Jock, A. A.
Jock, A. A.
Zang, C. U.1, Jock, A. A. 1*, Garba, I. H.2, Chindo, I. Y.2,
1Chemical & Petroleum Techniques, Department of Science Laboratory Technology, University of Jos-Nigeria
2Department of Chemistry, Abubakar Tafawa Balewa University, Bauchi-Nigeria
Wagner L Priamo(email@example.com)
Jock Asanja Alexander, Physicochemical and Phytochemical Characterization of Seed Kernel oil From Desert Date (Balanites Aegyptica)(2017)Journal of Chemical Engineering And Bioanalytical Chemistry 2(1)
The seed oil of Balanites aegyptiaca was extracted and its chemical, physical and phytochemical properties which accounts for the oil quality parameters were investigated. The elemental composition of the oil revealed highest metal concentration of sodium (5.9178±0.2 mg/g) in the seed kernel, while magnesium (2.2242±0.007 mg/g) and calcium (1.4643±0.5) in the seed oil. The chemical parameters evaluated include saponification value (200.02±0.12 mgKOH/g), acid value (2.14±0.28 (mgKOH/g), iodine value (104.39±0.00 100/g), peroxide value (2.95±0.00 (mEq/kg) and free fatty acid (0.82±0.01%).. The physical parameters determined were oil content, specific gravity, refractive index and moisture content. These were found to be 45.32±0.0026%, 0.90±0.03, 1.45 and 0.114±0.04%, respectively. The oil quality parameters indicate that the oil is of edible quality with highest %FFA content of 0.84 % and considered non-drying oil. The fatty acids composition of the oil was evaluated using GC-MS as FAME, the oil contains about 47.52 % unsaturated fatty acids. Phytochemical screening of the oil showed the presence of alkaloids, steroids, cardiac glycosides and carbohydrates. The seed kernel of Balanites Aegyptica is a good source of vegetable oil.
Balanites Aegyptica has multiple applications and almost every part of the plant is useful such as its flowers, leaves, bark, root and fruit. The tree is actually valued more for its fruits and seeds. The fruits and kernel are widely used in many countries including Nigeria especially during the dry season and drought periods (Lockett et al., 2000). Balanites aegyptica is used for firewood, charcoal, poles, timber, utensils, tool handle, food, fodder (Elseed et al., 2002), mulch, shade, windbreak and gum (Guinand et al., 2001). The plant may be grown for its fiber, oil and/or for medicinal values. It is also used in treatment of several diseases and disorders since ages.
Oils are heterogeneous biochemical substances which have in common, the property of being soluble in most organic polar solvents (chloroform, benzene, diethyl ether, etc) and insoluble in water (Author, 1995). Oils may be sourced from animal, vegetables or petrochemicals (Oxford, 2005). Oils are often divided into three categories according to their qualities and these categories are non-drying oils, semi drying oils and drying oils (Gunstone, 2002). Non-drying oils are slow to become oxidized, they remain liquid for a long time, and this quality makes them particularly useful as lubricants and as a fuel for lamps. Drying oils are quite quick to become oxidized and turn to solid after their oxidation. Thus, they are often used in paints and varnishes. A good example of a drying oil is linseed oil. Semi-drying oils have qualities intermediate between non-drying oils and drying oils (Mohammad and Syed, 2005).
Based on the source, there are two types of oils, organic oils and mineral oils. Organic oils are produced by plants, animals and other organisms through natural metabolic processes. Organic oils which are generally laid down by plants and animals are called triglycerides and are mainly energy reserves (Albert et al., 2002). Mineral oil is a type of oil which includes crude oil or petroleum and its refined components collectively termed petrochemicals (DiNardo, 2005).
The oil that is considered in this research is organic oil, a triglyceride extracted from a plant source (seed kernel of Balanites aegyptica) and is generally referred to as vegetable oil. These oils have been part of the human culture for millennia (McCullough, 2006). Vegetable oils apart from meeting the dietary needs of man, are also widely used in bioresources and biodiesel in recent times.
