DOI 10.1007/s12010-016-2379-y
Wuxi Chen 1 & Wei Guo 1 & Feng Gao 1 & Limei Chen 1 &
Shulin Chen 1 & Demao Li 1
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
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.
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].
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
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:
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].
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.
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
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.
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.
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
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.
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
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
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 4 The compositions of fatty acids (mol %) in PLs and modified PLs
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
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).
References
1. Kris-Etherton, P. M., Harris, W. S., & Appel, L. J. (2002). Fish consumption, fish oil, omega-3 fatty acids,
and cardiovascular disease. Circulation, 106, 2747–2757.
2. Simopoulos, A. P. (2002). Omega-3 fatty acids in inflammation and autoimmune diseases. Journal of the
American College of Nutrition, 21, 495–505.
1050 Appl Biochem Biotechnol (2017) 182:1037–1052
3. Morris, M. C., Evans, D. A., Bienias, J. L., Tangney, C. C., Bennett, D. A., Wilson, R. S., Aggarwal, N., &
Schneider, J. (2003). Consumption of fish and n-3 fatty acids and risk of incident Alzheimer disease.
Archives of Neurology, 60, 940–946.
4. Das, U. N. (2008). Folic acid and polyunsaturated fatty acids improve cognitive function and prevent
depression, dementia, and Alzheimer’s disease—but how and why? Prostaglandins, Leukotrienes and
Essential Fatty Acids, 78, 11–19.
5. Ren, L. J., Zhuang, X. Y., Chen, S. L., Ji, X. J., & Huang, H. (2015). Introduction of ω-3 Desaturase
obviously changed the fatty acid profile and sterol content of Schizochytrium sp. Journal of Agricultural
and Food Chemistry, 63, 9770–9776.
6. Galli, C., Sirtori, C. R., Mosconi, C., Medini, L., Gianfranceschi, G., Vaccarino, V., & Scolastico, C. (1992).
Prolonged retention of doubly labeled phosphatidylcholine in human plasma and erythrocytes after oral
administration. Lipids, 27, 1005–1012.
7. Ikeda, I., Sasaki, E., Yasunami, H., Nomiyama, S., Nakayama, M., Sugano, M., Imaizumi, K., & Yazawa,
K. (1995). Digestion and lymphatic transport of eicosapentaenoic and docosahexaenoic acids given in the
form of triacylglycerol, free acid and ethyl ester in rats. Biochimica et Biophysica Acta (BBA)-Lipids and
Lipid Metabolism, 1259, 297–304.
8. Dyerberg, J., Madsen, P., Møller, J. M., Aardestrup, I., & Schmidt, E. B. (2010). Bioavailability
of marine n-3 fatty acid formulations. Prostaglandins, Leukotrienes and Essential Fatty Acids, 83,
137–141.
9. Schuchardt, J. P., & Hahn, A. (2013). Bioavailability of long-chain omega-3 fatty acids. Prostaglandins,
Leukotrienes and Essential Fatty Acids (PLEFA), 89, 1–8.
10. Bhattacharya, S., & Biswas, J. (2009). Understanding membranes through the molecular design of lipids.
Langmuir, 26, 4642–4654.
11. Kidd, P., & Head, K. (2005). A review of the bioavailability and clinical efficacy of milk thistle phytosome:
a silybin-phosphatidylcholine complex (Siliphos®). Alternative Medicine Review, 10, 193–203.
12. Reddy, J., Vijeeta, T., Karuna, M., Rao, B., & Prasad, R. (2005). Lipase-catalyzed preparation of palmitic
and stearic acid-rich phosphatidylcholine. Journal of the American Oil Chemists’ Society, 82, 727–730.
13. Bi, Y. H., Duan, Z. Q., Li, X. Q., Wang, Z. Y., & Zhao, X. R. (2015). Introducing Biobased ionic liquids as
the nonaqueous media for enzymatic synthesis of phosphatidylserine. Journal of Agricultural and Food
Chemistry, 63, 1558–1561.
