Food Chemistry 147 (2014) 1724

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Microwave-assisted aqueous enzymatic extraction of oil

from pumpkin seeds and evaluation of its physicochemical
properties, fatty acid compositions and antioxidant activities
Jiao Jiao a, Zhu-Gang Li c, Qing-Yan Gai b, Xiao-Juan Li c, Fu-Yao Wei b, Yu-Jie Fu a,b,, Wei Ma a,d,
a

State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, PR China
State Engineering Laboratory of Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, PR China
Biotechnology Research Center, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, PR China
d
School of Pharmaceutical, Heilongjiang University of Chinese Medicine, Harbin 150040, PR China
b
c

a r t i c l e

i n f o

Article history:
Received 27 October 2012
Received in revised form 30 June 2013
Accepted 13 September 2013
Available online 1 October 2013
Keywords:
Microwave-assisted aqueous enzymatic
extraction
Pumpkin (Cucurbita maxima) seed oil
Physicochemical properties
Fatty acid compositions
Antioxidant activities

a b s t r a c t
Microwave-assisted aqueous enzymatic extraction (MAAEE) of pumpkin seed oil was performed in this
study. An enzyme cocktail comprised of cellulase, pectinase and proteinase (w/w/w) was found to be
the most effective in releasing oils. The highest oil recovery of 64.17% was achieved under optimal conditions of enzyme concentration (1.4%, w/w), temperature (44 C), time (66 min) and irradiation power
(419 W). Moreover, there were no signicant variations in physicochemical properties of MAAEEextracted oil (MAAEEO) and Soxhlet-extracted oil (SEO), but MAAEEO exhibited better oxidation stability.
Additionally, MAAEEO had a higher content of linoleic acid (57.33%) than SEO (53.72%), and it showed
stronger antioxidant activities with the IC50 values 123.93 and 152.84, mg/mL, according to DPPH radical
scavenging assay and b-carotene/linoleic acid bleaching test. SEM results illustrated the destruction of
cell walls and membranes by MAAEE. MAAEE is, therefore, a promising and environmental-friendly technique for oil extraction in the food industry.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Pumpkin (Cucurbita maxima), a very common member of Cucurbitaceae family, grows widely in temperate and subtropical regions
over the world. This plant has been employed in the food industry
for the production of purees, juices, jams and alcoholic beverages.
Pumpkin seeds as a rich source of bioactive compounds have been
used frequently as functional foods or medicines. Moreover, pumpkin seed oil has gained attention not only as edible oil, but also as a
potential nutraceutical. Pumpkin seed oil has been implicated in
providing a broad spectrum of health benets such as prevention
of prostate disease, retardation of the progression of hypertension,
mitigation of hypercholesterolemia and arthritis, reduction of
bladder and urethral pressure and improving bladder compliance,
alleviation of diabetes by promoting hypoglycaemic activity, lowering the levels of gastric, breast, colorectal and lung cancers, and
possessing good antioxidant potential (Fruhwirth, Wenzl, El-Toukhy, Wagner, & Hermetter, 2003; Fu, Shi, & Li, 2006; Rezig,

Corresponding authors at: State Key Laboratory of Tree Genetics and Breeding,
Northeast Forestry University, Harbin 150040, PR China. Tel./fax: +86 451
82190535 (Y.-J. Fu), +86 451 82193430 (W. Ma).
E-mail addresses: yujie_fu2011@yahoo.com (Y.-J. Fu), mawei@hljucm.net (W.
Ma).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.09.079

Chouaibi, Msaada, & Hamdi, 2012; Stevenson et al., 2007). Such

health benets mean that the effective extraction technologies
are required to ensure the quality of pumpkin seed oil.
Inert organic solvents are frequently used to extract oil from
seeds. However, regulatory problems associated with use and disposal of organic solvents have undesirable effects on oil quality and
costs. Environmental safety regulations and public health risks
necessitate the food industry consider alternatives to organic solvents in the oil extraction (Mitra, Ramaswamy, & Chang, 2009).
The development of non-hazardous alternative is vitally important
for the continued and sustainable growth of food enterprise. Aqueous-based solvent systems are increasingly preferred in the place
of traditional organic solvents. Aqueous enzymatic extraction
(AEE) technology is thought to be environmentally-friendly, cheap
and safe, and has been applied for the extraction of oils from seed
crops such as canola (Latif, Diosady, & Anwar, 2008), Kalahari melon (Nyam, Tan, Lai, Long, & Man, 2009), grape (Passos, Yilmaz, Silva, & Coimbra, 2009), sunower (Latif, & Anwar, 2009), olive
(Najaan, Ghodsvali, Khodaparast, & Diosady, 2009), sesame (Latif,
& Anwar, 2011), ax (Long et al., 2011), bayberry (Zhang et al.,
2012), peanut (Jiang, Hua, Wang, & Xu, 2010), wheat (Li, Song,
Zhou, Wang, & Cao, 2010), soybean (Towa, Kapchie, Hauck, & Murphy, 2010) and pumpkin (Qi, 2012; Wang, Xu, Yang, & Sun, 2011;
Wang, Zhang, Ji, & Yan, 2011). In previous studies, various enzymes

