IRON FORTIFICATION OF YOGURT AND PASTEURIZED MILK
By
Smith Gilliard Nkhata

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Food Science - Master of Science
2013

ABSTRACT
IRON FORTIFICATION OF YOGURT AND PASTEURIZED MILK
By
Smith Gilliard Nkhata
Both yogurt and pasteurized liquid milk was made from whole cow’s milk
which was fortified with ferrous bisglycinate, ferrous lactate and ferrous sulfate
microencapsulate. Yogurt was stored for 7 days and milk for 2 days before
consumer acceptance sensory test was done. Chemical analysis was done every
5 days for yogurt and every 3 days for pasteurized milk. Sensory mean scores
show that there were no significant differences between the control yogurt and
yogurt fortified with ferrous sulfate microencapsulate in appearance, flavor,
mouthfeel and overall preference. Significant differences (p<0.05) were observed
between control yogurt and yogurt fortified with ferrous bisglycinate and yogurt
fortified with ferrous lactate. The observation was different in milk where no
significant differences were observed in appearance and flavor in all treatments
while control milk and milk fortified with ferrous lactate showed no significant
differences in taste. Both thiobarbituric acid (TBA) and peroxide value (PV)
numbers were highest in yogurt and pasteurized milk fortified with ferrous sulfate
microencapsulate. Control yogurt had the lowest TBA value. PV was lowest in
yogurt fortified with ferrous lactate while pasteurized milk has low PV in control.
Therefore ferrous sulfate microencapsulate was the best option for fortifying both
yogurt and pasteurized liquid milk.

I would specifically dedicate this work to my beloved wife, Beatrice Chalinda
Nkhata and my lovely daughter Trinity Nkhata for enduring my absence when I
went for my studies abroad.

iii

ACKNOWLEDGEMENTS

My heartfelt appreciation should go to Dr. Zeynep Ustunol, professor in
dairy science in the Department of Food Science and Human Nutrition (FSHN) at
Michigan State University (MSU), for the technical support rendered to me when I
was at MSU for my course work and when I was back home for research. She
did not tire to provide technical support and guidance when I came home to
Malawi. She was always there for me. “Dr. U I really appreciate you and May God
richly bless you”
I would also extend my gratitude to Dr. Perry Ng, professor in cereal
science, for the timely assistance whenever he was called upon, throughout my
study. Dr. J. Harte for the Sensory Lab and her technical support and advice
before and during sensory evaluation, Dr. E. Ryser for the Microbiology lab. Dr. A
Mwangwela for allowing me to use Food and Nutrition Labs at Bunda College of
Agriculture.
I would also acknowledge all students who took part in helping me during sensory
evaluation both at MSU and Bunda College of Agriculture.
Lastly but not least I would also appreciate USAID for USAID Long-Term Training
and Capacity Building (ULTCB) in Malawi scholarship for the chance they gave me
to undergo my studies.
You all deserve a place in my heart.

iv

TABLE OF CONTENTS

LIST OF TABLES……………………………………..……………………………….........................vii
LIST OF FIGURES…………………………. ………………………………………………………...viii
KEY TO ABBREVIATIONS……….…………………………………………………………………….ix
1.0 INTRODUCTION .................................................................................................................. 1
2.0 LITERATURE REVIEW ........................................................................................................ 4
2.1. Milk Composition.............................................................................................................. 4
2.2. Heat Treatment of Milk ..................................................................................................... 5
2.2.1. Pasteurized Milk ...................................................................................................................... 6
2.2.2 Ultrahigh Temperature (UHT) Pasteurized Milk .................................................................. 6
2.3. Yogurt fortification ............................................................................................................ 7
2.4. Iron requirements by the body.......................................................................................... 7
2.5. Iron fortification compounds ............................................................................................. 8
2.6. Effects of Iron deficiency ................................................................................................ 11
2.7. Sensory effects ............................................................................................................. 15
2.8.Determination of Lipid Oxidation ..................................................................................... 16
2.8.1 Peroxide Value (PV) test....................................................................................................... 17
2.8.2 Thiobarbituric Acid (TBA) test .............................................................................................. 17
3.0.RATIONALE AND SIGNIFICANCE .................................................................................... 19
4.0. OBJECTIVES .................................................................................................................... 22
4.1. Long term objective: ....................................................................................................... 22
4.2. Specific objectives: ........................................................................................................ 22
5.0. MATERIALS AND METHOD ............................................................................................. 23
5.1. Materials: ....................................................................................................................... 23
5.2. Starter culture growth and activity .................................................................................. 23
5.3. Skim milk and yogurt titratable acidity (TA) .................................................................... 24
5.4. Yogurt manufacture ....................................................................................................... 25
v

5.5. Processing of pasteurized milk....................................................................................... 27
5.5. Determination of pH ....................................................................................................... 28
5.6. Peroxide value determination ......................................................................................... 29
5.7. Thiobarbituric acid (TBA) value ...................................................................................... 29
5.8. Sensory evaluation ........................................................................................................ 30
5.8.1. Yogurt ..................................................................................................................................... 30
5.8.2. Pasteurized Milk .................................................................................................................... 31
5.9 Statistical analysis........................................................................................................... 32
6.0 RESULTS AND DISCUSSIONS ......................................................................................... 33
6.1. Effect of iron salts on starter culture growth and activity ................................................. 33
6.2. Titratable acidity and pH for skim milk ............................................................................ 35
6.3. Sensory Analysis ........................................................................................................... 44
6.3.1. Sensory Evaluation of Yogurt. ............................................................................................ 44
6.3.2. Sensory evaluation of pasteurized milk. ............................................................................ 47
6.4. Chemical analysis of lipid oxidation ................................................................................ 49
6.4.1. PV test for yogurt during storage........................................................................................ 49
4.6.2. PV test for pasteurized milk in storage. ............................................................................. 51
6.4.3. TBA test for yogurt in storage. ............................................................................................ 53
7.0. CONCLUSION AND RECOMMENDATIONS .................................................................... 56
8.0. APPENDICES ................................................................................................................... 58
8.1. Milk flyer......................................................................................................................... 59
8.2. Yogurt flyer .................................................................................................................... 60
8.3. Questionnaire for pasteurized milk ................................................................................. 61
8.4. Questionnaire for yogurt................................................................................................. 64
8.5. Consent Form For Research Involving Human Subjects ................................................ 66
8.6. Application For Initial Review ......................................................................................... 68
9.0. REFERENCES .................................................................................................................. 70

vi

LIST OF TABLES

Table 1: Comparison of the mineral and vitamin components of cow’s milk and human
milk. Source: FSA 2002 ............................................................................................................................ 5
Table 2: Summary of iron salts used in food fortification. .................................................................. 11
Table 3: Sensory mean scores of yogurt fortified with different iron salt stored at 4 OC for 7
days ............................................................................................................................................................ 45
Table 4: Sensory mean scores of pasteurized milk fortified with different iron salt stored at
4 OC for 7 days. ........................................................................................................................................ 48
Table 5: Principal Investigator’s (PI) details................................................................................ 68

vii

LIST OF FIGURES

Figure 1: A comparison of the carbohydrate (dotted), protein (vertical) and fat (horizontal)
components of whole cow’s milk and human milk. Source: FSA, 2002. ........................................... 4
Figure 2: Flow diagram showing stages during processing of yogurt.............................................. 26
Figure 3: Flow diagram showing stages during processing of pasteurized milk ............................ 28
Figure 4: Effects of iron salts on growth and activities of Lactobacillus delbrueckii subsp.
bulgaricus and Streptococuss thermophilus in iron fortified reconstituted skim milk
incubated at 35 OC for 6 hours ............................................................................................................... 34
Figure 5: Changes of Titratable Acidity in skim milk fortified with different iron salts and
incubated at 35 OC for 6 hours ............................................................................................................... 36
Figure 6: Changes of pH in skim milk fortified with different iron salts and incubated at 35
C for 6 hours. .......................................................................................................................................... 37

O

Figure 7: Changes in pH for yogurt fortified with different iron salts during incubation at 43
O
C till pH 4.6 was reached ...................................................................................................................... 38
Figure 8: Changes in Titratable Acidity for yogurt fortified with different iron salts and
stored at 4 Oc for 30 days ....................................................................................................................... 42
Figure 9: Effects of different iron salts on pH for yogurt stored at 4OC for 30 days ....................... 43
Figure 10: Changes in PV in yogurt fortified with different iron salts and stored at 4oC for
30 days ...................................................................................................................................................... 50
Figure 11: Effects of iron fortification on lipid oxidation in pasteurized milk stored at 4 OC
for 18 days as determined by PV .......................................................................................................... 52
Figure 12: Effect of iron fortification on lipid oxidation in yogurt stored at 4 OC for 30 days
as determined by TBA test ..................................................................................................................... 55

viii

KEY TO ABBREVIATIONS

CFU:

Colony Forming Units

FDA:

Food and Drug Administration

g:

gram

GoM:

Government of Malawi

GRAS

Generally Regarded as Safe

Hb:

Hemoglobin

HTST:

High Temperature Short Time

ID:

Iodine deficiency

IDA:

Iron deficiency anemia

IDD:

Iodine deficiency disorder

LAB:

Lactic acid bacteria

MDA:

Malondialdahyde

MDHS:

Malawi Demographic Health Survey

Meq:

Milliequivalent

ml:

millimeter

MRS:

Man Rogosa and Sharpe

N:

Normality

NaFeEDTA: Ferric sodium ethylenediaminetetraacetic acid
NDM:

Non-fat dry milk

Nm:

nanometer (a billionth of a meter,10-9)

pH:

power of hydrogen
ix

PV:

Peroxide value

RDA:

Recommended daily allowance

TA:

Titratable acidity

TBA:

Thiobarbituric acid

TBARS:

Thiobarbituric reactive substances

UCRIHS:

University Committee on Research Involving Human Subject

UHT:

Ultra heat treatment

FAS:

Food Standard Agency

VAD:

Vitamin A Deficiency

WHO:

World health organization

Wt.

