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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 VEGETABLE OILS

Vegetable oils are obtained from plant seeds such as seeds such as sunflower, peanut, sesame, soy beans cotton seeds (Nzikou et al., 2010). They belong to a class of organic compounds called lipids. Vegetable oils contain a major component known as triglyceride which makes up 98% of its content and also the minor components (2%), comprising of free fatty acids, mono- and diglycerides, phosphatides, glycolipids, pigments, sterols, tocopherols, flavonoids, tannins, and trace metals (O’brien, 2018). Vegetable oils are widely used in food preparation, cosmetics and pharmaceutical industries, margarine, soap and detergent, paint and varnishes (Othman and Ngassapa, 2010).

Besides vegetable oils, other plant oils known as essential oils are obtained from plant materials such as flowers, leaves, bark, wood, roots or peels (Bansal, 2016).

2.2 FATTY ACIDS IN VEGETABLE OILS

Fatty acids are a class of aliphatic monocarboxylic acids composed of a carboxyl group and a hydrocarbon chain. Fatty acids are distinguished from one another by the nature of their hydrocarbon chain which can vary in length from 4 to 24 carbon atoms which can be saturated or unsaturated (one double bond, two or more double bonds). The most common fatty acids in vegetable oils are those containing 18 carbon atoms which includes stearic acid, oleic acid, linoleic, and linolenic acids (Ratnayake and Galli, 2009).

Figure 1: Structures of some C18 common fatty acids in vegetable oils (Ratnayake and Galli, 2009)

2.2.1 Nomenclature of Fatty Acids

The chemical nomenclature requires that carbon atoms be counted from the carboxyl end of the fatty acid (Davidson and Cantrill, 1985). However, for biological activity, carbon atoms are numbered from the terminal methyl group to the first carbon of the ethylenic bond. Such a classification is designated by the symbol ϖ-x, ϖx, or n-x, nx, where x denotes the position of the double bond closest to the terminal methyl group. Fatty acid abbreviations are made according to the number of carbon atoms in the molecule and the number of cis ethylenic double bonds. The general assumption is that all multiple double bonds are methylene interrupted (IUPAC-IUB commission on Biochemical nomenclature, 1978).

For example, linoleic acid with two double bonds, where one is located on the sixth carbon atom counted from the methyl group, will be abbreviated as C18:2n-6 (Mead and Fulco, 1976).

2.2.2 Formation of a Triglyceride in Vegetable Oils

A triglyceride (triacylglycerol) is an ester formed by combination of three fatty acid molecules and a glycerol alcohol. The reaction occurs when three hydroxyl (OH^–) groups of a single glycerol molecule react with the carboxyl group (COOH^–) of three fatty acids to form an ester bond. The fatty acids may be the same or different in structure.

Figure 2: Formation of triglyceride molecule (Boudreaux, 2020)

2.3 CLASSIFICATION OF FATTY ACIDS

Fatty acids may be classified according to the length of the hydrocarbon carbon chain as short, medium or long chained. Besides the hydrocarbon chain length, fatty acids may also be classified as unsaturated (Containing carbon-carbon double bond) and saturated (containing carbon-carbon single bond) (Turicka et al., 2011).

2.3.1 Saturated Fatty Acids

Saturated fatty acids contain only carbon-carbon single bonds in the hydrocarbon chain and naturally occurring saturated fatty acids usually have even number of carbon atoms (table 2.1) shows some major saturated fatty acids in vegetable oils.

Table 2.1:Major saturated fatty acids in vegetable oils (Gunstone, 1996)

(Source: Gunstone, 1996)

2.3.2 Unsaturated Fatty Acids

Unsaturated fatty acids contain one or more carbon-carbon double bond in the hydrocarbon chain and the double bonds may be located in the cis or trans configuration. Unsaturated fatty acids with only one double bonds are referred to as monounsaturated fatty acids (MUFA), for example, oleic acid (18:1n9). Whereas those with two or more double bonds are called polyunsaturated fatty acids (PUFA), for example, linoleic acid (18:2n6) and linolenic acid (18:3n3). Table 2.2 summarizes some major unsaturated fatty acids.

Table 2.2: Major unsaturated fatty acids in vegetable oils (Gunstone, 1996)

(Source: Gunstone, 1996)

2.3.3 Essential Fatty Acids (EFAs)

These are polyunsaturated fatty acids that are not biosynthesized by the human body, and therefore must be obtained from the diet. They are called essential because they are required for important biological processes like synthesis of prostaglandins and hormones (Glick and Fischer, 2013). Essential fatty acids have been reported to suppress production of pro-inflammatory compounds, and hence very important in control of diseases. Vegetable oils such as sunflower and sesame are some of the major and cheap sources of essential fatty acids, for example, alpha-linolenic acid (an omega-3 fatty acid) and linoleic (an omega-6 fatty acid).

2.3.3.1 Omega-3 Essential Fatty Acids

Vegetable oils are the main sources of omega-3 fatty acids especially alpha-linolenic (ALA). This fatty acid possesses health benefits such as anti-inflammatory, antiarrhythmic and antithrombotic properties (Weylandt et al., 2012). Fish and fish oil are rich sources of other omega-3 fatty acids specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

2.3.3.2 Omega-6 Essential Fatty Acids

Vegetable oils, seeds, and nuts are dietary sources of omega-6 fatty acids, for example, linoleic acid. Al-Khudairy et al. (2015) reported that omega-6 fatty acids are important in the body for reducing risk of heart disease, lowering total cholesterol levels, lowering “bad” (LDL) cholesterol levels, raising “good” HDL cholesterol levels, and reducing cancer risks.

The essential fatty acids in vegetable oils are nutritionally important, but most susceptible to oxidative degradation during storage, which may cause deterioration of oils leading to loss of nutritional value, alteration of sensory properties such as aroma, flavour, and colour of vegetable oils.

2.3.3.3 Previous studies on fatty acid composition

Various studies have reported fatty acid composition of different vegetable oils.

Musalima et al., (2019) reported fatty acid composition of peanut oil from Uganda contained 39.71-55.89% oleic acid, 20.21-35.59% linoleic and 11.91-17.16% palmitic acids.

Achola et al., (2017) reported a range of 14.61 to 18.61% palmitic acid, 26.79 to 33.44% linoleic acid 21.9-34.46, 2.19 to 3.46% stearic acid in peanut oils from Uganda.

Flagella et al., 2002 reported that standard sunflower oil contains approximately 15% saturated, 85% unsaturated fatty acid consisting of (14-43%) oleic acid and 44-75% linoleic acid.

In recent years, high quality sunflower oils have been produced with a range of composition via development of mid-oleic type (43.1 to 71.8%) and high oleic type (75 to 90.7%) sunflower varieties that has high oleic acid content than standard sunflower type (flagella et al., 2002).

