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Effect of microwave radiation on lipid oxidation of tilapia fish

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THESIS
SIGNATURE APPROVAL SHEET
Title of Thesis: E f f e c t o f M icrow ave R a d ia t io n on L ip id __________
O x id a tio n o f T i l a p i a F is h
Degree Candidate:.
K h aled A. A b o u -Z eid
Thesis and Abstract Approved
by Advisor: D r. Y o u s s e f s . H afez
Name
Human E c o lo g y
Department
♦Signature of Advisors
p-UDate:
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Advisory Committee:
Name:
Department:
Name*
Department:
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Human E c o lo g y
Dr* J a 9mohan J o s h i
A g r ic u ltu r e
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ABSTRACT
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Title o f thesis:
Effect o f Microwave Radiation on Lipid Oxidation o f Tilapia Fish
Degree candidate:
Khaled Abou-Zeid
Degree and year:
Master o f Science, 2002
Thesis directed by: Dr. Youssef S. Hafez, Professor, Department o f Human Ecology
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This study investigated the effect o f microwave cooking on the secondary oxidation of
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lipid in tilapia fillet treated with various antioxidants (ascorbic acid, a-tocopherol and
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BHT) during four weeks of storage at -10 °C. Lipid oxidation in tilapia fillet is thought
to initially occur after exposure to microwave radiation. Therefore, fillet were treated
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with three levels (0.01, 0.10 and 1.0%) o f each antioxidant as a primary model to
evaluate the optimum level required to protect lipids against oxidation induced by
microwave radiation. The optimum level o f the three antioxidants was found to be at
0.1% level. Therefore, fillet samples were treated with 0.1% o f each antioxidant before
frozen storage at -10 °C. The experiment was conducted with a two way factorial design
(4x4), four antioxidant treatments (none, ascorbic acid, a-tocopherol and BHT ) and four
storage periods (1,2, 3 and 4 weeks) before and after microwave cooking. The lipid
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oxidation damage in tilapia fillet was determined using fluorimetric thiobarbituric acid
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(TBA) test. The TBA values o f the fillet have significantly increased by either increasing
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microwave cooking or storage time. Addition o f antioxidant protected lipid against
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oxidation induced by these two factors. Among the antioxidants tested a-tocopherol
showed a superior effect of inhibition o f lipid oxidation to BHT, while ascorbic acid has
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no significant effect against lipid oxidation in most cases. In addition, ascorbic acid was
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found to act as a prooxidant rather than antioxidant after microwave cooking, particularly
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after 4 weeks o f storage at -10 °C. The result o f this study indicated that addition o f a-
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tocopherol and BHT was required to reduce lipid oxidation which was induced by either
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microwave cooking or frozen storage times.
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Effect of Microwave Radiation on Lipid Oxidation
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of Tilapia Fish
By
Khaled Abou-Zeid
Thesis submitted to the Graduate Faculty
o f the Department of Human Ecology
at the University of Maryland Eastern Shore
in partial fulfillment o f the requirement
for the degree of Master o f Science
2002
Advisory Committee members
Dr. Youssef Hafez, Professor, Chair
Dr. Jagmohan Joshi, Professor
Dr. Kisun Yoon, Assisstant Professor
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UMI Number: E P13876
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Acknowledgement
I would like to express my sincere thanks to Dr. Youssef Hafez, my advisor, for
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his kind guidance, encouragement and helpful criticism throughout the course o f my
graduate study and during the preparation o f this thesis. I extend my thanks to my
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committee members, Dr. Jagmohan Joshi and Dr. Kisun Yoon for their kind supports,
valuable suggestions and their efforts that contributed to the successful completion of this
research.
Acknowledgement is extended to Dr. Steve Hughes who provided tilapia for this
research. In addition, special thanks go to Mr. Matthew Whittiker for his assistance and
cooperation to help me to overcome many problems during my research.
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TA BLE OF CONTENTS
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Section
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List of Tables
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List o f Schemes
List o f Figures
C h ap ter 1: Introduction
Objectives
C h ap ter 2: Literature Review
2.1. Chemical and nutritional aspects o f lipid oxidation in foods
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2.1.1. Malonaldehyde: Formation and reactive species
2.2. Measurement techniques o f lipid oxidation
2.2.1. Determination of lipid oxidation by UV spectroscopy
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2.2.2. Determination of MDA by HPLC
2.2.3. Thiobarbituric acid (TBA) Test
2.2.3.1 Spectrofluorometric procedures o f lipid peroxidation
2.3. Effect of Microwave radiation on Different Food Components.
2.4. Detection o f Lipid Damage During Different Food Processing
2.5. Lipid Peroxides as a Cause o f Diseases.
C h ap ter 3: Materials and Methods
3.1. Chemicals and Reagents
3.2. Sample Preparations
3.3. Microwave Radiation
3.4. Determination of TBA Values as an Index o f Lipid Oxidation
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TABLE OF CONTENTS (C ont’d)
Page
Section
3.5. Water Content
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3.6. Statistical Analysis
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C hapter 4: Results & Discussion
4.1. Interactions between various antioxidants and storage times on lipid
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oxidation o f tilapia fillet before microwave cooking.
4.2. Interactions between various antioxidants and storage times on lipid
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oxidation of tilapia fillet cooked for two minutes in microwave oven.
4.3. Interaction between various antioxidants and storage times on lipid
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oxidation of tilapia fillet cooked for four minutes in microwave oven.
4.4. Interactions between microwave cooking times and various antioxidants
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and their effects on lipid oxidation o f tilapia fillet.
4.5 Effects o f microwave cooking and frozen storage on lipid oxidation o f
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tilapia fillet
4.6 Antioxidative activity of ascorbic acid, a-tocopherol and BHT towards
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lipid oxidation of tilapia fillet
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C hapter 5: Summary and Conclusion
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References
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Appendix I
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Appendix II
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Appendix III
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IV
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L IST O F TABLES
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Table
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1. Interactions between various antioxidants and storage times on lipid
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oxidation o f tilapia fillet as measured by TBA values before microwave
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cooking.
2. Interactions between various antioxidants and storage times on lipid
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oxidation o f tilapia fillet as measured by TBA values cooked for two
minutes in microwave oven.
3. Interaction between various antioxidants and storage times on lipid
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oxidation o f tilapia fillet as measured by TBA values cooked for four
minutes in microwave oven.
4. Interactions between microwave cooking times and various antioxidants
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and their effects on lipid oxidation as measured by TBA values o f tilapia
fillet.
5 Effects o f microwave cooking times on lipid oxidation o f tilapia fillet as
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measured by TBA values
6. Effect o f storage times on lipid oxidation o f tilapia fillet
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7. Antioxidative activity o f ascorbic acid, a-tocopherol and BHT towards lipid
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oxidation of tilapia fillet as measured by TBA values
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LIST OF SCHEMES
Scheme
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Scheme 1. Lipid hydroperoxides and TBA Reaction
Scheme 2. Conversion o f arachidonic acid to hydroperoxy acids and Leukotriene
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LIST O F FIG U RES
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Figure
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Figure 1. The changes in TBA o f the tilapia fillet after different microwave
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radiation times
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Figure 2. The changes in TBA o f the tilapia fillet during frozen storage at -10 °C
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CHAPTER 1
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Introduction
In the last decades, there has been an increase in the use o f microwave ovens
(MO) for food preparation which reflected in an increase in the number o f food products
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prepared specifically to defrost, or cook in MO at the market (Decareau, 1993).
Microwaves interact with water in food products and generate heat. Production o f heat
occurs primarily when ions accelerate and collide or dipoles rotate and line up in the
rapidly alternating electric field (Virtanen et al., 1997). Microwave energy has been also
used to pasteurize or sterilize foods at lower temperatures and needs shorter times than
necessary with conventional heating (Shin and Pyun, 1997).