Weight

x

1.0 INTRODUCTION
Iron is essential micronutrient in human nutrition. It is also a component of heme
in hemoglobin and myoglobin in which it plays important role in the transport, storage
and utilization of oxygen. Iron deficiency induces anemia, alters mental development,
decreases immunity (Gaucheron 2000) impairs cognitive scores in children and leads to
poor pregnancy outcome and lowers working capacity in adults (Martinez-Navarreta and
others, 2002). In cases in which anemia is severe and not corrected, blood transfusion
may become necessary. Anemia has been reported to contribute significantly to
maternal mortality and both maternal and fetal morbidity (Van de Broek and Letsky,
2000).The iron found in food can be highly bioavailable, as is the case with heme iron
which is found in red meat. However, the cost of these products is too high for many
people. The iron present in other products of vegetable origin, is non-heme and has the
disadvantage of interacting with substances in food that inhibit its absorption such as
tannins, phytates, and polyphenols hence it has low bioavailability. Much of this kind of
food is consumed by people in the lower socioeconomic classes, who thus cannot meet
their physiological needs for iron (Van de Broek and Letsky, 2000). Therefore it is
widespread in less industrialized countries as in developing countries. Iron deficiency is
also caused by either insufficient dietary intake of iron, poor absorption of iron or both
(Gaucheron 2000), insufficient absorbable iron, hookworm infection, malaria or Vitamin
A deficiency (Richard, 1997). Hence all these factors should be addressed
simultaneously in any food fortification strategy.
The best way to prevent problems associated with iron deficiency is through iron
fortification of food for the whole population or only for certain groups. Compounds used
in food fortification provide nonheme iron, so it is important to select fortification
1

compounds and foods vehicles that will not diminish iron bioavailability (Van de Broek
and Letsky 2000). Though food fortification may increase unit cost of food being
fortified, it is the most cost-effective technique than other interventions that have the
potential to achieve the same health or nutritional outcome, such as supplementation
(Allen and others, 2006). Iron fortification of flour, bread and cereals is practiced to
correct iron deficiency (ID). Iron fortification of milk and dairy product is considered as a
potential approach to prevent the ID disorder (Gaucheron 2000), since dairy foods are
an important part of the daily diet in most parts of the world; also, in the diets of those
most susceptible to iron deficiency primarily women and children.
Dairy products are an important group in human nutrition. Direct addition of iron
to milk or dairy product might be effective means of increasing the dietary intake of iron
to the general population. Malawi has a tropical climate and milk cannot be kept for
more than three hours at ambient temperature immediately after milking. Cooling
equipment are not available in many parts of the country and if available is not
affordable to rural people. The new scientific and efficient method to overcome this
problem is use of Ultra high temperature (UHT) pasteurization of milk. While yogurt
cannot store without refrigeration for long period of time aseptically processed UHT
pasteurized milk is shelf stable and can store for months without spoiling. Both
pasteurized milk and yogurt are excellent sources of vitamins, minerals and proteins but
like any other dairy product they contain very little iron (approximately 0.2mg/kg
(Gaucheron, 2000) which makes it impossible to meet iron Recommended Daily
Allowance (RDA). Therefore dairy products are logical vehicle for iron fortification (ElKholy and others, 2011) and considered as practical and cost-effective long term
solution (Abasi and Azari, 2011). Milk, either pasteurized or sterilized, is the most

2

commonly consumed dairy product in Malawi because of the relatively low cost,
availability and its readiness to drink. It was estimated that 80% of the total dairy
products consumed is pasteurized or sterilized liquid milk. Yoghurt and Chambiko are
also frequently consumed, taking up at least 15% of sales by volume. (Anonymous,
2004) These foodstuffs are distinguished as suitable vehicles because of their high
consumption by children, high risk group with regard to iron deficiency (Abbasi and
Azari, 2011)
Fortification with iron is technically more difficult than fortification with other
nutrients because iron is a prooxidant and therefore promotes lipid oxidation (El-Kholy
and others 2011). Therefore, the ideal iron compound for food fortification should be
one that supplies high bioavailability of iron and does not affect the nutritional value or
sensory properties of the food, should be stable during food processing and of low cost
(El-Kholy and others 2011). It is therefore proposed that iron salts should be
microencapsulated to reduce or prevent these negative effects. Microencapsulation is
the technology of packaging solid, liquid and gaseous materials in small capsules that
release their contents at controlled rates over prolonged period of time (Abbasi and
Azari, 2011). The choice of iron compounds also depends on its solubility in gastric juice
and on the presence of activators or inhibitors in the fortification food ( Boccio and
others 1997).

3

2.0 LITERATURE REVIEW
2.1. Milk Composition
Milk composition tends to differ from one species to another. There are also
variations within the same species depending on season, feeds, lactation stage,
intervals between milking, etc (O’Mahony 1988). The composition of milk varies
according to the animal from which it comes, providing the correct rate of growth and
development for the young of that species, thus for human infants, human milk is
obviously more suitable than cow’s milk. Indeed, the popular consensus among health
care professionals is that ordinary cow’s milk, goat’s milk, condensed milk, dried milk,
evaporated milk, or any other type of milk should not be given to a child under the age
of one as a breast milk substitute. (O’Mahony, 1988). This is because of differences in
.
the composition of milk that have been revealed by research over the last decade or so.
While cow’s milk and human milk contain a similar percentage of water, the relative
amounts of carbohydrate, protein, fat, vitamins and minerals vary widely (O’Mahony,
rate,
vitamins
1988)
Cow's milk

Human Milk

Figure 1: A comparison of the carbohydrate (dotted), protein (vertical) and fat
rison
(horizontal) components of whole cow’s milk and human milk. Source: FSA, 2002.
)

4

Table 1: Comparison of the mineral and vitamin components of cow’s milk and
human milk. Source: FSA 2002
Cow's Milk

Human Milk (per

(per 100g)

100g)

Sodium (mg)

43

15

Potassium (mg)

156

58

Calcium (mg)

120

34

Phosphorus (mg)

94

15

Iron (mg)

0.02

0.07

Zinc (mg)

0.4

0.3

Iodine (µg)

30

7

Folate (µg)

9

5

Vitamin C (mg)

2

4

Micronutrients

2.2. Heat Treatment of Milk
Heat treatment in the production of long life product is called sterilization. Product
is exposed to high heat treatment that the microorganism and most of the enzymes are
inactivated and the product can be kept several months under ambient temperature if
sterilized in the container or packaged under aseptic conditions. Although, products are
manufactured with UHT and aseptic processing in most countries, the market share of
UHT milk varies considerably by countries (Chavan and others, 2011). Aseptic
processing has great potential to increase dairy consumption in tropical countries as
there is a low milk trend due to high temperatures and limited refrigerated distribution
(Gedam and others, 2007). Production of long life milk can be done in two ways; a) in5

container sterilization with product in package being heated at about 115-120 0C for 20–
30 minutes. This can be stored at ambient temperature, b) UHT treatment with the
product heated at 135-150 0C for 4-15 seconds followed by aseptic packaging in
packages protecting the product against light and atmospheric oxygen and can be
stored at ambient temperature for long (Gedam and others 2007). However in most
cases milk is prepared and processed in the following ways:
2.2.1. Pasteurized Milk
Pasteurization is heat process that is designed to kill pathogenic bacteria in milk
and may cause spoilage of milk products. During pasteurization the milk is heated to a
F)
F)
minimum of 72OC (161° for a minimum of 15 seconds or 63 OC (145° for 30 minutes
and packaged under clean and sanitary conditions.The shelf-life of pasteurized milk
held under proper refrigeration, usually less than 7.2OC (45° can range from 12 to 21
F)
days post processing. Holding pasteurized milk at temperatures above 45° will shorten
F
the shelf life significantly. The majority of U.S. fluid milk is pasteurized using a high
temperature short time (HTST) continuous process of at least 161° (72° for 15
F
C)
seconds to be legally pasteurized.
2.2.2 Ultrahigh Temperature (UHT) Pasteurized Milk
UHT is the sterilization of milk by heating it for a short time135-150 0C for 4-15
seconds. Advantages of UHT processing include extended shelf life, lower energy
costs, and elimination of required refrigeration during storage and distribution if
aseptically packaged. Milk maybe packaged either before or after sterilization. Desirable
changes that take place during UHT processing of milk include destruction of
microorganisms and inactivation of enzymes, while undesirable changes include
browning reactions, loss of nutrients, sedimentation, fat separation, cooked flavor that
6

take place. Gelation of UHT milk during storage (age gelation) is a major factor limiting
its shelf life (Gedam and others, 2007). Based on sensory work, Oupadissakoon (2007)
reported butyric acid, sour aromatics, and lack of freshness as negative attributes with
UHT milk.
2.3. Yogurt fortification
Yogurt is a dairy product produced by use of lactic acid bacteria (LAB) through
the process of fermentation. Yogurt has gained widespread consumer acceptance. It is
an excellent source of calcium and protein but contains very little iron (El-Kholy and
others 2011). Therefore fortification of yogurt with iron would help solve this nutritional
need. However, before any such fortification is undertaken, the effects of iron addition
on microbial physiology during manufacture and shelf life of yogurt, oxidation of milk fat,
and the effect of iron on the taste and acceptance of a fortified yogurt must be
ascertained (Sharareh 1996). Yogurt fortified with 10, 20 and 40 mg of iron/kg showed
no difference in counts of Lactobacillus delbruckii ssp. Bulgaricus and Streptococcus
thermophilus after one day of storage from unfortified yogurt. But there was a significant
difference after 30 days of storage (Gaucheron 2000) but no increase in chemical
oxidation was detected. Many studies have been carried out on iron fortification of
yogurt. It is well known that major off-flavor is associated with fortified dairy product due
to catalyzed lipid oxidation by iron salts. Fresh yogurts fortified with iron from different
iron compounds are affected differently (El-Kholy and others 2011).
2.4. Iron requirements by the body
Recommended daily intake of dietary iron for normal infants are 1 mg iron per kg
per day and for children, male and female adolescents are 10, 12 and 15 mg per day
respectively, and adult men and postmenopausal women require only 10 mg per day
7

(Martinez-Navarrete and others, 2001). While pregnant and lactating mothers require
27mg and 10mg per day respectively. At birth, most term infants have 75 mg of
elemental iron per kilogram of body weight, found primarily as hemoglobin (75%), body
storage (15%) and tissue protein (10%) (Oski,1982). Infants of mothers with poorly
controlled diabetes and small-for-gestational-age infants have approximately 10% and
40% of normal storage iron, respectively, meaning that they may have less of a buffer
for protection from postnatal iron deficiency (Petry and others, 1992; Georgieff and
others, 1995). Because more than 80% of the iron of the newborn term infant is
accreted during the third trimester of gestation, infants born before term must accrete
more iron postnatally to catch up to their term counterparts during the first year. Thus,
the requirements for preterm infants range from 2 mg/kg per day for infants with birth
weights between 1500 and 2500 g (American Academy of Pediatrics, 1976) to 4 mg/kg
per day for infants weighing less than 1500 g at birth (Siimes and Jarvenpaa, 1982).
2.5. Iron fortification compounds
The most common iron fortification compounds can be classified into three
groups according to solubility: group 1, freely water-soluble iron (ferrous sulfate, ferrous
gluconate, ferrous lactate); group 2, poorly water-soluble iron or soluble in diluted acids
(ferrous fumarate, ferrous succinate); and group 3, water-insoluble iron or poorly soluble
in diluted acids (ferric orthophosphate, ferric pyrophosphate, elemental iron (Boccio and
others1997). Group 1 compounds can be completely dissolved and thus provide very
high bioavailable iron. However, they have the disadvantage of freely interacting with
the fortified food, which may alter its sensory properties. This can happen because iron
catalyzes oxidative processes and thus provokes fat rancidity. This catalytic oxidation
process may occur with other nutrients such as vitamins and amino acids, thus
8

decreasing the nutritional value of the food (Boccio and others 1997). Group 2
compounds have good solubility and thus good bioavailability. However, they have the
disadvantage of being used only in solid dehydrated food because they do not dissolve
in neutral liquids, in which they precipitate. In this last situation, the free fraction of iron
interacts with the constitutive elements of the food to decrease its nutritional value and
alter its sensory characteristics. Their advantage is that they cause far fewer negative
effects on food sensory attributes than freely water-soluble compounds and they still
readily enter the common iron pool during digestion. They have been suggested for use
in infant cereals (Hurrell, 1997a) and chocolate drink powders. Group 3 compounds
have a very low solubility. Thus, although they do not change the sensory properties or
nutritional value of the food, they have the disadvantage of having very low
bioavailability (Hurrell, 1997a; Boccio and others 1997).
Highly soluble compounds of iron like ferrous sulfate are desirable for food
fortification but cannot be used in many food vehicles hence less absorbed forms of iron
are commonly used in food fortification (Boccio and others 1997). Inorganic iron
compounds added to whole cow’s milk are poorly absorbed, because the compounds
attach extensively to whey proteins, casein micelles, salts, and fat droplets, reducing its
solubility. However, organic compounds of iron like ferrous lactate and ferrous
gluconate absorb more easily to the water phase of milk than ferrous sulfate
(Villalpando and others 2006). When the diet does not satisfy the body’s iron
requirements, nutritional deficiency of this element may occur. If this situation is not
reversed, anemia may result therefore it is important to select fortification compounds
and foods that will not diminish iron bioavailability to the body (Boccio and others 1997).
This necessitates the careful selection of both the food product to be fortified and the

