minor components and their roles on lipid oxidation in bulk oil that contains association colloids
TRANSCRIPT
University of Massachusetts - AmherstScholarWorks@UMass Amherst
Dissertations Dissertations and Theses
5-1-2012
Minor Components and Their Roles on LipidOxidation in Bulk Oil That Contains AssociationColloidsBingcan ChenUniversity of Massachusetts - Amherst, [email protected]
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Recommended CitationChen, Bingcan, "Minor Components and Their Roles on Lipid Oxidation in Bulk Oil That Contains Association Colloids" (2012).Dissertations. Paper 540.
MINOR COMPONENTS AND THEIR ROLES ON LIPID OXIDATION IN BULK OIL THAT CONTAINS ASSOCIATION COLLOIDS
A Dissertation Presented
by
Bingcan Chen
Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment
of the requirements for the degree of
DOCTOR OF PHILOSOPHY
MAY 2012
The Department of Food Science
© Copyright by Bingcan Chen 2012
All Rights Reserved
MINOR COMPONENTS AND THEIR ROLES ON LIPID OXIDATION IN BULK OIL THAT CONTAINS ASSOCIATION COLLOIDS
A Dissertation Presented
by
BINGCAN CHEN
Approved as to style and content by:
________________________________________ Eric A. Decker, Chair
________________________________________ D. Julian McClements, Member
________________________________________ Matthew A. Holden, Member
_______________________________________ Eric A. Decker, Department Head Department of Food Science
DEDICATIONS
To my parents and my wife,
who made all of this possible, for their endless encouragement and patience.
v
ACKNOWLEDGMENTS
I would like to express my heartfelt thanks to my advisor, Dr. Eric Decker, for this
greatest opportunity he has provided, for his guidance, enthusiasm, support and incredible
talent on this research and in my pursuit of a Doctoral degree. It was such a great honor
and best fortune for me to be his student. My entire life has been changed because of his
kindness. I would like to thank Dr. Julian McClements for his advice and support on this
research and in my pursuit of a Doctoral degree.
Also, I would like to extend thanks to Dr. Matthew Holden for being a member of
my committee. I appreciate his work and his constructive advice towards this dissertation.
I would like to express my appreciation to Jean Alamed for her continuous
assistance in my research as well as her friendship. Many thanks to Kla, Get, Ashley,
Jeffery, Caitlin, Mette, Ricard, Dao, Mickaël, and Giovanni for their continuing and
unconditional friendship and support over the years; I could not have done this without
their helps. I would also like to thank all faculty, staff and lab members with special
thanks to Dr. Xiao, Dr. Park, Dr. Goddard, Fran and Dan. In addition, I would like to
thank my friends outside of the University and/or country who continue to support my
endeavors.
Most importantly, I would like to thank my family, especially my Mom and Dad,
who love me so much, and my wife Jiajia for her endless love, support and
encouragement!
vi
ABSTRACT
MINOR COMPONENTS AND THEIR ROLES ON LIPID OXIDATION IN BULK OIL THAT CONTAINS ASSOCIATION COLLOIDS
MAY 2012
BINGCAN CHEN, B.S., SICHUAN UNIVERSITY OF SCIENCE AND ENGINEERING, SICHUAN, CHINA
M.S., CHONGQING UNIVERSITY, CHONGQING, CHINA
Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST
Directed by: Professor Eric A. Decker
The combination of water and surface active compounds found naturally in
commercially refined vegetable oils have been postulated to form physical structures
known as association colloids. This research studied the ability of 1,2-dioleoyl-sn-
glycerol-3-phosphocholine (DOPC) and water to form physical structures in stripped
soybean oil. Interfacial tension and fluorescence spectrometry results showed the critical
micelle concentration (CMC) of DOPC in stripped soybean oil was 650 and 950 μM,
respectively. Light scattering attenuation results indicated that the structure formed by
DOPC was reverse micelles. The physical properties of DOPC reverse micelles were
determined using small-angle X-ray scattering (SAXS) and fluorescence probes. These
studies showed that increasing the water concentration altered the size and shape of the
reverse micelles formed by DOPC.
The impact of DOPC reverse micelles on the lipid oxidation of stripped soybean oil
was investigated by following the formation of primary and secondary lipid oxidation
products. DOPC reverse micelles had a prooxidant effect, shortening the oxidation lag
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phase of SSO at 55 °C. It also was not able to change the lipid oxidation of stripped
soybean oil compared with DOPC reverse micelles at same concemtration ( i.e., 950 μM).
1,2-dibutyl-sn-glycerol-3-phosphocholine (DC4PC) which has the shorter fatty acid than
DOPC was not able to form association colloids and did not impact lipid oxidation rates.
This indicated that the choline group of the phospholipid was not responsible for the
increased oxidation rates and suggested that the physical structure formed by DOPC was
responsible for the prooxidant effect.
The impact of the DOPC reverse micelles on the effectiveness and physical location
of the antioxidants, α-tocopherol and Trolox was also studied. Both non-polar (α-
tocopherol) and polar (Trolox) were able to inhibit lipid oxidation in stripped soybean oil
in the presence of DOPC reverse micelles. Trolox was a more effective antioxidant than
α-tocopherol. Fluorescence steady state and lifetime decay studies suggested that both α-
tocopherol and Trolox were associated with DOPC reverse micelle in bulk oil. Trolox
primarily concentrated in the water pool of reverse micelle since it quenched NBD-PE
fluorescence intensity with increasing concentrations. A portion of α-tocopherol was also
associated with the aqueous phase of the DOPC reverse micelles but this was likely at the
oil-water interface since -tocopherol is not water soluble.
The addition of ferric chelator, deferoxamine (DFO) to stripped soybean oil
significantly prevented the lipid oxidation caused by DOPC reverse micelles as the lag
phase was extended from 2 to 7 days. DFO was also found to increase the antioxidant
activity of both Trolox and -tocopherol. Trolox and -tocopherol were found to be
rapidly decomposed by high-valence Fe(III) while low-valence-state (Fe (II) was much
less reactive. Fe(III) was also consumed by both hydrophilic Trolox and lipophilic α-
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tocopherol presumably though reduction to Fe (II). DOPC reverse micelles were able to
decrease antioxidants-iron interactions as evidence by a decrease in antioxidant depletion
by iron and a decrease in iron reduction by the antioxidants. These results suggested that
the ability of DFO to increase the antioxidant activity of -tocopherol and Trolox was
due to its ability to decrease free radical production and not its ability to decrease direct
iron-antioxidant interactions.
Overall, the results presented in this dissertation show phospholipids and water can
form reverse micelles in edible oils. These reverse micelles increase lipid oxidation rates
by increasing the prooxidant activity of iron. Free radical scavenging antioxidants can
inhibit oxidation promoted by the reverse micelles with polar Trolox being more effective
than non-polar tocopherol presumably because Trolox is more highly associated with
the reverse micelle. The reverse micelles produced by DOPC protected tocopherol and
Trolox from direct degradation by iron. The knowledge gained from this study will
improve our understanding of the mechanism of lipid oxidation in bulk oils which will
hopefully provide new technologies to improve the oxidation stability of edible oils. For
example, it may be able to use oil refining technologies to remove prooxidative minor
components that for physical structure in bulk oils.
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TABLE OF CONTENTS
Page ACKNOWLEDGMENTS ....................................................................................................... v ABSTRACT ............................................................................................................................ vi LIST OF TABLES ................................................................................................................ xiii LIST OF FIGURES............................................................................................................... xiv CHAPER 1. INTRODUCTION ....................................................................................................... 1 2. LITERATURE REVIEW ............................................................................................ 4
2.1 Oil refining procedure ........................................................................................... 4
2.1.1 Storage .................................................................................................... 4 2.1.2 Preparation ............................................................................................. 5 2.1.3 Solvent extraction .................................................................................. 5 2.1.4 Oil refining ............................................................................................. 6
2.2 Minor components in bulk oil............................................................................... 8
2.2.1 Free fatty acid ......................................................................................... 8 2.2.2 Monoacylglycerols and Diacylglycerols ............................................ 11 2.2.3 Phospholipids ....................................................................................... 13 2.2.4 Tocopherol ............................................................................................ 14 2.2.5 Pigments ............................................................................................... 15 2.2.6 Miscellaneous compounds .................................................................. 15
2.2.6.1 Nonsaponifiable minor components ................................... 15 2.2.6.2 Polar triacylglycerol polymers ............................................ 16 2.2.6.3 Oxidized triacylglycerols ..................................................... 17
2.2.7 Water ..................................................................................................... 18 2.2.8 Oxygen ................................................................................................. 19
2.3 The effect of minor components on bulk oil oxidation ..................................... 21
2.3.1 The effect of oxygen on the chemical stability of bulk oil ................ 21 2.3.2 The effect of water on the chemical stability of bulk oil ................... 22 2.3.3 The effect of free fatty acid on the chemical stability of bulk
oil ....................................................................................................... 23 2.3.4 The effect of MAG and DAG on chemical stability of bulk
x
oil ....................................................................................................... 24 2.3.5 The effect of phospholipids on the chemical stability of bulk
oil ....................................................................................................... 24 2.3.6 The effect of tocopherols on the chemical stability of bulk oil......... 25 2.3.7 The effect of pigments on the chemical stability of bulk oil ............. 26 2.3.8 The effect of miscellaneous compounds on the chemical
stability of bulk oil ............................................................................ 28 2.4 Association colloids and lipid oxidation in bulk oils ........................................ 29
2.4.1 Evidence for the presence of association colloids in bulk oil ........... 29 2.4.2 Do association colloids impact lipid oxidation? ................................ 30 2.4.3 The perspective researches on the mechanism of lipid
oxidation in bulk oil .......................................................................... 32 2.5 Methodologies to study the impact of minor components on the
physicochemical properties of bulk oil ........................................................ 33 2.5.1 Chemical analysis ................................................................................ 34
2.5.1.1 Primary lipid oxidation products ......................................... 34 2.5.1.2 Secondary lipid oxidation products ..................................... 34
2.5.2 Physical analysis .................................................................................. 35
2.5.2.1 Small/wide angle x-ray scattering (SAXS/WAXS)............ 35 2.5.2.2 Steady-state and time resolved fluorescence
measuremen .......................................................................... 36 2.5.2.3 Interfacial tension analysis .................................................. 37
3. THE IMPACT OF PHYSICAL STRUCTURES IN SOYBEAN OIL ON
LIPID OXIDATION .................................................................................................. 38 3.1 Introduction .......................................................................................................... 38 3.2 Materials and Methods ........................................................................................ 39
3.2.1 Materials .............................................................................................. 40 3.2.2 Preparation of stripped soybean oil .................................................... 40 3.2.3 Light scattering properties of oil samples ......................................... 41 3.2.4 Determination of critical micelle concentration (CMC) in
stripped soybean oil .......................................................................... 42 3.2.5 Small angle X-ray scattering (SAXS) ............................................... 43 3.2.6 Front-Face (FF) fluorimetric measurements ...................................... 44 3.2.7 Measurement of oxidation parameters ............................................... 45 3.2.8 Statistical analysis ................................................................................ 46
3.3 Results and discussion......................................................................................... 46
xi
3.3.1 Attenuation of the water/phospholipid/MCT system ......................... 46 3.3.2 Determination of the CMC of phospholipids in SSO and
MCT................................................................................................... 47 3.3.3 Detection and characterization of phospholipids physical
structures by small angle x-ray scattering ....................................... 51 3.3.4 Effect of water/phospholipid molar ratio on the physical
properties of DOPC association colloids ........................................ 54 3.3.5 Oxidation of stripped soybean oil containing association
colloids .............................................................................................. 58
4. ROLE OF REVERSE MICELLES ON LIPID OXIDATION IN BULK OILS: IMPACT OF PHOSPHOLIPIDS ON ANTIOXIDANT ACTIVITY OF α-TOCOPHEROL AND TROLOX .................................................................... 61 4.1 Introduction .......................................................................................................... 61 4.2 Materials and Methods ........................................................................................ 63
4.2.1 Materials .............................................................................................. 63 4.2.2 Preparation of stripped soybean oil .................................................... 64 4.2.3 Formation of DOPC reverse micelles in stripped soybean oil .......... 64 4.2.4 Measurement of oxidation parameters ............................................... 65 4.2.5 Small angle x-ray scattering (SAXS) study of bulk oil with
DOPC and antioxidants .................................................................... 66 4.2.6 Fluorescence measurement of bulk oil with DOPC and
antioxidants ....................................................................................... 67 4.2.7 Statistical analysis ................................................................................ 68
4.3 Results .................................................................................................................. 69
4.3.1 Impact of phospholipids on oxidative stability of SSO ..................... 69 4.3.2 The impact of phospholipids on the oxidative stability of
SSO in the presence of α-tocopherol ............................................... 71 4.3.3 The impact of phospholipids on the oxidative stability of
SSO in the presence of Trolox ......................................................... 75 4.3.4 The impact of antioxidants on the properties of DOPC
reverse micelles in SSO .................................................................... 79
4.4 Discussion ............................................................................................................ 83
5. IRON/ANTIOXIDANTS RELATIONSHIP IN BULK OIL OXIDATION .......................................................................................................... 87 5.1 Introduction .......................................................................................................... 87 5.2 Materials and Methods ........................................................................................ 89
5.2.1 Materials .............................................................................................. 89
xii
5.2.2 Formation of DOPC association colloids in stripped soybean oil or MCT......................................................................................... 89
5.2.3 Measurement of oxidation parameters ............................................... 90 5.2.4 Determination of Trolox and α-tocopherol concentrations ............... 91 5.2.5 Determination the concentration of ferric iron in MCT .................... 92 5.2.6 EPR spectroscopy ................................................................................ 92 5.2.7 Statistical analysis ................................................................................ 93
5.3 Results .................................................................................................................. 93
5.3.1 Effects of the metal chelator, Deferoxamine (DFO) on the
oxidative stability of stripped soybean oil ...................................... 93 5.3.2 Effects of Fe(III) and Fe(II) on the depletion of α-tocopherol
and Trolox in MCT ......................................................................... 100 5.3.3 Effects of Fe(III) and Fe(II) on the depletion of α-tocopherol
or Trolox in the presence of DOPC reverse micelles ................... 102 5.3.4 Measurement of Fe(III) loss by α-tocopherol and Trolox ............... 105 5.3.5 Effects of Fe(III) and Fe(II) on the formation of PBN spin
adducts in MCT .............................................................................. 109
5.4 Discussion .......................................................................................................... 110
6. CONCLUSION ................................................................................................... 113 BIBLIOGRAPHY ................................................................................................................ 117
xiii
LIST OF TABLES Table Page
2.1. Free fatty acid content in crude soybean oil as the function of bean storage time ......................................................................................................................... 9
2.2. Free fatty acid content in soybean oil as a function of processing step ................... 9
2.3. Free fatty acid content in retail oil ............................................................................ 11
2.4. Concentration of MAG and DAG in various oils as a function of refining ........... 12
2.5. Phospholipid composition (%) as extracted from commercial seeds ..................... 13
2.6. Phosphorous content (ppm) of soybean oil as a function of refining step ............. 14
2.7. Water content in freshly-opened, commercially available vegetable oils as determined by Karl Fischer Coulometer ............................................................ 18
2.8. Solubility of oxygen (ppm) in Marine oil ................................................................ 20
xiv
LIST OF FIGURES Figure Page
2.1. The schematic of oil refining process ......................................................................... 6
2.2. Schematic representation of the association colloids formed by minor constituents in bulk oil (A) a reverse micelle formed by oleic acid; (B) a reverse micelle formed by phospholipids; (C) Inverse lamella structure formed by monoacylglycerol; (D) a mixed reverse micelle formed by free fatty acids and phospholipids .................................................... 30
2.3. The essential parts of a small angle scattering system. The drawing shows the X-ray source X, the sample S, the scattering angle θ, the slits used to define the incident and scattered beams, and the detector D ........................ 35
3.1. Molecular structures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) ............................... 39
3.2. Attenuation of medium chain triacylglycerols (MCT) samples containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of water ....................................................................................... 47
3.3. Determination of critical micelle concentration (CMC) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in medium chain triacylglycerols (A)and stripped soybean oil (B) as determined by the absorbance (480 nm) of 7,7,8,8-tetracyano- quinodimethane....................................................... 49
3.4. Determination of critical micelle concentration of 1,2-dioleoyl-sn-glycero-3-phosphocholine in stripped soybean oil using interfacial tension ................ 51
3.5. Small angle x-ray scattering profiles of stripped soybean oil (SSO) and SSO + 950 μM 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3- phosphocholine (DC4PC) ..................................... 52
3.6. The pair distribution functions, p(r), calculated from the scattering profiles of stripped soybean oil (SSO) and SSO + 950 μM 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine(DC4PC) using GNOM analysis of the three small angle x-ray scattering profiles indicated in Figure 5. ........................................ 53
3.7. Small angle x-ray scattering profiles of stripped soybean oil with different water/1,2-dioleoyl-sn-glycero-3-phosphocholine molar ratios (WO). Inserted figures are the 2 dimensional small angle x-ray scattering pattern of each sample ......................................................................................... 55
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3.8. A postulated structural change of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as phospholipid concentrations are increased to above the critical micelle concentrations (CMC) form a reverse micelle and then the water:molar ratio is increased to form an anisotropic structure with a non-spherical shape ............................................... 57
3.9. The fluorescence intensity of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE) in stripped soybean oil with different water/1,2-dioleoyl-sn-glycero-3-phosphocholine molar ratios (WO). Inserted figure is the molecular structure of (NBD-PE) ....................... 58
3.10. Formation of lipid hydroperoxides (A), propanal (B) in stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) or 1,2-dibutyryl-sn-glycero-3- phosphocholine (DC4PC) and 200 ppm water at 37 °C. Data represent means (n =3) ± standard deviations. Some error bars are within data points .................................................................................................................... 59
4.1. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) during storage at 55 °C. Some of the error bars are within data points .................................................................................. 70
4.2. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with or with 10 or100 μM α-tocopherol during storage at 55 °C. Some of the error bars are within data points ........... 74
4.3. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 μM of 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) with or with 10 or100 μM α-tocopherol during storage at 55 °C. Some of the error bars are within data points ........... 75
4.4. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with or with 10 or 100 μM Trolox during storage at 55 °C. Some of the error bars are within data points ....................... 77
4.5. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 μM of 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) with or with 10 or100 μM Trolox during storage at 55 °C. Some of the error bars are within data points ....................... 78
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4.6. The neutralized fluorescence intensity of NBD-PE at stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with varying concentrations(0-100 μM) of α-tocopherol and Trolox ................................................................................................................... 81
4.7. The fluorescence lifetime decay of NBD-PE at stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with varying concentrations(0-100 μM) of α-tocopherol (A) and Trolox (B) concentrations ............................................................................ 82
5.1. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 2 mM deferoxamine (DFO) and/or 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) during storage at 55 °C. Some of the error bars are within data points ........................................ 95
5.2. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 100 M α-tocopherol, 2 mM deferoxamine (DFO) and/or 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) during storage at 55 °C. Some of the error bars are within data points ............................................................................................................ 97
5.3. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 100 M Trolox, 2 mM deferoxamine (DFO) and/or 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) during storage at 55 °C. Some of the error bars are within data points ........... 98
5.4. Depletion of α-tocopherol and Trolox (100 M) in medium chain triacylglycerols (MCT) in the presence of (A) Fe(III); and (B)Fe(II)............ 101
5.5. Depletion of α-tocopherol and Trolox (100 M) in medium chain triacylglycerols (MCT) containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) in the presence of (A) 40 ppm Fe(III); and (B) 100 ppm Fe(III) ........................................................................................... 103
5.6. Depletion of α-tocopherol and Trolox (100 M) in medium chain triacylglycerols (MCT) containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) in the presence of (A) 40 ppm Fe(II); and (B)100 ppm Fe(II) ............................................................................................. 105
xvii
5.7. Fe(III) development in medium chain triacylglycerols (MCT) containing (A) α-tocopherol or Trolox; (B) 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); (C) 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) after 24 h storage at room temperature. For each group, different letters on the top of columns represent significant differences (p < 0.05) ........................................................................................ 109
5.8. EPR detection of radical formations in the reaction consisting of (A) 0.1 M PBN in stripped soybean oil; (B) 0.1 M PBN in MCT; (C) 0.1 M PBN+100 μM α-tocopherol in MCT; (D) 0.1 M PBN+100 μM Trolox in MCT; (E) 0.1 M PBN+10 mM cumene hydroperoxides+40 ppm Fe(II) in MCT; (F) 0.1 M PBN+cumene hydroperoxides+40 ppm Fe(III) in MCT after 24 h storage at 55 oC ...................................................... 110
6.1. Proposed mechanism of bulk oil oxidation that contains reverse micelles ......... 115
LIST OF ABBREVIATIONS
xviii
BHA: Butylated hydroxyanisole
BHT: Butylated hydroxytoluene
CA: Calcein
CMC: Critical micelle concentration
DAG: Diacylglycerols
DC4PC: 1,2-dibutyl-sn-glycerol-3-phosphocholine
DFO: Deferoxamine
DOPC: 1,2-dioleoyl-sn-glycerol-3-phosphocholine
EPR: Electron paramagnetic resonance
FFA: Free fatty acid
MAG: Monoacylglycerols
MCT: Medium chain triacylglycerols
NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt
PAHs: Polycyclic aromatic hydrocarbons
PBN: N-t-butyl-phenylnitrone
PC: Phosphatidylcholine
PE: Phosphatidylethanolamine
PI: Phosphatidylinosital
PLs: Phospholipids
PS: Phosphatidylserine
SAXS: Small angle X-ray scattering
SSO: Stripped soybean oils
TAG: Triacylglycerols
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TBHQ: tert-Butylhydroquinone
TCNQ: 7,7,8,8-tetracyano-quinodimethane
TGP: Triacylglycerol polymers
TROLOX: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
1
CHAPTER 1
INTRODUCTION
Food oils from a wide variety of sources are important to human health and the food
industry since they are important components in a large variety of food products and
since they are an important cooking medium (1). However, most oils are susceptible to
oxidation as a result of their unsaturated fatty acids (2). The products formed in oxidized
oil include numerous free radical species, primary oxidation products like lipid
hydroperoxides and secondary oxidation products like aldehydes, hydrocarbons, ketones
and epoxides that negatively impact aroma (3). In addition, lipid oxidation can produce
toxic products that can adversely impact biological tissues. For instance, linoleic acid
hydroperoxides are toxic to wild type Saccharomyces cerevisiae at a low levels (0.2 mM)
(4). Oxidized palm oil has been shown to induce reproductive toxicity and organotoxicity
of the kidneys, lungs, livers, and heart of rats (5). High intake of a mixture of oxidized
lard and cod liver oil caused impaired fertility in female rats and an increased incidence
of morphologically abnormal spermatozoa in male rats (6). Oxidized lard, soybean oil,
and particularly sardine oil increased spontaneous liver tumor development and the
formation of 8-hydroxy-deoxyguanosine (8-OH-dG) in the liver DNA of mice (7). These
results along with others suggest that consumption of oxidized lipids should be avoided
whenever possible.
