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Keywords:

  • Candida rugosa;
  • Echium oil;
  • Esterification;
  • Rhizomucor miehei;
  • Stearidonic acid

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Conclusions
  7. References

Stearidonic acid (SDA) from echium oil was enriched substantially by a two-step lipase-catalyzed esterification using Lipase OF from Candida rugosa and Lipozyme RM IM from Rhizomucor miehei. During the first step, SDA was enriched in the fatty acid fraction via C. rugosa lipase-catalyzed esterification with lauryl alcohol. The optimum reaction conditions of water content, temperature, and enzyme loading were 0.25%, 30°C, and 2%, respectively, in terms of the content and yield of SDA. Under these conditions, SDA content was increased to 39.3 from 14.3% of the starting material. To further elevate SDA content, Lipozyme RM IM-catalyzed esterification was then conducted using the SDA enriched fatty acid from the first step and a maximum SDA content of 54.1% was obtained. Using this two-step lipase-catalyzed esterification, SDA content increased fourfold from 14.3 to 54.1% with a 74.8% yield. γ-Linolenic acid (GLA) was also enriched together with SDA.

Practical applications: SDA, which is one of ω-3 fatty acids, is considered as a source of health beneficial polyunsaturated fatty acids for vegetarians. SDA enrichment obtained in this study leads also to a high level of GLA. Therefore, this enrichment can be applied to produce a novel structured lipid containing a significant amount of SDA and GLA.

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Conclusions
  7. References

Stearidonic acid (SDA), all-cis-6,9,12,15-octadecatetraenoic acid, is one of n-3 PUFA and is the first metabolite in the conversion of ALA to longer chain highly unsaturated fatty acids [1]. SDA possesses similar biological properties to EPA and DHA which have been recognized for their health-promoting effects in human. There have been considerable and increasing levels of interest in SDA because of its broad range of physiological activities in humans including its application for the prevention of cardiovascular, inflammatory, and autoimmune diseases [1, 2]. In human bodies, ALA can be converted to SDA by Δ6-desaturase, and the resulting SDA can be turned into EPA and DHA via desaturation and elongation. However, due to the fact that Δ6-desaturase is rate limiting in humans, only 2–5% of ALA can be turned into EPA whereas 20–30% of SDA can be converted to EPA. Therefore, the level of interest in SDA is increasing because it provides efficient means of increasing EPA content.

SDA is a minor n-3 PUFA contributes to 0.5–2% of the total fatty acids in fish and other seafood, and is rarely found in commonly consumed vegetables, fruits, seeds, nuts, or commercial oils [2]. SDA can be found naturally, however, in blackcurrant seed oil and oils derived from some members of the Boraginaceae family [3]. Among these oils, echium oil is commercially available oil that is rich in SDA and also contains a large amount of GLA and ALA. Thus, this oil has a unique ratio of n-3 PUFA and n-6 PUFA that is not present in any other plant.

Several methods have been developed for enriching the PUFA from fish oil and plant oils including supercritical CO2, urea complexation, low temperature crystallization, preparative HPLC, and enzymatic methods. In particular, enzymatic method have been identified as one of the most attractive of these methods because they exhibit several distinct advantages over the other available methods such as mild reaction conditions, regiospecificity, and less formation of side reaction products. Many studies have been conducted regarding the enrichment of PUFA such as GLA [4], AA [5], EPA, and DHA [6-9] using lipase-catalyzed reactions, such as hydrolysis [10, 11], esterification [6, 12], alcoholysis [7], and a combination of hydrolysis and esterification [13].

To the best of our knowledge, there have not yet been any studies on the enrichment of SDA of natural plant origin using enzymatic method. In this study, enrichment of SDA from echium oil via a two-step lipase-catalyzed esterification was carried out. The first step was attempted to enrich SDA via Candida rugosa lipase-catalyzed esterification of the fatty acids (ca. 14% SDA) from echium oil with lauryl alcohol. The effects of several variables upon the enrichment of SDA were investigated including the water content, the reaction temperature, and the enzyme loading. In the second step, Lipozyme RM IM-catalyzed esterification of ethanol and the fatty acid (ca. 40% SDA) from the first step was carried out to further enrich the SDA. The effects of the water content and the reaction temperature were also investigated.