Hwang, (2005) reported that Fatty acid composition in Sesame seeds consist of abundant unsaturated fatty acid, like oleic acid (35.9-42.3%) and linoleic acid 41.5 – 47.9% from 80% of total fatty acids. Yoshida et al., (2000) reported fatty acid composition of seasame oil as 44% oleic, 34% linoleic and 10% palmitic and 7% stearic acids. High contents of linoleic acids and linolenic acids are another merit of sim sim oils as food source (Park, 2010)

Sesame seeds also contains less than 20% saturated fatty acids mainly palmitic (7.9 to 12% and stearic acid 4.8 to 6.1%. worldwide, fatty acid composition in sesame oil is variable among the different varieties of sesame seeds such as black brown.

Rehman et al., (2007) reported that Fatty acid composition of sesame seeds depends on different factors such as climatic situations soil conditions and ripeness of plants.

2.4 IMPACT OF FATTY ACID COMPOSITION ON HUMAN NUTRITION AND HEALTH

The ratio of unsaturated to saturated fatty acids in edible oils and fats is very important for human nutrition. While high levels of saturated fatty acids are desirable to increase oil stability, on the other hand, nutritionally they become undesirable, because high levels of saturated fatty acids are frequently considered to have an influence in increasing the concentration of low density lipoproteins (LDL), affecting the ratio of LDL to HDL (high density lipoproteins), a risk marker for Cardio Vascular Disease (CVD) (Barbour et al., 2015). Oleic acid, a monounsaturated fatty acid (MUFA) is however, thought to reverse the above effects. Oleic acid (C18:1n9) has also been associated with several human health benefits, including; decreased risk of cardio vascular disease (CVD) by reducing the levels of serum low-density lipoproteins (LDL) cholesterol; and maintaining the levels of high-density lipoproteins (HDL), without causing significant weight gain (Barbour et al., 2015).

Kris-Etherton (1999) reported that MUFAs decrease plasma triglyceride levels in comparison with carbohydrates. In addition, the MUFAs help in hindering the development of adrenoleukodystrophy (ALD) (Rizzo et al.,1986) and reversing inhibitory effects of insulin production (Vassiliou et al., 2009). MUFAs may also decrease platelet aggregation and increase ûbrinolysis, thereby protecting against thrombogenesis (Kris-Etherton, 1999). It also has anti-inflammatory properties that activate different pathways of immune competent cells (Carrillo et al., 2012).

Polyunsaturated fatty acids (PUFAs) such as linoleic (C18:2n6) are recognized for their susceptibility to oxidative rancidity because when heated at high temperatures makes it dangerous for human consumption (Isleib et al.,2006), and this instability leads to formation of trans-fatty acid, which has detrimental effect on human health as it causes cardiovascular disease (CVD) (Wang et al., 2015). In addition, linoleic acid is a metabolic precursor to arachidonic acid and eicosanoids, which has been associated with an increased risk of inflammation, cancers, CVD, and neurological disorders (Whelan, 2008).

2.4.1 Nutritional Index of Oils

The relationship between saturated and polyunsaturated FA content is expressed as nutritional index (P/S index). This value is an important parameter for determination of nutritional value of vegetable oils (Kostik et al, 2008). Vegetable oils with P/S index greater than 1 are considered to have a high nutritional value. Lawton et al. (2000) indicated that higher value of P/S index means a smaller deposition of lipids in the body. Other studies elsewhere have reported the P/S index value for some vegetable oils such as sunflower, peanut, soybean, and safflower as 6.76, 1.04, 4.26, and 10.55, respectively (Zambiazi et al., 2007; Daniewski, 2003).

2.5 BIOSYNTHESIS OF FATTY ACIDS

Fatty acid biosynthesis is a complex process due to compartmentalization of the biosynthesis pathway in different organelles in plant cells, and the extensive lipid movement from one organelle to another (Buchanan, 2000). An overview of various steps in the fatty acid biosynthesis pathway in plants is illustrated by Buchanan (2000) in (Figure3).

Figure 3: Fatty acid biosynthesis in plants (Buchanan, 2000)

Steps involved in fatty acid biosynthesis in plants; –

Step 1: Fatty acid biosynthesis begins with an ATP-dependent carboxylation process in which Acetyl-CoA carboxylase (also known as ACCase) catalyses the formation of malonyl-CoA (also known as activated Acetyl-CoA). Biotin carboxylase activates CO₂ by attaching it to nitrogen in the biotin ring of the biotin carboxyl carrier protein (BCCP). The flexible biotin arm of BCCP carries the activated CO₂ from the biotin carboxylase active site to the carboxyl transferase site and this enzyme transfers activated CO₂ from biotin to Acetyl-CoA, producing malonyl-CoA. ACCase is highly regulated enzyme which is active in light and inhibited in dark conditions. Also, being a part of the first step of lipid synthesis, this enzyme can be inhibited by a class of herbicides called ACCase inhibitors. The herbicide compounds affect meristem of the grasses and kill them by ceasing production of cell membrane.

Step 2: Malonyl-CoA transacylase (MT) exchanges the CoA for the ACP (Acyl Carrier Protein) which is an essential protein co-factor in fatty acid biosynthesis.

Step 3: The 3-ketoacyl-ACP synthase (KAS) enzyme has three isoforms – (KAS) I, II and III. KAS III is utilized during the first condensation reaction, which includes a carbon-carbon bond formation between C1 of an acetate primer and C2 of the malonyl-ACP. KAS I is active with C4-C14 acyl-ACPs and during production of C6:0 to C16:0. KAS II accepts only longer carbon chains and elongation of 16:0-ACP to C18:0-ACP requires KAS II.

Step 4: The 3-ketoacyl-ACP reductase (KR) enzyme reduces the keto group from 3-ketoacyl-ACP to form 3-hydroxyacyl-ACP.

Step 5: The 3-hydroxyacyl-ACP dehydratase catalyzes the removal of water from 3-hydroxyacyl-ACP to form the 2,3-trans-enoyl-ACP and in the next step, enoyl-ACP reductase converts 2,3-trans-enoyl-ACP to its corresponding saturated acyl-ACP.

Step 6: Enoyl-ACP reductase (ER) reduces the double bond and the repetitive condensation of 2 carbon units produces 16:0=ACP and 18:0-ACP. Each cycle of fatty acid synthesis adds two carbons to the acyl chain and the reaction stops at 16:0 or 18:0 when thioesterase terminates the biosynthesis cycle. 18:1-ACP is produced using 16.18:0-ACP using a stearoyl-ACP desaturase as a catalyst. 16:1-Δ9 is desaturated by using a soluble acyl-ACP Δ9 desaturase enzyme. Fatty acid elongation happens in the endoplasmic reticulum.

