EVALUATION OF ANTIOXIDANT POTENTIAL OF MONODORA
MYRISTICA (AFRICAN NUTMEG)
BY
IBOLO MARTHA NNEKA
BC/2006/076
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF NATURAL SCIENCES
CARITAS UNIVERSITY,
AMORJI-NIKE, ENUGU STATE.
AUGUST, 2010
1
TITLE PAGE
EVALUATION OF ANTIOXIDANT POTENTIAL OF MONODORA
MYRISTICA (AFRICAN NUTMEG)
BY
IBOLO MARTHA NNEKA
BC/2006/076
A PROJECT SUBMITTED TO THE DEPARTMENT OF
BIOCHEMISTRY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE
IN BIOCHEMISTRY
AUGUST, 2010.
2
APPROVAL PAGE
We hereby certify that the research work in this report is solely the work of IBOLO
MARTHA NNEKA with Registration Number BC/2006/076 for the award of Bachelor of
Science Degree (B.Sc.) in the Department of Biochemistry, Caritas University AmorjiNike, Enugu state.
…………………………...
……………………………
Dr. Charles N. Ishiwu
Date
(Supervisor)
…………………………..
……………………………
Mr. Moses Ezenwali
(Head of Department)
Date
……………………………
……………………………
External Examiner
Date
August, 2010
3
DEDICATION
This work is dedicated to Almighty God for His faithfulness to me and for seeing me
through, in this citadel of learning.
4
ACKNOWLEDGEMENT
I give joyful thanks and praise to Almighty God who in spite of all odds, made this project
research a reality. He has always been faithful to me. My special appreciation goes to my
supervisor, Dr. Charles N. Ishiwu for his warmly guidance and supervision. His unreserved
kindness and understanding not only inspired me but also encouraged me throughout the
trying moments of this work. I also wish to express my indebtedness to my Honourable
HOD, Mr. Moses O.Ezenwali for his unrelentful assistance throughout my laboratory work
and who contributed in no small measure to the completion of this work.My sincere
gratitude goes to my most learned and zealous lecturers, Mrs. Oluchi Ajemba, Mr. Ezesteven Peter, Dr. Ikpe, Mr. P. Ugwudike, and Mr. Omeh Yusuf.
My most profound gratitude goes to my loving and caring parents, Sir and Lady P. O. Ibolo
for their moral and financial support, whose love for education brought me to this citadel of
learning. My special thanks also goes to the managing director, Aniuzo international
limited (Palm Kernel Oil Mills Division), Emene, who source partly for the raw material
used for this research. My thanks also goes to my caring chancellor Rev. Sr. Kate Ikenga
for her advice and support to me. I thank my siblings: Blessing, Elizabeth, Anthony and
Christopher, who have been an everlasting source of love, strength and encouragement. I
wouldn’t forget to appreciate my colleagues, friends who have contributed inestimably
throughout the course of this work. In this respect, I am grateful to Patricia, Chidera, Kofi,
Chijioke, Tony, Chizoba, Peace, Modesta, and a host of others.
IBOLO MARTHA NNEKA
5
ABSTRACT
This work evaluates the antioxidant potential of Monodora myristica (African nutmeg).
Monodora myristica extract was obtained by solvent extraction using n-hexane and used as
treatment on freshly prepared crude palm kernel oil and palm oil. Equal volume of oil
samples were subjected to different concentration of extract treatment (0.2ml,0.4ml, 0.6ml,
0.8ml, 1.0ml using syringe. These oil samples were equally divided into two groups SS and
SR. Group SS was stored under the sun and group SR was stored in the room for three
weeks. These treated oil samples were analyzed on weekly basis at two different
parameters: Acid value (AV) of free fatty acid and thiobarbituric acid (TBA) value, using
standard methods. The main effect of extract was determined using ANOVA. For the two
varieties of oil, the acid value of free fatty acid increased significantly (P<0.05) as the
period extends for group SS without extract while those for group SR showed no
significant increase. But AV of oil samples treated with higher extract concentration
decreased significantly (P<0.05) for both groups SS and SR. TBA value also showed the
same trend of AV. Hence, monodora myristica extract yielded reducing effect in the
oxidative level of the oil varieties.
6
TABLE OF CONTENTS
Title page
----------------------------------------------------------
i
Approval page
---------------------------------------------------------
ii
Dedication
---------------------------------------------------------
iii
Acknowledgement
---------------------------------------------------------
iv
Abstract
---------------------------------------------------------
v
Table of content
---------------------------------------------------------
vi-viii
List of tables
--------------------------------------------------------
xi
List of figure
--------------------------------------------------------
x
Abbreviation
--------------------------------------------------------
xi
CHAPTER ONE
1.0
Introduction ---------------------------------------------------------
1
1.1
Significance of study -----------------------------------------------
6
CHAPTER TWO
2.0
Literature Review
------------------------------------------------
2.1
African nutmeg (Monodora myristica) ----------------------------
7
2.1.1
Scientific classification
------------------------------------------
7
2.1.2
Habitat/ ecology of Mondora myristica ----------------------------
8
2.1.3
Characteristics/morphology of monodora myristica -------------
8
2.2
Oil Palm
9
2.2.1
Scientific classification -----------------------------------------------
10
2.2.2
Origin and description of palm oil ---------------------------------
10
2.2.3
The Chemical composition of palm oil --------------------------
11
2.2.4
Physical characteristics of palm oil products ------------------
14
---------------------------------------------------
7
7
2.3
Palm kernel oil ------------------------------------------------------
14
2.3.1
The chemical composition of palm kernel oil-------------------
15
2.4
Modern uses of palm oil and palm kernel oil-------------------
16
2.5
Lipid oxidation-----------------------------------------------------
16
2.5.1
Lipid oxidation pathway ------------------------------------------
21
2.5.2
Mechanism of oxidation ------------------------------------------
22
2.6
General antioxidant action ---------------------------------------
24
2.6.1
Mechanism of antioxidant action ------------------------------
24
2.6.2
Antioxidant molecules --------------------------------------------
26
2.7
General review of photochemistry of monodra myristica --
27
2.7.1
Alkaloids ----------------------------------------------------------
27
2.7.2
Flavonoids --------------------------------------------------------
27
2.7.3
Glycosids --------------------------------------------------------
28
2.7.4
Saponins ----------------------------------------------------------
28
2.7.5
Tannins ------------------------------------------------------------
29
2.8
Application of vegetable oils -----------------------------------
29
2.8.1
Factors that cause oxidative rancidity in vegetable oil ------
30
2.9
Nutritional signification -----------------------------------------
33
CHAPTER THREE
3.0
Materials and methods --------------------------------------------
34
3.1
Equipment/apparatus ----------------------------------------------
35
3.2
procedurement of raw materials ---------------------------------
35
3.3
Study design ---------------------------------------------------------
37
3.4
Sample preparation ------------------------------------------------
37
3.5
Chemical analysis -------------------------------------------------
38
3.5.1
Determination of acid value (Av) -------------------------------
38
3.5.2
Determination of thiobarbituric acid number.------------------
38
8
3.6
Statistical analysis ------------------------------------------------
39
CHAPTER FOUR
4.0
Result and Discussion -------------------------------------------
40
4.1
Changes in Acid value of Palm Kernel and palm oil --------
46
4.2
Changes thiobarbituric acid value of palm kernel and palm oil---
46
4.3
Effect of monodora myristica extract on the
chemical indices of oil on storage
--------------------------------
47
CHAPTER FIVE
5.0
Summary and conclusion --------------------------------------------
48
5.1
limitations---------------------------------------------------------------
51
5.3
Future recommendation ----------------------------------------------
51
References -------------------------------------------------------------------------
53
Appendix I --------------------------------------------------------------------------
62
Appendix II -------------------------------------------------------------------------
63
Appenedix III ----------------------------------------------------------------------
65
9
LIST OFF TABLES
Table 1:
Fatty acid composition of palm oil (palm oil)
Table 2:
Fatty acid profile of palm kernel oil (palm kernel )
Table 3:
Acid value for palm kernel oil
Table 4:
Acid value for palm oil
Table 5:
Thiobarbituric acid value for palm kernel oil
Table 6:
Thiobarbituric acid value for palm oil
10
LIST OF FIGURES
Figure 1: African nutmeg seeds (Monodora myristica)
Figure 2: Africa Oil palm fruits (Elaeis guinesis)
Figure 3: Lipid oxidation pathway
Figure 4: Dried seed kernels of Afican nut meg.
Figure 5: Transverse section of palm fruit
Figure 6: n-Hexane extract of Monodora mystica
Figure 7: Crude palm oil
Figure 8: Crude palm kernel oil
11
ABBRERVIATIONS
AOCS:
Association of America Chemistry Society
AV:
Acid value
FFA:
Free fatty acid
PV:
Peroxide value
PKO:
Palm kernel oil
PO:
Palm oil
PUFA:
Polyunsaturated fatty acid
ROS:
Reactive oxygen specie
SR:
Storage in room
SS:
Storage in sun
TBA:
Thiobarbituric acid
12
CHAPTER ONE
1.0 INTRODUCTION
Lipid oxidation is one of the major reasons that food deteriorate and is caused by the
reaction of fat and oil with molecular oxygen, leading to off-flavours that are generally
called rancidity(Basturk et al., 2007). Exposure to light, pro-oxidants and elevated
temperature will accelerate the reaction (Kubow, 2009). Lipid oxidation and resultant
flavour impairment has seriously limited the storage potential of most fat containing foods
(Ihekoronye and Ngoddy, 1985).
Rancidity covers a wide range of biological activities where the effect is to “make
things worse” and thus adversely affect man’s economy. Free radicals and microorganisms
are known to cause chemical characteristics that lead to oxidation and deterioration in
quality of vegetable oils derived from the seeds or fruits pulps of plants (Basturk et al,
2007). The keeping quality of the oils is basically dependent on their chemical
compositions, for instance, the percentages of the degree of unsaturation. Rancidity is
associated with off-flavour and odour of the oil. There are two causes of rancidity. One
occurs when oil reacts with oxygen and is called oxidative rancidity. The other cause of
rancidity is by the combination of enzymes and moisture. Enzymes such as lipase liberate
fatty acids from the triglyceride to form di and/or monoglycerides and free fatty acids and
such liberation of fatty acid is called hydrolysis, hence hydrolytic rancidity.
13
The oxidation of fats is an important deteriorative reaction with significant
commercial implications in term of product value. The initial oxidation products that
accumulate are hydroperoxides, which may subsequently break down to form lowermolecular weight compounds such as alcohols, aldehydes, free fatty acids and ketones,
leading to autoxidative rancidity. The peroxide content present in alimentary fats attests to
its state of primary oxidation and thus its tendency to go rancid. Unsaturated fatty acids, in
fact, react with oxygen forming peroxides, which determine a series of chain reactions
whose end result is volatile substances having the characteristic smell of rancidness. These
reactions are accelerated by high temperatures and by exposure to light and oxygen (Yildiz
et al., 2002). The lower the peroxide and acid values, the better the quality of the
alimentary fats and their state of preservation.
