Food Chemistry 157 (2014) 132140

Contents lists available at ScienceDirect

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

Immobilized phospholipase A1-catalyzed modication

of phosphatidylcholine with n3 polyunsaturated fatty acid
TingTing Zhao a,e,1, Da Som No a,e,1, Byung Hee Kim b, Hugo S. Garcia c, Yangha Kim d, In-Hwan Kim a,e,
a

Department of Food and Nutrition, Korea University, Seoul 136-703, Republic of Korea
Department of Food Science and Technology, Chung-Ang University, Anseong 456-756, Republic of Korea
c
UNIDA, Instituto Tecnolgico de Veracruz, Veracruz, Ver. 91897, Mexico
d
Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 120-749, Republic of Korea
e
Department of Public Health Sciences, Graduate School, Korea University, Seoul 136-703, Republic of Korea
b

a r t i c l e

i n f o

Article history:
Received 19 August 2013
Received in revised form 24 December 2013
Accepted 5 February 2014
Available online 14 February 2014
Keywords:
Acidolysis
n3 Polyunsaturated fatty acid
Phosphatidylcholine
Phospholipase A1
Vacuum

a b s t r a c t
n3 Polyunsaturated fatty acids (n3 PUFA)-enriched phosphatidylcholine (PC) was successfully produced with fatty acid from sh oil and PC from soybean by immobilized phospholipase A1-catalyzed
acidolysis. Detailed studies of immobilization were carried out, and Lewatit VP OC 1600 was selected
as a carrier for preparation of immobilized phospholipase A1, which was used for modication of PC
by acidolysis. For acidolysis of PC with n3 PUFA, the effects of several parameters, namely, water content, temperature, and enzyme loading on the reaction time course were investigated to determine optimum conditions. The optimum water content, temperature, and enzyme loading were 1.0%, 55 C, and
20%, respectively. The highest incorporation (57.4 mol%) of n3 PUFA into PC was obtained at 24 h and
the yield of PC was 16.7 mol%. The yield of PC increased signicantly by application of vacuum, even
though a slight decrease of n3 PUFA incorporation was observed.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Phospholipids (PLs) are major constituents of cell membranes
and play crucial roles in the biochemistry and physiology of the cell
(Kidd & Head, 2005). PLs have been widely used in food, pharmaceutical, and cosmetic products as highly efcient emulsiers.
In recent decades, n3 polyunsaturated fatty acid (n3 PUFA),
especially eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA), have received signicant scientic attention because of their
health benets, which include improvement of immune function
and prevention of heart disease and cancer (Khandelwal et al.,
2013; Komprda, 2012; Kromhout, Bosschieter, & de Lezenne
Coulander, 1985; Larsson, Kumlin, Ingelman-Sundberg, & Wolk,
2004). Consumption of n3 PUFA has also been reported to provide
important benets with respect to functioning of the brain and retina, as well as accelerating the growth of preterm baby (Carlson,
Werkman, Peeples, Cooke, & Tolley, 1993; Lanting, Fidler, Huisman,
Touwen, & Boersma, 1994; Neuringer, Connor, Van Petten, &
Corresponding author at: Department of Food and Nutrition, Korea University,
Jeongneung-dong, Seongbuk-Gu, Seoul 136-703, Republic of Korea. Tel.: +82 2 940
2855; fax: +82 2 941 7825.
E-mail address: k610in@korea.ac.kr (I.-H. Kim).
1
TingTing Zhao and Da Som No contributed equally to this research.
http://dx.doi.org/10.1016/j.foodchem.2014.02.024
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

Barstad, 1984). Long-chain n3 PUFAs are characteristic of marine

oils and occur pervasively in the PLs of sh and marine species, with
EPA and DHA commonly accounting for up to 50% of their fatty acid
constituents (Haraldsson & Thorarensen, 1999). The presence of
long-chain and low-melting PUFA is believed to add uidity and
mobility to cell membranes and thus, they adjust membrane
integrity and function properly in lower ambient temperatures
(Haraldsson & Thorarensen, 1999). In addition, fatty acids are more
easily absorbed in the body as PL than as the corresponding triacylglycerols (TAGs) or ethyl esters (Galli et al., 1992). Because of the
positive inuence of n3 PUFAs on human health, there is a growing demand for them in the pharmaceutical industry in a PL form as
well as their natural TAG form (Carlson, 1991). Lemaitre-Delaunay
et al. (1999) have provided evidence for higher bioavailability of
DHA for incorporation into erythrocytes in human adults when
DHA is provided in PL rather than in TAG. Wijendran et al. (2002)
also reported that PL were about 2.1-fold more effective than TAG
as substrates for accretion of brain arachidonic acid in the development of neonatal primate brain.
Although PLs containing n3 PUFA is available in sh and marine products, renement procedures, including laborious extraction and separation, are required for it to be used industrially.
Thus, enzymatic modication using inexpensive plant lecithin

