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Appl Biochem Biotechnol (2017) 182:1037–1052

DOI 10.1007/s12010-016-2379-y

Phospholipase A1-Catalysed Synthesis of Docosahexaenoic


Acid-Enriched Phosphatidylcholine in Reverse
Micelles System

Wuxi Chen 1 & Wei Guo 1 & Feng Gao 1 & Limei Chen 1 &
Shulin Chen 1 & Demao Li 1

Received: 22 August 2016 / Accepted: 15 December 2016 /


Published online: 7 January 2017
# Springer Science+Business Media New York 2017

Abstract Phosphatidylcholine enriched with docosahexaenoic acid (DHA) was successfully


produced by phospholipase A1-catalysed acidolysis. Reverse micelles were firstly selected as
the reaction system to avoid the process of immobilization and to ensure the efficient catalysis
of phospholipase A1. Parameters were optimized for a high incorporation of phosphatidyl-
choline enriched with DHA (DHA-PC). A response-surface design with four factors, including
the water content, enzyme loading, pH and substrate-mass ratio were used to evaluate the
influence of the major factors and to predict the optimal reaction conditions. The results
indicated that the optimal reaction conditions for the production of DHA-PC were a water
content of 0.4%, an enzyme loading of 40%, pH 6.92 and a substrate-mass ratio of 2.13. Under
these optimized conditions, 20.90% of DHA content in DHA-PC was obtained.

Keywords Phosphatidylcholine . Docosahexaenoic acid . Phospholipase A1 . Catalysed


acidolysis . Reverse micelles

Introduction

Docosahexaenoic acid (DHA) is a ω-3 essential polyunsaturated fatty acid (PUFA), which
plays a significant role in improving human health and inhibiting human disease, such as
reducing blood viscosity, ameliorating rheumatoid arrhythmia and reducing the incidence of
diabetes and cancers [1–4]. Therefore, DHA is widely used as an additive in infant nutritional
products and the pharmaceutical industry [5].

* Demao Li
li_dm@tib.cas.cn

1
Tianjin Key Laboratory for Industrial Biological Systems and Bioprocess Engineering, Tianjin
Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin, No. 32, Xiqi Road,
Tianjin Airport Economic Park, Tianjin 300308, People’s Republic of China
1038 Appl Biochem Biotechnol (2017) 182:1037–1052

