Академический Документы
Профессиональный Документы
Культура Документы
By
MEIZHEN XIE
Bachelor of Science in Food Science
China Agricultural University
Beijing, China
2004
Thesis Approved:
Nurhan T. Dunford
Thesis Adviser
Carla Goad
Mark Wilkins
A. Gordon Emslie
Dean of the Graduate College
ii
ACKNOWLEDGMENTS
I want to thank my advisor Dr. Nurhan Dunford, for providing me with valuable
guidance, suggestions, encouragement and patience, which helped the completion of this
study.
Special gratitude is given to Dr. Goad whose valuable input is indispensible for
this study. I would like to thank Dr. Wilkins for being my committee member.
I also want to thank Oklahoma State University and the Robert M. Kerr Food &
Agricultural Product Center for providing a good learning atmosphere and excellent
research facilities.
Last but not least, I want to thank my family, friends and people in my research
laboratory for all the help, support, and encouragements.
iii
TABLE OF CONTENTS
Chapter
Page
I. INTRODUCTION ..................................................................................................... 1
1.1 Statement of problem ........................................................................................ 1
1.2 Hypothesis......................................................................................................... 2
1.3 Objective ........................................................................................................... 2
II. LITERATURE REVIEW ......................................................................................... 3
2.1 Wheat germ ....................................................................................................... 3
2.1.1 Wheat germ structure ....................................................................... 3
2.1.2 Wheat germ composition ................................................................. 4
2.2 Extraction of wheat germ oil............................................................................. 5
2.3 Aqueous enzymatic oil extraction ..................................................................... 5
2.3.1 Extraction process .............................................................................. 5
2.3.2 Forms of oil obtained by AEOE ........................................................ 6
2.3.3 Factors affecting AEOE yields .......................................................... 7
2.3.3.1 Enzyme type.......................................................................... 7
2.3.3.2 Particle size ........................................................................... 9
2.3.3.3 pH and temperature ............................................................. 10
2.3.3.4 Liquid: solid ratio................................................................ 11
2.3.3.5 Enzyme concentration ......................................................... 11
2.3.3.6 Extraction time .................................................................... 12
III. MATERIALS AND METHODS .......................................................................... 13
3.1 Source and preparation of wheat germ ........................................................... 13
3.2 Enzyme selection ............................................................................................ 13
3.2.1 Viscozyme L ...................................................................................... 13
3.2.2 Multifect CX GC................................................................................ 13
3.2.3 Multifect CX 13L............................................................................... 14
3.2.4 Alcalase 2.4L FG ............................................................................... 14
3.3 Enzyme screening tests ................................................................................... 14
3.4 Optimization by Response Surface Methodology .......................................... 15
3.5 Analytical methods ......................................................................................... 16
iv
LIST OF TABLES
Table
Page
vi
LIST OF FIGURES
Figure
Page
viii
NOMENCLATURE
ANOVA
Analysis of variance
v/w
Volume to weight
w/w
Weight to weight
rpm
Percentage
Degree centigrade
Gram
Hour
ml
Milliliter
min
Minute
Micrometer
mm
Millimeter
ix
CHAPTER I
INTRODUCTION
1.1 PROBLEM STATEMENT
Wheat germ is a byproduct of the grain milling industry. It contains about 10% oil.
The conventional wheat germ oil extraction method utilizes n-hexane as a solvent. This
method is very effective, with an oil yield of higher than 95%. However, n-hexane has
been proven to be an environmental pollutant. Hexane is a flammable solvent. Hence, it
can create an unsafe environment for the people working in the plants. In addition, health
conscious consumers may have concerns about potential solvent residue in hexane
extracted oil. Other alternatives to the hexane based extraction are mechanical pressing
and supercritical fluid extraction. Mechanical pressing completely avoids the use of
organic solvents. Thus the final product can be considered natural. However, the
efficiency of this method can be 50% or lower, depending on the germ pretreatment and
type of press used for extraction. Supercritical fluid extraction using non-toxic and
non-explosive carbon dioxide as a solvent produces a chemical-free final product. Carbon
dioxide is recycled to the system after oil separation. Hence supercritical carbon dioxide
extraction is environmentally benign and does not leave solvent residue in the oil.
However, the capital cost required for the supercritical fluid system set up is quite high.
New technologies need to be developed to overcome the disadvantages of the existing
extraction methods.
1
1.2 HYPOTHESIS
Aqueous enzymatic extraction, which utilizes water as the solvent and is performed
under mild conditions with the aid of enzymes, is a viable alternative technology for oil
extraction from wheat germ.
1.3 OBJECTIVE
The main objective of this study is to evaluate the efficiency of aqueous enzymatic
extraction of oil from wheat germ. The specific objectives are as follows:
i.
ii.
iii.
CHAPTER II
LIETRATURE REVIEW
2.1 WHEAT GERM
2.1.1 Wheat Germ Structure
Together with bran and starchy endosperm, wheat germ (WG) makes up the basic
structure of wheat grain (Figure 1) (Fennema 1985). WG is partly embedded in the
endosperm at the base of the wheat grain, and can be separated from bran and endosperm
by milling. Whole wheat grain moisture content is adjusted to 13-15% by tempering prior
to milling. This process softens the grain and toughens the bran making it easier to
separate it from the endosperm and germ (Atwell 2001). Also, tempering softens the
endosperm, which breaks apart with less force.
WG accounts for about 2-3% by weight of the grain (Atwell 2001), and consists of
embryo (or embryonic axis) and scutellum (Posner and Hibbs 1997). The embryo is about
1.2% by weight of the grain, and is the part that eventually develops into the first roots
and shoot of the new plant. The scutellum, which is located between the embryo and the
endosperm and accounts for about 1.5% by weight of the grain, is the part that transports
sugars hydrolyzed from endosperm to the embryo during germination (Barnes 1982).
In WG, oil is stored in a subcellular organelle called the oleosome, oil body or
spherosome, whose structure is presented in Figure 2 (Waltermann and Steinbuchel 2005).
As the main form of oil in plants, triacylglycerols (TAG) coalesce in the centre of the oil
3
bodies which are covered by a membrane made up of phospholipids (PLs). Unlike the
membranes of the other organelles in cells which possess a bi-layer structure of PLs, the
membrane of the oil body is a single-layer formed by PLs with protein embedded in it.
The hydrophobic end of the PLs is oriented toward the TAG matrix. The hydrophilic end
of PLs extends toward the aqueous cytoplasm. The proteins in the membrane of the oil
body are called oleosin (Huang 1992). The structure of the oil body serves two purposes:
maintaining lipids in the aqueous cytoplasm of the cell, and providing a large surface to
facilitate the action of lipases to break down lipids to produce energy during germination.
