Optimization of High EPA Structured Phospholipids Synthesis from ω-3 Fatty Acid Enriched Oil and Soy Lecithin

  D _{DAVID PUBLISHING} Optimization of High EPA Structured Phospholipids Synthesis from ω-3 Fatty Acid Enriched Oil and Soy Lecithin _{1 2 3 1} Teti Estiasih , Kgs Ahmadi , Erliana Ginting and Arya Ulil Albab

  1. Department of Food Science and Technology, Faculty of Agricultural Technology, Brawijaya University Jl. Veteran, Malang 65141, Indonesia

  2. Department of Agroindustrial Technology, Faculty of Agriculture, Tribhuwana Tunggadewi University Jl. Telaga Warna, Tlogomas, Malang 65144, Indonesia

  3. Indonesian Research Institute for Legumes and Tuber Crops (ILETRI), Kendalpayak, Malang, Indonesia Received: October 15, 2012 / Published: January 20, 2013.

  

Abstract: The molecular structure of phospholipids can be changed enzymatically to obtain different tailor-made phospholipids.

  Incorporation of -3 fatty acids into phospholipids structure increased their oxidative stability, suggesting more health beneficial phospholipids. This study aimed to optimize eicosapentaenoic acid (EPA) incorporation into phospholipids structure by acidolysis reaction using free lipase (EC 3.1.1.3) from Rhizomucor miehei. Deoiled soy lecithin from anjasmoro variety was used as phospholipids source, while -3 fatty acid enriched oil was used as acyl source. Oil enriched with -3 fatty acids was obtained from low temperature solvent crystallization of lemuru (Sardinella longiceps) by-product. Response surface methodology (RSM) was used in this study to determine the relationship between the three factors (enzyme concentration, reaction time and substrate ratio) and their effects on EPA incorporation into soy lecithin structure. The results showed that the relation between EPA content with three factors (reaction time, enzyme concentration and substrate ratio) was quadratic. The significant factors were substrate ratio and reaction time. Optimum conditions at a ratio of 3.77:1 between -3 fatty acids enriched oil and soy lecithin, 30% lipase concentration, and 24.08 h reaction time, gave 22.81% of EPA content of structured phospholipids.

  Key words: Structured phospholipids, enzymatic acidolysis, EPA, lipase, deoiled soy lecithin, -3 fatty acids enriched oil, lemuru.

  

  functional properties of lecithin. The molecular structure

  1. Introduction

  of phospholipids can be changed enzymatically or Soy lecithin or phospholipids (PL) is widely used as chemically to obtain different tailor-made technological an emulsifier in food industries. Soy lecithin comprises and/or physiological properties compared to those of of 18% phosphatidylcholine (PC), 14% natural substrate. Enzymatic modification being of phosphatidylethanolamine (PE), 9% interest as enzymes can be used to modify phospholipids phosphatidylinositol (PI), 2% phosphatidic acid (PA), in different ways [4]. other minor phospholipids, 11% glycolipid, and 27%

  Structured phospholipids with certain fatty acid neutral lipid [1]. Some studies reported that the major profile can be obtained by enzymatic synthesis [5]. This fatty acid in soy lecithin was linoleic acid [2, 3]. synthesis process gives benefits as the reaction

  Modification is usually performed to obtain desired conditions are not extreme and the enzyme is highly specific [6]. Incorporation of -3 fatty acids into

  Corresponding author: Teti Estiasih, PhD, research fields:

  phospholipids structure increases their oxidative lipid chemistry and technology. E-mail teties@yahoo.co.id and

• 5,8,11,14,17-eicosapentaenoic acid (EPA) and

  ω-3 fatty acids incorporation into phospholipids structure are enzyme concentration, reaction time, and ratio of acyl to phospholipids [15]. While enzymes used for structured phospholipids synthesis are lipase that may derive from

  2.2 Soy Lecithin Preparation from Anjasmoro Soybean Variety

  Crude soy lecithins were obtained from water degumming of soybean oil extracted from anjasmoro variety. Lemuru (Sardinella longiceps) oil as a by-product of fish meal processing was purchased from a local fish meal plant. ω-3 fatty acids enriched oil was obtained using low solvent crystallization of hydrolyzed lemuru oil. Other materials were lipase derived from Rhizomucor meihei, fatty acid methyl ester mixture, phospholipids standard, and BF3-methanol 14% (Sigma Co.), other chemical reagents, and TLC plate (silica gel G60 F254) (Merck).