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J. Jiao et al. / Food Chemistry 147 (2014) 1724

were used to hydrolyse structural polysaccharides of cell walls and

proteins associated with lipid bodies to enhance oils release. However, one major disadvantage associated with the process is the relatively low efciency, which can be overcome with the aid of
accelerated enzyme-catalysed reaction technologies. Microwave
irradiation, which has proved to be a clean, efcient and convenient energy source, has been widely utilised in accelerated enzyme-catalysed reactions for natural products and oils extraction
(Ji, Zhou, & Liu, 2010; Jiao et al., 2012; Yang et al., 2010; Zhang,
2012; Zhang et al., 2012). Herein, microwave-assisted aqueous
enzymatic extraction (MAAEE) is proposed for the extraction of
oil from pumpkin seeds.
The objective of this study was to investigate the extraction of
oil from pumpkin seeds using the MAAEE method. The physicochemical properties, fatty acid compositions and antioxidant activities of the extracted oil were evaluated and compared with those
extracted with an organic solvent. In addition, the microscopic
structures of seed samples before and after various extraction procedures were observed to clarify extraction mechanisms.
2. Materials and methods
2.1. Plant materials and reagents
Pumpkin seeds were provided by Heilongjiang Academy of
Agricultural Sciences, Harbin, Heilongjiang Province, China. The
seeds were dried in a vacuum drier at 75 C and 25 kPa for 24 h
to a moisture content of 2%, and ground into a powder. After passing through a series of standard-mesh sieves, the powders were
sealed in plastic containers and stored in a refrigerator at 4 C until
extraction.
Cellulase (E.C.3.2.1.1, P 400 U/mg), hemicellulase (E.C.3.1.1.73,
0.33.0 U/mg), pectinase (E.C.3.2.1.15, P 500 U/mg), b-glucosidase
(E.C.3.2.1.21, P 200 U/mg) and neutral proteinase (E.C.3.4.24.39,
50 U/mg) were purchased from Shanghai WeeBeyond Scientic &
Trade Co. (Shanghai, China). The HS-508 foam suppressor was
supplied by Shanghai Hengsheng Chemicals Co. (Shanghai, China).
Linoleic acid, b-carotene, p-anisidine and DPPH were obtained
from SigmaAldrich Co. (Steinheim, Germany). Additionally, other
reagents either of analytical or optical grades were purchased from
Beijing Chemical Reagents Co. (Beijing, China). Ultrapure water
was prepared from a Milli-Q system at the 18.3 MX resistance
(Millipore, Bedford, MA).
2.2. Soxhlet extraction (SE) process
10 g of ground pumpkin seeds (80-mesh) were extracted with
100 mL of n-hexane in a Soxhlet extractor at 90 C for 6 h, as described in the Soxhlet standard extraction method (AOAC, 1997).
The n-hexane was removed at 50 C under reduced pressure using
a rotary evaporator and the oil was dried at 105 C to a constant
mass under nitrogen in a drying oven. This technique produced
44.61 0.29 g of oil per 100 g of seeds, which was set as 100% oil
recovery for comparison. The recovered oil was stored at 4 C until
use.
2.3. Microwave-assisted aqueous enzymatic extraction (MAAEE)
process
A MAS-II (2450 MHz) microwave accelerated reaction system
manufactured by Shanghai Sineo Microwave Products Company
(Shanghai, China) was used in this study. It is a mono-mode open
microwave solvent extraction system, which includes a time controller, an infra-red temperature sensor, an electromagnetic stirrer
and a circulating water-cooling system. Time, temperature, power

