MQ84139 Ocr

PREPARATION OF SALT TRIPLE FORTIFIED WITH
VITAMIN A, IRON AND IODINE
KATARINA RUTKOWSKI
A thesis submitted in conformity with the requirements

for the Degree of Master of Applied Science
Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto
© Copyright by Katarina Rutkowski 2003

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Canada
Preparation of Salt Triple Fortified with Vitamin A, Iron and Iodine
MAsc, 2003
Katarina Rutkowski
Graduate Department of Chemical Engineering and Applied Chemistry
University of Toronto
Abstract
A triple fortified salt (TFS) containing vitamin A, iron and iodine was produced and studied
under two controlled climatic conditions: 40°C 60% and 100% relative humidity (RH) for three
months.
The highest vitamin A stability was observed in premixes made with Dry Vitamin A Palmitates
(250 CKCWD, 250 Food and 500; BASF Corporation, NJ, USA). In our best formulation TFS
retained of 65 + 2% vitamin at 40°C, 60% RH. The highest vitamin A retention at 40°C and
100% RH was 45 + 1%. The TFS containing vitamin A acetate retained a maximum of 48 + 5%
vitamin at 40°C, 60% RH. Introduction of all three micronutrients into one premix particle
resulted in reduced vitamin A retention to ~7% at 40°C, 60% RH.
Vitamin A was more stable with ferric NaEDTA than with any other iron compound. The source
of iodine did not significantly affect vitamin A retention.
li
Acknowledgements
I would like to express my sincerest gratitude to Professor Levente L. Diosady for all his kind
support, guidance and for giving me the opportunity to work on such an exciting project.
I would also like to thank Professor Toks Oshinowo for all his kind advice and valuable input on
the project.
I cannot forget Professor Otto Meresz and all my friends from the Food Engineering group,
especially Amanda Wright, Rizwan Yusufali, Mohina Sharma, Eylin Rodriguez, Olive Yao, Bih-
King Chen and Jane Lam for their input, moral support and friendship.
I would also like to thank my husband, Arthur, and my parents for their undying patience,
support, kind words of encouragement and love. I could not have done it without you.
iii
Table of Contents
Abstract ii
Acknowledgements ill
Table of Contents iv
List of Figures vii
List of Tables Xiv
1. Introduction 1
2. Theoretical Background §
2.1 Micronutrients 5
2.1.1 Physical and Chemical Properties 5
2.1.2 Physiology 9
2.1.3 Sources, Recommended Daily Intakes, Deficiencies 11
2.1.4 Nutrient — Nutrient Interactions 14

2.2 Criteria for Food Fortification 16
2.3 Designing Triple Fortified Salt 20
2.3.1 Premix Design 20
2.3.2 Granulation Technology 22
2.3.3 Microencapsulation Technology 29
2.3.4 Antioxidants 30
3. Experimental Techniques and Analytical Methods 34

3.1 Materials Used 34
3.2 Equipment Used 37
3.3 Procedure 38
3.3.1 Pan Agglomeration 38
3.3.2 Microencapsulation 38
3.4 Analytical Methods 39
3.4.1 The Sampling Method 39
3.4.2 Vitamin A Analysis 39
3.4.3 Iron Analysis 40
iv
3.4.4 Iodine Analysis 40
3.4.4.1 Determination of Potassium Iodide 40
3.4.4.2 Determination of Potassium Iodate 40
3.4.5 Moisture Content Determination 40
3.4.6 Particle Size Distribution Al
3.4.7 Packaging and Storage Conditions 42
4. Results and Discussions 43

4,1 Triple Fortified Salt 43
4.2 Premix Formulations 45
4.3 Stability of Vitamin A 50
4.3.1 Stability of Commercial Vitamin A Forms 50
4.3.2 Stability of Three Best Vitamin A Premixes 54
4.3.3 Vitamin A Acetate vs. Palmitate 60
4.3.4 Effect of Iron Source on Vitamin A Stability 65
4.3.5 Effect of lodine Source on Vitamin A Stability 70
4.3.6 Effect of the Type of the Binding Agent 74
4.3.7 Hydrophilic vs. Hydrophobic Coating Agents 75
4.3.8 Effect of Antioxidants 79
4.3.9 Effect of Humidity 82
4.3.10 Introducing All Three Micronutrients into One Particle 84
4.4 Reproducibility Test and Defects in Coating 86
4.5 Stability of Iron 90
4.6 Stability of Iodine 94
5. Conclusions 98
6. Recommendations 100
7. References and Bibliography 101
8. Nomenclature 108
9. Appendices 110
9.1 Analytical Methods 110
9.1.1 Vitamin A 110
9.1.2 Iron 112
9.1.3 Iodine from Potassium Iodate 113
9.2 Experimental Results 117
vi
List of Figures
Figure 2.1: Chemical structure of retinol and B-carotene. 5

Figure 2.2: Structure of Heme 7
Figure 2.3: Chemical structure of thyroxine, T, 8
Figure 2.4: A simplified cross sectional structure of a premix. 20
Figure 2.5: (a) Edge and face view of a pan granulator; (b) Stratification of particle size | 27
during rotation
Figure 2.6: Fluid bed 28

Figure 3.1: A Ro-Tap sieve shaker 41
Figure 4.1: Picture of TFS a4
Figure 4.2: The appearance of typical vitamin A, iron and iodine premixes 49
Figure 4.3: Stability of vitamin A premixes, granulated with dextrin, coated with 40% SS | 50
and stabilized with either BHA/BHT (marked with number 1) or TBHQ
(marked with number 2), in the presence of iron and potassium iodate premix.
The sample containing KR22i premix was prepared with iodized table salt. The
TFS samples were stored for three months at 40°C 60% RH.
and stabilized with either BHA/BHT (marked with number 1) or TBHOQ
(marked with number 2), in the presence of iron and potassium iodate premix.
The sample containmg KR22i premix was prepared with iodized table salt. The
TFS samples were stored for three months at 40°C ~100% RH.
and stabilized with either BHA/BHT or TBHQ, in the presence of different iron
compounds and potassium iodate premix. The TFS samples were stored for
three months at 40°C 60% RH.
compounds and potassium iodate premix. The TFS samples were stored for
three months at 40°C ~100% RH.
vii
Figure 4.7: Degradation rate of vitamin A premix KR 27-1 (BASF Dry VAP 500 53
granulated with dextrin, coated with 40% SS and stabilized with BHA/BHT) in
TES sample containing ferric NaEDTA. The TFS sample was stored for three
months at 40°C 60% RH and ~100% RH.
Figure 4.8: The stability of KR22 premix (BASF Dry VAP 250 CK CWD) mixed with all 55
four iron premixes and either potassium iodate premix or iodized salt. This
premix was granulated with dextrin, coated with 40% SS and stabilized with
TBHQ. The TFS samples were stored for three months at 40°C 60% RH.
Figure 4.9: The stability of KR22 premix (BASF Dry VAP 250 CK CWD) mixed with all 55
four iron premixes and either potassium iodate premix or iodized salt. This
premix was granulated with dextrin, coated with 40% SS and stabilized with
TBHQ. The TES samples were stored for three months at 40°C ~100% RH.
Figure 4.10: The stability of KR25 premix (BASF Dry VAP 250 Food) mixed with all four 57
iron premixes and potassium iodate premix. This premix was granulated with
dextrin, coated with 40% SS and stabilized with either BHA/BHT (KR25-1) or
TBHQ (KR25-2). The TFS samples were stored for three months at 40°C 60%
RH.
Figure 4.11; The stability of KR25 premix (BASF Dry VAP 250 Food) mixed with all four 58
iron premixes and potassium iodate premix. This premix was granulated with
TBHQ (KR25-2). The TFS samples were stored for three months at 40°C
~100% RH.
Figure 4,12: The stability of KR27 premix (BASF Dry VAP 500) mixed with all four iron 59
premixes and potassium iodate premix. This premix was granulated with
TBHQ (KR27-2). The TFS samples were stored for three months at 40°C 60%
RH.
Figure 4.13: The stability of KR27 premix (BASF Dry VAP 500) mixed with all four iron 59
premixes and potassium iodate premix. This premix was granulated with
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TBHQ (KR27-2). The TES samples were stored for three months at 40°C
~100% RH.
Figure 4.14: Stability of vitamin A acetate and palmitate premixes (granulated with dextrin, 62
coated with 40% SS and stabilized with either BHA/BHT or TBHQ), in the
presence of elemental iron and potassium iodate premix. The sample
containing KR22i premix (1-Palm) was mixed with iodized salt. The TFS
samples were stored for three months at 40°C 60% RH.
presence of ferrous fumarate and potassium iodate premix. The sample
presence of ferrous sulphate and potassium iodate premix. The sample
presence of ferric NaEDTA and potassium iodate premix. The sample
Figure 4.18: Effect of iron source on the stability of vitamin A in premix KR 22 (BASF Dry 65
VAP 250 CK CWD, granulated with dextrin, coated with 40% SS and
stabilized with TBHQ), in the presence of different iron compounds and

potassium iodate premix. The TFS samples were stored for three months at
40°C 60% RH.

Figure 4.19: Effect of iron source on the stability of vitamin A in premix KR 22 (BASF Dry 66
VAP 250 CK CWD, granulated with dextrin, coated with 40% SS and
stabilized with TBHQ), in the presence of different iron compounds and
ix
potassium iodate premix. The TFS samples were stored for three months at
40°C ~100% RH.
Figure 4.20: Effect of iron source on stability of premix KR 25-2 (BASF Dry VAP 250 68
Food, granulated with dextrin, coated with 40% SS and stabilized with TBHQ),
in the presence of different iron compounds and potassium iodate premix. The
TES samples were stored for three months at 40°C 60% RH.
Figure 4.21; Effect of iron source on vitamin A stability in premix KR 25-2 (BASF Dry 68
VAP 250 Food, granulated with dextrin, coated with 40% SS and stabilized
with TBHQ), in the presence of different iron compounds and potassium iodate
premix. The TFS samples were stored for three months at 40°C ~100% RH.
Figure 4.22: Effect of iron source on vitamin A stability in premix KR 27-2 (BASF Dry 68
VAP 500, granulated with dextrin, coated with 40% SS and stabilized with
TBHQ), in the presence of different iron compounds and potassium iodate

premix. The TFS samples were stored for three months at 40°C 60% RH.
Figure 4.23: Effect of iron source on stability of premix KR 27-2 (BASF Dry VAP 500, 69
granulated with dextrin, coated with 40% SS and stabilized with TBHQ), in the
presence of different iron compounds and potassium iodate premix. The TFS
samples were stored for three months at 40°C ~100% RH.
Figure 4.24: Effect of KIO3 premix and iodized salt (KI) on vitamin A stability (KR22 71
premix) in the presence of various iron compounds. The BASF Dry VAP 250
CK CWD was granulated with dextrin, coated with 40% SS and stabilized with
TBHQ. The TFS samples were stored for three months at 40°C 60% RH and
~100% RH.
Figure 4.25: Effect of iodine source on vitamin A stability (Watson VAP 250,000 [U/g 72
Powder) in the presence of various iron compounds. The vitamin A was
granulated with dextrin, coated with 40% SS and stabilized with TBHQ. The
Figure 4.26: Effect of iodine source on vitamin A stability (Watson VAP 250,000 IU/g 72
Powder) in the presence of various iron compounds. The vitamin A was
granulated with dextrin, coated with 40% SS and stabilized with TBHQ. The
TES samples were stored for three months at 40°C ~100% RH.
Figure 4.27: Stability of vitamin A premix (BASF Dry VAP 250 CK CWD) granulated with 74
two types of binder, coated with 40% SS, stabilized with TBHQ and stored at
40°C 60% RH for three months. 22: Casco Dextrin CAS: 9004-53-9 (yellow);
23-2: Casco Starch CAS: 9005-25-8 (white).
Figure 4.28: Stability of vitamin A premix (BASF Dry VAP 250 CK CWD) granulated with 74
two types of binder, coated with 40% SS, stabilized with TBHQ and stored at
40°C ~100% RH for three months. 22: Casco Dextrin CAS: 9004-53-9
(yellow); 23-2: Casco Starch CAS: 9005-25-8 (white).
Figure 4.29: Hydrophilic coating agents. The stability of two vitamin A forms (Watson 76
VAP 250,000 TU/g, KR 11 premix) and (BASF Dry VAP 250 Food, KR 12
premix) coated with 40% Shellac. The stability of vitamin A (Watson VAP
250,000 TU/g) coated with 30% MC/10% HEC (KR 15 premix) and 20%
MC/20% HEC (KR16 premix). These vitamin A premixes were granulated with
dextrin and stabilized with TBHQ. The TFS samples (containing iron and
potassium iodate premixes) were stored for three months at 40°C 60% RH.
Figure 4.30: Hydrophilic coating agents. The stability of two vitamin A forms (Watson 77
VAP 250,000 TU/g, KR 11 premix) and (BASF Dry VAP 250 Food, KR 12
premix) coated with 40% Shellac. The stability of vitamin A (Watson VAP
250,000 TU/g) coated with 30% MC/10% HEC (KR 15 premix) and 20%
MC/20% HEC (KR16 premix). These vitamin A premixes were granulated
with dextrin and stabilized with TBHQ. The TFS samples (containing iron and
potassium iodate premixes) were stored for three months at 40°C ~100% RH.
Figure 4.31: The stability of Watson VAP 250,000 IU/g coated with 40% SS (KR31-2 78
premix) and 30% SS (KR19-1 premix). The TFS samples (containing all four
iron premixes and potassium iodate premix) were stored for three months at
40°C 60% RH. These vitamin A premixes were granulated with dextrin and
stabilized with TBHQ.
Figure 4.32: The stability of Watson VAP 250,000 IU/g coated with 40% SS (KR31-2 78
premix) and 30% SS (KR19-1 premix). The TFS samples (containing all four
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iron premixes and potassium iodate premix) were stored for three months at
40°C ~100% RH. These vitamin A premixes were granulated with dextrin and
stabilized with TBHQ.
Figure 4.33: The effect of different antioxidants on the stability of vitamin A (BASF Dry 79
VAP 250 Food, KR 20 premix). The TFS samples (containing all four iron
premixes and potassium iodate premix) were stored for three months at 40°C
60% RH. This premix was granulated with dextrin, coated with 40% SS and
stabilized with either BHA/BHT (KR 20-1) or TBHQ (KR20-2).

Figure 4.34; The effect of different antioxidants on the stability of vitamin A (BASF Dry 80
VAP 250 Food, KR 20 premix). The TFS samples (containing all four iron
premixes and potassium iodate premix) were stored for three months at 40°C
~100% RH. This premix was granulated with dextrin, coated with 40% SS and
stabilized with either BHA/BHT (KR 20-1) or TBHQ (KR20-2).
Figure 4.35: Stability of premix containing all three micronutrients (Watson VAP 250,000 85
IU/g, potassium iodate and either ferrous fumarate or ferric NaEDTA) in one
particle, stored for three months at 40°C 60% RH. The premixes were
granulated with dextrin, coated with 40% SS and stabilized with TBHQ.
Figure 4.36: Stability of premix containing all three micronutrients (Watson VAP 250,000 85
IU/g, potassium iodate and either ferrous fumarate or ferric NaEDTA) in one
particle, stored for three months at 40°C ~100% RH. The premixes were
granulated with dextrin, coated with 40% SS and stabilized with TBHQ.
Figure 4.37: Comparison of stability of KR20-1 and KR25-1 premixes (BASF Dry VAP 250 86
Food), stored for three months at 40°C 60% RH. This vitamin A was
granulated with dextrin, coated with 40% SS and stabilized with BHA/BHT.
Figure 4.38: Microscope pictures of premixes KR20-1 and KR25-1 (BASF Dry VAP 250 87
Food). The vitamin A in both premixes was granulated with dextrin, coated
with 40% SS and stabilized with TBHQ.
Figure 4.39; Defects in coating of iron premixes, ferrous fumarate and ferric NaEDTA. 88
Figure 4.40: SEM images of vitamin A premixes KR20-1 and KR25-1 (BASF Dry VAP 250 89
Food). The vitamin A in both premixes was granulated with dextrin, coated
xii
with 40% SS and stabilized with TBHQ. Various magnifications.
Figure 4.41: | Microscope pictures (60x magnification) of elemental iron, ferrous fumarate, | 91
ferrous sulphate and ferric NaEDTA premixes. The iron compounds were
granulated with dextrin and coated with 40% SS.
Figure 4.42: | Microscopic images (60x magnification) of potassium iodide and potassium | 95
iodate premixes. The iodine compounds were granulated with dextrin and
coated with 40% SS.
Xiti
List of Tables
Table 2.1: The RDAs for vitamin A expressed in International Units (Us). il
Table 2.2: | Comparison of annual cost-effectiveness estimates between three countries. 17

Table 2.3: | Iron content and the bioavailability of some iron compounds previously used for 21
salt fortification.
Table 2.4: | The advantages and disadvantages of the methods used for agglomeration. 25
Table 2.5: | Common equipment used for granulation. 26
Table 2.6: | Food Antioxidants 31
Table 2.7: | Allowed Daily Intakes (ADIs) of some antioxidants permitted in foods. 32
Table 3.1: _ | List of used materials | 34
Table 3.2: | Commercial vitamin A forms tested 35
Table 3.3: | Handling of the six replicates prepared from each formulation. All replicates were | 42
put to the ovens at the same time.
Table 4.1: | Composition of the vitamin A premix formulations coated with 40% SS 46
Table 4.2: | Composition of the vitamin A premixes encapsulated with Shellac 46
Table 4.3: | Composition of the premixes containing all three micronutrients in one particle 47
Table 4.4: | Composition of the vitamin A premixes encapsulated with MC/HEC (level of 47
encapsulation was 40%)
Table 4.5: | Composition of the premixes and TFS samples containing various sources of 47
iodine. Components of premixes coated with 30% SS.
Table 4.6: | Retentions of vitamin A (BASF Dry VAP 250 CK CWD, KR 22 premix) with the | 56
standard deviations (based on minimum four replicates), the calculated first order
reaction rate constants and RSQ values. The TFS samples were stored for three
months at 40°C and both RH conditions (60% RH and ~100% RH). This premix
was granulated with dextrin, coated with 40% SS and stabilized with TBHQ.
Table 4.7: | Composition of the premixes used for vitamin A palmitate vs. acetate stability | 61
study (i=iodized salt, n=non-iodized salt)
Table 4.8: | The retention of vitamin A (BASF Dry VAP 250 Food, KR 20 premix) with the | 81
XiV
reaction rate constants (k) and RSQ values. The TFS samples were stored for three
months at 40°C ~100% RH. This premix was granulated with dextrin, coated with
40% SS and stabilized with either BHA/BHT (KR 20-1) or TBHQ (KR20-2).
Table 4.9: The retentions of vitamin A (BASF Dry VAP 500, KR 27 premix) with the 82
months at 40°C 60% RH. This premix was granulated with dextrin, coated with
Table 4.10: The retentions of vitamin A (BASF Dry VAP 500, KR 27 premix) with the 32
months at 40°C ~100% RH. This premix was granulated with dextrin, coated with
Table 4.11: Comparison of vitamin A stability in TFS stored at ambient conditions in 84
Mombassa and Nairobi (Kenya) and in U of T laboratory conditions (~25°C, 60%
RH) and environmental chamber (40°C ~100% RH) for three months.
Table 4,12: Composition of the premixes containing all three micronutrients (vitamin A, iron 84
and iodine) in one particle. The premix was designed so that it contained 25 000
TU/g of vitamin A, 100 mg/g of iron and 5 mg/g of iodine.
Table 4.13: The size distribution of microencapsulated premixes KR 20-1 and KR 25-1. 87
Table 4,14: Conversion of ferrous to ferric iron in the TFS samples containing BASF Dry VAP 92
250 CK CWD (KR22) premix stabilized with TBHQ and either iodized salt
(samples KA-18, 20, 22 and 24) or potassium iodate (samples KA-17, 19, 21 and
23). The samples were analyzed after two and three months of storage at 40°C
60% RH or 100% RH.
Table 4.15: Conversion of ferrous to ferric iron in the TFS samples containing potassium iodate 93
premix and BASF Dry VAP 250 Food (KR25) premix stabilized with either
BHA/BHT (samples KA-41 to 44) or TBHQ (samples KA-45 to 48). The samples
were analyzed after two and three months of storage at 40°C 60% RH or 100% RH.
Table 4.16: Conversion of ferrous to ferric iron in the TFS samples containing potassium iodate 93
XV
premix, and BASF Dry VAP 500 (KR27) premix stabilized with either BHA/BHT
(samples KA-57, 58, 59 and 60) or TBHQ (samples KA-61, 62, 63 and 64). The
samples were analyzed after two and three months of storage at 40°C 60% RH or
100% RH.
Table 4.17: Iodine retention in the TFS samples containing all four iron premixes and KR22 95
premix (BASF Dry VAP 250 CK CWD) stabilized with TBHQ. The samples were
analyzed after two and three months of storage at 40°C 60% RH or 100% RH.
Table 4.18: Iodine retention in TFS samples containing all four iron premixes and KR25 96
premix (BASF Dry VAP 250 Food) stabilized with either BHA/BHT (samples KA-
41, 42, 43 and 44) or TBHQ (samples KA-45, 46, 47 and 48). The samples were
Table 4.19: Iodine retention in TFS samples containing all four iron premixes and KR27 97
premix (BASF Dry VAP 500) premix stabilized with either BHA/BHT (samples
KA-57, 58, 59 and 60) or TBHQ (samples KA-61, 62, 63 and 64). The samples
Table 9.1: Summary of vitamin A stability results in TFS samples containing 40% soy 117
stearine encapsulated commercial vitamin A forms stabilized with BHA/BHT or
TBHQ and different iron and iodine compounds, stored at 40°C 60% RH for three
months. * All data + 2%.

Table 9.2: Summary of vitamin A stability results in TFS samples containing 40% soy 121
stearine encapsulated commercial vitamin A forms stabilized with BHA/BHT or
TBHQ and different iron and iodine compounds, stored at 40°C 100% RH for three
months. * All data + 2%.

Table 9.3: Summary of vitamin A stability results in TFS samples containing different vitamin 127
A premix formulations and different iron and iodine compounds, stored at 40°C
60% RH for three months. * All data + 2%.
Table 9.4: Summary of vitamin A stability results in TFS samples containing different vitamin 129
A premix formulations and different iron and iodine compounds, stored at 40°C
100% RH for three months. * All data + 2%.
XVi
1. Introduction
Micronutrient malnutrition is a serious threat to the health and productivity of more than two
billion people worldwide even though it is largely preventable [1]. Micronutrients are elements
or organic compounds, such as vitamins and minerals that are needed in relatively small amounts
(ug or mg per day) in the diet for normal body function. Inadequate intake of a micronutrient
results in health effects culminating in deficiency diseases.
The three micronutrient deficiencies of greatest public health significance are those of vitamin
A, iron and iodine. This is primarily because of their high prevalence and close association with
childhood illnesses and mortality. Women and children are more vulnerable to micronutrient
deficiencies because of increased micronutrient requirements for reproduction and growth,
respectively [2].
Vitamin A is essential for healthy growth, vision and development of a child. The body requires
vitamin A in small amounts but it plays an important role in strengthening a child's immune
system and integrity of epithelial cells. It is not produced by the body and therefore, must be
taken through breast-milk, foods rich in vitamin A or through supplementation.
In 1965, a Joint FAO/WHO Expert Group set a recommended intake of 750 ug of retinol per
day.
Vitamin A deficiency (VAD) is a serious problem in developing countries which can lead to
partial or total blindness. VAD has also been associated with a reduction of lymphocyte numbers
and increased risk of infectious morbidity and mortality.
More than 100 million children in 75 countries suffer from VAD. Today, more than 50%of
young children in countries where vitamin A deficiency is a health concern are receiving vitamin
A supplements - at a cost of 2 US cents per capsule. This in turn is estimated to have reduced the
childhood mortality rate by 23% to 34%, and cut measles mortality by as much as 50%. [3]
' Tron is required for normal formation of haemoglobin, iron enzymes, and other functioning iron
compounds and is present in all body cells. As a component of haemoglobin and myoglobin, it
functions as a carrier of oxygen in the blood and muscles.
Tron requirements for adult male are between 0.7 ~ 1.2 mg/day (about 14 ug/kg). Requirements
for adult female are between 1.2 ~— 2.5 mg/day (20 to 40 g/kg). The greater range is due to the
variability of menstrual blood loss.
fron Deficiency Anaemia (IDA) is associated with impaired work performance, developmental
delay and adverse pregnancy outcomes.
According to UNICEF, almost two billion people are estimated to be anaemic and millions more
are iron-deficient. Since the need for iron is greater during periods of rapid growth, children
from infancy through adolescence, as well as pregnant and menstruating women, may fail to
consume sufficient amounts of iron to meet their needs. It is estimated that 20% to 45% of
women of reproductive age, and approximately 70% of young children in developing countries
are anaemic. Low iron intake and poor bioavailability due to the high phytate, fiber and tea
consumption in diets, and malarial and parasitic infections contribute to IDA in the population in
Africa and Asia, where iron deficiency contributes to 20%of maternal deaths. [3]
Iodine is a component of two thyroid gland hormones, thyroxine and triiodothyronine, which are
necessary for normal growth and development. A person needs only a teaspoon of this
micronutrient, consumed in very small amounts over a lifetime.
The Recommended Daily Intake (RDI) for adult humans is 100 - 200 wg/day and the tolerable
Upper Intake Level (UIL) for adults is 1,100 ug/day.
Iodine Deficiency Disorders (IDD) are responsible for impaired physical and mental
development, including intellectual capacity in human beings. According to the World Health
Organization (WHO), iodine deficiency is the world's single most significant cause of
preventable brain damage and mental retardation. Nearly 20% of the population in the
developing world is iodine deficient and more than 830 million people have the telltale sign of
iodine deficiency - a swelling of the thyroid gland in the neck called goiter. [3, 4]
Historically, three main approaches have been used to solve the deficiency problem. One has
involved periodic dosing of affected persons with large doses of the micronutrients or regularly
with the required maintenance doses. Aid agencies, including Micronutrient Initiative (M1)
and Canadian International Development Agency (CIDA), are providing vitamin A and iron
supplements which are distributed through United Nations Children’s Fund (UNICEF) in
affected countries. This approach requires the consistent participation of families and
governments at the village level [5]. The second approach involves education of families on food
selection to improve the active nutrients’ status of children. This is the true long-term solution
but depends on an adequate supply of foods rich with these micronutrients and adequate income
to purchase or produce these foods. The third approach is food fortification.
Food fortification involves the addition of essential micronutrients to widely consumed food
products at levels above the natural state. It is a nutritional intervention program with a
specifically defined target, and the fortified foods are expected to become main source of the
added micronutrients. Consequently, food fortification is expected to help prevent nutritional
insufficiency in targeted population. Food fortification is the most cost-effective method of
fighting micronutrient deficiencies and has been widely and effectively used in the more
developed countries, e.g. iodized salt, milk and milk products fortified with vitamins A and D [6,
8].
The criteria used to select a food for fortification in developing countries include: use by the
targeted population; control of food production to confirm that fortification can be accomplished;
constant daily consumption to allow the dosage control and feasibility of fortification of the
locally produced food.
Some common types of food fortified with vitamin A are: sugar (Latin America), whole wheat,
monosodium glutamate, instant noodles, rice and other cereals, tea, fats and oils, milk and milk
powder and infant formulas.