How to prevent or retard lipid oxidation in food oil is a major focus of lipid research.
In the past six decades, a lot of attention has been drawn on finding effective ways to
extend the shelf life of oils. The addition of antioxidants is one effective way to retard
lipid oxidation (8). The most widely used antioxidants are free radical scavengers that
2
inactive free radicals formed in the initiation and propagation steps of lipid oxidation. A
number of natural or synthetic phenols can interact with hydroperoxyl (LOO) and
alkoxyl (LO) radicals, producing hydroperoxides and alcohols, respectively, and low
energy antioxidant radicals that do not readily promote the oxidation of unsaturated fatty
acids (3).
Among the free radical scavenging antioxidants, synthetic phenolic antioxidants
[e.g., butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-
Butylhydroquinone (TBHQ) normally exhibit higher antioxidative activity than natural
antioxidants. However, the utilization of many effective synthetic antioxidants is being
limited by consumer concerns of their negative impact on health. Research showed that
large doses of both BHA and BHT (500 mg/kg body weight/day) result in certain
pathological, enzyme, and lipid alterations in both rodents and monkeys, and in some
cases BHT has been reported to have teratogenic and carcinogenic effects in rodents (9).
Over the past several decades, food scientists have been mostly unsuccessful in their
search for natural antioxidants which could possess similar antioxidant activity as
synthetic antioxidants (10). The most common natural antioxidants in foods are
tocopherol and rosemary extracts. Interestingly, rosemary extracts are not actually
approved by the FDA for use as an antioxidant but instead as a flavor extract (FDA 21
CFR 182.(11)). Since the number of antioxidant options has not significantly increased in
the past 30 years and in fact may be decreasing since food companies are reluctant to
utilize synthetic antioxidants, new approaches are needed to fulfill the goal of retarding
oil and foods from oxidation. The development of novel approaches to inhibiting lipid
oxidation is highly reliant on our understanding of lipid oxidation mechanisms in variable
3
lipid systems.
The mechanism of lipid oxidation in bulk oil has been studied for many decades (12).
However, research has revealed that this reaction is extremely complex being a balance of
the activity of numerous prooxidative and antioxidative factors (12). On top of these
chemical effects, lipid oxidation is also impacted by physical properties of food and food
components that influence factors such as partitioning of antioxidants, diffusion of
oxygen and interaction of prooxidants with lipid substrates. The impact of physical
properties on lipid oxidation can be seen in heterogeneous food systems such as
emulsions where the characteristics of the emulsion droplet interface are an important
determinant in oxidative stability (13). Even bulk oils, which are often assumed to be a
homogenous liquid, have numerous physical structures that impact lipid oxidation. This
is because refined bulk oils contain numerous minor components that are amphiphilic,
such as monoacylglycerols (MAG), diacylglycerols (DAG), phospholipids, sterols, free
fatty acids (FFA), and polar products arising from lipid oxidation, such as lipid
hydroperoxides, aldehydes, ketones, and epoxides. These surface active compounds in
combination with water can form physical structures known as association colloids that
can physically impact lipid oxidation (14).
The objective of this research is therefore to better understand how these association
colloids impact the lipid oxidation in bulk oil. Besides, how the interactions between the
minor components naturally from bulk oils and association colloids could impact the lipid
oxidation of bulk oil will be investigated. By doing so, the results can not only enhance
the oxidative stability of bulk oil by avoiding the prooxidative minor components during
refining process, but also improve the antioxidative ability of antioxidants in the bulk oil.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Oil refining procedure
The progress of oil refining has been progressed from emphasizing one operation
independently in the early days toward the current integrating manufacturing facilities to
produce a more complete range of value-added products from the raw plant seeds to the
final products. Before getting the final oils, there are several procedures need to be
carried out, i.e., storage, preparation, mechanical extraction, solvent extraction,
degumming, deodorization, bleaching, etc.
2.1.1 Storage
The typical operation associated with storage includes receiving, sampling, drying,
storage, and cleaning prior to processing. All these procedures are to make sure the seeds
are suitable to extract oil. The following description is based on the industrial soybean oil
extraction and refining processing. Initially, soybeans received are sampled and analyzed
for moisture content, foreign matter, and damaged seeds. Then the seeds are weighed and
conveyed to large silo or metal tanks for storage prior to processing. When the facility is
ready to process the seeds, the seeds are removed from the containers and cleaned of
foreign materials and loose hulls if necessary. Screens typically are used to remove
foreign materials such as sticks, stems, pods, tramp metal, etc. An aspiration system is
used to remove loose hulls from the seeds. The seeds are passed through dryers to reduce
their moisture content to approximately 10 to 11 wt % and then are conveyed to process
bins for temporary storage and tempering for 1 to 5 days in order to facilitate dehulling.
5
2.1.2 Preparation
The preparation processing is fairly well standardized consisting four principal
operations: cracking, dehulling/hull removal, conditioning, and flaking. Soybeans are
conveyed from the process bins to the mill by means of belts or mass flow conveyors and
bucket elevators. In the mill, the beans may be aspirated again, weighed, cleaned of tramp
metal by magnets, and fed into corrugated cracking rolls. The cracking rolls “crack” each
bean into four to six particles, which are passed through aspirators to remove the hulls.
Then, the cracked beans and bean chips are conveyed to the conditioning area, where
they are put either into a rotary steam tubed device or into a stacked cooker and are
heated to “condition” them (i.e., make them pliable and keep them hydrated).
Conditioning is necessary to permit the flaking of the chips and to prevent their being
broken into smaller particles. Finally, the heated, cracked beans are conveyed and fed to
smooth, cylindrical rolls that press the particles into smooth “flakes”, which vary in
thickness from approximately 0.25 to 0.51 mm. Soybean flakes allow the soybean oil
cells to be exposed and the oil to be more easily extracted.
2.1.3 Solvent extraction
The extraction process consists of “washing” the oil from the soybean flakes with
hexane solvent in a countercurrent extractor. Then the solvent is evaporated from both the
solvent/oil mixture and the solvent-laden, defatted flakes. The solvent in oil is removed
by exposing the solvent/oil mixture to steam (contact and noncontact). Then the solvent is
condensed, separated from the steam condensate, and reused. Residual hexane not
6
condensed is removed with mineral oil scrubbers. The solvent removed oil, called “crude”
soybean oil, is stored for further processing or transportation.
2.1.4 Oil refining
Crude vegetable oils predominantly contain triacylglycerols and small amounts of
minor components that naturally occur in the plant component of their origin, such as
proteins, free fatty acids, phospholipids, metals, tocopherols, pigments and sterols. The
effective removal some of those minor components without sacrificing the loss of
antioxidants is necessary to achieve a finished oil quality with acceptable standards for
flavor, appearance and stability. The oil refining process which includes degumming,
neutralization, bleaching, and deodorization is therefore performed to produce oils
suitable for use by the food industry and the consumer (Figure 2.1).
Figure 2.1. The schematic of oil refining process
Oil seed extraction will produce a crude oil that contains lipids such as
phospholipids and sterols in addition to triacylglycerols. However, the extraction process
will also produce conditions where triacylglycerols can react with enzymes such as lipase
7
and lipoxygenase to form hydrolytic products of triacylglycerols (e.g. monoacylglycerols,
diacylglycerols and free fatty acids) and lipid oxidation products such as hydroperoxides.
Oil refining is performed to reduce the concentration of these minor components as they
can negatively impact the quality of the oil. For instance, caustic alkali is used to remove
free fatty acids in neutralization step since free fatty acids cause foaming and decreases
the smoke point of oils (15). Phosphoric or citric acid is used to aid the removal of
nonhydratable and hydratable phospholipids during the degumming step since
phospholipids cause cloudiness and can form brown colors during heating (16).
Bleaching is performed with activated bleaching earth at relatively high temperatures
around 100°C to decrease the concentration of pigments (e.g. chlorophyll), trace metals
and other polar compounds that can accelerate lipid oxidation. Finally, since the harsh
conditions of these steps can produce off-flavors due to oxidation and since the oil can
contain pesticides from the original oilseeds, the oils undergoes a deodorization step at
high temperatures (200-260°C) under reduced pressure. Deodorization processing under
high temperature not only removes volatile off-flavors and pesticides but also
decomposes lipid hydroperoxides, a potential lipid oxidation substrate. Overall, refining
is designed to remove minor components that negatively impact oil quality. However,
steps such as deodorization can also remove factors that improve oil quality such as
tocopherols and can cause the formation of trans fatty acids. While refining significantly
decreases the concentration of the minor components that impact the organoleptic quality
of oil, one should realize that even at these lower concentrations, minor components can
still impact oil chemistry such as lipid oxidation.
8
2.2 Minor components in bulk oil 2.2.1 Free Fatty Acid
The reaction between water and triacylglycerols (TAG) results in the formation of
free fatty acid (FFA) and diacylglycerol (DAG). TAG hydrolysis is accelerated by lipases,
extremes in pH and heat (17). The presence of FFA produces undesirable flavors,
foaming during mixing and heating and causes a decrease in the smoke/flash point of oil
thus reducing the maximum temperature to which they can be heated. FFAs in crude
vegetable oils will increase with the age of the oilseeds and can be abnormally high if the
oilseeds have been damaged or improperly stored. This is due to damage to cells in the
seeds allowing lipase to interact with TAG. For instance, in oilseeds stored from 0 to 47
days, the FFA content in the extracted crude oil increased from 0.88 to1.80 wt % (18).
Recent research from Alencar and coworkers showed that FFA concentrations will also
increase during crude oil storage (19).
There are several factors which can impact FFA concentrations in crude oils and
during the refining process, such as oilseeds storage time before extraction (Table 2.1),
the initial FFA content, water content and processing temperatures (Table 2.2), oil type,
etc. The presence of FFA can catalyze the further hydrolysis of TAG, thereby increasing
the total FFA concentration in oil.
9
Table 2.1 Free fatty acid content in crude soybean oil as the function of bean storage time
Bean storage time(d) FFA(%)
5 0.88
11 0.60
22 1.28
35 1.40
47 1.80
The moisture in soybean seeds is 13-15 %
Table 2.2 Free fatty acid content in soybean oil as a function of processing step
Processing step FFA (%) Crude 0.61 Degummed 0.31 Refined 0.05 Deodorized 0.02
Some earlier results showed the rate of TAG hydrolysis is proportional to the initial
level of FFA (20). Another important factor is the water concentration since this is one of
the required substrates for the hydrolytic reaction. Sarkadi and coworkers showed that
increasing of water content as a result of steam deodorization of peanut oil at 180oC
increased FFA formation (21). Considering that both FFA and water are important factors
in TAG hydrolysis, the following equation was used to estimate the formation of FFA
over time (t) in the bulk oils containing water (20).
2
2
[ ][ ]' [ ][ ]Sat
H Od FFAk FFA
dt H O
(1)
Here, k’ is the rate constant, [FFA] is the free fatty acid concentration, [H2O] is the water
concentration, [H2O]Sat is the water concentration at saturation.
The FFA content in deodorized food oils is required to less than 0.05 % in the
United States (22, 23). FFA are typically removed from oils by chemical neutralization
10
which normally involves the conventional alkali (sodium hydroxide) neutralization
process, precipitating the FFA as soap stock and being removed by mechanical separation
from the oil. Recently, some oil refineries are using physical refining. Physical refining
is a high temperature, vacuum process that can remove both FFA and phospholipids at the
same time thus effectively combining the neutralization and degumming steps. The
choice of physical or chemical refining is highly correlated to the initial FFA
concentration of the crude oil. When the FFA content is lower than 2.5 % in the crude oil,
physical refining can be employed without the neutralization step. In some cases physical
refining is used on oils with > 2.5 % FFA but neutralization must be performed first (12).
A major limitation of physical refining is that the high temperatures required can cause
the formation of trans fatty acids (24).
FFA are produced at almost each step of refining since they often involve high
temperatures and water (25). Despite this FFA concentrations as low as 0.005 wt % can
be obtained (26). However, one should realize that these FFA concentrations cannot be
maintained as even low FFA concentration can promote TAG hydrolysis. Therefore, the
FFA content of retail bottled edible oil is higher than the concentrations measured
immediately after refining process (Table 2.3). For example, retail canola oil has a FFA
concentration of approximately 0.1 wt%. Finally, free fatty acid concentrations can
increase in refined oils during operations such as frying where the oils are exposed to
high temperatures and water (27).
11
Table 2.3 Free fatty acid content in retail oil
Oil type Free Fat acids (wt %)
Canola 0.1
Coconut 3.93
Corn 0.12
Olive 0.30
Extra virgin olive 0.28
Peanut 0.32
Palm kernel 2.49
Palm olein 0.15
Rapeseed 0.04
Rice bran 0.08
Safflower 0.03
Sesame 2.37
Soybean 0.05
Sunflower 0.09
2.2.2 Monoacylglycerols and Diacylglycerols
Monoacylglycerols (MAG) are monoester of glycerol in which one of the hydroxyl
groups is esterified with fatty acids. There are two isomeric forms of MAG, i.e., 1-MAG
and 2-MAG, depending on the position of the ester bond on the glycerol group.
Diacylglycerols (DAG) are esters of the glycerol in which two of the hydroxyl groups are
esterified with fatty acids. They can exist in three structural isomers namely, 1,2-DAG,
2,3-DAG and 1,3-DAG (28). DAG is a common precursor for the synthesis of both TAG
and phospholipids in the oilseeds and oil bodies (28). Therefore, DAG that exists
naturally in crude vegetable oils can be derived from the incomplete biosynthesis of TAG
12
and phospholipids. Knowledge of the amounts of DAG naturally found in oilseeds is
scare due to the lack of in situ measurements.
MAG and DAG are also formed from the partial hydrolysis of TAG (29). The
amount of DAG and MAG reported in the crude oil are therefore composed by two parts,
inherent concentrations in the seeds and those formed after crushing the seeds and during
refining. The formation of DAG and MAG depends on oilseeds storage conditions,
temperature, and moisture as was discussed previously for FFA.
Table 2.4. Concentration of MAG and DAG in various oils as a function of refining (30, 31).
Refining
step
Soybean oil Palm oil
MAG(%) DAG(%) MAG(%) DAG(%)
Crude oil 0.11 1.10 0.26 6.60
Degummed 0.10 1.44 0.17 6.70
Bleached 0.06 1.25 0.17 6.70
Deodorized 0.07 1.05 0.08 6.9
Table 2.4 lists the amount of DAG and MAG in crude and refined soybean oil and
palm oil. After bleaching and deodorization, MAG concentrations decease while DAG
concentrations remain similar to crude oil or even increase (30-32). Farhoosh and
coworkers recently showed the DAG concentration in crude soybean oil (1.66 %)
decreased to 1.35 % after the neutralization step. However, after deodorization, the DAG
concentrations (1.74 %) increased and reached around the initial amount in the crude
soybean oils (25). They also observed the similar trend in the canola oil. Possible reasons
for the increased in DAG in these reports is the existence of lipase in the crude oil which
could form DAG or from TAG hydrolysis by high temperatures and water in the
deodorization step (33).
13
2.2.3 Phospholipids
Phospholipids (PLs) are integral elements of all cellular membranes in living
organism and possess unique chemical structures containing both lipophilic and
hydrophilic groups. Crude oil contains substantially high amount of PLs due to the
solvent extraction of cell membranes. Phospholipids in oils include phosphatidylcholine
(PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinosital (PI).
Different species of seeds have different compositions of phospholipids, but in general
PC is predominant (Table 2.5).
Table 2.5 Phospholipid composition (%) as extracted from commercial seeds
Soybean Sunflower Corn Rapeseed PC 21.9 25.4 30.4 24.6 PE 13.6 11.0 3.2 22.1 PI 12.0 19.4 16.3 14.7
The amount of phospholipids in the oilseeds is small comparing to animal tissues
(34). PLs in food oils cause foaming, cloudiness and darkening during thermal processing.
Normally, there are two types of PLs in crude oil, hydratable and non-hydratable. The
degumming step removes hydratable PLs by washing them into the water phase. Non-
hydratable PLs are not affected by water washing alone so citric or phosphoric acid are
often added to the wash water to bind divalent cations thus making non-hydratable PLs
hydratable. Physical refining can also be used to remove phospholipids as discussed
above. Like other minor components, only trace amount of PLs will remain in the final
refined oil (Table 2.6) with concentrations ranging from 20-2000 ppm with variations
among different oilseeds species and refining methods (35, 36).
Table 2.6 phosphorous content (ppm) of soybean oil as a function of refining step
14
Refining step Soybean Soybean palm Soybean corn
Physical Chemical Physical Chemical
Crude 600 510 9.1 327.8 33.2 109.7 7.5 Degummed 58 120 0.7 5.7 0.8 1.24 0.6 Refined 13.1 1 0.7 1.7 0.3 0.3 0.1 Deodorized 0.16 1 0.5 0.2 0.3 0.3 0.1
2.2.4 Tocopherols
Tocopherols are the major oil soluble vitamin in crude oil. When the oilseeds are
crushed, the tocopherols are extracted along with the oil. Natural tocopherols are
mixtures consisting of tocopherols. The volatility of tocopherols is above
free fatty acids but below triacylglycerols. Thus, during the refining processing,
tocopherols are partially removed during deodorization thus decreasing their
concentrations in the refined oil. Gogolewski and coworkers found that after the refining
of rapeseed oil the tocopherol losses amounted to 30 %. Two thirds of the loss resulted
from distillation and thermal degradation during deodorization, while one third was
caused by the combined effects of neutralization and bleaching (37). Rossi and coworkers
reported that steam deodorization removed 200-300 ppm of tocopherols out of the
original ~ 1000 ppm in crude palm oil (38).
The initial concentration and type of tocopherols in bulk oil vary with different
oilseeds species. For instance, soybean oil has a high tocopherol concentrations with
more than 65% -tocopherols and 20% -tocopherols while there is almost no -
tocopherols in grape seeds oil (39). During storage tocopherol concentrations decrease
which is accompanied by the generation of oxidized tocopherols. This is because
tocopherols act as hydrogen donors and react with free radicals.
15
2.2.5 Pigments
The intensity of the bulk oils color mainly depends on the presence of coloring
pigments such as carotenoids and chlorophyll. Vegetable oils with a minimum color
index are considered to be more suitable for edible and industrial purposes (40). Thus,
pigments in crude oil are partially removed during the degumming, refining, and
bleaching steps of the refining process.
Carotenoids are pigments in many vegetable oils and particularly in palm oil. They
contain a long conjugated polyene chains and are yellow/orange/red in color. Crude palm
oil normally contains 500-700 ppm of carotenes. These are mainly -carotene (24-42 %
of total carotene) and -carotene (50-60 %) along with low levels of several other
carotenes (41).
Chlorophylls are also found in vegetable oils. Chlorophyll includes chlorophyll a,
chlorophyll b, and the Mg-free chlorophyll derivatives pheophytin a and pheophytin b.
Some studies have reported no detectable chlorophylls in soybean oil after deodorization
(42). However, other groups showed the existence of chlorophyll and pheophytins in
refined oils (43). Pheophytin was reported to be the predominant chlorophyll, being about
0.06 and 0.10 ppm in refined soybean oil and corn oil, respectively (43).
2.2.6 Miscellaneous compounds
2.2.6.1 Nonsaponifiable minor components
Nonsaponifiable minor components in food oils include sterols, polycyclic aromatic
hydrocarbons (PAHs) and hydrocarbons (e.g. alkanes, alkenes and squalene)(44). Most
vegetable oils contain 700-1100 mg/100g of sterols, partly as free and partly as esterified
16
sterols. High levels are present in rapeseed oil and in corn oil (45). Sitosterol is generally
the major phytosterol (50-80% of total sterol) with campesterol, stigmasterol, and Δ5-
avenasterol also frequently attaining significant levels. Brassicasterol is virtually absent
from the major seed oils except for rapeseed oil where it comprises 10 % of the total
sterols.
Food oils can be contaminated with PAHs due to the wide distribution of PAHs in
the environment, their lipophilic nature, and the migration from contaminated packaging
materials (46). Sekeroglu and coworkers tested 40 vegetable oil samples from Turkey for
PAHs and found the total PAHs were above 25 ppb in most of the oils samples(47). More
significantly, total PAHs levels in virgin olive oil always were 54.4-110.8 ppb(48).
However, the refined food oils only occasionally exceeded the 25 ppb limit. Recently,
Guillen and coworkers confirmed the occurrence of PAHs during the sunflower oil
oxidation stored in the close container at room temperature for 112 months (49, 50).
Squalene (C30H50) is a highly unsaturated open-chain triterpene. Olive oil is the
major commercial oil with significant amounts of squalene (51). In refined food oils, new
nonsaponifiable compounds are also formed as a consequence of the reactions occurring
during the refining process (52). These compounds include steroidal, arising from the
dehydration of sterols, terpenic from terpenic alcohols, and other compounds deriving
from squalene isomerization.
2.2.6.2 Polar triacylglycerol polymers
Polar triacylglycerol polymers (TGP) are formed during vegetable oil refining (53,
54). TGP are generally formed during the high temperature refining steps, e.g. bleaching
17
and especially deodorization. An early study on the evolution of TGP in vegetable oils
during physical refining reported a 1-2 wt % increase of TGP concentrations at physical
refining temperatures less than 240 °C. When physical refining temperatures were
increased to 270 °C, a sharp increase in TGP cocnentration to 3.0 wt % was observed.