2 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Conclusions
  7. References

2.1 Materials

Echium oil was purchased from De Wit Speciality Oils (Texel, Holland). Lipase OF from C. rugosa was purchased from Meito Sangyo Co., Ltd. (Nagoya, Japan). Lipozyme RM IM from Rhizomucor miehei was purchased from Novo Nordisk Bioindustry, Ltd. (Seoul, Korea). Lauryl alcohol (purity, ≥99.0%) was purchased from Sigma Chemical Company (St. Louis, MO, USA). Silica gel 60 for column chromatography was purchased from Merck KGaA (Darmstadt, Germany). All of the other chemicals used in this study were purchased as the analytical grade unless otherwise noted.

2.2 Preparation of fatty acid from echium oil

Echium oil (150 g) was added to a solution of sodium hydroxide (60 g) in distilled water (150 mL) and ethanol (99%, 450 mL). The mixture was refluxed with stirring 500 rpm for 1 h. Water (300 mL) was added to the saponified mixture and the aqueous layer containing the saponifiable matter was acidified by adding an aqueous solution of 6 N HCl and adjusted to pH 1 to release the free fatty acids. The upper layer containing the fatty acid was extracted into n-hexane (300 mL) and washed twice with distilled water (150 mL). The hexane layer containing the fatty acid was then dried over anhydrous sodium sulfate. Then n-hexane was separated from the fatty acid by evaporation in a rotary evaporator at 40°C. The residual n-hexane in the fatty acid was removed completely by nitrogen flushing in a water bath of 40°C. The fatty acid composition of echium oil used in this study was shown in Table 1.

Table 1. Fatty acid composition (wt%) of echium oil, the fatty acid fractions obtained after Candida rugosa lipase-catalyzed esterification and Lipozyme RM IM-catalyzed esterificationa)
Fatty acidsInitialb)1st reactionc)2nd reactiond)
  • a)

    Tabular entries are the average of duplicate determinations from different experimental trials.

  • b)

    Echium oil used as a substrate.

  • c)

    Fatty acid fraction obtained after 2 h under the optimum condition for Candida rugosa lipase-catalyzed esterification.

  • d)

    Fatty acid fraction obtained after 8 h under the optimum condition for Lipozyme RM IM-catalyzed esterification.

C16:06.9 ± 0.36.1 ± 0.40.4 ± 0.0
C18:03.3 ± 0.06.5 ± 0.30.6 ± 0.0
C18:1n915.3 ± 0.45.4 ± 0.00.5 ± 0.0
C18:1n70.3 ± 0.20.3 ± 0.50.0 ± 0.0
C18:2n615.0 ± 0.13.6 ± 0.10.4 ± 0.0
C18:3n611.2 ± 0.230.9 ± 0.143.1 ± 1.0
C18:3n333.6 ± 0.17.8 ± 0.10.8 ± 0.1
C18:4n314.3 ± 0.339.3 ± 0.954.1 ± 1.0
SDA yield (wt%)100.0 ± 0.088.5 ± 1.074.8 ± 1.3

2.3 1st step – Candida rugosa lipase-catalyzed esterification

For the 1st step, the fatty acids from echium oil were selectively esterified with lauryl alcohol using C. rugosa lipase. The reactions were performed in a solvent-free system using a 50 mL water-jacketed glass vessel and the vessel was preheated to the desired temperature using a water circulator. Equimolar amounts of the fatty acid (3.0 g, 0.011 mol) and lauryl alcohol (2.0 g, 0.011 mol) were placed in the vessel together with the desired amount of deionized water. The reaction was subsequently initiated by the addition of the enzyme and the stirring of the reaction mixture with a magnetic stirrer at 500 rpm. Samples of 70 mg were withdrawn periodically and filtered through a 45 µm GHP Acrodisc syringe filter (Pall Corporation, Port Washington, NY, USA) to completely remove the enzyme. The samples were analyzed by TLC and GC.

2.4 Separation of the free fatty acids from the 1st step reaction mixture

A large-scale version of the first step was conducted under the optimum conditions. Equimolar amounts of the fatty acid (60.0 g, 0.22 mol) and lauryl alcohol (40.0 g, 0.22 mol) were placed in the 500 mL water-jacketed glass vessel together with 0.25% water of the total substrate and the vessel was preheated to 30°C. The reaction was subsequently initiated by the addition of 2% enzyme of the total substrate and the stirring of the reaction mixture with a magnetic stirrer at 500 rpm. And the SDA enriched fatty acid fraction was separated by the saponification of the 1st step reaction mixture which contained the free fatty acids, lauryl esters, and lauryl alcohol. Briefly, the reaction mixture (100 g) was dissolved in n-hexane (1 L) and the miscella was filtered through anhydrous sodium sulfate to remove any water and enzyme. A solution of sodium hydroxide (3.8 g) in distilled water (200 mL) and ethanol (95%, 200 mL) were added to the solution, and the mixture was placed into a 2 L separate funnel. The lower layer was collected and placed into another separate funnel and washed with n-hexane (1 L) to completely remove any residual lauryl esters. The layer was then acidified by the addition of concentrated hydrochloric acid (20 mL), and washed with n-hexane (200 mL) to allow for the recovery of the fatty acids. This fraction was then washed three times with distilled water (20 mL) and was filtered through anhydrous sodium sulfate. The solvent was evaporated with a rotary evaporator.

