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Enzymatic synthesis of lysophosphatidylcholine containing CLA from sn-glycero-3-phosphatidylcholine (GPC) under vacuum
Food Chemistry 129 (2011) 1–6
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Enzymatic synthesis of lysophosphatidylcholine containing CLA from sn-glycero-3-phosphatidylcholine (GPC) under vacuum Seung In Hong a,d, Yangha Kim b, Chong-Tai Kim c, In-Hwan Kim a,⇑ a
Department of Food & Nutrition, College of Health Sciences, Korea University, Jeongneung-Dong, Seongbuk-Gu, Seoul 136-703, Republic of Korea Department of Nutritional Science and Food Management, Ehwa Women’s University, Seoul 120-749, Republic of Korea c Korea Food Research Institute, Sungnam 463-746, Republic of Korea d Research Institute of Health Sciences, Korea University, Seoul 136-703, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 17 August 2010 Received in revised form 15 March 2011 Accepted 14 April 2011 Available online 20 April 2011 Keywords: Conjugated linoleic acid (CLA) sn-Glycero-3-phosphatidylcholine (GPC) Lysophosphatidylcholine (LPC) Novozym 435 Phosphatidylcholine (PC) Vacuum system
a b s t r a c t Lysophosphatidylcholine (LPC) was successfully synthesised by enzyme-catalysed esterification of snglycero-3-phosphatidylcholine (GPC) with the acid form of conjugated linoleic acid (CLA), using a vacuum system. GPC was prepared from phosphatidylcholine (PC) by alkaline deacylation using a methanolic tetra-butylammonium hydroxide solution. Five enzymes were tested as biocatalysts for direct esterification, and lipases were found to be more effective than phospholipases for production of LPC. Amongst the enzymes tested, Novozym 435, from Candida antarctica, gave the highest yield of LPC. The optimal temperature, molar ratio of substrate, and vacuum were 40 °C, 1:50 (GPC to CLA), and 1 mm Hg, respectively. The maximum yield (70 mol%) of LPC was obtained under optimal conditions after a 48 h reaction time. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The use of an enzymatic reaction has attracted attention for the synthesis of lysophospholipids (LPL). There are several advantages in using enzymatic reactions rather than chemical reactions. The first concerns the specificity of enzymes for their substrates and a particular regiospecific position; it is possible to incorporate a desired acyl group at a specific position on the backbone of a structure through enzymatic reactions, whereas chemical reactions do not possess this regiospecificity. The second advantage includes the mild reaction conditions and reduced amounts of chemical reagents required for synthetic reactions (D’Arrigo & Servi, 2010; Gunstone, 1999; Lee & Akoh, 1998; Servi, 1999). LPL and phospholipids (PL), containing specific fatty acids, have been synthesised by transesterification, and by direct esterification using an enzyme as a biocatalyst. In particular, the advantages of direct esterification include the production of purer reaction products and a shorter reaction time than is needed for transesterification (Adlercreutz, Budde, & Wehtje, 2002). sn-Glycero-3-phosphatidylcholine (GPC) is an ideal starting material for the preparation of various types of LPL or PL. However, GPC is well dissolved in water or polar solvents, whereas it is
⇑ Corresponding author. Tel.: +82 2 940 2855; fax: +82 2 941 7825. E-mail address: [email protected] (I.-H. Kim). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.04.038
immiscible with non-polar solvents. Although this characteristic has hampered its use in synthesis (D’Arrigo & Servi, 2010), some studies have been performed using cadmium chloride complexes of GPC to enhance its solubility in organic solvents (Baer & Kates, 1948; Chada, 1970). Other studies have been conducted, using fatty acid derivatives, such as fatty acid anhydrides and fatty acid vinyl esters, as acyl donors for production of lysophosphatidylcholine (LPC) from GPC (Blasi, Cossignani, Maurizi, Simonetti, & Damiani, 2008; Mazur, Hiler, Lee, Armstrong, & Wendel, 1991; Virto & Adlercreutz, 2000). However, reactions using natural fatty acids, rather than derivatives, are preferred because they avoid environmental pollution, and because the cost of fatty acid derivatives is higher than the cost of free acid substrates. Conjugated linoleic acid (CLA) was used as an acyl donor to produce LPC. CLA has received attention because of their ability to inhibit cancer, to lower blood cholesterol, to control diabetes and to aid in weight loss. The generic term ‘‘CLA’’ usually encompasses a number of conjugated linoleic acid isomers with double bonds at the 8c,10c-, 9c,11t-, 10t,12c-, and 11t,13t-positions (Sébédio, Christie, & Adlof, 2003). The present study describes (for production of LPC in a solventfree system) esterification of GPC with fatty acids using enzyme (Fig.1). A vacuum system was applied to control the water produced during esterification, because water in the reaction medium plays a crucial role for enzyme activity. When a minimal amount of water exists, the enzyme has a quasi-optimal configuration or
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Fig. 1. Reaction scheme for production of LPC from GPC and fatty acid.