Several desaturase enzymes catalyze the formation of cis-double bonds which causes kinks in the fatty acid chain (Figure 4) and generate a diverse variety of unsaturated fatty acids in plant membranes and storage reserves.

Figure4: Introduction of double bonds in plant lipids (Buchanan, 2000)

2.6 FACTORS AFFECTING THE FATTY ACID COMPOSITION IN VEGETABLE OILS

Fatty acid composition determines the physical and chemical characteristics of vegetable oils, and the fatty acid composition is not always constant but influenced by factors such as genetic, environmental, and oil extraction methods.

2.6.1 Genetic Factors

Fatty acid biosynthesis in plant cells is controlled by a group of genes identified as sad1, sad2, fad2a, fad2b, fad3a, and fad3b, which are collectively known as desaturase (Thambugala et al., 2013). The genes act by encoding enzymes that perform fatty acid synthesis in the plant cells.  Variation in fatty acid composition in the different vegetable oils could be due to the differential expression of the genes during seed development and maturation (Baud and Graham, 2006).

In peanut, sesame and sunflower seeds, it has been observed that increase in the oleic acid content leads to the reduction in the levels of palmitic, stearic, and linoleic acids, which suggests that there is close linkage of genes controlling these fatty acids (Barkley et al., 2013). Variations in fatty acid compositions among different crop varieties of the same species is due to the cumulative effect of several minor genes that modify the expression of fad3a and fad3b (Vrintel et al., 2005). Baud and Lepiniec (2009) reported that the genes expression programme related to the fatty acid synthesis are activated during the maturation phase of the seed, and most genes encoding fatty acid synthesis display a bell shaped pattern of expression during seed development. The fad2 genes are thought to be the rate limiting genes of the fatty acid biosynthesis pathway, and are highly influenced by environment (Fofana et al., 2006), and (Esteban et al., 2004) also reported that there is a significant gene environment interaction which determines the fatty acid composition differences.

2.6.2 Environmental Factors

Environmental growth conditions of plants such as temperature, soil, moisture, altitude, and light affects the expression of the alleles and relative activity of the genes responsible for fatty acid biosynthesis in plant cells. Therefore, vegetable oils obtained from plants seeds of the same species but from different localities may have different fatty acid compositions (Alizadeh, 2010).

2.6.2.1 Temperature

Varying temperatures during growth and maturation seasons of plants affects the level of gene expression which cause alteration in quantities of fatty acids of vegetable oil (Fofana et al., 2008). Byfield and Upchurch (2007) also confirmed that at high temperature during growth and maturation seasons of plant seeds, linoleic acid content reduces while oleic acid content increases because fatty acid desaturase enzymes, encoded by three genes converts linoleic acid to oleic acid, and hence altering the fatty acid composition in some seeds such as sunflower, soybeans, peanut, and sesame.

2.6.2.2 Soil

Soil contains nutrients such as nitrogen, potassium, and phosphorous which are used in the biosynthesis of fatty acids, and the concentration of these nutrients in the soils may influence the quality and composition of fatty acids in plant seeds (Stepien et al., 2017).

Kaptan et al. (2017) reported that the application of NPK fertilizers to the soils significantly increased the concentration of lignoceric and arachidic acids, whereas myristic and palmitic acids were decreased. On the other hand, fertilization had no significant change on unsaturated fatty acid content observed, except eicosenoic acid.

2.6.2.3 Water

Igbadun et al. (2006) showed that the crop yield response is very much dependent on the amount of water applied at different crop development stages than the overall seasonal water applied. Drought stress in the flowering stage of a plant leads to reduced availability of carbohydrates (glucosinolate) for fatty acid biosynthesis, and may cause reduction in the concentration of saturated fatty acids in the plant seeds (Geogel et al., 2007). Drought stress also leads to oxidation of polyunsaturated fatty acids in the plant seeds thus altering the fatty acid composition in plant seeds (Singh and Sinha, 2005).

2.7 FACTORS AFFECTING NUTRITIONAL QUALITY OF VEGETABLE OILS

There is still no standard defining the quality characteristics of vegetable oils for use as food and industrial applications, but there are a number of factors such as fatty acid compositions, oil extraction and processing methods, seed quality and storage systems that are used to evaluate the quality of a good vegetable oil (Okparanta et al., 2018; Abulude et al., 2007).

2.7.1 Fatty Acid Composition and Oil Oxidation

The quality of vegetable oils largely depends on the quantity of oleic and linoleic acids, which constitutes more than 80% of total fatty acids. Good quality vegetable oil contains high proportion of fatty acids like oleic, palmitic, and stearic acids which are resistant to autoxidation. The linoleic and linoleic acids are nutritionally important, but are susceptible to oxidation because their acyl residues are chemically reactive which adversely impacts oil stability and quality. Oxidative degradation of oil leads to loss of nutritional value, alteration of sensory properties like flavour, aroma, and colour. The degree of autoxidation and the potential for deterioration are important parameters of the vegetable oils. It has been reported that factors such as temperature, light, processing procedures, and oxygen concentration are the main causes of oxidation in vegetable oils (Kamal, 2006; Merrill et al., 2008).

2.7.2 Extraction and Processing Methods of Vegetable Oils

The traditional methods are crude, largely unscientific, inefficient, and yielding poor quality oil that contain substantial amount of contaminants.

The solvent extraction method is commonly applied to oilseeds with low oil content (< 20%), like soybean. This method has some disadvantages such as poor product quality caused by high processing temperatures, and a relatively high number of processing steps (Dawidowicz et al., 2008; Takadas and Doker, 2017).

Mechanical press methods are often used to extract vegetable oil from oilseeds having oil content higher than 20% (Sinha et al., 2015). There are two types of mechanical press method namely, cold-press and hot-press methods.

Cold-press or scarification method is carried out at low temperature below 50⁰C. Cold-pressed method are safer than hot-pressed method because high temperature is avoided in the cold press method. In cold-pressed oils, the purity and natural properties of oils are preserved (Azadmard- Damirchi et al., 2011; Bhatol, 2013).

The hot-press method is carried out at elevated temperature and pressure. This method results in decreased oxidative stability, degradation of valuable oil components and reduced oil shelf-life of the vegetable oil.

2.7.3 Refining and Storage

Inadequate handling and storage facilities for oilseeds and vegetable oils during refining and transportation may lead to contamination of oil products by soil, insects, rodents or microorganisms, and may cause loss or gain of moisture, odor or flavour (Ngassapa and Othman, 2001). The longer the seeds are stored, the higher the contamination and deterioration, and the higher the free fatty acid contents of the oil.