DHAs are classified into methyl esters, ethyl esters, triglycerides and phospholipids. DHA-
ethyl ester is decomposed into ethanol and DHA in the human body, and ethanol has an
irritating effect on embryos and infants. Via passive diffusion, DHA-ethyl ester is absorbed in
the human body at a rate of 20%. The absorption rate of DHA-triglyceride is only approxi-
mately 50%, which is much higher than that of DHA-ethyl ester [6, 7]. The absorption mode of
DHA-phospholipids (DHA-PLs) in the human body is primarily an initiative absorption
process, and the absorption rate is close to 100% [8, 9]. DHA-PLs can be hydrolyzed to
DHA and phospholipids by phospholipase in the body. Phospholipids are good emulsifiers that
can promote the formation of chyle particles and help to improve chyle’s stability and ability to
transport fatty acids. Therefore, they can contribute to the transport capacity of DHA, thus
increasing the absorption rate [10]. Phospholipids (PLs) are important components of cell
composition and play crucial physiological and biochemical roles [11]. PLs are highly efficient
emulsifiers, which have been widely used in food, healthcare products, drug manufacture and
cosmetic products [12, 13]. In recent years, DHA or eicosapentaenoic acid (EPA) in the form
of PLs has received more and more attention due to its beneficial physiological effects
[14–17]. In particular, DHA-PLs could improve the bioavailability of DHA and reduce the
tendency of obesity [18, 19]. Numerous animal and human experiments have shown that
DHA-PLs have positive biological function and great nutritional value [20, 21]. Indeed, it is
believed that DHA-PLs have anti-inflammatory properties and effectively prevent oxidation of
brain lipids, with reports that it enhances memory and learning and decreases lipids in blood
and tissue [22, 23].
The natural sources of DHA-PLs include primarily fish roe, krill oil and eggs.
However, their production is restricted by the production areas and raw materials, which
cannot meet the current demands [24]. Thus, more attention has been focused on the
synthesis of DHA-PLs [15, 25, 26]. The major productive preparation methods of DHA-
PLs are the chemical method and the enzymatic method. The chemical method results in
more by-products and affects the product safety due to the catalyst used and the
specificity and toxicity of the reagents [27]. The enzymatic methods for the production
of biomolecules have recently received substantial attention because the reaction condi-
tions are simple, by-products are reduced and the process is environmentally benign.
Since phospholipids and DHA are sensitive to oxidation in the manufacturing process,
biocatalytic production of DHA-PLs is more desirable than chemical methods, particu-
larly when regiospecific conformations of fatty acids are desired [28].
Reverse micelles are formed using a surfactant soluble in non-polar organic solvents that
spontaneously form inorganic nanostructures and are a W/O system that is optically transpar-
ent and thermodynamically stable [29]. Due to this unique structure, the reverse micelle system
not only provides a large oil-water interface for catalytic hydrolysis by enzymes but also
prevents direct contact between enzymes and the surrounding organic solvents, maintaining
the high activity of the enzyme. In addition, the polar nucleus within the reverse micelle has a
physiological environment. Most enzymes in the reverse micelle system can maintain high
activity and stability and sometimes can display super-activity.
Until now, the reaction system for the synthesis of DHA-PLs are an n-hexane and solvent-
free system in most of the literatures [30, 31]. Because the solvent-free reaction system is
relatively thick, which is unfavourable for mass transfer, the rate of reaction is slow, and if the
DHA is in a very high concentration, then the enzyme will lose its activity. In this study,
reverse micelles were applied to produce DHA-PC for the first time. Phospholipids derived
from soybean and DHA obtained from the saponification of alga oil were used as base material
Appl Biochem Biotechnol (2017) 182:1037–1052 1039

for the synthesis of DHA-PC by phospholipase A1-catalysed acidolysis. Then, DHA replaced
the fatty acids in the phosphatidylcholine sn-1 position. The objective of this study was to
improve the utilization rate and the incorporation of DHA in phosphatidylcholine and to
provide support for the theory for DHA-PC and its process products, which were beneficial for
food industrial and non-food applications.

Materials and Methods

Materials

Phospholipids (PC >70%) were purchased from Shanxisenfu Natural Products Co., LTD.
(Shanxi, China). The standard of PC (Purity >98%) was purchased from Beijing Wan Jia First
Chemical Technology Co., Ltd. (Beijing, China). The standards of LPC, GPC (purity >98%)
were purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. (Nanjing, China).
DHA-enriched fatty acids were extracted from Schizochytrium sp. in our lab (DHA ≥ 30%).
Phospholipase A1 was provided by Novozymes A/S (Bagsvard, Denmark). The standards of
fatty acids and sodium di-2-ethylhexyl sulfosuccinate (AOT) were purchased from Sigma-
Aldrich (St. Louis, MO, USA). All of the other chemicals used were of an analytical grade or
higher.

Preparation of the Reverse Micelle System

AOT (0.6–1.5 g) and 50 mL of isooctane were added to 250-mL flasks. In order to obtain a
transparent micelle solution, the mixtures were thoroughly stirred, then stored at room
temperature [32].