Balanites Aegyptica seed kernel is readily available in northern Nigeria and the seed which is obtained after it is being sucked is seen as a nuisance along markets and settlements in communities. This is because no suitable application for the seed kernel has been found in most developing countries. However, the seed kernel oil of Balanites aeyptiaca is a good source of raw material for food, cosmetic and pharmaceutical industries.
The objective of the present study is to quantitative determine the chemical composition, the physicochemical properties of Balanties aegyptiaca seed oil and to also investigate the antimicrobial and industrial potentials of the seed kernel oil.
2.1 Sample collection and preparation
The dried seeds of Balanites aegyptica were purchased from a vendors at Kwararafa market in Jos, Plateau State-Nigeria. The sample was spread under shade and sorted to remove unwanted materials. The seeds were manually crushed to remove the seed kernel from the husk shell and the kernel was then milled into powder using a ball mill.
2.2 Oil extraction
The seed kernel oil of Balanites aegyptica was extracted according to the method adopted by Mortadha et al. (2015). The solvent extraction was done using soxhlet apparatus and petroleum ether as the solvent. 200 g of the milled seed kernel was packed into a porous thimble and placed in a soxhlet extractor, using 250 mL of petroleum ether (with boiling point of about 60 – 80 oC) as extracting solvent for 8 hours repeatedly until the required quantity was obtained. A rotary evaporator was used to remove the excess solvent from the extracted oil. The oil was kept in the refrigerator without further treatment until needed for further analysis.
2.3 Ash content
The ash content was determined according to the method described by Pearson (1981). A clean dried crucible was weighed and 5 g of sample was placed on the empty crucible. The sample was heated in a Muffle furnace at 600 oC for 7 hours. The procedure was carried out in triplicate and mean was calculated. The ash content was determined as follows;
– %Ash content = w1 – w2
=Weight of crucible + sample before heating
=Weight of sample + crucible after heating
2.4 Elemental composition of oil and seed kernel
The composition of the elements were determined after the sample wet digestion with mineral acids by atomic absorption spectrophotometer (AOAC, 2006). 2 g of oil sample was transferred into 100 mL beaker and 5 mL each of conc. sulphuric and nitric acids were added. 2 mL of hydrogen peroxide was also added and the mixture was heated until a clear solution was obtained. The mixture was allowed to cool down to room temperature and was filtered. The resulting solution was transferred into a 50 mL volumetric flask and then made up to the mark point with distilled water prior to AAS analysis. Similarly, 5 g of the powdered seed kernel sample was heated at 700 °C for 8 hrs. The sample was removed from the furnace after 8 hrs and placed in the desiccator to cool. The ash was then dissolved in 50 mL 2 N HCl and transferred into 100 mL volumetric flask and made up to the mark point with distilled water for elemental analysis.
2.5 Moisture content
Seed moisture content, expressed as percentage by mass, was determined using 5 g of seeds by adapting the AOAC method 934.06 (AOAC, 1990). A clean dried petric dish was weighed empty and 5 g of the sample was put on it. The petric dish containing the sample was then placed in the oven at 105oC for 24 hrs. This also was done in triplicate and the mean was calculated.
%Moisture = a - b
Weight of petri dish + sample before drying in oven
Weight of dish + sample after drying.
2.6 Refractive index
The refractive index of the oil sample was determined according to the method of Cocks and VanRede (1997). The refractometer was connected to a thermostatically controlled water bath and the temperature maintained at 40oC. The oil sample was spotted onto the slide of the refractometer and viewed by rotating the knobs while the refractive index was recorded.