TGP formation also depends on the degree of saturation of the oils. For more saturated
oils, such as palm oil, TGP increases were restricted to 2 wt %, even at temperatures of
270 °C (55). Gomes used high-performance size exclusion chromatography to quantify
the amount of oligopolymer in refined oils and found that refined olive oil had 0.7 wt %
of oligopolymer twice of that in refined olive pomace oil (56). The final amounts of TGP
found in food oils are generally ~1 wt % depending on the fatty acid composition, initial
quality and refining technology used (57).
2.2.6.3 Oxidized triacylglycerols
Oxidized triacylglycerols (ox-TG), sometimes referred to as core aldehydes, are
generally nonvolatile oxidation products left in the oil after deodorization. Initial reports
suggested that ox-TG concentrations ranged from 4.55 wt % in crude oil to 9.29 wt %
after oil refining (57). However, more recently Hopia investigated the impact of oil
refining on the ox-TG contents in three vegetable oils, i.e., sunflower oil, soybean oil and
low erucic acid rapeseed oil (58). The results showed that the concentration of ox-TG
ranged from 0.53 to 0.90 % oil in the crude oils. After refining, ox-TG concentrations
were decreased in bulk oil and ranged from 0.29 % for sunflower oil to 0.78 wt % for
soybean oil. The loss was attributed to the adsorption of ox-TG by the activated earth
during bleaching. Farhoosh and coworkers found ox-TG concentrations in crude soybean
18
oil and canola oil at 4.2 and 2.8 wt %, respectively (25). After oil refining, ox-TG
decreased to 2.8 and 2.0 wt % in soybean oil and canola oil, respectively. They also
noticed the level of the ox-TG in the refined oils was proportional to its original level in
the crude oils.
2.2.7 Water
Since water and oil have opposite polarities and they tend to spontaneously phase
separate because of the hydrophobic effect one might not expect to find water in bulk oil
(59). However, as discussed in the proceeding sections, refined oils contain many surface
active compounds such as free fatty acids (FFA), monoacylglycerols (MAG),
diacylglycerols (DAG) and phospholipids (PLs). These surface active compounds have
the ability to emulsify water into bulk oils. The water content in freshly-opened,
commercially available vegetable oils range from 200-2000 ppm (Table 2.7) (14). Water
concentrations in oil can change after opening since oil can absorb water from
atmosphere or water in the oil can evaporate.
Table 2.7. Water content in freshly-opened, commercially available vegetable oils as determined by Karl Fischer Coulometer
Oil Water Content(ppm)
Sunflower 203
Canola 236
Peanut 221
Sesame 197
Extra virgin olive 865
Water in refined oil is derived in two possible ways. The first is from the original
water in the oilseeds. Sorption isotherms experiments showed water with three levels of
19
affinity in soybean seeds: (i) a region of strongly bound water at moisture concentrations
below 8%; (ii) a region of weakly bound water at moisture concentrations between 8 and
24%; and (iii) a region of very loosely bound water at concentrations greater than 24%
(60). When soybeans are dried for storage, the moisture content is ~ 10 wt %. That water
is likely the strongly bound water in the oilseed but it is possible that some of this water
ends up in the crude oil. The second possible source of water is the water used to wash
oils and remove the free fatty acids and phospholipids during the neutralization and
degumming steps. The majority of this water is removed by centrifugation. The
remaining water is removed by a vacuum dryer that controls the moisture content of the
washed oil to below 1000 ppm, most often in the range of 500 ppm (61).
2.2.8 Oxygen
Oxygen is a primary reactant in lipid oxidation reaction which fuels the fatty acid
decomposition pathway that causes rancidity. There are two types of oxygen in bulk oil
systems, dissolved oxygen (DO) and non dissolved oxygen. Dissolved oxygen is
incorporated into food while non dissolved oxygen stays in the headspace (62). The
solubility of oxygen and the rate of its diffusion in the bulk oil are relevant to the rate and
extent of lipid oxidation. Oxygen is as about 3-10 times more soluble in bulk oil than
water (saturation occurs at 5-10 ppm in pure water at 20 C) (63, 64). Schrader and
coworkers determined that the diffusion coefficient of oxygen in soybean oil was 0.6×10-
9 m2.s-1 at 20 °C which is lower than that of olive oil (65). The impact of temperature on
the solubility of oxygen in different types of oil is listed in Table 2.8. Some researchers
suggested that oxygen solubility was related to fatty acid chain length with oxygen
20
solubility decreasing with increasing the hydrocarbon chain length (66).
Table 2.8. Solubility of oxygen (ppm) in Marine oil
Oil type Temperature(oC)
20 40 60 80
Redfish oil 20.4 25.8 29.6 4.1
Capelin oil 19.5 26.6 34.4 8.5
Herring oil 35.8 42.2 45.2 25.8
Mackerel oil 13.6 25.6 28.7 3.7
Harp seal oil 29.1 35.7 37.9 10.1
Flounder oil 23.2 25.6 26.9 9.6
Cod liver oil 39.3 47.6 43.0 6.0
The pressure is 101.325 Kpa
Another form of oxygen associated with bulk oil is non dissolved oxygen or
headspace oxygen. The air space above the oil, for example in the oil tank or commercial
oil bottle, is where headspace oxygen located. The headspace oxygen content in olive oil
and marine oil at temperature lower than 60 °C increases with the increase of temperature,
which following the rule of the Henry’s Law, i.e., the solubility is proportional to the
partial pressure of headspace gas (67). In other words, as temperatures increase, oxygen
in the oil moves into the headspace. At temperatures higher than 60 °C there is a large
variation in the solubility of oil, especially in marine oil presumably due to heat
accelerated oxidation which consumes oxygen (Table 2.8.). The mass transfer between
headspace oxygen and oxygen in the oil is influenced by: (i) the rate of absorption; (ii)
the oxygen concentration in the headspace; (iii) the headspace surface area to volume
ratio; and (iv) agitation. With those factors being considered, an equation was developed
to describe the absorption rate of oxygen between bulk oil and oxygen in the headspace
21
(20).
( )O
dC KAC C
dt V
(2) Here, Co is the equivalent solubility of oxygen in the bulk oil; C is the oxygen content in
bulk oil after t times; K is the mass transfer coefficient; A/V is the area to volume ratio.
2.3 The effect of minor components on bulk oil oxidation
2.3.1 The effect of oxygen on the chemical stability of bulk oil
Lipid oxidation is not a spontaneous reaction. Thermodynamically, oxygen cannot
react directly with double bonds because the spin states are different. Ground state,
atmospheric oxygen is in a triplet state, whereas the double bond of unsaturated fatty
acids is in singlet state. Quantum mechanics requires that spin angular momentum be
conserved in reactions. Therefore, interactions between triplet oxygen and unsaturated
fatty acids demands that either the double bond be excited into a triplet state or oxygen is
converted to a singlet state. The former seems impossible due to the requirement of
prohibitive amounts of energy. The triplet oxygen also cannot convert to singlet states
itself thus direct reaction occurs between the oxygen and double bonds in oil do not occur
unaided (68). However, under the assistance of other minor components in bulk oil,
several reactions can happen. For example, photosensitizers such as chlorophylls can
produce singlet oxidation by mechanisms 3-5 outlined below.
Sen + hv → Sen* (3)
Sen* + 3O2 → 1O2* (4)
1O2* + LH → LOOH (5)
In addition, oxygen along with iron can generate alkyl radicals (L) as shown in
reactions 6 and 7. Schafer and coworkers proposed that iron-oxygen complexes ([Fe2+-
22
O2]) are one route by which iron can promote lipid oxidation in cell membrane (69).
Fe2+ + O2 → [Fe2+-O2] (6)
[Fe2+-O2] + LH → L (7)
Triplet oxygen is directly involved in lipid oxidation propagation through its ability
to react with alkyl radicals in diffusion controlled radical-radical reactions to form
peroxyl radicals as shown in reactions 8 and 9 (70):
L + O2 ↔ LOO (8)
LOO + RH → LOOH+ R (9)
Because of these multiple pathways that oxygen can be involved in lipid oxidation,
it is not surprising that many studies have shown that reduction of oxygen in packaged
bulk oil can extend shelf life (71, 72). An 18-months shelf life test, performed on virgin
olive oil indicated that the slowest oxidation rates occurred at the lowest initial dissolved
oxygen concentration (71). The same group also reported that removal of oxygen from
extra virgin olive oil by nitrogen purging lowered peroxide values (72). Packaging
technologies such as oxygen scavenging film can also improve the oxidative stability of
bulk oil (73, 74).
2.3.2 The effect of water on the chemical stability of bulk oil
Despite being present at relatively low levels, water could have an impact of the rate,
extent and mechanism of lipid oxidation in bulk oils since it may act as a solvent for
hydrophilic or amphiphilic antioxidants and prooxidants (such as transition metals, free
fatty acids, or lipid hydroperoxides
23
2.3.3 The effect of free fatty acid on the chemical stability of bulk oil
Generally, FFA with unsaturated bonds showed a lower oxidative stability than their
corresponding methyl esters and triacylglycerols (75). In addition, FFA themselves have
been reported to accelerate the oxidation of triacylglycerols in vegetable oils. FFA have
been shown to have several different prooxidative tendencies. Yoshida and coworkers
demonstrated that FFA (i.e., caprylic, capric, lauric, myristic, palmitic or stearic acid) in
microwaved stripped soybean oil increased oxidation with shorter the chain length and
the higher the levels of FFA being more prooxidative (76, 77). Aubourg and coworkers
later showed similar results in marine oil with a higher degree of oxidation seen in the
presence of short chain (lauric and myristic) than long chain (stearic and arachidic) fatty
acids at 30 oC (78). Paradiso and coworkers recently reported that low amounts of FFA
caused an increase in the formation of oxidized triacylglycerol and triacylglycerol
oligopolymers again showing the prooxidant activity of FFA (79). The prooxidant activity
of FFA is associated with its free acid group since methyl esters of fatty acids do not
accelerate oxidation. Proposed mechanisms of FFA include their ability to accelerate the
decomposition of hydroperoxides and bind metals to make them more prooxidative (75).
Obviously, it is very critical to control the levels and formation of FFA in crude, refined
and stored oils. While most commercial oils have FFA concentrations less than 0.05 wt %,
there may be addition benefits if these levels could be even lower. Minimizing FFA
formation during storage can be accomplished by decreased water exposure, minimizing
temperatures, preventing contact with lipases and avoiding exposure to extremes in pH.
2.3.4 The effect of MAG and DAG on chemical stability of bulk oil
24
Different impacts of MAG and DAG on the oxidative stability of bulk oils has been
reported. Min and coworkers found that 0-0.5 wt % of monostearin, distearin,
monolinolein, or dilinolein acted as prooxidants in soybean oil (80, 81). Colakoglu and
coworkers found that soybean oil containing 1 wt % monoolein increased the rate of
oxygen consumption (82). Wang and coworkers observed that randomized corn oil
contained higher levels of MAG and DAG (0.3 and 5.1 %, respectively) oxidized much
faster than natural corn oil with no detectable MAG and 1.4% of DAG(83). On the
contrary, Gomes et al found that1-3 wt % MAG decreased oxidation in stripped olive oil
at 60 °C (84). The effect of combinations of MAG or DAG with antioxidants in the bulk
oil has also been studied. MAG or DAG in combination with citric acid resulted in
greater antioxidant activity than the MAG or DAG itself (85). In addition, as the chain
length of the fatty acids on MAG or DAG was increased, the more effective citric acid
became.
2.3.5 The effect of phospholipids on the chemical stability of bulk oil
The impact of phospholipids (PLs) on bulk oil oxidation is also controversial.
Dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine
(DPPE) were reported to have poor antioxidant activity at 50oC and showed no
synergistic effect with α-tocopherol in methyl linoleate (86). Phosphatidylcholine (PC)
and phosphatidylethanolamine (PE) from egg yolk accelerated the oxidation of methyl
linoleate (86). Takenaka and coworkers also found that PC and PE promoted bonito oil
oxidation in the absence of α-tocopherol. However, they found that PE showed
synergistic antioxidant activity with α-tocopherol, while PC did not (87).
25
Alternatively, Bandarra and coworkers showed that 0.5 % PC was an effective
antioxidant in sardine oil at 40oC. In addition, a high synergistic effect was observed in
the same system with a mixture of α-tocopherol and PE (88). Others have reported
similar results that PLs can increase the activity of tocopherols in bulk oils (89-92). For
the researchers who observed the antioxidant activity of PLs in bulk oil, metal chelating,
free radical scavenging and formation of Maillard reaction products were given as the
mechanisms by which PLs inhibit oxidation (93-95).
2.3.6 The effect of tocopherols on the chemical stability of bulk oil
Tocopherols are the most common free radical scavenging antioxidants found in
vegetable oils. Vegetable oils normally contain tocopherols concentrations in the range of
200-1000 ppm which originate from the seeds. After refining, almost 70 % of the
tocopherols remain in the bulk oil with 30 % being removed during deodorization.
Overall, tocopherols are probably the most important antioxidants in vegetable oils (for
review see refs (96, 97)). The antioxidant mechanisms of tocopherols (TOC) are shown
below.
LOO + TOC → LOOH +TOC (10)
LOO + TOC → LOO-TOC (11)
However, under certain conditions tocopherols can also act as prooxidants. This is
thought to be due to their ability to reduce endogenous transition metals naturally
occurring in the oil or metals resulting from contamination during processing. These
reduced metals can then promote the decomposition of pre-existing lipid hydroperoxides
as shown in pathways 12 and 13. Yoshida and coworkers found that both α-tocopherol
26
(100 M) and α-tocotrienol (100 M) reduced cupric iron (300 M) and the
concomitant formation of α-tocopheryl quinone and α-tocotrienyl quinone was observed
by UV absorption spectrum and also by HPLC analysis in methyl linoleate. Interestingly,
the -, and -forms of tocopherols were not found to reduce cupric iron in the same
system (98)
TOC +Mn2+ → TOC + + Mn+ (12)
LOOH + Mn+ → LO +HO-+ Mn2+ (13)
The prooxidant activity of tocopherols has also been proposed to be due to the
ability of the tocopherol radical to promote fatty acid oxidation especially when
tocopherol radical concentrations are high (98-100).
LOOH + TOC → LOO + TOC (14)
LH + TOC → L + TOC (15)
Trace amounts of oxidized tocopherol exist in bulk oil after refining. Min and
coworkers reported that oxidized α-, γ- and δ-tocopherols promote the oxidation of
purified soybean oil in the dark at 55 °C (101, 102). Since it is possible that oxidized
tocopherols occur naturally in bulk oil and that tocopherol ingredients could contain
oxidized tocopherols, this could be a source of prooxidants in oils. More work is need to
determine if and how oxidize tocopherol could impact the antioxidant/prooxidant activity
of tocopherols.
2.3.7 The effect of pigments on the chemical stability of bulk oil
The two major pigments that can impact lipid oxidation in oils are cholorophyll and
carotenoids. Chlorophyll, a photosensitizer, is prooxidative when exposed to light due to
27
its ability to promote the formation of singlet oxygen (103). As described previously,
singlet oxygen is formed by chlorophyll photosensitization by energy transfer from light
to a sensitizer and then to triplet oxygen (104). Singlet oxygen can then initiate lipid
oxidation because it can directly react with the double bonds of unsaturated fatty acids to
form lipid hydroperoxides (105). In the bulk oil, it is important to remove as much
chlorophyll as possible during refining since most retail oils are stored in transparent
plastic packages.
Unexpectedly, chlorophyll and its degradation product, pheophytin, have also been
reported to inhibit lipid oxidation in the rapeseed and soybean oils at 30 °C during
storage in the dark. This has been proposed to be due to their ability to scavenge peroxyl
and other free radicals (106).
Carotenoids can be efficient singlet oxygen and excited photosensitizers quenchers
that reduce oxidation by converting excited photosensitizers or singlet oxygen to their
less reactive states (107). Burton also proposed that under low oxygen partial pressure
conditions, β-carotene can act as a lipid soluble chain breaking antioxidant (108). They
also found that at oxygen pressures of 20 kpa or higher, β-carotene and related
compounds are prooxidants in a methyl linoleate model system possibly due to the
formation of carotenoids oxidation products (109). During high temperature oil refining,
carotenoids can also become thermally degraded. Thermally degraded carotenoids were
reported to accelerate lipid oxidation in soybean oil at concentration of 50 ppm (110).
More research is needed to elucidate how chemical changes of carotenoids impact the
oxidative stability of oils.
28
2.3.8 The effect of miscellaneous compounds on the chemical stability of bulk oil
Small amounts of squalene (200 ppm) were reported to have a limited impact on the
oxidative stability of stripped olive oil 40 and 62 °C in the dark, whereas higher
concentration (7000 ppm) of squalene showed an antioxidative effect at the same system
(111). In rape seed oil, Malecka found that squalene (4000ppm) increased the oxidative
stability of oil heated at 170 °C for 10 h (112). No significant impact of squalene (0-
8000ppm) on stripped sunflower oxidation was observed by Mateos and coworkers (113).
Oppositely, squalene was found to accelerate oxidation of stripped olive oil in a
Rancimat apparatus at 100 °C (114).
The effect of sterols on the oxidation of olive oil was studied at 180°C by Gordon
(115). They found that Δ5-avenasterol and fucosterol were effective as antioxidants at
concentration of 0.1 wt %, while other sterols, including cholesterol and stigmasterol,
were ineffective. The proposed mechanism was that lipid free radicals could react rapidly
with sterols at unhindered allylic carbon atoms (115). Phytosterols have also been
reported to act as antioxidants in soybean, rice bran and sunflower (116-118).
Yoon and coworkers found that the oxidized triacylglycerol fractions obtained by
thermal oxidation of soybean oil acted as prooxidant when it was added to stripped
soybean oil (119). Gomes and coworkers found that oxidized triacylglycerol
oliogopolymers, a class of oxidation compounds present in refined vegetable oils, were
the most prooxidative in vegetable oil amongst all the oxidized triacylglycerol fractions
tested (120).
2.4. Association colloids and lipid oxidation in bulk oils
29
2.4.1 Evidence for the presence of association colloids in bulk oil
As discussed briefly above, one of the major issues that is often overlooked in bulk
oil oxidation is the physical properties of minor components (121). Bulk oil minor
components, such as monoacylglycerols (MAG) and diacylglycerols (DAG),
phospholipids (PLs), sterols, free fatty acids (FFA), and polar products arising from lipid
oxidation are surface active compounds. These surface active molecules have the ability
to form physical structures in the bulk oils in the presence of the small quantities of water
(~300 ppm) typically found in refined oils (14, 121). These structures are known as
association colloids. Recent research suggests that association colloids could be lipid
oxidation reaction sites in oils.
Previous studies have demonstrated that surface active molecules can self-assemble
in non-polar solvents (e.g. benzene, isooctane, cyclohexane, toluene, free fatty acid and
triacylglycerols) in the presence of water to form a variety of association colloids such as
reverse micelles, micro-emulsions, lamella structures, and cylindrical aggregates (Figure
3) (122-125). For instance, Sosaku and coworkers formed reverse micelle systems using
soybean phospholipids as a surfactant with fatty acids or fatty acid ethyl esters as the
organic solvent (126). Sinoj and coworkers recently formed nutrient delivery systems
consisting of water-in-oil nanoemulsions using oleic acid embedded in canola oil (127).
Chen and coworkers identified ordered lamellar structures in hazelnut oil in the presence
of monoglycerols (MAG) (128, 129). These studies suggest that the amphiphilic
molecules in refined vegetable oils have the ability to form association colloids (130).
30
Figure 2.2. Schematic representation of the association colloids formed by minor constituents in bulk oil (A) a reverse micelle formed by oleic acid; (B) a reverse micelle formed by phospholipids; (C) Inverse lamella structure formed by monoacylglycerol; (D) a mixed reverse micelle formed by free fatty acids and phospholipids. There are several lines of evidence that association colloids occur naturally in refined and
crude oils. The complete removal of water from refined oil is very difficult even at
temperatures up to 200 oC. This suggests that some of the water in oil is bound to polar
compounds possibly in association colloids. We have indirectly seen this in our research
since water content in oil is reduced by the removal of polar compounds. In addition, it
was observed by research groups using ultrafiltration (UF) that only a small portion of the
phospholipids in oils penetrates through UF membranes with a pore diameter of the order
of 4 nm (131-133). The proposed reason for the rejection of phospholipids by these non-
porous membranes was suggested to be due to the formation of phospholipids reverse
micelles, swollen in the presence of small quantities of water plus containing other minor
components such as pigments, which are then rejected by size exclusion. The amount of
rejection phsopholipids closely depended on the amount and the size of reverse micelles
formed during solvent extraction (134-136).
2.4.2 Do association colloids impact lipid oxidation?
31
Many of the compounds involved in lipid oxidation reactions such as lipid
hydroperoxides, free fatty acids and antioxidants are surface active. Previous research
with oil-in-water emulsions has shown that the physiochemical property of the water-oil
interface plays a very important role in lipid oxidation chemistry since it can impact the
location and reactivity of both prooxidants and antioxidants (137). This suggests that the
water-oil interface of association colloids could also impact lipid oxidation chemistry by
acting as nano-reactors. Some of the first research in this area was by Koga and Terao
who observed that the presence of phospholipids enhanced the antioxidant activity of α-
tocopherol in stripped oil containing a trace amount of water (1%) (92). This study found
that the presence of phospholipid increased the degradation of α-tocopherol in the
presence of the water soluble free radical generator, 2,2-azobis(2-amidinopropyl)
dihydrochloride (AAPH) suggesting that the phospholipids increased the exposure of α-
tocopherol to the water phase. This could also be due to the fact that the reduction
potential of α-tocopherol is lower in polar environments which could make tocopherol a
more efficient free radical scavenger (138). Degradation of α-tocopherol by the water-
soluble free radicals decreased as the phospholipid’s hydrocarbon tail group size was
decreased, and thus the ability of the phospholipid to form association colloids was lost.
