Food Chemistry 187 (2015) 53–57

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Food Chemistry
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Short communication

Effect of the refining process on Moringa oleifera seed oil quality

Dalia I. Sánchez-Machado a, Jaime López-Cervantes a,⇑, José A. Núñez-Gastélum a,b,
Gabriela Servín de la Mora-López c, Julia López-Hernández d, Perfecto Paseiro-Losada d
a
Departamento de Biotecnología y Ciencias Alimentarias, Instituto Tecnológico de Sonora, 5 de Febrero 818 Sur, 85000 Ciudad Obregón, Sonora, Mexico
b
Universidad Autónoma de Ciudad Juárez, Instituto de Ciencias Biomédicas, Departamento de Ciencias Químico-Biológicas, Anillo Envolvente del PRONAF y Estocolmo s/n,
Ciudad Juárez, 32310 Chihuahua, Mexico
c
Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Ciudad Universitaria, AP 354, 80000 Culiacán, Sinaloa, Mexico
d
Departmento de Química Analitica, Nutrición y Ciencia de Alimentos, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782, Spain

a r t i c l e i n f o a b s t r a c t

Article history:
Received 12 May 2014
Received in revised form 10 October 2014
Accepted 12 April 2015
Available online 18 April 2015
Chemical compounds studied in this article:
Oleic acid (PubChem CID: 445639)
Linoleic acid (PubChem CID: 5280450)
Linolenic acid (PubChem CID: 5280934)
Palmitic acid (PubChem CID: 985)
b-Carotene (PubChem CID: 5280489)
Malondialdehyde (PubChem CID: 10964)
We evaluated the physicochemical properties and oxidative stability of the oil extracted from the seeds of
Moringa oleifera during its refining process. Refining is accomplished in three stages: neutralization,
degumming, and bleaching. Four samples were analyzed, corresponding to each step of the processed
and crude oil. Increases in the density, viscosity, saponification value and oxidation of the oil were
detected during the refining, while the peroxide value and carotenoid content diminished. Moreover,
the refractive index and iodine content were stable throughout the refining. Nine fatty acids were
detected in all four samples, and there were no significant differences in their composition. Oleic acid
was found in the largest amount, followed by palmitic acid and behenic acid. The crude, neutralized,
and degummed oils showed high primary oxidation stability, while the bleached oil had a low incidence
of secondary oxidation.
Ó 2015 Elsevier Ltd. All rights reserved.

Keywords:
Fatty acids
Nonconventional oil
Carotenoids
Oxidative stability
Crude oil
Bleached oil
Neutralized oil
Degummed oil
Lipid oxidation

1. Introduction
Vegetable oils are an important part of human sustenance
worldwide. However, fats and oils, crude vegetable oils in particu-
lar, contain various kinds of minor constituents such as dirt,
moisture, gums, waxes, carbohydrates, proteinaceous materials,
pigments, flavoring substances, trace metals, antioxidants, and free
fatty acids (Gustone, 2005, chap. 6). The term crude oil is assigned
to oil that is not processed after being extracted from the animal or
plant material. Crude oil is usually refined to remove the majority
of these unwanted components and achieve the desired color and a
⇑ Corresponding author. Tel.: +52 644 4100900; fax: +52 644 4109001.
E-mail address: jaime.lopez@itson.edu.mx (J. López-Cervantes).
mild taste. In general, refined oil is clear, odorless, and resistant to
rancidity (Ranken, Kill, & Baker, 1997). The refining process gener-
ally includes the steps of degumming, neutralization, bleaching,
and vacuum deodorizing (Ortega-Nieblas & Vázquez-Moreno,
1993).
However, the lipids in both refined and crude oil may deterio-
rate over periods of prolonged storage and exposure to elevated
temperatures (Shahidi & Zhong, 2005, chap. 8). The problem is fur-
ther complicated if one takes into account that oxidation reactions
can be initiated, modified, or inhibited by factors such as tempera-
ture, light, pH, and the presence of enzymes, metals, and antioxi-
dants (Akoh & Min, 2008, chap. 12). However, oils rich in oleic
acid, which is a monounsaturated fatty acid, have a high oxidative
stability compared to oils containing polyunsaturated fatty acids.