Application of microwave heating for food processing provides potential
advantages such as, retention of product quality and improvement o f process efficiency.
As a result o f the unique penetrating nature and volumetric heat generation within the
food, rapid heating can be achieved. However, a problem that has often been encountered
is the occurrence of uneven temperature profiles within a product (Decareau, 1993). For
example, when high fat foods are cooked in microwave oven free radicals could be
formed according to the sharp increase o f temperature in a very short period (Lie-Ken-Jie
and Yan-Kit, 1998). This was attributed to the fact that fats have a great capacity for
storing microwave energy, although they have a small dielectric loss (Mohsenin, 1984).
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particularly on lipid oxidation and fatty acid decomposition in different kinds o f foods.
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Takagi et al. (1999) studied the effect of microwave roasting for 6 ,1 2 and 20 minutes at a
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frequency o f 2450 MHz on whole soybeans. The amounts o f triacyl-glycerols,
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Few studies have evaluated the effects o f microwave cooking on food,
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phospholipids and the percentages o f polyunsaturated fatty acids have significantly
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decreased (P<0.05) after 12 minutes o f microwave roasting.
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Aubourg (1999) demonstrated that fish quality decreases during frozen storage as
a result o f increasing time and temperature o f storage. At the same time, it has been
proven that accumulation o f free fatty acid in frozen fish is related to some extent with
the lack o f acceptability o f frozen fish because free fatty acid are known to cause texture
deterioration by interacting with proteins and have shown to be strongly correlated with
lipid oxidation (Aubourg, 1999)
Related studies pointed out that there is an intimate correlation between lipid
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peroxides and several diseases (Leaf, 1994). This is attributed directly to the fact that
lipid peroxide increase is related to the pathogenesis o f many degenerative diseases.
Atherosclerosis and coronary heart disease (CHD) are examples o f these diseases (Papas,
1999). Despite dramatic advances in treating atherosclerosis and CHD, their complete
prevention remains difficult. There are many factors that can negatively affect
atherosclerosis as well as CHD. Elevated plasma cholesterol and increased plasma
protein are believed to be the most prevalent factors. These factors could be provoked by
oxidative damage of lipids (Harris et al., 1993). Haberland et al. (1984) realized that
formation o f Schiff bases involving the amino groups o f lysine and malondialdehyde
(MDA), a product of lipid peroxidation, would result in the formation o f a net negative
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charge that is readily degraded by macrophages. This process enhances the uptake o f low
density lipids (LDL) by macrophages, which is the key for CHD and atherosclerosis
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development.
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Lately, it has been found that plasma lipids and platelet aggregation could be
affected by dietary fish oil; these beneficial effects include lowering plasma lipids and
platelet aggregation (Higdon et al., 2000; Lewis et al., 2000). Also, fish and particularly
fish oil have a clear protective effect on human health as compared to refined, enriched,
commercial supplementations that contains large amounts o f eicosapentaenoic and
docosahexaenoic acids (Baily, 2001; Muggli, 1994). Ackman (1989) reported that marine
fish can be broken down into four convenient categories: lean, low fat, medium fat and
high fat. These could contribute about 250,270, 1000 and 2000 mg o f total co-3 PUFA
per 100 grams, respectively. Dietary intake can compare favorably with the alternative o f
commonly available fish oil capsules. Thus, there is an urging demand for high quality
and longer shelf life foods, particularly seafood as well as a need to optimize processing
steps o f food to keep food nutritionally safe.
Over the past few years, many researchers have focused on the detrimental effect
o f microwave heating during cooking (Daglioglu, et al., 2000). From this point, fats,
among other food components, are vulnerable to be attacked by either oxygen free
radicals or hydroxyl radicals, particularly during microwave radiation. Protection o f fats
against different peroxidation factors required the presence o f a compound that is able to
dissipate free radicals away from the lipid oxidation cycle. Addition of different
antioxidants function as chain breaking species that terminate further initiation o f free
radical formation (Sean et al., 2001).
Synthetic and natural food antioxidants are used routinely in many foods,
especially those containing oils and fats. Phenolic antioxidants; such as, butylated
hydroxytoluene (BHT) and water soluble antioxidants such as ascorbic acid are used
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extensively in food as food additives. Their indirect benefits to health from inhibition of
lipids oxidation in foods are well documented (Papas, 1999).
Alpha-Tocopherol, a fat soluble compound, commonly known as vitamin E, has
been recognized as a strong antioxidant (Hansmann, 1997). Vitamin E defense against
oxidation is the key to preventing damage o f fats by attack from O 2 free radicals (Sean et
al., 2001). While there is a clear evidence for the requirement o f vitamin E in connection
with the amount and degree o f unsaturation o f polyunsaturated fatty acids (PUFA) in the
diet, the exact amount of vitamin E needed to compensate for this increased demand
caused by PUFA in the diet has not been systematically investigated in human being
(KulSs and Ackman, 2001).
Objectives:
Therefore, the objective o f this study was to determine the effect o f several
antioxidant treatments (none, ascorbic acid, a-tocopherol and BHT) and storage time (1,
2, 3 and 4 weeks) on lipid oxidation o f tilapia fillet stored at -10 °C before and after
microwave radiation.
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CHAPTER 2
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Literature Review
2.1. Chemical and Nutritional Aspects of Lipid Oxidation in Foods
One of the most important causes of meat deterioration is lipid oxidation, which
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affects fatty acids, particularly polyunsaturated fatty acids (PUFA) (Pearson et al., 1983).
This lipid autooxidative degradation produces by-products that change the food quality,
e.g. color, aroma, flavor, texture and even the nutritive value (Eriksson, 1982).
This modification o f fatty acids is principally carried out by an autocatalytic
mechanism of “free radicals”, called autooxidation, consisting o f three phases (Rahaijo
andSafos, 1993)
1. Initiation:
(a) RH + 0 2 -> R* + *OOH
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2. Propagation
(b) R* + 0 2 -» ROO •
(c) RH + ROO • -> ROOH + R •
(d) ROOH -» RO • + -OH
3. Termination
(e) R* + R* -> R -R
(f) R* + ROO • - > ROOR
(g) ROO • + ROO • -> ROOR + 0 2
Hydroperoxides (ROOH) are considered to be the most important initial reaction
products that are obtained from lipid oxidation; they are labile species o f a very transitory
nature which undergo changes and deterioration with the radicals (Groff and Gropper
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2000). Their breakage produces secondary by-products such as pentanal, hexanal, 4hydroxynonenal and malonaldehyde (MDA) (Raharjo and Safos, 1993).
2.1.1. Malonaldehyde: Formation and Reactive Species
Malonaldehyde is a three-carbon dialdehyde with carbonyl group at C-l and C-3
position. There are different theories about the possible mechanism o f MDA formation;
firstly, through hydroperoxides formed from PUFA, secondly, with three double bonds
(triene) or more, and thirdly, associated with phospholipids in animal food. Tarladgis et
al. (1960) postulated a mechanism for the formation o f MDA based on investigations
which showed that only peroxides which possessed a or P unsaturation to the peroxide
group were capable of undergoing cyclization to finally form MDA. However, some
studies pointed to the presence of small amounts o f MDA from:
(a) Fatty acids with less than three double bonds (Tarladgis et al., 1960); in this case,
MDA production is partially due to the secondary oxidation o f primary carbonyl
compounds (e.g. 2-nonenal)
(b) Endoperoxides involved in the synthesis o f prostaglandins, which could be non­
volatile MDA precursors capable of yielding MDA with heat or acids (Tarladgis
etal., 1960).