Although simple, procedures of
acid value (AV) or peroxide value (PV)
determination are cumbersome, destructive to the sample, costly, require potentially
hazardous solvents, substantial personnel time, glassware and accurate preparation of
reagents and are dependent on a visual endpoint (Ismail et al., 1993; Van de Voort et al.,
1994).
Oxidation is concerned mainly with unsaturated fatty acids. Oxidative rancidity is of
special interest as it leads to the development of off-flavour that can be detected early on in
the development of rancidity (Basturk et al., 2007)
Some slight deterioration at least is to expected in any commercial oil-bearing material and
is, in fact, inherent in the process by which fat is formed (Morel,1997). In the living plants
and animals, fats, carbohydrates and proteins are synthesized in a complicated series of
14
steps with the aid of certain enzymes. These enzymes are capable of assisting the reverse as
well as the forward reactions and hence under proper conditions may promote the oxidation
and degradation of the very substances that, they have previously been instrumental in
synthesizing (Basturk et al., 2007)
Oils in general are known to be susceptible to oxidation and microbial attack. The
composition of the various oils determines the extent of oxidation and type of organisms
likely to thrive in them (Chow et al., 2000). Several studies have demonstrated that
environment factors affect not only the fatty acid composition of vegetable oil, but also,
although apparently indirectly, the spatial arrangement of those acids on the triacylglycerol
molecule (Tay et al., 2002). Triacylglycerol composition and structure are important in the
areas of nutrition, oil stability and possible physiological effects.
Palm oil is extracted from the mesocarp of the fruit of the oil palm, Elaeis
guineensis. crude palm oil (CPO) has a deep orange-red colour due to the high content of
carotenoids and is a rich source of vitamin E consisting of tocopherols and tocotrienols
(Nesaretnam and Muhammad, 1999). Both beta carotenes and vitamin E are well known
nutritional antioxidants.
Palm oil is known to support the growth of fungi and bacteria especially when it
contains moisture (Cornellus, 2001).. Their lipolytic enzymes are so active that even under
unfavorable conditions palm oil is seldom produced with a free fatty acid content (FFA) of
less than 2% and under favorable conditions of processing, the free fatty acid content of this
oil reaches 20%and higher. When the fruit is bruised, lipolytic action occurs and a near
maximum FFA (8-10%) is reached within 40 minutes. The FFA of unbruised fruits may
increase only 0.2% or less in the course of 4 days (Cornellus, 2001).
15
The exposure in the sun is made under radiations of weak temperatures, varying
daily, creating an environment favourable to the chemical and enzymatic reactions of
hydrolysis and oxidation (Tan et al., 2002).
This study is aimed at examining the oxidative and biodeteriogenic effects of free radicals
contaminating the oils from the varieties of the oil palm (Elaeis guineensis) and palm
kernel oil and the chemical components of the oils and the effect of solvent extract of ehuru
(African nutmeg).
Oil palm is indigenous to the Nigerian coastal area. It was discovered by European
explorers in the early 1400’s and was distributed throughout tropical Africa by humans who
practiced shifting agriculture about 5000 years ago. The palm plant originated from the
jungle forest of East Africa and about 5000 years ago, palm oil was used by the pharaohs
for cooking and lighting. The cultivation of oil palm is restricted to the eastern sub zones
where its growth is favoured environmentally and climatically. Besides, it is a major cash
crop in this region. The first oil palm plantation was established at Sumatra in 1911 and at
Malaysia in 1917. About this time it was simultaneously established in West Africa and
tropical America.
Over the years, a little attention was paid to the industrial use of palm kernel oil.
Nevertheless, recent studies have indicated that apart from their domestic uses that they can
be used as engine lubricants, as replacement for biodiesel if their properties are enhanced.
Although high in saturated fats, it is a different oil to extract from the nut or kernel of palms
which has a yellowish white colour and a pleasantly mild flavor similar to coconut oil in
fatty oil acid composition and properties. Crude palm kernel oil (CPKO) is extracted from
palm kernels with palm kernel cake as a by-product. The physical and chemical properties
16
of the various palm oil products have been reviewed by Nesaretnam and Muhammad,
(1999).
Monodora myristica is a widespread and attractive small tree with very decorative
flowers appearing just before the leaves. The fruit is suspended on a long green stalk with
numerous seeds embedded in whitish sweet smelling pulp. The seed is oblong and pale
brown when fresh with a thin seed coat and hard kernel (Nesaretnam and Muhammad,
1999). The seed production is seasonal occurring between April to June. The fruits are
globular and ovoid; 3-4 inch long and about 3-5 inch diameter. The wood is hard. The seeds
are contained in a hard shell and have a very strong aroma . This plant is commonly called
Orchid flower tree in English, Ehuru Ofia in lgbo (Okafor, 2003). Monodora myristica is a
specie of calabash nutmeg, the edible seeds yield a nutmeg-flavoured oil which is used in
West Africa for cooking (Eggeling, 2002). Plants that belong to Annonaceae family are rich
in flavonoids and bioflavonoids and are known to have antioxidant activity (Shahidi et al.,
2009). Monodora myristica seed extract contains important pharmacological compounds,
alkaloids, flavonoids, and vitamins A and E as well as many important lipids; arhinolipids,
free fatty acids, glycolipids, phospholipids and sterols. The plant is widely used in
ethnomedicine, especially to relieve toothache as well as in the treatment of dysentery.
When roasted and ground, the seeds are rubbed on the skin for (unspecified) skin diseases
(Irvine, 2000). This suggests that the seeds of Monodora myristica plant could be
germicidal or antiseptic. The roasted ground seeds are chewed, then spat into the hand and
then rubbed across the forehead to relieve headache. The seeds are also crushed and used as
insecticide, while the root relieves toothache when crushed (Ogtinein unet al., 1999).
17
Monodora myristica seeds are also used for the treatment of constipation and as a stimulant
(Irvine, 2000). The essential oil from Monodora myristica seed is used in pharmaceutical
and dental preparation (Talalaji, 1999).
In this study, we have monitored characteristic parameter, namely acid value and
thiobarbituric acid value during storage of palm kernel oil and palm oil at different
environmental conditions treated with different concentration of seed extract of Monodora
myristica. Whereby, the acid value and thiobarbituric acid value, were assessed by the
conventional method and the UV-spectra were registered for each sample. Although
simple, procedures of acid value (AV) or peroxide value (PV) determination are
cumbersome, destructive to the sample, costly, require potentially hazardous solvents,
substantial personnel time, glassware and accurate preparation of reagents and are
dependent on a visual endpoint (Ismail et al., 1993; Van de Voort et al., 1994).
1.1
SIGNIFICANCE OF RESEARCH
The aim and objective of this research is to:
1.
To carryout solvent extraction of Monodora myristica
2.
To investigate the antioxidant effect of Monodora myristica extract on palm kernel
oil and palm oil at different environmental conditions.
18
CHAPTER TWO
2.0
LITERATURE REVIEW
2.1 AFRICAN NUTMEG
Figure 1: African nutmeg seeds (Monodora myristica)
2.1.1 Scientific Classification
Kingdom:
Plantae
Phylum/Division: Spermatophytae
(unranked):
Angiosperms
(unranked):
Magnoliids
Order:
Magnoliales
Family:
Annonaceae
Genus :
Monodora
19
Species:
myristica
Binomial name
Monodora myristica (African nutmeg)
2.1.2 Habitat/Ecology of Monodora myristica
Monodora myristica is tropically distributed. It is cultivated in East India, Malaysia, Sri
Lanka, West lndies and Africa. It could be propagated by stem culturing and budding
(Okafor, 2003). The Monodora species are also found in West Africa and are cultivated in
the southern parts of Nigeria. The trees are very common in Anambra, Abia, Delta, and
Enugu States.
Local Names
The plant is usually called Orchid flower and is also referred to as and called:
Ehuru - lgbo name
Ehinawosin - lkale name
Lakosin - Yoruba name
Uyenghen - Edo name
(Keay, 1989)
2.1.3 Characteristics/Morphology of Monodora myristica
Monodora myristica, commonly known as calabash nutmeg, ehuru, Jamaican nutmeg,
nuscade de Calabash, ariwo, airama, African nutmeg and African orchid nutmeg is a tropical
shrub of the Annonaceae or custard apple family of flowering plants (Okafor, 2003). The
flowers of Monodora myristica look very much like those of an orchid (hence the common
20
name of 'African orchid nutmeg'), and the fruit is a nearly spherical drupe about the same size
as an orange. The seeds and seed coats of the plant are used as a spice. The fruit contains a
number of these aromatic seeds embedded in a yellow pulp (Oguntinein et al., 1999).. The
seeds and their seed coat are removed and dried giving a heart-shaped spice some 3cm long
and 2cm broad at its widest part. Once dried these have an aroma reminiscent of nutmeg and
are sold whole to be grated as a nutmeg substitute (Talalaji, 1999).. At one time it was widely
sold as an inexpensive substitute for nutmeg, although this practice is less common today
outside its region of production (Nigeria). Calabash nutmeg has a nutmeg-like flavour with a
pungent overtone. The whole seed coat and seed is either ground and used as a seasoning for
West African soups or stews or is ground and used as a nutmeg-like flavouring in cakes and
desserts. As well as yielding calabash nutmeg the seed coat is often removed and the inner
true seed is sold as ehuru or ehiri (its name in igbo language, a Nigerian language).
2.2 OIL PALM
Figure 2: African Oil Palm fruits (Elaeis guineensis)
21
2.2.1 Scientific Classification
Kingdom:Plantae
Family:Arecaceae
Subfamily:Arecoideae
Tribe:Cocoeae
Genus:Elaeis
Jacq. Species
Elaeis guineensis
Elaeis oleifera
2.2.2 ORIGIN AND DISCRIPTION OF PALM OIL
The oil palms (Elaeis) comprise two species of the Arecaceae, or palm family. They are
used in commercial agriculture in the production of palm oil. The African Oil Palm Elaeis
guineensis is native to west Africa, occurring between Angola and Gambia, while the
American Oil Palm Elaeis oleifera is native to tropical Central America and South
America. The generic name is derived from the Greek for oil, elaion, while the species
name refers to its country of origin.
The palm fruit takes five to six months to mature from pollination to maturity. The palm
of fruit is reddish, about the size of a large plum and grows in large bunches. Each fruit is
made up of oily, fleshy outer layer (the pericarp), with a single seed (the palm kernel), also
rich in oil. When ripe, each bunch of fruit weigh 40-50 kilogrammes.
22
Oil is extracted from both the pulp of the fruit (palm oil, an edible oil) and the kernel (palm
kernel oil, used in foods and for soap manufacture). For every 100 kilograms of fruit
bunches, typically 22 kilograms of palm oil and 1.6 kilograms of palm kernel oil can be
extracted (Mauro et al., 2001).
Palm oil is the reddish-orange oil extracted from the fruit and kennel of a palm tree (Elaeis
Guineensis), a native to tropical West Africa. It is the most widely produced vegetable oil
in the world. This edible oil contains a very high percentage saturated fat and used in
making
soaps,
margarine,
and
lubricants,
besides
being
used
in
cooking.
