Preparation of Triacylglycerols Rich in Omega-3 FattyAcids from Sardine Oil Using a Rhizomucor miehei Lipase:Focus in the EPA/DHA Ratio

Paulo Bispo & Irineu Batista & Raul J. Bernardino &

Narcisa Maria Bandarra

Received: 20 March 2013 /Accepted: 30 October 2013 /Published online: 29 November 2013# Springer Science+Business Media New York 2013

Abstract The increasing evidence on the differential biochemical effects of eicosapentaenoicacid (EPA)/docosahexaenoic acid (DHA) raises the need of n-3 highly unsaturated fatty acidconcentrates with different amounts of these fatty acids. In the present work, physicochemicaland enzymatic techniques were combined to obtain acylglycerols, mainly triacylglycerols(TAG), rich in n-3 fatty acids. Sardine oil was obtained bywashing sardine (Sardina pilchardus)mince with a NaHCO3 solution, hydrolyzed in a KOH–ethanol solution, and concentrated withurea. The esterification reaction was performed in the stoichiometric proportion of substrates forre-esterification to TAG, with 10 % level of Rhizomucor miehei lipase based on the weight ofsubstrates, without any solvent, during 48 h. This procedure led to approximately 88 % ofacylglycerols, where more than 66 % were TAG and the concentration of n-3 fatty acids washigher than 60 %, the EPA and DHA ratio (EPA/DHA) was 4:1. The content of DHA in theunesterifed fraction (free fatty acids) increased from 20 to 54%,while the EPA level in the samefraction decreased from 33 to 12.5 % (EPA/DHA ratio ≈1:4). Computational methods (densityfunctional theory calculations) have been carried out at the B3LYP/6-31G(d,p) level to explainsome of the experimental results.

Keywords n-3 HUFA . EPA/DHA ratios . Triacylglycerols . Rhizomucormiehei lipase .

Sardine oil . Ab initio geometry optimization

Appl Biochem Biotechnol (2014) 172:1866–1881DOI 10.1007/s12010-013-0616-1

P. Bispo (*)Department of Biochemistry and CEDOC, Faculty of Medical Sciences, Universidade Nova de Lisboa(UNL), Campo Mártires da Pátria, 130, 1169-056 Lisbon, Portugale-mail: pauloffbispo@gmail.com

I. Batista :N. M. BandarraDivAv, IPMA, Av. Brasília, 1449-006 Lisbon, Portugal

R. J. BernardinoGIRM, Laboratory of Separation and Reaction Engineering, ESTM, Instituto Politécnico de Leiria,Santuário N.ª Sra. dos Remédios, Apartado 126, 2524-909 Peniche, Portugal

Introduction

The ingestion of n-3 highly unsaturated fatty acid (HUFA), particularly eicosapentaenoic acid(C20:5 n-3, EPA) and docosahexaenoic acid (C22:6 n-3, DHA), is associated withcardioprotective effects [1]. EPA is involved in eicosanoids biosynthesis, particularly prosta-glandins, leukotrienes, and thromboxanes, with an opposite effect of eicosanoids producedfrom arachidonic acid. DHA is fundamental in the newborn neurological system development,and it is esterified in retina membrane’s phospholipids. EPA and DHA have beneficial effectson immunological and inflammatory profile [2], some types of cancer [3], and in neurologicaland neurodegenerative disorders [4]. Both fatty acids can have in many physiopathologicconditions a complementary and/or synergistic function. However, some of the biochemicalprocesses regulated by n-3 HUFA, in vivo and in vitro, can be mediated by EPA and DHA bydifferent mechanisms, and some of these mechanisms can require an optimum ratio betweenEPA and DHA [5–8]. The different biological effects of EPA and DHA make the EPA/DHAratio important in the clinical practice [9] and also in the application of these fatty acids innutritional supplements and pharmaceutical products [10].