(c) Iron-dependent oxidative degradation o f amino acids (AA), complex
carbohydrates, pentoses and hexoses and from free radical products produced by
gamma-irradiation treatments “in vivo”. The amounts o f MDA like materials
produced depend on the energy o f irradiation (Yeo and Shibamoto 1992).
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The substances that react with TBA but which are not MDA, are called TBA-reactive
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substances (TBARS) (Igene et al., 1985) because:
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(a) They react with 2-thiobarbituric acid to give an adduct whose spectrum is
identical with that obtained from MDA standard;
(b) Their ultraviolet (UV) spectrum is identical o f that o f MDA standard at both pH
7.0 and pH 2;
(c) They co-elute with MDA standard when analyzed by HPLC
Polyunsaturated fatty acids in meats'are more likely to be found in esterified form
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with phospholipids than with triglycerides (Pikul and Kummerow, 1991). Therefore, the
phospholipid fractions have been identified as a primary substrate in the development of
the oxidative deterioration in muscle food. A study by Pikul and Kummerow, (1991)
revealed that relatively high levels of arachidonic acid as well as docatetraenoic acid,
dodecapentaenoic and dodecahexaenoic acid are found in phosphatidylserine and
phosphatidylcholine. These phospholipids were responsible for generation o f many of the
TBARS, including MDA in chicken liver, heart, plasma and egg yolk (Pikul and
Kummerow, 1991).
Pikul et al. (1989) illustrated factors which determine the extent and amount of
MDA formation from peroxidized polyunsaturated fatty acids as: the degree o f fatty acid
unsaturation, the presence o f metals, pH, temperature and duration o f heating. Iron
catalyses fatty acid hydroperoxide decomposition to MDA at physiological pH and
temperature. These studies suggest that the degradation products o f fatty acid
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hydroperoxides in living and post-mortem tissues may differ from the degradation
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products obtained by heat and acid treatment during the TBA test (Igene, et al., 1985).
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MDA is thought to be a carcinogenic initiator and mutagen, and therefore can
affect the safety o f food. It has been found that the type o f cooking (e.g. microwave,
roasting, etc.), time and temperature affected TBA content (Igene et al., 1979).
2.2. Measurement Techniques of Lipid Oxidation
Today, numerous methods are available to detect lipid peroxidation, but only a
few give the direct information about initial processes in lipid peroxidation, which is
highly relevant for shelf-life prediction. Determination o f thiobarbituric acid reactive
substances (TBARS) (Yagi, 1982), determination o f lipid peroxides (Asakawa and
Matsushita, 1980), ultraviolet and fluorescence measurement (Chio and Tappel, 1969;
Yagi, 1982) represent the most widely used methods in measurement o f lipid
peroxidation. Most of these methods measure secondary oxidation products and give
information about initial lipid peroxidation. Measurement o f oxygen consumption (Le
Tutour, 1990) has also been extensively used, but this method is not always appropriate
due to possible interfering oxygen consumption or production in complex systems
(Ozhogina and Kasaikina, 1995). Measurement o f hydroperoxides by simple iodometric
titration (AOCS, 1992), or by chromatographic separation using HPLC (AOCS, 1992) is
also used for measurement of initial lipid peroxidation. However, these methods do not
allow continuous measurement in reaction mixtures or in foods, and their use in quality
control is thus somewhat restricted (Baron et al., 1997). The techniques most widely used
to measure lipid oxidation are:
2.2.1. Determination of Lipid Oxidation by UV Spectroscopy
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This method uses the absorbance difference between acidified and basified MDA
solutions at 267 nm (Grau et al., 2000a). The ultraviolet absorption spectrum o f MDA is
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pH-dependent, and their absorption in this region between pH 3 and pH 7 shifts
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progressively. This behavior is attributed to the progressive dissociation o f the enolic
hydrogen with increasing pH: at pH 3 or lower, the compound is thought to have a cyclic
planer delta-cis-configuration, with an intramolecular hydrogen bond; above pH 7 it is
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completely dissociated and exists as a planar delta-trans-enolate anion (Kwon and Watts,
1963). The method has been successfully applied to assay o f MDA in distillates from
rancid foods. The test is simpler and rapid but less sensitive than TBA test.
2.2.2. Determination of MDA by HPLC
A number of recent studies have reported that HPLC method provided very
sensitive and selective detection o f lipid hydroperoxides. The measurement o f various
lipid hydroperoxides has been accomplished by normal-phase and reverse-phase HPLC
separation with various modes of detection. The method was found to produces a linear
correlation between TBA values and HPLC results (Henderson et al., 1999).
2.2.3. Thiobarbituric acid (TBA) Test
Thiobarbituric acid (TBA) reactions have been widely used to determine the
amount o f the lipid peroxides in the blood in various tissues (Izushi and Ogata, 1990). It
has been generally assumed that lipid peroxides undergo decomposition through several
possible intermediates to produce malonaldehyde (MDA) as shown in Scheme 1
(Hayaishi, 1982). MDA is thus produced and reacts with TBA to yield a pink colored
TBA pigment with characteristic absorption and fluorescence spectra (Yagi, 1982).
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CH
QOH
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VW v
R' Heat, H
+2
NH
TBA
TBA pigm ent
* X ex =
535 nm
Xem = 553 nm
* X ex
:excitation wavelength
A,em remission wavelength
Scheme 1 Lipid hydroperoxides and TBA reaction (reproduced from; Dahle et al.,1962).
TBA reactive material apparently decomposes under the conditions o f the TBA
test to produce MDA, which then reacts with TBA, but the identity o f these compounds
has not been established (Yagi, 1982).
2.2.3.I. Spectrofluorom etric Procedures of Lipid Peroxidation
This method is based on the fact that the excitation spectrum o f MDA-TBA
adduct shows its maximum at 532 nm. This method also requires a prior lipid extraction
(Jo and Ahn, 1998). Test on the whole sample or the extracted fat may be appropriate for
some samples. A recent study conducted by Yagi (1982) indicated that the fluorometric
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method had greater sensitivities than the spectrophotometric method, but was not
appropriate for meat. Later in 1998, Jo and Ahn, overcome this problem and developed a
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sensitive, simple, and reliable method that can determine lipid oxidation products in
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meat. Their results were reproducible and competitive to the other TBA tests.
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2.3. Effect of Microwave Radiation on Different Food Components.
The effects o f microwave roasting on phospholipids in soybeans were
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investigated in relation to moisture (Yoshida et al., 1997). Whole soybeans at different
moistures levels (9.6,38.2, and 51.9%) were roasted by exposure to microwaves at a
frequency o f 2,450 MHz. During microwave roasting, it was found that the lower the
moisture content the higher the internal temperature in soybeans. They also found that
phosphatidylcholine was the principal phospholipid in the extracted lipids from all
unroasted and roasted soybean samples. After microwave roasting, phospholipids
containing an amino group, especially phosphatidylethanolamine, was decreased
substantially (p<0.05) in lower-moisture soybeans. Increasing the moisture content,
however, depressed the rise in the internal temperature o f soybeans and prevented the
reduction in phospholipids and/or polyunsaturated fatty acids in the phospholipids. Based
on the changes in the composition and fatty acid distribution o f phospholipids in
soybeans during microwave roasting, it is necessary to consider the moisture content in
soybeans when roasting in a microwave oven (Yoshida et al., 1997).
In a similar study, whole soybeans were exposed to microwave roasting for 6, 12,
and 20 minutes at a frequency of 2,450 MHz whereas, phospholipid composition and
positional distribution o f the fatty acids were also studied (Yoshida and Takagi, 1997).
