Food Chemistry 170 (2015) 386–393

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Preparation of highly purified pinolenic acid from pine nut oil using a combination of enzymatic esterification and urea complexation Da Som No a,b, Ting Ting Zhao a,b, Yangha Kim c, Mi-Ra Yoon d, Jeom-Sig Lee d, In-Hwan Kim a,b,⇑ a

Department of Food and Nutrition, Korea University, Seoul 136-703, Republic of Korea BK21PLUS Program in Embodiment: Health–Society Interaction, Department of Public Health Sciences, Graduate School, Korea University, Seoul 136-703, Republic of Korea c Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 120-749, Republic of Korea d National Institute of Crop Science, Rural Development Administration, Gyeounggi-do, Suwon 441-857, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 8 November 2013 Received in revised form 26 May 2014 Accepted 15 August 2014 Available online 23 August 2014 Keywords: Candida rugosa lipase Lipase-catalysed esterification Pine nut oil Pinolenic acid Urea complexation

a b s t r a c t Pinolenic acid (PLA) is a polyunsaturated fatty acid of plant origin. PLA has been successfully enriched according to a two-step process involving lipase-catalysed esterification and urea complexation. For the first step, the fatty acids present in pine nut oil were selectively esterified with lauryl alcohol using Candida rugosa lipase. Under the optimum conditions of 0.1% enzyme loading, 10% additional water, and 15 °C, PLA was enriched up to 43 mol% from an initial value of 13 mol% in the pine nut oil. For the second step, the PLA-enriched fraction from the first step was subjected to a urea complexation process. In this way, PLA enrichments with purities greater than 95 mol% were obtained at urea to fatty acid ratios greater than 3:1 (wt/wt), and 100% pure PLA was produced at a urea to fatty acid ratio of 5:1 with an 8.7 mol% yield. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Pinolenic acid (PLA) is a polyunsaturated fatty acid of plant origin. It is formally designated as all-cis-5,9,12-18:3. Pine nut oil is the primary source of PLA. The fatty acid compositions of species belonging to the genus Pinus can vary significantly, especially in terms of their D5-olefinic acid content (Takagi & Itabashi, 1982; Wolff, Pédrono, Pasquier, & Marpeau, 2000). In general, pine nut species from warm regions contain significantly less D5-olefinic acids than those that grow at high altitude or in colder regions (Wolff, Comps, Deluc, & Marpeau, 1998). Pinus koraiensis is one of the most widely available commercial sources of pine nut oil, and consists of approximately 17–18% D5-unsaturated polymethylene-interrupted fatty acids (D5-UPIFAs), including 13–15% PLA. As a D5-UPIFA, PLA possesses certain properties and exhibits specific health benefits that distinguished it from several other 18:3 fatty acids. For example, PLA has lipid-lowering effects by altering the expression levels of various apo genes (Asset, Baugé, Wolff, Fruchart, & Dallongeville, 2001; Asset et al., 2000). It has also been reported that the ingestion of PLA reduces the serum levels of LDL by enhancing hepatic LDL uptake (Lee, Lee, Lee, Kim, & Rhee, ⇑ Corresponding author at: Department of Food and Nutrition, Korea University, Jeongneung-dong, Seongbuk-Gu, Seoul 136-703, Republic of Korea. Tel.: +82 2 940 2855; fax: +82 2 941 7825. E-mail address: [email protected] (I.-H. Kim). http://dx.doi.org/10.1016/j.foodchem.2014.08.074 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