Plant oil bodies usually contain 9498% (w/w) neutral lipids, 0.52% PLs and 0.53.5%
proteins (Huang 1992). In WG, oil is present in both scutellum and embryo. Oil bodies in
the cells of the epithelium and parenchyma in scutellum accumulate around protein
bodies and periphery of the cells (Morrson and others 1975; Murphy and others 1993).
The arrangement of oil bodies in the embryo is not well known.
2.1.2 Wheat Germ Composition
Wheat germ is rich in protein and lipid, but not in starch. The composition of WG
depends on the method used to separate it from the endosperm. In commercial WG, a
typical gross composition is as follows: moisture (6%), protein (26%), oil (10%), ash
(4%), starch (20%), crude fiber (3%) and other substances (15%) (Barnes 1982). The
occurrence of starch in commercial WG is mainly due to endosperm carry over during the
milling process. The oil content has been reported to be 25-30% in dissected WG which is
not contaminated by the bran and endosperm (Hargin and Morrison 1980). Linoleic acid
(18:2) is the predominant fatty acid (FA), which makes up 60% of the total FA in wheat
4
germ oil (WGO). Other important FAs present include palmitic acid (16:0) (15%), oleic
acid (18:1) (15%), and linolenic acid (18:3) (10%) (Barnes 1982).
2.2 EXTRACTION OF WHEAT GERM OIL
After separated from bran and endosperm, WG, which appears as yellow flake, is
subjected to an oil extraction process. Three methods are currently being used to produce
commercial WGO: organic solvent based extraction, mechanical pressing and
supercritical fluid extraction. In organic solvent based extraction, oil is extracted from
WG into hexane, and crude oil is obtained after the evaporation of the solvent (Barnes
and Taylor 1980). In mechanical pressing, oil is extruded out of WG by mechanical
pressure. Since the oil extraction yield is low, mechanical pressing is recommended when
WG is free of contaminations from endosperm and bran (Dunford 2009). Utilization of
supercritical fluid technology for WGO recovery eliminates the potential solvent residues
in the final product because solvent is completely removed from the product by reducing
the pressure of the system at a point that fluid returns back to gas phase (Eisenmenger and
others 2006).
2.3 AQUEOUS ENZYMATIC OIL EXTRACTION
2.3.1 Extraction Process
The steps involved in aqueous enzymatic oil extraction (AEOE) usually include
grinding (wet or dry) of the oil-bearing materials, mixing of the comminuted material
with aqueous solution, incubation with enzyme, separation of liquid and solid phases by
centrifugation or filtration and recovery of oil from liquid phase. Figure 3 provides a
flowchart of this process. Apart from the steps described above, other unit operations can
5
are usually difficult to separate, it is impossible to calculate the exact oil yield in one
particular form. As a result, oil extraction yield is usually calculated based on the total
amount of oil extracted into the liquid phase, which is the difference between the amount
of oil in the starting material and that in the solid residue after extraction (Bocevska and
others 1993; Rosenthal and others 1998; Sineiro and others 1998; Rosenthal and others
2001; Hanmoungjiai and others 2002; Lamsal and others 2006).
2.3.3 Factors Affecting AEOE Yields
AEOE yields are influenced by both the types of enzymes used and the processing
parameters employed.
2.3.3.1 Enzyme Type
The role of enzymes in AEOE from oil-bearing materials is to hydrolyze the
structural components of plant cells and facilitate the release of oil. Therefore, the
selection of enzymes is dependent on the composition of the cell wall and membrane that
form the boundary for oil bodies, and the cytoplasmic network where oil bodies are
imbedded. Plant cell wall is mainly comprised of cellulose, hemicelluloses and pectin
which all together account for 80-90% of the polysaccharides present (Dominguez and
others 1994). Protein as a dispensable structural component and other components are
present only at very low levels in cell wall (Keegstra and others 1973; Dominguez and
others 1994). Enzymes that are effective in breaking up the cell structure include but are
not limited to carbohydrases such as cellulase, hemicellulase and pectinase, and protease.
Commercial enzyme preparations including cellulase, hemicellulase, pectinase,
-glucanase, -amylase, and protease, have been utilized in AEOE. The individual or
7
combined effects of these enzymes on various oil-bearing materials have been studied.
These studies have shown that enzymes have a significant effect on enhancing the oil
extraction yield when compared to the control which is carried out without enzymes
(Barrios and others 1990; Sinerio and others 1998; Moreau and others 2004; Abdulkarim
and others 2006; Lamsal and others 2006; Latif and others 2008; Womeni and others
2008). For example, the use of -amylase for AEOE resulted in higher oil extraction yield
from avocado when compared to pectinase, protease, cellulase and different combinations
of these four enzymes (Buenrostro and Lopez-Munguia, 1986). Tano-Debrah and Ohta
(1994) reported that the highest increase in oil extraction yield from shea tree kernels was
achieved by a combination of protease and an enzyme with cellulase and hemicellulase
activities. Chen Man and others (1996) observed that in coconut oil extraction, a mixture
of cellulase, polygalacturonase, protease and -amylase was more effective than when
these enzymes were used individually. According to Womeni and others (2008) the
highest oil extraction yield from Irvinia gabonensis seeds (68%) was obtained with a
mixture of carbohydrases (cellulase, arabanase, -glucanase, hemicellulase and xylanase)
rather than protease or pectinase used individually. Protease was found to be superior to
-amylase, pectinase and cellulase in extracting oil from Moringa oleifera seeds, but a
mixture of these four enzymes was even more effective (Abdulkarim and others 2006).
For rice bran oil extraction, protease was found to give higher oil extraction yield when
compared to cellulase, hemicellulase, pectinase and a mixture of cell wall-degrading
enzymes (cellulase, arabanase, -glucanase, hemicellulase and xylanase) (Hanmoungjai
and others 2002). In rapeseed oil extraction pectinase, when used alone, was more
8
effective than cellulase, -glucanase and xylanase, but was less effective than the
combination of these 4 enzymes (Zhang and others 2007). A similar trend was observed
when Latif and others (2008) compared the effects of protease, pectinase, and mixtures of
cell wall-degrading enzymes on oil extraction yield from canola seeds. It was reported
that the use of protease for soybean (Lamsal and others 2006) and rice bran (Hanmougjai
and others 2002) oil extraction resulted in higher yields than those obtained from cell
wall-degrading enzymes. However, in corn germ oil extraction, protease resulted in lower
oil yield than that obtained by cell wall-degrading enzymes, especially cellulase (Moreau
and others 2004).
