Separation of saturated fatty acids from docosahexaenoic acid‐rich algal oil by enzymatic ethanolysis in tandem with molecular distillation

Abstract Algal oil, rich in docosahexaenoic acid (DHA) and an environmentally sustainable source of ω‐3 fatty acids, is receiving increasing attention. In the present study, a novel approach combining ethanolysis with a 1,3‐specific immobilized lipase (Lipozyme® TL IM) and molecular distillation was investigated to increase the DHA content of algal oil. Algal oil with a 45.94% DHA content was mixed with ethanol, pumped into a column filled with Lipozyme® TL IM, and then circulated for 4 hr at room temperature. The ethanol was then recycled by vacuum distillation. At an evaporator temperature of 150°C, the residue was separated by molecular distillation into a heavy component enriched with DHA glycerides (in the form of triglyceride (TG), diglyceride (DG), and monoglyceride (MG)) and a light component enriched with palmitic acid (PA) and DHA ethyl ester (EE). As a result, 76.55% of the DHA from the algal oil was present in the heavy component, whose DHA content was 70.27%. DHA‐MG was collected in the heavy component mostly in the form of 1‐MG. Lipozyme® TL IM appeared to specifically target PA rather than DHA at the sn‐1(3) position. The Lipozyme® TL IM allowed 90.03% of the initial DHA yield to be retained after seven reaction cycles. Therefore, an eco‐friendly and simple method for increasing the DHA content in algal oil has been developed.


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DHA enrichment is of great interest for the producers of functional foods, healthcare products, and drugs. Fish oil commonly contains 12% DHA and 18% eicosapentaenoic acid (EPA). Currently, the ethyl ester (EE) form is widely used to enrich ω-3 fatty acids in fish oil. The EE form is converted from the natural triglyceride (TG) form to obtain higher levels of EPA and DHA. However, DHA-EE might not be fully absorbed owing to its low bioavailability. The absorption rate of DHA-TG was found to be about 57% in humans, while that of DHA-EE was only about 21% (Lawson & Hughes, 1988). DHA-EE also produces ethanol as a metabolite, so is not suitable for children or adults with an alcohol allergy. DHA-EE is reportedly less stable and produces more harmful oxidative byproducts than DHA-TG (Ritter, Budge, Jovica, & Reid, 2015;Yoshii et al., 2002). In products derived from fish oil, it is difficult to avoid contamination with heavy metals, such as methylmercury (Silbernagel et al., 2011), and organic pollutants, such as polychlorinated biphenyls (Fernandes, Rose, White, Mortimer, & Gem, 2006). Furthermore, fish oil is not suitable for vegetarians. Therefore, DHA-rich algal oil has advantages over mainstream fish oil products on the market in terms of absorption rate, stability, and safety.
Through heterotrophic culture, n-3 biotechnological processes for DHA have been well established and been developed into industrial-scale production. Commonly, algal oil obtained from Schizochytrium sp. has a DHA content of about 45% and occupies an increasing market share. For nutritional and pharmacological purposes, it is vital to further increase the DHA concentration. Urea complexation (Hayes, Bengtsson, Van Alstine, & Setterwall, 1998), low-temperature crystallization (Brown, 1941), solvent extraction, and column chromatography, which require huge amounts of solvent, are widely used to concentrate or purify different forms of fatty acid.
The present study will use enzymatic ethanolysis techniques to avoid the large-scale use of toxic solvents. This environmentally friendly approach aims to combine enzymatic ethanolysis with molecular distillation to increase the DHA content of algal oil.

| Enzymatic ethanolysis of DHA algal oil
The DHA-rich algal oil was mixed well with ethanol then pumped into a column filled with Lipozyme ® TL IM and circulated using a peristaltic pump. The optimum conditions for algal oil ethanolysis were an algal oil/ethanol mass ratio of 1:2 at room temperature for 4 hr with 13% Lipozyme ® TL IM (w/w, relative to total reactants) as the catalyst, as determined by a previous study (He, Bai, Hong, Chen, & Yi, 2016). The product was then distilled under reduced pressure to recycle the ethanol. After each round of the ethanolysis reaction, the immobilized lipase was washed by pumping three column volumes of ethanol through the column. After removing the solvent, the recycled lipase was stored at 4°C before reuse in a new reaction.

