Table 6.4
Table 6.5
102
104
105
109
112
113
131
133
Folic acid retention in the Ultra Rice samples made with various
FePP sources and at different addition levels
147
xvi
135
138
LIST OF APPENDICES
Appendix 11.1.1
Appendix 11.1.2
Appendix 11.1.3
175
178
Appendix 11.1.4
Appendix 11.1.5
Appendix 11.1.6
Appendix 11.1.7
Appendix 11.1.8
Appendix 11.1.9
Appendix 11.1.10
172
176
177
178
179
183
185
Appendix 11.1.11
186
Appendix 11.1.12
187
Appendix 11.1.13
Appendix 11.1.14
Appendix 11.1.15
Appendix 11.1.16
Appendix 11.2.1
Appendix 11.2.2
xvii
192
Appendix 11.2.3
Appendix 11.2.4
199
Appendix 11.3.1
203
Appendix 11.3.2
Appendix 11.3.3
xviii
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, and
needs 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
1
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
2
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
3
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, CaCl2 solution 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 Rice technology. 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 Rice grains 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 Rice grains made by extrusion are actually encapsulated forms of the selected
4
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
7
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.
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.
8
OBJECTIVE
To develop microencapsulation-based technologies
for multiple nutrient fortification in staple foods
MICRONUTRIENTS
Vitamins: A, B1, niacin, folic acid
Minerals: iodine, iron, zinc
FOOD CARRIERS
Staple foods or food ingredients:
Salt, sugar, rice
APPROACHES
Microencapsulation: converting the selected vitamins and
minerals to appropriate forms which fit into the selected food
vehicles;
Multiple Fortification: incorporating several micronutrients in
their proper forms into the selected food vehicles.
Figure 2.1 Overview of the project scope
2.
3.
The detailed experimental design for each approach and the anticipated outcomes are
discussed in the following.
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
11
fortification with iron or other nutrients. Development of folic acid fortification of these
food vehicles using the technology platform was another focus of my research program.
Folic acid could be incorporated into the selected food vehicles either directly or using
microencapsulated premixes made by techniques developed for salt or rice fortification. The
study was to examine different fortification techniques and to identify appropriate
approaches. While it is preferable to use the simplest techniques to keep cost low and make
implementation easy, encapsulation processes may be needed to maintain vitamin stability
and consumer acceptability. To successfully combine folic acid addition with the delivery of
other nutrients, the understanding of the potential interactions between folic acid and other
micronutrients added in the same delivery systems had to be developed prior to designing an
effective fortification strategy.
The proposed research approaches will be discussed in the following chapters with relevant
results (Chapters 4 to 6).
12
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
13
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).
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,
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.
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.
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 the appearance 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
17
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.
pharmaceutical, food, cosmetic, chemical, and printing industries (Madene et al., 2006).
Microencapsulated actives
References
Pharmaceutical
Biological
Food
Agro-chemical
Pesticides, herbicides
Tsuji, 2001
Cosmetic and
personal care
Core ingredient
Wall material
Technique/Process
Figure 3.1 Schematic relationship between the core material, the wall material, and the
required 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
19
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.
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.
Matrix
Microsphere
Simple
Multi-wall
Multi-core
Microcapsules
Irregular
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 (<40%) and problems with heat-sensitive
materials (Gouin, 2004; Madene et al., 2006; Yuliani et al., 2004; Benita, 1996; SwRI
website).
Spray chilling/cooling is similar to spray drying, where the core material is emulsified in a
molten wall material then atomized to disperse droplets, which are immediately mixed with
21
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 coating involves 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).
22
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
23
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.
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
process can 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.
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).
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 have
great 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
26
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.
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;
27
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;
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.
28
Table 3.2 Microencapsulated food ingredients (Kirby, 1991; Gibbs et al. 1999, Gouin, 2004; Schrooyen et al. 2001; Desai, 2005)
Food ingredients Examples
Functions or properties
Applications
Acidulants
Adipic, fumaric,
citric, lactic and
ascorbic acids
Flavours
Sweeteners
Sugar, artificial
sweeteners such as
aspartame,
Prevent degradation by
Susceptible to heat, moisture, temperature and moisture, reduce
and other components
hygroscopicity, improve
flowability, control release
Chewing-gum, confectionery,
baking foods, mouthwash and
toothpaste
Colourants
Beta-carotene,
turmeric, annatto,
natural colours
Neutrase, lipase,
Enzymes and
lysozyme, pepsin,
microorganisms
amylases, proteases
Nutritional
ingredients
Vitamins, minerals,
amino acids,
essential oils
Miscellaneous
additives
Preservatives,
antioxidants,
levening agents
29
be non-sticky,
be impervious to water,
be inexpensive,
(Gouin, 2004; Schrooyen et al., 2001; Kirby, 1991; Gibbs et al., 1999, SwRI website)
Numerous coating materials have been used in food ingredient microencapsulation. Most of
them are natural or are derivatives of plant or animal food products, which have been
approved by FDA as GRAS (generally recognized as safe) materials. Table 3.3 lists some
commonly used coating materials for microencapsulation of food ingredients and their
applicable techniques.
30
developing countries. On the other hand, certain gums, e.g., sodium alginate, will be used in
the internal gelation for making extruded rice analogues. This polymer has been used
extensively in numerous applications due to its well-known gelling effect. The broad
availability and relative low cost of the materials will encourage the implementation of the
successful formulations developed in this study.
Table 3.3 Commonly used coating materials for microencapsulation of food ingredients
(Gouin, 2004; Schrooyen et al., 2001; Kirby, 1991; Gibbs et al., 1999, SwRI website)
Category
Coating materials
Applicable techniques
Carbohydrates
Cellulose and
derivatives
Gums
Lipids and
derivatives
fats, fatty acids, vegetable oils, mono-, di, or triglycerides, hydrophilic or lipophilic
waxes such as shellac, beeswax, PEG
(polyethylene glycol), paraffin
Proteins
31
Taylor & Davidson (2005) reviewed the applications of liposomes in the food industry and
indicated the potential ability of liposomes to stabilize and protect vitamins in food systems.
For example, liposome-entrapped vitamin A (retinal) demonstrated great stability in terms of
decreased light- or heat-induced degradation rates. Liposomes can be also used to
microencapsulate minerals, such as ferrous sulphate and calcium lactate. Lecithin liposomes
containing iron and calcium have been applied in the fortification of liquid foods, such as
milk (Schrooyen et al., 2001). The high cost of the formulated nutrients made liposomes
economically viable only in high-valued food products, rather than in inexpensive staples.
32
The research group headed by Hurrell and Zimmermann at ETH (Swiss Federal Institute of
Technology Zrich) has been working on iron delivery systems based primarily on ferric
pyrophosphate for years. They have also investigated simultaneous delivery of vitamin A,
iron, and iodine through fortified salt (Windhab et al., 2005). Recently, they proposed a
novel concept, Multi Microcapsule (MULMICAP), for food fortification, especially in salt.
The capsules were produced by continuous microprocessing, including multi-step milling,
dispersing/emulsification, cold spraying, and powder mixing operations. Hydrogenated palm
oil was used to pack iodine, micronized ferric pyrophosphate, and retinyl palmitate, and
form sub-capsules which were then integrated into spherical Multi Microcapsules with a
diameter of 20 to 100 microns. Such microcapsules were applied to African salt by
attachment to the surfaces of 1-2 mm salt crystals. The triple fortified salt (TFS) made this
way was reported to have excellent colour stability and relatively high stability of vitamin A
and iodine (87% and 84% retained, respectively) after 6 months storage. The clinical trials
showed that iron status and vitamin A status in the body were significantly improved in
goitrous children in Morocco after consumption of this TFS for 10 months (Windhab et al.,
2005).
The encapsulation process was later used to produce a coated iron premix, specifically
encapsulated ferrous fumarate (FeFum). A two-step encapsulation process was developed
(Yusufali, 2001), starting with particle agglomeration using a fluidized-bed followed by
pan-coating. Appropriate binders, encapsulants, and solvents for carrying the encapsulants
were carefully investigated. Dextrin was finally selected to mix with the iron compound and
33
the mixture was agglomerated into fine particles, which were then encapsulated by a
colour-masking agent, titanium dioxide, and a thin layer of soy stearine using methylene
chloride as the solvent carrier. The double-fortified salt prepared by such encapsulated iron
premix presented good stability (Yusufali, 2001).
The process was then 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 the organoleptic properties (Oshinowo et al., 2004 &
2007). The encapsulated iron and iodine premixes were then scaled up at Glatt Air
Techniques, NJ, from 5 kg to 60 kg, 120 kg, and finally to large commercial batches of 600+
kg using a 1200 L Wurster type agglomerator/coater (Diosady et al., 2004).
The encapsulated ferrous fumarate has also been tested in Ultra Rice. Improved colour and
unaffected iron bioavailability were observed (Li, 2005). The idea of microencapsulated
premix was also extended for making triple fortified salt with vitamin A, iron, and iodine.
Three Masters graduates attempted to develop formulations for delivering the three
micronutrients simultaneously in salt (Raileanu, 2002; Tay, 2002; Rutkowski, 2003). Single
and multiple premixes consisting of one or more selected nutrients were made by
microencapsulation and incorporated into blank or iodized salt to produce triple fortified salt
(TFS). Unfortunately, storage stability tests have revealed significant losses of vitamin A and
altered sensory properties of the TFS products. This approach will require more work before
it becomes commercially viable. Novel technical solutions are then required to make the
microencapsulation process more effective in terms of protecting the three active ingredients
and preventing their interactions.
Another research activity in our research group focused on rice fortification via Ultra Rice
technology. In this process, rice flour is extruded, producing reconstituted rice grains that
can be used as a carrier for micronutrients. It holds great promise for alleviating
micronutrient deficiencies in populations that consume rice-based diets. A unique advantage
of this technology is of its ability to protect the added micronutrients within the matrix.
34
So far, two stable formulations have been developed within our research group; one is
fortified with vitamin A (Lam, 2006) and the other contains iron, zinc, and several B
vitamins (Li, 2005). Technical feasibility, clinical effectiveness, and consumer acceptability
for the two formulations have been field-tested and achieved acceptable results (PATH,
2007).
In spite of the successes, we are also aware of problems remaining in the current systems of
double fortified salt (DFS) and Ultra Rice, which will be discussed in detail in the later
chapters. However, the research experience in salt and rice fortification projects has
confirmed that microencapsulation is a promising solution for ensuring the effectiveness of
food fortification in combating micronutrient deficiencies. The development of novel
technologies is always ongoing, and it is therefore desirable to adopt the recent advances in
other research fields into this area to make fortified foods better - with desired stability,
bioavailability, and sensory properties while maintaining low cost. This leads to another goal
of this study - to improve the current processes by incorporating new advances in other fields.
The improvements may result in advanced technologies for food fortification based on the
broad concept of microencapsulation. Ultimately, these efforts would extend the benefits of
the two successful interventions, double fortified salt (DFS) and Ultra Rice, in combating
global micronutrient deficiencies.
35
Surface defects observed on the microparticles (Figure 4.0) may cause the leakage of iron in
the presence of water or moisture, subsequently resulting in unwanted interactions and
sensory changes (Rutkowski, 2003). In addition, the porous iron particles are encapsulated
by a lipid material, e.g., soy stearine, which results in further reduced density and causes the
particles float on top of a liquid, readily separated from the salt. While under normal cooking
conditions the fat coating will melt, in cold water consumers would likely consider the
floating encapsulated premix particles as impurities and remove them (Lo, 2006). Moreover,
the coating material currently used, soy stearine, is susceptible to typical fat oxidation and
subsequent off-flavour. This was observed in the Ultra Rice made with the soy
stearine-coated FeFum premix (Li, 2005).
0.5 mm
0.27 mm
36
Extrusion
DoubleFortified Salt
(DFS)
Cutting
Drying
Colour-masking
Blending
IRON
PREMIX
Encapsulation
Coating
Coating
solution
solution
Iodized salt
Figure 4.1 Schematic process flow for making microencapsulated ferrous fumarate
Specifically, ferrous fumarate powder was mixed with binders, such as wheat or rice flour,
water and oil to form a moist dough. The dough was then extruded through an angel-hair die
to form the filaments with the diameter around 0.5 mm using a pasta extruder. These
filaments were then cut into 0.5 mm long cylindrical particles, which were then air-dried and
screened. The particles matching the size of typical salt grains (300 - 700 m) were then
covered with a thin layer of colour-masking agent (titanium dioxide), and encapsulated with
the selected shell materials.
The process is compatible with other unit operations, and is easy to control and operate,
In general, extrusion is a process used to mix, heat, and shear food ingredients to achieve
37
desired shape and texture. In our case, extrusion can agglomerate particles through effects of
compression and/or compaction. During the process, small particles are pressed close
together by mechanical means. The particle-particle adhesion is usually enhanced by the
addition of binders. Thus, the selection of appropriate binder materials was a key factor
considered in this study.
After extrusion the agglomerated particles must be coated to impart the desired chemical and
physical resistance. As discussed before, hydrophilic glassy polymers are extensively used in
oral drug delivery systems to achieve sustained or controlled release. Many hydrophilic
polymers have a high glass transition temperature and are glassy in appearance in their
dehydrated states. These glassy polymers provide an excellent physical barrier to the
diffusional transport of oxygen and small molecular-weight molecules. Upon water
penetration, a slow macromolecular relaxation process at the glass/rubbery swelling front
provides a swelling-controlled mechanism that can be used to regulate the drug release
kinetics (Pham & Lee, 1994). We planned to apply this concept to microencapsulating
micronutrients.
38
Supplier
Local supermarket
Local supermarket
Local supermarket
TM
Crisco (Procter & Gamble,
Shortening
Canada)
Corn flour
Local supermarket
Potato starch
Local supermarket
Dextrin
Casco, Canada
Ferrous fumarate
Almat Pharmachem Inc.
Butylated hydroxyanisole (BHA)
Sigma Chemicals, Canada
Butylated hydroxytoluene (BHT)
Sigma Chemicals, Canada
Sodium hexametaphosphate (SHMP) EM Sciences
Ascorbic acid
Sigma-Aldrich Chemicals
TM
Dow Chemicals Co.
Methocel E3 (HPMC)
Materials for coating
Titanium dioxide
Sigma Chemicals, Canada
BASF, Canada
Kollicoat IR White
TM
Dow Chemicals Co.
Methocel E3 (HPMC)
Ethanol
EM Sciences
Dichloromethane
EMD Chemicals Inc.
Soy stearine
Casco, Toronto, Canada
Chemicals for analysis
Potassium iodate
Sigma Chemicals, Canada
Potassium iodide
Fisher Scientific Co.
Sulphuric acid
EMD Chemicals Inc. USA
Hydrochloric acid
EMD Chemicals Inc. USA
Nitric acid
EMD Chemicals Inc. USA
1, 10-phenanthroline
Sigma Chemicals, Canada
Sodium thiosulphate
BDH Inc.
Potassium hydrophatalate
Sigma Chemicals, Canada
Starch (soluble)
Sigma-Aldrich Chemicals, USA
Hydroxylamine hydrochloride
Sigma Chemicals, Canada
39
Description
Food grade
R&D use
R&D use
R&D use
R&D use
ACS grade
R&D use
R&D use
Pharmaceutical grade
R&D use
ACS grade
R&D use
Food grade
ACS grade
ACS grade
R&D use
R&D use
R&D use
R&D use
0.1 N solution, ACS grade
R&D use
ACS grade
Reagent grade
4.2.2 Equipment
A KitchenAidTM Mixer, Ultra Power Series, Tilt-Head Stand (Figure 4.2), is used for
forming and pre-conditioning of the iron-containing mixture prior to extrusion. The mixer
produces a homogeneous dough-mass consisting of ferrous fumarate, the selected binder,
vegetable shortening, and water.
A La Monferrina P12 (Figure 4.3) pasta extruder was used. The machine is a single-screw,
cold-forming pasta extruder composed of several elements: a motor to drive the screw, two
tanks for dough preparation and extrusion, respectively, a screw, extruder barrel, and an
assembly of an angel-hair die and a four-blade cutter driven by a separate variable-speed
motor. The machine operates at low screw speed and low shear. The die and cutter assembly
defines the shape and size of the iron particles. The angel-hair die forms fine filaments with
a diameter of 0.5 mm, which are then cut longitudinally to form cylindrical particles with a
length of ~0.5 mm by controlling the flow rate and the cutter speed.
40
A smooth barrel,
After extrusion the iron agglomerates are dried to ~8% moisture content using a forced air
oven. The dried particles are screened through the Canadian Standard Sieve Series, to
remove fines and larger dough lumps. Particles with the size range of 300-700 m are
retained for coating.
An Intel PlayTM QX3 Computer Microscope was used to examine the surface characteristics
of the iron premix, while a Hitachi Scanning Electron Microscope (Model S-2500) was used
to examine the particle morphology at various scales.
A Uni-Glatt top-spray fluidized bed apparatus was used to apply glassy polymer coatings to
the extruded iron particles. As depicted in Figure 4.4, the aqueous coating solution was
pneumatically sprayed over the extruded iron particles, which were fluidized by pre-heated
air blown from the bottom of the bed. The detailed operating procedure is presented in
section 4.2.3 and Appendix 11.1.6.
41
Exhaust Air
Expansion chamber
with a filter bag
Column
Entry for pressurized air
Coating solution
Extruded particles
Coating chamber with a bed and an
air distribution plate
Support
Heater and air blower
Heated air
Figure 4.4 Schematic diagram for Uni-Glatt top spray fluidized bed assembly
Spray nozzle
Rotating pan
A laboratory pan coating assembly (Figure 4.5) was used to coat extruded particles with
hydrophobic coating materials, e.g., soy stearine, and Kollicoat IR, where pharmaceuticalgrade dichloromethane was used as the solvent carrier. The coater consists of a rotating pan
42
A CSZ model Z8 environmental chamber (Cincinnati Sub-Zero Co., USA) was used for
storage stability tests to obtain a controlled temperature (40oC) and relative humidity (60%
RH).
Particle Screening
Dried iron agglomerates were screened through a series of Canadian Standard Sieves with
sieve sizes of 1000, 710 and 300 m. Oversize particles and particles <300m in diameter
were discarded. Particles in the size range of 300 - 710 m were used for coating.
43
were off-white in colour with a greyish tint. Colour masking was followed by coating in
either a fluidized bed or drum (rotating pan) coater.
Pan Coating
The colour-masked sample was then placed in the tilted rotating pan. A motor drives the pan
to rotate in a counter-clockwise direction, while the coating solution contained in a Thin
Layer Chromatography (TLC) flask was sprayed over the tumbling particles by compressed
air at 5-6 psig. The detailed operating protocol is attached as Appendix 11.1.6.
44
Iron Analysis
Total iron was measured by atomic absorption spectrophotometry (AAS) using AOAC
method 3.6.1.2 (Fourteenth Edition, 1984). Details are presented in Appendix 11.1.1.
Ferrous iron content in the premix was determined by spectrophotometry (Harvey, Smart, &
Amis, 1955), as a complex with 1,10-phenanthroline. Details are presented in Appendix
11.1.1.
45
Iodine Analysis
Iodine content in the DFS samples was determined by iodometric titration (AOAC method
33.149). Details can be found in Appendix 11.1.1.
Statistical Analysis
Data from chemical assays were obtained from three to four replicates, and reported as the
mean value standard deviation (SD). One-way ANOVA was used to examine the statistical
significance between the sample performances, using the Origin Pro 7.5 Program Package.
46
Based on the results of the preliminary study, the best parameters for each step were
followed to prepare 12 formulations, which involved 7 process variables at 2 or 3 different
levels for each parameter. These formulations were then examined in terms of physical and
chemical properties.
Finally, the optimized formulations were added into iodized salt to form double fortified salt
(DFS). The DFS samples were subjected to a one-year stability test at 40oC and 60% RH.
During the storage test, iodine and ferrous iron retention were followed. The iodine
degradation kinetics and the interaction between iodine and ferrous iron in DFS samples
were analysed.
The initial phase of the study was aimed at proving the technical feasibility of using the
extrusion-based microencapsulation process for making FeFum premix for salt double
fortification and identifying operational parameters for detailed tests.
47
consistency of oil paint, which was not extrudable at all (Trueman, 2005; Lo, 2006). Clearly
some binding materials are required for forming a dough-like mass, which can be extruded.
The pasta extruder requires a feed that is plastic and uniform, since there is very little mixing
or shear in the barrel itself. The available power and the backpressure through the fine die
greatly limit the viscosity of the dough that can be extruded. Therefore the selection of
dough ingredients is critical for both the extrudability of the dough and the consistency of
the extrudate. The formulation requires a binder, water and a lubricant (shortening) in
addition to the active ingredient - ferrous fumarate. The required properties of the binder
edibility (accepted, non-toxic, safe food additive), wide availability, and low cost limited the
selection of acceptable binders to grain flours, proteins and starch derivatives.
In these preliminary investigations, we found that only natural grain flours were able to form
extrudable dough containing the iron compound in terms of ease of extrusion and desired
particle properties. Specifically, rice flour could be used on its own, but worked better with a
secondary binder such as dextrin. The maximum amount of FeFum in the dough was 60%
with careful adjustment of lipid and water content. Wheat flour could be used to form
perfectly extrudable dough on its own. The maximum amount of FeFum in the dough was
70%. The dough was harder to extrude than rice flour dough and the extrudates were bigger
in size. Dough prepared with durum wheat flour had a maximum FeFum content of 75%.
The dough was the easiest to extrude and the extrudates were of uniform size.
48
Potato starch, dextrin, and corm meal failed to bind the FeFum irrespective of the amounts
of water and oil added in the dough. This was likely due to the fact that the starch was not
gelatinized in this process. In industrial practice, wheat flour and rice flour are used to make
pasta or Asian noodle products by using cold-forming extruders, whereas corn meal\flour
and potato starch must be cooked during extrusion in high-shear cooking extruders. Without
heating externally or through shear, the ungelatinized starch from any source is inert like
sand, without any binding capacity.
It is clear that ungelatinized starch in itself was unsuitable for dough formation, and the
binding ability of the cereal flours was dependent, to a great extent, on their protein (gluten)
content. The mixture of pure vital gluten and starch with varied proportions were tested and
mostly resulted in too strong or too weak doughs (Yadava, 2008). The extruder used in the
study was a single-screw, cold-forming pasta extruder, with no controls for screw speed, feed
rate, and external heat input. Only two independent variables, the feed ingredient
composition and moisture content, could be adjusted for effective extrusion. With the limited
mixing and shear in the extruder, the mixture of gluten and starch could not form a
functional dough mass with the binding capacity of the natural combination of the two
biopolymers in grain flours.
Three common cereal flours, i.e., rice, wheat, and durum wheat flours, were further
investigated. The specifications of the materials are presented in Appendix 11.1.3.
We had extensive experience in extruding rice flour to produce re-formed rice grains using
the patented Ultra Rice technology. Therefore it was logical to initially use rice flour as the
binder for granulating FeFum. However, rice flour formed a relatively weak dough due to its
low gluten content (<7%). Accordingly, secondary binders were required to improve the
dough mass for extrusion. Dextrin and HPMC were investigated for this purpose, and the
observations are reported in Appendix 11.1.4.