EPA and DHA are mainly found in fish oils, which have been used in the production of n-3polyunsaturated fatty acids (n-3 PUFA) concentrates in the form of mainly triacylglycerols(TAG) or fatty acids ethyl esters. However, the bioavailability of n-3 PUFA depends on thetype of compounds present in the concentrates. Thus, acylglycerols, mainly TAG, are highlybioavailable, more “natural”, and easy to use in industrial formulations [11]. Recently, arandomized placebo-controlled study concluded that, after 6-month supplementation, theomega-3 Index (EPA+DHA) in red blood cell membranes increased to a greater extent whenconsumed in the form of TAG compared with FA-EE [12]. Having this into account, thenutritional and physiological advantages of TAG rich n-3 PUFA led to increasing interest fortheir production. TAG can be produced by physicochemical and catalytic/biocatalyticprocesses [13, 14]. The traditional physicochemical methods cause important losses ofn-3 PUFA mainly due to isomerization and oxidation reactions [15]. On the contrary,lipases offer many advantages as a result of the mild reaction conditions used as well aslow energy consumption. In addition, the limited formation of undesirable side productsreduces the separation and purification steps [16]. However, throughout (long-time)biocatalysis reaction, significant losses of these highly unsaturated fatty acids may occur.For instance, intermolecular and intramolecular bonds may lead to the formation ofdimers, which were found in commercial fish oils concentrates [17]. Fatty acid dimers maybe also formed in (bio)membranes to a considerable extent following radical generatedxenobiotics metabolism in vivo [18].

Sardine is the main pelagic resource of Portuguese coast when considering total annualcatches [19], and a total of 19,600 t was estimated for the sardine byproducts from canningindustry in 2009 [20]. In previous works, lipase enrichment of n-3 HUFA, mainly EPA andDHA, from sardine oil was used by means of, e.g., hydrolysis [21], transesterification [22],selective esterification with 1-butanol [23] or cholesterol [24], or by direct esterification withglycerol [25]. However, few studies have focused on the importance of biocatalysis as a meanto obtain triacylglycerides with different amounts of EPA to DHA in function of theirnutritional or pharmaceutical applications, and almost all these works were conducted overpure (or very pure) substrates and/or commercial (refined, bleached, and deodorized) fish oils,and/or made use of solvents in the reaction medium. For instance, Halldorsson et al. [25]produced acylglycerides, mainly diacylglycerols (DAG), with different EPA-to-DHA ratios,using various fish oils and RM IM lipase. Fernández-Lorente et al. [21] also attempt to producelipid fractions with different EPA/DHA ratios using sardine oil hydrolysis.

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The main objective of this work was the preparation of TAG, rich in n-3 HUFA, by using an-3 fatty acid concentrate obtained from non-commercial sardine oil, in a solvent-free system,using the immobilized Rhizomucor miehei lipase. Computational density functional theory(DFT) methods were used to explain some of the experimental results.

Materials and Methods

Materials

Sardine caught off in the Portuguese coast, in July, was purchased in the local market and kepton ice until used. All reactives used were of analytical grade.

Methods

Oil Recovery, Preparation of Free Fatty Acids, and Urea Complexation

Sardine oil was extracted from sardine mince by washing with a 2.5 % NaHCO3 aqueoussolution (3:1 v/v), followed by a mild heating (50–60 °C) to disrupt the emulsion andcentrifugation to separate the oil fraction.

Free fatty acids (FFA) were obtained after sardine oil hydrolysis (2 h at 65–70 °C) with a10 % KOH–ethanol aqueous solution (1:2 v/v) under a nitrogen atmosphere. A PUFAconcentrate was obtained by urea complexation as described by Bandarra et al. [26]. FFA (10 g)were mixed with urea (FFA/urea 1:3 w/w) in 95 % aqueous ethanol and heated (60–65 °C) withstirring until the whole mixture turned into a clear homogeneous solution. The mixture was left inthe dark at 2 °C for 24 h for crystallization. The crystals obtained (urea complexing fraction, UCF)were separated from the liquid fraction (non-urea complexing fraction, NUCF) by filtration with aWhatman no. 1 filter paper. The FFAswere recovered from both fractions, which were mixedwithwater, acidifiedwith concentrate HCl, and extractedwith hexane. The organic phasewas separatedfrom the aqueous layer and washed with distilled water to remove any remaining urea and thendried over anhydrous sodium sulfate. The solvent was subsequently removed at 40 °C underreduced pressure. The NUCF and UCF were weighed and the percentage of recovery calculated.The fatty acid composition of both fractions was determined by gas chromatographic procedure asdescribed elsewhere [26].

The yield of each fatty acid was calculated based on the equation:

Yield %ð Þ ¼ %FFAf x % W=%FFAi ð1Þ

Where FFAf is the percentage of fatty acid in UCF or NUCF, W is the percentage of theweight of the fraction, and FFAi is the percentage of fatty acid in the starting oil (as TAG).