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T. Zhao et al. / Food Chemistry 157 (2014) 132140

(PC, phosphatidylcholine) and a rich n3 PUFA source might be an

effective method for obtaining n3 PUFA-enriched PLs. Replacement of fatty acid residues present in a native PL by fatty acids
with benecial physiological effects can lead to tailored PLs that
offer intriguing marketing opportunities for manufacturers of
nutraceuticals.
Interest in the production of structured PLs containing specic
fatty acid residues has also increased signicantly in recent years.
Vikbjerg, Mu, and Xu (2007) have reported that phospholipase
A2-catalyzed synthesis of PL with caprylic acid by acyl modication of the sn2 position in PLs. Hossen and Hernandez (2005)
have reported that Lypozyme RM IM (from Rhizomucor miehei)
and Lipozyme TL IM (from Thermomyces lanuginosus) are effective
in the incorporation of conjugated linoleic acid into soybean PL.
Previous research concerning the incorporation of n3 PUFA into
PC by phospholipase A1 (PLA1) immobilized on Duolite A568 as
a carrier has been reported (Kim, Garcia, & Hill, 2010). However,
although a modied PC containing n3 PUFA can be produced successfully, there is a concomitant signicant decrease in PC yield as
a consequence of hydrolysis. Therefore, it is important to nd the
means to enhance the desired yield of modied PC.
In this study, detailed experiments were rst conducted regarding immobilization of PLA1, and then the immobilized enzyme was
employed for modication of PC. PC from soybean and highly
enriched n3 PUFA from sh oil were used as substrates for synthesis of structured PC by enzyme-catalyzed acidolysis. Although a
response surface methodology for optimizing reaction conditions
is preferred as it take into account interaction terms, to be consistent with our previous study about the modication of phospholipids, a one-factor-at-a-time approach was followed in this study.
Thus, the effects of water content, temperature, and enzyme loading on incorporation of n3 PUFA into PC were ascertained by monitoring reaction time course. In addition, the effect of vacuum was
examined with the goal of enhancing the yield of structured PC.

2.2. Immobilization of PLA1

For the immobilization of PLA1, a free type PLA1 was mixed
with sodium phosphate buffer solution (50 mM, pH 7.5) at various
ratios. The enzyme suspension was stirred at 300 rpm for 30 min
and its soluble protein concentration was determined according
to Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum
albumin as the standard. For hydrophobic carriers, one gram of
each carrier was wetted with 20 mL of ethanol for 2 h until the carrier sedimented. After decanting ethanol, the carrier was washed
with 40 mL sodium phosphate buffer and subsequently placed in
a ask with 10 mL of enzyme suspension. Immobilization was performed in a water bath shaker operating at 200 rpm and 30 C.
After shaking for 16 h, the suspension was ltered to recover the
resulting immobilized enzyme preparation, which was then rinsed
with 40 mL of sodium phosphate buffer and then dried at 45 C in a
vacuum oven overnight, and stored at 4 C until use. The xation
level (wt%) and the amount of protein bound in immobilized
enzyme (mg/g) were estimated by subtracting the protein remaining in the enzyme suspension after immobilization compared with
the initial protein concentration.
Fixation level (%) and amount of protein adsorbed onto the carrier (mg/g) were calculated by following equations:

Fixation level %

ab
 100
a

Protein amount in immobilized enzyme mg=g

ab
 100
cab

a: amount of initial protein in enzyme suspension (mg).

b: amount of unbound protein in enzyme suspension after
immobilization (mg).
c: amount of carrier used in immobilization (g).
2.3. Activity test for immobilized PLA1