Koga and Terao also found that the antioxidant activity of α-tocopherol could be
increased in bulk oil when it was conjugated to the polar head group of
phosphatidylcholine (PC) (95). This increase in activity was again thought to be due to
the increased partitioning of the reactive portion of α-tocopherol into the water phase of
the association colloids in bulk oil (95). This pioneering work was the first to suggest that
the presence of physical structure in bulk oils could impact lipid oxidation chemistry by
32
altering the activity of free radical scavenging antioxidants.
Wilailuk and coworkers evaluated the ability of water, cumene hydroperoxide, oleic
acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil
(n-hexadecane) system containing sodium bis(2-ethylhexyl) sulfosuccinate (AOT)
reverse micelles. This study found that water, cumene hydroperoxide, oleic acid, and
phosphatidylcholine can alter reverse micelle size and lipid oxidation rates (139, 140).
Kasaikina and coworkers found the decomposition of cumene hydroperoxides into free
radicals was accelerated by the existence of reserve micelle formed by cationic
surfactants in organic media (130, 141-144). In addition, they found the oxidation
stability of sunflower oil at 80oC in the presence of 0.1 mM 2,6-di-tert-butyl-4-
methylphenol (BHT) was decreased in the presence of fatty alcohols, such as 1-
tetradecanol, 1-octadecanol, and the MAG, 1-monopalmitoylglycerol. This result was
explained by the so-called "micellar effect": the relatively higher concentration of polar
species such as hydroperoxide and peroxyl radicals within and nearby the reverse
micelles formed in the presence of these fatty alcohols and MAG, leading to an increase
of the rate of chain initiation via an acceleration of the hydroperoxides decomposition
(145).
2.4.3 The perspective researches on the mechanism of lipid oxidation in bulk oil
Currently in the food industry, the trends of changing lipid profiles to contain more
polyunsaturated fatty acids and less hydrogenated fats result in food products with an
increased susceptibility to rancidity. In the meantime, the efficacy antioxidants available
in FDA “direct contact with food” category are limited. To overcome the challenge of
33
developing low trans fatty acids products with nutritionally significant amounts of
unsaturated fatty acids, the food industry must develop new antioxidant technologies. The
overall objective of this research will be to gain a better understanding of how the
chemical and physical properties of edible oils impact the deterioration of
polyunsaturated lipids. In particular, focus will be on how nano-sized structures in bulk
oil (association colloids) impact the oxidative stability of lipids since above mentioned
evidences indicate that these physical structures are the site of oxidation reactions.
Understanding how the physical nature of edible oils impacts rancidity could lead to the
development of new antioxidant technologies and to the more efficient use of existing
antioxidant ingredients.
The framework of this research include:(1) producing association colloids in
stripped soybean oil using inherent minor component(s) in bulk oil. The well-defined
structure will be characterized by all sorts of techniques; (2) how the physical properties
of association colloids impact the chemical properties of bulk oil in a more complex
system after the addition of minor oil components, including different phospholipids,
antioxidants and prooxidant (i.e.,transition metals); (3) elucidate the physical location of
these minor components in the association colloids. The relationship of the physical
location and chemical reactivity of the minor components will then be compared to
determine their relative overall ability to inhibit or promote lipid oxidation in bulk oil. By
better understanding how physical structures in bulk oils impact reactions that cause
rancidity, it is hoped that new antioxidant technologies can be developed.
2.5 Methodologies to study the impact of minor components on the physicochemical
34
properties of bulk oil
2.5.1 Chemical analysis
2.5.1.1 Primary lipid oxidation products
Lipid hydroperoxide in stripped soybean oil with or without association colloids will
be determined (146). Precisely weighted of oil will be mixed with 2.8 mL of methanol/1-
butanol (2:1). The reaction will be started by added 15 μL of 3.94 M ammonium
thiocyanate and 15 μL of ferrous iron solution. After 20 min of incubation at room
temperature, the absorbance will be measured at 510 nm using an UV-vis
spectrophotometer. Hydroperoxides concentrations will be determined using a standard
curve prepared from hydrogen peroxide.
2.5.1.2 Secondary lipid oxidation products
Headspace hexanal will be determined using a gas chromatograph (147). The
headspace conditions will be as follows:Sample (1 mL) in 10 mL glass vials capped with
aluminum caps with PTFE/silicone septa were pre-heated at 55 °C for 15 min in an
autosampler heating block. A 50/30 μmDVB/ Carboxen/PDMS solid-phase
microextraction (SPME) fiber needle from Supelco (Bellefonte, PA, USA) was injected
into the vial for 2 min to absorb volatiles and then was transferred to the injector port
(250 °C) for 3 min. The injection port was operated in split mode, and the split ratio was
set at 1:5. Volatiles were separated on a Supleco 30 m × 0.32 mm Equity DB-1 column
with a 1 μm film thickness at 65 °C for 10 min. A flame ionization detector was used at a
temperature of 250 °C. Concentrations will be determined using a standard curve made
from hexanal.
35
2.5.2 Physical analysis
2.5.2.1 Small/wide angle x-ray scattering (SAXS/WAXS)
X-ray scattering is powerful method for unraveling structural details and molecular
shapes (148). The physical principles of SAXS/WAXS are similar. Firstly, the electric
field of the incoming wave induces dipole oscillations in the atoms, then the accelerated
charges generate secondary waves that add at large distanced (far field approach) to the
overall scattering amplitude. All secondary waves have the same frequency but may have
different phases caused by different path lengths.
Figure. 2.3. The essential parts of a small angle scattering system. The drawing shows the X-ray source X, the sample S, the scattering angle θ, the slits used to define the incident and scattered beams, and the detector D.
X-ray from the source is formed into a fine beam, often by slits, and strike the
samples. A small fraction of this beam is scattered in other directions which forms an
angle with the direction of the incoming beam. A detector is used to record the scattering
intensity which depends on the scattering angle. The ordered structure information of the
sample can often be obtained from the analysis of the scattering intensity at a sequence of
scattering angles.
sin4q
(16)
36
Also, from the relation between the diffraction peaks, the nature of ordered materials,
such as liquid crystalline phase may be identified. When the composition of the sample is
known together with the nature of materials, X-ray diffraction data can be used to extract
information about the characteristic dimensions of materials. Also for systems without
long-range order, SAXS may provide useful information for micelles, liposomes and
other disperse systems, as well as on gel structure (149).
2.5.2.2 Steady-state and time resolved fluorescence measurement
Luminescence is the emission of light that can occur when a molecule excited to a
higher order electronic state relaxes back to the ground state. Depending on the electronic
nature of the excited state, singlet (S1) versus triplet (T1), luminescence is divided into
two categories, fluorescence and phosphorescence, respectively (150).
Fluorescence measurements can be typically made in two regimes, i.e., steady state
and time resolved measurements. Steady-state measurements are measured with constant
illumination and observation. Due to the nanosecond timescale of fluorescence, the
steady-state condition for a system is normally reached almost immediately after sample
is illuminated. In contrast, time-resolved measurements involve monitoring the temporal
dependence of a given fluorescence parameter, like emission intensity or anisotropy. For
these experiments the sample is exposed to a pulse of light that is shorter than the decay
time of the sample and measurements are then made using a high-speed detection system,
capable of making discrete observations within a nanosecond time regime. Although
time-resolved experiments are more complex, these measurements can provide novel
information that may not be observed in steady-state measurements due to averaging
37
processes. For example, anisotropy decays of fluorescent macromolecules are frequently
more complex than single exponential. The precise shape of the anisotropy decay
contains information about the shape of the macromolecule and its flexibility. Time-
resolved measurements of intensity decays may also contain information that is obscured
in steady-state measurements due to averaging processes (151, 152).
2.5.2.3 Interfacial tension analysis
Interactions occur between the molecules of a liquid and those of any liquid which is
not soluble in the liquid. These result in the formation of an interface. Energy is required
to change the form of this interface. The work required to change the shape of a given
surface is known as the interfacial tension.
Surfactants consist of a hydrophilic head and a hydrophobic tail. If a surfactant is
added to oil then it will initially enrich itself at the surface; the hydrophilic head projects
from the surface. Only when the surface has no more room for further surfactant
molecules will the surfactant molecules start to form agglomerates inside the liquid.
These agglomerates are known as reverse micelles. The interfacial tension of system will
decline as the increasing of surfactant concentration. The steady lowest interfacial tension
of the system will reach when reverse micelle formed after which will not change. The
surfactant concentrations at which reverse micelle formation is known as the reverse
critical micelle formation concentration (CMC). By doing interfacial tension analysis, the
surface active properties of minor components are able to be investigated.
38
CHAPTER 3
THE IMPACT OF PHYSICAL STRUCTURES IN SOYBEAN OIL
ON LIPID OXIDATION
3.1 Introduction
Bulk oil is often assumed to be a homogenous liquid, in contrast to more complex
multiphase systems, such as emulsions, which are regarded as heterogeneous liquids.
However refined oil, which is processed to remove many of the non-triacylglycerol
fractions, still contains numerous minor components that are amphiphilic, such as
monoacylglycerols, diacylglycerols, phospholipids, sterols, free fatty acids, and polar
products arising from lipid oxidation, such as lipid hydroperoxides, aldehydes, ketones,
and epoxides (121, 153, 154). In addition, refined oils also contain small but significant
amounts of water. Previous studies have demonstrated that surface active molecules can
self-assemble in the presence of water to form a variety of association colloids in non-
polar solvents (e.g. benzene, isooctane, cyclohexane, toluene, free fatty acid and
triacylglycerols), such as reverse micelles, micro-emulsions, lamella structures, and
cylindrical aggregates (122-124). For instance, Sosaku and coworkers formed reverse
micelle systems using soybean phospholipids as a surfactant in fatty acid or fatty acid
ethyl esters as the organic solvent (126). Sinoj and coworkers recently formed nutrient
delivery systems consisting of water-in-oil nanoemulsions using oleic acid embedded in
canola oil (127). These studies suggest that the amphiphilic molecules in refined
vegetable oils have the ability to form association colloids (130).
The mechanism of lipid oxidation in bulk oil has been studied for decades (155).
Research over the past two decades has shown that in emulsion systems, lipid oxidation
39
is strongly influenced by the physical properties of the emulsion droplets and their
interfaces, which can impact pro-oxidant/lipid interactions and the location of
antioxidants (156). Most previous research has treated bulk oil as a homogeneous system
without considering how lipid oxidation reactions could be influenced by their physical
structure. If bulk oils are actually micro-heterogeneous systems containing association
colloids, it is highly likely that these physical structures will impact lipid oxidation as
they do in emulsion systems.
To better understand how microstructures impact the oxidation of bulk oil, it will be
necessary to develop model association colloid systems that reflect the types of physical
structures that would be expected to exist in refined vegetable oils. Therefore, in this
study, a model system consisting of different concentrations and types of phospholipids
[1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dibutyryl-sn-glycero-3-
phosphocholine (DC4PC), Figure 3.1] and water was used to characterize association
colloids in stripped soybean oil and to evaluate their impact on lipid oxidation.
Figure 3.1.Molecular structures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC).
3.2 Materials and Methods
40
3.2.1 Materials
Avanti Polar Lipids, Inc (Alabaster, AL) was the source of 1,2-dioleoyl-sn-glycero-
3-phosphocholine (DOPC) and 1,2-dibutyryl-sn-glycero-3- phosphocholine (DC4PC). N-
(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-
phosphoethanolamine, triethylammonium salt (NBD-PE) was acquired from Invitrogen
(Carlsbad, CA). Soybean oil was purchased from a local store and stored at 4 °C. Silicic
acid (100-200 mesh), activated charcoal (100-400 mesh), 7,7,8,8-TCNQ (7,7,8,8-
tetracyano-quinodimethane) and hexane were purchased from Sigma-Aldrich Co. (St.
Louis, MO). Medium chain triacylglycerols (MCT, Miglyol®) were purchased from
Sasol North America Inc (Houston, TX). All other reagents were of HPLC grade or purer.
Distilled and deionized water was used in all experiments.
3.2.2 Preparation of stripped soybean oil
Stripped soybean oil was prepared according to the method of Boon et al (157). In
short, silicic acid (100 g) was washed three times with a total of 3 L of distilled water and
activated at 110 °C for 20 h. Activated silicic acid (22.5 g) and activated charcoal (5.625
g) were suspended in 100 and 70 mL of n-hexane, respectively. A chromatographic
column (3.0 cm internal diameter × 35 cm height) was then packed sequentially with 22.5
g of silicic acid followed by 5.625 g of activated charcoal and then another 22.5 g of
silicic acid. Thirty grams of soybean oil was dissolved in 30 mL of hexane, which was
then loaded onto the column and eluted with 270 mL of n-hexane. To retard lipid
oxidation during stripping, the collected soybean oil was held on ice and covered with
aluminum foil to eliminate the impact of light. The solvent in the stripped oil was
41
removed with a vacuum rotary evaporator (RE 111 Buchi, Flawil, Switzerland) at 37 °C,
and the remaining trace solvent was removed by flushing with nitrogen for at least one
hour. The colorless oil was kept at -80 °C for subsequent studies. Removal of minor
components was verified by spotting a sample on a silica gel G thin layer
chromatography (TLC) plate and developed in a tank with hexane/diethyl ether/acetic
acid (70:30:1 v/v/v) (158). Water content of the stripped oils was determined by Karl
Fisher (159).
3.2.3 Light Scattering Properties of Oil Samples
A series of samples were prepared to determine the DOPC and water concentrations
that produced association colloids without causing cloudiness in the oil. A stock solution
of DOPC was prepared at a concentration of 127 moles DOPC/mL chloroform, which
was blended into MCT over the concentration range of 0 to 1270 mole/kg lipid which is
in the range of phospholipid concentrations found in refined bulk oils (~ 2000 moles/kg
lipid). The mixture of MCT and DOPC was then titrated with water. The initial water
concentration in the samples was 0.67 mmoles/kg lipid (determined by Karl Fisher
analysis) and was increased to 560 mmoles/kg lipid by adding water. The vials were
tightly closed with Teflon-lined caps and placed on a stirrer motor at 25 °C for 24 hours
before measurements. The final water concentrations were verified by Karl Fisher
analysis. The clarity of the samples was measured by light scattering using an Agilent
7010 particle size spectrophotometer. The Agilent 7010 particle size spectrophotometer
measures the attenuation of a colloidal dispersion from 350-800 nm. The transparent
region was defined as samples with a measured attenuation 0.2 cm-1 (visibly clear),
42
while the cloudy region was defined as samples with attenuation > 0.2 cm-1.
3.2.4 Determination of critical micelle concentration (CMC) in stripped soybean oil
The procedure described by Kanamoto and coworkers (160) was followed to
spectrophotometrically determine the CMC of the phospholipids in stripped oil. In short,
varying concentrations of DOPC or DC4PC were dissolved in either SSO or MCT by
mixing for 12 h followed by addition of 7,7,8,8-tetracyano- quinodimethane (TCNQ,
5mg) to 5 g of oil/DOPC in a small conical flask and the mixture was agitated using a
magnetic stirrer for 5 h at room temperature. After sedimentation of excess TCNQ by
centrifugation at 2000 g for 20 min, absorbance measurements at 480 nm were recorded
(Shimazu 2014, Tokyo, Japan). SSO oil or MCT without DOPC was used as a control.
The CMC was taken as the intersection point of straight lines extrapolated from low and
high DOPC concentrations in the curve generated from absorbance and DOPC
concentrations on a semi-log plot.
The CMC of DOPC in SSO was also determined by interfacial tension (IFT)
measurements using Drop Shape Analysis (DSA100, Krüss GmbH, Hamburg, Germany)
equipped with a pendant drop module. A needle with a diameter of 1.830 × 10-3 m was
used to create a pendent drop (containing the stripped soybean oil and varying
concentrations of DOPC). The pendant drop was extruded from the syringe into a quartz
cell containing distilled water. Droplet images were taken every 2 s and the drop shape
analysis program supplied by the instrument manufacturer (based on the Young-Laplace
equation) was used to determine IFT values.
43
3.2.5 Small angle X-ray scattering (SAXS)
SAXS measurements were performed on the oil samples containing varying
concentrations of phospholipids and water using a Rigaku Molecular Metrology SAXS
instrument (Rigaku, Inc) at the W.M. Keck Nanostructures Laboratory at the University
of Massachusetts-Amherst. The instrument generates X-rays with a wavelength of 1.54 Å
and utilizes a 2-D multi-wire detector with a sample-to-detector distance of 1.5 m.
Samples were inserted into the 1 mm outer diameter quartz capillary (Hampton Research,
Aliso Viejo,CA, USA) which were then sealed by Duco® Cement and then enclosed in
an airtight sample holder. This assembly was then put in the X-ray beam path, and data
were collected on the samples for 3 hours.
The experimental SAXS intensity curves were corrected for background, sample
absorption and detector homogeneity, using MicroCal Origin 5.0™ software. The
scattering vector amplitude q is defined by equation (1), where θ is half of the scattering
angle and is the wavelength:
sin4q (1)
Fittings for the experimental SAXS curves were obtained using the GNOM program
(version 4.5, ATSAS, German) (161). For a set of monodisperse spherical particles
randomly distributed, the scattering intensity is given by the following equation (162):
(2)
where γ is a factor related to the instrumental effects; np corresponds to the number
of scattering species; Δρ is the electron density contrast between the scattering species
44
and the medium; V is the scattering species volume; P is the normalized particle form
factor (P(0)=1); and S is the structure factor of the particle system, its value being ≈ 1 for
non-correlated systems. In this particular case, the intensity function I depends solely on
the particle form factor and according to theory, the pair-distance distribution function
p(r), can be obtained by Fourier inversion of the intensity function (163):
(3)
This function provides information about the shape of the scattering particles as well
as their maximum dimension, Dmax accounted for by the r (pair-distance) value where p(r)
goes to zero.
3.2.6 Front-Face (FF) Fluorimetric Measurements
Front-face fluorescence with surface active fluorescent probes was used to verify the
ability of DOPC and water to form association colloids. The surface active fluorescent
probe used was NBD-PE which is a phospholipid analog with a fluorescent functional
groups covalently attached to the choline head group. Steady-state emission
measurements were recorded with a PTI Spectrofluorimeter (PTI, Ontario, Canada) with
the sample holder held at 22 °C. To minimize any reflection of the excitation beam by the
cell window and by the underlying liquid surface of the sample into the emission
monochromator, samples are stored in triangular suprasil cuvette. The emission was
observed at 90° to the incident beam, i.e., 22.5° with respect to the illuminated cell
surface. A 2.0 nm spectral bandwidth was used for both excitation and emission slits were
employed for the NBD-PE excitation at 468 nm. The integration time was 1 s, and the
wavelength increment during emission spectrum scanning was 1 nm. The intensity of the
45
spectra was determined as the emission signal intensity (counts per second) measured by
means of a photomultiplier. In order to avoid self quenching of probes, the concentrations
of NBD-PE was 9.5 M (1/100 of DOPC) (164).
3.2.7 Measurement of oxidation parameters
Lipid hydroperoxides were measured as the primary oxidation product using a method
adapted from Shanta and Decker (165). Accurately weighed oil samples were added to a
mixture of methanol/butanol (2.8 mL, 2:1, v:v) followed by addition of 15 μL of 3.94 M
thiocyanate and 15 μL of 0.072 M Fe2+. The solution was vortexed, and after 20 min the
absorbance was measured at 510 nm using a Genesys 20 spectrophotometer
(ThermoSpectronic, Waltham, MA). The concentration of hydroperoxides was calculated
from a cumene hydroperoxide standard curve.
Secondary oxidation products were monitored using a GC-17A Shimadzu gas
chromatograph equipped with an AOC-5000 autosampler (Shimadzu, Kyoto, Japan)
(147). Sample (1 mL) in 10 mL glass vials capped with aluminum caps with
PTFE/silicone septa were pre-heated at 55 °C for 15 min in an autosampler heating block.
A 50/30 μmDVB/ Carboxen/PDMS solid-phase microextraction (SPME) fiber needle
from Supelco (Bellefonte, PA, USA) was injected into the vial for 2 min to absorb
volatiles and then was transferred to the injector port (250 °C) for 3 min. The injection
port was operated in split mode, and the split ratio was set at 1:5. Volatiles were separated
on a Supleco 30 m × 0.32 mm Equity DB-1 column with a 1 μm film thickness at 65 °C
for 10 min. The carrier gas was helium at 15.0 mL/min. A flame ionization detector was
used at a temperature of 250 °C. Propanal concentrations were determined from peak
46
areas using a standard curve prepared from an authentic standard.
3.2.8 Statistical Analysis
Duplicate experiments were performed for all studies. All data shown represents
mean values standard deviations (n = 3). Statistical analysis of lipid oxidation kinetics
was performed using a one-way analysis of variance. A significance level of p<0.05
between groups was accepted as being statistically difference. In all cases, comparisons
of the means of the individual groups were performed using Duncan’s multiple range
tests. All calculations were performed using SPSS17 (http://www.spss.com; SPSS Inc.,
Chicago, IL).
3.3 Results and discussion
3.3.1 Attenuation of the water/phospholipid/MCT system
Commercial refined vegetable oils are optically clear and yet potentially contain
physical structures such as association colloids. This is possible because many
association colloids have dimensions below 100 nm and thus they do not strongly scatter
visible light (166). The objective of this initial study was to define the
phospholipid/water/lipid concentrations that produce optically transparent oils,
corresponding to commercial refined oils. To do this, a partial phase diagram of the three
component system water/phospholipid/MCT was constructed to determine the
phospholipid and water concentrations that did not form structures that scattered light
strongly as determined by light attenuation measurements (Figure 3.2) using a method
previously reported for phospholipids and water in olive oil (167). In the presence of
47
1.25, 2.5, and 5 mg DOPC/g MCT, a large increase in attenuation was observed at a water
concentration 20 mg/g MCT. At 20 mg/g MCT, the attenuation of the sample
containing the highest concentration of DOPC (5 mg DOPC/ g MCT) had the lowest
attenuation. This is likely due to the formation of a larger number of small non-light
scattering association colloids in the presence of high levels of DOPC. In the remainder
of the experiments we focused on those phospholipid/water/lipid concentrations that gave
optically transparent systems.
Figure 3.2. Attenuation of medium chain triacylglycerols (MCT) samples containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of water.