and stirring rate were controlled through an electronic control panel. Temperature was monitored by an infra-red temperature sensor and controlled by feedback to the regulator. The extraction
ask was placed in the microwave resonance cavity and connected
to the cooling system through a hole at the top of microwave accelerated reaction system.
A known amount of enzymes and 10 g of ground seed materials
(80-mesh) were introduced into the reaction ask (100 mL) with
60 mL water and pH was adjusted to 5 with citric acid. The reaction
ask was placed symmetrically in the microwave resonance cavity
and connected to the cooling system. A magnetic stirrer bar was
placed in the ask to ensure thorough mixing (200 rpm). Based
on the results of our preliminary experiments (data not shown),
the particle size 80-mesh, liquid/solid ratio 6:1 (mL/g), pH 5 and
stirring speed 200 rpm were optimal. The extraction process was
performed under a range of conditions according to the experimental design. After extraction, the solution was centrifuged at
9392g for 15 min to separate the oil-rich and emulsion phases.
The oil was withdrawn using a micropipette and the emulsion
demulsied with foam suppressor HS-508 and further centrifuged
to obtain any residual oil. The oils were combined, weighed and the
extraction yield expressed as percent ratio (%, g/g) MAAEE oil extracted: total oil obtained using SE. Control samples underwent
the same extraction process except for the addition of enzymes.
2.4. Experimental design of MAAEE process
Response surface methodology (RSM) was applied to identify
optimum levels of four key independent variables including
extraction temperature (C), enzyme concentration (%, w/w),
power (W) and time (min). On the basis of preliminary monofactor tests (data not shown), a BoxBehnken design (BBD) was
used to survey effects of independent variables at three levels on
the dependent variable (oil yield). A total of 30 randomized experiments including 24 factorial and 6 zero-point tests were designed.
The regression analysis was carried out to evaluate the response
function as a quadratic polynomial:

Y b0

k
X
j1

bj X j

k
X
XX
bjj X 2j
bij X i X j k 4
j1

i<j

where, Y is the predicted response; b0, bj, bjj and bij are the regression coefcients for intercept, linearity, square and interaction,
respectively; Xi and Xj are the independent coded variables; and k
represents the number of variables. The actual and coded levels of
the independent variables used in the experimental design are summarised in Table S1. The experiment data were analysed statistically with Design-Expert 7.0 (State-Ease, Inc., Minneapolis MN).
Analyses of variance (ANOVA) were performed to compare and
determine the optimal conditions for MAAEE.
2.5. Physicochemical properties
Specic gravity, acid, saponication, and iodine values of pumpkin seed oils were determined by AOCS Standard Methods (1997).
The refractive indexes of oils at 25 C were measured using an
ATAGO N3 hand-held refractometer (Atago Co. Ltd., Japan). Conjugated diene (CD), peroxides (PV) and p-anisidine (PAV) were measured as indexes for the oxidative stability of pumpkin seed oil
using IUPAC methods (Paquot & Hautfenne, 1987).
2.6. GCMS analyses of fatty acid compositions
The fatty acid compositions of pumpkin seed oils were analysed
by GCMS. Prior to injection, the extracted oils were converted to
their fatty acid methyl esters (FAMEs) according to the method

J. Jiao et al. / Food Chemistry 147 (2014) 1724

reported by Zhang et al. (2010). GCMS analysis of FAMEs were

performed by a Varian 450-GC/240-MS gas chromatography/mass
spectrometer (Varian, Santa Clara, CA, USA), equipped with a
DB-WAX capillary column (30 m  250 lm  0.25 lm lm thickness). The detail operating conditions were carried out as follows:
helium gas ow rate 3 mL/min; split ratio 1:10; injector temperature 250 C; injection volume 1 lL; oven temperature progress
from 150 to 210 C at the rate of 15 C/min, from 210 to 226 C
at the rate of 1 C/min; detector temperature 300 C; ion source
temperature 200 C; ionisation mode used at electronic impact
70 eV; mass range 50500 m/z. Identication of chemical constituents of pumpkin seed oils was based on the comparisons of their
retention indices and mass spectra with publish data and computer matching the mass spectra fragmentation patterns with
those stored in mass spectral library NIST05 provided by the software of GCMS system. Relative percentage compositions of oils
were calculated from the total ion chromatograms by a computerized integrator.

