Sudheera Polavarapu
June 2011
Abstract
Fish oil (FO) is rich in the polyunsaturated fatty acids, but sensitive to oxidation by many
factors including, heat, light and atmospheric oxygen. Microencapsulation of FO has been
reported to help protect FO from oxidation, especially in the presence of antioxidants. As
extra virgin olive oil (EVOO) is rich in oleic acid, and some antioxidant compounds, this
study aimed to investigate the potential of EVOO to protect FO from oxidation in the
presence of sugar beet pectin as a wall material. Ethylenediaminetetras-acetic acid (EDTA)
was also tested as a chelating agent to retard the lipid oxidation caused by the transition
metal ions present in the sugar beet pectin.
The oil-in-water emulsions were prepared and spray-dried to form microcapsules containing
fish oil alone (control) and a blend of FO/EVOO (1:1 weight ratio), at 25% (pH 3) and 50%
oil (pH 3 and pH 6) loadings. Fish oil and Fish oil- Extra virgin olive oil emulsions with
EDTA were prepared at 50% oil loading only at pH 6. The physicochemical characteristics
and oxidative stability of all FO and FO-EVOO emulsions and microcapsules were assessed.
The microcapsules showed low moisture content (<3% w/w) and water activity (~0.3). The
encapsulation efficiencies were >90% for all the formulations proving sugar beet pectin an
effective encapsulant.
Oxidative stability was assessed by testing propanal and hexanal in the head space using GC
analysis, fatty acid content, and oxidation stability using an Oxipress. It was concluded that
EVOO improved the oxidative stability of microencapsulated FO during accelerated storage
conditions (80C, 5 bar oxygen pressure), irrespective of oil loading. However, no effect
was detected at pH 3 during storage at room temperature (~27C). The addition of EDTA in
the formulation of the microcapsules significantly (P<0.05) increased the oxidative stability
of the microcapsules. It was also observed that microcapsules prepared from emulsions at
pH 6 were better in terms of long term storage when compared to the microcapsules
prepared from emulsions at pH 3.
II
Declaration
This is to certify that
(i) the thesis comprises only my original work towards the Masters
(ii) due acknowledgement has been made in the text to all other material used,
(iii) the thesis is 22,584 words in length as approved by the Graduate School, Faculty or
RHD Committee.
Sudheera Polavarapu
June 2011
III
Acknowledgements
My greatest gratitude to my supervisors Dr. Said Ajlouni, Dr. Maryann Augustin and Dr.
Christine Oliver who were abundantly helpful and offered invaluable assistance, support and
guidance from the very early stages of my Masters Project till the thesis submission. I
thank them for taking time to attend monthly meetings to discuss results and for the
constructive comments to the numerous reports I submitted. Many thanks for providing new
ideas and approaches to the project. Working under their supervision has triggered a passion
for research in me. I attribute my growth as a student to all of them and that I would benefit,
for a long time to come. I am grateful to them in every possible way. Also, my special thanks
to Dr. Christine Oliver for all her help and efforts in preparing the manuscripts for
publication.
I am grateful to all staff and students in the Department of Agriculture and Food Systems,
The University of Melbourne and CFNS (CSIRO Food and Nutritional Sciences) for their
assistance in my lab work, for sharing instruments and for creating a pleasant working
atmosphere. A warm thank you to the Senior Laboratory Manager Michelle Rhee at the
University for her assistance in ordering and supplying of chemicals and for all her support
while working in the lab. Furthermore, I convey my special acknowledgement to Li Jiang
Cheng, Zhiping Shen, Said Ajlouni, Rangika Weerakkody, Helen French, Christine Oliver
and Roderick Williams for their indispensable help and for being patient while teaching me
how to operate various lab instruments. I also appreciate their time and efforts for helping
me interpret the results obtained.
I am immensely thankful to Dr. Simon Crawford, School of Botany, The University of
Melbourne, Parkville for providing his expertise with the scanning electron microscope and
for helping me obtain the SEM images for all my samples.
Lastly, my parents deserve special mention for their endless love, encouragement and
prayers without which none of this would be of any significance. I thank my sister and
mother-in-law for always being a source of undying love, support and comfort. Words fail
IV
Table of Contents
ABSTRACT
II
DECLARATION
III
ACKNOWLEDGEMENTS
IV
TABLE OF CONTENTS
VI
ABBREVIATIONS
IX
LIST OF FIGURES
LIST OF TABLES
XII
Chapter 1- INTRODUCTION
2.1.1 Introduction
10
2.1.5 Bioavailability
11
2.2 Microencapsulation
12
12
14
16
17
19
19
21
23
23
23
23
3.1.3 Oxidation status of Fish oil and Extra virgin olive oil
24
25
29
30
30
30
3.4.3 Viscosity
30
30
31
31
31
31
32
32
32
32
32
33
33
storage conditions
3.5.10 Scanning electron microscopy (SEM)
3.6 Statistical analysis
33
34
35
35
35
35
4.1.3 Oxidation status of fish oil and extra virgin olive oil
37
4.2 Effect of partial substitution of fish oil with extra virgin olive oil
38
49
57
65
VII
66
79
86
References
88
Appendices
109
VIII
Abbreviations
SBP - Sugar beet pectin
FO - Fish oil
EVOO - Extra virgin olive oil
DGS - Dried glucose syrup
EDTA - Ethylenediaminetetras-acetic acid
EPA - Eicosapentaenoic acid
DHA - Docosahexaenoic acid
ALA- Alpha-linolenic acid
- CD cyclodextrin
PCL- Polycaprolactone
CMC Carboxy methyl cellulose
PEG Polyethylene glycol
PV- Peroxide value
IP- Induction period
IX
LIST OF FIGURES
Figure 1
14
production of microcapsules
Figure 2
22
Figure 3
25
using an oxipress
Figure 4
Process flow chart of the experimental plan for the production of spray
27
dried microcapsules
Figure 5
29
Figure 6
39
spray drying
Figure 7
Brightfield micrographs of (A) FO-25 (7.5% oil, wet basis) and (B)
40
Apparent viscosities of fish oil and fish oil-extra virgin olive oil (1:1 wt
41
ratio) emulsions (30% TS; 2% SBP, 20.5% DGS, 7.5% total oil and
30% TS; 2% SBP, 13% DGS, 15% total oil), intended for manufacture
of spray-dried microcapsules containing 25 and 50% oil, as a function
of shear rate
Figure 9
43
function of pH at 22 C
Figure 10
43
measured as a function of pH at 22 C
Figure 11
47
Scanning electron microscopy images of the fish oil (A, C) and fish
oil-extra virgin olive oil (B, D) spray-dried powders (25% oil loading),
prior (A, B) and after 3 month storage (C, D) under ambient conditions
(~25C, 0.5 aw)
48
Figure 13
50
50
Apparent viscosities of fish oil and fish oil-extra virgin olive oil (1:1 wt
51
ratio) emulsions (15% oil, wet basis) with and without EDTA at pH 6,
as a function of shear rate
Figure 16
56
(50% oil, dry basis), with and without EDTA at pH 6 after 0, 1, 2 and 3
month (AD) storage at room temperature (~25C)
Figure 17
57
oil spray-dried powders (50% oil, dry basis), with (A, C) and without
(B, D) EDTA, at pH 6, prior (A, B) and subsequent (C, D) to storage for
3 months at room temperature (~25C)
Figure 18
Particle size distributions of the original (fresh) fish oil and fish
58
Brightfield micrographs of (A) fish oil (pH 3) and (B) fish oil-extra
59
virgin olive oil (1:1 wt ratio) emulsions (pH 6) with 15% oil
Figure 20
Apparent viscosities of fish oil and fish oil-extra virgin olive oil (1:1 wt
60
XI
65
LIST OF TABLES
Table 1
18
Table 2
Formulation of the fish oil and fish oil-extra virgin olive oil (1:1 wt ratio)
28
emulsions at pH 3
Table 3
Formulation of the fish oil and fish oil-extra virgin olive oil (1:1 wt ratio)
28
36
Table 5
38
42
Table 7
44
and fish oil-extra virgin olive oil (1:1 wt ratio) (25 % & 50 % oil loading)
at pH 3
Table 8
46
46
fish oil and fish oil-extra virgin olive oil (1:1 wt ratio) during storage of
the powders in stoppered flasks at room temperature (~25C)
Table 10
53
oil-extra virgin olive oil blends (1:1 wt ratio) with and without EDTA at
pH 6, 50% oil (dry basis)
Table 11
55
55
XII
61
Table 14
Changes in moisture content and water activity of fish oil and fish
62
64
containing fish oil and fish oil-extra virgin olive oil (1:1 wt ratio) at pH 3
and pH 6 with 50% oil loading, during storage of the powders in stoppered
flasks at room temperature (~25C)
Table 16
68
olive oil (1:1 wt ratio) (25 % & 50 % oil loading) at pH 3 during storage at
room temperature (~25C, 03 month)
Table 17
68
olive oil (1:1 wt ratio) with and without EDTA (50% oil loading) at pH 6
during storage at room temperature (~25 C, 03 month)
Table 18
70
71
olive oil (1:1 wt ratio) (25 % & 50 % oil loading) at pH 3 during storage at
room temperature (~25C, 03 month)
Table 20
72
olive oil (1:1 wt ratio) with and without EDTA (50 % oil loading) at pH 3
and pH 6 during storage at room temperature (~25C, 03 month)
Table 21
72
75
Fatty acid composition of microencapsulated fish oil olive oil (25 % and
50 % oil loading) at pH 3 during storage at room temperature (~25C, 03
month)
XIII
76
Table 23 a
77
78
80
Oxidative
stability
of
emulsions
and
powders
containing
81
microencapsulated fish oil and fish oil-extra virgin olive oil (1:1 wt ratio)
(25% and 50% oil loading) at pH 3 during accelerated storage (80C,
oxygen pressure of 0.5 MPa)
Table 26
Oxidative
stability
of
emulsions
and
powders
containing
84
microencapsulated fish oil and fish oil-extra virgin olive oil (1:1 wt ratio)
made with and without EDTA (50% oil loading) at pH 3 during
accelerated storage (80C, oxygen pressure of 0.5 MPa)
Table 27
XIV
85
CHAPTER 1 - INTRODUCTION
This chapter provides the background and purpose to the research topic and discusses the
need for further research related to the area. The project hypothesis and specific aims of the
research are outlined.
Microencapsulation technology has been the central concept of this research and the two
other areas discussed in relation to it are the protection of bioactives (omega 3 fatty acids,
and polyphenols) and the application of sugar beet pectin as an encapsulant. Literature
review on the current scientific information/existing research in the above mentioned fields
was reviewed in the second chapter.
The third chapter includes all details about the raw materials (supplier details and
manufacturers specification), chemicals (supplier details), equipments (with the
manufacturers details and model numbers) used in the research project. It also describes
the procedures/method of analysis adopted for the characterization of emulsions and
microcapsules.
Chapter four describes the results obtained after analysis of the raw materials, emulsions
and microcapsules following the analytical methods described in Chapter 3 - Methods
(Section 3.1, 3.4, 3.5, 3.6). Conclusive inferences from the results obtained were also
presented.
Based on the results of the various analyses conducted and the comparison of results
performed between samples, the final conclusions of this study is summarised in fifth
chapter.
1.1 BACKGROUND
Consumer behaviour and attitudes toward healthy foods continue to grow especially in
developed countries. This has sparked a wide interest in foods, such as, functional foods
which contain ingredients with health promoting properties, and can offer specific health
benefits to consumers in addition to the anticipated nutritional value.
Functional foods have the potential to contribute to a healthier society and are important
components of an overall healthful lifestyle that includes a balanced diet and physical
activity. There have been mounting evidence in recent years that food choices are an
important factor in reducing the risk of developing heart disease, cancer, obesity, high
blood pressure, osteoporosis, and other unhealthy conditions (Australian Indigenous
Healthinfonet, 2008).
Fish oils are considered functional foods as they are a rich source of omega-3 fatty acids
such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Erkkila et al.,
2006). There has been mounting evidence in the recent years that omega-3 fatty acids have
positive long-term and current health benefits. Several prospective studies and clinical
trials have shown that omega-3 fatty acids can reduce the risk of developing heart disease
(Erkkila et al., 2006; GISSI-HF investigators, 2008). Various other studies have shown
that consumption of omega-3 fatty acids have been beneficial in treating neurological
disorders such as Alzheimers disease (Morris et al., 2003), in reducing bone loss (Griel et
al., 2007; Weiss, Barrett-Connor & von Muhlen, 2005) in lowering incidence of
depression and other related conditions (Lin & Su, 2007). However, the incorporation of
functional ingredients in a given food system and the processing and handling of such
foods are associated with nutritional challenges for their healthy delivery. The extreme
sensitivity of fish oils to oxidation can easily lead to the development of off-flavours and
cause significant loss of product quality, stability, nutritional value, and bio-availability
and the overall acceptability of the food product (Nawar, 1996; Watkins & German, 1998).
Consequently, microencapsulation has been successfully used to encapsulate omega-3 fatty
acids in order to prevent oxidation and to improve stability and bioavailability (Wakil et
al., 2010; Sanguansri & Augustin, 2006). Microencapsulation can be defined as a process
in which tiny particles or droplets are surrounded by a coating to give small capsules. The
material inside the microcapsule is referred to as the core, internal phase, or fill, whereas
the coating is sometimes called a shell, membrane, or wall material, as referred to
henceforth. Most microcapsules have diameters between a few micrometers and a few
millimetres (Microencapsulation, 2009). Microencapsulation is one example of technology
that has the potential to meet the challenge of successfully incorporating and delivering
functional ingredients into a range of food types.
The most common technology used for encapsulation has been spray drying because it is
efficient, cost effective, has readily available equipment and produces particles of
reasonably good quality (Dobry et al., 2009). Some of the encapsulant materials used in
the microencapsulation of foods are proteins, sugars, starches, gums, lipids and cellulosic
materials. Chitosan plus maltodextrin and liposomes have been reported in encapsulation
of omega-3 rich fish oils (Klaypradit & Huang, 2008; Reineccius, 1995). A considerable
number of research studies have been conducted on the oxidative stability of fish oil,
microencapsulated with different wall materials using one of the many chemical and
physical microencapsulation processes available. Literature review on the oxidative
stability of omega-3 fatty acids showed a variation of results due to the effectiveness of
wall materials used, changes in the process parameters, storage conditions and methods
employed for assessing the oxidative stability.
with the addition of antioxidants to improve the shelf life of the fish oil microcapsules
(Velasco, Dobarganes & Marquez ruiz, 2000; Heinzelmann et al., 2000).