http://dx.doi.org/10.1016/j.foodchem.2015.04.031
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
54 D.I. Sánchez-Machado et al. / Food Chemistry 187 (2015) 53–57
The increase in demand for food implies the need to increase
the overall production of alternative sources of edible oils
(Manzoor, Anwar, & Iqbal, 2007). Therefore, we focused our
attention on the Moringa oleifera tree, the fruit of which has many
health benefits. Tsaknis, Lalas, Gergis, and Spiliotis (1998) gave an
excellent description of the morphological generalities of the tree.
This plant has many potential uses in food, cosmetics, and other
industrial applications (Anwar, Latif, Ashraf, & Gilani, 2007). The
oil extracted from the seeds of M. oleifera is composed of 82%
unsaturated fatty acids, 70% of which is oleic acid. This oil contains
the same fatty acid profile as olive oil except for linoleic acid
(Tsaknis et al., 1998). But, currently, seeds from M. oleifera are
not used widely for extraction, processing, and marketing of edible
oil.
The main purpose of this work was to study the physicochemi-
cal properties of M. oleifera seed oil during the different stages of
refining and its oxidative stability throughout the process, and to
determine its fatty acid profile.
2. Materials and methods
2.1. Collection and sample preparation
Ripe pods of M. oleifera were collected from a field in South
Sonora, Mexico. The seeds were extracted manually and ground
in a hand mill, before being stored in plastic bags in the dark until
the extraction of the oil.
2.2. Extraction of the oil from the seeds of M. oleifera
A solvent extraction was performed using sonication.
Specifically, 50 g of sample was weighed into a 500 mL flask, and
250 mL hexane was added. The mixture was stirred until it was
homogeneous, and the flask was then sonicated for 15 min at room
temperature (Branson 1510, Danbury, CT, USA). Afterwards, the
mixture was filtered under vacuum, and the residue was subjected
to the same extraction procedure again. The oil-hexane mixture
was concentrated in a rotary evaporator (Büchi 124, Flawil,
Switzerland) at 40 °C to obtain the oil. In order to remove traces
of hexane, the oil was heated for 1 h at 60 °C under vacuum
(Ortega-Nieblas & Vázquez-Moreno, 1993).
2.3. Oil refining process
The refining process was conducted according to the report
from Ortega-Nieblas and Vázquez-Moreno (1993), with some
modifications. The crude oil (CO) was neutralized by adding the
required amount of 3 mol/L NaOH at room temperature with
stirring. Subsequently, the mixture was heated to 65 °C under
reduced pressure for 15 min, cooled in a separating funnel at room
temperature to facilitate decantation, and filtered. The neutralized
oil (NO) was washed with deionized water (20 wt%), and the water
was separated from the oil with the help of a separating funnel. For
the degumming process, the oil was heated in a water bath at
70 °C, boiling water (20 vol%) was added, and the mixture was
stirred for 10 min. After cooling the mixture, the degummed oil
(DO) was recovered by centrifugation at 3000 rpm for 15 min
(Harrier 18/80, Greenwich, CT, USA). Trace moisture was removed
with anhydrous sodium sulfate, and the oil filtered under vacuum
with Whatman No. 1 filter paper. The bleaching was performed by
adding 3 wt% activated carbon and heating to 110 °C for 10 min.
The charcoal and the bleached oil (BO) were separated by vacuum
