Ex Oxyscavenger

Clemson University
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12-2008
Development and characterization of moisture-

and heat- activated oxygen scavenging nanoparticle
Youngjae Byun
Clemson University, ybyun@clemson.edu
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DEVELOPMENT AND CHARACTERIZATION OF MOISTURE- AND HEAT-
ACTIVATED OXYGEN SCAVENGING NANOPARTICLE
A Dissertation
Presented to
the Graduate School of
Clemson University
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Food Technology
by
Young Jae Byun
December 2008
Accepted by:
William S. Whiteside, Ph.D., Committee Chair
Duncan Darby, Ph.D.
Kay Cooksey, Ph.D.
Paul L. Dawson, Ph.D.
i
ABSTRACT
α-tocopherol and a transition metal, iron, mixture was evaluated as a possible
oxygen scavenger. The effects of moisture, amount of transition metal, and thermal
processing on oxygen scavenging capability were also investigated. Results showed that
α-tocopherol and transition metal had 6.72 cc O2 per gram of oxygen scavenging capacity
and 0.11 cc O2 per gram per day of oxygen scavenging rate.
Nanoencapsulation technology was adopted for the possible oxygen scavenging
activation system. α-tocopherol-loaded poly ε-caprolactone (PCL) nanoparticles were
prepared by oil in water (O/W) emulsion solvent evaporation with ultrasonification. The
effects of PCL concentration, solvent in oil phase, and ultrasonification time on
encapsulation efficiency (%), particle mean size, and loading (%) were investigated.
Overall, 5% PCL in methylene chloride (DCM) as the solvent in the oil phase coupled
with 3 minute ultrasonification time showed the best encapsulation formulation.
Therefore, this formulation was selected for further research.
α-tocopherol-loaded nanoparticles and a transition metal in scavenger mixture were
evaluated as a heat activated oxygen scavenger. The effects of moisture, amount of
transition metal, and thermal processing on oxygen scavenging capability were also
studied. Results showed that α-tocopherol-loaded nanoparticle and transition metal had
6.44 cc O2 per gram of oxygen scavenging capacity and 0.21 cc O2 per gram per day of
oxygen scavenging rate. Heat was used as the activator to initiate the oxygen scavenging
reaction. However, adding moisture lowered the oxygen scavenging capability.
ii
An active fish gelatin film was developed by customized film applicator and its
physical properties and oxygen scavenging capabilities were investigated. The effects of
moisture and thermal processing on oxygen scavenging capability were also studied. α-
tocopherol-loaded nanoparticles and a transition metal was added into the warm water
fish gelatin and cast into a film. Agglomeration of the oxygen scavenger caused a rough
film surface, decreased tensile strength, increased elongation at break, and increased
oxygen permeability. The active fish gelatin film had an oxygen scavenging capacity of
1969.08 cc O2 per square meter per mil thickness with water as the activator which
triggered the oxygen scavenging reaction. However, thermal processing at temperature
above 140oF destroyed the oxygen scavenging ability of the film.
iii
DEDICATION
I dedicate this work to my parents, Boksoo Byun and Imsook Choi, my brother,
Youngwoo Byun, my father in law, Dongtae Choo, my mother in law, Baeksoon Lee, my
lovely wife, Hyojung Choo, and my baby, Shington, with great thanks and love.
iv
ACKNOWLEDGMENTS
At first and the foremost, I would like to thank my advisor, Dr. W. Scott
Whiteside, for providing me this opportunity and for his consistent support,
encouragement, and trust throughout doctoral studies. I would like to express my
gratitude to all of my committee members, Dr. Duncan Darby, Dr. Kay Cooksey, and Dr.
Paul Dawson. They always gave me great help and advice for the research.
I would like to special thanks Dr. Hyunjin Park, Dr. Youngteck Kim, Dr. Hojae
Bae, and Mr. Yongjae Lee for their friendship and unselfish assistance and help. They
sincerely helped me whenever I am having trouble with research and living in Clemson. I
would like to thank my colleagues, Ms. Bianca Stiller, Mr. Steffen Rau, Mr. Ebuel
Sirmats, Mr. Rijosh Cheruvathur, and Dr. Sunil Mangalassary. They were my good
memories in Clemson.
Finally, accomplishments are never achieved or celebrated alone. I share this with
all of my family and friends who have encouraged and supported me over the years,
especially my wife, Hyojung Choo, to whom I owe everything.
v
TABLE OF CONTENTS
Page
TITLE PAGE .................................................................................................................... i
ABSTRACT..................................................................................................................... ii
DEDICATION ................................................................................................................ iv
ACKNOWLEDGMENTS ............................................................................................... v
LIST OF TABLES ........................................................................................................viii
LIST OF FIGURES ........................................................................................................ ix
LIST OF SCHEMES...................................................................................................... xii
CHAPTER
I. GENERAL INTRODUCTION ...................................................................... 1
1.1. Overview of Active Packaging ......................................................... 1

1.2. Oxygen Scavenger ............................................................................ 3
1.3. Reactive Oxygen Species (ROS) .................................................... 11
1.4. Transition Metal .............................................................................. 14
1.5. Antioxidant ..................................................................................... 16
1.6. α-tocopherol .................................................................................... 20
1.7. Nanoencapsulation for Drug Delivery ............................................ 24
1.8. Gelatin ............................................................................................. 28
1.9. References ....................................................................................... 34
II. RESEARCH HYPHOTHESIS ................................................................... 40
III. RESEARCH OBJECTIVES ........................................................................ 42
IV. ALPHA-TOCOPHEROL AND A TRANSITION METAL

AS AN OXYGEN SCAVENGER ............................................................. 44
Abstract .................................................................................................. 44
4.1. Introduction ..................................................................................... 45
4.2. Materials and Methods .................................................................... 48
4.3. Results and Discussion ................................................................... 50
vi
Table of Contents (Continued)
Page
4.4. Conclusion ...................................................................................... 53

4.5. References ....................................................................................... 54
V. PREPARATION AND CHARACTERIZATION OF

ALPHA-TOCOPHEROL-LOADED NANOPARTICLES BY O/W
EMULSION SOLVENT EVAPORATION WITH
ULTRASONIFICATION ............................................................................ 65
Abstract .................................................................................................. 65
5.1. Introduction ..................................................................................... 66
5.3. Results ............................................................................................. 73
5.4. Discussion ....................................................................................... 76
5.5. References ....................................................................................... 79
VI. ALPHA-TOCOPHEROL-LOADED NANOPARTICLES AND

TRANSITION METAL AS A HEAT ACTIVATED
OXYGEN SCAVENGER............................................................................ 91
Abstract .................................................................................................. 91
6.1. Introduction ..................................................................................... 92
6.3. Results and Discussion ................................................................... 98
6.4. Conclusion .................................................................................... 101
6.5. References ..................................................................................... 102
VII. DEVELOPMENT AND CHARACTERIZATION OF A

MOISTURE ACTIVATED OXYGEN SCAVENGING
FISH GELATIN FILM ........................................................................... 109
Abstract ................................................................................................ 109

7.1. Introduction ................................................................................... 110
7.2. Materials and Methods .................................................................. 113
7.3. Results and Discussion ................................................................. 119
7.4. Conclusion .................................................................................... 123
7.5. References ..................................................................................... 124
VIII. GENERAL CONCLUSION .................................................................... 139
vii
LIST OF TABLES
Table Page
1.1. Selected commercial oxygen scavenger systems ......................................... 10
1.2. Nanoencapsulation method: advantages and drawbacks ............................. 25
1.3. Amino acid composition of gelatins, residues per 1000 residues ................ 29
1.4. Amino acid content in fish gelatins and pork gelatins ................................. 33
4.1. Batch compositions used for oxygen scavenging mixtures ......................... 57
4.2. Oxygen content (%) of different scavenging mixtures

during storage time ................................................................................ 58
5.1. Batch compositions used for constituting

α-tocopherol–loaded nanoparticle.......................................................... 82
5.2. Properties of α-tocopherol–loaded nanoparticle .......................................... 83
6.1. Batch compositions used for oxygen scavenging mixtures ....................... 104
6.2. Oxygen content (%) of different oxygen scavenging mixtures

during storage time .............................................................................. 105
7.1. Color (L, a, b) and Haze of

the unmodified gelatin and gelatin w/OS film ..................................... 127
7.2. Batch compositions used for the oxygen content (%) analysis.................. 128
7.3. Oxygen content (%) of different oxygen scavenging mixtures

during storage time .............................................................................. 129
viii
LIST OF FIGURES
Figure Page
1.1. Oxidation of ascorbic acid ............................................................................. 5
1.2. A structure of typical oxygen scavenging multilayer film............................. 9
1.3. A simplified version of bonding in the diatomic molecule

and its derivatives .................................................................................. 13
1.4. Transition metal or d block elements on periodic table ............................... 14
1.5. Synthetic phenolic antioxidant ..................................................................... 17
1.6. Natural antioxidant; tocopherol ................................................................... 18
1.7. Natural antioxidant; Flavonoids ................................................................... 19
1.8. Natural vitamin E analogue ......................................................................... 20
1.9. Mechanism of α-tocopherol action .............................................................. 22
1.10. Singlet oxygen oxidation of α-tocopherol ................................................... 23
1.11. The chemical structure of gelatin ................................................................. 30
4.1. Influences of the α-tocopherol, thermal processing, and transition metal

on oxygen scavenging capability (p < 0.05) .......................................... 59
4.2. Influence of moisture on oxygen scavenging capability.............................. 60
4.3. Influence of the amount of transition metal

on oxygen scavenging capability (p < 0.05) .......................................... 61
4.4. Structure of α-tocopherol and trimethylhydroquinone (TMHQ) ................. 62
4.5. Influence of moisture on oxygen scavenging capability of

TMHQ and transition metal ................................................................... 63
5.1. SEM images of nanoparticles (a) 3M1 (b) 3M2 (c) 3M3 ............................ 84
5.2. SEM images of nanoparticles (a) 5M1 (b) 5M2 (c) 5M3 ............................ 85
ix
List of Figures (Continued)
Figure Page
5.3. SEM images of nanoparticles (a) 3MA1 (b) 3MA2 (c) 3MA3 ................... 86
5.4. SEM images of nanoparticles (a) 5MA1 (b) 5MA2 (c) 5MA3 ................... 87
5.5. Effects of (a) PCL concentration, (b) solvent in the oil phase,
and (c) ultrasonification time on encapsulation efficiency (n=18 and
LSD = 7.61 for PCL concentration and solvent in the oil phase,
n=12 and LSD = 9.32 for ultrasonification time, p<0.05) ..................... 88
and (c) ultrasonification time on loading (n=18 and
LSD =0.2106 for PCL concentration and solvent in oil phase,
n=12 and LSD = 0.2579 for ultrasonification time, p<0.05) ................. 89
and (c) ultrasonification time on particle mean size (n=18 and
LSD =110.15 for PCL concentration and solvent in the oil phase,
n=12 and LSD =134.91 for ultrasonification time, p<0.05) .................. 90
6.1. Influences of the α-tocopherol, thermal processing, and transition metal

on oxygen scavenging capability (p < 0.05) ........................................ 106
6.2. Influence of the moisture on oxygen scavenging capability...................... 107
6.3. Influence of the amount of transition metal on oxygen

scavenging capability (p < 0.05) .......................................................... 108
7.1. Customized film applicator ........................................................................ 130
7.2. Surface morphology of the film by SEM

(a) unmodified gelatin (top view) (b) gelatin w/OS (top view)
(c) unmodified gelatin (side view) (d) gelatin w/OS (side view) ........ 131
7.3. Cross sectional image of the film by microscopy (a) unmodified gelatin
(b) gelatin w/OS ................................................................................... 133
7.4. Oxygen permeability of the unmodified gelatin and gelatin w/OS film
(p < 0.05).............................................................................................. 134
x
List of Figures (Continued)
Figure Page
7.5. Water vapor permeability of

the unmodified gelatin and gelatin w/OS film (p < 0.05) .................... 135
7.6. Mechanical properties (a) TS (b) %E of

7.7. The influence of the moisture on oxygen scavenging capability of

7.8. The influence of the thermal processing (140, 170, 200oF)

on oxygen scavenging capability of
the unmodified gelatin and gelatin w/OS film ..................................... 138
xi
LIST OF SCHEMES
Scheme Page
4.1 Oxygen scavenging reaction ........................................................................ 64
xii
CHAPTER ONE
GENERAL INTRODUCTION
1.1. Overview of Active Packaging
Traditionally, packaging has protected food from environmental conditions, such as
oxygen, moisture, light, microorganism, bacteria, and physical stress. Containment,
protection, and preservation are several major functions of packaging. In recent years,
there has been a huge demand for packaging that offers better and fresher food products.
Active packaging has been developed in response to many of these demands. Active
packaging is defined as “packaging that changes the conditions of the packed food to
extend shelf-life, to improve sensory properties, or to inhibit the growth of pathogenic
and spoilage microorganisms” (Ahvenainen, 2000). It involves the interactions between
package and packaged food or headspace atmosphere by the incorporation of certain
additives into packaging film or within packaging containers (Day, 2003).
Active packaging can be divided into four major categories; scavengers, absorbers,
emitters, and other systems (Ozdemir and Floros, 2004, Rooney, 1995, Vermeriren et al.,
1999). Scavenging systems remove oxygen, carbon dioxide, and ethylene in the
headspace. Absorbers remove excessive water and undesirable odors. Emitters release
ethanol, carbon dioxide, antioxidants, antimicrobial agent, flavor, and preservatives to the
packaged food or into the headspace of package. Other systems include self-heating, self-
cooling, and UV blocking.
The market for active packaging in 2004 was a modest $50 million worldwide
(Ozdemir and Floros, 2004). This market has been growing rapidly and recent reports
1
suggest that the active packaging markets will be an estimated $4.6 billion in 2008 and
reach $6.4 billion by 2013 (Dagliden, 2008).
2
1.2. Oxygen Scavenger
Some forms of food deterioration, such as development of off-flavor, color and flavor
changes, and nutritional losses, are caused by lipid, pigment, protein oxidation (Anklam
et al., 1997, Morales-Aizpurua and Tenuta-Filho, 2005). Additionally, excessive oxygen
in packaging can cause the growth of aerobic bacteria or proliferation of molds (Brown
and Williams, 2003). Modified atmosphere packaging (MAP), nitrogen gas flushing, and
vacuum packaging have been widely used to remove headspace oxygen. However, these
physical methods do not always remove the oxygen completely and cannot remove the
oxygen that permeates through the packaging film during storage. Oxygen scavengers
can reduce the residual oxygen to less than 0.1% and maintain an atmosphere with low
oxygen concentrations.
1.2.1. Definition
The terms absorbers and scavengers have been used to describe materials that remove
oxygen in the packaging system. Oxygen absorbers remove oxygen by physically
trapping the oxygen and not through a chemical reaction. Oxygen scavengers remove
oxygen by chemically combining the oxygen from the inner package environment (Brody
et al., 2001).
1.2.2. Tactics of oxygen scavenging technology
There are several principles on oxygen scavenging technology: iron oxidation
(Yamaji and Okitsu, 1980, Klein and Knorr, 1990), ascorbic acid oxidation (Graf, 1994,
3
Teumac et al., 2004), sulfite oxidation (Farrell and Tsai, 1987, Watson and Carney, 1977),
photosensitive dye oxidation (Floros et al., 1997), enzymatic oxidation (Labuza and
Breen, 1989, Andersson et al., 2001), unsaturated fatty acids (Inoue and Komatsu, 1990,
Inoue et al., 1994), unsaturated hydrocarbons (Speer and Roberts, 1994, Speer et al.,
1996), and immobilized yeast (Nezat, 1985, Nestorson et al., 2008).
The majority of currently available oxygen scavengers are based on the principle of
iron oxidation (Rooney, 1995, Vermeriren et al., 2003).
Fe Fe 2e

O H O 2e 2OH

Fe 2OH Fe OH

Fe OH O H O Fe OH (Equation 1.1)

Another scavenging technology is based on ascorbic acid oxidation. A diagram of
this reaction is shown in Figure 1-1. The initial step is the formation of dehydroascorbic
acid. This step is strongly dependent upon pH. Ascorbic acid can be regenerated by
reaction with reducing agents such as metallic sulfites (Rooney, 1995)
4
Figure 1.1. Oxidation of ascorbic acid (Rooney, 1995)
Another scavenging technology is based on sulfite oxidation. The sulfite reacts with
oxygen as in Equation 1.2 (Brody, 2001).
2SO O 2H O 2H SO (Equation 1.2)
5
A polymer film can be the medium for photosensitive dye oxidation. The oxygen
scavenging process is initiated when the film is exposed to ultraviolet (UV) light. The
reaction with oxygen is shown in Equation 1.3 (Rooney, 1995)

