Moringa oleifera belongs to the Moringaceae family and Moringa genus, the best known and most widely distributed species (Morton, 1991; Sengupta and Gupta, 1970). There are a few known varieties namely Jaffna, Chauakacheri Murunga, Chem, Kadu, Palmurungai, Periyakulam 1 (PKM 1) (Tsaknis et al., 1998) and Peregrina (Somali et al., 1984). The edible oil was extracted, where the tree is cultivated by boiling the seeds with water and collecting the oil from the surface of the water (Somali et al., 1984). The seed oil contains all the fatty acids contained in olive oil, except linoleic and was used as its acceptable substitute (Morton, 1991). Moringa oleifera Congo, Brazzaville is a selection of local types and is propagated only by seed. Until now a full characterization of the oil produced from the seeds of Moringa oleifera Congo-Brazzaville has not been reported. Additionally, the use of different methods of extraction and their effect on the composition and the characteristics of the oil has not been investigated. The oil was compared to virgin olive oil.
Also, the characteristics of Moringa oleifera seed oil can be highly desirable especially with the current trend of replacing polyunsaturated vegetable oils with those containing high amounts of monounsaturated acids (Corbett, 2003). High oleic acid vegetable oils have been reported to be very stable even in highly demanding applications like frying (Warner and Knowlton, 1997). The press cake obtained after oil extraction has positively charged protein molecules that have coagulant properties (Sutherland et al., 1994). These properties have been exploited in water clarification and wastewater treatments. Previous studies on Moringa oleifera have been focused on its medicinal uses and nutritional aspects of the tree parts (Lowell, 1999) and on the use of the seed in the clarification of waste-water during treatment (Folkard et al., 1993); however, little or no studies have been done on the oil properties, such as the triacylglycerol profiles and other physico-chemical properties apart from the fatty acid composition. In this study, some physical and chemical properties such as thermal behavior and triacyglycerol profile were determined following extraction using soxlhet and methanol-chloroform methods.
MATERIALS AND METHODS
Mature Moringa oleifera pods were collected from neighborhood gardens around University Campus Marien Ngouabi of Brazzaville. The seeds were removed from the pods, sorted and sun dried. Only seeds that were not damaged were chosen and stored under cool dry storage conditions until needed.
Proximate analysis of Moringa oleifera seed Moisture, crude protein (micro-Kjeldahl), crude fiber and oil (Soxhlet) contents were determined using the methods described by Pearson (1976), whereas, the ash content was determined using the method of Pomeranz and Meloan (1994) and total carbohydrate was determined by difference. All determinations were done in triplicate.
Oil extraction: Dried M. oleifera seeds were ground in a Moulinex model SeB PREP'LINE 850 (Moulin cafe). For solvent extraction (Soxlhet method), 50 g of ground seeds were placed into a cellulose paper cone and extracted using light petroleum ether (bp 40-60°C) in a 5 L Soxhlet extractor for 8 h (Pena et al., 1992). The oil was then recovered by evaporating off the solvent using rotary evaporator model N-1 (Eyela, Tokyo Rikakikal Co., Ltd., Japan) and residual solvent was removed by drying in an oven at 60°C for 1 h and flushing with 99.9% nitrogen. For methanol/chloroform extraction, 100 g of the ground seeds were homogenized with a chloroform mixture:methanol (1:1) and water. Two phases was obtained, aqueous layer (methanol-water) and organic layer (chloroform). Oil was recovered by evaporating off the solvent (chloroform) using rotary evaporator model N-1 (Eyela, Tokyo Rikakikal Co., Ltd., Japan) and residual solvent was removed by drying in an oven at 60°C for 1 h and flushing with 99.9% nitrogen All experiments were done in triplicates and the mean and standard deviations were calculated.
Physical and chemical analysis of crude oil
Thermal behaviour: The thermal property of the oil samples was investigated
by differential scanning calorimetry using a Perkin-Elmer Diamond DSC (Norwalk,
USA). The instrument was calibrated using indium and zinc. The purge gas used
was 99.99% nitrogen with a flow rate of 100 mL min-1 and a pressure
of 20 psi. Sample weights ranged from 5-7 mg and were subjected to the following
temperature program: frozen oil sample was heated at 50°C in an oven until
completely melted. Oil sample was placed in an aluminum volatile pan and was
cooled to -50°C and held for 2 min, it was then heated from -50 to 50°C
at the rate of 5°C min-1 (normal rate) (Che Man and Swe, 1995)
and 10°C min-1 (past rate) and held -50°C isothermally for
2 min and cooled from -50 to 50°C at the rate of 5°C min-1.
