Embed Size (px)
Citation preview
ORIGINAL PAPER
Extraction and Demulsification of Oil From Wheat Germ, BarleyGerm, and Rice Bran Using an Aqueous Enzymatic Method
Xuezhi Fang • Robert A. Moreau
Received: 7 August 2013 / Revised: 24 March 2014 / Accepted: 26 March 2014
� AOCS (outside the USA) 2014
Abstract An aqueous enzymatic method was developed
to extract oil from wheat germ. Wheat germ pretreatment,
effect of various industrial enzymes, pH, wheat germ to
water ratio, reaction time and effect of various methods of
demulsification, were investigated. Pretreatment at 180 �C
in a conventional oven for 4 min reduced the moisture
12.8–2.2 % and significantly increased the oil yield. Add-
ing a combination of protease (Fermgen) and cellulase
(Spezyme CP) resulted in a 72 % yield of emulsified oil
from wheat germ (both commercial and laboratory milled
wheat germ). Using the same oil extraction conditions
optimized for wheat germ, yields of 51 and 39 % emulsi-
fied oil were obtained from barley germ (laboratory mil-
led), and rice bran, respectively. Three physical
demulsification methods (heating, freeze-thawing, and pH
adjustment) and enzymatic methods (Protex 6L, Protex 7L,
Alcalase, Fermgen, Lysomax and G-zyme 999) were
compared. After demulsification with Protex 6L, free oil
yields of 63.8 and 59.5 % were obtained with commercial
wheat germ and with laboratory milled wheat germ,
respectively. Using the same demulsification conditions
optimized for wheat germ, yields of 45.7 % emulsified oil
and 35 % free oil were obtained for barley germ and rice
bran, respectively.
Keywords Wheat germ � Barley germ � Aqueous
enzymatic oil extraction � Demulsification
Introduction
Wheat is one of the most popular grain crops in the world,
with a production of about 600 million tons each year [1].
The countries with the highest wheat production are the
United States, China and Canada. When wheat is milled,
the main components of the wheat kernel are bran, germ
and endosperm [2]. Wheat germ (WG) is a nutritious
byproduct of the wheat milling industry and it contains
8–14 % oil and about 26 % protein. Wheat germ oil
(WGO) contains high levels of linoleic acid and vitamin E
[3, 4]. Linoleic acid is an essential fatty acid and vitamin E
is a valuable antioxidant that prevents the formation of free
radicals such as hydroxyl radical and superoxide radical.
On the basis of these characteristics, WGO has been uti-
lized in health-care foods, cosmetic products and other
applications.
Traditionally, there are three common processes for
recovery of oil from seeds: hydraulic pressing, expeller
pressing and solvent extraction. Organic solvent extraction
(mostly hexane) is regarded as an efficient oil extraction
method and is widely applied in the world. But hexane has
some undesirable safety and environmental properties [5].
In view of the safety and environmental concerns associ-
ated with the utilization of hexane, in 2001, the U.S.
Environmental Protection Agency issued stricter guidelines
for hexane emission during edible oil extraction [6].
Aqueous enzymatic extraction methods have received
X. Fang � R. A. Moreau (&)
Eastern Regional Research Center, Agricultural Research
Service, US Department of Agriculture, Wyndmoor, PA 19038,
USA
e-mail: [email protected]
X. Fang
e-mail: [email protected]
X. Fang
Research Institute of Subtropical Forestry, Chinese Academy of
Forestry, State Forestry Administration, Fuyang 311400,
People’s Republic of China
123
J Am Oil Chem Soc
DOI 10.1007/s11746-014-2467-5
consideration as safer and more environment friendly oil
extraction processes [7]. Aqueous enzymatic extraction
methods have been reported for soybean [8], peanut [9],
corn germ [10], rice bran [11] and several other oilseeds.
This process usually includes grinding or milling seeds of
oil-rich materials, churning, and enzymatic hydrolysis,
followed by centrifugation. Aqueous enzymatic extraction
can offer many advantages compared to conventional
extraction. For instance, it eliminates the use of organic
solvents and their associated health and safety concerns
and decreases costs and energy requirements. It also
enables simultaneous recovery of oil and valuable materi-
als such as protein and polysaccharides from oilseeds [12].
The conventional WGO extraction method utilizes
hexane as solvent. Two research groups investigated
aqueous enzymatic extraction to extract WGO. Xie et al.