Since palm oil has been consumed for its nutritional value and health benefits for more than
5,000 years, it is often said as nature's gift to the world. Today, it is the most widely
produced vegetable oil of the world. In some Asian countries, it is termed as ‘gold oil', for
its perfect balance of saturated and unsaturated fatty acids which do not adversely affect
cholesterol levels.
The purest form of palm oil is easily available in the tropical West Africa, Indonesia and
Malaysia, where it is widely cultivated. Besides being used in personal care products and
toiletries, it is also used to treat wounds and as a feedstock for biofuel.
2.2.3 The Chemical Composition Of Palm Oil
Palm oil consists mainly of glycerides made up of a range of fatty acids
including
triglyceries, mono and diglycerides. The oil palm gives its name to the 16 carbon saturated
fatty acid palmitic acid found in palm oil; monounsaturated oleic acid is also a constituent
23
of palm oil while palm kernel oil contains mainly lauric acid. Besides this, it is the largest
natural source of tocotrienol, part of the Vitamin E family. It also contains high
concentration of Vitamin K and dietary magnesium.
Triglycerides constitute the major component, with small proportions of diglycerides and
monoglycerides. Palm oil also contains other minor constituents, such as free fatty acids
and non-glyceride components. This composition determines the oil's chemical and
physical characteristics (Cornelius, 2001)..
The fatty acid composition of crude Malaysian palm oil is given in table 1. About 50% of
the fatty acids are saturated, 40% mono-unsaturated, and 10% polyunsaturated. It contains
adequate amounts of n-6, 18:2 essential fatty acid. In its content of monounsaturated 18:1
acid, palm oil is similar to olive oil, which is as effective as the more polyunsaturated oils
in reducing blood cholesterol and the risk of coronary heart disease.
Crude palm oil contains approximately 1% of minor components: carotenoids, vitamin E
(tocopherols and tocotrienols), sterols, phospholipids, glycolipids, terpenic and aliphatic
hydrocarbons, and other trace impurities . The most important are carotenoids and vitamin
E, both of which possess important physiological properties. The iodine value is between
50 and 56.
24
TABLE 1. Fatty acid composition of palm oil (PO)
% of total acids
Acid
Range
Mean
12:0
0.1-1.0
0.2
14:0
0.9-1.5
1.1
16:0
41.8-46.8 44.0
16:1
0.1-0.3
0.1
18:0
4.2-5.1
4.5
18:1
37.3-40.8 39.2
18:2
9,1-11.0 10.1
18:3
0.0-0.6
0.4
20:0
0.2-0.7
0.4
Source: (Pantzaris and Ahmad, 2004).
Since palm oil contains more saturated fats than canola oil, corn oil, linseed oil, soybean
oil, safflower oil, and sunflower oil, it can withstand extreme deep fry heat and is resistant
to oxidation.
25
2.2.4 PHYSICAL CHARACTERISTICS OF PALM OIL PRODUCTS
Palm oil is a semi-solid at room temperature (28◦C), the melting point range being
from 32–40◦C. The slip melting point method is commonly adopted for measuring this
parameter. By the DSC method the fat melts completely at 39–40◦C, when heated at
5◦C/min, from an oil cooled rapidly to −40◦C at 5◦C/min. The slip melting point is affected
by the content of free fatty acids and diacylglycerols. Thus crude oils have slightly higher
slip melting point than refined oils (Nyam et al., 2009).
Palm oil is produced from the fruit and kernel of the palm tree. The fruits are first
collected and pressed, yielding a rich, dark-red oil which is high in carotene (Pantzaris and
Ahmad, 2004). The oil thus obtained, is exposed to heat through processing and cooking
which turn its colour to pale creamy color. Conversion of crude palm oil to refined oil
involves removal of the products of hydrolysis and oxidation, colour and flavour. After
refining, the palm oil may be fractionated (separated) into liquid and solid phases by
thermo-mechanical means (controlled cooling, crystallization, and filtering).
2.3 PALM KERNEL OIL
Palm kernel oil (PKO) is obtained from processing the kernel from the fruit of the oil palm
tree (Elaies guineensis). Palm kernel oil has similar uses to coconut oil owing to their
similarity in composition (Pantzaris and Ahmad, 2004). Palm kernel oil (PKO) is gotten
from the kernel of the palm fruit and it is located inside the hard shell while the outer fleshy
mesocarp gives palm oil.
26
2.3.1 The Chemical Composition Of Palm kernel Oil
The major fatty acids in palm kernel oil are lauric acid (C12, 48%), myristic acid (C14,
16%) and oleic acid (C18, 15%) (Pantzaris and Ahmad, 2004).
The fatty acids mostly found in palm kernel oil are presented in Table 1 below.
Table 2. Fatty acid profile of palm kernel oil (PKO)
Type of fatty acid
Percentage
Lauric (C12:0)
48.2
Myristic (C14:0)
16.2
Palmitic (C16:0)
8.4
Capric (C10:0)
3.4
Caprylic (C8:0)
3.3
Stearic (C18:0)
2.5
Oleic (C18:1)
15.3
Linoleic (C18:2)
2.3
Others (unknown)
0.4
Source: (Pantzaris and Ahmad, 2004).
27
Palm kernel oil, coconut oil, and palm oil are three of the few highly saturated vegetable
fats. Palm kernel oil, which is semi-solid at room temperature, is more saturated than palm
oil and comparable to coconut oil. Like all vegetable oils, these three palm-derived oils do
not contain cholesterol (found in unrefined animal fats)] although saturated fat intake
increases both LDL and HDL cholesterol.
2.4 Modern Uses of Palm Oil and Palm Kernel Oil
As much as 90% of the palm oil produced finds its way into food products, while remaining
10% is consumed by various industries. It is widely used preparing margarine, shortening,
and vegetable cooking oil. In many parts of the world, it is still consumed in its unrefined
state to obtain a distinctive colour and flavour. Palm oil is extensively used in preparing dry
cake mix used for baking biscuits, cakes and sponge cakes, soaps, sauces, fat substitutes,
etc. Recently, palm and kernel oils have been increasingly used as biodiesel fuel.
Palm kernel oil is a common cooking ingredient; its increasing use in the commercial food
industry throughout the world is buoyed by its lower cost, the high oxidative stability
(saturation) of the refined product when used for frying, and its lack of cholesterol and
trans-fatty
acids,
both
viewed
as
being
heart-healthy
attributes.
.
2.5 LIPID OXIDATION
Lipid oxidation and resultant flavour impairment has seriously limited the storage potential
of most oil containing food (Ihekoronye and Ngoddy, 1985) Lipid oxidation generally
occurs after a long induction period. Once started it is generally a very rapid reaction. Lipid
oxidation
proceeds
by
a
28
free
radical
mechanism.
A free radical is a compound with an odd number of unpaired electrons.
Two free radicals were formed. These radicals are very reactive and generally do not have
long life times (Morel, 1997).. They enter into three main types of reactions:
1. Abstraction:
2. Addition:
29
3. Combination:
Lipid oxidation follows three main steps:
Initiation
Propagation
Termination
1.
Initiation
involves
the
formation
of
free
radicals.
Mechanism later:
In some cases may add oxygen directly to the double bond to form a biradical
Once the initial radicals have formed, the formation of other radicals proceeds rapidly..The
radicals can abstract H atoms from other lipids or react with other radicals to form alcohols
and ketones (Morel, 1997).
30
A.
B.
C.
Of great importance to the food industry is the splitting of C-C bonds and the formation of
aldehydes:
31
There can also be cleavage on the other side:
It is possible for two of the radicals formed to combine:
The change from the alcohol to the aldehyde is called a keto - enol shift. If the two free
radicals do not react with other molecules they may combine with each other.
32
2.5.1
LIPID OXIDATION PATHWAY
UNSATURATED FATTY ACIDS
FREE RADICALS
OXYGEN
Insolubilization of proteins
HYDROGENPEROXIDES
Breakdown products
Such as aldehydes, free
fatty acids, alchohol
Oxidation of pigments, flavours
, and hydrocarbons
and vitamins
Polymerization
(dark colour)
Source:
(Ihekoronye and Ngoddy, 1985)
Figure 3: lipid oxidation pathway
33
2.5.2 MECHANISMS OF OXIDATION
Autoxidation
This is a radical-chain process involving 3 sequences: Initiation, propagation and
termination.
1 – Initiation
In this stage, the molecule of unsaturated fatty acid loses a hydrogen atom leaving a free
radical which is required to start the propagation reaction (Ihekoronye and Ngoddy, 1985).
The reaction may be represented as follows:
RH
R+H
In a peroxide-free lipid system, the initiation of a peroxidation sequence refers to the attack
of a ROS (with sufficient reactivity) able to abstract a hydrogen atom from a methylene
group (- CH2-), these hydrogen having very high mobility (Morel, 1997). . This attack
generates easily free radicals from polyunsaturated fatty acids. .OH is the most efficient
ROS to do that attack, whereas O2.- is insufficiently reactive.
This peroxidation process is inhibited by tocopherols, mannitol and formate. The presence
of a double bond in the fatty acid weakens the C-H bonds on the carbon atom adjacent to
the
double
bond
and
so
makes
34
H
removal
easier.
Propagation
During the propagation stage, the free radical reacts with oxygen to form peroxidecontaining free radicals (Ihekoronye and Ngoddy, 1985).
R + O2
ROO
These in turn reacts with another mole of unsaturated compound to produce hydrogen
peroxides and new free radicals capable of continuing the chain reaction. As a peroxyl
radical is able to abstract H from another lipid molecule (adjacent fatty acid), especially in
the presence of metals such as copper or iron, thus causing an autocatalytic chain reaction.
The peroxyl radical combines with H to give a lipid hydroperoxide (or peroxide). This
reaction characterizes the propagation stage (Morel, 1997).
The hydroperoxides formed are unstable and willdecompose either to stable derivatives or
will split into two or more free radicals (Ihekoronye and Ngoddy, 1985).
Termination
When two radicals interact to form stable non-radical products, termination occurs:
R +R
RR
RO + R
ROR (Ihekoronye and Ngoddy, 1985)
35
Termination (formation of a hydroperoxide) is most often achieved by reaction of a
peroxyl radical with a-tocopherol which is the main lipophilic "chain-breaking molecule" in
the cell membranes. Furthermore, any kind of alkyl radicals (lipid free radicals) L. can react
with a lipid peroxide LOO. to give non-initiating and non-propagating species such as the
relatively stable dimers (Morel, 1997).LOOL or two peroxide molecules combining to form
hydroxylated derivatives (LOH). Some bonds between lipid peroxides and membrane
proteins are also possible.
2.6 GENERAL ANTIOXIDANT ACTIONS
Primary antioxidants are compounds that are able to donate hydrogen atom rapidly to a
lipid radical forming a new radical, more stable than the initial one (Murray et a/., 1990).
Monodora mystrica contains polyphenols which act as antioxidant.
2.6.1 MECHANISM OF ACTION OF ANTIOXIDANTS
The principle mechanism of action have been proposed for antioxidant is chain-breaking
mechanism, by which antioxidants donate an electron to the free radical present in the
system (Murray et al., 1990). .
Electron Donation
Primary antioxidants are compounds that are able to donate hydrogen atom rapidly to a
lipid radical forming a new radical, more stable than the initial one (Murray et a/., 1990).