2. Materials and methods

2.1. Materials
PLA1 (Lecitase Ultra) from T. lanuginosus/Fusarium oxysporum
was obtained from Novozymes (Franklinton, NC, USA) as an aqueous solution of 57.0% water (by vol.). Fatty acid standards were
purchased from SigmaAldrich, Inc. (St. Louis, MO, USA). Lewatit
VP OC 1600, Accurel MP 1000, and Duolite A568 were purchased
from Lanxess Energizing Chemistry (Leverkusen, Germany), Membrana GmbH (-Accurel System, Obernburg, Germany), and Rohm
and Haas France SAS (Chauny, France), respectively. Amberlite
XAD 7HP, Celite 545, Dowex 50w x8, Amberlite XAD4, and Octyl
silica were purchased from SigmaAldrich, Inc. Granulated PC
(purity 98%, from soybean) was obtained from Avanti Polar-Lipids, Inc. (Alabaster, AL, USA). The primary fatty acid residues in
the soybean PC were those of linoleic acid, 18:2n6
(64.9 mol%), palmitic acid, 16:0 (14.0 mol%), oleic acid, 18:1n9
(10.4 mol%), linolenic acid, 18:3n3 (6.2 mol%) and stearic acid,
18:0 (3.3 mol%). Highly enriched n3 PUFA from sh oil was
donated by Ilshinwells Co., Ltd. (Seoul, Republic of Korea). The
primary species in the highly enriched n3 PUFA from sh oil
were those of oleic acid (2.5 mol%), EPA, 20:5n3 (14.7 mol%),
docosapentaenoic acid, 22:5n3 (DPA, 6.0 mol%) and DHA,
22:6n3 (71.2 mol%). For the present purposes, we dened n3
PUFA as the sum of EPA, DPA, and DHA. The TLC plates were
coated with silica gel (layer thickness 0.25 mm, Kieselgel 60
F254, Merck KGaA, Darmstadt, Germany). All solvents and chemicals were analytical grade.

Enzymatic modication of PC was evaluated by performing

activity tests. A 1.2 g sample of highly enriched n3 PUFA from sh
oil (8 mol) and 2.8 g of PC (1 mol) were placed in a 50 mL waterjacketed glass vessel, mixed at 250 rpm and preheated to 55 C.
Then, 0.5 g of the enzyme (10% of total substrate weight) was
added. Samples (50 mg) of the product mixture were taken at regular intervals, dissolved in chloroform, and applied to silica-coated
preparative TLC plates and developed to obtain the PC fraction.
After saponication and methylation, the resulting fatty acid
methyl esters (FAMEs) were subjected to gas chromatographic
analysis.
The apparent activity was dened as the initial reaction rate
divided by the amount of immobilized enzyme. The specic activity was dened as the initial reaction rate divided by the amount of
protein. The acidolysis reaction involves concurrent decomposition
of PC and addition of fatty acid to the molecule. Since esterication
of fatty acid should be preceded by hydrolysis of PC, hydrolytic
activity of the enzyme was used to measure the initial reaction
rate. The apparent activity and the specic activity were calculated
as follows:

Apparent activity lmol=g particle=min

d
ef

Specific activity lmol=g protein=min

d
gf

d: hydrolyzed PC (lmol).
e: amount of immobilized enzyme used in the reaction (g).

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T. Zhao et al. / Food Chemistry 157 (2014) 132140

f: initial reaction time (min).

g: protein amount in the immobilized enzyme used in the reaction (g).

Table 1
Fixation level (%) and the specic activity (lmol/g protein/min) of immobilized PLA1
on different carriers.a
Carrier

Fixation level
(%)

Specic activity (lmol/g protein/

min)

Celite 545
Duolite A568
Amberlite XAD
7HP
Dowex 50w x8
Amberlite XAD4
Accurel MP 1000
Octyl silica
Lewatit VP OC
1600