In developed countries, wheat flour and cereal based foods have had significant success as
vehicles for iron. Other vehicles include salt, sugar, rice, curry powder, beverages, milk and
margarine.
Salt has been successfully used for over 70 years as a vehicle for iodine fortification. Other
vehicles of iodization have included bread, milk, flour and sugar. [7]
Sodium chloride (salt) is the most universal vehicle in the majority of developing countries
fitting the criteria above. Successful vitamin A, iron and iodine fortification of salt would have
applications in all countries affected by these deficiencies. Professor Diosady and the research
team from University of Toronto, together with Micronutrient Initiative (MI) have successfully
produced microencapsulated premix of iron and iodine and created a double fortified salt (DFS).
The DFS containing potassium iodide and ferrous fumarate was stable for up to 12 months
at
40°C and ~ 100% RH.
Unfortunately, finding a suitable technique for protecting vitamin A as a fortificant for salt is a
major task. In previous research conducted by our group, vitamin A demonstrated very good
stability in salt when humidity was moderate (laboratory conditions). However, when humidity
reached the level expected in the countries where the fortified salt will be used (60% or higher),
the stability of vitamin A was not sufficient for this program (the acceptable stability was set to
be 75% retention after three months in 60% RH).
This project is part of the ongoing research at the University of Toronto to examine the technical
feasibility of producing stable triple fortified salt (TFS) containing all three micronutrients. The
main objectives of the project were (a) to determine the technical feasibility of producing stable
triple fortified salt, (b) to develop processes for protecting vitamin A and producing TFS with
acceptable organoleptic and storage properties, (c) to test the processes using commercially
available vitamin A forms and (d) to investigate the effect of different types of iron and iodine
compounds on the retention of these vitamin A forms in TFS.
2. Theoretical Background
2.1 Micronutrients
2.1.1 Physical and Chemical Properties
Vitamin A
Vitamin A is a fat-soluble vitamin that is essential for the health of human beings. It comprises
of a family of molecules containing a 20 carbon structure with a methyl substituted cyclohexeny!
ting and a tetraene side chain with a hydroxyl group (retinol), aldehyde group (retinal),
carboxylic acid group (retinoic acid), or ester group (retinyl ester) at carbon 15.
The four double bonds in the polyene side chain of retinol theoretically permit 16 cis-trans
isomers but only three of these are the preferred structures found in nature.
Figure 2.1 shows the chemical structure of retinol and B-carotene.
Figure 2.1: Chemical structure of retinol and B-carotene.
B-carotene
The term vitamin A also includes provitamin A carotenoids that are dietary precursors of retinol.
The term retinoids refers to retinol, its metabolites and synthetic analogues that have a similar
structure.
Preformed vitamin A is a yellow crystalline powder or oil. Carotenoids are reddish-brown to
deep violet crystalline powders. Preformed vitamin A is found only in animal-derived food
products (e.g. liver, dairy products, eggs and fish), whereas dietary carotenoids are present
primarily in oils, fruits and vegetables.
Vitamin A is insoluble in water, soluble in alcohol, diethyl ether, petroleum, chloroform,
acetone, and fats and oils. B-carotene is also insoluble in water, scarcely soluble in alcohol, fats
and oils, ether, acetone and slightly soluble in chloroform and benzene.
All-trans-retinol exhibits ultraviolet (UV) absorption at 325nm. Retinol and retinyl esters
exhibit strong native fluorescence with excitation and emission maxima at wavelengths
of 325-
330 nm and 470-490 nm, respectively.
Vitamin A is extremely sensitive to oxygen (undergoes oxidation), light (promotes

cis-trans
isomerization), heat (catalyzes trans to cis isomerization), halogens (forms mixture
of cis-trans
isomers, particularly in the presence of light and high temperature), and is unstable
in acidic
environment (undergoes rearrangements of the double bonds and dehydrates).
In all fat-soluble vitamins, esters are significantly more stable than alcohols as the free
hydroxyl
group of their alcohol forms is highly sensitive to oxidation.
Vitamin A forms contain unsaturated carbon atoms and/or several double bonds, both highly
susceptible to oxidation. For example, retinol has both a free hydroxy group and 5 double bonds.
The esterification of retinol with acetic acid produces retinyl acetate, which has the hydroxy
group protected, but still has 5 double bonds susceptible to oxidation. [9]
Iron
Tron can exist in oxidation states ranging from -2 to +6. In biological systems, these oxidation
states occur primarily as ferrous (+2), ferric (+3), and ferryl (+4) states. Thanks to these
interconversions, iron can participate in electron transfer and reversibly bind ligands. The
common biological ligands for iron are oxygen, nitrogen and sulphur atoms.
In adult humans, haemoglobin is a macromolecule consisting of four peptide chains

(heteropolymers) and four heme complexes. A heme complex (figure 2.2) consists of an organic
ligand, porphyrin, with an iron atom coordinated to it through four nitrogen atoms.
Figure 2.2: Structure of Heme [14]
Sa, be ne Se
_
a
\
» if
if {proximal histidine)
Protein (globin}
The following physical-chemical properties should be taken into consideration when choosing
the source of iron fortificant:
e Ferrous salts can be utilized/bioabsorbed more efficiently than ferric salts
e Ferrous salts are more soluble than ferric salts
e Ferrous salts are more reactive than ferric salts in food systems
e Ferric iron generally has a greater tendency to form complexes than ferrous iron; the
formation of complexes will greatly reduce iron bioavailability
Ferrous salts are easily oxidized to ferric salts, which are usually orange or dark brown in colour.
Fe** (green) —»Fe** (orange/red) + e
This reaction is promoted in alkaline and high humidity conditions, in the presence of oxidizing
agents and common salt impurities such as magnesium chloride or sulphate. Salt containing
oxidized iron, especially ferric sulphate, exhibits metallic taste and discoloration. [10]
Electrolytic iron usually contains more than 96% of the total iron. It is almost insoluble in water
and has lower bioavailability than soluble iron salts. Other disadvantages are the dark grey
colour and high density which could lead to organoleptic problems and segregation.
Ferrous fumarate is a reddish orange to reddish brown powder that is odourless and
almost
tasteless. As for ferrous sulphate it is only slightly soluble in water. Ferrous sulphate
is the
cheapest and most widely used iron source in food fortification. It is used in two forms:
heptahydrate and anhydrous ferrous sulphate. The anhydrous ferrous sulphate is grayish white in
colour; it has a metallic and astringent taste. In contact with water it hydrates to form the light
green heptahydrate, which is sparingly soluble.
Ferric sodium EDTA is a pale yellow water-soluble powder with a high stability constant.
[11]
Many researchers indicate that while iron absorption from simple iron salts such as
ferrous
sulphate is very sensitive to inhibiting substances in food, the absorption from ferric NaEDTA
is
apparently not. [12, 13] While both ferric iron and iron complexes are typically poorly available,
the literature seems consistent in considering ferric EDTA to be bioavailable.
Iodine
fodine is a dark purple crystalline non-metallic element, which is insoluble in water, but soluble
in many organic solvents. In human beings it is a constituent of the thyroid hormones, thyroxine
T3 and Ty (figure 2.3) produced by thyroid gland.
Figure 2.3: Chemical structure of thyroxine, T, [24]

2
a in J No
- Ste J
lr ft NH;
Ty
I
Thyroxine
Two compounds of iodine are currently used for iodization: potassium iodate and iodide. The
Codex Alimentarius standard for food grade salt permits the use of their sodium and potassium
salts in the iodization program.
lodides are readily oxidized when in contact with oxygen in a high humidity environment
resulting in the formation and sublimation of free iodine. This process is accelerated in the
presence of acidic salt impurities, high temperature and sunlight. [10]
Oxidation: 27 —> I)+2e”
lodates are already oxidized thus they are more stable in tropical and subtropical climates.
However, presence of salt impurities also promotes reduction of iodate to form free iodine. [10]
Reduction: 2I°* + 10 e—> hb
A major consideration in the choice of the two compounds is the purity of the salt. The iodides
are more readily degraded in the presence of impurities and high humidity environment, whereas
the iodates remain stable in salt of lower quality.
Potassium iodate (KIO3) and potassium iodide (KI) were used for this study. Potassium iodide is
highly soluble in water (1440 g/L at 20°C) and the iodine content is 75%.
Most of the salt used in the affected areas is unrefined, thus the more stable KIO; is commonly
used there for iodization. Solubility of KIO; in water is very low (81.3 g/L at 20°C) and its
iodine content is 59.5%. [15]
2.1.2 Physiology
Vitamin A
The 11-cis-retinaldehyde (retinal) form of vitamin A is required by the eye for the transduction
of light into neural signals necessary for vision. It is also required for the integrity of epithelial
cells throughout the body. Retinoids are needed in the maintenance of the immune system.
Intestinal absorption of preformed vitamin A occurs following the processing of retinyl esters in
the lumen of the small intestine. The efficiency of absorption of preformed vitamin A is
generally high, in range of 70 to 90%. [16]
Absorbed B-carotene is enzymaticaly converted to vitamin A. The absorption efficiency of B-
carotene has been observed to range from 9 to 22%.
When vitamin A intake is adequate, over 90% of total body vitamin A is stored in the liver as
retinyl esters, where it is concentrated in the lipid droplets. [16]
Iron
The human body contains approximately 40 mg/kg (females) to 50 mg of iron /kg (males).
Almost 60% of it is found in haemoglobin and another 25% is in readily mobilizable iron store.
Most of the remaining 15% is in the myoglobin and as a component of enzymes. The iron
content of the body is highly conserved. In the absence of bleeding or pregnancy, only a small
quantity is lost or replaced each day.
The most important role of iron is the transport of oxygen (bound to haemoglobin) from the
environment to the tissues. The concentration of myoglobin in muscles is drastically reduced in
tissue iron deficiency, thus limiting the rate of diffusion of oxygen. [16]
Iron is absorbed in the upper small intestine by two pathways. One mediates the uptake of heme
iron derived primarily from haemoglobin and myoglobin in meat. The other allows for the
absorption of nonheme iron, primarily as iron salts from other sources such as plants and diary
foods.
Iodine
Iodine is an essential component of the thyroid hormones that are involved in the regulation of
various enzymes and metabolic processes. Major target organs are the developing nervous
system, muscles, heart and kidneys. Iodine occurs in the human body in only small amounts, 15
to 20 mg.
Iodine is ingested in a variety of chemical forms. Most ingested iodine is reduced to iodide in
the gut and absorbed almost completely. Once in the circulation, iodide is removed principally
by the thyroid gland and the kidneys. The thyroid selectively concentrates iodide in amounts
required for adequate hormone synthesis, and most of the remaining iodine is excreted in urine.
10
2.1.3 Sources, Recommended Daily Intakes, Deficiencies
Vitamin A
All sources of vitamin A are derived ultimately from provitamin A carotenoids, which
are
present in higher plants, and in lower forms of animal and plant life.
Common dietary sources of preformed vitamin A include liver, diary products, eggs and fish.
Major contributors of B-carotene are carrots, cantaloupe, broccoli, peas, and spinach. |
Vitamin A activity is expressed either in International Units (LU.) or in Retinol Equivalents
(RE).
One LU. is the activity of:
e@ 0.300 Lg of all-trans-retinol
® 0,344 wg of all-trans-retinyl acetate

e 0.55 pg of all-trans-retinyl palmitate
® 0.6 yg of all-trans-B-carotene
e 1.2 ug of other provitamin A carotenoids
One RE is equal to:
e 1 ug of all-trans-retinol
® 2 ug of supplemental all-trans-B-carotene
® 6 pg of dietary all-trans-B-carotene
In 1965, a Joint FAO/WHO Expert Group set a recommended daily intake of retinol to 750 pg
RE/day. The Recommended Dietary Allowances (RDA) for different age groups can be seen in
Table 2.1. [16]
Table 2.1: The RDAs for vitamin A expressed in International Units (TUs).
Age (years) | Children Men Women Pregnancy || Lactation

1-3 1000 IU
4-8 1333 IU
9-13 2000 IU
14-18 3000 IU 2330 IU 2500 TU 4000 IU
19+ 3000 IU 2330 1U 2565 IU 4335 TU
il
The most specific clinical effect of Vitamin A Deficiency (VAD) is xerophtalmia.
Night blindness is the first ocular symptom to be observed with VAD. VAD has been also
associated with a reduction of lymphocyte numbers, natural killer cells, and antigen-specific
immunoglobulin responses. A higher risk of respiratory infection and diarrhea has been reported
among children with mild to moderate VAD. [16]
It is estimated that 3 to 10 million children, mostly in developing countries, become
xerophtalmic and 250,000 to 500,000 go blind annually. [17]
Vitamin A is a fat-soluble vitamin and can be therefore stored by human bodies. Hence,
excessive intake can lead to hypervitaminosis. Hypervitaminosis A occurs at recurrent intakes
of 10 times RDI. Signs include bone and muscle pain, headache and visual impairment. Liver
damage is also possible. The positive fact is that vitamin A can be taken in large dose
supplements at wide intervals, e.g. every 3-5 months, without leading to hypervitaminosis.
Factors affecting the vitamin A requirements

Preformed vitamin A is found only in animal derived food products. Clinical signs of VAD are
prevalent in developing countries where animal and vitamin A fortified products are not
commonly available. |
The food matrix affects the intestinal absorption carotenoids. Increasing the level of fat in a low
fat diet has been shown to improve retinol and carotene absorption. [18]
Carotene bioavailability can differ with different processing methods of the same foods and
among different foods containing similar levels of carotenoids, due to variety differences, stage
of maturity, climatic conditions and processing and/or cooking. [19]
Diseases such as diarrhea and intestinal infections and parasitism seriously impede the
absorption of vitamin A.
Tron
Heme iron (derived from haemoglobin and myoglobin) is highly bioavailable and little affected
by dietary factors. Non-heme iron (derived mainly from cereals, vegetables and fruits)
absorption depends on the solubilization of predominantly ferric food iron in the acid
environment of the stomach and reduction to ferrous form by compounds such as ascorbic acid
12
or ferric reductase. Interactions with other meal components in the lumen also strongly influence
absorption of non-heme iron.
In general, iron requirements for adult males amount to 0.7 — 1.2 mg/day (about 14 g/kg).
Requirements for adult females are between 1.2 — 2.5 mg/day (20 to 40 pg/kg). The greater
range is due to the variability of menstrual blood loss.
Impaired physical work performance, developmental delay, cognitive impairment and adverse
pregnancy outcomes are typical consequences of Iron Deficiency Anemia (IDA). Common
causes of iron imbalance in humans are (1) increased iron loss which can be of physiologic
(increased menstruation, pregnancy, blood donation) or pathologic (bleeding, hemosiderinuria)
origin; (2) decreased absorption due to inadequate dietary iron and/or malabsorption. [16]
Factors affecting the iron requirements

The dietary iron absorption is determined by the iron requirements of the individual and by the
iron bioavailability based on the composition of the diet. The absorption mechanisms of heme
and nonheme iron are different. Diets containing no meat and little ascorbic acid have very
limited iron availability and can lead to iron deficiency. Decreased stomach acidity may also
lead to impaired iron absorption.
lodine
Most food sources contain low amounts of iodine (3 to 75 ug per serving). Foods of marine
origin have higher concentrations of iodine. Processed foods may also contain higher levels of
iodine because of the addition of iodized salt or additives such as calcium iodate and potassium
iodate.
The Recommended Daily Intake (RDI) for adult humans is 100 - 200 jg/day and the tolerable
Upper Intake Level for adults is 1,100 ug/day. [16]

The iodized salt is becoming the most common source of iodine, with about 70% of the salt
consumed being now iodized. The level of fortification that has been used ranges from 15-200
ppm. Numerous regional factors have to be considered when designing iodized salt, including
13
per capita salt consumption, degree of iodine deficiency, required shelf life, and climatic
conditions.
The Iodine Deficiency Disorders (IDD) include mental retardation, hypothyroidism, goiter,
cretinism, and varying degrees of other growth and developmental abnormalities.
Thyroid enlargement (goiter) is usually the earliest clinical feature of iodine deficiency.
Major international efforts such as the universal salt iodization program (USD have produced
dramatic improvements in the correction of iodine deficiency in the 1990s, mainly through the
use of iodized salt. [16]
Factors affecting the iodine requirements

Under normal conditions, the absorption of dietary iodine is more than 90%. Goitrogens,
components of some foods (e.g. cassava, soy flour), interfere with thyroid hormone production
and/or utilization through iodine absorption inhibition. [20]
The actual availability of iodine from iodized salt at the consumer level can vary over a wide
range as a result of [10]:
e Variability in the amount of iodine added during the iodization process;
e Uneven distribution of iodine in the iodized salt, within batches and individual bags;
e The extent of loss of iodine due to salt impurities, packaging, and environmental
conditions during storage and distribution;
e Loss of iodine due to food-processing, washing, and cooking processes in the household.
2.1.4 Nutrient —~ Nutrient Interactions
In micronutrient premixes containing vitamin A, and trace elements such as iron and iodine, the
dominant effects exerted on vitamin A are redox reactions. Trace minerals vary in redox
potential and iron is among the most reactive ones. The molecular structure of the trace mineral
has a significant impact on vitamin A stability. Free metal iron is the most reactive, followed by
sulphate, carbonate, oxide and the least reactive forms are chelated. Chelated forms become
incapable of initiating formation of free radicals. [9]
14
Vitamin A and Iron
A direct correlation between haemoglobin and serum retinol concentration has been observed.
Intervention studies among Indonesian girls have demonstrated that combining vitamin A with
iron supplementation was more effective in increasing haemoglobin concentrations than iron
alone. Various studies suggest that VAD impairs iron mobilization from body stores and
therefore vitamin A supplementation improves haemoglobin concentrations. [21, 22, 23]
Iron and Iodine

Ferrous salts are easily oxidized by iodates resulting in formation and sublimation of free iodine.
103+ 6Fe™* + 6H’ > I + 6Fe** + 3H,0
10; +1 +6H —> 3h+3H,0
Ferric salts can be reduced by iodides:

21+ 6Fe** —> I, + 2Fe**
The rate of these reactions depends mainly on the nature of the food and the environment. The
reactions are accelerated in the high humidity environment.
Iron and Vitamin C

Ascorbic acid was observed to strongly enhance the absorption of nonheme iron by reducing the
dietary ferric iron to ferrous iron. Ferrous iron then forms a soluble iron-ascorbic acid complex
in the stomach. This enhanced absorption effect is most noticeable when iron is consumed in
foods containing high levels of inhibitors like phytates or tannin. [25, 26]
Iron and Phytates, Polyphenols

Phytic acid is present in legumes, rice and grains. The inhibition of iron absorption from added
iron is related to level of phytate in a food. Polyphenols are found in tea, coffee, grain products,
herbs such as oregano and red wine and they markedly inhibit the absorption of nonheme iron.
Iron binds tannic acid forming an insoluble complex that results in impaired absorption. The
inhibitory effects are dose-dependent and reduced by the addition of ascorbic acid. [27]
15
fron and Calcium
Calcium inhibits the absorption of both heme and non-heme iron probably by interfering with
degradation of phytic acid. It has direct dose-related inhibiting effect such that iron absorption
was reduced by 50 — 60% at doses of 300 to 600 mg of calcium added to wheat rolls. [28]
2.2 Criteria for Food Fortification
The three main approaches to addressing micronutrient deficiencies are supplementation,

fortification, and dietary diversification.
Supplementation is periodic dosing of affected persons with micronutrients. More than one
nutrient may be provided, and they may be delivered in high quantities. [29]
The Codex Alimentarius definition of fortification and/or enrichment is “the addition of one or
more essential nutrients to a food whether or not it is normally contained in the food for the
purpose of preventing or correcting a demonstrated deficiency of one or more essential nutrients
in the population group”. [30]
Dietary diversification requires changes in eating habits of each individual with extensive
education and in agriculture policy with crop diversification.
Coverage through vitamin A supplementation is around 60% of children under five in about 30
developing countries. Iron supplementation is lacking, only 20 countries report more than 60%
coverage among pregnant women. However, oral supplementation programs are blighted by
problems ranging from an inadequate supply of supplements, which is often related to the low
priority attached to control of a deficiency, to poor compliance with their consumption. [17]
The major findings of UN/ACC/SCN recent research on iron anaemia [31] show that:
1. Both daily and weekly iron supplementation are efficacious, but weekly supplementation
is likely to be less effective than daily administration, except in situations where weekly
but not daily supervision is feasible
16
2. Weekly supplementation may be particularly disadvantageous during pregnancy and in
situations where the baseline prevalence of anemia is high
3. Unless ways are found to greatly improve compliance, neither daily nor weekly
supplementation is likely to be an effective approach to preventing and controlling
anemia in developing countries
4. Regardless of the degree of supervision that can be arranged, weekly iron administration
instead of daily is not recommended for pregnancy.
Supplementation may therefore be appropriate for dealing with severe deficiencies in the short-
term; however, the long-term solution of intervention should be a combination of food
fortification and dietary diversification. Applied research is required to effectively improve
utilizable micronutrient intakes by altered food usage and food fortification.
At present, iodine fortification is the most extensive fortification program in the World.
Table 2.2 shows the cost-effectiveness of fortification, supplementation and food diversification.
Food fortification has been found to be the most cost-effective method of fighting vitamin A
deficiency. [32]
Table 2.2: Comparison of annual cost-effectiveness estimates between three countries [32]
Annual Cost per Person (US$ in 1991)

. Guatemala 1979 | Philippines | Guatemala
Type of Intervention
Indonesia 1978 | 1980 1991
Philippines 1975
Per person 0.16 0.14 0.29

Fortification
Per high-risk person || 0.37 0.32 0.65
Capsule Per high-risk person || 0.48 0.32 1.52
distribution |
Per person 2.31 1.60