Not only do the types of enzymes, but also the specificity for pH and sources of the
same type of enzyme have different impacts on oil extraction yield. The order of
effectiveness of protease in improving oil extraction yield was found to be as follows:
alkaline protease > neutral protease > acid protease (de Moura and others 2008; Wu and
others 2009). It was also reported that fungal protease was superior to that extracted from
papaya (papain) (Hanmoungjai and others 2002). Cellulase secreted by Trichoderma
reesei showed higher efficacy in extracting oil than those produced by Trichoderma viride
and Aspergillus niger (Moreau and others 2004).
2.3.3.2 Particle Size
The particle size of the ground material is an important parameter for AEOE
efficiency. Smaller particle size indicates an efficient grinding which mechanically breaks
apart the cell structure of materials (Rosenthal and others 1996). In addition, smaller
particle size results in larger surface area which allows better contact between oil bearing
9
material and solvent and reduces resistance to the diffusion of oil, as well as other
components, from the solid matrix to the aqueous medium. The rate of enzyme diffusion
into the solid substrate is also improved with smaller particle size (Rosenthal and others
1996). Although the importance of smaller particle size has been well recognized, only a
few studies have been carried out to investigate its effect on oil extraction yield. A study
by Rosenthal and others (2001) on soybean oil extraction yield showed that the oil
extraction yield increased with a decrease in particle size from 1200 m to less than 200
m. Santamaria and others (2003) found that the oil extraction yield of Chilean hazelnut
was enhanced when the particle size of the material was reduced from 1.6 mm to 0.4 mm.
2.3.3.3 pH and Temperature
The pH and temperature of water employed in AEOE are usually dictated by the
enzymes used, so the effect of these two factors on oil extraction efficacy can be
considered as their impacts on enzyme activities. The pH of solvent could actually change
the solubility of some cell components, especially that of protein. In AEOE, protein
solubility is closely associated with oil extraction yield. In general a high oil extraction
yield is obtained when the solubility or extraction yield of protein is high, which happens
when the pH is away from its isoelectric point (pI) (Rosenthal and others 1996; Rosenthal
and others 1998; Hanmoungjai and others 2002). The pI of protein usually varies
depending on the source. For example, the pI for soybean protein is around 4.5, while that
for sunflower protein is 4.0 (Rosenthal and others 1996). Therefore, it is important that
the pH employed in AEOE is away from the pI of the protein for a given material, in
order to obtain high oil extraction yield.
10
additional improvement in extraction yield. In the extraction of Moringa oleifera seed oil,
Abdulkarim and others (2006) observed that the oil extraction yield was improved with
an increase in EC from 0.5 to 2.0% (v/w), and then decreased slightly at the concentration
of 2.5%. It is clear from above mentioned studies that there is an optimum EC that results
in maximum oil yield. However, economic feasibility of an AEOE process needs to be
determined by an optimization study that will balance EC, oil extraction yield and the
cost of enzyme (Zhang and others 2007).
2.3.3.6 Extraction Time
Action of enzyme on oil-bearing materials and mass transfer between the aqueous
medium and the material are time dependent processes. In most cases an increase in
extraction time (ET) results in an improvement in the oil extraction yield, but the
improvement slows down and eventually stops as time prolongs (Dominguez and others
1994; Sharma and others 2001; Sharma and others 2002; Abdulkarim and others 2006;
Zhang and others 2007). Hence time is a variable that needs to be included in process
optimization studies.
Several studies have reported the presence of significant interactions among ET, EC
and LSR indicating that these are not independent factors (Hanmoungjai and others 2001;
Zhang and others 2007; Womeni and others 2008). The optimum combination of these
factors needs to be investigated by employing an appropriate experimental design such as
factorial design or response surface methodology (RSM) in order to achieve the
maximum oil extraction yield.
12
CHAPTER III
MATERIALS AND METHODS
3.1 SOURCE AND PREPARATION OF WHEAT GERM
Full-fat WG used in this study was obtained from ADM Milling Co. (Enid, OK,
U.S.A.). The sample was ground using a laboratory mill (Model 3600, Perten Instruments,
Sweden) and kept in an airtight plastic container at -20 oC until further use for proximate
composition analysis and extraction tests.
3.2 ENZYME SELECTION
Initial enzyme screening tests were carried out by using 3 carbohydrases (Viscozyme
L, Multifect CX GC and Multifect CX 13L) and 1 protease (Alcalase 2.4L FG). Selection
of these enzymes was based on the chemical composition of wheat germ.
3.2.1 Viscozyme L
This enzyme was kindly provided by Novozymes (Bagsvaerd, Denmark). Viscozyme
L is produced from a selected group of Aspergillus strains. The enzyme contains a variety
of carbohydrases (arabanase, cellulase, -glucanase, hemicellulase and xylanase), and has
a declared activity of 100 FBG/g. One FBG is the amount of enzyme which releases
glucose or reducing sugar equivalent to 1 mol glucose from -glucans at pH 5.0 and 30
o
C in one minute. The optimum conditions for the activity of this enzyme are pH 3.3-5.5
13
3.2.2 Multifect CX GC
Multifect CX GC, an enzyme with cellulase activity, is provided by Genencor
(Rochester, NY, U.S.A). This enzyme is derived from a selected strain of Trichoderma
reesei, and has side activities including hemicellulase, xylanase, and -glucanase. The
declared activity is 3200 CMC/g. One CMC unit is defined as the amount of enzyme
which produces 1 mol glucose equivalent from carboxymethyl cellulose at pH 5.0 and
50 oC in one minute. This enzyme is effective at a pH of 2.7-5.7 and temperature of 35-70
o
C.
Ground WG (15 g) was mixed with 180 ml buffer in a 500 ml flask, to achieve a LSR
of 12: 1 (v/w). For Viscozyme L, Multifect CX GC and Multifect CX 13L 0.15M
citric-phosphate buffer at pH 5.0 was used. Two different buffers, 0.12M boric
acid-NaOH and 0.1M Tris-HCl, at pH 8.0 were used for Alcalase 2.4L FG. The mixture of
WG and buffer was placed in a water bath shaker (Model C76, New Brunswick Science,
Edison, NJ, USA), and heated to 50 oC with constant shaking at 200 rpm. Enzymes were
added at a concentration of 5% (w/w) based on the weight of the WG. Then the mixture
was incubated for 4 h. After the incubation, the mixture was centrifuged in a bench top
centrifuge (Sorvall RC 5C, Termo, Ashwville, NC, USA) at 13,000 rpm and 25 oC for 15
min. The liquid phase was drained off, and 180 ml deionized water was added to the
centrifuge tube containing wet residual solids to wash away the oil which may remain on
the wall of the centrifuge tube and in the solid matrix. The wet residue was well mixed
with the deionized water, and subjected to a second centrifugation under the same
conditions used before. The liquid phase was drained off once again. The wet residue was
dried in a forced-air oven (VWR Science, model 1370 FM, Bristol, CT) at 85 oC for 16 h.