filtration on a 0.45 lm membrane.
2.4. Physicochemical properties of the oils
The refractive index was determined according to method
Cc-7-25 of AOCS (1989). The density was estimated by weighing
a known volume of oil at 20 °C. The viscosity was analyzed with
a DV-E viscometer at 20 °C (Brookfield, Middleboro, MA, USA)
(Núñez-Gastélum et al., 2011). For determination of the acid value,
iodine value, and saponification, methods Ca 5a-40, Cd 1–25, and
Cd 3–25, respectively, were employed (AOCS, 1989). Carotenoids
were quantified spectrophotometrically at 450 nm using the
following equation:
mg of carotenoid A  V  FD  10
¼
g of sample g  E1%
1cm
where A is the absorbance at 450 nm, V is the volume of extract, FD
is the dilution factor, g is the sample mass, and E1%
1cm is the specific
extinction coefficient of b-carotene in hexane 3450 (Strati &
Oreopoulou, 2011).
2.5. Oxidation indexes
To establish the presence of primary oxidation in the samples
during the refining process, the peroxide value (Cd 8–53, AOCS,
1989) and the conjugated dienes content (Paquot & Hautfenne,
1987) were determined. Meanwhile, the method for p-anisidine
(Cd 18–90) was used to establish the presence of secondary oxida-
tion products of lipids (AOCS, 1989).
2.6. Determination of the fatty acid profile
The fatty acid profile was determined by gas chromatography in
accordance with the procedure of Núñez-Gastélum et al. (2011).
Specifically, 0.5 g dry sample was weighed in a tube with a screw
cap and treated with 2 mL of toluene and 3 mL 5 vol% methanolic
HCl. The mixtures were vortexed and placed into a water bath
for 2 h. After the samples had cooled to room temperature, 3 mL
6% K2CO3 solution and 2 mL toluene were added to the sample,
followed by agitation in the vortex. The samples were then
centrifuged for 5 min at 2400 rpm (Clay Adams Compact II
Centrifuge, Parsippany, NJ, USA). After the organic phase was sepa-
rated and dried with anhydrous Na2SO4, 1 mL of the organic phase
was filtered through a 0.45 lm membrane. All the samples were
analyzed in duplicate. The equipment consisted of a gas chro-
matograph 3800 with a flame ionization detector, a capillary
column CP-Sil 88 (60 m  0.25 mm i.d., thickness of 0.25 lm),
and a CP-8410 auto-injector, all from Varian Inc. (Palo Alto, CA,
USA). The injection volume was 1 lL (at 220 °C), the carrier gas
was helium (1 mL/min), and the detector temperature was held
constant at 235 °C. The column temperature was held at 120 °C
for 1 min and was then increased to 170 °C at a rate of 3 °C/min,
where it was held for 1 min, and then finally to 235 °C and main-
tained for 5 min. Peak identifications were based on comparing
the retention times with those of known standards obtained from
Sigma (St. Louis, MI, USA). The areas of the peaks were quantified
using the software Galaxie Workstation (Varian Inc., Palo Alto,
CA, USA). The relative amount of each fatty acid (% of a fatty acid
of the total fatty acids) was quantified by integrating the area
under the peak and dividing the result by the total area for all fatty
acids.
2.7. Oxidative stability of the oils
Tests on the oxidative stability of the oils were performed
according to the methodology proposed by Abuzaytoun and
Shahidi (2006), with minor modifications. Specifically, 0.5 g of oil
 D.I. Sánchez-Machado et al. / Food Chemistry 187 (2015) 53–57 55

