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Colloids and Surfaces B: Biointerfaces 106 (2013) 191– 197

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

nzymatic synthesis of phosphatidylserine using bile salt mixed micelles

lexandre Pinsollea,b, Philippe Royb, Corinne Buréc, Anne Thienpontd, Maud Cansell a,∗

Univ. Bordeaux, CBMN, UMR 5248, IPB, Allée Geoffroy Saint Hilaire, F-33600 Pessac, FrancePhosphoTech, ZAC de la Lorie, Rue Jan Palach F-44813 Saint-Herblain Cedex, FranceUniv. Bordeaux, CBMN, UMR 5248, Centre de Génomique Fonctionnelle, BP 68,146, rue Léo Saignat F-33076 Bordeaux Cedex, FranceUniv. Bordeaux, ISM, UMR 5255, 351 cours de la Libération, F-33405 Talence, France

r t i c l e i n f o

rticle history:eceived 24 October 2012eceived in revised form1 December 2012ccepted 3 January 2013vailable online xxx

eywords:hosphatidylserinearine lipids

hospholipase D

a b s t r a c t

Phosphatidylserine (PS) rich in polyunsaturated fatty acids of the n-3 series was obtained by enzymaticsynthesis with phospholipase D (PLD) and a marine lipid extract as substrate. Synthesis was performedusing mixed micelles composed of either sodium deoxycholate (SDC) or sodium cholate (SC). To limit theuse of surfactant and to monitor the performance of PLD, the mixed micelles were characterized both interms of bile salt/lipid molar ratio in the aggregates and of mean diameter. A fractional factorial experi-ment was selected to study the effect of pH, temperature, enzyme, L-serine concentrations, bile salt/lipidmolar ratio and Ca2+ content (in the case of SC only) on PS synthesis. The amount of L-serine was themain factor governing the equilibrium between transphosphatidylation and hydrolysis reaction. Increas-ing the bile salt/lipid molar ratio decreased PS synthesis yield. In contrast, pH (6.5–8) and temperature(35–45 ◦C) did not affect PLD activity in the tested conditions. This statistical approach allowed deter-

ransphosphatidylationood-grade emulsifiers

mining a combination of parameters (pH, temperature, bile salt/lipid molar ratio, enzyme and alcoholacceptor concentrations) for PS synthesis. After 24 h, the transphosphatidylation reaction led to 57 ± 2%and 56 ± 3% of PS in the phospholipid mixtures with SDC and SC, respectively. In both cases, about 10%of phosphatidic acid was present as a side-product. On the whole, this work provided fundamental basisfor a possible development of enzymatic PLD technology using food-grade emulsifiers to produce PScomplying with industrial constraints for nutritional applications.

. Introduction

Phospholipids (PL) are structural components providing stableiological membranes that isolate the organism from the outside.n addition, they also play key roles in metabolism-related andeurological diseases by participating or even generating cellularignals [1]. The biological functionalities of PL result from eitherheir fatty acid composition and/or their polar head group. Forxample, docosahexaenoic acid-containing phospholipids (DHA-L) induced differentiation and apoptosis in mouse M1 cells [2]. Inats, marine lipid-based liposomes (PL essentially) exerted a sim-lar hypotriglyceridemic effect to fish oil presenting a close fattycid composition, over the 2 weeks of the trial [3]. Among PL,hosphatidylserine (PS) is a major component of membranes and

s found in high concentration in neuronal cell membranes. PS ismplied in various brain functions such as cell signaling throughts interaction with phosphatidylkinase C [4], the enhancement of

∗ Corresponding author at: Université Bordeaux 1, Laboratoire CBMN, UMR 5248,PB, Allée Geoffroy Saint Hilaire, 33600 Pessac, France. Tel.: +33 05 40 00 68 19;ax: +33 5 56 37 03 36.

E-mail address: mcansell@enscbp.fr (M. Cansell).

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.01.026

© 2013 Elsevier B.V. All rights reserved.

Na+, K+-ATPase activity in the brain [5], or the increase of the brainglucose concentration [6]. In the central nervous system, PS couldalso act as a precursor for acetylcholine [7]. As a result, supple-mentation studies have been undertaken in aged rats suggestingthat orally administrated PS significantly ameliorated learning andmemory deficits [8,9]. Moreover, several intervention studies thatevaluated the positive effects of PS on dementia or impaired cog-nitive function have been published [10–13]. As a consequence,PS appeared to be an interesting candidate for the prevention andtreatment of age-associated cognitive diseases.