9

iron fortification compound to be added. Clearly, the iron compound must be first
optimized with respect to relative bioavailability. Ferrous bisglycinate, ferrous lactate
and ferrous sulfate microencapsulate have the following iron content ; 23%, 19% and
18% respectively. Ferrous bisglycinate (brown) and ferrous lactate (white) are found in
powdery form while ferrous sulfate (granulated cream white) is sometimes
microencapsulated with approximately 50% of vegetable fat. Microencapsulation is the
technology of packaging solid, liquid and gaseous materials in small capsules that
release their contents at controlled rates over prolonged period of time (Selaiman and
Sara, 2011). This keeps the iron from coming into contact with food, reducing the
chances of interactions that happens when conventional iron compounds are used
(Gaucheron 2000).
However, if the food vehicle contains potent inhibitors of absorption, the added
iron, like the native iron, will be poorly absorbed and will have little or no impact on the
iron status of the consumer. The success of a food fortification program thus depends
heavily on the absorbability of the added iron and its protection from major dietary
absorption inhibitors. For example, phytate, polyphenols and a satisfactory iron status in
an individual will diminish absorption, whereas vitamin C or low iron status will enhance
absorption (Hurrell 1997b). World Health Organization (WHO) Guidelines 2005
recommend the following fortificants in order of preference: ferrous sulfate, ferrous
fumarate, encapsulated ferrous sulphate, encapsulated ferrous fumarate, electrolytic
iron (added at twice the level of ferrous sulphate), ferric pyrophosphate (added at twice
the level of ferrous sulphate) and (ferric sodium ethylenediaminetetraacetic acid
(NaFeEDTA). The recent efficacy studies which have followed the guidelines have

10

demonstrated that prolonged consumption of iron fortified foods greatly improves iron
status of the consumers (Richard 1997b).

Table 2: Summary of iron salts used in food fortification.
classification

iron compound advantage

disadvantage
Freely interact with
fortified food altering

ferrous sulfate
1. Freely water
soluble

sensory properties

ferrous gluconate

because iron

ferrous lactate

high bioavailability

catalyzes oxidation.
suitable for solid

good solubility and
bioavailability,

soluble or

because they don’t

cause far fewer

2. Poorly water

dehydrated foods

dissolve in neutral

soluble in

ferrous fumerate

organoleptic

liquid, they

dilute acid

ferrous succinate

problems

precipitate

3. Water

ferric

insoluble or

orthophosphate

Don’t change

poorly

ferric

sensory or

soluble in

pyrophosphate

nutritional value of

low solubility and

dilute acids

elemental iron

the food.

bioavailability

2.6. Effects of Iron deficiency
Iron deficiency is most common in poor countries of the developing world. About
half of these iron deficient individuals develop iron deficiency anemia (IDA). Iron

11

deficiency is therefore a major health problem ranked by World Health Organization
(WHO) as 7th out of 10 major preventable risks for diseases and death that together
account for 40% of the 56 million deaths that occur world-wide each year (Hurrell
1997b). Iron fortification offers a more cost effective approach to providing additional
iron to some segment of the population. Studies of 3 month old infants have shown that
iron fortification of infant milk powder with ferrous sulfate significantly reduces anemia.
After 15 months the prevalence of anemia in a group receiving non-fortified milk was
35% and 13% in infant consuming fortified milk. Iron status was significantly improved
when ferrous bisglycinate was added to flavored milk in Saudi Arabia. Pilot fortification
trials in developing countries have given promising results but there are no major
success stories except for Chile due to lack of political commitment, insufficient funding,
too little technical support from local or multinational industry, poor distribution network,
or lack of nutrition education program for the consumers which are necessary for
successful fortification program (Hurrell, 1997a). There are also other factors that affect
the success of any fortification program. However if low bioavailability of food iron is the
determinant of iron deficiency anemia in developing countries, increasing the supply of
absorbable iron should decrease the prevalence of iron deficiency anemia (Hurrell and
Sean, 1997).
Many food vehicles for iron fortification contain substances that inhibit iron
absorption. Cereals contain phytic acids and polyphenols, milk contains calcium and
caseins while chocolate drinks contain polyphenols (Hurrell 1997a). The presence of
phytate, the major phosphorus storage compound in grain, has been associated with
reduced mineral absorption due to the structure of phyate which has high density of
negatively charged phosphate groups which form very stable complexes with mineral

12

ions causing non-availability for intestinal absorption (Walter and others, 2002).
Polyphenols also forms insoluble complexes with iron thereby reducing its bioavailability
to the body. The inhibitory effect of calcium on iron absorption was recognized many
years ago. Different studies have been conducted but they often give conflicting results
because several factors influence the interaction between calcium and iron absorption
(Allen, 1996). These factors include molar ratio of calcium to iron, forms of calcium and
iron and iron status of the subject. Calcium inhibits absorption of both heme and
nonheme. It enters into the mucosal cells by different pathways and leave in the same
form which implies that calcium inhibit the intracellular transport of iron (Hallberg 1992).
It also competes for iron binding sites in mobilferrin, a protein in the duodenal mucosa
that assists iron transport through the cell (Conrad and Umbreit 1993), and inhibits the
release of iron from mucosal cells into circulation (Wienk and others 1996). In addition,
many diets in developing countries to which fortified salt, sugar and other condiments
are added are high in phytic acid and polyphenols especially cereal and legume foods.
To ensure a level of absorption that is high enough to improve or maintain iron status, it
is necessary to prevent the fortification iron from reacting with the absorption inhibitors
that are inherently present in those foods. This can be accomplished by adding
absorption enhancers. The most common enhancer is vitamin C. Alternatives would be
bovine hemoglobin and NaFeEDTA where iron is in a protected form. Vitamin C can
increase absorption of both native iron and fortification iron several folds due to both its
reducing power and chelating action (Hurrel 1997). It can reduce ferric to ferrous iron
and/or maintain ferrous iron in the ferrous state and so prevent or decrease the
formation of insoluble complexes with absorption inhibitors or with hydroxide ion in the
gut. In addition, it can form soluble complexes with iron at low pH that remains soluble

13

and absorbable at the more alkaline duodenal pH. Layrisse and others (1974) reported
a sixfold increase in iron absorption (1.4% to 7.9%) by adult peasants in Venezuela who
consumed 100 g maize containing 2.8 mg iron and 70 mg added vitamin C. Similarly,
Cook and Monsen, (1976) reported that iron absorption in young men fed a liquid
formula meal containing 4.1 mg iron increased from 0.8% to 7.1% as vitamin C was
increased from 25 to 1000 mg. More recently, Siegenberg and others, (1991) reported
that the effect of vitamin C on phytate and polyphenols was dose dependent and that as
little as 30 mg vitamin C could completely overcome the effect of phytic acid (58 mg
phytate phosphorus) in maize bran added to white bread, whereas greater than 50 mg
vitamin C overcame the negative effect of meals containing greater than 100 mg
polyphenols added as tannic acid.
In a milk-based infant formula fortified with 15 mg iron as ferrous sulfate per liter,
iron absorption by infants was only 3% in the absence of vitamin C but increased to 5%
with 100 mg vitamin C per liter and to 8% with 200 mg per Liter (Sketel and others,
1986) The relatively low iron bioavailability from milk products can be assumed to be
due to the presence of two inhibitory factors, calcium (Hallberg and others, 1991) and
the milk protein casein (Hurrell and others, 1989). In a series of fortification trials in
Chile in which iron-fortified formulas were fed to infants, the improvement of iron status
was only modest in the absence of vitamin C but improved considerably when it was
added to formula (Walter and others, 1990). The widespread consumption of ironfortified and vitamin C-fortified formulas by infants in the United States is regarded as
the reason for the dramatic fall in the prevalence of anemia over the last 30 years (Yip
and others, 1987).

14

2.7. Sensory effects
Iron catalyzes lipid oxidation that results in rancidity and development of off
flavor. Fortification with FeCl3, FeSO4 or ferric/ferrous ammonium sulfate causes off
flavor and high TBA numbers (Gaucheron 2000). However these effects can be reduced
significantly. Ferric phosphate, ferric pyrophosphate or ferric ammonium citrate
produced slight flavor change when added to milk followed by pasteurization. Different
iron fortificants affect the food product differently. For example ferrous bisglycinate is
advantageous over NaFeEDTA because it has GRAS status. However it readily
promotes fat oxidation especially in cereals and undesirable color reactions occur in
some foods. Ferrous bisglycinate is suitable for fortification of commercial food products
such as liquid milk (Gaucheron 2000).
Many iron compounds are colored and cannot be used to fortify light-colored
foods. In addition, the more soluble iron compounds often react with substances in
foods, causing discoloration. It is reported that ferrous sulfate, ferrous lactate, ferrous
gluconate, and ferric ammonium citrate, as well as the less soluble ferrous fumarate and
ferric citrate, produce off-colors when added to a chocolate milk drink (Hurrell 1997b).
Whole milk could also be considered as a vehicle for iron fortification, but because of
the presence of calcium and casein, an absorption enhancer should be added to
improve absorption. Unfortunately, it is difficult to add vitamin C to fluid milk and it has
been reported to degrade rapidly to diketogluconic acid leading to changes in flavor
(Hegenauer and others, 1979).

Many soluble iron compounds rapidly produce off-

flavors when added to milk, owing to the promotion of lipolytic rancidity, oxidative
rancidity by the oxidation of free fatty acids, and the partial or complete loss of vitamins
A, C, and ß-carotene (Cocodrilli and others, 1985).

15

Pentane, a product of fat oxidation, is the major hydrocarbon formed by the
oxidative degradation of linoleic acid, and its formation correlates with the production of
off-flavors. Ferrous sulfate and ferrous gluconate rapidly generated pentane and were
judged unacceptable by a sensory panel after 4 to 6 weeks of storage. Ferric
pyrophosphate and reduced elemental iron generated far less pentane and still had
acceptable sensory characteristics after 7 weeks of storage (Hurrell 1997b).
Both ferrous sulfate and ferrous fumarate are available commercially in
encapsulated form. The coating usually made from partially hydrogenated oil from
soybean and cottonseed, or ethyl cellulose can prevent fat oxidation in infant formulas
fortified with the easily oxidized long chain polyunsaturated fatty acids (Hurrell
1997b).This keeps the iron from coming into contact with food, reducing the chances of
interactions that happens when conventional iron compounds are used (Gaucheron
2000).
2.8.