  2.1 Materials

  2. Materials and Methods

  ω-3 fatty acid enriched oil that was prepared from a by-product of Lemuru (Sardinella longiceps) canning processing using low temperature solvent crystallization. This study focused on the generation of soy lecithin that is highly enriched with EPA from inexpensive EPA source. Response surface methodology (RSM) was used in this study to determine the relationship between the three factors (enzyme concentration, reaction time and substrate ratio) and their effect on EPA incorporation into soy lecithin structure during lipase-catalyzed acidolysis.

  Anjasmoro is one of improved varieties cultivated in Indonesia that has high productivity (2.3 t/ha) and tolerant to puddle. As a acyl source, we used

  [5, 21], and phospholipase A ^{1 [12].} This study aimed to optimize EPA incorporation into soy lecithin from anjasmoro soybean variety.

  lanuginose

  [15, 17] or Thermomyces

  Rizhomucor miehei

  Incorporation of such fatty acid into phospholipids structure can be controlled during reaction [21]. Some factors that may affect acidolysis reaction in

  ω-3 Fatty Acid Enriched Oil and Soy Lecithin

  In general, enzyme used in structured phospholipids synthesis is specific 1,3-lipase and PLA ^{2 , which can} modify fatty acid in sn-1 and sn-2 positions.

  Almost all of the acidolysis studies in structured phospholipids synthesis used certain type of phospholipids, such as PC as a substrate [6, 15-20]. However, studies on structured phospholipids synthesis using a mixture of phospholipids such as soy lecithin are still limited. Therefore, there is a call for research in different properties of phospholipid forms in soy lecithin.

  regiospecific had been used by Haraldsson and Thorarensen [15] in PC containing EPA and DHA synthesis. Recent research suggested egg yolk was used as a source of PC and acyl source was -3 fatty acids concentrates. However, information on structured phospholipids synthesis using specific EPA incorporation is still lacking.

  Rhizomucor meihei (Lipozyme TM) with 1,3

  Some studies showed that enzymatic incorporation of -3 fatty acids into phopsholipids structure used lipase or phospholipase. A mobile lipase from

  The beneficial effects of EPA include reductions in heart diseases severity and various inflammatory disorders [10, 11]. EPA has been used in the prevention and treatment of atherosclerosis, arthritis, thrombosis, inflammation, diabetes, and cancer [12]. EPA also exhibits various physiological functions on being incorporated into membrane phospholipids [13, 14].

  cis -4,7,10,13,16,19-docosahexaenoic acid (DHA) [8, 9].

  cis

  PUFA are currently well known and have been almost exclusively attributed to

  phospholipids. The health beneficial effects of -3

  Lecithin extraction was performed according to Eshratabadi [22] with a minor modification. Lecithin was extracted from soybean oil using water degumming. About 20 g ± 0.1 mg of soybean oil was placed in a beaker glass and 10% (w/v reaction mixture) distilled water and 3 mL of hexane was added. The mixture was stirred and heated at 60 °C for 30 min. Oil and water phase were separated by centrifugation at 300 rpm for 30 min. Gum or crude lecithin was formed in the bottom layer (subnatant) and then dried in a vacuum oven at 25 °C and 1 atm for 1 h.

  This dried crude lecithin was then purified using the method employed by Nasir et al. [23] to remove neutral oil. Acetone was added into the crude lecithin with the ratio of 6:1 (v/w). The mixture was stirred for 1 h. The solvent was decanted to separate the lecithin. This treatment was repeated until the solvent was colorless. Lecithin was flushed with nitrogen to discard residual solvent.

  The mixture was shaked slowly at 300 rpm in a shaking water bath with temperature of 40 °C for different reaction times (Table 1). Afterwards, the mixture was centrifuged at 1,500 rpm for 15 min to precipitate structured phospholipids. Then the precipitate was washed with 1 mL hexane to discard residual free fatty acid. The optimized response in this study was EPA content in structured phospholipids. Data were analyzed using Design Expert 7.0 Trial Version. Structured phospholipids from optimum conditions were analyzed for Hydrophilic Lipophilic Balance (HLB) (saponification number and acid value) according to the method of AOCS [24], phospholipids profile, and fatty acid profile.