However, only few natural sources of PS are available. Amongthem, bovine brain cortex PS has been advocated but its use islimited in human nutrition due to the potential contamination byprotein prion. Phospholipase D (PLD) provides the opportunity forthe synthesis of novel PL species in particular because it is able tocatalyze the transphosphatidylation of serine [1,14,15]. Very often,soya lecithin is used as PL substrate. However, compared with PSof bovine origin, soya-PS lacks in n-3 long-chain polyunsaturatedfatty acids (PUFA). Recently, PS was obtained from squid skin [16]

and from krill source [9] matching the twofold purposes of highcontents in PS and in n-3 PUFA (more than 45% of total fatty acids).

Reaction media are very critical for the efficiency of enzymatictransphosphatidylation in particular due to the physicochemical

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92 A. Pinsolle et al. / Colloids and Surfa

ncompatibility of the hydrophilic enzyme and its hydrophobicubstrates [17]. In the literature, three strategies are proposed toacilitate the contact between these two entities: (i) creation ofn organic solvent/water interface resulting in a biphasic system.olvents such as diethyl ether [18–20], ethyl acetate [18,21,22] or-hexane [19,22] were often used although the amount of residualatter has to be carefully handled if PS is devoted to nutritional

pplications; (ii) use of solid particles in aqueous suspensionsike calcium sulfate or carbonate, silica, and diatomaceous earthlthough they often led to lower conversion ratio than the biphasicystems [22]; (iii) solubilization of the lipid species using surfactantolecules. Numerous surfactants have been used [23–26] but only

ery few of them were suitable for nutritional applications.This work aims at comparing the enzymatic synthesis of PS with

wo kinds of mixed micelles based on marine lipids and eitherodium deoxycholate (SDC) or sodium cholate (SC). The marineecithin used exhibited a high bioavailability [27] and interestingutritional properties owing to its fatty acid composition [3,28].o, the use of this substrate for the transphosphatidylation reac-ion would allow widening the sources of PS rich in n-3 PUFA. Thewo bile salts (BS), SDC and SC, are food-grade, allowing nutritionalpplications of the synthetized PS. Due to the importance of thenterface properties on PLD activity, mixed micelles were carefullyharacterized both in terms of composition and size. A fractionalactorial experiment was designed to study the effect of differenteaction parameters known to regulate PLD activity on the percent-ge of PS produced by transphosphatidylation. From this study,e chose a combination of parameters for PS synthesis that was

pplied to the kinetic study. The proportion of phosphatidic acidPA) due to phospholipid hydrolysis and the consumption of sub-trates (phosphatidylcholine (PC) and phosphatidylethanolaminePE)) were quantified.

. Materials and methods

.1. Materials

The marine lipids used for the mixed micelle preparation wereupplied by PhosphoTech (Nantes, France). The average lipid con-ent was: 69 wt% phospholipids, 27 wt% cholesterol and 4 wt%riacylglycerols [29,30]. More precisely, PL were constituted of8 wt% PC, 23 wt% PE, 2 wt% PS, 2 wt% phosphatidylinositol (PI) and

wt% sphingomyelin [30]. In PL, 56% of total fatty acids were PUFAmong which eicosapentaenoic acid (EPA) and DHA represented0% and 59%, respectively [27]. Bile salts, i.e. SDC or SC, L-serine andhosphoric acid were purchased from Sigma–Aldrich (Germany).hospholipase D (EC 3.1.4.4) from Actinomadura sp. was kindlyonated by Meito Sangyo Co (Tokyo, Japan). Solvents were of ana-

ytical grade. Acetonitrile, triethylamine (TEA), and methanol werePLC-grade solvents from Scharlau (Barcelona, Spain).

.2. Marine lipid solubilization by bile salts

Lipid micellization was performed by either SDC or SC additiono the marine lecithin using the same procedure described in [31].riefly, the lipid–bile salt mixtures were prepared by adding a smallolume of a concentrated SDC or SC solution (concentrations ran-ing from 5 to 70 mM, considering an average MW for of 414.57 and30.55 g/mol for SDC and SC, respectively) to the marine lipids (ran-ing from 2 to 10 mg, i.e. concentrations ranging from 3 to 16 mM,onsidering an average MW for the lipid mixture of 596 g/mol:

W calculated considering that phospholipids (70 wt%, MW of

75 g/mol) and cholesterol (30 wt%, MW of 386 g/mol) mainly influ-nced the solubilization mechanism). Micellization by bile saltsas followed by measuring the turbidity [optical density (OD) at