They found that during microwave roasting the greatest rate o f phospholipid losses (p<
0.05) was observed in phosphatidylethanolamine (PE), followed by phosphatidylcholine
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(PC) and phosphatidylinositol (PI), respectively. They also reported that the effects of
microwave roasting on the composition and positional distribution o f fatty acids were
likely clearer in PE than in PC or PI. However, the principal characteristics for the
positional distribution of fatty acids were still retained during microwave roasting:
unsaturated fatty acids, especially linoleic, were predominantly concentrated in the 2position, and saturated fatty acids, especially palmitic, primarily occupied the 1-position
after 12 or 20 minutes o f roasting. The results suggested that unsaturated fatty acids
located in the 2-position were significantly protected from microwave roasting.
The effects of microwave cooking and conventional baking on the fatty acid and
tr a n s
fatty acid compositions of puff pastries; which contain high amounts of
hydrogenated fat, were investigated by Daglioglu (2000). In addition, free fatty acids,
peroxide value, and induction time for oxidative stability by the Rancimat method were
also compared for microwaved and conventionally baked puff pastries. The data
indicated that there were considerable changes in acidity, peroxide value, and Rancimat
induction time in both microwaved and conventionally baked samples. Although the
content o f saturated fatty acids, such as palmitic and stearic and the ratio o f saturated to
unsaturated fatty acids did not change significantly, an apparent increase was determined
in
tra n s
oleic acid levels by both baking methods. In addition, a significant decrease in
linoleic acid content o f the samples was found by microwave baking.
Ramezanzadeh et al., (2000) studied the effect o f microwaves on proximate and
the fatty acid composition of rice bran during 16 weeks of storage. The moisture content
decreased significantly from an initial 8.4% to 6.4% in microwave-heated samples
regardless o f packaging methods and storage temperatures. Protein, fat, linoleic, and
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linolenic contents did not change significantly in all raw and microwave-heated samples
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during 16 weeks o f storage. Thus, it was concluded that fatty acids and proximate
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compositions were not changed drastically in microwave-heated rice bran compared with
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raw samples kept under similar storage conditions. These results indicate that the
stabilization o f rice bran by microwave heating can be employed without concern of
deleterious changes to major nutrient concentrations in the bran.
Takagi et al., (1999) exposed whole soybeans to microwave roasting for 6 ,1 2 or
20 minutes at a frequency o f 2,450 MHz. The seed coat, axis and sections o f cotyledons
separated from three soybean cultivars were analyzed for their fatty acid contents by gas
chromatography before and after microwave roasting. Significant decreases (P<0.05)
were observed not only in the amounts o f triacylglycerols and phospholipids but also in
the percentages o f polyunsaturated fatty acids in the seed compared with those o f the
cotyledons or the axis. These results imply that the effect o f microwave energy made
significant differences (P<0.05) in the distribution o f soybeans.
Agre and Hannine (1993) demonstrated the effects o f cooking on the fatty acid
concentrations and the stability o f long chain n-3 fatty acids in fish flesh. The cooking
methods used were boiling and baking in the conventional and microwave oven as well
as frying with sunflower and rapeseed oils. Three freshwater fish species (rainbow trout,
vendace and pike) with different sizes and lipid contents were studied. The
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concentrations o f long chain n-3 fatty acids in fish flesh increased in most cases due to
the cooking process. This was mostly caused by the loss o f moisture. However, in baked
and microwave-cooked vendaces their concentrations increased about 70% even when
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calculated on a dry weight basis. Frying oils were efficiently absorbed into lean vendace
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and pike, and to some extent into medium fatty rainbow trout. The absorption o f n-6 fatty
acids from cooking oils may interfere with the biological effects o f n-3 fatty acids.
2.4. Detection of Lipid Damage During Different Food Processes
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Lipid peroxidation has been measured in various kinds o f meat through 2-thiobarbituric acid (TBA) tests. Grau et al. (2000b) studied several variables such as kinds of
filter paper, amount of sample, antioxidant addition, stability of spectrophotometric
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measurements, handling and storage of samples which can influence TBA values in dark
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chicken meat using an acid aqueous extraction method with derivative
spectrophotometry. Filter paper with larger pore diameter led to lower TBA. Based on the
fact that protein binds MDA (especially through their free amino groups) and thus
prevent its reaction with TBA (Aubourg, 1999), filter papers with larger pores retain
smaller protein particles and so give filtrates with lower protein concentration than small
pore filter papers. Larger samples also led to lower TBA values. This could mainly be
due to a more incomplete extraction of MDA when higher amounts o f sample were used.
Addition o f the BHT/hexane solution immediately to the samples is useful to protect
ground meat from oxidation during analysis and to reduce variability of the method.
Moreover, it has been demonstrated that the addition of an antioxidant such as BHT prior
to the blending step was essential to prevent meat from oxidation. Furthermore, in cooked
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samples, addition o f BHT was not enough to avoid sample oxidation. It was necessary to
add ethylenediaminetetraacetic acid (EDTA) immediately after weighing in order to
chelate metal ions, especially free iron, which was released during the cooking procedure
and played a marked role in lipid oxidation. The thawing and refreezing o f a cooked
sample for three consecutive days led to a great increase in TBA values. Storage o f three
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cooked samples for 24 hour at 4 °C in a plastic zip-bag also led to a dramatic increase in
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TBA values. However, storage of vacuum-packed cooked samples for 7 months at -20 °C
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did not lead to an increase in TBA values.
Lipid damage was detected in blue whiting fillets frozen at -40 °C and stored at
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-30 °C and -10 °C for one year (Aubourg, 1999). At -30 °C most o f the lipid damage
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indices (free fatty acids, peroxide value, conjugated dienes, thiobarbituric acid index and
fluorescence detection) showed a significant correlation (p<0.05) with the storage time.
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However, as the fish damage increased (-10 °C damage) only the free fatty acid content
and fluorescence ratio showed a satisfactory correlation with the storage time. The
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reliability o f the remaining indices decreased in order to assess lipid damage and fish
deterioration changes. As an explanation, degradation products that are measured in such
indices can either be destroyed or interact with other constituents, so that the
determination cannot always provide an accurate method o f quality assessment. Kanatt et
al., (1998) studied the oxidation o f lipid in chicken meat during chilled storage as
affected by antioxidants combined with low-dose gamma irradiation. TBA values and
carbonyl content for irradiated samples o f ground chicken meat were higher than for non­
irradiated samples. Addition o f antioxidants tocopherol or BHT resulted in significant
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(p<0.05) retardation o f oxidative rancidity. Meat treated with antioxidant prior to
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irradiation had lower TBA values as compared to untreated irradiated counterparts. Free
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fatty acid values decreases after irradiation. Also a synergistic effect was observed in
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decreasing FAA content when antioxidants were added before irradiation. All the data
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demonstrated that irradiated meats were acceptable for consumption after 4 weeks o f
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storage.
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2.5. Lipid peroxides as a cause of diseases.
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There is an intimate correlation between lipid peroxides and certain diseases.
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Cardiovascular disease is considered to be one o f the common categories o f these
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diseases. In Eskimos, the low incidence o f mortality from coronary heart disease or CHD
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might be attributed to the relatively large intake o f ©3 fatty acids (Bang et al., 1979),
despite the excessive total fat from the high proportion o f marine vertebrates in their
diets.
Leaf (1994) found that every effect o f fish and marine mammal that has been
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tested on factors involved in CHD seemed replicable by two long-chain polyunsaturated
fatty acids, eicosapentaenoic (C20:5 ©3, EPA) and docosahexaenoic (C22:6 ©3, DHA)
which were present in oils o f fish and marine mammals. The action o f ©3 fatty acids may
account for the protection against CHD (Harris, 1989). A reduction o f total and low
density lipoprotein (LDL) cholesterol occurs only when ©3 fatty acids are replaced with a
high intake o f saturated fatty acids in the diet. Otherwise, the effects on total and LDL
cholesterol are also minimal and variable (Leaf, 1994). He also suggested that ©3 fatty
acids had an effect that produces a physically distinct form o f LDL particles which were
less atherogenic.