3.3.2 Determination of the CMC of phospholipids in SSO and MCT
Phospholipids can self-assemble into a variety of structures in water or oil phases
because of their amphiphilic nature (168). The nature of the structures formed depends on
the phospholipid’s chemical structure and solvent type (169). In non-aqueous media, such
as oil, phospholipids normally form reverse micelles when their concentration just
exceeds the CMC (critical micelle concentration), with the polar head groups pointing
0 5 10 15 20 25 30 35 40 45 50
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6 DOPC=1.25 mg DOPC=2.5 mg DOPC=5 mg
Att
en
uati
on
at
600 n
m (
%)
Water content (mg)
48
into the hydrophilic core of the reverse micelle and the non-polar tails pointing towards
the hydrophobic oil. Therefore, in the region of the phase diagram where the system is
clear (Figure 3.2), it is important to determine whether the DOPC concentration is above
or below its CMC so as to determine if any association colloid structures form. SSO can
rapidly oxidize resulting in the production of additional surface active oxidation products
which could alter the CMC. Therefore, the CMC of DOPC was measured in both SSO
and MCT to determine if SSO was a suitable solvent for analysis of association colloids.
One of the most important properties of reverse micelles is their ability to increase the
solubility of molecules. 7,7,8,8-TCNQ is not lipid soluble and does not absorb light at
480 nm when dispersed in non-polar solvents. 7,7,8,8-TCNQ can undergo charge-transfer
(170) interactions with surfactants when the surfactant concentration is above the CMC,
which can be observed by absorbance at 480 nm (160). The absorbance values of 7,7,8,8-
TCNQ solubilized in MCT were plotted as a logarithmic function of phospholipid
concentration (Figure 3.3 A). When the concentration of DOPC was increased, the
absorbance of TCNQ initially remained low, but there was a rapid increase in absorbance
at DOPC concentrations above 500 M. Based on the intercept of these two linear
regions, the CMC of DOPC was estimated to be ~ 550 M in MCT. This experiment was
repeated in SSO where the CMC of DOPC was found to be ~ 650 M (Figure 3.3 B).
Differences of the CMC in MCT and SSO could be due to differences in fatty acid chain
length and the presence of other minor surface active components in the oils that were not
completely removed by stripping (171). Since the CMC values for MCT and SSO were
fairly similar, SSO was used for all subsequent experiments. The CMC of DC4PC in the
SSO could not be determined by this method because DC4PC did not dramatically change
49
the absorbance of the 7,7,8,8-TCNQ before the DC4PC reached its solubility limit in the
SSO.
Figure 3.3. Determination of critical micelle concentration (CMC) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in medium chain triacylglycerols (A)and stripped soybean oil (B) as determined by the absorbance (480 nm) of 7,7,8,8-tetracyano- quinodimethane.
The CMC of DOPC in SSO was also determined by interfacial tension
10 100 10000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6A
b480
Phospholipid concentration (M)
550M
A
10 100 1000
0.0
0.5
1.0
1.5
2.0
2.5
Ab
480
Phospholipid concentration(M)
650M
B
50
measurements. At low DOPC concentrations, the interfacial tension decreased with
increasing DOPC concentration, but at higher DOPC concentrations the interfacial
tension reached a fairly constant value indicating that reverse micelles had been formed.
A semi-logarithmic plot between interfacial tension and the concentration of DOPC in
SSO is shown in Figure 3.4. Based on the intercept of the two linear regions a CMC of ~
950 M was calculated. The CMC value obtained by interfacial tension measurements is
about 300 M higher than that obtained with the spectrophotometric technique, which
could be due to some of the DOPC partitioning into the aqueous phase during the
interfacial tension measurements. Alternatively, the spectrophotometric and interfacial
tension methods may have given different CMC values because they are based on
different physical principles. For the purposes of this study, we considered the CMC of
DOPC in SSO to be in the range of 650-950 M. The normal concentration of
phospholipids in refined vegetable oil is higher than 950 M, suggesting that commercial
oils could have association colloids formed by phospholipids.
10 100 1000
0
2
4
6
8
10
12
14
16
IFT
(m
N/m
)
Phospholipid concentration (M)
950M
51
Figure 3.4. Determination of critical micelle concentration of 1,2-dioleoyl-sn-glycero-3-
phosphocholine in stripped soybean oil using interfacial tension.
3.3.3 Detection and characterization of phospholipids physical structures by small
angle x-ray scattering
As mentioned above, the specific structures formed by phospholipids in non-
aqueous solution depend on both concentration and phospholipid type. In consideration
of the complicated compositions of phospholipids in refined bulk oil, we first determined
how two types of phospholipids impacted association colloid structure in ternary systems
containing SSO, water, and either DOPC or DC4PC using. Figure 3.5 shows the SAXS
profiles in the SSO systems containing ~ 60 ppm water (i.e., intrinsic water content after
oil stripping) and either 950 M of DOPC or DC4PC. These phospholipid concentrations
were chosen as these samples did not strongly scatter light (Figure 3.2) but were equal to
or above the CMC of DOPC (Figure 3.3 and 3.4). As shown in Figure 3.5, the two
different types of phospholipids induced changes of the minor structures in stripped oil in
such a way that the scattering profiles are distinct and displaced towards different q
values and intensity. The Bragg space (d-spacing) and the intensity of the samples with
DOPC was similar to blank stripped oil, being 141 Å and 3 (a.u.) respectively. However,
the structures with DC4PC are quite different to the blank and the one with DOPC. Its
Bragg peak shifts towards a higher q value with Bragg space being 68 Å while the
intensity rises to 12 (a.u.). In addition, the structures with different types of phospholipids
are verified by pair distribution p(r) function analysis (Figure 3.6). The p(r) of DC4PC
had a long tail at high r, which is typical of a cylindrical structure. The r value at the
52
inflection (~20 nm) usually represents the diameter of the cylinder (172). The p(r) of
DOPC had a symmetrical shape which represents a spherical structure, presumably a
reverse micelle. For the bulk stripped soybean oil, there was only a flat line indicating the
presence of no structures.
Figure 3.5. Small angle x-ray scattering profiles of stripped soybean oil (SSO) and SSO + 950 M 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3- phosphocholine (DC4PC).
0.0 0.4 0.8 1.2 1.6 2.0
0
1
2
3
0
1
2
3
0
5
10
q (nm-1)
SSO
68Å
52.1 Å
141 Å
51.3 Å
Inte
nsit
y
DOPC
DC4PC
141 Å
52.6 Å
53
Figure 3.6. The pair distribution functions, p(r), calculated from the scattering profiles of stripped soybean oil (SSO) and SSO + 950 M 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine(DC4PC) using GNOM analysis of the three small angle x-ray scattering profiles indicated in Figure 3.5.
In the stripped oil, almost all the surface active minor components and the majority
of water had been removed by the silicic acid and activated charcoal sandwich
chromatography which is consistent to our, Karl Fisher, TLC and previous HPLC results
(173). Therefore it is reasonable that the bulk stripped oil did not contain any association
colloid structures (174). The structures formed by DC4PC are quite different to those
formed by DOPC. DOPC and DC4PC have the same phosphocholine hydrophilic head
group (Figure 3.1), but DOCP possesses cis- oleic fatty acids on sn-1 and sn-2 of
glycerol side chains, whereas DC4PC has 2 butyl fatty acids. As indicated, amphiphilic
molecules, like phospholipids, having a large tail area and a small head group area, can
self-assemble into reverse spherical micelles in highly non polar liquids due to a critical
packing parameter (CPP) values greater than 1 (175). There are no reported CPP values
of DC4PC. However, based on the critical packing parameter equation (176):
0 10 20 30 40 50
0.000
0.005
0.010
0.015
0.020
0.025
P(r)[
a.u
.]
r/nm
DOPC SSO DC4PC
54
(4)
(5)
(6)
where ν is the volume of the hydrocarbon tail and n is the number of carbons in the
hydrophobic chain, the CPP of DC4PC in SSO should be lower than that of DOPC,
approximately less than ½ assuming no difference of their effective area of the head
group. This is because the CPP is mainly dependent on the hydrocarbon tail length. A
low CPP often corresponds to the formation of cylindrical structures.
3.3.4 Effect of water/phospholipid molar ratio on the physical properties of DOPC
association colloids
Experimental and theoretical approaches have shown that the key structural
parameter of reverse micelles is the water/surfactant molar ratio (Wo) (177, 178). On one
hand, water will serve to bridge the phosphate head groups between neighboring
phospholipids through hydrogen bonds (179). On the other hand, the water content will
determine structure size as well as the amount of the water that is strongly associated
with the phospholipid head groups (164). Oil-rich phospholipid solutions with micellar
morphology can change to lamellar and hexagonal structures as the Wo is raised in the
system (24). In order to characterize the physical structure of stripped soybean oil with
DOPC (950 M) as a function of Wo, a combination of SAXS and Front-Face
Fluorimetry were used.
55
Figure 3.7. Small angle x-ray scattering profiles of stripped soybean oil with different water/1,2-dioleoyl-sn-glycero-3-phosphocholine molar ratios (WO). Inserted figures are the 2 dimensional small angle x-ray scattering pattern of each sample.
At high Wo, water is solubilized into phospholipid reverse micelles and structures
such as cylindrical micelles and spherical swollen micelles can form depending upon the
amount of water in the system (176). In our system there is a clear Bragg peak on all the
SAXS profiles, with d-space around 141 nm (Figure 3.7). The SAXS intensity is very
56
weak (i.e., 3 a.u.) for the system with Wo = 0 (no added water), suggesting that DOPC
alone forms very little structure. When the Wo increased from 0 to 35 and 47, the
scattering intensity increased from 3.0 to 8.0, and 13.0, respectively, presumably due to
the formation of association colloids by DOPC. Increasing Wo above 100 results in the 2-
D X-ray pattern of the samples becoming aligned, indicating a transformation from an
isotropic to an anisotropic system. At the same time, the SAXS intensity increased greatly
to 749.6 a.u. In all samples, no other Bragg peaks were observed (Figure 3.7), indicating
the absence of cylindrical micelles and spherical swollen micelles. Lack of
transformation form reverse micelles to cylindrical micelles and spherical swollen
micelles was also reported in a phospholipid in hexane and soybean oil system (180). The
fact that the reverse micelles did not transform to these other colloidal structures in our
system could be due to: (1) the increase in Wo was too small in our experimental design
so that the transformation did not occur; or, (2) the concentration of the DOPC was too
low to allow transformation. While the anistropic transition at Wo values greater than 100
indicates that the structures were changing symmetry. This could be due to a stretching
of the symmetrical structure into more of an oblong structure such as shown in Figure
3.8.
57
Figure 3.8. A postulated structural change of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as phospholipid concentrations are increased to above the critical micelle concentrations (CMC) form a reverse micelle.
Fluorescence intensity of selected probes can also be used to provide information
about the micro-environment of association colloids as a function of Wo. In this study, we
used NBD-PE as a fluorescence probe and Front Face Fluoremetery to detect changes in
its spectra. This probe is a phospholipid analog grafted with a fluorophore on the
phosphate head groups (Figure 3.9 insert) that has previously been used as an indicator
of changes in the interfacial properties of reverse micelle systems (164). Theoretically,
NBD-PE possesses similar properties as DOPC as both are amphiphilic molecules with
the ability to participate in the formation of association colloids and reside at the water-
oil interface (164). The probe is useful because its fluorescence decreases when it is
exposed to water. Figure 3.9 shows the fluorescence intensity of NBD-PE decreased with
increasing water concentrations suggesting that the exposure of the probe to water
increased. The exposure of the probe to the water phase can also be confirmed by a
change in the wavelength of maximum fluorescence intensity of NBD-PE with a red shift
58
occurring when the probe is exposed to a more polar environment (164). A red shift of 4
nm was observed for NBD-PE in the DOPC/stripped soybean system when the Wo was
increased from 11.7 to 46.8.
Figure 3.9. The fluorescence intensity of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE) in stripped soybean oil with different water/1,2-dioleoyl-sn-glycero-3-phosphocholine molar ratios (WO). Inserted figure is the molecular structure of (NBD-PE).
3.3.5 Oxidation of stripped soybean oil containing association colloids
Studies of lipid oxidation in bulk oils have been carried out for several decades, but
many of the mechanisms involved in this reaction are still unclear, including whether
physical structures in the oil can impact oxidation kinetics. The ultimate purpose of these
experiments was to produce soybean oil, water, and phospholipid systems with different
physical structures and determine if these physical structures impact lipid oxidation
kinetics. Therefore, the lipid oxidation kinetics of stripped soybean oil containing 1000
μM of either DOPC or DC4PC and ~ 200 ppm water was studied as determined by lipid
hydroperoxides and propanal during storage at 37 °C (Figure 3.10). In the presence of
0 20 40 60 80 1000.0
500.0k
1.0M
1.5M
2.0M
2.5M
3.0M
H
O
O
O
O+HN
P
O-O
O
ONH
NO
N
N+
O
O-
(CH3CH2)3
CH3(CH2)14
CH3(CH2)14
Inte
nsit
y (
Co
un
ts)
Wo
59
DOPC, the lag phase of lipid hydroperoxides (Figure 3.10A) and headspace propanal
(Figure 3.10B) were 8 and 13 days, respectively, compared to 13 and 17 days,
respectively, for the no phospholipid control. DC4PC had the same lag phase times for
lipid hydroperoxides and headspace propanal as the control (Figure 3.10A and B).
Figure 3.10. Formation of lipid hydroperoxides (A), propanal (B) in stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) or 1,2-dibutyryl-sn-glycero-3- phosphocholine (DC4PC) and 200 ppm water at 37 °C. Data represent means (n =3) ± standard deviations. Some error bars are within data points.
0 5 10 15 20 250
40
80
120
160
200
240
Lip
id h
yd
rop
ero
xid
es
(m
mo
l /K
g o
il)
Storage Time(day)
SSO SSO w/ 1000 microM DOPC SSO w/ 1000 microM DC4PC
A
0 5 10 15 20 250
40
80
120
160
200
240
Pro
pan
al
(m
ol
/Kg
oil
)
Storage Time(day)
SSO SSO w/ 1000 microM DOPC SSO w/ 1000 microM DC4PC
B
60
Phospholipids have previously been reported to have protective effects in bulk oils.
The mechanisms for these protective effects have been postulated to be due to the
phospholipid’s ability to chelate metals, decompose lipid hydroperoxides, directly
scavenge free radicals, and increase the antioxidant activity of tocopherols (181). In this
study, DOPC and DC4PC were chosen since they both have identical choline head groups
so any impact of the phospholipids on alteration in lipid oxidation kinetics by chemical
pathways would be similar. Therefore, the major differences in the oil systems containing
DOPC and DC4PC would be in how these phospholipids produced different physical
structures. As discussed above, 7,7,8,8-TCNQ was an effective tool to monitor the
formation of structures by DOPC but this probe did not detect structures formed by
DC4PC. Using small angle x-ray scattering, structures could be seen for both DOPC and
DC4PC but these structures were very different with DOPC forming spherical and DC4PC
forming cylindrical structures (Figure 3.6). Therefore these results suggested that the
spherical structures formed by DOPC were prooxidative while the cylindrical structures
formed by DC4PC had no impact on oxidation rates. These results suggest that
phospholipids have the ability to form colloidal structures in vegetable oils and that these
structures could have an impact on the oxidative stability of food oils.
61
CHAPTER 4
ROLE OF REVERSE MICELLES ON LIPID OXIDATION IN BULK OILS:
IMPACT OF PHOSPHOLIPIDS ON ANTIOXIDANT ACTIVITY OF
α-TOCOPHEROL AND TROLOX
4.1 Introduction
Lipid oxidation is one of the main factors limiting the shelf life of bulk oils, since it
adversely affects flavor and quality, and potentially produces toxic reaction products(182).
Preventing or inhibiting the oxidation of bulk oils is therefore of great importance to
consumers and the food industry. A variety of mechanisms have been proposed to be
responsible for the oxidation of bulk oils during processing and storage, with
photosensitized oxidation, metal-promoted and autoxidation being the most well-known.
Some factors that impact the oxidative stability bulk oils include: oil extraction and
processing conditions; light exposure; temperature; fatty-acid composition; antioxidant
composition; oxygen levels; and the presence of minor components (183). Manipulation
of these factors can be used to retard lipid oxidation in edible oils.
One of the most effective ways of inhibiting lipid oxidation in bulk oils is to
incorporate antioxidants (184). Depending on their mechanism of action, antioxidants can
be classified as either “primary” or “secondary” antioxidants (185). Tocopherols are the
most common primary antioxidant present in many vegetable oils, which may originate
naturally from the extracted oil itself or may be manually added after oil refining (186).
However, tocopherols may not be the most effective antioxidants in bulk oil systems.
62
Indeed, research has shown that hydrophilic antioxidants (Trolox or ascorbic acid)
possess better antioxidant activity than their hydrophobic analogues (tocopherol and
ascorbyl palmitate) in some bulk oil systems (156). The greater tendency for hydrophilic
antioxidants to accumulate at the air-water interface where oxidation may be expected to
begin is one of the mechanisms proposed to account for their better antioxidant activity in
bulk oils (156). However, other mechanisms have also been proposed to account for the
ability of hydrophilic antioxidants to act as better antioxidants than their hydrophobic
analogs in bulk oils since air is more hydrophobic than oil and thus there is no driving
force to concentrate hydrophilic antioxidants at the oil-air interface (181, 185). Studies
have shown that the addition of phospholipids to bulk oils increased the antioxidant
activity of tocopherol (181). It was postulated that the phospholipids formed
microstructures, known as association colloids, within the bulk oil, which caused the
tocopherol molecules to accumulate in the phospholipid microstructures where lipid
oxidation primarily occurred. Other researchers have also demonstrated the ability of
phospholipids to act as antioxidants in various kinds of bulk oils (90, 187). Several
mechanisms were proposed to account for the antioxidant activity of the phospholipids,
including their ability to chelate metals, decompose lipid hydroperoxides, and scavenge
free radicals. Nevertheless, there is still a poor understanding of the importance and
contribution of the combination of phospholipids and tocopherols to the oxidative
stability of bulk oils.
In the previous chapter, we characterized the formation of reverse micelles in bulk
oils, and their impact on lipid oxidation (188). These reverse micelles were formed by
adding phospholipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) into stripped
63
soybean oil (SSO) containing water levels similar to that found in commercial refined
oils. Using small angle x-ray scattering, this study showed that the combination of DOPC
and water resulted in the formation of reverse micelles in bulk oil. Alternately, when
phosphatidylcholine with short chain fatty acyl residues (1,2-dibutyryl-sn-glycero-3-
phosphocholine, DC4PC) was added to the same system, no reverse micelles were formed.
The lipid oxidation chemistry of these two systems was different with DOPC reverse
micelles demonstrating a prooxidative effect while DC4PC had no effect on oxidation
rates. This study suggests that the combination of phospholipids and water can form
physical structures in bulk oils and these structures can impact lipid oxidation chemistry.
In the present study, we examined if reverse micelles in bulk oil produced by
phosphatidylcholine(i.e., DOPC) were able to impact the activity of free radical
scavenging antioxidants. The antioxidants tested included alpha-tocopherol (non-polar)
and Trolox (polar), which are chemical analogs. In addition, the system had equal molar
concentrations of phospholipids with one system containing reverse micelles (DOPC) and
the other containing no measurable reverse micelles (DC4PC). We also measured how
the tocopherol analogs impacted the structure of the reverse micelles with the aim of
better understanding the location and properties of antioxidants in bulk oils. By better
understanding how physical structure in bulk oils impact the activity of antioxidants such
as tocopherols, it might be possible to design systems to improve the activity of these
important “natural” antioxidants.
4.2 Materials and methods
4.2.1 Materials
64
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dibutyryl-sn-glycero-3-
phosphocholine (DC4PC) were acquired from Avanti Polar Lipids, Inc (Alabaster, AL).
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-
phosphoethanolamine, triethylammonium salt (NBD-PE, Cat.No. N-360) was acquired
from Invitrogen. Soybean oil was purchased from a local store and stored at 4 °C. Silicic
acid, activated charcoal, hexane, α-tocopherol, Trolox were purchased from Sigma-
Aldrich Co. (St. Louis, MO). All other reagents were of HPLC grade or purer distilled
and deionized water was used as needed.
4.2.2 Preparation of stripped soybean oil
Stripped soybean oil with DOPC reverse micelles were prepared according to the
method of Chen et al (157). Briefly, formation of reverse micelles was accomplished by
pipetting DOPC (1000 μM, final concentration) in chloroform into an empty beaker and
then flushing with nitrogen until the chloroform was evaporated. The appropriate amount
of medium chain triacylglycerols (MCT) or stripped soybean oil (SSO) was then added
followed by double distilled water at a final concentration of 200 ppm. The oil samples
were then stirred in a beaker at 1000 rpm in a 20 °C incubator room for a 24 h.
4.2.3 Formation of DOPC reverse micelles in stripped soybean oil
The formation of DOPC reverse micelles was done using our previous procedure
(188). Briefly, DOPC (1000 μM) in chloroform was pipetted into an empty beaker and
then chloroform was removed by flushing with nitrogen. The appropriate amount of
stripped soybean oil was then added followed by double distilled water to a final amount
65
of ~ 200 ppm. The sample in beaker was magnetically stirred at the speed of 1000 rpm in
a 20 °C incubator room for a 24 hrs. Antioxidants were dissolved in ethanol, mixed with
stripped soybean oil and stirred for at least 12 hrs to obtain the homogenous samples. In
the lipid oxidation studies, a mixture of 75% of medium chain TAG and 25% SSO were
used due to the high amount of samples needed. Samples for the oxidation studies were
aliquoted into GC vials (1 mL/vial) stored at 55 °C in the dark. For fluorescence, the
NBD-PE probe was mixed into stripped soybean oil at the same time as DOPC and water.
4.2.4 Measurement of oxidation parameters
Lipid hydroperoxides were measured as the primary oxidation product using a method
adapted from Shanta and Decker (165). Accurately weighed oil samples were added to a
mixture of methanol/butanol (2.8 mL, 2:1, v:v) followed by addition of 15 μL of 3.94 M
thiocyanate and 15 μL of 0.072 M Fe2+. The solution was vortexed, and after 20 min the
absorbance was measured at 510 nm using a Genesys 20 spectrophotometer
(ThermoSpectronic, Waltham, MA). The concentration of hydroperoxides was calculated
from a cumene hydroperoxide standard curve.