catalytic activity and selectivity (Chae et al., 2009; Dordick, 1989; Han & Rhee, 1995; Rosu, Iwasaki, Shimidzu, Doisaki, & Yamane, 1998; Rosu, Yasui, Iwasaki, & Yamane, 1999; Zhao, Lu, Bie, Lu, & Liu, 2007). Lysophospholipids (LPL) are glycerophospholipids in which one acyl chain is lacking, and in which only one hydroxyl group of the glycerol backbone is acylated. Unlike phospholipids (PL), LPL are found only in small amounts in biological cell membranes. LPL are produced by phospholipases that exhibit diverse biological activities and serve as membrane-derived signalling molecules. They also serve as good emulsifying and solubilising agents, and as useful synthetic intermediates for the preparation of PLs, and they are applied in food, cosmetic, agrochemical and pharmaceutical industries. In particular, lysophosphatidylcholine (LPC) of LPL is abundantly found in nature (Adlercreutz et al., 2002; D’Arrigo & Servi, 2010; Han & Rhee, 1995; Virto, Svensson, & Adlercreutz, 1999). The purpose of this study was to optimise conditions for the efficient production of LPC containing CLA by enzyme-catalysed esterification under vacuum system. We prepared a GPC from phosphatidylcholine (PC) derived from soybean. The effects of several process parameters (enzyme screening, temperature, molar ratio and vacuum) on the yield of LPC containing a CLA were studied in a batch reactor. 2. Materials and methods 2.1. Materials Granulated PC (purity 95%, from soybean) was obtained from Avanti Polar-Lipids, Inc. (Alabaster, Al, USA). CLA (conjugated linoleic acid, C18:2, purity: 77.6%) was purchased from HK Biotech Co., Ltd. (Chinju, Republic of Korea). GPC, LPC and PC standards were purchased from Sigma Aldrich (St. Louis, MO, USA). TBIA methanolic solution (1.0 M methanolic tetra-butylammonium hydroxide solution) was purchased from Fluka (Sigma Aldrich, St. Louis, MO, USA). Lipozyme TL IM (the immobilised lipase from Thermomyces lanuginosus), Lipozyme RM IM (the immobilised lipase from Rhizomucor miehei), Novozym 435 (the immobilised lipase from Candida antarctica), Phospholipase A1 (LecitaseÒ Ultra) from
Thermomyces lanuginosus/Fusarium oxysporum, and phospholipase A2 (LecitaseÒ 10L) from porcine pancreas, were purchased from Novo Nordisk Bioindustry Ltd. (Seoul, Korea). Phospholipase A1 and phospholipase A2 were immobilised on DuoliteÒ A567 before use, according to Garcia, Kim, Lopez-Hernandez, and Hill (2008). The TLC plates used were coated with silica gel (layer thickness 0.25 mm, Kieselgel 60 F254, Merck, Darmstadt, Germany). All solvents and chemicals used were of analytical grade. 2.2. Preparation of GPC GPC was prepared from sn-1,2-PC by alkaline deacylation, as described by Brockerhoff and Yurkowski (1965), according to the following procedure. Briefly, 20 g of sn-1,2-PC were dissolved in 400 ml of diethyl ether, and TBAI methanolic solution (40 ml) was added. After magnetic stirring at room temperature for 2 h, an opalescent precipitate, composed of GPC, was formed. The solvent was removed and the precipitate was washed three times with 1 ml of diethyl ether, dissolved in 5 ml of methanol and transferred into a Teflon bottle. Methanol was removed by nitrogen flushing in a water bath of 50 °C. 2.3. Enzymatic synthesis of LPC Reactions were performed in a vacuum system, using a 50 ml water-jacketed glass vessel. GPC and CLA were used as the substrates for the synthesis of LPC by direct esterification. Prior to starting the reaction, the reaction vessel had been heated to the desired temperature using a water circulator. The weight of the total substrates was 15 g and immobilised lipases (10 wt.% based on total substrates, moisture-free) were added to a mixture of GPC and CLA. The reaction was performed by stirring with a magnetic stirrer at 600 rpm under a defined vacuum. The vacuum was controlled by a micrometering valve (Swagelok, Solon, OH, USA) and monitored by a digital vacuum gauge (Teledyne, Thousand Oaks, CA, USA). 2.4. Analysis of products Samples of 50 mg were dissolved in a mixture of chloroform and methanol (5:1, by vol), and then applied to silica-coated TLC
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50
(a)
LPC PC
40
Yield (mol%)
plates and developed with CHCl3/CH3OH/CH3COOH/H2O (75:40:8:3, by vol). LPC and PC were detected by spraying with 2,7-dichlorofluroscein solution (0.2% in 95% methanol solution). The bands corresponding to the LPC and PC were scraped off the TLC plate and methylated, using 14% BF3 in methanol as the catalyst. Heptadecanoic acid (0.2 mg) was used as an internal standard. The fatty acid methyl esters (FAME) were analysed by gas chromatography. A gas chromatograph (Varian 3800, Varian Inc., Walnut Creek, CA, USA), equipped with a Supelcowax 10 fused-silica capillary column (30 m 0.32 mm i.d.; Supelco, Bellefonte, PA, USA) and FID, was used. The column was held at 180 °C for 1 min and programmed to 230 °C for 10 min at the rate of 1.5 °C/min. The carrier gas was helium and the total gas flow rate was 50 ml/min. The injector and detector temperatures were 240 and 250 °C, respectively. FAME were identified by comparison with the retention times of the standards. The total amounts of LPC and PC were calculated from the total amount of FAME in each fraction.
30
20
10
0 1.0
(b)
Each reported value is the mean of determinations from duplicate samples, and the data were analysed by ANOVA and Duncan’s multiple range test. Statistical significance was accepted at a level of p < 0.05.
LPC/(LPC+PC)
0.8
2.5. Statistical analysis
0.6
0.4
0.2
3. Results and discussion 3.1. Enzyme screening
0.0 A
B
C
D
E
Enzyme Five enzymes were screened for their abilities to catalyse the direct esterification of GPC and CLA. The enzymes screened were Novozym 435, Lipozyme TL IM and Lipozyme RM IM, as lipases, and phospholipase A1 and phospholipase A2 as phospholipases. The trial for enzyme screening was performed for 12 h at a molar ratio of 1:10 (GPC to CLA), 50 °C, 10 mm Hg, and an enzyme loading of 10 wt.% based on total substrates. Yields of LPC and PC, and the ratio of LPC/(LPC + PC) obtained from the reactions catalysed by various immobilised enzymes are presented in Fig. 2a and b, respectively. Lipases were more effective than phospholipases for the production of LPC. A similar finding was reported by Hossen and Hernandez (2005). Amongst the enzymes tested in this study, Novozym 435 gave the highest yield of LPC. Thus, Novozym 435 was chosen as an appropriate enzyme for esterification of GPC with CLA. Several studies have described the reaction mechanism of GPC and fatty acids (Blasi et al., 2008; D’Arrigo & Servi, 2010; Virto, Svensson, & Adlercreutz, 1999; Xu, 2000). During esterification of GPC and fatty acid, it is thermodynamically easy to synthesise 2LPC, a regioisomer lacking the acyl chain at the sn-2 position, by enzymatic reaction. However, the fatty acid at sn-1 spontaneously migrates to sn-2, followed by the formation of PC through the second acylation of 1-LPC. Similar results were obtained in this study of the increase of PC during esterification of LPC. The amount of PC in the ratio of LPC/(LPC + PC), (Fig. 2b), was minimal compared to the production of LPC. Lipozyme RM IM and phospholipase A2, nevertheless, showed particularly low ratios of LPC/(LPC + PC). This suggests that both enzymes, by acyl migration and second esterification, accelerate the production of PC to a greater extent than other enzymes. Fatty acid compositions of LPC, produced by direct esterification using different enzymes, are shown in Table 1. Previous studies have reported the selectivity of enzymes for fatty acids, and this selectivity is related to chain length, and the configuration of the unsaturated fatty acid (Patel, Morrisett, & Sparrow, 1979; Rosu et al., 1998; Warwel & Borgdorf, 2000). The original CLA fraction
Fig. 2. Enzyme screening for production of LPC containing CLA; (a) yield of LPC and PC, (b) ratio of LPC/(LPC + PC). This experiment was performed at a molar ratio of 1:10 (GPC to CLA), temperature of 50 °C, a vacuum of 10 mm Hg, and enzyme loading of 10 wt.% based on total substrates for 12 h. (A) Novozym 435 (from Candida antarctica), (B) Lipozyme TL IM (from Thermomyces lanuginosus), (C) Lipozyme RM IM (from Rhizomucor miehei), (D) phospholipase A1 (from Thermomyces lanuginosus/Fusarium oxysporum), and (E) phospholipase A2 (from Porcine pancreas).