Several studies suggested that SBP could be a suitable wall material for manufacturing
lipophilic food ingredients due to its excellent emulsifying properties (Drusch, 2007;
Drusch et al., 2007; Leroux et al., 2003; Martinez-Dominguez, de la Puerta, &
Ruiz-Gutierrez, 2001). This functionality associated with SBP showed that it had potential
as a wall material for encapsulating w-3 acids in our study. Recent studies conducted by
Drusch (2007) also confer that SBP could be a promising alternative to the traditional
encapsulating agents like milk proteins and gum Arabic because of its ability to form
Oxidative stability of the microencapsulated fish oil (FO) in the presence of extra
virgin olive oil (EVOO).
2.1.1 INTRODUCTION
Bioactive compounds are biomolecules present in foods that exhibit the capacity to
modulate one or more metabolic processes, which promote better health. They indulge in
multiple metabolic activities and are beneficial for treating several diseases and target
tissues and are usually found in glycosylated, esterified, thiolyated, or hydroxylated forms.
Bioactive food components are found abundantly in most fruits and vegetables. However,
probiotics, conjugated linolenic acid, long-chain omega-3 polyunsaturated fatty acid, and
bioactive peptides are most commonly found in animal products such as milk, fermented
milk products and cold-water fish (Encyclopaedia of Food and Culture, 2003).
Numerous bioactive components from plant sources (e.g. phytosterol, carotenoids,
flavonoids, soluble fibre, polyphenols etc.) marine oils, milk and milk products (e.g.
peptides / proteins, pre and pro-biotics) are currently used in functional food formulations
(McClements et al., 2009; Engel & Schubert, 2006; Murphy, 2001; Lpez-Lpez et al.,
2009). However, a significant lack of understanding of bioactive ingredients availability,
mechanism of action and synergistic effects exists and hence, the acceptable levels of
specific bioactive ingredients to be used in different foods have to established to establish
the appropriate dosage of healthy bioactive components.
Significant research findings stated in the Encyclopaedia of Food and Culture (2003)
indicated that bioactive food components could act simultaneously at different or identical
target sites. For instance, in cardiovascular disease, the bioactive component isoflavone
may reduce circulating oxidized low-density lipoproteins in the plasma, bind cholesterol in
the intestinal tract and thereby reduces absorption of dietary cholesterol. Another example
is the lipid-lowering mechanism of dietary fibre and phytosterol/stanols. It occurs by
sequestering cholesterol in the intestinal tract and reducing cholesterol absorption. The
bioactive food components have thus been proven to have health-promoting characteristics
and reduce the risk of cancer, cardiovascular disease, osteoporosis, inflammation, type 2
diabetes, and other chronic degenerative diseases (Encyclopaedia of Food and Culture,
2003; Ferguson & Philpott, 2007; Pfeuffer & Schrezenmeir, 2000; Hooper & Cassidy,
2006; Shahidi, 2006).
Clearly, bioactive food components can play an important role in better health and disease
prevention. Consequently, there is a need to produce foods with optimal levels of
health-promoting bioactive food components that would assist consumers to make healthy
food choices for their diet.
2.1.2 OMEGA 3 FATTY ACIDS
Omega-3 fatty acids are considered essential fatty acids. They are essential to human
health but cannot be manufactured by the body. For this reason, omega-3 fatty acids must
be obtained from food. Also known as polyunsaturated fatty acids (PUFAs), omega-3 fatty
acids play a crucial role in brain function as well as normal growth and development (drsri,
2009).
Fish, plant, and nut oils are the primary dietary source of omega-3 fatty acids. Best food
sources containing both eicosapentaenoic acid (EPA), C20:5(n-3), and docosahexaenoic
acid (DHA), C22:6(n-3), are cold-water fatty fishes such as, salmon, mackerel, halibut,
sardines, tuna, and herring. Dietary sources of alpha-linolenic acid (ALA), C18:3(n-3) are
flaxseeds, flaxseed oil, canola (rapeseed) oil, soybeans, soybean oil, pumpkin seeds,
pumpkin seed oil, purslane, perilla seed oil, walnuts, and walnut oil. ALA is converted in
the body into EPA and DHA, which are more readily utilised in vivo (Drsri, 2009).
Extensive research indicates that omega-3 fatty acids have antiinflammatory properties and
reduce the risk associated with chronic diseases such as heart disease, cancer, and arthritis
(Geusens et al., 1994; Richardson, 2004; GISSI-prevenzione investigators, 1999). These
essential fatty acids are an important structural component in the brain and appear to be
particularly important for cognitive (brain memory and performance) and behavioural
function (Willat et al., 1998; Mellor, Langharne & Peet, 1995). Symptoms associated with
7
omega-3 fatty acid deficiency include extreme tiredness (fatigue), poor memory, dry skin,
heart problems, mood swings or depression, and poor circulation (Omega-3-fatty acids,
2010).
An appropriate balance of omega-3 and omega-6 in the diet is vital, as they work together
to promote health. Whilst omega-3 fatty acids help reduce inflammation, most omega-6
fatty acids tend to promote inflammation. An inappropriate balance of these essential fatty
acids contributes to the development of disease, while a proper balance helps maintain and
even improve health. A healthy diet should consist of roughly 2 - 4 times more omega-6
fatty acids than omega-3 fatty acids (Omega-3-fatty acids, 2010).
Omega-3 fatty acids are extremely susceptible to oxidative damage from heat, light, and
oxygen. When exposed to the air for long periods, the fatty acids in the oil become
oxidized and rancid. Rancidity not only gives the oil disagreeable off-flavour and smell,
but it also diminishes the nutritional value. More importantly, free radicals are produced in
this process and which are implicated in the development of cancer and other degenerative
diseases (Pham-Huy, He & Pham-Huy, 2008).
Rancidity arises when the oils are removed from their food package or are stored
improperly. Hence, oils rich in polyunsaturated fatty acids should be stored in dark glass,
tightly closed containers in the refrigerator or freezer.
2.1.3
Extra virgin olive oil (EVOO) comes from cold pressing of the olives, contains no more
than 0.8% acidity, and is judged to have a superior taste, delicate flavour and most
antioxidant benefits. Olive oil is composed mainly of oleic acid, C18:1(n-9), and palmitic
acid, C16:0, as well as other minor fatty acids, along with traces of squalene (up to 0.7%)
and sterols (about 0.2% phytosterol and tocosterols). The composition varies by cultivar,
region, altitude, time of harvest, and extraction process (Alarcon de la Lastra et al., 2001;
Olive oil, 2008). Olive oil also contains a group of related natural products with potent
antioxidant properties which give extra-virgin unprocessed olive oil its bitter and pungent
taste (Tripoli et al., 2005).
Diets rich in monounsaturated fats have been shown to reduce the risk of coronary heart
disease (Keys et al., 1986; Willet et al., 1995). This is particularly noteworthy, because
olive oil is extremely rich in monounsaturated fats, most notably oleic acid (Olive oil,
2008). Another health benefit of olive oil is its ability to displace omega-6 fatty acids,
while not having any impact on omega-3 fatty acids. This way, olive oil helps to build a
more healthy balance between omega-6 and omega-3 fatty acids (Haban et al., 2004).
Oleic acid is found in various animal and vegetable sources. It comprises 55-80% of olive
oil, and the saturated form of oleic acid is called stearic acid. Oleic acid shares the normal
reactions of fatty acids and alkenes. Oxidation at the double bond can split the molecule,
yielding chain ends with aldehyde, ketone or carboxylic acid groups. This reaction occurs
slowly in air, and it causes the oil in which it is present to go rancid (Oleic acid, 2008).
Polyphenols are a group of chemical substances found in plants, characterized by the
presence of more than one phenol unit or building block per molecule. Polyphenols are
generally divided into hydrolyzable tannins (gallic acid esters of glucose and other sugars)
and phenylpropanoids, such as lignins, flavonoids, and condensed tannins. Notable sources
of polyphenols include berries, tea, beer, grapes/wine, olive oil, chocolate/cocoa, coffee,
walnuts, peanuts, pomegranates, yerba mate, and other fruits and vegetables. High levels of
polyphenols can generally be found in the fruit skins (Polyphenols, 2008).
Based on the epidemiologic studies conducted by Arts & Hollman, (2005), it has been
observed that olive oil phenolic content, rather than its fatty acid profile is responsible for
at least some of its cardio protective benefits. There has been growing evidence that as
antioxidants, polyphenols may protect cell constituents against oxidative damage and,
therefore, limit the risk of various degenerative diseases associated with oxidative stress
(Luqman & Rizvi, 2006; Pandey, Mishra & Rizvi, 2009). Polyphenols also exert protective
effects in treating conditions like cancer, diabetes, aging, asthma and skin damages
(Pandey & Rizvi, 2009).
2.1.4
Bioactive compounds are susceptible to variations due to changes in their structure and
physicochemical properties as a result of processing and the possible interactions with
other food components and the intestinal microflora.
Functional foods rarely contain a single bioactive component and interactions between
bioactive components in the food is expected which could either be positive or negative.
For example, Vitamin C has been reported to reduce seleniums effectiveness against
chemically induced colon cancer (Ip, 1986). Selenium has been shown enhance the ability
of garlic to inhibit chemically induced mammary cancer in experimental animals
(Amagase, Schaffer & Milner, 1996).
There are very few research studies that can confirm or demonstrate the interaction or
effect of polyphenols with omega-3 fatty acids. A study conducted by Ren et al. (2008)
showed that homozygous sickle cell patients (HbSS) had reduced EPA and DHA in red
cells, platelets, and mononuclear cells when compared to their healthy counterparts
(HbAA). This difference in levels of omega-3 fatty acids was not because of reduced
omega 3 intake. Something else besides dietary deficiency was causing lower cell levels of
cellular omega-3 fatty acids in these patients. The study investigated, and concluded that
the lower levels of membrane EPA and DHA in blood cells of the HbSS patients could be
attributed to peroxidation resulting from a compromised antioxidant competence. Omega-3
fatty acids aid in cell membrane function and stability and polyphenols are known to
directly and indirectly prevent peroxidation of fats. In addition, the antioxidant status
affects cell membrane levels of omega-3 fatty acids in humans, and polyphenols were
clearly found to be superior dietary antioxidants to those studied in the above mentioned
research study (beta-carotene, tocopherol). Hence, there is a possibility that polyphenols
could act to protect omega-3 fatty acids in the cell membrane.
10
2.1.5
BIOAVAILABILITY OF NUTRIENTS
From a nutritional point of view nutrient bioavailability can be defined as the amount our
digestive system will be able to extract in a form that can be absorbed into the bloodstream
(Encyclopaedia of Food and Culture, 2003).
It has been established that during fat absorption, unsaturated long chain fatty acids are
esterified at a higher rate than saturated fatty acids of similar chain length. This
phenomenon has been attributed to differences in the binding affinity of fatty acids to a
cytosolic fatty acid-binding protein (Gang et al., 1980). Studies have been conducted to
evaluate the bioavailability of omega-3 fatty acids in microencapsulated fish-oil-enriched
foods compared with an equal amount of omega-3 PUFAs contained in fish oil capsules.
The results indicated that n-3 PUFA from microencapsulated fish-oil-enriched foods were
as bioavailable as n-3 PUFA in a capsule. Therefore, fortification of foods with
microencapsulated fish oil offers an effective way of increasing omega-3 PUFA intakes
and status in line with current dietary recommendations (Wallace et al., 2000; Volker,
Weng, Quaggiotto, 2005).
Singh et al. (2008) indicated that bioavailability and pharmacokinetics of polyphenolics
are governed by a number of factors; their native form (glycosylated/aglycone), the type of
sugar moiety present, and their physiochemical properties. Bioavailability of polyphenols
varies widely from one compound to another. It depends on their chemical structure, which
determines their absorption rate through the gastrointestinal tract, metabolism, and,
therefore, bioactivity (Manach, 2004). In nature, phenolic compounds occur as glucosides.
Phenolic acids like caffeic acid are easily absorbed through the gut barrier, whereas large
molecular weight polyphenols, such as proanthocyanidins are very poorly absorbed. Once
absorbed, polyphenols are conjugated to glucuronide, sulphate and methyl groups in the
gut mucosa and inner tissues. Non-conjugated polyphenols are virtually absent in plasma.
These reactions facilitate their excretion and limit their potential toxicity. The polyphenols
reaching the colon are extensively metabolised by the microflora into a range of low
molecular weight phenolic acids (Scalbert, 2002).
11
Polyphenols present as aglycones can be absorbed from the small intestine. However, most
of them are present in the form of esters, glycosides, or polymers and are not easily
absorbed in their natural form. Glycosylation influences chemical, physical, and biological
properties of the flavonoids and their absorption. It is generally accepted that the
breakdown of these conjugates to aglycones by acid hydrolysis in the stomach and by
microflora in the gut is required to produce the bioactive components that are readily
bioavailable to the body. However, relatively little is known about the ability of these
aglycone polyphenolics to reach the target cells or what the influence of further
metabolism in the body has on their spectra of biological activities. There are numerous
sites important for the metabolism of dietary polyphenols, including the gastrointestinal
tract, the liver, and various other organs such as the skin and brain (Singh et al., 2008).
The food matrix also plays a vital role in the bioaccessibility of a bioactive component in
functional foods. For example, a lipophilic food matrix is needed for bioavailability of
carotenoids whereas when phenolic compounds are in a food matrix the bioavailability of
certain polyhenols may be lowered depending on the matrix components (Parada &
Aguilera, 2007). Another study by Kean, Hamaker, & Ferruzzi, (2008) found that specific
food preparations of maize based food products could influence the bioaccessibility of
certain bioactive carotenoid species.
2.2 MICROENCAPSULATION
12
13
Identification of high
quality source of active
ingredient to be
encapsulated
Physical and
chemical properties
of the bioactive
Stability of the
bioactive
Development of
successful formulation
for a targeted
application
Physical and
chemical properties
of the bioactive
Stability of the
bioactive
Selection of
appropriate
encapsulant material
Selection of suitable
microencapsulation
process
Addition of
antioxidants,
chelating agents,
emulsifiers,
stabilizers and salts
Dry product or
liquid format
Figure 1: Illustration of the steps involved and the factors considered in the production of
microcapsules (Sanguansri and Augustin, 2006) with some modifications.