2.7 Specific gravity
The specific gravity of the oil was determined using a density bottle according to the methods described by (AOAC, 2000). The oil was vacuum filtered to remove any suspended particles. The weight of 50 mL empty density bottle was recorded and the density bottle filled with water recorded . Equivalent quantity of oil was replaced with the water in the same bottle and weighed. The specific density of the oil was determined as follows;
Specific density= w1-w0/w2-w0
= Weight of empty density bottle (g)
= weight of density bottle filled with water (g)
= weight of density bottle filled with oil (g)
2.8 Saponification value
Saponification was determined in accordance to the methods described by AOAC, 1990. 2 g of the oil sample was weighed into a 250 mL quick fit flask and 25 mL of 0.5 methanolic KOH was added. The flask was connected with an air condenser and boiled for 1 hour until all the fat was completely saponified. While the solution was still hot, it was then titrated with 0.5M HCl using phenolphthalein indicator to a colourless end point. A blank titration was carried out concurrently. The saponification value (SV) was calculated as;
SV = Saponification Value (mgKOH/g)
S = volume of HCl used (mL)
B = volume of HCl use for blank (mL)
N = nomality of HCl
wt = weight of oil sample (g)
56.10 = Molar mass of KOH
2.9 Acid value
The acid value was determined using titration and the method adopted was described by the British Standards Institution (BS EN ISO 660: 2009). 3 g of the oil sample was weigh into a conical flask and 50 mL of absolute alcohol added. This was heated on a water bath at 40 oC for half an hour to dissolve the oil completely. It was allowed to cool and then titrated against 0.1 M ethanolic KOH using phenolphthalein indicator until a pink colour which lasted for about 30 seconds was observed. A blank titration was carried out concurrently. The acid value was then calculated using the expression:
AV = (a-b)M x 56.1/W
AV = Acid Value (mgKOH/g)
a = volume of KOH in mL for blank
b = volume of KOH in mL for test
M= molarity of KOH
W= weight of the oil sample (g)
56.1 = molar mass of KOH
2.10 Iodine value
Iodine value was determined according to AOCS recommended practice (AOCS, 1998). 0.2 g oil sample was dissolved in 15 mL carbon tetrachloride in a conical flask and 25 mL WIJ’S solution was adde. The content was mixed vigorously when 20 mL of 10 % potassium iodide solution and 15 mL water were added. A blank was also prepared concurrently, both were placed in a dark room and allowed to stand for at least 1 hr. This was to allow for complete addition reaction between the double bonds of the oil and the liberated iodine to a pale yellow colour. At this point, a few drops of starch indicator solution were added and titrated against standard 0.1N Sodium thiosulphate to a blue end point. The iodine value was then determined as follows;
I V = M(a-b) x 126.9 x 100/1000 / W
= Iodine Value
126.9 = Molar mass of iodine.
M = Molarity of Sodium thiosulphate.
a = Volume (mL) of Sodium thiosulphate used for blank.
b = Volume (mL) of thiosulphate used for the test.
100/1000 = Multiplication factor as define for iodine value.
w = Weight of oil sample.