[3] used Alcalase 2.4L and obtained a 66.5 % yield of total
(emulsified) oil but they did not report a method to produce
free oil. Li et al. [4] reported a yield of 86 % total
(emulsified) oil, using a combination of four enzymes from
Novozymes (China) Investment Co., and they also did not
report a method to convert the emulsified oil to free oil.
Unlike hexane extraction, much of the oil extracted with
aqueous enzymatic extraction is present in the form of a
stable cream (oil-in-water emulsion). Many studies carried
out on aqueous enzymatic extraction have reported oil
extraction efficiencies, but few mentioned free oil recov-
ery. Several methods have been evaluated to break emul-
sions. The effects of enzymatic (phospholipase, protex 6L,
protex 7L) and nonenzymatic treatments (heating, freeze-
thawing, and pH) on free oil were compared [13–15].
Lamsal and Johnson [13] reported that the enzymatic
treatment achieved similar amounts of recoverable free oil
compared to freeze-thawing, but heating did not break the
emulsion. Zhang et al. [14] reported the highest free oil
yield using the endopeptidase Mifong 2709.
The current study was undertaken to investigate aqueous
enzymatic methods for wheat germ oil extraction and to
identify a method to demulsify the emulsified wheat germ
oil to produce free oil. We also applied our method to
barley germ and rice bran. There had been no previously
published methods for aqueous enzymatic extraction of
barley germ oil.
Materials and Methods
Materials
Commercial wheat germ (12.8 % moisture, 7.7 % oil,
particle size B1.68 mm), rice bran (4.6 % moisture,
18.5 % oil, particle size B0.35 mm) and hulless barley
(7.5 % moisture, 13.9 % oil, particle size B1.80 mm) were
purchased from Bob’s Red Mill Nature Foods (Milwaukee,
OR, USA). Spring wheat berries (Bronze Chief, 9.7 %
moisture, 6.7 % oil) were purchased from a local super-
market. Spring Wheat berries and hulless barley were
milled to produce a germ-enriched fraction using a Fitz-
patrick comminuting mill (Elmhurst, IL, USA), as previ-
ously described [16]. All of these samples were stored at -
4 �C. Alcalase 2.4L and Macerase were obtained from
Sigma (St. Louis, MO, USA) and EMD Biosciences (La
Jolla, CA, USA), respectively, and all other enzymes were
generously provided by DuPont Industrial Biosciences
(Wilmington, DE, USA).
Hexane Extraction
For hexane extraction, 1 g of material was weighed into a
55-mL glass screw-top tube, and 40 mL hexane was added.
The mixture was homogenized for 1 min with a Polytron
homogenizer (Brinkmann Instrument, Westbury, NY,
USA). The slurry was shaken with a wrist action shaker
(New Brunswick Scientific Inc, Edison, NJ, USA) for 1 h
at room temperature, then the mixture was filtered with a
Waterman Glass Microfiber filter (GF/A) and the solvent
was evaporated with nitrogen [7]. Hexane extraction of oil
from the three oil rich materials used in this study gave the
following yields: wheat germ (7.1 % oil), barley germ
(13.9 % oil) and rice bran (16.7 % oil). The aqueous
enzymatic extraction oil yields were reported as relative %
yields, based on the assumption that a 100 % oil yield was
obtained with hexane extraction.
Aqueous Enzymatic Extraction
The basic procedure for aqueous enzymatic extraction of
wheat germ is summarized in Table 1. To measure the
yields of emulsified oil, the floating emulsion layer was
carefully removed with a spatula and transferred to a
55-mL glass screw-top tube. Total oil in the emulsion was
extracted according to the method of Hara and Radin [17].
Table 1 Protocol for aqueous enzymatic extraction, adapted from
Moreau et al. [6]
1.Weigh 8.5 g wheat germ into a 55-mL polycarbonate centrifuge
tubes. Add 40 mL distilled water
2.Grind mixture with a Polytron homogenizer, 2 9 1 min
3.Adjust pH and add enzyme
4.Enzymatic reaction at 50 �C with shaking horizontally at
160 rpm for 20 h in a rotary shaker
5.Centrifuge at 4,000 rpm for 30 min in a BHG Hermele Z320
centrifuge
6.Carefully remove the top emulsion-interface into a 55-mL glass
screw top tube, extract emulsified oil with hexane/isopropanol,
and measure mass of emulsified oil
J Am Oil Chem Soc
123
The basic steps of the extraction include adding 24 mL of
hexane/isopropanol (3/2, v/v), followed by Polytron
homogenization for 2 9 1 min, and then shaking for 5 min
with a wrist action shaker. After that, 16 mL of sodium
sulfate solution (6.7 %, w/v) was added and then shaken
for 5 min. The mixture was centrifuged for 20 min at
200 rpm, the upper layer was carefully removed with a
pipette to a glass tube and the solvent was evaporated with
N2 [15]. The oil yield was calculated according to eq. (1).