Biointerfaces 106 (2013) 191– 197

400 nm] in a thermostated cell support using a Hitachi U-2810spectrophotometer (Tokyo, Japan). After stirring, the mixtures wereallowed to equilibrate until stable turbidity values were obtained.For each lipid concentration ([lip]tot), a solubilization curve wasobtained by plotting the evolution of OD as a function of total bilesalt concentration ([SDC]tot or [SC]tot). The solubilization point,corresponding to the bile salt amount required to completely sol-ubilize the lipids into mixed micelles, was determined as the pointat which further addition of bile salts only slightly affected thesuspension turbidity [32]. At this solubilization point, the concen-tration of SDC molecules not associated with the lipids ([SDC]bulk),and the molecular ratio of bile salt to lipid in the mixed micelles([SDC/lip]mic) were determined by the following equation [33]:

[SDC]tot = [SDC]bulk +[

SDClip

]mic × [lip]tot

A similar relationship was obtained for SC. Using linear regres-sion analysis, [SDC]bulk and [SC]bulk were deduced from theintercept of the extrapolated curves with the ordinate axis, and[SDC/lip]mic and [SC/lip]mic were deduced from the slopes. Sol-ubilizations were performed at 25 ◦C at different pH. They werecarried out in triplicate with a fresh lipid suspension.

NMR experiments on micellar structures were performed on aBruker Avance 400 spectrometer (Fremont, CA, USA) operating at162 MHz for 31P by means of a 5 mm QNP probe. 31P spectra wereacquired using a phase-cycled Hahn-echo pulse sequence withgated broadband proton decoupling [34] at 25 ◦C for all samples.Typical acquisition parameters were as follows: spectral window of50 kHz, 32 k data points, p/2 pulse width of 14 �s, interpulse delayof 50 �s, recycle delay of 5 s. Four thousand scans were recorded forthe micellar samples. Fourier transformations were applied to thespectra without any line broadening. Phosphorous chemical shiftswere obtained relatively to 85% phosphoric acid (0 ppm).

Mixed micelle size distribution, i.e. mean diameter and poly-dispersity index, was determined by dynamic light scattering withan multi-angle light scattering spectrophotometer (Malvern CGS-3, Southborough, MA, USA) consisting of a compact goniometersystem, an avalanche photodiode detector, and a digital correla-tor (ALV 5003, Langen, Germany). The light source was a HeNelaser, operating at a wavelength of 632.8 nm with a 22 mW out-put power. The scattering cell was immersed in a refractive indexmatching fluid whose temperature was controlled at 25 ± 0.1 ◦C.For each sample, data were acquired typically for 10 min, for 3 runsat angles ranging from 30 to 150◦. For each sample, at least threeseparate measurements were performed.

2.3. Experimental design for transphosphatidylation

Design of experiments (DOE) is the most efficient approach fororganizing experimental work. DOE selects a diverse and repre-sentative set of experiments in which all factors are independentof each other despite being varied simultaneously. The result is acausal predictive model showing the importance of all factors. DOEwas used in this study to screen the influence of factors implied inthe transphosphatidylation of the marine lecithin with PLD. Experi-ments were organized in a fractional factorial design [35]. Using thismethodology, a first-degree polynomial modelization was obtainedwith a minimized number of experiments and a high accuracy.Experiments were organized in an orthogonal matrix that was afraction of the full factorial design. Experiments were carried outat the summits of the 5- and 6-dimensions of SDC and SC experi-

mental domains, respectively. The fractional factorial experimentdesign consisted of 5 and 6 variables for SDC and SC, respectively:the enzyme (PLD), the molecular ratio of bile salt to lipid in themixed micelles (SDC/lip and SC/lip), the temperature (T), the acyl

A. Pinsolle et al. / Colloids and Surfaces B: Biointerfaces 106 (2013) 191– 197 193

Table 1Experimental settings of factors and responses used for the study of parameters effects and significance on the transphosphatidylation reaction using sodium deoxycholate(SDC) and sodium cholate (SC) mixed micelles.