2.11 Peroxide value
The peroxide value was also determined according to AOCS recommended practice (AOCS, 1998). 5 g of the oil sample was weighed into a 250 mL round bottom flask containing 20 mL of the solvent mixture of glacial acetic acid and chloroform in a ration 2:1 v/v. The content was swelled until the sample dissolved completely and then 0.5 mL of saturated potassium iodide solution was added followed by stirring with a glass rod for one minute. The resultant homogenous solution was allowed to stand in the dark room for about 1 min after which 30 mL of distilled water was added and titrated with standard 0.01 N Na2S2O3.5H2O. As soon as the yellow colour turns colourless, starch indicator 0.5 mL was added and titration continued until the blue colour changed to colourless. A blank titration was also carried out. The peroxide value was calculated by;
= Peroxide value (/kg)
W = weight of oil
= volume of Na2S2O3.5H2O used for test
= volume of Na2S2O3.5H2O used for blank
N = normality of the Na2S2O3.5H2O
2.12 Free fatty acid
The free fatty acid of the oil was determined using the method described by Harold et al. (1990). 50 mL of the oil sample was placed in a beaker, neutralized methylated spirit (0.1 M NaOH was added to methylated spirit to a pink colour and then heated) and heated on a hot plate for 1.0 min. Phenolphthalein indicator (3 drops) was added and then titrated against 0.1 N HCl to the appearance of the first permanent pink colour which lasted for at least 30 seconds, with the same intensity as that of the neutralized alcohol before the addition of sample. The %FFA is calculated as follows;
%FFA = Titrevalue x M x 28.3/W
28.2 = conversion factor in relation to oleic acid
W = Weight of sample
M = Molar concentration of HCl
2.13 Fatty acids composition of the oil
The percentage fatty acid composition of the oil was determined using the method adopted by Eqbal et al., (2011). Fatty acids methyl esters of the oil were prepared by dissolving 0.5 mL of the oil in 5 mL hexane and 5 mL sodium methoxide solution was added. The mixture was stirred vigorously using a vortex stirrer for 10 seconds. The solution was allowed to stand for 10 minutes to separate out the clear solution of fatty acids methyl esters from the cloudy aqueous layer. The upper layer was then collected carefully into a sample bottle and the fatty acids composition of the oil was determined by injecting its fatty acids methyl esters into a gas chromatographic mass spectrophotometer analyzer according to the conditions prescribed by Hale and Belgin (2011). A GCMS incorporated with an auto sampler and Real Time Analysis software system was used for analysing FAME. The split ratio was 1:20 and the flow-rate of carrier gas (helium) 2 mL/min. The injector and detector temperatures were fixed at 250°C. The temperature programme for the column was: hold at 60°C for 1 min increase by 13°C/min to 175°C, increase at 4°C/min to 215°C, and then hold at 215°C for 35 min., total run was 86 min. The mass spectrometer was operated in EI mode at 70 eV scanning the range 30-500 m/z in a 1 s cycle, in a full scan acquisition mode. Comparing with the MS self-contained chromatogram library, the species of every group were identified, and the relative proportion of each species (%) was obtained by dividing the individual peak area with the total peaks areas.
2.14 Phytochemical screening of the seed kernel oil
Phytochemical analysis of the oil was carried out using standard procedures prescribed by Vishnol (1979), Sofowora (1993) and Harborne (1998). The phytochemicals determined include; alkaloid, saponin, tannin, anthraquinone, cardiac glycoside, flavonoids, carbohydrate and steroid.
2.15 Bacteria Sensitivity Text
Two methods were used for the bacteria sensitivity test i.e, Well and Disk methods. In the Well method, the agar well dilution method was used as prescribed by Lino et al. (2006). A colony of individual organisms was emulsified in 5 mL of sterile nutrient broth and incubated at 37 oC for 3 hrs. This was diluted out to have a faint turbidity to avoid heavy inoculum and over growth. The inoculum was flooded on sterile Mueller Hinton Agar plates for each organism and was tested for with direct extract and solvent extract. The all four plates were used for each organism. The plates were then swirled to allow the inoculums to spread over the entire surface of the Agar. After allowing absorption of the inoculum onto the media, the Agar was allowed to dry in an incubator (Aboaba, et al., 2006). Wells of 6 mm were punched into the agar with a sterile corn borer for each organism. Using a sterile pasture pipette, each well was filled with 0.1 mL of the oil at dilution concentrations of 1/10, 1/100 and 1/100 mL. The same volumes of the undiluted oil and 10 ug/mL penicillin anti-biotic were filled into the respective holes bore; this was labeled at the back of the plates (Jorgensen and Turnidge, 2007). The entire preparation was allowed to stand on the bench for 3hrs for the extract to settle and possibly diffused into the medium. The plates were inverted and incubated at 37 oC for 24 hrs for bacterial growth and observation.
The Disc method was done using a filter paper which was perforated into small circles to make a paper disc. It was then sterilized by autoclaving. Each disc was soaked in the diluted extract and placed on the plate flooded with the inoculums according to the dilution factor labeled at the back of the plates. The same was done for the neat and the antibiotic. The plates were incubated immediately at 37 oC for 24 hrs for observation.