Phospholipase A1-Catalysed Acidolysis Reaction

The reactions between PLs and DHA-enriched fatty acids were accomplished in a tightly
capped 250-mL brown flask. The phospholipids and a proportion of DHA-enriched fatty acids
were added into the flask. Then, a solution of 50 mL of AOT/isooctane, PLA1 and water were
placed in the flask. Phosphate buffer solution was used to adjust the pH. The reaction mixture
was agitated in a shaking water bath operated at 200 rpm and 45 °C under a nitrogen
atmosphere for 24 h. The reaction mixture was concentrated by rotary evaporation at 40 °C
and washed three times using acetone when the reaction was finished. The supernatant was
discarded after centrifuging for 10 min under 3000 rpm at 4 °C, and the precipitate was stored
at −20 °C for analysis of DHA-PC.

Product Analysis

The compositions of PLs were analysed by high performance liquid chromatography


(Shimadzu LC-20 AD). A 250 × 4.6-mm Agilent XDB-C18 was employed. PLs and the
reaction products were dissolved in mixture of dichloromethane and methanol (2:1, v/v).
Methanol/acetonitrile/water (25:70:5) was the mobile phase for LC at a flow rate of 0.6 mL/
min, sample injection volume was 1 μL and the detector was set at 206 nm. Compared with the
retention times of standards, the PC, LPC and GPC were identified.
1040 Appl Biochem Biotechnol (2017) 182:1037–1052

Determination of DHA by GC

Samples (50 mg) were dissolved in chloroform and methanol (5:1) and then were applied to
preparative silica-coated TLC plates developed with chloroform/ethanol/acetic acid/water
(40:9:3:1.5, v/v/v/v). Bands corresponding to DHA-PC were recovered from the TLC plates,
extracted and methylated for DHA analysis. Nonadecanoic acid methyl ester was used as an
internal standard. GC conditions: GC-2010, Shimadzu, Japan; sp-2560 column
(100 m × 0.25 mm × 0.20 μm, Supelco, USA); initial column temperature 180 °C, increased
to 240 °C for 18 min at the rate of 30 °C/min; injector and detector (flame ionization detector,
FID) temperatures 250 and 260 °C; helium flow rate 63.7 mL/min; hydrogen flow rate 40 mL/
min; air flow rate 400 mL/min; sample injection volume 1 μL [33–36]. The incorporation of
DHA into PC (DHA-PC) was calculated as follows:

the incorporation of DHA into PC


Incorporation of DHA into PCð%Þ ¼  100
the incorporation of total fatty acid in PC

Experimental Design and Statistical Analysis

Experimental design and statistical data analysis were performed using Design-Expert Version
8.05b. The coefficient of determination R2 was used to determine whether the polynomial
model was adequate to fit the observed data. An F test was used to check its statistical
significance, while the significance of the regression coefficients was tested by a t test [37].

Results and Discussion

Effect of Substrate-Mass (DHA-Enriched Fatty Acids to Phospholipids) Ratio


on the Synthesis of DHA into PC

The quality of phospholipids was fixed by changing the DHA-enriched fatty acids to control
the substrate-mass ratio from 1 to 3 (DHA-enriched fatty acids /phospholipids, g/g). Other
reaction conditions were fixed as follows: water content of 0.8%, enzyme loading of 40%,
pH 6.8, reaction time of 24 h and reaction temperature of 45 °C.
The effect of the substrate-mass ratio on the incorporation of DHA-PC was shown in
Fig. 1a. The incorporation of DHA-PC appeared to increase initially and then decreased
slightly after reaching the peak value, with the increase in the substrate-mass ratio. The
incorporation of DHA-PC was increased when the substrate-mass ratio changed from 1 to 2
primarily because the probability that DHA-enriched fatty acids and the phospholipids would
combine increased, which promoted a positive reaction until the DHA-PC content increased to
the maximum with the increased DHA-enriched fatty acid content involved in the reaction.
The data showed an increase in fatty acid concentration, with the yield of both esterification
and transesterification reactions increasing gradually [38]. When the substrate-mass ratio was
changed from 2 to 3, it was observed that the content of DHA-PC slightly decreased. This
decline may have been caused by the increasing polarity of the reaction system with the
addition of the DHA-enriched fatty acids, which was not conducive to the spreading of
substrates and reduced the opportunity of molecule collisions among DHA-enriched fatty
Appl Biochem Biotechnol (2017) 182:1037–1052 1041

acids, phospholipids and PLA1. Similar results have been reported during PLA2-catalysed
esterification reactions with an increase in fatty acids. These reactions were traced to changes
that affect polarity or viscosity [39]. Therefore, the substrate-mass ratio of 2 was determined as
the most appropriate substrate-mass ratio for the incorporation of DHA-PC.