Gardening
Per high-risk person 3.63
17
Although fortification of staple foods has played a significant role in the more industrialized
nations, it has not been considered an option for less developed countries because of the lack of
centrally processed foods and poorly developed food-marketing systems. As food markets
expand, however, fortification options become increasingly available.
A fortified food has to meet several requirements:
e Commonly consumed by the target population

e Constant consumption pattern with a low risk of excess consumption
e Centrally processed with minimal stratification of the fortificant
e Good stability during storage
e No interactions between the fortificants and the carrier food

e Contained in most meals, with the availability unrelated to socio-economic status
e Linked to energy intake
e Relatively low in cost
In most cases food fortification is generally carried out at levels between 15-33% of the relevant
Recommended Daily Allowance (RDA) per daily serving. Fortification levels are limited by
taste, technological and economic considerations.
Salt is an attractive vehicle for fortification as it meets most of the aforementioned requirements.
lodized and iodated salts have long been available in developed countries where the problem of
IDD has been virtually eliminated. [54] The salt is iodized by the addition of fixed amounts of
potassium iodide or potassium iodate as either a dry solid or an aqueous solution at the point of
production. The salt iodization program’s success is mostly due to its relative technological
straightforwardness, proven efficacy and huge support from international organizations, such as
Kiwanis, ICC/IDD and UNICEF.
Advantages of salt as a vehicle for fortification

e Itis consumed by all segments of the population
e The consumption lies within narrow range of 10-15 g/day
e Most of it is centrally produced with minimal regional variation
18
Encouraged by the success of the salt iodization program and the positive progress made so far
with double fortification of salt using iodine and iron, the feasibility of the triple fortified salt
(TFS) containing iron, iodine and vitamin A is now being examined. The TFS would address
deficiencies of the three micronutrients at the same time in a cost effective manner.
Multiple fortification has been successful in the more developed countries, particularly in
fortification of cereals and infant-weaning foods. In some cases, fortification
two with
micronutrients (e.g., iron and vitamin A or iron and vitamin C) was used to enhance the effects
of fortification on micronutrient status. [25]
Double-fortified salt with potassium iodide or iodate and ferrous fumarate or ferrous sulphate
was previously developed by our group under supervision of Professor L.L. Diosady [33] and
with support from the Micronutrient Initiative (MI). Potassium iodide and potassium iodate
were encapsulated in modified starches, gelatin, sodium hexametaphosphate and purified sodium
chloride by spray drying and fluidized bed drying to produce microcapsules containing 0.3 to 2%
iodine. Salt mixed with ferrous fumarate or ferrous sulphate, and encapsulated iodine was then
stored at high temperature and humidity, 40°C with 60% RH or 100% RH. The salt containing
ferrous fumarate and potassium iodide retained more than 75% of the added iodine and
maintained its organoleptic properties for a year.
The MI is a not-for-profit organization based in Ottawa, Canada, which specializes in addressing

micronutrient malnutrition by promoting food fortification and supplementation programs. They
provide technical and operational support in the countries with most prevalent micronutrient
deficiencies.
19
2.5 Designing Triple Fortified Salt
2.3.1 Premix Design
The basic idea behind the design of a premix is to physically separate the active ingredients from
each other or a harmful environment. This has been done with iron and iodine in the preparation
of double fortified salt (DFS). The DFS prepared by the food engineering group of the
University of Toronto under Professor Diosady’s supervision and with support from MI was
stable for 12 months at 40°C 100% RH. [33]
The premix microcapsule consists of small particles that contain an active nutrient and binding
and, if needed, diluting agents surrounded by a moisture resistant coating or shell composed of
materials such as polymers, carbohydrates, fats and waxes. Microencapsulation is a useful tool
for protecting the integrity of pharmaceuticals and food ingredients such as vitamins, salts or
flavours from oxygen, water and light.
Figure 2.4 is a simplified drawing of an ideal premix particle, containing a micronutrient,
binders, fillers and a layer of coating.
Figure 2.4: A simplified cross sectional structure of a premix.
Structure of Premix
Encapsulant and
Antioxidant
Inert matrix (fillers

// and binders)
Micronutrients
When designing TFS the premix must be:
e Non-toxic and all constituents must be approved food additives in all targeted countries
20
e Physically and chemically stable under the targeted environmental conditions
e Bioavailable
e Uniform, with the same particle size as salt, to avoid segregation

e Sufficiently hard and tensile to avoid breaking during mixing with salt, storage and
distribution
e Inexpensive
© Without any colour or flavour
The premix has to be white/off white and tasteless so that the sensory properties of salt are not
_ changed.
Iron compounds used for fortification can be divided into two major groups: water soluble
compounds and insoluble compounds. Ferrous sulphate, ferrous fumarate, ferrous ammonium
sulphate, ferric ammonium sulphate, ferric ammonium citrate and ferric EDTA are examples of
soluble compounds which were previously used for salt fortification. Some insoluble compounds
are ferric phosphate, sodium iron pyrophosphate and reduced iron. [11]
A list of common iron compounds that have been previously used by our group in salt
fortification, their bioavailability and relative cost is summarized in table 2.3 [10]. The relative
costs were calculated on the basis of the iron compound, cost and bioavailability of each
compound.
Table 2.3: Iron content and the bioavailability of some iron compounds previously used for
salt fortification. [10]
Relative Bioavailability | Relative Cost

# Iron Compound “% Ke Content
Factor Factor
1 Electrolytic iron 98 0.13-0.90
2 Ferrous Fumarate 33 1.00 1.00
3 Ferrous Sulphate x 7H,O | 20 ~1.00 1.67

4 Ferric NaEDTA 13 2.50 2.57
21
The main problem in selecting an iron compound for fortification is that the ones with good
relative bioavailability are more likely to be unstable under storage conditions, cause
discoloration and changes in flavour or odour. Conversely, the lower the relative bioavailability,
the more iron needs to be given to achieve the required effect, thus increasing cost and the
probability of inducing undesirable side effects. [11, 53]
The RDIs for all three micronutrients are very low. The premix can not be produced by simple
encapsulation; dilution with fillers and binding agents is necessary for iodine and Vitamin A. In
addition, agglomeration has to be used to obtain a suitable size distribution that avoids
segregation of the premix in salt.
All three micronutrients are very unstable under targeted conditions thus microencapsulation is
required for their stabilization. Vitamin A can be easily oxidized thus it must be separated from
air and fat soluble antioxidants are needed to stabilize it.
The different types of granulation and coating materials and antioxidants that are available are
discussed in the following sections.
2.3.2 Granulation Technology
Granulation is a process by which a powder mixture is converted to larger free flowing particles
called granules. The reasons for granulation are:
e To obtain sufficiently large particles that will not segregate from the salt by increasing
their size
e To obtain the desired granule hardness and roughness

e To obtain the desired density
Granulation normally follows initial dry mixing of the necessary powdered ingredients so that a
uniform distribution of each ingredient through the premix is achieved.
For economic and technical reason premix should be mixed with salt in 1:100 ratio. The salt
crystals are between 300 jum and 710 jm large and the size of the premix needs to match the size
of salt to avoid segregation.
22
Segregation is primarily due to the differences in the size or density of the components of the
sample. The smaller and/or denser particles concentrate at the base of a container and the larger
and/or less dense ones above them. An ideal sample will contain all the ingredients in the correct
proportion and segregation will not occur. [34]
Excipients are materials used in the manufacturing or the compounding process for the purposes
of maintaining the appearance (colour and consistency), protecting the active species
(antioxidants), disguising any unpleasant taste/smell (sugar, flavouring agents), changing the
dissolution rates of active species (coating, matrix) and manufacturing (fillers/diluents, lubricants
and glidants).
Fillers and binders do not have pharmacological activity themselves and are used to dilute the
micronutrients and to bind them to form granules of the desired size. The binder has to allow the
micronutrient absorption after premix ingestion.
The function of a filler is to increase the bulk volume of the premix so that the final product has
the proper volume. The filler has to be inert, compatible, non-hydroscopic, soluble, compactable
and inexpensive. The most common fillers are: lactose, sucrose, glucose, mannitol, sorbitol,
calcium phosphate, calcium carbonate, and cellulose.
Lactose is the most widely used filler in the food industry. It offers good stability and fair
protection for micronutrients sensitive to moisture.
Calcium phosphate and calcium sulphate offer excellent protection against moisture. The draw-
back, though, is their high cost.
The function of a binder is to ensure that granules can be formed with required mechanical
strength. Binders may be added to the mixture prior to the granulation process as a dry powder,
or in the solution that is used for agglomeration in wet granulation processes
The most common binders are:
© Wet/Solution binders: acacia, gelatin, cellulose, cellulose derivatives, polyvinyl
pyrrolidone, starch, sucrose, polyethylene glycol.
e Dry binders: cellulose, methyl cellulose, polyvinyl pyrrolidone, polyethylene glycol
23
® Some compounds, e.g. sodium hexametaphosphate (SHMP) can be used as both a binder
and a stabilizer.
Starch, a complex carbohydrate, (CsHi90s),, is abundant in the seeds of cereal plants. It is an
inexpensive dilutent/binding agent widely used by commercial vitamin A producers. The
disadvantage of starch is that it has a tendency to absorb moisture.
Industrial dextrin is granular starch with lower molecular weight. Its molecules are reorganized
by roasting or by chemical modifications, causing the granules to be cold water-soluble.
Depending on the degree of roasting, dextrins are categorized as white and yellow dextrins
and
British gum.
Both, starch and yellow dextrin, will be used in the vitamin A, iron and iodine premix
preparations.
Description of Granulation Processes [34, 35]

Wet granulation — wet mixing of a powder mixture with liquid adhesives followed by wet
screening or granulation. The granulation liquid (usually water) may be used alone or, more
usually, as a solvent containing a dissolved adhesive (also referred to as a binder or binding
agent) that is used to ensure particle adhesion once the granule is dry.
Dry granulation or Slugging —preparing premix mixture without a wet binder or moisture. A
slug is formed and then compressed.
Direct compression — no granulation is involved in the process. It is becoming a desired
method in the pharmaceutical industry due to availability of excipients that enable this process to
be conducted.
Fluidized bed granulation — is a single step process of mixing the powders and developing
granules. This process requires only one piece of machinery that mixes the powder and granules
on a bed of air. It is becoming a commonly used method because of the high quality of its
products, convenience and the availability of suitable equipment.
Spray drying - is the most widely used industrial process involving particle formation and
drying. It is highly suited for the continuous production of dry solids in either powder, granulate
or agglomerate form from solutions, emulsions or pumpable suspensions.
Table 2.4 shows the advantages and disadvantages of the methods used for agglomeration.
24
Table 2.4: The advantages and disadvantages of the methods used for agglomeration.
[34]
Process Advantages Disadvantages
Wet Most reliable Need to prepare a uniform granulation
granulation | Highest probability of meeting the || Labor intensive
requirements for making a successful | Time consuming
particle Cannot be used for water sensitive
nutrients
Dry Useful for water and heat sensitive | Requires excipients with cohesive
Granulation | active ingredients properties
Fewer steps
Direct Can prepare a premix from powdered || Requires materials that can be directly
Compression || mixture without granulation compressed
Faster Excipients must have good flow
Useful for crystalline premixes properties
Useful for ingredients that are moisture || Can get non-uniform granules
sensitive Limited size range of particles
Limited number of excipients - optimal
bioavailability
Fluidized Requires only one piece of machinery | Expensive equipment

bed (saves labor cost, transfer losses and || The optimization of process requires
granulation | time) extensive research (there are many
Process can be automated after initial apparatus, process and product variables
process optimization which influence the granulation)
The method and conditions of granulation affect intergranular and intragranular pore structure by
changing the degree of packing within the granules. A simple bonding holds precompressed
granules, consisting of compressed micronutrients and binder particles, together during
compaction. Granules prepared by wet massing consist of intact active ingredients held together
in a sponge-like matrix of binder. Fluidized-bed granules are similar to those prepared by the wet
massing process, but possess greater porosity and a film of binding agent covers the granule
25
surface. With spray-dried systems the granules consist of spherical particles composed of an
outer shell and an inner core of particles. [34]
Common equipment used in the industry for granulation is listed in table 2.5.
Table 2.5: Common equipment used for granulation.
Wet Granulation Dry Granulation
Shear granulators Sluggers

High speed/ mixer granulators | Roller compactors
Fluidized bed granulators
Spray-driers
Spheronizers/ pelletizers
Extrusion / Spheronizes
Tumblers
Tumblers
The particles of a granular mass will cohere when they are tumbled and sprayed lightly with a
liquid binder. The growth may be due to agglomeration of small particles or layering of material
evaporated from the sprayed solution. Rotary kilns of the kind used for drying or chemical
reaction (cement or lime burning, for instance) are adapted to size enlarging service. Usually the
tumbling action is not very intense, only enough to expose the material to sprays. The sprays are
fine and are applied to the surface of the bed of particles. The tumbling action then distributes
the liquid uniformly through the mass. [36]
Spray Dryers
Spray drying involves the atomization of a liquid feedstock into a spray of droplets and
contacting the droplets with hot air in a drying chamber. The sprays are produced by either rotary
or nozzle atomizers. Evaporation of moisture from the droplets and formation of dry particles
proceeds under controlled temperature and airflow conditions. Powder is discharged
continuously from the drying chamber. Operating conditions and dryer design are selected
according to the drying characteristics of the product and powder specifications.
26
Pan granulators
The pan granulator consists of a tilted pan (inclined 30° to 60°) mounted on motor
drive unit.
The powder mixture is fed into the pan and the gravitational and centrifugal forces
force the
particles to move within the pan. Standard spray nozzle is employed to deliver small
quantities
of binding solution to create a basic adhesion between the particles. The granulation
is done by
the snowballing effect of the rotating disc. The rotation speed and angle depend on the size of
the mixture. Too high speed will force the mass to the pan rim due to centrifugal forces; with too
slow rotation sliding with no mixing will occur.
The required disc-rotation speed is given in terms of the critical speed, i.e., the speed at
which a
single particle is held stationary on the rim of the disc due to centripetal forces. The critical
speed
Nc is given by:
Ne = [(g sin 9)/ (2n°D)]"?
Where g is the gravitational acceleration, @ is the angle of the disc to the horizontal, and D is the
disc diameter. The typical operating range for discs is 50 to 75% of critical speed, with angles
of 45—55°. [37]
Figure 2.5 shows a diagram of rotating pan used for both agglomeration and pan coating.
Figure 2.5: (a) Edge and face view of a pan granulator; (b) Stratification of particle size
during rotation [36]
Concentrated « Hecyelg fines

solation ra
“Si _
mA 3
ie aK : Pd ra *s M
Roraries
Setutiat - badereivg
garage <OOTTT ER gg yagen
ae a 4
BResgioceting 9 Py oo ae
aeroner } ; e LN Bras
mutt
gH oY \
During agglomeration, the finer particles settle to the bottom and the largest remain at the top.
Because of the size stratification, the product of disk granulation is more uniform in size than of
drum granulators, which discharge a mixed product.
27
Fluid bed or Wiirster granulators
The fluid bed granulation process (also known as agglomeration) involves suspending
particulates in an air stream and spraying a liquid from the top down onto the fluidized
bed
(figure 2.6). Particles in the path become tacky, collide with other particles and adhere to them
to
form granules.
Figure 2.6: Fluid bed [38]
- <@———._ Binding solution inlet
Bed of suspended particles
Fluidizing air inlet
There are two different modes of fluid bed granulating: dry stage and wet stage. In dry stage
granulation, the particles only require a slight wetting to become tacky and stick to each other.
The granulating solution is applied at a rate less than or equal to the evaporation rate. Thus the
particles remain "dry" through the entire process. In wet stage granulation, the particles require
significant wetting before they become tacky enough to stick to each other. The granulating
solution is applied at a rate higher than the evaporation rate until the particles build up enough
moisture to granulate. The characteristics of the particles when wet and the type of granulating
solution that is being used will determine which mode of granulating is most appropriate. While
dry stage is more common, wet stage granulating allows for denser products. [36, 39]
28
2.3.3 Microencapsulation Technology
Microencapsulation is done in order to protect the micronutrients against deterioration by

environmental factors such as sunlight, temperature variations, moisture etc., to maintain
physical and chemical integrity of premix and to enhance product acceptance and appearance.
The choice of the coating material often depends on the purpose of microencapsulation and it is
often crucial to achieving these goals. There are over a hundred substances suited (and already
used) to form microcapsule films.
They can be put into roughly five categories as follows: [40]
e Polysaccharides/hydrocolloids, such as __ starch, algin/alginate, agar/agarose,

pectin/polypectate, carrageenan, and other gums.
e Proteins such as gelatin, casein, zein, soy, and albumin.
e Fats and fatty acids such as mono-, di- and triglycerides, and lauric, capric, palmitic and
stearic acid and their salts.
® Cellulosic derivatives such as methyl- and ethyl-cellulose.
e Hydrophilic and lipophilic waxes such as shellac, PEG (polyethylene glycol), or carnauba
wax or beeswax.
In several cases, mixtures of these compounds can be used to obtain better results.
Methocel, ethocel, shellac and soy stearine are commonly used coating agents which will also be
used for the premix preparations.
Methocel (methyl-cellulose, MC) is soluble in cold water, GI fluids and organic solvents.
Ethocel (ethyl-cellulose, EC) cannot be used alone because it is totally insoluble in water and GI
fluids. Its main purpose is to toughen the film. EC is soluble in ethanol and other organic
solvents.
Shellac is pH dependent, soluble in water (at pH > 7) and some organic solvents (isopropanol,
benzalcohol) and insoluble in GI fluids.
Soy stearine is fully hydrogenated soy oil with melting point ~ 60°C. It is insoluble in water,
soluble in organic solvents such as hexane, ether and dichloromethane.
29
The film coating is the most commonly used particle coating method in the pharmaceutical and
food industry. The advantages of film coating are that it adds a thin film or coat, the process is
quick, flexible and easily automated. [41]
Use of volatile solvents (e.g. dichloromethane) with toxic effects on worker safety and the
environment and the inability to hide the appearance of the granules are the main disadvantages
of film coating.
Film coating solutions can be either non-aqueous or aqueous. Two methods are commonly used:
spray pan coating and air suspension coating. The coat usually consists of a film former or
polymer, plasticizer, opacifier and colourant.
Film formers are used to produce smooth, thin film. Plasticizers give flexibility and elasticity to
the film. Opacifiers and colorants (e.g. titanium dioxide) are used to change the appearance of
the coated granule. [41]
Volatile organic solvents are commonly used to dissolve the coating materials. Water can be
also used; however its major disadvantage is the slow evaporation. This is especially unsuitable
with active nutrients sensitive to water.
The coating of solid particles can be done in a rotating pan and a fluidized bed.
2.3.4 Antioxidants
Antioxidants are substances capable of preserving food by delaying, retarding or preventing the
development of rancidity or other flavour deterioration due to oxidation. They can inhibit or
retard oxidation in two ways: either by scavenging free radicals via donation of an electron or a
hydrogen atom, in which case the compound is described as a primary antioxidant, or by
deactivation of metal ions and singlet oxygen, in which case the compound is a secondary
antioxidant. [42]
Table 2.6 shows antioxidants permitted in food applications.
30
Table 2.6: Food Antioxidants, [42, 43]
Galates
Phenols
Hydroquinone
BHA
Primary Antioxidants
BHT
“Hindered” Phenols
TBHQ
Tocopherols
Ascorbic Acid
Oxygen Scavengers | Ascorbyl Palmitate
Sulphites
Citric Acid
EDTA
_ Chelating Agents
Secondary Antioxidants Lecithin
Phytic Acid
Vitamin A
B-Carotene
Miscellaneous
Flavonoids
Nitrites
Antioxidants that are naturally occurring in food generally originate from plant-based materials.
The active components, namely phenolics and polyphenolics, including tocopherols, are
secondary plant metabolites and are first derived from phenylalanine and in certain cases and in
some plants from tyrosine. The resultant phenylpropanoids may then undergo further
transformation to yield benzoic acid derivatives as well as flavonoids, isoflavons, and other
complex polyphenols. Thus, natural food phenolics are present as a complex mixture of
compounds that provide a mixture of many active components present in the free, esterified,
glycosylated and bound forms [55]. The mode of action of natural antioxidants may involve
multiple mechanisms, depending on the source material and possible presence of synergists and
31
antagonists. Natural antioxidants are expensive and their use is restricted to some products. On
the other hand, they offer a wide range of antioxidant activity and solubility.
The most widely used synthetic antioxidants in food are butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), propyl gallate (PG), and tert-butylhdroquinone (TBHQ).
These
antioxidants may be added to food, individually or in combination, at a total level of up to
200
ppm on a fat basis. Synthetic antioxidants are inexpensive, widely applied and possess medium
to high antioxidant activity. However, they are only slightly water-soluble and their use is
banned in some countries due to increasing safety concerns.
Allowed Daily Intakes (ADIs) of some antioxidants permitted in food applications [42] are
shown in Table 2.7.
Table 2.7: Allowed Daily Intakes (ADIs) of some antioxidants permitted in foods [42]
Antioxidant ADI [ppm]

BHA 0-200*
BHT 0-200*
TBHQ 0-200*
Tocopherols 200
Phosphates 0-70 000
EDTA 75-300
Ascorbic acid Not limited
Ascorbyl palmitate or ascorbyl || 1250

stearate (or the sum of both)
*based on the fat content of the food
Tocopherols
Tocopherols are the most important radical-scavenging antioxidants that are found in plant
tissues. They have been found to be effective as antioxidants in a number of products, including
bacon, baked goods, butter, lard, margarine, rapeseed oil and sunflowerseed oil. Tocopherols
exhibit much lower antioxidant activity than synthetic antioxidants, e.g. BHA, BHT or TBHQ.
32
Some tocopherols are typically lost during refining, deodorizing, and processing operations
because of their heat liability.
As antioxidants, tocopherols exert their maximum effectiveness at relatively low levels,
approximately equal to their concentration in vegetable oil. Each molecule of a-tocopherol can
scavenge only two radicals without undergoing regeneration. At high concentrations and in
presence of traces of iron and copper salts, tocopherols may act as pro-oxidants. In general,
tocopherols with high vitamin E activity are less effective as antioxidants than those with low
vitamin E activity. The antioxidant activities range in the order: 5-tocopherol (most effective) >
y-tocopherol > B-tocopherol > a-tocopherol (least effective).
Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)

Both BHA and BHT are synthetic antioxidants and are widely used as food additives. They are
extremely soluble in fat and insoluble in water. When used together, BHA and BHT exhibit a
synergistic effect.
These antioxidants interrupt the free-radical chain of oxidative reactions by contributing
hydrogen from the phenolic hydroxy! groups, themselves forming stable free radicals, which do
not initiate or propagate further oxidation of lipids. The advantage of these synthetic compounds
_ over natural ones in the food industry is their stability and reliability in food systems.
Tertiary Butylhydroquinone (TBHQ)

TBHQ is regarded as the best antioxidant for protecting frying oils against oxidation and, like
BHA and BHT; it provides carry through protection to the finished fried product. TBHQ is
adequately soluble in oil and fat and does not complex with iron or copper ions, like propyl
gallate.
33
3. Experimental Techniques and Analytical Methods
3.1 Materials Used
All chemicals used, their suppliers and grades are listed in table 3.1.
Table 3.1: List of used materials
Material Producer Grade

1,10 Phenantroline Sigma Chemical Co., St. Louis, USA Laboratory
Butylated hydroxyanisole, BHA Sigma Chemical Co., St. Louis, USA Food
Butylated hydroxytoluene, BHT || Sigma Chemical Co., St. Louis, USA Food
Dextrin, yellow Casco, Toronto, ON Food
Dichloromethane EM Sciences, Gibbstown, NJ Laboratory
Electrolytic iron, reduced EM Sciences, Gibbstown, NJ ACS

Ethanol, 95% Commercial
Ethocell DOW Chemical Co., Midland, Michigan Drug

Ferric NaEDTA Aldrich Chemical Co Inc., Milwaukee, USA |} ACS
Ferrous Fumarate Sigma Chemical Co., St. Louis, USA ACS
Ferrous Sulphate Sigma Chemical Co., St. Louis, USA ACS
Hexane EM Sciences, Gibbstown, NJ Commercial, b.p. 60 - 68°C
Hydrochloric acid BDH Inc., Toronto, ON Laboratory
Isopropanol Aldrich Chemical Co Inc., Milwaukee, USA || Spectral
Methocell DOW Chemical Co., Midland, Michigan Drug
Potasstum Biphtalate Sigma Chemical Co., St. Louis, USA Laboratory
Potassium Hydroxide Aldrich Chemical Co Inc., Milwaukee, USA | Laboratory

Potassium Iodate Aldrich Chemical Co Inc., Milwaukee, USA || ACS
Potassium Iodide Aldrich Chemical Co Inc., Milwaukee, USA | ACS
Pyrogallic Acid Aldrich Chemical Co Inc., Milwaukee, USA | ACS