hv
D → *D
*
D → 3D
3
D + O2 → D + 1O2
A + 1O2 → AO2 (Equation 1.3)
When a photosensitive dye, D, absorbs light (hv), it is excited to a singlet state, *D,
which converts to the triplet excited state, 3D. This triplet excited state can convert the
oxygen to singlet oxygen. Once excited to its singlet state oxygen it can react with the
electron acceptors, A.
Another way to control the oxygen level is based on enzymatic oxidation. A
combination of two enzymes, glucose oxidase and catalase, has been used as an oxygen
scavenger (Vermeiren et al., 2003). In the presence of water, glucose was oxidized to
gluconic acid and hydrogen peroxide by glucose oxidase (Labuza and Breene, 1989). The
reaction is:
2 glucose 2O 2H O 2 gluconic acid 2H O (Equation 1.4)
Since hydrogen peroxide is an objectionable end product, catalase was applied
(Vermeiren et al., 1999). The reaction is:
2 H O catalase 2 H O O catalase (Equation 1.5)
6
1.2.3. Oxygen Scavenging Sachet
Iron powder is packaged in a small sachet to prevent direct contact with food. The
sachet material is highly oxygen permeable, so oxygen can permeate from the packaging
headspace inside the sachet, where it is then scavenged by iron powder (Ozdemir and
Floros, 2004). The sachet was clearly labeled “Do not eat”, to prevent accidental
consumption by customers. This product has been approved for use by the Japanese
Ministry of Health and the United States Food and Drug Administration (FDA). The U.S.
Department of Agriculture (USDA) has also approved iron’s use in indirect contact for
packaging beef jerky, dehydrated meat, and poultry products. As a general rule, one gram
of iron reacts with 300 cc of oxygen. In-package oxygen scavenger sachets are available
to react with 20 to 2,000 cc of oxygen (Brody, 2001).
However, there are several disadvantages to the use of iron-based oxygen scavenger
sachets. It interferes with the use of metal detectors in packaging lines and has the
potential risk of accidental ingestion by consumer. In addition, it cannot be used for
liquid products and there is a risk of leakage. To eliminate these problems, oxygen
scavengers have been incorporated into the packaging material (Vermeiren et al., 2003).
1.2.4. Oxygen Scavenging Film
Oxygen scavenging materials have also been incorporated into polymer films. This
technique can provide a uniform scavenging effect throughout the package and can
scavenge oxygen that passes through the package. Unlike oxygen scavenging sachets,
oxygen scavenging films need an activation system to prevent reaction with atmospheric
7
oxygen prior to use. There are several patents that use UV light as an activator to trigger
the oxygen scavenging reaction (Albert et al., 2004, Schmidt et al., 2006, Speer, 2006,
Yang et al., 2004). UV triggering oxygen scavenging films are usually comprised of an
oxygen scavenger, a transition metal catalyst, and a photoinitiator. The rate of oxygen
scavenging can be increased with a transition metal catalyst. The photoinitiator enhances
and facilitates the oxygen scavenging ability of the packaging film upon exposure to UV
light. The oxygen scavenging films are activated on the packaging line just before filling
and sealing, so that light never comes in contact with the food.
Multilayer oxygen scavengers are more effective at eliminating oxygen than single
layer scavenging systems. The structure of a typical multilayer oxygen scavenging
system is shown in Figure 1.2. The oxygen scavenging layer is highly oxygen permeable,
so oxygen can migrate from the inner packaging layer to the scavenging layer. However,
oxygen ingress from outside to the oxygen scavenging layer is limited by the oxygen
barrier layer. Migration of the oxygen scavenging substance or byproducts of the oxygen
scavenging reaction can be restricted by the inner layer.
8
Figure 1.2. A structure of typical oxygen scavenging multilayer film (Ozdemir and Floros,
2004)
9
Table 1.1. Selected commercial oxygen scavenger systems (Day, 2003, Vermeiren et al.,
2003)
Scavenger Packaging
Manufacturer Country Trade name
Mechanism form
Mitsubishi Gas Chemical Sachets and
Japan Ageless Iron based
Co. Ltd labels
Toppan Printing Co. Ltd Japan Freshilizer Iron based Sachets
Toagosei Chem. Industry
Japan Vitalon Iron based Sachets
Co. Ltd
Nippon Soda Co. Ltd Japan Seagul Iron based Sachets
Finetec Co. Ltd Japan Sanso-cut Iron based Sachets
Toyo Pulp Co. Japan Tamotsu Catechol Sachets
Toyo Seikan Kaisha Ltd Japan Oxyguard Iron based Plastic trays
Dessicare Ltd USA O-Buster Iron based Sachets
Multisorb Technologies USA FreshMax Iron based Labels
Ascorbate/
W.R. Grace Co. Ltd USA PureSeal Bottle crowns
metallic salts
Cryovac Sealed Air Co. USA OS1000 Light activated Plastic film
Photosensitive
Food Science Australia/
Australia ZERO2 dye/organic Plastic film
Visy Industries
compound
CMB Technologies France Oxbar Cobalt catalyst Plastic bottle
Standa Industrie France Oxycap Iron based Bottle crowns
EMCO Packaging
UK ATCO Iron based Labels
systems
Bioka Ltd Finland Bioka Enzyme based Sachets
Switzerla
Ciba Specialty chemicals Shelfplus O2 Iron based Plastic film
nd
10
1.3. Reactive Oxygen Species (ROS)
“Reactive oxygen species” (ROS) is a collective term that includes oxygen radicals
and non-radical derivatives of oxygen. A free radical may be defined as a molecule that
has one or more unpaired electrons (Valko et al., 2006). Radicals can be formed when a
covalent bond is broken if one electron from each of the pair shared remains with each
atom (Halliwell and Gutteridge, 1999a). If oxygen attempts to oxidize another atom or
molecule by accepting a pair of electrons from it, both of these electrons must be of
antiparallel spin so as to fit in to the vacant spaces in the π* orbitals (Figure 1.3). The
oxygen radicals include superoxide anion (O2·-), hydroxyl (HO·), peroxy (ROO·), alkoxy
(RO·), and hydroperoxy (HOO·) radicals. Non-radical derivatives include hydrogen
peroxide (H2O2), ozone (O3), and singlet oxygen (1O2) (Choe and Min, 2006).
1.3.1. Superoxide anion
Superoxide anion (O2·-) is considered the primary ROS, and can further interact with
other molecules to generate secondary ROS, either directly or prevalently through
enzyme- or metal-catalyzed processes (Hamilton et al., 1997). It can be generated in a
number of ways:
O2 + Fe (II) → O2·- + Fe (III) (Equation 1.5)
Reducing agents such as ascorbate contribute to the redox cycle.
Fe (III) + AsH- → Fe (II) + A·- + H+ (Equation 1.6)
Superoxide is depleted undergoing a dismutation reaction.
2O2·- + 2H+ → H2O2 + O2 (Equation 1.7)
11
Addition of another electron to O2·- will produce the peroxide ion (O22-) which is not
a radical. Since the extra electrons enter the orbital in O2·- and O22-, the strength of the
oxygen-oxygen bond decreases. Addition of another two electrons to O22- would
eliminate the bond entirely, so giving 2O2- species. Usually the two- and four-electron
products of O2 are hydrogen peroxide and water, respectively (Hallwell and Gutteridge,
1999a).
O2 + e- → O2·-
O2 + 2 e- + 2H+ → H2O2
O2 + 4 e- + 4H+ → 2 H2O (Equation 1.8)
1.3.2. Hydroxyl radical
Hydroxyl radicals are so reactive with all biological molecules that it is impossible to
evolve a specific scavenger of it. Homolytic fission of the O-O bond in H2O2 produces
two hydroxyl radicals (HO·). Homolysis can be achieved by heat or ionizing radiation. A
simple mixture of H2O2 and an iron (II) salt also forms the HO· radical (Halliwell and
Gutteridge, 1984).
Fe (II) + H2O2 → Fe (III) + HO· + OH-
Fe (III) + H2O2 → Fe (II) + O2·- + H+
HO· + H2O2 → H2O + H+ + O2·-
O2·- + HO· → 1O2* + OH-
O2·- + Fe (III) → Fe (II) + O2
HO· + Fe (II) → Fe (III) + OH- (Equation 1.9)
12
The overall sum of these, unless some other reagent is added to intercept HO·, is an
iron catalyzed decomposition of H2O2.
1.3.3. Singlet Oxygen
Singlet oxygen, 1O2*, can be generated by input of energy or by reaction with
superoxide and hydroxyl radical. It is not a radical because there are no unpaired
electrons. However, singlet oxygen is highly reactive.
Figure 1.3. A simplified version of bonding in the diatomic molecule and its derivatives
(Halliwell and Gutteridge, 1984).
13
1.4. Transition Metal
The International Union of Pure and Applied Chemistry (IUPAC) define a transition
metal as an element whose atom has an incomplete d sub-shell. Many of the chemical and
physical properties of the transition metals are based on their unfilled d orbital. In general,
transition metals, also called the d-block elements, are the elements that make up Groups
3 through 12 of the periodic table (Smith, 2005).
Figure 1.4. Transition metal or d block elements on periodic table (Clark, 2005)
Most transition metals have variable valence, meaning that they have more than one
possible oxidation state. Irons have two common oxidation states (+2, +3) in Fe2+ and
Fe3+ (Clark, 2005).
Fe: [Ar] 3d64s2
Fe2+: [Ar] 3d6
Fe3+: [Ar] 3d5
14
If a solution of an iron (II) salt is exposed to the air, it slowly oxidizes to the iron (III)
state. The Fe2+ undergoes one-electron oxidation, and O2 dissolved in the solution is
reduced to superoxide radical (Halliwell and Gutteridge, 1999b).
Fe2+ + O2
! " ### ! " · % O2·- + Fe (III) (Equation 1.10)
Intermediate complexes
15
1.5. Antioxidant
Antioxidants are compounds that react with free radicals and are themselves oxidized
to generate what are generally innocuous nontoxic compounds (Brody et al., 2001). In
food processing, antioxidants are added to foods to delay or prevent oxidation. In
package processing, antioxidants are incorporated into polymer resin to stabilize the
polymer.
1.5.1. Chain-breaking antioxidants
Chain-breaking antioxidants are sometimes called a free radical scavenging
antioxidants. They scavenge free radicals and inhibit chain initiation or break chain
propagation (Shi et al., 2001). Vitamin E, ubiquinol, carotenoids, and flavonoids are
major lipophilic chain-breaking antioxidants. Vitamin C, uric acid, bilirubin, and albumin
are major hydrophilic chain-breaking antioxidants.
1.5.2. Synthetic and natural antioxidant
Synthetic antioxidants are phenolic compounds such as butylated hydroxyanisol
(BHA), butylated hydrotoluene (BHT), and tertiary butylhydroquinone (TBHQ) (Figure
1.5). BHA and BHT are fairly stable to heat and are often used for stabilization of fats in
baked products. These synthetic antioxidants have been thoroughly tested for their
toxicological behaviors.
16
Figure 1.5. Synthetic phenolic antioxidant (Yanishlieva-Maslarova, 2001)
Natural antioxidants are vitamin C, vitamin E, and flavonoids (Yanishlieva-
Maslarova, 2001). Tocopherols are the best known and most widely used antioxidants
(Figure 1.6). It works as an antioxidant by donating the hydrogen from the hydroxyl
group to the lipid peroxyl radical. Flavonoids constitute a large group of naturally
occurring plant phenolics. The basic structure of these compounds consists of two
aromatic rings linked by a three-carbon aliphatic chain (Figure 1.7). Flavonoids may act
as antioxidants by scavenging radicals that include superoxide anions.
17
Figure 1.6. Natural antioxidant; tocopherol (Yanishlieva-Maslarova, 2001)
18
Figure 1.7. Natural antioxidant; Flavonoids (Yanishlieva-Maslarova, 2001)
19
1.6. α-tocopherol
Natural vitamin E comprises eight different forms, the α-, β-, γ-, δ-tocopherols and
the α-, β-, γ-, δ-tocotrienols (Figure 1.8). Among those compounds, α-tocopherol has the
highest chemical reactivity, free radical scavenging reactivity, singlet oxygen (1O2)
scavenging reactivity, and biological activity (Abidi, 2000, Lien et al., 1999, Mukai et al.,
1991).
Figure 1.8. Natural vitamin E analogue (Zingg, 2007)
20
α-tocopherol reacts with peroxy radicals as a free radical scavenger. It donates its
hydrogen atom at the 6- hydroxyl group on the chromanol ring to the free radicals. Alkyl
hydroperoxide and an α-tocopheryl radical are produced by this reaction. The α-
tocopheryl radical is stabilized through the resonance structure of the aromatic ring. This
radical can react with a peroxy radical to produces α-tocopherol peroxide or it can reacts
with another α-tocopheryl radical and produce a α-tocopherol dimers (Figure 1.9).
21
Figure 1.9. Mechanism of α-tocopherol action (Yanishlieva-Maslarova, 2001)
22
α-tocopherol also can react irreversibly with singlet oxygen and produce α-
tocopherol hydroperoxydienone, α-tocopherylquinone, and α-tocopherylquinone epoxide
(Figure 1.10).
α-tocopherol hydroperoxydienone
α-tocopherol
α-tocopherol endoperoxide
α-tocopheryl quinone α-tocopheryl quinone epoxide
Figure 1.10. Singlet oxygen oxidation of α-tocopherol (Choe and Min, 2006)
23
1.7. Nanoencapsulation for Drug Delivery
A nanoparticle is defined as solid, submicron-size drug carriers with diameter range
from 1 to 1000 nm. It is a collective term for both nanospheres and nanocapsules.
Nanospheres have a matrix type of structure. Drugs can be absorbed at the sphere surface
or encapsulated within the particle. Nanocapsules are vesicular systems in which the drug
is confined to a cavity consisting of an inner liquid core surrounded by a polymeric
membrane (Couvreur et al., 1995). The submicron size of nanoparticles offers a number
of distinct advantages over microparticles, including relatively higher intracellular uptake
(McClean et al., 1998). Many methods have been developed for preparing nanoparticles
(Table 1.2).
1.7.1. Emulsion polymerization
The emulsion polymerization method is classified into two groups, organic
continuous and aqueous continuous phase. The continuous organic phase method
involves the dispersion of monomer into an emulsion or into a material in which the
monomer is not soluble (Reis et al., 2006). In the aqueous continuous phase, the
monomer is dissolved in a continuous phase and surfactants or emulsifiers are not needed.
The polymerization process initiation occurs when a monomer molecule reacts with a
free radical or a monomer molecule transformed into a radical by radiation (Vauthier et
al., 2003). Chain growth starts when the initiated monomer radicals react with other
monomer molecules. Phase separation and formation of solid particles can take place
before and after termination of the polymerization reaction.
24
Table 1.2. Nanoencapsulation method: advantages and drawbacks (Reis et al., 2006)
Safety
Facility
Simplicity Need for of
Main group Method scaling- EE (%)
of procedure purification compou
up
nds
Emulsion
polymerization Low High NR Low Low
Nanoparticles (organic)
obtained by Emulsion
polymerization of polymerization High High High High Medium
a monomer (aqueous)
Interfacial
Low High Medium High Low
polymerization
Emulsion and
solvent High Low Low Medium Medium
evaporation
Nanoparticles
Nanoprecipitation High NR NR High Medium
obtained from
synthetic
Emulsification
preformed
and solvent Medium Medium High High Low
polymers
diffusion
Salting out with
synthetic High High High High Medium
polymers
Albumin
NR High NR Medium Low
nanoparticles
Gelatin
NR High NR Medium Low
Nanoparticles nanoparticles
obtained from Alginate
High Medium High High High
natural nanoparticles
macromolecules Chitosan
High Medium High High High
nanoparticles
Agarose
Medium High NR NR High
nanoparticles
Nanoparticles Techniques based
obtained by on supercritical or
NR High NR Low Low
desolvation of compressed fluids
macromolecules
1.7.2. Interfacial polymerization
Poly(alkylcyanoacrylate) nanoparticles were produced by interfacial polymerization.
Cyanoacrylate monomer and a drug product were dissolved into a mixture of an oil and
absolute ethanol. The mixture was then slowly dropped into the aqueous solution with
25
and without surfactant. Nanocapsules were formed spontaneously by polymerization of
cyanoacrylate after contact with initiating ions present in the aqueous solution. The
resulting colloidal suspension was concentrated by evaporation and vacuum (Al-Khour et
al, 1986).
1.7.3. Emulsion and solvent evaporation
The emulsion and solvent evaporation process is different from the nanoprecipitation
method in that the main phases stay immiscible at all times, only to be removed later by
evaporation (Mccarron et al., 2006). Biphasic (oil in water, o/w) and triphasic (water in
oil in water, w/o/w) emulsion systems have been investigated extensively. Emulsion and
solvent evaporation involves two steps. The first step requires emulsification of the
polymer solution into an aqueous phase. During the second step, the polymer solvent is
evaporated, inducing polymer precipitation as nanospheres. The size can be controlled by
adjusting the stir rate, type and amount of dispersing agent, the viscosity of organic and
aqueous phases, and the temperatures (Tice and Gilley, 1985).
1.7.4. Nanoprecipitation
In this method, the polymer and the drug are both dissolved in a water-miscible
solvent. Then this organic solution is poured into a continuously stirred aqueous phase in
the presence or absence of a stabilizer. Nanoparticles are formed instantaneously by rapid
solvent diffusion. The organic solvent is eliminated from the suspension by means of
reduced pressure (Mccarron et al., 2006). This method has been applied to various
26
polymers such as polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), and
polycaprolactone (PCL).
1.7.5. Salting out procedure
A salting out agent (electrolytes, such as magnesium chloride) is used to separate the
water-miscible solvent from the aqueous solution (Konan and Allemann, 2002). The
polymer and the drug are dissolved in acetone and emulsified into an aqueous phase
containing the salting out agent and stabilizer. This o/w emulsion is diluted with an
aqueous solution to enhance the diffusion of acetone into the aqueous solution, thus
inducing the formation of nanospheres.
27
1.8. Gelatin
Gelatin is derived from the fibrous protein collagen that is present in animal skin,
bone, and connective tissue (Schrieber and Gareis, 2007). Collagen molecules are
composed of a right handed triple-helix and these triple-helices are stabilized by inter-
chain hydrogen bonds (TeNijenhuis, 1997). Collagen denaturation causes separation of
the rods and total or partial separation of the chains due to destruction of the hydrogen
bond, causing loss of the triple-helix conformation.
1.8.1. Type of gelatin
Gelatin is produced from the collagen of animal skins and bones through either acid
(Type A) or alkaline (Type B) hydrolysis. Both gelatin types are composed of amino
acids, mainly alanine, glysine, hydroxyproline, and proline, with small quantities of other
amino acids (Table 1.3). In the case of type B gelatin, both asparagines and glutamine
are almost completely converted to aspartic and glutamic acids, respectively (Schrieber
and Gareis, 2007).
28
Table 1.3. Amino acid composition of gelatins, residues per 1000 residues (Rose, 1987)
Amino Acid Gelatin Type A Gelatin Type B

Alanine 112 117
Arginine 49 48
Asparagine 16 0
Aspartic acid 29 46
Glutamic acid 48 72
Glutamine 25 0
Glycine 330 335
Histidine 4 4.2
Hydroxyproline 91 93
Hyroxylysine 6.4 4.3
Isoleucine 10 11
Leucine 24 24.3
Lysine 27 28
Methionine 3.6 3.9
Phenylalanine 14 14
Proline 132 124
Serine 35 33
Threonine 18 18
Tyrosine 2.6 1.2
Valine 26 22
1.8.2. Properties and characterization of gelatin
Gelatin is a mixture of polymer chains of different lengths. Thus, real solutions are
not formed. Instead, colloidal solutions or sols are formed. On cooling, these sols convert
to gels, and on warming they revert to sols. If gelatin solution is cooled, the mobile
29
molecules aggregate to small clusters which continuously grow and subsequently form
gels. This unlimited reversibility of the gelling process is the most important
technological property of gelatin (Schrieber and Gareis, 2007).
Figure 1.11. The chemical structure of gelatin
1.8.3. Mechanical properties of gelatin film
Gelatin forms a three-dimensional network with intermolecular microcrystalline
junctions. Dehydration of this system may produce brittle films (Vanin et al., 2005).
Gelatin films always contain some water, normally 10-15%, and water acts as a necessary
plasticizer since gelatin films of low moisture (less than 5%) are too brittle to be useful.
In many applications, non-volatile plasticizers such as glycerol are added (Finch and
Jobling, 1977). Polyols are often cited as good plasticizers for protein-based materials
due to their ability to reduce intermolecular hydrogen bonding while increasing
intermolecular spacing (Audic and Chaufer, 2005). The addition of a plasticizer is
necessary to overcome the brittleness of gelatin films, to improve flow and flexibility,
and to increase toughness (Aydinli and Tutas, 2000).
30
Bradbury and Martin (1951) measured the elasticity, tensile strength and elongation
at break of gelatin films as a function of the temperature of drying the film and also the
relative humidity during measurement. Films were prepared in two ways: prepared from
the gel state and then drying with cool air, prepared from the sol state and then drying
above the gel point with air at 55-60℃. At all humidities, the film prepared from the gel
state had a greater tensile strength.
1.8.4. Fish gelatin
Gelatin from marine sources is a possible alternative to bovine gelatin (Yi et al.,
2006). The major advantages of fish gelatins are that they are not associated with Bovine
Spongiform Encephalopathy (mad cow disease) and that they are acceptable for various
religious dietary restrictions. Furthermore, it has been estimated that over a million tons
of fish byproducts are generated each year from the fish processing industry. This waste
stream could provide a valuable source of gelatin.
Gelatin has been extracted from the skins and bones of various cold water fish (e.g.
Alaska pollock, Cod, Hake, and Salmon) and warm water fish (e.g., Tuna, Catfish,
Megrim, Nile perch, shark, and Tilapia). Cold water fish gelatins have very low gel
modulus, low gelling temperature, and melting temperature compared to mammalian and
warm-water fish gelatins. Consequently, cold water fish gelatins behave as a viscous
liquid at room temperature, which limits their use in many applications. This is because
the cold water fish gelatins have lower concentration of imino acids, proline and
hydroxyproline, than the other species (Haug et al., 2004). The super-helix structure of
31
the gelatin gel is stabilized by steric restrictions. These restrictions are imposed by both
proline rings of the imino acids in addition to the hydrogen bonds formed between amino
acid residues (TeNijenhuis, 1997). Table 1.4 shows amino acid content in fish gelatin and
pork gelatin. Generally, collagens present in fish skins show a wider variety in amino
acid composition than those of mammalian collagens. The source and type of collagen
will influence the properties of the resulting gelatin. It has been observed that all fish
gelatins exhibits excellent film forming properties. In general, fish gelatin film has
similar tensile strength and elongation at break to that of bovine gelatin. However, fish
gelatin has lower water vapor permeability (WVP) than that of bovine gelatin because it
has much more hydrophobicity due to lower proline and hydroxyproline. Furthermore,
the WVP of cold water fish gelatin films was significantly lower than that of warm water
fish gelatin due to the reduced amount of proline and hydroxyproline (Avena-Bustillos et
al., 2006).
32
Table 1.4. Amino acid content in fish gelatins and pork gelatins (Karim and Bhat, 2008)
Alaska
Amino Acid Cod Hake Megrim Tilapia Pork
pollock
Alanine 96 108 119 123 123 112
Arginine 56 51 54 54 47 49
Aspartic acid 52 51 49 48 48 46
Glutamic acid 78 74 74 72 69 72
Glycine 344 358 331 350 347 330
Histidine 8 8 10 8 6 4
Hyroxylysine 6 6 5 5 8 6
Hydroxyproline 50 55 59 60 79 91
Isoleucine 11 11 9 8 8 10
Leucine 22 20 23 21 23 24
Lysine 29 26 28 27 25 27
Methionine 17 16 15 13 9 4
Phenylalanine 16 12 15 14 13 14
Proline 106 95 114 115 119 132
Serine 64 63 49 41 35 35
Threonine 25 25 22 20 24 18
Tyrosine 3 3 4 3 2 3
Valine 18 18 19 18 15 26
33
1.9. References
Abidi, S.L. 2000. Chromatographic analysis of tocol-derived lipid antioxidants.