14
the physical and chemical properties of the shell material can influence the functionality of
the final microcapsule and the processing technologies to be used for microencapsulation
(Sanguansri & Augustin, 2006). According to Augustin & Sanguansri (2007) a good
encapsulating material should have neutral taste and odour, low viscosity, good film
forming, gelling and barrier properties. It should also protect the core from degradation
during processing and storage and also mask any undesirable taste or odour associated with
the bioactive core when added into foods.
Some of the common encapsulant wall materials have been summarised by Vasishtha
(2003) and includes the following:
Fats and fatty acids such as mono-, di- and triglycerides, and lauric, capric, palmitic
and stearic acid and their salts.
Sugar derivatives.
Conjugates of casein and whey protein have been suggested as effective encapsulating
material for fish oil (Livney, 2010). Polymers such as -cyclodextrin (-CD) and
polycaprolactone (PCL) were used to encapsulate fish oil using aggregation and emulsion
diffusion methods and it was found that PCL protected FO better than (-CD) (Choia et al.,
2010). The use of complex coacervates composed of gelatin or -lactoglobulin, gum
arabic, starch, and crosslinked with glutaraldehyde have been reported to decrease
oxidation in fish oil (Lumsdon, Friedmann & Green, 2005). Other encapsulant materials
used for the encapsulation of omega-3 fatty acids were Maillard reaction products. Fish oil
here is emulsified with heated aqueous mixtures comprising of a protein source (sodium
caseinate, whey protein isolate, soy protein isolate, or skim milk powder) and
15
An emulsion
is said to be stable when there is no perceptible change in the size distribution of droplets,
its state of aggregation, or its spatial arrangement over the time-scale of observation, which
may vary from hours to months depending on the material (Vega & Roos, 2006;
Dickinson, 1994). Surfactants (e.g. polysorbates, phospholipids), proteins (e.g. milk
proteins) and/or thickening agents (gums, gelatin) can be used to increase the kinetic
stability of the emulsion (Dickinson & Woskett, 1989).
The particle size of emulsions is also a highly relevant factor as it influences the stability
of an emulsion prior to manufacture into spray-dried powder as well as the encapsulation
efficiency after spray drying. Results have shown that larger particles retained more oil
than smaller ones, but at the same time there was more unencapsulated oil at the surface of
big particles than the surface of small particles (Jafari et al., 2008; Jafari, He & Bhandari,
2007; Soottitantawat et al., 2005). The presence of unencapsulated oil on the surface of the
16
powder particles is the most undesirable property of encapsulated powders because of the
detrimental effects it has on the powder quality and stability (Vega et al., 2005).
The selection of a microencapsulation process depends on the properties of the core and
the wall materials, the release mechanisms desired, process type, and desired capsule
morphology and particle size. Most of the microencapsulation processes have been adapted
from the pharmaceutical and chemical industries (Augustin & Sanguansri, 2007). A range
of physical and chemical processes are available to encapsulate bioactive ingredients as
outlined in Table 1.
A process often used for encapsulation of oil and oil-soluble cores involves the use of
biopolymer gels as entrapment matrices. Examples include oil and oil-soluble cores
encapsulated in alginate beads by extrusion to form particles, followed by drying. For
efficient particle formation of this type methods like centrifugal extrusion and microject
methods have been developed (Benita, 1996).
Amongst the encapsulation processes mentioned above spray drying is the most commonly
used technique for microencapsulation. It is one of the oldest methods of encapsulation.
The process of spray drying is efficient, cost effective, has readily available equipment and
produces particles of reasonably good quality (Gharsallaoui, 2007). Food ingredients
microencapsulated by this method include fish oils, essential oils, vitamins, colourants,
flavours and other oil-soluble bioactives.
17
Mechanical Processes
Spray drying
Polymer-polymer incompatibility
Spray chilling
Fluidized bed
Electrostatic deposition
In situ polymerization
Centrifugal extrusion
In-liquid drying
suspension separation
Polymerization at liquid-gas
or solid-gas interface
Pressure extrusion or
spraying
The general process of spray drying involves the dispersion and homogenization of the
substance to be encapsulated and the encapsulating material to form a suspension in water
(slurry). The suspension is then fed into a spray drier. The solution is sprayed into the
drying chamber with the help of an atomiser or spray nozzle. There are different types of
nozzles available like rotary nozzles, single fluid pressure nozzles and ultrasonic nozzles.
Appropriate choice of the kind of nozzle to be used is based on the particle size required
for a specific application. The most common applications are in the 100 to 200 micrometre
diameter range. Hot drying gases are passed as either in the co-current or counter current
direction to the atomiser in the drying chamber. This hot air converts the suspension into
solid and the solvent into vapour. The dried particles are then collected in the particle
separator which is usually a cyclone separator (Spray drying, 2010). The dry powder is
often free flowing and the microcapsules are soluble in water.
Fish oil has been encapsulated by using various formulations, processing conditions and
encapsulation technologies (Gan, Cheng & Easa, 2008; Blatt et al., 2006; Heinzelmann &
Franke, 1999; Lumsdon, Friedmann & Green, 2005; Klinkesorn et al., 2005). According to
18
Sanguansri & Augustin (2006) the formulation and processing steps prior to spray drying
are considered important in the production of stable powder microcapsules. Processing
conditions such as, temperature, pH of the emulsion, total solids, drying rate and the order
of adding each of the components used during the manufacture of fish oil powders also
influence the microcapsule properties and stability.
19
materials coupled with appropriate formulation strategies have made possible the
production and stabilisation of nanoparticles that have potential applications in the food
and related industries (Sanguansri & Augustin, 2006).
Nanoencapsulation technology can be used to address some extraordinary needs of many
applications by producing nanosized particles and capsules. Nanocapsules in combination
with other microencapsulation methods can lead to new release characteristics (Persyn &
Oxely, 2008).
Nanotechnology and nanoscience involve the study of phenomena and materials, and the
manipulation of structures, devices and systems that exist at the nanoscale (<100 nm in
size). The properties of nanoparticles are not governed by the same physical laws as larger
particles, but by quantum mechanics. The physical and chemical properties of
nanoparticles for example, colour, solubility, strength, chemical reactivity and toxicity can therefore be quite different from those of larger particles of the same substance (Miller,
2008).
The altered properties of nanoparticles have created the possibility for many new profitable
products and applications. Engineered nanoparticles have been used to develop hundreds
of products and are available on supermarket shelves including transparent sunscreens,
light-diffracting cosmetics, penetration enhanced moisturisers, stain and odour repellent
fabrics, dirt repellent coatings, long lasting paints and furniture varnishes, and some food
products (Miller & Kinnear, 2007). It has been speculated that nanoencapsulation may gain
importance in the near future to develop designer probiotic bacterial preparations that
could be delivered to certain parts of the gastro-intestinal tract where they interact with
specific receptors (Paques & van Rijn, 2007). Biodegradable nano/microparticles of poly
(d,l-lactide-co-glycolide) (PLGA) and PLGA-based polymers have been widely explored
as carriers for controlled delivery of macromolecular therapeutics such as proteins,
peptides, vaccines, genes, antigens and some growth factors, etc (Raghavendra et al.,
2008).
20
Nanoencapsulation just like microencapsulation has many applications like protein, DNA
and RNA stabilization, small molecule delivery, extending circulatory half-life, modifying
drug transport, clear liquid formulations, stable colloid dispersions, controlled release,
targeted delivery, triggered release (Persyn & Oxely, 2008).
There has been a rise in concern in the use of nanotechnology as it involves the
manipulation of matter at the scale of atoms and molecules and hence, potentially
introduces some serious risks to human and environmental health. Toxicological literature
suggests that nanoparticles are more reactive, more mobile, and more likely to be toxic to
humans and the environment than larger particles. Preliminary scientific research has
shown that many types of nanoparticles can result in increased oxidative stress which can
result in the formation of free radicals that can lead to cancer, DNA mutation and even cell
death. Additionally, fullerenes, carbon nanoparticles, have been found to cause brain
damage in largemouth bass, a species accepted by regulatory agencies as a model for
defining ecotoxicological effects (Miller & Kinnear, 2007) thus, limiting the use of
nanotechnology extensively.
2.3 SUGAR BEET PECTIN AS WALL MATERIAL
Pectins are a family of plant-derived heteropolysaccharides comprised predominantly of a
linear chain of (14)-linked -D-galacturonic acid residues. These residues may be
partially esterified with a small percentage of rhamnose units to yield branches consisting
of neutral sugars, notably galactose and arabinose (Cho, 2001). Unlike citrus pectins, beet
pectins have ferulic acid groups esterified to some of the neutral sugars in the side-chains
of the so-called hairy regions (Guillon and Thibault, 1990; Micard and Thibault, 1999;
Saulnier and Thibault, 1999). Apples, guavas, quince, plums, gooseberries, oranges and
other citrus fruits, contain large amounts of pectin, while soft fruits like cherries, grapes
and strawberries contain small amounts of pectin (Cho, 2001).
The amount, structure and chemical composition of pectin differs between plants, within a
plant over time and in different parts of a plant (Cho, 2001). Pectins are generally
classified as high methyl ester (HM) pectins and low methyl ester (LM) pectins based on
21
the percentage of ester groups they possess or the degree of esterification (DE). If the DE
of the pectin is greater than 50% it is called as HM pectin, while if it is less than 50% it is
called a LM pectin (IPPA International Pectin Producers Association, 2001).
Pectins are produced commercially as a white to light brown powder, mainly extracted
from citrus fruits, and is used in food as a gelling agent particularly in jams and jellies. It is
also used in fillings, sweets, as a stabilizer in fruit juices and milk drinks and as a source of
dietary fiber (Pectin, 2010). In contrast to other pectins, sugar beet pectin (SBP) is more
interesting because of its unusual emulsifying properties. The emulsifying properties of
SBP have been attributed to its high protein content (up to 11%) and high level of
acetylation (up to 5%) (Leroux et al., 2003; Funami et al., 2007). SBP also contains the
known antioxidant, ferulic acid, which occurs esterified to the oxygen on C-2 of arabinose
or C-6 of galactose (Fry, 1983) (Figure 2). However, Drusch et al., (2007) and Katsuda
et al., (2008) showed that commercial SBP contains significant amounts of transition metal
ions, namely copper and iron, which promote lipid oxidation. Hence, antioxidants were
used in this research study to improve the oxidative stability of microencapsulated fish oil
with SBP as the wall material.
Rhamnose
Methyl groups
Galacturonic acid
Galactose
Arabinose
Figure 2 Schematic structure for generalised sugar beet pectin. Adapted from Morris et al.,
(2010) with some modifications made.
22
3.1.1.1PROTEIN CONTENT
The protein content of SBP was determined in triplicate using LECO (FP-2000 LECO
Corp., Michigan, USA) method, and the protein content was calculated using 6.25 as a
conversion factor.
23
The fatty acid composition of the oils prior to encapsulation was determined according to
Christie (2003) with some modification (Shen, 2010). The original and extracted oils were
derivatized using transmethylesterification and subsequently analyzed using a Varian 3400
gas chromatograph (Varian Associates Inc., USA) equipped with a BPX70 column (30 m
3.1.3 OXIDATION STATUS OF FISH OIL AND EXTRA VIRGIN OLIVE OIL
24
decline. The slope (-mbar hour-1) indicates the rate at which the sample is oxidized after
the initiation of oxidation. The slope was calculated by drawing a line at the IP point
parallel to the part of the curve recording the change in pressure (Figure 3).
P
t1
Pressure
(MPa)
etc),
proteins
(Sodium
Caseinate,
Whey
Proteins,
Gelatins
etc),
lipids (Hydrogenated fats, Vegetable oils, Palm Stearin etc) and waxes (Shellac or
Carnauba Wax etc) (Smith & Charter, 2010). In our study, DGS was used as a bulking
agent along with SBP to improve stability and microencapsulation of fish oil. Sugars in
association with emulsifying bioploymers have been shown to improve encapsulation
properties. Drusch (2007) successfully produced stable fish oil microcapsules using sugar
25
beet pectin as a wall material and glucose syrup as a bulk one. The same author reported
also that a pectin content of 1- 2% was sufficient for the formation of stable emulsions for
spray drying and proposed that the maximum oil loading in microcapsules containing FO
as the core may be limited to 50 % because of the amount of non-encapsulated fat obtained
in them. Hence, formulations were developed with 2% SBP along with DGS in the
preparation of FO and FO-EVOO emulsions with 25% and 50% oil loading at 30% TS.
SBP (2 g) was dispersed in distilled water at 70C and held at that temperature for a further
15 min before the addition of DGS. For samples with EDTA in the formulation, SBP (2 g)
was dispersed in 85 % of the total distilled water required at 70C and EDTA was added to
chelate the metal ions before the addition of DGS. The solution was subsequently cooled to
60C. The pH was then adjusted by adding 1M NaOH solution to samples whose pH had
to be altered to 6. The amount of NaOH solution was recorded to determine the remaining
water to be added to the pre-heated solution. The solution was subsequently cooled to
60C. FO and EVOO were pre-heated to 60C. A FO-EVOO blend was prepared by
combining the oils at a 1:1 weight ratio. The FO and the FO-EVOO blend were dispersed
into the pre-heated mixtures of SBP and DGS at 60C for 1-2 min using a high shear mixer
(Silverson, London, UK) at maximum speed to obtain a pre-emulsion. The pre-emulsions
were then homogenized at 60C using a high pressure two-stage homogenizer (APV
Rannie AS Homogenizer, Denmark) at 35 and 10 MPa. All emulsions were prepared at
30% total solids. The composition of the pre-emulsion with the percentage of each
component stated in terms of ingredient weight is outlined in Table 2 & 3. The process
flow chart of the experimental plan for the production of spray dried powders has been
presented in Figure 4. FO and FO-EVOO emulsions were prepared at 25% (pH 3) and 50%
(pH 3 & pH 6 ) oil loading in powder and emulsions with EDTA were prepared at 50% oil
loading in powder only at pH 6 yielding a total of 8 different emulsions (FO-25,
FO-EVOO-25, FO-50 (pH 3), FO-EVOO-50 (pH 3), FO-EDTA-50,FO-EVOO-EDTA-50,
FO-50 (pH 6), FO-EVOO-50 (pH 6)) (Figure 5 (A)). All emulsions were prepared in two
runs with duplicate samples in each run.