The dried residue was weighed and analyzed for oil content. The oil extraction yield is
calculated by the following formula:
* 100% (1)
Four different oil extraction runs without enzyme were conducted as controls. The
first control was carried out with deionized water (pH 6.6) as the aqueous solvent.
Citric-phosphate buffer at pH 5.0, boric acid-NaOH buffer at pH 8.0, and Tris-HCl buffer
15
at pH 8.0 were the other controls. Other processing parameters were kept the same as
those for the enzymatic process during the control experiments. A schematic flow
diagram of the extraction process is shown in Figure 4.
3.4 OPTIMIZATION BY RESPONSE SURFACE METHODOLOGY
Based on the results of the experiments described above, Alcalase 2.4L FG and
Multifect CX GC were chosen for the optimization experiments. In the case of Alcalase,
both boric acid-NaOH and Tris-HCl buffers were used to investigate the effect of
processing parameters on oil extraction yield under different buffer systems. Response
Surface Methodology (RSM) with central composite design was employed. Three
processing parameters, LSR, ET, and EC at 5 levels each were investigated as the
independent variables to estimate the oil extraction yield in the enzymatic process. The
actual and coded levels of the three independent variables are shown in Table 1. Control
experiments were also carried out for each enzyme. The levels of LSR and ET for the
control experiments were the same as those used in the enzymatic process. The
experimental designs for the non-enzymatic and enzymatic processes are presented in
Table 2 and 3, respectively. The extraction procedure and the other processing parameters
used in these optimization experiments were kept the same as those employed in the
experiments described in section 3.3.
3.5 ANALYTICAL METHODS
3.5.1 Moisture Content
The moisture content of the ground WG was determined according to AACC method
44-15A (AACC, 1995). The ground WG was brought to room temperature before use.
16
Aluminum moisture dishes were pre-dried in a forced-air oven (VWR Science, model
1370 FM, Bristol, CT) at 130 oC for 1 h prior to analysis, and then cooled in a desiccator
to room temperature. About 2 g of sample were weighed in the pre-dried aluminum dish,
and dried in the oven at 130 oC for 1 h. The moisture content of the sample was reported
as the loss in sample weight as percentage of the initial sample weight.
3.5.2 Ash Content
The ash content of the ground WG was measured by AOAC method 923.03 (AOAC,
1995). The ground WG was taken out of the freezer and cooled to room temperature prior
to use. Crucibles were pre-dried in a furnace (Fisher Science, Model 58 Isotemp Muffle
Furnace 600 Series, Fair Lawn, NJ) at 525 oC for 5 h, and then cooled down to room
temperature in a desiccator. Approximately 2 g of the ground wheat germ were weighed
into the pre-dried crucible and ashed at 525 oC for 5 h. The percentage residual weight
was reported as the ash content of the sample.
3.5.3 Oil Content
The oil contents of the ground WG and the dried residue after extraction were
measured according to AOAC method 2003.05 (AOAC, 2005). Samples were brought to
room temperature before analysis. About 1 g of sample was weighed in a cellulose
thimble, and extracted in a Soxtect extraction unit (Tecator, Model 1043 Extract Unit,
Sweden) with 40 ml of petroleum ether (Mallinckrodt, Paris, KE) for 1 hour. The amount
of oil extracted as the percentage of initial sample weight was reported as the oil content
in the sample.
3.5.4 Protein Content
17
The amount of protein in the ground WG was determined by the method of Forage
Analyses Procedures (1993). Protein was analyzed as nitrogen on a Leco Truspec
carbon-nitrogen analyzer (Truspec CN, Leco USA, St. Joseoh, MI). A factor of 5.7 was
used to convert the amount of nitrogen to the amount of protein in the sample.
3.5.5 Starch Content
Starch content of the ground wheat germ was determined according to AOAC
method 996.11 (AOAC, 2005), which is based on the use of thermostable -amylase and
amyloglucosidase.
3.5.6 Enzyme Activity Test
3.5.6.1 Viscozyme L
The activity of Viscozyme L was determined using the Somogyi-Nelson method
provided by the Joint Food and Agriculture Organization of the United Nations
(FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA),
and expressed in fungal beta-glucanase unit per gram (FBG/g) (JECFA 2000).
3.5.6.2 Multifect CX GC
The activity of this enzyme was measured in CMC/g according to the method
provided by JECFA (2003).
3.5.6.3 Multifect CX 13L
The method used to determine the activity of Multifect CX 13L was the same as that
for Multifect CX GC.
3.5.6.4 Alcalase 2.4L FG
The activity of Alcalase 2.4L FG was determined according to the method provided
18
by Megazyme (Wicklow, Ireland), and expressed as Unit/g (Megazyme 2006). One Unit
is defined as the amount of enzyme that produces the equivalent of 1 mol tyrosine per
minute from soluble casein at pH 8.0 and 40 oC.
3.6 STATISTICAL ANALYSIS
All analytical tests and extraction experiments were carried out at least in duplicate
and in randomized order with the mean values being reported. Analysis of variance
(ANOVA) of the results from enzyme screening and the analysis of RSM experiments
were performed using SAS 9.1 (SAS Institute Inc., 2003).