J. Chromatogr. a. 881, 197-216
Albert, C. F., Rooney, M. L., 2004. Triggered active packaging material,

US patent 6,821,482
Al-Khour, N., Robolt-Treupel, L., Fessi, H., Devissaquet, J. P., Puisieux, F., 1986.
Development of a new process for the manufacture of polysiobutylcyanoacrylate
nanocapsules. Int. J. Pharm. 28. 125-132
Ahvenainen, R., 2003. Active and intelligent packaging, in : Ahvenainen, R. (Eds),

Novel food packaging techniques, CRC Press, New York, USA, pp 5-21
Andersson, M., Andersson, T., Adlercreutz, P., Nielsen, T., Hornsten, E. G., 2002.
Toward an enzyme-based oxygen scavenging laminate. Influence of industrial
lamination conditions on the performance of glucose oxidase. Biotechnology and
bioengineering, 79 (1), 37-42
Anklam, E., Gaglione, S., Muller, A., 1997.

Oxidation behavior of vanillin in dairy products. Food Chemistry. 60, 43-51
Audic, J.-L., Chaufer, B., 2005. Influence of plasticizers and crosslinking on the
properties of biodegradable films made from sodium caseinate.
European Polymer Journal, 41, 1934–1942
Avena-Bustillos, R. J., Olsen, C. W., Chiou, B., Yee, E., Bechtel, P. J., McHugh, T. H.,
2006. Water vapor permeability of mammalian and fish gelatin films.
Journal of Food Science, 71. 202-207
Aydinli, M., Tutas, M., 2000. Water sorption and water vapour permeability properties of
polysaccharide (locust bean gum) based edible films.
Food Science and Technology, 33(1), 63–67
Bradbury, E., Martin, C., 1951. Mechanical properties and structure of sol-type and
gel-type gelatin films. Nature. 168, 837-838
34
Brody, A. L., Strpinsky E.P., Kline, L. R., 2001, Active food packaging for
food application, CRC Press, New York, USA, pp 11-30
Choe, E., Min, D. B., 2006. Chemistry and reactions of reactive oxygen species in foods.
Critical Reviews in Food Science and Nutrition, 46(1). 1-22
Clark, J., 2005. The general features of transition metal chemistry.

http://www.chemguide.co.uk/ inorganic/transition/features.html
Couvreur, P., Dubernet, C., Puisieux, F., 1995. Controlled drug delivery with
nanoparticles: current possibilities and future trends.
Eur. J. Pharm. Biopharm. 41. 2-13
Dagliden, J., 2008. Active, controlled and intelligent packaging for foods and beverages.
BBC research, London, UK
Day, B. P. F., 2003. Active packaging, in : Coles, R., Mcdowell, D., Kirwan, M.J. (Eds),
Food packaging technology, CRC Press, New York, USA, pp 282-302
Farrell, C. J., Tsai, B. C., 1987. Oxygen scavenger. US patent 4,702,966
Finch, C. A., Jobling, A., 1977. The physical properties of gelatin, in : Ward, A. G.,
Courts, A. (Eds), The science and technology of gelatin, Academic Press,
New York, USA, pp 249-294
Floros, J. D., Dock, L. L., Han, J. H., 1997. Active packaging technologies and
application. Food cosmetics and drug packaging, 20(1), 10-17
Graf, E., 1994. Oxygen removal. US patent 5,284,871
Hallwell, B., Gutteridge, M. C., 1984. Oxygen toxicity, oxygen radicals, transition metals
and disease. Biochem. J. 219. 1-14
35
Hallwell, B., Gutteridge, M. C., 1999a. Oxygen is a toxic gas- an introduction to oxygen
toxicity and reactive oxygen species, in: Hallwell, B., Gutteridge, M. C. (Eds),
Free radicals in Biology and Medicine. Oxford University Press,
New York. pp 1-35
Hallwell, B., Gutteridge, M. C., 1999b. The chemistry of free radicals and related
‘reactive species’, in: Hallwell, B., Gutteridge, M. C. (Eds), Free radicals in
Biology and Medicine. Oxford University Press, New York. pp 36-104
Hamilton, R. J., Kalu, C., Prisk, E., Padley, F. B., Pierce, H., 1997. Chemistry of free
radicals in lipids. Food chemistry, 60. 193-199
Haug, I. J., Draget, K. I., Smidsrod, O., 2004. Physical and rheological properties of fish
gelatin compared to mammalian gelatin. Food Hydrocolloid. 16. 25-34
Inoue, Y. Komatsu, T., 1990. Oxygen absorbent. US patent 4,908,151
Inoue, Y., Murabayashi, S., Fujinami, K., Yoshino, I., 1994. Oxygen absorbent
composition and method of preserving article with same. US patent 5,286,407
Klein, T., Knorr, D., 1990. Oxygen absorption properties of powdered iron.
Journal of food science. 55(3), 869-870
Konan, Y. N., Allemann, E., 2002. Preparation and characterization of sterile and
freeze-dried sub 200 nm nanoparticles. Int. J. Pharm. 233. 239-252
Labuza, T. P., Breene, W. M., 1989. Application of active packaging for improvement of
shelf-life and nutritional quality of fresh and extended shelf-life foods.
Journal of food processing and preservation, 13, 1-69
Lien, E., Ren, S., Bui, H., Wang, R., 1999. Quantitative structure-activity relationship
analysis of phenolic antioxidants. Free Radic. Biol. Med. 26, 285-294
36
Mccarron, P. A., Donnelly, R. F., Marouf, W., 2006. Celecoxib-loaded
poly(D,L-lactide-co-glycolide) nanoparticles prepared using a novel and
controllable combination of diffusion and emulsification steps as part of the
salting-out procedure. J. Microencapsule. 23, 480-498
McClean, S., Prosser, E., Meehan, E., O’Malley, D., Clarke, N., Ramtoola, Z., 1998.
Binding and uptake of biodegradable poly-lactide micro and nanoarticles in
intestinal epithelia. Eur. J. Pharm. Sci. 6. 153-163
Morales-Aizpurua I. C., Tenuta-Filho, A., 2005. Oxidation of cholesterol in mayonnaise

during storage. 89. 611-615
Mukai, K., Daifuku, K., Okabe, K., Tanigaki, T., and Inoue, K., 1991. Structure- activity
relationship in the quenching reaction of singlet oxygen by tocopherol
(Vitamin E) derivatives and related phenols. Finding of linear correlation between
rates of quenching of singlet oxygen and scavenging of peroxyl and phenoxyl
radicals in solution. J. Org. Chem. 56, 4188-4192
Nestorson, A., Neoh, K. G., Kang, E. T., Jarnstrom, L., Leufven, A., 2008. Enzyme
immobilization in latex dispersion coatings for active food packaging.
Packaging technology and science. 21, 193-205
Nezat, J. W., 1985. Composition for absorbing oxygen and carrier therof.
US patent 4,510,162
Ozdemir, M., Floros, J. D., 2004. Active food packaging technology.

Critical reviews in Food Science and Nutrition, 44. 185-193
Reis, C. P., Neufeld, R. J., Ribeiro, A. J., Veiga, F., 2006. Nanoencapsulation 1.
Methods for preparation of drug-loaded polymeric nanoparticles.
Nanomedicine: Nanotech. Biol. Med. 2, 8-21
Rooney, M. L., 1995. Overview of active food packaging, in : Rooney, M. L. (Eds),

Active food packaging, Blackie academic and professional,
37
Rose, P. I., 1987. Gelatin, in : Encyclopedia of polymer science and engineering,
Volume 3, Wiley & Sons, London, UK, pp 488-513
Schrieber, R., Gareis, H., 2007. From collagen to Gelatin, In: Schrieber, R.,
Gareis, H. (Eds), Gelatin Handbook, Wiley-VCH verlag GmbH & Co.
Weinheim, Germany, pp 45-117
Schmidt, R. P., Solis, J. A., Ching, T. Y., 2006. Transition metal carboxylates as catalysts
for oxygen scavenging, US patent 7,052,628
Shi, H., Noguchi, N., Niki, E., 2001. Introducing natural antioxidants, in: Pokorny, J.,
Yanishlieva, N., Gordon, M. (Eds), Antioxidant in food, CRC Press,
New York, pp 147-158
Smith, A., 2005. http://www.chemcases.com/cisplat/cisplat06.htm
Speer, D. V., Roberts, W. P., 1994. Improved oxygen scavenging compositions for low
temperature use. WO 94/07379
Speer, D. V., Morgan, C. R., Roberts, W. P., VanPutte, A. W., 1996. Multilayer structure
for a package for scavenging oxygen. US patent 5,529,833
TeNijenhuis, K., 1997. Thermoreversible networks: viscoelastic properties and structure

of gels. Advances Polymer Science, 130. 1-267
Teumac, F. N., Zenner, B. D., Ross, B. A., Deardurff, L. A., Rassouli, M. R., 2004.
Metal catalyzed ascorbate compounds as oxygen scavengers. US patent 6,709,724
Tice, T. R., Gilley R. M., 1985. Preparation of injectable controlled-release

microcapsules by solvent-evaporation process. J. Control. Release, 2. 343-352
Valko, M., Rhodes, C. J., Moncol, J., Izakovic, M., Mazur, M., 2006. Free radicals,
metals and antioxidants in oxidative stress-induced cancer.
Chemico-Biological Interactions, 160. 1-40
38
Vanin, F. M., Sobral, P. J. A., Menegalli, F. C., Carvalho, R. A., Habitante, A. M. Q. B.,
2005. Effects of plasticizers and their concentrations on thermal and functional
properties of gelatin-based films. Food Hydrocolloids, 19, 899–907
Vauthier, C., Dubernet, C., Fattal, E., Pinto-Alphandary, H., Convreur, P., 2003.
Poly(alkylcyanoacrylates) as biodegradable materials for biomedical application.
Adv. Drug Deliv. Rev. 55.519-548
Vermeiren, L., Devlieghere, F., Beest, M., Kruijf, N., Debevere, J., 1999,
Developments in the active packaging of foods.
Trends in food science and technology. 10, 77-86
Vermeiren, L., Heirlings, L., Devlieghere, F., Debevere, J., 2003. Oxygen, ethylene and
other scavengers, in : Ahvenainen, R. (Eds), Novel food packaging techniques,
CRC Press, New York, USA, pp 22-49
Watson, J. L., Carney, L. L., 1977. Oxygen scavenging methods and additives.
US patent 4,509,533
Yamaji, T., Okitsu, H., 1980. Oxygen scavenging and heat-generating compositions, and
deoxygenating and heat-generating structures. US patent 4,230,595
Yang, H., Ching, T. Y., Cai, K., Torres, L., 2004. Oxygen barrier copolymer,
US patent 6,818,151
Yanishlieva-Maslarova, N. V., 2001. Inhibiting oxidation, in: Pokorny, J., Yanishlieva,

N., Gordon, M. (Eds), Antioxidant in food, CRC Press, New York, pp 22-70
Yi, J. B., Kim, Y. T., Bae, H. J., Whiteside, W. S., Park, H. J., 2006.
Influence of transglutaminase-induced cross-linking on properties of fish
gelatin films. Journal of Food Science, 71(9), 376-383
Zingg, J., 2007. Vitamin E: An overview of major research directions.

Molecular Aspects of Medicine. 28. 400-422
39
CHAPTER TWO
RESEARCH HYPHOTHESIS
The following hypotheses are the basis of this study.
1. Oxygen free radicals may produced by non-enzymatic reactions of oxygen with a
transition metal. Then, α-tocopherol can eliminate the oxygen free radical by
donating its electrons to the free radicals. Therefore, oxygen content in package
headspace can be reduced by successive chemical reactions.
2. Thermal processing can accelerate the oxygen scavenging reaction outlined in
hypothesis 1.
3. Nanoparticles designed to exploit hypothesis 1 and 2 can be good oxygen
scavenging activators. Breakage of the nanoparticles can initiate the oxygen
scavenging reaction.
4. High quality α-tocopherol-loaded poly ε-caprolactone (PCL) nanoparticles can be
manufactured and qualified for application.
5. The encapsulated oxygen scavenger can be protected from moisture, heat, light,
and oxygen, thus preventing premature loss of scavenging capacity. The oxygen
scavenging nanopariticle may not react with atmosphere oxygen prior to use.
6. Breakage of the nanoparticle may be achieved by the application of heat,
exploiting the low melting point of PCL. α-tocopherol may be released from the
nanoparticle or oxygen free radicals which are produced by the transition metal
can penetrate into nanoparticle. Then, the oxygen content in the headspace can be
reduced by the successive chemical reaction.
40
7. Fish gelatin film can be manufactured and qualified for application.
8. The addition of α-tocopherol-loaded nanoparticles and transition metals into the
fish gelatin can modify the film properties.
9. Pure gelatin film is normally oxygen impermeable, but it is rendered oxygen
permeable upon exposure to water or water vapor. The headspace oxygen can
penetrate into the gelatin film structure when gelatin is rendered oxygen
permeable. Then, this oxygen can be consumed by the oxygen scavenger. The
oxygen scavenging fish gelatin without moisture may not react with oxygen.
41
CHAPTER THREE
RESEARCH OBJECTIVES
The overall objectives of this study were to develop a new oxygen scavenging
mechanism using a free radical scavenger and a transition metal and then develop new
activation system using moisture and heat activated oxygen scavenging nanoparticle.
The specific objectives of each chapter were:
Chapter IV
1. To determine the oxygen scavenging ability of α-tocopherol and transition metal
in concert
2. To investigate the combined effect of α-tocopherol, transition metal, thermal
processing, and moisture on oxygen scavenging capability
Chapter V
1. To develop the α-tocopherol-loaded poly ε-caprolactone (PCL) nanoparticle as an
oxygen scavenging activation system using emulsion solvent evaporation with
ultrasonification
2. To investigate the effect of PCL concentration, solvent in oil phase, and
ultrasonification time on encapsulation efficiency (%), particle mean size,
polydispersity, and loading (%)
3. To characterize the α-tocopherol-loaded nanoparticle by SEM
42
Chapter VI
1. To determine the oxygen scavenging ability of α-tocopherol-loaded nanoparticle
in conjunction with a transition metal
2. To investigate the effect of α-tocopherol-loaded nanoparticle, transition metal,
thermal processing, and moisture on oxygen scavenging capability
Chapter VII
1. To develop and characterize an active fish gelatin film containing α-tocopherol-
loaded nanoparticles and a transition metal developed in the previous objectives
as an oxygen scavenging layer
2. To investigate the effect of thermal processing and moisture on oxygen
scavenging capability of the active fish gelatin film
43
CHAPTER FOUR
ALPHA-TOCOPHEROL AND A TRANSITION METAL