was weighed in amber vials (2 mL) and placed uncovered in an Table 2

oven with a forced air stream at 60 °C (DKN402 Yamato, Tokyo, Oxidation indexes of the M. oleifera oil during the refining process.

Japan). The test was conducted for a week, taking a sample every
24 h. The primary oxidation was determined by the measuring
the content of conjugated dienes (IUPAC, 1987). Secondary oxida-
tion was evaluated by the thiobarbituric acid reactive substances
method (TBARS) (Khan & Shahidi, 2001).
2.8. Statistical analyses
The mean and standard deviation were calculated for the repli-
cates of each analysis. Analysis of variance was performed using
SPSS version 12. In addition, the Tukey test (P < 0.05) was used to
compare results from the different treatments.
3. Results and discussion
3.1. Physicochemical properties of the oils
Table 1 shows the values obtained from the physicochemical
analyses of the oil samples.
Significant difference was found in the density during the refin-
ing process; however this may be due to the small variation
between the repetition values for each measurement (Fraser &
Fogarty, 1989). The density of the major dietary fats varies from
0.91 to 0.97 g/mL, and the oils studied here fell within this range.
Manzoor et al. (2007) reported a density of 0.87 g/mL for Moringa
concanensis; the difference may be due to climatic aspects and spe-
cies variation. The average viscosities were 42.8 cP, 57.54 cP, 64.64
cP, and 62.62 cP for the CO, NO, DO, and BO, respectively. Tsaknis
et al. (1998) reported a viscosity of 91 cP for oil obtained from
the seeds of the Malawi variety of M. oleifera. In contrast,
Ogunsina et al. (2014) reported a viscosity of 43.6 cP for M. oleifera
oil cultivated in India. Clearly, the growing conditions, variety, and
method of extraction affect the physical properties of vegetable
oils.
The refractive index increased minimally during the processing
of the oil. The slight change in the refractive index was brought
about by the changes in the density and saponification. Ogunsina
et al. (2014) reported a refractive index of 1.47, similar to that
found in this study. The acid value in terms of oleic acid in the
CO was 1.28%, similar to that reported by Lalas and Tsaknis
(2002) in M. oleifera (1.12%). Firestone (2005, chap. 7) indicated
that the acidities or free fatty acids, related to lipid peroxidation,
of extra virgin olive oil and virgin olive oil do not exceed 0.8%
and 2%, respectively. No acidity in the oil was detected after the
neutralization step because the free fatty acids are removed by this
process (Ranken et al., 1997).
Analysis CO NO DO BO
*
PV (meq/kg) 1.00 ± 0.00 a
1.00 ± 0.00a
0.60 ± 0.00b
0.40 ± 0.00c
p-Anisidine 0.36 ± 0.02a 0.79 ± 0.04b 0.64 ± 0.01b 1.07 ± 0.02c
CD** 1.17 ± 0.01a 1.39 ± 0.05b 1.29 ± 0.02b 1.30 ± 0.07b
Values are reported as the mean of three replicates ± standard deviation. Means in a
line with different letters are significantly different according to the Tukey test
(P 6 0.05).
*
Peroxide value.
**
Conjugated dienes.
The determination of iodine is useful as a measure of the degree
of unsaturation of the oils and to give some idea of their oxidative
stability. The values obtained showed no significant difference
between treatments (Table 1). Tsaknis et al. (1998) and Anwar,
Nahid, and Rashid (2006) reported values of 66.83 and 65.86 cg I/
g oil, respectively, in M. oleifera, while Manzoor et al. (2007)
obtained a value of 67.00 cg I/g oil for M. concanensis. These values
are similar to those reported in this work of 64.63 cg I/g oil on
average.
The saponification value showed an upward trend during the
refining process, with a significant difference in each step. This
value describes the average molecular weight of the fatty acids
in the lipids. The increase could be due to low molecular weight
molecules being removed during refining, which would signifi-
cantly raise the average value. Anwar et al. (2006) and Lalas and
Tsaknis (2002) reported values of 186.30 and 188.36 mg KOH/g
oil, respectively, which are very close to that of the DO. Similarly,
the saponification value of olive oil has a range of 189.5–
191.5 mg KOH/g oil (Firestone, 2005).
3.2. Oxidation indexes
The amount of oxidation products in the oil of M. oleifera chan-
ged during the refining process (Table 2). The peroxides content
decreased, coupled with a slight increase in the p-anisidine value;
the conjugated dienes did not differ significantly between the
refining steps, except in the CO. This value indicates the degree
of primary oxidation; the effect of this oxidation is a rearrange-
ment of the double bond structure, promoted by light, heat,
enzymes, or metals (Shahidi & Zhong, 2005, chap. 8). The value
of conjugated dienes in the refined oil of the present study was
1.30, which is less than values reported by Tsaknis (1998) for
Moringa peregrina (1.66) and Anwar et al. (2006) for M. oleifera
(1.92), but similar to those of Anwar et al. (2007) for M. oleifera
seed oil (1.38).
The peroxide values show that there are significant differences
between the different stages of refining. The refining has a positive

Table 1
Physicochemical analysis of M. oleifera oil during the refined process.