Emulsified oil yield %ð Þ ¼ oil in emulsion ðgÞtotal oil in wheat germ ðg)
� 100
ð1Þ
The following enzymatic extraction parameters were
optimized: industrial enzymes, sequence of enzyme addi-
tion, amount of enzymes, substrate and water ratio (1:10,
1.5:10, 2:10, 2.5:10, dry weight of substrate), pH value
(4.0, 4.5, 5.0, 5.5, and 6.0), enzymatic reaction time (4, 8,
12, 16, and 20 h).
Two kinds of pretreatment methods (conventional oven
heating and microwave cooking) were evaluated. For
conventional oven heating, 200 g of wheat germ was put
onto a glass plate (20 cm 9 20 cm 9 5 cm) and heated at
180 �C for 6 min in a Thelco Model 130 DM Laboratory
Oven (Precision Scientific, Winchester, VA, USA).
Microwave cooking was carried out by placing 200 g germ
in a round Pyrex dish in an Amana Radar Range (1500 w,
2450 MHz) Microwave oven (Maytag, Inc., Newton, IA,
USA) for 3 9 25 s. All treatments were hydrolyzed at pH
5 for 20 h according to Table 1 without or with a enzy-
matic combination (5 % Spezyme CP and 5 % Fermgen).
After determining the optimal conditions for aqueous
enzymatic oil extraction and demulsification using com-
mercial wheat germ, experiments were conducted with the
same parameters to extract oil from laboratory milled
wheat germ, laboratory milled barley germ and commercial
rice bran.
Demulsification
For demulsification of wheat germ emulsion, the basic
steps are summarized in Table 2. For heating treatment, the
emulsion was heated at 95 �C for 3 h in oven. For freeze-
thawing treatment, the emulsion was frozen overnight at -
20 �C and thawed at room temperature [15]. For pH
adjustment, the pH of emulsion was adjusted to 3, 4, 6, 8
and 10 (add 0.5 M H2SO4 to decrease pH and 1 M NaOH
to increase pH). For enzymatic treatment, the emulsion was
adjusted to the appropriate pH for Alcalase 2.4L, Fermgen,
Lysomax, G-zyme 999, Protex 6L and Protex 7L, respec-
tively. The mixture was shaken at 50 �C for 1.5, 3, 6, 12,
and 20 h. Free oil yield was calculated by the following
equation.
Free oil yield %ð Þ ¼ free oil from emulsion ðgÞtotal oil in emulsion ðg)
� 100 ð2Þ
Statistical Analysis
All experiments were carried out at least in duplicate. The
values reported are the means ± SD. The data are analyzed
by ANOVA and Duncan’s multiple range test (Duncan’s
test). Significant differences were defined at p \ 0.05.
Results and Discussion
In the first experiment, eight individual commercial
enzymes and a control with no enzyme were screened for
their effect on the yields of emulsified oil. The three pro-
teases tested (Protex 6L, Fermgen and P4860) were the
only enzymes that successfully extracted oil from the
wheat germ (Table 3). Among the three proteases, the
highest oil yield (57.0 %) was achieved with Fermgen and
lower oil yields were achieved with P4860 (40.7 %) and
Protex 6L (12.8 %). Extraction of oil by proteases may
imply that the oleosins and other proteins surrounding the
oil-rich liposomes are the main barrier that hinders the
liberation and coalescence of oil from oil bodies. Xie et al.
[3.] also reported the highest yield of emulsified oil with a
protease (66.5 %) and oil yields of less than 10 % with
other enzymes and with no enzyme. In contrast, Li et al. [4]
reported oil yields of greater than 50 % with several types
of enzymes, including a protease and a cellulase, and an oil
yield of 31 % with no enzyme.