used as a substrate for the reaction was mainly composed of 9c,11t-CLA and 10t,12c-CLA, with similar compositions of 35.6 and 36.6 mol%, respectively. The concentration of 10t,12c-CLA in LPC produced by Novozym 435 was significantly greater than the concentration of 9c,11t-CLA. Meanwhile, Lipozyme TL IM, Lipozyme RM IM and phospholipase A1 exhibited higher selectivities for 9c,11t-CLA. However, phospholipase A2 was less effective for selective esterification of CLA with GPC. Warwel and Borgdorf (2000) reported that the lipases from Candida cylindracea and Mucor miehei exhibited a higher selectivity for 9c,11t-CLA than for 10t,12c-CLA. Wang, Li, Liang, Yang, and Zhang (2007) showed that Lipase AY30, from Candida rugosa, exhibited higher selectivity for 9c,11t-CLA than for 10t,12c-CLA, but a reverse trend was observed when Novozym 435, from Candida antarctica, was employed as a biocatalyst. Overall, our results showed that Novozym 435 was a very effective catalyst for the synthesis of LPC, and thus appropriate for its synthesis.
3.2. Temperature Reaction temperature is known to affect the esterification process catalysed by enzymes (Virto et al., 1999). Temperatures that are too high can induce irreversible denaturation of enzyme protein and shorten the half-life of enzyme activity, even though enzymes from microorganisms and immobilised enzymes are stable
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Table 1 Fatty acid composition (mol%) in LPC produced by different enzymes under vacuum *. CLAa
Fatty acids
c
C14:0 C16:0 C16:1(n7) C18:0 C18:1(n9) C18:1(n7) 9c,12c-C18:2 9c,11t-CLA 10t,12c-CLA Other CLAs *
0.1 ± 0.0 6.7 ± 0.7 0.1 ± 0.0 2.3 ± 0.1 11.9 ± 0.3 0.6 ± 0.0 1.1 ± 0.0 35.6 ± 0.2 36.6 ± 0.4 4.9 ± 0.2
Novozym 435b
Lipozyme TL IMb
Lipozyme RM IMb
Phospholipase A1b
Phospholipase A2b
0.3 ± 0.1 11.4 ± 1.0 0.1 ± 0.1 2.7 ± 0.2 9.8 ± 0.5 0.8 ± 0.1 1.1 ± 0.1 24.5 ± 0.1 40.7 ± 0.8 8.5 ± 2.4
0.2 ± 0.1 7.5 ± 0.9 0.1 ± 0.1 1.8 ± 0.1 10.6 ± 1.3 0.5 ± 0.1 1.2 ± 0.3 42.2 ± 2.4 29.3 ± 2.7 6.5 ± 2.3
0.2 ± 0.0 7.1 ± 0.2 0.4 ± 0.5 1.1 ± 0.2 10.6 ± 0.0 0.5 ± 0.0 1.2 ± 0.1 41.3 ± 2.9 28.6 ± 2.1 8.0 ± 4.4
0.0 ± 0.0 7.9 ± 0.5 0.0 ± 0.0 2.6 ± 0.8 11.7 ± 0.9 0.5 ± 0.2 1.2 ± 0.3 43.0 ± 2.5 27.0 ± 1.9 5.2 ± 3.3
0.0 ± 0.0 15.2 ± 0.4 0.0 ± 0.0 7.9 ± 0.8 16.3 ± 4.4 0.0 ± 0.0 3.2 ± 0.5 27.1 ± 4.1 27.7 ± 0.5 2.6 ± 0.1
The experiment was performed at a vacuum of 10 mm Hg and other detailed conditions were the same as Fig. 2. CLA (conjugated linoleic acid) used as substrate. b Immobilised enzymes used to produce LPC with GPC and CLA by direct esterification. c Means of duplicates ± standard deviations. a
100
80
Yield (mol%)
and active at high temperatures (Kim, Lee, Oh, & Kim, 2001; Xu, 2000). Moreover, excessively high temperatures accelerate side reactions, such as acyl migration and hydrolysis, due to the inhibition of reaction equilibrium (Rosu et al., 1999). The reaction temperature should be determined on an individual basis and be based on the melting points of the substrates (Kim & Kim, 2000) and products (Virto & Adlercreutz, 2000; Virto et al., 1999). The effect of reaction temperature on the production of LPC by direct esterification of GPC and CLA, using Novozym 435 as a biocatalyst, is shown in Fig. 3. For these trials, enzyme loading, substrate molar ratio, vacuum and reaction time were held constant at 10% of the total substrate weight, 1:10 (GPC to CLA), 10 mm Hg, and 12 h, respectively. The yield of LPC increased from 9.0 to 41.8 mol% when the temperature was increased from 20 to 40 °C. However, the yield of LPC remained constant when the temperature was further increased from 40 to 60 °C. Consequently, the optimal temperature to produce LPC through the esterification of GPC with CLA was 40 °C. Hence, in subsequent experimental trials, we employed 40 °C as the reaction temperature.
60
40 LPC PC
20
0 0
20
40
60
80
100
Molar ratio (CLA/GPC) Fig. 4. Effect of molar ratio on production of LPC and PC containing CLA. This experiment was performed at a vacuum of 10 mm Hg, temperature of 40 °C, and an enzyme loading (Novozym 435) of 10 wt.% based on total substrates for 48 h.
3.3. Molar ratio The effect of molar ratio on the production of LPC by direct esterification of GPC with CLA, using Novozym 435 as a biocatalyst, is shown in Fig. 4. The molar ratio of GPC to CLA varied from 1:6.25 to 1:100. For these trials, enzyme loading, vacuum and reaction time were held constant at 10% of the total weight of substrates,
50
Yield of LPC (mol%)
40
30
20
10
10 mm Hg and 48 h, respectively. The yield of LPC increased from 38.3 to 70.1 mol% when the molar ratio was increased from 1:6.25 to 1:50. Several studies have shown that a high excess of fatty acid over GPC is necessary to achieve sufficient conversion, and products of high purity (Adlercreutz, Lyberg, & Adlercreutz, 2003; Ichihara et al., 2005). However, if the concentration of the acyl donor is increased too much, a loss of lipase activity can occur by the inhibition of acidic substrate, which acidifies the microaqueous phase surrounding the lipase or causes desorption of water from the interface (Lee & Akoh, 1998; Yankah & Akoh, 2000). A similar result was obtained from our study. There was no significant increase in the yield of LPC when the molar ratio was changed from 1:50 to 1:75, and the yield of LPC decreased at a molar ratio of 1:100. For the yield of PC, a continuous increase with increased molar ratio was observed, although the PC yield was small, in the range of 0.2–2.2 mol%, as shown in Fig. 4. Virto and Adlercreutz (2000) reported that the formation of PC increases with the second acylation, when the concentration of acyl donor increases. These results point to the importance of determining the critical value of the acyl donor, CLA, in the reaction mixture. Hence, the best yield of LPC was obtained when the molar ratio of GPC and CLA was 1:50.