Experiment Run order pH Temperature (◦C) SDC/lip (mol/mol) L-serine/lip (mol/mol) PLD/lip (U/mmol) PSa (wt%)

Sodium deoxycholate (SDC)1 3 7 35 1 16 95 712 2 8 35 1 4 19 283 4 7 45 1 4 95 454 5 8 45 1 16 19 605 6 7 35 2 16 19 476 9 8 35 2 4 95 297 7 7 45 2 4 19 258 11 8 45 2 16 95 619 10 7.5 40 1.5 10 57 48

10 8 7.5 40 1.5 10 57 4711 1 7.5 40 1.5 10 57 46

Experiment Run order pH Temperature (◦C) [Ca2+] (mM) SC/lip (mol/mol) L-serine/lip (mol/mol) PLD/lip (U/mmol) PSa (wt%)

Sodium cholate (SC)1 3 6.5 35 5 1 16 95 722 2 7.5 35 5 1 4 19 253 4 6.5 45 1 1 4 95 424 5 7.5 45 1 1 16 19 595 6 6.5 35 1 2 16 19 506 9 7.5 35 1 2 4 95 257 7 6.5 45 5 2 4 19 198 11 7.5 45 5 2 16 95 639 10 7 40 3 1.5 10 57 49

10 8 7 40 3 1.5 10 57 481.5

dttwss(

itcwFHrtmtaotas

2

smtseTwgmo

11 1 7 40 3

a Percentage of phosphatidylserine (PS) relatively to the other phospholipids.

onor to substrate molecular ratio (L-serine/lip), the time (t) andhe activator concentration ([Ca2+]) (for SC, only). Each variable hadhree factor levels. The experiment at the center of the domainas performed in triplicate for statistical significance. The work-

heets for SDC or SC are shown in Table 1. Regression analyses andtatistical significance were obtained using MODDE 7.0 softwareUmetrics, Umeå, Sweden).

Transphosphatidylation was performed in screw-cap test tubesncubated in a water bath, under stirring. The reaction volume wasypically of 1 ml. The reaction was stopped by addition of 2 ml ofhloroform/methanol (2:1 (v/v)) to the reaction medium. Lipidsere recovered by centrifugation (Thermo Electron (Courtaboeuf,

rance), 2100 g, 10 min). Phospholipid classes were quantified byPLC and HPLC mass spectroscopy as described below. Using

eaction conditions chosen from the statistical design, kineticransphosphatidylation studies using the two types of mixed

icelles were performed for 24 h. In order to measure theransphosphatidylation capacity, two parameters were considereds described in [21]: the T% corresponding to the ratio between PSbtained divided by the sum of the remaining phospholipids andhe PS/PA ratio. Transphosphatidylation reactions were performedt least in triplicate with a fresh micellar solution. For each lipidpecies, values are means ± Standard Deviation (SD).

.4. Characterization and quantification of PS by HPLC

The initial marine lipid extract and the lipids obtained after PLDynthesis were dried under a nitrogen stream and dissolved in aixture of chloroform/methanol (2:1 (v/v)) to a final concentra-

ion of 5 mg/ml. The HPLC system consisted of an L-7100 Hitachiystem equipped with an automatic injector, a four-solvent deliv-ry system, a degasser, and a diode array detector (L-7455 Hitachi,okyo, Japan) with a detecting zone from 190 to 240 nm. The system

as connected to an EZ chrom-élite interface (Agilent Technolo-

ies, Tokyo, Japan) for control and analysis of chromatograms. Theethod for separation of phospholipid classes was a modification

f the method described by Mawatari and Murakami [36]. The HPLC

10 57 50

column was a ProtonSIL 120-5 Amino (250 × 4 mm, 5 mm, BischoffChromatography, Leonberg, Germany). Acetonitrile, methanol andan aqueous solution of 0.25% triethylamine (TEA) adjusted to pH 3.5with phosphoric acid were used in different proportions as mobilephases. The program used for the mobile phases was: acetoni-trile/methanol/TEA 71/21/8 (v/v/v) up to 3 min, replaced in 1 minby the mobile phase 68/21/11(v/v/v) used from 4 to 23 min, andfinally replaced by the mobile phase 71/21/8 (v/v/v) up to 30 min.The flow rate was 1 ml/min. The column temperature was 25 ◦C.The phospholipid classes were detected at 203 nm.