2.5 2nd step – Lipozyme RM IM-catalyzed esterification

To further enrich the SDA, the selective esterification using Lipozyme RM IM from R. miehei were performed with the fatty acid fractionated from the 1st step reaction mixture and ethanol. The fatty acids (3.0 g, 0.011 mol) from the 1st step reaction mixture and 99.9% ethanol (1.0 g, 0.022 mol) were placed in a 50 mL water-jacketed glass vessel and the reaction was initiated by the addition of the enzyme and stirring of the mixture with a magnetic stirrer at 250 rpm. Samples (70 mg) were withdrawn from the reaction mixture at appropriate time intervals for analysis.

2.6 Product analysis

Thirty milligrams of sample dissolved in a suitable amount of chloroform was loaded on a TLC silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany) and developed using a petroleum ether/diethyl ether/acetic acid (100:20:1, by volume) eluent. The free fatty acids and fatty acid esters were detected by spraying the TLC plate with a 2,7-dichlorofluoroscein solution (0.2% in 95% methanol). The bands corresponding to the free fatty acids and fatty acid esters were scrapped off from the TLC plate and methylated with 14% BF3 in methanol. The FAME were subsequently analyzed by a Varian 3800 GC (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 flame-ionization detector. The column was held at 180°C for 1 min and then heated to 210°C for 10 min at a rate of 1.5°C/min. Helium was used as the carrier gas with a total gas flow rate of 50 mL/min. The injector and detector temperatures were set at 240 and 250°C, respectively. The FAME were identified by comparison with the retention times of the standards. Heptadecanoic acid was used as an internal standard.

SDA content in the fatty acid fraction (wt%) and SDA yield in the fatty acid fraction (wt%) were calculated as follows:

  • display math
  • display math

where a, the weight of total fatty acid of the reaction product; b, the weight of SDA in the fatty acid of the reaction product; c, the weight of SDA in the fatty acid ester of the reaction product.

3 Results and discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Conclusions
  7. References

3.1 1st step – Candida rugosa lipase-catalyzed esterification

C. rugosa lipase-catalyzed esterification of fatty acids with lauryl alcohol has been suggested as an effective method for the enrichment of valuable fatty acids [5, 14]. Nagao et al. [14] reported that t10,c12-CLA was enriched in fatty acid, and c9,t11-CLA was recovered in lauryl esters when the esterification was performed with CLA mixture and lauryl alcohol using C. rugosa lipase as a biocatalyst. An attempt was also made to enrich AA from Mortierella single-cell oil via C. rugosa lipase-catalyzed esterification by Shimada et al. [5]. In this study, C. rugosa lipase-catalyzed esterification with fatty acids from echium oil and lauryl alcohol was examined as a first step and the effects of several important parameters on the enrichment of SDA, including the water content, temperature, and enzyme loading were studied.

3.1.1 Effect of the water content

Enzymatic reactions in non-aqueous media are critically sensitive to the level of water in the system. A minimal amount of water must be present for the enzyme to exist in its quasi optimum configuration in terms of its catalytic activity and selectivity [15]. Larger quantities of water, however, can be detrimental to enzymatic reactions because they could lead to a decrease in the catalytic activity of the enzyme, as well as undesired hydrolysis reactions [16, 17].