2004). Another health benefit of PLA is its appetite-suppressing effect (Ferramosca, Savy, Einerhand, & Zara, 2008; Pasman et al., 2008), where the presence of PLA in the gastro-intestinal tract effectively triggers the release of the satiety gut hormones cholecystokinin in the proximal small intestine (duodenum), and the glucagon like peptide-1 in the distal small intestine (ileum). Several attempts have been made to enrich value-added fatty acids using lipases, especially polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and c-linolenic acid (GLA). Numerous studies have been reported in the literature where polyunsaturated fatty acids were successfully enriched by the selective hydrolysis or alcoholysis of edible marine oils using lipases (Rakshit, Vasuhi, & Kosugi, 2000; Shimada, Fukushima et al., 1998; Wanasundara & Shahidi, 1998). The lipase-catalysed selective esterification of fatty acids with a variety of different alcohols has also been widely used for the enrichment of beneficial fatty acids (Schmitt-Rozieres, Vanot, Deyris, & Comeau, 1999; Shimada, Sugihara et al., 1998). Shimada et al. (1997) showed that it was possible to purify the DHA in tuna oil via the repeated selective esterification of the fatty acids in the oil with lauryl alcohol using Rhizopus delemar lipase. As another strategy, a combination of enzymatic and physical method has been investigated to fractionate and enrich functional fatty acids (Vali, Sheng, & Ju, 2003). The combination of enzymatic and physical methods is a practical and efficient way to fractionate fatty acids due to their different mechanisms, which can be applied to

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Scheme 1. Selective lipase-catalysed esterification of fatty acid with lauryl alcohol using Candida rugosa lipase.

different fatty acids. Vali et al. (2003) have suggested a practical method, which involves enzymatic reaction and crystallisation, to purify arachidonic acid from a single cell oil. Although we previously investigated the enrichment of PLA via the sn-1,3 regiospecific ethanolysis of pine nut oil triacylglycerol, using Novozym 435 from Candida antarctica, we were only able to achieve a final PLA content of 39 mol% (Lee et al., 2011; Zhao et al., 2012). In the current study, a lipase-catalysed esterification was used in combination with urea complexation to prepare highly purified PLA (purity > 99%). For the lipase-catalysed esterification, fatty acid from pine nut oil was esterified with lauryl alcohol using Candida rugosa lipase as a biocatalyst (Scheme 1). Several of the reaction parameters, including the enzyme loading, size of the additional water charge and reaction temperature were investigated to optimise the lipase-catalysed esterification. A urea complexation process was then conducted using the PLA enriched-fraction from the lipase-catalysed esterification to further increase the PLA content. The effect of the urea to fatty acid ratio on the enrichment of PLA was also studied. 2. Materials and methods 2.1. Materials Pine nuts (P. koraiensis) were purchased from the Agricultural Marketing Center (Seoul, Korea). C. rugosa lipase was purchased from Meito Sangyo Co., Ltd. (Nagoya, Japan). Lauryl alcohol (P99.0% purity) was purchased from Sigma Aldrich (St. Louis, MO). Extra pure urea was purchased from Samchun Pure Chemical Co. LTD (Kyungkido, South Korea). Silica gel 60 for column chromatography and silica gel TLC plates were purchased from Merck KGaA (Darmstadt, Germany). All of the other chemicals used in this study were purchased as the analytical grades, unless otherwise noted. 2.2. Methods 2.2.1. Preparation of fatty acid from pine nut oil The pine nuts (200 g) were homogenised using a coffee grinder, and the resulting homogenate was placed in a 2 L flask and stirred with 1 L of n-hexane for 2 h. The mixture was then filtered and the filtrate collected. The solvent was removed in vacuo, and the resulting pine nut oil (100 g) was added to a solution of sodium hydroxide (40 g) in distilled water (100 mL) and ethanol (99%, 300 mL). The mixture was heated at reflux with stirring at 500 rpm for 1 h before being cooled to ambient temperature and diluted with water (200 mL). The unsaponifiable matter was extracted into 300 mL of n-hexane and discarded. The aqueous layer containing the saponifiable matter was acidified by the addition of aqueous 6 N HCl to a pH of 1.0. The resulting lower layer was removed using a separating funnel and discarded, whereas the upper layer containing the fatty acid was extracted into n-hexane (200 mL) and washed with distilled water (2  100 mL). The hexane layer containing the fatty acid was then dried over anhydrous sodium sulphate before being distilled to dryness in vacuo. A stream of nitrogen was passed over the resulting product to remove any residual solvent.