In the next experiment, the best enzyme from Table 3,
Fermgen, was combined with other enzymes to evaluate
their cooperative effects on oil yield (Table 4). Four
combinations gave oil yields greater than 70 %, and the
combination of Fermgen ? Spezyme CP achieved the
Table 2 Protocol for demulsification of wheat germ emulsion
1.Enzymatic extraction of wheat germ using the optimized method
above
2.Decant the emulsion into a glass frit Buchner funnel to
concentrate the emulsion and remove and weigh the emulsion
3.Transfer 4.5 g emulsion into a 15-mL screw-top polycarbonate
tube
4.Treat emulsion using various demulsification methods (heating,
pH adjustment, freeze-thawing, enzymatic treatment)
5.Centrifuge at 4,000 rpm for 20 min or (when noted) 13,000 rpm
for 3 9 1 min
6.Transfer the free oil to a glass tube, rinsed the wall of centrifuge
tube with hexane and combined with free oil. Evaporate to
dryness with N2
J Am Oil Chem Soc
123
highest oil yield, 73.1 %. Various sequences of timing of
combining of Fermgen and Spezyme CP (adding one
enzyme first and the adding the other or adding both at the
same time) were evaluated but none of the different timings
caused a significant difference in oil yields (data not
shown). To try to simplify the process and make it more
scalable for pilot or industrial scale, and because the
enzymes have the similar optimal pH, Fermgen ? Spe-
zyme CP were selected for the remaining experiments, and
they were added simultaneously for the following
experiments.
The effects of pretreatment of wheat germ on oil yield
are compared in Table 5. Both microwave and oven-heat-
ing pretreatment caused a small but significant increase in
the oil yield of aqueous enzymatic extraction. It was pre-
viously suggested that for corn germ, oven heating and
microwave cooking may break down the barriers in the
lipid body membrane and cause the oil to be more effec-
tively extracted via aqueous enzymatic oil extraction [18].
It is possible that heating in the microwave oven increased
oil yields with wheat germ for the same reasons or perhaps
because it inactivates endogenous lipases. Although
microwave pretreatment caused a slightly higher oil yield,
for convenience and economic scale-up considerations,
180 �C oven-heating was chosen as the pretreatment
method for the following experiments. Moisture level and
heating time can be described by the power equation
y = 13.32X-1.302 (R2 = 0.9987). After 4 min of heating,
the oil yield and the moisture of wheat germ dropped
sharply (Fig. 1).
Figure 2 shows data achieved by either adding increas-
ing concentrations of Spezyme with a fixed 5 % concen-
tration of Fermgen or adding increasing concentrations of
Fermgen with a fixed 5 % concentration of Spezyme.
When the concentration of Spezyme CP was fixed at 5 %,
oil yield was 0 % when no Fermgen was added and it
Table 3 Comparison of the
yields of emulsified oil with
individual commercial enzymes
Data shown are means ± SD
Reactions were carried out at:
the ratio of substrate to water of
2:10, 10 % enzyme (V/W), and
a hydrolysis temperature of
50 �C and a duration of 20 h
Different letters in the same
column indicate significant
differences at the 5 % level
NA not applicable
*Also has hemicellulose activity
**An endopeptidase
Enzyme EC number Company Brand name Enzyme source pH Yield of
emulsified oil
(%)
No enzyme NA NA NA NA 5 0
No enzyme NA NA NA NA 8 0
Cellulase* EC 3.2.1.4 Genencor Multifect GC Trichoderma reesei 5 0
Cellulase* EC 3.2.1.4 Genencor Spezyme CP Trichoderma reesei 5 0
Protease** EC 3.21.14 Genencor Protex 6L Bacillus licheniformis 8 12.8 ± 2.1c
Protease EC 3.21.14 Genencor Fermgen Trichoderma reesei 5 57.1 ± 3.4a
Amylase EC 3.2.1.1 Genencor Spezyme FRED Aspergillus niger 5 0
Pectinase EC 3.2.1.15 Calbiochem Macerase Rhizopus 5 0
Cellulase* EC 3.2.1.4 Genencor Optiflow RC 2.0 Trichoderma reesei 5 0
Protease** EC 3.4.21.62 Sigma P4860 Bacillus licheniformis 8 40.7 ± 1.6b
Table 4 Effects of enzyme combinations on the yield of emulsified
oil of wheat germ
Treatments Yield of emulsified oil (%)
Fermgen (Control) 57.0 ± 2.0b,c
Fermgen ? Spezyme CP 73.1 ± 5.4a
Fermgen ? Multifect GC 71.7 ± 0.9a,b
Fermgen ? Macerase 70.8 ± 3.3a,b,c
Fermgen ? Optiflow RC 2.0 64.0 ± 6.9a,b,c
Fermgen ? Spezyme Fred 55.5 ± 1.0c
Fermgen/Multifect GC/Macerase 69.0 ± 2.3a