Effect of Water Content on the Synthesis of DHA into PC

Changes in the water content ranging from 0.4 to 1.4% (based on total substrate weight) were
examined to study the effect of water content on the modification of PC with DHA-enriched
fatty acids in reverse micelles. The substrate-mass ratio was 2:1, and the other reaction
conditions remained invariable, as above.
The incorporation of DHA-PC was increased by 10.58% when the water content was
increased from 0.4 to 0.8%, but it decreased gradually as the water content increased from 0.8
to 1.4% (Fig. 1b). It is well known that water content is crucial for the enzymatic reactions in
reverse micelles. In reverse micelles, the large or small diameter size of the reverse micelle
pool was determined by the high or low water content [29]. The water content was low, and the
diameter size of the reverse micelle pool was small. Therefore, it could not fully accommodate
the enzyme molecules, which caused part of the enzyme molecule inactivation in the organic
solvent. The water content was too high, the PLA1 could be completely soluble in the pool of
reverse micelles, but extra water could drop the stability of the reverse micelle system [40]. In
biocatalysis processing, the enzyme cannot maintain a basic catalytic activity below the critical
water, or Bessential water^ content, with surplus water content resulting in subsidiary reactions,
such as hydrolysis [15, 25]. Researchers have found that the water content can significantly
influence the incorporation of n-3 PUFA into PL, with reports that the highest incorporation of
EPA into PC was obtained with a water content of 5%, while the occurrence of hydrolysis side
reactions were threefold more than the acidolysis reaction when lipase containing 10% water
was used [26, 41, 42]. Zhao et al. (2014) reported that the optimum water content for
incorporation of the n-3 polyunsaturated fatty acid with phosphatidylcholine by immobilized
PLA1 was 1.0% (w/w) based on the substrate [15]. Therefore, to obtain a high
content of DHA-PC, it was recommended that the water content be 0.8% in this
study.

Fig. 1 Effects of substrate mass ratio (a) and water content (b) on the synthesis of DHA into PC in reverse
micelle system
1042 Appl Biochem Biotechnol (2017) 182:1037–1052

Effect of Enzyme Loading on the Synthesis of DHA into PC

Changes in the enzyme loading from 10 to 35% (wt.% based on substrate) were examined to
study the effect of enzyme loading on the modification of PC with DHA-enriched fatty acids in
reverse micelles. The substrate mass ratio was 2:1, water content was 0.8% and other reaction
conditions remained unchanged, as above.
The effect of PLA1 loading for this biocatalyst on the incorporation into DHA-PC
was shown in Fig. 2a. The loading level of PLA1 increased to 30% (as percentage of
total weight of substrates), while the incorporation into DHA-PC also increased
significantly. However, additional increases in the incorporation of DHA-PC were
minimal when the solution containing PLA1 was present at enzyme loading higher
than 30%. High enzyme loading was required for the effective incorporation of DHA
into PC by acidolysis in this system. Enzymatic reactions primarily consist of active
groups in the enzyme that acts with its substrate. With the increase in the amount of
PLA1, the increase in active genes accelerated the enzymatic reaction. However, for
loading of PLA1 above 30%, the content of DHA-PC decreased slightly. The use of a
high enzyme loading may have led to problems with agitation and decreased the mass
transfer. Another possible reason was that PLA1 contain a certain number of water,
which may lead to the reaction of hydrolysis [43]. Different enzyme loading levels
between 10 and 20% have been used in previous studies [15, 25]. Thus, 30% of
PLA1 loading will be used to synthesize DHA-PC.

