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Jiao 2014
Jiao 2014
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
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
18
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
19
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).
20
the second order polynomial model was applied to express the oil
yield as the following equation:
YO
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.
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.
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
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.
22
(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.
(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).
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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