Dextrin and hydroxypropyl methylcellulose (HPMC) are polymers derived from natural
carbohydrates - starch and cellulose - with relatively smaller molecular weights and simpler
49
chemical structures than their starting biopolymers. This in turns leaves dextrin and HPMC
with limited capability to bind small molecules, e.g., ferrous fumarate, to form an extrudable
dough mass. However, they can be used as secondary binders with small amounts added to
the primary binders, e.g., rice flour, to generate a dough mass with modified properties, such
as lower viscosity, greater strength, and better consistency.
The addition of dextrin or HPMC to rice flour-based FeFum mixture resulted in improved
dough texture. Dextrin made the extruded filaments smoother on both the axial and radial
surfaces, but the particles were brittle. HPMC provided extra strength to the original dough,
which in turn resulted in harder extrudates that were tough to cut, resulting in a rough
surface at the axial cut. Finally, a combination of rice flour and dextrin at the ratio of 80:20
was the most effective in terms of extrudability and particle properties (Appendix 11.1.4).
As indicated earlier, wheat flour and durum flour could be used on their own due to their
higher gluten content. The three cereal flours were used at a level of 25-30% of the total
weight of the dry mixture, which was the minimum range to form an extrudable dough with
the highest amount of FeFum. The detailed observations are presented in Appendix 11.1.5.
In general, durum wheat flour was the best in terms of making the extrusion easy with a
relatively fast production rate. Also, it was capable of carrying more active ingredient, up to
80% (w/w) FeFum in the dry mixture. In addition, the batches made with durum flour had
higher yields of particles with relatively uniform size distribution (300-710 m). However,
SEM images of surface morphology (Appendix 11.1.5) showed that the extrudates made
with durum flour had a more porous texture and rougher surface. This was probably due to
the relatively bigger particle size and higher protein content of durum flour, compared to rice
flour and soft wheat flour used in the study (Appendix 11.1.3).
Apparently, the difference in the particle size of the three raw materials had an impact on the
texture and density of the extrudates. The coarser durum flour was expected to require longer
mixing time, more moisture, and higher shear to be melted in forming a firm agglomerate
texture. As a result, under similar moisture content, mixing time, and extrusion temperature
50
and shear, the particles made with durum flour had more porous texture and relatively lower
bulk density (Appendix 11.1.5).
The key difference between the three binders is in their protein content. Defined as hard
wheat flour, durum flour contains more gluten than the others. During extrusion, gluten
forms a unique viscoelastic structure that works as a stretchable framework around starch
granules (Hui, 2006). Obviously, the higher gluten content in durum flour resulted in easier
operation and faster production rate, as presented in Appendix 11.1.5.
Another advantage of gluten is its high water-holding capacity. This made the dough mass
containing durum flour less dependent on the moisture content compared to the dough made
with rice flour, in terms of achieving similar machine operability. For example, in the case of
rice flour, with a dry mixture containing 30% of the binder and 70% of FeFum powder, the
amount of water added for making desired, extrudable dough became critical and was
limited within a narrow range of 19-20% (w/w).
dough became too viscous and the extruded filaments would stick together, whereas when
less than 18% of water was added, the dough became hard to extrude with a greatly reduced
flow rate and compromised quality of the product, i.e., brittle texture and lack of desired
consistency.
These observations are consistent with the literature and commercial practice, as durum
wheat flour is the usual raw material of most pasta products, whereas rice and corn flours
need to be pre-modified by heat and moisture treatments when used to produce gluten-free
pasta (Seiler, 2006).
To summarize, with the given extruder system, durum wheat flour, the typical ingredient for
pasta making, was the best binder for making extruded agglomerates rich in FeFum.
However, the resulting particle from the extrusion step is only the intermediate product that
requires further processes such as colour masking and encapsulation for making the final
product. It is then necessary to examine the selected binder materials on their compatibility
with colour-masking agents and coating polymers. Therefore, all three grain flours were
51
desirable dough
viscosity/consistency, which in turn affects the flow rate and residence time, as well as the
yield of extrudates with desired characteristics. Thus, effects of water and lubricating oil
addition levels were examined.
FeFum
50%
35%
30%
24% + 6% dextrin
50%
65%
70%
70%
~35%
~32%
~24%
19-20%
Lipids act as lubricants to reduce the friction between particles in the mixture and between
the screw/barrel surfaces and the fluid. Increased oil content was helpful in extruding
mixtures. For example, when the amount of shortening added to the wheat flour/FeFum
52
(70:30) formulation was increased from 2.5% to 5%, the extrusion flow rate dramatically
increased from 60 g/min to 100 g/min. However, this effect was not observed when rice flour
was used as the binder. When the shortening amount doubled from 2.5% to 5%, the wet
mixture of rice flour and FeFum (at the same ratio of 70:30) became non-extrudable with an
oily, slippery texture. This could be explained by the higher gluten content in wheat - the
protein imparted a higher tensile strength to the dough mass, which then required more
lubricant to reduce the friction in the barrel.
The shortening used in the study was semi-solid, hydrogenated soybean oil with a melting
point of 40-50oC. Typical 2.5% (w/w) shortening was added into the formulation together
with the appropriate amount of water. This idea was adopted from our previous study on
Ultra Rice. The pre-melt shortening added in the formulation could remain in its liquid
form during extrusion where a typical extrusion temperature of 50-55oC was attained. Once
the fluid passed through the die, the extrudates quickly cooled to room temperature. This
resulted in a rapid solidification of the lipid dispersed in the particles and a relatively dense
particle texture.
53
different cereal flours had different viscosities and consistencies, which in turn resulted in
different extrusion rates or residence times at the given extruder setting (~50 rpm screw
speed). For example, the formulation made with durum flour had about twice the flow rate
(~150 g/min) of that made with the combination of rice flour and dextrin (~80 g/min). Thus,
a faster cutter speed was required for durum flour formulations, i.e., at the level 4 of the
ten-position speed regulator or ~600 rpm, whereas level 3 (~400 rpm) was sufficient for rice
flour formulations. For a typical 1 kg batch approximately 15-20 minutes would be enough
for completing the extrusion including start-up and shutdown, while the period of time with
a relatively constant flow rate was only about 5 minutes. Close attention had to be paid to the
start-up and shutdown in order to obtain an acceptable yield of desired particles.
Post-extrusion drying
The FeFum dough normally contained ~20% moisture. After extrusion, this value dropped to
16-18% due to slight water evaporation at the die (at a dough temperature of 50-55oC). The
extruded agglomerates were dried in a forced air oven at 50oC for a few hours. Due to the
large surface area, the moisture content in the small particles dropped quickly to ~2% in 5
hours (Rabier, 2006). During the drying process, the bulk density of the particles remained
constant, while the particle density increased by >10%, which was expected as the particles
became denser when the entrapped water was evaporated.
Low residual moisture in the particles is preferred for a stable system and prolonged shelf
life. However, the colour-masking agents, e.g., TiO2, did not adhere well to particles made
with durum wheat flour with <2% moisture content. Apparently, the drying process had a
great impact on the particle surface properties that affected TiO2 adhesion. A drying test was
then carried to determine the optimal residual moisture in the extruded particles for proper
TiO2 adhesion, which will be discussed in the next section.
4.3.2 Investigation on Colour-Masking Process
The FeFum agglomerates made by extrusion were dark brown in colour due to the original
colour of the iron compound. A colour-masking process was necessary to ensure
acceptability of DFS. Titanium dioxide (TiO2) has been used in previous studies for this
54
Titanium dioxide is insoluble in water and only dissolves in hot concentrated H2SO4 and HF
(Merck Index). This greatly limits its applications since it can be only applied to solid
surface by adhesion or by suspension in a liquid carrier. An attempt to apply a wet paste of
TiO2 to the extruded FeFum particles was not successful, as the moisture within the TiO2
paste weakened the particles, which then readily crumbled.
A dry adhesion technique was therefore tested. Usually 25% (w/w, dry basis) TiO2 was
added to the extruded particles (300-710 m) and blended by continuous tumbling manually,
until no dark spots could be seen on the particles. The resulting particles were of a uniform
off-white colour. However, problems occurred when these particles were further
encapsulated with hydrophilic polymer coatings. The adhesion of TiO2 powder to the particle
surface was not strong enough to resist mechanical abrasion or shear force from the
fluidizing air during the next step of encapsulation, resulting in significant loss of TiO2. This
was especially severe when the coating was applied in the fluidized bed, where the
pre-heated air blew off much of the TiO2 before the coating was completed. Furthermore,
this loss of TiO2 made the coating process hard to control, and resulted in tiny open spots on
the surface of the final products (Table 4.3). Particles with this defect could be easily
discriminated in the salt.
55
Table 4.3 Surface defects on the final products caused by the loss of TiO2 during coating
(Pictures are at x60 magnification)
Extruded
Colour-masked with
25% TiO2
Different techniques and other colour-masking agents were then tested aiming to achieve
desirable colour on the iron particles.
4.3.2.1 Different techniques for applying TiO2
Incorporating TiO2 into the dough formulation
Rather than surface coating, TiO2 was incorporated into the dough before extrusion, with the
expectation of producing iron agglomerates with significantly lighter colour. As shown in
Table 4.4, 5% or 10% of TiO2 powder incorporated into the dough was somewhat helpful in
reducing the colour intensity of the iron agglomerates. However, these particles still required
~20% (w/w) of extra TiO2 to fully mask the dark colour. This would result in reduced iron
concentration in the final premix. Unfortunately, the incorporation of TiO2 made the dough
fragile and hard to extrude.
Table 4.4 Comparison of particle colour when TiO2 was incorporated in the dough
formulation before extrusion (Pictures are at x60 magnification)
No TiO2 added
in the dough
5% (w/w) TiO2
added in the dough
56
particles, especially to those made with durum flour. The effect of residual moisture level on
TiO2 adhesion was investigated. The results suggested that moisture was required for proper
coverage by TiO2. For the particles made with durum flour a minimum of 8% moisture was
required (Rabier, 2006). The effect of TiO2 dusting prior to drying the particles at 50oC was
investigated (Appendix 11.1.6).
TiO2 dusting after drying generally formed a thick, uniform layer on the extruded particles.
However, when the colour-masked particles were soaked in water, some of the TiO2 layer
peeled off, exposing the dark cores. Although much of the TiO2 powder dusted onto the
moist iron particles peeled off after drying, leaving only 10~20% on the particle surface,
which formed a thin film, that could survive the water washing. This was more pronounced
in the particles made with durum flour, which had a more porous texture and rougher surface
than those made with rice or wheat flour (Appendix 11.1.5, section 4.3.1.1). This may be due
to the significantly smaller particle size of the pigment (<10m) than the agglomerated
particles (~500 m), which allowed the tiny whitener particles to embed in the bumpy areas.
A small amount of TiO2 would be sufficient to fully cover the dark colour of the
agglomerates, due to the huge surface area of the colour-masking agent.
Since the tiny particles of TiO2 tended to aggregate, it was hard for the fine powder to spread
evenly onto the surface, which in turn required more materials to achieve proper coverage by
forming a thick layer. When subjected to external mechanical forces such as abrasion, this
thick layer of whitener powder tended to peel off.
A number of mechanisms may interact in TiO2 adhesion, including van der Waals force,
formation of solid or liquid bridges, electrostatic force, and mechanical interlocking due
mainly to the surface roughness (Yadava, 2008). By dusting the TiO2 powder onto moist iron
particles prior to drying could form liquid bridges by the interaction between the surface
moisture on the agglomerates and the TiO2 particles (Onwulata, 2005). When the
colour-masked iron particles were subjected to further drying, the liquid bridges (due to
capillary force) formed earlier could be solidified to form solid bridges, which might be even
stronger (Hanus & Langrish, 2007). In addition, there might be mechanical interlocking
57
between the fine pigment particles and the rough surface of the iron agglomerates. The
combined effects of these forces resulted in better TiO2 adhesion to the extrudates made with
durum flour when TiO2 was applied to the moist particles right after extrusion but before
drying. On the other hand, dusting the TiO2 powder onto the dried agglomerates mainly
involved electrostatic forces, which were weaker than other surface binding forces. Still, this
approach seemed to result in better TiO2 adhesion on particles made with rice flour when
~10% of dextrin was used in the extrusion formulation. Dextrin was reported to interact with
titanium dioxide, enhancing the retention of TiO2 on paper coated with dextrin-based
adhesives (Rogols & Hyldon, 1963). In addition, the scientific literature indicated that many
polysaccharides, including starch, have strong interactions with titanium dioxide (rutile) at
pH range of 5~6, which is the isoelectric point of rutile (Liu et al. 2000). Our previous study
of simulated Ultra Rice by extrusion showed that the extruded rice grains had a pH range of
5~6 in the 1% (w/v) solution made of ground grain powder (Li, 2005). Based on these
considerations, one possible explanation would be that the residual moisture at the surface of
the iron extrudates made with the rice flour-dextrin formulation might enhance the titanium
atom hydroxylation at pH 5~6 (also its isoelectric point), which in turn interacted with starch
through an acid/base-like reaction. This interaction model has been used to explain several
metal solid-polymer adsorption and adhesion processes (Liu et al. 2000)
In general, TiO2 dusting before drying was better in forming a stronger, thin film of TiO2 on
the iron particles made with durum flour, which was confirmed by a later study (Yadava,
2008); whereas TiO2 adhesion to the dried agglomerates resulted in better coverage in the
iron extrudates made of rice flour and wheat flour. This was taken into account when
designing the final formulations for storage stability test.
4.3.2.2 Alternative colour-masking agents
Zinc oxide and talc were compared to TiO2 as colour masking agents. Zinc oxide is also
commonly used in the food industry as white pigment; while talc is a common inert filler
and food whitening agent. The results (Table 4.5) showed that more materials were required
when ZnO and Talc were used for proper coverage. This is mainly due to their relatively
lower refractive indices compared to TiO2. Nevertheless, ZnO formed better films featuring
a uniformly thin layer compared to the rough surface produced by Talc.
58
Refractive index
Minimal amount needed to fully cover
the dark colour of the core with a size
of 300-710 m
TiO2
ZnO
Talc (hydrous
magnesium silicate)
2.4-2.9
2.02
1.59-1.60
25%
35%
30%
Surface morphology
(x60 magnification)
59
polymers are extensively used in oral drug delivery systems to achieve sustained or
controlled release. The glassy polymers provide excellent physical barriers to protect the
core ingredient and regulate its release in a swelling-controlled mechanism upon water
penetration. This part of the work was then aimed to investigate the applicability of the
pharmaceutical concept in developing a stable, bioavailable microencapsulated iron premix
for food fortification. It was anticipated that the resulting product would have improved
physical characteristics, desired stability and subsequently reduced interactions with other
food components and/or added micronutrients, as well as desirable release properties when
absorbed in the body.
Two typical glassy polymers were chosen in this study, MethocelTM E3 from Dow Chemicals
Co. and Kollicoat IR White from BASF, due to their availability and success in film coating
of oral drug tablets. MethocelTM is a brand name for hydroxypropyl methylcellulose
(HPMC), which is a cellulose derivative formed by the reaction of propylene oxide and
methyl chloride with alkali cellulose. Depending on the different ratios of hydroxypropyl
and methyl substitution, HPMC is available as various commercial products with different
physical/chemical properties, such as water or organic solubility, viscosity, and thermal
gelation temperature. Variations in the structure and properties make HPMC suitable for a
broad range of applications in drug formulations, food and beverages, household cleaners
and paints, paper and textile products, etc. In these applications, HPMC plays various
functions such as film former, filler/excipient, thickener, lubricant, and emulsifier. In this
study, MethocelTM E3 made by Dow Chemicals Co. was used as encapsulant. It contains
28-30% methyl substitution, 7-12% hydroxypropyl substitution, and has an average viscosity
of 2.4-3.6 mPa.s (in a 2% water solution). MethocelTM E3 was selected because it has been
successfully used as a film-forming agent in the application of oral tablet coatings.
Kollicoat is a brand name of BASF for a full range of excipients used for instant release,
sustained release, and enteric coatings, which share polyvinylalcohol-polyethylene glycol
(PVA-PEG) graft-copolymer as the major polymer component. Kollicoat IR white was
chosen specifically due to its rapid dispersibility in water and low viscosity of the resulting
solution even at polymer concentrations of up to 25%. In addition, it contains a white
60
pigment and a surfactant that enables the embedding of the pigment within the polymer
without segregation or precipitation, which is a drawback of simpler mixtures. Kollicoat IR
White is composed of 45-74% PVA-PEG copolymer, 5-10% polyvinylpyrrolidone, 10-20%
TiO2, 10-20% kaolin, and 1-5% sodium lauryl sulphate. It was expected to enhance
colour-masking of particles during the encapsulation process.
61
The preliminary results showed that both techniques were viable for making encapsulated
products with desired physical characteristics, regardless of the coating materials used. The
resulting iron premixes using either equipment were reproducible even with varied coating
material levels and particle loads. In general, the pan coater was more suitable for coating
with Kollicoat and soy stearine, while the fluidized bed could produce uniform distribution
of MethocelTM forming a viscous, transparent film.
Nonetheless, both techniques had some drawbacks. The agglomerated particles experienced
a great loss of TiO2 within the fluidized bed. This whitener layer was readily blown off by
the fluidizing air stream, and some TiO2 powder was then lodged in the filter bag at the top
of the coating column and could not be recycled to the fluidized bed. This loss of TiO2
resulted in exposed dark spots on the finished products, which further darkened the overall
appearance of the DFS. In the disc coater, the TiO2 powder rubbed off at the beginning of a
typical batch could be eventually reattached to the particles by continuous tumbling.
However, due to the simplicity of this open apparatus, some finished or partially finished
products could fly off the rotating pan, resulting in particle loss as high as 20%. In addition,
when aqueous solutions for hydrophilic polymers were used, the slow evaporation rate at
room temperature caused the coated particles to stick together and form big clumps. Based
on these observations, standardized operation protocols for achieving acceptable,
reproducible results were developed for both machines (Appendix 11.1.8). These standard
protocols were carefully followed later in the preparation of the 12 final formulations for the
stability test.
62
Options
Binder material
Antioxidants
TiO2 adhesion
(25% of the extrudates)
Coating material
Coating technique
Table 4.7 Formulation design of microencapsulated FeFum premixes made by extrusion and
polymer coatings
As shown in Table 4.7, the tested formulations included 6 iron extrudates, 8 colour-masked
particles, and 12 iron premixes. While the 12 premixes were the end products for preparing
double fortified salt, the other iron particles were intermediate products which could also
provide important information with respect to the effects of the selected materials/techniques
on the particle properties. The detailed composition of the 12 final formulations is presented
in Appendix 11.1.9.
63
Table 4.8 Total iron and ferrous iron contents in the 12 final formulations
Extruded
formulation
Total Fe
(% w/w)
Colour-masked
formulation
Total Fe
(% w/w)
E-1
21.5 1.1
C-1
16.9 0.6
E-2
E-3
E-4
21.4 0.9
Total Fe
(% w/w)
Ferrous Fe
(% Fe2+/FeTotal)
P-1
19.0 0.4
94.0 0.3
P-2
P-3
17.7 0.6
96.2 1.1
P-4
19.2 0.8
96.5 1.2
P-5
16.8 0.4
90.9 1.0
P-6
20.1 0.8
95.8 0.6
P-7
17.0 0.5
94.7 0.6
C-2
C-3
16.7 0.8
C-4
17.7 0.5
C-5
17.2 0.1
P-8
18.4 0.5
97.9 1.4
C-6
17.7 0.1
P-9
16.7 0.7
95.8 1.3
C-5
17.2 0.1
P-10
16.9 0.4
95.9 1.4
21.3 0.4
21.4 1.8
Encapsulated
premix
E-5
20.9 1.1
C-7
P-11
17.3 0.4
95.5 0.6
E-6
20.8 0.6
C-8
17.9 0.9
P-12
16.7 0.7
93.9 0.8
Note: the results are mean standard deviation, which were obtained from three or four
replicates. Some formulation data were missing due to the loss of the samples or the
analytical errors.
64
The reason for the higher iron content obtained in the newly lab-processed premixes was due
to the fact that the extrusion produced denser particles with fewer pores, which in turn
required less colour-masking and coating materials. Over 90% of the original FeFum added
in the premixes remained in its ferrous form, regardless of the formulation used. This
indicates
that
the
selected
combinations
of
binder/coating
materials
and
Figure 4.6 Effect of binder materials on iron digestibility of the extruded particles (Note 1:
Data were from the extruded formulations: E-1, E-3, and E-6, which had the same ingredient
composition, only varied in binder types.) (Note 2: the error bars represent the standard
deviations from three or four replicates of the measurements on the same samples.)
65
As shown in Figure 4.7, the coating materials used in the study had a more pronounced
effect on iron digestibility. The premixes coated with Kollicoat seemed to have a relatively
faster dissolution rate, over 85% of the iron dissolved within the first half hour, whereas
<80% of iron was released from the formulations made with MethocelTM. This may be due
to the fact that Kollicoat, made by BASF, contained some emulsifiers, which likely resulted
in a quicker coating disintegration and iron dissolution. On the other hand, Formulation P-5,
which was coated with 10% of soy stearine, showed retarded iron release. The dissolution
rate for this formulation was significantly slower than that of other premixes coated with
hydrophilic polymers (p<0.05).
Figure 4.7 Effect of different coating materials on iron digestibility of the microencapsulated
premixes (Note 1: data were from the encapsulated premixes: P-4, P-5, and P-12, which had
the same ingredient composition and used the same colour-masking/coating methods, but
varied in binder and coating materials.) (Note 2: the error bars represent the standard
deviations from three or four replicates of the measurements on same samples.)
Nonetheless, all 12 formulations released essentially all iron within 2 hours. The detailed
dissolution profiles for all 12 formulations are presented in Appendix 11.1.10. This was
expected, as the coating materials used for most formulations were hydrophilic with high
water solubility.
66
As expected, each step of the encapsulation process had a cumulative effect against iron
dissolution. As shown in Figure 4.8, the amount of iron dissolved in 2 hours was not affected
by any of the processing steps, while the dissolution rate was slowed somewhat with each
processing step. The extrusion formed the particles which were sufficiently porous not to
impede the rate of iron release. Both the TiO2 colour-masking step and the encapsulation
process seemed to slow down the initial iron dissolution rate, but this difference was not
statistically significant (p>0.05) after 2 h when essentially all of the iron in the premixes was
released.
Figure 4.8 Effect of each step of the encapsulation process on iron digestibility (Iron
dissolution profiles in the formulations made with 30% of durum flour as the binder and
coated with 25% of TiO2 and 15% of MethocelTM, specifically with the data from the
formulations of E-3, C-3, and P-4, respectively.) (Note 2: the error bars represent the
standard deviations from three or four replicates of the measurements on same samples.)
67
weak acid solution after 2 hours. However, when the binder content was reduced to 25% in
the durum formulation, the leakage of iron increased. This suggested that a minimum of 30%
binder material might be required to form agglomerates with acceptable integrity.