2.5. Characterization and quantification of PS by massspectrometry (MS)

The methods used in mass spectrometry were adapted from[37]. The initial marine lipid extract and the lipids obtained afterPLD synthesis were diluted to a final concentration of 0.05 mg/mlin a mixture of chloroform/methanol (2:1 (v/v)) containing 7.5 mMammonium acetate. For Shotgun MS, each sample was infused intothe TurboV electrospray source of a QTRAP 5500 mass spectrome-ter (AB Sciex, Concord, Ontario, Canada) at a flow rate of 7 �l/min.Electrospray ionization mass spectrometry (ESI-MS)/MS experi-ments (Precursor Ion scans) were performed in the negative (PE,PS, PA, PI, phosphatidylglycerol (PG)) and positive ion modes (PC)with fast polarity switching (50 ms) and a scan rate of 200 Da/s.MS/MS experiments included one positive mode precursor ion scanand 45 negative mode precursor ion scans. Phospholipid specieswere identified using Lipid View software (v1.0, AB Sciex). Forliquid chromatography (LC)/MS/MS (multiple reaction monitoring(MRM) mode) analyses, the same 5500 QTRAP instrument was usedcoupled to a LC system (Ultimate 3000, Dionex). Two �l of lipidextracts were added to 2 �l of internal standards (PE 17:0/17:0,PS 17:0/17:0, PC 17:0/17:0, PA 17:0/17:0, PI 17:0/14:1 and PG

17:0/17:0, all at 10 �M concentration) and dissolved in 16 �l ofsolvent A consisting of isopropanol/methanol/water (5:1:4) (v/v))with 0.2% formic acid (v/v) and 0.028% ammonium hydroxide (v/v).Analyses were achieved in the negative (PE, PS, PA, PI, PG) and

194 A. Pinsolle et al. / Colloids and Surfaces B: Biointerfaces 106 (2013) 191– 197

Fig. 1. Typical solubilization curves of marine lipid suspensions by sodium cholate atpat

penouwrFmca(p4

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Table 2Bile salt/lipid molecular ratio in mixed micelles ([BS/lip]mic) at equilibrium and cor-responding bile salt concentration in the continuum medium ([BS]bulk) for sodiumdeoxycholate and sodium cholate as a function of pH.

Bile salt pH [BS/lip]mica [BS]bulk (mM)a

Sodium deoxycholate 7.0 0.92 ± 0.09 2.6 ± 0.48.0 0.79 ± 0.09 1.3 ± 0.2

Sodium cholate 6.5 0.90 ± 0.11 7.3 ± 1.07.0 0.90 ± 0.12 9.2 ± 1.4

H 8, followed by the variation of the optical density (OD) at 400 nm with surfactantddition. (Total lipid concentration 3.2 (♦), 6.4 (�), 9.6 (©), and 12.9 (�) mM). Theotal lipid concentration remained constant upon sodium cholate addition.

ositive modes (PC) with fast polarity switching (50 ms). MS/MSxperiments were performed by 20 positive MRM scans and 196egative MRM scans according to the phospholipid identificationsbtained by shotgun MS. Phospholipid species were identifiedsing Lipid View software (v1.0, AB Sciex) and the area of LC peaksere determined using MultiQuant software (v2.1, AB Sciex). In

eversed phase, separations were carried out at 40 ◦C on a Synergiusion-RP, 80 A pore size, 150 × 1 mm, 4 �m particles (Pheno-enex, Le Pecq, France). The gradient elution program was a

ombination of eluent A and eluent B (isopropanol with 0.2% formiccid and 0.028% ammonium hydroxide) with 30% B (0 min); 50% B0–5 min); 80% B (6–30 min); 95% B (31–41 min); 5% B (42–47 min)rogressively replaced by 30% B (52–62 min). The flow rate was0 �l/min. Three �l sample volumes were injected.

. Results and discussion

Global properties of the substrate aggregates play an importantole for modulating enzyme activity. Like most lipolytic enzymes,LD is more active to substrate molecules in aggregated form thann monomeric form, as evidenced by the typical increase in activityo micelle-forming substrates above the critical micelle concentra-ion [17]. In this work, mixed micelles were carefully characterizedn order, first, to determine the minimum amount of detergent nec-ssary to get the mixed aggregates. This is important especiallyf the surfactant has to be removed after synthesis. Secondly, thisharacterization allowed discussing the possible effect of the bilealt type used on the PLD transphosphatidylation capacity.