Determination of Lipid Oxidation
Lipid oxidation is a complex process following free radical chain reactions. Lipids

do oxidize by a radical chain mechanism through initiation, propagation, and termination
stages. This reaction can be catalyzed my metals like iron and copper, light, heat,
enzymes like lipoxygenase and other factors. The initiation stage is characterized by
formation of highly reactive free radicals. The free radicals react with oxygen to form
hydroperoxides (ROOH) and more free radicals. Hydroperoxides formed degrade and
form aldehydes, secondary compounds of lipid oxidation. Some hydroperoxide branch
and form more free radicals during propagation stage. In the final part of the reaction
free radicals react with each other to form polymers, non-radical monomer products like

16

ketones, ethers, alkanes (Schaich 2005). This lipid auto-oxidative degradation results in
products’ change in food quality, e.g. aroma, flavor, texture and also the nutritive value.
Lipid oxidation can be detected using different ways. There is no single test
available to measure all oxidative events at once, at all stages of oxidative process and
applicable to all types of foods. Some of the most commonly used methods to
determine lipid oxidation are Peroxide Values (PV) and Thiobarbituric Acid (TBA) tests.
2.8.1 Peroxide Value (PV) test.
Peroxide Value (PV) is one of the most commonly used methods to determine
lipid oxidation due to its simplicity. It is based on the reduction of hydroperoxide group
hence it is more sensitive in detecting early stages of oxidation. The amount of iodine
liberated is proportional to the amount of peroxide present in food sample. Released
iodine (I ) is assessed by titration against a standardized solution of Na S O using a
2

2 2

3

starch indicator. The major disadvantage of this method is that it does not measure low
PV due to difficulty in determining end point in a titration procedure.
2.8.2 Thiobarbituric Acid (TBA) test
Hydroperoxides are labile species which undergo changes and deterioration with
the radicals. Their breakage causes secondary products such as pentanal, hexanal, 4hydroxynonenal and malondialdehyde (MDA) (Fernandez and others 1997). MDA is a
three-carbon dialdehyde with carbonyl groups at the C-1 and C-3 positions and is
produced during lipid oxidation in oils containing linolenic or arachidonic acid.
MDA production is partially due to the secondary oxidation of primary carbonyl
compounds e.g. 2-nonenal (Sinnhuber & Yu, 1977). TBA test is based on the MDA
reaction with TBA reagent to obtain a red/pink pigment (chromagen), which results from

17

the condensation of two molecules of TBA with one molecule of MDA and the probable
elimination of two molecules of water with absorbance at 530 nm. The major
disadvantage of this method is that it measures later stages of oxidation as MDA is one
of the byproducts of hydroperoxides breakdown. TBA can also be interfered with a
number of compounds like amino acids and carbohydrates in the presence of iron
(Fernandez and others 1997).
The reaction with TBA occurs by attack of the monoenolic form of MDA on the active
methylene groups of TBA. The intensity of color is a measure of MDA concentration and
has been organoleptically correlated with the rancidity. The mechanism of
malonaldehyde liberation from linolenic acid has been suggested. However secondary
products from linoleic acid also form a red pigment with the TBA test (Asakawa and
Matsushita, 1979)

18

3.0.

RATIONALE AND SIGNIFICANCE

Iron deficiency is one of the three major micronutrient deficiency disorders in Malawi.
Others are Vitamin A deficiency (VAD) and Iodine deficiency (ID). The Government of
Malawi (GoM) is currently implementing fortification of sugar and encouraging use of
iodized salt to reduce VAD and ID respectively. There are reported cases of anemia in
Malawi. Nationally prevalence of anemia in both children and mother are still high.
Malawi Demograpic Health Survey (MDHS) 2010 reveals that 64 percent of children
ages 6-59 months are anemic; 24 percent have mild anemia, 37 percent have moderate
anemia, and 3 percent have severe anemia. Children in rural areas (65 percent) have a
higher anemia prevalence compared with children in urban areas (53 percent). Among
the districts, anemia prevalence ranges from a high of 77 percent in Chikhwawa to a low
of 46 percent in Chiradzulu. It also indicates that 29 percent of women are anemic; 22
percent have mild anemia, 7 percent have moderate anemia, and 1 percent has severe
anemia. Although there is moderate variation by urban-rural residence and region,
differences vary greatly by district, ranging from a high of 51 percent having anemia in
Mangochi to a low of 18 percent in Chitipa. This has been a serious problem for a long
time as indicated by one study that was conducted more than 10 years earlier and
showed a similar trend. It showed that between July 1997 and June 1998, the
prevalence of all anemia (Hb < 11g/dl) in a population of urban women (n=4708)
attending antenatal clinic at Queen Elizabeth Hospital in Blantyre was 57.1% and the
prevalence of severe anemia (Hb< 7g/dl) was 3.6%. In a rural area (Namitambo Health
Centre in Chiradzulu District) prevalence of anemia and severe anemia in pregnant
women (n=2293) was 72% and 4%, respectively. A second study specifically measuring
prevalence in an unselected group of women attending rural (Chiradzulu) and semi-

19

urban (Mangochi) antenatal clinics reported a prevalence of 58% (n=729) (Munasinghe
and Van deBroek 2006). Other interventions like promotion of dietary diversification and
iron supplementation, provision of iron tablets to pregnant women at antenatal clinic,
has been used for a long time but prevalence of iron deficiency still remains high
especially in young children, pregnant and lactating women (MDHS, 2004). Iron found in
human milk is far more bioavailable, resulting in much lower rates of iron-deficiency
anemia in children that are exclusively breastfed compared to children that rely on lowiron cow milk formula. Nevertheless, 6% to 20% of exclusively breastfed infants remain
at risk for reduced iron stores. A higher rate (20%–30%) of iron deficiency has been
reported in breastfed infants who were not exclusively breastfed (Anonymous, 1976)
MDHS 2010 reported 53.2 % and 65.2% of urban and rural children respectively and
25.3% and 30.0% of urban and rural women respectively were anemic. This recent
report shows that anemia is still a serious problem in Malawi despite all the
interventions taking place.
Iron nutrition is particularly important during the weaning period, when the infant is
growing rapidly and has a high demand for iron. Cereal porridges are common
complementary foods during the weaning period and often provide much of the dietary
iron intake because the iron contribution from human milk is low (Hurrell and others,
2003). Because of the high phytate content of cereal porridges, iron absorption of native
iron and fortification iron may be very low (Hurrell and others, 2003; Lorenz and others,
2007)). One mole of phytic acid binds 6 moles ferric irons so that even relatively small
quantities of residual phytate are still strongly inhibitory (Hurrell and others, 2003).
Studies indicated that adding 10 mg/100 g phytic acid to bread rolls decreased iron
absorption by 20% and that adding 20 mg/100 g decreased iron absorption by 40%

20

(Hurrell and others, 2003). Phytate: iron molar ratios greater than 0.15 are regarded as
indicative of poor iron bioavailability.
Iron supplementation is the main strategy used in developing countries to combat
IDA. Although supplementation is a reliable strategy to prevent anemia, the problems
with poor compliance, low bioavailability, and the often poorly managed distribution
systems have reduced the effectiveness of this approach (Bovell-Benjamin and Guinard
2003). The recommended daily allowance (RDA) for iron for a normal infant is 1 mg per
kg per day and for children and male and female adolescents are 10, 12, 15 mg per
day. A pregnant mother requires 27mg iron per day while breast feeding mother
requires 10mg iron per day. In the United States of America use of iron fortified infant
formulas from 1970s to the late 1980s was a success. During this period formulas were
fortified with 10-12mg/L of iron. The rate of iron deficiency anemia dropped from 20% to
less than 3% (Anonymous, 1989). Determination of acceptable range of iron
concentration depends on standard used to assess iron sufficiency. In the US, iron
concentration of iron fortified formulas range from 10mg/L to 12mg/L. the US Food and
Drug Administration (FDA) recommends that iron fortification should not be less than
6.7mg/L. In Europe, infant formula tends to contain 4mg/L to 7mg/L (Anonymous, 1999).
Malawi has experienced an increasing number of people consuming milk and milk
products both in urban and rural areas with 80% consumed in the form of pasteurized
liquid milk (Anonymous, 2004). However, the milk contains little iron with no impact on
reducing iron deficiency. Therefore fortifying milk with iron will increase iron intake to the
populace which may, in the long run, contribute to reducing the prevalence of iron
deficiency disorder (IDD). Therefore the outcome of this study will help the GoM and its
development partners to strategize ways to reduce iron deficiency through fortification of

21

milk. Its seeks to increase the iron content of iron fortified pasteurized liquid milk and
yogurt by 20-30% per 200mL serving which has a potential to increase dietary iron
intake.
There have been extensive studies on iron fortification of both fermented and
unfermented milk and milk products. Different iron compounds, their advantages and
disadvantages have been clearly documented. Most of the iron fortification that have
been done focused much on solid and semi-solid milk products. Little had been done on
fortification of pasteurized liquid milk and yogurt. This research seeks to establish how
the quality of pasteurized liquid milk and yogurt fortified with ferrous bisglycinate, ferrous
lactate and ferrous sulfate microencapsulate will change over a given storage time.

4.0. OBJECTIVES
This study hypothesizes that fortification of pasteurized milk and yogurt will
enhance milk and yogurt iron content and result in increased dietary iron intake and
consequently reduce prevalence of iron deficiency.
4.1. Long term objective:
To increase dietary iron intake through increased consumption of iron fortified milk and
yogurt.
4.2. Specific objectives:
1.

To determine effects of microencapsulated ferrous sulfate, ferrous
bisglycinate, and ferrous lactate on yogurt culture growth.

2.

To evaluate the sensory quality and storage stability of iron fortified
pasteurized milk and yogurt.

22

5.0. MATERIALS AND METHOD
5.1. Materials:
Whole cow’s milk (Michigan Milk Producers Association, Ovid, MI, USA) for
yogurt production was provided by Michigan State University Dairy Plant, East Lansing,
MI. Milk for production of pasteurized milk was purchased from Bunda College Student
Farm, Lilongwe, MW. Ferrous bisglycinate, ferrous lactate

and microencapsulated

ferrous sulfate with 50% vegetable fats were provided by Dr. Paul Lohmann Inc, US.
Commercial Hansen’s DVS yogurt culture Yo-fast containing Lactobacillus delbruckii
ssp Bulgaricus and Streptococcus thermophilus was purchased from Chris Hansen
Milwaukee, WI. MRS agar (Difco, USA) was obtained from Microbiology Laboratory at
Michigan State University, MI USA. Chloroform, glacial acetic acid, Thiobarbituric acid
(TBA), starch, sodium hydroxide, toluene, sodium thiosulfate (Sigma-Aldrich, USA) were
also used. However some of chloroform and glacial acetic acid used for milk analysis
were purchased from Lab Enterprises, Blantyre, MW. Additional ingredients that were
used to manufacture yogurt were sucrose from Michigan Sugar Company (Saginaw, MI,
USA), stabilizer (Continental Custom Ingredients, Chicago, IL, USA), non-fat dry milk
solids (Michigan Milk Producers Association, Ovid, MI, USA) and strawberry puree
(Kraus & Co., Walled Lake, MI, USA)
5.2. Starter culture growth and activity
Reconstituted skim milk (12% w/v) was fortified with ferrous bisglycinate
(63mg/kg), ferrous lactate (79mg/kg) and sulfate dried microencapsulate (83mg/kg) (Dr
Paul Lohmann Inc, US). The fortification levels were different because ferrous
bisglycinate, ferrous lactate and ferrous sulfate microencapsulate have different iron
content so there was a need to harmonize iron in the final products. Ferrous
23

bisglycinate has approximately 23% iron. Ferrous lactate and ferrous sulfate
microencapsulate have approximately 19% and 18% iron respectively. In order to obtain
a product that has approximately 15mg/kg of iron, which can contribute 20-30% towards
RDA for different categories of people when 200ml of fortified product is consumed,
there was a need to compute how much of each iron salt should be used for fortification.
Using same levels of fortification for all iron salts would mean that more iron would be
present in one sample and less iron in the other sample. This would undoubtedly affect
both the sensory and chemical analysis results.