The results of the concentration of trace metals in seed kernel and seed kernel oil of Balanites aegyptica are shown in Table 1. The presence of trace metals is an important factor as far as the quality of edible oil is concerned. Metallic elements such as Na, K, Ca, Mg, Fe, Cu, Zn and Mn are essential human nutrients mainly for growth. Cd, Cr, Co, Ni and Pb pose detrimental effects on health of plants and animals even in relatively small amounts (Pehlivan et al., 2008). The metal with highest concentration in the seed kernel was sodium (5.92±0.20 mg/g), followed by zinc (4.19±0.2 mg/g) while the highest in the seed kernel oil was magnesium (2.220±0.007 mg/g) and followed by calcium (1.46±0.5 mg/g). These metals are required in the body for certain metabolic functions. Lead has the lowest concentration 0.0021±0.0002 mg/g and 0.0022±0.0001 mg/g in the seed kernel and seed kernel oil respectively. Lead is one of the most toxic heavy metals. The toxicity of lead to human is well known: It replaces calcium and consequently, can accumulate in the skeletal system. Exposure of human to lead is associated with various neurodevelopmental effects, cardiovascular diseases, impaired renal function and fertility, hypertension and adverse pregnancy outcomes (Jock et al., 2016). However, lead concentration in the seed kernel and seed kernel oil was within admissible limit of 0.01 mg/g by World Health Organization (Gebrekidan and Samuel, 2011).
The quality assessment of Balanites aegyptica seed kernel oil was analyzed by evaluating physicochemical properties such as oil content, specific gravity, refractive index, free fatty acid, peroxide, iodine and saponification values as presented in Table 1. The oil content was found to be 45.32% above the limit for most oil seed crops (Pritchard, 1991). This shows that the oil content is high and is a factor that is favourable for industrial application.
The specific gravity of the seed kernel oil of Balanites aegyptica was 0.90±0.00 while the refractive index was 1.45. These values are in close agreement with the FAO/WHO international standard for edible oil shown in Table 2.
The Free fatty acids (FFA), acid value and peroxide values are important parameters in evaluating the quality of fats and oils with respect to rancidity and oxidation. The acid value of Balanites aegyptica seed kernel oil was 2.14±0.28 mg KOH/g while the free fatty acids value was 0.82±0.01 mg KOH/g. Acid value was determined to quantify the fatty acid found in the oil as it measures the free fatty acids of oil. The acid value was low (2.14±0.28 mg KOH/g) and this shows that the oil is stable (Haftu, 2015). Oils with high acid value, also implied high % FFA and will undergo rancidity due to the hydrolysis of the free fatty acids on storage. The acid value and % FFA of Balanites aegyptica seed kernel oil are lower than FAO/WHO standard for edible oils (Table 2). The low %FFA reduces the tendency of the oil to undergo hydrolytic activities. In most oils, the level of free fatty acid which causes deterioration is noticed when the %FFA calculated as oleic acid falls within the range of 0.5 - 1.5% (Manji et al., 2013).
Saponification value is an index of average molecular mass of fatty acid in the oil sample. The SV value obtained for Balanites aegyptica seed kernel oil was 200.02mgKOH/g. The value is within the range of 195–205 mg KOH/g for edible palm oils (Erum, et al., 2017). The saponification value of oil serves as an important parameter in determining the suitability of the oil for soap making.
Peroxide value is used as a measure of the extent to which rancidity reactions have occurred during storage it could be used as an indication of the quality and stability of fats and
Oils. The peroxide value determined for the seed kernel oil of Balanites aegyptica is 2.95 mEq/g and is lower than FAO/WHO standard shown in Table 2. A low peroxide value in the present study increases the suitability of the oil for a long storage due to low level of oxidative and lipolytic activities (Adegbe et al., 2016).