26
Pre-emulsion
Homogenization
(35+10 MPa)
Spray Drying
Powder Microcapsules
Figure 4 Process flow chart of the experimental plan for the production of spray-dried
microcapsules.
27
Table 2 Formulation of the fish oil and fish oil-extra-virgin olive oil (1:1 wt ratio)
emulsions at pH 3.
FO-50
FO-EVOO-25
FO-EVOO-50
SBP
DGS
20.5
13
20.5
13
FO
7.5
15
3.75
7.5
EVOO
3.75
7.5
Water
70
70
70
70
FO-EDTA-50
FO-EVOO-EDTA-50
FO-50
FO-EVOO-50
SBP
EDTA(Na
0.05
0.05
DGS
12.95
12.95
13
13
FO
15
7.5
15
7.5
EVOO
7.5
7.5
Water
70
70
70
70
salt)
28
29
3.4.3 VISCOSITY
The viscosity of the emulsions was measured using a cup and bob geometry (Paar Physica
Rheometer, MCR 300, Anton Paar, Austria) at 25C within 2 h of preparation. The shear
rate () was increased in 30 steps from 0.2 to 291 s1. The duration of measurement at each
shear rate was 10 s.
30
United
Kindom).
Titrations
were
performed
by
31
The spray-dried powders (10% w/v) were reconstituted at 70C for 3 h under constant
stirring, and then rested for 1 h at room temperature (~25C). Particle size was determined
as described above (Section 3.4.1).
32
equilibrated at 60 C for 15 min in a water bath. An aliquot (1 ml) of the headspace was
then analyzed using a Varian 3400 gas chromatograph (Varian Associates,Inc., USA)
equipped with a BPX-70 fused silica capillary column (30 m
film thickness) and a FID (flame ionisation detector). The injector and detector
temperatures were 250 C and 275 C, respectively. The oven temperature was
programmed at 60 C and held for 5 minutes. The temperature was then increased to 175 C
C at 4C min .
-1
3.6 STATISTICS
All analytical determinations were carried out at least in duplicate. The results were
reported as the mean standard deviation (SD) of these measurements. The analysis of
variance (One way ANOVA) was performed at 95% confidence level using SPSS 18
(SPSS Inc, Chicago, USA) and LSD (least significance) was used to separate the means.
33
34
Results of fatty acid composition of FO and EVOO was quantified using GC are shown in
Table 4. The major fatty acids found in FO were palmitic acid (C16:0) (21%), oleic acid
(C18:1) (~14%), eicosapentaenoic acid, EPA (C20:5n-3) (6.6%), and docosahexaenoic
acid, DHA (C22:6n-3) (26.5%).The fatty acid composition of FO is highly dependent on
the age, sex, spawning cycle, season, species and origin of the fish (Donmez, 2009). In this
study, the FO was derived from tuna. The EPA and DHA content of the FO were found to
be very close to that of the manufacturers specification. Gotoh et al., (2006) quantified
fatty acids present in big eye tuna oil, and the reported EPA and DHA percentages were
6.5% and 24% respectively.
Results of fatty acids analysis in EVOO were in agreement with those reported in the
literature. The major fatty acid in EVOO is oleic acid, which can vary from 5583%. It
was present at a level of 80% in the EVOO used in this work. The other major fatty acid
present is palmitic acid and normally its content ranges between 7.520% (Olive oil,
2008). It was 7% in the EVOO used in this work.
Table 4 Fatty acid composition (%) of the initial oils
Fatty Acida
Palmitic acid, C16:0
13.51 1.79
79.54 0.12
6.64 0.92
ND
26.54 0.53
ND
Others
32.21 4.12
13.47 0.01
35
4.1.3 OXIDATION STATUS OF FISH OIL AND EXTRA VIRGIN OLIVE OIL
4.1.3.1 ASSESSMENT OF OXIDATIVE STABILITY USING THE OXIPRES
The Oxipres results of the original FO, EVOO and their 1:1 blend are given in Table 5.
The IP for these oils under the accelerated conditions (80C, 0.5 bar oxygen pressure) used
in Oxipres assessments were 11.85 0.07 h, >100 h and 16.3 0.14 h, respectively, for
FO, EVOO and the 1:1 mixture of FO-EVOO. The uptake of oxygen after the IP was -210
9.19 mbar h-1 and -119 4.24 mbar h-1, respectively, for FO and a 1:1 mixture of
FO-EVOO. EVOO was monitored for up to 105 h, during which time no detectable
oxidation was observed. Hence no oxidation rate (slope) for this sample was provided.
This confirmed that olive oil was more stable to oxidation than FO. That might be
expected based on the fatty acid composition alone as the omega-3 fatty acids in FO are
very prone to oxidation. In addition, olive oil contains phenolic compounds, which are
known to be antioxidative (Psomiadou & Tsimidou, 2002).
36
Table 5 Oxidative stability of original oils during accelerated storage (80C, oxygen
pressure of 0.5 bar)
a
FO
11.85 0.07
-210 9.19
EVOO
>100 0.00
16.3 0.14
-119 4.24
Sample
OLIVE
OIL
ON
EMULSION
AND
MICROCAPSULE
CHARACTERISTICS
The average particle size (d3,2) of the different emulsions ranged between 0.410.43 m
(Figure 6). These values were comparable to those reported for oil-in-water emulsions
made by emulsifying 20% w/w orange oil with 2% SBP (Leroux et al., 2003) and
homogenised with three passes at 200 bars. SBP dissolved in water alone had a (d3,2) of
2.38 0.31 m, and contributed to the particle size of the emulsions. This observation also
accounts for the minor peak present in the particle size distribution graph of the emulsions.
There was no significant difference (P > 0.05) in the average particle sizes between the
emulsions containing 25% and 50% oil loading. This indicated that the presence of 2%
SBP was sufficient emulsifier to produce physically stable fish oil-in-water emulsions
containing 15% oil (wet basis). Similarly, other studies (Drusch, 2007; Drusch et al., 2007;
Leroux et al., 2003; Nakauma et al., 2008; Siew & Williams, 2008) have reported that
1.52% SBP was sufficient to produce oil-in-water emulsions containing up to 18% oil
(wet basis). For example, Nakauma et al., (2008) made stable oil-in-water emulsions with
1.5% SBP and 15% w/w oil (medium chain triglyceride) homogenised with two passes at
50 MPa.
37
7
6
Volume (%)
5
4
3
2
1
0
0.01
0.1
10
100
1000
Figure 6 Particle size distributions of the original (fresh) emulsions, prior to spray drying.
Values are the averages of triplicate measurements SD;
(FO-EVOO-50);
(FO-50);
(FO-25);
(FO-EVOO-25).
38
Figure 7 Brightfield micrographs of (A) FO-25 (7.5% oil, wet basis) and (B)
FO-EVOO-50 (15% oil, wet basis) emulsions at pH 3.
The apparent viscosities of the emulsions as a function of shear rate are shown in Figure 8.
All the formulations displayed shear thinning or pseudo-elastic behaviour, which is a
common feature of oil-in-water emulsions (McClements, 1999).
Emulsions containing 15% oil (wet basis) had greater apparent viscosities than
corresponding emulsions containing 7% oil, as expected. However, emulsions containing
either FO or a 1:1 FO-EVOO mixture with the same gross formulation had different
viscosities. It is possible that the factor responsible for these differences was the presence
of phenolic compounds in the olive oil. For example, polyphenols are known to complex
with protein (Spencer et al., 1988). An interaction between polyphenols and residual
protein in SBP can alter the interfacial properties of the oil droplet or the properties of the
bulk phase and also affect viscosity of the emulsion. Other studies showed that
polyphenol--casein complexes alter the properties of an air/water interface and also exist
as complexes in the bulk solution (Aguie-Beghin et al., 2008).
39
1800
1600
Viscosity (mPa/s)
1400
1200
1000
800
600
400
200
0
0
50
100
150
200
250
300
Figure 8 Apparent viscosities of fish oil and fish oil-extra virgin olive oil (1:1 wt ratio)
emulsions (30% TS; 2% SBP, 20.5% DGS, 7.5% total oil and 30% TS; 2% SBP, 13%
DGS, 15% total oil), intended for manufacture of spray-dried microcapsules containing 25
and 50% oil, as a function of shear rate. Values are the average of duplicate measurements
SD;
(SBP-DGS-FO-50);
(SBP-DGS-FO-25);
(SBP-DGS-FO-EVOO-50);
(SBP-DGS-FO-EVOO-25).
Zeta potential measurements of the FO and FO-EVOO emulsions (2% SBP, 13% DGS,
15% total oil) intended for the manufacture of spray-dried microcapsules with 50% oil
loading, as a function of pH, were determined using an autotitrator to predict the stability
of the emulsions (Table 6). The emulsions were measured at pH values between 6 and 1.
The zeta potential of a particle can be defined as the overall charge that the particle
acquires in a particular medium. Depending on the zeta potential of the particle, the
stability of an emulsion can be explained. If the particles in a suspension have a large
negative or positive zeta potential then they will tend to repel each other and resist the
formation of aggregates. Conversely, particles of opposite charge tend to associate. The
dividing line between stable and unstable suspensions is generally taken at either +30 mV
40
or -30 mV. Particles with zeta potentials more positive than +30 mV or more negative than
-30 mV are normally considered stable (Zeta Potential Measurement Using an Autotitrator
from Malvern Instruments and the Effect Of pH, 2005). The autotitration plots revealed
that both FO and FO-EVOO emulsions were most stable at pH greater than 5 (Figure 9 &
10). Both emulsions would be less stable as pH is reduces below 5, and would be least
stable at pH ~1.5 as the iso-electric point range for the emulsions was between 1.2 and 1.5.
It has been observed that at pH less than 5 the zeta potential of the emulsions decreases
rapidly and eventually reached an iso-electric point after pH 1.5.
Table 6 Zeta potential values of FO and FO-EVOO emulsions measured at 22 C.
a
Sample
FO-50 (pH 3)
-12.12 0.39
FO-50 (pH 6)
-39.8 0.51
FO-EVOO-50 (pH 3)
-13.56 0.47
FO-EVOO-50 (pH 6)
-41.4 0.66
After manufacture, the moisture content of the spray-dried microcapsules made with SBP
and containing 25% and 50% oil loadings ranged from 1.61.8%, with a water activity of
~0.27 (Table 7).The maximum moisture specification for most spray-dried powders in the
food industry was between 34 % (Masters, 1991). The encapsulation efficiency of the
spray-dried microcapsules made with SBP containing 25% and 50% of FO and FO-EVOO
blend was >90%. A significant difference (P < 0.05) in the encapsulation efficiencies of
the microcapsule powders containing different oil loadings was recorded (Table 7). The
microcapsule powders with 50% oil loading had lower encapsulation efficiency (~90%)
than those with 25% oil loading (encapsulation efficiency ~98%). In concurrence with the
solvent-extractable fat contents, encapsulation efficiency was not affected by the
composition of the microencapsulated oil.
41
10
Zeta Potential (m V)
0
0
-10
-20
-30
-40
-50
pH
Figure 9 Autotitration curve for FO emulsion with zeta potential measured as a function of
pH at 22 C.
5
0
Zeta Potential (m V )
-5 0
-10
-15
-20
-25
-30
-35
-40
-45
pH
Figure 10 Autotitration curve for FO-EVOO emulsion with zeta potential measured as a
function of pH at 22 C.
42
Table 7 Properties of spray-dried powders containing microencapsulated fish oil and fish
oil-extra virgin olive oil (1:1 wt ratio) (25% and 50% oil loading) at pH 3.
a
Property
Microcapsule
FO-25
FO-EVOO-25
FO-50
FO-EVOO-50
Moisture content
1.83 0.02
1.73 0.01
1.66 0.01
1.62 0.03
0.27 0.02
0.26 0.01
0.27 0.02
0.27 0.00
24.39 0.06
24.38 0.06
49.09 0.14
49.22 0.12
Solvent-extractable oil
(% of free oil in
powder)
Encapsulation
efficiency (%)
0.53 0.01
0.52 0.00
4.99 0.01
5.00 0.01
97.85 0.04a
97.87 0.04a
90.43 0.09b
90.42 0.08b
0.42 0.00
0.43 0.00
0.41 0.00
0.43 0.00
Mean particle
diameter, d32, prior to
spray drying
a
Means within the row followed by different superscript letters differ significantly at P = 0.05
The amount of solvent-extractable fat in the spray-dried powders with the same oil loading
but different oil composition was comparable (Table 7). The FO and FO-EVOO powders
gave ~2% solvent-extractable fat as a percentage of total fat in powder at 25% oil loading,
and ~10% solvent-extractable fat at 50% oil loading. The amount of free oil in the powders
can generally be described as the amount of oil that can be found on the surface of the
powder particles and can include oil that is within cracks in the powder particle that is
readily accessible to solvent (Buma, 1971). Several studies had been conducted to evaluate
the factors affecting the oxidative stability of spray-dried powders containing encapsulated
oil (Velasco, Dobarganes & Mrquez-Ruiz, 2003; Drusch et al., 2007; Mrquez-Ruiz,
Velasco & Dobarganes, 2003). However, due to the different matrix and core materials
used, and the various methods and conditions employed to monitor oil oxidation, no
consensus has yet been reached. Velasco, Dobarganes and Marquez- Ruiz (2003) stated
that lipid distribution in a microcapsule is an important factor influencing oxidation in
encapsulated oil. They discussed various studies conducted to evaluate the oxidation of
43
44
Microcapsule
0 month
3 month
0 month
3 month
FO-25
1.83 0.02
2.18 0.05
0.27 0.02
0.49 0.00
FO-50
1.66 0.01
2.06 0.03
0.27 0.02
0.50 0.01
FO-EVOO-25
1.73 0.01
1.95 0.01
0.26 0.01
0.47 0.01
FO-EVOO-50
1.62 0.03
1.93 0.02
0.27 0.00
0.48 0.00
Table 9 Particle size of the reconstituted spray-dried microcapsules containing fish oil
and fish oil-extra virgin olive oil (1:1 wt ratio) during storage of the powders in stoppered
flasks at room temperature (~25C).