Effect of pH on the Synthesis of DHA into PC

As we know, pH had great effects on the enzyme behaviour. Particularly in reverse


micelle system, the pH of the core water determines the charges on the enzyme
molecules and the membranes of reverse micelles [44]. PH determines the confor-
mation for enzyme catalysis and the dissolving capacity in reverse micelle system.
Moreover, its change can cause a change in the interactions between enzyme
molecules and the membrane of reverse micelles, which affects the catalytic activ-
ities of the enzyme [45]. In the reverse micelle microenvironment, pH measurements
have technically been infeasible until now. Therefore, the formation of micelle is
generally determined by the pH of the initial buffer solution during the experiment.
In this study, the pH was adjusted with different phosphate buffers. As shown in
Fig. 2b, with the increase in the initial pH value, the incorporation of DHA into PC
appeared to increase from 5.5 to 7.0 and then decreased gradually after reaching the

Fig. 2 Effects of enzyme loading (a), pH (b) and temperature (c) on the synthesis of DHA into PC in reverse
micelle system
Appl Biochem Biotechnol (2017) 182:1037–1052 1043

peak value. There were many acidic and alkaline side-chain groups of amino acids
in PLA1. The ionogenic groups of PLA1 were affected by the different pH values,
which the state of ionization greatly influences the interaction with matrix. In the
following experiments, a pH value of 6.98 was chosen for the synthesis of DHA-
enriched PC in reverse micelle system.

Effect of Temperature on the Synthesis of DHA into PC

In general, an increase in the reaction temperature results in an increase in reaction rates as


defined by Arrhenius’s law. For endothermic reactions, a higher temperature is beneficial to
obtain higher yields due to a shift in the thermodynamic equilibrium.
However, excessively high temperatures can reduce the amount of active enzyme
due to degradation of the enzyme and more rapid loss of activity [46, 47]. In
addition, excessive temperature increases the occurrence of side reactions, such as
acyl migration or hydrolysis [48]. So, the optimal temperature of a reaction should be
designed on an individual basis, taking into consideration the temperature stability of
both the substrates and products [49–51].
In this experiment, temperatures ranging from 35 to 55 °C were tested for the
production of DHA-PC, with the maximum productivity occurring at 45 °C
(Fig. 2c). When the temperature was increased to 55 °C, the incorporation was
reduced from 18.07% at 45 °C to 13.64%. This finding illustrated that the activity
of PLA1 in the reverse micelle system was more sensitive to high temperatures.
Generally, the rate of all chemical reactions increases with the rise of temperature,
including enzyme-catalysed reactions. However, it simultaneously increases the de-
naturation rate of the enzyme. Higher temperature also increases the lipid-oxidation
rate, particularly if PUFAs are used as acyl donors [46, 47]. The decrease in DHA-
PC production at temperatures above 45 °C may occur for two reasons: structural
instability in the reverse micelles or reduction of the enzyme activity with the
increasing temperature. In a previous study from Park et al., the maximum reaction
rate and yield occurred at 50 °C when phospholipase A2 was used for incorporation
of PC into EPA in organic solvent [52]. Peng et al. also reported that maximal
incorporation was observed at 57.5 °C for production of structured phospholipids by
Lipozyme TLIM [53]. Hence, we choose 45 °C as the optimal temperature for
following experiments, since it resulted in maximum incorporation under these
experimental conditions.