Fermgen/Optiflow RC2./Macerase 71.8 ± 6.1a
Fermgen/Spezyme CP/Macerase 57.2 ± 1.5a
Data shown are means ± SD
Different letters in the same column indicate significant differences at
the 5 % level
The reaction conditions were as follows: ratio of material to water of
2:10, pH 5, 5 % enzyme (V/W), a hydrolysis temperature of 50 �C
and a time of 20 h, respectively
Table 5 Effects of pretreatment on the yield of emulsified oil of
wheat germ using aqueous enzymatic extraction
Pretreatments Yield of emulsified oil (%)
Oven-heating, no enzyme 0c
Microwave, no enzyme 6.0 ± 1.0c
Enzyme treated without pretreatment 67.9 ± 1.2b
Microwave, with enzyme 75.7 ± 2.0a
Oven-heating, with enzyme 72.4 ± 4.8a,b
Data shown are means ± SD
Different letters in the same column indicate significant differences at
the 5 % level
The reaction conditions were as follows: (1) Oven heating: 180 �C for
6 min, (2) For microwave cooking: microwave 3 9 25 s, (3) Enzy-
matic extraction: ratio of material to water of 2:10, pH 5, 5 %
Fermgen and Spezyme CP (V/W), respectively, a hydrolysis tem-
perature of 50 �C and a time of 20 h
J Am Oil Chem Soc
123
increased significantly when 0.5 % of Fermgen was added,
and continued to increase with the increasing amounts of
Fermgen. It was found that increasing the concentration of
Fermgen beyond 2 % caused no significant increase in oil
yield. When the concentration of Fermgen was fixed at
5 %, there was a slight increase in oil yield at 0.5 and
1.0 % Spezyme and no significant change at 2–6 % Spe-
zyme CP. To try to reduce the cost of enzymes, in the
following experiments, the combination of 2 % Fermgen
and Spezyme were chosen for the remainder of the
experiments.
A comparison of the oil yields obtained when varying
the wheat germ and water ratio on oil yield is shown in
Fig. 3. The oil yield increased when the ratio increased
from 1:10 to 2:10 and there was no significant oil yield
increase when the ratio was increased to 2.5:10. At higher
wheat germ/water ratios, the mixture was very viscous and
difficult to homogenize, and the oil yield dropped dra-
matically (data not shown).
The effects of pH on oil yield were investigated (Fig. 4).
The oil yields were nearly constant between pH 4.0 and 5.0
(with the highest oil yield 71.9 % at pH 4.5), and then
dropped dramatically at pH 5.5 and higher. Li et al. [4]
found that 5.25 is the optimal pH for wheat germ. Optimum
pH conditions for oil extraction found in other studies were
pH 4.0 for corn germ [6] and 10.0 for rapeseed [19]; this
may be due to different composition of material and the
enzyme used. We then investigated the effect of reaction
time on the oil yield of wheat germ (Fig. 5). The oil yield
was very low when the reaction time was less than 4 h. The
oil yield improved significantly when the reaction time was
increased from 4 h to 8 h, and achieved the highest oil
B
A
62
64
66
68
70
72
74
Yie
ld o
f E
mul
sifi
ed O
il (%
)
y =R²=0.9987
0
2
4
6
8
10
12
14
16
0 2 4 6 8
0 2 3 4 5 6 7 8
Moi
stur
e (%
)
Oven-heating minutes (min)
Fig. 1 Effect of duration of conventional oven heating pretreatment
of wheat germ on a yield of emulsified oil and b moisture content.
The reaction conditions were: oven temperature of 180 �C, the ratio
of wheat germ to water was 2:10, the amount of Fermgen and
Spezyme CP was each 5 % (V/W), the reaction temperature and time
were 50 �C and 20 h
Fig. 2 Comparison of oil yields achieved by: a Adding increasing
concentrations of Spezyme with a fixed 5 % concentration of
Fermgen, and b Adding increasing concentrations of Fermgen with
a fixed 5 % concentration of Spezyme. The reaction conditions were
as follows: wheat germ to water ratio was 2:10, pH was 5, and the
amount of fixed enzyme was about 5 % (V/W), the reaction
temperature and time were 50 �C and 20 h, respectively
a
b
cc
30
35
40
45
50
55
60
65
70
1:10 1.5:10 2:10 2.5:10
Yie
ld o
f E
mul
sifi
ed O
il (%
)
Ratio of Wheat Germ to Water
Fig. 3 Effects of varying the ratio of wheat germ to water on the
yield of emulsified oil. The other reaction conditions were: pH was
5.0, the enzyme was 2 % (V/W) Spezyme CP and 2 % (V/W)
Fermgen, the reaction temperature and time were 50 �C and 20 h,
respectively
J Am Oil Chem Soc
123
yield (71.8 %) when treated for 12 h, followed a slight
drop for longer reactions times.