Properties CO NO DO BO
a b c
Viscosity (Pa s) 42.8 ± 2.99 57.54 ± 2.17 64.64 ± 1.32 62.62 ± 0.57c
Density (g/mL) 0.89 ± 0.01a 0.91 ± 0.02b 0.92 ± 0.01b 0.94 ± 0.00b
RI* 1.464 ± 0.00a 1.464 ± 0.00a 1.465 ± 0.00ab 1.466 ± 0.00b
FFA** (%) 1.29 ± 0.01 ND ND ND
IV  (g I/100 g) 63.94 ± 0.10a 64.51 ± 0.44a 64.92 ± 0.75a 65.16 ± 0.49a
SN   (mg KOH/g) 160.62 ± 0.42a 188.43 ± 1.69b 190.58 ± 0.88bc 195.68 ± 4.01c
Carotenoids 6.5 ± 0.50c 2.0 ± 0.02b 3.1 ± 0.10c 1.2 ± 0.15b

Values are reported as the mean of three replicates ± standard deviation. Means in a line with different letters are significantly different under the test of Tukey (P 6 0.05).
Viscosity and density were measured at 20 °C.
CO: crude oil; NO: neutralized oil; DO: degummed oil; BO: bleaching oil.
*
Refractive index.
**
Free fatty acids.
 
Iodine value.
  
Saponification number.
56 D.I. Sánchez-Machado et al. / Food Chemistry 187 (2015) 53–57

effect on the oxidative stability of the oil. Nzikou et al. (2009) and in a-linolenic acid together with a decrease in saturated fatty acids
Anwar and Rashid (2007) reported peroxide values of 1.67 and reduces platelet aggregation, triglyceride levels, and blood pres-
1.27 meq/kg of oil, respectively; these values are higher than those sure (Wijendran & Hayes, 2004). The refining process does not
obtained in the present study. Shahidi and Zhong (2005, chap. 8) affect the essential fatty acids in the oil, leaving them available
noted that the increase in this value might be caused by low for consumption and maintaining the nutritional value.
temperatures during the last phase of ripening in seeds or by the
use of high temperatures in the process of oil extraction.
3.5. Oxidative stability of the of M. oleifera seed oils
The p-anisidine value increased from 0.36 to 1.07 during the
refining process (Table 2). p-Anisidine reacts with secondary oxida-
The primary and secondary oxidations of the oils subjected to
tion products, i.e., aldehydes and ketones, so it serves as a standard
heat treatment under a forced air stream were examined (Fig. 1).
test for the rapid characterization of oils. A value of less than 1.07
The CO, NO, and DO samples showed high primary oxidation sta-
was obtained for M. oleifera. Manzoor et al. (2007) obtained a value
bility, as measured by the concentration of conjugated dienes.
of 1.84 for M. oleifera, while Latif and Anwar (2008) reported a
For the secondary oxidation, BO had the lowest values.
value of 1.82 for M. concanensis. The different values may be due
No tests were performed with the addition of an external
to the influence of various agents or oxidation catalysts as air
antioxidant. Carotenoids and natural antioxidants, such as pheno-
and light (UV). Oxidative deterioration can often be prevented or
lic compounds, present in the CO, NO, and DO samples helped
minimized by adding antioxidants such as butylated hydrox-
reduce free radicals, thereby protecting the unsaturated fatty acids
yanisole (BHA) and butylated hydroxytoluene (BHT). However,
(Akoh & Min, 2008, chap. 12). Abuzaytoun and Shahidi (2006)
adding these compounds increases the cost of processing
mentioned that the oils treated with adsorbents are devoid of
(Gustone, 2005, chap. 6). Also consumers do not want these
minor components such as pigments, tocopherols, and phenolic
synthetic antioxidants in the premium products.
compounds. For this reason, the BO presented the greatest value
for conjugated dienes.
3.3. Carotenoid content
Furthermore, the BO showed greater stability in terms of the
secondary oxidation. The other oils underwent oxidation that
The concentrations of carotenoids found in the oils were 6.5,
culminated in the production of malondialdehyde, as detected by
3.1, 2.0, and 1.2 g/kg oil for the CO, NO, DO, and BO, respectively.
the TBARS test, after oxidation of the substances that protected
The pigment concentration in the oil was reduced after each step
the fatty acids from primary oxidation (Khan & Shahidi, 2001).
of the refining, and a large change in the amount of carotenoids
was noted after the neutralization process. The last step of refine-
ment reduced the amount of carotenoids because bleaching
removes these pigments (Ranken et al., 1997). The crude oil shows
the typical absorption spectrum of carotenoids in solution.
Rodriguez-Amaya (2001, chap. 2) reported the maximum absorp-
tion of these pigments to be approximately 450 nm.