In this study, preliminary trial reactions were conducted in an attempt to establish a suitable range for the water content. C. rugosa lipase-catalyzed esterifications within a wide range of different water contents (from 0 to 25%) were studied. However, no differences were detected between the SDA contents obtained at water contents >3% (data not shown) even though high amount of water (ca. 20%) as an optimum water content for concentration of DHA or AA using C. rugosa lipase was reported by previous studies [5, 14]. The optimum water content may be different depending on the amount of enzyme loading, substrates, and reaction systems. The effect of water content on the SDA content and yield in the fatty acid fraction following C. rugosa lipase-catalyzed esterification was thus investigated in the range of 0–3% of total substrate weight (Fig. 1). For these experiments, the molar ratio of fatty acid to lauryl alcohol, temperature, and enzyme loading were 1:1, 30°C, and 0.3% of the total substrate weight, respectively.

image

Figure 1. Effects of the water content on the SDA content (A) and the yield (B) in the fatty acid fraction after Candida rugosa lipase-catalyzed esterification at 30°C with a 0.3% enzyme loading, and a fatty acid to lauryl alcohol ratio of 1:1 (mol/mol) as a function of reaction time. Filled circle, 0%; open circle, 0.1%; filled reverse triangle, 0.25%; open triangle, 0.5%; filled square, 0.75%; open square, 1%.

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For the trial reaction involving no added water (0%), although the yield of SDA was higher (95%) than that of any of the reactions containing water, increases in the SDA content rarely occurred. During the first 6 h of reaction, there was a significant increase in the SDA content when the water content was increased from 0 to 0.25%, but the differences achieved between the SDA contents at water contents >0.25% were negligible. Furthermore, a maximum SDA content of 35.9% was achieved after only 8 h, with a water content of 0.25%. For the yield of SDA, although similar yields were obtained at water contents of 0.1–0.25% after 8 h, the SDA content from the reaction containing a water content of 0.25% was higher than that with a water content of 0.1%. Too much water gave an undesired effect on the enzymatic activity, which can have an adverse impact on the selectivity of an enzyme. Furthermore, several reports have demonstrated that the catalytic activity and internal flexibility of the enzyme are dependent on the amount of water bound to the enzyme rather than the enzyme itself [18, 19]. Consequently, 0.25% was selected as the optimum water content to study the effects of the other process variables.

3.1.2 Effect of temperature

Reaction temperature can have a significant impact on the rate of an enzymatic reaction, and the temperature therefore represents an important process parameter on enzymatic reactions. Most of studies on C. rugosa lipase were performed at near room temperature [5, 14]. The effects of different temperatures on the SDA content and yield in the fatty acid fraction following C. rugosa lipase-catalyzed esterification as a function of reaction time are shown in Fig. 2. Temperatures in the range of 20–50°C were evaluated in present study. For these experiments, the molar ratio of fatty acid to lauryl alcohol, water content, and enzyme loading were 1:1, 0.25% of the total substrate weight, and 0.3% of the total substrate weight, respectively. After 2 h of the reaction, the SDA content increased as the temperature increased from 20 to 30°C. Further increase in the temperature, however, led to a significant decrease in the SDA content. The highest SDA content (37.9%) was obtained at 30°C and 12 h with 87.4% yield. It is worth mentioning that the melting point of lauryl alcohol substrate is 24°C, and that the reduction in the selectivity of C. rugosa lipase at 20°C compared with 30°C may therefore have occurred as a consequence of the melting point of lauryl alcohol being lower than that of the reaction temperature. Most of the studies on C. rugosa lipase-catalyzed esterification were performed at room temperature or above and provided similar results to those obtained at 30°C in the present study [5, 14]. Overall, 30°C was selected as the optimum temperature for the enrichment of SDA via the lipase-catalyzed esterification of the fatty acids from echium oil with lauryl alcohol in terms of the SDA content and the yield.

image

Figure 2. Effects of the temperature on the SDA content (A) and the yield (B) in the fatty acid fraction after Candida rugosa lipase-catalyzed esterification with a 0.25% water content, 0.3% enzyme loading, and fatty acid to lauryl alcohol ratio of 1:1 (mol/mol) as a function of reaction time. Filled circle, 20°C; open circle, 30°C; filled reverse triangle, 40°C; open triangle, 50°C.

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3.1.3 Effect of enzyme loading