Mean particle diameter, d3,2 (m) a
Microcapsule
0 month
1 month
a
2 month
b
3 month
a
2.91 0.10b
FO-25
0.50 0.01
FO-50
0.70 0.00 c
0.78 0.00c
2.06 0.08c
3.06 0.05c
FO-EVOO-25
0.50 0.00 a
0.50 0.00a
0.90 0.01a
1.62 0.01a
FO-EVOO-50
0.69 0.00 b
0.79 0.01d
1.69 0.03b
1.69 0.02a
0.50 0.00
0.92 0.00
Means within the row followed by different superscript letters differ significantly at P = 0.05
45
5
4
3
2
0
0.01
10
100
3
2
0.1
III
0.01
1000
6
Volume (%)
Volume (%)
0.1
4
3
2
1
10
100
1000
IV
5
4
3
2
1
0
0.01
II
6
Volume (%)
Volume (%)
0
0.1
10
100
1000
0.01
0.1
10
100
1000
and 3 month (AD) stored under room temperature conditions (~25C, exposed to light).
Values are the averages of triplicate measurements SD;
(FO-EVOO-50);
(FO-50);
(FO-25);
(FO-EVOO-25).
Visual inspection of the microcapsule powders revealed that the samples stored for 2 and 3
months contained clumps that were not evident in those of the fresh microcapsule powders
(0 month), or those stored for 1 month. These observations suggested the development of
cohesive interactions between particles over the period of storage time. A number of
variables are known to contribute to the time consolidation effects on food powders.
Storage temperature, exposure of powders to moisture content in air, physical and chemical
changes in the powder sample during storage time, and variations in powder bulk density
have all been found to contribute significantly to the time-consolidation effects of powders
(Teunou, 2000; Fitzpatrick 2004; Onwulata 2005).
46
Figure 12 Scanning electron microscopy images of the fish oil (A, C) and fish oil-extra
virgin olive oil (B, D) spray-dried powders (25% oil loading), prior (A, B) and after 3
month storage (C, D) under ambient conditions (~25C, 0.5 aw).
47
The average particle size (d3,2) of the FO and FO-EVOO emulsions, with and without
EDTA, ranged between 0.350.36 m. The particle size distributions of the emulsions
revealed a multimodal distribution of particles, with the majority of particles ranging in
size from ~0.21 m in diameter, and a minor proportion ranging from 110 m (Figure
13). As mentioned in (section 4.2), SBP dissolved in water had an average particle size
(d3,2) of ~ 2.5 m and contributed to the particle size of the emulsions. A slight difference
in mean particle size distributions was observed in FO and FO-EVOO-EDTA when
compared to FO-EVOO and FO-EDTA emulsions. That indicated that the addition of
EDTA as chelating agent had very little influence on the mean particle size of emulsions.
The average particle size (d3,2) of all the emulsions were determined after 1 month of
storage at 22C, and ranged between 0.35-0.36 m. That indicated that all emulsions were
stable to droplet aggregation, as evidenced by the lack of change in the average particle
size values, (d3,2) of the fresh emulsions and corresponding emulsions after 1 month
storage. The absence of droplet aggregation could be attributed to the strong repulsive
forces between droplets which were large enough to overcome the various interactions
(mainly Van der Waals forces and hydrophobic interactions) between droplets. It was also
suggested that as pH was increased from the natural pH of pectin solution (pH 3) to pH 6,
the ratio of protonated and deprotonated carboxyl groups present on pectin molecule would
decrease, thereby increasing the negative charge of the pectin molecules. These changes in
pH values increased repulsion between the emulsion droplets and stabilises the droplets
against aggregation.
Visual examination of the emulsions by brightfield microscopy showed that all emulsions
were homogeneous without any visible particle aggregation or any marked presence of
free (nonencapsulated) oil (Figure 14). Based on the results and literature findings
discussed in section 4.2, those results again established that 2% SBP was sufficient to
produce fine and stable oil-in-water emulsions containing at least 15% w/w oil.
48
10
9
8
Volume (%)
7
6
5
4
3
2
1
0
0.01
0.1
10
100
1000
Figure 13 Particle size distributions of the original (fresh) emulsions (15% oil, wet basis),
with and without EDTA, at pH 6. Values are the averages of triplicate measurements SD;
(FO-EDTA-50);
(FO-EVOO-EDTA-50);
(FO-50);
(FO-EVOO-50).
49
rate range measured. The presence of high concentrations of metal ions in SBP (Drusch et
al., 2007; Katsuda et al., 2008) and phenolic compounds in EVOO (Cicerale et al., 2009;
Frankel, 2010) could be the underlying cause for the differences in the flow properties of
FO emulsions containing both EVOO and EDTA when compared to FO emulsions
containing either EVOO or EDTA. Also, the differences in interfacial properties of FO and
FO-EVOO emulsions stabilised with SBP have already been established and discussed in
relation to polyphenol-protein complexation (section 4.2).
1000
900
Viscosity (mPa/s)
800
700
600
500
400
300
200
100
0
0
50
100
150
200
250
300
Figure 15 Apparent viscosities of fish oil and fish oil-extra virgin olive oil (1:1 wt ratio)
emulsions (15% oil, wet basis) with and without EDTA at pH 6, as a function of shear rate.
Values are the average of duplicate measurements SD;
(FO-EVOO-EDTA-50);
(FO-50);
(FO-EDTA-50);
(FO-EVOO-50)
The moisture content of the spray-dried powders made with SBP (with and without EDTA)
at pH 6 with 50% oil loadings ranged from 2.832.96 %, with an aw of ~0.3 (Table 10).
The moisture content values were typical of that for most spray-dried powders. The
maximum moisture specification for most spray-dried powders in the food industry is
between 34 % (Masters 1991). This is in order to achieve microbiological stability. The
moisture content and aw of the spray-dried powders made with SBP (with and without
50
EDTA) at pH 6 with 50% oil loadings was also determined after 3 months of storage
(Table 11). An increase in moisture content and aw was observed in all microcapsules and
the percent increase in the moisture content and aw was found to be highest in
microcapsules which did not contain EDTA. That increment in moisture content was
expected as the sample containers in which the powders were stored were permeable to
moisture and the aw of the atmosphere (~0.5) was greater than that of the freshly
manufactured powders.
The spray-dried powders with and without EDTA at pH 6 and 50% oil loading gave ~10%
solvent-extractable fat of total fat in powder. The effect of solvent-extractable fat on lipid
oxidation has been investigated and it has been concluded that it cannot be used to predict
the shelf-life of microencapsulated oils (Drusch & Berg, 2008; Velasco, Dobarganes &
Mrquez-Ruiz, 2003). Furthermore, since the amount of solvent-extractable fat was
considerably small, it was unlikely to correlate with the oxidative stability of the
microencapsulated oils. Also, the spray-dried powders were found to be free-flowing,
indicating that the powders did not have high free fat on the surface.
The encapsulation efficiency of the spray-dried microcapsules with and without EDTA at
pH 6 with 50% oil loading was ~90%. There was no difference in the encapsulation
efficiencies of microcapsules without EDTA and microcapsules with EDTA. Thus, it could
be concluded that neither the addition of EDTA nor the oil composition in the core of the
microcapsule had any effect on either solvent-extractable fat or encapsulation efficiency.
The particle size distribution and d3,2 values of the reconstituted spray-dried microcapsules
at 03 month storage under room temperature conditions was determined (Figure 16 &
Table 12). A general increase in particle size was observed in all microcapsules over
storage time at room temperature. The particle size values for FO and FO-EVOO
microcapsules over storage time were similar indicating no influence of type of oil/oils in
the core of the microcapsule on particle size.
51
Table 10 Properties of powders containing microencapsulated fish oil and fish oil-extra
virgin olive oil blends (1:1 wt ratio) with and without EDTA at pH 6, 50% oil (dry basis).
a
Property
Microcapsule
FO-EDTA
FO-EVOO-
-50
EDTA-50
2.83 0.01
2.89 0.02
2.98 0.03
2.96 0.04
0.30 0.01
0.30 0.00
0.29 0.00
0.29 0.00
49.27 0.12
49.22 0.13
49.29 0.17
49.29 0.03
Solvent-extractable
4.73 0.00
4.73 0.00
4.72 0.00
4.72 0.00
90.41 0.08
90.40 0.09
90.41 0.12
90.42 0.02
0.36 0.00
0.35 0.00
0.35 0.00
0.35 0.00
Moisture content
FO-50
FO-EVOO
-50
(%)
oil
(% of free oil in
powder)
Encapsulation
efficiency (%)
Mean particle
diameter, d3,2,
prior to spray
drying
a
The average oil droplet size (d3,2) of all the reconstituted microcapsules at zero month was
~0.80.9 m. This showed that the particle size of zero month powders were slightly
greater than the particle size of the corresponding emulsions prior to drying. This increase
in particle size may be due to changes to the interfacial structure of SBP, causing
coalescence of oil droplets and also, some pectin aggregation during the spray drying
process. According to Rees (1969), the pectin chain interactions are promoted under low
aw conditions. Kirby, MacDougall, & Morris (2006) used atomic force microscopy (AFM)
52
SEM of the microcapsules indicated that the stored powders had an increase in
agglomeration in the powder particles when compared to the SEM images of the fresh
powders (Figure 17). The microcapsules were regular in shape with wrinkled surface and
no cracks or pores. This surface wrinkling has been associated to be caused by the
mechanical stress caused during spray drying process and has been discussed in section
4.2. Most images had microcapsules as discrete units with no incomplete or damaged shell.
53
microcapsules (50% oil, dry basis) (with and without EDTA) at pH 6 on storage at room
temperature (~25C).
a
Microcapsule
0 month
3 month
0 month
3 month
FO-EDTA-50
2.83 0.01
3.25 0.01
0.30 0.01
0.49 0.01
FO-EVOO-EDTA-50
2.89 0.02
3.01 0.06
0.30 0.00
0.48 0.00
FO-50
2.98 0.03
3.53 0.06
0.29 0.00
0.49 0.01
FO-EVOO-50
2.96 0.04
3.19 0.12
0.29 0.00
0.49 0.00
Table 12 Particle size values of the reconstituted spray-dried microcapsules (50% oil, dry
0 month
1 month
2 month
3 month
FO-EDTA-50
0.87 0.01b
0.88 0.01a
1.30 0.03ab
1.46 0.03a
FO-EVOO-EDTA-50
0.84 0.00a
0.89 0.00a
1.35 0.04b
1.44 0.03a
FO-50
0.89 0.00b
0.89 0.00a
1.28 0.06a
1.44 0.04a
FO-EVOO-50
0.87 0.01c
0.90 0.02a
1.27 0.04a
1.46 0.03a
Means within the row followed by different superscript letters differ significantly at P = 0.05
54
5
4
3
2
1
0
0.01
5
4
3
2
1
0.1
10
100
0
0.01
1000
10
100
1000
6
5
4
3
2
6
Volume (%)
Volume (%)
0.1
5
4
3
2
1
1
0
0.01
6
Volume (%)
Vo lum e (% )
0.1
10
100
1000
0
0.01
0.1
10
100
1000
dry basis), with and without EDTA at pH 6 after 0, 1, 2 and 3 month (AD) storage at
room temperature (~25C). Values are the averages of triplicate measurements SD;
(FO-EDTA-50);
(FO-EVOO-EDTA-50);
(FO-50);
55
(FO-EVOO-50).
Figure 17 Scanning electron microscopy images of the fish oil-extra virgin olive oil
spray-dried powders (50% oil, dry basis), with (A, C) and without (B, D) EDTA, at pH 6,
prior (A, B) and subsequent (C, D) to storage for 3 months at room temperature (~25C).
4.4
EFFECT
OF
pH
ON
EMULSION
AND
MICROCAPSULE
CHARACTERISTICS
The mean particle diameters (d3,2) of FO and FO-EVOO emulsions at pH 3 with 50% oil
loading were 0.41 0.00 m and 0.43 0.00 m, respectively. In comparison, both the FO
and FO-EVOO emulsions at pH 6 with 50% oil loading had a slightly smaller mean
particle diameter of 0.35 0.0 m. The difference in particle size observed for emulsions
at pH 3 and pH 6 indicated a slight influence of pH on the emulsion particle size. It has
already been discussed in section 4.2 that 2% SBP was sufficient to prepare stable FO
emulsions with 50% oil loading. The small difference in mean particle diameters of the
56
emulsions made at pH 3 and pH 6 suggests that there was sufficient emulsifier present to
prepare finely-dispersed emulsions and any change in conformation / charge of the SBP as
pH is raised from pH 3 to 6 did not have a marked effect on the emulsifying capacity of
SBP.
The particle size distributions of FO and FO-EVOO emulsions at pH 3 and pH 6 showed a
non-Gaussian, largely uni-modal distribution (Figure 18). It was observed that there was a
shoulder on the major peak in the distribution curve in all the 4 emulsions, corresponding
to particles having a mean diameter between 110 m, and this was more prominent in the
emulsions prepared at pH 3. This shoulder could be because of the presence of small
aggregates caused by the interaction between particles. When the pH of a solution is
altered the proteins present in the mixture may experience some conformational changes
and as they approach their isoelectric point they precipitate (Vaclavik & Christian, 2008).
This precipitation may cause the proteins to loose their emulsifying capacity as the
precipitated particles collide, stick and break apart until a stable mean particle size is
reached and in the due process causes some aggregation.
9
8
V o l u m e (% )
7
6
5
4
3
2
1
0
0.01
0.1
10
100
1000
Figure 18 Particle size distributions of the original (fresh) fish oil and fish oil-extra virgin
olive oil (1:1 wt ratio) emulsions made at pH 3 and 6. Values are the averages of triplicate
measurements SD;
57
The brightfield micrographs of the emulsions are shown in Figure 19. The micrographs of
pH 3 and pH 6 emulsions were comparable and appeared to have no large aggregates of
undissolved SBP. Additionally, all the emulsions were homogeneous with no indication of
any unencapsulated oil.
Figure 19 Brightfield micrographs of (A) fish oil (pH 3) and (B) fish oil-extra virgin olive
having
15% oil (wet basis) that were adjusted to pH 6 were less viscous than the FO and
FO-EVOO emulsions at pH 3, indicating the influence of pH. The zeta potential data
showed that the SBP-stabilised oil droplets had an isoelectric point of ~!1.5. Thus the
droplets were more negatively charged at pH 6 (-potential = -41.5 2.12 mV) than pH 3
(-potential = -14.0 1.41 mV). As the pH is increased to pH 6, the carboxyl groups on the
pectin molecule (as well as protein) lose H+ and therefore the negative charge on the SBP
will increase, resulting in increased electrostatic repulsion. At pH 3, there is less charge
(smaller zeta potential) and therefore the tendency for the particles to aggregate is
increased. The increased interaction between particles lead to an increase in viscosity of
the emulsions made at pH 3.