Oil refining processes may lead to contamination of vegetable oils with heavy metals which accelerates oxidation reaction.  Oil refining removes antioxidants which leads to acceleration of oxidation reaction that reduces shelf-life. Addition of synthetic antioxidants such as BHT, BHA and BTHQ may also lower oil quality. To retain oil quality, care must be taken while storing vegetable oils for a period of time to prevent their deformation as they easily undergo oxidative deterioration, hence shortening their shelf life.

Bukola et al. (2015) reported that oil stored in a refrigerator has a greater nutritional quality than that of a cupboard and shelf upon long storage because the peroxide and acid values obtained from such oils are lower than the other means of storage.

2.7.4 Pro-oxidants

Pro-oxidants in oil have a detrimental effect on oil stability. Metals act as pro-oxidants by electron transfer whereby they liberate radicals from fatty acids or hydroperoxides as in the following reactions (Gordon, 1990):

M^(n+l)+ + RH              Mⁿ⁺ + H⁺ R·

ROOH + Mn+                      RO· + OH- + M^(n+l)+

ROOH + M^(n+l)+              ROO· + H+ + Mⁿ⁺

Two of the more active metals to induce oxidation are copper and iron of which copper is the most pro-oxidative (Garrido, Frias, Diaz and Hardisson, 1994).

2.8 VEGETABLE OIL EXTRACTION METHODS

Most vegetable oils are extracted from plant seeds, and the oil yield will depend on seed variety, soil, and environmental conditions around the oil-bearing plant, as well as on pretreatment procedure, and on the particular extraction method (s) used (Mariana et al., 2013). Selection of suitable extraction method(s) is a key factor, and may depend on whether small or large scale oil extraction is intended. Vegetable oil producers widely use methods like traditional, solvent extraction, and mechanical expression to extract vegetable oils from plant seeds. The percentage yield and percentage oil recovery are evaluated using the expressions;

% Oil yield (1),

% Oil recovery =  (2),

Where Moil = mass of oil (kg),

Mseed= mass of oilseed (kg),

X = oil content of oil seed.

2.8.1 Traditional Method

Oluwole et al. (2012) reported that the traditional method of vegetable oil extraction involves, collection of seed pods, shelling of the pods/winnowing, roasting/drying of seeds to reduce moisture, grinding the roasted seeds by mortar and pestle or in between two stones to form a paste. The paste is mixed with water, and boiled to obtain the oil by floating and skimming. The oil is then dried by further heating.

The traditional method is tedious, time consuming, energy sapping, largely unscientific, inefficient, and yielding poor quality oil (Olaniyan and Yusuf, 2012). This method is mainly practiced among the rural communities to obtain vegetable oils on a small scale from peanut and sesame seeds, and also sheanut.

2.8.2 Solvent Extraction Method

In soxhlet extraction, normally a solid material containing the desired compound is placed inside a thimble made from thick filter paper, which is loaded into the main chamber of the Soxhlet extractor. The soxhlet extractor is placed onto a flask containing the extraction solvent-hexane and is then equipped with a condenser, and the solvent is heated to reflux. The solvent vapour travels up a distillation arm and floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the solid material.

The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound will then dissolve in the warm solvent. When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle may be allowed to repeat many times, over hours or days.

During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles, the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled.

After extraction, the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. The non-soluble portion of the extracted solid remains in the thimble and is usually discarded.

The solvent extraction method is commonly applied to plant seeds with low oil content (<20%). This method is considered as one of the most efficient methods in vegetable oil extraction, with less residual oil left in the cake or meal (Tayde et al., 2011). The choice of solvent is based mainly on the maximum leaching characteristics of the desired solute substrate (Dutta et al., 2015). Solvents commonly used are hexane, diethyl ether, petroleum ether, and ethanol. Other considerations are high solvent-solute ratio, relative volatility of solvent to oil, oil viscosity and polarity, and cost (Muzenda et al., 2012; Takadas and Doker, 2017). Hexane a hydrocarbon, with chemical formula C₆H_14, boiling and melting points 68.7°C and -95.3°C respectively, has become the solvent of choice for solvent extraction (liquid-liquid extraction) because of its high stability, low evaporation loss, low corrosiveness, little greasy residue, and better odor and flavour of the extracted products (Muzenda et al., 2012). Solvent extraction using hexane has several drawbacks, including high capital equipment cost and operational expenditures, the perpetual hazard of fire and/or explosion, as well as the residual solvent associated with both oil and meal.

The primary prerequisite for solvent extraction of oils is the rupturing of the seed or feed material to render the cell wall more porous, and complete rupturing of the cell wall is necessary for rapid extraction. Soxhlet based solvent extraction process is the primary means of extracting vegetable oil from oleaginous materials. The Soxhlet process is also widely used in laboratory scale oil extraction (Abdelaziz et al., 2014), but large scale operation of this process would require a commercial solvent extractor (Ogunniyi, 2006). The major advantage of the Soxhlet process is solvent recycling (over and over) during extraction. However, disadvantages of the Soxhlet method include high solvent requirement, time, and energy consumption (Takadas and Doker, 2017), as well as sample being diluted in large volumes of solvent (Rassem et al., 2016).

2.8.3 Mechanical Expression Method

Mechanical expression involves the application of pressure using hydraulic or screw presses to force oil out of an oil-bearing material (Arisanu, 2013). Mechanical press methods are often used to extract vegetable oil from plant seeds having oil content higher than 20% (Sinha et al., 2015). There are two types of mechanical press methods namely, cold-press and hot-press methods. Cold-press or scarification method is carried out at low temperature (below 50^oC). In cold-pressed oils, the purity and natural properties of seed oils are preserved (Azadmard- Damirchi et al., 2011; Bhatol, 2013), and this includes the retention of valuable nutraceuticals like phytosterols and tocopherols in the extracted oil (Kittiphoom et al., 2015). Because of these attractive qualities, there is growing global demand for cold-pressed oils. Cold-pressed oils are safer than hot-pressed seed oils because adverse effects caused by high temperatures are avoided. Some of the likely adverse effects of hot pressed oils are decreased oxidative stability, degradation of valuable oil components, and reduced oil keeping quality (Matthaus, and Brühl, 2003).

The hot-press method is carried out at elevated temperature and pressure and results into higher oil yield largely due to decreased oil viscosity at high temperatures. This enhances oil flow during extraction, thus high temperature increases the efficiency of the extraction process and oil yields of up to 80% possible (Patel et al., 2016).

Mechanical expression methods have the advantage of low operation cost and producing high quality light colored oil with low concentration of free fatty acids. However, it has a relatively low yield compared to solvent extraction, and is therefore comparatively inefficient, often with a large portion of oil left in the cake or meal after extraction (Bhuiya et al., 2015).