| Molecular distillation
KDL-2 short path distillation equipment (UIC Corporation, Alzenau, Germany) was used in the present study. The algal oil was placed in the feed vessel, which was surrounded by a heating jacket. A temperature of 40°C was used to ensure that the oil flowed well. The vacuum pump was switched on after liquid nitrogen had been added to the cold trap. The evaporation pressure fell to below 1 × 10 −3 mPa after the diffusion pump was turned on. The wiper basket was switched on at a low speed when the evaporator temperature rose. When the desired temperature had been reached, the speed of the wiper basket was set to 350 rpm. The feed vessel was then cautiously opened, and the oil allowed to flow dropwise into the evaporation system through the roller wiper system at a feed rate of approximately 80 ml/h. The light components were quickly vaporized and cooled on the condenser surface, and the distillate was collected. Almost nothing was found in the cold trap cooled with liquid nitrogen. Any heavy components that could not be vaporized were collected in a collecting bulb. After collection, all samples were kept below 4°C in the refrigerator.

| Gas chromatography (GC) analysis
The FAMEs were prepared based on the AOCS method Ce-1b 89 (2007). A 37-component FAME mix from Sigma-Aldrich was used as the FAME external standard, which was run in parallel with the samples.
The injection port and detector temperatures were both set at 250°C.
The column temperature was initially ramped to 210°C at a rate of 3°C/min and held for 1 min, then ramped to 219°C at a rate of 0.5°C/ min and held for 1 min, and finally ramped to 240°C at a rate of 10°C/ min and held for 13.5 min. The final temperature was 250°C, which was held for 1 min. The GC peaks were identified by comparing the retention times with those of the corresponding standards, with the relative contents then calculated, and expressed in mol%.
The PA-EE was analyzed under the same GC conditions. The concentrations of PA-EE were calculated from the peak areas from GC using the corresponding linear regression equation in an external standard calibration method.

| High-performance liquid chromatography (HPLC) analysis
The analytes were separated on a Waters 2695 Alliance HPLC system with a 2998 PDA detector (Waters) using an Agilent C18 column

| Analysis of fatty acid composition at sn-2 position
The fatty acid composition at the sn-2 position of the algal oil was determined by pancreatic lipase-catalyzed hydrolysis (Luddy, Barford, Herb, Magidman, & Riemenschneider, 1964;Pina-Rodriguez & Akoh, 2009). Briefly, 1 M Tris-HCl buffer (2 ml), 0.05% sodium cholate solution (0.5 ml), 2.2% calcium chloride solution (0.2 ml), and pancreatic lipase (40 mg) were mixed with algal oil (100 mg) then incubated at 40°C for 3 min. The mixture was then vortexed for 2 min, and 6 M HCl (1 ml) was added to stop the reaction. The hydrolyzed product was extracted twice with diethyl ether (2 ml), which was then removed by blowing with nitrogen gas. The concentrated extract was separated on a silicic acid 60 F 254 TLC plate (Yantai Chemical Industry Research Institute) impregnated with a solution of boric acid in methanol (5%, w/v) to prevent isomerization, developed using hexane/diethyl ether/acetic acid (50:50:1, v/v/v), and visualized under UV light (254 nm). The band corresponding to 2-MG was scraped off the TLC plate then converted to the corresponding FAME. The composition was then analyzed by GC.