58
1800
1600
Viscosity (mPa/s)
1400
1200
1000
800
600
400
200
0
0
50
100
150
200
250
300
Figure 20 Apparent viscosities of fish oil and fish oil-extra virgin olive oil (1:1 wt ratio)
emulsions with 15% oil at pH 3 and pH 6, as a function of shear rate. Values are the
average of duplicate measurements SD;
(FO-50 (pH 6));
The moisture content of the FO and FO-EVOO powders at pH 6 with 50% oil loading was
2.98 0.03% and 2.96 0.04%, respectively at 0 month storage (Table 13). The moisture
content of the FO and FO-EVOO powders at pH 3 with 50% oil loading was 1.66
0.01% and 1.62 0.03%, respectively. A water activity of ~0.3 was found for all the
powders. The higher moisture content in pH 6 microcapsules was directly related to the
smaller particle size diameter. Smaller particle size provided the larger surface area and
facilitated entrapping more water molecules between particles.
Moisture content and aw of FO and FO-EVOO powders made at pH 3 and pH 6 with 50%
oil loading was determined after 3 months of storage under ambient conditions to assess
their physical stability. Both properties increased with increased storage duration. It was
observed that the percent increase in moisture content and aw was greater in microcapsules
made at pH 3 (Table 14) and as pointed out previously (section 4.3), that was probably due
to absorption of moisture from the surrounding environment (aw ~0.5) as the samples were
stored in (plastic) containers that were permeable to moisture. The moisture content
59
measurements were in agreement with the results of aw, with the aw increasing with the
increase in moisture content.
FO and FO-EVOO microcapsules prepared at pH 3 and pH 6
had ~10% solvent-extractable fat and encapsulation efficiencies of ~90% (Table 13). Thus,
it can be concluded that neither pH nor the type of oil/oils in the core of microcapsule had
any effect on either solvent-extractable fat or encapsulation efficiency.
It can be collectively concluded that there were no differences among the microcapsules at
pH 3 and pH 6 when total oil percentage, extractable oil percentage and encapsulation
efficiency was compared.
Table 13 Properties of powders containing microencapsulated fish oil and fish oil-olive oil
Property
Microcapsule
FO-50
(pH 3)
1.66 0.01
FO-EVOO-50
(pH 3)
1.62 0.03
FO-50
(pH 6)
2.98 0.03
FO-EVOO-50
(pH 6)
2.96 0.04
0.27 0.02
0.27 0.00
0.29 0.00
0.29 0.00
49.09 0.14
49.22 0.12
49.29
49.29 0.03
0.17
Solvent-extractable oil (%)
4.99 0.01
5.00 0.01
4.72 0.00
4.72 0.00
Encapsulation efficiency
90.43 0.09
90.42 0.08
90.41
90.42 0.02
(%)
Mean particle diameter,
d3,2, prior to spray drying
(m)
a
0.12
0.41 0.00
0.43 0.00
60
0.35 0.00
0.35 0.00
Table 14 Changes in moisture content and water activity of fish oil and fish oil-extra
virgin olive oil (1:1 wt ratio) microcapsules at pH 3 and pH 6 with 50% oil loading over
storage time at room temperature (~25C).
Microcapsule
0 month
3 month
0 month
3 month
FO-50 (pH 3)
1.66 0.01
2.06 0.03
0.27 0.02
0.5 0.01
FO-EVOO-50 (pH 3)
1.62 0.03
1.93 0.02
0.27 0.0
0.48 0.0
FO-50 (pH 6)
2.98 0.03
3.53 0.06
0.29 0.0
0.49 0.01
FO-EVOO-50 (pH 6)
2.96 0.04
3.19 0.12
0.29 0.0
0.49 0.0
at room temperature. The increase in particle size for FO and FO-EVOO microcapsules
over storage time were similar, indicating only a minor influence of the type of oil/oils in
the core of the microcapsule. The particle size of all zero month powders at pH 3 and pH 6
with 50% oil loading were slightly greater than the particle size of the corresponding
emulsions prior to spray drying. As stated previously in section 4.3, this could be attributed
to the destabilisation of emulsion droplets during the spray drying process as well the
formation of pectin aggregates.
A significant (P < 0.05) increase in particle size of all the microcapsules at pH 6 with 50%
oil loading was observed after spray drying. However, the increase in particle size of the
microcapsules over storage was comparatively small in comparison with the particle size
of the FO and FO-EVOO microcapsules at pH 3 with 50% oil loading during storage.
61
4
3
2
5
4
3
2
1
1
0
0.01
Volum e (% )
V olum e (% )
0.1
10
100
0
0.01
1000
0.1
100
1000
Volum e (% )
V olu m e (% )
10
5
4
3
2
1
0
0.01
5
4
3
2
1
0.1
10
100
1000
0
0.01
0.1
10
100
1000
Figure 21 Particle size distributions of reconstituted spray-dried fish oil and fish oil-extra
virgin olive oil microcapsules prepared from emulsions at pH 3 and 6 with 50% oil loading
prior (A), and subsequent to storage for 1 (B), 2 (C) and 3 (D) month under room
temperature conditions (~25 C, aw 0.5, exposed to light). Values are the averages of
triplicate measurements SD;
(pH 6));
62
(FO-50
fish oil and fish oil-extra virgin olive oil (1:1 wt ratio) at pH 3 and pH 6 with 50% oil
loading, during storage of the powders in stoppered flasks at room temperature (~25C).
Mean particle diameter, d3,2, (m)
a
Microcapsule
0 month
1 month
FO-50 (pH 3)
0.70 0.00c
0.78 0.00c
2.06 0.08c
3.06 0.05c
FO-EVOO-50 (pH 3)
0.69 0.00b
0.79 0.01d
1.69 0.03b
1.69 0.02a
FO-50 (pH 6)
0.89 0.00b
0.89 0.00a
1.28 0.06a
1.44 0.04a
FO-EVOO-50 (pH 6)
0.87 0.01c
0.90 0.02a
1.27 0.04a
1.46 0.03a
2 month
3 month
Means within the row followed by different superscript letters differ significantly at P = 0.05
This
phenomenon of lumping can be attributed to the change in moisture content and aw that
occurred in the microcapsules over storage time (Table 14).
63
A
A
C
C
Figure 22 Scanning electron microscopy images of fish oil spray-dried microcapsules
from emulsions prepared at pH 3 (A, C) and pH 6 (B, D) with 50% oil loading, prior (A,
B) and subsequent (C, D) to storage at ambient conditions (~25C, aw 0.5, 3 months).
Lipid oxidation is a major issue when dealing with oils and powders encapsulated with oils
because of the thermal and oxidative reactions that take place during storage. The primary
products of lipid oxidation are the hydroperoxides which are colourless, tasteless and
odourless. These hydroperoxides are unstable and can further degrade into various low
molecular weight compounds with distinctive colour, odour and flavour characteristics.
These low molecular weight compounds include alkanes, alkenes, alcohols, ketones,
aldehydes, acids and esters. Some of these compounds impart off-flavor/taste to the
powders and drastically decrease the shelf-life of the product (Tamime, 2009).
64
Many secondary products have been identified from the radical and photosensitized
oxidations of polyunsaturated lipids. These secondary products mainly consist of
oxygenated monomeric materials including epoxy-hydroperoxides, oxo-hydroperoxides,
hydroperoxy
epidioxides,
dihydroperoxides,
hydroperoxy
bis-epidioxides,
and
Propanal and hexanal are the main volatiles formed by oxidative decomposition of
omega-3 fatty acids and omega-6 fatty acids, respectively (Frankel et al., 1994). Hence, the
propanal and hexanal contents measured at different storage times in the microcapsule
powders can be used as indicators of oxidative stability.
Propanal was detected in all the fresh (0 month) FO and FO-EVOO microcapsules made
with 25% and 50% oil loading at pH 3 (Table 16) indicating a possibility of lipid oxidation
taking place either during the emulsion preparation or spray drying process. An increase in
the propanal content in all powders was positively correlated with the increase in storage
time. The amount of propanal detected in the headspace of all the fresh (0 month)
microcapsules were similar to amounts detected by Drusch et al., 2007; Serfert, Drusch &
Schwarz, 2009 for SBP-, caseinate-, n- octenylsuccinate starch and gum arabic stabilised
FO-in-water emulsions, but only in the absence of added antioxidants. The highest content
of propanal in the microcapsule powders was recorded after 3 months of storage in FO-50
(32.35 0.92 g/g powder) and FO-EVOO-50 (28.70 4.53 g/g powder) powders made
at pH 3. Significant differences in propanal content (P < 0.05) amongst all the samples
stored for up to 2 months were noted. The propanal content in microcapsule powders
65
stored 02 months was higher in microcapsule powders containing 50% oil loading than
powders containing 25% oil loading. This is expected as a more robust interface is
anticipated for the 25% microcapsules, resulting in reduced porosity causing less oxygen
diffusion and hence, less oxidation on storage. The results also showed that the FO-EVOO
powders generally had slightly higher propanal concentrations compared to FO powders
alone, irrespective of oil loading, at most times during storage. However, there were no
significant differences (P > 0.05) in the propanal content in the FO and FO-EVOO samples
at 3 months. The continuous increment of propanal in all powders during storage indicated
the steady oxidation and degradation of omega-3 fatty acids. These observations revealed
that the addition of EVOO was not able to protect FO from oxidation. One of the reasons
for EVOO not being able to increase the oxidative stability of microencapsulated FO could
be the presence of transition metal ions (e.g. Fe, Cu) present in SBP and also, the low pH
environment that led to rapid oxidation.
No propanal was detected in all the fresh (0 month) FO and FO-EVOO powders made with
and without EDTA (50% oil loading) at pH 6 (Table 17). However, a general increase in
propanal content was observed with increase in storage time. The microcapsules with
EDTA had less propanal content compared to the microcapsules without EDTA indicating
that EDTA exerted a significant (P < 0.05) protective effect on the microcapsules with
regard to their oxidative stability.
The results also showed that the FO-EVOO powders had propanal contents significantly (P
< 0.05) lower than FO powders alone indicating that the antioxidant compounds in EVOO
were effective at pH 6. For example, addition of EVOO at 50% oil loading decreased
propanal content by 2.13 fold compared to microcapsules without EDTA after 3 months of
storage (Table 17).
66
Table 16 Propanal content in microencapsulated fish oil and fish oil-extra virgin olive oil
(1:1 wt ratio) (25% and 50% oil loading) at pH 3 during storage at room temperature (~25
C, 03 month) .
Propanal content (g g-1 powder)
a
Microcapsule
0 month
1 month
2 month
3 month
FO-25
1.49 0.09ab
2.63 0.25a
8.50 0.14a
25.85 2.62 a
FO-50
1.66 0.03b
4.55 0.21b
19.15 1.63 b
28.70 4.53 a
FO-EVOO-25
1.41 0.02a
3.97 0.24b
9.28 0.16 a
24.60 3.25 a
FO-EVOO-50
1.58 0.03ab
6.35 0.12c
20.30 0.14 b
32.35 0.92 a
Means within columns followed by different superscript letters differ significantly at 5% level
Table 17 Propanal content in microencapsulated fish oil and fish oil-extra virgin olive oil
(1:1 wt ratio) with and without EDTA (50% oil loading) at pH 6 during storage at room
temperature (~25 C, 03 month).
Propanal content (g g-1 powder)
a
Microcapsule
0 month
1 month
2 month
3 month
FO-EDTA-50
0a
1.90 0.42a
1.95 1.01a
18.90 0.00 b
FO-EVOO-EDTA-50
0a
1.06 0.08a
1.19 0.08a
7.68 0.40 a
FO-50
0a
2.37 0.01a
9.15 0.92 b
27.55 0.07 c
FO-EVOO-50
0a
1.88 0.21a
7.50 0.85 b
8.80 0.71 a
Means within columns followed by different superscript letters differ significantly at 5% level
Data reported in literature established the fact that metals like Fe and Cu act as catalysts for
the formation of highly reactive hydroxyl radicals which initiate a chain reaction for lipid
oxidation (Benedt & Shibamoto 2007; Goldstein, Meyerstein & Czapski 1993). The
significant (P < 0.05) differences in the propanal content between microcapsules with and
without EDTA confirmed EDTAs efficiency in inhibiting oxidation and also that metal
67
ion-induced oxidation reactions was one of the major reasons for the oxidative
deterioration of the FO and FO-EVOO microcapsules during storage. Jacobsen (2010)
performed some studies to retard lipid oxidation in foods enriched with omega-3-fatty
acids. It was found that the addition of EDTA (5 mg/kg milk) to skimmed milk
significantly retarded the oxidation when the emulsions were enriched with 1.5% FO.
Data gained from those studies also indicated that EDTA was effective in preventing
oxidation in omega-3 fatty acid-enriched mayonnaises and salad dressings when compared
to fitness bars. The article also illustrated that the effectiveness of EDTA might also
depend on the initial level of lipid hydroperoxides in the FO or the emulsion.
At pH 6, improved oxidative stability can be expected as the solubility of iron in water
increases with decreasing pH (Donnelly et al., 1998; Graf et al., 1984; Mancuso et
al.,1999). Therefore, the high propanal contents of FO and FO-EVOO microcapsules at pH
3 as compared to FO and FO-EVOO microcapsules at pH 6 could be attributed to the
increased solubility of iron. Indeed, some amount of propanal was found in FO and
FO-EVOO microcapsules at pH 3 prior to storage (zero month), whereas no detectable
propanal was observed in FO and FO-EVOO microcapsules at pH 6 at zero month. The
propanal content of FO and FO-EVOO with 50% oil loading at pH 6 were significantly
lower than the FO and FO-EVOO samples with 50% oil loading at pH 3 (P < 0.05),
irrespective of the storage duration (Table 18). This also showed that there was an effect of
pH on the oxidative stability of the powders. The results revealed that the FO-EVOO
powders at pH 6 had propanal contents (8.8 0.71 g/g powder) significantly (P < 0.05)
lower than FO powders (27.55 0.07 g/g powder) alone after 3 months of storage.