0 20
30
40
50
60
Temperature ( oC) Fig. 3. Effect of temperature on production of LPC containing CLA. This experiment was performed at a vacuum of 10 mm Hg, a molar ratio of 1:10 (GPC to CLA), and an enzyme loading (Novozym 435) of 10 wt.% based on total substrates for 12 h.
3.4. Vacuum It has been noted that water is one of the important factors for the equilibrium of esterification, and that enzymes require a
S.I. Hong et al. / Food Chemistry 129 (2011) 1–6
certain amount of surface water to maintain their structure and activity (Adlercreutz et al., 2003; Han & Rhee, 1995; Rosu et al., 1999). Too much water, however, shifts the reaction towards hydrolysis, rather than synthesis. A number of methods to control the water content of the reaction mixture have been introduced, such as the utilisation of nitrogen (Rosu et al., 1999), molecular sieves (Blasi et al., 2008), saturated salt solution (Rosu et al., 1998; Virto et al., 1999) and vacuum (Rosu et al., 1998). Amongst these methods, the use of vacuum is an effective way to hinder lipid oxidation as well as to control by-products such as water, and ethanol (Rosu et al., 1998). Fig. 5 shows the yields of LPC and PC obtained using various vacuum conditions, as a function of reaction time. In the absence of vacuum, the yield of LPC was minimal throughout the observed reaction time of esterification. In a vacuum system, the highest yield, up to ca. 70 mol%, was achieved at 48 h, with a high vacuum in the range of 1 mm Hg, and with reaction conditions near equilibrium. It has been found that esterification of lysophospholipid is more difficult to accomplish than is the esterification of normal alcohols (Adlercreutz et al., 2003). Some studies have shown that an acid form was less effective for direct esterification of fatty acids with GPC than fatty acid derivatives, such as fatty acid anhydrides or fatty acid vinyl esters (Blasi et al., 2008; Mazur et al., 1991; Virto & Adlercreutz, 2000). Other studies have demonstrated the need of co-solvents, such as t-butanol or DMF (dimethylformamide) (Blasi et al., 2008; Kim & Kim, 1998). In the present study, however, the vacuum system achieved a high efficiency for direct esterification using GPC, and the acid form of CLA. As shown in Fig. 5a, although 0.5 mm Hg is a greater vacuum than 1 mm Hg, equilibrium was not approached with an LPC yield
(a)
Yield of LPC (mol%)
60 50 40
0.5 mmHg 1 mmHg 10 mmHg 50 mmHg 100 mmHg 300 mmHg 760 mmHg
30 20 10 0 4.0
(b)
Yield of PC (mol%)
3.5
of 63 mol% during a reaction time of 96 h. The probable reason is that the reaction mixture possessed insufficient water at the vacuum of 0.5 mm Hg (Virto & Adlercreutz, 2000). At vacuum levels of 100 and 300 mm Hg, the reaction rates were slow and yield of LPC fell compared to the yields produced under conditions of high vacuum. The yield of LPC was significantly increased during a reaction time of 24 h and, during the following 24 h, there was a slow but steady increase in the yield of LPC. However, the yield of LPC decreased after a reaction time of 48 h. It is assumed that this result was due to the retention of too much water to produce LPC at a vacuum of lower than 100 mm Hg. The water formed during esterification cannot effectively be removed from the reaction mixture under weak vacuums such as 100 and 300 mm Hg. Thus, the decreased yield is attributed to the fact that the rates of the hydrolysis reactions increase as the water content in the reaction mixture increases (Kim & Kim, 2000). Fig. 5b shows that the yield of PC increased very slowly, but PC was continuously being formed if the reactions were maintained. The rate of increase of PC yield differed according to vacuum level. The fastest production of PC occurred at vacuum levels of 1 and 10 mm Hg, and a less-rapid increase was shown at 0.5 mm Hg, and at vacuums lower than 50 mm Hg. Thus, the effects of insufficient water and hydrolysis were also demonstrated in the reaction rate for PC production. Hence, the optimal vacuum level and reaction time for synthesis of LPC by direct esterification of GPC and CLA were 1 mm Hg, and 48 h, respectively. 4. Conclusions This study showed that it is possible to synthesise LPC in a vacuum system through direct esterification using GPC and CLA as an acid form. Lipases were more effective than phospholipases for the production of LPC and, of the various lipases, the highest yield of LPC was achieved by using Novozym 435. Also, this individual enzyme was identified as having a selectivity that depended on the configuration of CLA isomers. Additional optimal conditions were a temperature of 40 °C, and a molar ratio of 1:50 (GPC to CLA). The application of vacuum was one of the most important parameters, because the water formed as a by-product was controlled during esterification. The effect of vacuum on the synthesis of LPC was that a high product yield was achieved under conditions of high vacuum; the best yield was obtained at 1 mm Hg, and reaction equilibrium was achieved at a reaction time of 48 h. Consequently, this study shows that the vacuum system is an effective method for the production of LPC from GPC and CLA using enzyme.
80 70
5
Acknowledgement
3.0
This study was supported by Food High Pressure Technology Development Project, Korea Food Research Institute, Korea.
2.5 2.0
References
1.5 1.0 0.5 0.0 0
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Time (h) Fig. 5. Effect of vacuum on production of LPC (a) and PC (b) containing CLA as a function of reaction time. This experiment was performed at temperature of 40 °C, and an enzyme loading (Novozym 435) of 10 wt.% based on total substrates.