19
CHAPTER IV
RESULTS AND DISCUSSION
4.1 CHARACTERIZATION OF WHEAT GERM
The proximate composition of the ground WG used for the optimization tests is
shown in Table 4. The moisture content of the sample was about 11%. The high moisture
content of WG is due to the fact that whole wheat grain moisture content is adjusted to
about 13 to 15% prior to milling. The reasons for the moisture adjustment were discussed
earlier in section 2.1.1 of this thesis. The oil content of the WG extracted by petroleum
ether was about 11% (w/w, as is basis) (Table 4). This result is in agreement with the data
reported in the literature for commercial WG (Barnes 1982; Dunford and Zhang 2003;
Zhu and others 2006), but is much lower than that obtained from dissected WG, 25-30%
(Hargin and Morrison 1980). The lower oil content in commercial WG is because of the
contamination from bran which usually contains less than 5% oil and significant amount
of starchy endosperm (Barnes 1982). Indeed, about 9% starch was detected in our
samples (Table 4). This is much lower than that reported by Barnes (1982), 20%. WG is
well known as a rich source of plant protein. According to the literature, WG contains 26
to 36% protein (Barnes 1982; Ge and others 2000; Claver and Zhou 2005; Zhu and others
2006). WG used in this study had about 34% protein (Table 4) which is within the range
reported in literature. The level of ash in WG was similar to that reported in literature
(Barnes 1982; Zhu and others 2006). Other components, which account for about 30% of
20
WG, may comprise mainly non-starch carbohydrates including free sugars such as
sucrose and raffinose, fiber and pentosans (Dubois and others 1960; Amado and Arrigoni
1992).
The particle size distribution of the ground WG used in this study is presented in
Table 5. About 89% of the ground WG had a particle size between 150 and 500 m. The
size of the ground WG is much smaller than those reported in literature, 1 mm or larger
(Dominguez and others 1995; Sengupta and Bhattacharyya 1996; Hanmoungjai and
others 2001; Santamaria and others 2003), indicating a sufficient grounding of the WG.
4.2 EFFECT OF ENZYME TYPE ON OIL EXTRACTION YIELD
The activities of the enzymes measured prior to screening tests as well as their
declared activities by the suppliers are presented in Table 6. There were discrepancies
between measured and the declared activities for Viscozyme L, Multifect CX 13L and
Multifect CX GC. This is because of the slight variations in analytical protocols used for
the activity measurements.
The oil extraction yields obtained from aqueous extraction processes with and
without enzymes (the controls) are shown in Figure 5. It was found that the control tests
carried out at pH 8.0 (controls 3 and 4) resulted in significantly higher oil yields than
those conducted at pH 6.6 and 5.0 (controls 1 and 2). The effect of pH on oil extraction
yield may be due to their influence on the solubility of proteins. The pI of WG protein is
around 4.0 (Ge and others 2000). Although pH 5.0 and 6.6 are higher than pI of WG, it
appears that most proteins still remain insoluble at these pH values. Indeed, a preliminary
protein analysis on the residual WG after extraction indicated that at low pH protein
21
extraction yields were lower than those at pH 8 (unpublished data). At pH 8.0, which is
far away from the pI 4.0, the solubility of WG protein is enhanced greatly. Since oil
bodies are embedded in the matrix of proteins in the cytoplasmic network of the cell, the
increased solubilization of protein into the aqueous medium consequently results in the
increased oil release from the WG. As can be seen from Figure 5, oil extraction yield
obtained with boric acid-NaOH buffer at pH 8.0 (control 3) was significantly lower (p <
0.05) than that obtained with Tris-HCl buffer at pH 8.0. This might be due to the
interaction of buffer components with proteins. A comparison of the oil extraction yields
with the four controls indicates that both pH and buffer system play an important role in
the aqueous extraction of WGO.
This study demonstrated that Alcalase 2.4L FG gave significantly higher oil yield
than Viscozyme L, Multifect CX 13L and Multifect CX GC (Figure 5). This finding is
consistent with the results obtained with rice bran (Hanmoungjai and others 2002),
coconut (Chen Man and others 1996), Moriga oleifera seed (Abdulkarim and others
2006), and soybean (Rosenthal and others 2001), where proteases and carbohydrases
were compared for their effects on oil extraction yield. No significant difference was
observed among the oil extraction yields obtained by Viscozyme L, Multifect CX 13L and
Multifect CX GC.
The effects of Alcalase 2.4L FG in two different buffer systems were also
investigated (Figure 5). The experimental results showed that Alcalase 2.4L FG produced
a significantly higher oil yield in Tris-HCl buffer than that in boric acid-NaOH buffer. As
mentioned in the first paragraph of this section, a similar trend was observed during
22
The experimental data obtained from the RSM design for processes with and without
enzymes are shown in Tables 7 and 8, respectively. For the non-enzymatic process in
boric acid-NaOH buffer at pH 8.0, the oil extraction yields ranged from 15.62 to 47.7%,
both of which were obtained at ET of 0.5 h, and LSRs of 4 and 20, respectively (Table 7).
The range of the oil yield was broader when Tris-HCl buffer at pH 8.0 was used for WGO
extraction, 3.97 - 48.07%. The lowest and highest yields were observed at LSR of 12, and
ETs of 24 and 0.5 h, respectively. In citric-phosphate buffer at pH 5.0, the non-enzymatic
process resulted in oil yields ranging from 2.07 to 17.07%, which were at LSR of 20 and
ET of 0.5 h, and LSR of 4 and ET of 12.25 h, respectively.
For the enzymatic process with Alcalase 2.4L FG in boric acid-NaOH buffer at pH
8.0 (Alcalase-B), the lowest oil yield of 2.79% was obtained at LSR, EC and ET of 4,
2.55% and 12.25 h, respectively. The highest oil yield of 36.32% was at LSR, EC and ET
of 16.5, 4% and 5.25 h, respectively (Table 8). In Tris-HCl buffer at pH 8.0, the oil
extraction yields obtained by Alcalase 2.4L FG ranged from 10.63 to 66.45%. The
extraction conditions which resulted in the lowest and highest oil yields were the same in
both buffer systems. It appears that Tris-HCl buffer is a better choice for WGO extraction
than boric acid-NaOH. As mentioned earlier in this thesis, the effect of buffer on
extraction yield might be due to difference in interaction of buffer components with WG
proteins.
The oil yields varied between 2.57 and 22.99% when Multifect CX GC was used for
WGO extraction (Table 8). The lowest value was observed at LSR of 16.5, EC of 4 and
ET of 5.25. The highest value was obtained at LSR of 4, EC of 2.55%, and ET of 12.25 h.
24
In summary, the non-enzymatic processes resulted in higher oil yield at pH 8.0 than at pH
5.0. The enzymatic process with Alcalase 2.4L FG in Tris-HCl (Alcalase-T) buffer gave
the highest oil yield.
The following quadratic models were developed to explain the relationship between
the oil extraction yield and processing parameters in the six extraction processes.
(2)
(3)
(4)
(5)
(6)
(7)
YB, YT, and YC represent the estimated oil extraction yields for the non-enzymatic
processes in boric acid-NaOH at pH 8.0, Tris-HCl at pH 8.0 and citric-phosphate buffer at
pH 5.0, respectively. Y
Alcalase-B,
Alcalase-T,
and
extraction yields in the enzymatic processes with Alcalase-B, Alcalase-T, and Multifect
CX GC, respectively. R, C and T are the processing parameters LSR, EC and ET,
respectively.