.1. Characterization of marine lipid–bile salt mixed micelles

Mixed micelles that were used for transphosphatidyl reactionere accurately characterized. First, the micellar state was con-rmed by the narrow and intense NMR signals of the cholinend ethanolamine head groups (Results not shown). Second, theizes of the mixed micelles obtained with SDC and SC were foundoughly the same (i.e. 10 nm) in agreement with others [38].hird, [SC]bulk and [SC/lip]mic were determined from solubili-ation curves obtained for various marine lipid concentrationsFig. 1). Similar curve patterns were obtained with SDC (resultsot shown). Values obtained for mixed micelles at equilibrium areeported in Table 2 for the two bile salts studied. ExperimentalH values were chosen to be equal to or higher than the critical

icellization pH (i.e. 7.08 for SDC and 6.65 for SC [39]). Increas-

ng the pH from 6.5 to 8.0 did not significantly modify [BS/lip]micalues nor [BS]bulk values. Irrespective of the pH, [BS]bulk valuesere in the order of the critical micellar concentration (cmc) of

7.5 0.87 ± 0.10 8.8 ± 1.3

a Values are means ± SD (n = 3).

the main acids present in bile (11 mM for cholic acid, 3 mM fordeoxycholic acid) [39]. This suggested that the solubility of bile saltmonomers was not dramatically modified in the pH range studied.Higher [BS/lip]mic values than that obtained with pure PC bilay-ers ([SC/PC] = 0.4 [38]) were observed. This possibly accounted forthe presence of cholesterol preventing cholate insertion into lipidassemblies [40].

3.2. Experimental design for transphosphatidylation

In this work, PLD from Actinomadura sp. was used. This microbialenzyme has been isolated and identified for a long time [41]. Sev-eral parameters, i.e. pH, temperature, metal ions, and lipid substratesignificantly influenced the activity of this microbial PLD [42]. Theenzyme activity was also highly dependent on the nature of theinterface provided by the presence and/or the nature of the sur-factants [42]. Nevertheless, taking into account the experimentalconditions, optimal pH values ranged from 5.5 to 7.0. The addi-tion of calcium (1 mM) slightly enhanced PLD activity. Likewise,anionic surfactants like sodium deoxycholate and cholic acid werefound to be effective activators of PLD from Actinomadura sp. [42].It is worth noting that PLD activity was determined using cholinequantification, corresponding to the hydrolysis reaction and not thetransphosphatidylation one. In this work, experimental parameterswere investigated for their influence toward the transphosphatidy-lation reaction, i.e. pH (6.5 to 8.0 depending on the bile salt used),temperature, the bile salt nature (SC and SDC) and calcium con-centration (from 1 to 5 mM, only with SC). The amount of acceptoralcohol, the substrate to enzyme ratio, and the dilution of the sub-strate into the micellar aggregates, i.e. the variation of the bilesalt/lipid ratio, were also studied.

The percentage of PS synthesized within the experimental con-ditions varied from 19 to 72% (Table 1). The results were fitted toa first-order polynomial model by multiple linear regression andbackward elimination. Using ANOVA, for SDC and SC micellar sys-tems, the fraction of the variation of the response explained by themodel (R2) and the fraction of variation of the response explainedby the model adjusted for degrees of freedom (R2adj.) were bothhigher than 0.95, proving the accuracy of the model for the designexperiments. Moreover, the residual analysis exhibited a linear dis-tribution and the residuals were normally distributed indicatingthat our assumption of normality of model errors was valid (datanot shown).

Fig. 2A and B show the effect of reaction parameters on PS syn-thesis using mixed micelles based on SDC and SC. Varying the pHbetween 6.5 and 8 (depending on the bile salt used) had no signif-icant effect on PLD activity in the case of both SC and SDC systems.Raising the temperature up to 45 ◦C slightly stimulated the PLDactivity only in the case of SDC system. With SC, the Ca2+ concentra-tion in the range between 1 and 5 mM did not affect the enzymatic

activity. Calcium addition was not tested with SDC because it pro-moted micelle aggregation. Aggregation would modify the surfaceprovided to PLD, preventing accurate comparison of the two bilesalt micellar systems. In general, metal ions are not essential for

A. Pinsolle et al. / Colloids and Surfaces B: Biointerfaces 106 (2013) 191– 197 195

Fig. 2. Multiple regression coefficients measuring the effect of the change level ofthe experimental parameters tested on PS synthesis using mixed micelles based ons ◦

SaS

mpbatpilanfmo(f1p6cPkd

3

p

Fig. 3. Time-dependence of PC (©) and PE (�) consumption and accumula-tion of reaction products, PS (�) and PA (�), using mixed micelles based onsodium deoxycholate (SDC/lip = 1 mol/mol; phosphate buffer pH 6.5) (A) andsodium cholate (SC/lip =1 mol/mol; phosphate buffer pH 7) (B). In both cases,

odium deoxycholate (A) and sodium cholate (B). Temperature ( C); SDC/lip andC/lip (mol/mol); L-serine/lip (mol/mol); PLD/lip (U/mmol), Ca2+ (mM). Error barsre calculated with a 0.95 confidence interval and 5 and 4 degrees of freedom forDC and SC, respectively. Coefficients with P-value lower than 0.05 are significant.