Over the past few years, literature has extensively covered the physiological and
pharmacological effects o f fish oils as major source o f ©3 fatty acids. These effects are
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summarized as follow:
. Decreases blood pressure in normal and moderately hypertensive subjects (Frenoux
etal., 2001).
. Decreases blood viscosity (Simopoulos, 1999)
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. Decreases microvascular albumin leakage in insulin dependent diabetics (Barcelli,
et al., 1990).
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. Decreases plasma triglycerides (Vidon et al., 2001)
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. Decreases vascular response to norepinephrine (Wince and Rutledge 1982).
. Decreases vascular ventricular fibrillation from ischemia (Kang and Leaf, 2000).
. Decreases cardiac toxicity of cardiac glycosides in vitro (Leaf, 1994).
. Deceases platelet adhesion (Ignarro,1989).
. Deceases leukocyte/endothelial interactions (De Caterina et al., 2000)
. Increases vascular compliance (Chin-Dusting et al., 1998)
. Increases Platelet survival (Pirich et al., 1999)
Unsaturated fatty acids such as arachidonic acid are known to be converted
enzymatically to various hydroperoxy acids such as 5-, 12-, and 15-hydroperoxy
arachidonic acids (Hayaishi, 1982). Five-hydroperoxy arachidonic acid is further
converted to leukotriene A, B or C, which causes asthmatic attacks in the case o f
overproduction as shown in Scheme 2 (Bailey, 2001). These hydroperoxy unsaturated
fatty acids exhibit various biological functions such as chemotaxis and vasodilation.
Meanwhile, they cause some deteriorative reactions in various degenerative disorders,
and have generally been considered to be the major TBA reactive materials in various
tissues and blood (Yagi, 1982).
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ooh N
.
,OOH
Arachidonic acid (AA)
COOH
.COOH
COOH
15-Hydroperoxy AA
5-Hydroperoxy AA
/V COOH
OH
HOO...
_ Leukotriene A
COOH
COOH
OH
C = /\/V
OH
I2-Hydroperoxy AA
Leukotriene B
Leukotriene C
Scheme 2 Conversion of arachidonic acid to hydroperoxy acids and Leukotriene
(reproduced from; Bailey, 2001).
Infra-red results demonstrate the occurrence o f lipid peroxides in the human
atheromatous cell wall (Leaf, 1994). This was followed by the isolation o f
hydroperoxides o f cholesterol Iinolate from lipids o f advanced ahtreosceloritic plaques o f
the human aorta (Harland et al., 1971). Therefore, oxidation o f lipids plays a major role
in the genesis o f atherosclerosis as well as cellular signaling. Cellular phospholipids and
amino acids play an important role in signaling events and in transformation. Exposure to
eicosapentanenoic acid (EPA) and docosahexaenoic acid (DHA) produces a remodeling
o f cellular phospholipids so that they are substantially reduced in amino acid-containing
species. Such changes would reduce eicosanoid synthesis, modulate signal transduction
pathways and attenuate the activation o f protein kinase C (Carmia, 1994).
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C hapter 3
M aterials and M ethods
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3.1. Chem icals and Reagents
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Trichloroacetic (TCA) acid, HC1, and pyridine were purchased from VWR
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Scientific Products (Bridgeport, NJ). n-Butanol, thiobarbituric acid, ascorbic acid,
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butylated hydroxytoluene (BHT) and sodium dodecylsulphate (SDS) were
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purchased from Sigma Chemical Co. Ethanol, 1,1,3,3-tetramethoxypropane (TMP),
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and a-tocopherol were purchased from Sigma Chemical Co. (St Lous, MO).
3.2. Sam ple P reparation
Tilapia fish used in this study were obtained from Aquaculture Research and
Demonstration Lab at the University o f Maryland Eastern Shore, Princess Anne,
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Maryland. Thirty-two tilapias weighing approximately 200 grams each were filleted
and trimmed of skin and adhering fat. After washing the fillet with chilled water,
fillets were grounded in a large plastic container using Waring blender (model
3 1BL91). Four grams of the ground fillet were packed in 288 zip-lock freezer bags.
Samples were treated with three levels (0.01,0.10 and 1.00%) o f three antioxidants
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(ascorbic acid, a-tocopherol and BHT) to evaluate the optimum level o f antioxidants
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required to exhibit lower lipid oxidation level. The antioxidants were added to tilapia
fillet before storage at -10 °C. The experiments were conducted with a two way
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factorial design (3x3), three levels of antioxidants (0.01, 0.10 and 1.00%) and three
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MWR periods (0,2, and 4 minutes). This model o f study shows that 0.1 % is the
optimum level required to protect lipid against oxidation for the three antioxidants
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(Appendix II). Therefore, the zip-lock bags were divided into 4 groups; the first,
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second, and third group were treated with 0.1 % ascorbic acid, 0.1 a-tocopherol, and
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0.1% BHT, respectively, while the fourth group was kept as the non-treated samples.
All bags were placed in a foam box and stored at -10°C for 4 weeks.
3.3. Microwave Radiation
Lipid oxidation in tilapia fillet was initially thought to occur after exposure to
microwave radiation (MWR). Therefore, oxidation o f fillet at three assigned MWR
periods (0 ,2 , and 4 minutes) was used as a model to initially evaluate the potential
level required to protect lipids against oxidation by three antioxidants (ascorbic acid,
a-tocopherol and BHT). Samples were taken at 1 week intervals up to 4 weeks for
determination of lipid oxidation. At the corresponding week, samples were thawed
for 10 minutes in room temperature and then were placed in 50 ml beaker and
covered with PVC film. Each sample was placed at the middle o f the rotary plate o f
the microwave oven (Panasonic Dimension4, Model 7107163) and heated for either
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2 or 4 minutes with 1100 W effective power and 2450 MHz frequency. Analysis o f
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the TBA values was carried out immediately after microwave radiation.
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3.4. Determination of TBA Values as an Index of Lipid Oxidation
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Y agi’s original fluorometric method (Yagi, 1987) was used with some
modification (Jo and Ahn, 1998) to determine the TBA values o f lipid peroxides; the
ground fillet was blended with 50 pi o f BHT (7.2 %) in a mortar (Pikul, et al., 1983)
and then was homogenized in Eberbach homogenizer (Cat. No. 7265, Ann Arbor,
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Michigan) with 9 ml of deionized distilled water (DDW) for 20 minutes. One ml o f
homogenated sample with 400 p.1 o f 8.1% SDS, 3.0 ml o f HC1, 3.0 ml o f 20mM
TBA, and 500 pi o f deionized water were placed into a test tube. The samples were
vortexed and then heated at 90 °C in a water bath for 15 min. After cooling for 10
min, 2 ml o f deionized water and 10 ml of n-butanol-pyridine solution were added to
the mixture. Next, the samples were mixed thoroughly and centrifuged at 9,800 x g
for 20 minutes. After centrifugation, the mixture’s upper layer (the organic phase)
was separated by using a 25 ml separatory funnel. The fluorescence intensities of the
organic phase (Appendix I) were measured using, RF-5301Spectrofluorophotometer (Shimadzu Scientific Instruments) at an excitation
wavelength o f 520 nm and an emission wavelength o f 550 nm.