All the response surface models shown above were significant (p < 0.05) (Tables 9
25
and 10). The models developed for the non-enzymatic processes (Equations 2-4)
satisfactorily explain the oil extraction yield in relation to the processing parameters,
which is evident from the high coefficients of determination (R2 > 0.94) (Table 9). For the
enzymatic processes, the models established for oil yield from Multifect CX GC and
Alcalase-T treated WG had the highest and lowest R2 (0.9267 and 0.49), respectively
(Table 10). A significant lack of fit is detected for all the models. However, the residual
analysis showed that for each model, there are only one or two outliers contributing to the
significant lack of fit. Although there is a significant lack of fit, the high R2 values for
most of the models, except for the one for Alcalase-T, support the adoption choice of the
models. In these cases, the information about the effects of the processing parameters can
still be investigated from the analysis.
The variables that had significant effects on oil extraction yield in boric acid-NaOH
buffer were the quadratic terms of LSR and ET, and the interaction term of these two
parameters (Table 11). In the case of Tris-HCl buffer, the variables with significance were
the linear and quadratic terms of LSR, and EC. The interaction term for LSR/ET was not
significant. For the citric-phosphate buffer system, the linear terms of LSR and ET were
significant. No interaction was observed between LSR and ET.
The estimated coefficients of the quadratic models for enzymatic processes are
shown in Table 12. The terms for linear EC, and EC/ET interaction for WG treated with
Alcalase-B were significant (Table 12). When Alcalase-T system was used for WGO
extraction, the EC/ET interaction term was the only variable that showed significance.
The linear and quadratic terms of LSR and the linear term of ET were significant for
26
response surface. The change in the shape is due to the interaction between EC and ET. At
low EC an increase in ET resulted in an enhancement in oil yield (Figures 14 and 15). At
medium ECan increase in ET did not have a significant effect on oil yield (Figures 16
and 17). A decrease in oil yield with the increase in ET was observed at high EC (Figures
18 and 19). The increase in LSR lead to an improvement in the oil yield regardless of the
EC, which means there is no interaction between LSR and EC. In the Alcalase-B process
at low EC (0.1%) high oil extraction yields were obtained at high ET (Figures 14 and 15).
However at high EC (5%) high oil yield was obtained at low ET (Figures 18 and 19).
Figures 20-25 are the response surfaces and the contours for oil yield obtained with
Multifect CX GC as a function of LSR and ET at EC of 0.1, 2.5 and 5%. An increase in
EC did not improve the oil yield, nor did it change the shape of the response surface,
indicating no interaction between EC, LSR and ET. The oil extraction yield increased
with the increasing ET and decreased with increasing LSR. This trend is similar to that
observed with the corresponding non-enzymatic process.
The response surfaces and contours for oil yield obtained with Alcalase-T, as a
function of LSR and ET at EC of 0.1, 2.5 and 5%, are presented in Figures 26-31.
However, the relationship between the oil yield and LSR, ET and EC in this process is not
discussed in this thesis because of the low R2 (0.49) of the model developed and lack of
fit. It is obvious that within the experimental range, the processing parameters interact
with each other in a more complex manner in Alcalase-T process than in other processes
investigated. For Alcase-B the highest predicted oil yield was about 40%. In general oil
yield from Multifect CX GC was lower than the other two enzyme systems examined in
28
this thesis.
Although the highest predicted oil yield for Alcalase-B was about 40%, this value is
much lower than that for the corresponding non-enzymatic process (about 70%). The
highest predicted oil yield for both the enzymatic process with Multifect CX GC and the
corresponding non-enzymatic process (citric-phosphate buffer at pH 5.0) was very low,
about 20%, indicating that this enzyme did not have a significant effect on the oil
extraction yield. This result is in contrast with that obtained with corn germ (Moreau and
others 2004) where Multifect CX GC improved the oil extraction yield significantly as
compared to the control. This might be because of the differences in chemical
composition of WG and corn germ. Fiber content of corn germ (11.4%) (Ronyai and
others 1998) is much higher than that of WG (3%) (Barnes 1982). Since fiber is a
substrate for cellulase, Multifect CX GC works better with high fiber content material.
Cellulase alone is probably not enough to destroy the WG cell structure and release the oil
because of its low fiber and high protein contents.
29
CHAPTER V
CONCLUSION
In this study four enzymes, Viscozyme L, Multifect CX 13L, Multifect CX GC and
Alcalase 2.4L FG, were screened for their efficacies to extract oil from WG. Two different
buffer systems (boric acid-NaOH and Tris-HCl) were employed for Alcalase 2.4L FG
(protease). The results from the screening experiments showed that all four enzymes
failed to improve oil extraction yield when compared to the corresponding controls under
the conditions chosen for the screening studies. Alcalase 2.4L FG and Multifect CX GC
were selecetd for optimization of processing parameters for maximum oil extraction yield
by using RSM. The corresponding non-enzymatic processes were also optimized. In
general, the models developed for oil extraction yield satisfactorily explained the
relationship between oil yield and processing parameters in most processes, expect for the
process with Alcalase T. The results showed that the effects of processing parameters
and their interactions varied depending on both enzyme type and buffer system. The
highest predicted oil yield was about 40% when Alcalase-B was used for extraction.
However, this yield was much lower than that of the corresponding non-enzymatic
process (about 70%). The highest predicted oil yield with Multifect CX GC was about
20%, which was not significantly different from that of the corresponding non-enzymatic
process. In conclusion, AEOE was not effective in extracting oil from WG within the
range of processing parameters investigated in this study.
30
FUTURE RESEARCH
In this study, the experimental data from the RSM design showed that the Alcalase-T
process resulted in the highest oil extraction yield observed. However, the model
developed for this system had a low R2 (0.49). Further research should be conducted to
refine experimental range within which a model can satisfactorily explain the relationship
between oil extraction yield and the processing parameters, and thus maximizing the oil
extraction. Since the non-enzymatic process at pH 8.0 can produce an oil yield as high as
70%, an extraction process involving a non-enzymatic process followed by an enzymatic
treatment should be considered with the objective of achieving higher oil yields. Mixtures
of protease and cellulase should also be examined for their combined efficiency to
recover oil from WG.