25.8
73.2
78.2

1.0  103
6.3  103
6.6  103

29.2
28.9
38.0
39.0
79.5

2.2  103
2.2  103
5.8  103
0.7  103
6.7  103

2.4. Immobilized PLA1-catalyzed modication of PC

The enzymatic modication of PC with n3 PUFA was accomplished using a two-step approach. In the rst step, acidolysis
between PC and n3 PUFA was carried out under atmospheric
pressure in a solvent-free system using a 50 mL water-jacketed
glass vessel. For all reactions, a ratio of fatty acid to PC was 8:1
(mol/mol), which was optimized from our previous study (Garcia,
Lopez-Hernandez, Hill, & Kim, 2008; Kim, Garcia, & Hill, 2007;
Kim et al., 2010). Prior to initiating the reaction, the reaction vessel
had been heated to the desired temperature using a water circulator. The weight of total substrate was 3 g, with various amounts of
water and various amount of immobilized PLA1 (0.21.4% and 2.5
30%, respectively, based on total substrate weight) added to a mixture of PC and fatty acids. The reaction was initiated by enzyme
addition while stirring with a magnetic stirrer at 250 rpm under
nitrogen of atmosphere. In the second step, the same reactor was
connected to a vacuum pump at various points in the reaction time
course to produce a pressure of 0.7 kPa. Vacuum was controlled by a
micrometering valve (Swagelok Co., Solon, OH, USA) and monitored
by a digital vacuum gauge (Teledyne Technologies Inc., Thousand
Oaks, CA, USA).
2.5. Analysis of products
Samples (50 mg) of reaction mixtures were dissolved in chloroform, and then applied to preparative silica-coated TLC plates and
developed
with
chloroform/methanol/acetic
acid/water
(75:40:8:3, by vol.). Lysophosphatidylcholine (LPC) and PC were
detected with a 2,7-dichlorouroscein spray (0.2% by wt in 95%
methanol/water, by vol.). Bands corresponding to LPC and PC were
recovered from TLC plate, extracted, and methylated; heptadecanoic acid was used as an internal standard. The resulting FAMEs
were analyzed by a Varian 3800 gas chromatograph (Varian, Inc.,
Palo Alto, CA, USA) using a Supelcowax 10 fused-silica capillary
column (30 m  0.25 mm i.d.; Supelco, Inc., Bellefonte, PA, USA)
and ame ionisation detector. The column was held at 180 C for
1 min and programmed to 230 C for 10 min at the rate of 5 C/
min. The carrier gas was helium and total gas ow rate was
50 ml/min. The injector and detector temperatures were 240 and
250 C, respectively. FAMEs were identied by comparison with
the retention times of standards. The incorporation of n3 PUFA
into PC and the yield of PC were calculated as follows:

Incorporation of n  3 PUFA into PC

the mole of EPA DPA DHA in PC

 100
the mole of total fatty acid in PC

Yield of PC

the mole of PC after the reaction

 100
the mole of initial PC

3. Results and discussion

3.1. Immobilization of PLA1
3.1.1. Carrier screening
Immobilization of an enzyme yields the enzyme reusable, minimizes product contamination by the enzyme, and simplies separation of product and enzyme. The type of carrier is a crucial factor
in such an enzyme immobilization process. Table 1 shows the
effect of the selected carriers on the xation level (%) and specic

a
Tabular entries, the average of duplicate determinations from different experimental trials.

activity (lmol/g protein/min) of the immobilized enzyme. Among

the eight carriers examined, Lewatit VP OC 1600, Accurel MP 1000,
Amberlite XAD 4 and Octyl silica were hydrophobic. Since hydrophobic carriers are not easily suspended in the enzyme suspension,
pre-wetting hydrophobic carriers with ethanol prior to immobilization was suggested to solve this problem in the previous studies
(Vikbjerg et al., 2007; Zhang, Hellgren, & Xu, 2007). In this study,
Pre-wetting indeed made it possible to suspend these carriers in
the enzyme suspension, resulting also in increased xation level
of PLA1. Lewatit VP OC 1600 (xation level, 79.5%) exhibited the
highest xation, followed by Amberlite XAD 7HP (78.2%) and Duolite A568 (73.2%).
The catalytic activities of the various immobilized PLA1 were
compared by measuring the specic activities based on its hydrolytic activity, according to Vikbjerg et al. (2007). Lewatit VP OC
1600 (specic activity, 6.7  103 lmol/g protein/min), Amberlite
XAD7HP (6.6  103 lmol/g protein/min) and Duolite A568
(6.3  103 lmol/g protein/min) as carriers resulted not only in
signicantly higher xation levels but also higher specic activities, compared with the other carriers tested. However, low xation did not necessarily lead to low specic activity of the
immobilized enzyme. For instance, in case of Accurel MP 1000,
even though xation level was low, specic activity was signicantly higher, compared with Amberlite XAD 4, which showed very
similar xation level to Accurel MP 1000. Among the carriers,
Lewatit VP OC 1600 had the highest protein xation as well as specic activity. Thus, Lewatit VP OC 1600 was judged a suitable carrier for further experimentation.
3.1.2. Protein concentration of initial enzyme suspension
The initial enzyme concentration is also an important factor to
be considered for the efciency of the immobilization. The initial
PLA1 suspension, whose protein concentration ranged from 2.9
to 29.6 mg/mL, was tested for the immobilization of PLA1.
Increased protein concentration in the PLA1 suspension increased
the amount of protein immobilized (mg/g, Fig. 1a). Meanwhile,
the xation level remained constant up to a protein concentration
of 14.9 mg/g, but deceased signicantly with further increased protein concentration. This indicated that, after the carrier was saturated with loaded protein, binding ability of the carrier decreased
with the increasing protein concentration of the initial PLA1
suspension.
The apparent activity increased steadily when protein amount
in the carrier was increased from 16.9 to 147.9 mg/g, which corresponded to a protein concentration of 2.920.6 mg/mL, (Fig. 1b).
However, there was no signicant difference in apparent activity
as the protein amount in the carrier increased further. Meanwhile,