14. Rosseto, R., & Hajdu, J. (2014). Synthesis of phospholipids on a glyceric acid scaffold: design and
preparation of phospholipase A2 specific substrates. Tetrahedron, 70, 3155–3165.
15. Zhao, T. T., Kim, B. H., Garcia, H. S., Kim, Y., & Kim, I. H. (2014). Immobilized phospholipase A1-catalyzed
modification of phosphatidylcholine with n-3 polyunsaturated fatty acid. Food Chemistry, 157, 132–140.
16. Sakai, K., Okuyama, H., Yura, J., Takeyama, H., Shinagawa, N., Tsuruga, N., Kato, K., Miura, K., Kawase,
K., & Tsujimura, T. (1992). Composition and turnover of phospholipids and neutral lipids in human breast
cancer and reference tissues. Carcinogenesis, 13, 579–584.
17. Pepping, J. (1999). Phosphatidylserine. American journal of health-system pharmacy: AJHP: official
journal of the American Society of Health-System Pharmacists, 56(2038), 2043–2034.
18. Hiratsuka, S., Ishihara, K., Kitagawa, T., Wada, S., & Yokogoshi, H. (2008). Effect of dietary
docosahexaenoic acid connecting phospholipids on the lipid peroxidation of the brain in mice. Journal of
Nutritional Science and Vitaminology, 54, 501–506.
19. Hiratsuka, S., Koizumi, K., Ooba, T., & Yokogoshi, H. (2009). Effects of dietary docosahexaenoic acid
connecting phospholipids on the learning ability and fatty acid composition of the brain. Journal of
Nutritional Science and Vitaminology, 55, 374–380.
20. Tang, X., Li, Z. J., Xu, J., Xue, Y., Li, J. Z., Wang, J. F., Yanagita, T., Xue, C. H., & Wang, Y. M. (2012).
Short term effects of different omega-3 fatty acid formulation on lipid metabolism in mice fed high or low
fat diet. Lipids in Health and Disease, 11, 70.
21. Rossmeisl, M., Jilkova, Z. M., Kuda, O., Jelenik, T., Medrikova, D., Stankova, B., Kristinsson, B.,
Haraldsson, G. G., Svensen, H., & Stoknes, I. (2012). Metabolic effects of n-3 PUFA as phospholipids
are superior to triglycerides in mice fed a high-fat diet: possible role of endocannabinoids. PloS One, 7,
e38834.
22. Awada, M., Meynier, A., Soulage, C. O., Hadji, L., Géloën, A., Viau, M., Ribourg, L., Benoit, B., Debard, C.,
& Guichardant, M. (2013). N-3 PUFA added to high-fat diets affect differently adiposity and inflammation
when carried by phospholipids or triacylglycerols in mice. Nutrition & Metabolism (London), 10, 23.
23. Vigerust, N. F., Bjørndal, B., Bohov, P., Brattelid, T., Svardal, A., & Berge, R. K. (2012). Krill oil versus fish
oil in modulation of inflammation and lipid metabolism in mice transgenic for TNF-α. European Journal of
Nutrition, 52, 1315–1325.
24. Burri, L., Hoem, N., Banni, S., & Berge, K. (2012). Marine omega-3 phospholipids: metabolism and
biological activities. International Journal of Molecular Sciences, 13, 15401–15419.
Appl Biochem Biotechnol (2017) 182:1037–1052 1051
25. Vikbjerg, A. F., Mu, H., & Xu, X. (2005). Parameters affecting incorporation and by-product formation
during the production of structured phospholipids by lipase-catalyzed acidolysis in solvent-free system.
Journal of Molecular Catalysis B: Enzymatic, 36, 14–21.