Based on the above experiments, the optimal reaction
conditions achieved were: Pretreatment by heating at
180 �C for 4 min in a conventional oven, the ratio of wheat
germ to water was 2.5:10, pH was 4.5, 2 % Spezyme CP
and 2 % Fermgen were added to the slurry and incubated
12 h at 50 �C.
Aqueous enzymatic extraction produces an oil emulsion
which needs to be broken to get free oil. Several different
demulsification methods have been reported for soybean [20,
21], and coconut [22]. To the best of our knowledge, there is no
previous literature about the demulsification of wheat germ
emulsion since only emulsified oil yields were reported in the
two previous wheat germ aqueous enzymatic oil extraction
studies [3, 4]. In an attempt to break the wheat germ oil
emulsion, we tried heating, freezing-thawing, changing pH,
but no free oil was obtained. Enzymatic demulsification was
compared using four commercial proteases and two com-
mercial phospholipases (Table 6). After demulsification for
6 h, only Protex 6L caused measurable levels of free oil. When
the reaction time was increased to 20 h, Protex 6L and Alcalase
2.4L obtained 12.72 and 7.57 % free oil yield, respectively.
This may imply that these two enzymes have the capacity to
release some of the emulsified oil, but most of the oil remains in
the emulsion. A higher centrifuge speed (13,100 rpm) was
applied to the treatments of Protex 6L and Alcalase 2.4L,
which was able to significantly increase free oil yield and
achieved about 63 % oil yield with Protex 6L. This indicates
that higher speed centrifugation improves demulsification. It
was reported that some types of phenolic compounds in wheat
germ are rapidly oxidized due to the presence of high levels of
endogenous polyphenol oxidase and perhaps other enzymes
[23, 24]. We observed that the color of slurry became dark
brown during aqueous enzymatic extraction, probably due to
the oxidation of phenols. We speculated that phenolics or their
quinone products may inactivate some of the protease and
thereby decrease oil yields. On the other hand, wheat germ
contains about 20 % carbohydrates [1], and some of the oli-
gosaccharides produced by cellulase during aqueous enzy-
matic extraction may further emulsify the oil and reduce the
free oil yield.
In all of the previous experiments, oil was extracted from
commercial wheat germ and the process was optimized for
this material. In the final experiment fresh wheat germ and
fresh barley germ were prepared using a recently-reported
bench-scale process [16]. When compared to commercial
wheat germ, bench scale wheat germ resulted in nearly
identical yields of emulsified oil and yields of free oils
(Table 7). When bench scale barley germ and rice bran
were evaluated, the yield of emulsified oil was about 30 and
45 % lower than the yield with commercial wheat germ and
the yield of free oil was about 28 and 45 % lower than that
for commercial wheat germ. The method gave consistent oil
yield results for wheat germ, but if one intends to apply the
method for other materials such barley germ and rice bran,
further optimization studies will be needed.
Conclusion
Unlike the previous two wheat germ papers that only
reported yields of emulsified oil [3, 4], the current study is
the first to report a combined aqueous enzymatic oil
extraction and demulsification process that enables the
production of free oil from wheat germ. This report is also
the first one to publish aqueous enzymatic extraction of oil
0
10
20
30
40
50
60
70
80
4 4.5 5 5.5 6
Yie
ld o
f E
mul
sifi
ed O
il (%
)
pH
Fig. 4 A comparison of various pH values on total oil yield. The
reaction conditions were: ratio of wheat germ to water was 2:10, the
enzyme was 2 % (V/W) Spezyme CP and 2 % (V/W) Fermgen, the
reaction temperature and time were 50 �C and 20 h, respectively
0
10
20
30
40
50
60
70
80
4h 8h 12h 16h 20h
Yie
ld o
f E
mus
lfie
d O
il (%
)
Reaction Time (h)
Fig. 5 Effect of reaction time on yield of emulsified oil. The reaction
conditions were: wheat germ and water ratio was 2.5–10, pH was 5,
the enzyme was 2 % (V/W) Spezyme CP and 2 % (V/W) Fermgen,
the reaction temperature and duration were 50 �C, and 20 h
respectively
J Am Oil Chem Soc
123
from barley germ. Although the yields of emulsified and
free oil were modest, it should be noted that the parameters
were optimized for wheat germ and a higher oil yield would
be expected when optimized for barley germ and rice bran.