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Mazur, A. W., Hiler, G. D., Lee, S. C., Armstrong, M. P., & Wendel, J. D. (1991). Regioand stereoselective enzymatic esterification of glycerol and its derivatives. Chemistry and Physics of Lipids, 60(2), 189–199. Patel, K. M., Morrisett, J. D., & Sparrow, J. T. (1979). A convenient synthesis of phosphatidylcholines: Acylation of sn-glycero-3-phosphocholine with fatty acid anhydride and 4-pyrrolidinopyridine. Journal of Lipid Research, 20(5), 674–677. Rosu, R., Iwasaki, Y., Shimidzu, N., Doisaki, N., & Yamane, T. (1998). Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum. Journal of Molecular Catalysis B: Enzymatic, 4(4), 191–198. Rosu, R., Yasui, M., Iwasaki, Y., & Yamane, T. (1999). Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solventfree system. Journal of the American Oil Chemists’ Society, 76(7), 839–843. Sébédio, J.-L., Christie, W. W., & Adlof, R. (2003). Advances in conjugated linoleic acid research. Champaign, Illinois: AOCS Press. Servi, S. (1999). Phospholipases as synthetic catalysts. Topics in Current Chemistry, 200, 127–158. Virto, C., & Adlercreutz, P. (2000). Lysophosphatidylcholine synthesis with Candida antarctica lipase B (Novozym 435). Enzyme and Microbial Technology, 26(8), 630–635. Virto, C., Svensson, I., & Adlercreutz, P. (1999). Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid. Enzyme and Microbial Technology, 24(10), 651–658. Wang, Y., Li, X., Liang, Y., Yang, B., & Zhang, S. (2007). Enzymatic fractionation of conjugated linoleic acid isomers by selective esterification. Journal of Molecular Catalysis B: Enzymatic, 46(1–4), 20–25. Warwel, S., & Borgdorf, R. (2000). Substrate selectivity of lipases in the esterification of cis/trans-isomers and positional isomers of conjugated linoleic acid (CLA). Biotechnology Letters, 22(14), 1151–1155. Xu, X. (2000). Production of specific-structured triacylglycerols by lipase-catalyzed reactions: A review. European Journal of Lipid Science and Technology, 102(4), 287–303. Yankah, Vivienne, & Akoh, Casimir (2000). Lipase-catalyzed acidolysis of tristearin with oleic or caprylic acids to produce structured lipids. Journal of the American Oil Chemists’ Society, 77(5), 495–500. Zhao, H., Lu, Z., Bie, X., Lu, F., & Liu, Z. (2007). Lipase catalyzed acidolysis of lard with capric acid in organic solvent. Journal of Food Engineering, 78(1), 41–46.
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Enzymatic synthesis of lysophosphatidylcholine containing CLA from sn-glycero-3-phosphatidylcholine (GPC) under vacuum Seung In Hong a,d, Yangha Kim b, Chong-Tai Kim c, In-Hwan Kim a,⇑ a
Department of Food & Nutrition, College of Health Sciences, Korea University, Jeongneung-Dong, Seongbuk-Gu, Seoul 136-703, Republic of Korea Department of Nutritional Science and Food Management, Ehwa Women’s University, Seoul 120-749, Republic of Korea c Korea Food Research Institute, Sungnam 463-746, Republic of Korea d Research Institute of Health Sciences, Korea University, Seoul 136-703, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 17 August 2010 Received in revised form 15 March 2011 Accepted 14 April 2011 Available online 20 April 2011 Keywords: Conjugated linoleic acid (CLA) sn-Glycero-3-phosphatidylcholine (GPC) Lysophosphatidylcholine (LPC) Novozym 435 Phosphatidylcholine (PC) Vacuum system
a b s t r a c t Lysophosphatidylcholine (LPC) was successfully synthesised by enzyme-catalysed esterification of snglycero-3-phosphatidylcholine (GPC) with the acid form of conjugated linoleic acid (CLA), using a vacuum system. GPC was prepared from phosphatidylcholine (PC) by alkaline deacylation using a methanolic tetra-butylammonium hydroxide solution. Five enzymes were tested as biocatalysts for direct esterification, and lipases were found to be more effective than phospholipases for production of LPC. Amongst the enzymes tested, Novozym 435, from Candida antarctica, gave the highest yield of LPC. The optimal temperature, molar ratio of substrate, and vacuum were 40 °C, 1:50 (GPC to CLA), and 1 mm Hg, respectively. The maximum yield (70 mol%) of LPC was obtained under optimal conditions after a 48 h reaction time. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The use of an enzymatic reaction has attracted attention for the synthesis of lysophospholipids (LPL). There are several advantages in using enzymatic reactions rather than chemical reactions. The first concerns the specificity of enzymes for their substrates and a particular regiospecific position; it is possible to incorporate a desired acyl group at a specific position on the backbone of a structure through enzymatic reactions, whereas chemical reactions do not possess this regiospecificity. The second advantage includes the mild reaction conditions and reduced amounts of chemical reagents required for synthetic reactions (D’Arrigo & Servi, 2010; Gunstone, 1999; Lee & Akoh, 1998; Servi, 1999). LPL and phospholipids (PL), containing specific fatty acids, have been synthesised by transesterification, and by direct esterification using an enzyme as a biocatalyst. In particular, the advantages of direct esterification include the production of purer reaction products and a shorter reaction time than is needed for transesterification (Adlercreutz, Budde, & Wehtje, 2002). sn-Glycero-3-phosphatidylcholine (GPC) is an ideal starting material for the preparation of various types of LPL or PL. However, GPC is well dissolved in water or polar solvents, whereas it is
⇑ Corresponding author. Tel.: +82 2 940 2855; fax: +82 2 941 7825. E-mail address: [email protected] (I.-H. Kim). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.04.038
immiscible with non-polar solvents. Although this characteristic has hampered its use in synthesis (D’Arrigo & Servi, 2010), some studies have been performed using cadmium chloride complexes of GPC to enhance its solubility in organic solvents (Baer & Kates, 1948; Chada, 1970). Other studies have been conducted, using fatty acid derivatives, such as fatty acid anhydrides and fatty acid vinyl esters, as acyl donors for production of lysophosphatidylcholine (LPC) from GPC (Blasi, Cossignani, Maurizi, Simonetti, & Damiani, 2008; Mazur, Hiler, Lee, Armstrong, & Wendel, 1991; Virto & Adlercreutz, 2000). However, reactions using natural fatty acids, rather than derivatives, are preferred because they avoid environmental pollution, and because the cost of fatty acid derivatives is higher than the cost of free acid substrates. Conjugated linoleic acid (CLA) was used as an acyl donor to produce LPC. CLA has received attention because of their ability to inhibit cancer, to lower blood cholesterol, to control diabetes and to aid in weight loss. The generic term ‘‘CLA’’ usually encompasses a number of conjugated linoleic acid isomers with double bonds at the 8c,10c-, 9c,11t-, 10t,12c-, and 11t,13t-positions (Sébédio, Christie, & Adlof, 2003). The present study describes (for production of LPC in a solventfree system) esterification of GPC with fatty acids using enzyme (Fig.1). A vacuum system was applied to control the water produced during esterification, because water in the reaction medium plays a crucial role for enzyme activity. When a minimal amount of water exists, the enzyme has a quasi-optimal configuration or
2
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Fig. 1. Reaction scheme for production of LPC from GPC and fatty acid.