The thiobarbituric acid (TBA) value (mg/kg sample) was determined using
Yagi’s standard equation (Yagi, 1987). By taking the fluorescence intensities o f 1 ml
o f the sample as f and 3 ml o f standard solution o f TMP, which obtained by reacting
0.5 nmol of tetramethoxypropane with TBA as F, the lipid peroxide level (Lp) can be
expressed in terms o f malonaldehyde as follow:
f 3.0 f
L„ = 0.5 x —x — = —x 1.5 (nmol/ml sample)
p
F 1.0 F
3.5. Water Content
Water content was determined by the weight difference o f fish fillet (2-3 g)
before and after 24 h at 105 °C according to standard methods o f the Official and
Tentative Methods o f the American Oil Chemists Society (AOAC, 1992).
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3.6. Statistical Analysis.
Data were analyzed using the General Linear Models procedure for analysis
o f variance (Statistics®, 1998). The experiment was conducted on a completely
randomized design (CRD) with a two way factorial design (4x4), four types o f
antioxidants (none, ascorbic acid, a-tocopherol, and BHT) and four storage times (1,
2,3, and 4 weeks). Significant differences between the means o f the treatments were
determined using Tukey’s (HSD) at a 5% level o f probability.
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CHAPTER 4
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Results & Discussion
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4.1. Effect of interaction between various antioxidant treatments and storage time
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on lipid oxidation of tilapia fillet before microwave cooking.
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Secondary lipid oxidation products such as malonaldehyde (MDA) were
measured using thiobarbituric acid (TBA) test (Table 1). A progressive increase o f TBA
values as an index o f lipid oxidation was observed for all samples after 1, 2, and 3 weeks
o f storage. No significant differences in TBA value were obtained during the fourth week
o f storage for samples treated with a-tocopherol and BHT as compared to the third week
(2.1373 vs. 2.0635 and 2.8373 vs. 2.6623, respectively). On the other hand, significant
increases (p<0.01) in TBA values were obtained after four week o f storage for non­
treated samples and samples treated with ascorbic acid as compared to those after three
weeks (3.2037 vs. 2.9480 and 3.3745 vs. 3.0370) (Table 1). These results suggest that a tocopherol and BHT have a higher activity than ascorbic acid; therefore they terminate
further lipid oxidation o f tilapia after 3 weeks o f storage at -10 °C. Changes in lipid
oxidation were detected fluorimetrically and were converted into TBA value according to
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Yagi’s method (Yagi, 1982). Fluorescence detection was found sensitive enough to
provide great differences during storage (Pikul et al., 1989), because any changes in
MDA formed will immediately produce different fluorescence intensities o f MDA-TBA
adduct, even at very low concentrations. Previous work on blue whiting fillet (Aubourg et
al., 1998) showed an increase in TBA values after 6 days o f storage, which is in
accordance with the present results.
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Table 1
Interactions between various antioxidants and storage times on lipid oxidation o f tilapia
fillet as measured by TBAXvalues before microwave cooking
Antioxidant
Storage
None
Ascorbic acid
a-tocopherol
BHT
Time (wk)
each value represents a mean o f six replicates y
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1
1.1680gh
1.3615fg
0.9813h
1.1057gh
2
2.1550s
2.2005°
1.3473fg
1.6035f
3
2.9480bc
3.0370bc
2.0635°
2.6623d
4
3.2037a
3.3745a
2.1373°
2.8373cd
ANOVA
P
Storage (A)
<0.01
Antioxidant (B)
<0.01
A*B
<0.01
TBA value is expressed as mg malonaldehyde/kg meat
y means with different superscripts are significantly different (p<0.01)
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Despite the strong antioxidant potency of ascorbic acid, no significant differences
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were observed between samples treated with 0.1% ascorbic acid and non-treated samples
over 4 weeks o f storage period. In this study, this might be attributed to the low contact
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between fillet lipids and ascorbic acid as a water soluble vitamin. On the other hand, after
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1 week o f storage, there were insignificant differences in TBA values between the three
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treatments (none, ascorbic acid and BHT). After 2 weeks of storage, significant (p<0.01)
inhibition o f lipid oxidation was observed in samples treated with either a-tocopherol or
BHT through out the storage. However, a-tocopherol shows a superior inhibitory effect
of lipid oxidation over BHT after three weeks o f storage. This might also be attributed to
the strong activity of a-tocopherol as an antioxidant. Similar results have been reported
by Kulas and Ackman (2001). Their results indicated that a-tocopherol has a strong
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affinity to donate hydrogen and terminate radical formation during storage o f unsaturated
fatty acids. Their study was also consistent with the mechanism demonstrated by Groff &
Grapper (2000) which define the role o f a-tocopherol as a chain breaking species in the
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peroxidation of fatty acids.
The water contents of samples were almost constant (77.2±0.2) during the 4
weeks of storage at -10 °C because samples were stored in zip-lock bags. Therefore, it is
evident that zip-lock packaging inhibit moisture lose o f fillet during the 4 weeks of
storage. The results are consistent with the study report by Aubourg et al. (1998). Their
results showed that water content o f frozen blue whiting ranged between 80 and 85% in
all o f their samples and no significant changes have occurred after 12 days o f storage.
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4.2. Effect of interaction between various antioxidant treatments and storage time
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on lipid oxidation of tilapia fillet cooked for two minutes in microwave oven.
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When tilapia fillet was cooked in microwave oven for two minutes, the TBA
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values were relatively higher than those of the raw fillet. A progressive increase o f TBA
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value was observed for non-treated samples and fillet treated with ascorbic acid (Table
2). After two weeks of storage, cooking in microwave oven revealed sharp increases in
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TBA values for non-treated sample and fillet treated with ascorbic acid (Table 2). The
TBA value increased from 1.7903 to 3.1875 and 1.8263 to 3.1898 for non-treated and
ascorbic acid samples, respectively. These results indicate that lipid oxidation of the
fillets stored for 2 weeks at -10 °C were doubled after two minutes o f microwave
cooking. After three weeks o f storage, ascorbic acid treatment has no significant effect
on lipid oxidation as compared to non-treated samples, while there was a significant
increase in TBA value for samples treated with ascorbic acid after 4 weeks. This might be
explained by its low activity as compared to the other antioxidants such as a-tocopherol.
It is believed that ascorbic acid is completely oxidized after four weeks o f storage as well
as the its pro-oxidative properties in food systems containing fatty acids. These results are
in agreement with earlier observation o f the pro-oxidative activity o f ascorbic acid in
food system (Nielsen et al., 2000; Nissen et al., 1999). They reported that ascorbic acid is
able to release Fe (II) from phosvatin-Fe(II) complex found in egg yolk. Since Fe (II) is
known to catalyze heterolytic cleavage o f lipids hydroperoxides resulting in free radical
formation, it could subsequently propagates lipid oxidation processes. On the other hand,
after 2 weeks o f storage, no significant increase in lipid oxidation was observed for
samples treated with a-tocopherol However, there was a significant increase in lipid
oxidation in samples treated with BHT as compared to the first week. This indicates that
a-tocopherol shows a superior antioxidative potency to BHT.
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Table 2
Interactions between various antioxidants and storage times on lipid oxidation o f tilapia
fillet as measured bv TBAXvalues after 2 minutes o f microwave cooking
Antioxidant
Storage
Time (wk)
Ascorbic
none
a-tocopherol
BHT
Acid
each value represents a mean of six replicates
1
1.7903s
1.8263s
1.0868'
1.2455hi
2
3.1875d
3.1898d
1.4108hi
1.8065s
3
3.6757°
3.7687°
2.1748f
2.8030°
4
4.1752b
4.5878a
2.2565f
2.8743°
ANOVA
P
Storage (A)
<0.01
Antioxidant (B)
<0.01
A*B
<0.01
x TBA value is expressed as mg malonaldehyde/kg meat
y means with different superscripts are significantly different (p<0.01)
The change in water content o f fillet was observed after 2 minutes o f microwave
cooking. A decrease in the total water content ranges from 49.4-50.2% in all samples was
detected after 2.0 minutes of microwave radiation (MWR). Similar results have been
observed after 4 weeks o f storage. The decrease o f water content may be attributed to the
absorption process of microwave energy by the fish fillet. This process elevates the
temperature o f the fillet and consequently enhances the vaporization o f water. Although
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some vaporized water condenses as the fillet cool down, the change in water content was
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substantial.