Optimization of the Reaction System Using Response-Surface Model Fitting

Experiments were designed with four factors, each varied at three levels and conduct-
ed in triplicate. The four factors chosen were the substrate-mass ratio (g/g, DHA-
enriched fatty acids /PLs), enzyme loading (wt.% based on substrate), water content
(wt.% based on total substrate) and pH. The experimental design is presented in
Table 1. The test of significance for the response-surface coefficient and the analysis
of variance (ANOVA) for the response-surface model in Table 2 indicated that the
model was significant at P < 0.0001. After comparing the R2 value and the results of
the lack-of-fit test, the constructed model was found to be adequate to describe the
observed data. According to the results of variability in response, the lack of fit was
1044 Appl Biochem Biotechnol (2017) 182:1037–1052

Table 1 Experimental setup for surface response design and corresponding results (the response)

Exp number Water content (%) Enzyme loading (%) pH Substrate mass ratio (g/g) The incorporation
of DHA into PC (%)

1 0.8 40 7 3 15.422
2 0.4 30 7 3 15.251
3 1.2 30 6 2 11.07
4 0.8 20 6 2 10.386
5 0.4 20 7 2 15.886
6 0.8 20 7 1 12.460
7 0.4 30 7 1 12.973
8 0.8 30 7 2 19.753
9 0.8 30 6 1 9.323
10 0.8 40 7 1 14.722
11 0.8 30 8 1 12.780
12 0.8 40 8 2 15.009
13 1.2 20 7 2 16.232
14 0.4 40 7 2 20.743
15 1.2 30 8 2 14.173
16 0.4 30 8 2 16.369
17 0.8 30 7 2 19.750
18 1.2 40 7 2 16.187
19 1.2 30 7 3 15.407
20 1.2 30 7 1 14.427
21 0.4 30 6 2 16.007
22 0.8 40 6 2 15.435
23 0.8 30 7 2 18.253
24 0.8 30 8 3 12.857
25 0.8 30 6 3 12.385
26 0.8 30 7 2 18.812
27 0.8 20 7 3 14.169
28 0.8 20 8 2 12.484
29 0.8 30 7 2 18.459

Table 2 The test of significance for the response surface coefficient and the analysis of variance (ANOVA) for
the response surface model

Sum of squares df Mean square F p (p > F)

Model 202.081 4 14.43 13.68 < 0.0001


A. Water content 7.89 1 7.89 6.71 0.0213
B. Enzyme loading 21.12 1 21.12 17.97 0.0008
C. pH 6.85 1 6.85 5.83 0.0301
D. Substrate mass ratio 6.49 1 6.49 5.52 0.0340
AB 6.01 1 6.01 5.11 0.0403
AC 1.88 1 1.88 1.60 0.2269
AD 0.42 1 0.42 0.36 0.5591
BC 1.59 1 1.59 1.35 0.2639
BD 0.24 1 0.24 0.21 0.6554
CD 2.23 1 2.23 1.89 0.1903
A2 2.10 1 2.10 2.47 0.1382
B2 10.39 1 10.39 10.29 0.0063
C2 97.41 1 97.41 87.18 < 0.0001
D2 74.34 1 74.34 67.02 < 0.0001
Residual 16.46 14 1.18
Lack of fit 14.45 10 1.44 2.87 0.1609
Pure error 2.02 4 0.50
Cor Total 226.24 28
Appl Biochem Biotechnol (2017) 182:1037–1052 1045

not obvious. The correlation coefficient was 0.93, and the relativity between the
predicted value and the measured value was approximately 93.19%, which indicated
that the model could well reflect the changing regulation.

Optimization of the Reaction System

Based on the results of the response-surface experiment, response-surface figures were