Secondary oxidation products were monitored using a GC-17A Shimadzu gas
chromatograph equipped with an AOC-5000 autosampler (Shimadzu, Kyoto, Japan)
(147). Sample (1 mL) in 10 mL glass vials capped with aluminum caps with
PTFE/silicone septa were pre-heated at 55 °C for 15 min in an autosampler heating block.
A 50/30 μmDVB/ Carboxen/PDMS solid-phase microextraction (SPME) fiber needle
from Supelco (Bellefonte, PA, USA) was injected into the vial for 2 min to absorb
volatiles and then was transferred to the injector port (250 °C) for 3 min. The injection
66
port was operated in split mode, and the split ratio was set at 1:5. Volatiles were separated
on a Supleco 30 m × 0.32 mm Equity DB-1 column with a 1 μm film thickness at 65 °C
for 10 min. The carrier gas was helium at 15.0 mL/min. A flame ionization detector was
used at a temperature of 250 °C. Propanal concentrations were determined from peak
areas using a standard curve prepared from an authentic standard.
4.2.5 Small angle x-ray scattering (SAXS) study of bulk oil with DOPC and
antioxidants
SAXS measurements were performed on the oil samples containing varying
concentrations of phospholipids and water using a Rigaku Molecular Metrology SAXS
instrument (Rigaku, Inc) at the W.M. Keck Nanostructures Laboratory at the University
of Massachusetts-Amherst. The instrument generates X-rays with a wavelength of 1.54 Å
and utilizes a 2-D multi-wire detector with a sample-to-detector distance of 1.5 m.
Samples were inserted into the 1 mm outer diameter quartz capillary (Hampton Research,
Aliso Viejo,CA, USA) which were then sealed by Duco® Cement and then enclosed in
an airtight sample holder. This assembly was then put in the X-ray beam path, and data
were collected on the samples for 3 hours.
The experimental SAXS intensity curves were corrected for background, sample
absorption and detector homogeneity, using MicroCal Origin 5.0™ software. The
scattering vector amplitude q is defined by equation (1), where θ is half of the scattering
angle and is the wavelength:
sin4q (1)
67
Fittings for the experimental SAXS curves were obtained using the GNOM program
(version 4.5, ATSAS, German) (161). For a set of monodisperse spherical particles
randomly distributed, the scattering intensity is given by the following equation (162):
(2)
where γ is a factor related to the instrumental effects; np corresponds to the number of
scattering species; Δρ is the electron density contrast between the scattering species and
the medium; V is the scattering species volume; P is the normalized particle form factor
(P(0)=1); and S is the structure factor of the particle system, its value being ≈ 1 for non-
correlated systems. In this particular case, the intensity function I depends solely on the
particle form factor and according to theory, the pair-distance distribution function p(r),
can be obtained by Fourier inversion of the intensity function (163):
(3)
This function provides information about the shape of the scattering particles as well
as their maximum dimension, Dmax accounted for by the r (pair-distance) value where p(r)
goes to zero.
4.2.6 Fluorescence measurement of bulk oil with DOPC and antioxidants
Front-face fluorescence with surface active fluorescent probes was used to verify the
ability of DOPC and water to form association colloids. The surface active fluorescent
probe used was NBD-PE which is a phospholipid analog with a fluorescent functional
groups covalently attached to the choline head group. Steady-state emission
measurements were recorded with a PTI Spectrofluorimeter (PTI, Ontario, Canada) with
68
the sample holder held at 22 °C. To minimize any reflection of the excitation beam by the
cell window and by the underlying liquid surface of the sample into the emission
monochromator, samples are stored in triangular suprasil cuvette. The emission was
observed at 90° to the incident beam, i.e., 22.5° with respect to the illuminated cell
surface. A 2.0 nm spectral bandwidth was used for both excitation and emission slits were
employed for the NBD-PE excitation at 468 nm. The integration time was 1 s, and the
wavelength increment during emission spectrum scanning was 1 nm. The intensity of the
spectra was determined as the emission signal intensity (counts per second) measured by
means of a photomultiplier. In order to avoid self quenching of probes, the concentrations
of NBD-PE was 9.5 M (1/100 of DOPC) (164).
The fluorescence decay of NBD-PE in the systems was carried out at 22 °C using a
PTI Laserstrobe fluorescence lifetime instrument with a PTI GL-3300 nitrogen laser and
a GL-302 tunable dye laser with C-500 laser dye, exciting the oil samples at 500 nm.
Each data point on a lifetime decay curve represents five laser flashes, and each decays
represents 100 of these data points spaced out over the collection time interval.
Data were analyzed using the commercial PTI software. Fluorescence decays were
fitted to sums of two and three exponentials, and the average lifetime was calculated.
4.2.7 Statistical analysis
Duplicate experiments have been performed with freshly prepared samples. All data
shown represents the mean values ± standard deviation of triplicate measurements.
Statistical analysis of lipid oxidation kinetics was performed using a one-way analysis of
variance. A significance level of p<0.05 between groups was accepted as being
69
statistically difference. In all cases, comparisons of the means of the individual groups
were performed using Duncan’s multiple range tests. All calculations were performed
using SPSS17 (http://www.spss.com; SPSS Inc., Chicago, IL).
4.3 Results
4.3.1 Impact of phospholipids on oxidative stability of SSO
Commercial soybean oil is a highly complex liquid system containing a number of
different components that could potentially alter the rate and extent of lipid oxidation
such as tocopherols, chlorophyll, phospholipids, fatty acids, and water (185).
Consequently, stripped soybean oil (SSO) was used as a model oil in this study to reduce
any effects associated with these minor components. Previous research using small angle
x-ray diffraction showed that the combination of DOPC and water resulted in the
formation of reverse micelles in bulk oil while DC4PC did not form structures10. Initially,
we examined the influence of phospholipid type (DOPC and DC4PC at 1000 μM) on the
formation of lipid hydroperoxides and headspace hexanal in SSO during storage at 55 °C
(Figure 4.1A and B). In the absence of added phospholipids, the lag phase for both lipid
hydroperoxides and headspace hexanal lasted 2 days. Addition of DC4PC had little
impact on the lipid oxidation profile, but the addition of DOPC reduced the lag-period by
1 day, confirming that it acted as a prooxidant.
70
Figure 4.1. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 μM of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) during storage at 55 °C. Some of the error bars are within data points.
Previous studies of the oxidative stability of bulk oils containing phospholipids
suggest that phosphatidylethanolamine (PE) improves their oxidative stability (189, 190).
On the other hand, studies on the impact of phosphatidylcholine (PC) have indicated
seemingly contradictory results. Prooxidant, antioxidant, and no effects of PC in bulk oil
0 1 2 3 4 5 6 70
100
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400
500
600
Lip
id H
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rop
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Blank (SSO) SSO w/ 1000 microM DOPC SSO w/ 1000 microM DC4PC
A
0 1 2 3 4 5 6 70
100
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Hexa
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mo
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B
71
oxidation have been reported in the literature (191, 192). In agreement with our study, a
prooxidant activity of DOPC was reported by Takenaka, et al (192) in bonito oil, which
was attributed to the unsaturated hydrocarbon chain of the PC used. In our study we used
phosphatidylcholine with oleic acid. DOPC was chosen because of its ease of handling
and dispersion into bulk oil due to its low melting point and due to its high oxidative
stability. In general, the oxidative stability of oleic acid is 10-20 times greater than the
linolenic and linolenic acids found in SSO and thus the fatty acyl residues in the added
DOPC would not be expected to decrease the oxidative stability of the oil. In addition, Le
Grandios, et al (193) showed that oleyl and linolenyl residues in PC are more oxidatively
stable than the same residues in triacylglycerols (TAG) again suggesting that the oleyl
residues in the DOPC added to the SSO would not be responsible for the observed
increase in oxidation rates. These data suggest that the prooxidant activity of DOPC in
comparison to DC4PC is not due to its unsaturated fatty acids.
It is possible that the impact of the phospholipids on the oxidation stability could be
due to the polar head groups. However, since both phospholipids tested had the same
head group at equal molar concentrations this is not likely. One major difference between
DOPC and DC4PC in SSO is their ability to form physical structure with DOPC forming
reverse micelles and DC4PC forming cylindrical structures (188). In addition, this study
showed that DOPC could form physical structures at much lower concentrations than
DC4PC due to its lower critical micelle concentration. Overall, these data suggested that
the prooxidant activity of DOPC could be related to its ability to form reverse micelles.
4.3.2 The impact of phospholipids on the oxidative stability of SSO in the presence
72
of α-tocopherol
Previous studies of the influence of antioxidant polarity on lipid oxidation concluded
that polar antioxidants inhibit lipid oxidation in bulk oil systems more effectively than
non-polar antioxidants (156, 194, 195). A number of studies have previously examined
the effects of combinations of phospholipids and antioxidants on lipid oxidation in bulk
oils, such as α-tocopherol, flavonoids, etc (90, 196, 197). Some of these studies used
lecithin with a high proportion of PE as the phospholipid source, some of them used
commercial oils with unknown intrinsic antioxidant levels, and some used model systems
like triolein, methyl linoleate, or methyl laurate which may not accurately reflect
commercial bulk oil compositions (197). For this reason, we used a model system that
consisted of stripped soybean oil to mimic commercial oil triacylglycerol compositions
and well-defined phospholipids and antioxidants to provide a more fundamental
understanding of the complex physicochemical phenomenon involved.
The formation of lipid hydroperoxides (LH) and headspace hexanal in SSO
containing different levels of phospholipid (0 and 1000 μM DOPC) and the non-polar
antioxidant α-tocopherol (0, 10 and 100 μM) was measured during incubation at 55 °C
(Figures 4.2). As discussed earlier, in the absence of phospholipid and antioxidants the
lag phase of lipid hydroperoxides (LH) and hexanal formation for the control was 3 days
for the SSO. The incorporation of 1000 μM DOPC reduced the lag phase of LH and
hexanal formation to 2 days, again indicating that this phospholipid was prooxidative. In
the absence of DOPC, the duration of the lag phase increased with increasing α-
tocopherol concentration compared to the control. The addition of 10 and 100 μM α-
tocopherol increased the lag phase for LH to 9 and 27 days, respectively and headspace
73
hexanal formation to 7 and 27 days, respectively. When DOPC was present, the impact of
α-tocopherol on lipid oxidation was more complex (Figures 4.2 A and B). DOPC
enhanced the antioxidant activity of 10 μM α-tocopherol by extending the lag phase of
LH formation from 9 to 11 days (Figure 4.2 A), and hexanal formation from 7 to 11 days
(Figure 4.2 B). The opposite trends were observed when 100μM α-tocopherol and
DOPC were added to the SSO. In this case, the lag time was shorter (less oxidatively
stable) in the presence of DOPC than in its absence (Figure 4.2 A and B). For example,
DOPC decreased the lag phase of LH formation from 27 to 23 days (Figure 4.2 A) and
hexanal formation from 27 to 20 days (Figure 4.2 B) in the presence of 100 μM α-
tocopherol.
74
Figure 4.2. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with or with 10 or100 M α-tocopherol during storage at 55 °C. Some of the error bars are within data points.
The impact of DC4PC on the antioxidant activity of α-tocopherol was also
determined (Figure 4.3A and B). As observed previously, DC4PC did not impact with
lipid hydroperoxide or hexanal formation rates compared to the control. α-Tocopherol
again decreased oxidation rates but in this case DC4PC decreased the effectiveness or α-
0 5 10 15 20 25 30 35 400
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700
800
900 Blank (SSO) SSO w/ 1000 uM DOPC SSO w/ 1000 uM DOPC +10 uM TOC SSO w/ +10 uM TOC SSO w/ 1000 uM DOPC +100 uM TOC SSO w/ +100 uM TOC
Lip
id H
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0 5 10 15 20 25 30 35 400
500
1000
1500
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3500
4000 Blank (SSO) SSO w/ 1000 uM DOPC SSO w/ 1000 uM DOPC +10 uM TOC SSO w/ +10 uM TOC SSO w/ 1000 uM DOPC +100 uM TOC SSO w/ +100 UM TOC
He
xa
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l (m
icro
mo
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il)
Storage time (day)
B
75
tocopherol at both 10 and 100 µM unlike DOPC which enhances antioxidant activity only
at 10 µM.
Figure 4.3. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 M of 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) with or with 10 or100 M α-tocopherol during storage at 55 °C. Some of the error bars are within data points.
4.3.3 The impact of phospholipids on the oxidative stability of SSO in the presence
0 5 10 15 20 25 300
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800
900 Blank (SSO) SSO w/ 1000 uM DC4PC SSO w/ 1000 uM DC4PC +10 uM TOC SSO w/ +10 uM TOC SSO w/ 1000 uM DC4PC +100 uM TOC SSO w/ +100 uM TOC
Lip
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1500 Blank (SSO) SSO w/ 1000 uM DC4PC SSO w/ 1000 uM DC4PC +10 uM TOC SSO w/ +10 uM TOC SSO w/ 1000 uM DC4PC +100 uM TOC SSO w/ +100 UM TOC
He
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76
of Trolox
The impact of different concentrations of Trolox (10 and 100 μM), the polar analog
of α-tocoherol, on the oxidative stability of SSO with or without 1000 μM DOPC was
also investigated. Again, the formation of LH and hexanal were determined during
storage at 55 °C (Figure 4.4 A and B). Trolox at 10 µM increased the lag phase for both
lipid hydroperoxides and headspace hexanal formation from 3 (control) to 14 days. When
10 µM Trolox was added to the SSO in combination with 1000 μM DOPC the lag phase
for lipid hydroperoxides increased from 14 to17 days while headspace hexanal formation
was similar in the presence and absence of DOPC. Increasing the concentration of Trolox
to 100 μM further decreased lipid oxidation rates with no appreciable increase in lipid
hydroperoxides or hexanal after 37 days of storage (again more effective thanα-
tocopherol). However, like α-tocopherol, the presence of 1000 μM of DOPC decreased
the antioxidant activity of 100 µM Trolox with the lag phase of LH and hexanal
formation decreasing to 27 and 20 days, respectively.
0 5 10 15 20 25 30 35 400
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800
900 Blank (SSO) SSO w/ 1000 uM DOPC SSO w/ 1000 uM DOPC +10 uM Trolox SSO w/ +10 uM Trolox SSO w/1000 uM DOPC +100 uM Trolox SSO w/ +100 mciroM Trolox
Lip
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77
Figure 4.4. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with or with 10 or 100 M Trolox during storage at 55 °C. Some of the error bars are within data points.
In the presence of DC4PC, the antioxidant activity of Trolox decreased (Figure 4.5A
and B). At a concentration of 10 M Trolox, DC4PC decreased the lag phase for
hydroperoxide formation from 9 to 7 days and hexanal formation from 9 to 5 days. At a
concentration of 100 M Trolox, DC4PC decreased the lag phase for hydroperoxide and
hexanal formation to 25 days (no LH or hexanal formation was observed for Trolox
alone).
0 5 10 15 20 25 30 35 400
500
1000
1500
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2500
3000
3500
4000 Blank (SSO) SSO w/ 1000 uM DOPC SSO w/ 1000 uM DOPC +10 uM Trolox SSO w/ +10 uM Trolox SSO w/1000 uM DOPC +100 uM Trolox SSO w/ +100 uM Trolox
Hexa
nal (m
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Storage time (day)
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78
Figure 4.5. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 1000 M of 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) with or with 10 or100 M Trolox during storage at 55 °C. Some of the error bars are within data points
Overall, both α-tocopherol and Trolox inhibited lipid hydroperoxide and hexanal
formation with increasing concentrations from 10 to 100 µM increasing antioxidant
activity. The more polar Trolox was more effective than α-tocopherol which is similar to
trends reported by other researchers (156).
0 5 10 15 20 25 30 35 40
0
100
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500
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700
800
900 Blank (SSO) SSO w/ 1000 uM DOPC SSO w/ 1000 uM DC4PC +10 uM Trolox SSO w/ +10 uM Trolox SSO w/1000 uM DC4PC +100 uM Trolox SSO w/ +100 mciroM Trolox
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79
4.3.4 The impact of antioxidants on the properties of DOPC reverse micelles in SSO
Aqueous self-assembly of amphiphilic surfactants to form association colloids are
recognized to be driven primarily by hydrophobic interactions (198). In non aqueous
systems, uncertain effects could also drive the self-assembly of amphiphiles into
association colloids and this is referred to as reverse self-assembly (199). Our previous
research illustrated 1000 µM of amphiphilic DOPC plus 200 ppm water resulted in the
formation of reverse micelles, a type of association colloid. Here, the physical structures
of the DOPC/water reverse micelles containing different concentrations of antioxidants
were measured using small angle x-ray scattering (SAXS) and the fluorescence steady
state and lifetime decay of the surface active fluorescent probe, NBD-PE. These studies
were not carried out with DC4PC as this phospholipid does not form association colloids
at the concentrations used in this study.
SAXS is a unique technique which has been widely used to distinguish structure
transitions in reverse micelles. In our previous study, SAXS was able to determined that
DOPC and water formed reverse micelles and that the size and shape of the reverse
micelles changed with varying water concentrations. The same technique was employed
in this study to determine if α-tocopherol and Trolox had any impact on the structure of
DOPC reverse. SAXS patterns revealed that the scattering patterns of DOPC reverse
micelles were not altered by either α-tocopherol or Trolox (data not shown) indicating
that the antioxidants did not have a major impact on the size and shape of the reverse
micelles. The only difference of SAXS between the samples was scattering intensity;
however, this difference did not increase with increasing antioxidants concentration
Others reported the phospholipids aliphatic chain is the main factor that impacts the
80
scattering intensity of phospholipids structures (200). Therefore, the intensity differences
caused by the antioxidants could be due to their association with the phospholipid acyl
chains thus causing attenuation of the x-ray scattering.
Fluorophores have been reported to provide valuable insights into
microenvironmental changes in reverse micelles (164). Previously, we selected NBD-PE,
a phospholipid analog grafted with a fluorophore on the phosphate head groups to study
the interfacial properties of DOPC reverse micelle systems. NBD-PE emission intensity
increases in a polar environment. The lowest recorded emission of NBD-PE was
observed in the SSO control. The addition of DOPC and water in SSO increased the
emission to 8.5 ×105 counts, 30% higher than in SSO alone. Neither α-tocopherol nor
Trolox caused a shift in the fluorescence emission peak wavelength (data not shown). The
addition of 10 µM α-tocopherol decreased the emission intensity of NBD-PE in the
DOPC reverse micelles but further increases in α-tocopherol did not further decrease
NBD-PE fluorescence (Figure 4.6). Conversely, a concentration dependent decrease in
NBD-PE fluorescence occurred in the presence of Trolox.
81
Figure 4.6. The neutralized fluorescence intensity of NBD-PE at stripped soybean oil containing 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) with varying
concentrations(0-100 M) of α-tocopherol and Trolox.
The low NBD-PE emission in the SSO control is likely due to the water
concentrations (< 60 ppm in pure SSO). When DOPC and water were added to the SSO,
NBD-PE was incorporated into the reverse micelles thus placing it in a more polar
environment thus increasing its emission intensity. The ability of increasing
concentrations of the water soluble Trolox to decrease fluorescence intensity suggests to
that Trolox can interact with water in the reverse micelle thus decreasing NBD-PE-water
interactions and thus fluorescence intensity. Additionally, the polar Trolox molecules
could directly interact with NBE-PE thus decreasing fluorescence intensity by decreasing
the ability of NBD-PE to become excited. Overall, this data suggests that both the NBD-
PE probe and Trolox are incorporated into the DOPC reverse micelles.
0 10 20 30 40 50 60 70 80 90 1000.5
0.6
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0.8
0.9
1.0
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Antioxidant concentration(M)
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82
Figure 4.7. The fluorescence lifetime decay of NBD-PE at stripped soybean oil containing 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) with varying concentrations(0-100 M) of α-tocopherol (A) and Trolox (B) concentrations.
Information about the properties of the antioxidants in the reverse micelles can also
be gained by measuring the lifetime decay of NBD-PE. Overall, neither α-tocopherol not
Trolox had a major impact on the fluorescence lifetime of NBE-PE (Figure 4.7A and B).
125 130 135 140 145 150 155 160
0
50
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Inte
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(A
.U.)
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A
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.)
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50M Trolox 100M Trolox IRF
B
83
However, there was a slight trend of Trolox decreasing the lifetime of NBE-PE in DOPC
reverse micelles. This again suggests that both the NBD-PE probe and Trolox are
incorporated into the DOPC reverse micelles.
4.4 Discussion
The main motivation of investigating the impact of DOPC based reverse micelles in
stripped soybean oil on the physicochemical properties of antioxidants was to better
understand the underlying phenomena of lipid and antioxidant interactions in bulk oil.
The parameters evaluated were phospholipids with the same polar head groups and
different fatty acyl chains (DC4PC and DOPC) that would result in formation of different
types of physical structures. Our previous study showed that DOPC but not DC4PC
formed reverse micelles in SSO10. In addition, DOPC reverse micelles accelerated lipid
oxidation while DC4PC, which does not form physical structure had no impact on lipid
oxidation rates. This data suggest that lipid oxidation in bulk oils is not only influenced
by the traditional chemical factors, such as lipid compositions, transition metals and
antioxidants, but is also related to the existence of physical structures of. Since one of the
most effective techniques employed for retarding the lipid oxidation is the addition of
free radical scavengers, this study was conducted as an extension of the previous study to
determine how physical structure impact the activity of antioxidant in bulk oils. In this
study, α-tocopherol and Trolox were chosen as hydrophobic and hydrophilic antioxidants
that have similar free radical scavenging functional groups (e.g same chromanol ring)
with different hydrocarbon tails (201).
DOPC by itself promoted lipid oxidation while DC4PC had no effect. This would
84
suggest that the physical structure formed by DOPC increased lipid oxidation rates since
both DOPC and DC4PC have the same polar head groups and thus would impact the
chemistry of lipid oxidation in a similar manner. This is supported by the previous
observation that DOPC below its critical micelle concentration, where no reverse
micelles are formed, does not promote lipid oxidation (188). The prooxidative effect of
DOPC suggest that reverse micelles facilitate lipid oxidation.