catalytic activity and selectivity (Chae et al., 2009; Dordick, 1989; Han & Rhee, 1995; Rosu, Iwasaki, Shimidzu, Doisaki, & Yamane, 1998; Rosu, Yasui, Iwasaki, & Yamane, 1999; Zhao, Lu, Bie, Lu, & Liu, 2007). Lysophospholipids (LPL) are glycerophospholipids in which one acyl chain is lacking, and in which only one hydroxyl group of the glycerol backbone is acylated. Unlike phospholipids (PL), LPL are found only in small amounts in biological cell membranes. LPL are produced by phospholipases that exhibit diverse biological activities and serve as membrane-derived signalling molecules. They also serve as good emulsifying and solubilising agents, and as useful synthetic intermediates for the preparation of PLs, and they are applied in food, cosmetic, agrochemical and pharmaceutical industries. In particular, lysophosphatidylcholine (LPC) of LPL is abundantly found in nature (Adlercreutz et al., 2002; D’Arrigo & Servi, 2010; Han & Rhee, 1995; Virto, Svensson, & Adlercreutz, 1999). The purpose of this study was to optimise conditions for the efficient production of LPC containing CLA by enzyme-catalysed esterification under vacuum system. We prepared a GPC from phosphatidylcholine (PC) derived from soybean. The effects of several process parameters (enzyme screening, temperature, molar ratio and vacuum) on the yield of LPC containing a CLA were studied in a batch reactor. 2. Materials and methods 2.1. Materials Granulated PC (purity 95%, from soybean) was obtained from Avanti Polar-Lipids, Inc. (Alabaster, Al, USA). CLA (conjugated linoleic acid, C18:2, purity: 77.6%) was purchased from HK Biotech Co., Ltd. (Chinju, Republic of Korea). GPC, LPC and PC standards were purchased from Sigma Aldrich (St. Louis, MO, USA). TBIA methanolic solution (1.0 M methanolic tetra-butylammonium hydroxide solution) was purchased from Fluka (Sigma Aldrich, St. Louis, MO, USA). Lipozyme TL IM (the immobilised lipase from Thermomyces lanuginosus), Lipozyme RM IM (the immobilised lipase from Rhizomucor miehei), Novozym 435 (the immobilised lipase from Candida antarctica), Phospholipase A1 (LecitaseÒ Ultra) from
Thermomyces lanuginosus/Fusarium oxysporum, and phospholipase A2 (LecitaseÒ 10L) from porcine pancreas, were purchased from Novo Nordisk Bioindustry Ltd. (Seoul, Korea). Phospholipase A1 and phospholipase A2 were immobilised on DuoliteÒ A567 before use, according to Garcia, Kim, Lopez-Hernandez, and Hill (2008). The TLC plates used were coated with silica gel (layer thickness 0.25 mm, Kieselgel 60 F254, Merck, Darmstadt, Germany). All solvents and chemicals used were of analytical grade. 2.2. Preparation of GPC GPC was prepared from sn-1,2-PC by alkaline deacylation, as described by Brockerhoff and Yurkowski (1965), according to the following procedure. Briefly, 20 g of sn-1,2-PC were dissolved in 400 ml of diethyl ether, and TBAI methanolic solution (40 ml) was added. After magnetic stirring at room temperature for 2 h, an opalescent precipitate, composed of GPC, was formed. The solvent was removed and the precipitate was washed three times with 1 ml of diethyl ether, dissolved in 5 ml of methanol and transferred into a Teflon bottle. Methanol was removed by nitrogen flushing in a water bath of 50 °C. 2.3. Enzymatic synthesis of LPC Reactions were performed in a vacuum system, using a 50 ml water-jacketed glass vessel. GPC and CLA were used as the substrates for the synthesis of LPC by direct esterification. Prior to starting the reaction, the reaction vessel had been heated to the desired temperature using a water circulator. The weight of the total substrates was 15 g and immobilised lipases (10 wt.% based on total substrates, moisture-free) were added to a mixture of GPC and CLA. The reaction was performed by stirring with a magnetic stirrer at 600 rpm under a defined vacuum. The vacuum was controlled by a micrometering valve (Swagelok, Solon, OH, USA) and monitored by a digital vacuum gauge (Teledyne, Thousand Oaks, CA, USA). 2.4. Analysis of products Samples of 50 mg were dissolved in a mixture of chloroform and methanol (5:1, by vol), and then applied to silica-coated TLC
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50
(a)
LPC PC
40
Yield (mol%)
plates and developed with CHCl3/CH3OH/CH3COOH/H2O (75:40:8:3, by vol). LPC and PC were detected by spraying with 2,7-dichlorofluroscein solution (0.2% in 95% methanol solution). The bands corresponding to the LPC and PC were scraped off the TLC plate and methylated, using 14% BF3 in methanol as the catalyst. Heptadecanoic acid (0.2 mg) was used as an internal standard. The fatty acid methyl esters (FAME) were analysed by gas chromatography. A gas chromatograph (Varian 3800, Varian Inc., Walnut Creek, CA, USA), equipped with a Supelcowax 10 fused-silica capillary column (30 m 0.32 mm i.d.; Supelco, Bellefonte, PA, USA) and FID, was used. The column was held at 180 °C for 1 min and programmed to 230 °C for 10 min at the rate of 1.5 °C/min. The carrier gas was helium and the total gas flow rate was 50 ml/min. The injector and detector temperatures were 240 and 250 °C, respectively. FAME were identified by comparison with the retention times of the standards. The total amounts of LPC and PC were calculated from the total amount of FAME in each fraction.
30
20
10
0 1.0
(b)
Each reported value is the mean of determinations from duplicate samples, and the data were analysed by ANOVA and Duncan’s multiple range test. Statistical significance was accepted at a level of p < 0.05.
LPC/(LPC+PC)
0.8
2.5. Statistical analysis
0.6
0.4
0.2
3. Results and discussion 3.1. Enzyme screening
0.0 A
B
C
D
E
Enzyme Five enzymes were screened for their abilities to catalyse the direct esterification of GPC and CLA. The enzymes screened were Novozym 435, Lipozyme TL IM and Lipozyme RM IM, as lipases, and phospholipase A1 and phospholipase A2 as phospholipases. The trial for enzyme screening was performed for 12 h at a molar ratio of 1:10 (GPC to CLA), 50 °C, 10 mm Hg, and an enzyme loading of 10 wt.% based on total substrates. Yields of LPC and PC, and the ratio of LPC/(LPC + PC) obtained from the reactions catalysed by various immobilised enzymes are presented in Fig. 2a and b, respectively. Lipases were more effective than phospholipases for the production of LPC. A similar finding was reported by Hossen and Hernandez (2005). Amongst the enzymes tested in this study, Novozym 435 gave the highest yield of LPC. Thus, Novozym 435 was chosen as an appropriate enzyme for esterification of GPC with CLA. Several studies have described the reaction mechanism of GPC and fatty acids (Blasi et al., 2008; D’Arrigo & Servi, 2010; Virto, Svensson, & Adlercreutz, 1999; Xu, 2000). During esterification of GPC and fatty acid, it is thermodynamically easy to synthesise 2LPC, a regioisomer lacking the acyl chain at the sn-2 position, by enzymatic reaction. However, the fatty acid at sn-1 spontaneously migrates to sn-2, followed by the formation of PC through the second acylation of 1-LPC. Similar results were obtained in this study of the increase of PC during esterification of LPC. The amount of PC in the ratio of LPC/(LPC + PC), (Fig. 2b), was minimal compared to the production of LPC. Lipozyme RM IM and phospholipase A2, nevertheless, showed particularly low ratios of LPC/(LPC + PC). This suggests that both enzymes, by acyl migration and second esterification, accelerate the production of PC to a greater extent than other enzymes. Fatty acid compositions of LPC, produced by direct esterification using different enzymes, are shown in Table 1. Previous studies have reported the selectivity of enzymes for fatty acids, and this selectivity is related to chain length, and the configuration of the unsaturated fatty acid (Patel, Morrisett, & Sparrow, 1979; Rosu et al., 1998; Warwel & Borgdorf, 2000). The original CLA fraction
Fig. 2. Enzyme screening for production of LPC containing CLA; (a) yield of LPC and PC, (b) ratio of LPC/(LPC + PC). This experiment was performed at a molar ratio of 1:10 (GPC to CLA), temperature of 50 °C, a vacuum of 10 mm Hg, and enzyme loading of 10 wt.% based on total substrates for 12 h. (A) Novozym 435 (from Candida antarctica), (B) Lipozyme TL IM (from Thermomyces lanuginosus), (C) Lipozyme RM IM (from Rhizomucor miehei), (D) phospholipase A1 (from Thermomyces lanuginosus/Fusarium oxysporum), and (E) phospholipase A2 (from Porcine pancreas).