Salt, iodized Sifto Canada Inc, Mississauga, ON Food
34
Material Producer Grade
Salt, non-iodized Cargill Food Inc., Mineapolis, USA Top-flo® uniodized salt
Shellac A.S. Paterson Co. Ltd, Toronto, ON Drug
Sodium Tiosulphate BDH Inc., Toronto, ON ACS
Soy Stearine Casco, Toronto, ON Fully hydrogenated soy oil
Starch, white Casco, Toronto, ON Food
Sulphuric acid BDH Inc., Toronto, ON Laboratory
Tert-Butyl Hydroquinone, TBHQ || Sigma Chemical Co., St. Louis, USA Food
Titanium Dioxide EM Sciences, Gibbstown, NJ Food
Vitamin A acetate BASF Corporation, New Jersey, USA Food
Vitamin A palmitate BASF Corporation, New Jersey, USA Food
Vitamin A palmitate Watson Food Co., Inc., West Haven, USA Food
Vitamin A palmitate Roche Vitamins Inc., Parsippany, USA Food
Vitamin A, USP standard USP Rockville MD, USA Analytical
For vitamin A premix, nine commercial vitamin A forms in two different forms (palmitate and
acetate) were tested. Six of them are produced by BASF Corporation, two by Roche Vitamins
Inc. and one by Watson Foods Co., Inc. The vitamin A forms differ in the composition of their
matrix, bioactivity and the type of antioxidant they are stabilized with. Detailed description can
be found in table 3.2.
Table 3.2: Commercial vitamin A forms tested
Bioactivity
# | Vitamin A Matrix Product Number
[1.U./g]
BASF Dry VAP | 250 000 E 414, sucrose, starch, E 307, E 341 Prod:303081
1 | 250 Food Lot:200719YVYO &
483111U0
BASF Dry VAP | 250 000 E 414, sucrose, starch, BHT, E 301, E 341 } Prod:311740
3
250 CK CWD Lot:739795TO
35
Bioactivity
Vitamin A Matrix Product Number
[1.U./g] .
BASF Dry VA ] 325 000 E 414, sucrose, starch, E 307, E 341 Prod:30569401
Acetate 325 GFP Lot:029119YO
BASF Dry VAP | 250 000 gelatin, sucrose, starch, BHT, E 554 Prod:306325
250 CWD Lot:11344182
BASF Dry VAP } 500 000 gelatin, sucrose, modified starch, BHT, || Prod:30306901
500 E554 Lot:183027TO
BASF Dry VA | 500 000 gelatin, sucrose, modified starch, BHT, E || Prod:303064
Acetate 500 554 Lot:20830174
Roche Dry VAP | 500 000 corn starch, gelatin, sucrose, BHT, Prod: 0483834 004
500 Lot:WB00104197
Roche Dry VAP | 250 000 modified starch, sucrose, fractionated | Prod:50 00718004
250 S/N coconut oil, E 301, sodium benzoate, | Lot:WB11104541
sorbic acid, silicon dioxide
Watson VAP 250 || 250 000 modified starch, coconut oil, BHT, silica || Prod:F240114
Powder flour Lot:01542
Note: E 301-sodium ascorbate (antioxidant), E 307-DL-alpha-tocopherol (USP/FCC, antioxidant), E 341- tricalcium
phosphate (anti-caking agents), E 414 - gum Arabic, E554- sodium aluminium silicate (anti-caking agents)
Products 1, 2, 3 and 4 are dispersible even in cold water (10°C) and form a stable milky
emulsion. [44]
Products 5 and 6 are dispersible in warm water (35-40°C) and form a milky emulsion. [44]
Roche Dry VAP 500 is insoluble in water. [45]
Roche Dry VAP 250 S/N disperses quickly and completely in cold water. High concentrations
produce cloudy dispersions, which, however, remain uniform for relatively long periods. [46]
Vitamin A palmitate from Watson dissolves in cold water resulting in a cloudy dispersion. [57]
36
Four forms of iron used in this study were electrolytic iron, ferrous fumarate, ferrous
sulphate
and ferric NaEDTA. These sources were chosen based on their positive stability results in
double and triple fortified salt, previously studied by our group.
Potassium iodate and potassium iodide are most commonly used for salt iodization and
were also
used in this study.
White starch and yellow dextrin were used as binders to granulate the micronutrients.
The ability of three hydrophilic (Methocel, Ethocel and Shellac) and one hydropho
bic (soy
stearine) coating agents to protect the active ingredients from the hi gh humidity environm
ent was
also studied.
BHA, BHT and TBHQ are fat soluble antioxidants which were used to stabilize the vitamin A
premix.
3.2 Equipment
Two stainless steel rotating pans were used for both wet agglomeration and spray coating. The
pans were 26 cm and 13 cm in diameters and 6 cm deep. A 250 mL hand sprayer bottle from ©
VWR was used as an atomizer to spray the binding and coating solutions.
Blending of both, solid powders prior to granulation and salt with premixes, was done in a glass
or plastic jar by shaking for 10 minutes on a shaking apparatus (Burrell wrist action shaker
model #75, Burrell Corp., Pittsburg, PA, USA). Grinding was done using a Braun coffee
grinder.
UV-Vis spectroscopy was done with a UV-Vis Spectrophotometer, Beckman Coutler, model
DU-7U. The fluorescent emission was measured with a Perkin Elmer Luminiscence
Spectrophotometer, model LS 50B.
37
3.3 Experimental Procedure
3.3.1 Pan Agglomeration
Wet agglomeration was used to granulate the premix.
Steps involved in granulation

e blending
e adding liquid
® granulating
e drying
® screening
Well blended material consisting of fillers and active ingredients was placed in a rotating pan.
The pan was angled at approximately 45 degrees and rotated at about 50 rpm. The speed
depended on the size of the pan used and the amount of material granulated.
_ A hand sprayer was used to spray the binding solution, which was usually water or water-
ethanol solution.
Once the material in the pan was free flowing and the size of the particles increased sufficiently,
the material was dried in a Blue M oven from Blue M Electric Company, Blue Island, IL, model
number OV-490A-3 at 60°C. After drying, the material was sieved using the Ro-Tap sieve
shaker. Two Tyler standard US Mesh sieves with sizes 300 um and 710 lum were used,
Particles falling into this range were used in the coating process.
3.3.2 Microencapsulation
The microencapsulation was also done in the pan agglomerator. The granulated and screened
particles were placed into the rotating pan and the angle and rotation speed were set to
approximately the same values as for the agglomeration. The granules were coated with coating
agents dissolved in hot solvents (either ethanol-water solution or dichloromethane) using the
hand sprayer. The air inlet was set at approximately 3 ~ 5 psig. The concentration of the coating
solution was dependent on the coating agent used. In case of soy stearine, 20% w/v solution was
needed. For other coating agents, 30-40% solutions were sufficient.
38
Dichloromethane evaporated practically immediately and no additional drying step was needed.
When ethanol-water solution was used as solvent, an additional drying step was required (~15
min at 60°C). The level of encapsulation was determined by weighing the particles before and
after each run.
3.4 Analytical Methods
3.4.1 The Sampling Method

The sampling technique relied on the concept of random sampling. A set of nine fractions was
built from a bulk of sample so that each fraction contained all the characteristics, including
particle size distribution, as the bulk from which they all came.
Each sample was built from these fractions by a process of multiple additions so that a
statistically representative image of the bulk existed in each of the newly created sample
fractions.
3.4.2 Vitamin A Analysis

A standard fluorometric method [47] was used for vitamin A analysis. This method is based on’
measurement of fluorescence of retinol and retinol esters in hexane after appropriate cleanup
which separates the vitamin A from other lipids in the sample matrix. The hexane extract is
excited by radiation at 330 nm and the fluorescent emission is measured at 480 nm. The
fluorescence of retinol is a linear function of concentration from zero to 0.25 g/mL. At higher
concentrations self-absorption becomes significant. [48]
The method consists of saponification (also called alkaline hydrolysis), extraction and
measurement of fluorescence.
During saponification, 70% potassium hydroxide solution and 1% ethanolic pyrogallol are used
to free the vitamin A from fat and to convert the retinol esters into retinol (vitamin A alcohol).
After saponification, most of the saponifiable material becomes soluble in polar solvents.
Vitamin A can be then extracted with hexane.
The fluorescence was measured with a Perkin-Elmer Luminescence Spectrometer, Model LS
SOB, PE Shelton, Connecticut. The details can be found in Appendix 9.1.1.
39
3.4.3 Iron Analysis
Spectrophotometric determination of iron developed by Harvey, Smart and Edwards (1955) was
used [49]. When 1, 10-phenantroline is added to a solution containing both ferrous and ferric
irons, a reddish orange ferrous complex and a yellow ferric complex form immediately. The iron
(ff) complex has an absorbance maximum at 512 nm. The two complexes have identical
extinction coefficients at 396 nm. The absorbance was determined with a Beckman Coulter
model DU-7U, UV Spectrometer. The details are presented in Appendix 9.1.2.
3.4.4 lodine Analysis
3.4.4.1 Determination of Potassium Iodide
The iodine content was determined using epithermal neutron activation analysis (ENAA), using
the slowpoke reactor at the Royal Military College in Kingston, Ontario.
1 to 2 g of sample was weighed into a polyethylene vial. The sample vials were then shielded in
cadmium and irradiated at 1 kW for 3 minutes using a neutron flux of 5.0 x 10'' cm®sec”!. The
samples were rested for 6 minutes and the gamma emissions were measured at 443 keV using a
hyperpure germanium based gamma ray spectrometer. The concentration of iodine was
calculated from the calibration curve obtained using series of standards covering a range of 0 to.
1000 ppm. [50]
3.4.4.2 Determination of Potassium Iodate

lodate was determined by iodometric titration using AOAC standard method 33.149 [51]. This
method is based on reduction of iodate to free iodine, which forms a deep blue color with starch.
The free iodine is then titrated with standardized solution of sodium tiosulphate. The details are
presented in Appendix 9.1.3.
3.4.5 Moisture Content Determination

Moisture content was determined gravimetrically. Approximately 20g samples (w;) were dried
at 105°C for 24 hours. After cooling down the samples were weighed (w;). The weight
difference is assumed to be the moisture content.
Moisture [%] = (w; — wp) * 100 % / w;
40
wj — initial weight, before drying [g]
wy — final weight [g]
3.4.6 Particle Size Distribution

The analysis was performed using the Ro-Tap sieve shaker (figure 3.1). A stack of three Tyler
standard US Mesh sieves with sizes 300 j1m, 500 um and 710 um was used.
Figure 3.1: A Ro-Tap sieve shaker [52]
Approximately 50g samples were shaken for 10 ~ 15 minutes. The separated parts were wei ghed
and the fraction (% by weight) of the particles accumulated between two neighboring sieves was
determined.
Mean particle diameter retained by a screen is the sum of the aperture of the screen on which the
material is retained, plus the aperture of the next largest screen, divided by 2.
Mean particle diameter of a sample is the sum of the mass fractions retained on each screen
multiplied by the mean diameter of particles retained by that screen.
4]
3.5 Packaging, Storag e Conditions and Sample Handling
Each sample formulation was prepared in six 150g replicates, packed in polyethylene “Ziploc”
bags, 18 x 15 cm. The samples were stored under two conditions for three months (hence the
replicates, table 3.3):
e high temperature, high humidity (40°C, 100% RH)
e high temperature, medium humidity (40°C, 60% RH)
The 40°C and 60% RH setting is a good estimate of the average conditions in tropical
environment. A relative humidity of 100% was chosen as the extreme condition that TES is
expected to encounter during storage, transportation and distribution.
The high temperature and high humidity conditions were maintained using controlled
temperature oven. The approximately 100% relative humidity was achieved by placing a tray of
water into the oven thus obtaining approximately saturated air.
The high temperature and medium humidity conditions were maintained in an environmental
chamber type ZH-8-1-H/AC, manufactured by Cincinati Sub-Zero Co., Cincinnati, OH, USA.
Table 3.3: Handling of the six replicates prepared from each formulation. All replicates
were put to the ovens at the same time.
Total Time Spent in an Oven Stability

Environment | Replicate #
1 month |2 months | 3 months | Results
1 X 1" mo
60% RH 2 x 2™ mo
3 x 3™ mo
4 X 1 mo
100% RH 5 x 2™ mo
6 X 3™ mo
This was done to keep the conditions constant throughout the storage period for each replicate.
The stability of the micronutrients was determined monthly.
42
4, Results and Discussions
This thesis involved the study of three main areas.

First, processes for protecting vitamin A and producing TFS with acceptable organoleptic and
storage properties were developed and tested using commercially available vitamin A forms.
Secondly, the effect of different types of iron and iodine compounds on the retention of these
vitamins in triple fortified salt was investigated. Four iron and two iodine compounds were used
for this study.
Lastly, several binders, coating agents and antioxidants were tested for their suitability to protect
the vitamin A against deterioration by environmental factors such as high temperature, moisture
and sunlight and their ability to maintain the physical and chemical integrity of the matrix.
4.1 Triple Fortified Salt
Triple fortified salt (TFS) was successfully prepared using three micronutrients: vitamin A, iron
and iodine. The targeted concentrations in the TFS were:
@ 250 IU of vitamin A/g of salt
e 1000 ppm of iron
e 50 ppm of iodine
These values were selected for several reasons:

e The average daily consumption of salt is estimated to be around 10g per person.
® Considering the recommended daily intake for vitamin A (2500 IU/day), assuming a 30%
bioavailability and retention during storage and distribution, the targeted concentration in
TFS needed to be 250 IU/g of salt.
43
e The TFS is designed to provide one third of the 3 mg/day RDI for iron. Thus based on
the iron content in different compounds and a 10% typical bioavailability, a level of 1000
ppm of iron in salt, equivalent to 10mg iron per day is desired.
e The level of iodization is not uniform throughout the world, although many countries use
50 ppm. Therefore the TFS samples will contain 50 ppm of iodine. If we assume the per-
capita daily requirement of iodine is 200 ug, the level of iodine required in the TFS is 20
ppm. Assuming that only 50% of I, is retained during transit and storage, the level of
iodization required is 52 ppm as KI or 67 ppm as KIOs.
All micronutrients were granulated and coated to form the respective premixes, which were
blended with non-iodized salt. In some cases iodized table salt was used instead of iodine
premix. The average size of salt crystals is between 300 um and 710 uum. The granulation was
used to increase the size of the premix to match the size of salt. The main purposes of the
granulation were to prevent the segregation, to dilute the micronutrients and to achieve a
desirable granule shape.
Figure 4.1 shows a microscopic image of salt fortified with all three micronutrients (60 x
magnification). The micronutrients were granulated with dextrin to match the size range of salt
crystals and coated with soy stearine to protect them from a high temperature (40°C) and high
humidity (60% RH or ~100% RH) environment. The vitamin A and iodine premixes were
slightly yellow in colour, the ferric NaEDTA was off-white.
Figure 4.1: Picture of TFS
3 ae
= . : Iron premix (ferric NaE DTA)
Vitamin A premix (KR22)
Iodine premix (potassium iodate)
Salt crystals
one
44
4,2 Premix Formulations
From the nine commercial vitamin A sources, 36 premixes were prepared and tested both alone
and after being mixed with the salt. The targeted vitamin A concentration in each of them was
25 000 IU/g of premix which would give the desired 250 IU/g salt at 1% addition level.
Three main types of vitamin the A premix were produced:

1. Premix containing only vitamin A as the active ingredient.
2. Premix containing vitamin A and iodine (either as iodide or as iodate).
3. Premix containing all three micronutrients in one particle.
The premix containing source vitamin A and iron in one particle was studied previously by our
group [56], and the retention of vitamin A was between 80% (ferric NaEDTA) and 20% (ferrous
sulphate) after two months of storage in 40°C 60% RH.
All vitamin A sources were granulated with either Casco Dextrin CAS: 9004-53-9 (yellow) or
Casco Starch CAS: 9005-25-8 (white) and encapsulated with Soy Stearine (30% or 40%),
Shellac (40%) or Methyl Cellulose (MC) and Hexaethyl Cellulose (HEC). The antioxidants used
to stabilize the vitamin A premix were either BHA and BHT (in 1:1 ratio) or TBHQ.
The composition of all the premixes used in this study is shown in Tables 4.1 to 4.5. Table 4.1
shows the premix formulations which were coated with 40% soy stearine. These vitamin A
premixes were mixed with potassium iodate or iodized salt and one of the four iron premixes to
form the TFS. Vitamin A premixes coated with Shellac can be found in Table 4.2. Premixes
containing all three micronutrients in one particle are listed in Table 4.3. The composition of the
vitamin A premixes encapsulated with MC/HEC (level of encapsulation is 40%) are shown in
Table 4.4. Lastly, Table 4.5 lists the composition of various premix and TFS formulations: (a)
TFS formed with vitamin A premix mixed with KI and iron premixes, (b) vitamin A granulated
with a source of iodine, and (c) vitamin A coated with 30% of soy stearine.
45
Table 4.1: Composition of the vitamin A premix formulations coated with 40% SS
Sample # | Vitamin A Binder | Antioxidant | Encapsulant

KR20-1 | BASF Dry VAP 250 Food Lot:200719YO | Dextrin | BHA/BHT | 40%SS
KR20-2 | BASF Dry VAP 250 Food Lot:200719YO | Dextrin | TBHQ 40%SS
KR21 BASF Dry VAP 250 Food Lot:200719YO | Starch TBHQ 40%SS
KR22 BASF Dry VAP 250 CK CWD Dextrin | TBHQ 40%SS
KKR23-1 | BASF Dry VAP 250 CK CWD Starch BHA/BHT | 40%SS
KR23-2, | BASF Dry VAP 250 CK CWD Starch TBHQ 40%SS
KR24-1 | BASF Dry VA Acetate 325 GFP Dextrin | BHA/BHT | 40%SS
KKR24-2 | BASF Dry VA Acetate 325 GFP Dextrin | TBHQ 40%SS
KR25-1 | BASF Dry VAP 250 Food Lot:483111UO | Dextrin | BHA/BHT | 40%SS
KR25-2 | BASF Dry VAP 250 Food Lot:483111U0 | Dextrin | TBHQ 40%SS
KR26-1 | BASF Dry VAP 250 CWD Dextrn | BHA/BHT | 40%SS
KR26-2. | BASF Dry VAP 250 CWD Dextrin | TBHQ 40%SS
KR27-1 | BASF Dry VAP 500 Dextrin | BHA/BHT | 40%SS
KR27-2 | BASF Dry VAP 500 Dextrin | TBHQ 40%SS
KR28-1 | BASF Dry VA Acetate 500 Dextrin | BHA/BHT. | 40%SS
KR28-2 | BASF Dry VA Acetate 500 Dextrn | TBHQ 40%SS
KR29-{ | Roche Dry VAP 500 Dextrin | BHA/BHT | 40%SS
KR29-2 Roche Dry VAP 500 Dextrin | TBHQ 40%SS
KKR30-1 | Roche Dry VAP 250 S/N Dextrin | BHA/BHT | 40%SS

KR30-2 | Roche Dry VAP 250 S/N Dextrin | TBHQ 40%SS
KR31-1 | Watson VAP 250,000 IU/g Powder Dextrin | BHA/BHT | 40%SS

KR31-2 | Watson VAP 250,000 IU/g Powder Dextrin | TBHQ 40%SS
Table 4.2: Composition of the vitamin A premixes encapsulated with Shellac
Sample # | Vitamin A Binder Antioxidant | Encapsulant
KRII Watson VAP 250,000 IU/g Powder | Dextrin TBHQ 40% Shellac
KRIi2 BASF Dry VAP 250 Food Dextrin TBHQ 40% Shellac
46
Table 4.3: Composition of the premixes containing all three micronutrients in one particle
Sample # | Vitamin A Binder | Antioxidant | Encapsulant | note

KRI13-1 | Watson VAP | Dextrin | TBHQ 40%SS with FeFum and
250,000 IU/g Powder KIO; in one particle
KRI13-2 | Watson VAP | Dextrin | TBHQ 40%SS with Fe NaEDTA and
KR14-1 | Watson VAP | Dextrin | TBHQ 30%SS with FeFum and
KR14-2 | Watson VAP | Dextrin | TBHQ 30%SS with Fe NaEDTA and
Table 4.4: Composition of the vitamin A premixes encapsulated with MC/HEC (level of
encapsulation was 40%)
Sample # | Vitamin A Binder | Antioxidant | Encapsulant
KRI15 Watson VAP 250,000 IU/g Powder | Dextrin | TBHOQ 30% MC/ 10 %HEC
KRI6 Watson VAP 250,000 IU/g Powder {| Dextrin | TBHQ 20% MC/ 20 “HEC
Table 4.5: Composition of the premixes and TFS samples containing various sources of
iodine. Components of premixes coated with 30% SS.
Sample# | Vitamin A Binder | Antioxidant | Encapsulant | Source of

lodine in TFS
KRI7 Watson VAP 250,000 IU/g || Dextrin | TBHQ 40%SS KI premix
Powder
KR18-1 Watson VAP 250,000 [U/g || Dextrin | TBHQ 40%SS KIO; and vit A
Powder in one particle
KR18-2 | Watson VAP 250,000 IU/g || Dextrin | TBHQ A0%SS KI and vit A in
Powder one particle
47
Sample # | Vitamin A Binder | Antioxidant | Encapsulant | Source of
Iodine in TFS
KRI9-1 | Watson VAP 250,000 IU/g | Dextrin | TBHO 30%SS KIO3 premix
Powder
KRI19-2 BASF Dry VAP 250 Food _ | Dextrin | TBHQ 30%SS KIO3 premix
The coated vitamin A premixes were slightly yellow in colour. These premixes were mixed with
an iron premix and an iodine premix or iodized salt. Total of 120 samples were prepared for
each test condition (40°C 60% RH and 40°C ~100% RH). When iodine premix or the vitamin A
premix containing source of iodine (either in the form of potassium iodide or potassium iodate)
was used, the non-iodized salt was used for sample preparation.
Four iron premixes were prepared using one of the following iron compounds:
e Electrolytic iron, reduced