One would expect the prooxidant activity of DOPC reverse micelles to decrease the
activity of the antioxidants. However, at low antioxidant concentrations, DOPC increased
the activity of both Trolox and α-tocopherol. Koga and Terao (181) have postulated that
physical structures formed by phospholipids can increase the antioxidant activity of
tocopherols by allowing them to concentration at the oil-water interface where lipid
oxidation is most prevalent. However, increasing α-tocopherol and Trolox concentrations
to 100 µM in the presence of a constant DOPC concentrations resulted in a reduction in
the effectiveness of the antioxidant. Antioxidants such as tocopherols in bulk oils have
been shown to lose effectiveness as their concentrations increase and this could help to
explain this observation. However, loss of effectiveness with increasing concentrations is
not typically seen at the α-tocopherol and Trolox concentrations used in this study (202).
A simple explanation for this observation is not available but it could be due to
differences in the number and/or type of association colloids found in this simple model
system compared to commercial oils that would contain a much greater variety of
endogenous surface active molecules. Another possible explanation is that the DOPC
reverse micelles used in this study would produce a negatively charged interface that
could attract prooxidative transition metals. Partitioning of high concentrations of α-
85
tocopherol and Trolox into the same physical location as the transition metals could allow
the antioxidants to reduce the metals into their more prooxidative state and thus increase
lipid oxidation rates which would be seen as a decrease in the effectiveness of the
antioxidants. This pathway could be further increased by the accumulation of lipid
hydroperoxides, the substrate for metal promoted lipid oxidation, into these same
structures since they have been reported to partition into reverse micelle structures in oils
and act as the co-surfactant (145). The presence of DC4PC only decreased the activity of
α-tocopherol and Trolox indicating that it was not able to enhance the activity of the
antioxidants.
Trolox was a more effective antioxidant in the presence of the DOPC micelles than
α-tocopherol which is in agreement with previous research (156). The fluorescence of
NBD-PE was affected to a much greater extent by Trolox than α-tocopherol suggesting
that Trolox was partitioning into the DOPC reverse micelles differently than α-tocopherol.
This could be due to Trolox’s higher water solubility which would allow it to partition
into the water phase while α-tocopherol has virtually no water solubility and thus at best
would align at the oil-water interface. Differences in the physical location of Trolox and
α-tocopherol could alter the overall impact on lipid oxidation by influencing their
antioxidant (free radical scavenging) or prooxidant (reduction of transition metals)
activity.
This study showed that the antioxidant activity of both Tolox and α-tocopherol were
influenced by physical structures in bulk oils. Work such as this can provide important
data on how physical structures in bulk oils impact the activity of antioxidants. Since few
new antioxidants are available for food applications, this information might provide
86
information on how to alter the physical structures in bulk oils such that we can utilize
the available food antioxidants more effectively.
CHAPTER 5
87
IRON/ANTIOXIDANTS RELATIONSHIP IN BULK OIL OXIDATION
5.1 Introduction
Lipid oxidation is one of the major factors limiting the shelf life of bulk oils, since it
adversely affects flavor and quality by forming a very complex mixture of lipid
hydroperoxides, fatty acid chain-cleavage products, and polymeric materials (203,204).
In addition, lipid oxidation presents food safety concerns as low concentrations of lipid
oxidation products, such as 4-hydroxynonenal (4-HNE) and malonaldehyde (MDA) can
promote inflammation, atherosclerosis, neurodegenerative diseases and cancer (205).
The current dietary trends to consume healthier oils, such as omega-3 fatty acids is a
major challenge for food scientists since these oils are extremely susceptible to lipid
oxidation (206). Therefore, new strategies to prevent or inhibit the oxidation of bulk oils
are of major importance to consumers and the food industry. Based on the current
knowledge of lipid oxidation mechanisms, some approaches have been developed to
fulfill this goal. The application of antioxidants can retard lipid oxidation (8). But in
many foods the currently approved antioxidant are not sufficient to prevent rancidity.
Since very few new antioxidants are available to the food industry, new technologies are
needed to enhance the effectiveness of currently available food-grade antioxidants.
Bulk oil is a deceptively complex food system since it contains numerous minor
components, such as free fatty acid, sterols, antioxidants, phospholipids,
monoacylglycerol, and water (121,207). Recent studies from our group found that in bulk
oils some of these minor components are able to form physical structures known as
association colloids. These physical structures can increase lipid oxidation reactions and
alter the effectiveness of antioxidants, such as α-tocopherol, Trolox, and chlorogenic acid
88
esters (188,208,209). For example, the more polar Trolox was able to associate with
association colloids more effectively than nonpolar -tocopherol. Since association
colloids increase lipid oxidation rates, the association of Trolox with these association
colloids could help explain by it’s a more effective antioxidant than -tocopherol.
The role of transition metals on lipid oxidation in food systems has been intensively
studied (210,211). Low valence-state metal ions, such as ferrous and cuprous, can
participate in the initiation and propagation steps of lipid oxidation by abstracting
hydrogen to directly form free radicals and decomposing lipid hydroperoxides into free
radicals such as the alkoxyl radical (212). The prooxidative effect of high-valence-state
metal ions is less clear although there is some evidence that they can promote oxidation
when they are reduced by antioxidants to form the more prooxidative low valence state of
the metal ions (213,214). For example, ascorbic acid and gallic acid can promote lipid
oxidation by reducing iron to its low valence-state (215,216).
Vegetable seed oils inevitably contain transition metals which originate from the
seed and/or manufacturing equipment and ingredients suggesting that they could be
important prooxidants in bulk oils. Tocopherols are important antioxidants in bulk oils
but they have been reported to have prooxidant activity in vegetable oils at high
concentrations (217,218). While association colloids in the bulk oil seem to act as
important nano-reactors in bulk oils, it is unclear what promotes this oxidation. In
addition, since molecules such as Trolox and tocopherols can also have prooxidative
activity, it is possible that association colloids could negatively impact lipid oxidation by
enhancing the prooxidant activity of antioxidants. Therefore, the present investigation
was undertaken to explore if iron was an important prooxidant in bulk oil containing
89
association colloids. In addition, the ability of high or low valence state iron to interact
with α-tocopherol and Trolox was also determined. Iron-antioxidant interactions were
determined in a model system containing non-oxidizable medium chain triacylglycerol
(MCT) by measuring the depletion of antioxidants and the formation of free radicals.
5.2 Materials and methods
5.2.1 Materials
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was acquired from Avanti
Polar Lipids, Inc (Alabaster, AL). Soybean oil was purchased from a local store and
stored at 4 °C. Medium chain triacylglycerols (MCT, Miglyol®) were purchased
from Sasol North America Inc (Houston, TX). Silicic acid, activated charcoal,
calcein (CA), deferoxamine (DFO), methanol, hexane, alpha-tocopherol, Trolox,
and N-t-butyl-phenylnitrone (PBN) were purchased from Sigma-Aldrich Co. (St.
Louis, MO). Anhydrous ferric chloride and anhydrous ferrous chloride beads
(99.998 % purity) were purchased from Sigma-Aldrich Co.,(St. Louis, MO). All
other reagents were of HPLC grade or purer distilled and deionized water was used
as needed.
5.2.2 Formation of DOPC association colloids in stripped soybean oil or MCT
Stripped soybean oil with DOPC reverse micelles were prepared according to the
method of Chen et al (188). Briefly, formation of reverse micelles was accomplished by
pipetting DOPC (1000 μM, final concentration) in chloroform into an empty beaker and
then flushing with nitrogen until the chloroform was evaporated. The appropriate amount
90
of medium chain triacylglycerols (MCT) or stripped soybean oil (SSO) was then added
followed by double distilled water at a final concentration of 200 ppm. The oil samples
were then stirred in a beaker at 1000 rpm in a 20 °C incubator room for a 24 h.
5.2.3 Measurement of oxidation parameters
For lipid oxidation kinetics studies antioxidants were incorporated into stripped
soybean oil by stirring at 300 rpm for at least 12 h at room temperature to obtain the
homogenous samples. Then, samples were aliquoted (1 mL/vial) into GC headspace vials
and stored at 55 °C in the dark.
Lipid hydroperoxides were measured as the primary oxidation products using a
method adapted from Shanta and Decker (165). Oil samples were weighed and recorded
before adding to a mixture of methanol/butanol (2.8 mL, 2:1, v:v) followed by addition of
15 μL of 3.94 M thiocyanate and 15 μL of 0.072 M Fe2+. The solution was vortexed, and
after 20 min the absorbance was measured at 510 nm using a Genesys 20
spectrophotometer (ThermoSpectronic, Waltham, MA). The concentration of
hydroperoxides was calculated from a cumene hydroperoxide standard curve.
The secondary oxidation product, hexanal, was monitored using a GC-17A
Shimadzu gas chromatograph equipped with an AOC-5000 autosampler (Shimadzu,
Kyoto, Japan). Samples (1 mL) in 10 mL glass vials capped with aluminum caps with
PTFE/silicone septa were heated at 55 °C for 15 min in an autosampler heating block
before measurement. A 50/30 μm DVB/ Carboxen/PDMS solid-phase microextraction
(SPME) fiber needle from Supelco® (Bellefonte, PA, USA) was injected into the vial for
2 min to absorb volatiles and was then transferred to the injector port (250 °C) for 3 min.
91
The injection port was operated in split mode, and the split ratio was set at 1:5. Volatiles
were separated on a Supleco 30 m × 0.32 mm Equity DB-1 column with a 1 μm film
thickness at 65°C for 10 min. The carrier gas was helium at 15.0 mL/min. A flame
ionization detector was used at a temperature of 250°C. Hexanal concentrations were
determined from peak areas using a standard curve prepared from an authentic standard.
5.2.4 Determination of Trolox and α-tocopherol concentrations
For HPLC analysis, 100 μL of each sample was dissolved in 0.9 ml methanol and
then was passed through a 0.45 μm filter. A 20 μl aliquot of this sample solution was
separated using a Shimadzu HPLC system equipped with a Shimadzu diode array
detector (DAD), a Waters 474 Scanning Fluorescent Detector and a 250 mm × 4.6 mm
i.d., 5 μm, Inertsil C18 analytical column. The mobile phase consisted of 4 % purified
water with 3 mM phosphoric acid at pH 2.6 (solvent A) and 96 % methanol (solvent B)
using isocratic gradient at a flow rate of 1 ml/min. Column temperature was set at 38 °C.
Trolox was detected using DAD at a wavelength of 280 nm. Detection of α-tocopherol
was conducted using both DAD at 295 nm and fluorescence at an excitation wavelength
of 290 nm and an emission wavelength of 330 nm. Trolox and α-tocopherol in the
samples were identified by comparing their relative retention times and UV spectra with
authentic compounds and were quantitated using an external standard method.
Depletion of antioxidants was determined using At/A0×100%
Where Ao and At were the peak area at time zero and t, respectively.
92
5.2.5 Determination the concentration of ferric iron in MCT
Ferric iron content was determined by UV-visible spectrophotometric method using
calcein (CA) dye method developed by Thomas et al (219). Briefly, calcein solution was
prepared by adding 0.75 g of calcein in 400 g MCT and stirred overnight to dissolve the
dye. The solution was centrifuged at 10,000 rpm for 10 min and upper clear orange color
solution was collected and defined as the dye solution. Equal volume of dye solution was
added directly to bulk oil samples containing ferric iron and the absorbance was
measured at 352 nm using a Shimadzu UV-2101 PC UV−vis scanning spectrophotometer
(Shimadzu, Kyoto, Japan).
Remaining ferric iron in MCT was determined using Abt/Ab0×100%
Where Abo and Abt were the absorbance at time zero and t, respectively.
5.2.6 EPR spectroscopy
EPR measurements were carried out at room temperature using a Bruker EPR
Elexsys-500 spectrometer operating at the X-band. The samples were placed in 707-SQ-
250 M, thin wall quartz EPR sample tubes (Wilmad Glass Co., Buena, NJ, USA) and
inserted into the ER 4122-SHQE high sensitivity TE102 cylindrical mode single cavity
(Q ~ 3000) optical window of the EPR system. Instrument settings were: center field,
3470 G; scan range, 100 G; gain, 60; time constant, 128 ms; modulation amplitude, 1 G;
phase 0°; microwave power, 20 mW. Data collection was performed using the
computerized program Xepr.
93
5.2.6 Statistical analysis
Duplicate experiments were performed with freshly prepared samples. All data
shown represents the mean values ± standard deviation of triplicate measurements.
Statistical analysis of lipid oxidation kinetics was performed using a one-way analysis of
variance. A significance level of p<0.05 between groups was accepted as being
statistically different. In all cases, comparisons of the means of the individual groups
were performed using Duncan’s multiple range tests. All calculations were performed
using SPSS17 (http://www.spss.com; SPSS Inc., Chicago, IL).
5.3 Results
5.3.1 Effects of the metal chelator, Deferoxamine (DFO) on the oxidative stability of
stripped soybean oil
Transition metals are implicated in many lipid oxidation pathways as they are able to
generate free radicals that initiate and propagate lipid oxidation in food systems
(220,221). The different redox states of iron have different pathways by which they can
promote oxidation (222). For example, low-valence transition metals can promote the
decomposition of lipid hydroperoxides into free radicals such as the alkoxyl radical
(reaction 1). The high-valence state of transition metals can also decompose lipid
hydroperoxide (reaction 2) but this pathway is very slow and may not be very important
in foods. However, compounds such as antioxidants can reduce metals resulting in the
formation of the highly prooxidative low-valence state thus promoting oxidation
(reaction 3) (207,223).
Fe2+ + LOOH → Fe3+ + LO•+-OH (reaction 1)
94
Fe3+ + LOOH → Fe2+ + LOO•+H+ (reaction 2)
Fe3+ + AH→ Fe2++A•+H+ (reaction 3)
Bulk oil naturally contains transition metals. Therefore, gaining a better
understanding of the chemical role of transition metals in bulk oil oxidation is necessary
to develop effective prevention strategies to extend the shelf-life of oils. To gain a better
understanding of the role of iron on lipid oxidation in bulk oils, we studied the effect of
deferoxamine (DFO), a specific Fe(III) chelator, on the oxidative stability of stripped
soybean oil (SSO) in the absence and presence of phospholipid reverse micelles.
The formation of lipid hydroperoxides (LH) and headspace hexanal in SSO
containing different levels of phospholipids (0 and 1000 μM DOPC) and DFO (0 and 2
mM) was measured during incubation at 55 °C (Figures 5.1 A and B). The lag phase for
both lipid hydroperoxides and headspace hexanal for SSO lasted 4 days. The addition of
DOPC reduced the lag phase by 2 days, which is in agreement with our previous study
indicating that DOPC reverse micelles act as a prooxidant (209). The incorporation of
DFO had no significant effect on the lag phase of SSO (p>0.05), but extended the lag
phase of SSO containing DOPC from 2 to 7 days (p<0.05).
95
Figure 5.1. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 2 mM deferoxamine (DFO) and/or 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) during storage at 55 °C. Some of the error bars are within data points.
In order to understand if iron played a role in lipid oxidation in the presence of free
radical scavenging antioxidants, different combinations of DFO, α-tocopherol and Trolox
were added to the SSO in the presence and absence of phospholipids reverse micelles
0 2 4 6 8 10 12 140
200
400
600
800
Lip
id H
ydro
per
oxid
es (
mm
ol/
Kg
oil)
Storage time (day)
SSO SSO + DFO SSO + DOPC SSO + DOPC +DFO
A
0 2 4 6 8 10 12 14
0
200
400
600
800
1000
B
Hex
an
al (
mol
/Kg o
il)
Storage time (day)
SSO SSO + DFO SSO + DOPC SSO + DOPC + DFO
96
during incubation at 55 °C (Figures 5.2 and 5.3). The lag phase of lipid hydroperoxides
(LH) and hexanal formation was 25 days for SSO containing 100 μM α-tocopherol
(Figures 5.2 A and B). In the presence of DOPC reverse micelles and -tocopherol, the
lag phase decreased to 16 days for both LH and hexanal formation, again showing that -
tocopherol was less effective in the presence of DOPC reverse micelles (188). DFO
increased the lag phase of LH and hexanal formation in the presence of -tocopherol
both in the presence and absence of DOPC reverse micelles. In the absence of DOPC
reverse micelles, the lag phase was increased by 5 days while in the presence of DOPC
reverse micelles the lag phase was increased by 10-11 days. Unlike the previous
experiment, DFO was able to inhibit lipid oxidation in the absence of DOPC micelles
suggesting that -tocopherol was somehow increasing the prooxidant activity of iron.
0 5 10 15 20 25 30 35
0
100
200
300
400
500
600
700
Lip
id H
ydro
per
oxid
es (
mm
ol/
Kg
oil)
Storage time (day)
SSO+Toc SSO+Toc+DFO SSO+DOPC+Toc SSO+DOPC+Toc+DFO
A
97
Figure 5.2. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 100 M α-tocopherol, 2 mM deferoxamine (DFO) and/or 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) during storage at 55 °C. Some of the error bars are within data points.
The impact of DFO on the antioxidant activity of Trolox was also tested in the
presence and absence of DOPC reverse micelles (Figure 5.3 A and B). The lag phase of
lipid hydroperoxides (LH) and hexanal formation was 40 days for the SSO containing
100 μM Trolox showing that the more polar Trolox was a more effective antioxidant than
-tocopherol as predicted by the antioxidant polar paradox (224). However, unlike -
tocopherol, the incorporation of DFO did not increase the lag phase of LH and hexanal
formation in the absence of DOPC micelles. As observed previously, DOPC again
diminished the effectiveness of Trolox decreasing the lag phase to 21 days for both LH
and hexanal formation. Even though DOPC decreased the activity of both -tocopherol
and Trolox, the more hydrophilic Trolox was still the most effective of the two. When
DOPC micelles, Trolox and DFO were present in combination, DFO helped partially
offset the prooxidative effect of DOPC by increasing the lag phase for LH and hexanal
0 5 10 15 20 25 30 35
0
100
200
300
400
500
600
700
B
Hex
anal
(
mol
/Kg
oil
)
Storage time (day)
SSO+Toc SSO+Toc+DFO SSO+DOPC+Toc
SSO+DOPC+Toc+DFO
98
formation to 29 days.
Figure 5.3. Formation of lipid hydroperoxides (A) and hexanal (B) in stripped soybean oil containing 100 M Trolox, 2 mM deferoxamine (DFO) and/or 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) during storage at 55 °C. Some of the error bars are within data points.
DFO, a powerful iron chelator, has been successfully used in clinical settings to treat
patients with acute and chronic iron overload syndromes (225). DFO has a remarkably
high affinity for ferric ion allowing it to inhibit the initiation and propagation of lipid
0 5 10 15 20 25 30 35 40 450
100
200
300
400
500
600
700
Lip
id H
ydro
per
oxi
des
(m
mol
/ K
g o
il)
Storage time (day)
SSO+Trolox SSO+Trolox+DFO SSO+DOPC+Trolox SSO+DOPC+Trolox+DFO
A
0 5 10 15 20 25 30 35 40 450
100
200
300
400
500
600
700
B
Hex
anal
(
mol
/Kg
oil)
Storage time (day)
SSO+Trolox SSO+Trolox+DFO SSO+DOPC+Trolox SSO+DOPC+Trolox+DFO
99
oxidation (226). Therefore, it was anticipated that oxidative stability of bulk oil would be
improved by DFO if iron was an important prooxidant. In this study, DFO was not an
effective antioxidant in soybean oil stripped of its minor components including
endogenous antioxidants and phospholipids. However, DFO was effective in the
presence of DOPC reverse micelle indicating that the iron was an important prooxidant in
the micelles. This could occur since phospholipids can bind iron (227) and thus could
concentrate iron at the lipid-water interface of reverse micelles. Lipid hydroperoxides are
also surface active and could accumulate at the water-oil interface of the reverse micelles
(14). Thus, the prooxidant activity of DOPC could be due to their ability to concentrate
both endogenous iron and lipid hydroperoxides at the water-lipid interface of reverse
micelles thereby increasing the ability of iron to decompose lipid hydroperoxides.
The combination of the free radical scavengers, -tocopherol and Trolox, and DOPC
reverse micelles also impacted the ability of DFO to inhibit lipid oxidation. In the
absence of DOPC reverse micelles, DFO was a weak antioxidant in the presence of -
tocopherol (increased lag phase approximately 5 days) and did not inhibit oxidation in the
presence of Trolox. However, in the presence of DOPC reverse micelles and -
tocopherol or Trolox, DFO was a strong antioxidant increasing the lag phase of oxidation
to 11 and 13 days for -tocopherol and Trolox, respectively. This observation could be
due to the ability of DFO to inhibit iron-promoted generation of free radical that would
deplete the antioxidants in the DOPC reverse micelles system. However, it is also
possible that DFO could improve the ability of -tocopherol or Trolox by inhibiting
direct degradation by iron.
100
5.3.2 Effects of Fe(III) and Fe(II) on the depletion of α-tocopherol and Trolox in
MCT
In order to better understand interactions between high and low-valence iron with -
tocopherol and Trolox, a medium chain triacylglycerides (MCT) model bulk oil system
was used. MCT was used in these experiments since is does not contain unsaturated fatty
acids and thus there would be no fatty acid oxidation that could alter α-tocopherol or
Trolox concentrations. Figure 5.4A shows the depletion of α-tocopherol and Trolox
during storage at 55 oC in the presence of high-valence Fe(III). The concentration of both
α-tocopherol and Trolox were relatively constant in the absence of Fe(III). Incubation of
α-tocopherol or Trolox with 150 M Fe(III) resulted in a similar trend in antioxidant
depletion with 15 and 12 % of α-tocopherol and Trolox consumed, respectively, after one
hour storage. α-Tocopherol and Trolox concentrations in the presence of 150 M Fe(III)
reached equilibrium (~50% consumed) after 12 h. When 600 M of Fe(III) was added,
the pattern of antioxidants consumption was different with α-tocopherol being completely
consumed after 8 h of storage. Increasing the Fe(III) concentration to 600 M had much
less effect on Trolox consumption with a maximum of approximately 60% depletion of
Trolox after 24 h. The observed consumption of -tocopherol and Trolox in the presence
of Fe(III) could be due to the ability of these antioxidants donate an electron to iron thus
converting ferric to ferrous ions. Since Fe(III) was more reactive with -tocopherol than
Trolox, this could help explain why DFO was able to improve the antioxidant activity of
-tocopherol but not Trolox in the absence of DOPC reverse micelles (Figure 5.2 and
5.3) as DFO could decrease -tocopherol’s ability to reduce ferric ions to the more
prooxidative ferrous ions and could also decrease Fe(III) promoted -tocopherol
101
consumption which would help maintain a higher concentrations of -tocopherol thus
more effectively inhibiting lipid oxidation.