135

T. Zhao et al. / Food Chemistry 157 (2014) 132140

the specic activity of immobilized enzyme increased when the

protein amount of the immobilized enzyme was increased from
16.9 to 56.9 mg/g and the specic activity remained constant when
the protein amount of the immobilized enzyme was increased up
to 147.9 mg/g. However, the specic activity decreased with
further increase of protein amount in immobilized enzyme. The
decline in specic activity was related to the increased steric
restrictions and the mass-transfer limitations of the enzyme. Due
to the lack of surface area, the enzyme tends to be packed into
more than a monolayer on the surface, causing decrease in activity
(Madoery, Gattone, & Fidelio, 1995). Therefore, an initial enzyme
concentration of 20.6 mg/g was selected for the immobilization
of PLA1 and the resulting immobilized enzyme preparation was
used for subsequent acidolysis reactions.

3.2. Modication of PC with n3 PUFA using immobilized PLA1

3.2.1. Water content
Water content is a very important variable that affects both the
reaction rate and the thermo-equilibrium of reactions catalyzed by
phospholipases. In the acidolysis reaction, water plays a complex
role in terms of compromising the activity, hydrolysis side reactions, reaction rate, and extent of incorporation of fatty acids. Since
excess water might act exclusively as a nucleophilic substrate for
hydrolysis rather than for esterication of desired fatty acids,
water content in the reaction mixture is crucial for optimization
of the acidolysis reaction in terms of yield.
In this study, water contents ranging from 0.2% to 1.4% (based
on total substrate weight) were tested for modication of PC with

(a)

80

Fixation level (%)

160
75
120

70
65

80

60
40
55

Protein amount
in immobilized enzyme (mg/g)

200

85

50
0

10

15

20

25

30

0.0008

(b)

0.005

0.0006

0.004

0.003

0.0004

0.002
0.0002
0.001

0.000

0.0000
0

20

40

60

80

100

120

140

160

Apparent activity (ug/g particle/min)

Speciic activity (ug/g protein/min)

Protein concentration in PLA1 solution (mg/mL)

180

Protein amount in immobilized enzyme (mg/g)

Fig. 1. The effect of protein concentration in the PLA1 suspension on the immobilization process. Immobilization procedure and the activity test of immobilized enzyme are
described in Section 2; (a) xation level (j) and protein amount of immobilized enzyme (d); and (b) specic activity (d) and apparent activity (j).