AS AN OXYGEN SCAVENGER
Abstract
α-tocopherol and a transition metal, iron, in an oxygen scavenger mixture were
evaluated as a possible oxygen scavenger in this study. The effects of α-tocopherol,
transition metal, moisture, and thermal processing on oxygen scavenging capability were
also investigated. The initial oxygen content in the cup headspace was 20.90% and was
decreased to 18.03% after thermal processing and 60 days of storage when the oxygen
scavenger mixture contained α-tocopherol and transition metal. α-tocopherol and
transition metal were required for optimum oxygen scavenging capability. Otherwise, the
oxygen scavenging capability decreased. Furthermore, the oxygen content was decreased
to 17.10% when the amount of transition metal increased from 100 to 150 mg.
Trimethylhydroquinone (TMHQ) and transition metal as an oxygen scavenger was also
investigated. Oxygen content (%) in the cup headspace was decreased from 20.90% to
18.37% after thermal processing and 60 days of storage when the oxygen scavenger
mixture contained TMHQ, transition metal, and moisture. Results indicated that α-
tocopherol or TMHQ and a transition metal in oxygen scavenger mixture coupled with
thermal processing had oxygen scavenging capability.
44
4.1. Introduction
Food deterioration, such as the development of off-flavor, color and flavor changes,
and nutritional losses, can be often caused by oxidation (Anklam et al., 1997, Morales-
Aizpurua and Tenuta-Filho, 2005). Additionally, excessive oxygen in the packaging can
cause the growth of aerobic bacteria or proliferation of molds (Brown and Williams,
2003).
Vacuum packaging has been widely used to eliminate oxygen in the packaging
headspace. However, the oxygen that permeates from outside to headspace through
packaging cannot be removed by this technique. Oxygen scavengers can remove the
oxygen that permeates from outside into the headspace using chemical reactions. On the
other hand, oxygen absorbers remove oxygen by physical trapping (Brody et al., 2001).
Oxygen scavengers have been commercialized in the food packaging industries
during recent decades. It is used in various forms; sachet, plastic film, labels, plastic trays,
and bottle crowns (Rooney, 1995). There are several principles of oxygen scavengers, but
the most widely used concepts are iron oxidation, ascorbic acid oxidation, and
photosensitive dye oxidation (Ahvenainen, 2003). The most common and effective form
of oxygen scavengers are based on the principle of iron oxidation (Rooney, 1995,
Vermeriren et al., 1999). Iron-based oxygen scavengers are mostly used in sachet form,
but future trends in active packaging are focused on the use scavenging compounds
incorporated in the packaging film (Ahvenainen, 2003). Moreover, consumers do not like
the term “iron-based”.
45
α-tocopherol is a natural chain-breaking antioxidant (Hamilton et al., 1997) with a
positive consumer perception. It has been incorporated into the polymer materials as a
stabilizer (Al-Malaika et al, 1994. Al-Malaika et al., 1999) and as an antioxidant in
controlled release packaging to reduce the oxidation in food (Lacoste et al., 2005, Siro et
al., 2006, Wessling et al., 2000).
In this research, α-tocopherol in the presence of a transition metal, iron, was
investigated as an oxygen scavenger. Oxygen free radicals are derived from the non-
enzymatic reactions of oxygen with transition metals (Bagchi and Puri, 1998). The
transition metal can activate oxygen to the singlet electron state oxygen. Then, this
activated oxygen undergoes subsequent reduction to reactive oxygen species (ROS),
which is an oxygen free radical. α-tocopherol is a strong free radical scavenger. It also
can react irreversibly with singlet oxygen and produce tocopherol hydroperoxydienone,
tocopherylquinone, and quinine epoxide (Choe and Min, 2006).
The principle of the oxygen scavenger in this study is that oxygen free radicals can
be produced by the transition metal. Then, α-tocopherol can donate its electrons to
scavenge the oxygen free radical. When the free radical gains the electron from α-
tocopherol, it returned to its ground state and the free radical is eliminated. Therefore, the
oxygen content in the headspace will be reduced by these successive chemical reactions.
The objective of this study was to develop a new oxygen scavenger using α-
tocopherol and a transition metal, iron. Additionally, trimethylhydroquinone (TMHQ), a
precursor to the synthetic α-tocopherol, and a transition metal were also examined as an
46
oxygen scavenger. The influences of moisture, thermal processing, and amount of
transition metal on oxygen scavenging capability were also investigated.
47
4.2. Materials and Methods
4.2.1. Materials
α-tocopherol (96.2%, Mw 430.7) was purchased from EMD Bioscience (CA.USA).
Trimethylhydroquinone (97%, TMHQ) was purchased from Sigma-Aldrich (MO, USA).
Iron chloride (II), tetrahydrate was purchased from J.T.Baker (NJ, USA). Retortable
plastic cups with 3% EVOH barrier were provided from Printpack (GA, USA). 48 ga
PET/60 ga Foil/3 mil CPP film was used as cup lids.
4.2.2. Sample preparation
α-tocopherol with different amounts of transition metal (0, 50, 100, 150 mg) and with
or without water were placed inside a high oxygen barrier retortable cup with 115 cc of
ambient air (20.90% O2). The cup was sealed with PET/Foil/CPP film using Lab sealer
(Packaging Technologies, IA, USA). The sealing temperature was 470oF with 60 psi
pressure at 1 second dwell time.
4.2.3. Thermal processing
A pilot-scale rotary, single cage, water spray retort in static mode was employed in
this research. Samples were thermally processed for 30 minute at 170oF using a Surdry
Model APR-95 Rotary Pilot Retort (Stock America, NC, USA)
48
4.2.4. Oxygen Content Analysis
The oxygen content in the cup headspace was analyzed by a headspace
oxygen/carbon dioxide analyzer (model 6600, Illinois Instrument, IL, USA). A sampling
needle with a 0.45 µm PTFE filter was inserted and 15 cc headspace gases were sampled
through a septum. Calibration of headspace analyzer was done using ambient air after
each sample measurement. All of the samples were measured in triplicate at day 0, 1, 4,
7, 14, 30, and 60.
4.2.5. Statistical analysis
Statistics on a completely randomized design were performed with the analysis of
variance (ANOVA) using SAS (version 9.1, SAS Institute Inc., Cary, NC, USA) and
differences among mean values were processed by Duncan’s multiple range test.
Significance was defined at a level of P < 0.05.
49
4.3. Results and Discussion
4.3.1. Oxygen content (%) reduction during storage time
This study was designed to assess the influence of mixture variables on oxygen
content (%) reduction. Nine batches with different composition in the scavenger mixture
were prepared (Table 1). Eleven batches are shown on Table 1, but batch A-T-R and A-R
were used twice with different name of A-T100-R and A-T0-R, respectively. The oxygen
content (%) in the headspace of all batches decreased during the storage time (Table 2).
However, there was no oxygen content (%) reduction on batch A-T until day 4. In this
case, a lag time can be seen before the scavenger starts to reduce oxygen content (%).
Batch A-T150-R showed the highest oxygen content (%) reduction and batch A-R (A-T0-
R) showed the lowest reduction in oxygen content (%).
4.3.2. Effects of α-tocopherol, transition metal, thermal processing, and moisture on
oxygen scavenging capability
In this study, oxygen content (%) in the headspace may have been reduced by a
successive chemical reaction. There are two chemical reaction steps (Scheme 1 (b)). In
first step, oxygen free radicals are produced in the presence of a transition metal (Bagchi
and Puri, 1998). In the second step, the oxygen free radicals are eliminated by receiving
electrons from α-tocopherol (Smirnoff, 2005). Therefore, the presence of both the
transition metal and α-tocopherol are essential conditions for the oxygen scavenger
mixture. Furthermore, thermal processing can accelerate oxygen scavenging reaction.
50
The effects of α-tocopherol, transition metal, and thermal processing on the oxygen
scavenging capability have been investigated (Fig 1). The oxygen content (%) on batch
A-T-R was significantly lower than the others over the entire storage time. This suggests
that α-tocopherol and transition metal with thermal processing eliminate oxygen
effectively. Batch T-R and A-T had higher oxygen content than batch A-T-R. There was
no free radical scavenging reactions and no acceleration in initiation step on batch T-R
and A-T, respectively (Scheme 1 (b)). α-tocopherol and thermal processing were required
for optimum oxygen scavenging capability and for the acceleration of the oxygen
scavenging reaction, respectively. Otherwise, the oxygen scavenging capability decreased.
The oxygen content (%) on batch A-R was significantly higher than the other batches at
day 60, indicating that the oxygen scavenger mixture without transition metal had the
lowest oxygen scavenging capability. There was no initiation step on batch A-R (Scheme
1 (b)). Therefore, a transition metal was required for optimum oxygen scavenging
capability. In addition, the effect of moisture on oxygen scavenging capability has been
investigated (Fig. 2). There was no significant difference in oxygen content (%) between
batch A-T-R and A-T-R-H during storage time, except for day 30. Thus, the addition of
moisture did not affect the oxygen scavenging capability.
4.3.3. Effect of the amount of transition metal
It was observed that batch A-T150-R showed the highest oxygen scavenging
capability (Fig 3). The oxygen scavenging capability was increased when the amount of
transition metal in scavenger mixture was increased from 0 to 150 mg at day 60.
51
4.3.4. TMHQ with transition metal as an oxygen scavenger
The free radical scavenging activity of α-tocopherol is derived from a chromanol ring
structure (Smirnoff, 2005). The synthetic vitamin E is produced by a chemical reaction of
trimethylhydroquinone (TMHQ) with isophytol (Bonrath et al., 2006). TMHQ has two
hydroxyl groups on its ring structure whereas α-tocopherol has one hydroxyl group on its
chromanol ring structure (Fig. 4). In this research, TMHQ and a transition metal as an
oxygen scavenger was also investigated (Fig. 5). In addition, the effect of moisture on
oxygen scavenging also investigated. The oxygen content (%) reduction on batch TM-T-
R-H was significantly higher than that on batch TM-T-R until day 14. TMHQ with
moisture showed better oxygen scavenging capability than one without moisture.
However, there were no significant differences in oxygen scavenging capability between
batch TM-T-R and TM-T-R-H after day 30.
52
4.4. Conclusion
The oxygen content (%) in the headspace of all batches decreased during storage
time. α-tocopherol and transition metal were required for optimum oxygen scavenging
capability. Otherwise, the oxygen scavenging capability decreased. The oxygen
scavenging capability was increased when the amount of transition metal was increased.
Thermal processing can accelerate the oxygen scavenging reaction. Adding moisture did
not affect the oxygen scavenging capability.
Generally, the scavenging capacity can be as low as 1 cc O2 per gram and the
scavenging rate can be 0.1 cc O2 per gram per day (Jerdee et al., 2003, Speer et al., 1994).
In this research, the oxygen scavenging capacity and rate were calculated by initial
oxygen content and oxygen content at day 60. α-tocopherol and transition metal had 6.72
cc O2 per gram of oxygen scavenging capacity and 0.11 cc O2 per gram per day of
oxygen scavenging rate. In addition, TMHQ was tested instead of α-tocopherol in the
oxygen scavenger mixture. This scavenging mixture had 4.86 cc O2 per gram of oxygen
scavenging capacity and 0.10 cc O2 per gram per day of oxygen scavenging rate. These
results demonstrate that the oxygen scavenger mixture containing α-tocopherol or TMHQ
and transition metal with thermal processing can be used as an oxygen scavenger. TMHQ
is somewhat less effective than α-tocopherol.
53
4.5. References

Al-Malaika, S., Ashley, H., Issenhuth, S., 1994, The antioxidant role of α-tocopherol in
polymers. I. The nature of transformation products of α-tocopherol formed during
melt processing of LDPE. Journal of Polymer science:
Part A: Polymer chemistry. 32, 3099-3113
Al-Malaika, Goodwin, C., Issenhuth, S., Burdick, D., 1999. The antioxidant role of
α-tocopherol in polymers II. Melt stabilizing effect in polypropylene.
Polymer degradation and stability. 64, 145-156
Anklam, E., Gaglione, S., Muller, A., 1997. Oxidation behavior of vanillin in dairy
products. Food Chemistry. 60, 43-51
Bagchi, K., Puri, S., 1998. Free radicals and antioxidants in health and disease.
Eastern Mediterranean Health Journal. 4, 350-360
Bonrath, W., Haas, A., Hoppmann, S., Netsher, T., Pauling, H., 2006.
Manufacture of alpha-tocopherol. US patent 7,153,984
Brody, A. L., Strpinsky E.P., Kline, L. R., 2001, Active food packaging for food
application, CRC Press, New York, USA, pp 11-30
Brown, H. and Williams, J., 2003, Packaged product quality and shelf life, in : Coles, R.,
Mcdowell, D., Kirwan, M.J. (Eds), Food packaging technology, CRC Press,
Choe, E., Min, D. B., 2006. Chemistry and reactions of reactive oxygen species in foods.
Critical Reviews in food science and nutrition. 46, 1-22
Day, B. P. F., 2003, Active packaging, in : Coles, R., Mcdowell, D., Kirwan, M.J. (Eds),
Food packaging technology, CRC Press, New York, USA, pp 282-302
54
Hamilton, R. J., Kalu, C., Prisk, E., Padley, F. B., Pierce, H., 1997.
Chemistry of free radicals in lipids. Food Chemistry. 60, 193-199
Jerdee, G. D., Leonard, J. P., Ching, T. Y., Goodrich, J. L., Rodgers, B. D.,
Schmidt, R. P., 2003. Oxygen scavenging packaging.US patent 6,569,506
Lacoste, A., Schaich, K. M., Zumbrunnen, D., Yam, K. L., 2005, Advancing
controlled release packaging through smart blending. Packaging Technology
and Science. 18, 77-87
Morales-Aizpurua I. C., Tenuta-Filho, A., 2005. Oxidation of cholesterol in

mayonnaise during storage. 89. 611-615
Rooney, M. L., 1995. Active packaging in polymer film, in : Rooney, M. L. (Eds),

Active food packaging, Blackie academic and professional, New York, USA,
pp 74-110
Siro, I., Fenyvesi, E., Szente, L., Meulenaer, B. D., Devlieghere, F., Orgovanyi, J.,
Senyi, J., Barta, J., 2006. Release of alpha-tocopherol from antioxidative low
density polyethylene film into fatty food simulant: influence of complexation
in beta-cyclodextrin. Food additives and contaminants. 23, 845-853
Smirnoff, N., 2005. Ascorbate, tocopherol and carotenoids: metabolism, pathway

engineering and functions, in : Smirnoff, N. (Eds), Antioxidants and
Reactive oxygen species in plants. Blackwell publishing, New York, USA,
pp 53-86
Speer, D. V., Morgan, C. R., Roberts, W. P., Vanputte, A. W., 1994. Multilayer
structure for a package for scavenging oxygen. US patent 5,350,622
Teumac, F. N., Zenner, B. D., Ross, B. A., Dearduff, L. A., Rassouli, M. R., 2004.
Metal catalyzed ascorbate compounds as oxygen scavengers. US patent 6,709,724
Vermeiren, L., Devlieghere, F., Beest, M., Kruijf, N., Debevere, J., 1999, Developments
in the active packaging of foods. Trends in food science and technology. 10,
77-86
55
Wessling, C., Nielsen, T., Leufven, A., 2000. The influence of α-tocopherol
concentration on the stability of linoleic acid and the properties of low density
polyethylene. Packaging technology and science. 13, 19-28
56
Table 4.1. Batch compositions used for oxygen scavenging mixtures
Code α-tocopherol Transition metal Thermal Processing Water

(A, mg) (T, mg) (R, ℉) (H, ul)
A-T-R 500 100 170 -
T-R - 100 170 -
A-R 500 - 170 -
A-T 500 100 - -
A-T-R-H 500 100 170 50
A-T0-R 500 - 170 -
A-T50-R 500 50 170 -
A-T100-R 500 100 170 -
A-T150-R 500 150 170 -
Code TMHQ Transition metal Thermal Processing Water

(TM, mg) (T, mg) (R, ℉) (H, ul)
TM-T-R 500 100 170 -
TM-T-R-H 500 100 170 50

a
A: α-tocopherol, T: Transition metal, R: Thermal Processing, H: Water
b
T0: 0 mg of transition metal, T50: 50 mg of transition metal, T100: 100 mg of transition
metal, T150: 150 mg of transition metal

c
TM: Trimethylhydroquinone (TMHQ)
57
Table 4.2. Oxygen content (%) of different scavenging mixtures during storage time
Day 0 Day 1 Day 4 Day 7 Day 14 Day 30 Day 60

A-T-R 20.90± 20.37± 20.20± 19.97± 19.93± 19.17± 18.03±
0.00a 0.06b 0.00bc 0.12cd 0.32d 0.06e 0.06f

T-R 20.90± 20.40± 20.90± 20.47± 20.47± 19.83± 18.83±
0.00a 0.10b 0.00a 0.06b 0.06b 0.29c 0.15d

A-R 20.90± 20.47± 20.53± 20.43± 20.43± 20.40± 20.43±
0.00a 0.06bc 0.06b 0.06bc 0.06bc 0.00c 0.12bc

A-T 20.90± 20.90± 20.90± 20.47± 20.23± 19.63± 18.40±
0.00a 0.00a 0.00a 0.06ab 0.15b 0.12c 0.70d

A-T-R-H 20.90± 20.33± 20.13± 19.90± 19.60± 18.53± 17.83±
0.00a 0.12b 0.15b 0.26bc 0.52c 0.12d 0.35e

A-T0-R 20.90± 20.47± 20.53± 20.43± 20.43± 20.40± 20.43±
0.00a 0.06bc 0.06b 0.06bc 0.06bc 0.00c 0.12bc

A-T50-R 20.90± 20.40± 20.33± 20.20± 20.03± 19.23± 19.43±
0.00a 0.10ab 0.06ab 0.10ab 0.31bc 0.47d 0.90cd

A-T100-R 20.90± 20.37± 20.20± 19.97± 19.93± 19.17± 18.03±
0.00a 0.06b 0.00bc 0.12cd 0.32d 0.06e 0.06f

A-T150-R 20.90± 20.40± 20.20± 19.87± 19.33± 18.13± 17.10±
0.00a 0.00b 0.00b 0.06c 0.06d 0.29e 0.20f

TM-T-R 20.90± 20.37± 20.30± 20.37± 20.37± 20.00± 19.57±
0.00a 0.15b 0.10b 0.15b 0.15b 0.17c 0.15d

TM-T-R-H 20.90± 19.60± 18.67± 18.30± 18.03± 18.30± 18.37±
0.00a 0.26ab 0.60b 0.46b 0.58b 1.47b 2.14b

a
Results are expressed as the mean ± SD (n=3)
b
a-f: The different letters within same row differ significantly (p < 0.05)
58
21.5
aaaa a aa
21.0 b
bb b c
aaa a a
ab a a
20.5 b
Oxygen content (%)
b b
20.0 b
19.5 c A-T-R
b
19.0
bc T-R
18.5 c
A-T
18.0
17.5 A-R
17.0
16.5
0 1 4 7 14 30 60
Storage time (day)
Figure 4.1. Influences of the α-tocopherol, thermal processing, and transition metal on
oxygen scavenging capability (p < 0.05)
59
A-T-R A-T-R-H
21.50
21.00
Oxygen content (%)
20.50
20.00
19.50
19.00
18.50
18.00
17.50
17.00
0 10 20 30 40 50 60 70
Storage time (day)
Figure 4.2. Influence of moisture on oxygen scavenging capability
60
25.00
aaaa aaaa abcc a b
abbc ab c a a
bb b
20.00 c c
Oxygen content (%)
15.00 A-T0-R
A-T50-R
10.00
A-T100-R
5.00 A-T150-R
0.00
0 1 4 7 14 30 60
Storage time (day)
Figure 4.3. Influence of the amount of transition metal on oxygen scavenging capability
(p < 0.05)
61
α-tocopherol
Trimethylhydroquinone (TMHQ)
Figure 4.4. Structure of α-tocopherol and trimethylhydroquinone (TMHQ)
62
TM-T-R TM-T-R-H
22.00
21.00
Oxygen content (%)
20.00
19.00
18.00
17.00
16.00
15.00
0 10 20 30 40 50 60 70
Storage time (day)
Figure 4.5. Influence of moisture on oxygen scavenging capability of TMHQ and
transition metal
63
(a) Oxygen scavenging reaction
(b) Summarized oxygen scavenging reaction
Initiation step
Oxygen + Transition metal
Thermal processing
Oxygen free radical
Scavenging step
α-tocopherol + Oxygen free radical
Dimer or tocopheryl qunione
Scheme 4.1. Oxygen scavenging reaction
64
CHAPTER FIVE
PREPARATION AND CHARACTERIZATION OF ALPHA-TOCOPHEROL-