used as a substrate for the reaction was mainly composed of 9c,11t-CLA and 10t,12c-CLA, with similar compositions of 35.6 and 36.6 mol%, respectively. The concentration of 10t,12c-CLA in LPC produced by Novozym 435 was significantly greater than the concentration of 9c,11t-CLA. Meanwhile, Lipozyme TL IM, Lipozyme RM IM and phospholipase A1 exhibited higher selectivities for 9c,11t-CLA. However, phospholipase A2 was less effective for selective esterification of CLA with GPC. Warwel and Borgdorf (2000) reported that the lipases from Candida cylindracea and Mucor miehei exhibited a higher selectivity for 9c,11t-CLA than for 10t,12c-CLA. Wang, Li, Liang, Yang, and Zhang (2007) showed that Lipase AY30, from Candida rugosa, exhibited higher selectivity for 9c,11t-CLA than for 10t,12c-CLA, but a reverse trend was observed when Novozym 435, from Candida antarctica, was employed as a biocatalyst. Overall, our results showed that Novozym 435 was a very effective catalyst for the synthesis of LPC, and thus appropriate for its synthesis.
3.2. Temperature Reaction temperature is known to affect the esterification process catalysed by enzymes (Virto et al., 1999). Temperatures that are too high can induce irreversible denaturation of enzyme protein and shorten the half-life of enzyme activity, even though enzymes from microorganisms and immobilised enzymes are stable
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Table 1 Fatty acid composition (mol%) in LPC produced by different enzymes under vacuum *. CLAa
Fatty acids
c
C14:0 C16:0 C16:1(n7) C18:0 C18:1(n9) C18:1(n7) 9c,12c-C18:2 9c,11t-CLA 10t,12c-CLA Other CLAs *
0.1 ± 0.0 6.7 ± 0.7 0.1 ± 0.0 2.3 ± 0.1 11.9 ± 0.3 0.6 ± 0.0 1.1 ± 0.0 35.6 ± 0.2 36.6 ± 0.4 4.9 ± 0.2
Novozym 435b
Lipozyme TL IMb
Lipozyme RM IMb
Phospholipase A1b
Phospholipase A2b
0.3 ± 0.1 11.4 ± 1.0 0.1 ± 0.1 2.7 ± 0.2 9.8 ± 0.5 0.8 ± 0.1 1.1 ± 0.1 24.5 ± 0.1 40.7 ± 0.8 8.5 ± 2.4
0.2 ± 0.1 7.5 ± 0.9 0.1 ± 0.1 1.8 ± 0.1 10.6 ± 1.3 0.5 ± 0.1 1.2 ± 0.3 42.2 ± 2.4 29.3 ± 2.7 6.5 ± 2.3
0.2 ± 0.0 7.1 ± 0.2 0.4 ± 0.5 1.1 ± 0.2 10.6 ± 0.0 0.5 ± 0.0 1.2 ± 0.1 41.3 ± 2.9 28.6 ± 2.1 8.0 ± 4.4
0.0 ± 0.0 7.9 ± 0.5 0.0 ± 0.0 2.6 ± 0.8 11.7 ± 0.9 0.5 ± 0.2 1.2 ± 0.3 43.0 ± 2.5 27.0 ± 1.9 5.2 ± 3.3
0.0 ± 0.0 15.2 ± 0.4 0.0 ± 0.0 7.9 ± 0.8 16.3 ± 4.4 0.0 ± 0.0 3.2 ± 0.5 27.1 ± 4.1 27.7 ± 0.5 2.6 ± 0.1
The experiment was performed at a vacuum of 10 mm Hg and other detailed conditions were the same as Fig. 2. CLA (conjugated linoleic acid) used as substrate. b Immobilised enzymes used to produce LPC with GPC and CLA by direct esterification. c Means of duplicates ± standard deviations. a
100
80
Yield (mol%)
and active at high temperatures (Kim, Lee, Oh, & Kim, 2001; Xu, 2000). Moreover, excessively high temperatures accelerate side reactions, such as acyl migration and hydrolysis, due to the inhibition of reaction equilibrium (Rosu et al., 1999). The reaction temperature should be determined on an individual basis and be based on the melting points of the substrates (Kim & Kim, 2000) and products (Virto & Adlercreutz, 2000; Virto et al., 1999). The effect of reaction temperature on the production of LPC by direct esterification of GPC and CLA, using Novozym 435 as a biocatalyst, is shown in Fig. 3. For these trials, enzyme loading, substrate molar ratio, vacuum and reaction time were held constant at 10% of the total substrate weight, 1:10 (GPC to CLA), 10 mm Hg, and 12 h, respectively. The yield of LPC increased from 9.0 to 41.8 mol% when the temperature was increased from 20 to 40 °C. However, the yield of LPC remained constant when the temperature was further increased from 40 to 60 °C. Consequently, the optimal temperature to produce LPC through the esterification of GPC with CLA was 40 °C. Hence, in subsequent experimental trials, we employed 40 °C as the reaction temperature.
60
40 LPC PC
20
0 0
20
40
60
80
100
Molar ratio (CLA/GPC) Fig. 4. Effect of molar ratio on production of LPC and PC containing CLA. This experiment was performed at a vacuum of 10 mm Hg, temperature of 40 °C, and an enzyme loading (Novozym 435) of 10 wt.% based on total substrates for 48 h.