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4.3. Effect of interaction between various antioxidant treatments and storage time
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on lipid oxidation of tilapia fillet cooked for four minutes in microwave oven.
Tilapia fillet was cooked in microwave oven for 4 minutes to further evaluate the
influence of microwave cooking time on lipid oxidation. The thiobarbituric acid (TBA)
values suggests that lipid oxidation o f samples treated with ascorbic acid were not
significantly different from those of non-treated samples during the first and second week
o f storage (Table 3), however, a sharp increase after 3 weeks o f storage was observed for
samples treated with ascorbic acid. In addition to the low activity and pro-oxidant nature
o f ascorbic acid, this sharp increase might be attributed to the synergetic effect o f storage
time with the four minutes of microwave radiation. On the other hand, after either 2 or 4
weeks o f storage, the TBA values of samples treated with a-tocopherol or BHT were not
significant difference then those after 1 week. This suggests that both a-tocopherol and
BHT inhibit lipid oxidation after 4 minutes of microwave cooking.
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A decrease in the total water content ranging from 70.1 to 70.7% in all samples
was detected after 4 minutes o f microwave radiation (MWR). Similar results have been
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observed after 4 weeks o f storage.
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Table 3
Interactions between various antioxidants and storage times on lipid oxidation o f tilapia
fillet as measured by TBAXvalues after 4 minutes o f microwave cooking
Antioxidant
Storage
none
Time (wk)
Ascorbic
a-tocopherol
BHT
Acid
each value represents a mean o f six replicates y
1
2.0720efg
2.2015ef
1.1863h
1.4067gh
2
3.3450c
3.3995bc
1.6095fgh
1.9923cfg
3
4.0875b
5.17453
2.3622e
3.2293cd
4
4.8653a
5.5462a
2.5778de
3.4255bc
ANOVA
P
Storage (A)
<0.01
Antioxidant (B)
<0.01
A*B
<0.01
x TBA value is expressed as mg malonaldehyde/kg meat
y means with different superscripts are significantly different (p<0.01)
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4.4. Interactions between microwave cooking times and various antioxidants and
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their effects on lipid oxidation of tilapia fillet.
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The secondary lipid oxidation o f tilapia fillet was collectively measured before
and after microwave cooking during frozen storage. As shown in Table 4, the TBA value
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o f non-treated samples was significantly different (p<0.01) from the corresponding TBA
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value o f the fillet after 2 and 4 minutes o f microwave cooking. This indicates that the
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amount o f malonaldehyde formed after microwave cooking is relatively higher than that
before cooking. Similar results were observed for samples treated with ascorbic acid.
This suggests that ascorbic acid has no protective effect on lipid oxidation o f tilapia fillet
after microwave cooking. On the other hand, a-tocopherol and BHT were more effective
against lipid oxidation in tilapia fillet after microwave cooking. No significant differences
in TBA values were observed for samples treated with a-tocopherol and BHT after 2 or 4
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minutes of microwave cooking as compared to the non-microwave treated raw fillet.
Thus, it is evident that a-tocopherol and BHT have a strong ability to inhibit lipid
oxidation in frozen tilapia fillet during microwave cooking.
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30
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Table 4
Interactions between microwave cooking times and various antioxidants and their effects
on lipid oxidation as measured bv TBA* values o f tilapia fillet.
Antioxidant
Microwave
none
Time (min)
Ascorbic
a-tocopherol
BHT
Acid
each value represents a mean o f twenty four replicates
0
2.3687ef
2.4934de
1.6324f
2.0522ef
2
3.2072bcd
3.3432abc
1.7322ef
2.1823ef
4
3.5925ab
4.0804a
1.9340ef
2.5135cde
ANOVA
P
Radiation (A)
<0.01
Antioxidant (B)
<0.01
A*B
<0.01
x TBA value is expressed as mg malonaldehyde/kg meat
y means with different superscripts are significantly different (p<0.01)
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31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4.5 The effect of microwave cooking and frozen storage on lipid oxidation of tilapia
fillet
There was a highly significant (p<0.01) effect o f microwave cooking on the mean value
o f the TBA of tilapia fillet (Table 5) regardless o f the antioxidants and microwave
cooking time. After 2 minutes of microwave cooking, lipid oxidation increased
significantly from 2.1367 to 2.61162. Furthermore, lipid oxidation increased significantly
(p<0.01) from 2.136 to 3.0301 after 4 minutes o f microwave radiation. The total TBA
values and microwave radiation times were .'strongly correlated
{ tL=
0.998) as shown in
figure 1. Thus, increasing microwave cooking periods significantly increase the oxidation
level o f tilapia fillet. On the other hand, there was a significant (p<0.01) storage effect on
the lipid oxidation o f tilapia fillet (Table 6). Irrespective o f antioxidants, there was a
successive increase in the TBA values as an index o f lipid oxidation during 4 weeks o f
storage at -10 °C (Figure 1). After 4 weeks o f storage, lipid oxidation was almost three
folds higher than that o f the corresponding TBA during the first week. This indicates that,
lipid oxidation increased significantly (p<0.01) during frozen storage as a result of
increasing malonaldehyde formation. High r2 values between the TBA and storage times
indicated a linear development of the secondary lipid oxidation o f tilapia fillet after 1, 2,
3 and 4 weeks o f frozen storage.. However, there was no significant interaction between
microwave radiation and storage times.
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 5
Effect o f microwave cooking times on lipid oxidation o f tilapia fillet as measured by
TBA value
Microwave Time (min.)
TBAX
0
2.1367c
2
2.6162b
4
3.0301s
x TBA values (n = 96) is expressed as mg malonaldehyde/kg meat;
means with different superscripts are significantly different (p<0.01)
3.5
Y = 0 .2 2 3 4 (x) + 2.1476
r2= 0.9982
3.0
O)
O)
j*
E
<
m
i2.5
2.0
0.0
j
2.0
3.0
4.0
Microwave cooking tim e (min.)
I
j
Figure 1. The changes in TBA o f the tilapia fillet after different microwave radiation
times
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33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 6
Effect o f Storage time on lipid oxidation o f tilapia fillet as measured by TBA value
Storage Time (wk)
TBAX
1
1.4527d
2
2.2706°
3
3.1655b
4
3.4885a
x TBA values (n = 72) is expressed as mg malonaldehyde/kg meat;
means with different superscripts are significantly different (p<0.01)
4.0
Y = 0.70023 (X )+ 0.84375
" f2= 0.9675
3 .5
3.0
o>
o>
E
2.5
3
I2.0
0.0
1.0
2.0
3 .0
4 .0
Storage time (wk)
Figure 2. The changes in TBA o f the tilapia fillet during frozen storage at -10 °C
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34
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4.6 Antioxidative activity of ascorbic acid, a-tocopherol and BHT towards lipid
oxidation of tilapia fillet
The antioxidative potency o f the three tested antioxidants (ascorbic acid, a tocopherol and BHT) was compared with the non-treated sample (Table 7). Ascorbic
i
!
acid has no significant effect against lipid oxidation in tilapia fillet as measured by TBA
values as compared to the non-treated samples (3.0561 vs. 3.3057). Addition o f a -
|t
tocopherol and BHT significantly inhibit lipid oxidation (p<0.01) as compared to the
j
non-treated sample. However, a-tocopherol showed a superior effect on lipid oxidation to
|
i
BHT (1.7662 vs. 2.2493, respectively).