icrobial PLD activity [17] although some studies showed that theresence of calcium in transesterification reaction was beneficialoth in terms of selectivity and reaction rates [21]. For both SCnd SDC systems, the main factors influencing PLD selectivity werehe L-Serine/lip ratio and the PLD/lip ratio that positively acted onhospholipid conversion. This was in agreement with the fact that

ncreasing L-serine concentration favored the transphosphatidy-ation instead of the hydrolysis pathway as already observed withqueous suspension systems [22]. The fact that the bile salt/lip ratioegatively influenced PLD transphosphatidylation may account

or a dilution of the phospholipid substrate in the mixedicelles. This effect was also observed with mixed micelles based

f 3-[3-Cholamidopropyl)dimethylammonio]-1-propanesulfonateCHAPS) and PC [43]. From our statistical approach, we chose theollowing reaction conditions for the kinetic study: (i) with SDC,00 mg of marine lipids and 67 mg of SDC (SDC/lip = 1 mol/mol),hosphate buffer pH 6.5; (ii) with SC, 100 mg of marine lipids and8 mg of SC (SC/lip =1 mol/mol), phosphate buffer pH 7. In bothases, 250 mg of L-serine (L-serine/lip = 16 mol/mol) and the lowerLD/lip level (2 mg of PLD, i.e. 19 U/mmol) were used. Becauseinetic study was performed for 24 h, the reaction temperature wasecreased to 25 ◦C to prevent PUFA oxidation.

.3. Kinetic study

Fig. 3A and B show the kinetic evolution of phospholipid com-osition during PS synthesis using SDC and SC mixed micelles,

L-serine/lip = 16 mol/mol and PLD/lip = 19 U/mmol were used. The reaction was con-ducted at 25 ◦C. Values are means ± SD (n = 3).

respectively. Similar patterns were obtained for both systemsshowing the formation of PS from the transphosphatidylation reac-tion, the consumption of PC and PE substrates, and PA appearanceas a hydrolysis product. Both systems were effective, generatingnearly 60% PS in 24 h. The fact that PC was not entirely converted(about 10% remaining in the reaction media) could be due to anaccumulation of choline. Indeed, this byproduct of the transpho-sphatidylation reaction was shown to easily accumulate in theimmediate vicinity of the enzyme resulting in a lower conver-sion [44]. The conversion yield of the starting phospholipids intoPS remained almost constant for all kinetics and was nearly thesame for the two bile salts: 42 ± 1% and 44 ± 2%, for SDC and SCsystems, respectively. The PS/PA ratio is a good indicator of theselectivity between transphosphatidylation and hydrolysis. Inter-estingly, the prolonged incubation did not promote hydrolysis ofthe product or the substrate since the PS/PA ratio remained almostthe same all along the kinetic study (5.7 ± 0.4 and 5.0 ± 0.2, for SDCand SC systems, respectively). PLD showed preferential substrateselectivity toward PC compared with PE as evidenced by the dras-tic decrease in PC in the 2 first hours of the kinetic study while PEproportion remained almost constant (Fig. 3A and B). This was inagreement with other studies showing that PC was the best sub-strate for transphosphatidylation among PE and PG [21,42]. Thiswas also confirmed by investigating the rate of PLD hydrolysis usingphospholipids with different head groups [45,46]. It is worth not-ing that the relatively high conversion rate may be attributed tothe high serine concentration in the water phase (250 mg/ml) used

in this work. This was also observed with an aqueous suspensionsystem with silica-adsorbed lecithin, i.e. increasing L-serine by afactor of 2 reduced the formation of PA by almost a factor of 4 [22].

196 A. Pinsolle et al. / Colloids and Surfaces B: Biointerfaces 106 (2013) 191– 197

F f PS (Cc

3

l(McDm(ammtwsuntPPtulf

dependent on their molecular distribution in the nutritional lipidsource [27].

On the whole, this study provided suitable experimental con-ditions for the synthesis of a lecithin enriched both in PS and EPA

Table 3Molecular species of PS synthetized using the SDC and SC micellar systems.