26. Kim, I. H., Garcia, H. S., & Hill Jr., C. G. (2010). Synthesis of structured phosphatidylcholine containing n-
3 PUFA residues via acidolysis mediated by immobilized phospholipase A1. Journal of the American Oil
Chemists’ Society, 87, 1293–1299.
27. Lyberg, A. M., Adlercreutz, D., & Adlercreutz, P. (2005). Enzymatic and chemical synthesis of phospha-
tidylcholine regioisomers containing eicosapentaenoic acid or docosahexaenoic acid. European Journal of
Lipid Science and Technology, 107, 279–290.
28. Schmid, A., Dordick, J., Hauer, B., Kiener, A., Wubbolts, M., & Witholt, B. (2001). Industrial biocatalysis
today and tomorrow. Nature, 409, 258–268.
29. Sharma, S., Yadav, N., Chowdhury, P. K., & Ganguli, A. K. (2015). Controlling the microstructure of
reverse micelles and their templating effect on shaping nanostructures. The Journal of Physical Chemistry B,
119, 11295–11306.
30. Hossen, M., & Hernandez, E. (2005). Enzyme-catalyzed synthesis of structured phospholipids with
conjugated linoleic acid. European Journal of Lipid Science and Technology, 107, 730–736.
31. Yamamoto, Y., Mizuta, E., Ito, M., Harata, M., Hiramoto, S., & Hara, S. (2014). Lipase-catalyzed
preparation of phospholipids containing n-3 polyunsaturated fatty acids from soy phospholipids. Journal
of Oleo Science, 63, 1275–1281.
32. Hong, S. C., Park, K. M., Son, Y. H., Jung, H. S., Kim, K., Choi, S. J., & Chang, P. S. (2015). AOT/
isooctane reverse micelles with a microaqueous core act as protective shells for enhancing the thermal
stability of Chromobacterium viscosum lipase. Food Chemistry, 179, 263–269.
33. Chi, Z. Y., Hu, B., Liu, Y., Frear, C., Wen, Z. Y., & Chen, S. L. (2007). Production of ω-3 polyunsaturated
fatty acids from cull potato using an algae culture process. Applied Biochemistry and Biotechnology, 137,
805–815.
34. Zhang, Y., Min, Q. S., Xu, J., Zhang, K., Chen, S. L., Wang, H. J., & Li, D. M. (2016). Effect of malate on
docosahexaenoic acid production from Schizochytrium sp. B4D1. Electronic Journal of Biotechnology, 19, 56–60.
35. Chen, W. X, Wang, H. J, Zhang, K., Gao, F., Chen, S. L., & Li, D. M. (2016). Physicochemical properties
and storage stability of microencapsulated DHA-rich oil with different wall materials. Applied Biochemistry
and Biotechnology, 1–14.
36. Liu, B., Liu, J., Sun, P. P., Ma, X. N., Jiang, Y., & Chen, F. (2015). Sesamol enhances cell growth and the
biosynthesis and accumulation of docosahexaenoic acid in the microalga Crypthecodinium cohnii. Journal
of Agricultural and Food Chemistry, 63, 5640–5645.
37. Heck, J. X., Flôres, S. H., Hertz, P. F., & Ayub, M. A. Z. (2005). Optimization of cellulase-free xylanase
activity produced by Bacillus coagulans BL69 in solid-state cultivation. Process Biochemistry, 40, 107–112.
38. Adlercreutz, D., Budde, H., & Wehtje, E. (2002). Synthesis of phosphatidylcholine with defined fatty acid in
the sn-1 position by lipase-catalyzed esterification and transesterification reaction. Biotechnology and
Bioengineering, 78, 403–411.
39. Egger, D., Wehtje, E., & Adlercreutz, P. (1997). Characterization and optimization of phospholipase A 2
catalyzed synthesis of phosphatidylcholine. Biochimica et Biophysica Acta (BBA)-Protein Structure and
Molecular Enzymology, 1343, 76–84.