Biological organs contain many polyunsaturated fatty acids (PUFA), such as linoleic, lenic
36
and arachidonic acid, mainly in the form of ester with cholesterol. These PUFA can
undergo lipid peroxidation that can be interrupted by the primary antioxidant by the
donation of electrons.
The whole process can be depicted as follows.
RH + O2 (Singlet oxygen) - - - - -ROOH
ROOH + ~ e ' ~- - - - - RO. + HO- + ~ e ' ~
ROOH + ~ e ' ~- - - - ROO. + H' + ~ e ' ~
ROO.+a-TO. - - - - Non radical products.
RH = Polyunsaturated fatty acid (PUFA)
ROOH = PUFA hydroperoxide
RO. = Alkoxyl radical
ROO. = Peroxyl radical
a - TO. = Tocopheryl radical
Metal Chelation
This can be accomplished by deactivation of high-energy species, absorption of UV light,
scavenging of oxygen and thus reducing its concentration (Omenn et al, 1996). Chelation
of metal catalyzes free radical reaction or inhibits peroxidase. The ability of antioxidant to
chelate transition metal ions can be followed spectroscopically. High molecular weight
proteins bind directly or indirectly to redox active metals and thus inhibit the production of
metal-catalyzed free radicals. Some low molecular weight compounds, such as
polyphenols, in addition to their ability to donate hydrogen atom and thus act as chain-
37
breaking antioxidant, can also chelate transition metal ions and hence inhibit free radical
formation (Omenn et al., 1996).
2.6.2 ANTIOXIDANT MOLECULES
Antioxidants are a group of substances, which when present at low concentrations, in
relation to oxidizable substrates, significantly inhibit or delay oxidation and oxidative
processes, while often being oxidized themselves (Kanner et al., 1999). The application of
antioxidants are widespread, in industries theyare used in preventing polymer from
oxidative degradation, rubber and plastic from losing strength, gasoline from autooxidation,
synthetic and natural pigments from discolouration and as additives to cosmetics, food
(especially food with high fat content) beverages and baking products
(Kanner et a/, 1999).
Vitamin E - The Tocotrienols: Super Anti-Oxidants
Vitamin E is one of the most important phytonutrients in edible oils. It consists of eight
naturally occurring isomers, a family of four tocopherols (alpha, beta, gamma and delta)
and four tocotrienols (alpha, beta, gamma and delta) homologues. While most Vitamin E
supplements on the market today are composed of the more common tocopherols,
tocotrienols are believed to be a much more potent antioxidant than tocopherols.
Tocotrienols are naturally present in most plants, however they are found most abundantly
in palm oil extracted from palm fruits.
38
2.7 GENERAL REVIEW OF PHYTOCHEMISTRYOF MONODORA MYRISTICA
2.7.1 Alkaloids
They are a group of basic secondary plant substance, which usually possesses an ncontaining heterocyte. Alkaloids exist in plants as salts, amine or n-oxides. Dicotyledonous
plants are the real producers of alkaloids (Evans, 1989). They appear in large members and
in many variation in these plants. They are bitter to taste, so when present in plants, insects
and predators tend to move away from such plants. They also protect the plant from the
effect of singlet oxygen (Bonner and Varner, 1965). Alkaloids at high concentration,
produces a variety of toxic effects on animals. Their pharmaceutical and medicinal
importance can be seen to act on the cardiovascular system and some have been resorted to
be antihypertensive. Alkaloids also contribute to liver disease and hepatocellular tumor
(Antoniodes and Owen, 1982). Alkaloids of Catharanthus roseus are used in cancer
chemotherapy.
2.7.2 Flavonoids
The origin of the names is from a Latin word "FLAVUS" meaning yellow. They are a
series of related water soluble phenolic glycosides having in common a basic structural
unit. The CI5 skeleton of flavones. The flavones are sap-soluble (Bonner and Varner,
1965). The phonetic compound contributes to the colour of soft fruits, which are scarlet,
crimson and purple anthocyamins e.g. cyamidin-3-rutinoside. They are widely distributed
in nature but are more common in the higher plants and in young tissues, where they occur
in the cell sap. Flavonoids contribute to the taste and flavour of foodstuffs (Bonner and
39
Varner,..1965). Flavonoids when consumed in certain quantity could lead to serious
disorder in the system.
2.7.3 Glycosides
These are the products obtained after condensation of sugar with different types of organic
hydroxyl compounds. These are referred to as the cardiac-active or cardio-tonic glycosides
examples include amygdalin (Stryer, 1975). In small doses, glycosides promote mild gastric
irritation causing a reflux from the bronchioles. This can be attributed to its wide
usage but in larger dose, they lead to vomiting (Evans, 1989). A larger number of
glycosides and their aglycone have antimicrobial activities.
2.7.4 Saponins
Saponins are useful in the production of soft drinks, beers, confectioneries, shampoos,
soaps, fibre extinguishers and beverages and this is attributed to its foaming ability (Liener,
1972). They are quite toxic when injected into the bloodstream and are harmless when
taken by mouth since the sarsaparilla is rich in saponins but is used in the preparation of
non-alcoholic beverages (Evans, 1989). The highest sapogenin concentration occurs in the
reproductive parts of the plants, the seeds containing 18% trigonenin (Bonner and Varner,
1965). Saponin have some medicinal properties, since it has beenreported to have antiinflammatory, anti-fungal, antimycolic, bacteriostatic and other biological activities. When
ground in a powdering form, causes violent sneezing
40
2.7.5 Tannins
The word "tannin" signifies substances present in plant extracts, which are able to combine
with protein of animal hides, prevent their Putrefaction and the conversion to leather
(Evans, 1989). Those tannins are responsible for the taste qualities of wines, tea and coffee.
They are astrigent and styptic (i.e. the dry sensation felt in the mouth). Tannins
due to their antiseptic properties prevent fungal attacks (Bonner and Varner, 1965; Evans,
1989). They also have tumorigenic and carcinogenic effects.
2.8 APPLICATIONS OF VEGETABLE OILS
Many forest trees produce seeds that contain fatty oils; these can be processed into
vegetable oils for use in cooking, food industry and soap-making, and also as fuel.
Producing fixed oils is a simple process and can be done locally, with locally made
equipment. In the first stage, the oil is extracted from the seeds by dry expression or by
boiling the crushed raw material in water.
Vegetable oils also provide inputs to the more complex detergent industry, which uses fatty
alcohol derivatives of lauric oils, which currently come mainly from palm kernels primarily coconut (Cocos nucifera) and African oil palm (Elaeis guineensis), with smaller
amounts from wild stands of babassu palm (Orbignya sp.) (De Silva and Atal, op. cit.).
Palm oil is processed to produce edible fats (margarine), soaps and candles and is used in
pharmacy and cosmetics and as an important raw material in oleochemistry (fat chemistry).
Palm kernel oil (PKO) is more unsaturated and hence can be hydrogenated to a wider range
of products which could be used either alone or in blends with other oil for biscuit dough,
filling creams, cake icing, ice cream, imitation whipping cream, substitute chocolate and
41
other coatings, sharp melting and melting margarines etc. Lauric oil (CNO, PKO) is very
important in soap making and a good soap must contain at least 15% lauric acids for quick
lathering while soap made for use in sea water is based on virtually100% lauric oils. Mostly
palm kernel oil are now used for the manufacture of short chain fatty acids, fatty alcohols,
methyl esters, fatty amines, for use in detergents, cosmetics and many other cosmetic
products but less consideration is given it for other purpose. Monodora myristica seed are
used as condiment in West Africa, a decoction of the seed is used to treat guinea worm
infection. The seeds are used as a remedy for constipation, when mixed with palm oil.
Roasted and powdered seeds of the plant are very effective in curing stomach ache. The
seeds are rubbed on the forehead to cure headache (Gill, 1992).
2.8.1 FACTORS THAT CAUSE OXIDATIVE RANCIDITY IN VEGETABLE OIL
Many factors can affect the tendency of an oil to become rancid. The first is too much
exposure to air. Since oxidative rancidity is the most likely kind of rancidity to affect your
food oils, you always want to keep those oils in bottles that are tightly capped. (A tightly
capped bottle will help prevent your oil from being unnecessarily exposed to oxygen.)
The next factors are heat and light. Since both of these factors can also speed up the
rancidity process, protection from heat and light are also important when it comes to your
food oils. With respect to light, your best bet is to purchase oils in bottles made from darker
(tinted) glass (usually dark brown or dark green glass). You'll also want to store your oils in
a cabinet that is lightproof. With respect to heat, many oils are best kept in the refrigerator
where the temperature remains continuously low. (I will explain in a moment why I do not
42
believe refrigeration is necessary for extra virgin olive oil, but why I still believe it is very
important to store this oil in a cool spot.) Protecting your food oils from light and heat is a
moment-by-moment process. For example, it means paying attention to the spot you place a
bottle of oil when using it in a recipe. You never want to place it directly next to or above a
stove that is turned on due to the increased risk of damage from heat. You also want to take
the trouble of capping the bottle whenever you are not pouring oil from it.
The chemical composition of an oil is also a key factor in the risk of rancidity. Here the
basic principles involve saturated and unsaturated fat. The more saturated fat contained in
an oil, the less susceptible it is to rancidity. The greater the amount of unsaturated fat in an
oil, the more likely it is to become rancidity. Since the healthiest plant oils are all highly
unsaturated, they are especially susceptible to rancidity.
Some unsaturated oils, like extra virgin olive oil, are a little less susceptible to rancidity
because a larger amount of their unsaturated fat falls into a special category called
"monounsaturated." Extra virgin olive is about 75% monounsaturated, which is somewhat
unusual for a plant oil. Plant oils usually have more polyunsaturated fat than
monounsaturated fat, and that is one reason why they are particularly susceptible to
rancidity. While the highly monounsaturated nature of extra virgin olive oil doesn't mean
that you can forget about the issue of rancidity, it does mean that this unique oil is a little
more stable than oils that have much fewer monounsaturates.
Both omega-3 and omega-6 fatty acids are always polyunsaturated. When it comes to plant
oils, if you are trying to make sure that your diet contains an ample supply of omega-3s,
you are always at the greatest risk for rancidity. Flaxseed oil, for example, contains about
43
15 grams of alpha-linolenic acid per ounce. Alpha-linolenic acid is a polyunsaturated
omega-3 fatty acid not found in a wide variety of foods, and it's the basic building block for
all other omega-3 fatty acids. Many food scientists look upon the alpha-linolenic acid found
in flaxseeds oil as the most delicate part of its composition that needs to be protected from
oxidative rancidity. In a case like flaxseed oil, where the chemical composition of the oil
places it at great risk for rancidity, it's best to avoid any type of heating at temperatures
above 150°F (66°C) and to store the oil in the refrigerator.
Free radicals
Every cell has chemical reactions involving the oxidation and reduction of molecules.
These reaction or redox pathways can lead to the production of free radicals. A free radical
is any chemical species capable of independent existence possessing one or more unpaired
electrons. Biological free radicals are thus highly unstable molecules that have electrons
available to react with various organic substrates (Sahart, 2001).