Figure 5.4. Depletion of α-tocopherol and Trolox (100 M) in medium chain triacylglycerols (MCT) in the presence of (A) Fe(III); and (B)Fe(II)
Since -tocopherol and Trolox consumption reached an equilibrium and did not
continue to decrease in concentrations, this suggests that Fe(II) did not cause antioxidant
consumption. To test this hypothesis, Fe(II) was also added to the MCT model system
0 10 20 30 40 50
0
20
40
60
80
100D
eple
tion
of
anti
oxid
an
t (%
)
Storage time(hrs)
100M Trolox+MCT 100M Toc+MCT
150M Fe3+
+100M Trolox
150MFe3+
+100M Toc
600M Fe3+
+100M Trolox
600M Fe3+
+100M Toc
A
0 10 20 30 40 50
0
20
40
60
80
100
B
Dep
leti
on o
f an
tiox
ida
nt
(%)
Storage time(hrs)
100M Trolox+MCT
100M Toc+MCT
150M Fe2+
+100M Trolox
150M Fe2+
+100M Toc
600M Fe2+
+100M Trolox
600M Fe2+
+100M Toc
102
(Figure 5.4B). Initial experiments were performed with reagent grade ferrous chloride
powder but rapid -tocopherol and Trolox consumption was observed (data not shown).
It was thought that this could be due to contaminating Fe(III) so experiments were
repeated using a high purity form of anhydrous ferrous supplied under vacuum. In these
experiments, 150 M Fe(II) did not significantly decrease -tocopherol and Trolox for
the first 12 h of storage compared to a 42 and 50% decrease in -tocopherol and Trolox,
respectively, during the same time period in the presence of 150 M Fe(III) (Figure
5.4A). In the presence of 600 M Fe(II), Trolox consumption was more rapid than -
tocopherol. In all cases, -tocopherol and Trolox consumption increased during
prolonged storage but the level of consumption was always significantly lower than
Fe(III) (p>0.05, Figure 5.4A). The depletion of antioxidants during the later stages of
incubation may be due to the oxidation of ferrous to ferric upon reaction with oxygen.
5.3.3 Effects of Fe(III) and Fe(II) on the depletion of α-tocopherol or Trolox in the
presence of DOPC reverse micelles
Previous results showed that DFO more effectively increased the antioxidant activity
of α-tocopherol and Trolox in the presence of DOPC micelles (Figure 5.2 and 5.3). This
could be due to DFO’s ability to decrease iron-promoted decomposition of lipid
hydroperoxides into free radicals, a factor that would decrease -tocopherol and Trolox
consumption and thus increase their ability to scavenge free radicals. However, it is also
possible that the improved activity of DFO in DOPC reverse micelles could be due to its
ability to inhibit the direct consumption of -tocopherol and Trolox by iron which again
would increase antioxidant concentrations and would also decrease the reduction of ferric
103
into the more prooxidative ferrous ions. To test this possibility, the depletion of α-
tocopherol and Trolox by Fe(III) or Fe(II) in MCT in the presence of DOPC reverse
micelles was determined (Figure 5.5 and 5.6). These experiments were also conducted
with DC4PC, a phosphatidylcholine with butyric acid. DC4PC does not form reverse
micelles at the concentrations tested (188), so experiments could be conducted with the
same concentration of the choline head group in the absence of association colloids.
Figure 5.5. Depletion of α-tocopherol and Trolox (100 M) in medium chain
0 10 20 30 40 500
20
40
60
80
100
Dep
leti
on o
f a
nti
oxid
an
t (%
)
Storage time(hrs)
150M Fe3+
+100M Trolox+DOPC
150M Fe3+
+100M Toc+DOPC
150M Fe3+
+100M Trolox+DC4PC
150M Fe3+
+100M Toc+DC4PC
A
0 10 20 30 40 50
0
20
40
60
80
100
Dep
leti
on o
f an
tiox
idan
t (%
)
Storage time(hrs)
600M Fe3+
+100M Trolox+DOPC
600M Fe3+
+100M Toc+DOPC
600M Fe3+
+100M Trolox+DC4PC
600M Fe3+
+100M Toc+DC4PC
B
104
triacylglycerols (MCT) containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) in the presence of (A) 40 ppm Fe(III); and (B) 100 ppm Fe(III)
In the absence of Fe(III), neither DOPC nor DC4PC promoted the consumption of -
tocopherol and Trolox and (Figure 5.4A and Band -tocopherol and Trolox were
stable in the absence of added iron (data not shown). In the presence of 150 M Fe(III)
and DOPC reverse micelles, the consumption of both -tocopherol and Trolox was less
than in the absence of DOPC (Figure 5.5A). For example, after 24 h of incubation, -
tocopherol concentrations were 10% lower than time 0 in the presence of DOPC reverse
micelles compared to 40% lower in the absence of DOPC reverse micelles. In the
presence of 600 M Fe(III), Trolox was depleted more rapidly than -tocopherol in the
presence of DOPC reverse micelles while the opposite was true in the absence of DOPC.
Trolox has been found to more highly associated than -tocopherol with water in DOPC
micelles (212). This means that it could have greater contact with phosphatidylcholine
bound iron at the water-lipid interface and thus could be more rapidly depleted.
In the presence of DC4PC which does not form reverse micelles, Fe(III) was rapidly
consumed by both α-tocopherol and Trolox. At low (150 M) and high (600 M) Fe(III)
concentrations, DC4PC slightly increased both α-tocopherol and Trolox consumption
compared to the absence of phospholipid (Figure 5.6A and B). DC4PC would be
expected to bind iron as would any phosphatidylcholine. The ability of DC4PC to slightly
promote the consumption of α-tocopherol and Trolox could be due to its ability to
increase the solubility of iron in the oil by acting as a lipid soluble metal chelator and
thus making the iron more reactive if the iron chelated to DC4PC would still be redox
105
active. Since DC4PC does not form reverse micelles at the concentrations tested, this
suggests that the ability of DOPC to inhibit both α-tocopherol and Trolox depletion was
not due to the phospholipid head group but instead was due to the presence of reverse
micelles.
Figure 5.6. Depletion of α-tocopherol and Trolox (100 M) in medium chain triacylglycerols (MCT) containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) in the presence of (A) 40 ppm Fe(II); and (B)100 ppm Fe(II)
5.3.4 Measurement of Fe(III) loss by α-tocopherol and Trolox
0 10 20 30 40 50
0
20
40
60
80
100
Dep
leti
on
of
an
tioxid
an
t (%
)
Storage time(hrs)
150M Fe2+
+100M Trolox+DOPC
150M Fe2+
+100M Toc+DOPC
150M Fe2+
+100M Trolox+DC4PC
150M Fe2+
+100M Toc+DC4PC
A
0 10 20 30 40 500
20
40
60
80
100
Dep
leti
on o
f an
tiox
idan
t (%
)
Storage time(hrs)
600M Fe2+
+100M Trolox+DOPC
600M Fe2+
+100M Toc+DOPC
600M Fe2+
+100M Trolox+DC4PC
600M Fe2+
+100M Toc+DC4PC
B
106
Many researchers have determined iron concentration in bulk oil (228-230). For
example, Mendil and coworkers measured iron content in bulk oil with a flame atomic
absorption spectrometer (FAAS) and total iron was found to be 139, 127, 105, and 52
ppm (i.e., 200-800 M) in olive oil, hazelnut oil, sunflower oil and corn oil, respectively
(230). While this experiment is helpful to verify that iron exist in commercially refined
oils, it does not provide any information on the redox state of the iron. This is not
uncommon since analytic techniques to measure iron redox state in bulk oil are limited.
Verifying the existence of iron recycling pathway by measuring the redox form of iron
has been extremely helpful in understanding the role of iron redox form in lipid oxidation
in both in vivo systems and oil-in-water emulsion (231,232). Therefore, in this study we
initially attempted to track the concentration of Fe(III) in MCT with calcein
acetoxymethyl ester (calcein-AM), an Fe(III) specific probe used successfully in cells and
biological fluids (233). However, calcein-AM solubility in MCT was quite limited so
calcein was successfully substituted due to its higher solubility in hydrophobic solvents
(219). Preliminary studies showed that calcein had strong absorbance at 365nm in MCT
which was quenched by ferric chloride. It also showed a good linear relationship (r>0.99)
with Fe(III) in MCT over the range of 0-600 M Fe(III) (data not shown). Room
temperature was chosen in these experiments since the 55 °C used in the previous
experiments caused too rapid of a reaction between ferric ions and the antioxidants.
The ability of -tocopherol and Trolox to reduce Fe(III) was determined at 600 M
Fe(III) and varying concentrations of the antioxidants (Figure 5.7A). After 24 h of
incubation, Fe(III) concentrations only decreased slightly (<5%) in the absence of added
antioxidants. -Tocopherol significantly decreased Fe(III) at concentrations as low as 20
107
M. Maximal decrease in Fe(III) concentrations was observed at -tocopherol
concentrations of 60 M. Trolox exhibited a similar trend on Fe(III) concentrations
although its maximal decrease in Fe(III) concentrations was 40 M. At 100 M, the
decreased in Fe(III) concentrations was slightly greater for Trolox than -tocopherol
(p<0.05).
DOPC reverse micelles by themselves caused a greater decrease in Fe(III) (Figure
5.7B) than in the absence of DOPC (Figure 5.7A) suggesting that the DOPC by itself
could reduce Fe(III) (Figure 5.7B). DOPC reverse micelles decreased the ability of both
-tocopherol and Trolox to reduce iron as significant decreases in Fe(III) were not seen
until -tocopherol and Trolox concentrations were greater than 40M. As in the absence
of DOPC reverse micelles, at 100 M, the decreased in Fe(III) concentrations was
slightly greater for Trolox than -tocopherol (p<0.05).
Overall, DC4PC (Figure 5.7C) changed the concentration of Fe(III) in a similar
manner to the absence of phospholipid (Figure 5.7A). For example, DC4PC had very
little impact on Fe(III) concentrations in the absence of antioxidants. In addition, both -
tocopherol and Trolox were able to significantly decreased Fe(III) at concentrations as
low as 20 M as seen in the absence of phospholipid. However, DC4PC decreased the
maximal decrease in Fe(III) concentrations by -tocopherol from 60 to 40 M.
108
0
20
40
60
80
100 600M Fe
3+
600M Fe3+
+Toc
600M Fe3+
+Trolox
Rem
ain
ing o
f F
e3+(%
)
Antioxidant concentration(M)
0 20 40 60 80 100
aA
bb
c
c c
B
CC
C
C
A
0
20
40
60
80
100 600M Fe3+
+DOPC
600M Fe3+
+Toc+1000M DOPC
600M Fe3+
+Trolox+1000M DOPC
Rem
ain
ing
of
Fe3+
(%)
Antioxidant concentration(M)0 20 40 60 80 100
B
aAa
a
bb b
A A
B B B
109
Figure 5.7. Fe(III) development in medium chain triacylglycerols (MCT) containing (A) α-tocopherol or Trolox; (B) 1000 M of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); (C) 1,2-dibutyryl-sn-glycero-3-phosphocholine (DC4PC) after 24 h storage at room temperature. For each group, different letters on the top of columns represent significant differences (p < 0.05)
5.3.5 Effects of Fe(III) and Fe(II) on the formation of PBN spin adducts in MCT
Since both α-tocopherol and Trolox can scavenge free radicals generated in oils, one
may argue that the depletion of antioxidants in this research could be due to the presence
of free radicals. In order to clarify this potential pitfall and provide more evidence for the
different role of Fe(III) and Fe(II) in bulk oil, an EPR study was employed in an effort to
detect the formation of free radicals in our MCT model system under different conditions.
A comparison of the EPR signals of SSO (Figure 5.8A) and MCT (Figure 5.8B)
after 24 h storage at 55 oC showed that PBN trapped free radicals were only observed in
SSO indicating that there were no detectable free radicals in our MCT model system.
When antioxidants and iron were added to MCT, the formation of α-tocopheroxyl
(Figure 5.8C) or Trolox (Figure 5.8D) radicals were not detected. This was true in the
0
20
40
60
80
100 600M Fe3+
+DC4PC
600M Fe3+
+Toc+DC4PC
600M Fe3+
+Trolox+DC4PC
0
Rem
ain
ing
of F
e3+(%
)
Antioxidant concentration(M)20 40 60 80 100
aA
bB b
B
c C cC c C
C
110
presence or absence of DOPC or DC4PC (data not shown). To determine if iron was
reactive in the MCT model, it was added in combination with cumene hydroperoxide. In
this system, Fe(II) was observed to form PBN trapped radicals (Figure 5.8E) but Fe(III)
was not (Figure 5.8F). This confirms that Fe(II) could be an active prooxidant in bulk
oils and that Fe(III) is much less reactive as has been observed oil-in-water emulsions.
Figure 5.8. EPR detection of radical formations in the reaction consisting of (A) 0.1 M PBN in stripped soybean oil; (B) 0.1 M PBN in MCT; (C) 0.1 M PBN+100 μM α-tocopherol in MCT; (D) 0.1 M PBN+100 μM Trolox in MCT; (E) 0.1 M PBN+10 mM cumene hydroperoxides+40 ppm Fe(II) in MCT; (F) 0.1 M PBN+cumene hydroperoxides+40 ppm Fe(III) in MCT after 24 h storage at 55 oC
5.4 Discussion
Cumene Peroxides+Fe3+
at 24 hrsF
C
PBN in Oxidized SSO (24 hrs)
Cumene Peroxides+Fe2+
at 24 hrs
B
A
E
PBN in MCT
PBN in MCT containing Fe3+
+TOC
D PBN in MCT containing Fe3+
+TROLOX
Field (G)
111
DFO was able to inhibit lipid oxidation in SSO in the presence of DOPC reverse
micelles indicating that iron was an active prooxidant in the presence of association
colloids. Since refined oils contain association colloids due to the presence of surface
active minor components (e.g. phospholipids, free fatty acids, lipid hydroperoxides, etc.)
and water (121), this could help explain why the metal chelator citric acid is an effective
antioxidant in commercial oils. DFO was even more effective at increasing the oxidative
stability of oils with DOPC reverse micelles in the presence of -tocopherol and Trolox.
This could be due to the decreased iron-promoted generation of free radicals or direct
iron-promoted decomposition of antioxidants.
Many phenolic antioxidants, such as the chlorogenic, caffeic, sinapic and ferulic acid, can
reduce Fe(III) to Fe(II) in aqueous environments resulting in the consumption of the
antioxidant. For example, 1 molecule of caffeic acid was reported to reduce 9 atoms of
Fe(III) (234,235). Our result showed that -tocopherol and Trolox can reduce Fe(III) in
bulk oil as seen by both the ability of Fe (III) to decrease antioxidant concentrations
(Figure 5.4) and the ability of antioxidants to decrease Fe(III) concentrations (Figure
5.7B). In this study, DOPC reverse micelles decreased interactions between iron and -
tocopherol or Trolox as seen by both a decrease in iron-promoted -tocopherol and
Trolox decomposition and a decrease in the ability of -tocopherol and Trolox to
decrease Fe(III) concentrations. This suggests that DFO did not decrease the iron-
promoted decomposition of -tocopherol and Trolox. Therefore, it is more likely that the
ability of DFO to enhance the activity of -tocopherol and Trolox in DOPC reverse
micelles was due to its ability to inhibit iron-promoted generatation of free radicals
through pathways such as hydroperoxides decomposition. This could occur if iron
112
concentrated at the water-lipid interface of the DOPC reverse micelles and increased its
ability to decompose surface active lipid hydroperoxides. Decrease production of free
radicals from decomposing lipid hydroperoxides would decrease the consumption of the
antioxidants thus increase their effectiveness.
CHAPTER 6
113
CONCLUSION
This research has shown that 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) can
self-assemble in stripped soybean oil (SSO) to form thermodynamically stable
association colloid structure, most likely reverse micelles. Interfacial tension and
fluorescence spectrometry results showed the critical micelle concentration (CMC) of
DOPC in stripped soybean oil was 650 and 950 μM, respectively which are well within
the phospholipid concentrations found in refined vegetable oils. Light scattering
attenuation results showed water concentrations was a critical factor in DOPC reverse
micelle structure with a 500 ppm water threshold above which association colloids
altered the optical properties of stripped soybean oil. Small-angle X-ray scattering (SAXS)
and fluorescence probes again showed that water had a very strong impact on the
properties of the association colloids formed by DOPC.
Measurement of primary and secondary lipid oxidation products revealed that
reverse micelles formed by DOPC reverse micelles had a pro-oxidant effect, shortening
the lag phase of SSO at 55 °C. This research also showed that 1,2-dibutyl-sn-glycero-3-
phosphocholine (DC4PC) did not form association colloid in stripped soybean oil, nor did
it change lipid oxidation kinetics of stripped soybean oil indicating that it was not the
choline head group that responsible for the observed accelerated lipid oxidation but
instead was likely the reverse micelle itself. The addition of ferric ion chelator,
deferoxamine (DFO) in stripped soybean oil significantly prevented the lipid oxidation
caused by DOPC reverse micelles extending lag phase from 2 to 7 days. This indicated
that iron was a strong prooxidant in the DOPC reverse micelles. Lipid hydroperoxides
are also surface active and therefore likely concentrate in the DOPC reverse micelles.
114
Therefore the proposed mechanism of oxidation in DOPC reverse micelles is iron
promoted lipid hydroperoxide decomposition into free radicals.
DOPC reverse micelles can also impact the physical location of antioxidants such as
non-polar α-tocopherol and its polar analog Trolox. The steady state fluorescence
intensity of the probe, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-
glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE), revealed that both α-
tocopherol and Trolox were associated with DOPC reverse micelle in bulk oil. Trolox
primarily concentrated in the water pool of reverse micelle since it quenched the NBD-
PE intensity with increasing concentrations. A portion of α-tocopherol was also
associated with the aqueous phase as evidence by the fact that it did not impact NBD-PE
intensity when its concentration was increased from 50 to 100 μM. The shortened life
time of NBD-PE fluorescence by Trolox but not -tocopherol was provided evidence that
Trolox was more highly associated with the water pool of the DOPC reverse micelles.
Increasing the concentration of both -tocopherol and Trolox increased the stability of
bulk stripped soybean oil containing DOPC reverse micelles. Hydrophilic Trolox had
better antioxidant activity than hydrophobic α-tocopherol. This could be due to Trolox’s
ability to more highly associate with the water phase of the reverse micelles where lipid
oxidation is prevalent.
Since -tocopherol and Trolox both associate with the DOPC reverse micelles, it is
possible that they could also interact with iron at the oil-water interface as proposed in
Figure 6.1. High-valence (FeIII) and low-valence-state (FeII) iron could interact with
Trolox and α-tocopherol) causing their depletion or increasing the reactivity of iron if the
antioxidant caused iron reduction. However, DOPC reverse micelles minimized iron-
115
antioxidant interactions as seen by their ability to decrease iron-promoted -tocopherol
and Trolox degradation as well as decrease the ability of the antioxidants to reduce Fe(III)
to Fe(II). It is unclear how the DOPC reverse micelles decrease interactions between iron
and -tocopherol or Trolox. It does not seem to be due to physical hindrance since the
reverse micelles protected both hydrophilic Trolox which would concentrate in the
aqueous phase and hydrophobic -tocopherol which would concentrate in the lipid phase.
Therefore, protection could be due to the phosphate group that would bind iron and
decrease its ability to interact with the antioxidants.
Figure 6.1. Proposed mechanism of bulk oil oxidation that contains reverse micelles
Overall, this research identified several important new insights into potential lipid
116
oxidation mechanisms in bulk oil. First, it showed that DOPC can form physical
structures such as reverse micelles in vegetable oils at the concentrations of
phospholipids and water found in commercially refined oils. These physical structures
increase lipid oxidation rates especially oxidation promoted by iron. Antioxidants were
also shown to associate with the DOPC reverse micelles with the more hydrophilic
Trolox associating with the association colloids than -tocopherol. The evidence of
acceleration of oxidation by DOPC reverse micelles suggests that these physical
structures could be a major site for oxidation reactions which could help explain by
hydrophilic antioxidants are often more effective than hydrophobic antioxidants in bulk
oils.
The results of this research also identified a number of areas where future
experiments would be beneficial in developing knowledge of how association colloids
impact bulk oil oxidation mechanisms and could present new opportunities for inhibiting
lipid oxidation:
(i) Investigation the impact of free fatty acids on bulk oil oxidation containing
association colloids;
(ii) Detection dissolved oxygen in oil and its role on iron redox recycling;
(iii) Identification the reaction products between α-tocopherol or Trolx and Fe(III)
and their role on bulk oil oxidation;
(iv) Investigation the relationship between lipid hydroperoxides generation,
decomposition and the iron recycling in bulk oil system.
(v) Investigation the impact of other phospholipids, such as phosphatidylserine (PS)
and phosphatidylethanolamine (PE) on bulk oil oxidation, specifically on the physical
117
structure formation and chemical stability.
The knowledge gained from this study will improve our understanding of causes and
prevention of lipid oxidation in bulk oils. It suggests that DOPC reverse micelles can
alter the microenvironment where chemical degradation reactions occur, such as lipid
oxidation. In addition, lipid oxidation in bulk oils is not only influenced by the traditional
chemical factors, such as fatty acid composition, transition metals and antioxidants, but is
also related to the existence of physical structures. Understanding how these physical
structures impact lipid oxidation and antioxidant mechanisms could lead to the
development of new antioxidant technologies and/or methods to use existing antioxidants
more effectively.
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