  ω-3 Fatty Acid Enriched Oil and Soy Lecithin

2.3 Acidolysis Reaction of Oil Enriched with

• -3 Fatty Acids and Deoiled Soy Lecithin

  -3 fatty acids and soy lecithin (phospholipids) were put into a number of reaction tubes at different ratios as shown in Table 1. Free enzyme of Lipozyme (Rhizomucor miehei) at different concentrations (Table 1) were subsequently added.

  23.84

  20

  20.39

  20.39 12 3.5:1

  20

  24.26

  24.26 13 3.5:1

  20

  23.84 14 3.5:1

  23.38

  20

  19.34

  19.34 15 1.818:1 20 14.16 -1.682

  14.16 16 5.182:1 20 16.07 +1.682

  16.07 17 3.5:1 3.18 18.88 -1.682 0

  18.88 18 3.5:1 36.82 22.02 +1.682 0

  22.02 19 3.5:1 20 12.28 -1.682 12.28 20 3.5:1 20 13.75 +1.682 13.75

  23.38 11 3.5:1

  20

  This activity was referred to Haraldsson and Thorarensen [15] with a slight modification. Oil enriched with

  15.84 2 2.5:1 10 18.46 -1 -1 +1

  2.4 Analysis of Phospholipids

  Phospholipids analysis was performed using TLC according to Nzai and Proctor’s method [25] with

  Table 1 Second ordo central composite design with three factors to optimize EPA content of structured phospholipids.

  No.

  Actual variable Coded variable Response of EPA content (%)

  Substrate ratio* Enzyme concentration** Reaction time (h)

  X ₁ X ₂ X ₃ 1 2.5:1 10 15.84 -1 -1 -1

  18.46 3 2.5:1 30 19.78 -1 +1 -1

  19.70 10 3.5:1

  19.78 4 2.5:1 30 16.77 -1 +1 +1

  16.77 5 4.5:1 10 21.24 +1 -1 -1

  21.24 6 4.5:1 10 15.51 +1 -1 +1

  15.51 7 4.5:1 30 19.38 +1 +1 -1

  19.38 8 4.5:1 30 21.57 +1 +1 +1

  21.57 9 3.5:1

  

  19.70

  20

  ω-3 Fatty Acid Enriched Oil and Soy Lecithin

  3. Results and Discussions

  (1:1), and quantification by TLC scanner at 500 nm wavelength (Dual Wave Length Chromato Scanner CS-930 and Data Recorder DR-2 Shimadzu).

  slight modification. About 1 mg of sample or standard was dissolved in chloroform:methanol (95:5 v/v) and 10 µL of sample or standard solution was spotted into TLC plate. The TLC plate was activated by heating for 10 min at 100 C prior to analysis. The plates were developed for 40 min in a mobile phase solution containing chloroform, methanol, and distilled water at a ratio of 75:25:3 (v/v/v). Visualization was performed by charring using H ^{2 SO 4 :distilled water}

  The predominant fatty acids in -3 fatty acids enriched oil were EPA and DHA (Table 2). In original lemuru oil, the EPA and DHA content were 15.87% and 13.58%, respectively. Meanwhile, EPA and DHA content in enriched oil were 1.79 and 1.72 times higher than the original oil. Lemuru oil is a good source of EPA rather than DHA. According to Howe et al. [28], fish oil generally contains higher EPA than DHA, except for tuna oil. Hence, in this study, we used lemuru oil as the source of EPA in the preparation of high EPA structured phospholipids from soy lecithin.

2.5 Fatty Acid Analysis

  C, 250 C and 230 C, respectively. Samples and standard were injected at the volume of 2 L. Methylation of phospholipids was performed according to Park and Goin’s method [26]. Analysis of fatty acid composition of phospholipids was conducted after phospholipids separation using TLC. Each spot was scrapped off and extracted according to Christie [27]. Derivatization prior to GC analysis was performed using Park and Goin’s method [26].

  4.80 5.00 nd Oleic acid (C18:1

  28.49

  51.74

  7.82 Eicosapentaenoic acid/EPA (C20:5 -3) nd 28.36 24.36 Docosahexaenoic acid/DHA (C22:6 -3) nd 23.38 4.13 EPA + DHA nd

  Docosanoic acid (C22:0) 3.59 nd nd Docosaenoic acid (C22:1) 1.40 nd

  3.94 Arachidonic acid (C20:4 -3) 1.37 nd nd

  54.52 Linolenic acid (C18:3 -3) 5.26 1.90

  1.23 Linoleic acid (C18:2 -6) 48.64 2.79

  -9) 11.86 15.68

  0.80 Stearic acid (C18:0)

  Nitrogen was used as a gas carrier with a pressure of 200 kg/m ² , while for supporting and burning gas, air and hydrogen were used with a pressure of 0.15 kg/cm ² and 0.6 kg/cm ² , respectively. Injector, column, and detector temperature was 230