References
1. Dunford NT (2005) Germ oils from different sources. In: Shahidi F
(ed) Bailey’s industrial oil and fat products. John, Hoboken,
pp 195–230
2. De Vasconcelos M, Bennett R, Castro C, Cardoso P, Saavedra
MJ, Rosa EA (2013) Study of composition, stabilization and
processing of wheat germ and maize industrial by-products. Ind
Crop Prod 42:292–298
3. Xie MZ, Dunford NT, Goad C (2011) Enzymatic extraction of
wheat germ oil. J Am Chem Soc 88:2015–2021
4. Li HJ, Song CL, Zhou HM, Wang N, Cao DW (2011) Optimization
of the aqueous enzymatic extraction of wheat germ oil using
response surface methodology. J Am Oil Chem Soc 88:809–817
5. Amarasinghe B, Kumarasiri MPM, Gangodavilage NC (2009)
Effect of method of stabilization on aqueous extraction of rice
bran oil. Food Bioproduct Process 87:108–114
6. Moreau RA, Johnston DB, Powell MJ, Hicks KB (2004) A
comparison of commercial enzymes for the aqueous enzymatic
extraction of corn oil from corn germ. J Am Oil Chem Soc
81:1071–1075
7. Dominguez H, Nunez MJ, Lema JM (1994) Enzymatic pretreat-
ment to enhance oil extraction from fruits and oilseeds—a
review. Food Chem 49:271–286
8. Campbell KA, Glatz CE (2009) Protein recovery from enzyme-
assisted aqueous extraction of soybean. Biotechnol Prog
26:488–4959
9. Zhang SB, Lu QY, Yang HS, Li Y, Wang SA (2011) Aqueous
enzymatic extraction of oil and protein hydrolysates from roasted
peanut seeds. J Am Oil Chem Soc 88:727–732
10. Moreau RA, Dickey LC, Johnston DB, Hicks KB (2009) A
process for the aqueous enzymatic extraction of corn oil from dry
milled corn germ and enzymatic wet milled corn germ (E-Germ).
J Am Oil Chem Soc 86:469–474
11. Taha FS, Mourad RM, Mohamed SS, Hashem AI (2012) Enzy-
matic pretreatment of stabilized rice bran with mixed enzymes:
evaluation of oil. Am J Food Technol 7:452–469
12. Rosenthal A, Pyle DL, Niranjan K, Gilmour S, Trinca L (2001)
Combined effect of operational variables and enzyme activity on
aqueous enzymatic extraction of oil and protein from soybean.
Enzyme Microb Tech 28:499–509
13. Lamsal BP, Johnson LA (2007) Separating oil from aqueous
extraction fractions of soybean. J Am Oil Chem Soc 84:785–792
14. Zhang SB, Liu XJ, Lu QY, Wang ZW, Zhao X (2007) Enzymatic
demulsification of the oi-rich emulsion obtained by aqueous
extraction of peanut seed. J Am Oil Chem Soc 90:1261–1270
15. de Moura JMLN, Campbell K, Mahfuz A, Jung S, Glatz CE,
Johnson L (2008) Enzymatic-assisted aqueous extraction of oil
and protein from soybeans and cream de-emulsification. J Am Oil
Chem Soc 85:985–995
Table 6 Free oil yield using six
commercial enzymes for
demulsification at enzyme
concentration of 2 % (to convert
emulsified oil to free oil)
NA not applicable
Enzymes Brand name Enzyme source pH Free oil yield
(4,000 rpm, 30 min)
Free oil yield
(13,100 rpm, 3 min)
No enzyme 8 0 NA
No enzyme 5 0 NA
Protease Protex 6L Bacillus
licheniformis
8 12.7 ± 2.4 63.8 ± 4.9
Protease Fermgen Trichoderma reesei 5 7.6 ± 3.8 42.0 ± 2.4
Protease Protex 7L Bacillus
licheniformis
8 0 NA
Protease Alcalase 2.4L Bacillus
licheniformis
8 0 NA
Phospholipase
A1
G-zyme 999 5 0 NA
Phospholipase
A2
Lysomax 5 0 NA