19

3. Results and discussion

3.1. Selection of appropriate enzyme for MAAEE process
As presented in Fig. 1, more oil was recovered using hydrolytic
enzymes (29.4259.88%) compared with the control (28.43%, no
enzyme). Cellulase, pectinase and proteinase increased oil recovery
signicantly whilst hemicellulase and b-glucosidase had only a
weak effect. A cocktail comprised of cellulase, pectinase and proteinase (1/1/1, w/w/w) was used for subsequent work and yielded
59.88 1.27%. The effective action of the selected enzyme cocktail
during aqueous extraction can be attributed to degradation of cellulose, pectin and protein, breaking down the structural integrity
of the seed cell walls (Pericin, Radulovic, Trivic, & Dimic, 2006).
In addition, the enzymatic treatment may breakdown the protein
networks of cotyledon cells and oleosin-based membranes that
surround lipid bodies, which liberates more oils.
3.2. Optimisation of MAAEE process

2.7. Total tocopherols (TT) and total phenolics (TP) contents

The TT content of pumpkin seed oil was determined according
to the colorimetric method described previously by Wong, Timms,
and Goh (1988). A calibration curve of a-tocopherol in toluene was
performed in the concentration ranges of 0240 mg/mL. Results
were expressed in mg of a-tocopherol per kilogram of oil. The TP
content was determined spectrophoto-metrically using the Folin
Ciocalteaus reagent according to the previous method reported
by Capannesi, Palchetti, Mascini, and Parenti (2000). A calibration
curve of gallic acid in methanol was carried out in the concentration ranges of 0.040.40 mg/mL. Results were expressed in mg of
gallic acid per kilogram of oil.

2.8. Determination of antioxidant activities

Scavenging activities of oils towards DPPH radicals were determined according to the pervious method reported by Long et al.
(2011). The b-carotene/linoleic acid bleaching test was carried
out as described by Liu et al. (2009). Antioxidant activities of the
tested pumpkin seed oils are reported as IC50 values, which were
calculated using logarithmic regression curves for DPPH radical
scavenging or b-carotene bleaching inhibition percentage (%) versus the concentration of tested oils (mg/mL).

MAAEE parameters including extraction temperature, enzyme

concentration, power and extraction time were optimised by
RSM using BBD to obtain the maximum yield of pumpkin seed oils.
All the experimental data and the predicted data from the established response surface analysis model are shown in Table S1.
3.2.1. Fitting the mathematical model
As the results of ANOVA for the quadratic model shown in
Table S2, the p-value of model (p < 0.0001) implies the model is
signicant, and the p-value of lack of t model (p = 0.1531) implies
the lack of t model is not signicant relative to the pure error, and
the determination coefcient of model (R2 = 0.9644) suggests the
mode is applicable. Moreover, the signicance of each coefcient
of the model was also determined using the t-test and p-value.
As the results in Table S2 illustrate, factors with signicant effects
on oil yield (p < 0.05) were linear terms of X1, X2, X3 and X4, interaction term of X3X4 and quadratic terms of X 21 ; X 22 ; X 23 and X 24 , while
other ve interaction terms (X1X2, X1X3, X1X4, X2X3 and X2X4) were
not signicant (p > 0.05). In stating the obvious, signicant might
be expected to have greater impact and insignicant less. Hence,

2.9. Scanning electron micrographs (SEM)

A Quanta-200 environmental scanning electron microscope system (FEI Company, USA) was used to examine morphological alterations of pumpkin seed samples before and after extraction. The
samples were xed on a specimen holder with an aluminium tape
and sputtered with a thin layer of gold prior to examination under
high vacuum condition at an accelerating voltage of 12.5 kV
(20 lm, 1000  magnication).

2.10. Statistical analyses

Results were expressed as means standard deviations. The
data were statistically analysed using the SPSS statistical software,
version 17.0 (SPSS Inc., Chicago, IL, USA). Differences between
means were determined by analysis of variance (ANOVA) with Tukeys HSD post hoc test on the level of signicance declared at
p < 0.05.

Fig. 1. The effects of different enzymes on the oil yield by MAAEE. Con: control; Cel:
cellulase; Pec: pectinase; Hem: hemicellulase; Glu: b-glucosidase; Pro: proteinase;
and EC: enzyme cocktail (cellulase/pectinase/proteinase = 1/1/1, w/w/w). The other
parameters were set as follows: enzyme concentration 1.0%, irradiation power
400 W, temperature 40 C and extraction time 60 min. Control was the samples
prepared and treated identically with MAAEE process except for enzymes addition.
Mean SD values not sharing the same lowercase letters are signicantly different
(p < 0.05).