The heating and cooling thermogram for the normal and the fast (hyper DSC) scan
rates were recorded and the onset, peak and offset temperatures were tabulated.
These values provide information on the temperature at which the melting process
starts, the temperature at which most of the TAG have melted and the complete
melting temperature of the oil, respectively.
Viscosity measurements: A rheometer as described by Nzikou et al. (2007) was used to measure the different oil viscosities. By this procedure, a concentric cylinder system is submerged in the oil and the force necessary to overcome the resistance of the viscosity to the rotation is measured. The viscosity value (mPas) is automatically calculated on the basis of the speed and the geometry of the probe. Temperature (20°C) was controlled with a water bath connected to the rheometer. The experiment was carried out by putting 3 mL of sample in a concentric cylinder system using 100 sec as shear rate.
Chemical analysis: Determinations for peroxide, iodine and saponification
values, unsaponifiable matter and free fatty acid contents were carried out
using Pena et al. (1992) standard analytical methods. The fatty acid
composition was determined by conversion of oil to fatty acid methyl esters
prepared by adding 950 μL of n-hexane 50 mg of oil followed by 50 μL
of sodium methoxide using the method of Cocks and Van Rede (1966). The mixtures
were vortex for 5 sec and allowed to settle for 5 min. The top layer (1 μL)
was injected into a gas chromatograph (model GC-14A, Shimadzu Corporation, Kyoto,
Japan) equipped with a flame-ionization detector and a polar capillary column
(BPX 70 0.25), 0.32 mm internal diameter, 60 m length and 0.25 μm film
thickness (SGE Incorporated, USA) to obtain individual peaks of fatty acid methyl
esters. The detector temperature was 240°C and column temperature was 110°C
held for 1 min and increased at the rate of 8°C min-1 to 220°C
and held for 1 min. The run time was 32 min. The fatty acid methyl esters peaks
were identified by comparing their retention time with those of standards. Percent
relative fatty acid was calculated based on the peak area of a fatty acid species
to the total peak area of all the fatty acids in the oil sample. The minerals
were determined by atomic absorption spectrophotometry. One gram samples in
triplicate, were dry ashed in a muffle furnace at 550°C for 8 h until, a
white residue of constant weight was obtained. The minerals were extracted from
ash by adding 20.0 mL of 2.5% HCl, heated in a steam bath to reduce the volume
to about 7.0 mL and this was transferred quantitatively to a 50 mL volumetric
flask. It was diluted to volume (50 mL) with deionised water, stored in clean
polyethylene bottles and mineral contents determined using an atomic absorption
spectrophotometer (Perkin-Elmer, model 2380, USA). These bottles and flasks
were rinsed in dilute hydrochloric acid (0.10 M HCl) to arrest microbial action,
which may affect the concentrations of the anions and cations in the samples.
The instrument was calibrated with standard solutions.
Statistical analysis: Values represented are the means and standard deviations for three replicates. Statistical analysis was carried out by Excel version 8.0 software. Significance was defined at p<0.05.
RESULTS AND DISCUSSION
Proximate analysis of Moringa oleifera seed oil: Results obtained showed that the seeds contained 5.3% moisture, 39.3% crude oil, 37.6% crude proteins, 13.6% carbohydrate (by difference), 3.2% crude fiber and 4.2% ash (Table 1). The high percentage of oil makes this seed a distinct potential for the oil industry. According to Benthall (1946), Burkill (1966), Irvine (1961), Makkar et al. (1997), Duke and Atchley (1984) and Abdulkarim et al. (2005), the mature seed yields 22-38% oil. Jamieson (1939) reported a 40% yield by weight of the seed. Variation in oil yield may be due to the differences in variety of plant, cultivation climate, ripening stage, the harvesting time of the seeds and the extraction method used.
Minerals: It is of interest to note that the most prevalent mineral element in M. oleifera seeds is magnesium, which is a high as 251.30±0.02 mg/100 g dry mater (Table 2). Mg plays a significant role in photosynthesis, carbohy-drate metabolism, nucleic acids and binding agents of cell walls (Russel, 1973). Calcium (83.75±0.01 mg/100 g dry matter) is also the major component of bone and assists in teeth development (Brody, 1994).