® Ferrous Fumarate
e Ferrous Sulphate, heptahydrate

e Ferric Sodium EDTA
Both iron and iodine premixes were granulated with dextrin and coated with 40% soy stearine.
The colour of the iodine premixes was yellowish. Elemental iron gave grey premix, ferrous
fumarate gave white premix with brown spots (showing defects in coating), ferrous sulphate was
off white and the premix containing Fe NaEDTA was sandy in colour. Figure 4.2 shows the
appearance of typical vitamin A, iron and iodine premixes.
48
Figure 4.2: The appearance of typical vitamin A, iron and iodine premixes
KIO; Fe NaEDTA
: REGS
Fe Fum Fe SO4x 7H,O FeElem
ae a. | oe
oe oe ss . : —
- S en
on,
a x ,, s
» — oeay
Peet a i
Soca
eee. Fe slants
Vitamin A Premix (KR22) Vitamin A Premix (K25-1)

ggg “ap iat :
a
aoe”
a ‘ oe,
4.3 Stability of Vitamin A
4.3.1 Stability of Commercial Vitamin A Forms
The complete results for the vitamin A stability tests are shown in Appendix 9.2. A minimum of
four replicates of each sample were analyzed and the standard deviations were calculated.
The results (Figures 4.3. and 4.4) show that overall vitamin A is highly unstable under the
studied conditions. The average retention of vitamin A in TFS after three months at 40°C 60%
RH and 40°C ~100% RH was 39 + 1% and 31 + 1%, respectively. Although there is a wide
range within both RH studies, seven of the commercially available forms of vitamin A in 40°C
60% RH and five in 40°C ~100% RH demonstrated above average stability. These included:
BASF Dry VAP 250 Food (premix KR20 in both RH and KR25 in 40°C 60% RH)
@
° BASF Dry VAP 250 CK CWD (premix KR22 in 40°C and both RH)
¢ BASF Dry VA Acetate 325 GFP (premix KR24 in 40°C 60% RH)
e BASF Dry VAP 500 (premix KR27 in 40°C and both RH)
e Roche Dry VAP 500 (premix KR29 in 40°C and both RH)
e Roche Dry VAP 250 S/N (premix KR30 in 40°C 60% RH)
e Watson VAP 250,000 IU/g Powder (premix KR 31 in 40°C ~100% RH)
The instability of some vitamin A samples in particular demonstrates the importance of choosing
an appropriate source of commercial vitamin A. It is clear that the processes used for
commercial stabilization of vitamin A have a major effect on the vitamin’s stability in the triple
fortified salt.
Figure 4.3: Stability of vitamin A premixes, granulated with dextrin, coated with 40% SS
and stabilized with either BHA/BHT (marked with number 1) or TBHQ (marked with
number 2), in the presence of iron and potassium iodate premix. The sample containing
50
KR22i premix was prepared with iodized table salt. The TFS samples were stored for
Stability of vitamin A with different iron compounds

after 3 months; 60% RH
60.00
% Vitamin A Retention
50.00
40.00
30.00
20.00
40.00
and stabilized with either BHA/BHT (marked with number 1) or TBHQ (marked with
number 2), in the presence of iron and potassium iodate premix. The sample containing
KR22i premix was prepared with iodized table salt. The TFS samples were stored for
Stability of vitamin A with differentiron compounds

after three months; 100% RH
50.00
40.00
30.00
20.00
40,00
0.00
Figures 4.5 and 4.6 show the vitamin A retention after three months of storage in 40°C 60% RH
and 40°C ~100% RH respectively, for formulations containing different iron compounds.
In 40°C 60% RH vitamin A was most stable when ferric NaEDTA was used. In contrast, the
lowest stability was observed under these conditions when ferrous fumarate was used. The same
trends were not observed at 40°C ~100% RH. At 40°C ~100% RH the reverse was true: the best
stability of vitamin A was achieved with ferrous fumarate and the worst with ferric NaEDTA.
51
Because the difference between these results is less than 5%, the source of iron was not a major
factor affecting the stability of vitamin A. Nonetheless, as shown in the previous research done
by our group [56], iron is a major contributor to the instability of TFS.
compounds and potassium iodate premix. The TFS samples were stored for three months
at 40°C 60% RH.
Stability of vitamin A with each iron compound

after three months; 60% RH
Fe elem Fe Fum Fe Suiph Fe NaEDTA
compounds and potassium iodate premix. The TFS samples were stored for three months
at 40°C ~100% RH.
Stability of vitamin A with each iron compound

after three months: 100% RH
40.00
fe
2
§& 30.00
o
Va
< 20.00
&
—E
£ 10.00
>
oe
0.00
Fe elem Fe Fum Fe Sulp Fe NaEDTA
52
An ANOVA was performed to evaluate the effects of iron and commercially produced vitamin A
forms on the stability of vitamin A in TFS.
There was significant effect of vitamin A source on the vitamin’s stability in TFS (P < 0.05) or
(P=1.77E-5) stored at 40°C 60% RH. In contrast, vitamin A stability was not significantly
influenced by its source (P=0.157) when stored at 40°C ~100% RH. In addition, the ANOVA
indicated that the stability of vitamin A is not significantly affected by the source of iron for
either the 40°C 60% RH or the ~100% RH, (P>0.05) or (P=0.315 for 40°C 60%RH and P=0.173
for 40°C ~100%RH).
The decrease in vitamin A in TFS as a function of time was found to be consistent with a first
order reaction rate. The first order reaction rate constants (k) were calculated to be (Figure 4.7)
0.15/month (5.8x10™ s"') at 40°C 60% RH and 0.3/month (1.2 x107 s") at ~100% RH for sample
containing KR 27-1 premix (BASF Dry VAP 500) and ferric NaEDTA (R* (RSQ) 0.94 and 0.97,
respectively). The results confirm that, as expected, the rate of degradation of vitamin A was
slower in the lower humidity environment.
Figure 4.7; Degradation rate of vitamin A premix KR 27-1 (BASF Dry VAP 500 granulated
with dextrin, coated with 40% SS and stabilized with BHA/BHT) in TFS sample containing
ferric NaEDTA. The TFS sample was stored for three months at 40°C 60% RH and
~100% RH.
Degradation rate of KR27-1 premix in the

presence of Fe NaEDTA
NM ©
.
fF
InA/Ao
:
©
:
©
to.
—-~
time (mo)
|@ 60% RH Me 100% RH Gmmmmml
ine ar (60% RH) Sas near (100% RH) |
53
Based on these results, premixes KR22, 25 (which is identical to KR 20), and 27 were selected
for further study as they each demonstrated promising vitamin A stability under one or both
environmental conditions studied.
4.3.2 Stability of Three Best Vitamin A Premixes
Premixes KR 22, 25 and 27 gave the best stability under both conditions studied (40°C 60% RH
and ~100% RH). 13 formulations gave stability greater than 45% at 40°C 60% RH and four
formulations retained more than 40% of vitamin A at 40°C ~100% RH. In addition, these
samples did not change color even after three months of storage at 40°C ~100% RH. This is a
desirable characteristic as one of the requirements for any fortified food is that the organoleptic
characteristics are maintained.
The most stable premixes will now be discussed in greater detail.
KR22
The BASF Dry VAP 250 CK CWD was specially developed for sugar fortification and for use in
vitamin mixtures for the food industry. The product contains a minimum of 250,000 I.U. of
vitamin A palmitate per gram.
For inclusion in TFS this vitamin A product was granulated with dextrin, coated with soy
stearine and stabilized with TBHQ. Eight different salt samples were produced by mixing the
premix with either KIO3 premix or iodized salt and all four iron sources. The decrease in
vitamin A concentration during storage at 40°C 60% RH and ~100% RH for three months are
presented in Figures 4.8 and 4.9, respectively.
54
Figure 4.8: The stability of KR22 premix (BASF Dry VAP 250 CK CWD) mixed with all
four iron premixes and either potassium iodate premix or iodized salt. This premix was
granulated with dextrin, coated with 40% SS and stabilized with TBHQ. The TFS samples
were stored for three months at 40°C 60% RH.
60% RH, KR22 [Elist mo [%] W2nd mo [%] El3rd mo [%] |

100,00
80.00
60.00
40.00
20.00
0.00
Figure 4.9: The stability of KR22 premix (BASF Dry VAP 250 CK CWD) mixed with all
four iron premixes and either potassium iodate premix or iodized salt. This premix was
granulated with dextrin, coated with 40% SS and stabilized with TBHQ. The TFS samples
were stored for three months at 40°C ~100% RH.
100% RH, KR22 [Eist mo [%] H¥2nd mo [%] C3rd mo [%]|

This premix (KR22) showed acceptable stability with four out of eight samples retaining more
than 50% of this vitamin A after three months (40°C 60% RH). At 40°C ~100% RH, only two
samples retained more than 40% of vitamin A.
This vitamin A was most stable in the samples which contained ferrous fumarate and ferric
NaEDTA mixed with KIO; premix, 56 + 4% and 54 + 3% respectively. However, the best
55
stability at 40°C ~100% RH for three months was observed in samples containing elemental iron
with KIO; (46 + 7%) and ferrous fumarate with iodized salt (45 + 1%).
The retentions of vitamin A in premix KR22, the calculated first order reaction rate constants and
RSQs for those samples which retained more than 40% of the original vitamin A after three
months at 40°C and both RH conditions (60% RH and ~100% RH) are presented in Table 4.6.
The premix was blended with all four iron premixes and either potassium iodate premix and non-
iodized salt or iodized table salt.
Table 4.6: Retentions of vitamin A (BASF Dry VAP 250 CK CWD, KR 22 premix) with the
standard deviations (based on minimum four replicates), the calculated first order reaction
rate constants and RSQ values. The TFS samples were stored for three months at 40°C
and both RH conditions (60% RH and ~100% RH). This premix was granulated with
dextrin, coated with 40% SS and stabilized with TBHQ.
KR22_ | Sample Initial 1" mof2"™ mof3™ molk RSQ

premix | constituents conc. [%] | [%] [%] [“%o] [mo™]
60% | Fe elem 100 84412 178+7 |55+6 0.19 0.923
RH” | FeFum/KIO; 100 85+6 6341 15644 [0.21 0.977
FeNaEDTA/KIO; | 100 7t11 |69+4 |5443 [0.19 0.963
FeNaEDTA 100 8646 |76+5 [5043 | 0.22 0.909
~100% | Fe elem/KIO; 100 7446 [6342 |46+7 10.25 0.986
RH FeFum 100 76+2 [6045 [4541 0.27 0.998
Table 4.6 shows that the rate of degradation of vitamin A in premix KR22 is relatively slow (the
k values are low) in both RH environments. The first order reaction behavior is supported by the
fact that the value for RSQ was greater than 0.9 in all cases.
KR 25
BASF Dry VAP 250 Food was developed for fortifying dry or powdered food products that must
be dissolved in water prior to use. This product is said to be suitable for the production of
56
hypoallergenic foods, as it contains no protein. The product contains at least 250,000 LU. of
vitamin A pre gram.
BASF Dry VAP 250 Food was also granulated with dextrin, coated with 40% soy stearine and
stabilized with either TBHQ or a mixture of BHA and BHT (1:1 ratio).
e 25-1: BHA/BHT
e 25-2: TBHQ
Eight different samples were produced by mixing the vitamin A premix with a KIO; premix,
non-iodized salt and one of four iron sources. The stability results are presented in F igures 4,10
(40°C 60% RH) and 4.11 (40°C ~100% RH).
Figure 4.10: The stability of KR25 premix (BASF Dry VAP 250 Food) mixed with all four
iron premixes and potassium iodate premix. This premix was granulated with dextrin,
coated with 40% SS and stabilized with either BHA/BHT (KR25-1) or TBHQ (KR25-2).
The TFS samples were stored for three months at 40°C 60% RH.
fo
60% RH, KR25 |Etst mo [% } Ei2nd mo [%] L13rd mo [%]
100.00
80.00
60.00 ee
40.00 -
20.00 4
0.00 +b
57
Figure 4.11: The stability of R25 premix (BASF Dry VAP 250 Food) mixed with all four
iron premixes and potassium iodate premix. This premix was granulated with dextrin,
coated with 40% SS and stabilized with either BHA/BHT (KR25-1) or TBHQ (KR25-2).
The TFS samples were stored for three months at 40°C ~100% RH.
i oe
| 100% RH, KR25 [Elist mo [%] B¥2nd mo [%] 13rd mo [%] |

100.00
80.00
60.00 +
40.00 -
20.00 -
0.00 LES
Five out of the eight premixes showed stability higher than 48% after three months at 40°C, 60%
RH. The highest stability of 55 + 2% was achieved in the sample which contained elemental
iron and vitamin A stabilized with BHA/BHT.
In 40°C ~100% RH, vitamin A loss was high. None of the samples retained more than 35% of
vitamin A after three months. The lowest rate of degradation (k) in 40°C 60% RH was
0.19/month (sample Fe elem-2). In 40°C ~100% RH the k value was found to be almost twice as
high as the value for 40°C 60% RH environment, 0.36/month (sample FeFum-2). While the
ferric NaEDTA~1 sample offered very good stability in the 40°C 60 % RH (53 + 6%), in 40°C
~100% RH the lowest amount of vitamin A (11 + 4%) was observed after the same time period
after three months of storage. The sample containing ferric NaEDTA and vitamin A stabilized
by TBHQ showed very good stability under both conditions (51 + 2% in 60% RH and 34 + 7%
in 40°C ~100% RH).
KR 27
BASF Dry VAP 500 is recommended by the manufacturer particularly for products with high
moisture content, and for preparations that are to be stored in climates with high relative
humidity. The stability of this vitamin A in the dry powder is supposed to be very good even in
the presence of minerals. The product contains ~ 500,000 I.U./g of vitamin A.
58
This vitamin A product was granulated with dextrin, coated with 40% soy stearine and stabilized
with 27-1: BHA/BHT (1:1 ratio) or 27-2: TBHQ. Reasonable stability was observed in both RH
environments. Figures 4.12 and 4.13 show the stability of this premix for storage period of three
months under 40°C 60% RH and ~100% RH, respectively.
Figure 4.12: The stability of R27 premix (BASF Dry VAP 500) mixed with all four iron
premixes and potassium iodate premix. This premix was granulated with dextrin, coated
with 40% SS and stabilized with either BHA/BHT (KR27-1) or TBHQ (KR27-2). The TFS
60% RH, KR27 | mist mo 1%] §82nd mo [%] Hlsré mo 1%1 |

Figure 4.13: The stability of KR27 premix (BASF Dry VAP 500) mixed with all four iron
premixes and potassium iodate premix. This premix was granulated with dextrin, coated
with 40% SS and stabilized with either BHA/BHT (KR27-1) or TBHQ (KR27-2). The TFS
100% RH, KR27 [EA ist mo [%] B2nd mo (%] El3rd mo [%] |
100.00
80.00
Hl
60.00 +
a Ee
40.00 ao
20.00 - -
&& <e
59
The samples containing elemental iron showed high vitamin A retention, with 65 + 2% retained
(sample Fe elem-1) at 40°C 60% RH and as much as 44 + 6% (sample Fe elem-2) at 40°C

~100% RH. Fe NaEDTA-1 sample retained 61 + 5% of vitamin A at 40°C 60% RH and just
under 40% at 40°C ~100% RH.
Overall, four out of the eight TFS samples containing KR22 premix (BASF Dry VAP 250 CK
CWD) gave vitamin A retentions greater than 50% after two months of storage at 40°C 60% RH.
In addition, two samples retained more than 40% of this vitamin A at 40°C ~100% RH after the
same period of time.
In the case of premix KR25 (BASF Dry VAP 250 Food), three TFS samples showed retention
greater than 50% at 40°C 60% RH, but none of them retained more than 40% of vitamin A at
40°C ~100% RH after three months of storage.

TFS samples containing premix KR27 (BASF Dry VAP 500) showed very good stability under
both RH conditions. At 40°C 60% RH, two samples retained more than 60% of vitamin A and at
40°C ~100% RH three samples showed retention higher than 40% after three months of storage
under these conditions.
All three vitamins (BASF Dry VAP 250 CK CWD, BASF Dry VAP 250 Food and BASF Dry
VAP 500) should be investigated further as they show promising stability in TFS samples.
Altering the composition of their matrix and coating agents could promote their stability even
more.
4.3.3 Vitamin A Acetate vs. Palmitate
The effect of different vitamin A esters on the vitamin’s stability in TFS was evaluated by
comparing the stability of vitamin A acetate and palmitate. In general, vitamin A palmitate
showed better stability than the acetate form under all experimental conditions. This is most
likely due to the differences in chemical structure between the acetate and palmitate forms.
60
Six vitamin A palmitate and four vitamin A acetate premixes were studied. The premixes were
granulated with dextrin, coated with 40% SS and stabilized with either BHA/BHT or TBHQ.
Their composition is shown in Table 4.7. The TFS samples consisted of vitamin A premix, one
of four iron premixes and either iodized salt (salt samples containing KR22i premix), non-
iodized salt (salt samples containing KR22n premix) or potassium iodate premix (all remaining
salt samples).
At 40°C 60% RH, the most stable TFS sample containing vitamin A palmitate was obtained in
the presence of elemental iron and potassium iodate, using BASF Dry VAP 500 premix. 64.86 +
1.92% of vitamin A was retained in this case. In addition, thirteen out of 24 samples showed
retention greater than 45% over the three month period. In comparison, using the vitamin A
acetate a maximum of only 48 + 5% vitamin A was retained (in TFS containing ferrous sulphate
and potassium iodate premix) under the same conditions, when the BASF Dry VA Acetate 325
GFP was used. A similar trend was observed for the 40°C ~100% RH.
Despite the increased stability of vitamin A palmitate over the acetate form, this ester does
possess a lower bioavailability than vitamin A acetate. One IU is equal to 0.300 pg of all-trans-
retinol, only 0.344 jg of all-trans-retinyl acetate but almost double the amount of the palmitate
form (0.55 pg of all-trans-retinyl palmitate). In addition to stability, bioavailability must be

considered when choosing the appropriate form of vitamin A for fortification.
Table 4.7: Composition of the premixes used for vitamin A palmitate vs. acetate stability
study (=iodized salt, n=non-iodized salt)
Premix # Label Vitamin A Antioxidant

KR22i 1- Palm BASF Dry VAP 250 CK CWD TBHQ
KR22n 2- Palm BASF Dry VAP 250 CK CWD TBHQ

KR25-1 3- Palm BASF Dry VAP 250 Food BHA/BHT
KR25-2 4- Palm BASF Dry VAP 250 Food TBHQ
KR27-1 5 - Palm BASF Dry VAP 500 BHA/BHT
IKR27-2 6 - Palm BASF Dry VAP 500 TBHQ

KR24-1 1 - Acet BASF Dry VA Acetate 325 GFP BHA/BHT
61
Premix # Label Vitamin A Antioxidant
KR24-2 2 - Acet BASF Dry VA Acetate 325 GFP TBHQ
KR28-1 3 - Acet BASF Dry VA Acetate 500 BHA/BHT
KR28-2 4~- Acet BASF Dry VA Acetate 500 TBHQ
The following Figures (4.14. to 4.17) show the retention of vitamin A palmitate and acetate with
all four iron compounds (elemental iron, ferrous fumarate, ferrous sulphate and ferric N8EDTA)
stored for three months at 40°C 60% RH. Figure 4.14 presents the stability of vitamin A acetate
and palmitate premixes in TFS samples containing elemental iron. The stability of these vitamin
A compounds with ferrous fumarate can be seen in Figure 4.15. Figure 4.16 shows the stability
of these vitamin A forms TFS samples containing ferrous sulphate. Finally, the stability of
vitamin A acetate and palmitate premixes in TFS samples containing ferric NaEDTA can be
found in Figure 4.17.
Figure 4.14: Stability of vitamin A acetate and palmitate premixes (granulated with
dextrin, coated with 40% SS and stabilized with either BHA/BHT or TBHQ), in the
presence of elemental iron and potassium iodate premix. The sample containing KR22i
premix (1-Palm) was mixed with iodized salt. The TFS samples were stored for three
months at 40°C 60% RH.
60% RH, 40C, Fe elem [C¥ist mo [%] @2nd mo (%] Gard mo [% |] |

100.00
80.00
60.00 +
40.00 -
20.00 +
0.00 4
In the presence of elemental iron, four out of six vitamin A palmitate premixes gave stability
higher than 45% after three months of storage at 40°C 60% RH. The highest stability was
obtained with sample 5-Palm (premix number KR27-1), which demonstrated 65 + 2% retention.
62
This is also the highest retention of vitamin A obtained during this study. None of the vitamin A
acetate premixes showed retention higher than 40% after three months of storage under the same
conditions. Out of all vitamin A acetates, BASF Dry VA Acetate 325 GFP (2 — Acet) showed
the highest stability with 38 + 6% retention after three months storage at 40°C 60% RH.
presence of ferrous fumarate and potassium iodate premix. The sample containing KR22i
66% RH, 40C, FeFum Bist mo [%) 2nd mo [%] 3rd mo (%]|
Sample # - -
Three vitamin A palmitate forms out of six gave retention over 40% in salt samples containing
ferrous fumarate. The highest retention of vitamin A palmitate was 56 + 4%, obtained with
sample 1-Palm (premix KR22n) after three months of storage at 40°C 60% RH. All vitamin A
acetate forms showed retention greater than 30%, although one demonstrated greater than 40%
retention in the presence of ferrous fumarate, after three months of storage under the same
conditions. This was obtained with BASF’s Dry VA Acetate 325 GFP (2 —Acet) with 45 + 4%
retention after three months of storage at 40°C 60% RH.
presence of ferrous sulphate and potassium iodate premix. The sample containing KR22i
63
60% RH, 40C, FeSuiph [Eiistmo (%] B2nd mo [%] O3rd mo [%]|
100.00
80.00
60.00
40.00 --e
20.00
0.00 ~
4-Palm 2-Palm 3-Palm 4-Paim 5-Palm 6§-Palm 1-Acet 2-Acet 3-Acet 4-Acet
Sample#
In the presence of ferrous sulphate, vitamin A acetate showed considerably higher stability than
with any other iron source. Two premixes retained greater than 40% of vitamin A after three
months of storage at 40°C 60% RH. Under these conditions, the 2-Acet (KR28-2 premix) was
the most stable of the vitamin A acetates (48 + 5% vitamin A retained after three months). Only
BASF Dry VAP 250 Food (3-Palm, premix # KR25) was as stable, with 48 + 6% retention.
Three of the vitamin A palmitate premixes studied gave retentions greater than 40% under the
same conditions.
presence of ferric NaEDTA and potassium iodate premix. The sample containing KR22i
60% RH, 40C, FeNaEDTA Gistmo[%}] 82nd mo [%} 3rd mo [%] |

100.00
80.00
1-Paim 2-Paim 3-Paim 4-Palm 5-Palm 6-Paim 1-Acet 2-Acet 3-Acet 4-Acet
Sample#
64
While five of the six samples containing vitamin A palmitate gave over 50% retention in the
presence of ferric NaEDTA, vitamin A acetate samples were not as stable with ferric NaEDTA
as with the other iron sources. The highest stability (36 + 4%) was demonstrated again by
sample 2-acet. This premix retained as much as 48 + 5% of its vitamin A in TFS blended with
ferrous sulphate and 45 + 4% when blended with elemental iron.
4.3.4 Effect of Iron Source on Vitamin A Stability
The influence of iron source on vitamin A stability was tested using the three most stable
premixes, KR22 (BASF Dry VAP 250 CK CWD), KR25 (BASF Dry VAP 250 Food) and KR27
(BASF Dry VAP 500). Only TFS samples containing potassium iodate and vitamin A stabilized
with TBHQ were studied.
KR 22
This premix, containing BASF Dry VAP 250 CK CWD, gave very good stability in both
conditions (40°C 60% RH and ~100% RH). Figures 4.18 and 4.19 show the retention of this
vitamin A premix with all four sources of iron over a period of three months.
Figure 4.18: Effect of iron source on the stability of vitamin A in premix KR 22 (BASF Dry
VAP 250 CK CWD, granulated with dextrin, coated with 40% SS and stabilized with
TBHQ), in the presence of different iron compounds and potassium iodate premix. The
i :
Effect
of iron source on vitamin
A stability,
| KR 22, 60% RH
|| og
3 80.00
i e
I £
2 60.00
<=
&
| = 40.00
=
| 2 20.00 |&FeNaEDTA
\@@FeSutph
0.00 + =
0 1 2 3
Time (months)
65
Figure 4.18 shows that vitamin A was most stable in the presence of ferrous fumarate and ferric
NaEDTA after three months of storage in 40°C 60% RH. The lowest stability was observed in
the presence of elemental iron. No major difference in the stability of vitamin A with any iron
compound was observed after two months of storage in 40°C 60% RH.
Figure 4.19: Effect of iron source on the stability of vitamin A in premix KR 22 (BASF Dry
VAP 250 CK CWD, granulated with dextrin, coated with 40% SS and stabilized with
TBHQ), in the presence of different iron compounds and potassium iodate premix. The
TFS samples were stored for three months at 40°C ~100% RH.
Effect of iron source on vitamin A stability,

KR 22,100% RH
100.00
is 80.00
1
© 60.00 ie
ft
= 40.00
E ‘ @Fe elem
2 20.00 @iFeFum
. &FeNaEDTA
0.00 : [Se FeSulph
0 1 2 3
Time (months}
In contrast to 40°C 60% RH, at 40°C ~100% RH the highest vitamin A stability was achieved by
fortifying the salt with elemental iron. In addition, after two months of storage, the difference in
stability between TFS with elemental iron and the other iron sources tested was approximately
20%.
KR 25-2
The results of vitamin A degradation in samples containing premix KR 25-2 (BASF Dry VAP
250 Food stabilized with TBHQ) are presented in Figures 4.20 (40°C 60% RH) and 4.21 (40°C
~100% RH).
At 40°C 60% RH (Figure 4.20), this premix showed the best stability in the presence of
elemental iron and ferric NaEDTA with 54 + 2% and 51 + 2% retention after three months,
respectively. The samples containing ferrous fumarate retained only 31% of vitamin A after three
66
months. The ferrous sulphate proved to be the most reactive source of iron in this case, resulting
in the retention of only 28% vitamin A after the same period of time. The 1° order reaction rate
constant for vitamin A degradation in TFS containing ferrous sulphate was found to be
0.42/month (RSQ=0.963).
Figure 4.20: Effect of iron source on stability of premix KR 25-2 (BASF Dry VAP 250
Food, granulated with dextrin, coated with 40% SS and stabilized with TBHQ), in the
presence of different iron compounds and potassium iodate premix. The TFS samples were
stored for three months at 40°C 60% RH.
Effect of iron source on vitamin A stability,

KR 25-2,60% RH
890.00
$
% vitamin A retention
¢&
60.00
aR
40.00 @Fe elem
@BFeFum
20.00 @&FeNaEDTA
SFesSulph
0.00 i + 4
0G 4 2 3
Time (months)
Conversely, samples containing ferrous fumarate were very stable with close to 60% retention
after two months at 40°C ~100% RH (Figure 4.21). Samples with ferric NaEDTA showed
decreased stability compared to samples with other iron compounds during the first two months
of storage. After three months of storage, salt samples containing elemental iron, ferrous
fumarate and ferric NaEDTA demonstrated retention ~ 30%, whereas samples with ferrous
sulphate contained only 16% of vitamin A.
67
Figure 4.21: Effect of iron source on vitamin A stability in premix KR 25-2 (BASF Dry
VAP 250 Food, granulated with dextrin, coated with 40% SS and stabilized with TBHQ),
in the presence of different iron compounds and potassium iodate premix. The TFS
| Effect
of iron source on vitam
A stability,
in
i KR 25-2,100% RH
|
|
'& 80.00
12
, 2 60.00
<q
|g
/E 40.00
ae [@Fe elem
2 20.00 SBF eFum
| 0.00
@&FeNaEDTA
. , |e Fesuiph
| 0 1 2 3
i Time (months)
I<R 27-2
KR 27-2 contains BASF Dry VAP 500 coated with 40% soy stearine and stabilized with T BHQ.
Figures 4,22 and 4.23 show the degradation of vitamin A in this premix with time.
Figure 4.22: Effect of iron source on vitamin A stability in premix KR 27-2 (BASF Dry
VAP 500, granulated with dextrin, coated with 40% SS and stabilized with TBHQ), in the
presence of different iron compounds and potassium iodate premix. The TFS samples were
stored for three months at 40°C 60% RH.
_
Effect ofiron source on vitamin A stability,
KR 27-2,60% RH
100,00
|
& 80.00
i| 3&
!
g@ 60.00
(o: < 40.00
ot el
@Fe elem
£ oe) GRE
e F um
: a 20.00 [|d@eFeNaEDTA
(ME eSulph
0.00 : :
0 1 2 3
| Time (months)
68
The TFS samples containing the ferric NAEDTA premix were again the most stable. It is worth
noting that all three vitamin A premixes (KR22, 25 and 27) stored in the 40°C 60% RH showed
highest stability with this type of iron.
Figure 4.23: Effect of iron source on stability of premix KR 27-2 (BASF Dry VAP 500,
granulated with dextrin, coated with 40% SS and stabilized with TBHQ), in the presence of
different iron compounds and potassium iodate premix. The TFS samples were stored for
Effect of iron source on vitamin A Stability,