2.2.2. Lipase-catalysed esterification The lipase-catalysed esterification of the fatty acids from pine nut oil with lauryl alcohol was carried out using C. rugosa lipase. A portion of the fatty acids from the pine nut oil (3.0 g, 11 mmol) and lauryl alcohol (2.0 g, 11 mmol) were placed in a 50 mL water-jacketed glass vessel and the vessel reactor was preheated to the desired temperature using a water circulator. Distilled water was then added to the vessel, and the esterification process was initiated by the addition of the enzyme into the substrate mixture with stirring at 400 rpm. Samples (150 lL) were withdrawn periodically and mixed with chloroform (600 lL). These sample mixtures were then filtered through a 0.45 lm GHP Acrodisc syringe filter (Pall Corporation, Port Washington, NY) to remove the enzyme and analysed by TLC and GC.

2.2.3. Separation of the reaction product following the lipase-catalysed esterification To allow for further enrichment of the PLA, the unesterified fatty acid fraction was separated from the reaction product following the lipase-catalysed esterification by saponification and subsequent acidification as follows. Following a scaled-up lipase-catalysed esterification reaction, a portion of the reaction mixture (100 g) was dissolved in n-hexane (1 L), and the resulting solution was filtrated through anhydrous sodium sulphate to remove the enzyme and dry the solvent. The filtrate was then collected and placed in a separating funnel together with a 1.9% (w/v) NaOH solution (200 mL) and 95% ethanol (200 mL). The resulting mixture was agitated before being allowed to settle. The lower layer was collected and placed in a second separating funnel where it was washed with n-hexane (1 L). A 6 N HCl solution (40 mL) was then added to the mixture to acidify the mixture, and the fatty acid fraction was recovered by extraction with n-hexane (200 mL). The n-hexane solution containing the fatty acid fraction was washed with water several times before being filtrated through anhydrous sodium sulphate. The solvent was then removed in vacuo to give the fatty acid product and a stream of nitrogen gas was passed over the product mixture to remove any residual solvent.

2.2.4. Urea complexation A portion of the PLA-enriched fatty acid (6.0 g) from the lipasecatalysed esterification process was dissolved in the desired amount of methanol (urea/methanol ratio of 0.3 g/mL) together with urea [6, 12, 18, 24, or 30 g of urea for urea/fatty acid ratios of 1:1, 2:1, 3:1, 4:1 and 5:1 (wt/wt), respectively], and the resulting mixture was refluxed until it became clear in appearance. The solution was then cooled to a temperature in the range of 0–4 °C where it was held overnight to allow for crystallisation to occur. The resulting crystals were collected by filtration and the filtrate was concentrated in vacuo to give a residue, which was treated with a hot 0.1 N HCl solution (urea/HCl ratios of 0.5 g/mL). The acidified solution was then extracted with n-hexane (urea/hexane ratios of 0.25 g/mL) twice to allow for the recovery of the PLA concentrate. The combined hexane extracts were washed with water (urea/hexane ratios of 0.5 g/mL) before being dried over anhydrous sodium sulphate and distilled to dryness in vacuo to give the PLA concentrate, which was then stored at 40 °C.

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2.2.5. Analysis of products Thirty microlitre samples were dissolved in chloroform (120 lL) and loaded on to TLC plates and the plates were developed using a mixture of petroleum ether, diethyl ether and acetic acid (100:20:1 – v/v/v). The plates were subsequently developed with a 2,7dichlorofluoroscein solution (0.2% in 95% methanol) to allow for the fatty acid and fatty acid lauryl ester fractions to be visualised. The corresponding methyl esters were prepared using 14% BF3 in methanol. One microlitre samples of the extract were injected into a Varian 3800 chromatograph (Varian Inc., Walnut Creek, CA) fitted with a Supelcowax 10 fused-silica capillary column (30 m  0.32 mm i.d.; Supelco, Bellefonte, PA). The injector and FID temperatures were set at 240 and 250 °C, respectively. The oven temperature was held at 180 °C for 1 min and then heated to 210 °C at a rate of 1.5 °C/min. The fatty acid methyl esters were identified by comparing their retention times with those of the known standards. Heptadecanoic acid (0.2 mg) was used as an internal reference standard. The degree of conversion (mol%), PLA content in the fatty acid fraction (mol%) and PLA yield in the fatty acid fraction (mol%) were calculated as follows:

Degree of conversion ðmol%Þ ¼

b  100; aþb

PLA content in fatty acid ðmol%Þ ¼

c  100; a

c PLA yield in the fatty acid ðmol%Þ ¼  100; cþd where a is the number of moles of fatty acid in the reaction product, b is the number of moles of synthesised fatty acid lauryl ester in the reaction product, c is the number of moles of PLA in the fatty acid of the reaction product, and d is the number of moles of PLA in the fatty acid lauryl ester of the reaction product. 3. Results and discussion 3.1. Lipase-catalysed esterification 3.1.1. Effect of enzyme loading The enzyme loading is a crucial factor in terms of controlling the rate of an enzymatic reaction, with larger charges of enzyme generally affording faster reaction rates compared with smaller charges. The presence of too much enzyme, however, can be inefficient from an economic perspective and can also have an adverse impact on the level of selectivity delivered by the enzyme itself (Šabeder, Habulin, & Knez, 2006; Villeneuve et al., 2005). An enzyme loading in the range of 0.01–0.5% of the total substrate weight was investigated in the current study. The molar ratio of fatty acid to lauryl alcohol, reaction temperature and additional water charge were set at 1:1, 25 °C and 15% of the total substrate weight, respectively. The effect of the enzyme loading on the degree of conversion is shown in Fig. 1a. A faster initial reaction rate was observed when a higher enzyme loading was used in all cases. Furthermore, the PLA content in the fatty acid fraction increased significantly during the initial stage of the reaction as the enzyme loading was increased from 0.01% to 0.1% (Fig. 1b). Although an enzyme loading of 0.5% gave the fastest initial reaction rate of all of the loadings tested during the first 4 h, in terms of the PLA content of reaction, this loading level gave a maximum PLA content of only 38.0 mol%. This low level of PLA enrichment was attributed to the specificity of C. rugosa lipase being suppressed by the fast reaction rate at an enzyme loading of 0.5% (Vázquez, Kleiner, & Akoh, 2012). The maximum PLA content of ca. 40 mol% was observed with enzyme loadings of 0.1% and

100

(a) 80

60

40

20

45

(b)

40 35 30 25 20 15

(c) 100

80

60

40

0

5

10

15

20

25

Fig. 1. Effects of enzyme loading on the degree of conversion (a), PLA content in the fatty acid (FA) fraction (b), and PLA yield in the FA fraction (c) following the Candida rugosa lipase-catalysed esterification as a function of reaction time. The reaction was performed at 25 °C with an additional water charge of 15% (of the total substrate weight) and a fatty acid to lauryl alcohol ratio of 1:1 (mol/mol).

0.25%. At enzyme loadings greater than 0.1%, the PLA content effectively reached its maximum point before being slowly reduced. Even though the highest PLA content could be obtained with enzyme loadings of 0.1% and 0.25%, the PLA yield obtained from the former was higher than that of the latter at the highest PLA content (Fig. 1c). Taken together, these results indicated that an enzyme loading of 0.1% represented an appropriate compromise between these two factors. 3.1.2. Effect of additional water It is known that reactions involving lipases require water to maintain the enzyme structure and achieve catalytic activity

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(Watanabe et al., 2003a; Zaks & Klibanov, 1988). The use of too much water, however, can result in a shift in the reaction towards hydrolysis, rather than esterification, and lead to a decrease in lipase activity and an increase in by-product formation (He & Shahidi, 1997; Yang, Xu, He, & Li, 2003). An additional charge of water in the range of 0 to 15% of the total substrate weight was added to each reaction at the beginning of the process. The molar ratio of fatty acid to lauryl alcohol, reaction temperature and the enzyme loading were set at 1:1, 25 °C and 0.1% of the total substrate weight, respectively. The effect of

100

(a)

80

60

40

20

45

(b)

40 35 30 25 20 15

(c) 100

80

60

40 0

5

10

15

20

25

Fig. 2. Effect of the additional water charge on the degree of conversion (a), PLA content in the fatty acid (FA) fraction (b), and PLA yield in the FA fraction (c) following the Candida rugosa lipase-catalysed esterification as a function of reaction time. The reaction was performed at 25 °C with an enzyme loading of 0.1% (of the total substrate weight) and a fatty acid to lauryl alcohol ratio of 1:1 (mol/mol).