Figure 4.9 Effect of binders on particle integrity when dissolved in pH 4 HCl solution (Data
from formulations E-1, E-3, E-4, E-6)
Table 4.9 Effect of coating materials on premix integrity in pH 4 HCl solution
Formulation
P-1
P-2
P-3
P-4
P-5
P-6
P-7
P-8
P-9
P-10
P-11
Binder
30% rice/dextrin
30% rice/dextrin
30% durum
30% durum
30% durum
30% durum
30% durum
25% durum
25% durum
25% durum
30% wheat
Coating material
10% Methocel
15% Kollicoat
15% Methocel
10% Methocel
10% soy stearine
10% Methocel
15% Kollicoat
5% Methocel
15% Kollicoat
5% Kollicoat
15% Methocel
% Iron dissolved
after 30 minutes
% Iron dissolved
after 2 hours
5
4
3
3
4
5
8
10
9
6
13
19
12
20
17
22
23
24
27
15
30% wheat
10% Kollicoat
8
21
P-12
The detailed dissolution profiles for all 12 formulations are presented in Appendix 11.1.11.
68
As seen in Table 4.9, the hydrophilic coatings seemed to enhance iron release at pH 4. After
2 h, the majority of formulations released over 20% of the original iron, compared to only
~10% in uncoated agglomerates (Figure 4.9). This suggested that encapsulation with the
hydrophilic polymers accelerated the iron leakage. Conversely, the insoluble TiO2 layer
seemed to make the particles more resistant to water penetration, as shown in Figure 4.10.
50
% Fe dissolved
40
30
20
10
30
60
90
120
Figure 4.10 Effect of each step of the encapsulation process on iron premix integrity in pH 4
HCl solution (Data from the formulations starting with the same extrudates, followed by
same TiO2 colour-masking, but varied in coating materials, specifically E-3, C-3, P-4, P-5,
P-7)
Compared to MethocelTM, Kollicoat coatings seemed to make the premixes less resistant to
water penetration when dispersed in the weak acid solution (Figure 4.10). This was
consistent with the results from iron in-vitro bioavailability tests, where the premixes coated
with Kollicoat had a relatively faster dissolution rate in the pH 1 HCl solution. Again, the
small amount of emulsifier contained in the Kollicoat formulation made the polymer film
(PVA-PEG) formed on the iron particle surface more water-friendly. On the other hand,
the film of soy stearine protected the core well at the beginning, due to its hydrophobic
nature, while after 2 h the premix coated with 10% of the lipid still released ~20% of iron
content. This may be due to poor film-forming by the lipid. Clearly, the coating level (10%)
in this formulation (P-5) was not enough for the lipid to form a continuous
water-impermeable film around the iron extrudates.
69
Table 4.10 Bulk density changes in iron particles after each processing step
Bulk
Extruded
density
formulation
(g/mL)
E-1
1.054
E-2
E-3
E-4
E-5
E-6
1.182
+12.1
C-2
0.942
1.056
1.042
C-3
1.112
+18.0
P-1
1.024
-13.4
P-2
P-3
1.005
-9.6
P-4
1.005
-9.6
P-5
0.937
-15.7
P-6
0.986
-8.0
P-7
0.962
-10.3
C-4
1.072
+13.8
C-5
1.232
+16.7
P-8
1.134
-8.0
C-6
1.161
+9.9
P-9
1.065
-8.3
C-5
1.232
+16.7
P-10
1.155
-6.3
1.169
+12.2
P-11
1.102
-5.7
P-12
1.019
-12.8
C-7
C-8
It is worth noting that formulations P-6 and P-7 were prepared with TiO2 adhesion before
drying. The colour-masked particles were further dried and the extra, loose TiO2 powder was
removed prior to coating with hydrophilic polymers. Thus, these two formulations showed a
lower bulk density of <1 g/mL. Except these two, all formulations using hydrophilic
polymers had a bulk density of >1 g/mL, which was one of the goals for improved premix
properties. Soy stearine coated particles had the lowest density (the formulation P-5), which
was consistent with previous observations (Lo, 2005).
The results for particle density were even better. As seen in Table 4.11, premixes prepared
70
with the new process, i.e., extrusion followed by hydrophilic polymer coating, had much
higher particle densities than the premix made using fluidized-bed agglomeration and lipid
coating. At 1.70 to 1.85 g/mL, the particle density of the extrusion-based premixes was
similar to that of Kenyan iodized salt. This would ensure uniform distribution of the
encapsulated iron premix in the iodized salt when making DFS samples. Therefore,
high-quality DFS samples could be prepared with minimal subsequent particle segregation.
Table 4.11 Comparison of particle densities of various premixes and raw ingredients
71
shared a similar surface morphology showing fine TiO2 particles fully or partially embedded
in the gelled polymer. Conversely, in the case of MethocelTM, when some TiO2 was blown
away during the fluidized bed coating, the exposed areas were later covered with a
transparent film of gelled HPMC polymer, leaving dark spots on the particles.
The uncoated, extruded particles also caused significant iodine loss, varying from ~50% to
75%, depending on the binder materials and the amounts used. There was a slight difference
between binder materials; however, the amount of the binder used in the extrusion
formulation had a greater impact. When less binder was used, the iron was more exposed in
the particles, which resulted in more iodine loss in the fortified salt samples. For example,
E-3 and E-4 containing 30% and 25% durum flour, respectively, the iodine retention in the
DFS made with E-4 was approximately half of that made with E-3 (~25% vs ~50% iodine
retained). Clearly, an effective coating is needed to prevent iodine loss.
which resulted in higher iodine retentions, varying from 56% to 63%. There was no obvious
effect from the TiO2 adhesion techniques used. Whether the pigment was dusted onto the
moist iron particles right after extrusion or onto the dried particles, the use of same amount
of the whitener (25%, w/w on dry basis of the extrudates) could provide equivalent
protection for iodine in the DFS.
Eight of twelve encapsulated FeFum premixes retained >75% of the iodine in DFS after 1
year storage. Exceptions were in formulation P-7 made with 30% of durum flour as the
binder and 15% of Kollicoat as the coating material, and the three samples made with 25%
of durum flour with various coatings. Apparently, the extruded iron particles with less binder
could not provide sufficient protection even with TiO2 and polymer film coatings. There was
no clear difference in the performance of MethocelTM HPMC (Dow Chemicals Co.) and
Kollicoat IR white (BASF).
Since the designed formulations differed in many operating variables, it was difficult to
generate a general conclusion with respect to the effects of different materials and techniques
used in each process step. When the data were grouped as extruded, colour-masked, and
encapsulated, trends became clearer, as shown in Figure 4.11.
One-way ANOVA test was used to analyze the results. Clearly, each step of the process, i.e.,
extrusion, colour-masking, and polymer coatings, had a significantly positive effect, which
added up together to result in desired iodine stability in the DFS samples containing the
newly lab-processed FeFum premixes. The new premixes had virtually no interaction with
iodine since similar iodine retention was obtained compared to that in the iron-free iodized
salt.
Over the storage period, large iodine losses were observed in all uncoated samples.
73
Figure 4.11 Iodine stability in DFS samples containing various FeFum forms powder,
extruded, and colour-masked particles, as well as encapsulated premixes, after one-year
storage at 40oC and 60% RH. (Note: the results are the mean values obtained from three or
four replicates, and the error bars represent the standard deviations.)
A parallel experiment was performed to compare microencapsulated FeFum premix with
other iron sources in DFS. So far, the two most successful approaches to the development of
DFS, one has focused on increasing the bioavailability of less reactive iron compounds, such
as ferric pyrophosphate (FePP), and the other on microencapsulation of reactive,
bioavailable iron compounds, such as ferrous sulphate and ferrous fumarate.
The iron bioavailability of the chemically very stable ferric pyrophosphate can be enhanced
by reducing its particle size, i.e., micronization. It is reported that the bioavailability (RBV)
of micronized ferric pyrophosphate (FePP) increased from 50% to 95% when the average
particle size was reduced from 21 m to 0.5 m (Wegmller et al., 2004). Several studies
have been conducted using micronized FePP for salt dual fortification, and improved iron
status was achieved in African children (Zimmermann et al. 2004; Wegmller et al. 2006).
However, micronization dramatically increases the surface area available for reaction, and
thus the increased reactivity of micronized FePP with iodine causes substantial iodine loss
when added into iodized salt (Andersson et al. 2008).
74
On the other hand, encapsulation of reactive iron compounds in an inert but digestible
coating material results in effectively fortified foods with high iron bioavailability, while
reducing chemical interactions with other micronutrients and minimizing sensory changes
(Hurrell, 2002; Zimmermann et al., 2004; Andersson et al., 2008). With careful formulation
design, the encapsulated iron fortificants could achieve a good balance between reactivity
and functionality in fortified foods.
As shown in Figure 4.12, the sample containing FeFum powder retained only 46% of iodine
after 6 months, which was still better than any of the FePP containing samples. This was
somewhat surprising since FeFum was supposed to be more reactive than FePP.
Nevertheless, the micronized pyrophosphates (25, 2.5, and 0.5 m in particle size for
Fortitech, Dr. Paul Lohmann, and SunActive, respectively) had larger surface area than
FeFum (50~100 m), which resulted in increased interaction between iron and iodine, and
subsequently higher iodine losses.
Figure 4.12 Relative iodine retention in the DFS samples containing different sources of
FePP during 6 months storage at 40oC and 60% RH (Note: the results are the mean values
obtained from three or four replicates, and the error bars represent the standard deviations.)
75
As presented in Appendix 11.1.15, the extrusion, colour-masking, and polymer coatings, had
little impact on ferrous oxidation, with >90% of the iron retained in its ferrous form at the
beginning of the storage test. After 10 months storage at ambient conditions, little ferrous
loss occurred in the absence of iodine, with up to 5% of loss in extruded particles, 1-4% of
loss in colour-masked particles, and <2% of loss in most of the encapsulated samples.
However, when these iron particles were added into iodized salt and stored at higher T and
RH, the ferrous iron losses were more pronounced: up to ~6% in the encapsulated premixes,
~11% in the colour-masked particles, and 13% in the extruded particles. Again, it is clear
that each process step had cumulative effect in the protection of not only iodine, but also
ferrous iron. The results confirmed that interaction between iodine and ferrous iron occurred
in the DFS samples during storage, which will be discussed further in a later section.
To better understand the effects of the key parameters of the process, the data were grouped
by process stages extruded, colour-masked, and encapsulated - and analyzed using
one-way ANOVA test, as shown in Figure 4.13. Not surprisingly, the two encapsulated forms,
newly lab-processed premixes coated with hydrophilic polymers and the Glatt premix made
by pilot scale fluidized bed agglomeration and soy stearine coating, had significantly higher
76
ferrous iron retentions (p<0.05) than the other iron particles. It confirms that all coatings
played an important role in protecting the stability of ferrous iron. Similarly, the
colour-masking layer of TiO2 also provided a physical barrier to the iron extrudates, which
resulted in a less pronounced, but significant reduction in ferrous loss. Although the
averaged value of ferrous retention in the extruded particles was greater than that of the iron
powder, the difference between these two groups was not statistically significant (p>0.05).
Figure 4.13 Ferrous iron stability in various FeFum forms in DFS samples, after 10 months
storage at the ambient condition and one-year storage under 40oC and 60%RH, respectively.
(Note: the results are the mean values obtained from three or four replicates, and the error
bars represent the standard deviations.)
When the ferrous retention values were compared within each group over the storage period,
it was noticed that ferrous iron was stable at the ambient conditions in the absence of iodine.
In contrast, when added into the iodized salt and stored at higher T and RH, the FeFum
powder, the extruded, and colour-masked particles experienced significant ferrous losses
over the year. Clearly, both the storage conditions and the presence of iodine contributed to
the ferrous loss. However, it can be concluded that the formulation composition and
processing techniques in the steps of extrusion and colour-masking had greater impacts than
variations in the encapsulation process.
77
2+
3+
2 I + 10 Fe I2 + 10 Fe
The microencapsulation was aimed at minimizing the direct contact of the two species in a
single food matrix by keeping them in separate phases. The effectiveness of the
microencapsulation system developed in this study in achieving this goal was then examined
by a correlation analysis between the losses of the two nutrients in the same DFS samples
during the storage.
Based on the results of iodine and iron retention in the DFS samples after one year of storage
at 40oC and 60% RH, the losses of the two minerals were calculated as shown in Figure 4.14.
Ferrous losses not due to the interaction with iodine were subtracted from the values of
ferrous loss in DFS to calculate the actual ferrous loss due to the interaction with iodine.
(Detailed data analysis is presented in Appendix 11.1.16).
Similarly, the actual iodine losses due to the interaction with iron were calculated by
subtracting the iodine loss in the control the original iodized salt - from the losses of iodine
in the DFS samples containing various particles. A plot was then generated using the iodine
loss values against the ferrous losses (Figure 4.14). The points represented the experimental
data, while the straight line was calculated from the stoichiometric equation. The molar ratio
between iodate and ferrous iron is 1:5. However, the experimental data were reported as the
losses in mass. Therefore, the atomic weights of iodine and iron, 127 and 56, were used to
correct the ratio between the two elements by a factor of 1:2.2 (iodine vs. ferrous iron).
78
Iodine-iron interaction
Stoichiometric ratio
100
80
Control - FeFum
powder
60
Extruded
40
Colour-masked
20
Newly Encapsulated
0
0
50
100
150
200
250
Figure 4.14 Iodine-iron interaction in DFS correlation analysis between iodine and ferrous
iron losses in the DFS samples containing various iron particles after one year storage under
40oC and 60% RH
The experimental results matched the theoretical value very well, confirming that interaction
between iodine and iron occurs in the DFS samples. Each step of the microencapsulation
process had positive effects on preventing the direct interaction, as shown with a
simultaneously progressive reduction in losses of both iodine and ferrous iron. When
examining the individual effect of each step, it is clear that the extrusion process had the
greatest impact on reducing the loss of both nutrients due to the significant reduction in
surface area when the iron powder was agglomerated into a more compacted form. The
coating of TiO2 enhanced this effect further due to the solid physical barrier formed, which is
highly water-insoluble. The actual coatings were also critical in stabilizing both nutrients.
79
The experimental data were then plotted as ln (% iodine retention) vs. the storage time, as
shown in Figure 4.16. A generally good correlation between the data and the linear
regression lines (with the correlation coefficients >0.9 for all sample groups) confirmed that
100
Corr. Coef.
a
80
0.9974
Newly encapsulated
0.9871
0.9996
Colour-masked
0.9720
Extruded
0.9951
0.9930
a
60
b
c
40
20
0
d
0
12
15
Figure 4.15 Correlation of iodine degradation in the DFS samples with a first-order
degradation pattern. (Note 1: the values of the regression lines with different superscripts are
statistically significant at p<0.05). (Note 2: the results are the mean values obtained from
three or four replicates, and the error bars represent the standard deviations.)
The statistical analysis on the degradation trend lines suggested there was no significant
difference (p>0.05) between the two encapsulated groups and the control the original
iodized salt. It indicated the incorporation of well-coated iron premixes in the iodized salt
did not cause changes to iodine degradation kinetics. However, the incorporation of uncoated
and partially processed FeFum had significant impacts on iodine degradation parameters,
with progressively increased first order rates and decreased half-lives, as shown in Table
4.12.
80
Figure 4.16 Apparent first order degradation kinetics of iodine in the DFS samples made
with various iron particles during one-year storage under 40oC and 60% RH
Table 4.12 Iodine first order degradation rate constant (k) and the storage half-life estimated
for the DFS samples containing various FeFum particles when stored at 40oC and 60% RH
Iodine degradation rate
constant (k) (month-1)
Half-life
(months)
0.0185
38
0.0202
35
0.0228
31
0.0376
19
0.0567
12
FeFum powder
0.1202
DFS samples
81
82
Dough formation
Mixing rice flour with a binding matrix including micronutrients, antioxidants,
moisture barrier agents, binding agents, cross-linking agents
The process starts with blending the selected micronutrients into rice flour and other
components, including alginate, shortening, and antioxidants. The homogeneous wet mixture
is formed into a dough mass, which is then extruded to produce rice-shaped kernels. After
extrusion, calcium chloride solution is sprayed on the extruded rice grains so that a
83
crosslinking reaction between calcium and alginate would occur to harden the surface of the
simulated rice grains. This crosslinking/hardening process is the core of the Ultra Rice
technology.
Ultra Rice grains consist of highly fortified, reconstituted rice grains manufactured by
extrusion, which are then blended into market rice. It is a promising novel technology for
rice fortification, which has the unique advantage of protecting the added micronutrients
within the manufactured grains (PATH, 2007). Previously, two stable formulations
containing selected micronutrients have been developed within our research group under the
sponsorship of PATH. These formulations have been field-tested and achieved acceptable
results in terms of production feasibility and product stability.
However, the process created some problems: although the reconstituted grains have the
shape and size of actual rice, they can crack and disintegrate during washing, soaking, and
cooking. This may be caused by the failure of the rigid surface structure, which is too weak
or too rigid to resist or stretch with the rice flour core when the starch absorbs water and
gelatinizes under high temperature and pressure. The formation of the thin Ca-alginate
surface network is highly dependent on the sufficient, uniform covering of the surface with
calcium chloride spray. This coating process is hard to control and rather unreliable, as
demonstrated in recent large-scale field tests in Brazil (PATH report, 2008). These
constraints have greatly hindered the commercialization of the technology. Therefore, it is
necessary to explore other techniques for making the reformed rice grains with improved
structural strength.
Converting all of the Na alginate to the cross-linked Ca alginate would provide a complete
three-dimensional cage structure that would enclose and protect the flour and the added
micronutrients. To achieve this, the Ca alginate structure must be developed throughout the
grain after extrusion forming. The objective of this phase of the program was to investigate
techniques for forming the cross-linked alginate structure in-situ after extrusion, and to
develop viable commercial technology for the production of Ultra Rice based on internal
gelation. This approach required that both the alginate (as Na salt) and Ca be present in the
84
dough in an inactive form, which would be released to allow the reaction to proceed under
controlled conditions preferably right after the extrusion step. I proposed to incorporate
calcium in the form of a salt into the dough mixture before extrusion, which could be
released by either heat or by a chemical reaction. However, it was anticipated that premature
internal gelation would make extrusion difficult due to the high viscosity of the system, or
would result in the destruction of the newly formed cross-linked structure by shear in the
extruder. Thus, the initial objective of the work was to find techniques for the slow,
controlled release of calcium and completion of the crosslinking reaction.
Alginate is a linear co-polymer composed of two monomeric units, D-mannuronic acid and
L-guluronic acid. The D-mannuronic acid in the polymer is connected in the -configuration
through 1,4 linkages, while the L-guluronic acid is -1,4- linked (Draget et al., 2005). The
monomers form different regions, which are made up exclusively of one unit or the other
(M-blocks or G-blocks), or regions with two monomers in an alternating sequence (Figure
5.2). Because of the particular shapes of the monomers and their modes of linkage in the
polymer, the geometries of the G-block, M-block, and alternating regions are substantially
different. Specifically, the G-blocks are buckled whereas the M-blocks have a ribbon shape.
When two G-blocks are aligned side by side, a diamond shaped hole will form, which has
85
dimensions ideal for the cooperative binding of calcium ions. As depicted in Figure 5.2,
when calcium ions are added into an alginate solution, a Ca-induced G-block alignment
occurs, and the calcium ions are bound between two polymer chains like eggs in an egg box,
resulting in a so-called egg-box structure (Draget et al., 2005).
Alginate monomers
Chain conformation
Block distribution
Figure 5.2 Structural characteristics of alginate (on the left, adapted from ISP Product User
Guide) and the egg-box structure formed by alginate-Ca gelation (on the right, adapted from
FMC Technical Brochure)
The overall composition of M/G residues and their distribution patterns vary with seaweed
species. Together with the length of the blocks, these factors determine the physical and
chemical properties of the alginate molecules. Specifically, G-blocks provide gel-forming
capacity, while MM and MG provide flexibility to the uronic acid chains with flexibility
increasing in the order GG<MM<MG (FMC Technical Brochure). In terms of gel formation,
high G alginates (with an M/G ratio <1) provide strong, brittle gels that are heat stable, while
high M alginates (M/G ratio >1) form weaker, more elastic gels that have less heat stability
(ISP Product User Guide).
The Ca/G-block stoichiometric ratios for egg-box dimers and multimers are estimated as 1/4
and 1/2, respectively (Fang et al., 2007). The stoichiometry of the gelation process can be
expressed by the following ion exchange reaction (Khairou et al. 2002):
2(Na - Alg)n + Ca2+ = (Ca Alg2)n + 2Na+
where Na-Alg denotes the sodium alginate (C5H7O4COONa), Ca-Alg2 is the Ca-alginate gel
complex. Based on the molecular weights of 216 g/mol and 110.98 g/mol for sodium
alginate and CaCl2, the amounts of CaCl2 and alginate used in the original formulation of
86
Ultra Rice (0.5 wt% and 1.5 wt%, respectively) provide a molecular ratio of 1/1.54, which
is sufficient for the complete conversion of Na-alginate in the system to its cross-linked Ca
form.
In practice, alginate-Ca gelation is obtained through two approaches: diffusion setting and
internal setting (Draget et al., 2005). The diffusion method is characterized by letting a
crosslinking ion (e.g. Ca2+) diffuse from an outer reservoir into an alginate solution. Internal
setting (sometimes also referred to as in situ gelation) differs from the former process, as the
Ca2+ ions are released in a controlled fashion from an inert calcium source within the
alginate solution. The rate of release is usually controlled by pH change and/or through the
limited solubility of the calcium salt source (Draget, 2000).
In the case of internal setting, Ca salts including the insoluble CaCO3 or the slightly soluble
CaSO4 may be used as a Ca2+ source. Alternately the Ca2+ ions may be complexed in a
chelating agent (EDTA, citrate, etc.). The activation of the crosslinking ions is usually linked
to a change in pH caused by the addition of organic acids or lactones (Poncelet, 2001).
Lowering of the pH readily releases Ca2+ from CaCO3 or other complexing compounds.
Generally, the rate at which the calcium is made available to the alginate molecules depends
primarily on pH in addition to the amount, particle size, and intrinsic solubility
characteristics of the calcium salt (Draget et al., 1991; ISP Product User Guide).
In most situations, the release of Ca during the ingredient mixing is so rapid that a
sequestrant is required to control the reaction by competing with the alginate for binding Ca
ions (Draget et al., 2005). Commonly used food-approved sequestrants include sodium
hexametaphosphate (SHMP), sodium tripolyphosphate (STPP), tetrasodium pyrophosphate
(TSPP), ethylenediaminetetraacetic acid (EDTA), and sodium citrate. Since the ultimate
distribution of the Ca ions between the alginate and the sequestrant favours the latter
progressively, an optimum level of sequestrant in the system needs to be carefully
investigated (McHugh, 1987).