KR 27-2,100% RH
5 80.00
=
2
o 60.00
t
c
€ 40.00
s @Fe elem
S 20.00 G}FeFum
i &FeNaEDTA
0.00 ~ ; SFeSulph
0 1 2 3
Time (months)
All TFS samples, except after two months of storage, showed similar stability of vitamin A
during the three month study in 40°C ~100% RH (Figure 4.23). After two months of storage the
sample containing ferrous sulphate showed significantly higher retention of vitamin A (67 + 2%)
than any other TFS sample.
Overall, vitamin A stability in salt fortified with ferric NaEDTA was high. The highest retention
was 61 + 5% after three months at 40°C 60% RH with premix KR27-2 (Figure 4.22). In
addition, stabilities higher than 50% after three months at 40°C 60% RH were observed for six
premixes out of the 36 studied. Vitamin A was less stable with all the other iron sources studied.
The increased stability observed with ferric NaEDTA is probably due to the fact that the EDTA
complex binds iron and makes it less reactive. In addition, iron is in the ferric form which is not
susceptible to oxidation.
In general, these three vitamin A premixes described above demonstrated high stability with
ferric NaEDTA premix. Five out of six samples retained more than 50% of the initial
69
concentration after storage at 40°C 60% RH for three months. Therefore, from stability point of
view it seems that the ferric NaEDTA is the best choice for triple salt fortification. In addition,
ferric NaEDTA is highly bioavailable (with relative bioavailability factor around 2.50) and its
bioabsorption is believed to be unaffected by some inhibiting substances found in food [3, 30].
However, the iron content in this compound is only 13% and the relative cost factor is very high,
2.57 (Table 2.3).
Three out of six TFS samples containing elemental iron retained more than 50% of the vitamin A
under the same conditions as ferric NaEDTA. Elemental iron contains approximately 98% of
iron. However, the elemental iron is almost insoluble in water and its relative bioavailability
factor is believed to be between 0.13 and 0.90 (Table 2.3).
Only one TFS sample blended with ferrous fumarate retained more than 50% of its vitamin A
after three months of storage at 40°C. The advantages of ferrous fumarate over the previous two
compounds is that it has a higher iron content (33%), it is less expensive (relative cost factor is
1.00) than ferric NaEDTA, it is more bioavailable than elemental iron (~1.00), and it is tasteless,
odourless, and slightly soluble in water (Table 2.3).
None of the salt samples mixed with ferrous sulphate retained more than 50% of their vitamin A
after three months of storage. The iron content of this compound is less than ferrous fumarate
(20%), its relative bioavailability and solubility are similar to fumarate and the relative cost
factor is 1.67 (Table 2.3).
It is difficult to conclude whether or not the obvious advantages of using ferric NaEDTA in
terms of vitamin A stability would prevail over its high cost. In general, vitamin A is more
stable in TFS containing this iron compound than with any other source of iron.
4.3.5 Effect of Iodine Source on Vitamin A Stability
The influence of iodine source on vitamin A stability was also studied by testing the following
sources of iodine:
© KIO; premix (TFS samples containing vitamin A premixes KR22 and KR31-2)
e KI premix (TFS samples containing vitamin A premix KR17)
70
e Vitamin A with KIO; in one particle (KR18-1 premix) )
© Vitamin A with KI in one particle (KR18-2 premix)
e lodized salt (TFS samples containing vitamin A premix KR22)
Two commercial vitamin A sources were used in this study:

® BASF Dry VAP 250 CK CWD (KR22 premix)
e Watson VAP 250,000 IU/g Powder (premixes KR31-2, 17, 18-1 and 18-2)
The vitamin A premixes were granulated with dextrin, coated with soy stearine (40%) and
stabilized with TBHQ. In the case of the vitamin A premixes which contain iodine (either as
potassium iodate or as potassium iodide) in the same particle, the iodine compound was first
dissolved in distilled water. This solution was then used to granulate the vitamin A.
Figure 4.24 shows the stability of vitamin A (BASF Dry VAP 250 CK CWD, premix KR22) in
the presence of potassium iodate and iodized salt (containing potassium iodide) after three
months of storage at 40°C and at both RH conditions (60% RH and ~100% RH).
Figure 4.24: Effect of KIO; premix and iodized salt (KI) on vitamin A stability (KR22
premix) in the presence of various iron compounds. The BASF Dry VAP 250 CK CWD
was granulated with dextrin, coated with 40% SS and stabilized with TBHQ. The TFS
samples were stored for three months at 40°C 60% RH and ~100% RH.
| @&K1IO03 60% RH
. &KIO3 100% RH
KIO3 vs. lodized Salt, @lodized Salt60% RH
3rd month Bilodized Sait 100% RH
60.00
40.00
20.00
7 t 7
Fe elem FeFum FeNaEDTA FeSuitph
Source of tron
'
The TFS samples containing iodized salt showed stability comparable to the KIO; premix,
especially in the presence of ferric NaEDTA. Samples containing KIO; premix and elemental
71
iron showed higher retention of vitamin A in 40°C ~100% RH than in 40°C 60% RH after three
months of storage. Similarly, the TFS samples blended with iodized salt and ferrous fumarate
premix gave higher retention at 40°C ~100% RH. The TFS with ferrous fumarate showed higher
stability in 40°C 60% RH than in 40°C ~100% RH during the first two months of storage.
The effect of iodine source on vitamin A stability (Watson VAP 250,000 IU/ g Powder) after
three months of storage is shown in Figure 4.25 and 4.26. The complete stability results for all
three months can be found in Appendix 9.2. The premixes KR18-1 (containing vitamin A with
KIO3 in one particle) and KR18-2 (containing vitamin A with KI in one particle) were studied
only in the presence of ferrous fumarate and ferric NaEDTA.
Figure 4.25: Effect of iodine source on vitamin A stability (Watson VAP 250,000 TU/g
Powder) in the presence of various iron compounds. The vitamin A was granulated with
dextrin, coated with 40% SS and stabilized with TBHQ. The TFS samples were stored for
| @Kipremix |
Effect
of lodine Source KIO3 premix |
60% RH @vitA+ KIO3 |
mivitA
+ Ki |
| Fe elem FeFum FeNaEDTA FeSutph

lron Source
The results show no significant difference in vitamin A stability when these different iodine and
iron compounds are present in the TFS samples stored in 40°C 60% RH for three months. The
retention of vitamin A in all TES samples was approximately 35%.
Figure 4.26: Effect of iodine source on vitamin A stability (Watson VAP 250,000 IU/g
Powder) in the presence of various iron compounds. The vitamin A was granulated with
72
dextrin, coated with 40% SS and stabilized with TBHQ. The TFS samples were stored for
1
{ K] premix
Effe
of ct
lodine Source ; KiO3 premix
i 100% RH i @vitA
+ KIO3
i,Mvit
Ae KE
le 40.00
Sss
S
S 30.00
ja
aD
< 20.00
ls
is
'§ 10.00
i>
'S 9.00
Fe elem FeFum FeNaEDTA FeSulph
fron Source
At 40°C ~100% RH all iodine sources demonstrated similar vitamin A stability. The highest
retention (35%) was achieved in TFS with the ferrous fumarate premix and with the premix
containing vitamin A and KI in one particle (KR18-2 premix),
The finding that iodine source had no significant effect on vitamin A stability is very important.
It means that it is possible to combine vitamin A and iodine in one premix, instead of using two
separate premixes. Production of triple fortified salt containing vitamin A and iron premixes and
iodized salt seems to be affordable as well. The salt iodization program is well established in
most targeted countries. Production of only two premixes, instead of three would be less
expensive and reduce processing significantly.
Based on previous studies conducted by our group, potassium iodide is more stable in the
presence of added ferrous iron than potassium iodate. Therefore potassium iodide in any form
(iodized salt, KI premix or KI in one particle with vitamin A) can be used for the production of
triple fortified salt. The production of a particle containing all three micronutrients will be
explored in Section 4.3.13.
73
4.3.6 Effect of the Type of the Binding Agent
Vitamin A (BASF Dry VAP 250 CK CWD) was granulated with two types of binder in this
study:
@ KR22 - Casco Dextrin CAS: 9004-53-9 (yellow)

e KR23-2 - Casco Corn Starch CAS: 9005-25-8 (white)
The difference between these two binders is that dextrins are starch hydrolysis products obtained
in a dry roasting process either using starch alone or with trace levels of acid catalyst. The
yellow dextrin is cold water soluble, stickier than the white starch and only about 1/3 the amount
of water is required to granulate it compared to the white starch.
The premixes were coated with 40% SS, stabilized with TBHQ and stored at 40°C and both RH
conditions (60% RH and ~100% RH) for a period of three months. Figures 4.27 and 4.28
demonstrate the differences in vitamin A stability using the two binding agents.
Figure 4.27: Stability of vitamin A premix (BASF Dry VAP 250 CK CWD) granulated with
two types of binder, coated with 40% SS, stabilized with TBHQ and stored at 40°C 60%
RH for three months. 22: Casco Dextrin CAS: 9004-53-9 (yellow); 23-2: Casco Starch CAS:
9005-~-25-8 (white)
60% RH, Type of Binder Used = [aist mo [%] 2nd mo [%] G3rd mo [%] |
100.00
80.00
60.00
40.00 -
20.00
0.00
Vv :
tron Source -~
Figure 4.28: Stability of vitamin A premix (BASF Dry VAP 250 CK CWD) granulated with
two types of binder, coated with 40% SS, stabilized with TBHQ and stored at 40°C ~100%
74
RH for three months. 22: Casco Dextrin CAS: 9004-53-9 (yellow); 23-2: Casco Starch CAS:
900S-25-8 (white)
|
100% RH, Type of Binder Used (“tstmo [%] @2nd mo [%] G3rd mo [%]
400.00
80.00
60.00 +
40,00 +
20.00 +
0.00 -
Iron Source ed
The results show that vitamin A granulated with the dextrin was considerably more stable than
the same vitamin A granulated with the white starch. Premixes KR21, KR23-1 and KR23-2 were
granulated with the white starch and showed the lowest vitamin A stability (~7%) after two
months of storage under 40°C and both 60% and ~100% RH.
Therefore, dextrin is the preferred binding agent for production of vitamin A premix used in
TES.
4.3.7 Hydrophilic vs. Hydrophobic Coating Agents
Two commercial vitamin A forms (Watson VAP 250,000 IU/g and BASF Dry VAP 250 Food)
were granulated with dextrin, coated with various coating agents and stabilized with TBHQ.
Three hydrophilic coating agents (Shellac, Methylcellulose (Methocel, MC) and Hexaethyl
Cellulose (Ethocel, HEC)) and one hydrophobic agent (soy stearine (SS)) were used to coat these
vitamins.
Two commercial vitamin A forms were coated with shellac (level of coating was 40%):
e KR11- Watson VAP 250,000 TU/g
e KRI12- BASF Dry VAP 250 Food
75
Methocel (MC) and Ethocel (HEC), hydrophilic coating agents, were used in two different
proportions to coat vitamin A (Watson VAP 250,000 IU/g Powder):
® KRI15 - 30% MC/ 10% HEC
e KRI16 - 20% MC/ 20% HEC
All of the coating agents are commonly used as former of aqueous films in the pharmaceutical
and food industries. The vitamin premixes were granulated with dextrin and stabilized with
TBHQ. The TBHQ was first dissolved in dichloromethane, and then sprayed on the granulated,
dried and sieved particles. The coating was subsequently applied.
Figures 4.29 and 4.30 show the stability of vitamin A observed when the above coating agents
were used. The TFS samples were stored for three months at 40°C and both RH conditions
(60% RH and ~100% RH).
Figure 4.29: Hydrophilic coating agents. The stability of two vitamin A forms, Watson
VAP 250,000 TU/g (KR 11 premix) and BASF Dry VAP 250 Food (KR 12 premix) coated
with 40% Shellac. The stability of Watson VAP 250,000 [U/g coated with 30% MC/10%
HEC (KR 15 premix) and 20% MC/20% HEC (KRI16 premix). These vitamin A premixes
were granulated with dextrin and stabilized with TBHQ. The TFS samples (containing
iron and potassium iodate premixes) were stored for three months at 40°C 60% RH.
60% RH |E1st
mo [%] MA2nd mo [%]} El3rd mo [%]|
80.00
60.00 +—
40.00 LE
20.00 -
0.00 4
760
Figure 4.30: Hydrophilic coating agents. The stability of two vitamin A forms, Watson
VAP 250,000 TU/g (KR 11 premix) and BASF Dry VAP 250 Food (KR 12 premix) coated
with 40% Shellac. The stability of Watson VAP 250,000 ITU/g coated with 30% MC/10%
HEC (KR 15 premix) and 20% MC/20% HEC (KRI16 premix). These vitamin A premixes
were granulated with dextrin and stabilized with TBHQ. The TFS samples (containing
iron and potassium iodate premixes) were stored for three months at 40°C ~100% RH.
_
|
100% RH [Elist
mo (%] M2nd mo [%] EA3rd mo [%]
The stabilities of premixes KR11 (Watson VAP 250,000 IU/g coated with 40% Shellac) and
KR12 (BASF Dry VAP 250 Food, also coated with 40% Shellac) were very similar and varied
between 30% and 42% after 3 months of storage at 40°C 60% RH. This results are comparable
to several vitamin A premixes coated with 40% SS.
In the case of MC/HEC the vitamin A stability was a maximum of 26% (KR15 with FeFum)
after 2 months. Both coating agents showed little ability to protect vitamin A from the high
humidity and temperature environment. Therefore, they are not suitable for use as coating agents
alone.
For the sake of comparison, vitamin A (Watson VAP 250,000 IU/g) was granulated with dextrin
and coated with a fat-soluble coating agent, soy stearine (SS). Two different levels of coating
were studied:
@ 40% soy stearine (KR 31-2)
© 30% soy stearine (KR 19-1)
77
Figures 4.31 (40°C 60% RH) and 4.32 (40°C ~100% RH) show the stability of vitamin A stored
for three months at 40°C and at both RH using two different levels of coating with SS. Both
premixes were stabilized with TBHQ (0.02% per fat weight).
Figure 4.31: The stability of Watson VAP 250,000 IU/g coated with 40% SS (KR31-2
premix) and 30% SS (KR19-1 premix). The TFS samples (containing all four iron premixes
and potassium iodate premix) were stored for three months at 40°C 60% RH. These
vitamin A premixes were granulated with dextrin and stabilized with TBHQ.
60% RH, Coated with SS | 11st mo [%] 2nd mo [%] 3rd ro (%] |
=
23
e
2
oe
a
9
&
—E
&
>
P4 T * 7 - T T T
31-2 Feelem 31-2 FeFum 31-2 31-2 FeSulph 19-1 FeFum 19-1
FeNaEDTA FeNaEDTA
Figure 4.32: The stability of Watson VAP 250,000 IU/g coated with 40% SS (KR31-2
premix) and 30% SS (IKR19-1 premix). The TFS samples (containing all four iron premixes
and potassium iodate premix) were stored for three months at 40°C ~100% RH. These
vitamin A premixes were granulated with dextrin and stabilized with TBHQ.
| 100% RH, Coated with SS Bist mo [%] 2nd mo [%] 13rd mo [%]
= 80.00 :
s
| &o 60.00
1, &
| <f 40.00 4
| 2 a
| $ 20.00 |
>
| 8 0.00 ti : = he :
| 31-2Feelem 31-2 FeFum 34-2 31-2FeSuiph 19-1 FeFum 19-1
FeNaEDTA FeNaEDTA
78
The stability of vitamin A coated with 40% SS was considerably higher than in the case of 30%
SS coating after three months, under both RH conditions. The retention of KR31-2 premix
(coated with 40% SS) with ferrous fumarate was 31 + 2% and 35 + 8% with ferric NaEDTA in
40°C 60% RH after three months of storage. Premix KR19-1 retained only 25 + 4% in 40°C
60% RH after the same duration of study. Similar trends were observed in 40°C ~100% RH.
Therefore, the higher level of SS coating is desirable as the premixes coated with 40% SS
showed higher stability than the ones coated with 30% SS
The results obtained using Shellac (maximum 42% after three months in 40°C 60% RH, Figure
4.29) are very similar to the ones obtained with 40% SS in Figures 4.31 and 4.32. The stability
of shellac coated vitamin A premix in the presence of ferrous fumarate was 30 + 3% and with
ferric NaEDTA was 35 + 2% at 40°C 60% RH after three months. Based on these results, the
utilization of Shellac as a coating agent for vitamin A should be further investigated.
4.3.8 Effect of the Type of Antioxidant
Three fat-soluble artificial antioxidants were investigated for their ability to enhance vitamin A
stability in the premixes. These included BHA, BHT and TBHQ. The maximum concentration
of these antioxidants allowed in foods is 0.02% on fat weight. Because BHA and BHT are
believed to have synergistic interactions [29], they were used together in a 1:1 ratio.
The following Figures (4.33 and 4.34) show the retention of vitamin A (BASF Dry VAP 250
Food) stabilized with these antioxidants after three months of storage at 40°C 60% RH and 40°C
~100% RH, respectively. The samples marked with number 1 contained BHA/BHT antioxidants
(premix KR20-1). The samples marked under 2 were stabilized with TBHQ (premix KR20-2).
Figure 4.33: The effect of different antioxidants on the stability of vitamin A (BASF Dry
VAP 250 Food, KR 20 premix). The TFS samples (containing all four iron premixes and
potassium iodate premix) were stored for three months at 40°C 60% RH. This premix was
79
granulated with dextrin, coated with 40% SS and stabilized with either BHA/BHT (KR 20-
1) or TBHQ (KR20-2).
60% RH, Antioxidant [Eitstmo [%] Bi2nd mo [%] E¥3rd mo [%] |
100.00
80.00
60.00
40.00
20.00
x tron Source v
Figure 4.34: The effect of different antioxidants on the stability of vitamin A (BASF Dry
VAP 250 Food, KR 20 premix). The TFS samples (containing all four iron premixes and
potassium iodate premix) were stored for three months at 40°C ~100% RH. This premix
was granulated with dextrin, coated with 40% SS and stabilized with either BH.A/BHT
(KR 20-1) or TBHQ (KR20-2).
100% RH, Antioxidant [fist

mo [%] BB2nd mo [%] El3rd
mo [%] |
100.00
80.00
60.00 -
40.00
20.00
0,00 4
Several samples stored at 40°C ~100% RH retained more vitamin A than the same samples
stored in 40°C 60% RH. This can be explained by the fact that the humidity in the
environmental chamber temporarily (for three weeks) decreased under 60% RH due to a
malfunction.
80
The samples stored at 40°C ~100% RH followed a logarithmic degradation dependence (RSQ >
0.9) so the k values were calculated to compare the effects of the antioxidants on the stability of
vitamin A. The results are presented in Table 4.8.
Table 4.8: The retention of vitamin A (BASF Dry VAP 250 Food, KR 20 premix) with the
rate constants (k) and RSQ values. The TFS samples were stored for three months at 40°C
~100% RH. This premix was granulated with dextrin, coated with 40% SS and stabilized
with either BHA/BHT (KR 20-1) or TBHQ (KR20-2).
Sample # Initial 1 mol2™ mof3™ molk RSQ

cone. [%] | [%] [%] [%] {mo}
Fe elem -1 100 7044 5342 44+4 0.27 0.981
Fe Fum -1 100 63+1 42+] 32+8 0.38 0.987
Fe NaEDTA -1_ || 100 68 +3 55+0 3145 0.38 0.964
FeSO,x7H.0 -1 || 100 7O+11 5241 48 +3 0.25 0.942
Fe elem -2 100 59 +3 39 +4 31+6 0.40 0.974
Fe Fum -2 100 78 +5 55+2 46+4 0.26 0.988
Fe NaEDTA -2_ || 100 61+1 42 +8 30+7 0.40 0.992
FeSO4x7H;0 -2 || 100 66 +2 44+] 3141 0.39 0.997
Based on the k values, there was no significant difference observed between the rate of
degradation of the samples stabilized with BHA/BHT and TBHQ. The k value deviates between
~0.25/ month and ~0.4/ month in both cases. The stability of vitamin A is more influenced by
the type of the iron compound than by the type of the antioxidant used.
81
4.3.9 Effect of Humidity
The rate of vitamin A degradation in premix KR27 (BASF Dry VAP 500) at 40°C and both 60%
RH and ~100% RH was compared by fitting the data to a first order reaction rate model. In
sample KR27-1, the BHA/BHT antioxidants were used to stabilize the vitamin A while in KR27-
2 the TBHQ was used. These reaction rates and RSQ values for both 40°C 60% RH and 40°C
~100% RH are presented in Tables 4.9 and 4.10.
All samples except Fe NaEDTA-2 (40°C 60% RH) showed a logarithmic relationship of vitamin
A degradation with time. The ferrous sulphate sample (40°C ~100% RH) could not be analyzed
as only the initial concentration and the third month stability is known.
Table 4.9: The retentions of vitamin A (BASF Dry VAP 500, KR 27 premix) with the
60% RH. This premix was granulated with dextrin, coated with 40% SS and stabilized
Sample Initial 1 = mo}2™ ~— mo | 3™ mo Tk RSQ

constituents cone. [(%] 1 [%] [%] [%] [mo™
Fe elem -1 100 89 +8 8047 6542 0.14 0.969
FeFum -1 100 7643 58 +1 4A9x8 0.24 0.989
FeNaEDTA -1 100 82+17 78 +2 61+3 0.15 0.948
Fe SQ4x7H,0 -1 100 3345 44+2 36+1 0.32 0.900
Fe elem -2 100 60 +6 AT+7 38 +5 0.32 0.952
FeFum -2 100 61412 4124 3444 0.36 0.969
FeNaEDTA -2 100 84 + 20 71 +13 3547 0.33 0.862

Fe 804x7H,20 -2 100 77 +12 58+4 46+ 11 0.26 0.999
Table 4.10: The retentions of vitamin A (BASF Dry VAP 500, KR 27 premix) with the
82
~100% RH. This premix was granulated with dextrin, coated with 40% SS and stabilized
Sample Initial 1“ mof2™ mol3™ mol k RSQ

constituents conc. [Yo] | [%] [%] [%] [mo"]
Fe elem -1 ~ 1100 55+2 [3642 |28+3 042 | 0.963
FeFum -1 100 79+7 61+1 47+5 0.25 0.999
FeNaEDTA -1 100 7849 6243 3942 0.30 0.974
Fe elem -2 100 72 +10 5248 44+6 0.28 0.980
FeFum -2 100 6248 4242 37 £8 0.34 0.951
FeNaEDTA -2 100 69 +2 45+2 30 +3 0.40 0.999
Fe SO4x7H20 -2 100 78£0 6742 42+14 0.28 0.949
The highest vitamin A losses were observed at 40°C ~100% RH. This is expected in systems
where mass transfer is not highly restricted. The rate of degradation is much faster in. 40°C
~100% RH with reaction rate constants varying from 0.25/ month to 0.42/ month. The reaction
constants in 40°C 60% RH fluctuated between 0.14/ month and 0.36/ month.
The results confirm that the high humidity environment had a negative impact on vitamin A
stability. The best retention at 40°C ~100% RH was 47 + 5%, obtained by two premixes: KR20-
1 (BASF Dry VAP 250 Food coated with 40% SS and stabilized with BHA/BHT) and KR29-2
(Roche Dry VAP 500 coated with 40% SS and stabilized with TBHQ). The average retention
was only 31%. However, the studies done by our group under field conditions in Kenya and
Nigeria showed that these laboratory conditions were harsher compared to those experienced by
samples in the field. The stability of vitamin A in TFS was at least 20% higher in the local
environment than in our 40°C ~100% RH laboratory environmental chambers (some of the
results are shown in Table 4.11).
83
Table 4.11: Comparison of vitamin A stability in TFS stored at ambient conditions in
Mombassa and Nairobi (Kenya) and in U of T laboratory conditions (~25°C, 60% RH) and
environmental chamber (40°C ~100% RH) for three months.
Retention of vitamin A after three months [%]