136

T. Zhao et al. / Food Chemistry 157 (2014) 132140

n3 PUFA. For these trials, reaction temperature, enzyme loading

and molar ratio of PC to fatty acid were held at 55 C, 10% (based
on total substrate weight) and 1:8, respectively. The effect of water
content on acidolysis reaction was shown in the Fig. 2. n3 PUFA
was dened as the sum of EPA, DPA, and DHA and was not detected
in the initial soybean PC. During the initial 3 h reaction time, radical increase in the incorporation of n3 PUFA into PC occurred, but
the yield of PC decreased in all the water contents tested. Although
longer reaction times led to greater incorporation of n3 PUFA in
the PC there was a concurrent signicant decrease in PC yield as
a consequence of hydrolysis.
The incorporation of n3 PUFA into PC increased drastically as
the water content increased from 0.2% to 1.0%, but there was no
signicant difference between 1.0% and 1.4%. Meanwhile, a significant decrease in the yield of PC was observed as the water content
increased from 0.2% to 0.6%. However, when water content was
further increased from 0.6 to 1.4, the magnitude of the decreases
in the yield of PC was relatively small.
The precise role water plays to increase incorporation of fatty
acid to PC remains to be elucidated and has been the subject of
extensive speculation. A possible explanation could be that essential water is needed for the immobilized PLA1 to have catalytic
activity. Essential water is known to be needed to maintain the

Incorporation of n-3 PUFA (mol%)

into PC

50

(a)

40

30

20

10

0
0

10

15

20

25

Reaction time (h)

100

(b)

PC yield (mol%)

80

60

40

20

0
0

10

15

20

25

Reaction time (h)

Fig. 2. Effect of water content on the incorporation of n3 PUFA into PC (a) and the
yield of PC (b) by acidolysis as a function of reaction time shown by: 0.2% (d); 0.6%
(s); 0.8% (.); 1.0% (D); 1.2 (j). Reaction performed at a molar ratio of 1:8 (PC:n3
PUFA), 55 C reaction temperature, and enzyme loading at 10% (based on total
substrate weight); n3 PUFA is dened as sum of EPA, DPA, and DHA; and bar,
mean pooled SD (n = 2).

three-dimensional conguration necessary for catalytic activity

(Zhao, Lu, Bie, Lu, & Liu, 2007). Since free type PLA1 is in a form
of aqueous solution, the activity of PLA1 could be closely related
to essential water. Another explanation is that water facilitates
the acidolysis reaction by promoting hydrolysis. Since hydrolysis
of PC should precede esterication of fatty acid, water can contribute in the hydrolysis reaction as a substrate.
Similar effects of adding water to the reaction mixture have
been evaluated previously by different authors. Haraldsson and
Thorarensen (1999) have reported that 5% water addition resulted
in the highest incorporation of EPA into PC using PLA1 as a biocatalyst, but also gave the highest degree of hydrolysis as a side reaction. Vikbjerg et al. (2007) have reported that greater extents of
acidolysis of PC with caprylic acid at water contents of 24% with
Lipozyme TL IM (from T. lanuginosus). In the present study, water
content of 1.0% or higher showed the highest incorporation of
n3 PUFA (33.9 mol%) with a 30.9 mol% yield. Therefore, 1.0%
water was selected as optimum for further experimentation.
3.2.2. Temperature
In general, an increase in the reaction temperature of enzymecatalyzed reactions results in increased reaction rates, according
to Arrheniuss law and barring enzyme denaturation. A higher temperature favours higher yields for endothermic reactions owing to
a shift in thermodynamic equilibrium. A reactions optimal temperature should be determined on an individual basis and based
on the melting point of the substrates and products (Kim & Kim,
2000; Virto & Adlercreutz, 2000; Virto, Svensson, & Adlercreutz,
1999).
In this study, temperatures ranged from 35 to 65 C were tested
for the modication of PC with n3 PUFA, because at less than
35 C, the substrate mixture could not be stirred sufciently due
to high viscosity of the mixture. For these trials, water content,
enzyme loading and molar ratio of PC to fatty acid were maintained
at 1.0% (based on total substrate weight), 10% (based on total substrate weight) and 1:8, respectively. The effect of temperature on
the acidolysis reaction is shown in Fig. 3. During the rst 6 h of reaction, the incorporation of n3 PUFA into PC increased signicantly
when the temperature was increased from 35 to 55 C. However,
when the temperature was further increased from 55 to 65 C, the
incorporation of n3 PUFA into PC decreased in the same time
frame. Moreover, at 55 C, the maximum incorporation of ca.
32.0 mol% was achieved after only 6 h. Meanwhile, at all temperatures tested, no signicant differences in the incorporation of n3
PUFA were observed after 24 h. A slightly higher yield of PC was obtained with the lower temperatures, but there were no signicant
differences observed in the temperature range tested.
In general, enzyme stability is inuenced by temperature; a
high temperature will greatly reduce the enzyme stability and its
half-life through denaturation. A higher temperature will also
increase the lipid oxidation rate, especially if PUFAs are used as
acyl donors (Kim, Lee, Oh, & Kim, 2001; Xu, 2000). In a previous
study from our research group, the highest incorporation was
achieved at 55 C when a free type PLA1 was used (Kim, Garcia,
& Hill, 2007; Peng, Xu, Mu, Hy, & Adler-Nissen, 2002). In addition,
Peng et al. (2002) have reported a maximal observed incorporation
at 57.5 C in batch reactions for the same enzyme and substrate.
These previous reports validate the optimal temperature determined in the present study. Hence, 55 C was selected as the optimal temperature and used in subsequent experimental trials,
representing the maximum incorporation with the fastest reaction
time achieved at this temperature.
3.2.3. Enzyme loading
Enzyme loadings ranging from 2.5% to 30% (based on total substrate weight) were tested for the modication of PC with n3