As suggested by Figure 5.3, several factors play important roles in alginate-Ca gel formation
and its properties. Potential strategies for internal setting thus include using different Ca
87
sources with limited solubility; using appropriate sequestrants to compete with alginate for
binding Ca ions; changing some environmental factors such as pH and temperature to alter
the solubility of selected Ca sources and/or the activity of the sequestrants.
Figure 5.3 Factors affecting alginate-Ca reaction (adapted from ISP Product User Guide)
5.3 Experimental Strategy Towards Formulation Design
Based on the literature review, three mechanisms of controlled or delayed gelation of
alginate and calcium were proposed, including 1) the use of calcium salts with limited
solubility and appropriate sequestrants at a desired ratio, 2) changing the pH of the system to
trigger slowed release of calcium from an insoluble salt, and 3) changing the temperature to
alter the activity of added sequestrants in the system so as to free some bound calcium ions
slowly.
88
Supplier
Description
Alginate sources
Danisco FD170
Food grade
Danisco FD175
Danisco, Denmark
Danisco FD120
Danisco FD155
Food grade
Food grade
Food grade
FMC RF6650
FMC LF20/40
TIC HG600F
TIC 900
ISP DMF
ISP GMB
Food grade
Food grade
Food grade
Food grade
Food grade
Food grade
Calcium sources
Calcium chloride
Sigma-Aldrich Chemicals
R&D use
Food grade
Calcium carbonate
Food grade
Calcium iodate
Sigma-Aldrich Chemicals
R&D use
Calcium lactate
Food grade
Sigma-Aldrich Chemicals
R&D use
Sigma-Aldrich Chemicals
R&D use
Sodium citrate
Sigma-Aldrich Chemicals
R&D use
Sodium EDTA
Sigma-Aldrich Chemicals
R&D use
Sigma-Aldrich Chemicals
R&D use
Sequestrant sources
Rice flour
Local supermarket
Local supermarket
Shortening
Sigma-Aldrich Chemicals
R&D use
Sigma-Aldrich Chemicals
R&D use
Citric acid
Sigma-Aldrich Chemicals
R&D use
Ascorbic acid
BASF, Canada
Pharmaceutical grade
Sodium ascorbate
Sigma-Aldrich Chemicals
R&D use
Erythorbic acid
Sigma-Aldrich Chemicals
R&D use
89
Sodium erythorbate
Sigma-Aldrich Chemicals
R&D use
BASF, Canada
Pharmaceutical grade
Thiamin mononitrate
BASF, Canada
Pharmaceutical grade
Folic acid
BASF, Canada
Pharmaceutical grade
R&D use
Zinc oxide
R&D use
MethocelTM HPMC
Rice flour and soybean shortening were procured from local markets. Sodium alginates were
obtained through PATH from 4 suppliers, Danisco, ISP, FMC Biopolymers, and TIC Gums.
They vary in particle size, viscosity, and M/G composition. Their detailed specifications are
discussed in the results section. Several calcium salts were obtained from Sigma-Aldrich and
Dr. Paul Lohmann as food-grade, including CaCl2, CaSO4 dihydrate, calcium lactate, CaCO3,
and Ca(IO3)2. Metal chelating agents including TSPP, STPP, SHMP, EDTA, and sodium
citrate were obtained from Sigma-Aldrich.
Moisture content
Residual moisture content in the finished grains was determined gravimetrically after drying
at 105oC to constant weight.
The determination of grain cooking integrity was done by a similar measurement. ~5 g of the
grain sample was placed in a small aluminum bakery dish with 10 mL of water added,
resulting in a rice/water ratio of 1:2. The container was then placed in the steaming basket on
a rice cooker and steamed for 10 min after boiling. With 10 min cooling, the cooked rice was
observed and ranked on the scale described above. The microscope pictures of all
formulations were also taken to permit unbiased comparisons between different formulations
later.
The detailed ranking scheme is presented in Appendix 11.2.1, with relevant microscopic
pictures as references.
91
Computer Microscope, and the colour was measured using the Hunter L*a*b*
spectrophotometric system. In the system, L* stands for the lightness (black=0, white=100),
a* stands for the gradation from red (+) to green (-), and b* stands for the gradation from
yellow (+) to blue (-). During the measurement, a beaker containing a sample of
approximately 100 g with a constant depth of 100 mm was placed under the light source of
the Hunter colorimeter, and the L*, a*, and b* values were measured. The integrated colour
difference (E*) was then calculated as the sum of the differences of the L*, a* and b*
values of the sample from those of the reference, e.g., the native rice, based on the following
equation:
E* = L *2 + a *2 + b *2
Texture measurement on cooked rice grains
A single compression test was used to measure the texture of cooked rice grains (Sesmat &
Meullenet, 2001). This instrumental measurement was used to complement visual
observations of grain integrity. Specifically, a Texture Analyzer (model TA-XT2i, Texture
Technologies Corp., Scarsdale, N.Y., U.S.A.) was used to perform compression tests,
courtesy of Professor Drick Rousseaus Food Research Group at Ryerson University,
Toronto, Canada. The detailed procedure is presented in Appendix 11.2.2
92
The use of sparingly soluble calcium salts, such as CaSO4, and appropriate sequestrants
seemed more promising for our purpose. This strategy was investigated with respect to the
effects of individual components on the internal gelation process.
93
Based on these observations, the best choices of alginates were Danisco FD 175 and ISP
DMF, which were used for further tests on internal gelation.
Danisco FD170
Danisco FD120
Danisco FD175
Danisco FD155
FMC RF6650
FMC LF20/40
TIC HG600F
TIC 900
ISP DMF (high M)
ISP GMB (High G)
No alginate
Extrusion performance
Yield of
Residual
Viscosity Particle size Extrusion grains with
moisture after
(mPa.s)
(m)
rate (g/min) desired size
drying (%)
(%)
20-50
20-50
350-550
350-550
400-600
100-200
Medium
High
200-350
110-270
-
100
620
100
200
100
400
200
100
100-150
200-355
-
94
~200
~230
~235
~265
~206
~240
~200
~240
~307
~278
~210
87
87
89
87
88
88
85
86
80
85
92
9.3
11.9
7.7
10.1
10.1
9.7
11.3
12.9
7.7
7.5
13
Table 5.3 Grain integrity and sensory properties of the reconstituted rice made with different
alginate sources
Alginate
1
2
3
4
5
6
7
8
9
10
Control
Danisco FD170
Danisco FD120
Danisco FD175
Danisco FD155
FMC RF6650
FMC LF20/40
TIC HG600F
TIC 900
ISP DMF (high M)
ISP GMB (High G)
No alginate
Rinsing/Cooking
integrity (ranking
with 0-5 scale)
5
5
5
5
5
5
5
5
5
5
0
Citric acid was added at the level of 0.5% (w/w) in the trial of CaCO3, since it was originally
used in stable Ultra Rice formulations as an antioxidant. In this case, it was expected to
alter the pH of the wet mixture, which in turn should result in an increased release of the free
calcium ions from the insoluble salt. The idea of using calcium iodate was based on a unique
property of this salt: it has an increased solubility at increased temperature, i.e., 0.17 wt% at
room temperature and 1.38 wt% at 60oC. It was expected the salt would release more free Ca
ions during the extrusion as the temperature of the operation varied from ~35oC at the
beginning of mixing to ~55oC at the exit of the die.
95
Unfortunately, none of the trials could produce the desired grain integrity, although the
results were slightly better in trials using CaSO4. An attempt to incorporate some
sequestrants in the internal gelation system was then made.
Table 5.4 Effects of different Ca sources on extrusion operability and grain integrity using
the internal gelation process in the absence of sequestrants
Solubility
at 25oC
CaCl2
74.5 g/100 mL
CaSO4
0.24 g/100 mL
CaCO3
(+ citric acid)
Ca(IO3)2
Extrusion Yield of
Residual
Grain
Ca/Alginate
grains with moisture after
rate
integrity
ratio
(g/min) desired size drying (%) (ranking with
(%)
0-5 scale)
1:3
1:3
1:1
~240
~200
~325
87
85
72
7.1
7.8
6.3
0
2
2
Insoluble
1:3
~288
81
7.4
0.17 g/100 mL
1:1
~244
71
5.2
As shown in Table 5.5, the use of STPP resulted in improved grain integrity, probably due to
the potentially delayed alginate-Ca gelation in the system. Specifically, the combination of
CaSO4 with appropriate amounts of sequestrants resulted in much better grain integrity
during soaking/cooking.
The chosen ratio between of the three components for the internal gelation was based on the
composition of the current Ultra Rice formulations, in which alginate was added at 1.5%
(w/w), calcium chloride was sprayed at 0.5% (w/w) on the surface of the extruded grains,
and STPP was used in a combined antioxidant system at the level of 0.3% (w/w).
Among the selected Ca sources, CaSO4 dihydrate (gypsum) was the best in terms of
providing delayed gelation and subsequently resulting in better grain integrity. Clearly, this
is due to its limited solubility (0.24g/100 mL), whereas CaCO3 is water insoluble and CaCl2
has a high solubility (74.5 g/100 mL). Ca lactate also has moderate solubility (3.9~6.4 wt%
at 25oC) and gave acceptable results, which were worth further investigation.
96
Table 5.5 Comparison of different Ca sources using the internal setting process in the
presence of STPP as a sequestrant
Yield of
Residual
Grain integrity
Alginate/Ca/ Extrusion rate grains with
moisture
(ranking with
STPP ratio
(g/min)
desired size after drying
0-5 scale)
(%)
(%)
CaCl2
CaSO4
CaCO3 (+ citric acid)
Ca lactate
3:1:0.6
3:1:0.6
3:1:0.6
~227
~305
~335
88
80
81
10
6.2
6.3
1
3
3
3:1:0.6
3:1:0.6
3:1:0.6
~225
~318
~300
83
78
81
8.9
4.4
6.5
1
3
3
Table 5.6 Comparison of different sequestrants in the internal setting process using same
alginate and Ca sources (i.e., ISP DMF alginate and CaSO4)
STPP
SHMP
Na citrate
EDTA
Alginate/CaSO4
/sequestrant
ratio
Extrusion rate
(g/min)
3:1:0.4
3:1:0.6
3:1:0.8
3:1:0.8
3:1:0.8
3:1:0.4
~305
~335
~302
~271
~291
~312
Yield of grains
Residual Grain integrity
with desired moisture after (ranking with
drying (%)
size (%)
0-5 scale)
82
75
85
82
86
77
97
8.3
6.4
7.1
6.4
4.1
9.5
4
3
3
2
2
4
As shown in Table 5.7, no STPP or too much of the sequestrant had negative impacts on the
grain integrity. The best results were obtained with the ratios of 1:0.2 and 1:0.4. This result
was confirmed by repeated trials using EDTA.
CaSO4/STPP ratio
Yield of grains
Residual
Grain integrity
Extrusion rate
with desired size moisture after (ranking with
(g/min)
(%)
drying (%)
0-5 scale)
1:0
~200
85
7.8
1:0.2
~338
76
5.5
4-5
1:0.4
~305
82
8.3
1:0.6
~335
75
6.4
1:0.8
1:1
~302
85
7.1
~287
79
8.1
1:0.2
~290
77
4.3
4-5
1:0.4
~311
77
9.5
CaSO4/EDTA ratio
98
This might be because that alginate dispersion in the melted shortening could slow down its
hydration, while the dispersion of CaSO4/STPP in the oil could result in fewer free Ca ions
available for interaction at the beginning of extrusion. Thus, the separate addition of these
ingredients would be effective in delaying the spontaneous interaction between alginate and
free Ca ions in the system.
99
together with alginate into the rice flour dough. The addition levels of these ingredients were
based on the stable formulation of the multiple-fortified Ultra Rice. It was found that the
iron/zinc formulation produced acceptable core and surface integrity without further CaCl2
over spray after extrusion. This suggested that a soft gel might be slowly formed during
extrusion between ferrous/zinc ions and alginate. However, the incorporation of ferrous
fumarate powder resulted in an unacceptable dark brownish colour. This may restraint the
use of this approach.
Based on the preliminary investigation, it seemed feasible to incorporate both alginate and
calcium into rice flour mixtures in the presence of sequestrant prior to extrusion, wherein a
calcium salt with limited solubility, such as CaSO4, was used. The internal gelation actually
enhanced the extrusion operation to some extent with a faster flow rate. The newly prepared
grains also had a better core integrity as no grain cracking was observed during soaking and
cooking tests. However, these grains had not yet achieved the surface integrity of grains
made with the original diffusion setting method. During the soaking test the newly prepared
grains were likely to exude starch from the core into the water, resulting in a cloudy solution,
which subsequently caused the grains to stick together during cooking. Clearly, fine-tuning
of the system was required prior to generating useful formulations.
which the ratio of CaSO4/STPP = 1%:0.2% gave the best grain integrity. The maximum
concentration of alginate tested previously was 3%, which did not adversely affect the
extrusion process. The objective of the design was to determine the ranking of the factors in
terms of their impact on extrusion and grain integrity, as well as to find the best
concentration level for each factor. The results could ultimately enable us to find the best
formulation using these ingredients.
Level
1
2
3
Factor
B
CaSO4
STPP
3.0%
1.5%
0.5%
3.0%
1.5%
0.5%
0.6%
0.3%
0.1%
Formula
Alginate
ISP DMF
CaSO4
STPP
1
2
3
4
5
6
7
8
9
A1 B1 C1
A1 B2 C2
A1 B3 C3
A2 B1 C2
A2 B2 C3
A2 B3 C1
A3 B1 C3
A3 B2 C1
A3 B3 C2
3.0%
3.0%
3.0%
1.5%
1.5%
1.5%
0.5%
0.5%
0.5%
3.0%
1.5%
0.5%
3.0%
1.5%
0.5%
3.0%
1.5%
0.5%
0.6%
0.3%
0.1%
0.3%
0.1%
0.6%
0.1%
0.6%
0.3%
Nine batches (Table 5.9) of grains were produced by extrusion. In all cases, the same basic
formulation was used, including rice flour, shortening, and water at the same concentrations.
Other operating variables, such as the blending sequence and extrusion parameters, were
also kept the same. The samples produced were tested for grain integrity during soaking and
cooking.
101
The results (Table 5.10) from the integrity tests were analysed based on the statistical
concept of orthogonal experiment, as shown in Table 5.11. The analysis indicated that the
calcium compound was the most important factor among the three ingredients, while the
sequestrant had the least impact on grain integrity. Further analyses on the best level for each
ingredient (Figure 5.4) showed that the highest addition level (3%) for both alginate and
CaSO4 resulted in better grains, whereas the addition level of STPP was best at the
intermediate level, i.e., 0.3%.
Table 5.10 Results of the grain integrity tests in the formulations prepared by the orthogonal
design
Cooking integrity
(1-5)
5
4
1
2
1
0
0
1
1
Trial
1
2
3
4
5
6
7
8
9
Soaking integrity
(1-5)
5
4
0
4
3
0
2
1
1
Total ranking
(1-10)
10
8
1
6
4
0
2
2
2
Extrusion rate
(g/min)
234
260
267
250
260
219
309
279
355
Formula
1
2
3
4
5
6
7
8
9
A1 B1 C1
A1 B2 C2
A1 B3 C3
A2 B1 C2
A2 B2 C3
A2 B3 C1
A3 B1 C3
A3 B2 C1
A3 B3 C2
Tj1 (level 1)
Tj2 (level 2)
Tj3 (level 3)
Rj (difference)
Alginate
CaSO4
STPP
3.0%
3.0%
3.0%
1.5%
1.5%
1.5%
0.5%
0.5%
0.5%
3.0%
1.5%
0.5%
3.0%
1.5%
0.5%
3.0%
1.5%
0.5%
0.6%
0.3%
0.1%
0.3%
0.1%
0.6%
0.1%
0.6%
0.3%
19
18
12
10
14
16
13
15
ISP DMF
102
Figure 5.4 Statistical analysis of the best level for each factor
Therefore, the best ratio deduced from the orthogonal experiment would be
alginate/CaSO4/STPP (3%:3%:0.3%). This formulation was not tested in the original 9 trials,
in which the best formulation was trial #1 with the ratio of 3%:3%:0.6%. Both formulations
were re-tested using alternative sources of the three ingredients.
Alginate/CaSO4/STPP = 3/3/0.3
Alginate/CaSO4/STPP = 3/3/0.6
(The best ratio deducted statistically (The optimal ratio chosen from the 9
from the orthogonal experiment)
trials of the orthogonal experiment)
Cooking integrity
(Ranking 0-5)
Cooking integrity
(Ranking 0-5)
ISP DMF
Danisco 175
4.5
Danisco 155
4.5
103
Table 5.13 Verification of the optimal ratio (alginate/CaSO4/STPP = 3/3/0.6) using various
alginate sources
Extrusion rate
Cooking integrity
Alginates
(g/min)
(Ranking 0-5)
ISP DMF
Danisco 175
Danisco 155
FMC RF6650
FMC LF20/40
TIC HG600F
TIC 900
ISP GMB (high G)
234
288
312
265
335
296
249
5
4
4
5
5
4.5
4.5
309
4.5
All samples had equally good grain integrity, which confirmed the best formulation was
viable. In addition, the results confirmed that the alginate was not as important as the
calcium compound in the internal gelation system. Variations in the physical/chemical
properties of the alginate sources did not have an obvious impact on grain integrity, as long
as the best concentration level (3%) was used.
As shown in Table 5.14, when the addition levels of both ingredients were increased to 5%
and 8%, respectively, the grains retained the similar cooking integrity to that of the sample
made with 3% of both materials. On the other hand, the increased amounts of both
ingredients caused obvious difficulty in the extrusion process, slowing the extrusion rates.
Considering the increased product cost associated with the higher addition levels, the
optimal ratio determined in the orthogonal study was followed in all later tests.
104
3/3/0.6
5/5/1.0
8/8/1.6
234
228
198
Cooking integrity
(ranking at 0-5)
5
5
4
Table 5.15 Verification of the optimal ratio with various calcium compounds
Ca compound
Cooking integrity
(Ranking 0-5)
CaSO4
Ca lactate
CaCl2
CaCO3
Ca(IO3)2
234
267
310
298
336
5
1
0
0
0
CaO
Not extrudable, the strong alkali tended to break down rice flour
and formed a yellowish dough.
105
ratio of 3%:3% produced acceptable grains. Again, it was confirmed that the sequestrant was
necessary but it was the least important factor among the three ingredients.
Table 5.16 Verification of the optimal ratio with various sequestrant compounds
Sequestrant
None
392
STPP
234
TSPP
293
4.5
SHMP
352
EDTA
316
4.5
Na citrate
339
Despite its name, glutinous rice does not contain dietary gluten, thus can be consumed as
"gluten-free". The feature that distinguishes it from other types of rice is that it has almost no
amylose (linear starch) but is very high in amylopectin (branched starch). This leads to the
unique characteristics of glutinous rice, i.e., lower gelatinization point and soft, sticky
texture after cooking.
The idea of using glutinous rice flour in place of regular rice flour was primarily due to its
whiter colour. During the extrudability test, using dough made of glutinous rice flour made
extrusion easier. It also resulted in smoother surface of the grains that were whiter in colour,
as shown in Figure 5.5.
106
Rice flour-based
Multi-Fe formula
Glutinous flour-based
Multi-Fe formula
Rice flour-based
Vit A formula
Glutinous flour-based
Vit A formula
Figure 5.5 Comparison of grain appearance made with glutinous rice flour in place of regular
rice flour
During the soaking and cooking tests, no cracking or checkering was noted on the grains
made with glutinous flour, but the grains were likely to allow some starch to seep out,
resulting in cloudy water. Not surprisingly, without addition of any alginate/Ca, the cooked
grains had much softer and stickier texture; however, with the addition of the three
ingredients at the best ratio, the cooked grains showed much better texture, resembling
polished rice, as shown in Figure 5.6 and Table 5.17. With further addition of HPMC (5%wt
of MethocelTM E3 from Dow Chemical Co.), the grains showed enhanced hardness and
cohesiveness, producing the best formulation this far.
With the best ratio of alginate/CaSO4/STPP used and addition of HPMC (5%)
Glutinous flour
Figure 5.6 Comparison between glutinous rice flour and regular rice flour using the best
ratio and addition of HPMC
107
Table 5.17 Effect of using glutinous rice flour on the grain properties
Trials
Taste test
No alginate/Ca involved
305
Very sticky
288
Sticky, brittle
227
275
Showing best
combination of
harness/springiness
348
4.5
Cooking integrity
(Ranking 0-5)
296
280
315
5
5
HMPC source
108
Table 5.19 Comparison of alternative blending processes on grain integrity with the best
ratio used
Extrusion rate
(g/min)
Cooking integrity
(Ranking 0-5)
Texture in taste
(Ranking1-5)
CaSO4/STPP in melted
shortening and added in later
265
Softer (1)
305
315
Soft (2)
234
Hard (3)
Blending sequence
109
Next, the successful internal gelation systems were used in preparing reconstituted rice
containing vitamin A and the previously optimized antioxidant system and in a multiplefortified formulation containing iron and B vitamins.
The detailed formulation compositions are presented in Appendix 11.2.3. In addition to the
internal gelation system, 1% TiO2 was added to the multiple-fortified formulation since we
have found, in a parallel study (section 6.5.2.1), that TiO2 improved grain colour. Sodium
erythorbate was used in the vitamin A formulation in place of the original antioxidant,
ascorbic acid, since the use of sodium erythorbate prevented the formation of a yellowish
hue observed with the use of ascorbic acid (Diosady & Li, 2008). All other components were
kept the same as in the original commercial formulations.
Two other formulations were also produced by replacing regular rice flour with glutinous
flour and with addition of HPMC at the level of 3%, since this combination had enhanced
the grain integrity. The detailed compositions of these two batches are also presented in
Appendix 11.2.3. After the four formulations were prepared, the physical properties, grain
integrity, and the retentions of the added micronutrients were measured.
The measurements of grain colour using Hunter L*a*b* system showed that the four
formulations made with the best gelation systems had much improved colour over the
original formulations (Figure 5.7). However, this improvement was probably not the
contribution of the gelation system, but rather it was due to the other formulation
refinements described above. These improvements resulted in a closer match to the
appearance of actual rice.
The grain integrity test results are presented in Figure 5.8. It is clear that the use of glutinous
rice flour and HPMC made the extrusion process easier, resulting in higher extrusion rates.
However, during the cooking test, the grains in the two samples made with glutinous flour
completely stuck together. In contrast, the grains made with regular rice flour could retain
their shape/intactness reasonably well after cooking; still, the grain integrity was not as good
110
as that of the grains made without the active ingredients. Clearly, the other components,
especially the antioxidants (acidic materials), weakened the gelation structure to some extent.
A better understanding of the potential interactions between the gelation materials and other
ingredients in the system is required for the development of industrially viable formulations.