Components of the TSF
Mombassa | Nairobi | U of T Lab | 40°C ~100% RH
Watson VAP (granulated with HPMC

and coated with 40% SS) + KIO; + | 27 37 42 10
ferrous fumarate
Watson VAP (granulated with HPMC

and coated with 40% SS) + KIO; + |} 33 43 51 2
reduced iron
4.3.10 Introducing All Three Micronutrients into One Particle
The best option for TFS production would be to design a stable premix containing all three
micronutrients. Thus a premix containing Watson VAP 250,000 IU/g Powder, potassium iodate
and ferrous fumarate or ferric NaEDTA was prepared, mixed with non-iodized salt and stored for
three months at 40°C and both RH conditions (60% RH and ~100% RH). Each premix was
granulated with dextrin, coated with 40% (KR13) or 30% (KR14) soy stearine and stabilized
with TBHQ (0.02% per fat weight). Table 4.12 shows the composition of the premixes.
The premixes were added to non-iodized salt in a ratio of 1:100. The stability of vitamin A in
these four TFS samples is shown in Figures 4.35 and 4.36.
Table 4.12: Composition of the premixes containing all three micronutrients (vitamin A,
iron and iodine) in one particle. The premix was designed so that it contained 25 000 IU/g
of vitamin A, 100 mg/g of iron and 5 mg/g of iodine.
Number | Composition Antioxidant Coating
KR13-1 | Watson VAP 250,000 IU/g + Fe Fum + KIO; + Dextrin 0.02% TBHQ | 40% SS
KKR13-2 | Watson VAP 250,000 IU/g + Fe NaEDTA + KIO; + Dextrin | 0.02% TBHQ | 40% SS
84
Number | Composition Antioxidant | Coating
KR14-1 |) Watson VAP 250,000 IU/g + Fe Fum + KIO; + Dextrin 0.02% TBHQ | 30% SS
KR14-2 | Watson VAP 250,000 IU/g + Fe NaEDTA + KIO; + Dextrin | 0.02% TBHQ [30% SS
Figure 4.35: Stability of premix containing all three micronutrients (Watson VAP 250,000
[U/g, potassium iodate and either ferrous fumarate or ferric NaEDTA) in one particle,
stored for three months at 40°C 60% RH. The premixes were granulated with dextrin,
coated with 40% SS and stabilized with TBHQ.
60% RH, AI! 3 Micronutrients in One

13 FeFum 13 FeNaEDTA
[ENist mo [%]} B§2nd mo [%] Ed3rd mo [% ] |
Figure 4.36: Stability of premix containing all three micronutrients (Watson VAP 250,000
TU/g, potassium iodate and either ferrous fumarate or ferric NaEDTA) in one particle,
stored for three months at 40°C ~100% RH. The premixes were granulated with dextrin,
coated with 40% SS and stabilized with TBHQ.
100% RH, AIL 3 Micronutrients in One

= 40.00
8
& 30.00
@
a
< 20.00 -
E
E
# 10.00
>
3e
0.00 4 :
13 FeFum 13 FeNaEDTA 14 FeFum 14 FeNaEDTA
[E¥1stmo [%] nd mo (%] Ed3rd mo [%]|
85
Both graphs show that the vitamin A retention after three months was less than 10% in both
environments. The samples containing ferric NaEDTA did not change color; however the
samples containing ferrous fumarate turned sandy.
Based on our results it can be concluded that the interaction between the three micronutrients
decreased vitamin A retention significantly.
4.4 Reproducibility Test and Defects in Coating
Two premixes, KR20 and KR25 were prepared using the same vitamin A, BASF Dry VAP 250
Food, had the same composition (both contained dextrin as a binding agent, were coated with
40% SS and stabilized with BHA/BHT (KR20-1 and KR25-1) or TBHQ (KR20-2 and KR25-2))
and were produced in the same way (pan granulation and pan encapsulation). All samples were
stored at 40°C 60% RH and 100% RH during the same period of time. This was done to test the
reproducibility of the premix preparation, especially of the coating operation. Figure 4.37
compares the stability of vitamin A in these two premixes stored at 40°C 60% RH.
Figure 4.37: Comparison of stability of KR20-1 and KR25-1 premixes (BASF Dry VAP 250
Food), stored for three months at 40°C 60% RH. This vitamin A was granulated with
dextrin, coated with 40% SS and stabilized with BHA/BHT.
Reproducibility Test 11st mo [%] Gnd mo [%] A3rd

mo [%]
100.00
80.00
60.00
40.00 -
20.00 -
9.00 4
As can be seen from the graph, there is significant variation in stability between the two
premixes. The salt samples with the same iron compound are identical and should show similar
86
stability. However, KR25-1 with ferric NaEDTA gave 53 + 6% retention after three months, but
KR20-1 with the same iron source showed only 32 + 4% retention after the same period of time.
On the other hand, vitamin A retention in both premixes using ferrous fumarate was 41 + 3% and
43 + 7%. Similar results were observed for the premixes KR 20-2 and KR25 -2, both stabilized
with TBHQ.
Both premixes were sieved after granulation to obtain particles with size between 300 jum and
710 pm. After this step the granules were coated with 40%soy stearine and an antioxidant. The
size distributions of these two premixes after microencapsulation are shown in Table 4.13.
Figure 4.38 shows microscopic images (60x magnification) of both vitamin A premixes (KR20-1
and KR25-1).
Table 4.13: The size distribution of microencapsulated premixes KR 20-1 and KR 25-1.
Particle Size [um] | KR 20-1 [%] ] KR 25-1 [%|

>710 14 13
>500 & <710 40. 68

>300 & <500 43 17
<300 3 2
Figure 4.38: Microscope pictures of premixes KR20-1 and KR25-1 (BASF Dry VAP 250
Food). The vitamin A in both premixes was granulated with dextrin, coated with 40% SS
and stabilized with TBHQ.
KR25-1 KR20-1
87
The size distribution and the pictures confirm that in general the KR25-1 premix had bigger,
more uniform granules than the premix KR20-1.
Defects in coating can be caused by:

e Process conditions,
e Both process conditions and coat formulation,

e All previous reasons plus the premix matrix.
Figure (4.39) shows microscopic images of ferrous fumarate and ferric NaEDTA encapsulated
with 40% soy stearine. These pictures were taken with 60x magnification and the defects in
coating are easily visible. The defects are more visible in case of ferrous fumarate due to its dark
colour.
Figure 4.39: Defects in coating of iron premixes, ferrous fumarate and ferric NaEDTA.
Defects in Defects in
coating, Ferrous coating, Ferric
Fumarate NaEDTA
Scanning Electron Microscope (SEM) images of vitamin A premixes KR20-1 and KR25-1
(Figure 4.40) were obtained using Hitachi SEM S-2500 in order to see the defects in the premix
coating. The images show that the film around the premix particles was not entirely impervious
to water.
88
Figure 4.40: SEM images of vitamin A premixes KR20-1 and KR25-1 (BASF Dry VAP 250
Food). The vitamin A in both premixes was granulated with dextrin, coated with 40% SS
and stabilized with TBHQ. Various magnifications.
KR20-1
aK
89
The coatings are fairly homogeneous, however a closer look at the images shows some voids
and, surprisingly, some crystalline material. This material could be possibly crystallized vitamin
A or an antioxidant (BHA, BHT, TBHQ).
The results prove that this method of premix preparation has many variables (speed of spraying,
speed of rotating pan, amount of solids to be coated, particle size distribution etc.) and it requires
a lot of practice from the person preparing the premix. In addition, the pictures and the stability
results indicate that our problems with coating are caused by both the process conditions and the
coat formulations. The film characteristics may improve at the production level since better
equipment and more control (less person to person variability) would be available.
4.5 Stability of Iron
The reactive nature of iron makes it difficult to use in food fortification. According to the
literature, among the 4 iron compounds studied, the Ferric Na EDTA should be the most stable
since it doesn’t undergo oxidation. The chelating EDTA ties up iron thus greatly reduces its
reactivity. Interestingly, the bioavailability is believed to be elevated and not reduced in the
chelated form [3, 30]. The other three iron compounds can be easily oxidized by potassium
iodate.
Images of all four iron premixes (elemental iron, ferrous fumarate, ferrous sulphate, ferric
NaEDTA) are shown in Figure 4.41. Ferrous sulphate, ferrous fumarate and ferric NaEDTA
form granules easily. It is very difficult to granulate elemental iron to the desired size (300 um
to 710 um). When wetted, it forms nice granules with both binding agents, dextrin and starch.
However, the particles easily crumble after drying.
90
Figure 4.41: Microscope pictures (60x magnification) of elemental iron, ferrous fumarate,
ferrous sulphate and ferric NaEDTA premixes. The iron compounds were granulated with
dextrin and coated with 40% SS.
Ferrous Fumarate premix
Some samples fortified with ferrous fumarate turned brown after three months of storing.
Several ferrous sulphate samples turned yellow and green after prolonged storing. Although the
color was very nice and could perhaps be used by salt producers to attract new clients, the taste
was an awful metallic taste. Fortification with elemental iron resulted in slightly grey coloration
of the samples. However, the samples that retained vitamin A reasonably well (on average more
than 30%) did not show significant discoloration.
91
The iron content in TFS samples was analyzed as the 1,10 — Phenanthroline complex using the
UV/Vis Spectrophotometry, as discussed in section 3.4.3. The standard deviation of this method
was found to be + 2%, based on the minimum of four measurements.
Since ferric iron has low bioavailability (with exception of ferric NaEDTA), the conversion rate
of ferrous to ferric iron was studied. Stability results for iron in the TFS samples containing the
three most stable vitamin A premixes (KR22, 25 and 27) are shown in Tables 4.14 to 4.16. The
salt samples were stored at 40°C 60% RH or 100% RH for three months and analyzed after the
second and third months of storage.
Table 4.14 shows the iron retention in TFS samples containing KR22 premix (BASF Dry VAP
250 CK CWD) as a source of vitamin A. The samples KA-17, 19, 21 and 23 were mixed with
potassium iodate premix and non-iodized salt. Samples KA-18, 20, 22 and 24 contained iodized
salt. The TFS samples (Table 4.15) form KA-41 to KA-44 contained KR25-1 premix (BASF
Dry VAP 250 Food stabilized with BHA/BHT). The other four salt samples contained KR25-2
premix (BASF Dry VAP 250 Food stabilized with TBHQ). The first four TFS samples (KA-57
to KA-60) listed in Table 4.16 contained KR27-1 premix (BASF Dry VAP 500 stabilized with
BHA/BHT) while the rest (Ka-61 to KA-64) contained KR27-2 (BASF Dry VAP 500 stabilized
with TBHQ) premix. |
Table 4.14: Conversion of ferrous to ferric iron in the TFS samples containing BASF Dry
VAP 250 CK CWD (KR22) premix stabilized with TBHQ and either iodized salt (samples
KA-18, 20, 22 and 24) or potassium iodate (samples KA-17, 19, 21 and 23). The samples
Sample |Source of | Initial % | 2"' month % Fe" 3" month % Fe**

i iron Fe" 60% RH |~100% RH | 60% RH | ~100% RH
KA-17 Fe elem 95 93 93 86 82
KA-18 Fe elem 95 94 92 91 92
KA-19 Fe Fum 93 91 90 88 92
KA-20 Fe Fum 93 94 92 90 90
KA-21 Fe NaEDTA [5 10 9 5 10
KA-22 Fe NaEDTA {5 7 10 9 10
92
Sample | Source of | Initial % | 2" month % Fe’* 3" month % Fe?*
# iron Fe” 60% RH | ~100% RH | 60% RA | ~100% RH
KA-23 Fe Sulph 97 96 97 96 97
KA-24 | Fe Sulph 97 97 95 93 93
Table 4.15: Conversion of ferrous to ferric iron in the TFS samples containing potassium
iodate premix and BASF Dry VAP 250 Food (KR25) premix stabilized with either
BHA/BHT (samples KA-41 to 44) or TBHQ (samples KA-45 to 48). The samples were
Sample | Source of | Initial = % | 2"¢ month % Fe”* 3" month % Fe"

# iron Fe™ 60% RH | ~100% RH | 60% RH | ~100% RH
KA-41 Fe elem 95 90 88 89 90
KA-42 Fe Fum 93 91 90 88 89
KA-43 Fe NaEDTA | 5 7 11 13 9
KA-44 | FeSulph | 97 86 95 94 94
KA-45 Fe elem 95 94 91 89 92
KA-46 Fe Fum 93 91 89 87 90
KA-47 Fe NaEDTA [5 5 5 8 9
KA-48 Fe Sulph 97 93 94 94 95
Table 4.16: Conversion of ferrous to ferric iron in the TFS samples containing potassium
iodate premix, and BASF Dry VAP 500 (KR27) premix stabilized with either BHA/BHT
(samples KA-57, 58, 59 and 60) or TBHQ (samples KA-61, 62, 63 and 64). The samples
Sample |Source of | Initial % | 2"! month % Fe” 3™ month % Fe**

# iron Fe” 60% RH | ~100% RH | 60% RH | ~100% RH
KA-57 Fe elem 95 93 94 92 92
KA-58 || Fe Fum 93 92 92 92 91
93
Sample | Source of | Initial % | 2™ month % Fe"* 3" month % Fe”
# iron Fe” 60% RH | ~100% RH | 60% RH | ~100% RH
KA-59 |FeNaEDTA [5 5 5 10 9
KA-60 | Fe Sulph 97 96 91 96 96
KA-61 Fe elem 95 91 90 93 92
KA-62 | Fe Fum 93 92 91 96 91
KA-63 |FeNaEDTA | 5 4 5 4 6
KA-64 | Fe Sulph 97 94 96 96 97
In all three cases, the iron premixes showed very good stability with, on average, only 5%
conversion to ferric iron. The ferric NAEDTA showed maximum 8% conversion to ferrous iron
in the presence of BASF Dry VAP 250 Food (KR25) premix.
4.6 Stability of Iodine
Two iodine compounds were used in this study: potassium iodate (KIO3) and potassium iodide
(KI). Iodine instability is due to the fact that KIO; can be readily reduced to free iodine in the
presence of ferrous salts. KI can be oxidized in the presence of oxidizing agents. In both cases
elemental iodine is volatile.
Microscopic images of both iodine premixes (potassium iodide and potassium iodate) are shown
in Figure 4.42. Since the daily requirement of iodine is so small (100-200 j1g/day), the iodine
compound has to be dissolved in granulation solution (distilled water) and sprayed on the binder
(dextrin or starch) during the granulation process. Both iodine compounds form granules easily.
94
Figure 4.42: Microscopic images (60x magnification) of potassium iodide and potassium
iodate premixes. The iodine compounds were granulated with dextrin and coated with
40% SS.
Potassium Iodide premix Potassium Iodate premix
The stability of iodine in the salt fortified with the three most stable premixes (KR22, 25 and 27)
was studied. Premix KR22 was blended with both potassium iodate and iodized salt (fortified
with potassium iodide). The other two premixes were mixed only with potassium iodate. The
samples containing KIO; premix were analyzed using iodometric titration. The samples made
with iodized salt (containing KI) were analyzed using epithermal neutron activation analysis
(ENAA). Both analytical methods are discussed in section 3.4.4. Figures 4.17 to 4.19 show
iodine retention in these three premixes after three months of storage at 40°C 60% RH and 100%
RH.
Table 4.17: Iodine retention in the TFS samples containing all four iron premixes and
KR22 premix (BASF Dry VAP 250 CK CWD) stabilized with TBHQ. The samples were
analyzed after two and three months of storage at 40°C 60% RH and 100% RH.
Sample | Source of | Initial [%] | 2" month [%] 3™ month [%]

# iron iodine 60% RH | ~100% RH | 60% RH | ~100% RH
KA-21_ | Fe elem 100 87 80 89 74
KA-22 Fe Fum 100 93 90 89 88
KA-23 | Fe NaEDTA | 100 98 98 94 95
95
Sample | Source of | Initial [%] | 2" month [%] 3° month [%]
# iron iodine 60% RH | ~100% RH | 60% RH | ~100% RH
KA-24 | Fe Sulph 100 90 91 83 79
KA-25 || Fe elem 100 98 98 99 96
KA-26 || Fe Fum 100 97 95 97 95
KA-27. | Fe NaEDTA | 100 101 100 99 99
KA-28 | Fe Sulph 100 98 94 95 92
Table 4.18: Iodine retention in TFS samples containing all four iron premixes and KR25
premix (BASF Dry VAP 250 Food) stabilized with either BHA/BHT (samples KA-41, 42, 43
and 44) or TBHQ (samples KA-45, 46, 47 and 48). The samples were analyzed after two
and three months of storage at 40°C 60% RH and 100% RH. .
Sample | Source of | Initial [%] | 2" month [%] 3" month [%]
# iron KIO; 60% RH | ~100% RH | 60% RH ~100% RH
KA-41 Fe elem 100 86 80 80 72
KA-42 Fe Fum 100 97 94 98 87
KA-43 Fe NaEDTA | 100 100 97 99 92
KA-44 Fe Sulph 100 87 87 76 70
KA-45 Fe elem 100 82 74 82 70
KA-46 Fe Fum 100 100 91 93 88
KKA-47 FeNaEDTA | 100 100 96 99 94
KA-48 Fe Sulph 100 95 95 85 83
96
Table 4.19: Iodine retention in TFS samples containing all four iron premixes and KR27
premix (BASF Dry VAP 500) premix stabilized with either BHA/BHT (samples KA-57, 58,
59 and 60) or TBHQ (samples KA-61, 62, 63 and 64). The samples were analyzed after two
and three months of storage at 40°C 60% RH and 100% RH.
Sample | Source of | Initial [%] | 2" month [%] 3 month [%]

# iron KIO; 60% RH | ~100% RH | 60% RH | ~100% RH
KA-57 |Feelem | 100 90 90 82 83

KA-58 Fe Fum 100 99 97 88 81
KA-59 Fe NaEDTA | 100 97 98 94 89
KA-60 Fe Sulph 100 90 85 79 71
KA-61 Fe elem 100 87 78 78 69
KA-62 Fe Fum 100 95 95 90 72
KA-63 Fe NaEDTA |] 100 100 100 99 96
KA-64 Fe Sulph 100 88 78 86 74
In all three cases, the highest stability of iodine was achieved in samples which contained ferric
NaEDTA. This is due to the fact that ferric NaEDTA is in its oxidized state as is potassium
iodate. There is probably no reducing agent strong enough to promote the reduction of iodate to
free iodine. Excellent stability of iodine was also observed in the presence of ferrous fumarate.
Ferrous sulphate is more soluble and more reactive than ferrous fumarate and thus the retention
of iodine was lower in the sulphate containing salts.
97
5. Conclusions
1. The feasibility of producing triple fortified salt (TFS) with acceptable stability of the
three added micronutrients, namely vitamin A, iron and iodine, has been demonstrated.
2. Microencapsulation of vitamin A, iron and iodine protects the TFS from micronutrients
loss even under severe storage conditions for at least three months. The highest retention
of vitamin A at 40°C, 60% relative humidity (RH) was 65% after three months of storage
(TFS sample containing BASF Dry Vitamin A Palmitate 500 premix stabilized with
BHA/BHT, potassium iodate and elemental iron premixes).
3. The study clearly showed that moisture plays a critical role in the stability of these
micronutrients. All of them showed better stability in the lower humidity environment.
4, The commercial stabilization technique of vitamin A preparation greatly influenced the
Vitamin A stability. The three commercial vitamin A forms with the highest stability
were: BASF Dry Vitamin A Palmitate 250 CK CWD, BASF Dry Vitamin A Palmitate
250 Food and BASF Dry Vitamin A Palmitate 500.
5. Vitamin A Palmitate was significantly more stable than vitamin A Acetate.. The best
retention of vitamin A Acetate in TFS was only 48 + 5% after three months of storage at
40°C, 60% RH.
6. In general, vitamin A was more stable in samples containing ferric NaEDTA premix than
in samples containing premixes made with other iron compounds.
7. When vitamin A, iron and iodine were combined in one premix, the retention of vitamin
A was only about ~7%, after three months of storage at 40°C, 60% RH. It is therefore
not practical to produce a single premix containing all three nutrients.
8. The stability of vitamin A is significantly influenced by the type of binder used. White
comstarch is more hygroscopic than the yellow dextrin; consequently it negatively
influenced the stability of vitamin A.
9. Hydrophobic coating agents improved the stability of the commercial vitamin A forms
more significantly than hydrophilic agents. Vitamin A coated with 40% of soy stearine
was more stable than the same vitamin A coated with any other coating agent tested.
98
10. As expected, defects in coating negatively influenced stability of vitamin A. Two
samples of vitamin A premix (based on BASF’s Dry Vitamin A Palmitate 250 Food)
showed significantly different stability even though they were stabilized in the same
matrix, produced the same way and stored under the same conditions during the same
period of time.
11. No considerable difference was observed between the rate of degradation of vitamin A in
premixes stabilized with BHA/BHT antioxidants (ratio 1:1) and those stabilized with
TBHQ.
12. In all of the iron premixes studied, ferrous iron was stable (~5% conversion rate of
ferrous to ferric iron in the TFS samples containing the three most stable vitamin A
premixes at both 40°C, 60% RH and 100% RH). Salt discolouration was only observed
in TFS stored at 40°C, 60% RH when the vitamin A retention was less than ~30%.
13. The type of iodine compound, potassium iodate or potassium iodide, used for salt
fortification did not have any significant effect on vitamin A stability. Therefore, it is
possible to produce TFS by adding vitamin A and iron premixes to existing iodized or
1odated refined salt.
99
6. Recommendations
1. The technical feasibility of TFS production was shown. The process optimization should
be investigated further and the economical feasibility should be determined.
2. Light promotes cis-trans isomerization of vitamin A and consequently decreases its
stability. The effect of light on stability of vitamin A should be investigated.
3. Vitamin A Acetate should not be further considered in the salt fortification project as it
is
highly unstable.
4. Commercial vitamin A forms are all already stabilized with antioxidants and different
coating agents. The possibility of stabilizing pure retinol palmitate and developing a new
formulation especially for TFS should be investigated.
5. The vitamin A premix is too light and it was difficult to blend it with salt as it kept
segregating. This most probably influenced the stability results. Using titanium dioxide
could add some weight to the premix as well as mask its yellow colour.
6. During the vitamin A analysis starch formed a gel layer, which interfered with the
extraction. Use of enzymes such as amylase and/or taka-diastase to digest the starch
should be explored.
7. Itis difficult to control the coating process in the pan agglomerator. The use of fluidized
bed should be further explored as it offers better process control and more even coating
film could be achieved. Better coating would also result in improved retention of all
micronutrients.
8. As demonstrated, the type of binder can strongly influence the stability of vitamin A.
Therefore additional binders should be investigated.
9. The last step in commercial production of salt is drying, which heats it to > 60°C just
before packaging. If the soy stearine is to be used, coat hardeners will have to be
explored to avoid its melting and the clumping together of the salt.
100
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International Food Policy Research Institute, 1995
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Ottawa: Micronutrient Initiative/International Development Research
Centre/International Agricultural Centre, 1996
Nestel P.; Food fortification in developing countries. New York: US Agency for
International Development/Vitamin A Field Support Project (VITAL), 1993.
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children in southern India receiving a small weekly dose of vitamin A. N Engl J Med
1990;323:929-935
Muhilal, Permeisih D., Idjradinata Y.R. et al.; Vitamin A fortified monosodium

glutamate and health, growth and survival of children: a controlled field trial. Am J
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Food Fortification: Science, Technology and Policy; Food and Nutrition Bulletin, vol.
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Deficiencies: Tools for Policymakers and Public Health Workers; Committee on

Micronutrient Deficiencies, Institute of Medicine, 1998
Coelho M.; Vitamin Stability in Premixes and Feeds; A Practical Approach in