2.9 ANALYTICAL METHODS OF FATTY ACIDS AND HEAVY METALS

Worldwide, the most commonly employed techniques for analysis of fatty acid profiles of vegetable oils are gas chromatography (GC) and high performance liquid chromatograph (HPLC) (Botinestean et al., 2012).

HPLC is the most commonly used tool for the analysis of pharmaceutical products, but it is less successful in the quantification of fatty acids due to the absence of chromophores or fluorescent functional groups. Compared with the standard GC method, this method achieves acceptable separation and precision, but has poor sensitivity (Tsuyama et al., 1992).

GC is the most commonly used technology for the analysis of fatty acids which require derivatization to fatty acid methyl esters (FAME) due to the high boiling points of fatty acids, which are difficult to evaporate (Laakso et al., 2002).

2.9.1 Principles of Gas Chromatography

Chromatography is a separation technique based on the affinity difference between two phases, the stationary and mobile phases. A sample is injected into a column, either packed or coated with the stationary phase, and separated by the mobile phase based on the difference in interaction (distribution or adsorption) between compounds, and the stationary phase. Compounds with a low affinity for the stationary phase move faster through the column, and elute earlier. The compounds that elute from the end of the column are determined by a suitable detector. The mobile phase is always composed of an inert gas such as Nitrogen, Hydrogen or Helium which does not interact with the components in a mixture, but carries or transports them through the column, and the stationary phase is a “solid” which is either a packed column or a capillary column ( Al-Bukhaiti et al., 2017).

This technique enables the separation, identification, and purification of components of a mixture for qualitative and quantitative analysis. However, the use of Gas Chromatography is limited to volatile and thermally stable compounds or molecules that may undergo derivatization reactions to form volatile and thermally stable products (Lehotay and Hajslova, 2002).

During a Gas Chromatography separation process, the sample is vaporized and carried by the mobile gas phase (i.e., the carrier gas) through the column. The quality of separation (resolution) depends on how long the components to be separated stay in the stationary phase, and on how often they interact with this phase. The type of interaction between component and phase (selectivity) is determined by the functional groups. The polarity of the phase is a function of stationary phase substituents (Grob and Eugene, 2004).

To measure a sample with unknown concentration, a standard sample with a known concentration is injected into the Gas Chromatography. The standard sample peak retention time and area are compared to the test sample to calculate the concentration.

Major components of GC include; –

Gas supply – involves two types of gases, carrier gas to carry the sample through the column, and a detector support gas to support the flame in the flame ionization detector.

Introduction of sample – can be by hand or auto sampler. The most common type of sample introduction involves syringe injection of the sample into a heated injection port.

Column – separation of components of the sample takes place in the heated column, a long tube running from the sample injector to the detector.

Detector – detector function involves detection of sample components as they pass through the end of the column. The most common detector is a Flame Ionization Detector (FID), in which molecules of the sample are burned in the flame with produced ions detected.

A computer system – is used by most GC systems to collect and analyze the data. Signals from the detector are collected and converted into user friendly information. Generally, the system generates a chromatogram which is plotted using information on the amount of the component as peak area with elution time as retention time. Contents and concentration of various components of the sample are determined by comparing various chromatograms.

Chromatography/Flame Ionization Detector (GC/FID) is widely used analytical technique in quantitative analysis of fatty acids in vegetable oils, petrochemical, pharmaceuticals, and natural gas. The FID detector typically uses hydrogen/air flame into which the separated sample is oxidized and produce electrically charged particles (ions) which are collected and produce an electrical signal which is then measured. The GC/FID technique is robust, and of low cost compared to the Mass Spectrometry. This system has a challenge because the FID is extremely sensitive to hydrocarbon impurities from the hydrogen and air supply for the flame. The impurities can cause increased baseline noise and reduce the detector sensitivity.

Gas Chromatography/Mass Spectrometry (GC/MS) is a hyphenated technique developed from the coupling of GC and MS. The two instruments are highly compatible with each other, however, GC operates at high pressure (760 torrs) while the MS operates at a vacuum (5-l0torr). During the identification of compounds in the MS, the mass spectra acquired by this hyphenated technique offer more structure related information on the interpretation of fragments of the ions. The fragments ions with different relative abundances can be compared with the library spectra. Nowadays, GC/MS is integrated with various online MS databases for several reference compounds with search capabilities that could be useful for spectra match for the identification of separated components. Compounds that are adequately volatile, small, and thermostable in GC conditions can be easily analyzed by GC/MS. Sometimes polar compounds, with a number of hydroxyl groups, need to be derivatized prior to any analysis. The most common derivatization technique is the conversion of the analyte to its trimethylsilyl derivative. It is suitable for analysis of both volatile and nonpolar compounds.

GC/MS technique is limited in that, the compounds analyzed must be sufficiently volatile to allow transfer from liquid phase to mobile carrier gas and thus to elute from analytical column to the detector. Hence many compounds are too polar or too large to be analyzed with this technique. GC/MS has positive attributes in that it offers high efficiency separation with numerous columns, excellent limit of quantification, and also allows use of mass spectral library for identification of samples. During GC/MS analysis, vaporized sample is carried through the GC column with the help of heated carrier gas through the column where the components are separated. The separated components then enter MS through an interphase, and this is followed by ionization, mass analysis, and detection of mass-to-charge ratios of ions generated from each component by the mass spectrometer. The process of ionization not only ionizes the molecule, but also break the molecule into the positive or negative modes (Ruchira et al., 2012).

2.9.1.1 Selection of the Chromatographic Column

An appropriately selected column can produce a good chromatographic separation which provides an accurate and reliable analysis. An improperly used column can often generate confusion, inadequate, and poor separations which can lead to results that are invalid or complex to interpret. There are over 10,000 compounds that can be analyzed by Gas Chromatography, over 400 Gas Chromatography capillary columns (Jennings, 1990). The selection of proper capillary column for any application should be based on four significant factors which are stationary phase, column internal diameter, film thickness, and column length.

The differences in the chemical and physical properties of injected organic compounds, and their interactions with stationary phase are the basis of separation process. When strength of the analyte-phase interactions differs significantly for two compounds, one is retained longer than the other. How long they are retained in the column (retention time) is a measure of these analyte-phase interactions. Two compounds that co-elute (do not separate) on a particular stationary phase might separate on another phase of different physical and chemical property (Kupiec, 2004).

As a general rule, it is advisable to use similar polarities for phase and target compounds, for example, nonpolar molecules require nonpolar polysiloxane phases in the column. For FAME analysis, for example a capillary column with DB-23, 50% Cyanopropyl 50% methylpolysiloxane or HP-88, 88% Cyanopropyl 12% arylpolysiloxane can be used as a stationary phase because they are polar (Rahman et al., 2015).