| Analysis of glyceride composition in heavy component
To analyze the glyceride species in the heavy component, the sample was separated on a silicic acid 60 F 254 TLC plate, developed using hexane/diethyl ether/acetic acid (80:20:1, v/v/v), and visualized under UV light (254 nm). The bands corresponding to MG, DG, and TG were scraped off the TLC plate, converted to the corresponding FAME, and then analyzed by GC. The total peak areas were calculated for each glyceride. The relative content of each glyceride in the heavy component was then estimated by comparison with the total peak areas.  Table 1 shows that the predominant fatty acid of algal oil was DHA, accounting for 45.94% of the total fatty acids. The percentage of DHA located at the sn-2 position of algal oil was 55.43%. The fatty acid composition of the sn-1(3) position was calculated based on the total TG and sn-2 position fatty acid compositions, according to the formula (3 × TG-sn-2 MG)/2 (Turon, Bachain, Caro, Pina, & Graille, 2002).

| Total and sn-2 fatty acid compositions of algal oil
Palmitic acid (PA) was also an abundant fatty acid in algal oil, accounting for 37.40% of the total fatty acids, mostly distributed at the sn-1 or sn-3 positions. Algal oil also contained a high level of n-6 docosapentaenoic acid (DPA, 8.93%), a polyunsaturated fatty acid, which was also found to be preferentially located at the sn-2 position.

| Reusability of Lipozyme ® TL IM in ethanolysis
The reusability of the immobilized lipase was crucial for the feasibility of this bioprocess and the practical application of lipase.
To evaluate the potential of the lipase for industrial application, the operational stability of Lipozyme ® TL IM was determined by TA B L E 1 Total, sn-2, and sn-1(3) fatty acid compositions of DHA-rich algal oil (n = 3)

| Molecular distillation
The PA, DPA, and DHA percentages in the light and heavy components under different molecular distillation conditions were determined by GC (   (Table 3).  (Table 4).

| Glyceride composition in the heavy component
As shown in Figure 4, the glycerides in the heavy component con-

| D ISCUSS I ON
As saturated fatty acids were found to be located mainly at the sn-1 or sn-3 position of the glycerol backbone of TG (Table 1) DHA-MG than sn-2 DHA-MG was found in the distilled sample ( Figure 2). Acyl migration from the sn-2 position to the sn-1(3) position has been reported to occur spontaneously, reaching a molar proportion of about 9:1 between 1-MG and 2-MG (Boswinkel, Derksen, Riet, & Cuperus, 1996;Sanchez, Tonetto, & Ferreira, 2016).
A high temperature during molecular distillation also favors this migration (Ferreira & Tonetto, 2017;Poisson, Devos, Godet, Ergan, & Pencreac'h, 2009 position of the glycerol backbone. Lipozyme ® 435 can reportedly be recycled 5-10 times without an obvious loss in activity (Zhang et al., 2018). As Lipozyme ® TL IM is destroyed when stirred, a fixed bed reactor was selected for the present study which has shown that it can be used up to seven times with only a 10% loss in its   (Table 3). Therefore, an evaporator temperature of about 150°C was found to be efficient for separating the EE and MG forms of DHA. Therefore, 150°C was set as the optimum temperature in the present study for excluding the EE form of DHA from the heavy component.
When the evaporator temperature was higher than 150°C, although more saturated fatty acids would be evaporated and collected in the light component, some DHA-MG would also be evaporated.
This explained the inefficiency of further raising the DHA content by increasing the evaporator temperature above 150°C. Our findings showed that enzymatic ethanolysis in tandem with molecular distillation can also be used for the synthesis and separation of DHA-EE and DHA-MG by altering the evaporator temperature. In theory, DG and TG can be separated by further increasing the evaporator to a higher temperature but when the temperature was higher than 180°C, we found that the color of the DHA algal oil became dark rather than remaining a light yellow color, making it necessary to use other methods, such as column chromatography, to further separate DG and TG.
In conclusion, the present study has developed an eco-friendly and simple method combining ethanolysis and molecular distillation to separate saturated oils from algal oil thereby increasing its DHA content from 45.94% to 70.27%.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.