One of the main factors effecting the enrichment of SDA during a lipase-catalyzed esterification is the selection of an appropriate enzyme loading. At a fixed amount of substrate, the rates of enzymatic reaction are dependent on enzyme loading up to a maximum. The number of active sites present for a reaction is proportional to the enzyme loading and is one of the most important factors to be considered in the study of any enzymatic reaction. However, the use of too much enzyme can have an adverse impact on the selectivity of the enzymes [20, 21]. It is therefore of crucial importance that the optimum enzyme loading is carefully determined for reasons of economical feasibility and efficiency. The effects of different enzyme loadings on the SDA content and yield in the fatty acid fractions resulting from the C. rugosa lipase-catalyzed esterification as a function of reaction time are shown in Fig. 3. Enzyme loadings in the range of 0.1–3% of the total substrate weight were evaluated in the current study. For these experiments, the molar ratio of fatty acid to lauryl alcohol, water content, and temperature were set at 1:1, 0.25% of the total substrate weight, and 30°C, respectively. A marked increase in the SDA content of the fatty acid fraction was observed as the enzyme loading was increased from 0.1 to 2%. Further increase in the enzyme loading from 2 to 3%, however, did not lead to further increases in the SDA content. The maximum SDA content (ca. 40%) was achieved after a reaction time of only 2 h with an enzyme loading of 2% with 88.5% yield. Although, a similar maximum SDA content was obtained using an enzyme loading of 1%, a fourfold increase in the reactions time was required compared with an enzyme loading of 2%. Based on these results, an enzyme loading of 2% was selected as the optimum value in terms of the reaction time, as well as the enrichment and yield of SDA.

image

Figure 3. Effects of the enzyme loading on the SDA content (A) and the yield (B) in the fatty acid fraction after Candida rugosa lipase-catalyzed esterification at 30°C with a water content of 0.25% and a fatty acid to lauryl alcohol ratio of 1:1 (mol/mol) as a function of reaction time. Filled circle, 0%;open circle, 0.3%; filled reverse triangle, 0.6%; open triangle, 1%; filled square, 2%; open square, 3%.

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3.2 2nd step – Lipozyme RM IM-catalyzed esterification

PUFA was effectively enriched by alcoholysis using Lipozyme RM IM from R. miehei as a biocatalyst [4, 10, 13]. In the present study, a maximum SDA content of ca. 40% was achieved from the first step which was performed via C. rugosa lipase-catalyzed esterification. To further enrich the SDA, Lipozyme RM IM-catalyzed esterification was carried out based on the idea that this lipase would selectively target short chain and saturated fatty acids. The SDA enriched fraction (ca. 40%) obtained from the first step was used in the second step with ethanol to enrich the SDA in the fatty acid fractions. In the 2nd step, variations in the water content and temperature were investigated to determine the optimum reaction parameters.

3.2.1 Effect of water content

Lipase-catalyzed esterifications require small amounts of water to keep the enzyme hydrated and achieve good catalytic activity. The water content of these reaction systems can also have a significant impact on both the rate of the reaction and the position of the equilibrium. The activities of most lipases increase with increasing water content, although too much water can lead to the aggregation of certain lipases, which can have an adverse impact on their activity [22, 23].

The effects of different water contents on the SDA content and yield in fatty acid fractions following Lipozyme RM IM-catalyzed esterification as a function of reaction time are shown in Fig. 4. Water contents in the range of 0–3% of the total substrate weight were evaluated in the current study. For these experiments, the molar ratio of fatty acid to ethanol, enzyme loading, and temperature were set at 1:2, 2%, and 30°C, respectively. No significant increase in the SDA content was observed when water content was increased, even though the initial reaction rate increased. The highest SDA content of 54.1% was obtained after 8 h when no extra water was added (0%). These results indicated that the fast reaction rate led to a reduction in the selectivity of Lipozyme RM IM. In present study, water was produced during the reaction as a reaction product during the esterification and subsequently accumulated in the reaction mixture. Water can influence the molecular configuration of enzymes in organic media, which can also affect the activity and selectivity of the enzymes, although the details of the ways in which these changes occur remain unclear [24-27]. Lipozyme RM IM from R. miehei can also be affected in the activity as well as selectivity by water content and this enzyme displays good activity in low water environments [28].

image

Figure 4. Effects of the water content on the SDA content (A) and the yield (B) in the fatty acid fraction after Lipozyme RM IM-catalyzed esterification at 30°C with a 2% enzyme loading and a fatty acid to ethanol ratio of 1:2 (mol/mol) as a function of reaction time. Filled circle, 0%; open circle, 0.2%; filled triangle, 0.6%; open triangle, 1%; filled square, 3%.

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The reductions observed in the SDA content after the reactions reached equilibrium under the different water content conditions may have been caused by an increase in the rate of the corresponding hydrolysis reactions, which are the reverse reaction of the esterification. Therefore, controlling the water content is an important factor in terms of inhibiting the hydrolysis reaction, which could weaken the selectivity of the lipase. Taken together these results indicated that an additional water would not be effective for the 2nd reaction of Lipozyme RM IM-catalyzed esterification.