Enzymatic Esterification

The lipase, R. miehei “Lipozyme IM”, immobilized in macroporous anion exchange resin fromNovo Industries, Denmark, was purchased from Fluka. The specific activity of this lipase was48.3 U per milligram of preparation as reported by the supplier, with a sn-1,3 specificity. Theesterification reaction was performed in open tubes at the stoichiometric proportion ofsubstrates (FFA/glycerol 3:1) for esterification to TAG, with 10 % level of lipase based onthe weight of fatty acids in NUCF. The mixture was heated in a water bath at 55 °C, with

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gentle stirring by bubbling nitrogen, and maintained in the dark for 48 h. The reaction wasstopped at pre-defined time intervals by adding hexane. The enzyme was removed bycentrifugation, the solvent evaporated under a stream of nitrogen, and the recovered samplestored at −20 °C until further analysis.

Evaluation, Yield, and Identification of the Degree of Esterification

The lipid mixtures were separated by thin layer chromatography (TLC) to determine thepercentage of the different lipid classes. Silica gel plates 60F-254 (Merck, Germany) with20×20 cm and 0.25mm thickness were used. The plates were previously activated at 100 °C for1 h. The oil samples were solubilized in chloroform to a 10 mg/ml concentration and 10-μlaliquots were applied in each TLC point. A mixture of chloroform/acetone/acetic acid(96:3.8:0.2 v/v/v) was used to elute the TLC plates. The developed plates were sprayed witha 10% ethanolic solution of phosphomolibdic acid and dried in the oven (Bioblock Durocell) at120 °C for 10 min. The identification of FFA, monoacylglycerols (MAG), DAG, and TAGwasdone by comparison with Fluka standards (oleic acid,α-monolein,α,β-dipalmitin, and triolein)and the quantification was performed using a scanner and the version 2.4 of Quantity Onesoftware from PDI Imageware Systems (USA).

In order to characterize the fatty acid profile of FFA, MAG, DAG, and TAG, somedeveloped plates (silica gel 60 20×20 cm and 0.50 mm thickness) were sprayed with a2,7-dichlorofluoresceine ethanolic solution (0.2 %). The bands were revealed under ultravioletlight, scraped from the plates and the FFA, MAG, DAG, and TAG fractions extracted withchloroform.

Fatty Acid Analyses

Fatty acid methyl esters (FAMEs) were prepared according to [27] as modified by [28]. TheFAMEs preparation was carried out using 300 mg of lipids and 5 ml of the acetylchloride/methanol mixture (1:19, v/v). The transesterification was carried out at 80 °C for1 h. After cooling, 1 ml of water and 2 ml of n-heptane were added to the mixture, which wasstirred and centrifuged at 2,150×g for 10 min. The organic phase was collected, filtered, anddried over anhydrous sodium sulfate. The solvent was removed under nitrogen and the FAMEsdissolved in 0.1 ml of n-heptane. The analyses were performed using a Varian CP-3800(Walnut Creek, CA, USA) gas chromatograph equipped with an autosampler and fitted witha flame ionization detector at 250 °C.

The separation was achieved using a polyethylene glycol capillary column DB-WAX(30 m length, 0.25 mm i.d., and 0.25 μm film thickness) from Hewlett Packard (Albertville,MN, USA). After holding at 180 °C for 5 min, the temperature was ramped at 4 °C/min to220 °C and maintained at 220 °C for 25 min with the injector at 250 °C. The split ratio was100:1. The identification of the sample peaks was made by comparison of the retention timeswith the standards from Sigma. The fatty acid profile was obtained by calculating the relativearea percent of the chromatographic peaks.

The percentage of EPA, DPA, and DHA in the TAG and FFA was calculated by thefollowing equation:

% FAYield ¼ %Wt x %FAwt=%FAið Þ ð2Þ

WhereWt is the percentage of each fraction (TAG or FFA) at time t, FAwt is the percentage offatty acid in TAG or FFA fraction at time t, and FAi is the percentage of fatty acid in the NUCF.

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DFT Geometry Optimization

Computational DFT methods were used to analyze the structure and electronic properties of allthe fatty acids and their derivatives studied. All the structures have been fully optimizedwithout symmetry constraints using the Becke three-parameter hybrid functional combinedwith the Lee, Yang, and Parr correlation functional (B3LYP) [29–31] along with the split-valence double-zeta Pople basis set 6-31G(d,p), which is well known to produce accurategeometries. There is evidence that this functional theory provided more accurate energies thanother computationally more demanding methods [32].

The vibrational frequency calculations were performed at the same level of theory to checkthat all structures were global minima of the potential energy surface (denoted by an absence ofnegative vibrational frequencies) and to correct the computed energies for zero-point energiesas well as translational, rotational, and vibrational contributions to the enthalpy. All compu-tational methods were done using Gaussian 03 program package [33]. All the figures andelectrostatic potential surfaces were constructed using the Molekel software [34].