Ruminant Diets, Proceedings 13 Annual Florida Ruminant Nutrition Symposium, pp
127-145; BASF Corporation;
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10. Diosady L.L., Alberti J.O., Ramcharan K., Venkatesh Manar M.G.; Iodine Stability in
Salt Double Fortified with Iron and Iodine. Food and Nutr Bull 2002; 23; 2: 196-207
11. Clydesdale F. M., Wiemer K. L.; [ron Fortification of Foods; Academic Press Inc.,
1985
12. MacPhail, A.P. et all; Factors affecting the absorption of iron from ferric NaEDTA;
Br. J. Nutr., 45, 215 — 227, 1981 .
13. Martinez-Torres, C., et all; Ferric EDTA complex as iron fortificant; Am. J. Clin.
Nutr., 32, 809 — 816, 1979
14. Ferni G., Perutz M.F.; Haemoglobin and Myoglobin; Oxford: Claredon Press, 1981
15 . Windholz, M et al (editors); The Merck’s Index, 9" Ed. Merck & Co. Inc, NJ, USA
16. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium,
Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and
Zine; Panel on Micronutrients, Subcommittees on Upper Reference Levels of Nutrients
and of Interpretation and Use of Dietary Reference Intakes, and the Standing Committee
on the Scientific Evaluation of Dietary Reference Intakes, 2002.
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17. Mason J.B., Lofti M., Dalmiya N., Sethuraman K., Diechler M.; The micronutrient
report: current progress and trends in the control of vitamin A, iron, and iodine
deficiencies / Ottawa: International Development Research Centre, c2001
18. Livrea M.A. (editor); Vitamin A and retinoids : an update of biological aspects and
clinical applications. Basel ; Boston : Birkhuser Verlag, c2000
19, Scott, K.J, Rodriquez-Amaya, D.; Pro-vitamin A carotenoid conversion factors:
retinol equivalents — fact or fiction?, Food Chemistry, Vol.: 69, Issue: 2, May 1, 2000,
pp. 125-127
20. Divi R.L., Doerge D.R.; Anti-Thyroid Isoflavones from Soybean, Biochemical
Pharmacology, Vol. 54, Issue 10, Nov 1997, pp. 1087 — 1096
21. Mejia L.A., Chew F.; Hematological effect of supplementing anemic children with
vitamin A alone and in combination with iron. Am J Clin Nutr 1988; 48:595—600
22. Bloem M.W.; Interdependence of vitamin A and iron: an important association for
programmes of anaemia control. Proc Nutr Soc 1995; 54:501-8.
23, Roodenburg A. J. C., West C. E., Beynen A. C.; Vitamin A status affects the efficacy of
iron repletion in rats with mild iron deficiency; Journal of Nutritional Biochemistry,
Vol: 7, Issue: 2, February, 1996; pp. 99-105
24, Greer M.A.; The Thyroid Gland; New York: Raven Press, c1990
25. Hallberg, L.; Effect of vitamin C on the bioavailability of iron from food; Vitamin C
Ascorbic Acid, , Appl. Sci. Publ., London, 1981; pp 49 — 61
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26. Nieves Garcia-Casal, M., Layrisse, M., Pablo Pefia-Rosas, J., Ramirez, J., Leets, L.,
Matus, P.; [ron absorption from elemental iron-fortified corn flakes in humans. role
of vitamins A and C; Nutrition Research, Vol: 23, Issue: 4, April, 2003, pp. 451-463
27, Heath, A.M., Fairweather-Tait, Susan J.; Clinical implications of changes in the
modern diet: iron intake, absorption and status; Bailliére's Best Practice and Research
in Clinical Haematology, Vol: 15, Issue: 2, June 2002, pp. 225 — 241
28. Bendich A.; Calcium supplementation and iron status of females; Nutrition, Vol: 17,
Issue: 1, January, 2001, pp. 46-51
29. Bender’s Dictionary of Nutrition and Food Technology, 7" Ed.; Woodhead
Publishing Ltd, 1999
30. Codex Alimentarius / Jot FAO/WHO Food Standards Program, Codex Alimentarius
Commission, 2"' Ed.; Rome: Food and Agriculture Organization of the United Nations :
World Health Organization, c1992
31 . UN/ACC/SCN; 4th Report on the World Nutrition Situation, January 2000;

Switzerland: UN ACC Sub-Committee on Nutrition, 2000.
http://www.sph.emory.edu/PAMM/ironupdate.htm
32. Phillips M., Sanghvi T., Suarez R., McKigney J., Vargas V., Wickham C.; The costs and
effectiveness of three vitamin A interventions in Guatemala. Washington, DC: US
Agency for Int. Develop., 1994
33, Diosady, L.L., Alberti, J.O., Venkatesh Mannar, M.G.; Microencapsulation for iodine
stability in salt fortified with ferrous fumarate and potassium iodide. Food Research
International 35, 2002, 635-642
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34 . Summers M., Aulton M.; Granulation; http://www.elsevier-international.com/e-
books/pdf/473
pdf
35 . D. M. Parikh, Handbook of Pharmaceutical Granulation Technology, New York:

Dekker, c1997
36. Stanley M. Walas; Chemical Process Equipment — Selection and Design; Butterworth-
Heinemann, 1990
37, Perry R. H., Green D. W. (editors); Perry’s Chemical Engineers’ Handbook, 7" Ed.,
The McGraw-Hill Companies, Inc., New York 1997
38. Glatt Ingenieurtechnik GmbH, Batch Fluid Bed Systems; http://www.glatt-

weimar.de/documents_e/al .htm
39, Fluid Air Inc.; Fluidized Bed; http:/Avww.fluidairinc.com/fb_theory_2.htm#1
40. Karsa D. R., Stephenson R. A.; Encapsulation and Related Drug Processes,
[Cambridge]: Royal Society of Chemistry, c1993
4}. Benita S.; Microencapsulation: Methods and Industrial Applications, New York :
Marcel Dekker, c1996
42. Pokorny J., Yanishlieva N., Gordon M.; Antioxidants in Food, Practical Applications;
Woodhead Publishing Ltd, 2001
43. Food Chemicals Codex, 4° Ed., Committee on Food Chemicals Codex, Institute of
Medicine, 1996
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44. BASF Products for the Food and Pharmaceutical Industry, Technical Information,
2001; http://www.basf.com/static/OpenMarket/Xcelerate/Preview_cid-
993571524076_pubid-974236728470_c-Article.html
45. Product Data and MSDS for Roche Dry Vitamin A Palmitate 500;
https://www.nutraaccess.com/main.asp?pageID=62
46. Product Data and MSDS for Roche Dry Vitamin A Palmitate 250 S/N;
https://www.nutraaccess.com/main.asp?pageID=62
47. Jorg, A., Methods of Vitamin Assays, New York, 1985
48. Ball G.F.M.; Fat-Soluble Vitamin Assays in Food Analysis, Elsevier Science Publisher
Ltd., 1988.
49, Harvey A.E., Jr., Smart J.A., Amis E.S., Simultaneous Spectrophotometric
Determination of Iron (ID) and Total Iron with 1,10-Phenantroline. Analytical
Chemistry, vol. 27, No.1, 1955
50. Heydorn K.; CRC’s Neutron Activation Analysis for Clinical Trace Element
Research. Boca Raton, FL, USA: CRC Press, 1984; 1:84-112
51. Association of Official Analytical Chemists; Official Method of Analysis. 14" Ed.,
Method 33.149, Arlington VA: AOAC 1984, pg 637
52. Laval Lab Inc.; Tyler Ro-Tap Sieve Shakers; http://www.lavallab.com/particle-

size/rotap_sieve_shaker.htm
53. Narasing Rao B.S., Vijyasarathy C.; Fortification of Common Salt with Iron: Effect of
chemical Additives on stability and Bioavailability. Am J Clin Nutr 1975; 28:1395-
1140
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54. Venkatesh Mannar M.G.; Control of Iodine Deficiency Disorders by lodization of
Salt: Strategy for Developing Countries. In: Hertzel B.S. et al eds; The prevention and
Control of lodine Deficiency Disorders. Amsterdam, Elsevier; 1987:111-125
55. Shahidi F. and Naczk M.; Food Phenolics: Sources, Chemistry, Effects and
Applications. Technomic Publishing Company, Lancaster, PA. 1995
56. Raileanu I.; Triple Fortification of Salt with Iodine, [ron and Vitamin A. M.A.Sc.
Thesis: University of Toronto, Canada, 2002.
57. Watson Foods Co., Inc.; Vitamin A Palmitate 250,000 IU/gram Product Information,
Included in Appendices
107
8. Nomenclature
Abs Absorbance
BHA Buhtylated hydroxyanisole
BHT Buhtylated hydroxytoluene
°C Degrees Celsius
cm centimeters
CWD Cold water dispersible
EC Ethylcellulose
FAO Food and Agricultural Organization
Fe Chemical symbol for iron
Fe elem Short for elemental iron
Fe Fum Short for ferrous fumarate
Fe NaEDTA Short for ferric sodium ethylenediaminetetraacetic acid salt
Fe Sulph Short for ferrous sulphate
g gram
GI Gastrointestinal
Hydroxypropylmethylcellulose
Chemical symbol for iodine
Iron Deficiency Anemia
Iodine Deficiency Disorders
International Unit
Chemical symbol for potassium iodide
Chemical symbol for potassium iodate
Methylcellulose
Micrometer (10° meter)
Parts per million
Recommended Daily Intake
Retinol Equivalents
Relative humidity
Residual sum squares
108
Revolutions per minute
Ss Soy stearine
TBHQ tert-butyl hydroquinone
UNICEF United Nations Children’s Foundation
USP United States Pharmacopeia
VAA Vitamin A acetate
VAD Vitamin A Deficiency
VAP Vitamin A palmitate
WHO World Health Organization
109
9. Appendices
9.1 Analytical Methods

9.1.1 Vitamin A
All glassware used was covered by aluminum foil to protect the vitamin A from light. The
reagents and standards were prepared on the day of analysis. The samples were ground using a
coffee grinder.
1. Chemicals:
6 95 %ethanol
e Pyrogallol
® Potassium Hydroxide Solution
e USP Vitamin A Reference Solution
e Hexane (commercial grade, boiling range 60 - 68°C)

e Isopropanol (spectral grade)
2. Solution Preparation
© Pyrogallol: 1% (w/v) pyrogallic acid in alcohol (95% ethanol)
e 70% potassium hydroxide solution: 1 g of KOH/ mL of distilled water.
3. Procedure
The method consists of saponification, extraction and measurement of fluorescence.
3.1 Saponification (also called alkaline hydrolysis) is used to free the vitamin A from fat and to
convert the retinol esters into retinol (vitamin A alcohol). After saponification, most of the
saponifiable material becomes soluble in polar solvents. The procedure is as follows:
3.1.1 To approximately 1g of ground sample add
e 1 mL of potassium hydroxide solution

® 4mL of 1% (w/v) ethanolic pyrogallol
e 1mZL of distilled water
110
3.1.2 Mix thoroughly by swirling and shaking or use vortex mixer
3.1.3 Heat the sample in water bath at 60°C for 30 minutes
3.1.4 Cool sample to room temperature
Prepare a reagent blank with 1 mL of potassium hydroxide solution, 4 mL of ethanolic pyrogallol

and 1 mL of distilled water.
3.2 Extraction.
During extraction, the vitamin A is extracted from the sample with non-polar solvent.
3.2.1 To cooled sample add
e 4mL of distilled water
© 10 mL of hexane
3.2.2 Mix for 1 minute on Vortex mixer or 10 minutes on wrist action shaker
3.2.3 Centrifuge at about 1000 rpm to separate and clear hexane layer
3.2.4 Remove upper layer with a transfer pipette and transfer it to 25 mL volumetric flask
3.2.5 Add 10 mL of hexane and repeat steps 2, 3 and 4
3.2.6 Make to 25 mL with hexane
3.3 Preparation of standards

3.3.1 Warm capsule of USP vitamin A reference standard to room temperature.
3.3.2 Cut top, express content into a small beaker and weight accurately
3.3.3 Transfer to 100 mL volumetric flask, dilute to mark with hexane and mix thoroughly
3.3.4 Pipette 2 mL to 25 mL volumetric flask, dilute to mark with isopropanol and mix
thoroughly
3.3.5 Make more than 4 solutions for standardization by diluting series of 1-5 mL to 25 mL
volumetric flasks with isopropanol. Targeted concentrations: from 0.3 g/mL to 1.2 ug/mL.
3.4 Measurement of fluorescence

3.4.1 Read hexane (background) in cuvette
3.4.2, Read standards, starting with the most concentrated solution
3.4.3 Read blank
111
3.4.4 Read samples
The standards and or the blanks should be re-read regularly i.e. every 5 or 10 samples.
4. Calculation of vitamin A content

Calculate concentrations of standards from data on the USP standards’ bottle.
Correct all fluorescence reading of samples by subtracting the reagent blank reading.
Lg vitamin A/ g of sample =c x V/ w
c — Concentration of vitamin A in ,1g/mL of solution read from the calibration curve

V — Final dilution volume of extract analyzed
w — Weight of sample in grams
9.1.2 Iron
1. Chemicals
® concentrated sulphuric acid
e 1,10-phenanthroline solution
e buffer solution (pH 3.98)
e Reagent Solution (3% w/v): Dissolve reagent grade 1,10-phenanthroline monohydrate in
distilled water which has been heated just to boiling. After cooling, dilute the solution to
desired volume (varies with the quantity of samples).
e Buffer: Prepare a 0.2 M solution of potassium biphtalate.
3. Procedure
3.1 grind sample with a coffee grinder and weigh approximately 5 g using an analytical balance.
3.2 transfer the sample to a 100 mL volumetric flask and add
® Approximately 50 mL of distilled water to dissolve the salt.
112
e Add 2 mL of concentrated sulphuric acid to the flask to digest the iron compounds in the
sample.
3.3 place the volumetric flask on a hot plate in a fume hood to melt the water insoluble coating.
3.4 dilute the solution to the 100 mL mark using distilled water after being cooled, and transfer 5
mL of the solution to a 25 mL volumetric flask.
3.5 to the same 25 mL volumetric flask add
@ 5 mL of 1,10-phenanthroline solution
® 10 mL of buffer solution (pH 3.98)
° top with distilled water
3.6 measure the absorbencies of the solution under wavelength 396nm and 512 nm, using a
Cary® UV-Visible spectrophotometer
4. Calculation
Compare the results to the calibration curves prepared by standard ferric and ferrous iron
solution and calculate the concentration of each iron.
1. Total iron concentration [ug/mL] = absorbance at 396nm / slope of the calibration curve at
396 nm
2. 1" Estimate of ferrous iron concentration [ug/mL] = absorbance at 512nm / slope of the
calibration curve for Fe (I) at 512 nm
3. Ferric iron concentration [j1g/mL] = total iron conc. — 1" est. of ferrous conc.
4. Ferric iron contribution to abs. = ferric iron concentration x slope of the calibration curve for
Fe (ID at 512 nm
5. Subtract the ferric iron contribution to abs.
6. Ferrous iron concentration [tg/mL] = subtracted ferric iron contribution to abs. / slope of the
calibration curve for Fe (11) at 512 nm
113
7. Ferrous iron remaining [%] = ferrous iron conc. / total iron conc.
8. Total iron [ppm] = total iron concentration [ug/ml] x final dilution volume analyzed [mL] /
weigh of sample [g]
9.1.3 Iodine from Potassium Iodate
1. Chemicals:
e Potassium iodate
e Potassium iodide
® Sodium thiosulphate
e Starch
e Sulfuric acid
2.1. Solution A:
2.1.1. 0.1 N sodium thiosulphate stock solution:
Weight out 6.2 g of Na2S.O3 and dissolve in 250 mL distilled water. Store in a rubber
stoppered bottle kept away from direct heat and light.
2.1.2. Working solution (0.005 N)

Dilute 12.5 mL of stock solution to 250 mL with distilled water. Use working solution for
titration
2.2. Solution B:
2.2.1. Starch solution:
Boil 250 mL distilled water in a beaker. Triturate approximately 2.5 g of soluble starch in
15mL cold water. Add the starch paste to the boiling water and continue to boil for
approximately 1 minute. Let the solution cool to room temperature. Store the solution in a
114
stoppered bottle. Prepare fresh after 1 week or if mould appears. Sodium benzoate can be
added to increase the shelf life of this solution.
2.3. Solution C:
2.3.1. KIO 3 stock solution:
Weigh 1.0 g of KIOs, recording weight to the nearest 0.1mg. Dissolve the KIO; in 100mL
distilled water in a volumetric flask. Store the solution in a stoppered bottle.
2.3.2. KIO; working standard:

Using a pipette, transfer 5 mL of stock solution into a 100 mL volumetric flask. Dilute
to the mark with distilled water. Use standard to check strength of thiosulphate solution.
2.4. Solution D:
2.4.1. 2 ™N sulfuric acid:
To 200 mL distilled water, add slowly 12.5 mL concentrated H,SO,. Make up to

volume with distilled water. Store solution is a glass bottle in a cool dry environment.
. Procedure
3.1. Standardization of sodium thiosulphate

With a pipette, measure exactly 2 mL of standard KIO3 solution into a 250 mL Erlenmeyer
flask. Add approximately 150 mL of distilled water, 3-4 drops of sulfuric acid solution, a
pinch of KI crystals. Titrate the liberated iodine with the thiosulphate solution until the
solution turns pale yellow. Add ImL starch indicator and continue titration until the solution
just turns from purple to colorless. Record the milliliters of thiosulphate used.
3.2. Determination of iodine content in salt sample

Weigh out 5-10 g of iodized salt into a 250 mL Erlenmeyer flask, recording exact weight
used. Add approximately 150 mL of distilled water, 3-4 drops of sulfuric acid solution, a
pinch of KI crystals. Titrate the liberated iodine with the thiosulphate solution until the
solution turns pale yellow. Add 1 mL starch indicator and continue titration until the solution
just turns from purple to colorless. Record the milliliters of thiosulphate used.
115
4. Calculations
4.1. Strength of thiosulphate
conc. of KIO; standard (ugI/mL) * 2mL / mL of thiosulphate = pg I/mL thiosulphate
4.2. Iodine content

strength of thiosulphate (ug I/mL thiosulphate) * mL. thiosulphate / weight of sample (g) =
ug I/g sample = ppm I[
* to convert ppm I to ppm KIOs3: multiply ppm I by 1.685
116
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€ 87 L¥ rr LFr9 001 r8t | weld rl um.j2 LYMM | Z-L DT
1 ¥SZ L¥@ L¥79 001 v3 | Wer I ura] a LOI CLR
tFY L¥€1 ZFEE 001 ILI | Wea | COM | VLGaeNew 9M | 7-9 RD
LFL € FEL ZF SZ 001 ILI | Weld | COI wIn{a4 ORD | 1-9 RDI
76 ZF L¥8€ 001 ost [yea | cora| viawened| sram | 7s
1¥6 L¥ vz OL F€€ 001 IL'sst | wera | com wn.J9.J SRDI SRI
TFS L¥L CFT 001 POOL! | Wed | COM | VLGaeNer PRs | CP RDI
CFP L¥Z1 € FLT 001 roll | Wea | cor wan.J2 pra | T-PR
L¥FE€ yFs L¥ ST 00] 961 | Wed | COLT | V.LGAeNed era | -e RDI
OFS CFI CF 001 961 | Wed | ODI wnJoq ely ERD
€ FOE L# Sp 0 #96 001 6LL | Weg | COrd | VLGAeNe TR | 7-7 RD
1 ¥ 62 OF IP b FZ 001 6LI | Wea | cont wuna.J TMM | -7R
L¥LZ Fre ZF 8P 001 gol | Weld | cont | VLdaeNer LUA | 7-1 Ro
€FLZ L¥ pe vtSp 001 gol | Weg | COM wn.f-f IROL} -iRot
im] as ¥ [%] as ¥ [%] as ¥ [om] | bres 3 py Wa Srl] xpumeid | AY %00T
“OU03 GUL pal “SHO ODF pul “2200 OF yk enruy e "SHOS TERT WES | supe] How | V¥ HA # aduass
"YT F BIE [TV , “SHIWOUT 99.14) 10} HA %GOOT Doh I¥ peiojs ‘spunoduros oulpor pue wor 3Ue.19jj1p
PUL SHONVINULIO] XMMeId VY URULIIA JUAAITJIP SULNTe}UOD sopdures SAT Ul sypMsea AVIquIs VW UIUTERIA Jo ArvuTUIMNS :p°6 O1qB L
OeT
L¥re L¥9r TF S9 001 OL | Wuela | COTM | VLGaeNed | 7-GIUM | POI-VI
L¥7E vib 7 ¥99 001 OLI | Weg | corms une. | 2-6INM | €Ol-W
ZF rl EFIE FIP 001 681 | We | COMM | VLGSeNed | I-61 | ZOI-WH
€FZI TFIE 6 ¥8E 001 681 | Weg | COM wuntey | I-61MM | 1OI-WoL
pF Le Gam ad 7 + 6S 001 ELI | Url It | VIGAeNed | 2-8 | O0T-WI
L¥S€ L$ 8p L #19 001 ELL | MUR ml umnjot | c-8R | 660-VI
i%] as 9 iy] ds im] aS ¥ [eo] | [res 3 py 344 Bri] xrmoid | AY %O00T
*SHOD OU, | “OUOS OM ,,7 | 9U0D OW , [| [VHT] , su09 [EDIE | aes | sulpey HO HA | #aydures
VITAMIN A PALMITATE
250,000 1U0/Gram
Watson Code: RI 70000/F 240114
Description:
Vitamin A Palmitate 250,000 1U/uram is a free flowing, light yellow. fine particle powder
consisting of spherical particles. The particles consist of vitamin A palmitate oil distributed in
droplets of 1 - 2 microns micrvencapsulated in a food grade modified starch matrix with BHT
{FCC, E321) as an antioxidant and silica flour as a flow agent.
Specification:
Vitamin A Potency: 250,000 IU per gram
Loss on Drying maximum 5% (105 C; 4h)

Solubility: Dissolves in cold water resulting ina cloudy
dispersion.
Stability: This product is sensitive to humidity, air, heat
and light.
Storage: Store in a cool and dry place in the original
packing.
Ingredient Statement: Modilied starch, Vitamin A Palmitate.

Coconut Oil, BHT (antioxidant), Silica Flour
(flow agent) 4
The modified starch, coconut ofl, BH'T and silica flour are considered processing raids and do not
require labeling on the finished product application.
Technical Support:
For technical assistance, please contact Watson Foods Company Technical Serviee Departme
nt
al $00/388-348 |,
Watson] Foods8 Co, ine.

301 Heffernan Drive, West Haven, CT 06516, Telephone (203)
932-3000
TOTAL FP. at

MQ84139 Ocr

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PREPARATION OF SALT TRIPLE FORTIFIED WITH

VITAMIN A, IRON AND IODINE

A thesis submitted in conformity with the requirements

Graduate Department of Chemical Engineering and Applied Chemistry

© Copyright by Katarina Rutkowski 2003

Acquisitions and Acquisisitons et

395 Wellington Street 395, rue Wellington

Your file Votre référence

The author retains ownership of the L'auteur conserve la propriété du

resulted in reduced vitamin A retention to ~7% at 40°C, 60% RH.

2.1.4 Nutrient — Nutrient Interactions 14

3. Experimental Techniques and Analytical Methods 34

4. Results and Discussions 43

Figure 2.1: Chemical structure of retinol and B-carotene. 5

Figure 2.6: Fluid bed 28

three months at 40°C ~100% RH.

stabilized with TBHQ), in the presence of different iron compounds and

40°C 60% RH.

stabilized with TBHQ), in the presence of different iron compounds and

TBHQ), in the presence of different iron compounds and potassium iodate

stabilized with either BHA/BHT (KR 20-1) or TBHQ (KR20-2).

Table 2.2: | Comparison of annual cost-effectiveness estimates between three countries. 17

months. * All data + 2%.

months. * All data + 2%.

60% RH for three months. * All data + 2%.

Upper Intake Level (UIL) for adults is 1,100 ug/day.

powder and infant formulas.

2.1.1 Physical and Chemical Properties

Figure 2.1: Chemical structure of retinol and B-carotene.

Vitamin A is extremely sensitive to oxygen (undergoes oxidation), light (promotes

In adult humans, haemoglobin is a macromolecule consisting of four peptide chains

Fe** (green) —»Fe** (orange/red) + e

Figure 2.3: Chemical structure of thyroxine, T, [24]

Oxidation: 27 —> I)+2e”

Reduction: 2I°* + 10 e—> hb

® 0,344 wg of all-trans-retinyl acetate

Age (years) | Children Men Women Pregnancy || Lactation

Factors affecting the vitamin A requirements

Factors affecting the iron requirements

Upper Intake Level for adults is 1,100 ug/day. [16]

Factors affecting the iodine requirements

2.1.4 Nutrient —~ Nutrient Interactions

incapable of initiating formation of free radicals. [9]

Iron and Iodine

Ferric salts can be reduced by iodides:

Iron and Vitamin C

Iron and Phytates, Polyphenols

2.2 Criteria for Food Fortification

The three main approaches to addressing micronutrient deficiencies are supplementation,

Annual Cost per Person (US$ in 1991)

Per person 0.16 0.14 0.29

Per person 2.31 1.60

A fortified food has to meet several requirements:

e Commonly consumed by the target population

e No interactions between the fortificants and the carrier food

Advantages of salt as a vehicle for fortification

The MI is a not-for-profit organization based in Ottawa, Canada, which specializes in addressing

stable for 12 months at 40°C 100% RH. [33]

Figure 2.4: A simplified cross sectional structure of a premix.

Inert matrix (fillers

When designing TFS the premix must be:

e Uniform, with the same particle size as salt, to avoid segregation

Fe (green) —»Fe (orange/red) + e