Table 7 Comparison of the yield of emulsified oil and yield of free
oil using aqueous enzymatic extraction for commercial wheat germ
and laboratory milling wheat germ and barley germ
Wheat germ Moisture
(%)
Oil
content (%
DW)
Yield of
emulsified
oil (%)
Yield of
free oil (%)
Commercial
wheat germ
12.8 ± 0.1 7.7 ± 0.7 71.9 ± 1.4c 63.8 ± 4.9c
Laboratory
milled
wheat germ
9.7 ± 0.0 6.7 ± 0.1 71.6 ± 1.8c 59.5 ± 3.4c
Laboratory
milled
barley germ
7.5 ± 0.1 13.9 ± 0.5 51.0 ± 1.1b 45.7 ± 2.6b
Commercial
rice bran
4.6 ± 0.0 18.5 ± 0.4 39.3 ± 0.8a 34.5 ± 1.3a
Data shown are means ± SD
The reaction conditions are as follows: (1) For enzymatic extraction,
the ratio of material to water was 2.5:10, the material was heated in an
oven for 4 min at 180 �C, pH was 4.5, the amount of single enzyme
was about 2 % (V/W), the hydrolysis temperature and reaction time
were 50 �C and 12 h, respectively. (2) For demulsification, 2 % (V/
W) Protex 6L was added and hydrolyzed for 20 h at 50 �C, suspen-
sion was centrifuged 3 9 1 min at 13,100 rpm
Different letters in the same column indicate significant differences at
the 5 % level
J Am Oil Chem Soc
123
16. Moreau RA, Hicks KB (2013) Removal and isolation of germ-
rich fractions from hull-less barley using a Fitzpatrick commi-
nuting mill and sieves. Cereal Chem 90:546–551
17. Hara A, Radin NS (1978) Lipid extraction of tissues with a low-
toxicity solvent. Anal Biochem 90:420–426
18. Dickey LC, Cooke PH, Kurantz MJ, McAloon A, Parris N,
Moreau RA (2007) Using microwave heating and microscopy to
estimate optimal corn germ oil yield with a bench-scale press.
J Am Oil Chem Soc 84:489–495
19. Zhang SB, Wang Z, Xu SY (2007) Optimization of the aqueous
enzymatic extraction of rapeseed oil and protein hydrolysates.
J Am Oil Chem Soc 84:97–105
20. Chabrand RM, Kim HJ, Zhang C, Glatz CE, Jung S (2008)
Destabilization of the emulsion formed during aqueous extraction
of soybean oil. J Am Oil Chem Soc 85:383–390
21. Chabrand RM, Glatz CE (2009) Destabilization of the emulsion
formed during the enzyme-assisted aqueous extraction of oil from
soybean flour. Enzyme Microb Tech 45:28–35
22. Raghavendra SN, Raghavarao K (2011) Aqueous extraction and
enzymatic destabilization of coconut milk emulsions. J Am Oil
Chem Soc 88:481–487
23. Gasztonyi MN, Farkas RT, Berki M, Petroczi IM, Dadood HG
(2011) Content of phenols in wheat as affected by varietal and
agricultural factors. J Food Compos Anal 24:785–789
24. Jiang HX, Martin J, Okot-Kotber M, Seib PA (2011) Color of
whole-wheat foods prepared from a bright-white hard winter
wheat and the phenolic acids in its coarse barn. J Food Sci
76:846–852
25. Sengupta R, Bhattacharyya DK (1996) Enzymatic extraction of
mustard seed and rice bran. J Am Oil Chem Soc 73:687–692
26. Sharma A, Khare SK, Gupta MN (2001) Enzyme-assisted aque-
ous extraction of rice bran oil. J Am Oil Chem Soc 78:949–951
27. Hanmoungjai P, Pyle DL, Niranjan K (2001) Enzymatic process
for extracting oil and protein from rice bran. J Am Oil Chem Soc
78:817–821
28. Hernandez N, Rodriguez-Alegria ME, Gonzalez F, Lopez-Mun-
guia A (2000) Enzymatic treatment of rice bran to improve
processing. J Am Oil Chem Soc 77:177–180
J Am Oil Chem Soc
123