Table 7
Antioxidative activity o f ascorbic acid, a-tocopherol and BHT towards lipid oxidation o f
tilapia fillet as measured bv TBA values
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|
1]
Antioxidant
TBAX
None
1056?
Ascorbic acid
3.3057a
a-tocopherol
1.7662°
BHT
2.2493b
___________________________________________
x TBA values (n = 72) is expressed as mg malonaldehyde/kg meat;
means with different superscripts are significantly different (p<0.01)
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35
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t
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CH APTER 5
Sum m ary and Conclusion
The purpose of this study was to evaluate lipid oxidation o f tilapia induced by
microwave radiation over 4 weeks of storage at -10 °C. Fluorimetric thiobarbituric acid
test was used to determine lipid oxidation product (malonaldehyde) of tilapia fillet in
absence and presence o f the three assigned antioxidants (ascorbic acid, a-tocopherol and
|
i
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BHT). A preliminary study was conducted to evaluate the optimum level that is required
to protect lipid against oxidation induced by either microwave radiation or storage times.
The experiments were conducted with a two way factorial design (3x3), three levels o f
antioxidants (0.01, 0.10 and 1.00%) and three MWR periods (0, 2, and 4 minutes). It was
found that 0.1% level o f the tested antioxidants is the optimum level at which lipid is
highly protected against microwave radiation. Therefore, the effects o f storage times (1,
2 ,3 , and 4 weeks) and microwave radiation (0,2, and 4 minutes) on tilapia fillet was
evaluated using 0.1 % level o f ascorbic acid, a-tocopherol, BHT, respectively. Lipid
!
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oxidation increased significantly (p<0.01) after either 2 or 4 minutes o f microwave
cooking when compared to raw samples. Fillet treated with a-tocopherol showed
significantly (p<0. 01) less lipid oxidation than non-treated fillet. In addition, atocopherol showed a superior effect on lipid oxidation to BHT. On the other hand,
samples treated with ascorbic acid have no significant effect on lipid oxidation after
3i
i
microwave cooking in most cases.
It is concluded that addition of a-tocopherol or BHT to tilapia fillet delayed the
onset o f lipid oxidation induced by frozen storage times at -10 °C and microwave
radiation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Appendix I
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500.0
Wavelength (nm)
Excitation spectra o f raw samples after 4 minutes o f microwave cooking: (a) 1st week, (b) 2nd week, (c) 3 rd week, and (d) 4th week
Appendix II
Effects o f different concentrations o f ascorbic acid (0.01,0.1,1.0 %) and three
microwave times (0,2, and 4 minutes) on the lipid oxidation of tilapia fillet
Level
%
0.01
0.10
1.00
mean
mean
Microwave radiation time
2
4
0
2.2032
1.3646
1.8293
2.2015
1.5763
0.9543
1.5774°
1.3831
2.2477
2.5142
2.0483s
1.2340°
1.8844b
2.3063s
1.7990b
Level
%
0.01
0
0.9567de
Microwave radiation time
4
2
1.6428s
1.2808b
0.10
0.9813d°
>—
*
©
oo
o\
ooo
Q.
Effects o f different concentrations of a-tocopherol (0.01,0.1, 1.0 %) and three
microwave times (0, 2, and 4 minutes) on the lipid oxidation o f tilapia fillet
1.1863bc
1.00
0.8417°
1.0412cd
1.1063bcd
Effects o f different concentrations o f BH T (0.01, 0.1,1.0 %) and three microwave times
(0,2, and 4 minutes) on the lipid oxidation o f tilapia fillet
Microwave radiation time
4
2
1.7987s
1.4525b
Level
%
0.01
0
1.1483°
0.10
1.1057°
1.2455cd°
1.4067bc
1.00
1.0790°
1.2162d°
1.3868bcd
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Appendix III
Effects o f different concentrations of ascorbic acid (0.01, 0.1,1.0 %) and three
microwave times (0,2, and 4 minutes) on the lipid oxidation o f tilapia fillet
A N A L Y SIS OF VARIANCE TABLE FOR TBA
SS
SOURCE
DF
RAD (A)
LEVEL (B )
A*B
RESIDUAL
2
2
4
45
10.5050
1.99834
0.47496
3.22867
TOTAL
53
16.2069
MS
5.25249
0.99917
0.11874
0.07175
F
73.21
13.93
1 . 65
P
0.0000
0.0000
0.1772
Effects o f different concentrations of a-tocopherol (0.01, 0.1,1.0 %) and three
microwave times (0,2, and 4 minutes) on the lipid oxidation o f tilapia fillet
A N A L Y SIS OF VARIANCE TABLE FOR TBA v
SS
MS
SOURCE
DF
RAD (A )
LEVEL (B )
A* B
RESIDUAL
2
2
4
45
1.33945
0.83750
0.42875
0.42804
TOTAL
53
3.03375
0.66973
0.41875
0.10719
0.00951
F
70.41
44.02
11.27
P
0.0000
0.0000
0.0000
Effects o f different concentrations of BH T (0.01, 0.1,1.0 %) and three microwave times
(0,2, and 4 minutes) on the lipid oxidation of tilapia fillet
A N A L Y SIS OF VARIANCE TABLE FOR TBA
DF
SS
SOURCE
RAD (A)
LEVEL (B )
A*B
R ESIDUAL
2
2
4
45
1.58863
0.62153
0.23960
0.43079
TOTAL
53
2.88055
MS
0.79431
0.31076
0.05990
0.00957
F
82.97
32.46
6.26
P
0.0000
0.0000
0.0004
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Effects o f different antioxidants and storage time on lipid oxidation products o f tilapia
fillets before microwave radiation
A N A L Y SIS OF VARIANCE TABLE FOR TBA
SS
SOURCE
DF
STOR (A )
A N T I (B )
A* B
RESIDUAL
3
3
9
80
46.0579
10.6202
1.88711
1.85310
TOTAL
95
60.4183
MS
F
15.3526
^3. 54 0 0 7
0.20968
0.02316
662.79
152.83
9.05
P
0 . 0000
0.0000
0. 0000
Effects o f different antioxidants and storage time on lipid oxidation products o f tilapia
fillets after 2 minutes of microwave radiation
A N A L Y SIS OF VARIANCE TABLE FOR TBA
SOURCE
DF
SS
STOR (A )
A N T I (B )
A* B
RESIDUAL
3
3
9
80
55.1087
44.3359
5.44598
1.69360
TOTAL
95
106.584
MS
F
18.3696
14 . 7 7 8 6
0.60511
0.02117
867.72
698.09
28.58
P
0. 0000
0.0000
0.0000
Effects o f different antioxidants and storage time on lipid oxidation product
fillets after 4 minutes of microwave radiation
A N A L Y SIS OF VARIANCE TABLE FOR TBA
SOURCE
DF
SS
STOR (A )
A N T I (B )
A* B
R ESIDU AL
3
3
9
80
84.9942
69.3086
9.09024
9.78291
TOTAL
95
173.176
MS
F
28.3314
23.1029
1.01003
0.12229
231.68
188.92
8.26
p
0 . 0000
0.0000
0 . 0000
Influence o f different times o f microwave cooking on lipid oxidation products for all
storage times
A N A L Y SIS OF VARIANCE TABLE FOR TBA
SS
SOURCE
DF
RAD (A )
A N T I (B )
A *B
R ESIDU AL
2
3
6
276
38.3824
109.731
14.5338
215.914
TOTAL
287
378.561
MS
19.1912
36.5770
2.42230
0.78230
F
24.53
46.76
3.10
P
0.0000
0.0000
0 . 0060
52
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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