  1.79 Palmitooleic acid (C16:1 -7) 1.05 19.57

  3.33

  Fatty acid profile of -3 fatty acids enriched oil, deoiled soy lecithin, and structured phospholipids from optimum conditions of acidolysis reaction were analyzed using gas chromatography (Shimadzu GC

  0.39 Palmitic acid (C16:0)

  0.53 Myristic acid (C14:0) 1.29 nd

  High EPA structured phospholipids Lauric acid (C12:0) 0.60 nd

  Fatty acid De-oiled soy lecithin -3 fatty acids enrihed oil

  Linoleic acid was a predominant fatty acid obtained in soybean lecithin derived from Anjasmoro variety (Table 2). Normally, soybean oil has high linoleic acid Table 2 Fatty acids profile of -3 fatty acids enrihed oil, deoiled soy lecithin, and high EPA structured phospholipids.

  8A). The column used for separation was capillary CBP20 0.25 m bonded silica column with dimension of 50 mm in length, i.d. 0.22 mm and o.d. 0.33 mm.

  26.97

  ω-3 Fatty Acid Enriched Oil and Soy Lecithin

  content that may comprise 55.83% of the total fatty acids [29]. Soybean lecithin consisted of 32.63% PI, 29.18% PC, 27.15% PE and 11.01% PA with the order PI > PC > PE > PA (Table 3). Wu and Wang [1] reported that soy lecithin had 18% PC, 14% PE, 9% PI, 2% PA, 2% minor PL, 11% glycolipid, 5% sugar complex and 37% neutral lipid. Commercial lecithin has different phospholipids composition with a predominant phospholipids is PC or PE [30]. Higher PC or PE concentration in commercial lechitin is due to fractionation treatment. Wang [3] reported that predominant fatty acid in PE or PC was linoleic acid, followed by palmitic acid.

  The results of optimization revealed that the suitable model for acidolysis reaction with three factors (enzyme concentration, reaction time, ratio of oil enriched with -3 fatty acids to soy lecithin) was quadratic. However, the model was not significant. Factors that affected the response in quadratic manner were the ratio of -3 fatty acids enriched oil to soy lecithin and reaction time, while the effect of enzyme concentration was not significant.

  The effect of substrate ratio and enzyme concentration was quadratic (Fig. 1). At high enzyme concentration, enzyme was sufficient for hydrolysis of fatty acid from original phospholipids, and subsequently esterification of -3 fatty acid enriched oil into phospholipids structure. At low enzyme concentration, it was supposed that enzyme would hydrolyze fatty acid from phospholipids structure, however, the synthesis was limited. This led to low EPA content of structured phospholipids. Similarly, increasing substrate ratio also enhanced EPA content, but then it decreased. According to Vikbjerg et al. [5], fatty acid composition of structured phospholipids depended on the substrate ratio (mole of acyl donor/mole of phospholipids) and incorporation of acyl increased a long with the availability of acyl or mole substrate ratio. This study showed that increasing acyl donor decreased incorporation of EPA into

  Table 3 Phospholipids profile of deoiled soy lecithin and structured phospholipids.

  Phospholipids Deoiled soy lecithin

  High EPA structured phospholipids Phosphatidylinositol 32.63 22.96 Phosphatidylcholine 29.18 25.85 Phosphatidylethanolamine 27.15 22.32 Phosphatidic acid

  11.01

  28.87 through different ratios of PC to palmitic acid as substrate (1:2 to 1:10) and 20% enzyme used, the maximum incorporation obtained at the ratio of 1:5. Increasing substrate ratio from 1:5 to 1:10, however, did not increase the incorporation level [6].

  This study showed that high amounts of acyl donor did not always increase the level of incorporation. This was due to the preference of enzyme to hydrolyze and esterify acyl from and into phospholipids structure. At high acyl concentration, reaction tended to synthesize structured phospholipids. However, an increase of acyl availability could limit the hydrolysis due to inhibition effect of acyl [31].