LOADED NANOPARTICLES BY O/W EMULSION SOLVENT
EVAPORATION WITH ULTRASONIFICATION
Abstract
α-tocopherol-loaded poly ε-caprolactone (PCL) nanoparticles were prepared by
emulsion solvent evaporation with ultrasonification technique. The influences of PCL
concentration (3 and 5%), solvent in the oil phase (methylene chloride (DCM) and
methylene chloride: acetonitrile = 50:50 (DCM: ACN)), and ultrasonification time (1, 2,
3 minute) were investigated. The statistical evaluation of the results showed that
encapsulation efficiency was decreased when ultrasonification time was increased from 1
to 3 minute. DCM as a solvent in the oil phase and 5% PCL showed better encapsulation
efficiency than DCM: ACN and 3% PCL, respectively. Particle mean size was decreased
when ultrasonification time was increased from 1 to 3 minute. Nanoparticles with DCM
as a solvent in the oil phase had larger particle mean size than the particle with DCM:
ACN. There were no significant differences in particle mean size between two PCL
concentrations. PCL with 3% concentration had higher α-tocopherol loading (%) than 5%
PCL. There were no significant differences in α-tocopherol loading (%) with different
ultrasonification times or with different solvent in the oil phase. Overall, 5% PCL in
DCM as solvent in the oil phase with 3 minute ultrasonification time showed the best
encapsulation formulation.
65
5.1. Introduction
Free radicals or, more generally, reactive oxygen species (ROS) are products of
normal cellular metabolism. They are the molecules or molecular fragments containing
unpaired electrons. The unpaired electron usually gives a considerable degree of
reactivity to the free radical. ROS are well recognized for playing a dual role as both
deleterious and beneficial species. The harmful effect of free radicals occurs in biological
systems when there is an overproduction of ROS on one side and a deficiency of
enzymatic and non-enzymatic antioxidants on the other. Overproduction of ROS can be
an important mediator of damage to cell structures, including lipids and membranes,
proteins, and DNA (Valko et al., 2007).
Vitamin E has a fundamental role in the normal metabolism of all cells. It has
biological antioxidant activity capable of terminating chain reactions and it can
chemically prevent lipid oxidation (Comb, 2008). α-tocopherol is the most abundant
lipid-soluble, chain-breaking antioxidant and it is the most biologically active form of
vitamin E compounds. The free radical scavenging reactivity of the four tocopherols has
been measured, with the order of α-tocopherol > γ-tocopherol > β-tocopherol > δ-
tocopherol (Lien et al., 1999). α-tocopherol can also act as an efficient scavenger of
singlet oxygen (1O2). It has been shown that α-tocopherol scavenges 1

O2 by a
combination of physical quenching and chemical reaction. The chemical reactivity with
singlet molecular oxygen has been found, with the order of α-tocopherol > β-tocopherol >
γ-tocopherol > δ-tocopherol (Mukai et al., 1991). The physical quenching ability of
singlet molecular oxygen has been measured, with the order of α-tocopherol >> β-
66
tocopherol > γ-tocopherol > δ-tocopherol (Kaiser et al., 1990). The general trend of
biological activity has been reported as follows: α-tocopherol > β-tocopherol > γ-
tocopherol > δ-tocopherol (Abidi, 2000).
Many methods have been developed for preparing nanoparticles. Commonly used
methods of preparing nanoparticles from biodegradable polymers include emulsion
solvent evaporation (Scholes et al., 1993, Davada and Labhasetwar, 2002, Sahoo et al.,
2002), nanoprecipitation (Fessi et al., 1989, Govender et al., 1999, Leo et al., 2004),
salting out procedure (Allemann et al., 1992, Konan et al., 2002), and a combined method
(Mccarron et al., 2006). The differences between emulsion solvent evaporation and
nanoprecipitation is that the main phases in the emulsion solvent evaporation stay
immiscible at all times, only to be removed later by evaporation. Emulsion solvent
evaporation method involves two steps. The first step requires emulsification of the
polymer solution into an aqueous phase. During the second step, the solvent used in the
polymer solvent is evaporated, inducing polymer precipitation as nanospheres (Reis et al.,
2006).
In this research, poly ε-caprolactone (PCL) was used to encapsulate α-tocopherol
within nanospheres. PCL is semi-crystalline biodegradable and biocompatible polyester
with low glass transition temperature and melting point (Pitt, 1990). It has been
investigated for drug delivery for several years and it is non-toxic and non-mutagenic.
Moreover, it is considerably lower cost than other biodegradable polyesters such as
polyglycolide, polylactide, and their copolymers.
67
The aim of this work was to entrap α-tocopherol within PCL nanoparticles by O/W
emulsion solvent evaporation with ultrasonification method and to optimize the
encapsulation formulation. To achieve this goal, this study was designed to assess the
influence of formulation variables on the characteristics of nanoparticles such as
encapsulation efficiency, α-tocopherol loading, particle size, zeta potential, and
morphology. The formulation variables were as follows: (1) concentration of PCL; (2)
solvent in the oil phase; (3) ultrasonification time.
68
5.2.1. Materials
Poly ε-caprolactone (PCL, Mn 42,500 and Mw 65,000) was purchased from Sigma-
Aldrich (MO, USA). Polyvinyl alcohol hydrolyzed 88% (PVA, Mw 22,000) was
purchased from Acros organics (NJ, USA). Methylene chloride (DCM), methanol, and
acetonitrile (ACN), all HPLC grade were purchased from J.T.Baker (USA). Phosphate
Buffered Saline (PBS, 10x liquid concentrate) was purchased from EMD Bioscience
(CA.USA).
5.2.2. Preparation of α-tocopherol-loaded PCL nanoparticle
In this study, α-tocopherol-loaded PCL nanoparticles were prepared by emulsion
solvent evaporation method. Three factors were statistically examined, PCL
concentration, solvent in the oil phase, and ultrasonification time.
5.2.3. Formulation of nanoparticles containing α-tocopherol
Nanoparticles were prepared using an oil-in-water emulsion solvent evaporation with
ultrasonification technique. In this procedure, specific amount of PCL (300 or 500 mg)
was dissolved in 10 mL of solvent (methylene chloride or methylene chloride:
acetonitrile = 50: 50) containing 10mg of α-tocopherol. A 2% PVA (w/v) solution was
prepared in PBS solution. The PCL solution was added to 40 mL of the PVA solution.
The total mixture was then placed in an ice bath and emulsified using a Branson Digital
69
Sonifier (model 250, Connecticut USA) with 55 W of energy output for a specific time (1,
2, 3 minute) to obtain an oil-in-water emulsion. Another 40 mL of the PVA solution was
then added to the emulsion. The final solution was stirred for 12 hour on a magnetic stir
plate to allow the evaporation of methylene chloride and acetonitrile and to allow the
formation of the nanoparticles. The suspension was then centrifuged at 12000 rpm for 20
minute. The pellet was resuspended in distilled water and centrifuged three more times at
3000 rpm for 20 minute each. These washing steps were performed to remove
unencapsulated PVA and α-tocopherol. The nanoparticles were collected and frozen at -
80oC for at least 2 hour and subsequently freeze dried for 2 days. The freeze dried
nanoparticles were stored at 4oC.
5.2.4. α-tocopherol encapsulation efficiency (%) and loading (%)
Encapsulation efficiency was determined by an extraction method. Dried
nanoparticles (10 mg) were dissolved in 5 mL of methylene chloride and 5 mL of
distilled water. The mixture was vigorously vortexed for 1 minute and sonicated 5 minute
in order to extract the α-tocopherol into the organic solution. Then, methylene chloride
was evaporated and replaced with methanol. The 2 mL of organic solution was filtered
and the α-tocopherol content of the solution was analyzed by HPLC (Waters 1525 Binary
HPLC pump, USA).
The experiment was performed in triplicate and the encapsulation efficiency was
calculated using the ratio of the mass of α-tocopherol determined analytically to the mass
of α-tocopherol added during the formation process, as shown in Equation 1. The α-
70
tocopherol loading (%) was calculated using the ratio of the mass of α-tocopherol
determined to the mass of total nanoparticle, as shown in Equation 2.
-.// 01 α 2030456708 96267:;<69 :=

Encapsulation ef)iciency % , > 100 (Equation 1)
-.// 01 α 2030456708 .9969 :=
-.// 01 α 2030456708 96267:;<69 :=

α ! tocopherol loading % ,
-.// 01 202.8 <.<04.72;386 :=
> 100 (Equation 2)
5.2.5. Particle mean size and zeta potential
A dilute suspension of nanoparticles was prepared in distilled water. Particle mean
size and size distribution were determined by Zetasizer (Nano-ZS, Malvern Instrumet,
UK). The surface charges on nanoparticles were also examined by measuring their zeta
potentials using the Zetasizer.
5.2.6. Scanning electron microscopy
The morphology of the nanoparticles was examined by scanning electron microscopy
(S-4800 UHR FE-SEM, Hitachi high technologies America, Inc.). Surfaces were
prepared using platinum coating.
71
Statistics on a completely randomized block design were performed with the analysis
of variance (ANOVA) using SAS (version 9.1, SAS Institute Inc., Cary, NC, USA).
Differences among mean values were processed by Duncan’s multiple range tests and the
least significant difference (LSD). Significance was defined at a level of P < 0.05.
72
5.3. Results
5.3.1. Morphology of PCL nanoparticle
The shape of nanoparticles was spherical and regular as visualized in the SEM
photographs (Figure 1-4). There were agglomerates of fragments when DCM: ACN was
used as a solvent in the oil phase, especially at 3 minute ultrasonification time.
5.3.2. Encapsulation efficiency (%)
Encapsulation efficiency ranged from 25 to 96% depending on the different factors
(Table 2). 5M1 and 5MA1 showed the best encapsulation efficiencies (96 and 95%,
respectively) and 5M2 and 5M3 showed around 90% of encapsulation efficiency. Other
formulations showed less than 80% of encapsulation efficiency.
The effects of PCL concentration, solvent in the oil phase, and ultrasonification time
on encapsulation efficiency were evaluated (Fig. 5). It was observed that all three
variables significantly affected the encapsulation efficiency. The encapsulation efficiency
of nanoparticles with 5% PCL was higher than that of nanoparticles with 3% PCL.
Higher encapsulation efficiency also occurred when DCM was used as the solvent in the
oil phase. The encapsulation efficiency was decreased when ultrasonification time
increased from 1 to 3 minute.
73
5.3.3. α-tocopherol loading (%)
α-tocopherol loading (%) in the nanoparticles ranged from 2.12 to 3.51% (Table 2). It
was observed that α-tocopherol loading (%) of 5M3 was higher than that of 5M1, 5M2,
and 5MA1. The effects of PCL concentration, solvent in the oil phase, and
ultrasonification time on α-tocopherol loading (%) were also evaluated (Fig. 6). It was
observed that there were significant differences in α-tocopherol loading (%) between two
PCL concentrations. Particularly, the loading (%) in nanoparticles with 3% PCL was
higher than that with 5% PCL. There were no significant differences in α-tocopherol
loading (%) with different ultrasonification time or with different solvent in the oil phase.
5.3.4. Particle mean size, zeta potential, and polydispersity
In this research, less than 500 nm of particle mean size was considered as an
adequate particle mean size. Polydispersity is the information about the size distribution
of nanoparticles and is ranged from 0 (good size distribution) to 1 (poor size distribution).
Thus, more than 0.5 polydispersity was considered as a poor size distribution in this
research. Particle mean size ranged from 247 to 1070 nm (Table 2). 5M3 had 369 nm
particle mean size with 0.27 polydispersity and 5M2 had 448 nm particle mean size with
0.40 polydispersity. On the other hand, 5M1 and 5MA1 had particle mean size above 700
nm with 0.60 polydispersity. Therefore, 5M1 and 5MA1 showed inadequate size
distribution. Zeta potential is the information about the surface charge of nanoparticles.
All the nanoparticles from all formulations showed negative zeta potential in this research.
74
The effects of PCL concentration, solvent in the oil phase, and ultrasonification time
on particle mean size were also evaluated (Fig. 7). There were no significant differences
in particle mean size between two PCL concentrations. However, the other factors such
as solvent in the oil phase and ultrasonification time exhibited significant effect on the
particle mean size of nanoparticle. Particularly, DCM: ACN as the solvent in the oil
phase showed smaller particle mean size than DCM as the solvent in oil phase. Particle
mean size was decreased when ultrasonification time was increased from 1 to 3 minute.
75
5.4. Discussion
The solvent in the oil phase dissolves into the aqueous phase and then, evaporates in
the O/W emulsion solvent evaporation method. The extent and speed of solvent transfer
from the oil phase into the aqueous phase depends on the solubility. The solvent
composition can be a key factor in controlling the solvent removal rate and the final size
of the microspheres (Maia et al, 2004). In this study, the particle mean size of the
nanoparticles prepared by DCM: ACN as the solvent in the oil phase was smaller than
that prepared by DCM only (Fig. 7). The water-miscible solvent quickly diffused out of
the polymer solution. Then, DCM was removed leading to hardening of the capsule
(Luong-Van et al, 2007). The water solubility of ACN was greater than DCM and
therefore the rate of precipitation was higher than the rate with DCM. Due to the high
water solubility of ACN, it had higher diffusion rate before hardening. This was the
major reason for the smaller particle mean size prepared by mixture of DCM and ACN
(Kim et al, 2005). In this study, encapsulation efficiency was increased when DCM was
used as the solvent in the oil phase (Fig. 5). By increasing the particle mean size, the α-
tocopherol diffusion into the aqueous solution decreased because there was a longer
distance to α-tocopherol travel. Consequently, higher encapsulation efficiency was
associated with an increase in the particle mean size. There were no significant
differences in α-tocopherol loading (%) between the two solvents (Fig. 6).
Polymer concentration is also a key factor. In this study, encapsulation efficiency was
increased by increasing PCL concentration from 3 to 5% (Fig. 5). By increasing the
polymer concentration in the organic phase, the viscosity of the solution was increased
76
(Ito et al, 2007). Increasing viscosity can decrease the α-tocopherol diffusion into the
aqueous phase and thus increase the α-tocopherol incorporation into the nanoparticles
(Song et al, 2008). Consequently, the encapsulation efficiency of nanoparticles was
increased by increasing the polymer concentration. Conversely, α-tocopherol loading (%)
was decreased by increasing PCL concentration (Fig. 6). Increasing viscosity increased
the total mass of PCL in the nanoparticles. This decreased the ratio of α-tocopherol to the
total mass of nanoparticles, thus decreasing α-tocopherol loading (%). It was observed
that there were no significant differences in particle mean size between two PCL
concentrations (Fig. 7). The solvent in the oil phase and ultrasonification time more
significantly affected particle mean size than PCL concentration.
Ultrasonification time is another key factor. In this study, particle mean size was
decreased by increasing ultrasonification time from 1 to 3 minute (Fig. 7). The increased
time of ultrasonification led to the formation of smaller nanoparticles. It was also
observed that encapsulation efficiency was decreased by increasing ultrasonification time
from 1 to 3 minute (Fig. 5). Increasing the ultrasonification time resulted in a reduction in
the encapsulation efficiency due to the decreasing particle mean size (Song et al, 2008).
Again, encapsulation efficiency is associated with the particle mean size. There were no
significant differences in α-tocopherol loading (%) with respect to ultrasonification time
(Fig. 6).
Only four formulations showed over 90% of encapsulation efficiency; 5M1, 5M2,
5M3, and 5MA1 (Table 2). However, 5M1 and 5MA1 showed poor polydispersity, 0.59
77
and 0.60, respectively. 5M3 showed relatively higher loading (%), smaller particle mean
size, and better polydispersity than other formulations.
In conclusion, α-tocopherol-loaded PCL nanoparticles were prepared by O/W
emulsion solvent evaporation with ultrasonification technique. It has been shown that
PCL concentration, solvent in the oil phase, and ultrasonification time all significantly
affected the encapsulation efficiency (%). In contrast, solvent in the oil phase and
ultrasonification time did not significantly affect α-tocopherol loading (%) and PCL
concentration did not significantly affect particle mean size. Overall, 5% PCL in DCM as
the solvent in the oil phase with 3 minute ultrasonification time showed good
encapsulation efficiency (%), smaller particle mean size with good polydispersity, well-
shaped particle, and high α-tocopherol loading (%). Due to these results, this formulation
was selected for further research.
78
5.5. References
Abidi, S.L. 2000. Chromatographic analysis of tocol-derived lipid antioxidants.

J. Chromatogr. a. 881, 197-216
Allemann, E., Gurny, R., Doelker, E., 1992. Preparation of aqueous polymeric
nanodispersions by a reversible salting out process, influence of process
parameters on particles size. Int. J. Pharm. 87, 247-253
Combs, G. F., The vitamins: Fundamental aspects in nutrition and health. 3rd Ed.
Elsevier academic press, London, UK, pp. 181-212
Davada, J. and Labhasetwar, V., 2002. Characterization of nanoparticle uptake

by endothelial cells. Int. J. Pharm. 233, 51-59
Fessi, H., Puiseux, F., Devissaguet, J.P, Ammoury, N. and Benita, S., 1989.
Nanocapsule formation by interfacial polymer deposition following
solvent displacement. Int. J. Pharm. 55, R1-R4
Govender, T., Stolnik, S., Garnett, M. C., Illum, L., Davis, S. S., 1999. PLGA
nanoparticles prepared by nanoprecipitation: drug loading and release studies of
a water soluble drug. J. Control. Release. 57, 171-185
Ito, F., Fujimori, H., Makino, K., 2007. Incorporation of water-soluble drugs in PLGA
microspheres. Colloids Surf. B: Biointerfaces. 54, 173-178
Kaiser, S., Di Masico, P., Murphy, M.E., Sies, H., 1990. Physical and chemical
scavenging of singlet molecular oxygen by tocopherols,
Arch. Biochem. Biophys. 277, 101-108
Kim, B. K., Hwang, S. J., Park, J. B., Park, H. J., 2005. Characteristics of felodipine-
located poly (ε-caprolactone) microspheres. J. Microencapsul. 22, 193-203
Konan, Y. N., Gurny, R., Allemann, E., 2002. Preparation and characterization of sterile
and freeze-dried sub-200 nm nanoparticles. Int. J. Pharm. 233, 239-252
79
Leo, E., Brina, B., Forni, F., Vandelli, M. A., 2004. In vitro evaluation of PLA
nanoparticles containing a lipophilic drug in water-soluble or insoluble form.
Int. J. Pharm. 278, 133-141
Lien, E., Ren, S., Bui, H., Wang, R., 1999. Quantitative structure-activity relationship
analysis of phenolic antioxidants. Free Radic. Biol. Med. 26, 285-294
Luong-Van, E., Grondahl, L., Nurcombe, V., Cool, S., 2007. In vitro biocompatibility
and bioactivity of microencapsulated heparan sulfate.
Biomaterials. 28, 2127-2136
Maia, J. L., Santana, M. H. A., M.I.R., 2004. The effect of some processing conditions on
the characteristics of biodegradable microspheres obtained by an emulsion solvent
evaporation process. Braz. J. Chem. Eng. 21, 1-12
Mccarron, P. A., Donnelly, R. F., Marouf, W., 2006. Celecoxib-loaded poly(D,L-lactide-