389

the additional water charge on the degree of conversion is shown in Fig. 2a. When the size of the additional water charge was increased up to 10%, the degree of conversion increased significantly. There was no significant difference, however, in the level of conversion after 4 h, when the size of the additional water charge was increased from 10% to 15%. In contrast, the degree of conversion was small at an initial water content of 0%, with the conversion being only 7.5% at 24 h. The effect of the size of the additional water charge on the PLA content is shown in Fig. 2b. A significant increase in the PLA content in the fatty acid fraction was observed when the size of the additional water charge was increased from 0% to 5%. Furthermore, a slight increase in the PLA content was observed when the size of the additional water charge was increased further from 5% to 10%. No significant difference was observed, however, in the PLA content or yield when the size of the additional water charge was increased from 10% to 15% throughout the entire reaction (Fig. 2b and c). The results revealed that a maximum PLA content of 41 mol% could be obtained following 8 h of the reaction using an additional water charge of 10% or 15% with a yield of approximately 78 mol%. It has been reported that C. rugosa lipase exhibits high activity towards the hydrolysis of triacylglycerol (Shimada, Fukushima et al., 1998; Virto et al., 1994). C. rugosa lipase has also been reported to possess the ability to discriminate between fatty acids containing double bonds at different positions during an esterification reaction (Nagao et al., 2002; Vázquez et al., 2012). To take advantage of these mutually exclusive behaviours, it would be necessary to vary the amount of water initially present in the system depending on the type of reaction or type of substrate being used. In terms of the difference between esterification and hydrolysis reactions catalysed by C. rugosa lipase, an increase in the size of the additional water charge could potentially lead to a shift in the thermodynamic equilibrium in favour of hydrolysis. Numerous studies have demonstrated, however, that the initial water contents of C. rugosa lipase-catalysed esterification reactions can vary from 0% to 30% (López-Martínez, Campra-Madrid, RamírezFajardo, Esteban-Cerdán, & Guil-Guerrero, 2006; Nagao et al., 2002). Vázquez et al. (2012) reported the use of a C. rugosa lipase-catalysed esterification for the enrichment of stearidonic acid (18:4D6) that was conducted without water with a relatively large enzyme loading. In contrast, Shimada et al. (2003) successfully achieved the enrichment of Mead acid (20:3D5) via a C. rugosa lipase-catalysed esterification reaction with a large additional water charge of 20% and a small enzyme loading. Similar to the latter, in the current study, the esterification activity of C. rugosa lipase was increased significantly when a large additional charge of water was employed. This increase in the extent of the esterification reaction, even in the presence of a large amount of water, has been explained by Shimada et al. (2001). In general, fatty alcohols are good substrates for lipases, whereas the corresponding esters (waxes) are not. The esterification of fatty acids with a fatty alcohol therefore proceeds efficiently, even in the presence of a large amount of water, because the esterification products (fatty alcohol esters) are poor substrates for hydrolysis by the lipases. In addition, it is well known that water exerts its influence in enzymatic reactions by forming hydrogen bonding interactions with functional groups within the protein molecule, thereby ‘‘unlocking’’ the enzyme structure. In this way, water effectively behaves as a lubricant or plasticizer to provide the enzyme molecule with the flexibility that it needs for enzymatic catalysis (Kurkal et al., 2005; Kurplus & McCammon, 1983; Zaks & Klibanov, 1988). On the basis of the PLA content and yield values achieved for additional water charges of 10% and 15%, 10% was selected as the optimum initial water level because the inclusion of a smaller amount of water was deemed to be more effective in terms of the downstream process.