137

T. Zhao et al. / Food Chemistry 157 (2014) 132140

Incorporation of n-3 PUFA (mol%)

into PC

40

(a)

30

20

10

10

15

20

25

Reaction time (h)

100

(b)

Yield of PC (mol%)

80

60

40

20

10

15

20

25

Reaction time (h)

Fig. 3. Effect of temperature on the incorporation of n3 PUFA into PC (a) and the yield of PC (b) by acidolysis as a function of reaction time shown by: 35 C (d); 45 C (s);
55 C (.); 65 C (D). Reaction performed at a molar ratio of 1:8 (PC:n3 PUFA), water content at 1.0% (based on total substrate weight), and enzyme loading at 10% (based on
total substrate weight); n3 PUFA is dened as sum of EPA, DPA, and DHA; and bar, mean pooled SD (n = 2).

PUFA. For these trials, water content, temperature, and molar ratio
of PC to fatty acid were kept at 1.0% (based on total substrate
weight), 55 C, and 1:8, respectively. The effect of enzyme loading
on the acidolysis reaction is shown in Fig. 4 The incorporation of
n3 PUFA into PC increased signicantly when enzyme loading
increased up to 20%. However, the differences between the n3
PUFA contents achieved at enzyme loading of 20% and 30% were
minimal. For example, at 12 h, the incorporation of n3 PUFA increased by 22.5 mol%, as enzyme loading was increased from 10%
to 20%. However, the difference in the n3 PUFA contents achieved
between enzyme loading of 20% and 30% was only 4.3 mol%. It is
important to reduce the enzyme loading for economic feasibility,
although reactions, such as acidolysis and esterication, are proportional to enzyme loading (Blasi et al., 2009; Karabulut, Durmaz,
& Hayaloglu, 2009).
The yield of PC decreased sharply during the rst 3 h of reaction
and remained constant after 6 h for all enzyme loadings examined.

With the higher enzyme loading, the lower yield of PC was

obtained. Previous studies have also reported that the extent of
hydrolysis increased along with increased incorporation of fatty
acid residues into PC when higher enzyme loadings were employed
(Doig & Diks, 2003; Vikbjerg, Mu, & Xu, 2005). Overall, during the
acidolysis reaction, the yield of PC decreased signicantly although
the incorporation of n3 PUFA into PC increased. For enzyme loading, 20% was selected as the optimum for further study, when the
incorporation of n3 PUFA into PC was primarily taken into consideration. The total n3 PUFA content in modied PC obtained
after 24 h at this condition was 57.4 mol% and the yield of PC
was 16.7 mol%. In addition, vacuum was applied in the next step
to enhance the yield of PC.
3.2.4. Vacuum
It is well known that enzymatic reactions in non-aqueous media are critically sensitive to the water content of the system. There

T. Zhao et al. / Food Chemistry 157 (2014) 132140

Incorporation of n-3 PUFA (mol%)

into PC

138

(a)

70
60
50
40
30
20
10
0

10

15

20

25

Reaction time (h)

100

(b)

Yield of PC (mol%)

80

60

40

20

10

15

20

25

Reaction time (h)