co-glycolide) nanoparticles prepared using a novel and controllable combination
of diffusion and emulsification steps as part of the salting-out procedure.
J. Microencapsul. 23, 480-498
Mukai, K., Daifuku, K., Okabe, K., Tanigaki, T., and Inoue, K., 1991. Structure-
activity relationship in the quenching reaction of singlet oxygen by tocopherol
(Vitamin E) derivatives and related phenols. Finding of linear correlation between
rates of quenching of singlet oxygen and scavenging of peroxyl and phenoxyl
radicals in solution. J. Org. Chem. 56, 4188-4192
Pitt, C.G., 1990. Poly ε-caprolactone and its copolymer. In: Chasin, M., Lange, R. (Eds),
Biodegradable polymers as drug delivery systems, Marcel Dekker, New York,
pp. 71-120
Reis, C. P., Neufeld, R. J., Ribeiro, A. J., Veiga, F., 2006. Nanoencapsulation 1. Methods
for preparation of drug-loaded polymeric nanoparticles. Nanomedicine:
Nanotech. Biol. Med. 2, 8-21
Sahoo, S. K., Panyam, J., Prabha, S., Labhasetwar, V., 2002. Residual polyvinyl alcohol
associated with poly (D, L-lactide-co-glycolide) nanoparticles affects their
physical properties and cellular uptake. J. Control. Release. 82, 105-114
80
Scholes, P. D., Coombes, A. G. A., Illum, L., Davis, S. S., Vert, M., Davies, M. C., 1993.
The preparation of sub-200nm poly(lactide-co-glycolide) microspheres for
site-specific drug delivery. J. Control. Release. 25, 145-153
Song, X., Zhao, Y., Wu, W., Bi, Y., Cai, Z., Chen, Q., Li, Y., Hou, S., 2008.
PLGA nanoparticle simultaneously loaded with vincristine sulfate and verapamil
hydrochloride: systematic study of particle size and drug entrapment efficiency.
Int. J. Pharm. 350, 320-329
Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., Telser, J., 2007.
Free radicals and antioxidants in normal physiological functions and human
disease. Int. J. Biochem. Cell Biol. 39, 44-84
81
Table 5.1. Batch compositions used for constituting α-tocopherol–loaded nanoparticle
Code PCL concentration Solvent in oil phase Ultrasonification
(%) time (min)
3M1 3 DCM 1
3M2 3 DCM 2
3M3 3 DCM 3
5M1 5 DCM 1
5M2 5 DCM 2
5M3 5 DCM 3
3MA1 3 DCM : ACN 1
3MA2 3 DCM : ACN 2
3MA3 3 DCM : ACN 3
5MA1 5 DCM : ACN 1
5MA2 5 DCM : ACN 2
5MA3 5 DCM : ACN 3

a
DCM: Methylene chloride, ACN: Acetonitrile
b
DCM: ACN; DCM: ACN = 50: 50
82
Table 5.2. Properties of α-tocopherol–loaded nanoparticle
Code Encapsulation α-tocopherol Total amount Particle Polydispersity Zeta

efficiency (%) loading (%) of particle mean size (PI) potential
(mg) (nm) (mV)
3M1
79.57±10.32bc 3.01±0.38ab 264.33±2.52d 1070 ±55.75a 0.70±0.04a -14.73±0.61g
3M2
65.22±6.78cd 3.21±0.22a 203.00±16.64fg 908.23±4.71b 0.70±0.01a -12.83±0.29f
3M3
54.53±5.22d 3.06±0.25a 178.33±2.89gh 370.23±14.35e 0.28±0.04ef -10.57±0.21bc
5M1
96.42±4.27a 2.26±0.14c 426.33±10.02a 767.83±46.30c 0.59±0.02b -11.97±0.49ef
5M2
90.72±14.15ab 2.12±0.42c 430.67±22.14a 448.87±0.85d 0.40±0.02c -11.77±0.81de
5M3
90.95±6.15ab 2.50±0.28bc 365.67±33.86b 368.03±3.47e 0.27±0.03ef -12.13±0.23ef
3MA1
79.65±7.89bc 3.51±0.35a 226.67±2.89ef 399.50±11.07e 0.37±0.01cd -10.87±0.72cd
3MA2
50.39±4.06d 3.20±0.20a 158.00±20.22h 316.20±3.92f 0.25±0.01f -9.83±0.12b
3MA3
24.91±1.83e 3.19±0.17a 78.33±7.64i 247.70±1.65g 0.10±0.04g -7.70±0.16a
5MA1
95.29±12.52a 2.41±0.49c 400.33±35.70a 733.17±27.80c 0.60±0.04b -16.23±0.74h
5MA2
63.18±9.54d 2.12±0.34c 298.67±13.58c 392.77±12.42e 0.33±0.02de -15.23±1.10gh
5MA3
59.41±9.08d 2.31±0.32c 257.33±5.03de 376.10±7.08e 0.29±0.08ef -15.70±0.26gh
a
b
a-i: The different letters within same row differ significantly (p < 0.05)
83
(a)
(b)
(c)
Figure 5.1. SEM images of nanoparticles (a) 3M1 (b) 3M2 (c) 3M3
84
(a)
(b)
(c)
Figure 5.2. SEM images of nanoparticles (a) 5M1 (b) 5M2 (c) 5M3
85
(a)
(b)
(c)
Figure 5.3. SEM images of nanoparticles (a) 3MA1 (b) 3MA2 (c) 3MA3
86
(a)
(b)
(c)
Figure 5.4. SEM images of nanoparticles (a) 5MA1 (b) 5MA2 (c) 5MA3
87
90 a
80
(a)
Encapsulation efficiency (%)

70
b
60
50
40
30
20
10
0
3% 5%
PCL (%)
90
80
a (b)
70 b
60
50
40
30
20
10
0
DCM DCM:Ace
Solvent in oil phase
100
90
a (c)
80
b
70
c
60
50
40
30
20
10
0
1 min 2 min 3 min
Ultrasonification time (min)
Figure 5.5. Effects of (a) PCL concentration, (b) solvent in the oil phase, and (c)
ultrasonification time on encapsulation efficiency (n=18 and LSD = 7.61 for PCL
concentration and solvent in the oil phase, n=12 and LSD = 9.32 for ultrasonification
time, p<0.05).
88
3.5 a
(a)
3
2.5 b
Loading (%)
2
1.5
0.5
0
3% 5%
PCL (%)
3 a
a (b)
2.5
2
Loading (%)
1.5
0.5
0
DCM DCM:Ace
3 a a
(c) a
2.5
2
Loading (%)
1.5
0.5
0
1 min 2 min 3 min
ultrasonification time on loading (n=18 and LSD =0.2106 for PCL concentration and
solvent in the oil phase, n=12 and LSD = 0.2579 for ultrasonification time, p<0.05).
89
600 a
(a) a
500
Particle mean size (nm)

400
300
200
100
0
3% 5%
PCL (%)
700 a
(b)
600
500
b
400
300
200
100
0
DCM DCM:Ace
800 a
(c)
700
600
b
500
400 c
300
200
100
0
1 min 2 min 3 min
ultrasonification time on particle mean size (n=18 and LSD =110.15 for PCL
concentration and solvent in the oil phase, n=12 and LSD =134.91 for ultrasonification
time, p<0.05).
90
CHAPTER SIX
ALPHA-TOCOPHEROL-LOADED NANOPARTICLES
AND TRANSITION METAL AS A HEAT ACTIVATED
OXYGEN SCAVENGER
Abstract
α-tocopherol-loaded nanoparticles and a transition metal, iron, as a heat activated
oxygen scavenger were evaluated in this study. The influences of transition metal,
moisture, and thermal processing on oxygen scavenging capability were investigated. The
initial oxygen content (%) in the cup headspace was 20.90% and was decreased to
20.37% after thermal processing and 30 days of storage when the oxygen scavenger
mixture contained α-tocopherol-loaded nanoparticles, moisture, and a transition metal.
Furthermore, the oxygen content (%) in the cup headspace was decreased to 19.50%
when moisture was eliminated from the mixture and was decreased to 20.13% when the
amount of transition metal increased from 100 to 150 mg. α-tocopherol-loaded
nanoparticles and a transition metal in the oxygen scavenger mixture with thermal
processing had 6.44 cc O2 per gram of oxygen scavenging capacity and 0.21 cc O2 per
gram per day of oxygen scavenging rate. Results showed that α-tocopherol-loaded
nanoparticles and a transition metal in the oxygen scavenger mixture with thermal
processing had the best oxygen scavenging capability.
91
6.1. Introduction
During the past 30 years, oxygen scavengers in packaging have been investigated
(Farrell and Tsai, 1984, Foltynowicz et al., 2002, Klein and Knorr, 1990, Speer et al.,
1994). Oxygen scavengers can prevent microbial growth, development of off-flavors,
color and flavor changes, and nutritional losses. The most powerful and effective oxygen
scavenger is based on iron oxidation (Rooney, 1995). Iron powder is packaged in a small
sachet to prevent direct contact with food. The sachet material is highly oxygen
permeable, so oxygen can permeate from the packaging headspace inside the sachet,
where it is then scavenged by iron powder (Ozdemir and Floros, 2004). However, there
are several disadvantages to the iron-based oxygen scavenger sachet. They cannot pass
metal detectors on a packaging line and they pose a potential risk of accidental ingestion
by consumers. In addition, it cannot be used for liquid products and it could leak. To
eliminate these problems, oxygen scavengers can be incorporated into the packaging
material (Vermeiren et al., 2003). Additionally, a more uniform scavenging effect can be
achieved by incorporation of an oxygen scavenger into the packaging material.
Scavenging activation systems are needed on oxygen scavenging films to prevent the
reaction with atmospheric oxygen prior to use. Many patents have been issued utilizing
UV light as an activator to trigger oxygen scavenging reaction (Albert et al., 2004,
Schmidt et al., 2006, Speer, 2006, Yang et al., 2004). UV triggered oxygen scavenging
films are usually comprised of an oxygen scavenger, a transition metal catalyst, and a
photoinitiator. The rate of oxygen scavenging can be increased by the transition metal
catalyst. The photoinitiator enhances and facilitates the oxygen scavenging by the
92
packaging film upon exposure to UV light. However, there is a significant price increase
placed on oxygen scavenging films due to the high cost of photoinitiators.
Nanoencapsulation has been widely used in the medical and food industries.
Encapsulated materials can be protected from moisture, heat, light, and oxygen. Several
biodegradable polyesters, such as polyglycolide, polylactide, and polycaprolactone, are
used as wall materials. Polycaprolactone (PCL) is considerably cheaper than others and
has been investigated for drug delivery several years (Pitt, 1990).
Previous research has shown that α-tocopherol and a transition metal have oxygen
scavenging capability. Oxygen free radicals are produced in the presence of the transition
metal and these free radicals were eliminated by receiving electrons from α-tocopherol.
Therefore, the transition metal and α-tocopherol can scavenge oxygen by this successive
chemical reaction. Furthermore, thermal processing has been shown accelerate the
oxygen scavenging reaction.
In this research, α-tocopherol loaded PCL nanoparticles were prepared by emulsion
solvent evaporation. Then, an oxygen scavenging mixture including the α-tocopherol-
loaded nanoparticles and a transition metal were prepared. Breakage of the nanoparticles
may be achieved by the application of heat due to the low melting point of PCL (Albert et
al., 2004, Pitt, 1990). α-tocopherol may be released from the nanoparticles or oxygen free
radicals produced by transition metal can penetrate into the inside nanoparticles.
93
The aim of this study was to develop a heat triggered oxygen scavenger. The
influences of moisture, heat, and the amount of transition metal on oxygen scavenging
capability were also investigated.
94
6.2.1. Materials
Polycaprolactone (PCL, Mn 42,500 and Mw 65,000) was purchased from Sigma-Aldrich
(MO, USA). Polyvinyl alcohol hydrolyzed 88% (PVA, Mw 22,000) was purchased from
Acros organics (NJ, USA). Iron chloride (II), tetrahydrate was purchased from J.T.Baker
(NJ, USA). Retortable plastic cups with 3% EVOH barrier were provided from Printpack
(GA, USA). 48 ga PET/60 ga Foil/3 mil CPP film was used as cup lid. Methylene
chloride (DCM) with HPLC grade was purchased from J.T.Baker (USA). Phosphate
Buffered Saline (PBS, 10x liquid concentrate) was purchased from EMD Bioscience
(CA.USA).
ultrasonification technique. In this procedure, 300 mg of PCL was dissolved in 10 mL of
methylene chloride containing 10 mg of α-tocopherol. A 2% PVA (w/v) solution was
prepared in PBS solution. The PCL solution was added to 40 mL of the PVA solution.
Sonifier (model 250, Connecticut USA) with 55 W of energy output for 3 minute to
obtain an oil-in-water emulsion. An additional 40 mL of the PVA solution was added to
the emulsion. The final solution was stirred for 12 hour on a magnetic stir plate to allow
the evaporation of methylene chloride and to allow the formation of the nanoparticles.
95
The suspension was then centrifuged at 12000 rpm for 20 minute. The pellet was
resuspended in distilled water and centrifuged three more times at 3000 rpm for 20
minute each. The resulting nanoparticles were collected and frozen at -80oC for at least 2
hour and subsequently freeze dried for 2 days. The freeze dried nanoparticles were stored
at 4oC.
6.2.3. Sample preparation
α-tocopherol-loaded nanoparticles with differing amounts of transition metal (0, 25,
50, 100 mg) and with or without water were placed inside a high oxygen barrier
retortable cup with 115 cc of ambient air (20.90% O2). Then the cup was sealed with
PET/Foil/CPP film with a Lab sealer (Packaging Technologies, IA, USA). The sealing
temperature was 470oF with 60 psi pressure at 1 second dwell time.
6.2.4. Oxygen content analysis
each sample measurement. All of the samples were measured in triplicate at day 0, 1, 4,
7, 14, and 30.
96
97
6.3.1. Oxygen content (%) analysis during storage time
This study was designed to assess the influence of mixture variables on oxygen
scavenging capability of α-tocopherol-loaded nanoparticles and a transition metal. Eight
batches with different compositions of scavenger mixtures were prepared (Table 1). Ten
batches are shown on Table 1, but batch N-T-R-H and N-R-H were used twice with
different name of N-T50-R-H and N-T0-R-H, respectively. The oxygen content (%) in
the headspace of all batches except batch N decreased during the storage time (Table 2).
6.3.2. Effects of α-tocopherol-loaded nanoparticle, transition metal, moisture, and
thermal processing on oxygen scavenging
Transition metals react with oxygen and then produce oxygen free radicals (Bagchi
and Puri, 1998). However, the encapsulated α-tocopherol was prevented from reacting
with the oxygen free radicals by the wall of nanoparticles in this study. Without breakage
of the nanoparticles, there was a little opportunity for the oxygen free radicals to react
with α-tocopherol. Disruption of the nanoparticle was achieved by thermal processing in
this research. α-tocopherol could be released from the nanoparticle or the oxygen free
radicals could penetrate into the inside nanoparticle after thermal processing.
The effects of α-tocopherol-loaded nanoparticles, transition metal, and thermal
processing on the oxygen scavenging capability have been investigated (Fig 1). The
oxygen content (%) on batch N-T-R-H was significantly lower than the others after day
14. α-tocopherol-loaded nanoparticles and a transition metal in the scavenger mixture
98
with thermal processing reduced the oxygen in the headspace effectively. However, there
were no free radical productions on batch N-R-H owing to lacking of a transition metal,
no breakage of nanoparticles on batch N-T-H owing to lacking of thermal processing, and
no free radical scavenging reaction on batch T-R-H owing to lacking of α-tocopherol-
loaded nanoparticles. Due to those reasons, batch N-R-H, N-T-H, and T-R-H had higher
oxygen content (%) than batch N-T-R-H. α-tocopherol-loaded nanoparticles, a transition
metal, and thermal processing should be used altogether, because oxygen scavenging was
less effective without these components. There was no oxygen content reduction on batch
N over the entire storage time, thus showing that α-tocopherol-loaded nanoparticles
without transition metal, moisture, and thermal processing had no oxygen scavenging
effect at all. Therefore, the α-tocopherol-loaded nanoparticles may be stable for storage
under ambient conditions.
The effects of moisture on the oxygen scavenging capability have been investigated
(Fig 2). The oxygen content (%) of batch N-T-R was significantly lower than that of
batch N-T-R-H after day 14. Therefore, oxygen scavenging capability was lowered by
adding moisture into the oxygen scavenger mixture. α-tocopherol and PCL are
hydrophobic materials, so α-tocopherol may not diffuse through the nanoparticle if the
outside environment was hydrophilic, or it may not be totally released from PCL
nanoparticles. Therefore, there was less chance for oxygen free radicals to react with α-
tocopherol in the presence of moisture. However, oxygen scavenging capability of batch
N-T-R-H was still higher than that of batch N-R-H, N-T-H, and T-R-H at day 30 (Fig. 1).
99
This suggests that the oxygen scavenging mixture with moisture still can scavenge
oxygen well.
6.3.3. Effect of the amount of transition metal
The effect of the amount of transition metal in the scavenger mixture on oxygen
content (%) reduction has been investigated (Fig. 3). The oxygen content (%) in the
headspace was decreased when the amount of transition metal in the scavenger mixture
was increased from 0 to 150 mg during storage time. It was observed that batch N-T100-
R-H showed the highest oxygen scavenging capability. However, there was no significant
difference in oxygen content (%) between batch N-T25-R-H and N-T50-R-H at day 30.
100
6.4. Conclusion
α-tocopherol-loaded nanoparticles, transition metal, and thermal processing were
required for optimum oxygen scavenging capability. Otherwise, the oxygen scavenging
capability decreased. Transition metals and α-tocopherol-loaded nanoparticles are
required for producing oxygen free radicals and for eliminating the radicals, respectively.
In addition, thermal processing is required for disrupting the nanoparticle structure to
initiate the oxygen scavenging reaction. Therefore, heat is the activator which triggers the
oxygen scavenging reaction in this system. Oxygen scavenging capability increased as
the amount of transition metal increased. However, the addition of moisture lowered the
oxygen scavenging capability. α-tocopherol-loaded nanoparticles without transition metal,
moisture, and thermal processing had no oxygen scavenging effect. The α-tocopherol-
loaded nanoparticles may be stable for storage under ambient conditions.
Generally, the scavenging capacity can be as low as1 cc O2 per gram and the
scavenging rate can be 0.1 cc O2 per gram per day (Jerdee et al., 2003, Speer et al., 1994).
In this research, the oxygen scavenging capacity and rate were calculated by initial
oxygen content and oxygen content at day 30. α-tocopherol-loaded nanoparticle and
transition metal had 6.44 cc O2 per gram of oxygen scavenging capacity and 0.21 cc O2
per gram per day of oxygen scavenging rate. These results demonstrate that the oxygen
scavenger mixture containing α-tocopherol-loaded nanoparticle and a transition metal can
be used as a heat activated oxygen scavenger. This oxygen scavenger can be used in both
dry and wet conditions but it would be expected to be more effective if used with dry
products.
101
6.5. References

US patent 6,821,482
Bagchi, K., Puri, S., 1998. Free radicals and antioxidants in health and disease.
Eastern Mediterranean Health Journal. 4, 350-360
Farrell, C. J., Tsai, B. C., 1985. Oxygen scavenger. US patent 4,536,409
Foltynowicz, Z., Kozak, W., Fiedorow, R., 2002. Studies of oxygen uptake on
O2 scavengers prepared from different iron-containing parent substances.
Packaging Technology and Science, 15, 75-81
Klein, T., Knorr, D., 1990. Oxygen absorption properties of powdered iron.
Journal of Food Science, 55, 869-870
Labuza, T. P., 1987. Oxygen scavenger sachets. Food Research, 32. 276-277
Ozdemir, M., Floros, J. D., 2004. Active food packaging technologies.