2.9.1.2 Fatty Acids Methyl Ester Preparation (Derivatization)

Methyl esters are the favorite derivatives for Gas Chromatography analysis of fatty acids because they are more volatile than fatty acids (Christie, 1998). Derivatization is a process of converting fatty acids into fatty acid methyl esters, and this can be carried out through two possible approaches;

1. By a two-steps reaction involving saponification followed by esterification.
2. By a single step reaction known as transesterification.

The two main chemical reactions that occur during methylation are hydrolysis and esterification.

2.9.1.3 Hydrolysis of Fatty Acids

Hydrolysis results in a mixture of fatty acids and glycerol from triglycerides as indicated by the reaction in figure 5. Where R is a linear carbon chain.

Figure 5:  Hydrolysis of fatty acids (Sigma, 2008)

2.9.1.4 Esterification (Methylation) of Fatty Acids

The esterification of fatty acids to fatty acid methyl esters is performed using an alkylation derivatization reagent. The esterification reaction involves the condensation of the carboxyl group of an acid, and the hydroxyl group of an alcohol in the presence of a catalyst. The catalyst protonates an oxygen atom of the carboxyl group making the acid much more reactive. An alcohol then combines with the protonated acid to yield an ester with the loss of water. The catalyst is removed with the water. Esterification is illustrated in reactions (2) and (3). The alcohol that is used determines the alkyl chain length of the resulting esters, for example, the use of methanol will result in the formation of methyl esters, whereas the use of ethanol will result in ethyl esters.

Figure 6: Methylation of fatty acids (Sigma, 2008)

2.9.1.5 Methylation Methods

2.9.1.5.1 Base Catalyzed and Acid Catalyzed (Two-Step) Method

In this procedure, a known amount of oil sample is placed into centrifuge tubes to which potassium hydroxide (10M) solution, and methanol are added. The reaction is performed at 55°C for 1.5h with mixing for 5s every 20min. After cooling to room temperature, 0.5mL sulphuric acid (10M) solution is added, and the reaction is continued at 55°C for 1.5h with mixing for 5s every 20min. After cooling to room temperature, n-hexane is added, and the mixture centrifuged for 5min. The hexane phase from the top layer of the solution is extracted, and transferred to screw cap for Gas Chromatography (GC) analysis (Christie, 1998).

2.9.1.5.2 Borontrifluoride/Methanol (BF₃/CH₃OH)

An aliquot of lipid extract is mixed with Borontrifluoride –methanol mixture and heated to a maximum temperature of 100⁰C for 2–90 min depending on the type of lipid. After cooling to ambient temperature, water and a non-polar solvent are added, vortexed, and the two phases separated by centrifugation. The upper organic phase containing the methyl esters is carefully removed to a new vial where it is removed under a stream of nitrogen gas (N₂). The remaining residue containing fatty acid methyl esters is dissolved in n-hexane prior to GC analysis.

2.9.1.5.3 Sodium Methoxide/Methanol (NaOCH₃/CH₃OH)

Aliquot (20–40mg) is dissolved in dry toluene, 2ml 0.5M sodium methoxide (metallic sodium in anhydrous methanol) added and the samples heated at 60 ^oC for 20 min. This esterification step can also be carried out at room temperature (20⁰C). A few drops of glacial acetic acid are added followed by 2ml saturated NaCl solution to eliminate excess methoxide. With 50ppm butylated hydroxy toluene (BHT) 2 ml n-hexane are added, and vortexed vigorously. The upper organic phase containing the extracted methyl esters is taken, dried over anhydrous sodium sulphate or 100 μl of a water scavenger such as 2, 2 dimethoxypropane added, and dried under a gentle stream of N₂ at 40 ⁰C. The fatty acid methyl esters are resuspended in 2 ml of n-hexane with 50ppm BHT and injected for GC analysis. Dilution of the sample in n-hexane may be necessary, depending on the GC response.

2.9.1.5.4 Potassium hydroxide/Methanol (KOH/CH₃OH)

The oil sample is dissolved in n-hexane and KOH methanolic reagent added and the samples heated at 50⁰C for 10–15min. A few drops of glacial acetic acid are added followed by water and n-hexane. The solution is vortexed vigorously and the upper organic phase taken and dried over anhydrous sodium sulphate prior to injection for GC analysis.

2.9.1.5.5 Sulphuric Acid/Methanol (H₂SO₄/CH₃OH)

An aliquot is added to a 0.1M H₂SO₄ in methanol and the tubes heated to 100 ^oC for 30–60min. Sodium bicarbonate solution is added to neutralize the reagent and n-hexane to extract the FAMEs. The organic layer is carefully removed, then dried over anhydrous sodium sulphate. Successive n-hexane extracts are added and evaporated under a stream of N₂. The residue is re-dissolved in n-hexane for GC analysis. (Wang et al., 2014).

2.10 ANALYTICAL INSTRUMENTS USED IN FATTY ACID ANALYSIS.

GC is extensively used in food analysis for routine qualitative and/or quantitative determination of components such as fatty acids, sterols, alcohols, oils, and low mass carbohydrates. Further applications in food analysis are devoted to the detection and quantification of food contaminants such as pesticides, environmental pollutants, natural toxins, veterinary drugs, and packaging materials. GC is reliable, efficient, and cost effective method of separation which requires small amount of sample, and also provides fast and highly accurate quantitative analysis.

2.11 THE PHYSICOCHEMICAL PROPERTIES OF VEGETABLE OILS

Physical and chemical properties of vegetable oil is determined in order to evaluate the nutritional quality and stability of the vegetable oils. These properties include viscosity, density, acid value, saponification value, iodine value, and peroxide value.

2.11.1 Viscosity

Oil viscosity is defined in two ways, either based on its absolute viscosity or its kinematic viscosity. The absolute viscosity is the resistance of oil to flow and shear due to internal friction, and it is measured in SI unit of Pa*s. Other units include (1poise = 100cP = 1 g*cm-¹*s-¹ = 0.1Pa*s).

The kinematic viscosity is the resistance of oil to flow and shear due to gravity, and it is measured in SI unit of m²/s. kinematic viscosity of oil can be obtained by dividing the oil absolute viscosity with its corresponding density (Singh and Heldman, 2001).

Viscosity of oil is used to determine the degree of unsaturation and molecular weight of the fatty acid. Viscosity increases with molecular weight of fatty acids and decreases with unsaturation (Kumar et al., 2010).

Viscosity is an important factor in selecting a good vegetable oil for food frying. The higher viscosity of frying oils the greater content of oil in the fried foods (Dana and Saguy, 2006), and this is because, high viscosity allows the oils to be accumulated more easily on the surface of fried foods, and enter inside the food during the cooling period (Maskan, 2003).

It has been well established that temperature has a strong influence on viscosity of vegetable oils, with viscosity generally decreasing with increase in temperature.  The absolute viscosity of oil is an important parameter in determining quality of oils with regard to its fatty acid composition.