Iodine value measures the degree of unsaturation in a fat or vegetable oil. It determines the stability of oils to oxidation and allows the overall unsaturation of the fat to be determined qualitatively. The iodine value of the seed kernel oil is 104.39 gI/100g and within the range of FAO/WHO standard as shown in Table 2. Generally, oils having iodine value below 100 are non-drying, those having values between 100 -130 are semi-drying and those having values above 130 are termed drying oils (Asuquo, 2008). The oil of Balanites aegyptica will then serve as semi-drying oil which is also comparable to that of crude fish oil 105.5g/100g as reported by the International Union of Pure and Applied Chemistry (1990).
Table 3 shows comparison of the physical characteristics of seed kernel oil of Balanite aegyptica by different authors and present studies. The seed contains low moisture content (0.114±0.04 %) as compared to other studies (Table 3). This means, the seed kernel of Balanites aegyptica can be stored for an extended time under suitable condition without deterioration. The moisture content of the oil is low, an indication that its stability is guaranteed. Oil seeds deteriorate as a result of high moisture content; this happens when heat is generated by oxidation reaction and increases the temperature of the stored seed thereby accelerating deterioration even to the point of charring the seed. Moisture content gives an indication of a food shelf life and nutritive value, hence low moisture content is a requirement for long storage life (Aurand et al., 1987).
The ash content of the seed kernel is 4.77±0.01 % and is similar with previous studies shown in Table 3. This is the measure of the total amount of minerals present in a food. The mineral content of a food is a measure of the amount of specific inorganic components present and ash is the inorganic residue remaining after the water and organic matter have been removed. Balanites aegyptica seed kernel ash content is lower when compared with cotton seed (4.56%) as reported by Muhammad et al., (2012). This shows a reasonable amount of mineral content in the seed kernel.
Table 4 shows GCMS and identities of the fatty acids present in the oil. The most prominent of the fatty acids are palmitic acid (16:0) (20.51%), oleic acid (18:1) (28.32%), linoleic acid (18:2) (19.20%) and stearic acid (18:0) (15.97%). The oil contains about 47.52% unsaturated fatty acid which makes it a good edible oil. Moreover, there are some compounds not included identified in minor and traced amounts while others cannot be identified due to complication of their chemical nature. However, oils with high degree of un-saturation are considered good for the heart because it decreases total cholesterol and low density lipoproteins. A high dietary intake of saturated fatty acids (SFAs) is a risk factor for development of obesity and cardiovascular disease (Gillian et al., 2008).
The result of phytochemical screening test of the seed kernel oil of Balanite aegyptica is shown in Table 5. The oil revealed strong presence of steroids. Carbohydrates, cardiac glycosides and alkaloids were also present, while anthraquinones, saponins, tannins and flavonoids were absent in the seed kernel oil. These phytochemicals may be responsible for the application of the oil in treating certain skin infections (Daya et al., 2011).
The bacteria and fungi sensitivity test carried out on the four fungi and bacteria is summarized in Table 6. There was no inhibition of any of the microorganism by the seed kernel oil of Balanites aegyptica. Daya and Vaghasiya (2011) reported the use of this oil in the treatment of some bacteria and the lack of inhibition shown in the analysis could be as a result of the difference in the method of oil extraction and variation in the properties of the seed kernel oil of Balanites aegyptica as reported by Elfeel (2010). Elfeel et al. (2006) also reported a morphological variation and chemical characteristics of fruits and seeds of Balanite aegyptica within different geographical source in Sudan. Variation in diosgenin level was also reported by Bishnu et al., (2005) within different province.
The present study on the chemical composition, physicochemical properties, and phytochemical characterization of oil from Balanites aegyptica seeds suggest that the seed of this plant has high oil content and it can be a good source of raw material for many oil based products (soap, shampoo, bio-diesel, lubricants, etc). The oil could also be utilized successfully as a source of edible oil for human consumption as it makes a good dietary source of magnesium and calcium. Generally, the seed oil exhibited good physio-chemical properties and could be useful for industrial applications
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