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J. Jiao et al. / Food Chemistry 147 (2014) 1724

the second order polynomial model was applied to express the oil
yield as the following equation:

YO

63:34  2:11X 1 3:27X 2  2:07X 3 1:45X 4  2:42X 3 X 4

12:99X 21  1:97X 22  6:51X 23  2:01X 24

where Yo is the oil yield (%); X1 is the extraction temperature (C);

X2 is the enzyme concentration (%, w/w); X3 is the irradiation power
(W); and X4 is the extraction time (min).
3.2.2. Analysis of the response surface
As shown in Fig. 2a, temperature and irradiation power had
signicantly effect on oil recovery, and it demonstrated that
extraction temperature around 45 C and irradiation power around
420 W resulted in high oil yield. As shown in Fig. 2b, at a xed
enzyme concentration, oil yield increased with rising temperature
as the rate of enzyme-catalysed reactions increased with

increasing temperature. However, at higher temperatures (45

55 C), the yield decreased signicantly as enzymes, which are proteins, became denatured at higher temperatures. A similar trend in
oil recovery was observed for microwave power (Fig. 2c). Higher
power led to localised increased temperatures, deactivating the
enzymes. It was observed the medium enzyme concentration
around 1.25% favored extraction, as shown in Fig. 2d. With
increasing time, at higher enzyme concentrations (1.251.50%),
recovery increased slightly. Generally, the more enzymes used,
the faster the extraction process. However, the enzyme concentration had to be determined carefully to achieve the optimal
balance between oil recovered and cost. As observed in
Fig. 2e and f, at a given extraction temperature or microwave
power, oil recovery increased with extended time initially but
the yield stabilized with prolonged extraction, which might be
due to depletion of the substrates and/or product inhibition of
enzymes.

Fig. 2. Response surface plots presenting correlative effects of irradiation power and temperature (a), temperature and enzyme concentration (b), enzyme concentration and
irradiation power (c), time and enzyme concentration (d), temperature and time (e), and irradiation power and time (f) on the oil yield by MAAEE.

J. Jiao et al. / Food Chemistry 147 (2014) 1724

21

Based on the mathematical model built, the optimal experimental conditions were as follows: extraction temperature 43.96 C,
enzyme concentration 1.40% (w/w), irradiation power 419.13 W
and extraction time 65.63 min. Considering the actual operation,
extraction temperature, power and extraction time were modied
to 44 C, 419 W and 66 min, respectively.
3.2.3. Verication of the predictive model
The reliability of the theoretical model was veried under optimal parameters. A yield of 64.17 0.42% was obtained from these
experiments, which was a good t for the value forecasted (65.33%)
by the regression model. Therefore, the oil extraction conditions
achieved by RSM were reliable and practical.
3.3. Physicochemical properties
The physicochemical properties of MAAEE-extracted oil
(MAAEEO) and SE-extracted oil (SEO) are illustrated in Table 1.
No signicant differences (p > 0.05) were observed for the refractive index, specic gravity, acid or saponication values from
either method. The values were similar to those reported by Rezig
et al. (2012). Additionally, MAAEE yielded an oil higher (p < 0.05) in
iodine value (116.26 0.47 g I2/100 g oil) compared with SE
(113.81 0.32 g I2/100 g oil), which suggests a higher content of

Table 1
Physicochemical properties of pumpkin seed oils obtained by different methods.

Refractive index (25 C)

Specic gravity (25 C, g/mL)
Acid value (mg KOH/g oil)
Saponication value (mg KOH/g oil)
Iodine value (g I2/100 g oil)
Conjugated dienes (CD) (%)
Peroxide value (PV) (meq/kg)
p-Anisidine value (PAV)

MAAEEO

SEO

1.47 0.00
0.91 0.00
6.97 0.09
183.37 0.96
116.26 0.47
3.12 0.07
2.46 0.03
1.07 0.01

1.46 0.00
0.92 0.01
7.08 0.13
184.41 1.70
113.81 0.32
3.57 0.06
2.84 0.08
1.37 0.04

MAAEEO, MAAEE-extracted oil; SEO, SE-extracted oil.

Fig. 4. Antioxidant activities of the oils assessed by DPPH radical scavenging assay
(a) and b-carotene/linoleic acid bleaching test (b). MAAEEO: MAAEE-extracted oil;
SEO: SE-extracted oil.