constructed regarding the response value with four factors using Design Expert 8.05b
(Figs. 3a–e and 4a–e). The relationship and the interaction between any two param-
eters were demonstrated by analysing the contour plots (Fig. 3b–f and 4b–f). The
results showed the same tendency for the parameters as described above. The optimal
design parameters were obtained by the software via interactive computing between
the four factors.
Figure 3a, b showed the effects of water content and enzyme loading on the
synthesis of DHA-PC. In Figs. 3 and 4, the synthesis of DHA-PC gradually
increased with increased enzyme loading, reaching a final plateau value near 40%,
whereas the water content had little effect on the synthesis of DHA-PC. Figure 3b
identified the optimal reactions under which the highest incorporation of 20% was
predicted. The effect of water content and pH was shown in Fig. 3c, d when
enzyme loading was set at 30%, and the substrate-mass ratio was 2:1 as the central
point. The figure showed that the maximum and minimum incorporation of 19.75
and 11.07% could be achieved by conducting experiments with water contents of
0.8 and 1.2% for pH values of 7 and 6, respectively. The interplay of these sets of
parameters was significant; other groups were not significant. The optimum condi-
tions for the synthesis of DHA-PC were predicted using Design Expert 8.05b. The
model prediction of the synthesis of DHA-PC was 20.42% under the conditions that
the water content was 0.4%, the enzyme loading was 40%, the pH was 6.92 and the
substrate-mass ratio was 2.13.
The compositions of the initial PLs and modified PLs obtained under the optimal
conditions were showed in Fig. 5 and Table 3. The content of PC was 71.24% in the initial PL,
and the LPC was 3.07%. After phospholipase A1-catalysed hydrolysis, a part of PC was
gradually transformed to LPC and GPC, and the content of PC, LPC and GPC were
46.82, 14.15 and 5.52%, respectively. Hydrolysis reaction was inevitable during the acid
hydrolysis reaction and transesterification, which had led to major hydrolysis of PC [42,
43]. But, LPC rich in DHA also has the function of healthcare, which can also be used in
medicine and cosmetics industry. Therefore, we will further investigate to get more
optimized conditions to reduce the generation of GPC as far as possible in the next study.
The compositions of fatty acid in PLs and DHA-PLs were detected by gas
chromatography (GC) and the results were shown in Table 4. Palmitic acid C16:0
(21.79 mol%) and linoleic acid C18:2 (54.09 mol%) were the major components of
the fatty acid compositions of PLs. After being modified by PLA1-catalysed
acidolysis, the palmitic acid C16:0 (30.27 mol%) of DHA-PLs appeared to increase
slightly, while linoleic acid C18:2 was reduced from 54.09 to 31.10 mol%. The
reason for this result could be guessed to the complex reaction kinetics, which
consisted of interesterification and hydrolysis of PC [30]. Eicosapentaenoic acid,
C20:5 (1.26 mol%) and docosapentaenoic acid C22:5 (4.83 mol%) and DHA C22:6
(21.07 mol%) were detected in the final products. This result showed that DHA was
1046 Appl Biochem Biotechnol (2017) 182:1037–1052

Fig. 3 Contour plots and 3D response surface of the synthesis of DHA into PC catalysed by PLA1 in a reverse
micelle system. a Water content vs. enzyme loading; b water content vs. pH; c water content vs. substrate mass ratio

successfully incorporated into phospholipids by phospholipase A1-catalysed


acidolysis.
Table 5 showed some recently published studies on the synthesis of structured
phospholipids. Although the synthesis of DHA-PC was 20.90% in our study, we
chose DHA as the only evaluation index instead of -3 unsaturated fatty acids.
Appl Biochem Biotechnol (2017) 182:1037–1052 1047

Fig. 4 Contour plots and 3D response surface of the synthesis of DHA into PC catalysed by PLA1 in reverse micelle
system. a Enzyme loading vs. pH; b enzyme loading vs. substrate mass ratio; c pH vs. substrate mass ratio

This study was the first to choose the reverse micelle system to produce DHA-PC,
which could not only avoid the process of immobilized PLA1 but also keep highly
catalytic activities of PLA1.
1048 Appl Biochem Biotechnol (2017) 182:1037–1052

Fig. 5 High performance liquid chromatography of a phospholipids and b modified phospholipids