3.4. Fatty acid profile of the M. oleifera seed oils

The composition of fatty acids in the M. oleifera oil at different

stages of refining is shown in Table 3. In general, the oils exhibit
a fatty acid profile with oleic acid (C18:1n9) as the most prevalent
fatty acid, while a-linolenic acid (C18:3n3) was found in a smaller
proportion (Lalas & Tsaknis, 2002). Among the oils obtained, sig-
nificant differences were found only in the stearic acid (C18:0)
and a-linolenic acid contents. Gustone (2005, chap. 6) noted that
oleic acid represents 92% of cis-monounsaturated fatty acids in
vegetable oils, and Chan, Bruce, and McDonald (1991) mentioned
that this acid, together with a-linolenic acid and linoleic acid, is
responsible for reducing heavy cholesterol (LDL). In this way, the
M. oleifera seed oil can be considered a nutraceutical food.
Likewise, it has been reported that the combination of an increase

Table 3
Fatty acid profiles of the oils from M. oleifera during the refining process.

Fatty acid CO NO DO BO
C16:0 7.87 ± 0.27a 7.55 ± 0.16a 7.55 ± 0.18a 7.69 ± 0.19a
C16:1n7 2.03 ± 0.14a 1.98 ± 0.03a 1.94 ± 0.07a 1.96 ± 0.07a
C18:0 6.84 ± 0.14a 7.07 ± 0.05a,b 7.44 ± 0.18b,c 7.23 ± 0.14c
C18:1n9 71.09 ± 0.90a 71.84 ± 0.47a 71.39 ± 0.40a 71.22 ± 0.71a
C18:2n6 0.92 ± 0.31a 0.70 ± 0.02a 0.63 ± 0.08a 0.76 ± 0.17a
C18:3n3 0.57 ± 0.18a 0.36 ± 0.09a,b 0.30 ± 0.05b 0.34 ± 0.05a,b
C20:0 4.62 ± 1.99a 3.48 ± 0.19a 3.58 ± 0.14a 3.64 ± 0.20a
C20:1n9 1.65 ± 0.03a 1.59 ± 0.08a 1.53 ± 0.04a 1.60 ± 0.07a
C22:0 5.40 ± 0.32a 5.44 ± 0.30a 5.63 ± 0.34a 5.58 ± 0.47a

Values are reported as the mean of three replicates ± standard deviation. Means in a
line with different letters are significantly different according to the Tukey test Fig. 1. Oxidative stability of the M. oleifera oils as determined by the conjugated
(P 6 0.05). dienes content and the TBARS method.
 D.I. Sánchez-Machado et al. / Food Chemistry 187 (2015) 53–57
Thus, oil with a greater degree of refining showed a significant
increase in oxidative stability during typical storage (Abuzaytoun
& Shahidi, 2006).
4. Conclusions
A simple refining process improved the storage characteristics
and shelf life of M. oleifera oil. The fatty acid profile of the oil is
not affected by the various refining stages. The bleached oil
showed an improved oxidative stability. However, the DO pre-
sented similar values. The results indicate that a refining process
that includes only the steps of degumming and neutralization
allows a good edible oil to be obtained.
Acknowledgments
This research was financed under Project no PROFAPI-00334
from Instituto Tecnológico de Sonora.
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