Iron compound was omitted from

control treatment. The reconstituted milk was then sterilized at 121 0C for 5 minutes.
Each flask was cooled to 35 0C and inoculated with 1% (wt/wt) commercial Hansen’s
yogurt culture Yo-fast 10, containing Lactobacillus delbruckii ssp bulgaricus and
Streptococus thermophilus, and incubated at 35 0C for 6 hours. Growth of lactic acid
bacteria was determined by sampling at 1.5 hour interval and plating on de Man
Rogosa, and Sharpe (MRS) agar (Difco). Plates were incubated at 350C, 48 hours
under aerobic conditions. After incubation the colonies formed were counted and results
expressed as CFU/ml.
5.3. Skim milk and yogurt titratable acidity (TA)
Prior to determining titratable acidity (TA) the samples were thoroughly mixed.
Nine grams of each sample was placed in Erlenmeyer flask to which 4 drops of
phenolphthalein indicator was added. The mixture was titrated with 0.1N NaOH to the
first permanent shade of pink.
Percent TA was calculated as follows:

24

%TA = (ml of NaOH × N of NaOH × meq.wt lactic acid)/ wt of

sample

Where ml = milliliter of sodium hydroxide used, meq = milliequivalent weight in
which 1ml of 0.1N NaOH =0.009008g C3H6O5 , wt = weight of sample.
5.4. Yogurt manufacture
Four batches of yogurt were manufactured at Michigan State University Dairy
Plant

using the following formulation; 80.5% whole milk, 4.0% nonfat dry milk (NDM)

(Michigan Milk Producers Association, Ovid, MI, USA), 0.5% stabilizer (Continental
Custom Ingredients, Chicago, IL, USA) and 5.0% sucrose (Michigan Sugar Company,
Saginaw, MI, USA). The whole milk was fortified with ferrous bisglycinate (63 mg/kg),
ferrous sulfate microencapsulate (83 mg/kg) or ferrous lactate (79 mg/kg). Control
yogurt had no iron salts added. The yogurt mix was warmed to 60oC, homogenized dual
stage 2000, 500 psi and pasteurized at 850C for 30 min then cooled to 43 0C. It was
inoculated with 1% (wt/wt) commercial Hansen’s yogurt culture Yo-fast 10, containing
Lactobacillus delbruckii ssp bulgaricus and Streptococus thermophilus and incubated at
43 0C until pH 4.6 was reached. Finally, 10% strawberry puree (Kraus & Co., Walled
Lake, MI, USA) was added as flavorant.
Yogurt for each treatment was divided and stored into two separate containers.
One part was used for sensory analysis while the other was used for chemical analysis
of lipid oxidation. Yogurt for sensory analysis was separated and stored at refrigeration
temperature for one week before sensory evaluation was done in order to give time for
the iron to fully interact with milk component. It was feared that if the sensory analysis
25

was done immediately after manufacturing yogurt the sensory results would not reflect
sensory
refl
the actual sensory attributes of stored yogurt as commercial yogurt spend some time in
spen
storage or on shelves before it is bought and consumed by consumers. Yogurt for
chemical analysis was also stored at refrigeration temperature and the analysis was
done after every 5 days till 35 days storage period was reached. This period was
.
chosen because it approximates the shelf life of yogurt under refrigeration.
mix fortified milk blend, stabilizer and sweetener

homogenize dual stage 2000, 500 psi at 60 oC

heat treatment 85 oC, 30 min

cool to 43 oC

inoculate
(1.0% Hansen’s yogurt culture Yo-fast 10, Lactobacillus delbruckii ssp
Bulgaricus and Streptococus thermophilus

incubate at 43 OC until pH 4.6

blend strawberry puree (10%)

package in 8 oz cup, cool and store at 4 OC

Figure 2: Flow diagram showing stages during processing of yogurt.
:

26

5.5. Processing of pasteurized milk
Whole milk (Bunda College Student Farm, Lilongwe, MW) was fortified with
ferrous bisglycinate (63 mg/kg), ferrous lactate (79 mg/kg) and ferrous sulfate (83
mg/kg). No iron salts was added to control treatment. Batch pasteurization was used to
pasteurize milk from each treatment separately where milk was heated in a container to
63OC and held at this temperature for 30 minutes. Thermometer was used to detect
temperature changes. While milk was being pasteurized bottles were being heated at
temperature around 100OC to reduce recontamination of pasteurized milk. When the
pasteurization temperature and time was reached, the milk was then cooled and packed
in bottles

and stored under refrigeration temperature. Ten bottles were prepared for

each treatment; therefore 40 bottles were prepared for all the four treatments. At each
testing interval a single bottle from each treatment was used and the remaining contents
were discarded.

27

Whole milk

Iron salt fortification

Batch pasteurization
(63 OC ) for 30min

Cool to room temperature (25 oC) packaged in heated containers
(100 oC) pasteurization

Bottled and stored at 4 oC

Figure 3: Flow diagram showing stages during processing of pasteurized milk
:

5.5. Determination of pH
The pH meter was used to measure the pH of the samples. The tip of the pH
meter sits in a buffer which is standardized to a neutral pH 7. Small samples of yogurt
were taken from each treatment for measure
measurements. The electrode was inserted in
.
yogurt for few minutes and was read when it stabilized. This was repeated for each
stabilized.
sample and cleaning the electrode with distilled water after using on each sample and
finally the tip for the electrode was dipped in a buffer solution.
28

5.6. Peroxide value determination
Yogurt was diluted with distilled water to 20% solution. Five grams of 20% yogurt
solution was put in 250 ml Erlenmeyer flask to which 30 ml of acetic /chloroform solution
was added and swirled in order to dissolve the sample. 0.5 ml of saturated KI was
added by pipette and the solution was allowed to stand while occasionally shaken for 1
minute. Thirty mL of distilled water and 1 mL starch indicator (1% starch) was added.
0.01 N sodium thiosulphate solution was titrated immediately until the brown (or yellow)
color disappeared representing end point. This procedure was repeated for pasteurized
liquid milk where 5g sample was used in place of 20% yogurt. The determination on the
blank was done on 30 mL acetic acid/chloroform solution + 0.5 mL KI solution +30 mL
distilled water + 1mL starch indicator using 0.001N sodium thiosulfate as a titrant. The
peroxide values were expressed as milliequivalents of peroxide per 1000g sample
using.
PV was calculated as follows:

PV = [(S-B)×N×1000] / weight of samples in grams

where S = mL of thiosulfate required for titration, B= mL of thiosulphate required for the
blank and N= normality of thiosulphate solution.
5.7. Thiobarbituric acid (TBA) value
One gram yogurt was diluted with 100ml distilled water then 3.0 g diluted yogurt
was put in Erlenmeyer flask (125-300ml) separately, and 10 ml toluene was added to
samples separately. Then 10 ml of TBA reagent which was prepared by dissolving 1g
29

of TBA powder in 75ml of 0.1N NaOH and diluted to 100ml with distilled water according
to Sinnhuber and Yu, (1977), was pipetted (using a pipette bulb) into the flask. The
flasks were swirled and shake frequently for four minutes. The entire content was
transferred into 250 ml separatory funnel. The layers was allowed to separate and the
lower layer was collected in a screw cap test tube (18 ×145 mm or 25 ×200mm). The
tube was heated in boiling water for 30 minute. The test tube was cooled under running
water and transferred the portion of the sample to the Spectronic 20 cuvette. The
absorbance of the sample at 530nm was read using distilled water as the blank.
Average absorbance ×100 as the TBA values is reported with the result normalized per
g of sample.
TBA was calculated as follows:
TBA = Absorbance × 100)/g of sample

5.8. Sensory evaluation
5.8.1. Yogurt
Consumer acceptance sensory test was carried out in which four samples of
yogurt fortified with ferrous bisglycinate (63mg/kg), ferrous lactate (79mg/kg), ferrous
sulfate microencapsulated (83 mg/kg) and a control treatment were presented to 100
untrained panelists upon approval by University Committee on Research Involving
Human Subject (UCRIHS). The panelists were recruited by posting flyers around
Michigan State University (MSU) campus and by sending emails containing the flyer to
different departments at MSU. Ninety eight panelists consisting mainly graduate
students, undergraduate students and faculty at MSU. Sensory evaluation was

30

conducted in individual booths in the sensory laboratory in the Department of Food and
Human Nutrition at MSU. Upon arrival at the sensory laboratory each subject read an
explanation of the study and gave their informed consent. Yogurt samples were put into
2 oz plastic cups labeled with randomly selected three digit numbers and refrigerated till
time of evaluation. The samples were presented in a randomized manner across
subjects to ensure that the order did not introduce bias into the results. Subjects were
asked to taste and evaluate all four samples and indicated their degree of liking on a
nine point hedonic scale from 1=dislike extremely to 9=like extremely and 5=neither like
nor dislike. The panelist evaluated each sample for likeability of the appearance, body
texture, flavor and overall acceptance. Panelists were provided with purified water at
room temperature for rinsing between samples.
5.8.2. Pasteurized Milk
Pasteurized milk was manufactured at Bunda College Food Laboratory in the
Department of Home Economics and Human Nutrition. The milk was stored for 2 days
at refrigeration temperature before consumer acceptance sensory test was done. The
milk was evaluated using consumer panel. The panelists were recruited by flyer pasted
on different notice boards around Bunda College campus. One hundred panelists
consisting primarily of undergraduate students, graduate students and staff members
participated in the sensory evaluation. Upon arrival at the food laboratory each subject
read an explanation of the study and gave their informed consent. There was not a
separate approval for this evaluation because the UCRIHS approval was for both yogurt
and pasteurized milk. Fifty millimeters of each sample were put in a hundred millimeters
cup labeled with randomly selected three digit numbers. The order of presentation was
randomized. Subjects were asked to taste all the four samples and indicate their degree
31

of liking on a nine point hedonic scale from 1=dislike extremely to 9= like extremely and
5=neither like nor dislike. The panelists evaluated the samples based on appearance,
mouth feel, taste, flavor and overall acceptance. The panelists were provided with water
for rinsing between samples
5.9 Statistical analysis
Statistical parameters that were generated were mean, standard deviation and
one way analysis of variance (ANOVA) for sensory, TA, PV and TBA data. Statistical
software IBM SPSS Statistics 20 was used for statistical analysis of the results. For
yogurt sensory analysis SIMS 2000 for windows was used to generate means and
statistical differences. Least Significant Difference (LSD) at p<0.05 was used to
determine significant differences between means from all the treatments. All
experiments were replicated two times except for starter culture growth and activity
experiment which was done in one replicate with two analyses.

32

6.0 RESULTS AND DISCUSSIONS
6.1. Effect of iron salts on starter culture growth and activity
Figure 4 shows that the growth of commercial Hansen’s yogurt culture Yo-fast
10 containing Lactobacillus delbruckii ssp Bulgalicus and Streptococus thermophilus
were different from each other amongst treatments. Though at time 0 hr the bacterial
count was almost the same for all treatments there was steady increase in counts as
time progressed. After 6 hours both ferrous bisglycinate and ferrous sulfate had the
highest bacterial population indicating that they supported bacterial growth the best.
None of the iron salts were inhibitory to the growth of the yogurt culture.
However, the growth of yogurt starter culture was affected differently. In the
control treatment the growth was slower but after three hours the growth rate increased
significantly which shows that there was rapid increase in number of bacteria. Growth of
yogurt starter culture in the presence of ferrous lactate was rapid in the first one and a
half hours and later the graph flattens before rapid increase after three hours. This
shows that in the first 1.5 hours ferrous lactate enhanced bacterial growth more than
any other treatment. At the same time this behavior as compared to a control treatment
shows that ferrous lactate was not as supportive of yogurt starter culture growth as the
rest of the treatments. There was more growth in ferrous bisglycinate which is indicative
of a more supportive effect of ferrous bisglycinate. The general picture from Figure 4 is
that the three iron salts, ferrous bisglycinate, ferrous lactate and ferrous sulfate
microencapsulate were more favorable to the growth of yogurt culture growth as shown
by having shortest lag phase.