Many free radicals are generated from naturally occurring processes such as oxygen
metabolism and inflammatory processes. For example, when cells use oxygen to generate
energy, free radicals are created as a consequence of ATP production by the mitochondria
(Sahart, 2001). Exercise can increase the levels of free radicals as can environmental
stimuli such as ionizing radiation (from industry, sun exposure, cosmic rays, and medical xrays), environmental toxins, altered atmospheric conditions (e.g. hypoxia and hyperoxia),
ozone and nitrous oxide (primarily from automobile exhaust). Lifestyle stressors such as
cigarette smoking and excessive alcohol consumption are also greater levels of oxidative or
nitrosative stress.
44
2.9 NUTRITIONAL SIGNIFICANCE
Hydrolytic rancidity caused by the release of free fatty acids from glycerides, is
significantly important in terms of flavour production but is of little consequence in terms
of nutrition as the fats are enzymically hydrolysed in the small bowl before they are
absorbed by the body. (Hydrolytic rancidity gives strong cheeses like stilton their sharp
burning taste).
Oxidative rancidity leads to the formation of both unpalatable and toxic compounds. Three
distinct classes of substance occuring in oxidised fat have been shown to be toxic:
a) Peroxidised fatty acids (peroxidised fatty acids destroy both vitamin A and E in foods).
b) Polymeric material (under normal food processing conditions these appear in small
enough quantities to be insignificant).
c) Oxidised sterols (thought to be involved in the causation of artherosclerotic disease).
45
CHAPTER THREE
3.0
MATERIALS AND METHODS
3.1 Equipments / Apparatus
Maker
weighing balance (triple beam balance)
Larle ®
Analytical balance,
Ohaus scale corporation
Labouratory dry oven (hot air oven)
DHG 9101 Model
Uv-Vis Spectrophotometer
Lemfield
Water bath
801A Model
Volumetric flask
Permagold
Measuring cylinder, Beaker,
Simax
Cornical flask, burette
Soxhlet extractor, Heating mantle,
Pyex
Extraction thimble, Condenser,
pH meter
Hannah microprocessor
Filter paper
Whatman
Mortar and pestle
46
3.2 Procedurement Of Raw Materials
The selected indigenous spice, African nutmeg (Monodora myristica) was purchased
from ogbete main market in Enugu state of Nigeria and identified by Dr. B.C. Ndukwu (a
plant taxonomist) of department of plant science and biotechnology, University of Port
Harcourt and was furtherly authenticated by Dr Charles N. Ishiwu of Department Of Food
Science And Technology, University of Nigeria, Nsuka and also a senior lecturer in
Biochemistry Department of Caritas University, Enugu state.
One purchase, the spices (seeds) were collected into sterile glass desiccators and stored in
an oven maintained at 70ºC until use.The spices (dried samples) were estimated to have
been in the market for 6-7days before purchase.
The varieties of vegetable oil used palm kernel oil and palm oil were processed and
obtained from Aniuzo International Limited ( Palm Kernel oil Mills Division), Emene in
Enugu state and Anieke Palm oil Mills, Ubulu-uku in Delta state. These were done in large
quantity to minimize chances of variation and to maintain experimental homogeneity in
sample seletion.
.
47
Figure 4: African nutmegs: dried seed kernels, which have been removed from their fruit
husk, seed covering and hard seed coat.
Figure 5: Transverse section of palm fruit.
48
3.3 STUDY DESIGN
This study was conducted on two different varieties of vegetable oil (palm kernel oil
and palm oil). Each of the varieties was divided into two groups A and B. Group A was
exposed to adverse tropical condition under the sun, while Group B was kept indoors.
There were 6 samples of equal volume for each group of each variety of oil (five(5) of the
samples contain different concentrations of extract African nutmeg, and one(1) contains no
extract). Therefore, there were total of 12 samples for each variety.The study was
conducted for 0, 1, 2, and 3 weeks. Each sample was analyzed in triplicates and the mean
was used in final content calculation. All experimental procedures were carried out
simultaneously under the same condition used for storage.
3.4 SAMPLE PREPARATION
The method of AOAC (1990) was used. The African nutmeg seeds were heated in
an oven (hot air) at 105ºC (for easy extraction of the oil ). They were then weighed in the
digital weighing balance and grounded using spiral grinder. The freshly collected seeds of
the Monodora myristica were sun dried and powdered using a pistle and mortar. The
powder was defatted with n-hexane (65 - 69°C) using soxhlet apparatus. The whole filtrate
was allowed to evaporate at room temperature leaving the oil.
49
3.5 CHEMICAL ANALYSIS
3.5.1 Determination Of Acid Value(AV)
The free fatty acid content of a fat/oil is the number of milligrammes of KOH
required to neutralize lg of FFA present in fat/oil sample.The acid value is the number of
mg of KOH necessary to neutralize the free acid in lg of sample. The acid values
(MgKOH/g) of the oil samples were determined according to Polish Standard (PN-EN ISO
660:2005). Weighed samples of around 20 g were dissolved in 100 cm3 of ethanol: diethyl
ether mixture (1:1, v/v) and titrated with 0.1 N potassium hydroxide solution using
phenolphthalein as an indicator. Analyses were carried out in triplicate the acid value is the
mg KOH used to neutralize 1.0 g of each oil sample. Results were used as reference data
for model building.
The acid value is given by T – B x 5.61/W0.1M KOH contains 5.6mg/ml or 5.6g/l where
T=Titre value of the sample; B=Titre value of a blank. The blank was provided as a control
by titrating 2.5ml of the neutral alcohol without sample. The free fatty acid (FFA) is
normally determined as oleic acid where by the acid value = 2 x FFA.
NaOH may be used and a generalized formula may be used (for palm oil and fractions):
25.6 x MNaoH x V/W where V= Volume of NaOH solution used in ml; W=Weight of
sample
3.5.2 Determination Of Thiobarbituric Acid Number (TBA)
The thiobarbituric acid value TBA was determined by modification of method described by
Odo and Ishiwu, (1999), the PORIM Test Method. Thiobarbituric acid value TBA is the
50
intensity of pink pigment formed between 2-tiobarbituric acid and the oxidized lipid
measured optically in a colorimeter. This has been found to increase as oxidation advanced.
Malonaldehyde is probably involved in the reaction (Odo and Ishiwu, 1999)
.
10g of the sample is added into 50ml distilled water in a distillation flask. 2.5ml of 4M
HCl is added to raise the pH to about 1.5. Then antibumping granules are added and the
distillation kit is set up. The mixture is heated in a heating mantle such that 50ml distillate
is collected in 10minutes from the time boiling started. 5ml of the distillate and 5ml of TBA
reagent (0.288g/100ml) of glacial acetic acid were added into a stoppered tube and heated
in a boiling water bath for 35minutes. Blank determination was made using 5ml of distilled
water and 5ml reagent. The tubes were cooled in running water and the reading of the
absorbance against blank was taken at 538nm.
3.6 STATISTICAL ANALYSIS
All statistics were performed using MICROSOFT EXCEL version 2007 software.
51
CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
The mean acid value (AV) for free fatty acid and thiobarbituric acid (TBA) values of tested
oil sample are shown in Table 3 - 6, respectively. Data for the tested oil samples were
obtained by measuring samples from the same producer in triplicate. Mean values in Table
3 - 6 are followed by lists those pairs of weeks, between which statistically significant
difference exists.
Tables 3 and 4 respectively showed the Acid Values (AV) of free fatty acid, while tables 5
and 6 showed the thiobarbituric acid values (TBA) of crude palm kernel oil and palm oil
stored with varying concentration of 0.2%-1.0% of n-hexane extract of Monodora myristica
seed, stored in the sun and in the room. The trend observed above for AV was also the same
with that of TBA in all the storage conditions only that the AV values were higher than that
of TBA. Ihekoronye and Ngoddy (1985) reported that the AV of any lipid were both
measure of hydrolytic rancidity and that the lower their values, the slower was the rate of
hydrolytic rancidity. Hence crude palm oil stored with varying concentration of 0.2%-1.0%
extract of Monodora myristica seeds were less prone to oxidative rancidity. This showed
that the extracts at varying concentration demonstrated high antioxidant activity. However
the antioxidant activity was higher as concentration of Monodora myristica extract
increases at both environmental conditions.
52
Table 3
ACID VALUE FOR PKO (MgKOH/g)
CONCN.
PRE-
WEEK 1
OF
STORAGE SS
WEEK 1
WEEK 2
WEEK 2
WEEK 3
WEEK 3
SR
SS
SR
SS
SR
EXTRACT DAY
(ML)
Control
(0.00)
4.75±0.00
5.92±0.36 4.89±0.31 8.24±0.20 5.38±0.08 14.72±10
9.70±0.51
0.2
4.70±0.15
5.66±0.28 4.74±0.36 8.06±0.32 5.14±0.13 14.68±0.31
7.74±0.26
0.4
4.45±0.12
5.30±0.28 4.52±0.12 7.86±0.37 5.00±0.31 13.56±0.48
7.22±0.21
0.6
3.92±0.41
4.96±0.31 4.08±0.26 7.32±0.41 4.84±0.26 13.18±0.26
6.26±0.21
0.8
3.44±0.32
4.54±0.29 3.94±0.31 6.92±0.51 4.62±0.31 13.06±0.26
6.18±0.26
1.0
3.06±0.91
4.08±0.34 3.70±0.31 6.40±0,28 4.28±0.31 13.10±0.13
5.99±0.31
Changes in mean ± SD acid value content of Palm kernel oil stored in sun and Palm kernel
oil stored in the room
KEY
SS = Storage in Sun;
SR = Storage in Room
AV = Acid Value;
TBA = Thiobarbituric Acid
PKO = Palm Kernel Oil;
PO = Palm Oil
SD = Standard deviation
53
Table 4:
ACID VALUE FOR PO (MgKOH/g)
CONCN.
PRE-
WEEK 1
OF
STORAGE SS
EXTRACT
DAY
WEEK 1
WEEK 2
WEEK 2
WEEK 3
WEEK 3
SR
SS
SR
SS
SR
(ML)
Control
14.27±0.26
15.71±0.23
15.42±0.18
17.75±0.13
16.26±0.08
21.02±0.12
18.74±0.36
0.2
14.13±0.11
15.24±1.24
15.21±1.13
17.32±0.52
16.06±0.19
20.83±0.26
18.22±0.34
0.4
13.92±0.26
15.01±0.08
14.73±0.52
16.63±0.08
16.00±1.10
19.92±0.52
16.88±0.26
0.6
13.53±1.02
14.94±0.15
14.71±0.91
16.26±0.15
15.72±0.90
19.61±0.81
16.32±0.31
0.8
13.27±1.31
14.51±0.92
13.81±0.71
15.84±0.12
15.48±1.21
18.91±1.00
16.02±0.81
1.0
13.06±1.42
14.39±1.10
13.36±1.02
14.81±1.10
14.13±1.40
17.26±1.23
15.72±0.92
(0.00)
Changes in mean ± SD acid value content of Palm oil stored in sun and Palm oil stored in
the room.