In addition to its influence on the solubilisation of transition metals, pH also affects oxygen
solubility and mobility, and the rate of non-enzymatic browning reaction in foods . There
have been studies where FO-enriched mayonnaise showed increased oxidation with
decreasing pH during storage and also that metal ions significantly promoted oxidation
(Tong et al., 2000; Jacobsen, Timm & Meyer, 2001). In another study, polyoxyethylene 10
lauryl ether was used as an emulsifier in making model emulsions and it was found that
iron showed a highly increased pro-oxidative effect at pH 3 compared to that at pH 7
68
because of the increased solubility of iron at low pH (Cho et al., 2003). However, in the
current study the data indicated that the effect of pH was not significant (P > 0.05) in the
case of FO without added EVOO. Consequently, it could be concluded that the antioxidant
effect of EVOO was readily available at pH 6.
Table 18 Propanal content in microencapsulated fish oil and fish oil-extra virgin olive oil
(1:1 wt ratio) with 50% oil loading at pH 3 and pH 6 during storage at room temperature
(~25C, 03 month) .
Propanal content (g g-1 powder)
a
Microcapsule
0 month
1 month
FO-50 (pH 3)
1.66 0.03
FO-EVOO-50 (pH 3)
2 month
b
3 month
4.55 0.21
19.15 1.63
28.70 4.53bc
1.58 0.03ab
6.35 0.12c
20.30 0.14b
32.35 0.92c
FO-50 (pH 6)
0a
2.37 0.01a
9.15 0.92a
27.55 0.07b
FO-EVOO-50 (pH 6)
0a
1.88 0.21a
7.50 0.85a
8.80 0.71a
Means within columns followed by different superscript letters differ significantly at 5% level
FO-EVOO
microcapsules (50% oil loading) with EDTA at pH 6 (Table 20). FO and FO-EVOO
microcapsules (50% oil loading) without EDTA at pH 6 had some amount of detectable
hexanal in the 2nd and 3rd month and no detectable hexanal prior to 2 months (Table 21).
The later development of hexanal compared to propanal may be expected as hexanal and
propanal are secondary oxidation products of omega-6 (e.g. C18:2) and omega-3 fatty
acids (e.g. DHA, EPA, C18:3), respectively. It is well known that omega-3 fatty acids are
more prone to oxidation than omega-6 fatty acids. It has, however, been reported that
hexanal cannot be used as an adequate marker for the beginning of oxidation in case of
69
virgin olive oil, although it has been successful with refined vegetable oils (Snyder et al.,
1988; Warner et al., 1988).
Table 19 Hexanal content in microencapsulated fish oil and fish oil-extra virgin olive oil
Microcapsule
FO-25
1.4 0.21a
FO-50
1.6 0.36a
FO-EVOO-25
2.1 0.42a
FO-EVOO-50
1.8 0.71a
Means within columns followed by different superscript letters differ significantly at 5% level
No significant difference (P > 0.05) in the hexanal content between FO and FO-EVOO
microcapsules (25% and 50% oil loading) at pH 3 microcapsules after 3 months of storage
was observed. FO and FO-EVOO microcapsules with EDTA had significantly less hexanal
content compared to the samples without EDTA (P < 0.05). It was also observed that
FO-EVOO microcapsules with and without EDTA had hexanal contents significantly
lower than FO microcapsules with and without EDTA (P < 0.05). Also, the hexanal
content of FO and FO-EVOO with 50% oil loading at pH 6 were significantly lower than
the FO and FO-EVOO samples with 50% oil loading at pH 3 (P < 0.05).. That was in
agreement with the propanal results and confirmed that pH was also an influencing factor
in the oxidation of the microencapsulated oil.
Objectionable rancid odour was noted in all microcapsule powders on sniffing the samples
stored in containers for 3 months at ambient conditions .There are a wide range of volatile
compounds present in FO and EVOO and these volatiles bring out different tastes and
odours. Each of these compounds have different thresholds of perception. It is anticipated
that propanal and hexanal give rise to a rancid odour.
70
Table 20 Hexanal content in microencapsulated fish oil and fish oil-extra virgin olive oil
(1:1 wt ratio) with and without EDTA (50% oil loading) at pH 6 during storage at room
temperature (~25C, 03 month) .
Microcapsule
0 month
1 month
2 month
3 month
FO-EDTA-50
0.00 0.00a
0.00 0.00a
0.00 0.00a
1.26 0.02c
FO-EVOO-EDTA-50
0.00 0.00a
0.00 0.00a
0 .00 0.00a
0.18 0.04a
FO-50
0.00 0.00a
0.00 0.00a
0.12 0.01a
0.51 0.01b
FO-EVOO-50
0.00 0.00a
0.00 0.00a
0.41 0.28a
0.44 0.06b
Means within columns followed by different superscript letters differ significantly at 5% level
Table 21 Hexanal content in microencapsulated fish oil and fish oil-extra virgin olive oil
(1:1 wt ratio) with 50% oil loading at pH 3 and pH 6 during storage at room temperature
(~25C, 03 month).
Hexanal content (g g-1 powder)
a
Microcapsule
0 month
1 month
2 month
3 month
FO-50 (pH 3)
0.00 0.00a
0.00 0.00a
0.00 0.00a
1.60 0.36a
FO-EVOO-50 (pH 3)
0.00 0.00a
0.00 0.00a
0 .00 0.00a
1.80 0.71a
FO-50 (pH 6)
0.00 0.00a
0.00 0.00a
0.12 0.01a
0.51 0.01b
FO-EVOO-50 (pH 6)
0.00 0.00a
0.00 0.00a
0.41 0.28a
0.44 0.06b
Means within columns followed by different superscript letters differ significantly at 5% level
The major fatty acids present in the oils (FO and EVOO) and all encapsulated oils were
palmitic acid (C16:0), oleic acid (C18:1), EPA (C20:5) and DHA (C22:6). Hence, the
71
percentage composition of each of the major fatty acids mentioned above was determined
[Table 22 (a & b), Table 23 (a & b) and Table 24 (a & b)] after extraction of the oil
from the microcapsules. The ratio of EPA to C16:0 and DHA to C16:0 were also provided.
Other minor fatty acids present in each of the oils were calculated as a percentage and
represented as others in the tables. The data has been expressed as the unsaturated fatty
acids to C16:0 because the absolute value of C16:0 did not change, only its % relative to
the unsaturated fatty acid changed as oxidation proceeded.
As expected, EPA and DHA, being polyunsaturated fatty acids had significant changes in
their composition over the storage period (P > 0.05). The amounts of EPA (C20:5n-3) and
DHA (C22:6n-3) in the oil extracted from all fresh powders containing microencapsulated
FO were slightly lower than that of the pure FO. The extreme susceptibility of
polyunsaturated fatty acids to oxygen, light and temperature is widely known (Garg et al.,
2006; Kolanowski & Laufenberg, 2006) and hence, the oxidation of the omega-3-fatty
acids in the oil must have occurred during the preparation of emulsions and/or the spray
drying process. The monounsaturated fatty acid composition was comparable. As
expected, the polyunsaturated omega-3 fatty acids EPA and DHA in all microcapsules
decreased over the storage period. In contrast, there was no significant change (P > 0.05)
in the oleic acid content with storage time. This result might be explained in terms of the
structure of the fatty acids. Generally, the greater the number of double bonds in the fatty
acid, the more unstable the fatty acid and the greater the ease of its oxidation (e.g. by
oxygen, light) (Olive oil source, 2010).
The percentage decrease in EPA and DHA was found to be greater in microcapsules
having 50% oil loading when compared to microcapsules with 25% oil loading, at pH 3
(Tables 22, a & b). The highest stability of EPA and DHA was found to be in FO
microcapsules (25% oil loading) with the EPA decreasing from 6.4% at 0 month to 6.2% at
the 3rd month, and DHA from 23.6% at 0 month to 21.8% at the 3rd month. FO-EVOO-50
microcapsules and FO-50 microcapsules were found to be the least stable after 3 months of
storage. The EPA content decreased from 3.3% to 2.7% in FO-EVOO-50 and from 6.7%
to 5.6% in FO-50, and DHA from 12% to 9.3% in FO-EVOO-50 and from 24% to 18% in
72
FO-50 microcapsules. A percentage decrease of 16.31% and 22.84% in EPA and DHA
was observed in FO-EVOO-50 microcapsules over 3 months of storage whereas, a
percentage decrease of 16.49% EPA and 25.55% DHA was recorded in FO-50
microcapsules indicating that EVOO had a slight antioxidative effect on controlling lipid
oxidation of microencapsulated FO. On the contrary, FO-EVOO-25 had a greater
percentage decrease of EPA and DHA, 12.04% and 17.75%, respectively, when compared
to 2.82% EPA and 7.83% DHA in FO-25. That indicated that the concentration of
antioxidative components present in EVOO was not sufficient to protect the FO from
oxidation or other mechanisms come into play which offset or (negated) the antioxidant
effects of EVOO components.
FO and FO-EVOO microcapsules without EDTA at pH 6 in the formulation were found to
be least stable when compared to the microcapsules with EDTA (Table 23, a & b). That
suggested that the metal ions present in SBP were one of the underlying factors responsible
for lipid oxidation in FO and FO-EVOO microcapsules prepared at pH 3. FO-EVOO
microcapsules were less oxidised than FO microcapsules made with 50% oil loading at pH
6. EPA and DHA contents decreased by 0.33% and 1.99%, respectively, in FO-EVOO-50
microcapsules and 0.74% for EPA and 4.46% for DHA in FO-50 microcapsules.
Consequently, it could be concluded that EVOO was as effective in preventing lipid
oxidation in FO microcapsules as EDTA during long term storage. That might be
attributed to the high antioxidant content and radical scavenging activity of EVOO.
FO and FO-EVOO microcapsules (50% oil loading) at pH 3 were found to be less stable
when compared to FO and FO-EVOO microcapsules (50% oil loading) at pH 6 [Table 22
(a & b) and Table 23 (a & b)]. EPA and DHA contents decreased by 16.49% and
25.55%, respectively, in FO-50 microcapsules and EPA by 16.31% and DHA by 22.84%
in FO-EVOO-50 microcapsules at pH 3. EPA and DHA contents decreased by 12.71% and
19.16%, respectively in FO-50 microcapsules and 10.41% and 15.89%, respectively, in
FO-EVOO-50 microcapsules at pH 6. This shows that FO microcapsules were less stable
than FO-EVOO microcapsule at both pH 3 and pH 6.
73
Table 22 a Fatty acid composition (%) of microencapsulated fish oil (25% and 50% oil loading) at pH 3 during storage at room
temperature (~25C, 03 months).
Microcapsule
FO-25
Fatty acid
FO-50
0 month
1 month
2 month
3 month
0 month
1 month
2 month
3 month
C16:0
19.78 1.25
21.14 0.13
22.16 0.01
23.05 0.29
20.78 0.15
21.53 0.49
23.58 0.08
25.34 0.08
C18:1
14.07 0.86
15.07 0.39
14.72 0.13
16.11 0.17
14.55 0.12
14.690.65
16.03 0.05
15.31 0.04
C20:5n-3
6.39 0.14
6.36 0.17
6.42 0.05
6.21 0.07
6.67 0.29
6.14 0.12
5.40 0.01
5.57 0.11
C22:6n-3
23.63 0.17
22.55 0.92
21.66 0.22
21.78 0.08
24.23 1.24
21.07 0.26
18.35 0.11
18.04 0.12
Others
37.13 0.67
35.05 0.57
33.77 0.42
36.56 0.61
33.77 0.57
36.56 0.21
24 0.24
24.46 0.19
3.5 0.01
3.63 0.03
3.44 0.02
3.34 0.01
3.24 0.04
DHA:EPA
3.7 0.11
3.54 0.05
3.4 0.00
EPA:C16
0.32 0.01
0.30 0.01
0.29 0.00
0.27 0.00
0.32 0.01
0.29 0.01
0.23 0.00
0.22 0.00
DHA:C16
1.20 0.08
1.07 0.05
0.98 0.01
0.94 0.01
1.17 0.05
0.98 0.01
0.78 0.01
0.71 0.01
74
Table 22 b Fatty acid composition (%) of microencapsulated fish oilextra virgin olive oil (1:1 wt ratio) (25% and 50% oil loading) at
pH 3 during storage at room temperature (~25C, 03 months).
Microcapsule
FO-EVOO-25
Fatty acid
FO-EVOO-50
0 month
1 month
2 month
3 month
0 month
1 month
2 month
3 month
C16:0
14.71 0.08
15.02 0.04
14.87 0.21
15.72 0.01
14.57 0.17
15.01 0.05
14.94 0.05
15.52 0.16
C18:1
46.05 0.27
46.89 0.09
46.59 0.37
48.52 0.07
45.41 0.15
46.18 0.10
46.98 0.06
48.62 0.30
C20:5n-3
3.24 0.03
3.15 0.01
3.07 0.03
2.85 0.10
3.25 0.06
3.09 0.07
2.92 0.01
2.72 0.03
C22:6n-3
12.00 0.23
11.30 0.13
11.02 0.03
9.87 0.03
12.04 4.4
10.74 0.32
10.26 0.03
9.29 0.17
Others
24.00 0.61
24.4 0.26
24.73 0.11
24.91 0.01
24.73 0.45
24.91 0.34
24.91 0.15
23.84 0.34
DHA:EPA
3.70 0.04
3.61 0.01
3.6 0.01
3.46 0.13
3.71 0.06
3.48 0.04
3.50 0.00
3.42 0.03
EPA:C16
0.22 0.00
0.21 0.00
0.21 0.00
0.18 0.00
0.22 0.01
0.21 0.00
0.19 0.00
0.18 0.00
DHA:C16
0.82 0.01
0.75 0.01
0.74 0.00
0.63 0.00
0.81 0.05
0.72 0.02
0.69 0.00
0.60 0.02
75
Table 23 a Fatty acid composition (%) of microencapsulated fish oil (50% oil loading) with and without EDTA at pH 6, during
storage at room temperature (~25C, 03 months).