Vitamin A formula
Multi-Fe formula
L*
a*
b*
L*
a*
b*
Rice flour-based
76.0
0.7
21.3
77.1
0.6
16.8
Glutinous flour-based
83.0
-1.1
19.2
72.5
3.5
20.1
Controls
L*
a*
b*
L*
a*
b*
72.9
4.7
15.6
67.4
1.8
21.4
Native grains
L*
a*
b*
77.4
2.0
17.6
Rice flour-based
L*
75.8
a*
0.5
b*
14.9
Glutinous flour-based
10.7
12
9
6
6.7
5.7
4.1
5.6
3.5
1.6
Internal gelation
Vit A
Internal gelation
Multi-Fe
Blank simulated
rice
Original Vit A
Original multi-Fe
Figure 5.7 Colour measurements of the four new formulations made with the best internal
gelation systems
Figure 5.8 Micronutrient fortified Ultra Rice formulations made with the best internal
gelation systems
111
The potential interactions of the gelation system with the added micronutrients were
investigated using four new formulations. As shown in Table 5.20, folic acid seemed stable
in the new formulations made with the internal gelation systems, with ~95% retained after
the extrusion process. This was comparable to the formulations made with the original
external calcium diffusion setting, where an average folic acid retention of 96% was
observed (results from the later chapter work). In contrast, vitamin A stability was greatly
reduced by the internal gelation systems, with ~40% of the vitamin lost during processing.
Conversely, the original surface crosslinking technique could retain >90% of the added
vitamin A (Lam, 2006). Clearly, technical challenges still remain in the application of the
best gelation systems, particularly in obtaining appropriate grain integrity and nutrient
stability.
Table 5.20 Micronutrient retentions in the four new formulations made with the best internal
gelation systems
With the best gelation
system
Vitamin A retention
(Designed target - 1800 IU/g)
Rice flour-based
Glutinous flour-based
1046.5 34.1
1108.2 45.7
284.6 4.9
292.0 3.7
Note: mean standard deviation, which were obtained from three or four replicates for each sample
measurement.
As indicated earlier, the visual observations of grain integrity during soaking and after
cooking were used as a reasonably consistent and reliable qualitative measurement to assess
the formulations. Ideally a standardized analytical method should be adapted for quantitative
measurement of grain integrity. Attempts were made to use an instrumental texture
measurement for quantifying grain integrity.
Although texture measurement is commonly used in the food industry for determination of
sensory properties of food products, little information was found in the literature relevant to
reconstituted rice grains. There are two types of methods available - instrumental methods
and sensory methods. Particularly in the research area of rice quality and sensory properties,
numerous studies have shown instrumental methods could generate equally reliable data as
112
sensory tests (Deshpande & Bhattacharya 1982; Juliano et al., 1981; Okadome, 2005). Also,
the instrumental methods on texture measurement have many advantages over sensory
methods, not the least of which is the fact that a group of well trained panelists is not
required as in sensory tests.
Springiness
(the initial slope)
Ratio of hardness
and springiness
0.73 0.19
0.12 0.04
6.08
0.78 0.08
0.15 0.04
5.20
0.97 0.08
0.15 0.03
6.47
0.97 0.15
0.12 0.07
8.08
0.92 0.06
0.99 0.18
1.32 0.27
0.92 0.13
1.12 0.16
0.84 0.07
0.83 0.15
0.91 0.25
0.74 0.08
0.14 0.08
0.12 0.04
0.15 0.04
0.10 0.03
0.14 0.04
0.19 0.03
0.22 0.06
0.19 0.05
0.14 0.07
6.57
8.25
8.80
8.82
8.01
4.42
3.77
4.79
5.29
1.25 0.10
0.20 0.03
6.24
1.33 0.31
0.21 0.11
6.32
0.35 0.07
0.06 0.01
5.83
0.56 0.08
0.09 0.01
6.53
113
0.78 0.07
0.095 0.01
8.16
0.28 0.02
0.03 0.003
9.33
0.35 0.05
0.03 0.002
11.67
Note: the experimental data are presented as mean value standard deviation, which were obtained from
three or four replicates for each sample measurement.
As shown in Table 5.21, the Ultra Rice grains made with the original process had similar
hardness and springiness to the native rice. This is in good agreement with the previous
observation of the grain integrity of the two formulations, which retained reasonable
shape/intactness after cooking. The multiple-fortified formulation was slightly harder than
the vitamin A containing sample, which may be related to the presence of the iron compound
in the matrix.
The results for reconstituted rice without any alginate or calcium also gave similar hardness
and springiness values as the native rice grains. However, the previous grain integrity test
indicated that the alginate-free sample did not retain its integrity after cooking. Clearly, the
values of hardness and springiness on their own may result in misleading assessment.
Therefore, the ratio of hardness over springiness may be considered as a more valuable
parameter. Based on the literature (Perez et al., 1996), a higher value in hardness means the
material is probably hard and brittle, while greater values of springiness may suggest the
material is softer and/or stickier. An optimal combination of both could represent desirable
texture, such as a hardness-springiness (H-S) ratio of 6 calculated for cooked native rice.
Based on this consideration, the results of the alginate-free control was re-examined and it
was found that the sample had a higher value for hardness and lower number for springiness,
which means the cooked grain was harder but stickier. This seems in agreement with the
visual observations, wherein the grains disintegrated to some extent during soaking with
some starch seeping out, forming a sticky rice-cake like texture after cooking. Moreover, the
calculation of the H-S ratio showed that the sample had a value of >8, much higher than that
of the native rice.
114
The H-S ratio of all tested samples was then calculated. The ratios had a good correlation
with visual observations. The formulations made with the best gelation systems had H-S
ratios close to 6, while the samples with poor grain integrity had H-S ratios either much
smaller or greater than 6. Particularly in the sample made with the best glutinous flour
system, even though the values of hardness and springiness were much different from those
of the native rice, the H-S ratio was in the similar range, which was in agreement with the
visual observation that this sample had the best grain integrity/intactness after cooking.
Clearly the single compression test could be used to obtain valuable quantitative information
that correlates well with qualitative sensory assessments of grain integrity.
5.5.5 Mechanism of Alginate-Calcium Interaction in the Ultra Rice System
The gelation would normally form an egg-carton-like network, as follows, in which the
calcium ions are embedded in the two chains of the alginate polymer.
Specifically, the original technique of calcium over-spray on the extruded rice grains
resulted in a network at the grain surface similar to a fish-net with a pore size of ~ 1 m
(Liu et al., 2002), which could prevent the bigger starch granules leaking out. However, this
rigid structure could not resist, or stretch with, the core expansion when rice starch absorbed
water and gelatinized during cooking, resulting in grain cracking. In contrast, the internal
115
Alginate-calcium network
throughout the grain
Figure 5.9 Two proposed models for alginate-calcium gelation in Ultra Rice: fish-net
model for external/diffusion setting (left) and inter-lock model for internal setting (right)
Figure 5.10 Alginate-calcium gel structure made with external (left) and internal (right)
gelation processes (from Liu et al., 2002 with permission)
Other studies also found similar effects during the preparation of alginate-calcium beads in
116
an aqueous system (Hills et al., 2000). As shown in Figure 5.10, external gelation resulted in
a denser network at the surface of the bead, with decreasing calcium concentration towards
the core, while internal or in-situ gelation resulted in a looser, porous network with
apparently constant calcium concentration throughout the bead (Liu et al. 2002).
Quick examinations of Ultra Rice grains using XPS (X-ray Photoelectron Spectroscopy)
and ToF-SIMS (Time-of-Flight Secondary Ion Mass Spectroscopy) confirmed that the grains
made by the external crosslinking method had a higher Ca concentration at the grain surface,
while the grains made by internal gelation had a similar Ca distribution in both the core and
the surface layer. Detailed results are presented in Appendix 11.2.4.
Overall, the best ratio between alginate, CaSO4, and STPP obtained in the orthogonal
experiment could result in controlled in-situ gelation through extrusion process, which was
mainly due to the competitive effect between alginate and the sequestrant in binding free
Ca2+ slowly released from CaSO4, which has limited solubility.
1.
The original process for making reconstituted rice grains by Ca-alginate external
crosslinking led to grain cracking during cooking, due to the weak, rigid surface
structure.
2.
3.
The crosslinking between Ca and alginate was attempted by slowly releasing Ca2+ ions
based on increasing temperature or decreasing pH. This approach was not successful.
Using calcium salts with limited solubility in combination with sequestrants has proven
to be promising. The competition between the sequestrant and the alginate for the free
calcium ions in the system delayed internal gelation until the extrusion process was
almost completed, producing reconstituted rice grains with acceptable core integrity and
appearance. CaSO4 was an effective Ca2+ source. This approach actually enhanced the
extrusion operation to some extent with a faster flow rate.
4.
It has been determined, using an orthogonal experimental design, that the calcium salt
117
plays the most important role in the internal gelation process. The appropriate ratio of
the three key components, i.e., alginate, calcium, and sequestrant, is critical for forming
an effective internal gelation system. The best results were obtained with DMF alginate
from ISP, CaSO4, and STPP.
5.
Reconstituted rice with grain properties similar to those of the native rice could be
consistently produced with formulations in which the alginate, CaSO4 and STPP
concentration was maintained at 3%, 3%, and 0.6%, respectively.
6.
When active ingredients were added to the rice matrix, the integrity of the extruded
grains deteriorated somewhat. This was especially noticeable with acidic antioxidants.
Still, the fortified grains prepared by internal setting were superior in appearance to
those made by calcium overspray.
7.
The Ultra Rice production could be greatly simplified by adapting the internal gelation
system, and based on the laboratory tests pilot tests and commercialization should be
investigated.
8.
The gelation system developed in this study provides a robust structural platform for
delivering minor active components in matrices on a 1-10mm scale, not only in
micronutrient fortification, but also potentially for other applications, such as drug,
nutraceuticals, and active ingredient delivery in functional foods, animal feeds, and
agro-chemicals.
1. Further improvements to either the internal gelation system or the antioxidant system
should be attempted in order to generate robust systems which are unaffected by the
added active ingredient systems.
2. Quantitative analytical methods need to be established for measuring physical properties
of the reconstituted rice grains, including grain integrity after soaking and cooking,
overall hardness and surface hardness. In addition, it would be better to develop a valid
method for characterizing gel formation and/or gel strength during all stages of the
process.
3. It may be prudent to re-examine the other approaches for achieving controlled or delayed
internal gelation in the presence of the antioxidant systems and the selected
118
119
To maximize the value of the current food fortification interventions aimed at eliminating
micronutrient deficiencies in poor populations of developing countries, it seems logical to
incorporate other micronutrients into existing or planned programs for the fortification of
sugar, salt and rice. Folic acid was chosen in this study due to the increased awareness
worldwide of the dietary deficiency of this vitamin.
Previously, folate deficiency was considered rare due to the adequate presence of folate in a
wide variety of foods. However, dietary/nutrition surveys had revealed that most adults had
inadequate serum folate levels, and folate deficiency was common, particularly in elderly
populations (Buttriss, 2005). Poor dietary habits such as those of chronic alcoholics can lead
to folate deficiency. The predominant causes of folate deficiency in non-alcoholics are
impaired absorption or metabolism caused by certain diseases or medications (including
kidney and liver diseases and/or interference of drugs), or an increased need for the vitamin,
e.g., during pregnancy and lactation.
With more focused studies, inadequate folate intake has been linked with development of
birth defects, several chronic diseases including vascular disease and certain cancers, and
brain disorders such as depression, reduced cognition, and Alzheimers disease (Cho et al.,
2002; Rampersaud et al., 2002; Choi & Manson, 2000; Miller, 2004). The most widely
known problems related to folate insufficiency are neural tube defects (NTD), characterized
by abnormal neural tube closure, which is estimated to affect about 1~5 pregnancies/1000
(Caudill, 2004). In the early 1990s, it was demonstrated that periconceptional consumption
of folic acid supplements (400 g/day) could dramatically reduce the risk of NTD by up to
72%. Other symptoms of folate deficiency are megaloblastic anemia in adults (a condition
characterized by fewer, and enlarged red blood cells with a decreased ability to carry oxygen)
and slow growth rate in infants and children. More subtle signs include digestive disorders
(such as diarrhea and loss of appetite), weight loss, weakness, sore tongue, headaches, heart
palpitations, irritability, forgetfulness, and behaviour disorders. The understanding of the role
120
of folates in birth defects led to a series of public health policy changes worldwide, aimed at
reducing folic acid deficiency (Bailey et al., 2003).
Based on the fact that folate is a cofactor in the regulation of normal plasma homocysteine
concentrations through a key remethylation reaction, sub-optimal folate status was linked to
hyperhomocysteinemia, which is considered to be a significant risk factor for atherosclerotic
vascular disease in the coronary, cerebral and peripheral vessels (Bailey et al., 2003). More
intervention trials are ongoing to further understand the mechanisms behind these
correlations and determine whether increased folic acid intake through either
supplementation or food fortification could result in a reduction of disease incidence and
morbidity.
Many developed countries have introduced mandatory folic acid fortification of wheat flour.
This intervention has been highly successful (Grosse et al., 2006). Thus, it would be
worthwhile to investigate the possibility of incorporating folic acid within a broader
intervention that could provide other critical micronutrients simultaneously. There is a need
for other vehicles as poor people in developing countries do not have access to industrially
processed food ingredients, such as wheat flour. Depending on the dietary habits specific to a
region, other food carriers, such as salt, sugar or rice could be considered as folic acid
vehicles. Particularly, iodized salt has been successfully implemented in many countries, and
vitamin A fortified sugar has long been available in many Latin American countries.
Previously our research group has developed a stable Ultra Rice formulation that could
effectively deliver iron, zinc, and several B vitamins in a fortified rice product. It was then
logical to test the incorporation of folic acid into this stable rice formulation.
Folic acid could be incorporated directly into the selected food vehicles or added in a
protected form, alone or in combination with other micronutrients. In this study I examined
different fortification techniques. While it would be preferable to apply folic acid directly,
encapsulation processes may be needed to protect the vitamin in food. The objective of this
phase of the work was to identify potential, undesirable interactions between folic acid and
other micronutrients. It was hoped that folic acid fortification could be incorporated into the
121
Folic acid (pteroyl glutamic acid) (Figure 6.1) is the parent compound for a series of related
chemical substances that show similar biochemical properties and are known collectively as
the folates. Generally, folate (pteroylglutamate, Figure 6.2) is used to describe the naturally
occurring vitamin in food, while folic acid is the synthetic form that is found in supplements
and added in fortified foods. Naturally occurring food folate differs from folic acid in that
the pteridine moiety exists in the reduced form, while the folate molecule is polyglutamated.
pteridine
p-aminobenzoic acid
glutamic acid
Figure 6.1 Chemical structure of folic acid (C19H19N7O6, MW = 441.4) (adapted from
IUPAC-IUB website)
122
Natural folates found in foods are in a conjugated form, which reduces their bioavailability
by as much as 50% (Gregory, 1997). In addition, they are much less stable, with rapid loss of
activity over periods of days or weeks. In contrast, the synthetic folic acid is stable for
months or even years in fortified foods (Ball, 2006). Chemically pure folic acid is a
yellow/orange, odourless, tasteless microcrystalline substance (MW 441.41) (Ball, 2006),
which chars above 250C. The UV spectrum of L-folic acid at pH 13 shows absorptions at
256 nm (e = 30,000), 282 nm (e=26,000), and 365 nm (e=9800) (Francis, 1999). It is
practically insoluble in cold water and sparingly soluble in boiling water (20 mg/100ml). It
is moderately stable to heat, humidity and atmospheric oxygen, but it will lose its activity in
the presence of light, oxidizing or reducing agents, and acidic or alkaline environments
(Roche brochure).
Dietary folates are usually converted during absorption from the intestinal tract so that only
tetrahydrofolic acid (THF) enters the blood and is subsequently transported to the tissues.
The function of THF is to carry and transfer various forms of one-carbon units during
biosynthetic reactions. Folate helps produce and maintain new cells, which is especially
important during periods of rapid cell division and growth such as infancy and pregnancy
(Kamen, 1997). 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 et al., 1998). Both adults and
children need folate to make normal red blood cells and prevent anemia. In addition, folate is
essential for the metabolism of homocysteine, and helps maintain normal levels of this
amino acid. Elevated blood levels of homocysteine are associated with coronary heart
disease and stroke (Bailey et al., 2003). The study of folate deficiency has pointed to a
number of critical functions of folates in the development of serious birth defects and
chronic diseases.
Leafy green vegetables such as spinach and turnip greens, citrus fruits and juices, dried
beans and peas, are all natural sources of folate, but most of these are rare or absent in the
diet of the poor in developing countries. Liver is also rich in folate. Folic acid fortification of
123
flour in many developed countries made fortified cereals important contributors to the folate
intake of their general population.
g.
Numerous studies (Cho et al., 2002; Caudill et al., 2001; Choumenkovitch et al., 2001;
Cuskelly et al., 1999; Dietrich et al., 2005; Grosse et al., 2006; Quinlivan & Gregory, 2003;
Rader, 2002 & 2005; Rader et al., 2000; Yetley & Rader, 2004) have demonstrated that
mandatory fortification of enriched cereal-grain products with folic acid within the U.S. led
to a significant improvement of blood folate status of the overall adult, non-supplement
using U.S. population. After fortification, the category bread, rolls, and crackers became
the single largest contributor of folate to the American diet, accounting for >15% of total
intake, surpassing vegetables, which were the number one folate food source prior to
fortification (Grosse et al., 2006).
124
be effective in preventing NTD (Castilla et al., 2003; Hertrampf & Cortes, 2004; Hertrampf
et al., 2003).
As of September 2003, 38 other countries, including Australia and New Zealand, had
introduced or agreed to introduce folic acid fortification of flour, at a levels of 140-220
g/100 g. Interestingly, no countries in the EU are among these (Buttriss, 2005). Most EU
countries still rely on folic acid supplementation to address the issue of NTD, despite
suggestions to introduce compulsory fortification to balance the requirements of increasing
folic acid intake in certain populations and avoiding adverse effects in other groups of the
population (Buttriss, 2005).
There has been some concern that levels of folic acid in the diet exceed the mandated target
levels (Gregory, 2004; Quinlivan & Gregory, 2003a; Choumenkovitch et al., 2002). This is
due to the underestimation of naturally occurring folate in the current food composition
database, higher bioavailability of folic acid than most naturally occurring forms of food
folate, and the addition of excess folic acid by manufacturers under good manufacturing
practice (GMP) to ensure that the mandated levels be achieved despite losses during
processing (Gregory, 2004; Rader et al., 2000; Whittaker et al., 2001).
The primary concern related to excessive folic acid intake from either fortified foods or
supplements is the possibility of masking B12 deficiency anemia in the elderly. Folic acid
prevents megaloblastic anemia and potentially masks the diagnosis of vitamin B12 deficiency
by correcting the anemia while allowing neurological damage to continue (Quinlivan &
Gregory, 2003a). The only study to date focused on this problem provides some reassurance
that no measurable harm has occurred from this phenomenon at the current levels of folic
acid intake (Mills et al., 2003).
There is still a high degree of uncertainty in the bioavailability of food folates and folic acid,
which is needed to calculate the desired fortification level. To address folate insufficiency,
125
the U.S. Institute of Medicine (IOM) has identified an Estimated Average Requirement
(EAR) of 320 g/day, which is expressed in a novel unit, Dietary Folate Equivalent (DFE). It
is defined as:
DFE = g natural food folate + 1.7 x g synthetic folic acid
where the multiplier of 1.7 was determined from the ratio of the experimentally determined
bioavailability of added folic acid (85% available) and natural dietary folate (50% available)
(Caudill, 2004).
Based on these figures, the IOM has developed the Dietary Reference Intakes (DRIs) for
folic acid, which is a set of reference values used for planning and assessing the vitamin
intake for healthy people. DRIs consist of three important parameters, including
Recommended Dietary Allowances (RDA), Adequate Intakes (AI), and Tolerable Upper
Intake Levels (UL). The RDAs for folate are suggested by the IOM to be 150 to 300 g/day
for children depending on age, 400 g/day for adults, but increased amounts, 600 g/day and
500 g/day for women during pregnancy and lactation, respectively.
An AI is set when there is insufficient scientific data available to establish a RDA, and it
meets or exceeds the amount required to maintain a nutritional state of adequacy in nearly all
members of a specific age and gender group. Thus, the AI for folate has been set at 65
g/day for infants aged 0-6 months and 80 g/day for those 7-12 months in age.
The UL is the maximum daily intake unlikely to result in adverse health effects. The IOM
has established the ULs for synthetic folic acid for all age groups as 1000 g/day. There
appears to be no health risk associated with possible levels of natural food folate, thus no UL
has been set for folates naturally present in food.
The bioavailability of naturally occurring folate is still debated, as results reported in the
literature are highly variable (Finglas et al., 2006). The bioavailability of synthetic folic acid
(both supplemental and in fortified foods) is accepted to be higher than that of natural dietary
folate. However, the exact ratio of the bioavailabilities of these two forms remains debatable,
mainly due to the limitations in existing analytical methods for accurately determining folic
126
acid and natural dietary folate (Rader et al., 2000). Therefore, further studies are required in
terms of gaining a better understanding of the factors governing folate bioavailability and
improving protocols for its determination in humans. This will lead to a better food
composition database with more accurate values for natural folates and their bioavailabilities
in individual foods and representative mixed diets (Finglas et al., 2006). These data will be
critical in refining the optimal fortification levels.
Unlike synthetic folic acid, which is a fully oxidized, naturally occurring food folates are
reduced at the 5, 6, 7, and 8 positions of the pteridine ring and so are prone to oxidative
cleavage. The breakdown at the C9-N10 bond results in two degradation products, which are
inactive and cannot be biologically converted to any active folate form (Figure 6.3)
(McKillop et al., 2002; Suh et al., 2001). Generally, native folate is unstable during cooking
and food preparation. The vitamin losses are the result of a combination of thermal
degradation and leaching into the cooking water (Ball, 2006). The degree of loss can be
influenced by environmental factors, such as pH, oxygen, metal ion concentrations,
antioxidant levels, cooking duration, and product/water ratio (McKillop et al., 2002).
Extensive losses of dietary folate have been reported in boiled vegetables and baked meats,
ranging from 55% to 85% (Ball, 2006; McKillop et al., 2002).
On the other hand, synthetic folic acid is more stable. In its monoglutamate form, folic acid
is stable to heat, but becomes heat labile in acid solution. In aqueous solution, folic acid is
stable at 100oC for 10 h at pH range 5.0-12.0 when protected from light, but becomes
increasingly unstable as the pH decreases below 5.0 (Ball, 2006). Treatment of folic acid
with alkaline potassium permanganate cleaves the molecule at the C9-N10 bond, yielding
127
Figure 6.3 Oxidative cleavage products of folates (adapted from McKillop et al., 2002)
Figure 6.4 Cleavage products of folic acid under acidic and alkaline conditions (adapted
from Saxby et al., 1983)
Folic acid is photosensitive, and is degraded in aqueous solution to various products by
visible or ultraviolet light. Akhtar et al. (2003) have reported that when folic acid is
irradiated with ultraviolet light, it is first converted to 2-amino-4-hydroxy-6-formyl pteridine
(pterine-6-carboxaldehyde) and a diazotizable amine (p-aminobenzoyl-L-glutamic acid). On
further irradiation, the aldehyde is converted to the corresponding 6-carboxylic acid
128
Besides the relatively high stability of pure, synthetic folic acid under normal storage
conditions, numerous studies have demonstrated the acceptable stability of added folic acid
in fortified breads, vitamin-mineral premixes, fortified flours and grains, during typical
storage and baking conditions, as well as in high moisture environments (Ball, 2006; Rader
et al., 2000; Gujska & Majewska, 2005; Albala-Hurtado et al., 2000; de Jong et al., 2005).