We can note that unsaturated fatty acid content drops with the time of addition of extract
then is stabilized from the first week. This fall is felt much in the case of the crude palm
kernel oil. It would be due to the fact that when the oil samples are exposed to the sun and
in the free air, their unsaturated fatty acids fix oxygen and oxidize (Blumenthal and Stier,
1991).
54
Table 5:
THIOBARBITURIC ACID VALUE FOR PKO
CONCN.
PRE-
WEEK 1
OF
STORAGE SS
WEEK 1
WEEK 2
WEEK 2
WEEK 3
WEEK 3
SR
SS
SR
SS
SR
EXTRACT DAY
(ML)
Control
3.12±0.08
4.14±0.22 4.00±0.26 6.92±0.84 5.50±1.10
9.10±0.81
6.30±0.75
0.2
3.06±0.01
4.09±0.16 3.86±0.29 6.69±1.04 5.32±1.24
8.68±0.50
6.00±0.35
0.4
3.08±0.05
4.12±0.05 3.51±0.33 6.21±1.13 5.01±1.21
8.53±0.13
5.54±0.26
0.6
3.19±0.06
3.81±0.08 3.06±0.36 5.90±1.19 4.78±1.26
8.30±0.26
5.08±0.10
0.8
2.30±0.12
3.15±0.13 2.87±0.13 5.42±0.91 4.43±1.00
7.83±0.26
4.80±0.13
1.0
2.02±0.17
2.72±0.19 2.31±0.21 4.91±0.75 3.90±1.20
7.35±0.26
4.22±0.13
(0.00)
Changes in mean ± SD TBA value content of Palm kernel oil stored in sun and Palm kernel
oil stored in the room.
From the values deduced from thiobarbituric acid TBA evaluation, the trend is similar to
that of acid value
55
Table 6:
THIOBARBITURIC ACID VALUE FOR PKO
CONCN.
PRE-
WEEK 1
OF
STORAGE SS
WEEK 1
WEEK 2
WEEK 2
WEEK 3
WEEK 3
SR
SS
SR
SS
SR
EXTRACT DAY
(ML)
Control
5.20±0.35
6.57±0.26 5.50±0.21 9.20±0.19 6.61±0.20
10.11±0.17
7.00±0.11
0.2
5.01±0.22
6.29±0.13 5.35±0.29 8.92±0.19 6.36±0.17
9.888±0.15
6.72±0.08
0.4
4.90±0.13
6.01±0.21 5.10±0.31 8.41±0.21 6.10±0.34
9.52±0.11
6.51±0.14
0.6
4.72±0.52
5.74±0.41 4.81±0.26 7.00±0.26 5.90±0.26
9.07±0.13
6.32±0.14
0.8
4.49±0.34
5.42±0.32 4.52±0.26 6.81±0.16 5.71±0.21
8.77±0.25
6.01±0.21
1.0
4.11±0.26
5.10±0.19 4.15±0.21 6.38±0.19 5.20±0.34
8.41±0.27
5.52±0.19
(0.00)
Changes in mean ± SD TBAvalue content of Palm oil stored in sun and Palm oil stored in
the room.
For the two varieties of oil, the acid value of free fatty acid increased significantly
(P<0.05) as the period extends for group SS without extract while those for group SR
showed no significant increase. But AV of oil samples treated with higher extract
concentration decreased significantly (P<0.05) for both groups SS and SR. TBA value also
showed the same trend of AV. Hence, monodora myristica extract yielded reducing effect
in the oxidative level of the oil varieties.
56
From the study, it is evident that the extract of seeds of Monodora myristica has
promising antioxidant activity. In the present study, the percentage (%) yield of the extract
was found to be 18.9%, which is relatively low when compared to a previous study on the
plant. In a previous study (Esuoso et al., 2000) reported that Monodora myristica seed
extract had a yield of 34.7-68.8%. The possible difference in the yield could be as a result
of geographical and climatic factors, which has been found to affect plant constituents, or
time of collection of the seed, method of storage, the variety of the parent plant and the
nature of the soil on which it is planted (Alam et al., 1982).
In the fatty acid content of palm kernel oil and palm oil, stored in the sun, values in the
same column, bearing different superscripts differ significantly P<0.05.hence there was
significant increase in oxidation which also reduced as the extract increased.
Monodora myristica seed has been found to contain a lot of secondary plant metabolites
namely: alkaloids, carbohydrates, flavonoids, glycosides, proteins, saponins, and tannins.
Alkaloids at high concentration, has been found to produce a variety of toxic effects on "
animals. They also protect the plant from the effect of singlet oxygen (Bonner and Varner,
1965). These plant constituents are known to be biological active, eliciting a variety of
actions such as antioxidant effects (Bauer et al., 1996). It can also be concluded that the
antioxidant activity of the extract could be attributed to flavonoids, which are fourid in
antioxidant plant such as Aspidium cacutarium (Ghoghari et al., 2006), Phyllanthus debilis
klein ex Willd (Kumaran and Karunakaran, 2006) as well in Tephrosia purpurea (Jain et al.,
2006).
57
4.1
Changes In Acid Value of PKO and PO
The variation of the percentage of free fatty acid of crude palm kernel oil and palm
oil placed in the sun is shown in Table 3-4. At equal volume of oil, the little decrease in
free fatty acid content was observed during the first 3 weeks. Whereas,the group kept in the
dark room showed significant decrease in the free fatty acid content. But the higher the
concentration of extract treatment in each sample, the lesser the free fatty acid value.
Higher value of percentage free fatty acid content was observed in crude palm oil.
It would be due to the fact that when PKO and PO are exposed to the sun and in the free air,
their unsaturated fatty acids fix oxygen and oxidize (Blumenthal and Stier, 1991). The acid
value of an oil may be used as a measure of quality. However, the acid value of the oil must
not be too high, as this denotes an excessively high content of free fatty acids, which causes
the oil to turn sour. Discoloration may also occur. Palm kernel oil should have an acid value
of at most 0.1 - 1.0% [1], or 5%. Oils and fats spoil by readily becoming rancid. Rancidity
is promoted by light, atmospheric oxygen and moisture and leads to changes in odor and
taste.
4.2
Changes in Thiobarbituric acid value of PKO and PO
The variation of the thiobarbituric acid number of crude palm kernel oil and pam oil placed
in the sun is shown in Table 5-6 . At equal volume of oil, the signicant decrease in content
was observed during the first 3 weeks of storage at different environmental conditions. But,
the group kept in the dark room showed significant decrease than those kept under the sun.
But the higher the concentration of extract treatment in each sample, the lesser the TBA
value. Higher value was observed in crude palm oil.
58
It would be due to the fact that when PKO and PO are exposed to the sun and in the free air,
their unsaturated fatty acids fix oxygen and oxidize (Blumenthal and Stier, 1991).
Most any food can technically become rancid. The term particularly applies to oils.
Oils can be particularly susceptible to rancidity because their chemistry which makes them
susceptible to oxygen damage. When food scientists talk about rancidity, they are often
talking about a specific type of rancidity involving oxygen damage to foods, and this type
of rancidity is called "oxidative rancidity." During the process of oxidative rancidity,
oxygen molecules interact with the structure of the oil and damage its natural structure in a
way that can change its odour, its taste, and its safety for consumption.
Spices contain phenols and essential oils, which are inhibitory to microorganisms
(Nakatani, 1999). It was reported that fat and proteins bind or solubilize phenolic
compounds thereby reducing their availability for antimicrobial activity (McMance and
Widdowson, 1993; McNeil and Schmidt,1993).
4.3
Effect Of Monodora Myristica Extract On The Chemical Indices Of Oil On
Storage
The main objective of this study was to evaluate the antioxidant potential of
monodora myristica. Two different varieties of oil were treated with monodora myristica
extract and tested to determine the antioxidant potentials of monodora myristica using
standard methods. The analysis results demonstrated that the extract treatment of the oil
samples enhanced antioxidation.
59
CHAPTER FIVE
5.0
SUMMARY AND CONCLUSION
The study results favoured the highest concentration of treatment and storage of the
tested oil samples at the different environmental conditions.
Recently, the determination of PV in commercial oils was assessed by the modern Infrared
Spectroscopy (IR) (Yildiz et al., 2002); which can also be extended to the determination of
AV in butter. The theoretical principle of IR had been reported earlier (Koczoñ et al., 2001,
2003, 2006) and the technique finds application in the food analysis (Ismail et al., 1993;
Chippie et al., 2002; Guillen and Cabo, 2002; Tay et al., 2002; Van de Voort et al., 2004)
and significantly less number for monitoring of chemical changes in foods (Quilitzsch et
al., 2005).
Fats and oils are quite unstable substances. When stored for any considerable length
of time, especially when the temperature is high and the air has free access to them, they
deteriorate and spoil. In this respect different fats differ markedly. Some spoil very much
more rapidly than others. Among the various fats, spoilage takes the form of rancidity. The
fat acquires a peculiarly disagreeable odor and flavor. A vast amount of scientific research
has been carried on to determine the cause and nature of rancidity, but investigators are far
from agreement on the subject. For present purposes it is sufficient to point out that
spoilage of a fat, usually identical with rancidity, is accompanied by partial splitting of the
fat into glycerin and fatty acids. The glycerin disappears, or at any rate is unobjectionable,
but the fatty acids remain dissolved in the fat, give it an acid reaction, and contribute to its
objectionable rancid flavor. The rancidity of a given parcel of fat is not necessarily the
60
result of long storage under unfavorable conditions. The fat may have been spoiled and
rancid from the moment of its production. This will inevitably be true when the materials
from which it was produced have undergone decomposition. Thus the fat obtained from
putrefying carcasses will be rancid and so will the oil expressed from fermented cottonseed.
In other words, to obtain a sound and sweet fat, the raw material must be sound and sweet;
it must be worked up speedily before it has had time to decompose; and this must be done
under clean and sanitary conditions. The fat thus obtained must be stored under favorable
conditions and its consumption cannot be too long delayed. These conditions it is difficult
to obtain in many of the less civilized portions of the world, especially in the tropics, where
many fat- and oil-yielding raw materials are produced. Hence fats and oils made at the
source of the raw materials may be less sound than those produced at or near the place of
consumption.
All oils are fats, but not all fats are oils. They are very similar to each other in their
chemical makeup, but what makes one an oil and another a fat is the percentage of
hydrogen saturation in the fatty acids of which they are composed. The fats and oils which
are available to us for culinary purposes are actually mixtures of differing fatty acids so for
practical purposes we'll say saturated fats are solid at room temperature (20C) and
unsaturated fats we call oils are liquid at room temperature. For dietary and nutrition
purposes fats are generally classified as saturated, monosaturated and polyunsaturated, but
this is just a further refinement of the amount of saturation of the particular compositions of
fatty acids in the fats.
Connoisseurs of good edible palm oil know that the increased FFA only adds ‘bite’ to the
oil flavour. At worst, the high FFA content oil has good laxative effects. The free fatty acid
61
content is not a quality issue for those who consume the crude oil directly, although it is for
oil refiners, who have a problem with neutralization of high FFA content palm oil.