3.3. Molar ratio The effect of molar ratio on the production of LPC by direct esterification of GPC with CLA, using Novozym 435 as a biocatalyst, is shown in Fig. 4. The molar ratio of GPC to CLA varied from 1:6.25 to 1:100. For these trials, enzyme loading, vacuum and reaction time were held constant at 10% of the total weight of substrates,
50
Yield of LPC (mol%)
40
30
20
10
10 mm Hg and 48 h, respectively. The yield of LPC increased from 38.3 to 70.1 mol% when the molar ratio was increased from 1:6.25 to 1:50. Several studies have shown that a high excess of fatty acid over GPC is necessary to achieve sufficient conversion, and products of high purity (Adlercreutz, Lyberg, & Adlercreutz, 2003; Ichihara et al., 2005). However, if the concentration of the acyl donor is increased too much, a loss of lipase activity can occur by the inhibition of acidic substrate, which acidifies the microaqueous phase surrounding the lipase or causes desorption of water from the interface (Lee & Akoh, 1998; Yankah & Akoh, 2000). A similar result was obtained from our study. There was no significant increase in the yield of LPC when the molar ratio was changed from 1:50 to 1:75, and the yield of LPC decreased at a molar ratio of 1:100. For the yield of PC, a continuous increase with increased molar ratio was observed, although the PC yield was small, in the range of 0.2–2.2 mol%, as shown in Fig. 4. Virto and Adlercreutz (2000) reported that the formation of PC increases with the second acylation, when the concentration of acyl donor increases. These results point to the importance of determining the critical value of the acyl donor, CLA, in the reaction mixture. Hence, the best yield of LPC was obtained when the molar ratio of GPC and CLA was 1:50.
0 20
30
40
50
60
Temperature ( oC) Fig. 3. Effect of temperature on production of LPC containing CLA. This experiment was performed at a vacuum of 10 mm Hg, a molar ratio of 1:10 (GPC to CLA), and an enzyme loading (Novozym 435) of 10 wt.% based on total substrates for 12 h.
3.4. Vacuum It has been noted that water is one of the important factors for the equilibrium of esterification, and that enzymes require a
S.I. Hong et al. / Food Chemistry 129 (2011) 1–6
certain amount of surface water to maintain their structure and activity (Adlercreutz et al., 2003; Han & Rhee, 1995; Rosu et al., 1999). Too much water, however, shifts the reaction towards hydrolysis, rather than synthesis. A number of methods to control the water content of the reaction mixture have been introduced, such as the utilisation of nitrogen (Rosu et al., 1999), molecular sieves (Blasi et al., 2008), saturated salt solution (Rosu et al., 1998; Virto et al., 1999) and vacuum (Rosu et al., 1998). Amongst these methods, the use of vacuum is an effective way to hinder lipid oxidation as well as to control by-products such as water, and ethanol (Rosu et al., 1998). Fig. 5 shows the yields of LPC and PC obtained using various vacuum conditions, as a function of reaction time. In the absence of vacuum, the yield of LPC was minimal throughout the observed reaction time of esterification. In a vacuum system, the highest yield, up to ca. 70 mol%, was achieved at 48 h, with a high vacuum in the range of 1 mm Hg, and with reaction conditions near equilibrium. It has been found that esterification of lysophospholipid is more difficult to accomplish than is the esterification of normal alcohols (Adlercreutz et al., 2003). Some studies have shown that an acid form was less effective for direct esterification of fatty acids with GPC than fatty acid derivatives, such as fatty acid anhydrides or fatty acid vinyl esters (Blasi et al., 2008; Mazur et al., 1991; Virto & Adlercreutz, 2000). Other studies have demonstrated the need of co-solvents, such as t-butanol or DMF (dimethylformamide) (Blasi et al., 2008; Kim & Kim, 1998). In the present study, however, the vacuum system achieved a high efficiency for direct esterification using GPC, and the acid form of CLA. As shown in Fig. 5a, although 0.5 mm Hg is a greater vacuum than 1 mm Hg, equilibrium was not approached with an LPC yield
(a)
Yield of LPC (mol%)
60 50 40
0.5 mmHg 1 mmHg 10 mmHg 50 mmHg 100 mmHg 300 mmHg 760 mmHg
30 20 10 0 4.0
(b)
Yield of PC (mol%)
3.5
of 63 mol% during a reaction time of 96 h. The probable reason is that the reaction mixture possessed insufficient water at the vacuum of 0.5 mm Hg (Virto & Adlercreutz, 2000). At vacuum levels of 100 and 300 mm Hg, the reaction rates were slow and yield of LPC fell compared to the yields produced under conditions of high vacuum. The yield of LPC was significantly increased during a reaction time of 24 h and, during the following 24 h, there was a slow but steady increase in the yield of LPC. However, the yield of LPC decreased after a reaction time of 48 h. It is assumed that this result was due to the retention of too much water to produce LPC at a vacuum of lower than 100 mm Hg. The water formed during esterification cannot effectively be removed from the reaction mixture under weak vacuums such as 100 and 300 mm Hg. Thus, the decreased yield is attributed to the fact that the rates of the hydrolysis reactions increase as the water content in the reaction mixture increases (Kim & Kim, 2000). Fig. 5b shows that the yield of PC increased very slowly, but PC was continuously being formed if the reactions were maintained. The rate of increase of PC yield differed according to vacuum level. The fastest production of PC occurred at vacuum levels of 1 and 10 mm Hg, and a less-rapid increase was shown at 0.5 mm Hg, and at vacuums lower than 50 mm Hg. Thus, the effects of insufficient water and hydrolysis were also demonstrated in the reaction rate for PC production. Hence, the optimal vacuum level and reaction time for synthesis of LPC by direct esterification of GPC and CLA were 1 mm Hg, and 48 h, respectively. 4. Conclusions This study showed that it is possible to synthesise LPC in a vacuum system through direct esterification using GPC and CLA as an acid form. Lipases were more effective than phospholipases for the production of LPC and, of the various lipases, the highest yield of LPC was achieved by using Novozym 435. Also, this individual enzyme was identified as having a selectivity that depended on the configuration of CLA isomers. Additional optimal conditions were a temperature of 40 °C, and a molar ratio of 1:50 (GPC to CLA). The application of vacuum was one of the most important parameters, because the water formed as a by-product was controlled during esterification. The effect of vacuum on the synthesis of LPC was that a high product yield was achieved under conditions of high vacuum; the best yield was obtained at 1 mm Hg, and reaction equilibrium was achieved at a reaction time of 48 h. Consequently, this study shows that the vacuum system is an effective method for the production of LPC from GPC and CLA using enzyme.
80 70
5
Acknowledgement
3.0
This study was supported by Food High Pressure Technology Development Project, Korea Food Research Institute, Korea.
2.5 2.0
References
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Time (h) Fig. 5. Effect of vacuum on production of LPC (a) and PC (b) containing CLA as a function of reaction time. This experiment was performed at temperature of 40 °C, and an enzyme loading (Novozym 435) of 10 wt.% based on total substrates.
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