31
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38
Factor level
-1.68
-1
+1
+1.68
7.5
12
16.5
20
0.1
1.1
2.55
0.5
5.25
12.25
19.25
24
39
Run
Extraction time
-1
-1
-1
-1
-1.68
1.68
1.68
-1.68
10
40
Run
Enzyme concentration
Extraction time
-1
-1
-1
-1
-1
-1
-1
-1
-1
-1
-1
-1
-1.68
10
-1.68
11
-1.68
12
1.68
13
1.68
14
1.68
15
16
41
g/100g germ *
Moisture
11.01 0.19
Oil
11.17 0.12
Protein
33.79 0.32
Starch
9.08 0.08
Ash
4.86 0.02
30.09
42
>500
5.85
150-500
88.58
<150
3.86
43
a
b
Enzyme
Activity a
Activityb
Viscozyme L
170 FBG/g
100FBG/g
Multifect CX 13L
2294 CMC/g
3900CMC/g
Multifect CX GC
4338 CMC/g
3200CMC/g
Alcalase 2.4L FG
12.7 U/g
2.4 AU/g
44
Table 7: Observed and predicted oil extraction yield (%) for non-enzymatic processes.
Run
pH 8.0 (Tris-HCl)
Observed
Predicted
value
value
pH 5.0 (Citric-phosphate)
Observed
Predicted
value
value
45
19.10
17.72
35.54
30.97
10.10
16.38
18.50
19.28
9.64
6.32
17.03
16.29
42.83
42.99
38.56
36.38
4.83
3.18
25.66
27.97
7.64
6.71
9.06
7.39
15.62
16.34
17.42
20.5
17.07
17.31
23.27
21.65
3.97
5.17
11.39
12.25
47.70
46.54
25.27
25.66
2.61
3.89
31.99
32.94
48.07
50.77
2.07
2.91
22.48
22.38
6.77
8.72
8.55
9.17
10
22.89
22.38
7.05
8.72
8.21
9.17
45
Table 8: Observed and predicted oil extraction yield (%) for enzymatic processes.
Run
1
Alcalase-B
Observed
Predicted
value
value
8.09
5.16
Alcalase-T
Observed
Predicted
value
value
41.41
14.67
Multifect CX GC
Observed
Predicted
value
value
9.47
11.04
46
8.55
9.42
20.44
24.7
13.06
14.98
20.77
17.38
56.94
49.72
8.97
10.13
19.46
17.38
48.89
40.15
4.43
3.87
5.61
1.72
22.16
20.99
14.40
15.92
27.58
25
59.00
56.32
7.57
7.38
36.32
29.49
66.45
52.28
2.57
1.62
20.23
17.19
12.87
29.7
7.59
6.98
2.79
5.1
10.63
23.11
22.99
19.99
10
11.94
13.03
13.97
29.25
9.23
8.08
11
11.16
17.36
12.49
41.6
3.34
3.09
12
24.48
29.72
53.46
53.5
3.90
5.67
13
9.49
16.75
37.77
36.37
7.18
6.98
14
8.36
10.62
46.11
31.07
12.02
10.9
15
17.14
16.14
19.88
20.46
8.51
8.73
16
15.66
16.14
21.90
20.46
8.86
9.38
46
Table 9: Analysis of variance for response surface quadratic models for the non-enzymatic processes.
Non-enzymatic
processes
Source
Sum of squares
Degree of freedom
F-value
P>F
5
14
3
11
19
401.82
2.78
8.85
1.12
144.61
<0.0001
pH 8.0
(Boric acid-NaOH)
Model
2009.12
Residual
38.9
Lack of fit
26.56
Pure error
12.34
Total
2048.02
coefficient of variance=6.17%, R2=0.9810
7.89
0.0044
882.23
11.29
41.59
3.03
78.14
<0.0001
pH 8.0 (Tris-HCl)
Model
4411.16
5
Residual
158.07
14
Lack of fit
124.77
3
Pure error
33.3
11
Total
4569.23
19
2
coefficient of variance=16.81%, R =0.9654
13.74
0.0005
92.99
2.01
7.35
0.55
46.27
<0.0001
pH 5.0
(Citric-Phosphate)
Model
464.95
5
Residual
28.13
14
Lack of fit
22.04
3
Pure error
6.09
11
Total
492.08
19
2
coefficient of variance=15.59%, R =0.9429
13.26
0.0006
47
Mean square
47
Table 10: Analysis of variance for response surface quadratic models for the enzymatic processes.
Enzymatic
processes
Source
Alcalase-B
Model
1951.57
Residual
493.93
Lack of fit
466.64
Pure error
27.29
Total
2445.5
coefficient of variance=30.62%, R2=0.7980
Sum of squares
Degree of freedom
F-value
P>F
9
22
5
17
31
216.84
22.45
93.33
1.61
9.66
<0.0001
58.14
<0.0001
9
22
5
17
31
605.63
259.8
1127.9
4.48
2.33
0.0509
Alcalase-T
Model
5450.64
Residual
5715.66
Lack of fit
5639.49
Pure error
76.17
Total
11166
coefficient of variance=47.38%, R2=0.4881
251.74
<0.0001
9
22
5
17
31
78.27
2.53
10.51
0.19
30.9
<0.0001
Multifect CX GC
Model
704.39
Residual
55.73
Lack of fit
52.53
Pure error
3.2
Total
760.12
coefficient of variance=17.67%, R2=0.9267
55.75
<0.0001
48
Mean square
48
Table 11: Estimated coefficients of the quadratic models for non-enzymatic processes.
pH 8.0 (Tris-HCl)
pH 5.0 (Citric-phosphate)
49
Variable
Parameter
Estimate
p Value
Variable
Parameter
Estimate
p Value
Variable
Parameter
Estimate
p Value
intercept
12.02
0.0113
intercept
76.01
<0.0001
intercept
12.72
0.0027
Ratio
0.1
0.8451
ratio
-4.58
0.0005
ratio
-1.11
0.0211
ratio*ratio
0.14
<0.0001
ratio*ratio
0.22
<0.0001
ratio*ratio
0.022
0.174
Time
0.22
0.4832
time
-4.88
<0.0001
time
0.94
0.0033
time*time
0.035
0.0009
time*time
0.14
<0.0001
time*time
-0.012
0.1356
ratio*time
-0.13
<0.0001
ratio*time
-0.04
0.3091
ratio*time
-0.021
0.1994
Ratio and time represent liquid: solid ratio and extraction time, respectively.
49
Table 12: Estimated coefficients of the quadratic models for enzymatic processes.