3.2.2 Effect of temperature

The use of high reaction temperatures can lead to an increase in the reaction rate in accordance with the Arrhenius law [29]. However, too high temperature could lead to the irreversible denaturation of the enzyme protein and a reduction in the activity of the enzyme [30]. Furthermore, the optimum reaction temperature can differ according to the reaction conditions, substrate, and enzyme [31, 32]. The effects of different temperatures on the enrichment of the SDA in the fatty acid fraction following Lipozyme RM IM-catalyzed esterification as a function of reaction time are shown in Fig. 5. At temperature in the range of 20–40°C, the SDA content reached its maximum value after 8 h and then decreased slightly except in the case of the experiment conducted at 10°C. Overall, the increasing rate of SDA content at 10°C was the slowest of all of the temperature conditions evaluated. The maximum SDA content was 54.1% after 8 h at 30°C in the range of 20–40°C. This result appeared to be different from those of the other studies about esterification using Lipozyme RM IM that were carried out at higher temperature than 30°C [32, 33]. Rahmatullah et al. [4] reported the successful enrichment of GLA from fatty acids of borage oil via Lipozyme RM IM-catalyzed selective esterification of the fatty acids with 1-butanol in the presence of n-hexane as a solvent, with 50°C being established as the optimum temperature for the process. Even though the same enzyme was used in this particular case to affect a similar reaction, a different optimum temperature was obtained. This difference in the optimum temperature could be attributed to the fact that they conducted their investigation in a solvent system, whereas our reaction was conducted in a solvent-free system. On the basis of these results, a reaction temperature of 30°C was selected as the optimum temperature for the process, in terms of SDA content and yield.

image

Figure 5. Effects of the temperature on the SDA content (A) and the yield (B) in the fatty acid fraction after Lipozyme RM IM-catalyzed esterification with an enzyme loading of 2%, water content of 0%, and a fatty acid to ethanol ratio of 1:2 (mol/mol) as a function of reaction time. Filled circle, 10°C; open circle, 20°C; filled reverse triangle, 30°C; open triangle, 40°C.

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3.3 Fatty acid specificity of lipases on SDA enrichment

The fatty acid specificities of lipases can be affected by the individual characteristics of different fatty acids, such as the length of carbon chain, and the number and position of their double bonds. C. rugosa lipase is known to be weakly active towards several PUFA, including AA, EPA, and DHA [5, 8]. In this study, SDA was enriched by a two-step lipase-catalyzed esterification using different lipases, and the fatty acid compositions of SDA enriched fatty acid fractions from the 1st and 2nd reaction are shown in Table 1. C. rugosa lipase exhibited a strong preference towards Δ9-unsaturated fatty acids such as oleic acid, linoleic acid, and ALA, whereas it discriminated strongly against both GLA and SDA. Through C. rugosa-catalyzed esterification, the SDA and GLA contents increased from 14.3 and 11.2% in the starting material to 39.3 and 30.9% in the fatty acid fraction, respectively. In contrast, no significant differences were observed between the saturated fatty acids contents of the echium oil fatty acid and fatty acid fractions after the 1st reaction. The SDA could therefore be further enriched by the removal of the saturated fatty acids from the SDA enrichment obtained from the 1st reaction. Hills et al. [34] demonstrated that Lipozyme RM IM strongly discriminated against GLA and DHA, so GLA content of evening primrose oil could be enriched seven- to ninefold. In another study, it was established that Lipozyme RM IM preferred fatty acids with a cis double bond at the Δ9-position [35]. In this study, Lipozyme RM IM appeared to have a high selectivity towards saturated fatty acids as well as fatty acids containing double bonds at the Δ9-position. Therefore, SDA could be further enriched in the fatty acid fraction via Lipozyme RM IM-catalyzed esterification with ethanol as a second step reaction. Given that Lipozyme RM IM acted on GLA as weakly as on SDA like C. rugosa, these two fatty acids were enriched in the fatty acid fraction. Finally, these two fatty acids were identified as the major peaks in the chromatograms of the fatty acid fraction following the two-step lipase-catalyzed esterification. The maximum SDA content and total content of SDA and GLA was 54.1 and 97.2% after the two-step lipase-catalyzed esterification.

4 Conclusions

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Conclusions
  7. References

SDA enrichment was successfully achieved by a two-step lipase-catalyzed esterification using Lipase OF from C. rugosa and Lipozyme RM IM from R. miehei. In both reactions, SDA was enriched in the fatty acid fraction of reaction mixture and GLA was also enriched together with SDA. The total content of SDA and GLA in the starting material and in the final enrichment were 25.5, and 97.2%, respectively.

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).

The authors have declared no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Conclusions
  7. References
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