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3.1.3. Effect of temperature The reaction temperature represents a critical parameter in an enzymatic reaction because it effectively controls the activity and selectivity of the enzyme, and an increase in the reaction temperature usually leads to an increase in the rate of the reaction. Reaction temperatures that are too high, however, can induce higher lipase deactivation rates (Martinek & Mozhaev, 1993; Yang et al., 2003). The optimum temperature for an enzymatic reaction depends on the type of reaction, melting points of the reactants, and the nature of the enzyme, and several other reaction parameters. In general, lipases exhibit their best activity at room temperature or above (Senanayake & Shahidi, 1999; Shimada et al., 2001). There are several lipases, however, known as cold active enzymes, which operate most effectively at temperatures below room temperature, such as those from Pseudomonas sp. and Penicillium camembertii (Kojima, Sakuradani, & Shimizu, 2006; Pyo, Hong, Kim, Kim, & Kim, 2012). The effect of temperature was investigated in the current study at temperatures in the range of 0–55 °C. For these trial reactions, the enzyme loading and additional water charge were held constant at 0.1% and 10% of the total substrate weight, respectively. The effect of different temperatures on the degree of conversion is shown in Fig. 3a. As the reaction temperature was decreased from 55 to 35 °C, the degree of equilibrium conversion increased significantly. Even though the maximum conversion varied depending on the temperature, the esterification reaction reached equilibrium around 4 h for all of the temperatures tested. In contrast, the PLA content in the fatty acid fraction increased drastically as the temperature was decreased from 55 to 15 °C (Fig. 3b). At reaction temperatures higher than 5 °C, the PLA content either increased continuously or remained constant after it reached its equilibrium. At temperatures of 0 and 5 °C, the PLA contents decreased after they reached their peak. The maximum PLA content of 42.6 mol% was observed at temperatures of 5 and 15 °C after 12 h of reaction time. Furthermore, at the maximum PLA content, the PLA yield and temperature values overlapped (Fig. 3b and c). The optimum temperature can vary depending on the reaction system or substrates being used, even when the same enzyme is being employed. In several different studies, similar esterification reactions have been carried out at temperatures in the region of room temperature (Nagao et al., 2003; Schmitt-Rozieres et al., 1999; Shimada, Sugihara et al., 1998; Vázquez et al., 2012). However, Watanabe et al. (2003b) reported that the esterification of a free fatty acid containing CLA with glycerol to produce the monoacylglycerol of CLA proceeded more efficiently at 5 °C than it did at 30 °C. In addition, Yasufuku and Ueji (1995) demonstrated that lipase AY (C. rugosa) exhibited a higher enantioselectivity at 10 °C than it did at 37 °C in an esterification reaction with a suitable amount of water. In accordance with these previous studies, the C. rugosa lipase used in the current study was revealed to exhibit higher levels of activity and selectivity at temperatures below room temperature. Based on these results, a temperature of 15 °C was selected as the optimum temperature following consideration of the energy costs associated with the cooling process. 3.2. Urea complexation Urea complexation is a classical way in which to concentrate polyunsaturated fatty acids. Urea forms tetragonal crystal by itself and spiral hexagonal shaped crystals with a fatty acid. The urea molecules bond together via hydrogen bonding interactions, whereas strong van der Waals interactions exist between the urea and fatty acid molecules which are maintained in the crystalline form. Fatty acids with shorter chain lengths (i.e., less than 6–8) or containing double bonds are less likely to form urea complexes

100

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(b)

40 35 30 25 20 15

(c) 100

80

0oC 5oC 15oC 25oC 35oC 45oC 55oC

60

40

0

5

10

15

20

25

Fig. 3. Effect of temperature on the degree of conversion (a), PLA content in the fatty acid (FA) fraction (b), and PLA yield in the FA fraction (c) following the Candida rugosa lipase-catalysed esterification as a function of reaction time. The reaction was performed at an enzyme loading of 0.1% (of the total substrate weight) with an additional water charge of 10% (of the total substrate weight) and a fatty acid to lauryl alcohol ratio of 1:1 (mol/mol).