Molecular species a (mol%) SDC micellar system SC micellar system

PS 16:0/16:0 0.02 ± 0.00 b 0.12 ± 0.07PS 16:0/18:1 4.65 ± 0.05 3.86 ± 0.05PS 16:0/20:4 2.02 ± 0.33 2.10 ± 0.07PS 16:0/20:5 16.87 ± 0.35 21.65 ± 0.19PS 16:1/20:4 0.00 ± 0.00 0.01 ± 0.01PS 14:0/22:6 0.00 ± 0.00 2.41 ± 0.01PS 18:0/20:5 2.25 ± 0.19 2.70 ± 0.24PS 18:1/20:4 0.21 ± 0.06 0.09 ± 0.01PS 16:0/22:6 53.86 ± 1.32 48.15 ± 1.02PS 16:1/22:5 0.00 ± 0.00 0.01 ± 0.00PS 18:0/20:6 0.00 ± 0.00 1.19 ± 0.00PS 18:1/20:5 2.45 ± 0.10 2.13 ± 0.17PS 18:0/22:6 5.02 ± 1.03 4.69 ± 0.82PS 20:1/20:5 12.99 ± 0.45 10.84 ± 0.61PS 18:1/24:5 0.00 ± 0.00 0.04 ± 0.01

ig. 4. Molecular distributions of PC (A) and PE (B) in the initial marine lipids and oholate (�).

.4. Molecular characterization of the synthetized PS

The marine lecithin used as substrate for the transphosphatidy-ation with PLD was mainly composed of PC (65 wt%) and PE25 wt%) (Fig. 3A and B). These phospholipids were analyzed by

S. It was previously shown that the marine lecithin was mainlyomposed of EPA (22:6; 33 wt%), palmitic acid (16:0; 31 wt%) andHA (20:5, 15 wt%) [27]. These fatty acids were distributed in theain species of PC (PC 36:5 and PC 38:6) (Fig. 4A) and PE (PE 36:5)

Fig. 4B). The high proportion of PE 40:6 suggested that gadoleiccid (20:1; 4 wt%) and stearic acid (18:0; 4 wt%) present in thearine lecithin were concentrated in PE. PS prepared from thearine lecithin exhibited a similar fatty acid pattern to the ini-

ial PC and PE substrates (Fig. 4 C). The high proportion of PS 38:6as consistent with the fact that PS issued from both PC and PE

pecies. This indicated that: (i) PDL acted on the different molec-lar species of both PC and PE and (ii) the reaction conditions didot alter PUFA. Table 3 presents in details the molecular distribu-ion of fatty acids in PS species. PS 38:6 was essentially composed ofS 16:0/22:6 while PS 18:1/20:5 was present in very low amount.S 36:5 corresponded to PS 16:0/20:5 whereas PS 40:6 reflected

he presence of PS 18:0/22:6 and PS 20:1/20:5. In all these molec-lar species, it was worth noting that EPA and DHA were mainly

ocated at the sn-2 position. These data were of special relevanceor the in vivo bioavailability of these fatty acids that was highly

) synthetized using mixed micelles based on sodium deoxycholate (�) and sodium

a Notation of phospholipid species: In PS 16:0/18:1, palmitic acid (16:0) and oleicacid (18:1) are known to be located in the sn-1 and sn-2 positions, respectively.

b Values are mean ± SD corresponding to three different LC/MS/MS (MRM mode)analyses.

ces B:

aycwSi

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R

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A. Pinsolle et al. / Colloids and Surfa

nd DHA. No significant difference on the transphosphatidylationield was pointed out when using SDC or SC mixed micelles. Thisould be related to the fact that the two types of mixed micellesere very similar both in terms of size and molecular composition.

ince only food-grade detergents were used, PS obtained could findnteresting applications as nutraceuticals.

cknowledgements

The authors acknowledge the French National Association ofechnical Research (ANRT) for its financial support through ah. D. research grant for A. P. We are indebted to Anne Marieorthe for her help in the fractional factorial experiment designnd the statistical interpretation of the data. The authors are alsorateful to Axelle Grélard for NMR analysis and Reiko Oda for herssistance for micelle size measurements. Samples of enzymesere generously donated by Meito Sangyo Co (Tokyo, Japan). We

cknowledge funding of the Région Aquitaine and of platforms Pro-éome (http://www.pgfb.u-bordeaux2.fr/proteome/index.html),nd Métabolome-Lipidome-Fluxome of Bordeaux (https://ww.bordeaux.inra.fr/umr619/RMN index.htm) for contribu-

ion to mass spectrometry equipment. We thank Pr Fernandoeal-Calderon for his careful reading of the manuscript.

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