Critical reviews in food science and nutrition, 44. 185-193
Pitt, C.G., 1990. Poly ε-caprolactone and its copolymer. In: Chasin, M., Lange, R. (Eds),
Biodegradable polymers as drug delivery systems, Marcel Dekker, New York,
pp. 71-120

pp 74-110
Schmidt, R. P., Solis, J. A., Ching, T. Y., 2006. Transition metal carboxylates as catalysts
for oxygen scavenging, US patent 7,052,628
102
Speer, D. V., Morgan, R., Roberts, W. P., VanPutte, A. W., 1994. Multilayer structure for
a package for scavenging oxygen. US patent 5,350,622
Speer, D. V., 2006. Photoinitiator blends for high speed triggering, US patent 7,153,891
Vermeiren, L., Heirlings, L., Devlieghere, F., Debevere, J. 2003. Oxygen, ethylene and
US patent 6,818,151
103
Table 6.1. Batch compositions used for oxygen scavenging mixtures
Nanoparticle Transition metal Thermal Water

Code
(mg) (mg) processing (℉) (µl)
N-T-R-H 200 50 170 50
N-R-H 200 - 170 50
N-T-H 200 50 - 50
T-R-H - 50 170 50
N-T-R 200 50 170 -
N 200 - - -
N-T0-R-H 200 - 170 50
N-T25-R-H 200 25 170 50
N-T50-R-H 200 50 170 50
N-T100-R-H 200 100 170 50

a
N: α-tocopherol-loaded nanoparticle, T: Transition metal, R: Thermal Processing, H:
Water
b
T0: 0 mg of transition metal, T25: 25 mg of transition metal, T50: 50 mg of transition
metal, T100: 100 mg of transition metal
104
Table 6.2. Oxygen content (%) of different oxygen scavenging mixtures during storage
time
Day 0 Day 1 Day 4 Day 7 Day 14 Day 30

N-T-R-H 20.90 ± 20.50 ± 20.47 ± 20.33 ± 20.37 ± 20.37 ±
0.00 a 0.00 b 0.06 b 0.06 c 0.06 c 0.06 c
N-R-H 20.90 ± 20.60 ± 20.53 ± 20.43 ± 20.57 ± 20.53 ±
a b b c b b
0.00 0.00 0.06 0.06 0.06 0.06
N-T-H 20.90 ± 20.90 ± 20.50 ± 20.40 ± 20.50 ± 20.50 ±
0.00 a 0.00 a 0.00 b 0.00 c 0.00 b 0.00 b
T-R-H 20.90 ± 20.50 ± 20.53 ± 20.43 ± 20.43 ± 20.53 ±
0.00 a 0.00 bc 0.06 b 0.06 c 0.06 c 0.06 b
N-T-R 20.90 ± 20.60 ± 20.57 ± 20.43 ± 20.10 ± 19.50 ±
a b b c d e
0.00 0.00 0.06 0.06 0.00 0.10
N 20.90 ± 20.90 ± 20.90 ± 20.90 ± 20.90 ± 20.90 ±
0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a
N-T0-R-H 20.90 ± 20.60 ± 20.53 ± 20.43 ± 20.57 ± 20.53 ±
0.00 a 0.00 b 0.06 b 0.06 c 0.06 b 0.06 b
N-T25-R-H 20.90 ± 20.50 ± 20.50 ± 20.43 ± 20.50 ± 20.27 ±
0.00 a 0.00 b 0.00 b 0.06 c 0.00 b 0.06 d
N-T50-R-H 20.90 ± 20.50 ± 20.47 ± 20.33 ± 20.37 ± 20.37 ±
0.00 a 0.00 b 0.06 b 0.06 c 0.06 c 0.06 c
N-T100-R-H 20.90 ± 20.50 ± 20.30 ± 20.13 ± 20.07 ± 20.13 ±
a b bc c c c
0.00 0.00 0.00 0.06 0.12 0.38
a
b
a-c: The different letters within same row differ significantly (p < 0.05)
105
21.2
aaaaa a a a a a a
20.8 b b
b b b bc bc bbb
c c b b b d cd
Oxygen content (%)
c c
20.4
N-T-R-H
20.0 N-R-H
N-T-H
19.6 T-R-H
N
19.2
18.8
0 1 4 7 14 30
Storage time (day)
Figure 6.1. Influences of the α-tocopherol, thermal processing, and transition metal on
oxygen scavenging capability (p < 0.05)
106
N-T-R-H N-T-R
21.2
20.8
Oxygen content (%)
20.4
20.0
19.6
19.2
18.8
0 5 10 15 20 25 30 35
Storage time (day)
Figure 6.2. Influence of the moisture on oxygen scavenging capability
107
21.2
aaaa
20.8 a
bbb aaa aa a
aa
Oxygen content (%)
b a b ab
20.4 ab
b c b
N-T0-R-H
20.0
N-T25-R-H
19.6 N-T50-R-H
N-T100-R-H
19.2
18.8
0 1 4 7 14 30
Storage time (day)
Figure 6.3. Influence of the amount of transition metal on oxygen scavenging capability
(p < 0.05)
108
CHAPTER SEVEN
DEVELOPMENT AND CHARACTERIZATION OF

A MOISTURE ACTIVATED OXYGEN SCAVENGING
FISH GELATIN FILM
Abstract
An oxygen scavenger was added into warm water fish gelatin and cast into films.
Surface morphology, cross sectional images, tensile strength (TS), elongation at break
(%E), oxygen permeability, water vapor permeability, color and haze of the oxygen
scavenging gelatin (gelatin w/OS) film were compared to those of the unmodified gelatin
film. Results indicated that the oxygen scavenger was well incorporated into the film
structure on gelatin w/OS film. Gelatin w/OS film had rough surfaces, decreased TS,
increased %E, and increased oxygen permeability due to the incorporation and
agglomerations of the oxygen scavenger. The addition of the oxygen scavenger also
caused color and haze alterations to the gelatin film. There were no significant
differences in water vapor permeability between unmodified gelatin and gelatin w/OS
film. In addition, the effects of moisture and thermal processing on oxygen scavenging
capability of gelatin w/OS film were also investigated. The initial oxygen content (%) in
the cup headspace was 20.90% and it was decreased to 4.56% after 50 days of storage
upon moisture exposure. Gelatin w/OS film had a good oxygen scavenging capacity,
1969.08 cc O2 per square meter per mil thickness, and water used an activator to trigger
the oxygen scavenging reaction. However, thermal processing abolished the oxygen
scavenging ability of gelatin w/OS film due to the low melting point of gelatin.
109
7.1. Introduction
Some applications of active packaging have been developed and commercialized
during the past decade. Oxygen scavenging is one of the most popular functions of active
packaging. It can be used to remove the oxygen that permeates from outside into the
package headspace through a plastic film by chemical reaction (Brody et al., 2001).
Oxygen scavengers effectively prevent oxidative damage of food constituents such as oils,
fats, flavors, pigment, and nutritive components. They can also prevent proliferation of
molds and aerobic bacteria (Ahvenainen, 2003). There are various formats used for
oxygen scavenging systems: sachets, plastic films, labels, plastic trays, and bottle crowns
(Rooney, 1995). The most effective and commonly used oxygen scavenger is the iron-
based oxygen scavenger in sachet form, but it has several disadvantages. It has a potential
risk of accidental ingestion by the consumer and it cannot be used for liquid products. To
eliminate these problems, oxygen scavengers have been incorporated into packaging
films (Vermeiren et al., 2003). Recently, several researchers added an oxygen scavenger
into a PET or LDPE matrix. Those active films effectively scavenged oxygen and are
very useful in food packaging (Galdi et al., 2008, Miltz and Perry, 2005, Sacchi et al.,
2008).
Oxygen scavenging films that are activated by UV light are commonly used as
oxygen scavengers (Albert et al., 2004, Speer, 2006, Yang et al., 2004). However, there is
a substantial price increase due to the high cost of the photoinitiator used in UV triggered
oxygen scavenging films. Breaking open nano- or microparticles may be another good
activating strategy. Oxygen scavengers could be activated by the breakage of a nano- or
110
microparticle. Heat, UV light, water, or physical pressure can rupture the nano- or
microparticle structure. A previous study showed that α-tocopherol-loaded nanoparticles
and transition metals in an oxygen scavenging mixture have oxygen scavenging
capability.
Biopolymers have been considered as a substitute for commercial synthetic polymers.
They are derived renewable resources and some biopolymers have the potential for
recyclability. One of the protein based biopolymers is gelatin. Gelatin has been widely
used in the food and pharmaceutical industries due to its high film forming ability and
low melting point. Generally, gelatin films exhibit good oxygen barrier properties at low
relative humidity. However, they have poor oxygen barrier properties at high relative
humidities due to their hydrophilic properties (Mchugh and Krochta, 1994). This
characteristic has limited the application of gelatin as a packaging material, but this
property may be useful if gelatin is to be used as moisture activated an oxygen
scavenging film. Oxygen scavenging films should have poor oxygen barrier properties to
react with the headspace oxygen (Ozdemir and Floros, 2004). Gelatin film is normally
oxygen impermeable, but it can be rendered oxygen permeable by application of water or
water vapor. Therefore, water could be used as an activator which triggers oxygen
scavenging reaction on gelatin film.
Mammalian gelatin films have superior mechanical properties and most research on
gelatin film is focused mainly on mammalian gelatin film. However, there are religious
restrictions and bovine spongiform encephalopathy (BSE) concerns for mammalian
gelatin films (Choi and Regenstein, 2000). Fish gelatin film has been studied as an
111
alternative to mammalian gelatin film (Bae et al., 2008, Karim and Bhat, 2007, Yi et al.,
2006). There are two types of fish gelatin; cold water fish gelatin and warm water fish
gelatin. Utilization of cold water fish gelatin has been limited due to its low gelation
temperature and low gel strength due to lower concentration of proline and
hydoxyproline (Leuenberger, 1991). Warm water fish gelatin has physical properties
more similar to mammalian gelatin since it has more proline and hydroxyproline than
cold water fish gelatin (Piez and Gross, 1960, Sarabia et al., 2000).
In this research, an oxygen scavenging system (α-tocopherol-loaded nanoparticle and
transition metal) was incorporated into the warm fish gelatin film. The gelatin film with
the oxygen scavenger was thermally processed in a retort chamber to make the film more
oxygen permeable, assuming that oxygen would transfer from the package headspace into
film structure. Oxygen free radicals would be produced by the reaction of oxygen with
the transition metal after ingress of oxygen into the structure. Then, the α-tocopherol-
loaded nanoparticles could consume the oxygen free radicals. Therefore, the oxygen
content (%) in the headspace might be reduced by oxygen scavenging gelatin film.
The aim of this study was to develop moisture activated oxygen scavenging fish
gelatin film. The influences of moisture and thermal processing on oxygen scavenging
capability were also investigated.
112
7.2.1. Materials
Polycaprolactone (PCL, Mn 42,500 and Mw 65,000) was purchased from Sigma-Aldrich
(MO, USA). Polyvinyl alcohol hydrolyzed 88% (PVA, Mw 22,000) was purchased from
Acros Organics (NJ, USA). Iron chloride (II), tetrahydrate was purchased from J.T.Baker
(NJ, USA). Retortable cups with 3% EVOH barrier were provided from Printpack (GA,
USA). 48 ga PET/60 ga Foil/3 mil CPP film was used as cup lid. Methylene chloride
(DCM) with HPLC grade was purchased from J.T.Baker (USA). Phosphate Buffered
Saline (PBS, 10x liquid concentrate) was purchased from EMD Bioscience (CA.USA).
Gelatin 200 bloom-fish-8 mesh was purchased from Vyse Gelatin Company (Illinois,
USA). Glycerol, Anhydrous was purchased from J. T. Baker (New Jersey, USA)
ultrasonification technique. Initially, 300 mg of PCL were dissolved in 10 mL of
methylene chloride containing 10 mg of α-tocopherol. A 2% PVA (w/v) solution was
prepared in the PBS solution. The PCL solution was added to 40 mL of the PVA solution.
Sonifier (model 250, Connecticut USA) with 55 W of energy output for 3 minute to
obtain an oil-in-water emulsion. Another 40 mL of the PVA solution was added to the
emulsion. The final solution was stirred for 12 hour on a magnetic stir plate to allow the
113
evaporation of methylene chloride and to allow the formation of the nanoparticles. The
suspension was then centrifuged at 12000 rpm for 20 minute. The pellet was resuspended
in distilled water and centrifuged three more times at 3000 rpm for 20 minute each. The
nanoparticles were collected and frozen at -80oC for at least 2 hour and subsequently
freeze dried for 2 days. The freeze dried nanoparticles were stored at 4oC.
7.2.3. Solution Preparation
Warm-water fish gelatin was used for solution preparation because cold-water fish
gelatins behave as a viscous liquid at room temperature (Avena-Bustillos et al., 2006),
which limits their application for film casting. Fifty grams of fish gelatin and 10 g of
glycerol were dissolved in 200 mL of 60oC degassed, distilled, and deionized water and
stirred for 2 hour at 60±5oC. Then, two grams of α-tocopherol-loaded nanoparticles were
added to the solution. After 10 minute stirring, 400 mg of transition metal added. The
final solution was stirred for 1 minute.
7.2.4. Film Casting
Approximately 35 mL of the prepared film solution was cast onto a BYTAC®
(Norton Performance Plastics Corporation, Wayne, NJ, USA) coated 8” x 16” glass plate
which was formed utilizing a custom designed film applicator as shown in Figure 1. After
12 hr drying, the films were peeled off from the glass plates and cut into test specimens.
The test specimens were immediately placed into a constant temperature and humidity
chamber (25oC, 50% RH) and held for 48 hr prior to testing.
114
7.2.5. Scanning Electron Microscopy (SEM)
The surface morphology of the film was examined by scanning electron microscopy
(S-4800 UHR FE-SEM, Hitachi high technologies America, Inc.). Surfaces were
prepared using platinum coating.
7.2.6. Microscopy observation
The cross section of the film was observed using Nikon OPTIPHOT (Nikon,
Japan) equipped with a color camera (NC-8 CCD, NEC, Japan) and Video Micro Scaler
IV-550. A 40X objective lens was used for the observation of the cross section of the
film.
7.2.7. Oxygen Permeability
The oxygen transmission rate (OTR) of the films was measured according to ASTM
standard method D 3985 using an OX-TRAN 2/20 (Mocon, Inc., Minneapolis, MN,
USA). Samples were exposed to 50% relative humidity (RH) and tested at 23±1°C.
Oxygen permeability (cc·mil/100in2·day·atm) was calculated by multiplying the oxygen
transmission rate by the film thickness. The test was done in triplicate and the mean value
was reported.
7.2.8. Water Vapor Permeability (WVP)
The cup method (ASTM standard method E96-80, 1987) was used to determine
water vapor permeability (WVP) in a constant temperature and humidity chamber at 25oC
115
and 50% RH. The WVP values were corrected using the WVP Correction Method
(Mchugh et al., 1993). Poly methyl methacrylate cups (Piedmont Plastics, Inc.,
Greenville, S.C., U.S.A.) sealed with O-rings was filled with water. Film samples (7cm x
7cm) were mounted over the cups. Weight loss from the cup was measured as a function
of time for 12 hour. The test was done in triplicate and the mean value was reported.
7.2.9. Mechanical Properties
The tensile strength (TS) and elongation (%E) at break of the films were measured
using an Instron Universal Testing Machine (Model 4201, Instron Corp., Canton, MA,
U.S.A.) according to the ASTM standard method D882-88. Specimen samples, 10 cm x
2.54 cm, were cut from film samples prepared on glass plates (8” x 16”). Samples were
conditioned for 48 hour at 25oC and 50% relative humidity (RH) in a constant
temperature and humidity chamber before the measurement. Initial grip separation and
cross-head speed were set at 5 cm and 25 mm/min, respectively. The values presented
were the average of seven measurements.
7.2.10. Optical properties
Haze, lightness (L), redness (a), and yellowness (b) of films were measured using
Hunterlab ColorQuest II Spectrophotometer (Hunter Associates Laboratory, Inc., Reston,
VA, USA). The values presented were the average of three measurements.
116
7.2.11. Sample preparation for oxygen content
Specimen samples, 5 cm x 5 cm,℉were cut from film samples prepared on glass
plates (8” x 16”). The samples were placed inside a high oxygen barrier retortable plastic
cup with 115 cc of ambient air (20.90% O2). Cups were prepared with or without 0.50
mL of water. Then, the cups were sealed with PET/Foil/CPP film by a Lab sealer
(Packaging Technologies, IA, USA). The seal temperature was 470oF with 60 psi
pressure at 1 second dwell time.
7.2.12. Thermal processing
A pilot-scale rotary, single cage, water spray retort in static mode was employed for
thermal processing. Samples were processed for 30 minute at 140, 170, and 200oF using a
Surdry Model APR-95 Rotary Pilot Retort (Stock America, NC, USA).
7.2.13. Oxygen content analysis
each sample measurement. All of the samples were measured at day 0, 1, 3, 5, 7, and 50.
The test was done in triplicate and the mean value was reported.
117
118
7.3.1. Surface morphology and cross sectional image of the film
The surface morphology images were taken using SEM. Imaging was performed on
the top and side view of the unmodified gelatin film and the gelatin film with oxygen
scavenger (gelatin w/OS) film (Fig. 2). The unmodified gelatin film had a smooth surface
morphology without any particles (Fig. 2a). On the other hand, there were many particles
on the surface of gelatin w/OS film (Fig. 2b). It was also observed that the particles were
incorporated into the film structure on gelatin w/ OS film (Fig. 2d). However, the
unmodified gelatin film had no incorporated particles (Fig. 2c) visible in the side view.
The cross sectional image of the unmodified gelatin and gelatin w/OS film were also
taken using an optical microscope (Fig. 3). There was agglomeration of nanoparicles
inside the gelatin w/OS film (Fig. 3b). This suggests that mechanical stirring does not
sufficiently distribute the nanoparticles in the gelatin structure.
Given the SEM and an optical microscope images, it was concluded that the oxygen
scavengers were successfully incorporated into the film structure in the gelatin w/OS film.
The gelatin w/OS film had a rough surface due to the incorporation and agglomeration of
the nanoparticles.
7.3.2. Oxygen permeability and Water vapor permeability
In related research, it was found that gelatin films with lipid additions had increased
oxygen permeability. This was caused by the hydrophobic nature of the lipid component
facilitating the oxygen transfer (Bertan et al., 2005). The oxygen permeability of the
119
unmodified gelatin and gelatin w/OS film were measured (Fig. 4). The addition of α-
tocopherol-loaded nanoparticles into the film structure caused an increase in the oxygen
permeability of gelatin w/OS film. This may be due to the hydrophobic nature of the α-
tocopherol-loaded PCL nanoparticles. Agglomeration of nanoparticles might also have
contributed to the increased oxygen permeability by interrupting the gelatin film matrix.
Water vapor permeability of the unmodified gelatin and gelatin w/OS film were also
measured (Fig. 5). There were no significant differences in water vapor permeability
between the unmodified gelatin and gelatin w/OS film.
7.3.3. Mechanical Properties
Other researchers (Wang et al., 2009) have reported that tensile strength of gelatin
films increased linearly with increasing corn oil addition. When oil was added into the
gelatin film, a more compact film matrix was observed with increased oil content.
However, the tensile strength was decreased when hydrophobic nanoparticles were added
into the gelatin film in this research (Fig. 6). As previously stated, it is possible that the
agglomeration of α-tocopherol-loaded nanoparticles interrupted the gelatin film matrix
and, consequently, decreased tensile strength. Due to the same reasons, elongation at
break (%) slightly increased by adding α-tocopherol-loaded nanoparticles into the gelatin
film.
120
7.3.4. Optical properties
The color and haze value of the unmodified gelatin and gelatin w/OS films are
showed in Table 1. In this observation, all of the color values of the gelatin w/OS film
were significantly different from those of the unmodified gelatin film. The lightness (L)
and red value (a) of the film were decreased by adding oxygen scavenger. On the other
hand, yellow values (b) and haze values of the film were increased by adding oxygen
scavenger.
7.3.5. Oxygen content (%) analysis during storage time
Ten film batches with different processing methods and mixture compositions were
prepared (Table 2). A comparison of Batches G-H and G/OS-H suggest that, given the
proper conditions, the gelatin w/OS film can be an effective scavenger (Table 3). For
example, there was significant oxygen content (%) reduction for Batch G/OS-H. Initial
oxygen content (%) on Batch G/OS-H was 20.90% and it was decreased considerably to
4.56% at day 50. While Batch G-H also showed some reduction in oxygen content (%),
Batch G/OS-H was an order of magnitude higher in oxygen removal. The small
reduction in headspace oxygen shown for batch was believed to be due to a physical
entrapment of oxygen by swollen gelatin film.
121
7.3.6. Effect of moisture on oxygen scavenging capability
The effect of moisture on the oxygen content (%) reductions was investigated and the
results can be seen in Figure 7. Batch G, the unmodified gelatin film in a dry
environment, exhibited no oxygen scavenging capability. Batch G/OS, the gelatin w/OS
film in a dry environment, also exhibited no oxygen scavenging properties. However, the
data for Batch G/OS-H, the gelatin w/OS film in a wet environment, had oxygen content
(%) that was significantly lower than the other samples at day 50. Thus, gelatin w/OS
film in the presence of water can scavenge the oxygen more effectively. Water triggers
the oxygen scavenging reaction in the gelatin w/OS film.
7.3.7. Effect of thermal processing on oxygen content reductions
The effect of thermal processing on oxygen content (%) reduction was also
investigated (Fig. 8). Three different processing temperatures, 140, 170, and 200oF, were
used. It was observed that there were no significant differences in oxygen content (%)
reductions between the unmodified gelatin and gelatin w/OS film after thermal
processing. Also, there were no significant differences in oxygen content (%) reductions
with different processing temperature. Therefore, gelatin w/OS film lost its oxygen
scavenging capability after thermal processing due to low melting point of gelatin.
122
7.4. Conclusion
In this research, an active fish gelatin film containing oxygen scavenger nanoparticles
was developed. The results gathered by SEM and optical microscope observations
confirmed that the oxygen scavenger nanoparticles were incorporated into the gelatin
film structure. Agglomerations of the oxygen scavenger nanoparticles caused a rough
film surface, decreased tensile strength, increased elongation at break (%), and increased
oxygen permeability. Color and haze values were also altered after adding oxygen
scavenger into the gelatin film. The active fish gelatin film with moisture can scavenge
oxygen effectively. Water is an activator which triggers the oxygen scavenging reaction.
However, thermal processing eliminates the oxygen scavenging ability of the active fish
gelatin film.
Generally, the scavenging capacity of an oxygen scavenging layer can be 250 cc O2
per square meter per mil thickness and more often at least 500 cc O2 per square meter per
mil thickness (Jerdee et al., 2003, Speer et al., 1994). In this research, the oxygen
scavenging capacity was calculated by initial oxygen content and oxygen content at day
50. The active fish gelatin film had 1969.08 cc O2 per square meter per mil thickness of
oxygen scavenging capacity. These results demonstrate that the active fish gelatin film
can be used as a moisture activated oxygen scavenging layer.
123
7.5. References