Oil extraction: Characteristics of the oil were compared with M.
oleifera varieties others country, described by Tsaknis et al. (1998),
Dahot and Memon (1985), Ferrao and Ferrao (1970) and Abdulkarim et al.
(2005). The extracted oils were liquid at room temperature. The oil content
of M. oleifera Congo-Brazzaville seeds and the level at which, the differences
are significant are shown in Table 3. The oil extraction with
the Soxlhet method had the highest yield, due to the increased ability of the
solvent to overcome forces that bind lipids within the sample matrix (Lumley
and Colwell, 1991). The Blye and Dyer method, showed the low yield due to losses
during the separation of the two phases, aqueous layer (methanol-water) and
organic layer (chloroform).
||Proximate analysis of Moringa oleifera oil seed
|ND: Not Determined; a:Mean±SD; b:
Abdulkarim et al. (2005), cCrude protein = N (%)x6.25;
dCarbohydrate obtained by difference
||Mineral elemental composition of Moringa oleifera seeds
|Values are mean±SD of triplicate determinations
||Physical and chemical properties of Moringa oleifera
seed oil extracted using solvent process
|Means for the determined values in the same row followed by
the same superscript letter are not significantly different (p<0.05);
a:Abdulkarim et al. (2005); bOil = Weight of
extracted oilx100/weight of seed; PV: Peroxide Value; FFA: Free Fatty Acid;
IV: Iodine Value
The results of the above researchers agree with those of the present research.
Physical and chemical properties of oil
Differential Scanning Calorimetry (DSC): DSC is suitable to determine these
physical properties. Results obtained from the heating with the DSC showed slight
differences in both melting behaviour for the two oil samples when temperatures
scanning (5°C min-1 and 10°C min-1) were used.
The heating profiles using the scan rate (5 and 10°C min-1) for
the 2 extractions methods showed that there is two major peaks (2) and (2'),
4 small shoulder peaks 1,1' and 3, 3', respectively (Fig. 1
and 2). The shoulder peaks 1 and 1' represented the melting
temperature of unstable crystals of the low melting TAG that pre-maturely melted.
The more stable low melting unsaturated TAG crystals melted at a higher temperature
shown as peaks 2 and 2'.
||Heating profiles of 2 M. oleifera oils extracted by
two methods (Blye and Dyer; Soxlhet), at 5°C min-1 scan rate
||Heating profiles of two Moringa oleifera oils extracted
by two methods (Blye and Dyer;Soxlhet), at 10°C min-1 scan
The higher melting, more saturated TAG peaks (3 and 3') appeared at higher
temperatures. According to cooling/heating rates, the DSC makes it possible
to highlight the existence of various crystalline forms called polymorphism.
According to cooling/heating rates, the DSC makes it possible to highlight the
existence of various crystalline forms called polymorphism. However in the case
of the study, of mixed triglycerides saturate-unsaturated, at the speed of 5°C
min-1, this polymorphism can be particularly rich since at the same
temperature corresponding to the two major peaks 2 and 2' (Fig.
1), it seems to have the existence of another peak on the peaks 2 and 2'
(Fig. 1), which is probably due to the coexistence of two
crystalline varieties: the forms α and β, which is thermodynamically
unstable. This crystalline form β' disappears, when the speed increased
at 10°C min-1 (Fig. 3). The form β', existing
like a state of transition.
||Heating profiles of M. oleifera oil extracted by soxlhet
method, at 5 and 10°C min-1 scan rate
Viscosity: Viscosity is a measure of resistance of a fluid to deform
under shear stress. It is commonly perceived as thickness, or resistance to
pouring. Viscosity describes a fluids internal resistance to flow and
may be thought of as a measure of fluid friction. In optics to know, the rheological
proprieties of these oils, we studied the influence of temperature on viscosity.
Activation energies of the various classes of fatty acids contained in these
oils were shown Table 3. When, the temperature increases,
viscosity decreases exponentially (Table 4) some is the extraction
method (Arslan et al., 2005; Nzikou et al., 2007). Viscosity
varies between 66.82 and 46.08 mPa.s when temperature decreases of 45-5°C
by Blye and Dyer method. By Soxlhet method, the viscosity of oil decreases of
62.51-43.78 mPa.s (Table 4). The viscosity of the oil obtained
by Blye and Dyer method was highest, possibly because of the water that was
absorbed by the gums (phospholipid) during extraction. This calculator calculates
the effect of temperature on reaction rates using the Arrhenius equation.