The relative dimerization energies (ΔEc corresponding to the process 2 × HUFA→[HUFA −HUFA], where HUFA represents each carboxylic acids studied) were calculated as the energydifference at 298 K between the final energy of the dimers and the sum of the energies of twomonomers, at the same theoretical level, an approximation previously used [35].

Statistical Analysis

Statistical analysis was carried out with SPSS software (SPSS Inc., USA), version 19.0, forWindows. Two-tailed paired sample t test was used to compare measures. The results arepresented as a mean ± SD. The significance was considered at p <0.05.

Results and Discussion

Urea Complexation

Urea complexation was used for the preparation of n-3 PUFA concentrates because it is a simplemethod, it can be performed under mild conditions, generally gives good yields, and is easy toscale up. The sardine oil extracted from sardine mince was essentially constituted by TAGaccording to TLC analysis (data not shown). The FA profile of sardine oil (Table 1) was typicalof this species [36] with 26 % of saturated fatty acids (SFA), 28 % of monounsaturated fatty acids(MUFA), and 40 % of n-3 PUFA. The yields of the NUCF and UCF were ≈33 % and ≈49 %(based on starting fish oil), respectively. These yields depend on the relative percentages of thedifferent fatty acids present in the starting oil. As it can be seen in Table 1, the n-3 PUFA level in theNUCF is almost double of that in the starting oil. A similar increase of EPA and DHA percentagewas observed in NUCF. In a previous workwith salmon oil, a concentrate with around 70% of n-3PUFAwas obtained [26]. In parallel, an important decrease (≈86 %) of SFA in NUCF occurred.MUFA were also complexed by urea which was more effective with long-chain fatty acids(20:1 and 22:1). Similar results were reported by Ratnayake et al. [37] using different fish oils.

Synthesis of Acylglycerols By Lipase

Figure 1 shows the evolution of acylglycerols and FFA during the esterification reaction. Anoticeable decrease of FFAwas recorded after 4 h of reaction with the consequent formation of

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acylglycerols. After this period, a gradual decrease of FFA percentage at a mean rate of≈0.58 %/h was observed, which is quite similar to the increasing rate of acylglycerolsformation recorded (approximately 0.60 %/h). TAG increased from ca. 38 % after 4 h to morethan 66 % at the end of the reaction, which represents a medium rate of 0.95 %/h. At the end ofthe reaction, TAG represented 75 % of total acylglycerols. The high reaction rate achieved canbe due to the absence of solvent in the reaction medium as reported by Selmi et al. [38].On the other hand, Halldorsson et al. [25], working with RM IM lipase, at 40 °C, indirect esterification reaction of various fish oils (including sardine oil FFA) and a 2:1FFA/glycerol ratio, mainly obtained DAG and MAG. Thus, the high percentage of TAGformed during the reaction may be explained by the spontaneous acyl migration occurringin the reaction medium from 1(3)-MAG to 2-MAG and 1,3-DAG to 1(3),2-DAG such asreferred by several authors [39–41].

During the reaction period, the percentage of MAG detected was always very low (0–2 %).However, a rapid formation of DAG was recorded and its percentage was about 37 % after 4 h.However, in the next period until 28 h, a slight decrease of DAG percentage was observedtogether with a small increase of TAG. This evolution indicated that apparently only a verysmall amount of FFA was incorporated into acylglycerols. The esterification of FFA to formTAG seemed to be more effective at the final period (between 42 and 48 h) of the experiment.

Table 1 Fatty acid profile (% of total fatty acids) of starting oil, NUCF, and UCF