than those containing longer chain lengths of saturated fatty acids (Hayes, Bengtsson, Alstine, & Setterwall, 1998). Several factors, including the temperature, urea to fatty acid ratio and type of solvent, can affect the inclusion of fatty acids in the urea complex. Following the C. rugosa lipase-catalysed esterification, the PLAenriched fatty acid fraction was separated from the reaction product as described above in the materials and methods section. Urea complexation was then carried out to allow for further enrichment of the PLA. Urea to fatty acid ratios in the range of 1:1–5:1 were tested in the current study, with methanol being used as the

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solvent. The variations in the PLA content and PLA yield in the PLAenriched filtrate fractions are shown in Table 1. As the urea to fatty acid ratio increased from 1:1 to 5:1, the PLA content in the filtrate increased, although the PLA yield decreased drastically. At a ratio

of 1:1, the PLA was enriched from an initial value of 42.6% following the lipase catalysed esterification to 57.3 mol%, with a PLA yield of 62.5 mol%. At a ratio of 3:1, the complexation process afforded a PLA content of 94.6 mol% with a PLA yield of 38.2 mol%. The PLA

Table 1 Fatty acid composition (mol%) and PLA yield (mol%) of the initial pine nut oil, the unesterified fatty acid fraction following the lipase-catalysed esterification under the optimum conditions and PLA enrichments following the urea complexation at various urea to fatty acid ratiosa. Fatty acids

Original pine nut oilb

PLA enrichment after esterificationc

PLA enrichments after urea complexationd 1:1e

2:1

3:1

4:1

5:1

C16:0 C18:0 C18:1(D9) C18:1(D11) C18:2(D5,9) C18:2(D9,12) C18:3(D5,9,12) C20:0 C20:1(D11) C20:2(D11,14) C20:3(D5,11,14)

5.3 ± 0.0 2.3 ± 0.1 27.1 ± 0.1 0.4 ± 0.0 2.2 ± 0.0 46.4 ± 0.1 13.3 ± 0.0 0.3 ± 0.0 1.1 ± 0.0 0.6 ± 0.0 1.0 ± 0.0

8.4 ± 1.5 7.5 ± 0.3 8.4 ± 0.6 – 8.1 ± 0.4 13.3 ± 0.1 43.0 ± 0.7 1.3 ± 0.1 3.9 ± 0.3 2.0 ± 0.0 4.1 ± 0.3

0.4 ± 0.1 – 8.9 ± 0.5 0.5 ± 0.0 9.4 ± 0.0 14.1 ± 0.1 57.3 ± 0.5 2.0 ± 0.1 0.5 ± 0.0 2.3 ± 0.0 4.7 ± 0.1

– – 0.1 ± 0.1 – 5.0 ± 0.5 10.7 ± 0.6 78.4 ± 1.8 – – 0.3 ± 0.3 5.5 ± 0.0

– – – – 0.3 ± 0.4 1.8 ± 0.3 94.6 ± 0.7 – – – 3.3 ± 0.0

– – – – – – 99.1 ± 0.0 – – – 0.9 ± 0.0

– – – – – – 100.0 ± 0.0 – – – –

PLA yieldf

100

77.8 ± 1.2

62.5 ± 1.7

55.2 ± 0.8

38.2 ± 0.1

18.2 ± 0.4

8.7 ± 0.4

a

Values represent the average of duplicate determinations from different experimental trials. b Fatty acid composition (mol%) of original pine nut oil. c Unesterified fatty acid fraction after 8 h of lipase-catalysed esterification under the optimum conditions of 1:1 (fatty acid to lauryl alcohol), 15 °C, 0.1% enzyme loading and 10% additional water. d Filtrate after urea complexation at various urea to fatty acid (wt/wt) ratios, detailed protocols are indicated in Section 2.2.5. e The ratios of urea to fatty acid (wt/wt). f PLA yield (mol%) in the PLA enrichment following the lipase-catalysed esterification and urea complexation processes.