Microcapsule
FO-EDTA-50
Fatty acid
FO-50
0 month
1 month
2 month
3 month
0 month
1 month
2 month
3 month
C16:0
19.50 0.66
19.66 0.35
20.13 0.50
19.71 0.44
19.10 0.57
19.48 0.65
20.37 0.11
20.16 0.38
C18:1
14.71 0.72
13.56 0.28
15.06 0.85
15.36 1.93
17.38 2.29
16.95 2.88
18.16 2.70
18.74 2.29
C20:5n-3
6.06 0.24
6.44 0.33
5.89 0.21
5.48 0.35
5.82 0.06
5.64 0.02
5.36 0.05
5.08 0.06
C22:6n-3
24.13 0.18
25.05 0.33
22.52 0.16
20.57 1.33
23.28 0.15
22.30 0.37
20.43 0.60
18.82 0.18
Others
35.52 0.36
35.30 0.27
36.65 0.29
38.88 1.90
34.42 1.66
34.98 1.85
35.84 1.94
37.20 2.00
DHA:EPA
3.99 0.13
3.89 0.04
3.83 0.16
3.76 0.02
4.00 0.06
3.95 0.06
3.78 0.08
3.71 0.03
EPA:C16
0.31 0.00
0.33 0.01
0.29 0.00
0.28 0.02
0.31 0.01
0.29 0.01
0.26 0.00
0.25 0.00
DHA:C16
1.22 0.03
1.29 0.04
1.12 0.04
1.04 0.06
1.22 0.03
1.16 0.02
1.00 0.02
0.93 0.01
76
Table 23 b Fatty acid composition (%) of microencapsulated fish oilextra virgin olive oil (1:1 wt ratio) (50% oil loading) with and
without EDTA at pH 6, during storage at room temperature (~25C, 03 months).
Microcapsule
FO-EVOO-EDTA-50
Fatty acid
FO-EVOO-50
0 month
1 month
2 month
3 month
0 month
1 month
2 month
3 month
C16:0
15.30 1.02
16.31 0.25
16.39 0.55
16.56 0.78
15.76 0.23
15.86 0.13
16.21 0.21
16.25 0.08
C18:1
35.88 2.09
40.40 0.68
39.80 0.37
40.70 2.01
40.26 1.21
41.18 1.54
42.19 0.99
42.10 0.90
C20:5n-3
3.69 0.08
3.22 0.24
3.22 0.14
3.19 0.04
3.17 0.28
3.04 0.26
2.84 0.15
2.84 0.12
C22:6n-3
14.39 0.25
12.66 0.68
12.60 0.03
12.11 0.11
12.52 0.68
11.88 0.61
10.77 0.08
10.53 0.38
Others
30.74 2.78
27.99 0.51
27.99 0.33
27.44 2.86
28.30 0.09
28.04 0.54
27.99 0.55
28.28 0.96
DHA:EPA
3.90 0.02
3.90 0.09
3.92 0.17
3.79 0.06
3.96 0.14
3.91 0.14
3.80 0.17
3.71 0.06
EPA:C16
0.24 0.02
0.24 0.01
0.20 0.00
0.19 0.01
0.18 0.02
0.18 0.01
0.16 0.01
0.16 0.01
DHA:C16
0.92 0.08
0.77 0.03
0.77 0.03
0.73 0.03
0.79 0.03
0.75 0.03
0.66 0.00
0.65 0.02
77
78
Table 24 Oxidative stability of bulk oils (FO, EVOO, FO-EVOO (1:1 wt ratio) during
accelerated storage (80C, oxygen pressure of 0.5 bar).
a
FO
11.85 0.07
-210 9.19
EVOO
>100 0.00
16.3 0.14
-119 4.24
Sample
There was no significant (P > 0.05) difference in the IP values between emulsions made
for manufacture of powders with 25% and 50% oil loading at pH 3 (Table 25). That also
suggested that there was no effect of oil composition or oil loading on the IP values of
emulsions. Similarly, FO microcapsules (25% and 50% oil loading) revealed the same IP
values (6.95 0.07 h). However, the IP value increased from 7.90 h to 11.90 h when the
oil load was increased from 25% to 50% in FO-EVOO microcapsules. The results
indicated that spray-dried FO-EVOO microcapsules had greater oxidative stability than
spray-dried FO microcapsules under the accelerated storage conditions.
The enhanced oxidative stability of the FO-EVOO microcapsules could be due to the
presence of olive oil, which contains large amounts of oleic acid (5583%) and much
smaller amounts of saturated fatty acids (14%). That interpretation was in agreement with
the findings of Parkanyiova et al., (2000) who reported that triacylglycerols containing
bound linoleic acid were oxidised several times faster than triacylglycerols containing only
oleic and saturated fatty acids. However, the Oxipres data were in direct contrast to those
obtained for powders prepared at pH 3 and stored under ambient conditions, as determined
by propanal and hexanal analysis and fatty acid composition data. This could be attributed
to a variety of auto oxidative mechanisms that take place with respect to the change in
temperature of oxidation. It has been widely reported that the antioxidative potential of
virgin olive oil is affected by both storage and thermal treatment due to modifications in
79
oxidative/hydrolytic reactions. Other studies have shown that thermal treatment of virgin
olive oil resulted in an increase in the formation of hydroxytyrosol and tyrosol from virgin
olive oil secoiridoids (Beter et al., 2008; Brenes et al., 2002; Lerma-Garcia et al., 2009;
Sacchi et al., 2002). Another study by Carrasco-pancorbo et al., (2005) demonstrated that
hydroxytyrosol contributed to the oxidative stability of virgin olive oil.
Table 25 Oxidative stability of emulsions and powders (25% and 50% oil loading)
containing fish oil and fish oil-extra virgin olive oil (1:1 wt ratio) at pH 3 during
accelerated storage (80C, oxygen pressure of 0.5 bar).
FO-25
4.90 0.14a
-28.00 4.24b
FO-50
4.60 0.00a
-38.00 1.41a
FO-EVOO-25
5.00 0.14a
-21.50 0.71b
FO-EVOO-50
4.65 0.07a
-27.00 1.41b
Sample
(A) Emulsion
(B) Microcapsule
FO-25
FO-50
FO-EVOO-25
FO-EVOO-50
0.07
6.95 0.07
7.90 0.00
6.95
-225.00 14.14d
-2350.50 7.78a
11.95 0.35c
-612.50 3.54c
-792.00 1.41b
As expected, the addition of antioxidant i.e EDTA, in the formulation of the microcapsules
significantly increased the IP of FO and FO-EVOO microcapsules and emulsions with
EDTA, offering better oxidative stability. The FO-EVOO emulsions and microcapsules
showed greater oxidative stability with greater IPs as compared to FO microcapsules at
pH 6 with 50% oil loading (Table 26B). The enhanced stability of FO-EVOO
microcapsules could be attributed to the absence of polyunsaturated fatty acids.
Monounsaturated fatty acids possess only one single double bond making them more
80
stable than polyunsaturated fatty acids yet not as stable as saturated fats (Unsaturated fats
2010; Extra virgin olive oil 2010). All emulsions (FO and FO-EVOO) made with and
without EDTA at pH 6 had relatively small slope values indicating a slow rate of oxidation
in the emulsions, whereas these values were much larger in all the microcapsules (Table
26). This difference in oxidative stability between emulsions and microcapsules may be
due to the orientation of the EDTA and the phenolic compounds present in EVOO in an
aqueous phase and in a dried state. Transition metal ions, such as iron and copper, would
be located in the aqueous phase of oil-in-water emulsions and oriented in the oil/water
interface (Frankel et al., 1994; Jacobsen et al., 2008). EDTA, omega-3 hydroperoxides and
the polar phenolics would also be present in the aqueous phase of the liquid emulsions.
Therefore, it is possible that the shared location and high level of contact between the proand anti- oxidants in the aqueous phase of the liquid emulsions was more effective in
preventing lipid oxidation than in dried state, due to the water restricted environment in the
latter. Other factors that could possibly cause differences in lipid oxidation in emulsions
and microcapsules would be factors affecting the antioxidant activity and rate of oxidation
in both systems. The effectiveness of antioxidants in oil-in-water emulsions is dependent
on several parameters, such as their structure, polarity, location, radical scavenging and
metal-chelating attributes (Mattia et al., 2010; Paiva-Martins & Gordon, 2002,
Paiva-Martins et al., 2006). The rate and extent of lipid oxidation may also be influenced
by numerous variables like pH, type of emulsifier, oxygen availability and structure,
thickness and composition of the interface (Coupland & McClements, 1996; McClements
& Decker, 2000; Velasco et al., 2003, 2006).
The FO and FO-EVOO emulsions had greater IP values at pH 3 than FO and FO-EVOO
emulsions at pH 6 (Table 27). No significant difference (P > 0.05) in IP values was
observed between FO and FO-EVOO emulsions made with 50% oil loading at pH 3. The
FO-EVOO emulsion had a similar IP value to FO emulsion made with 50% oil loading at
pH 6. All emulsions made with 50% oil loading at pH 3 and pH 6 had lower slope values
than the microcapsulated oil, indicating a slow rate of oxidation. No significant difference
(P > 0.05) between the spray-dried FO and FO-EVOO microcapsules at pH 3 with 50% oil
loading when compared to the corresponding spray-dried FO and FO-EVOO
81
microcapsules at pH 6 (Table 27). Data revealed that the FO-EVOO microcapsules were
better in terms of oxidative stability when compared to FO microcapsules both at pH 3 and
pH 6. In case of FO-EVOO and FO emulsions, the IP values were very small and did not
vary significantly (P > 0.05) from each other irrespective of oil composition and pH. This
result indicated that microencapsulation was significantly (P < 0.05) effective in
preventing lipid oxidation than in liquid emulsions. It has been previously discussed
(section 4.5.2) as to how the effectiveness of the antioxidants may be affected by numerous
variables like structure, polarity, location (e.g. water, oil, interface) and levels of metals
(Fe & Cu) availability. It is possible that the efficacy of antioxidants present in EVOO has
been affected by one of these factors and that could have caused these differences in
microcapsules and emulsions lipid oxidation.
82
Table 26 Oxidative stability of emulsions and powders (50% oil loading) containing fish
oil and fish oil-extra virgin olive oil (1:1 wt ratio) made with and without EDTA (50% oil
loading) at pH 6 during accelerated storage (80C, oxygen pressure of 0.5 bar).
FO-EDTA-50
13.90 0.14b
-16.50 2.12ab
FO-EVOO-EDTA-50
23.65 0.49c
-11.50 4.95c
FO-50
3.20 0.28a
-24.00 7.07ab
FO-EVOO-50
3.90 0.28a
-31.50 3.54a
Sample
(A) Emulsion
(B) Microcapsule
FO-EDTA-50
10.00
0.28
-421.50 6.36b
FO-EVOO-EDTA-50
19.65 1.06d
-131.50 3.54d
FO-50
7.35
0.21
-480.50 4.95a
FO-EVOO-50
12.70
0.14
-97.00 16.97c
83
Table 27 Oxidative stability of emulsions and powders (50% oil loading) containing fish
oil and fish oil-extra virgin olive oil (1:1 wt ratio) at pH 3 and pH 6 during accelerated
storage (80C, oxygen pressure of 0.5 bar).
FO-50 (pH 3)
4.60 0.00a
-38.00 1.41a
FO-EVOO-50 (pH 3)
4.65 0.07a
-27.00 1.41b
FO-50 (pH 6)
3.20 0.28a
-24.00 7.07ab
FO-EVOO-50 (pH 6)
3.90 0.28a
-31.50 3.54a
Sample
(A) Emulsion
(B) Microcapsule
0.07
-2350.50 7.78a
FO-50 (pH 3)
6.95
FO-EVOO-50 (pH 3)
11.95 0.35c
-792.00 1.41b
FO-50 (pH 6)
7.35
0.21
-480.50 4.95a
FO-EVOO-50 (pH 6)
12.70
0.14
-97.00 16.97c
84
At pH 3
- EVOO did not improve the oxidative stability of microencapsulated FO during
storage at room temperature.
- In contrast, EVOO improved the oxidative stability of microencapsulated FO
during accelerated storage conditions.
At pH 6
- FO-EVOO microcapsules had greater oxidative stability than FO microcapsules
both during storage at room temperature and under accelerated storage conditions.
The main focus of this work was to produce physically stable powders containing
microencapsulated FO with EVOO added for its natural antioxidants and additional health
benefits. Results demonstrated that EVOO did contribute to some antioxidative effect
when microcapsules were prepared at pH 3 and 6 under accelerated storage conditions.
85
However, the same could not be said about the protective effect of EVOO on FO-EVOO
microcapsules prepared at pH 3 when stored under ambient conditions. A significant and
positive effect on oxidative stability of FO microcapsules was observed with the addition
of EDTA in the microcapsule formulation. Similarly, SBP was effective as a wall material
but, for long term stability of FO microcapsules it required EDTA (a metal-chelating
additive) to counteract the pro-oxidative effects caused by the metal ions (iron and copper)
associated with it.
The results obtained through this research project helped to improve understanding of
o the protection requirements to minimise oxidation of FO during processing of
FO microcapsules.
o how and where to add the microencapsulated omega 3 fatty acids in a product.
o the possible interactions of omega-3-fatty acids with other ingredients.
o the physical and shelf-life properties of the FO-EVOO powders.
FUTURE DIRECTIONS
This study identified the potential application of SBP as a food matrix, and tried to
protect FO with the natural antioxidants present in EVOO by optimising the process
parameters to stabilize microcapsules. However, it could be suggested that testing SBP
after depolymerising could yield good results. It is recommended also for further
studies that concentrate on natural food ingredients rich in antioxidants such as
phytosterols could be useful to protect FO from oxidation. Furthermore, the in vitro
analysis of FO-EVOO powders could be examined to examine the susceptibility of the
microcapsule to simulated gastric and intestinal digestion.
Finally, testing the delivery of omega-3 fatty acids in the form of FO-EVOO powder
into various food products (cereal products e.g. breakfast cereals, bakery products e.g.
bread or into cake mixes and fruit nut bars) could have significance nutritional value
and possible industrial applications.
86
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107
APPENDICES
1. Percentage protein present in the SBP sample.
The nitrogen content in SBP sample (triplicate analysis) found by LECO was:
SBP replicate 1 = 3.887 mg
SBP replicate 2 = 3.871 mg
SBP replicate 3 = 3.950 mg
Average of replicate 1 +
2 + 3= 3.9 mg
108
Density of propanal is 0.81 g/ml, therefore we need to convert the propanal concentration
to ml/g to have the same units, so have to multiply by 10-3
so 0.0020 x 10-3 ml (0.81 g/ml)
is 1.62 * 10-6 g
which is 1.62 g/g of powder.
Substituting the value of y (absorbance value of the extract at 725 nm) in the above
equation,
x = 0.257- 0.0759 / 3.8617
= 0.047 mg/ ml caffeic acid in 0.2 ml
Therefore, in 5.2 ml of the extract there is 0.244 mg caffeic acid.
0.244 mg caffeic acid is present in 2.5 g oil and hence, there is 97.5 mg/kg oil.
110