Alcalase-B
Alcalase-T
Multifect CX GC
50
Variable
Parameter
Estimate
p Value
Variable
Parameter
Estimate
p Value
Variable
Parameter
Estimate
p Value
intercept
-13.11
0.3259
intercept
20.7
0.6454
intercept
21.36
<0.0001
ratio
0.75
0.5932
ratio
-3.15
0.508
ratio
-2.26
<0.0001
ratio*ratio
0.2
0.6935
ratio*ratio
0.28
0.114
ratio*ratio
0.064
0.0009
conc
7.88
0.0493
conc
13.19
0.317
conc
0.85
0.509
conc*conc
-0.21
0.6963
conc*conc
2.06
0.2618
conc*conc
-0.2
0.2695
time
1.03
0.2066
time
-1.41
0.603
time
0.57
0.0441
time*time
-0.016
0.5019
time*time
0.11
0.1523
time*time
-0.013
0.1152
ratio*time
0.027
0.4857
ratio*time
0.049
0.7069
ratio*time
-0.0035
0.7872
ratio*conc
-0.0044
0.9809
ratio*conc
-0.88
0.1691
ratio*conc
-0.052
0.4072
conc*time
-0.49
0.0004
conc*time
-0.95
0.025
conc*time
0.046
0.2575
Ratio, conc and time represent liquid: solid ratio, enzyme concentration and extraction time, respectively.
50
Endosperm
Bran
Germ
Embryo
51
52
Oil-bearing material
Grinding
Enzyme
Incubation
Centrifugation
Solid phase
Recovering of oil
53
Figure 4: A schematic of aqueous enzymatic oil extraction procedure employed in this study.
Oil-bearing material
Grinding
Enzyme
Incubation
Centrifugation
Solid phase
Centrifugation
Liquid phase
Solid phase
54
Oil Oil
Extraction
Yield
Extraction
Yield(%)
(%
60.0
c
50.0
b
40.0
30.0
20.0
a
10.0
C
ct
CX
-T
M
ul
tif
e
lc
al
as
e
-B
lc
al
as
e
M
ul
tif
e
ct
CX
oz
ym
isc
13
L
eL
l4
V
Co
nt
ro
l3
Co
nt
ro
l2
nt
ro
Co
Co
nt
ro
l1
0.0
55
Activity (U/g)
Week 1
Week 2
Week 3
56
Week 6
Week 7
Activity (CMC/g)
4800
4600
4400
4200
Week 1
Week 4
57
Week 5
Figure 8: Response surface for oil extraction yield by non-enzymatic process in boric
acid-NaOH buffer at pH 8.0.
58
Figure 9: Response surface contour for oil extraction yield by non-enzymatic process in
boric acid-NaOH buffer at pH 8.0.
59
Figure 10: Response surface for oil extraction yield by non-enzymatic process in Tris-HCl
buffer at pH 8.0.
60
Figure 11: Response surface contour for oil extraction yield by non-enzymatic process in
Tris-HCl buffer at pH 8.0.
61
Figure 12: Response surface for oil extraction yield by non-enzymatic process in
citric-phosphate buffer at pH 5.0.
62
Figure 13: Response surface contour for oil extraction yield by non-enzymatic process in
citric-phosphate buffer at pH 5.0.
63
Figure 14: Response surface for oil extraction yield by enzymatic process with Alcalase-B,
as a function of liquid: solid ratio and extraction time at enzyme concentration of 0.1%.
64
Figure 15: Response surface contour for oil extraction yield by enzymatic process with
Alcalase-B, as a function of liquid: solid ratio and extraction time at enzyme concentration of
0.1%.
65
Figure 16: Response surface for oil extraction yield by enzymatic process with Alcalase-B,
as a function of liquid: solid ratio and extraction time at enzyme concentration of 2.5%.
66
Figure 17: Response surface contour for oil extraction yield by enzymatic process with
Alcalase-B, as a function of liquid: solid ratio and extraction time at enzyme concentration of
2.5%.
67
Figure 18: Response surface for oil extraction yield by enzymatic process with Alcalase-B,
as a function of liquid: solid ratio and extraction time at enzyme concentration of 5%.
68
Figure 19: Response surface contour for oil extraction yield by enzymatic process with
Alcalase-B, as a function of liquid: solid ratio and extraction time at enzyme concentration of
5%.
69
Figure 20: Response surface for oil extraction yield by enzymatic process with Multifect CX
GC, as a function of liquid: solid ratio and extraction time at enzyme concentration of 0.1%.
70
Figure 21: Response surface contour for oil extraction yield by enzymatic process with
Multifect CX GC, as a function of liquid: solid ratio and extraction time at enzyme
concentration of 0.1%.
71
Figure 22: Response surface for oil extraction yield by enzymatic process with Multifect CX
GC, as a function of liquid: solid ratio and extraction time at enzyme concentration of 2.5%.
72
Figure 23: Response surface contour for oil extraction yield by enzymatic process with
Multifect CX GC, as a function of liquid: solid ratio and extraction time at enzyme
concentration of 2.5%.
73
Figure 24: Response surface for oil extraction yield by enzymatic process with Multifect CX
GC, as a function of liquid: solid ratio and extraction time at enzyme concentration of 5%.
74
Figure 25: Response surface contour for oil extraction yield by enzymatic process with
Multifect CX GC, as a function of liquid: solid ratio and extraction time at enzyme
concentration of 5%.
75
Figure 26: Response surface for oil extraction yield by enzymatic process with Alcalase-T, as
a function of liquid: solid ratio and extraction time at enzyme concentration of 0.1%.
76
Figure 27: Response surface contour for oil extraction yield by enzymatic process with
Alcalase-T, as a function of liquid: solid ratio and extraction time at enzyme concentration of
0.1%.
77
Figure 28: Response surface for oil extraction yield by enzymatic process with Alcalase-T, as
a function of liquid: solid ratio and extraction time at enzyme concentration of 2.5%.
78
Figure 29: Response surface contour for oil extraction yield by enzymatic process with
Alcalase-T, as a function of liquid: solid ratio and extraction time at enzyme concentration of
2.5%.
79
Figure 30: Response surface for oil extraction yield by enzymatic process with Alcalase-T, as
a function of liquid: solid ratio and extraction time at enzyme concentration of 5%.
80
Figure 31: Response surface contour for oil extraction yield by enzymatic process with
Alcalase-T, as a function of liquid: solid ratio and extraction time at enzyme concentration of
5%.
81
VITA
MEIXHEN XIE
Candidate for the Degree of
Master of Science
Thesis: AQUEOUS ENZYMATIC EXTRACTION OF WHEAT GERM OIL
82