An acceptable average loss during processing of 10-20% of added folic acid has been
reported.
As the vitamin is heat sensitive in acid, it has been suggested that high pressure treatments
are better for native folate retention in vegetables, fruit juices, soups, and beverages than
traditional sterilization processes (Finglas et al., 2006). The addition of proper antioxidants is
also helpful in stabilizing added folic acid (Ball, 2006). Another possible way to increase the
bioavailability of naturally occurring food folates is through processing, e.g., by breaking the
matrix or deconjugating glutamate residues from the polyglutamate chain (Finglas et al.,
2006).
129
(FAO, 1995).
The review of the vitamin chemistry shows that folic acid is reasonably robust, and under
appropriate environmental conditions remains stable and effective. Based on the available
literature, application of folic acid to sugar should result in a stable system. However,
addition in combination with vitamin A is not clearly predictable, as antioxidants such as
vitamin A can rapidly degrade folic acid, and be degraded also as a consequence. Whether
this is a significant problem had to be determined by experimentation.
The addition of folic acid to salt is not addressed by the literature. While it is expected that
folate should be stable in a dry refined salt, this may not be the case in impure salt, or where
the carrier for attaching folic acid to the salt adversely influences pH or ionic composition.
Salt creates a highly corrosive environment, and the presence of metallic impurities can lead
to significant folic acid loss. As folic acid is sensitive to pH in several pH regions (either <5
or >12), depending on the composition of the system, it would be prudent to do a simple
experimental confirmation of the vitamin stability in specific salts to be used in the field.
The case of rice is similar. On its own, folic acid will be stable on or in rice. The stability in
the presence of iron is unclear. It may be necessary to encapsulate the iron compound, adjust
the pH or add an antioxidant. Ultimately this should not be a major problem, but fortification
technology may have a major effect on the success of fortification. Our previous work with
130
Ultra Rice indicated that thiamine is stable in the presence of iron. We also expect that
folate would be equally resistant to degradation. Ultimately, the fortification of rice will
require a confirmatory test.
Fortification of sugar, salt or rice seems feasible, perhaps requiring fine-tuning of the system
in terms of stabilizers or binders. The simple experimental series initially proposed in this
program was expected to confirm the suitability of this approach at least in sugar and salt, by
testing the effect for a limited number of simple stabilizers.
Guatemalan iodized salt and vitamin A fortified sugar used in this study were obtained
through the Micronutrient Initiative (MI). Four commercial forms of food grade FePP used
in the Ultra Rice formulations were obtained through the Program for Appropriate
Technology in Health (PATH). The other formulation materials and analytical reagents for
the determination of folic acid, vitamin A, and iodine are listed in Table 6.1.
Table 6.1 List of Chemicals used in the study of folic acid fortification
Material / Chemical Name
Supplier
Description
Local supermarket
Glatt Air Techniques, NJ, USA
Almat Pharmachem Inc.
through PATH
through PATH
Dr. Paul Lohmann GmbH KG,
Germany
131
R&D use
Food grade
Food grade
Food grade
p-Aminobenzoylglutamic acid
(p-ABGA)
3-Aminophenol
Sodium nitrite
Sodium hydroxide
Sulfamic acid
Zinc (granule)
Food grade
R&D use
Pharmaceutical grade
Pharmaceutical grade
R&D use
ACS grade
ACS grade
R&D use
R&D use
0.1 N solution, ACS
BDH Inc.
grade
Sigma-Aldrich Chemicals, USA ACS grade
Sigma Chemicals, Canada
ACS grade
Sigma Chemicals, Canada
ACS grade
Spectrophotometric
Sigma Chemicals, Canada
grade
Sigma Chemicals, Canada
ACS grade
Folic acid was incorporated into the Guatemalan iodized salt and vitamin A fortified sugar
by various addition methods, as listed in Table 6.2.
The salt or sugar samples containing folic acid powder were prepared by directly blending
the desired amounts of folic acid into the samples. Each mixture was placed in a capped
glass bottle and shaken for ~5 min, and then transferred to plastic zipbags for storage
testing.
The samples made by spraying folic acid in either water solution or oil suspension were
132
prepared in a similar way to prepare the polymer coated iron premix as described in Chapter
4. Specifically, the salt or sugar samples were first loaded in the rotating pan. The pre-stirred
aqueous solution or oil suspension containing desired amounts of folic acid was slowly
sprayed on the samples with constant rotation of the disc and agitation using a plastic spoon
to prevent particle agglomeration. Compressed air at 3~5 psig was used as the atomization
agent.
Table 6.2 Experimental design for incorporating folic acid in Guatemalan salt and sugar
Refined Guatemalan salt
fortified with folic acid
Sample 1: segregation
plain & iodine interaction
Sample 2: segregation &
iodine interaction
Sample 3: segregation by
particle size
Sample 4: segregation by
particle size & iron
interaction
Sample 5: segregation &
iron interaction
Sample 6: blank iodized
salt
Coarse Guatemalan
vitamin A fortified sugar
Sample 1: segregation
plain & Vit A interaction
Sample 2: segregation &
Vit A interaction
Sample 3: segregation by
particle size
Sample 4: segregation &
Vit A interaction
Encapsulated FeFum
or current Glatt FeFum
formulation
As presented
in the original
salt sample
Incorporated in the
encapsulated FeFum
Folic acid
agglomerates by
extrusion
133
The folic acid premix was prepared using the similar extrusion protocol for making
microencapsulated FeFum premix. The desired amount of folic acid was blended into rice
flour and converted to extrudable dough by adding melted shortening and water. The wet
dough mixture was then extruded to form tiny cylindrical particles with a diameter and a
length of ~0.5 mm. The particles were then air-dried at 40oC and screened to collect with the
desired size ranging from 500 m to 700 m. The folic acid premix was then blended into
the salt or sugar samples with the desired target concentrations.
The folic acid/FeFum premix was prepared by using the extruded FeFum premix and
incorporating folic acid through the colour-masking process. Specifically, folic acid powder
was pre-blended with TiO2 and then used as the colour-masking agent for the extruded
FeFum particles. The colour-masked particles containing both folic acid and FeFum were
further encapsulated with desirable amount of hydrophilic polymer, i.e., HPMC. The premix
was then added into the salt or sugar samples to meet the target concentrations.
Six salt and six sugar samples were prepared and then stored at 40oC and 60% RH. The
retentions of added micronutrients, i.e., iodine, vitamin A, and folic acid, were measured
after 1, 2, 3, and 9 months storage and reported as the percentage of the original levels.
Folic acid fortified Ultra Rice
In parallel with the project of folic acid fortification of salt and sugar, the feasibility of
incorporating folic acid into Ultra Rice was also investigated.
As shown in Table 6.3, ten formulations were prepared using the standard laboratory
protocol for making the extruded Ultra Rice. The detailed procedure is reported in the
previous chapter. The samples were designed to test the effects of various FePP forms at
different addition levels on folic acid stability as well as on grain appearance. These samples
were then subjected to a 3 month storage test at 40oC and 60% RH.
Four commercial forms of food grade FePP were tested. The Colombian sample and the
sample from Fortitech had average particle sizes of ~25 m, while FePP supplied by Dr.
134
Paul Lohmann A.G. and the SunActive products were micronized with particle sizes ranging
from 1 to 10 m. The three regular FePPs have an average iron content of 25%, while the
SunActive FePP contains much lower iron, i.e., 8.16% or one third of the iron presented in
the other sources, due mainly to the incorporation of large amount of emulsifiers in this
commercial formulation. The designed addition level of the iron forms (e.g., 3.68 wt% of the
three regular FePPs and 11.04% of the SunActive) would result in an iron concentration of
~9 mg/g of rice premix. When the extruded rice is diluted with market rice at a ratio of 1:200,
this would lead to a daily iron intake of ~9 mg based on a daily consumption of 200g of rice.
TiO2 was added as colour-masking agent for improved grain appearance, i.e., whiter colour.
The design of formulation 10 was aimed to explore whether high concentrations of folic acid
and iron could be added into the system without adverse effects on grain properties and
nutrient stability.
Table 6.3 Experimental design for preparing folic acid fortified Ultra Rice formulations
FePP sources
FePP (%)
TiO2 (%)
1 Fortitech
3.68
2 Colombian
3.68
3 SunActive
11.04
3.68
3.68
3.68
3.68
(ppm)
None
300
700
300
None
8.82
135
ZnO (%)
600
0.42
Before the formulations were developed, viable analytical methods for folic acid were
standardized. The standardized analytical protocols are reported in Appendix 11.3.1.
Two spectrophotometric methods were adapted for folic acid determination. The direct
extraction method was used for salt and sugar samples, while the coupling reaction method
was used for Ultra Rice samples.
Vitamin A analysis
Vitamin A determination method was adapted from our previous protocol for Ultra Rice
(Lam, 2006). The Association of Official Analytical Chemists method 43.003 (AOAC
Fourteenth Edition), and Methods of Vitamin A Assays (Parrish, et al., 1985) were used to
develop the extraction procedure of vitamin A from the samples. The vitamin A
concentration in the extract was then determined by spectrofluorometry using an excitation
wavelength of 330 nm, and an emission wavelength of 480 nm. USP Vitamin A Reference
Solutions were used to calibrate the method.
Iodine analysis
Iodine content in the Guatemalan double or triple fortified salt was measured using the
titration method described in the previous chapter.
136
Physical properties, e.g., colour stability, of prepared samples were measured visually using
an Intel PlayTM QX3 Computer Microscope, and using Hunter L*a*b* spectrophotometric
system, as described in the previous chapter.
137
Table 6.4 Folic acid (FA) concentration in the final formulations of the Guatemalan fortified
salt and sugar samples for storage stability test under 40oC and 60% RH
Guatemalan salt samples Target FA Conc.
(g/g)
Iodine 42.9 +1.95 ppm
Experimental FA Conc.
(g/g)
Spraying
efficiency (%)
FA powder blended in
20
20.4 + 0.2
FA sprayed on batch 1
20
6.2 + 0.4
31
FA sprayed on batch 2
100
34.1 + 0.2
34
FA extruded premix
20
19.1 + 1.0
20
10.4 + 0.2
FeFum/FA premix
40
37.6 + 0.6
20
8.0 + 0.2
40
Target FA Conc.
(g/g)
Experimental FA Conc.
(g/g)
Spraying
efficiency (%)
20
19.1 + 0.3
20
12.5 + 0.6
62
200
111.5 + 1.7
55
FA extruded premix
20
25.1 + 1.9
20
4.3 + 1.2
23
200
88.3 + 2.9
44
FeFum/FA premix
40
66.7 + 6.6
FA powder blended in
Canadian sugar
200
183.0 + 0.7
FA sprayed on Canadian
blank salt
Guatemalan sugar
samples
Vitamin A
16.44 +1.09 ppm
FA powder blended in
FA aqueous spray on
batch 1
FA aqueous spray on
batch 2
50
Note: the experimental data are mean value standard deviation, which were obtained from three or four
replicates for each sample measurement.
As shown in Table 6.4, all addition methods except spraying were able to incorporate folic
138
acid into the samples at the expected fortification levels. Several batches of salt and sugar
samples made by the spraying process gained only one third to one half of the originally
designed amount of folic acid, which indicated that the process with the lab-scale rotating
pan lost 40% to 70% of the added folic acid into the atmosphere or on the surface of the pan.
Stability Tests
Six samples of either salt or sugar were prepared and stored at 40oC and 60% RH. Originally
the stability test was designed for 3 months; however, after 3 months storage, the trends of
some nutrient loss were unclear, and the test was thus extended to 9 months.
6.5.1.1. Folic acid stability
As shown in Figures 6.5 and 6.6, folic acid was generally stable in the fortified samples after
9 months storage at high T and RH, with >80% retained in all salt samples and ~75%
retained in all sugar samples except the sample made by spraying folic acid in oil suspension.
Fat oxidation as indicated by rancid smell in this particular sample would have produced
9 months
100
80
60
40
20
0
1
FA powder
2
FA spray
3
FA premix
4
FA spray +
Glatt Fe
premix
5
FA/FeFum
premix
Figure 6.5 Folic acid retentions in the fortified Guatemalan salt samples during 9 months
storage at 40oC and 60% RH. (Note: the results are the mean values obtained from three or
four replicates, and the error bars represent the standard deviations.)
139
In the control samples, blank Canadian salt and sugar, loss of folic acid was observed only in
sugar (>10%). This suggested that there might be interaction between folic acid and the
sugar grains. This is consistent with the results of Verlinde et al. (2006), who proposed a
non-enzymatic glycation reaction between folic acid and reducing sugars.
2 months
3 months
9 months
100
80
60
40
20
0
1
FA powder
2
FA water spray
3
FA premix
4
FA oil spray
5
FA/FeFum
premix
Figure 6.6 Folic acid retentions in the fortified Guatemalan sugar samples during 9 months
storage at 40oC and 60% RH. (Note: the results are the mean values obtained from three or
four replicates, and the error bars represent the standard deviations.)
Among the different incorporation techniques, the addition of folic acid premix prepared by
extrusion showed the best results, with virtually no vitamin loss in either salt or sugar
samples. The direct addition of folic acid either in its powder form or by spraying in water
solution or oil suspension resulted in the most losses of the vitamin, regardless of the food
vehicles used. When incorporated into the encapsulated iron premix and then added into the
food vehicles, the stability of folic acid was affected to some extent, probably due to the
interaction of the active iron compound and/or the colour-masking agent, TiO2, with the
vitamin.
As shown in Figure 6.7, iodine in the original Guatemalan salt was very stable, with ~95%
retained after 9 months storage. The incorporation of folic acid, regardless of the techniques
used, resulted in extra losses of iodine, up to 40%. The best result was obtained in the
140
sample containing folic acid premix made by extrusion, with >88% iodine retention. The
greatest losses were in the samples made by spraying folic acid as a water solution, as shown
in samples 2 and 4, where only ~60% of the original iodine was retained after 9 months. It is
interesting to note that ~15% of iodine loss occurred right after sample preparation and
within the first month, which might be related to the manner of spraying folic acid solution
onto the salt surface, where some of the iodine could have been washed away.
The fact that all salt samples had some iodine degradation suggested that there might be
interactions between iodine and folic acid, as well as other formulation ingredients. This will
be discussed further.
2 months
2
FA spray
3
FA premix
3 months
9 months
100
80
60
40
20
0
1
FA powder
4
FA spray +
Glatt Fe
premix
5
FA/FeFum
premix
Control Guatemalan
blank iodized
salt
Figure 6.7 Iodine retentions in the fortified Guatemalan salt samples during 9 months storage
at 40oC and 60% RH. (Note: the results are the mean values obtained from three or four
replicates, and the error bars represent the standard deviations.)
6.5.1.3.Vitamin A stability in Guatemalan vitamin A fortified sugar
As shown in Figure 6.8, up to 60% of the vitamin A in the original Guatemalan sugar was
lost during 9 months of storage at 40oC and 60% RH, even in the absence of any other
micronutrients. This confirmed that vitamin A is most vulnerable among the studied
micronutrients. Not surprisingly, incorporation of folic acid in the premix did not cause
much extra loss of vitamin A, with 35% retained in the sample made with folic acid premix
and 31% retained in the sample containing the combined premix of folic acid and FeFum.
141
The other three samples made by direct addition either as powder or as sprayed solution or
suspension lost additional vitamin A. Particularly, the sample containing folic acid powder
experienced rapid vitamin A degradation within the first three months. It indicated that
reactions might occur between folic acid and vitamin A. The addition of active ingredients,
such as folic acid, accelerated the vitamin A degradation rate to some extent, whereas the
inert agents, including the encapsulated premixes, had little impact on vitamin A stability.
2 months
3 months
9 months
4
FA oil spray
5
FA/FeFum
premix
100
80
60
40
20
0
1
FA powder
2
FA water
spray
3
FA premix
Control Guatemalan
blank vit A
fortified sugar
Figure 6.8 Vitamin A retentions in the fortified Guatemalan sugar samples during 9 months
storage at 40oC and 60% RH. (Note: the results are the mean values obtained from three or
four replicates, and the error bars represent the standard deviations.)
Digital pictures of the salt and sugar samples before and after 3 months storage are presented
in Appendix 11.3.2.
Most salt and sugar samples retained their colour, except the two sugar samples made by
either water spray or oil spray of folic acid, in which the sugar grains became irregular and
tended to stick to each other with rough surface. Both salt and sugar are very hygroscopic.
However, the Guatemalan salt had a smaller particle size than the sugar; thus, it had a much
larger surface area and required higher moisture content to reach the flow moisture point.
When the samples were prepared by spraying the same amount of water solution of folic
acid, the sugar sample was more likely to turn to lumps.
142
It is worth noting that folic acid premixes either made with folic acid alone or incorporated
in the FeFum premix using the process as described before, presented a similar physical
appearance to the salt and sugar grains, which enabled the premixes to fit into the carrier
foods well. This also confirmed that the microencapsulation process discussed in Chapter 4
could be also used for developing other micronutrient delivery systems for food fortification.
6.5.2 Folic Acid Fortification in Multiple Fortified Ultra Rice
6.5.2.1 Ultra Rice appearance and colour stability
The digital pictures of the multiple fortified Ultra Rice samples are presented in Appendix
11.3.3.
The Ultra Rice grains made by extrusion could resemble the native rice in terms of shape
and grain size, but lacked the translucent and shiny appearance, subsequently resulting in
darker colour. The addition of folic acid and FePP imparted a yellowish hue to the grains. On
the other hand, the incorporation of TiO2 gave the grains a bright white colour and opaque
texture, due to its very high refractive index.
The visually observed colour differences were also confirmed by the Hunter L*a*b*
measurements. As shown in the Figures 6.9 to 6.12, all samples made with FePP and folic
acid had greater positive b* values, ranging from 18 to 22, than the native rice (17). With the
addition of TiO2 at different levels, this b* value decreased to 12~16, which indicated the
whitener could cover the yellow shade.
Various sources of FePP imparted slightly different colour to the Ultra Rice grains. As
shown in Figure 6.9, the Colombian FePP gave the lightest colour with the greatest L*
and/or the smallest E*. The sample made with Fortitech FePP had the second lightest
colour. Both micronized FePP forms, from Dr. Paul Lohmann and SunActive, imparted a
greenish hue (smaller a* values) and darker shade (smaller L* values and greater E*) to the
Ultra Rice grains. After 3 months storage at high T and RH, not surprisingly, all samples
were darker, as indicated by the reduced L* values or increased E* numbers.
143
FePP source
Dr. Paul Lohmann
Fortitech
Colombian
SunActive
Integrated colour difference from the
reference - native rice
Original t=0
L*
a*
FePP (%)
3.68
3.68
3.68
11.75
b*
67.35
1.76
21.42
66.69
1.06
23.92
68.10
2.11
21.78
68.08
1.66
23.38
68.82
3.18
18.88
67.60
2.60
22.58
65.87
1.21
18.78
65.92
-0.04
20.92
t=0
t=3 months
12.4
11.6 12.1
11.0
10.2 10.9
10.7
12
t= 3 months
L*
a*
b*
8.7
9
6
3.5
3
0
Dr. Paul
Lohmann
Fortitech
Colombian
SunActive
Blank simulated
rice
Figure 6.9 Colour stability of the Ultra Rice grains made with various FePP sources
FePP source
1
2
3.68
None
Original t=0
L*
a*
FePP (%)
b*
t= 3 months
L*
a*
b*
71.97
70.93
67.35
0.96
2.23
1.76
19.71 70.38
21.14 70.80
21.42 66.69
77.37
2.03
17.06
75.76
0.51
14.87
t=0
0.78
1.79
1.06
21.00
21.16
23.92
t=3 months
12.4
10.7
12
7.9
7.4
7.5
5.9
6
3.5
3
0
FePP 1%
FePP 2%
FePP 3.68%
Blank simulated
rice
Figure 6.10 Colour stability in the Ultra Rice grains made with Dr. Paul Lohmann FePP at
different addition levels
144
TiO2 (%)
FePP source
None
None
1
2
3
Original t=0
L*
a*
67.35
1.76
74.97
0.36
78.24
-0.06
78.55
-0.36
b*
21.42
15.92
14.12
12.80
77.37
2.03
17.06
75.76
0.51
14.87
t=0
t= 3 months
L*
a*
b*
66.69 1.06 23.92
78.70 0.05 16.72
79.86 -0.33 14.66
79.92 -0.58 12.98
t=3 months
12.4
12
10.7
9
6
3.4
4.2 4.5
5.5 5.9
3.5
2.5
0
None
TiO2 1%
TiO2 2%
TiO2 3%
Blank simulated
rice
Figure 6.11 Colour stability of Ultra Rice grains with addition of TiO2 as the
colour-masking agent at different levels
Folic acid Lohmann
(ppm)
FePP (%)
Higher overage
Lower overage
Control - Native rice
Control - Blank
simulated rice
300
600
3.68
8.82
None
None
Original t=0
L*
a*
b*
60.91
66.22
2.99
2.24
22.38 59.37
21.73 66.30
77.37
2.03
17.06
75.76
0.51
14.87
t=0
20
17.2
t= 3 months
L*
a*
b*
2.66
2.01
22.53
22.57
t=3 months
18.7
15
11.9
12.1
10
3.5
5
0
Higher overage
Lower overage
Figure 6.12 Colour stability of Ultra Rice grains made with higher levels of folic acid and
FePP
145
As indicated by Figure 6.10, increased addition levels of FePP resulted in more pronounced
colour changes in the Ultra Rice grains. With 1% FePP from Dr. Paul Lohmann in the
formulation the Ultra Rice grains were closer in colour to rice, while an increase in iron
content caused darker colours to develop, especially the sample containing 3.68% of the iron
compound had a greatly reduced L* value and/or increased E* value. Again, storage over 3
months darkened the grains.
The effect of TiO2 on colour was promising. As shown in Figure 6.11, improvements in the
lightness were observed in the samples containing different levels of TiO2, as indicated by
greatly increased L* values. Also, the grain chromaticity was likely to shift from red to green
(with reduced a* values) and from yellow to blue (with reduced b* values). This trend was
further enhanced by the increase in the addition level of TiO2 up to 3%. However, when
looking into the integrated colour difference - E* value, it is found that the increase in the
TiO2 addition level did not always cause positive effects. Actually the addition of 3% TiO2 in
the formulation resulted in a sample with much higher E* value from the reference the
native rice, compared to the sample made with 1% TiO2. This was confirmed by visual
observation (Appendix 11.3.3): the sample containing 3% TiO2 was totally opaque and
eliminated the semi-translucent pearlescence seen in the native rice and the original Ultra
Rice grains. Overall, grains with 1% of TiO2 had the smallest difference E* value.