33

160

140

120

cfu/ml (*100000)

100

80

Control

60

Ferrous
Bisglycinate
Ferrous
Lactate

40

Ferrous
Sulfate
20

0
0.0

1.5
3.0
4.5
Time (hours)

6.0

Figure 4: Effects of iron salts on growth and activities of Lactobacillus delbrueckii
subsp. bulgaricus and Streptococuss thermophilus in iron fortified reconstituted
skim milk incubated at 35 OC for 6 hours

34

The control treatment had the longest lag phase. This shows that fortifying skim milk
with iron enhanced bacterial growth to a larger extent in ferrous bisglycinate and ferrous
sulfate microencapsulated than in ferrous lactate enriched yogurt. This contradicts what
Hekmat and MacMahon (1997) reported. They reported that counts of L. delbrueckii ssp
bulgaricus and S. thermophilus after one day of storage in iron fortified skim yogurt were
not significantly different from counts in unfortified yogurt. It was therefore concluded
that starter culture growth was independent of whether or not the milk had been fortified
with iron. However this conclusion was based on casein-chelated iron and whey proteinchelated iron compounds which are different from those used in the present study.
6.2. Titratable acidity and pH for skim milk
Results presented in Figure 6 show that there was a steady increase in TA in all
the treatments as time progressed though TA increases were not significantly different
among treatments at each testing interval. This means that there was increase in
microbial activities and microbial growth which resulted in more lactic acid being
produced by lactic acid bacteria. This is especially true of Lactobacillus delbruckii ssp
bulgalicus and Streptococus thermophilus. Hatkins and Nannen (1993) observed that
during growth and fermentation the pH of the medium decreases due to accumulation of
organic acid primarily lactic acid. This therefore increased TA in the sample.

35

1.2

1

0.8

Titratable
Acidity (%)
0.6

Control

Ferrous
Bisglycinate
0.4
Ferrous
Lactate

0.2

Ferrous
Sulfate

0
0

1

2
3
Time (hours)

4

5

Figure 5: Changes of Titratable Acidity in skim milk fortified with different iron
salts and incubated at 35 OC for 6 hours
36

6

5.5

5

Control
pH 4.5
Ferrous
Bisglycinate
4
Ferrous
Lactate

Ferrous
Sulfate

3.5

3
1.5

3
4.5
Time (hours)

6

Figure 6: Changes of pH in skim milk fortified with different iron salts and
incubated at 35 OC for 6 hours.

37

7

6.5

6

5.5
pH
Control
5
Ferrous
Bisglycinate
4.5
Ferrous
Lactate
4

Ferrous
Sulfate

3.5

3
4

4.5

5
5.5
Time (hrs)

6

6.5

Figure 7: Changes in pH for yogurt fortified with different iron salts during
incubation at 43 OC till pH 4.6 was reached

38

When yogurt batches were put in the incubator there were noticeable differences
in reaching the desirable pH of 4.6 which is the isoelectric point of caseins. There were
significant differences in decrease of pH amongst treatments. Figure 7 shows that the
treatment fortified with ferrous sulfate was the first to reach the desirable pH, after 4.5
hours. This agrees well with the results in Figure 4 where ferrous sulfate
microencapsulates fortified yogurt shows rapid microbial growth and therefore rapid
production of lactic acid by LAB which lowers the pH. This shows that ferrous sulfate
microencapsulate was more supportive of yogurt culture growth. Ferrous lactate was
the last to reach pH 4.6 due to low lactic production. Likewise in Figure 4 ferrous lactate
showed less supportiveness to yogurt culture growth than any other iron salts. Control
reached pH 4.6 after 5 hours. Ferrous bisglycinate and ferrous lactate took more than
6.5 hours to reach desirable pH. These differences in reaching the desirable pH were
due to the difference in production of lactic acid by Hansen’s yogurt culture Yo-fast 10,
Lactobacillus delbruckii ssp bulgaricus and Streptococus thermophilus in the presence
of different iron salts. This disagrees with what Hekmet and MacMahon (1997) and Elnagar and Shenana (1998) found. They reported that iron fortification had no effect on
the incubation time required for yogurt mixes. Hekmet and MacMahon (1997) used
casein-chelated iron and whey protein-chalated iron to fortify yogurt and determined the
effects of iron on bacterial and sensory qualities of yogurt. It was found that all batches
reached pH 4.3 after 5 hours. The pH values of control and fortified samples reached
pH 4.2 after 1 day. In this present study ferrous bisglycinate, ferrous lactate and ferrous
sulfate microencapsulated were used. All treatments reached pH 4.2 more than 5 hours
which also contrast their findings. Unlike Hekmet and MacMahon (1997) study whose

39

desired pH was 4.3 the present study’s desired pH was the isoelectric point of caseins
(pH 4.6). Yogurt fortified with ferrous bisglycinate and ferrous lactate took a longer time
to reach a desirable pH due to slow production of lactic acid by lactic acid bacteria while
the control treatment and yogurt fortified with ferrous sulfate reached pH 4.6 earlier.
Based on these results the incubation period were different for each treatment which
clearly indicates that iron compounds affected the time yogurt reached desired pH
during incubation. Another observation on the results in this present study revealed that
pH change was more rapid in skim milk than in yogurt when incubated for the same
length of time. For example pH after 4.5 hours for iron fortified skim milk are as follows;
control 4.31, ferrous bisglycinate 4.22, ferrous lactate 4.28 and ferrous sulfate
microencapsulated 4.28. After the same time in yogurt the pHs were; control 4.67,
ferrous bisglycinate 5.21, ferrous lactate 5.22 and ferrous sulfate microencapsulated
4.57. The reason for this behavior was well explained by King and others (1959) who
reported that all the added iron is associated with skim milk. When Iron is added it binds
to the colloidal phase of caseins at about 80-90% and hence reduces the pH of skim
milk upon addition. This decrease in pH is related to the acidities of iron solutions and to
exchanges between iron ions and micellar bound H+ (Gaucheron 2000). Therefore the
combined effect of exchanges between iron ion and micellar bound H+ and LAB
activities resulted in rapid decrease in pH for skim milk.
Results in Figure 8 shows that there was a general trend in all the treatments in
regard to the TA in stored yogurt. There was an increase in lactic acid expressed as
%TA up to first 15 days. Thereafter there was a decrease in TA. During growth and
fermentation, the pH of the medium decreases because of the accumulation of organic

40

acids, primarily lactic acid (see Figure 9). Growth of lactic acid bacteria continues as
long as there are enough growth nutrients, no toxic or inhibiting compounds and the
hydrogen ion concentration is maintained above the level that specific strain can
tolerate (Hutkins and Nannen, 1993). The lowest pH was at day 15 which was due to
high lactic acid accumulation (high TA). This high lactic acid became the growth limiting
factor of bacteria hence bacterial growth was retarded and eventually the production of
lactic acid decreased. Consequently the pH started increasing at day 20, see Figure 9.
Not only do most lactic acid bacteria grow more slowly at low pH, but acid
damage and loss of cell viability may also occur in cells held at low pH. Moreover,
inhibition of the starter culture by lactic acid and low pH acts to prevent, in part, overacidification of yogurt (Hutkins and Nannen, 1993). This decrease in pH in fermented
food is advantageous in the sense that organic acids produced during yogurt
fermentation can potentially enhance iron and zinc absorption via the formation of
soluble ligands (Gibson and others, 2006). Especially in weaning foods, such
physicochemical properties of fermented foods is highly desirable, for the fact that
children are most of the time highly vulnerable for food pathogens due to their
physiological conditions (Jay, 2000; Wambugu and others, 2002). According to Elyas
and others (2002), the increased acidity and low pH as a result of fermentation
enhances the keeping quality of fermented foods, by inhibiting microbial growth and
also contributing to the flavor of the processed food.

41

2.5

2

1.5
Titratable
Acidity (%)

Control

Ferrous
Bisglycinate

1

Ferrous
Lactate
0.5

Ferrous
Sulfate

0
0

5

10

15
Days

20

25

30

Figure 8: Changes in Titratable Acidity for yogurt fortified with different iron salts
and stored at 4 Oc for 30 days
42

4.6

4.5

4.4

Control

pH 4.3

Ferrous
Bisglycinate
Ferrous
Lactate

4.2

Ferrous
Sulfate
4.1

4
0

5

10
15
20
Time (Days)

25

30

Figure 9: Effects of different iron salts on pH for yogurt stored at 4OC for 30 days
43

The pH was lowest in yogurt fortified with ferrous sulfate microencapsulated. This was
due to high lactic acid produced indicative of highest culture activity. This disagrees with
what El-Kholy and others (2011) found. They reported that fortification of yogurt with
different iron salts has no effect on the total lactic acid bacteria when fresh and during
cold storage. The differences in pH for different iron salts suggest that bacterial
activities in stored yogurt were different in each of the treatments. In all cases the pH for
yogurt fortified with ferrous bisglycinate was highest except on day zero. The highest pH
meant that yogurt culture growth was still more active as low pH was not yet a limiting
factor. No wonder TA values were also high (see Figure 8).

6.3. Sensory Analysis
6.3.1. Sensory Evaluation of Yogurt.
Data in Table 2 shows that there were significant differences in sensory attributes
between samples. There was no significant differences between the control yogurt and
yogurt fortified with ferrous sulfate microencapsulated in all the attributes studied
(p<0.05, n =98). This shows that fortifying yogurt with ferrous sulfate microencapsulate
does not affect the consumer acceptability of the sensory properties of yogurt. This is a
welcome idea considering that the average relative bioavailability of ferrous sulfate
when used as a fortificant is 100% (Hurrell 1997). Both ferrous bisglycinate and ferrous
lactate were significantly lower than control treatment in appearance, body texture,
flavor and overall acceptance (p<0.05, n=98). This means that they both altered
consumer acceptability of all sensory properties of yogurt evaluated. . However the
effects on yogurt were not significantly different from each other. The difference in
44

appearance could be overcome by using flavor or coloring like chocolate that will mask
any change in color due to iron salt. Lack of significant differences between control
yogurt and yogurt fortified with ferrous sulfate is due to microencapsulation of ferrous
sulfate. Microencapsulation keeps the iron from coming in contact with the food vehicle
thereby preventing undesirable interaction that happen when conventional ferrous
sulfate is used (Boccio and others, 1997).

Table 3: Sensory mean scores of yogurt fortified with different iron salt stored at
4 OC for 7 days
Ferrous
Attribute

Ferrous

Ferrous

P-

Control

Sig
sulfate

Appearance

6.92a

bisglycinate

lactate

Value

6.72a

5.03b

5.24b

0.0001
***

Body texture

6.95a

7.04a

6.33b

6.46b

0.0001
***

Flavor

6.67a

6.66a

5.63b

6.00b

0.0001
***

Overall
6.58a

6.68a

5.54b

5.84b

0.0001

***

acceptance
a-b

Means with different superscript within a row are significantly different at p<0.05, n =
98

Yogurt fortified with ferrous bisglycinate had the lowest flavor and appearance
acceptability scores because ferrous bisglycinate easily oxidizes to ferric form [Fe3+]

45

which then cause off-color development and fat oxidation (Haile, 2006). Yogurt fortified
with ferrous bisglycinate and ferrous lactate scored significantly lower than control
yogurt and yogurt fortified with ferrous sulfate because panelists said the former were
grayish in color which was unpleasant to the panelists. The grayish appearance was
due to the gray to dark color of ferrous bisglycinate. While the color of ferrous lactate is
white the brown appearance of ferrous lactate fortified yogurt maybe due to other
reactions

between

ferrous

lactate

and

milk

components.