Fatty acid (%) Starting oil NUCF Yield UCF Yield

C14:0 6.0±0.21 0.4±0.03 2.0 10.0±0.29 80.8

C16:0 14.7±0.15 0.3±0.02 0.7 26.8±0.39 88.9

C18:0 2.9±0.05 0.1±0.00 0.9 5.4±0.09 90.5

Other SFAa 2.7 2.7 2.3

ΣSFA 26.3 3.5 44.5

C16:1 9.0±0.14 4.4±0.22 15.9 11.9±0.20 64.8

C18:1 13.6±0.13 2.0±0.03 4.8 22.9±0.31 82.6

C20:1 2.7±0.05 0.9±0.03 10.8 4.5±0.17 82.8

C22:1 1.0±0.05 0.1±0.00 2.3 1.9±0.10 92.8

Other MUFAb 1.3 1.8 0.6

ΣMUFA 27.5 9.2 41.8

C18:2n-6 1.1±0.01 1.4±0.03 39.7 0.9±0.01 40.0

C18:3n-3 1.0±0.00 1.4±0.02 44.8 0.7±0.01 32.6

C18:4n-3 4.2±0.02 8.3±0.14 65.2 0.4±0.02 5.2

C20:5n-3 17.6±0.27 34.5±0.23 64.6 3.7±0.22 10.2

C22:5n-3 1.7±0.07 3.0±0.05 58.5 0.9±0.13 24.9

C22:6n-3 10.4±0.33 22.0±0.43 69.6 2.0±0.19 9.3

Other PUFAc 6.8 11.8 2.1

ΣPUFA 42.8 82.3 10.7

Σn-6 3.0 4.3 1.9

Σn-3 39.5 77.4 8.6

Σn-3/Σn-6 13.3 18.0 4.4

a iso14:0, 15:0, 17:0, 19:0, 20:0b 17:1c 16:2n-4, 16:3n-3, 16:4n-3, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6, 20:3n-3, 20:4n-3, 21:5n-3, 22:5n-6

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The percentages of the most important fatty acids in the NUCF and in the different lipidsfractions after 48 h of esterification are presented (Table 2). Despite the low level of SFA in theNUCF, the R. miehei lipase showed a high specificity for these fatty acids (FA) that were largelyincorporated in the acylglycerols (mainly DAG) fraction (data not shown). Similarly, MUFAwere also easily esterified so that after 48 h, 20:1 and 22:1 FA were only found in theacylglycerol fractions (Table 2).

The evolution of the percentage of EPA in different fractions (Fig. 2a) indicates its goodincorporation both in DAG and TAG and its consequent decrease in the FFA fraction. Theincorporation of docosapentaenoic acid (DPA, 22:5 n-3) in DAG and TAG was also quiteefficient as can be observed in Fig. 2b where a complete esterification of this fatty acid wasobserved at the end of the experiment. A completely different pattern was observed for theincorporation of DHA in the acylglycerols (Fig. 2c), where a gradual increase of its percentagein the FFA fraction was recorded. This rise evidences the difficulty of esterification of DHA incomparison with other fatty acids. The level of DHA in TAG and DAG was almost constantduring the first 24 h of reaction and attained around 10 %. As indicated in Fig. 2c, in the last24 h, DHA increased gradually and achieved a level of incorporation of 16 % in DAG and closeto 10 % in TAG (Table 2).

After 28 h of reaction time, despite the low specificity of R. miehei lipase for DHA, theincorporation of DHA in DAG and TAG occurred, although more pronounced in DAG(Fig. 2c). This slow increase of DHA incorporation only took place when the concentrationof DHA in the reaction mixture was relatively high.

%

0

20

40

60

80

100

0 10 20 30 40 50

Time (hours)

TAG DAG FFA MAG ACYLGLYCEROLS

***1 **2 **3 NS NS NS *4

Fig. 1 Evolution of FFA, MAG, DAG, TAG and total acylglycerols during the esterification reaction. 1 p=0.001,between TAG T0 and T4h; 2 p<0.01, between TAG T4h and T16h; 3 p<0.01, between TAG T16h and T20h; 4

p<0.05, between TAG T41h and T48h; NS: not significant

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The low specificity of this lipase to esterify DHA could be a mean for DHA enrichment ofFFA fraction in this fatty acid. In the present work, the final percentage of DHAwas 2.7 higherthan its initial value after 48 h of reaction.

Similar results on the low specificity of this lipase for DHAwere reported [25, 42, 43]. Thedifferent behaviors of these n-3 HUFA towards esterification may be related to the positionof the nearest double bond of the carboxylic group [26, 43] (Fig. 3). The carbon atom ofthe first double bond is located in positions 4, 5, and 7 for DHA, EPA, and DPA,respectively, which means that the closer the double bond of carboxylic group, the higheris the stereochemical hindrance, which makes more difficult the adaptation of the substrateto the active centre of the lipase.

Haman and Shahidi [44] found that the incorporation of n-3 PUFA into triolein catalyzed byLipozyme RM IM in the presence of hexane followed the order EPA > DPA > DHA. A similarpattern was also observed in the present work.