Interestingly, this E* value was further improved after storage, indicating the sample
looked more natural after storage, in contrast to the observations of all other samples in the
study.
As seen in Figure 6.12, increased additions of both FePP and folic acid resulted in much
darker grain colours, which deteriorated further during storage.
146
Folic acid retention in these samples was followed for three months. As indicated in Table
6.5, the vitamin was stable with >80% folic acid retained in all rice formulations. The
samples made with the two micronized FePPs retained slightly more folic acid. The addition
levels of the iron compound had the expected effect of reducing folic acid stability - with
more vitamin retained in the formulation containing less iron. Nevertheless, all samples
containing 1% to 3.68% of the iron could retain >90% of the original folic acid, regardless of
the iron source used. Surprisingly, the incorporation of TiO2 at 2% in the formulation seemed
to have an adverse impact on folic acid stability, resulting in >13% loss of the vitamin after 3
months storage. This may be related to the light sensitivity of folic acid. TiO2, as indicated in
the literature with a photocatalytic effect (Hashimoto, et al., 2005), might speed up the
vitamin degradation.
Table 6.5 Folic acid retention in the Ultra Rice samples made with various FePP sources
and at different addition levels
Folic acid relative retention (%)
Formulation variations
1 month
2 months
3 months
1
Fortitech 3.68%
98.8 1.4
93.4 1.8
93.1 1.5
Colombian 3.68%
101.6 2.2
99.8 4.5
93.1 4.8
SunActive 11.04%
101.5 1.2
96.9 2.2
98.2 1.6
97.9 3.1
95.7 1.9
96.6 2.0
96.3 1.6
98.0 2.3
96.4 4.2
99.7 2.9
101.3 2.4
100.2 1.6
98.5 2.5
95.3 1.9
93.5 4.3
94.3 1.4
92.8 1.8
87.4 2.5
102.1 2.4
93.9 2.1
91.8 6.0
10
92.1 2.0
90.7 1.1
82.3 8.9
Note: the results are reported as mean value standard deviation, which were obtained from
three or four replicates for each sample measurement.
147
As discussed earlier in reference to Guatemalan salt and sugar samples, folic acid was
generally stable when incorporated into salt, and there was no apparent interaction between
folic acid and salt. The early results from an ongoing study in our research group seemed to
confirm this observation. When folic acid was added into potassium iodate solution and
sprayed together onto iodized salt, there was no obvious loss of folic acid after one month
(Zheng & Saunders, 2008). However, when folic acid was sprayed on sugar, it seemed less
stable, perhaps due to non-enzymatic glycation of folic acid by reducing sugars as reported
by Verlinde et al. (2006).
The potential interactions between added micronutrients were examined. In salt samples,
increased iodine losses were associated with greater losses of folic acid, particularly in the
samples made with folic acid added as a powder or by sprayed on. However, there was no
clear trend attributable to the different addition techniques. The data were inconclusive and
we will not have a clear understanding of the nature and extent of interactions until a current
parallel study is completed.
A clearer picture of interaction between vitamin A and folic acid in the sugar samples was
observed. Vitamin A losses were correlated with losses of folic acid, irrespective of the
techniques used to prepare the samples. However, this correlation was not strong enough to
establish a casual relationship. The literature indicates that vitamin A degradation products
may react with amino acids (Kim, et al. 2000), whereas folic acid contains a glutamate
moiety. However, there has been no explanation as to how this reaction may affect vitamin A
stability.
Based on these results, it seems prudent to incorporate folic acid into salt or sugar as a
separate premix, since this resulted in improved folic acid stability and unaffected iodine and
vitamin A stability. The addition of folic acid by incorporating it into the encapsulated
FeFum premix to achieve triple fortified foods, such as salt simultaneously fortified with
iodine, iron, and folic acid is also promising. In this series of tests, folic acid was added to
TiO2 in the colour-masking step. The vitamin stability was decreased somewhat by TiO2
148
149
1. The storage tests have shown that folic acid was generally stable in the three fortified
foods studied: iodized salt, vitamin A fortified sugar, and iron fortified Ultra Rice. After
3 months storage, >90% of the added vitamin was retained in most rice samples, >80%
were retained in most salt samples, and ~75% in most sugar samples after 9 months
storage at high T and RH.
2. The incorporation technique had a pronounced impact on the stability of folic acid as
well as the other added micronutrients. The best results were obtained when folic acid
was incorporated as a separate premix, which protected the vitamin and prevented the
interactions with other micronutrients present in the systems. Specifically, there was
virtually no folic acid loss when the vitamin was added to the salt and sugar samples in
the form of a single extruded premix even after 9 months storage at 40oC and 60% RH.
3. Examination of interactions between folic acid and the delivery systems revealed that
salt and rice flour-based matrices did not react with the vitamin, while it may have
reacted with reducing sugars through a non-enzymatic glycation in sugar samples.
Therefore, salt and rice were confirmed as suitable food vehicles for folic acid
fortification.
4. In some salt samples, folic acid might react with iodine, resulting in losses of both
nutrients. A parallel study in our research group will explore this further. Some
interaction between folic acid and vitamin A was observed in sugar samples as well as
between folic acid and TiO2 in the rice system.
5. This part of work has also confirmed that extrusion followed by polymer coating is a
feasible microencapsulation technology for making various delivery systems containing
selected micronutrients. In addition, it backs up the hypothesis that proper encapsulation
may be needed to ensure the stability in foods fortified with multiple nutrients.
1. Based on the promising results from this part of work, it is recommended that detailed
studies on formulation design and process development for salt double or triple fortified
with iodine, iron, and folic acid should be initiated.
2. The interactions between the added micronutrients should be explored further.
150
3. Adding folic acid through the industrial salt iodization process seems promising and
should be pilot tested. As no extra equipment or salt processing is required, the effective
formulation and process developed could be readily incorporated into the universal salt
iodization program to bring immediate human health benefits.
151
7 CONCLUSIONS
A microencapsualtion-based technology platform for the effective delivery of micronutrients
has been developed. The applicability of the technology has been demonstarted in typical
staple foods: salt, sugar, and rice. The process is comprised of agglomeration by extrusion
followed by colour-masking and encapsulation by polymer coating. With appropriate
combinations of different unit operations, this flexible technology platform is adaptable to
broader applications, not only in food fortification, but also in active ingredient delivery in
functional foods, oral drugs, and agrochemicals.
1.
2.
Salt double fortified with iodine and iron using the microencapsulated FeFum premix
made by the extrusion-based agglomeration process had the desired sensory properties
and was stable for up to a year.
3.
152
4.
It is feasible to incorporate folic acid into the existing fortification programs using the
microencapsulation technology platform developed in this study. The results indicate
that folic acid fortification in conjunction with industrial salt iodization processes should
be readily commercialized.
The positive results warrant further examination of this approach, with a vision to achieve
commercially viable solutions to active ingredient delivery. Specific opportunities are
presented in the following recommendations.
achieved within a semi-solid rice starch matrix through extrusion. This evidence greatly
advances the knowledge in this field and opens up opportunities for broader applications.
3. Through investigating the developed microencapsulation-based technology platform in
and cooking was developed. This method can be used as an indirect assessment of the
gel formation within the semi-solid starch matrix.
In addition to the above contributions to the scientific field, the study resulted in the
development of successful technologies/processes for food fortification, which are expected
to bring immediate benefits in human health and social development.
153
8 RECOMMENDATIONS
The processes and stable formulations developed in this program are ready for testing on a
larger scale. On our suggestion, PATH initiated a commercial scale test using our
recommended internal gelation systems for producing Ultra Rice in Brazil.
The incorporation of folic acid fortification into current salt iodization technology seems
feasible and should be tested in a small salt plant, once the current long-term stability tests
are completed.
The field tests of microencapsulating ferrous fumarate using the optimized process and
formulation obtained from the study have been initiated in India. The initial results were
inconclusive, due to the unavailability of the right equipment (Diosady, 2008). The
economical feasibility of using the advanced technologies in the current fortification
programs should be examined once the pilot-scale trials are completed.
potential interactions between the acidic antioxidants with the internal gelation system could
be prevented through the use of the salt forms of the acidic antioxidants or through
microencapsulating one or more of the active ingredients prior to extrusion and Ca/alginate
cross-linking. This approach should be explored further.
Binders
Figure 8.1 Model premix system for salt fortification made by the extrusion agglomeration
followed by polymer coating, containing multiple micronutrients such as iron, zinc, and
vitamin A
Figure 8.2 Model Ultra Rice premix made by extrusion using internal gelation and followed
by polymer coatings for delivering multiple micronutrients including iron and vitamin A
155
2 mm
Alginate-calcium network
throughout the grain
7~10 mm
Figure 8.3 Model Ultra Rice premix made by extrusion using internal gelation, containing
sub-capsules of microencapsulated premixes of iron and vitamin A made by extrusion-based
technology platform
In summary, the results of this thesis work are very promising, and should be tested on a
series of systems used to deliver one or more reactive ingredients through food and other
matrices.
156
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167
10 NOMENCLATURE
m
Micrometer
AAS
AI
Adequate Intakes
AOAC
BHA
Butylated hydroxyanisole
BHT
Butylated hydroxytoluene
DFE
DFS
Double-fortified salt
DRI
EAR
EDTA
Ethylenediaminetetraacetic acid
FAO
FDA
FeFum
Ferrous fumarate
FePP
Ferric pyrophosphate
GDL
Glucono-delta-lactone
GMP
GRAS
HPMC
Hydroxypropyl Methylcellulose
IDA
IDD
MI
Micronutrient Initiative
MSG
Monosodium glutamate
NTD
pABG
p-aminobenzoylglutamic acid
PATH
PEG
Poly(ethylene glycol)
ppm
PVA
168
RBV
RDA
RH
Relative Humidity
RSD
SEM
SHMP
Sodium hexametaphosphate
STPP
Sodium tripolyphosphate
Temperature
TFS
THF
Tetrahydrofolic acid
ToF-SIMS
TPP
Tripolyphosphate
TSPP
Tetrasodium pyrophosphate
VAD
Vitamin A deficiency
WHO
XPS
169
Appendix 11.1.1
LIST OF APPENDICES
Analytical methods used in research approach 1
172
Appendix 11.1.2
Appendix 11.1.3
Appendix 11.1.4
Appendix 11.1.5
Appendix 11.1.6
Appendix 11.1.7
Appendix 11.1.8
Appendix 11.1.9
Appendix 11.1.10
175
176
177
178
178
Appendix 11.1.11
186
Appendix 11.1.12
187
Appendix 11.1.13
Appendix 11.1.14
Appendix 11.1.15
Appendix 11.1.16
Appendix 11.2.1
Appendix 11.2.2
170
192
Appendix 11.2.3
Appendix 11.2.4
199
Appendix 11.3.1
203
Appendix 11.3.2
Appendix 11.3.3
171
11 APPENDICES
Appendix 11.1 Detailed Analytical Methods and Results from the Study of
Microencapsulated FeFum Premix for Salt Double Fortification
Appendix 11.1.1 Analytical methods used
Bulk Density (DB) was measured by weighing a known volume of iron particles in a
graduated cylinder and compacted by gently tapping the flask. The weights of the empty
flask and sample-filled flask were recorded. The same operation was repeated by filling the
flask with distilled water. The bulk density of the sample was then calculated by the
following equation, for an average of four replicates:
DB =(W2-W1)/(W3-W1) x Dw,
W1 is the weight of the empty flask; W2 is the weight of the sample-filled flask; W3 is the
weight of water-filled flask; Dw is water density.
Particle Density (DP) was measured by the weight of the mass per unit volume of the solid
only. Specifically, after the measurement of the bulk density as described before, the void
volume in the sample-filled flask was determined by dropwise addition of hexane. The
weight of the flask was recorded as W4, and the particle density of the sample was calculated
by the following equation:
DP = (W2-W1)/[(W3-W1)/Dw (W4-W2)/DH],
W1, W2, W3, and DW are defined as above; W4 is the weight of the flask filled with both
the sample and hexane; DH is hexane density.
Iron Analysis
Total iron was measured by atomic absorption spectrophotometry (AAS) using AOAC
method 3.6.1.2 (Fourteenth Edition, 1984). Approximately 60 mg iron premix is accurately
weighed and digested in 20 mL concentrated HNO3 and HCl (1:1, v/v) until approximately
25% of the liquid retained. The digested samples were cooled and then diluted to 100 mL
with distilled water. The absorbance at 248.3 nm was measured with a Perkin-Elmer AA100
atomic absorption spectrophotometer, and the concentration of iron was calculated based on
a calibration curve obtained using standard iron reference solution.
Ferrous iron content in the premix was determined by spectrophotometry (Harvey, Smart, &
Amis, 1955), as a complex with 1,10-phenanthroline. Approximately 300 mg of iron premix
was accurately weighed and dispersed in ~40 mL of distilled water, followed by acidifying
with 1 mL of concentrated sulphuric acid. The sample solution was boiled for 5 minutes for
digestion. After cooling, the digested sample was diluted to 100 mL with distilled water. A
172
173
Appendix 11.1.2
Preliminary observations on suitability of different binder materials
Binder
Observations
could form an extrudable dough by itself with
addition of ~5% lipid and ~40% water; the
extrudates were crumble; the addition of 50%
Dextrin
FeFum could form a sticky dough which made
extrusion hard to proceed and the extrudates were
very rough in surface.
could form a sticky dough by itself, yet hard to
extrude; could not form an extrudable dough with
HPMC the addition of FeFum regardless of the ratios
between the binder and FeFum, and the amounts of
water and lipid added.
could not form an extrudable dough by itself or
Potato
by a combination with FeFum, regardless of water
starch
and lipid contents.
could not form an extrudable dough by itself or
Corn
by a combination with FeFum, regardless of water
meal
and lipid contents
a perfectly extrudable dough could be formed by
itself; with the addition of FeFum up to 70% (w/w)
Rice
an extrudable dough could be formed, while lipid
flour
and water contents needed to be carefully adjusted;
the extrudates were sticky and rough in the surface
when cut at the axial direction.
Comments
could not be used as the
primary binder; might be used
as a secondary binder.
174
Appendix 11.1.3
Differences between three cereal flours used as binders in the study of
microencapsulated FeFum premix for salt fortification
Rice flour
Wheat flour
< 300 m
< 300 m
0.937 0.041
0.832 0.020
0.880 0.006
1.427 0.074
1.310 0.016
1.320 0.016
Starch %
80.1
76.2
71.4
Protein (gluten) %
6.4
9.4
13.2
Fat %
0.8
1.3
1.8
2.0
2.1
3.1
Water %
11.8
12.0
12.0
$30/bag of 45 lbs
$6/bag of 22 lbs
$6/bag of 22 lbs
Retail price
Note: The composition information, such as the contents of starch, protein, fat, and fiber
were obtained from the suppliers specification sheets, other technical data were measured in
the laboratory. The experimental data are presented as mean value standard deviation,
which were obtained from three or four replicates for each sample measurement.
175
Appendix 11.1.4
Preliminary investigation of dextrin and HPMC as secondary binders
(Lo, 2006; Mourret, 2006)
Formulation
70% FeFum, 30% rice flour
70% FeFum,
30% rice flour:dextrin (80:20)
70% FeFum,
30% rice flour:dextrin (70:30)
70% FeFum,
30% rice flour:dextrin (50:50)
70% FeFum,
30% rice flour:HPMC (90:10)
Observations
Hard to extrude, and the extrudates also
stuck together
Extrudable, with non-sticky extrudates,
the best combination so far
Extrudable, but the extrudates were
stickier than the above formulation
Ranking on
extrudability
(0-3)
1
3
2
Not extrudable
80% FeFum,
Hard to extrude
1
20% rice flour:HPMC (85:15)
80% FeFum,
Not extrudable
0
20% rice flour:HPMC (75:25)
Note: the ranking scales are: 0 not extrudable at all; 1 hard to extrude with very low flow
rate; 2 extrudable but the particles collected were not of desired properties in appearance
and/or surface smoothness; 3 easy for extrusion with desired particle properties.
176
Appendix 11.1.5
Comparison of extrudability and product characteristics between three cereal flours
used in the study as binders
177
Appendix 11.1.6
Comparison on the effectiveness of TiO2 adhesion before and after drying (pictures
were taken from optical microscopy at x60 magnification)
Formulation
After extrusion
TiO2 dusted
after drying
Washed and
filtered
TiO2 dusted
before drying
Washed and
filtered
70% FeFum
30% rice/dextrin
70% FeFum
30% wheat flour
70% FeFum
30% durum flour
75% FeFum
25% durum flour
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 operation using the fluidized bed
and the pan coater
A Uni-Glatt laboratory fluidized bed coater was used for hydrophilic polymer coating. It was
observed that the quality of developed polymer film could be greatly affected by the
operation conditions (Table 11.1.8.1). Thus, it was necessary to examine the operation
parameters and find the optimal combinations.
Table 11.1.8.1 Examples of unsuccessful coatings with different materials and techniques
10% Methocel - Fluidized bed
There are several parameters in the fluidized bed system which greatly affect the process and
quality of the final products, including flow-rate and pressure of the spraying liquid,
composition and rheology of the coating solution, flow-rate and temperature of the fluidizing
air. These parameters could be controlled and adjusted on the machine.
The results from a preliminary investigation showed that the coating chamber needed to be
pre-heated and the temperature of the process air had to be high enough to ensure a rapid
evaporation of the coating solution, thus preventing the coated particles sticking together.
The flow rate of the fluidizing air was dependent on the particle size/density and the loaded
weight, where proper fluidization and recirculation of the loaded particles were the key
requirements. Air flap of the instrument, which has five positions from 15% to 75%, was
used to control the air flow rate. The concentration of the spray solution was crucial to the
ease of operation and time consumed. High concentrations made the polymer solution too
viscous and caused difficulties in operation, whereas too diluted solutions would take
prolonged time for the operation. The optimal concentration of the coating solution was 2%
for MethocelTM and 5% for Kollicoat. The flow rate of the spray solution was controlled by
a peristaltic pump attached to a variable speed regulator. It was adjusted in accordance with
solvent evaporation rate (controlled by the system temperature) and the degree of particle
fluidization (controlled by the air flap opening). The desired flow rate of the spray solution
should be as fast as possible to minimize the operation time and provide sufficient wetting
effect on the particles, but not so much that the suspended particles would stick together
(Figure 11.1.8.1). The nozzle opening and the air pressure for the spray nozzle jointly
formed the atomization angle, which integrated with the spray flow rate and the nozzle
position in the bed to generate the dynamic air/water/coating contact angle (Figure 11.1.8.2),
which played the most important role in coating efficiency.
179
Overall, an optimal combination of the parameters was obtained from the preliminary study
as shown below (Barquin, 2006):
Temperature of the fluidizing air: 70-85oC
Air flow rate for the fluidizing air: 37-45% of flap opening
Flow rate of coating solution: < 60 mL/hour or <1mL/min
Air pressure for the spray nozzle: 1.5 2 bars
Vertical position of the spray nozzle: at 1/3 to the top of the coating chamber
Figure 11.1.8.1 Flow rate of the spray solution for proper wetting that ensures the process
turn to coating rather than particle agglomeration (adapted from Guignon, et al. 2002)
Coating solution + compressed air
Iron particles
Hot fluidizing air
Coating solution
Air
Spray droplet
Atomization angle
Figure 11.1.8.2 Dynamic air/water/coating contact angle that is responsible for effective
coating within fluidized beds (adapted from Glatt website; Guignon, et al. 2002; Dowling
website)
180
181
5. Mix the melted soy stearine with dichloromethane and water in the 250 mL beaker in a
fume hood. Ensure the mixture is uniform.
6. Transfer the contents of the 250 mL beaker into the TLC flask.
7. Attach the nozzle attachment to the TLC flask.
8. Place the glass flask on a hot plate at around 50oC so that the soy stearine does not
solidify.
9. Set up the pan coating apparatus. This apparatus must be placed in a fume hood to remove
the dichloromethane vapours. Set the rotating pan at a 45o angle and set the rotation speed to
be approximately 50 rpm.
10. Place the colour-masked particles in the rotating pan and test if the particles move freely
across the pan.
11. Attach the hose connected to the compressed air supply to the nozzle of the TLC flask.
Turn on the air supply to about 3 psig.
12. Take the flask off the hot plate and start spraying the coating solution onto the particles
in the rotating pan. Keep the flask at least 30 cm away from the pan and make sure the
spraying occurs uniformly. The spray should be fine so that a uniform layer of coating is
formed on the particles.
Note: To spray, place your thumb on the small hole at the bottom of the nozzle attachment.
To stop spraying, remove your thumb.
CAUTION: Wear protective equipment while spraying dichloromethane. Wear a mask,
gloves and face shield. Keep the TLC flask at arm's length under the fume hood.
13. Once the particles start to become coated, they may stick to each other. In this case, use
a plastic spoon to ensure agglomeration of particles does not occur. Place the spoon at the
top of the pan at the 90o angle to the pan while it is rotating. The spoon will gently scrape the
surface of the pan and remove any stuck particles.
Note: If the coating solution seems to be solidifying, place it on the hot plate and let it melt
before continuing the spraying process. If the nozzle is clogged, remove the nozzle
attachment and lay it on the hot plate to melt the soy stearine.
182
Appendix 11.1.9
Detailed composition of the 12 final microencapsulated FeFum premixes
Extrusion
Formulation
Binder
FeFum
Colour
masking
Encapsulation
Shortening
Antioxidant
TiO2
Polymer
17.09%
49.83%
rice flour
(16.61% Fe)
4.27% dextrin
1.78%
0.07% BHA,
0.07% BHT
17.80%
Methocel
9.09%
16.34%
47.67%
rice flour
(15.89% Fe)
4.09% dextrin
1.70%
0.07% BHA,
0.07% BHT
17.02%
Kollicoat
13.04%
20.46%
durum flour
47.74%
(15.91% Fe)
1.71%
None
17.05%
Methocel
13.04%
21.36%
durum flour
49.83%
(16.61% Fe)
1.78%
0.07% BHA,
0.07% BHT
17.80%
Methocel
9.09%
21.36%
durum flour
49.83%
(16.61% Fe)
1.78%
0.07% BHA,
0.07% BHT
17.80%
Soy stearine
9.09%
21.36%
durum flour
49.83%
(16.61% Fe)
1.78%
0.78% BHA,
0.07% BHT
17.80%
Methocel
9.09%
20.43%
durum flour
47.67%
(15.89% Fe)
1.70%
0.07% BHA,
0.07% BHT
17.02%
Kollicoat
13.04%
18.53%
durum flour
55.59%
(18.53% Fe)
1.85%
Methocel
4.76%
16.92%
durum flour
50.75%
(16.92% Fe)
1.69%
Kollicoat
13.04%
10
18.53%
durum flour
55.59%
(18.53% Fe)
1.85%
Kollicoat
4.76%
11
20.46%
wheat flour
47.74%
(15.91% Fe)
1.71%
None
17.05%
Methocel
13.04%
12
21.36%
wheat flour
49.83%
(16.61% Fe)
1.78%
0.07% BHA,
0.07% BHT
17.80%
Kollicoat
9.09%
183
Drying
During drying, the extrudates were dried until the moisture content could be assumed to be
zero. Therefore, the amount of water was not taken into consideration when calculating the
final premix composition.