Fig. 4. Effect of enzyme loading on the incorporation of n3 PUFA into PC (a) and the yield of PC (b) by acidolysis as a function of reaction time shown by: 2.5% (d); 10% (.);
20% (D); 30% (j). Reaction performed at a molar ratio of 1:8 (PC:n3 PUFA), water content at 1.0% (based on total substrate weight), and 55 C reaction temperature; n3
PUFA is dened as sum of EPA, DPA, and DHA; and bar, mean pooled SD (n = 2).

is a critical lower limit in water content below which enzymes will

not be able to maintain their catalytic activities. However, too
much water can lead to undesired enzyme-catalyzed side reactions
such as hydrolysis (Kim, Garcia, & Hill, 2010). In the acidolysis
reaction, excess hydrolysis can adversely affect the yield of PC.
Application of vacuum is known to favour esterication in terms
of thermodynamics, by removing produced water (Rosu, Iwasaki,
Shimidzu, Doisaki, & Yamane, 1998). Thus, vacuum was applied
here to enhance the yield of PC via immobilized PLA1-catalyzed
esterication of n3 PUFA and LPC, the latter of which was initially
released by acidolysis. For these trials, water content, temperature,
enzyme loading, and molar ratio of PC to fatty acid were maintained at 1.0% (based on total substrate weight), 55 C, 20% (based
on total substrate weight), and 1:8, respectively. Under these reaction conditions, a sharp increase in incorporation of n3 PUFA was
observed whereas the yield of PC decreased signicantly during
the rst 3 h of reaction under atmospheric pressure. Meanwhile,

there were no signicant differences in the incorporation and yield

after 9 h of reaction. Therefore, 3 and 9 h from the start of reaction
were selected as two different points for vacuum application. In
addition, as a reference, vacuum was applied continuously
throughout an entire reaction course. At each point of vacuum
application, the pressure of the reaction system was controlled at
0.7 kPa. The effect of vacuum on acidolysis reaction is shown in
Fig. 5. For reference trials in which vacuum was applied continuously from the start of the reaction, the incorporation of n3 PUFA
into PC was negligible even though the yield of PC remained over
90 mol% throughout the reaction. Meanwhile, for the trials in
which vacuum was applied after 3 and 9 h from the start of
reaction, the yields of PC increased drastically as reaction time
increased whereas the extent of incorporation of n3 PUFA somewhat decreased after vacuum was applied. For example, for the trials in which vacuum was applied after 3 h from the start of
reaction, the incorporation of n3 PUFA, and yield of PC were

139

Incorporation of n-3 PUFA (mol%)

into PC

T. Zhao et al. / Food Chemistry 157 (2014) 132140

(a)

50

40

30

20

10

20

40

60

80

100

Reaction time (h)

100

(b)

Yield of PC (mol%)

80

60

40

20

0
0

20

40

60

80

100

Reaction time (h)

Fig. 5. Effect of vacuum on the incorporation of n3 PUFA into PC (a) and the yield of PC (b) by acidolysis as a function of reaction time. Reaction under atmospheric pressure,
dash lines and under vacuum, solid lines; vacuum applied after 0 h (d), 3 h (s) and 9 h (.) of reaction under atmospheric pressure; vacuum pressure, 0.7 kPa; reaction at a
molar ratio of 1:8 (PC:n3 PUFA), water content at 1.0% (based on total substrate weight), 55 C reaction temperature, and enzyme loading at 20% (based on total substrate
weight); n3 PUFA is dened as sum of EPA, DPA, and DHA; and bar, mean pooled SD (n = 2).

32.6 mol%, and 61.8 mol%, respectively, after 96 h of reaction.

Meanwhile, for trials in which vacuum was applied after 9 h from
the start of reaction, at 102 h, the incorporation of n3 PUFA was
35.8 mol%, but the yield of PC was 45.8 mol%.

Acknowledgement
This work was supported by Food Technology Development
Program, Ministry for Food, Agriculture, Forestry and Fisheries,
Republic of Korea.

4. Conclusion
PC from soybean was successfully modied by incorporation of
residues of n3 PUFA derived from sh oil using immobilized
PLA1. Water content, temperature and enzyme loading were
shown to have signicant effects on both the incorporation of
n3 PUFA into PC and yield of PC. Reaction conditions were taken
into consideration in terms of an appropriate balance between the
extent of incorporation of n3 PUFA residues and the yield of PC.
Although a slight decrease in n3 PUFA incorporation occurred,
vacuum application was highly effective for increasing the yield
of PC.

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