Yalcin et al. (2012) reported the viscosities of different vegetable oils from Turkey as olive (61.2mPa*s), hazelnut (59.7mPa*s), cottonseed (57.3mPa*s), canola (53.5 mPa*s), soybean (48.7mPa*s) and sunflower (48.2mPa*s) respectively.

The Principles of Viscosity Measurement is based on Ostwald’s viscometer, also known as U-tube viscometer or capillary viscometer, which is a device used to measure viscosity of a liquid with a known density. The method of determining viscosity with this instrument consist of measuring the time for a known volume of the liquid to flow through the capillary under influence of gravity. The instrument must first be calibrated with material of known density such as pure (deionized) water. Knowing the value of viscosity of one liquid, viscosity of other liquids can then be calculated.

=

η₁ = is viscosity of liquid 1. η₂ = is viscosity of liquid 2.

t₁ = flowtime of liquid 1.  𝑡₂ = 𝑓𝑙𝑜𝑤𝑡𝑖𝑚𝑒 𝑜𝑓 𝑙𝑖𝑞𝑢𝑖𝑑 2.

d₁ = density of liquid 1.  𝑑₂= 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑙𝑖𝑞𝑢𝑖𝑑 2.

Figure 7:  Ostwald’s Viscometer (Generali, 2018)

2.11.2 Density

Density of oil is defined as mass per unit volume at specified pressure and temperature.  The oil relative density or specific gravity of oil is defined as the ratio of the density of oil to that of water at the same specified pressure and temperature. Density of vegetable oils is temperature dependent, and decreases in value when temperature increases. The range is from 0.91 to 0.93g/cm³ between the temperature of 15^oC and 25^oC.

Ackman and Eaton (1977) indicated that a different proportion of fatty acids could be a major factor for the increase in density of vegetable oil. For example, the high density of soybean oil 0.9193g/cm³ reported was attributed to the higher content of linoleic acid (Ackman and Eaton, 1977).

Density is an important factor which influences oil absorption as it affects the drainage rate after frying, and also the mass transfer rate during the cooling stage of frying. The codex standard for density of vegetable oils range between 0.919 to 0.925gm/cm³ (Stahidi, 2005).

2.11.3 Acid Value (AV)

Acid value represents the mass of potassium hydroxide (KOH) in milligrams required to neutralize the free fatty acid in 1g of oil sample. Acid value (AV) is a good indicator of oil degradation caused by hydrolysis or enzymes (Othman and Ngassapa, 2010). Acid value increases with days of storage under ambient conditions. Acid value of oils determines the purity of oils.

The maximum recommended acid value for all vegetable oils is 4mgKOH/g of oil (FAO/WHO, 2019). A low acid value indicates the oil is stable and pure for human consumption (Orthoefer, 2007).

2.11.4 Saponification Value (SV)

Saponification value represents the number of milligrams of potassium hydroxide (KOH) required to saponify (hydrolyze) one (1g) of fat under the conditions specified. Saponification value is used as a measure of the average molecular weight (Chain Length) of all fatty acids present in the vegetable oil sample (Mohammed and Alli, 2015; Fazal et al., 2015).

The long chain fatty acids found in fats have a low saponification value, while the short chain fatty acids have a high saponification value (Musa et al., 2012). The recommended saponification values for different vegetable oils range between 187-196mgKOH/g for peanut, 185-193mgKOH/g for sesame, 188-194mgKOH/g for sunflower (FAO/WHO, 1999). Ezeagu et al. (1998) reported that a saponification value of 200mgKOH/g indicates a high proportion of short chain fatty acid of low molecular weight in a vegetable oil, and this property makes the vegetable oil potential for use in soap making, cosmetic industry, and sources of essential fatty acids in the body.

2.11.5 Peroxide Value (PV)

The peroxide value is defined as the weight of active oxygen contained in one gram of oil or fat (Horwitz, 1975), and It determines the degree of oxidation of oil which is an indication of the level of deterioration of oils and fats (Okechalu et al., 2011). A freshly refined oil should have nil peroxide value.

WHO/FAO (2019) recommended maximum peroxide value of 10meq/kg for all vegetable oils.  A high peroxide value indicates high level of oxidative rancidity of the oil which may be caused by increase in storage time, temperature, and air contact with the oil (Kamau and Nanua, 2008). Oils exposed to both atmospheric oxygen and light may show a much larger increase in peroxide value during storage.

Low peroxidative value are indicative of low levels of oxidative rancidity of the oils and also suggest strong presence or high levels of antioxidant (Adelaja, 2006).  Low peroxide value may also be used as a measure of shelf-life and freshness of the oil.

2.11.6 Iodine Value (IV)

Iodine value is the mass of iodine in grams that is required to react with 100g of fat or oil sample. The iodine value is used as a measurement of the total unsaturation of vegetable oils, and also as an indicator of their susceptibility to oxidation (Knothe, 2006). Vegetable oils can be divided into four major categories depending on their iodine values namely, saturated oils (iodine value between 5 and 50), mono-unsaturated oils (50 and 100), di-unsaturated oils (100 and 150), and tri-unsaturated oils (over150).

The higher the iodine value, the greater degree of unsaturation, and the more the oil becomes susceptible to oxidation (Onyeike, 2003). Vegetable oils with low iodine values have a high proportion of saturated fatty acids which are stable to oxidative degradation. The recommended iodine value for different vegetable oils range between 7.7 – 107 mg of I₂/100 g for peanut oil, 105 – 120mg of I₂/100 g for sesame oil, and 110 – 143mg of I₂/100 g for sunflower oil respectively.

2.12 HEAVY METALS IN VEGETABLE OILS

Heavy metals are defined as metallic elements that have a relatively high density compared to water (Ferguson, 1990). Heavy metals can be classified as potentially toxic metals such as Arsenic (As), cadmium (Cd), and lead (Pb) which can be very harmful even at a low concentration when ingested over a long time period (Unak et al., 2007).

Micro essential elements such as iron (Fe), copper (Cu), and zinc (Zn) are required in small quantities for various biochemical and physiological functions. The essential elements can also produce toxic effects when the metal intake is excessively elevated (Gopalani et al., 2007).

2.12.1 Sources of Heavy Metals in Vegetable Oils

All heavy metals are naturally occurring elements that are found throughout the earth’s crust. Anthropogenic activities such as mining and smelting operations, industrial production and use, domestic and agricultural use of metals, and metal-containing compounds cause environmental contamination by heavy metals (He, 2005; Shallari et al., 1998). Environmental contamination can also occur through metal corrosion, atmospheric deposition, soil erosion of metal ions, and leaching of heavy metal ions, sediment re-suspension and metal evaporation from water resources to soil and ground water (Nriagu, 1989). Natural phenomena such as weathering and volcanic eruptions have also been reported to significantly contribute to heavy metal pollution (Ferguson, 1990; Bradl, 2002; He, 2005).