Ferrous

sulfate

microencapsulate is brownish in color but due to its encapsulation there were no
significant differences in appearance with control treatment. Use of strawberry puree
lessened the degree of browning in all yogurt but it was not enough to mask everything
as color of ferrous bisglycinate and ferrous lactate were not fully masked.
Encouragingly, all yogurts were rated above average sensory mean score of 5. As a
result it inferred that lipid oxidation that was detected by PV and TBA resulting from iron
fortification had a negligible effect on how well the yogurts were liked. This was also
observed by Hekmat and MacMahon (1997) where casein-chelated and whey proteinchelated iron fortified yogurts were all scored above average.
Yogurt fortified with ferrous bisglycinate had significantly higher pH at each
testing interval. Coincidentally yogurt fortified with ferrous bisglycinate had the lowest
sensory mean scores in all the sensory attributes that were evaluated suggesting a
relationship existed between pH and sensory acceptability of yogurt. However PV
showed that there were no significant differences amongst treatments at day 7 when
sensory evaluation was done on yogurt. Results in Figure 8 and Figure 9 suggest that
pH did not affect PV.

46

6.3.2. Sensory evaluation of pasteurized milk.
Sensory data in Table 4 shows no significant differences in color among samples
at p<0.05. This disagrees with the finding from sensory evaluation of iron fortified yogurt
above (see Table 3) where appearance acceptability were not significantly different in
control yogurt and yogurt fortified with ferrous sulfate microencapsulate but were scored
significantly lower in yogurt fortified with ferrous bisglycinate and yogurt fortified with
ferrous lactate. The same was the case with body texture, flavor and overall preference.
There were no significant differences in flavor in pasteurized liquid milk across all
treatments. Milk fortified with ferrous sulfate microencapsulate had significantly lower
sensory mean score on taste than any other treatment (p<0.05). There were significant
differences in overall preference between milk fortified with ferrous sulfate
microencapsulate, which had the lowest sensory mean score, and the rests of the
treatments.
Data

from

Figure

11

shows

that

milk

fortified

with

ferrous

sulfate

microencapsulate had significantly higher PV than milk fortified with ferrous bisglycinate
and control milk at the time sensory evaluation was done (after day 2). However PV for
milk fortified with ferrous sulfate was not significantly different from milk fortified with
ferrous lactate at the time of sensory evaluation. As expected, sensory mean score for
milk fortified with ferrous sulfate shows that it was least preferred. This, therefore,
suggests that acceptability of pasteurized milk fortified with ferrous sulfate was based
also on effects of lipid oxidation in the milk among other factors that influenced scores
for sensory attributes that were evaluated.

47

Table 4: Sensory mean scores of pasteurized milk fortified with different iron salt
stored at 4 OC for 7 days.
Attributes

Control

Ferrous

Ferrous

Ferrous

Bisglycinate

Lactate

Sulfate

7.48 ± 1.4a

7.49 ± 1.42a

7.20 ±1.73a

7.30 ± 1.83a

6.74 ± 1.78a

6.58 ± 1.65a

6.67 ± 1.76a

6.52 ± 1.89a

6.88 ±1.74a

7.08 ± 1.63a

6.92 ±1.59a

6.29 ± 2.25b

6.52 ± 1.88a

7.01 ±1.72b

6.82 ± 1.78b

6.59 ± 2.0a

6.92 ± 1.48a

6.99 ± 1.51a

6.84 ± 1.51a

6.45 ± 2.12b

Appearance

Flavor

Taste

Texture

Overall
preference
a-b

Means with different superscript in a row are significantly different to each other
(p<0.05), n = 100

Overall milk fortified with ferrous sulfate microencapsulate was least preferred
especially due to its taste. The panelists were unable to detect significant differences in
color and flavor. This is an interesting observation since ferrous bisglycinate is dark in
color and it was expected to impact color changes in milk fortified with it. However
results indicates that this was not the case as it did not significantly impact on color of
the fortified milk (p<0.05). All milk were rated from like slightly to like moderately in all
sensory attributes under study on the hedonic scale.

48

6.4. Chemical analysis of lipid oxidation
6.4.1. PV test for yogurt during storage.
There is a general trend of increasing the PV as storage time increases as
shown in Figure 10. But after 10 days of storage PV increased rapidly in yogurt fortified
with ferrous sulfate microencapsulated an indication that there was high lipid oxidation.
However there was a sharp decrease after 20 days of storage. This decrease is due to
decomposition of hydroperoxides into secondary products of lipid oxidation like
aldehyde, ketones and alcohol. These changes coincide with an increase in TBA
(Figure 10) that measures MDA, secondary product of lipid oxidation. Yogurt fortified
with ferrous lactate had the least lipid oxidation. After 23-25 days the control had the
highest PV. This may be due to the delay in decomposition of hydroperoxides into
secondary products of oxidation as there was no iron to catalyze oxidation process. This
may also be due to relatively slow peroxidation in the early days and this had an effect
on the time hydroperoxide disintegrated into secondary products.

49

600

500

400

Control

300
PV
(meq/kg)

Ferrous
Bisglycinate
200
Ferrous
Lactate
Ferrous
Sulfate

100

0
0

5

10

15
Days

20

25

30

Figure 10: Changes in PV in yogurt fortified with different iron salts and stored at
4oC for 30 days
50

4.6.2. PV test for pasteurized milk in storage.
PV results for pasteurized milk shows that there were significant differences
(p<0.05) between treatments even after just 2 days. Control has the lowest PV values
throughout the testing period seconded by milk fortified with ferrous sulfate
microencapsulate. Milk fortified with ferrous bisglycinate had high PV values after 12
days. Overall there were no significant differences in mean PV between milk fortified
with ferrous bisglycinate and milk fortified with ferrous lactate. This trend differs with
results obtained on yogurt PV values within the same storage time (see figure 10)
where there were no significant differences till after 10 days.
After

10

days

of

storage

time

yogurt

fortified

with

ferrous

sulfate

microencapsulate gave the highest PV values. However the trend remains the same
that after some time PV values in all treatments start to decrease due to degradation of
hydroperoxide into secondary products. There was rapid degradation of hydroperoxides
in milk fortified with ferrous bisglycinate and milk fortified with ferrous lactate than in
control and milk fortified with ferrous sulfate microencapsulate. Control had a least PV
values throughout the testing period. This does not agree with the finding in yogurt
where there was no difference in PV between control yogurt and yogurt fortified with
ferrous lactate, see Figure 10. The prooxidant effects of ferrous bisglycinate and
ferrous lactate were more pronounced throughout the testing period. This present study
clearly indicates that iron fortification significantly reduces shelf life of pasteurized milk
and if pasteurized liquid milk is to be fortified there is a need to encapsulate iron so that
interaction between iron and milk lipids is significantly reduced.
51

20

18

16

14

PV (meq/kg)

12

Control

10

8

Ferrous
Bisglycinate

6

Ferrous
Lactate

4

Ferrous
Sulfate

2

0
0

2

4

6

8 10
Days

12

14

16

18

Figure 11: Effects of iron fortification on lipid oxidation in pasteurized milk stored
at 4 OC for 18 days as determined by PV
52

This finding differs with the result obtained in yogurt where highest PV values were
observed in yogurt fortified with ferrous sulfate microencapsulate. The major
disadvantage of the PV method is that iodine can be absorbed by unsaturated double
bonds of fatty acids, and also oxygen present in potassium thiosulfate solution may also
liberate iodine. This may result into higher PV.
Another important observation is that in both cases, PV and TBA tests, ferrous
sulfate microencapsulate shows that it enhances lipid oxidation more than other iron
salts. The reason behind this behavior is not clear from present study. However it may
be concluded that microencapsulation did not provide enough barriers between the iron
and milk lipids such that oxidation occurred. This may be due to loss of
microencapsulation during homogenization, incubation and microbial growth and
activities. This is not conclusive and more studies have to be done on this. This
behavior was observed in yogurt and not in pasteurized milk.
6.4.3. TBA test for yogurt in storage.
Data in Figure 12 show that control yogurt had the lowest TBA values seconded
by yogurt fortified with ferrous lactate. There was steady increase in TBA values for all
the

treatments

except

in

the

control.

Yogurt

fortified

with

ferrous

sulfate

microencapsulates and yogurt fortified with ferrous bisglycinate had the highest TBA
values. TBA reagent when reacted with MDA, a secondary product of lipid oxidation
resulting from degradation of hydroperoxide, forms a pink color complex called
chromagen which has absorbance at 530nm. As more of MDA are produced as
oxidation continues more chromagen are formed and higher TBA numbers are obtained

53

at absorbance 530nm. This also shows that different iron salts had different effects on
formation of chromagen reflected by different TBA numbers.
Low TBA in control may be due to low hydroperoxide formation in the early
stages of lipid oxidation which consequently resulted into low MDA produced and the
subsequent low TBA numbers. If there is high rate of oxidation the MDA produced will
be high and more chromagen is formed. This will increase absorbance and
subsequently results into high TBA numbers. In yogurt fortified with ferrous bisglycinate,
ferrous lactate and ferrous sulfate microencapsulate the steady increase in TBA may be
due to increased breakdown of hydro peroxides to MDA which resulted into increased
TBA values when reacted with TBA reagent. Since control had the lowest TBA therefore
it can be concluded that all the iron salts under study enhanced lipid oxidation in yogurt.
However there are other TBA reactive substances (TBARS) that affect the results of
TBA test. Fernandez and others 1997 noted that iron salts affect TBA values because it
catalyzes the breakdown of hydroperoxides to MDA and catalyzes degradation of amino
acids to sugars (deoxyribose, hexoses, pentoses) in the presence of air to yield MDA.
This will definitely give high TBA which may be misinterpreted as resulting from
oxidation.

54

0.35

0.3

0.25

0.2
Control

TBA/g

Bisglycinate

0.15

Lactate
Sulfate
0.1

0.05

0
0

5

10

15
Days

20

25

30

Figure 12: Effect of iron fortification on lipid oxidation in yogurt stored at 4 OC for
30 days as determined by TBA test
etermined
55

7.0. CONCLUSION AND RECOMMENDATIONS
All the iron salts studied supported the growth and activities of yogurt starter
culture studied though their effects were different for each iron salt. Ferrous bisglycinate
supported culture growth the best after 6 hours of incubation. More lactic acid was
produced as incubation time increased to 6 hours for iron fortified reconstituted skim
milk as shown by an increase in TA. In yogurt there was an increase in TA during early
days of storage and a decrease in TA towards the end of 30 days storage time.
Consumer acceptance sensory analysis indicated that yogurt fortified with ferrous
sulfate microencapsulated were not statistically different from unfortified yogurt in all
sensory attributes under study. However, it was statistically higher in sensory
acceptability than yogurt fortified with ferrous bisglycinate and ferrous lactate as both
gave a darker color which could have been masked by using a more dark food coloring
like chocolate.
Pasteurized milk showed statistically insignificant differences in color for all the
treatments an indication that the panelist were unable to detect color change as was the
case with yogurt. Of all the attributes under study in pasteurized milk, taste was scored
the lowest in ferrous sulfate microencapsulated which presumably led to it being
preferred the least by panelist.
In both yogurt and pasteurized liquid milk lipid oxidation increased as storage
time increased as measured by PV and TBA. Ferrous sulfate fortified yogurt was similar
in consumer acceptability to unfortified yogurt in all sensory attributes. However, TBA
and PV tests showed higher values in yogurt fortified with ferrous sulfate

56

microencapsulate than in yogurt fortified with ferrous bisglycinate and yogurt fortified
with ferrous lactate. This may be attributed to increased lipid oxidation as
microencapsulation was done using vegetable oil which may also have been oxidized
leading to high PV and TBA numbers. Or microencapsulation was destroyed during
yogurt mixes homogenization and incubation such that there was free interaction
between ferrous sulfate and milk fats leading to high PV and TBA. Therefore, there is a
need to establish a reason why ferrous sulfate microencapsulated gave higher PV and
TBA (an indication of oxidation) and higher mean sensory score (an indication better
sensory quality) because, as would be expected, higher PV and TBA was supposed to
go with lower sensory means scores. This study has shown that microencapsulation
does not reduce oxidation in yogurt especially when fortification was done before
homogenization, pasteurization and incubation but it does reduce oxidation in
pasteurized liquid milk.
In this present study bioavailability of fortified iron in yogurt when consumed has
not been examined. Therefore another study, in-vitro or in-vivo, would be important to
help find out the bioavailability of iron in the body after consuming iron fortified yogurt
and milk using animal subjects.