EPA and DHA Ratio in FFA and TAG Fractions

Figure 4 shows the EPA/DHA ratio in FFA and TAG fractions during the esterificationreaction. A gradual decrease in the EPA/DHA ratio was observed in the FFA fraction, whichwas 1.6 on the beginning of the reaction. The low incorporation of DHA in acylglycerolsfraction was responsible for the final ratio of ≈0.24. A gradual increase of the EPA/DHA ratioin the TAG fraction was recorded until 28 h of reaction, where it achieved a value slightly

Table 2 Fatty acid profile (% oftotal fatty acids) of sardine oil con-centrate by urea complexation(NUCF) and different lipid fractionsafter 48 h of esterification usingR. miehei lipase

n.d. not detecteda15:0, 20:0b20:4n-6, 20:4n-3, 21:5n-3, 22:4n-6

Fatty acid (%) NUCF (%) Lipid fractions (%)

FFA TAG

C14:0 0.9±0.14 0.5 1.0

C16:0 0.8±0.15 2.9 2.0

C18:0 1.3±0.09 1.7 2.2

Other SFAa 0.9 0.7 0.7

ΣSFA 3.9 5.8 5.9

C16:1 7.7±0.06 1.2 9.9

C18:1 8.4±0.12 4.1 11.0

C20:1 1.9±0.03 n.d. 2.7

C22:1 0.2±0.02 n.d. 0.2

ΣMUFA 18.2 5.3 23.9

C18:2n-6 5.7±0.05 5.2 5.0

C18:3n-3 0.3±0.02 n.d. 0.4

C18:4n-3 8.5±0.05 13.3 7.0

C20:5n-3 33.3±0.10 12.7 39.2

C22:5n-3 2.9±0.06 n.d. 3.2

C22:6n-3 20.2±0.16 53.7 9.7

Other PUFAb 5.3 3.8 4.3

ΣPUFA 76.2 88.7 68.8

Σn-6 7.6 5.2 6.4

Σn-3 68.6 83.4 62.4

Σn-3/Σn-6 9.0 15.9 9.8

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EPA(%)

0

10

20

30

40

50

0 10 20 30 40 50

Time (hours)

TAG DAG FFA

DPA(%)

0

1

2

3

4

5

0 10 20 30 40 50

Time (hours)

TAG DAG FFA

DHA(%)

0

10

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60

0 10 20 30 40 50Time (hours)

TAG DAG FFA

a

b

c

Fig. 2 Evolution of the percent-age of EPA, DPA, and DHA inDAG, TAG, and FFA fractionsduring the esterification reaction

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higher than 5. The easier incorporation of EPA in TAG is also responsible for this increment ofthe EPA/DHA ratio. In the next 20 h, there was a decrease in the EPA/DHA ratio until a valueclose to 4.0, mainly due to the slightly incorporation of DHA in TAG fraction.

A higher EPA/DHA ratio (over 7) was achieved in DAG fraction after 16 h of reaction(data not shown). A regular decrease of this ratio was then observed attaining less than 3

Monomer Dimer Ester Dimer

DHA

EPA

Fig. 3 OptimizedDFTstructures of the n-3HUFAmonomers, dimers, and ester dimers derivatives of DHA and EPA

0

1

2

3

4

5

6

7

8

9

10

4 16 20 24 28 41 48

Reaction time(h)

EPA/DHA Ratio

FFA TAG

Fig. 4 EPA/DHA ratio in FFAand TAG fractions during the es-terification reaction

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after 48 h, reflecting the incorporation of DHA in DAG during this period. Halldorsson et al.[25] also obtained in DAG fraction a low EPA/DHA ratio (0.26) in an attempt to separate EPAand DHA using sardine oil.

In the present work, these experimental conditions can lead to the production of a largerange of EPA-to-DHA ratios considering all fractions (acylglycerols and FFA). In fact, in thelast years some studies highlight the differential biologic effects of EPA and DHA, e.g., ascardioprotective fatty acids or immunomodulators [9, 45, 46]. A recent study conducted inWistar rats to evaluate the effect of different EPA and DHA ratios, on the endpoint of oxidativedamage (protein redox state), indicated that the EPA/DHA ratio 1:1 showed the lowestoxidative score. They concluded that such antioxidant activity may differ among differentfish oil sources in face of the variations of EPA/DHA content [47].

Percentage of EPA and DHA in FFA, DAG, and TAG Fractions

Figure 5 shows the percentage (calculated according to Eq. 2) for EPA and DHA in FFA andTAG fractions during the reaction. The maximum percentage of EPA (30 %) in FFA wasrecorded after 4 h and then continuously decreased until the end of the reaction when it wasless than 4 %. The percentage of DHA in the FFA fraction also attained its maximum (57 %)after 4 h and its decrease was considerably slower compared with that of EPA. On the otherhand, DHA percentage was always higher in the FFA fraction, except at 48 h, when similarpercentages (ca. 32 %) were recorded in FFA and TAG fractions. Higher EPA percentage wasrecorded in the DAG fraction (data not shown) than in TAG until 24 h. But later on, the TAGfraction was richer in this fatty acid (Fig. 5). For DPA, the percentage in TAG fraction wasclose to 75 % at the end of reaction (data not shown).