40. Hakoda, M., Shiragami, N., Enomoto, A., & Nakamura, K. (2003). Measurements of hydrodynamic
diameter of AOT reverse micelles containing lipase in supercritical ethane and its enzymatic reaction.
Bioprocess and Biosystems Engineering, 25, 243–247.
41. Haraldsson, G. G., & Thorarensen, A. (1999). Preparation of phospholipids highly enriched with n-3
polyunsaturated fatty acids by lipase. Journal of the American Oil Chemists’ Society, 76, 1143–1149.
42. Li, D. M., Qin, X. L., Wang, W. F., Li, Z. G., Yang, B., & Wang, Y. H. (2016). Synthesis of DHA/EPA-rich
phosphatidylcholine by immobilized phospholipase A1: effect of water addition and vacuum condition.
Bioprocess and Biosystems Engineering, 39, 1305–1314.
43. Kim, I. H., Garcia, H. S., & Hill, C. G. (2007). Phospholipase a 1-catalyzed synthesis of phospholipids
enriched in n−3 polyunsaturated fatty acid residues. Enzyme and Microbial Technology, 40, 1130–1135.
44. Menger, F. M., & Yamada, K. (1979). Enzyme catalysis in water pools. Journal of the American Chemical
Society, 101, 6731–6734.
45. Petersen, S. B., Jonson, P. H., Fojan, P., Petersen, E. I., Petersen, M. T. N., Hansen, S., Ishak, R. J., &
Hough, E. (1998). Protein engineering the surface of enzymes. Journal of Biotechnology, 66, 11–26.
46. Kim, J., Lee, C. S., Oh, J., & Kim, B. G. (2001). Production of egg yolk lysolecithin with immobilized
phospholipase A2. Enzyme and Microbial Technology, 29, 587–592.
47. Xu, X. (2000). Production of specific-structured triacylglycerols by lipase-catalyzed reactions: a review.
European Journal of Lipid Science and Technology, 102, 287–303.
1052 Appl Biochem Biotechnol (2017) 182:1037–1052
48. Rosu, R., Yasui, M., Iwasaki, Y., & Yamane, T. (1999). Enzymatic synthesis of symmetrical 1,3-diacyl-
glycerols by direct esterification of glycerol in solvent-free system. Journal of the American Oil Chemists’
Society, 76, 839–843.
49. Virto, C., Svensson, I., & Adlercreutz, P. (1999). Enzymatic synthesis of lysophosphatidic acid and
phosphatidic acid. Enzyme and Microbial Technology, 24, 651–658.
50. Kim, J., & Kim, B. G. (2000). Lipase-catalyzed synthesis of lysophosphatidylcholine using organic
cosolvent for in situ water activity control. Journal of the American Oil Chemists’ Society, 77, 791–797.
51. Virto, C., & Adlercreutz, P. (2000). Lysophosphatidylcholine synthesis with Candida antarctica lipase B
(Novozym 435). Enzyme and Microbial Technology, 26, 630–635.
52. Park, C. W., Kwon, S. J., Han, J. J., & Rhee, J. S. (2000). Transesterification of phosphatidylcholine with
eicosapentaenoic acid ethyl ester using phospholipase A2 in organic solvent. Biotechnology Letters, 22,
147–150.
53. Peng, L., Xu, X., Mu, H., Høy, C. E., & Adler-Nissen, J. (2002). Production of structured phospholipids by
lipase-catalyzed acidolysis: optimization using response surface methodology. Enzyme and Microbial
Technology, 31, 523–532.
54. Xi, X., Feng, X. M., Shi, N. R., Ma, X. X., Lin, H., & Han, Y. Q. (2016). Immobilized phospholipase A1-
catalyzed acidolysis of phosphatidylcholine from Antarctic krill (Euphausia superba) for docosahexaenoic
acid enrichment under supercritical conditions. Journal of Molecular Catalysis B: Enzymatic, 126, 46–55.