US patent 6,821,482

Avena-Bustillos, R. J., Olsen, C. W., Olson, D. A., Chiou, B., Yee, E., Bechtel, P. J.,
and Mchugh T. H., 2006. Water Vapor Permeability of Mammalian and
fish gelatin films, J Food Sci, 71(4), 202-207
Bae, H. J., Darby, D. O., Kimmel, R. M., Park, H. J., Whiteside, W. S., 2008.
Effect of transglutaminase-induced crosslinking on properties of
fish gelatin-nano clay composite film. Food Chemistry, Article in Press
Bertan, L.C., Tanada-Palmu, P.S., Siani, A.C., Grosso, C.R.F., 2005.

Effect of fatty acids and ‘‘Brazilian elemi” on composite films based on gelatine.
Food Hydrocolloids 19, 73–82
Brody, A. L., Strpinsky E.P., Kline, L. R., 2001. Active food packaging for food
application, CRC Press, New York, USA, pp 11-30
Choi, S. S., Regenstein, J. M., 2000. Physicochemical and sensory characteristics of fish
gelatin. Journal of Food Science, 65(2). 194-199
Galdi, M. R., Nicolais, V., Maio, L. D., Incarnato, L., 2008. Production of Active PET
film: Evaluation of scavenging activity. Packaging Technology and Science, 21.
257-268
Karim, A. A., Bhat, R., 2008. Fish gelatin: properties, challenges, and prospects as
an alternative to mammalian gelatins. Food hydrocolloids, Article in press.
124
Leuenberger, B.H., 1991. Investigation of viscosity and gelation properties of
different mammalian and fish gelatins. Food hydrocolloids, 5(4). 353-361
Mchugh, T. H., Avena-Bustillos, R., and Krochta, J. M., 1993. Hydrophillic edible films:
Modified procedure for water vapor permeability and explanation of thickness
effects. J Food Sci, 58. 899-903
Mchugh, T. H., Krochta, J. M., 1994. Permeability properties of edible films, in ;

Krochta, J. M., Baldwin, E. A., Nisperos-Carriedo, M. O. (Eds), Edible
coatings and films improve food quality. CRC Press, New York, USA,
pp 139-188
Miltz, J., Perry, M., 2005. Evaluation of the performance of iron-based oxygen
scavengers, with comments on their optimal applications.
Packaging Technology and Science, 18. 21-27
Ozdemir, M., Floros, J. D., 2004. Active food packaging technology.

Critical reviews in Food Science and Nutrition, 44. 185-193
Piez, K. A., Gross, J. G., 1960. The amino acid composition of some fish collagens:
The relation between composition and structure. Journal of Biological Chemistry,
235(4), 995–998.

pp 74-110
Sacchi, R., Savarese, M., Regno, A. D., Paduano, A., Terminiello, R., Ambrosino, M. L.,
2008. Shelf life of vegetable oils bottled in different scavenging
polyethyleneterephthalate (PET) containers. Packaging Technology and Science,
21. 269-277
Sarabia, A. I., Go´mez-Guille´n, M. C., & Montero, P. (2000). The effect of added salt on
the viscoelastic properties of fish skin gelatin. Food Chemistry, 70, 71–76.
Speer, D. V., Morgan, C. R., Roberts, W. P., Vanputte, A. W., 1994. Multilayer structure
for a package for scavenging oxygen. US patent 5,350,622
125
Vermeiren, L., Heirlings, L., Devlieghere, F., Debevere, J. 2003. Oxygen, ethylene and
Wang, L., Auty, M. A. E., Rau, A., Kerry, J. F., Kerry, J. P., 2009. Effect of pH and
addition of corn oil on the properties of gelatin-based biopolymer films.
Journal of food engineering, 90. 11-19
US patent 6,818,151
Yi, J. B., Kim, Y. T., Bae, H. J., Whiteside, W. S., Park, H. J., 2006. Influence of
Transglutaminase-Induced Cross-Linking on Properties of Fish Gelatin Films.
J Food Sci, 71(9). 376-383
126
Table 7.1. Color (L, a, b) and Haze of the unmodified gelatin and gelatin w/OS film
L a b Haze
a a b b
Unmodified gelatin 96.66±0.13 -0.01±0.01 0.58±0.07 5.71±0.25
b b a a
Gelatin w/OS 96.00±0.14 -0.19±0.05 1.95±0.38 9.19±0.90
a
Results are expressed as the mean ± SD (n=3).
b
a-b: The different letters within same column differ significantly (p < 0.05)
127
Table 7.2. Batch compositions used for the oxygen content (%) analysis
Thermal processing temperature Water

Film
(oF) (1 mL)
G Unmodified gelatin - -
G/OS Gelatin w/OS - -
G-H Unmodified gelatin - O
G/OS-H Gelatin w/OS - O
G-140-H Unmodified gelatin 140 O
G/OS-140-H Gelatin w/OS 140 O

a
OS: Oxygen scavenger,
b
G: Gelatin, G/OS: Gelatin w/OS, H: Water
128
Table 7.3. Oxygen content (%) of different oxygen scavenging mixtures during storage
time
day 0 day 1 day 3 day 5 day 7 day 50
G 20.90±0.00a 20.90±0.00a 20.90±0.00a 20.90±0.00a 20.90±0.00a 20.90±0.00a
G/OS 20.90±0.00a 20.90±0.00a 20.90±0.00a 20.90±0.00a 20.90±0.00a 20.90±0.00a
G-H 20.90±0.00a 20.47±0.06a 19.43±0.25b 18.60±0.40bc 17.93±0.76cd 17.63±0.80d
G/OS-H 20.90±0.00a 20.50±0.00ab 19.77±0.06b 18.80±0.10c 17.87±0.06c 4.56±1.29d
G-140-H 20.90±0.00a 20.47±0.06c 20.50±0.00c 20.60±0.00b 20.50±0.00cd 20.50±0.00c
G-170-H 20.90±0.00a 20.43±0.06d 20.50±0.00c 20.60±0.00b 20.50±0.00c 20.50±0.00c
G-200-H 20.90±0.00a 20.40±0.10e 20.47±0.06de 20.60±0.00b 20.57±0.06bc 20.50±0.00cd
G/OS-140-H 20.90±0.00a 20.43±0.06d 20.50±0.00c 20.57±0.06b 20.50±0.00c 20.50±0.00c
G/OS-170-H 20.90±0.00a 20.43±0.06d 20.50±0.00c 20.60±0.00b 20.50±0.00c 20.50±0.00c
G/OS-200-H 20.90±0.00a 20.40±0.10c 20.50±0.00b 20.50±0.00b 20.50±0.00b 20.50±0.00b
a
Results are expressed as the mean ± SD (n=3).
b
a-e: The different letters within same raw differ significantly (p < 0.05)
129
Figure 7.1. Customized film applicator
130
(a)
(b)
Figure 7.2. Surface morphology of the film by SEM (a) unmodified gelatin (top view) (b)
gelatin w/OS (top view) (c) unmodified gelatin (side view) (d) gelatin w/OS (side view)
131
(c)
(d)
Figure 7.2. Continued
132
(a)
(b)
Figure 7.3. Cross sectional image of the film by microscopy (a) unmodified gelatin (b)
gelatin w/OS
133
0.0012
a
0.001
(cc·mil/100in2·day·atm)
Oxygen Permeability
0.0008
b
0.0006
0.0004
0.0002
0
Unmodified gelatin Gelatin w/OS
Figure 7.4. Oxygen permeability of the unmodified gelatin and gelatin w/OS film (p <
0.05)
134
2
a
1.6 a
Water vapor permeability
(cc·mil/100in2·day·atm)
1.2
0.8
0.4
0
Figure 7.5. Water vapor permeability of the unmodified gelatin and gelatin w/OS film (p
< 0.05)
135
35.00 a
(a)
30.00
Tensile Strength (MPa)
25.00
20.00 b
15.00
10.00
5.00
0.00
35.00 a
(b)
30.00
b
Elongation at Break (%)
25.00
20.00
15.00
10.00
5.00
0.00
Figure 7.6. Mechanical properties (a) TS (b) %E of the unmodified gelatin and gelatin
w/OS film (p < 0.05)
136
25.00
a a a a a ab a a a a a a a a
b cb
20.00 bb bb b
Oxygen content (%)
15.00
G
G/OS
10.00
G-H
G/OS-H
c
5.00
0.00
0 1 3 5 7 50
Storage time (day)
Figure 7.7. The influence of the moisture on oxygen scavenging capability of the
unmodified gelatin and gelatin w/OS film (p < 0.05)
137
21.00
20.90
20.80
Oxygen content (%)
20.70 G-140-H
G-170-H
20.60
G-200-H
G/OS-140-H
20.50
G/OS-170-H
G/OS-200-H
20.40
20.30
20.20
0 10 20 30 40 50 60
Storage time (day)
Figure 7.8. The influence of the thermal processing (140, 170, 200oF) on oxygen
scavenging capability of the unmodified gelatin and gelatin w/OS film
138
CHAPTER EIGHT
GENERAL CONCLUSION
An oxygen scavenger mixture containing α-tocopherol and a transition metal was
developed as an oxygen scavenger. The oxygen scavenger mixture eliminates the
headspace oxygen effectively by a successive chemical reaction, not by the physical
entrapment of oxygen. Oxygen free radicals produced by the reaction of oxygen with a
transition metal and α-tocopherol eliminates the oxygen free radical by donating their
electrons to the free radical. In addition, thermal processing accelerates the oxygen
scavenging reaction, while the addition of moisture did not affect the oxygen scavenging
capability.
Nanoencapsulation technology was successfully adopted to create a heat activated
oxygen scavenging system. α-tocopherol-loaded nanoparticles with a transition metal
scavenge the headspace oxygen effectively upon thermal processing. Heat was used to
trigger the oxygen scavenging reaction in this system. The addition of moisture decreased
oxygen scavenging capability of α-tocopherol-loaded nanoparticles and transition metal.
This oxygen scavenger system can be used in both dry and wet conditions but proved to
be more effective if used in dry products. α-tocopherol-loaded nanoparticles without
transition metal, moisture, and thermal processing had no oxygen scavenging effect. The
α-tocopherol-loaded nanoparticles were stable for storage under normal conditions.
An active fish gelatin film has been developed as a moisture activated oxygen
scavenging layer. The α-tocopherol-loaded nanoparticles and transition metal were
incorporated into the film structure. Agglomerations of the oxygen scavenger caused a
139
rough surface, decreased tensile strength, increased elongation at break (%), and
increased oxygen permeability. Color and haze values were also altered significantly after
adding oxygen scavenger into the gelatin film. Water was used to trigger the oxygen
scavenging reaction in this scavenging layer. The active fish gelatin film demonstrated a
good oxygen scavenging capacity.
140

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Clemson University

Development and characterization of moisture-

Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations

α-tocopherol and a transition metal, iron, mixture was evaluated as a possible

and 0.11 cc O2 per gram per day of oxygen scavenging rate.

Nanoencapsulation technology was adopted for the possible oxygen scavenging

activation system. α-tocopherol-loaded poly ε-caprolactone (PCL) nanoparticles were

effects of PCL concentration, solvent in oil phase, and ultrasonification time on

with 3 minute ultrasonification time showed the best encapsulation formulation.

Therefore, this formulation was selected for further research.

α-tocopherol-loaded nanoparticles and a transition metal in scavenger mixture were

evaluated as a heat activated oxygen scavenger. The effects of moisture, amount of

reaction. However, adding moisture lowered the oxygen scavenging capability.

triggered the oxygen scavenging reaction. However, thermal processing at temperature

above 140oF destroyed the oxygen scavenging ability of the film.

encouragement, and trust throughout doctoral studies. I would like to express my

especially my wife, Hyojung Choo, to whom I owe everything.

TITLE PAGE .................................................................................................................... i

LIST OF TABLES ........................................................................................................viii

LIST OF FIGURES ........................................................................................................ ix

LIST OF SCHEMES...................................................................................................... xii

I. GENERAL INTRODUCTION ...................................................................... 1

1.1. Overview of Active Packaging ......................................................... 1

II. RESEARCH HYPHOTHESIS ................................................................... 40

III. RESEARCH OBJECTIVES ........................................................................ 42

IV. ALPHA-TOCOPHEROL AND A TRANSITION METAL

4.4. Conclusion ...................................................................................... 53

V. PREPARATION AND CHARACTERIZATION OF

VI. ALPHA-TOCOPHEROL-LOADED NANOPARTICLES AND

VII. DEVELOPMENT AND CHARACTERIZATION OF A

Abstract ................................................................................................ 109

VIII. GENERAL CONCLUSION .................................................................... 139

1.1. Selected commercial oxygen scavenger systems ......................................... 10

1.2. Nanoencapsulation method: advantages and drawbacks ............................. 25

4.1. Batch compositions used for oxygen scavenging mixtures ......................... 57

4.2. Oxygen content (%) of different scavenging mixtures

5.1. Batch compositions used for constituting

5.2. Properties of α-tocopherol–loaded nanoparticle .......................................... 83

6.2. Oxygen content (%) of different oxygen scavenging mixtures

7.1. Color (L, a, b) and Haze of

7.3. Oxygen content (%) of different oxygen scavenging mixtures

1.1. Oxidation of ascorbic acid ............................................................................. 5

1.2. A structure of typical oxygen scavenging multilayer film............................. 9

1.3. A simplified version of bonding in the diatomic molecule

1.4. Transition metal or d block elements on periodic table ............................... 14

1.5. Synthetic phenolic antioxidant ..................................................................... 17

1.6. Natural antioxidant; tocopherol ................................................................... 18

1.7. Natural antioxidant; Flavonoids ................................................................... 19

1.8. Natural vitamin E analogue ......................................................................... 20

1.9. Mechanism of α-tocopherol action .............................................................. 22

1.10. Singlet oxygen oxidation of α-tocopherol ................................................... 23

1.11. The chemical structure of gelatin ................................................................. 30

4.1. Influences of the α-tocopherol, thermal processing, and transition metal

4.2. Influence of moisture on oxygen scavenging capability.............................. 60

4.3. Influence of the amount of transition metal

4.4. Structure of α-tocopherol and trimethylhydroquinone (TMHQ) ................. 62

4.5. Influence of moisture on oxygen scavenging capability of

6.1. Influences of the α-tocopherol, thermal processing, and transition metal

6.2. Influence of the moisture on oxygen scavenging capability...................... 107

6.3. Influence of the amount of transition metal on oxygen

7.1. Customized film applicator ........................................................................ 130

7.2. Surface morphology of the film by SEM

7.5. Water vapor permeability of

7.6. Mechanical properties (a) TS (b) %E of

Fe 2OH Fe OH

Fe OH O H O Fe OH (Equation 1.1)

2SO O 2H O 2H SO (Equation 1.2)

2 glucose 2O 2H O 2 gluconic acid 2H O (Equation 1.4)

2 H O catalase 2 H O O catalase (Equation 1.5)