57

8.0 APPENDICES

58

8.1. Milk flyer

Do you like milk?
Welcome to Sensory Evaluation of Iron Fortified
Pasteurized Liquid Milk
It will not take you much time! Just 5 – 10 minutes!!!

Venue: HE/HN Food Laboratory
Date:
Time:

Friday, December 7, 2012

9:00 am till 100 panelists have participated
Everybody is welcome!!!

Incentives shall be provided to participants as soon
s/he finishes the Sensory Test.
First Year Students are also encouraged to participate

For more information call 0999 746 608/0882 943 912

See you there everybody!!!!!!!

59

8.2. Yogurt flyer

DO YOU LIKE STRAWBERRY YOGURT?
PLEASE JOIN US FOR A SENSORY STUDY FOR
STRAWBERRY YOGURT!!!!!

TIME: 10:00 AM – 1:00 PM or Until 75 panelists have
participated

Date: Tuesday July 3rd, 2013

Where: Sensory Lab (Room 102 G. Malcolm Trout Building).
Corner of Wilson and Farm Lane

The test will take approximately 10-15 min.
You will receive a FREE MSU Dairy Store Coupon (2
scoops)

60

8.3. Questionnaire for pasteurized milk
You will be provided with 4 samples. Please evaluate each sample in the order
presented. Remember to rinse your mouth with water provided when moving to the next
sample.
1. Sample 537
Take the sample and sensory evaluate it. Put the number that corresponds to
your degree of liking (in the first column) below the attribute being evaluated.
mouth Overall
appearance flavor taste feel
preference
9.
Like
extremely
8. like very
much
7.
like
moderately
6.
like
slightly
5.
neither
like or dislike
4.
dislike
slightly
3.
dislike
moderately
2.
dislike
very much
1.
dislike
extremely

61

2. Sample 674
Rinse you mouth with water and evaluate the sample as in 1 above
mouth Overall
preference
Appearance flavor taste feel
9.
Like
extremely
8.
like
very
much
7.
like
moderately
6. like slightly
5. neither like or
dislike
4.
dislike
slightly
3.
dislike
moderately
2. dislike very
much
1.
dislike
extremely
3. Sample 317
Rinse your mouth and evaluate the sample as in 1 above
mouth Overall
Appearance flavor taste feel
preference
9.
Like
extremely
8. like very
much
7.
like
moderately
6. like slightly
5. neither like
or dislike
4.
dislike
slightly
3.
dislike
moderately
2. dislike very
much
1.
dislike
extremely

62

4. Sample 413
Rinse your mouth and evaluate the sample as in 1 above
mouth Overall
appearance flavor taste feel
preference
9.
Like
extremely
8. like very
much
7.
like
moderately
6. like slightly
5. neither like
or dislike
4.
dislike
slightly
3.
dislike
moderately
2. dislike very
much
1.
dislike
extremely

How many times do you drink milk a month? _______________________________
Is
milk
always
_______________________________________________________

Thank you for your participation!

63

available?

8.4 Questionnaire for Yogurt
5. Sample 537
Take the sample and sensory evaluate it. Put the number that corresponds to your
degree of liking (in the first column) below the attribute being evaluated.

appearance

Body
texture

flavor

Overall
preference

9.
Like
extremely
8.
like
very
much
7.
like
moderately
6. like slightly
5. neither like
or dislike
4.
dislike
slightly
3.
dislike
moderately
2. dislike very
much
1.
dislike
extremely

6. Sample 674
Rinse you mouth with water and evaluate the sample as in 1 above
Body
Overall
appearance
texture
flavor
preference
9. Like extremely
8. like very much
7. like moderately
6. like slightly
5. neither like or
dislike
4. dislike slightly
3.
dislike
moderately
2. dislike very
much
1.
dislike
extremely

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7. Sample 317
Rinse your mouth and evaluate the sample as in 1 above
Body
appearance texture flavor
9.
Like
extremely
8. like very
much
7.
like
moderately
6. like slightly
5. neither like
or dislike
4.
dislike
slightly
3.
dislike
moderately
2. dislike very
much
1.
dislike
extremely

8. Sample 413
Rinse your mouth and evaluate the sample as in 1 above
Body
appearance texture flavor
9.
Like
extremely
8. like very
much
7.
like
moderately
6. like slightly
5. neither like
or dislike
4.
dislike
slightly
3.
dislike
moderately
2. dislike very
much
1.
dislike
extremely

65

Overall
preference

Overall
preference

8.5. Consent Form for Research Involving Human Subjects
IRON FORTIFICATION OF PASTEURIZED MILK AND YOGURT
Invitation to participate
You are invited to participate in a research study, which compares sensory properties of UHT milk and
yogurt fortified with different iron salts
Purpose of the study
To evaluate sensory attributes and overall acceptability of iron fortified UHT milk and yogurt
Basis for subject selection
They will be selected based on their ability to detect differences in sensory attributes. Those with cold or
allergies to a specific ingredient will not be asked to participate. Participants must be at least 18 year old.
Potential risks
The UHT milk to be evaluated will be sterilized milk with no any other ingredients added other than the
iron salts (ferrous sulfate microencapsulated, ferrous Bisglycinate or ferrous lactate). All these salts are
USDA/FDA approved for use in foods intended for human consumption at approved levels. Yogurt will
contain the following ingredients in addition to iron; milk, culture, sugar, Non-fat dry matter, stabilizer,
cream, water. All these are FDA approved for yogurt production. These products sample pose no adverse
health risk upon ingestion, provided the subject has not been identified as being susceptible to allergic
reaction. If you believe there is a potential of an allergic reaction upon ingesting the test product or you
believe that participating will violate your religious or cultural belief, notify the on-site sensory evaluation
coordinator or principal investigator immediately. You will be released from participating in the study.
Potential benefit
There are no direct benefits gained from participating in this study. However your participation provides
valuable data for the development of iron fortified milk and yogurt. Information obtained from this study
will be published in appropriate scientific journals to expand our current knowledge in enhancing the
health value of fortification in dairy products
Explanation of procedure
You will be provided with four coded samples and a questionnaire. You will be provided with water for
rinsing your mouth between samples. The testing excersize will take a maximum of 25 minutes of your
time depending upon your speed of testing
Assurance of confidentiality

66

Any information obtained in connection with this study that will be identified with you will be kept
confidential by ensuring that all consent forms are securely stored and your privacy will be protected to
the maximum extent allowable by law. All data analyzed will be reported in an aggregate format that will
not permit associating with specific responses or findings.
Withdrawal from this study
Participating from this study is voluntary. Your decision to refuse participation will not affect your
present or future relationship with the principal investigator or MSU. You are also free to withdraw or
stop participating at any time you feel it is necessary to do so.
Compensation for participation
After you have completed your sensory testing session and turned in your sensory ballot, you will be
offered an ice cream coupon for your time and effort.
Offer to answer questions
If you have any questions, please do not hesitate to contact the onsite evaluation leader or principal
investigator. You are voluntarily making a decision to participate in this study today. Your signature
certifies that you have decided to participate after having read the information provided above and that
you had an adequate opportunity to discuss this study with principal investigator and have had your
questions answered to your satisfaction. You will be given a copy of this consent form to keep upon
request. In case you have question you may email Smith Nkhata on nkhatasm@msu.edu.

SIGNATURE OF SUBJECT: ___________________

DATE: _______________________

In my judgment the subject is voluntarily and knowingly giving informed consent and possesses the legal
capacity to give informed consent to participate in this research study.
SIGNATURE OF INVESTIGATOR: _______________

67

DATE: ________________________

8.6. Application For Initial Review

APPROVAL OF A PROJECT INVOLVING HUMAN SUBJECTS
Biomedical, Health Sciences Institutional Review Board (BIRB)
Social Science, Behavioral, Education Institutional Review Board (SIRB)
207 Olds Hall, Michigan State University
East Lansing, MI 48824-1047
Phone: (517) 355-2180
Fax: (517) 432-4503
E-mail: irb@msu.edu
Office Hours: M-F (8:00 A.M.-5:00 P.M.)

IRB#: x12-614
APPLICATION ID#: i041351

Title of Project: Iron Fortification of UHT milk and yogurt
Table 5: Principal Investigator’s (PI) details

Responsible Project Zeynep Ustunol
Investigator:

Mailing
Address:

2105 S. Anthony
Hall
MSU

Identification
Number:

XXX-XX-1560

Phone:

5-7713 EXT. 184

Department:

Food Science & Human
Nutrition

Fax:

517-353-1676

College:

AGRICULTURE AND
NATURAL RESOURCES

Email:

ustunol@msu.edu

Academic Rank:

Professor

The Human Research Protection Program (HRPP) has deemed this project as exempt, in accord with
federal regulations for projects exempt from Institutional Review Board (IRB) review. As an exempt
protocol, the appropriate IRB will not be further involved with the review or continued review of the
project, as long as the project maintains the properties that make it exempt.
•

Since the HRPP is no longer involved in the review and continued review of this project, it is the
Principal Investigator who assumes the responsibilities for protection of human subjects in this

68

•

•

•

•

•
•

•

•

project and ensures that the project is performed with integrity and within accepted ethical
standards, particularly as outlined by the Belmont Report (see exempt educational materials).
The Principal Investigator assumes responsibility for ensuring that the research subjects be
informed of the research through a documented or undocumented consent process, if
appropriate.
The Principal Investigator assumes the responsibility to maintain confidentiality of the subjects
and the data, and maintain the privacy of the subjects and protection of the data through
appropriate means. If data is anonymous, the investigators will make no attempt to identify any
individuals.
The Principal Investigator assumes the responsibility that co-investigators and other members of
the research team adhere to the appropriate policies to protect human subjects, maintain
confidentiality and privacy, and adhere to accepted ethical standards.
If the Principal Investigator adds additional investigators to an exempt protocol, he/she may
inform the HRPP of the additions. This may be of particular importance to graduate students if
the Graduate School requires proof of IRB approval.
Any complaints from participants regarding the risk and benefits of the project must be reported to
the HRPP.
Since the Principal Investigator and co-investigators are charged with human subject protection
and adhering to ethical principles in exempt research, it is appropriate that investigators be
trained in human subject principles. The Principal Investigator and all members of the research
team are required to complete MSU IRB educational requirements or equivalent.
Any change in the protocol which may raise the project from exempt to an expedited or full review
category must be presented to the HRPP. If there is any question about a change in protocol the
Principal Investigator should consult the Director of the HRPP. Failure to submit changes which
raise the protocol out of the exempt category will be considered non-compliance and will be
subject to investigation and action by the HRPP.
I accept responsibility for conducting the proposed research in accordance with the protections of
human subjects as specified by the IRB, including the supervision of faculty and student coinvestigators. There will be adequate resources and facilities to carry out the research.

By signing below, the Principal Investigator assures that he/she will abide by the terms of this assurance
and the HRPP exempt policy.

SIGN
HERE:

____________________________________________________________

Date:

____________________________________________________________

69

9.0 REFERENCES

70

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