Fig. 4. Chromatograms of the initial pine nut oil (a), fatty acid fraction following the Candida rugosa lipase-catalysed esterification at 15 °C with an enzyme loading of 0.1% (of the total substrate weight), additional water charge of 10% (of the total substrate weight), and fatty acid to lauryl alcohol ratio of 1:1 (mol/mol) (b), and the filtrate fractions following the urea complexation at a urea to fatty acid ratio of 5:1 (wt/wt) (c).

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contents obtained at ratios of 4:1 and 5:1 were 98.6 and 100 mol% with the PLA yields of 16.7 and 8.7 mol%, respectively. 3.3. Fatty acid compositions of initial pine nut oil and after each step The fatty acid compositions of the initial pine nut oil, fatty acid fraction after the enzymatic esterification at the optimum condition, and filtrates obtained from the urea complexation are shown in Table 1. There were two major fatty acid products in the initial pine nut oil sample, including oleic acid (C18:1D9) and linoleic acid (C18:2D9,12). Both of these acids are D9-unsaturated fatty acids and their levels were found to decrease drastically throughout the reaction. In contrast, the levels of several saturated fatty acids, including palmitic acid, stearic acid and D-5 UPIFA, increased significantly. Overall, C. rugosa lipase appeared to exhibit its highest level of selectivity towards D9-unsaturated fatty acids, especially linoleic acid, followed by oleic acid. This lipase also discriminated strongly against PLA, to the extent that PLA could be enriched in the unesterified fatty acid fraction. In accordance with our result, Nagao et al. (2002) have efficiently fractionated c9,t11-CLA and t10,c12-CLA using the selectivity of C. rugosa lipase. Highly purified PLA fractions were subsequently obtained by the urea complexation process. By the urea complexation, remaining fatty acids, especially saturated and mono unsaturated ones, were efficiently removed. Overall, our strategy to combine the selective enzymatic esterification and the urea complexation was suitable for the enrichment of PLA in that the pine nut oil contains high levels of D9-unsaturated fatty acids and saturated fatty acids. Fig. 4 shows the chromatograms of samples of the initial pine nut oil, PLA-enriched fatty acid fraction following enzymatic esterification, and PLA-enriched filtrate fraction following urea complexation at a 5:1 ratio. These results clearly demonstrate that 100% pure PLA was produced following the urea complexion process at a ratio of 5:1. In addition, the resulting PLA enrichment (100% purity) was verified by EI mass spectrometry (data not shown) and the result corresponded with the mass spectrum reported by Dobson and Christie (2002). 4. Conclusion PLA was successfully enriched in its fatty acid form using a combination of lipase-catalysed esterification and urea complexation. Since C. rugosa lipase discriminates against PLA, PLA was enriched from initial value of 13.3–42.6 mol% in the unesterified fatty acid fraction following esterification. High purity PLA (i.e., greater than 95 mol%) was obtained following a subsequent urea complexation process with urea to fatty acid ratios of 3:1 and 4:1. The use of a urea to fatty acid ratio of 5:1 afforded 100% pure PLA, although the yield was only 8.7 mol%. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (2013R1A1A2006050). References Asset, G., Baugé, E., Wolff, R. L., Fruchart, J. C., & Dallongeville, J. (2001). Effects of dietary maritime pine seed oil on lipoprotein metabolism and atherosclerosis development in mice expressing human apolipoprotein B. European Journal of Nutrition, 40(6), 268–274. Asset, G., Leroy, A., Bauge, E., Wolff, R. L., Fruchart, J.-C., & Dallongeville, J. (2000). Effects of dietary maritime pine (Pinus pinaster)-seed oil on high-density

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Preparation of highly purified pinolenic acid from pine nut oil using a combination of enzymatic esterification and urea complexation.

Pinolenic acid (PLA) is a polyunsaturated fatty acid of plant origin. PLA has been successfully enriched according to a two-step process involving lip...
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