  The effect of substrate ratio and reaction time on the EPA incorporation into structured phospholipids was quadratic (Fig. 2). Maximum incorporation obtained at certain reaction time. Longer reaction time did not increase EPA incorporation into phospholipids structure. Reddy et al. [6] observed that incorporation of palmitic acid and stearic acid into PC was maximum at reaction time of 24 h. Extended reaction time did not increase the level of incorporation. That finding was similar to thoseresults obtained in this study. After maximum reaction time, longer reaction time was supposed to lead the hydrolysis, including hydrolysis of incorporated EPA from structured phospholipids. Enzymatic reaction was hydrodynamic process and lipase was able to hydrolyze and synthesize depending on particular factors. One of them was water availability that stimulated reaction into hydrolysis. The availability of water in this study was limited to 10% (w/w substrate) that aimed to promote esterification reaction.

  ω-3 Fatty Acid Enriched Oil and Soy Lecithin

  a b

  

Fig. 1 Response surface (a) and contour (b) of enzyme concentration and substrate ratio in acidolysis reaction of high EPA

structured phospholipids synthesis.

  a b

  

Fig. 2 Response surface (a) and contour (b) of reaction time and substrate ratio in acidolysis reaction of high EPA

structured phospholipids synthesis.

  The effect of reaction time and enzyme Among the three factors, ratio of substrate and concentration on EPA incorporation into structured reaction time gave significant effect on EPA content phospholipids was also quadratic (Fig. 3). High and the relation was quadratic (Fig. 2). The quadratic incorporation of EPA tended to occur at high enzyme equation to predict the response was Y = -36.76 –

  X

  concentration. At low enzyme concentration, 9.059X ^{1 + 2.05X 2 + 3.78X} ₂ ^{3 – 0.033X} ₂ ^{1 2 + 0.19X} ₂ ^{1 3} X increasing reaction time enhanced EPA incorporation. 0.031X ^{2 3 + 0.70X 1 – 0.029X 2 – 0.056X 3 with X 1 =} However, longer reaction time decreased incorporated ratio of oil enriched with -3 fatty acids to soy EPA of structured phospholipids. It was supposed that lecithin, X ^{2 = enzyme concentration, X 3 = reaction} at a low enzyme concentration, hydrolysis of fatty time, and Y = EPA content (%) of structured acid from original phospholipids occurred at the phospholipids. Optimum conditions were obtained at a beginning of reaction, followed by esterification of -3 fatty acids to soy ratio of oil enriched with

• – X

  EPA. At long reaction time, incorporated EPA can be lecithin 3.77:1, enzyme concentration 30%, and hydrolyzed from the structured phospholipids. reaction time 24.08 h. At these optimum conditions, Optimum incorporation of EPA was supposed to be the response was predicted to be 22.81% of EPA. higher than enzyme concentration of 30% and reaction Verification showed that the response of EPA content

  ω-3 Fatty Acid Enriched Oil and Soy Lecithin

  a b

  

Fig. 3 Response surface (a) and contour (b) of reaction time and enzyme concentration in acidolysis reaction of high EPA

structured phospholipids synthesis.

  was supposed to replace palmitic acid in phospholipid There was an interesting phenomenon in fatty acid structure. Optimum condition was achieved at ratio of composition of structured phospholipids (Table 2). oil enriched with -3 fatty acids to soy lecithin 3.77:1,

  Incorporation of EPA decreased palmitic acid content enzyme concentration 30%, and reaction time 24.08 h. sharply, while linoleic acid increased. This reflected

  At these optimum conditions, the response was that palmitic acid was replaced by EPA in predicted to be 22.81% of EPA, meanwhile, phospholipids structure. Acidolysis reaction using verification showed 24.23%. Further work is needed to lipase was reversible [32]. At the beginning, lipase know degree of EPA incorporation into phospholipids hydrolyzed fatty acids from phospholipids structure. classes as well as physiological effects of high EPA

  Afterwards, EPA from -3 fatty acids enriched oil structured phospholipids ingestion. was esterified. Therefore, the increase of linoleic acid content was due to hydrolysis of other fatty acids

  Acknowledgments rather than linoleic acid from phospholipids structure.

  The authors thank to Directorate General of Higher HLB value of structured phospholipids was 8.24,

  Education, Ministry of Education and Culture, while original soy lecithin had HLB of 5.0. This Republic of Indonesia for the funding of this work suggested that high EPA structured phospholipids was through Strategic National Research Grant with Grant suitable for oil in water emulsion. Fatty acid Contract No. 404/SP2H/PL/Dit.Litabmas/IV/2011. composition of structured phospholipids would influence the HLB value [33]. Increasing desaturation

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