Oxygen is eight times more soluble in fats than in water and it is the oxidation
resulting from this exposure that is the primary cause of rancidity. The more
polyunsaturated a fat is, the faster it will go rancid. This may not, at first, be readily
apparent because vegetable oils have to become several times more rancid than animal fats
before our noses can detect it. An extreme example of rancidity is the linseed oil (flaxseed)
that we use as a wood finish and a base for oil paints. In just a matter of hours the oil
oxidizes into a solid polymer. This is very desirable for wood and paint, but very
undesirable for food.
Antioxidants are often added to fat-containing foods in order to retard the
development of rancidity due to oxidation. Natural anti-oxidants include flavonoids,
polyphenols, ascorbic acid (vitamin C) and tocopherols (vitamin E).
Synthetic antioxidants include butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT), propyl 3,4,5-trihydroxybenzoate also known as propyl gallate and ethoxyquin.
The natural antioxidants tend to be short-lived, so synthetic antioxidants are used when a
longer shelf life is preferred.
62
The effectiveness of water-soluble antioxidants is limited in preventing direct oxidation
within fats, but is valuable in intercepting free radicals that travel through the watery parts
of foods.
A combination of water-soluble and fat-soluble antioxidants is ideal, usually in the ratio of
fat to water.
In addition, rancidification can be decreased, but not completely eliminated, by
storing fats and oils in a cool, dark place with little exposure to oxygen or free radicals,
since heat and light accelerate the rate of reaction of fats with oxygen. (Oxidative rancidity
or autooxidation is a chemical reaction with a low activation energy consequently the rate
of reaction is not significantly reduced by cold storage).
5.1
LIMITATIONS
There were limitations to the present study which were barriers in achieving ideal
experimental conditions.
The current study was conducted on limited parameters of tested intervals and constant
temperature, the ranges for the interval for test at the tropical environmental conditions may
have been varied. Therefore the present study did not show the significant stability of the
tested oil samples.
5.2
FUTURE RECOMMENDATIONS
1.
PalmOilTester which is a fast, user-friendly and reliable testing system for crude and
refined palm oil is recommended as it enables the determination of acidity (FFA),
63
DOBI & Carotene content, the values of Peroxide (PV), anisidine (AnV) and iodine
(IV) in few minutes.With its simplicity, PalmOilTester is ideal to performe analysis
during every production stages in palm oil industry to monitor the quality of oil in
real time, from the oil mill to the refinery plant, during the acceptance and storage
phases, as well as during trading of finished products.Several comparative studies
have demonstrated that the analytical accuracy of PalmOilTester matches that of
AOCS/MPOB reference methods, with the advantages that PalmOilTester is easier
to use and outputs results much faster.
2.
The use of apparatus called Rancimat is recommended to calculate effect of
antioxidant on oil and fat, though other methods like used for determination of
rancidity are Peroxide value ( Primary Oxidation) and Anisidine value( Secondary
Oxidation) in fat or oil .Peroxide value provides the extent of rancidity present in
the oil.Peroxi de value is found by formation of iodine when oil or fat are
reacts with iodine ion. Totox value are also used to check the quality of oil and fat .
Totox Value = Anisidine value + 2x Peroxide value. Thiobarbituric acid value also
provides useful information on oxidative level of rancid oil.
64
BIBLIOGRAPHY
Akpanabiatu, M. I. Ekpa, O. D. , Mauro, A. and Rizzo, R. (2001). Nutrient Composition of
Nigerian Palm Kernel from Dura and Tenera Varieties of the Oil Palm (Elaeis
guineensis). Food Chem. 72:173-177.
Aubourg, S. (1999). Recent advances in assessment of marine lipid oxidation by using
fluorescence. Journal of American Oil Chemistry society. 76: 409-419.
Aubourg, S. (2001). Fluorescence study of the pro-oxidant effect of free fatty acids on
vegetable oils. Journal of science Food Agriculture. 81:385-390.
Banso, A. and Ayodele, P.O. (2005). Effect of two tropical spice extracts on the growth of
fungi in fruit juices. Nigerian Journal of Applied Arts and Sciences (NIJAAS) 1: 3542.
Banso, A. and Sani, A . (2003). Antimicrobial effects of leaf extract of Ricinus communis.
African Scientist. 4(3): 129-133.
Basturk, A., Javidipour, I., and Boyaci, I. H. (2007). Oxidative stability of natural and
chemically interesterified cottonseed, palm and soybean oils. Journal of Food
Lipids. 14 (2):170-188.
Bonner, J. and Varner, J. E. (1965). Plant Biochemistry. Academic Press, London. pp. 252703.
Chow, M. C. and Honley, C. C. (2000). Surface active properties of palm oil with respect to
the processing of palm oil. Journal of Oil Palm Research. 12(1): 107-116.
65
Cornelius, J. A. (2001) Comparison of Traditional and Industrial Palm Oil. Oil Palm News;
No.28.Tropical Development and Research Institute, London.
Coursey, D. G. (1963). The Deterioration of Palm Oil during Storage. Journal of West
African Science Association. 7:101-13.
Cutler, R. G. (1984). Antioxidants, aging, and longevity. In Free Radicals in Biology,
Academic, Orlando, Florida. I: 371-428.
Damenn, G. S., Goodman, G. E., Thornquist, M. D., and Cullen, M. R. (1996). Free
radicals and Antioxidants. Bio. Med., 334: 11 50-11 55.
Demarco, E., Savarese, M., Parisini, C., Battimo, I., Falco, S., Sacchi, R. (2007). “Frying
performance of a sunflower/palm oil blend in comparison with pure palm oil”.
European Journal of Lipid Science and Technology 109: 237.
Egan, H., Kirk, R. S. and Sawyer, R. (1981). Pearson’s Chemical Analysis Food. 8th ed.
Churchill Livingstone, New York, Pp 507-546.
Eggeling, W. J. (2002). The Indigenous Trees of the Uganda protectorate (Revised and
enlarged by Ivan R. Dale). Government printer, Entebbe Uganda, Crown Agents
for the Colonies, London. Pp. xiii and 491.
Ekeke G.I, Uwakwe A.A. and Nwaoguikpe, R.N. (2000). Edible legumes as nutritionally
beneficial antisickling agents Nig. J. Biochemistry Mol. Biol. 16: 200-201.
Ekpa, O. D. an kpe, U. J. (1996). Effect of Coconut Oil Concentration on the Melting point
profile and Free Fatty Acid Formation of Palm Oil. Nigerian Journal 0f Chem. Res.
1 8-12.
66
Ekpa, O. D., Fabara, E. P. and Morah, F. N. I. (1994) Variation in Fatty Acid Composition
of Palm Oils from two Varieties of the Oil Palm (Elaeis guineensis). J. Sci. Food
Agric. 64: 483-486.
Ekpa, O. D., Akpanabiatu, M. I., Amelio, M. and Rizzo, R. (2001). A Comparative Study
of the Triglyceride and Fatty Acid Compositions of Palm Oil from Plantations in
South-Eastern Nigeria. Global J. Pure & Applied Sci. 7:61-65.
Ekpa, O. D., Akpanabiatu, M. I., Amelio, M. and Rizzo, R. (2001). A Varietal Differences
and Polymorphism in Palm Oil: A Case Study of Palm Oil Blended with coconut
Oil. Global J. Pure & Applied Sci. 7:277-283. European Journal of Lipid Science
and Technology :Lipid -
Fett Volume 109 Issue 4, Pages 373 – 379 Special Issue:
Palm oil Published Online: 15 Mar 2007 Copyright © 2010 WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim
Esuoso, K. O., Lutz, H., Bayer, E., Kutubuddin, M. (2000). Unsaponifiable lipid
constituents of some underutilized tropical seed oil. Journal of Agricultural
Food.Chem. 48(2): 231-234.
Evans, R. W. (1989). Pharmacognosy (13'~ ed); Baillere Tindall, London. pp. 527-665.
Ghoghari, A. M., Bagul, M.S., Anandjiwala, S., Chauhan, M. G. and Rajani, M. (2006).
Free radical scavenging activity of Aspidium cicutarium rhizome. J. Natural
Remedies. 6(2): 1 31 -1 34
Gibon, V. (2007). Palm oil refining. European Journal of Lipid Science and Technology
109(4).
67
Gill LS (1992). Ethnomedical uses of plants in Nigeria. University of Benin Press, Benin
city, Nigeria. p. 165, 248.`
Goh, S.H., Choo, Y.M., Ong, A.S. (2005). Minor constituents of palm oil. Journal of
American Oil Chem Soc 1985;62:237-40.
Ihenkoronye A.I. and Ngoddy P.O. (1985). Tropical Fruits and Vegetables. In: Integrated
food Science and Technology for the Tropics. Macmillan Publisher Ltd, London. pp
63-75.
Irvine, F. R. (2000). Woody Plants of Ghana with special reference to their uses. Oxford
University Press, London pp. 13-23.
Ismail, A.A., Van de Voort, G. Emo and Sedman, J. (1993). Rapid quantitative
determination of
free fatty acids in fats and oils by Fourier transform infrared
spectroscopy. Journal of American Oil Chem Soc., 4: 335-341.
Jacobsberg B. (2000). Palm oil characteristics and quality. In: O. S. Chai, and A.
Awalludin, (Eds). Proceedings of the 1st MARDI Workshop on Oil Palm
Technology. Kuala Lumpur, Malaysia: Malaysia Agricultural Research and
Development Insitute (MARDI), 1974:48-70.
Jain, A., Singhai, A. K. and Dixit, V. K. (2006). In vitro evaluation of Tephrosia purpurea
pers for antioxidant activity. Journal of Naturalremedies. 6(1): 162-1 64.
Kanner, P. H., Guyton, K. E., and Gorospe, M. (1999). Oxidative stress and the
molecular biology of antioxidant defense (J. G. Scandolios, (Ed). Cold Spring
Harbor Laboratory Press, New York, pp. 247-272.
68
Kapoor, I. P. S., Bandana Singh, Gurdip Singh, Carola S. De Heluani, M. P. De Lampasona
and Cesar, A. N. C. (2009). Chemistry and in Vitro Antioxidant Activity of Volatile
Oil and Oleoresins of Black Pepper (Piper nigrum) Journal of Agricultural and
Food Chemistry;57 (12), 5358-5364
Kubow, S. (2009). Lipid Oxidation Products in Food and Atherogenesis. Nutrition Reviews
51(2).
Kuku, F. O. (1976). Some Mould-induced Changes in Palm Kernels. Nig Stored Products
Res Inst Tech Rep 9:62-72.
Kumaran, A. and Karunakaran, J. (2006). Antioxidant activity of polyphenols from
phyllantus debilis
klein ex Willd. Journal NaturalRemedies. 6(2): 141 -146
Matthäus, Bertrand (2007). "Use of palm oil for frying in comparison with other highstability oils". European Journal of Lipid Science and Technology 109: 400.
Mielke, S. (2001). Present and future position of palm and palm kernel oils in world supply
and trade. Journal of American
Oil Chemistry Society.,1985;62:193-97.
Morel, C. 0. (1997). Mechanisms of Fatty Acid Oxidation. http:llwww.atozofhealth.com.
Downloaded on the 1lth June 2005.