Halldorsson et al. [25] reported, at 28 h of reaction time, for sardine oil, that the percentageof DHA into the FFA fraction and EPA into the acylglycerols (DAG and MAG) products wasvery high, 78.4 % and 85.9 %, respectively. In the present work, at 28 h, the yield of EPAincorporation in acylglycerols (TAG and DAG) was similar, 86 %, although the yield of DHAin FFA fraction was only near 50 %.

0

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4 16 20 24 28 41 48

Reaction time(h)

EPA (%)

FFA TAG

0

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20

30

40

50

60

70

80

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4 16 20 24 28 41 48

Reaction time(h)

DHA (%)

FFA TAG

Fig. 5 Percentage (calculated according to Eq. 2) for EPA and DHA in FFA and TAG fractions during the reaction

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Ab Initio Geometry Optimization

In the present work, the total percentage of DHA in FFA, DAG, and TAG fractions amounted≈81 % after 48 h of reaction time, whereas it was about ≈97 % for EPA. In order to explain thisdifference between DHA and EPA, a theoretical study was conducted by DFT. Thus, thesearch of the minimum energy conformations using several initial geometries was donefollowing this theory. As shown in Fig. 3, geometric optimization B3LYP/6-31G (d,p) datafor EPA and DHA indicated the preferential bent structure as an isolated molecule. Theminimum energy obtained (Table 3) indicated that the conformation adopted by DHA wasmore stable when compared with EPA, probably due to intramolecular bonds stabilization.EPA and DHA showed great tendency to generate radicals (and radical stabilization), with nosignificant differences between both fatty acids (Table 3). No significant differences were alsofound on the net atomic charge between DHA and EPA of the analyzed hydrogen atoms usingthis theoretical model (Table 4). As indicated in Table 4, the hydrogens in C21 and C18 inDHA and in C19 and C16 in EPA had the highest net atomic charges, which are one of thepossible locations for H-abstraction and radical formation. In case of DHA, Lyberg andAdlercreutz [48] showed that C20-monohydroperoxy-DHA provided the largest contributionto the total amount of monohydroperoxides. These preliminary data allowed concluding thatboth DHA and EPA showed identical capacity to form radicalar structures, e.g., by oxidativemechanisms, as isolated molecules.

During the biocatalysis reaction, FFA fraction was gradually enriched with DHA, achievingmore than 53 % in this fraction after 48 h, while EPA increased mainly in acyglycerol fractions(TAG and DAG). The presence of polar compounds, like FFA, MAG, or DAG, even at verylow concentrations, can change the structural and organizational state of lipid core. Underthese conditions, it is assumed that the true state of bulk oil resembles that of micellar/lamellarorganization, as previously described [49]. In micellar system, the fatty acids (and also DAG)may be in the water-in-oil interface. FFA, in anionic state (depending on pH), are orientated tothe water core [49], which meant that they are more prone to oxidation and radical generation,e.g., by the presence of transition metals. Once generated, the radical (alkyl radical) can react,e.g., with oxygen or with the neighbor fatty acid. However, in the absence of oxygen, PUFAcross-linked bridges may lead to formation of dimers by C–C linkage, mediated by radicalformation [18, 50, 51]. Kosugi and Azuma [42], working with pure n-3 HUFA and RM lipasein a direct esterification with glycerol, indicated that free DHA and EPA can producepolymeric compounds. It is accepted that dimeric fatty acids can be linked by peroxide bonds,ether bonds, ester bonds, or carbon–carbon bonds, depending on oxidative conditions [51].

The biocatalytic reaction was conducted in the dark and nitrogen bubbling. Under theseexperimental conditions, strong limitation to the oxygen-mediated oxidative pathways andperoxide formation were expected. On the other side, it is known that the primary and

Table 3 Total energies E (a.u.) andcomplexation energies ΔEc(kcal mol−1) for the zsystemsstudied

DHA EPA

E ΔEc E ΔEc

FFA −100.800 −93.061FFA dimer −201.603 −12.02 −186.123 −10.94Radical dimer −100.857 −355.98 −93.117 −356.20EE −108.664 −100.924EE dimer −217.328 −0.51 −201.848 6.83