The presence of heavy metals and their chemical forms in vegetable oils depend on several factors. The metals might originate from the soil and fertilizers, and be absorbed by the plant roots. Heavy metals might be introduced during the production processes such as bleaching, hardening, refining, and deodorization. The metals may also be introduced into the vegetable oils by contamination through metal processing equipments (Leonardis et al., 2000).

For many years, it has been discovered that vegetable oil may contain heavy metals such as iron, nickel, lead, cadmium, copper, and Arsenic (Mendil et al, 2009). Various studies on vegetable oils consumed in different countries such as India, China, Nigeria, and others reported presence of heavy metals such as lead, cadmium, arsenic, nickel in vegetable oils sold in markets (Juszczak 2008).

Cadmium: This toxic element is easily transferred from sosil to plants, which are increasingly contaminated by cadmium from phosphate-based fertilizers. Cadmium can also be present in vegetable oils, as a result of contamination through refining process, the storage tank or the packing material such as a colorant or stabilizer in plastics (Mendil et al., 2009).

Copper: Copper is known to be both vital and toxic for many biological systems, and may enter the food materials from soil through mineralization by crops, food processing and environmental contamination such as application of copper based pesticides which are in common use in some countries (Onianwa et al., 2001; Koc et al., 2008).

Zinc: The presence of zinc in vegetable oil could be due to absorption by plants from soil or contamination of the vegetable oils during refining process, storage and packaging materials). According to WHO (2003) the Maximum Permissible Limit (MPL) of zinc in vegetable oils is 10000µg/g or mg/g.

Reported zinc levels in different vegetable oils from other parts of the world are in the ranges of 0.04-0.70 µg/g and 0.0484-0.2870 mg/kg (Garrido et al., 1994; Pehlivan et al., 2008).

Iron: Iron may enter vegetable oils from the soil through uptake of minerals by the roots, and also the reaction between the relatively high-unsaturated portion of the oil with the surface of iron containers used during transportation, storage, and processing of vegetable oils.

Lead: research study shows that the presence of lead in vegetable oil could be attributed to deposition or bioaccumulation from soil containing phosphate base fertilizer that were used in the plantation or may be due to water used for irrigation (Ansari et al., 2009). Other reports also reveal that contamination of vegetable oil with lead may also be due to industrial emission, combustion of fuel in refinery process, and from packaging material such as stabilizer and colorant in plastic (La Pera et al., 2004).

2.12.2 Effects of Heavy Metals on Human Health

Various studies around the world have confirmed the presence of heavy metals such as cadmium, lead, copper, iron, nickel, zinc, chromium, cobalt in vegetable oils, and reports indicate that toxic elements such as cadmium and lead can be very harmful even at low concentration when ingested over a long time period (Unak et al., 2007). The essential metals like zinc, copper, iron, and nickel can also produce toxic effects when the metal intake is excessively elevated (Gopalani et al., 2007).

Zinc: Zinc is an essential functional and structural element in most metabolic pathways in humans, and is regarded as cofactor for many enzymes. However, excessive levels of zinc in the body harms some physiological processes like breathing. Zinc deficiency can lead to growth retardation and immunological abnormalities (Tahsin and Yankov, 2007). According to WHO (2003) the Maximum Permissible Limit (MPL) of zinc in vegetable oils is 10000μg/g.

Cadmium: Cadmium is a highly toxic heavy metal with very low absorption levels in humans (3-5%) after exposure with contaminated foodstuffs. Cadmium accumulates in the human body and damages mainly the kidneys and liver. Cadmium is retained in the human body specifically in liver and kidney with a long biological half-life (10-30yrs). Joint expert committee on food additives of FAO and WHO offers the authorized index of provisional tolerable weekly intake for cadmium as 7μg/kg per body weight.

Iron: Iron deficiency is frequently associated with anemia, and thus reduces working capacity and impairs intellectual development (Schumann et al., 2007). It is known that adequate iron in diet is very important for decreasing the incidence of anemia. WHO (2003) established a Maximum Permissible Limit (MPL) for iron as 1.5mg/kg of body weight.

Lead: Lead is similar to cadmium because it has no beneficial role in human metabolism, and mainly producing progressive toxicity. The presence of lead in the body can lead to toxic effects, regardless of exposure pathway. Some researchers have suggested that lead continues to contribute significantly to socio-behavioral problems such as juvenile delinquency and violent crime (Needleman et al., 2002). WHO (1993) has established a provisional tolerable weekly intake (PTWI) for lead of 0.025mg/kg of body weight.

2.12.4 Determination of Heavy Metals in Vegetable Oils

2.12.4.1 Atomic Absorption Spectrometry (AAS)

Atomic absorption spectrometry (AAS), is an analytical technique that is used to determine the concentration of metals in samples. It is based on the principle that an atom in the ground state absorbs the light of wavelengths that are characteristic to each element when light is passed through the atoms in the vapour state, and since this absorption of light depends on the concentration of atoms in the vapour, the concentration of the target element in the sample is determined from the measured absorbance. The Beer-Lambert law describes the relationship between concentration and absorbance. Absorbance is directly proportional to concentration of the sample.

A = ɛbc,

Where A = absorbance (no unit),

ɛ = molar absorptivity constant (Lmol^-1cm^-1),

b = Path length of the sample (cm),

c = Concentration of sample in solution (molL^-1).

Analyzing a sample to see if it contains a particular element means using light from that element. For example, with lead, a lamp containing lead emits light from excited lead atoms that produce the right mix of wavelengths to be absorbed by any lead atoms. Some of the radiation is absorbed by the lead atoms in the sample, and the greater the number of atoms in the vapour, the more radiation is absorbed.

The amount of light absorbed is proportional to the number of lead atoms. A calibration curve is constructed by running several samples of known lead concentration under the same conditions as the unknown.

The amount the standard absorbs is compared with the calibration curve and this enables the calculation of the lead concentration in the unknown sample. Consequently, an atomic absorption spectrometer needs the following three components: a light source; a sample cell to produce gaseous atoms; and a means of measuring the specific light absorbed.

2.12.4.1.1 Flame Atomic Absorption Spectrometry (FAAS)

In flame atomic absorption spectrometry, a sample is aspirated into a flame and atomized. A light beam from a hollow cathode lamp of the same element as the target metal is radiated through the flame, and the amount of absorbed light is measured by the detector. This method is much more sensitive than other methods, and free from spectral or radiation interference by co-existing elements. Pretreatment is either unnecessary or straightforward. However, it is not suitable for simultaneous analysis of many elements, because the light source is different for each target element.