Fig. 3. GCMS analyses of fatty acid compositions (% of total fatty acids) of the oils obtained by different means. MAAEEO: MAAEE-extracted oil; SEO: SE-extracted oil.

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J. Jiao et al. / Food Chemistry 147 (2014) 1724

(a)

(b)

(c)

(d)

Fig. 5. SEM of pumpkin seed samples: non-extraction (a), SE (b), MAEE (c), and MAAEE (d). MAEE: microwave-assisted aqueous extraction was performed identically with
MAAEE under optimal conditions except for the enzyme addition.

unsaturated fatty acids. Moreover, 3.12 0.07% of CD,

2.46 0.03 meq/kg of PV and 1.07 0.01 of PAV from MAAEEO
were found to be signicantly (p < 0.05) lower than 3.57 0.06%
of CD, 2.84 0.08 meq/kg of PV and 1.37 0.04 of PAV from SEO,
respectively. CD, PV and PAV are widely employed indices for the
measurement of oxidative degradation products in oils. The oxidative stability of MAAEEO in terms of CD, PV and PAV was signicantly better than SEO, which can probably be attributed to the
elevated temperature and prolonged duration of SE processing.
Consequently, the results of physicochemical properties demonstrated that MAAEE produced an oil of higher quality than the conventional method.
3.4. Fatty acid compositions
As shown in Fig. 3, nine main components, ve saturated fatty
acids (SAFA), two monounsaturated fatty acids (MUFA) and two
polyunsaturated fatty acids (PUFA) were identied. Among them,
palmitic acid C16:0 (12.0513.71%) and stearic acid C18:0 (5.72
5.99%) were the most predominant SAFA, oleic acid C18:1
(23.9024.63%) was the principal MUFA, and linoleic acid C18:2
(53.7257.33%) was the main PUFA. These values are close to those
reported by Liu, Zhang, and Cui (2003), Mitra et al. (2009) and
Rezig et al. (2012). However, there are slight differences in fatty
acid compositions between these results and those reported by
Fan, Yuan, Liu, Feng, and Mao (2010) and Wang et al. (2011). This
might due to different cultivars, climatic conditions and post
harvest treatments.
Differences in the amounts of the four fatty acids between
MAAEEO and SEO were found to be statistically insignicant

(p > 0.05) except for linoleic acid (p < 0.05). The relatively greater
proportion of linoleic acid (57.33 0.41%) was produced by
MAAEE. It is worth mentioning that linoleic acid possesses favourable nutritional and physiological effects in the prevention of coronary heart diseases and cancers (Oomah, Ladet, Godfrey, Liang, &
Girard, 2000). Therefore, MAAEE offers health benets in addition
to those of reduced costs and increased yield over that of SE.
3.5. TT and TP contents
Plants are rich sources of natural antioxidants of which tocopherols and phenolics are considered to be the best-known components. They are considered to be primary chain breaking
antioxidants in free radical chain reactions or converting lipid radicals to more stable products, thus improving antioxidant potentials of plant oils (Fruhwirth et al., 2003; Rezig et al., 2012). In
this work, signicantly (p < 0.01) higher contents of TT
(856.81 9.27 mg a-tocopherol/kg oil) and TP (128.84 1.36 mg
gallic acid/kg oil) were determined in MAAEEO compared with
SEO (772.31 12.64 mg a-tocopherol/kg oil for TT and
73.32 0.77 mg gallic acid/kg oil for TP), respectively. The hydrolysis of pumpkin seed cell walls by enzymatic preparations during oil
extraction may cause release of greater amounts of tocopherols
and phenolics resulting in a much higher availability of such bioactive components into oils (Chiacchierini, Mele, Restuccia, & Vinci,
2007; Latif & Anwar, 2009, 2011; Ranalli, Pollastri, Contento, & Iannucci, 2003). Moreover, enzymatic hydrolysis can reduce the interaction of antioxidants with the seed polysaccharides, proteins and
pectins, enhancing their release into the oil phase (Ranalli, Malfatti,
Lucera, Contento, & Sotiriou, 2005).