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secondary oxidation compounds can inhibit lipase activity. However, lipase continuouslyproduced acylglycerols, and in the last 7 h, the TAG content increased from 51 % to morethan 66 % (p<0.05). Thus, one can assume that such dimers mainly occurred via C–Cmolecular bonds. The total and complexation energies of dimers from EPA and DHA, theirradicals, and ethylic esters were calculated (Table 3). It was taken into account that in each n-3HUFA, intra- and intermolecular interactions could take place, which may have influence onthe dimer formation. After optimizing these initial possibilities, the global minimum config-urations were used for the final optimization study. Interestingly, the EE of both DHA and EPA

Table 4 Partial Mulliken charges(a.u.) for the identified protons ofthe systems studied

DHA EPA

FFA EE FFA EE

C21/C19 0.1031 0.1034 0.0953 0.0953

0.0966 0.0966 0.1033 0.1032

C20/C18 0.0705 0.0707 0.0704 0.0702

C19/C17 0.0765 0.0764 0.0717 0.0717

C18/C16 0.1028 0.1027 0.1106 0.1106

0.1023 0.1027 0.0972 0.0970

Electrostatic Potential

0.162

0.107

0.0329

-0.0415

-0.116

Electrostatic Potential

Electrostatic PotentialElectrostatic Potential

-0.137

-0.0496

0.0376

0.212

0.125

0.0767

0.0260

-0.132

-0.0794

-0.0267

-0.0735

0.0589

0.0146

-0.0293

-0.116

a b

c d

Fig. 6 Electrostatic potential for DHA and derivatives. The electrostatic potential (a.u.) is represented over aconstant electronic isodensity r (Å−3) surfaces of volume Vs (Å−3). All figures correspond to r=0.05 Å−3. a DHAmonomer, b DHA dimer, c DHA ester, d DHA ester dimer

1878 Appl Biochem Biotechnol (2014) 172:1866–1881

showed a more stable conformation compared with the free fatty acid form (Table 3). As shownin Table 4, no significant differences were observed in the net atomic charge, determined byMulliken method, for hydrogens in C18, C19, C20, and C21 of EE-DHA compared with DHA.However, the distance between, e.g., the C20–C20 in the diDHA–dimer is less than half (9.5 Å)of that for diEE–DHA–dimer (21.5 Å) (Fig. 6). This may indicate that EE-DHA are not in theright conformation to dimerize, by C–C cross links, compared with free DHA. The dimerizationenergy of diDHA, diEPA, and ethyl-ester fatty acids (EE-FA) (Table 3) shows the preferentialdimer formation of diDHA (−12.02 kcal/mol) compared with diEPA (−10.94 kcal/mol) andespecially diEE-EPA (6.83 kcal/mol). DHA and EPA showed great tendency, with no significantdifferences, for radical–radical dimer formation, −355.98 kcal/mol and −356.20 kcal/mol,respectively. However, as indicated above EPA is mainly found in acylglycerol fractions,whereas DHA increased in FFA fraction, thus creating conditions for the dimerization ofthis fatty acid. Conversely, assuming the micellar/lamellar model, it is known that DHA ismore stable in this system compared with the bulk oil [52–54]. In the present reactionconditions, DHA may act as antioxidant [55] that facilitates the production of radicals and thedimer formation.

Conclusions

The urea complexation of sardine oil allowed obtaining a concentrate with EPA percentageranging between 33.3 and 34.5, and DHA percentage between 20.2 % and 22.0 %, which werethe double of their respective percentages in the starting oil.

The utilization of these enzymatic conditions [10 % (w/w) of immobilized RM lipase, 3:1FFA/glycerol ratio, 55 °C, without solvent] led to obtaining 88 % of acylglycerols, where TAGrepresented more than 66 %. In this fraction, the percentage of n-3 PUFA was higher than62 %, in which n-3 HUFA represented approximately 52 % (39.2 % EPA and 9.7 % DHA,ratio EPA/DHA of 4:1). The incorporation of n-3 HUFA in TAG followed the order EPA≥DPA>>DHA. The low percentage of DHA in the acylglycerols put into evidence the lowspecificity of this lipase to catalyze the esterification of DHA. The low specificity of the R.miehei lipase led to an increase of DHA percentage in FFA, which was almost triple of itsinitial value at the end of the reaction time (ratio EPA/DHA near 1:4). This enrichment of theFFA fraction in DHA suggests that the lipase from R. miehei may be used as a means toprepare fatty acids concentrates rich in DHA.

The theoretical results are consistent with the experimental findings observed in thebiocatalysis reaction. DHA showed great tendency to radical and dimer formation by radical–radical linkage (C–C bond). With these experimental conditions, it is important to definestrategies to protect the n-3 HUFA, mainly DHA in the FFA fraction, by using specificantioxidants and/or radical traps.

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