Murray, P. E., Vaya, J. and Aviran, M. (1990). Nutritional Antioxidant: Mechanisms .
of Action, Analyses of Activities and Modern Application. Bentham Science
Publishers Ltd. New York, pp. 99-110.
Naczk, M., J. Pink, R. Zadernowski and D. Pink, (2002). Multivariate model for the
prediction of total phenolic acids in crude extracts of polyphenols from canola and
rapeseed meals: A preliminary
study. Am. Oil Chem. Soc., 8: 759-762.
69
Nakatani N (1999). Antioxidative and antimicrobial constituents of herbs and spices. In:
Spices, Herbs and Edible Fungi. G. Charalambous (Ed). Elsivier, London. pp. 251271.
Nasirullah, (2005). Physical refining: electrolyte degumming of nonhydratable gums from
selected vegetable oils. Journal of Food Lipids 12(2).
Nesaretnam, K and Muhammad, B. (1993). Nutritional properties of palm oil. Selected
Readings on the Palm Oil and its Uses. p. 57-67
Nielsen S. and Suzanne S. (2002). Introduction to chemical analysis of foods. Daryaganj,
Indian, Pp 181-202
Nwachukwu, N. (2000). Nutritional and Antinutritional substances in some selected
indigenous spices PhD Thesis, FUTO, Nigeria.
Nyam, K.L., Tan, C.P., Lai, O.M., ., Long, K., Che Man, Y.B.(2009). Physicochemical
properties and bioactive compounds of selected seed oils LWT - Food Science and
Technology. 42 (8):1396-1403
Odo, F. O. and Ishiwu, C. N. (1999). Experimental procedures for food and water analysis.
Enugu: Computer edge. Pp 25-35.
Oguntimein, B., Ekundayo, O., Laasko, I. and Hitunen, R. (1999). Constituents of the
essential oil of Monodora tenuifolia. Flav. And Fragr. J., 4: 193-1 95.
Ojiako, O. A. and Akubugwo, E. I. (1997) An Introductory Approach to Practical
Biochemistry CRC Publications, Owerri, Pp. 132.
70
Okafor, J. C. (2003). Some useful Tropical Plans in Health Care Delivery, Forestry
Commission HQ, Enugu. pp. 3-9.
Okiy, D. A. (1979) Aspects on Quality of Palm Oil. NIFOR Newsletter No. 18 Pp 7-8.
Omenn, G. S., Goodman, G. E., Thornquist, M. D. and Cullen, M. R. (1996). Free radicals
and Antioxidants. Bio. Med., 334: 11 50-11 55.
Palm Oil Research Institute of Malaysia, (PORIM). (1990). The PORIM Test Method.
Palm Oil Research Institute of Malaysia.
Pantzaris T.P and Mohammed J. A. (2000): Palm Kernel oil article. Palm Oil Research
Institute of Malaysia (PORIM), 2000.
Pantazari TP, Ahmad MJ (2004). Palm Kernel oil. Palm oil processing technology report.
Res. Instit. of Malaysia. p.220.
Pearson, D. (1976) The Chemical Analysis of Foods. 7th Edition, Edinburgh (Churchill
Livingstone).
Quilitzsch, R., M. Baranska, H. Schulz and E. Hoberg, (2005). Fast determination of carrot
quality by spectroscopy methods in the UV-VIS, NIR and IR range. J. Applied Bot.
Food Qual., 79: 163-167.
Renuka, D., Jayalekshmy R. (2007). Antioxidant efficacy of phytochemical extracts from
defatted rice bran in the bulk oil system Food Chemistry 104 (2), pp. 658-664.
Rutowski, A. (1983). Traditional Palm Oil Processing in Western Africa. Fette Seifen
Anstrichmittel 85:262 – 267.
Sahart, M. H. (2001). Free radicals and Antioxidant. Biol. Med., 28:1685-1 696.
71
Shahidi, F. and Zhong, Y. (2009). Measurement of antioxidant activity in food and
biological systems ACS
Symposium Series 956, pp. 36-66.
Simeh, A., Tengku A.and Tengku M. A. (2001). "The Case Study on the Malaysian Palm
Oil" Oil palm - the backbone of economic growth Global Oils and Fats business
magazine. 6(2): 6-8.
Stevenson, T. ( 2006). "Malaysia targets alternative fuels market". The Daily Telegraph
(London).http://www.telegraph.co.uk/finance/2952784/Malaysia-targets-alternativefuels-market.html. Retrieved 22 September 2009.
Stryer, L. (1988). Biochemistry 3rd ed. W. A. Freeman and Company, New York pp. 331
and 470.
.Talalaji, S. J. (1 999). Essential oil from Monodora myristica grown in Ghana. West
African Pharmacist, 4: 64-65.
Tan, B. K., Ohih, F. C. (1997). Malaysian palm oil chemical and physical characteristics.
PORIM Technology 1981;3. New findings on palm oil. Nutr Rev 1987;45:205-07.
Tan, C.P., Selamat, J. and Yusoff, M. S. (2002). Comparative studies of oxidative stability
of edible oils by differential scanning colorimeter and oxidative stability index
methods. Food Chemistry.76(3), pp.378-385.
Tay, A., Singh, S.S., Krishnan, A. and Gore, J.P. (2002). Authentication of olive oil
adulterated with vegetable oils using fourier transform infrared spectroscopy.
Lebensm. Wiss. u. Technol., 35: 99-103.
72
Van de Voort, F.R., Ismail, J. Sedman, J. Dubois and Nicodemo, T. (1994). The
determination of peroxide value by Fourier transform infrared spectroscopy. J. Am.
Oil Chem. Soc., 9: 921-926.
Yamane, K., Kawasaki, K.,Sone, K., Hara, T., Prakoso, T. (2007). Oxidation stability of
biodiesel and its effects on diesel combustion and emission characteristics.
International Journal of Engine
Research 8(3).
Yesil-celiktas, O. (2009). Influence of supercritical carbon dioxide and methanolic extracts
of rosemary on oxidation and sensory properties of wheat germ oil. Journal of Food
Quality 8: 885-898.
Yildiz, G., R.L. Wehling and Cuppett, S. L. (2002). Monitoring PV in corn and soybean
oils by NIR Spectroscopy. Journal of American Oil Chemistry Society., 11: 10851089
73
APPENDIX I
PREPARATION OF REAGENT AND SAMPLES
CRUDE FAT CONTENT OF MONODORA MYRISTICA
Sample: Monodora myristica (minced) 90g
Reagents: n-hexane (750ml).
(Extract concentration at equal volume of oil: 0.2, 0.4, 0.6, 0.8, 1.0 (ml))
ACID VALUE DETERMINATION (FREE FATTY ACID (FFA))
Sample: palm kernel oil (1g for each concentration)
Palm oil (1g for each concentration)
Reagents: Ethanol 95%v/v
Potassium hydroxide solution (0.1N)
Phenolphthalein indicator
Fat solvent- (ethanol and ether in 1:1 ratio)
Oxalic acid (0.1N)
Acid value =MgKOH/g
THIOBARBITURIC ACID DETERMINATION
Sample: palm kernel oil (10g for each concentration)
Palm oil (10g for each concentration) Reagents: HCl (4M)
Thiobarbituric reagent (0.288g/100ml of glacial acetic acid); Distilled water.
74
APPENDIX II
CALCULATIONS
CRUDE FAT CONTENT
%Fat = C-A × 100
B
Where A= Weight of empty flask
B= weight of the sample
C= Weight of flask + oil after drying
ACID VALUE
The acid value is the number of milligrams of potassium hydroxide required to neutralize
the acidity of one gramme of the oil or fat.
Acid value =
5.61× T
W
(MgKOH/g)
Where T= volume of the milliliters of 0.1N required.
W= weight in grams of the oil sample
5.61 = Equivalent weight of KOH ( constant).
75
THIOBARBITURIC ACID NUMBER
TBA= 7.8 × O.D. mg malonaldehyde/kg
Where O.D= optical density (absorbance unit)
7.8= constant (k)
Wavelength = 538nm (Odo and Ishiwu , 1999).
Average for each parameter qualitative index was calculated thus;
Mean X= X/n
Where X= sum of the number of acid
n= total number of acid values
Standard deviation of qualitative index for each sample at different concentration of extract
and different environmental condition was calculated using the formular,
S.D= ∑(x- X) 2
n-1
Where x = qualitative index for each sample
X = average mean; n = total number of qualitative index.
76
APPENDIX III
SAMPLES USED FOR THE STUDY
Figure: 6
n- Hexane extract of monodora myristica (African nut meg)
Figure: 7
Crude Palm Oil (CPO)
Figure: 8
Crude palm kernel oil (CPKO)
77
APPENDIX IV
Palm Oil Specifications
STANDARD
PORAM
&MEOMA SPECIFICATIONS
COLOUR CL
CODE
PRODUCT DESCRIPTION
FFA MNI
IV
MP
DOBI
R/Y
2.3OTSB1040 CRUDE PALM OIL
(CPO)
5%
0.0025
2.4
OTSB1041 RBD PALM OIL AS PLAMITIC
0.10% 0.10% 50-55 33-39
OTSB1042 RBD PALM OLEIN
0.10% 0.10%
56
3R
24
3R
MIN MAX
48
OTSB1043 RBD PALM STEARINE
44
0.20% 0.15%
3R
MAX MIN
PALM FATTY ACID DISTILLATE
70 %
OTSB1044
1.0%
(PFAD)
MIN
70 %
OTSB1045 PALM ACID OIL
(PAO)
3.0%
MIN
CRUDE PALM KERNEL OIL AS
OTSB1046
19
0.10% 0.10%
LAURIC
1R
MAX
78
PT
(CPKO)
24
OTSB1047 RBD PALM KERNEL OIL
0.10% 0.10% 17-18
1R/10Y
MIN
22
OTSB1048 RBD PALM KERNEL OLEIN
24
0.10% 0.10%
1R/10Y
MIN MIN
OTSB1049 RBD PALM KERNEL STEARINE
RBD PALM KERNEL FATTY ACID
OTSB1050
DISTILLATE
(PKFAD)
OTSB1051 MARGARINE
0.10% 16.00%
33-40
YELLOW
OTSB105 VEGETABLE GHEE
OTSB1053 VEGETABLE SHORTENINGS
0.10% 0.10% 40-50 40
2.5R/25Y
OTSB1054 DOUGH FATS
0.10% 0.10% 33-39 46-51
3R
PORAM
PALM OIL REFINERS ASSOCIATION MALAYSIA
MEOMA
MALAYSIAN EDIBLE OILS MANUFACTURERS ASSOCIATIO
FFA
FREE FATTY ACID
MNI
MOISTURE & IMPURITIES
IV
IODINE VALUE
MP
MELTING POINT
DOBI
DETERIORATION OF BLEACHIBILITY INDEX
79
R/Y
RED/YELLOW
SAP
SAPONIFIABLE MATTER
TFM
TOTAL FATTY MATTER
PPM
PERMITTED ANTIOXIDANTS
http://www.oilpac.com/palmoilspec.htm
SPECIFICATIONS
Parameter
Specifications
% FFA
4.78
% MVM
0.08
Peroxide Value
2.57
80