η = Axexp (-Ea/RxT)
||The activation energy (KJ moL-1)
||The universal gas constan
||The temperature (°C)
R has the value of 8.314x10-3 KJ moL K-1. We should use this calculator to investigate the influence of temperature on viscosity. Linear regression analysis was applied to the logarithmic form of Arrhenius equation in order to determine the parameters of the relation (Table 5). ln η against 1/T,-Ea/RT is the slope from, which Ea was evaluated. Activation energies of oils are shown in
||Oil viscosity at various temperature in degree celsuis
||Energie plot derived from the Arrhenius equation
Table 3. The highest value of activation energy is obtained by Blye and Dyer method (6.80 KJ moL-1) and 6.57 KJ moL-1 by Soxlhet method.
Chemical properties: The chemical properties of oil are amongst the most important properties that determines the present condition of the oil. Free fatty acid and peroxide values are valuable measures of oil quality. The iodine value is the measure of the degree of unsaturation of the oil. The free fatty acid and the unsaponifiable matter content of the Soxlhet method were significantly higher (p<0.05) than those of the Blye and Dyer method (Table 3). There was no significant difference in the iodine and saponification values in the two extraction methods (p>0.05). The slightly higher value of unsaponifiable matter in the Soxlhet method may be due to the ability of the Solvent to extract other lipid associated substances like, sterols, fat soluble vitamins, hydrocarbons and pigments (Bastic et al., 1978; Salunke et al., 1992).
Fatty acid composition: The major saturated fatty acids in Moringa
oleifera seed oil were palmitic, stearic, arachidic and behenic acids and
the main unsaturated fatty acid is oleic acid (74.68%) with small amounts of
palmitoleic, linoleic, linolenic and eicosenoic acids (Table 6
and 7). There was no significant difference (p>0.05) in
the amounts of the major fatty acids in the two oil samples. The two oil samples
of Moringa oleifera contained a substantial amount of behenic acid (5.22
and 5.33%), respectively. The oil can, therefore, be used as a natural source
of b ehenic acid, which has been used as an oil structuring and solidifying
agent in margarine, shortening and foods containing semi-solid and solid fats,
eliminating the need to hydrogenate the oil (Abdulkarim et al., 2005).
||Relative percent composition of fatty acid in Moringa oleifera
|Means for the determined values in the same row followed by
the same superscript letter are not significantly different (p<0.05),
aSunga and Whitby (1995), Dahot and Menon (1985), Ferrao and
Ferrao (1970) and Abdulkarim et al. (2005)
||Melting behaviour of Moringa oleifera seed oil using
different scan rates. Experimental conditions: temperature program set at
-50°C for 10 min, rising to 50°C at rate of 5 and of 10°C min-1
The high percentage of oleic acid in the oil makes it desirable in terms of
nutrition and high stability cooking and frying oil. Many circumstances have
focused attention on high-oleic vegetable oils. It has been demonstrated that
a higher dietary intake of bad fats (saturated and trans fatty acids) is associated
with an increased risk of coronary heart disease caused by high cholesterol
levels in the blood (Mensink and Katan, 1990; Siguel and Lerman, 1993) whereas,
a higher intake of good fats (monounsaturated/oleic) is associated with decreased
risk (Corbett, 2003). High oleic-acid vegetable oils such as high-oleic corn,
sunflower and canola have been found to have enough oxidative stability to be
used in demanding applications such as frying (Petukhov et al., 1999;
Warner and Knawlton, 1997). In addition, high-oleic oils have low saturated
fatty acid levels. Therefore, high-oleic oils can be viewed as a healthy alternative
to partially hydrogenated vegetable oils (Abdulkarim et al., 2005).
Moringa oleifera seed oil has the potential to become a new source of
high-oleic acid oil and its full potential should be exploited. It contains
high monounsaturated to saturated fatty acids ratio and might be an acceptable
substitute for highly monounsaturated oils such as olive oil in diets. Moringa
oleifera is a tree growing rapidly even in poor soil and is little affected
by drought (Sengupta and Gupta, 1970; Morton, 1991) and can be easily grown
in poor third world countries. The production of useful oil from its seeds could
be of economic benefit to the native population of the areas, where the tree