Conclusions

In this study, the phosphatidylcholine was successfully modified by the synthesis of DHA
through PLA1-catalysed acidolysis in reverse micelles. Several compromises were presented
to obtain a high content of products, because many of the parameters facilitating the incorpo-
ration resulted in the increased synthesis of DHA-PC in the reaction system. The response
models satisfactorily expressed the incorporation of DHA-PC regarding the water content,
enzyme loading, pH and substrate-mass ratio in the batch reactor, which were shown to have
significant effects on the incorporation of DHA-PC. The highest incorporation of DHA-PC
was 20.90% under the optimized conditions: a water content of 0.4%, an enzyme loading of

Table 3 The composition of PLs and modified PLs

Compositions PLs Modified PLs

PC (%) 71.24 46.82


LPC (%) 3.07 14.15
GPC (%) – 5.52

Table 4 The compositions of fatty acids (mol %) in PLs and modified PLs

Fatty acids PLs Modified PLs

C 12:0 – 3.18 ± 0.05


C 16:0 21.79 ± 0.32 30.27 ± 0.19
C 18:0 3.76 ± 0.08 2.39 ± 0.03
C 18:1 4.11 ± 0.02 0.71 ± 0.01
C 18:2 54.09 ± 0.16 31.10 ± 0.26
C 18:3 7.49 ± 0.03 4.55 ± 0.02
C 20:5 – 1.26 ± 0.03
C 22:5 – 4.83 ± 0.11
C 22:6 – 21.07 ± 0.23

PLs were purchased from Shanxisenfu Natural Products Co., LTD. Modified PLs were the final products by
phospholipase A1-catalysed acidolysis. Reaction conditions were fixed as follows: substrate-mass ratio of 2.13,
water content of 0.4%, enzyme loading of 40%, pH 6.92, reaction time of 24 h and reaction temperature of 45 °C
Appl Biochem Biotechnol (2017) 182:1037–1052 1049

Table 5 Some of recent studies on the synthesis of structured phospholipids

PUFA Solvent Enzyme or Conditions References


system chemical
catalyst

PUFA- incorporation Hexane Lipase OF 2.0 mmol of Soy-PL, a Yamamoto


(47.1 ± 2.1 wt%) phospholi PUFA-Et /Soy-PL acyl et al. [31]
pase A2 ratio of 7, 13 mL of
hexane, 2.2 × 105 U of
lipase OF, a temperature
of 37 °C, and 72 h of
reaction time.
Conjugated linoleic Solvent-free Immobilized Reaction temperature, Vikbjerg
acid (30%) and system phospholipase 45 °C; substrate ratio, et al. [25]
docosahexaenoic A2 9 mol/mol caprylic
acid(20%) acid/PC; water addition
of 2%; 30 wt.%
immobilized enzyme;
and reaction time, 48 h.
n-3 PUFA (21 mol%) Solvent-free Immobilized Temperature, 55 °C; Loading Kim et al.
system phospholipase of the enzyme: 10% of the [43]
A1 total weight of substrates;
reaction time, 6 h.
n-3 PUFA (57.4 mol%) Solvent-free Immobilized The optimum water content, Zhao et al.
system phospholipase temperature, and enzyme [15]
A1 loading were 1.0%, 55 °C,
and 20%, respectively.
Incorporation of DHA/EPA Solvent-free Immobilized Temperature 55.7 °C, water Li et al. [42]
(30.31%) system phospholipase addition 1.1 wt% and
A1 substrate mass ratio
(ethyl esters/PC) 6.8:1.
DHA (59.0 mol%) SCCO2 as the Immobilized FA to PC was 10, under Xi et al.
solvent phospholipase 12 MPa and 50 °C [54]
medium A1 SCCO2, immobilized
enzyme loading rate of
15 wt% of the total
substrates.

40%, a pH of 6.92 and a substrate-mass ratio of 2.13. The optimum technical conditions were
determined on the view point of process cost, and they provided theoretical guidance for the
production of DHA-PC.

Acknowledgements This study was supported by the International Cooperation Project (grant no.
2014DFA61040), the Youth Innovation Promotion Association CAS, and the Hi-Tech Research and Develop-
ment Program (863) of China (grant no. 2014ARA021701).

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