J. Jiao et al. / Food Chemistry 147 (2014) 1724

3.6. Antioxidant activities

Antioxidant activities of oils prepared with MAAEE and SE were
assessed using DPPH radical-scavenging assay and b-carotene/linoleic acid bleaching test. DPPH is commonly used to test antioxidant capacity. As illustrated in Fig. 4a, DPPH radical-scavenging
activities rose with increasing concentration of the extracted oils.
However, MAAEEO exhibited superior efcacy in scavenging the
DPPH radicals (p < 0.05, IC50 123.93 1.85 mg/mL) compared with
SEO (150.38 1.07 mg/mL). The b-carotene/linoleic acid bleaching
method is commonly used to evaluate inhibition of lipid peroxidation. As shown in Fig. 4b, inhibition of b-carotene bleaching was
dose dependent. The lipid peroxidation inhibitory activity of
MAAEEO (152.84 2.34 mg/mL) was signicantly (p < 0.05) higher
than SEO (183.26 3.79 mg/mL). Thus, oil extracted by MAAEE
exhibited superior antioxidant activities compared with oil obtained using SE in both aqueous and lipid-based systems.
Several studies have demonstrated that the antioxidant potentials of plant oils can be attributed to the PUFA, tocopherols and
phenolics (Fruhwirth et al., 2003; Latif & Anwar, 2011; Rezig
et al., 2012; Zhang et al., 2010). MAAEEO exhibited higher antioxidant activity than SEO, which can be partly explained by the higher PUFA (57.65%) in MAAAEO than that in SEO (53.90%). In
addition, the novel MAAEE method can enhance the release of
phenolics and tocopherols from the pumpkin seed into oils, thus
also contributing to the superior antioxidant activities to such oils
as against that of conventional SEO. These results are consistent
with the data for aqueous enzymatic extraction of sesame and axseed oils (Latif & Anwar, 2011; Long et al., 2011).
3.7. SEM observation
To better understand the mechanism for the MAAEE of oils from
pumpkin seeds, structural changes in samples before and after
extraction were examined by SEM. As illustrated in Fig. 5a, the intact oil cells were presented on the surface of seed tissues before
extraction. These oil cells disappeared following SE (Fig. 5b), but
the external surface of seed tissues was still intact and smooth.
In the case of microwave-assisted aqueous extraction (MAAE)
without addition of enzymes (Fig. 5c), a partial destruction of the
morphological structure of seed tissues was observed. However,
MAAEE caused signicantly structural changes to the seed tissues,
where most cells were crimped, disorganised and broken (Fig. 5d).
With SE, the mass transfer was affected by the chemical afnity between organic solvents and oils. In contrast, MAAEE of oils is based
on the insolubility of oils in water. Therefore, the destruction of cell
walls and oleosin-based membranes with the help of hydrolytic
enzymes and microwave irradiation was benecial for releasing
oils, which were previously bound in the plant cell structure.
4. Conclusions
In the present work, MAAEE technique was optimised to
achieve high-performance extraction of pumpkin seed oil. The
highest oil recovery of 64.17% was obtained when pumpkin seeds
were treated with 1.4% (w/w) of an enzyme cocktail (cellulase/pectinase/proteinase = 1/1/1, w/w/w) for 66 min, at 44 C and 419 W.
Comparisons of the physicochemical characteristics of MAAEEO
with those of SEO revealed similar refractive indexes, specic gravity, acid and saponication values but MAAEEO demonstrated better oxidation stability with lower CD (3.12%), PV (2.46 meq/kg) and
PAV (1.07). In addition, GCMS results showed that MAAEE increased amounts of valuable linoleic acid (57.33%) compared with
SE (53.72%). MAAEEO also exhibited superior efcacy in scavenging
DPPH radicals and inhibiting b-carotene bleaching, showing lower

23

IC50 values (123.93 and 152.84, mg/mL) than SEO (150.38 and
183.26, mg/mL) due to higher contents of PUFA (57.65%), tocopherols (856.81 mg a-tocopherol/kg oil) and phenolics (128.84 mg gallic acid/kg oil). Moreover, SEM results demonstrated that MAAEE
technique breakdown cell walls and membranes efciently significantly increasing the release of oils. These results suggest MAAEE
produces high-quality edible oil for the pharmaceutical and
healthy food elds. Overall, MAAEE is a promising environmental-friendly technology for oil extraction in the food industry.
Acknowledgements
The authors gratefully acknowledge the nancial supports by
Special Fund of National Natural Science Foundation of China
(31270618) and Project for Distinguished Teacher Abroad, Chinese
Ministry of Education (MS2010DBLY031).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2
013.09.079.
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