Colour-masking
Mass of titanium dioxide: 25% (w/w) of the dried extrudates
The total mass of the colour-masked extrudates was 127.52 grams.
Microencapsulation
Mass of encapsulants: 10% (w/w) of the colour-masked iron particles
100 grams of the above colour-masked particles were used for encapsulation, and 10 grams
of the coating material was applied to the particles, assuming no coating loss.
The final mass is 110 grams.
The final premix composition was calculated based on the final mass as follows.
% ferrous fumarate = 70 / 127.52*100/110*100
% rice flour
= 24 / 127.52*100/110*100
% dextrin
= 6 / 127.52*100/110*100
% shortening
= 2.5 / 127.52*100/110*100
% titanium dioxide = 25 / 127.52*100/110*100
% encapsulants
= 10 / 110*100
= 49.83%
= 17.09%
= 4.27%
= 1.78%
= 17.80%
= 9.09%
184
Appendix 11.1.10
Iron in vitro bioavailability test results of the optimized formulations of
microencapsulated FeFum premixes
Formulation
0 min
P-12
0.56
30 min
60 min
90 min
120 min
69.1
33.3
89.3
81.7
56.3
99.9
93.1
101.1
102.2
101.1
101.0
96.6
101.9
102.7
102.8
100.9
101.2
100.4
102.8
103.4
73.8
60.6
75.0
86.6
66.2
74.7
89.9
87.2
86.1
94.0
85.6
84.3
90.2
87.7
86.5
94.1
88.2
85.2
90.5
89.6
86.7
95.1
88.3
85.7
69.3
69.0
62.3
70.4
69.1
84.7
93.0
93.9
86.8
86.9
72.8
94.8
87.2
94.1
101.1
96.4
96.0
96.2
94.3
100.9
87.8
97.8
95.3
87.3
97.9
99.2
97.6
101.1
95.6
96.2
101.8
88.6
100.7
94.9
96.5
97.4
97.5
98.9
99.6
95.7
96.5
102.6
90.3
100.4
87.1
99.8
98.8
98.6
Note: the values are % of the iron dissolved in the digestion solution (pH1 HCl) at different
time points during the 2h dissolution test
185
Appendix 11.1.11
Particle integrity dissolution test results of the optimized formulations of
microencapsulated FeFum premixes
Formulation
0 min
30 min
60 min
90 min
120 min
5.1
3.1
11.2
3.1
7.2
5.1
17.2
5.5
9.9
5.8
20.7
7.8
12.1
8.1
25.4
9.8
1.8
6.4
5.5
7.6
3.6
9.5
12.0
12.5
4.4
14.0
16.1
16.2
6.1
17.5
21.2
21.3
4.9
7.2
5.9
3.1
4.2
4.8
7.8
5.7
9.3
6.0
7.8
6.8
10.0
7.0
6.6
9.9
8.5
12.3
9.7
15.9
9.1
12.0
8.7
13.0
9.4
12.6
13.6
12.0
17.8
19.4
21.4
11.9
17.1
13.3
19.4
11.8
19.9
17.4
22.3
22.7
24.2
26.6
15.1
21.0
Note: the values are % of the iron released in the weak acid solution (pH4 HCl) at different
time points during the 2h dissolution test.
186
Appendix 11.1.12
Physical characteristics of the final 12 microencapsulated FeFum premixes (digital
pictures are at x60 magnification)
187
Appendix 11.1.13
SEM images of the final 12 microencapsulated FeFum premixes (at ~5000
magnification)
188
Appendix 11.1.14
Relative iodine retention in DFS samples containing various FeFum particles during
one year storage under 40oC and 60% RH
Note: the results are mean value standard deviation, which were obtained from three or
four replicates for each sample measurement.
189
Appendix 11.1.15
Ferrous iron retention in formulated FeFum particles and in DFS samples, when
stored at the ambient condition and the higher conditions of 40oC & 60% RH,
respectively
Ferrous iron retention (%)
t=0 at the beginning
of storage
93.04 0.71
92.90 1.56
94.02 0.34
94.18 3.06
89.54 2.55
91.86 1.42
93.53 2.97
93.66 2.61
84.69 1.42
91.38 0.81
89.01 0.95
92.77 2.18
95.32 1.92
94.12 4.07
86.05 2.52
96.17 1.09
95.37 5.74
94.29 1.58
91.79 0.76
93.28 1.23
94.46 1.82
88.56 5.05
89.29 1.46
92.09 2.62
85.69 2.96
83.47 0.45
87.89 1.85
96.53 1.20
90.88 1.04
95.77 0.64
95.23 2.87
90.86 1.05
93.72 1.72
93.83 0.79
88.66 2.74
90.19 1.35
94.73 0.59
92.03 3.08
92.86 1.96
95.43 0.32
96.44 0.44
95.34 0.31
97.92 1.44
95.93 1.39
95.76 1.25
90.03 6.51
93.58 1.15
94.89 3.39
96.15 2.84
91.84 1.29
94.21 2.64
82.43 1.89
85.44 4.56
85.74 2.52
97.69 7.24
95.30 1.15
94.62 1.07
92.50 1.02
92.15 2.05
85.82 1.29
95.52 0.55
93.83 4.52
91.93 1.33
93.77 2.08
90.01 1.84
81.08 0.53
93.45 3.15
92.15 2.43
90.84 1.41
93.86 0.81
89.63 0.87
91.05 1.61
97.88 0.11
95.69 1.31
94.13 2.02
99.58 0.42
98.81 0.87
79.35 5.16
Note: the results are mean value standard deviation, which were obtained from three or
four replicates for each sample measurement.
190
Appendix 11.1.16
Detailed data processing for analysing iodine-iron interaction in DFS
191
Appendix 11.2 Detailed Analytical Methods and Results in Ultra Rice Study
Appendix 11.2.1 Ranking scheme for measurement of grain integrity during soaking
and cooking
Grain
integrity
during
soaking
(Ranking)
192
Microscopic pictures
(x10 magnification)
Reference
- natural
rice
193
Grain
integrity
after
cooking
(ranking)
Observation
or
Evaluation criteria
194
195
Appendix 11.2.2 Texture measurement on cooked Ultra Rice grains for grain integrity
The cooked rice samples were prepared as described in the section of cooking integrity.
Specifically, fifteen grams of the grains were placed in an aluminum bakery tray to a depth
of 5 cm and diameters of 15 cm at the top and 10 cm at the bottom. Thirty mL of water was
added. The container was placed in the steam basket of a rice cooker and cooked for 10 min
after boiling. The sample was left to stand for another 10 min before being removed from the
cooker, and was further cooled to room temperature before the texture measurement. To
obtain comparable data, the same aluminum container and cooking conditions were used for
all tests.
The container of cooked Ultra Rice grains was positioned at the center of the platform of
the texturometer. A 5-kg load cell was used, and the compression plate traveled for a set
distance of 5 mm at a pretest/posttest speed of 1 mm/s and a test speed of 0.1 mm/s. Force in
N required to compress the sample was recorded as a function of the distance traveled by the
plunger (mm). Three or four replications were performed for each sample on different spots.
An example of a typical force-distance curve is shown in Figure 11.2.2.1
1.2
Hardness = peak height
Force (N)
0.8
0.6
0.4
Springiness =
initial slope
0.2
-6
-5
-4
-3
-2
0
-1
0
-0.2
Distance (mm)
Figure 11.2.2.1 Example of force-distance curve of a compression test for cooked Ultra Rice grains
Within this instrumental test, two test stages were defined: compression and adhesion. The
compression stage was defined as the stage where the flat plate compression contacted the
surface of the rice sample and traveled until it reached the maximum distance, 5 mm. The
adhesion stage of the curve was defined as the stage from the point at which the flat plate
started to travel back to its original position until it returned to the starting point.
Data collected from this single compression test (Sesmat & Meullenet, 2001) could be
processed using various regression methods to generate substantial information about
sensory properties of the test sample, including hardness, stickiness, cohesiveness, adhesives,
etc (Perez et al., 1996). However, in this study a simplified interpretation of the curve was
used to obtain two major parameters, hardness and springiness, as they are important factors
affecting palatability and expected to have direct correlation with the grain integrity.
Specifically, the hardness was obtained as the peak force (N) for each profile, and the
springiness was deducted as the initial slope of the curve up to a distance of 1 mm.
196
Appendix 11.2.3
Detailed compositions of the final 4 formulations used for verifying the optimal internal
gelation systems in the actual nutrient-fortified formulations
Table 11.2.3.1 Formulation composition with the best ratio of the internal gelation system
used in the vitamin A fortified Ultra Rice formula
Vitamin A formula using the optimal ratio of
alginate/CaSO4/STPP
(%)
Note
0.72
Selected nutrient
BHA
0.01
BHT
0.01
0.52
Citric acid
0.50
0.60
3.00
CaSO4
3.00
Shortening
2.50
Rice flour
89.14
32
100
Antioxidant system
Structural ingredients
Table 11.2.3.2 Formulation composition with the best ratio of the internal gelation system
used in the multi-iron Ultra Rice formula
Multiple-iron formula using the optimal ratio of
alginate/CaSO4/STPP
(%)
Thiamine mononitrate
0.07
Folic acid
0.03
3.68
0.42
BHA
0.01
BHT
0.01
Citric acid
0.50
0.60
3.00
CaSO4
3.00
1.00
Shortening
2.50
Rice flour
85.18
32
100
197
Note
Selected nutrients
Antioxidant system
Colour-masking agent
Structural ingredients
Table 11.2.3.3 Formulation composition with the best ratio of the internal gelation system
used in the vitamin A fortified formula with addition of HPMC as the texture enhancer and
glutinous rice flour in place of regular rice flour
Vitamin A formula using the optimal ratio of
alginate/CaSO4/STPP and glutinous flour
(%)
Note
0.72
Selected nutrient
BHA
0.01
BHT
0.01
0.52
Citric acid
0.50
0.60
3.00
CaSO4
3.00
3.00
Shortening
2.50
86.14
32
100
Antioxidant system
Structural ingredients
Table 11.2.3.4 Formulation composition with the best ratio of the internal gelation system
used in the multi-iron formula with addition of HPMC as the texture enhancer and glutinous
rice flour in place of regular rice flour
Multi-iron formula using the optimal ratio of
alginate/CaSO4/STPP and glutinous flour
(%)
Thiamine mononitrate
0.07
Folic acid
0.03
3.68
0.42
BHA
0.01
BHT
0.01
Citric acid
0.50
0.60
3.00
CaSO4
3.00
3.00
Shortening
2.50
83.18
32
100
198
Note
Selected nutrients
Antioxidant system
C1s
9.00E+04
8.00E+04
7.00E+04
Counts / s
6.00E+04
O1s
5.00E+04
4.00E+04
3.00E+04
Ca2p
2.00E+04
1.00E+04
0.00E+00
1100
1000
900
800
700
600
500
400
300
200
100
Peak BE
FWHM eV
At. %
SF
285.03
2.50
337978.72
87.42
1.000
Ca2p
346.14
0.00
-165.05
0.00
5.070
O1s
532.78
3.03
124012.23
12.58
2.930
C1s
C1s
6.00E+04
Counts / s
5.00E+04
4.00E+04
O1s
3.00E+04
2.00E+04
Ca2p
1.00E+04
0.00E+00
1100
1000
900
800
700
600
500
400
300
200
100
Peak BE
FWHM eV
At. %
SF
C1s
285.03
2.50
241154.11
87.36
1.000
Ca2p
346.93
3.32
2681.98
0.20
5.070
O1s
532.58
3.12
87596.30
12.44
2.930
199
Survey
1 Scan, 58.1 s, 400m, CAE 200.0, 1.00 eV
1.60E+05
C1s
1.40E+05
1.20E+05
Counts / s
1.00E+05
8.00E+04
O1s
6.00E+04
4.00E+04
Ca2p
2.00E+04
0.00E+00
1100
1000
900
800
700
600
500
400
300
200
100
Peak BE
FWHM eV
At. %
SF
284.93
2.51
423687.31
89.13
1.000
Ca2p
348.09
1.28
1950.11
0.08
5.070
O1s
532.63
3.13
130769.21
10.78
2.930
C1s
Figure 11.2.4.2a XPS profile of the reconstituted grains made with CaCl2 over-spray process
- outside layer
Survey
1 Scan, 58.1 s, 400m, CAE 200.0, 1.00 eV
6.00E+04
C1s
5.00E+04
O1s
Counts / s
4.00E+04
3.00E+04
2.00E+04
Ca2p
1.00E+04
0.00E+00
1100
1000
900
800
700
600
500
400
300
200
100
Peak BE
FWHM eV
At. %
SF
284.97
2.74
223073.24
84.07
1.000
Ca2p
348.44
0.00
1223.70
0.09
5.070
O1s
532.68
3.04
107203.13
15.84
2.930
C1s
Figure 11.2.4.2b XPS profile of the reconstituted grains made with CaCl2 over-spray process
- cross-section
200
Survey
1 Scan, 58.1 s, 400m, CAE 200.0, 1.00 eV
1.00E+05
C1s
9.00E+04
8.00E+04
7.00E+04
Counts / s
6.00E+04
O1s
5.00E+04
4.00E+04
3.00E+04
Ca2p
2.00E+04
1.00E+04
0.00E+00
1100
1000
900
800
700
600
500
400
300
200
100
Peak BE
FWHM eV
At. %
SF
C1s
285.04
2.52
307129.94
88.58
1.000
Ca2p
347.71
1.32
4157.97
0.24
5.070
O1s
533.21
3.29
98867.76
11.18
2.930
Figure 11.2.4.3a XPS profile of the reconstituted grains made with internal gelation - outside
Survey
1 Scan, 58.1 s, 400m, CAE 200.0, 1.00 eV
6.00E+04
C1s
5.00E+04
Counts / s
4.00E+04
3.00E+04
O1s
2.00E+04
Ca2p
1.00E+04
0.00E+00
1100
1000
900
800
700
600
500
400
300
200
100
Peak BE
FWHM eV
At. %
SF
285.01
2.49
178356.87
87.86
1.000
Ca2p
347.03
1.70
1836.69
0.18
5.070
O1s
533.14
3.41
61878.55
11.95
2.930
C1s
Figure 11.2.4.3b XPS profile of the reconstituted grains made with internal gelation cross-section
201
Sample Parameter:
Sample: Sample A - outer surface
Origin:
File:
Sample Parameter:
Sample: sample A
Origin:
Spectrum Parameter:
Polarity:
positive
Area / m: 100x100
Time / s:
30
PI dose:
0.00E+000
A-OS_2P.dat
File:
Spectrum Parameter:
Polarity:
positive
Area / m: 100x100
Time / s:
30
PI dose:
0.00E+000
A-XS_2P.dat
x101
x101
6.0
C3H4
1.0
C3H4
Ca
5.0
Intensity
Intensity
0.8
0.6
0.4
4.0
3.0
2.0
Ca
0.2
1.0
39.6
39.7
39.8
39.9
40.0
40.1
40.2
40.3
40.4
39.6
39.7
39.8
39.9
40.0
40.1
40.2
40.3
40.4
mass / u
mass / u
Figure 11.2.4.4 ToF-SIMS profile of the Ca-free grains (left: outside layer; right:
cross-section)
Sample Parameter:
Sample: Sample B - outer surface
Origin:
File:
Spectrum Parameter:
Polarity:
positive
Area / m: 100x100
Time / s:
30
PI dose:
0.00E+000
B-OS_2P.dat
Sample Parameter:
Sample: sampleB
Origin:
File:
Spectrum Parameter:
Polarity:
positive
Area / m: 100x100
Time / s:
30
PI dose:
0.00E+000
B-XS_2P.dat
x101
6.0
x101
6.0
C3H4
5.0
5.0
4.0
4.0
Intensity
Intensity
Ca
3.0
3.0
C3H4
2.0
2.0
1.0
1.0
39.6
39.7
39.8
39.9
40.0
40.1
40.2
40.3
40.4
39.6
mass / u
39.7
39.8
39.9
40.0
40.1
40.2
40.3
40.4
mass / u
Figure 11.2.4.5 ToF-SIMS profile of the grains made with CaCl2 overspray (left: outside
layer; right: cross-section)
Sample Parameter:
Sample: Sample C - outer surface
Origin:
File:
Spectrum Parameter:
Polarity:
positive
Area / m: 100x100
30
Time / s:
PI dose:
0.00E+000
C-OS_2P.dat
Sample Parameter:
Sample: sampleC
Origin:
File:
Spectrum Parameter:
Polarity:
positive
Area / m: 100x100
30
Time / s:
PI dose:
0.00E+000
C-XS_2P.dat
x102
x102
Ca
C3H4
1.4
1.5
1.2
Intensity
Intensity
1.0
1.0
0.8
0.6
0.5
0.4
Ca
C3H4
0.2
39.6
39.7
39.8
39.9
40.0
40.1
40.2
40.3
40.4
mass / u
39.6
39.7
39.8
39.9
40.0
40.1
Figure 11.2.4.6 ToF-SIMS profile of the grains made with internal gelation
layer; right: cross-section)
202
40.2
40.3
40.4
mass / u
(left: outside
Appendix 11.3 Detailed Analytical Methods and Results in Folic Acid Tests
The literature indicates there are various methods available for the determination of folic
acid in different systems, including official methods based on microbiological assays, liquid
chromatography, HPLC, radiobiological assay, fluorimetry, electroanalytical techniques, and
spectrophotometric methods. Although the spectrophotometric methods have been reported
with several shortcomings, such as narrow range of determination and requiring heating or
extraction, they still remain as the choice of simple, rapid, and sensitive assays for the
determination of folic acid in pure form or concentrated tablets.
Two spectrophotometric methods were compared for their abilities to measure folic acid in
the fortified staple foods salt, sugar and rice samples, which had generally low fortification
levels.
Coupling Reaction Method
The coupling reaction method was modified from the work conducted by Nagaraja et al.
(2002) and Hutchings et al. (1947). Folic acid was first reductively cleaved to
p-aminobenzoylglutamic acid (p-ABGA) in the presence of zinc and HCl. The p-ABGA was
diazotized. This was followed by coupling with iminodibenzyl (IDB) or 3-aminophenol
(3-AP) or sodium molybdatepyrocatechol (MoPC). The absorbance of the coloured
complexes could be readily measured by spectrophotometer. The chemical reactions
involved in the protocol are presented bellow.
Folic Acid
p-ABGA
Diazotization
Figure 11.3.1.1 Chemical reactions involved in the coupling reaction method for folic acid
determination (adapted from Nagaraja et al. 2002)
203
From the three coupling reagents 3-aminophenol (3-AP), iminodibenzyl, and sodium
molybdate-pyrocatechol, 3-AP was chosen due to its complex having longer stability and
greater range for detection. Also it has no interference with other vitamins existing in the
same sample system. Nevertheless, it is a common chemical with little hazard to human
health.
Direct Extraction Method
The direct extraction method was adopted from Ghasemi & Vosoughs work (2002), in
which folic acid and other B vitamins were directly extracted into diluted NaOH solution
and measured by uv/vis spectrophotometry at different wavelengths. Specifically, 0.01 N
NaOH was used to extract folic acid in our study, and then measured at the wavelength of
284 nm.
Results & Discussion
The initial scans of the standard solutions prepared by two methods showed similar folic
acid absorbance spectra to the ones indicated in the literature. The calibration curves derived
are shown in Figures 11.3.1.2 and 11.3.1.3. Clearly, good correlations between the folic acid
(FA) concentration and the absorbance values were obtained for the both methods, with the
values of correlation coefficient close to 1. However, the slope of the calibration curve from
the coupling method was 0.0377, which was lower than the value given in the literature
(0.0701). This lead to a detection limit of ~2.5 g/mL for the method since the absorbance
reading was below 0.1 AU at this point. From the calibration curve of the direct extraction
method it seemed that this method could accurately measure the folic acid concentration as
low as 1 g/mL.
Based on the results, the direct extraction method was chosen for determination of folic acid
in fortified salt and sugar, due to its simple protocol, better time efficiency, and relatively
higher sensitivity to lower concentrations of folic acid, whereas the coupling method was
used in the determination of folic acid in the reconstituted Ultra Rice, due mainly to its
higher specificity which was expected to generate reliable results in the presence of other
components in the relatively complex rice flour matrix.
204
0.2
y = 0.0377x + 0.0069
0.15
R = 0.9978
0.1
0.05
0
0
2
3
FA Concentration (ug/mL)
Figure 11.3.1.2 Calibration curve for folic acid determination with the coupling reaction
method
y = 0.0535x + 0.0142
1.2
R = 0.9999
Abs
1
0.8
0.6
0.4
0.2
0
0
10
12
14
16
18
20
Figure 11.3.1.3 Calibration curve for folic acid determination with the direct extraction
method
205
7.
8.
Stock solution (500 g/mL): 0.0500 g of folic acid was dissolved in 50 mL of 0.1N NaOH
and diluted to 100 mL with the same solvent.
Reductive cleavage of folic acid: 1 g Zn and 10 mL of 5N HCl were added to 2 mL of the
stock solution in a tube.
The tube was shaken for 20 minutes by hand and the contents were filtered.
Intermediate solution (20 g/mL): After filtration, the solution was diluted to 50 mL with
dH2O.
Working solutions (1.6 4.8 g/mL): 2, 3, 4, 5, and 6 mL of the intermediate solution
were placed into tubes.
Diazotization: 2 mL of 5N HCl, 2 mL of 1% NaNO2, and 2 mL of 2% sulfamic acid were
added to each of the working solution, waiting for 5 minutes after each addition of reagent
solution.
Coupling reaction: 5 mL of 1% 3-AP was added to each of the working solution and
heated for 5 minutes in a boiling water bath. The solutions were orange-yellow in colour.
The working solutions were cooled, 3 mL of 5N HCl was added, and diluted to 25mL with
dH2O. The final coloured complex was stable for about 13 days (Nagaraja et al. 2002).
206
5.
The sample solution was heated in a boiling water bath for 5 min, and cooled to the room
temperature. The resulting sample solution was diluted with dH2O to 25 mL for the
spectrometry measurement.
207
208
Appendix 11.3.2
Colour stability of the double or triple fortified Guatemalan salt or sugar samples after
3 months storage under 40oC and 60% RH (Microscopic pictures were taken at x60
magnification)
209
Appendix 11.3.3
Folic acid-containing multiple fortified Ultra Rice appearance (microscopic pictures
were taken at x10 magnification)
Native rice
Colombian FePP
Fortitech FePP
Lohmann FePP
SunActive FePP
Figure 11.3.3.1 Comparison of Ultra Rice grains in colour made with various FePP sources
2% FePP
3% FePP
1% FePP
1% TiO2
2% TiO2
Native rice
3% TiO2
Figure 11.3.3.2 Comparison of Ultra Rice grains in colour made with different addition
levels of FePP (source of Dr. Lohmann) and TiO2
210