Purification of 1,2-Diacylglycerols by a Two-Step Crystallization

Feb 8, 2017 - 1,2-Diacylglycerols (1,2-DAGs) have wide applications in food, medicine, and chemical industries. In this study, a two-step solvent crys...
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Purification of 1,2-Diacylglycerols by a Two-Step Crystallization Yuxiao Zhu,† Qingzhe Jin,† Xingguo Wang,† and Xiaosan Wang*,† †

State Key Laboratory of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, P. R. China ABSTRACT: 1,2-Diacylglycerols (1,2-DAGs) have wide applications in food, medicine, and chemical industries. In this study, a two-step solvent crystallization was designed to purify 1,2-DAGs. First, the crude alcoholysis product was crystallized in a nonpolar solvent. Under the optimized conditions (a 1:10 substrate ratio of crude product to hexane; −40 °C for 18 h), fatty acid ethyl esters and triacylglycerols were fully removed. Subsequently, the solid fraction obtained in the first step purification was crystallized further in a polar solvent. Under the optimized conditions (1:12 substrate ratio of product to methanol; −20 °C for 6 h), pure 1,2-DAGs were obtained in 77.3% overall yield. Compared to previous studies, our method for the separation of 1,2-DAGs is readily scalable, much simpler, and very effective. Additionally, the process is much milder and avoids acyl migration of 1,2-DAGs toward 1,3DAGs, which favors the stability of the target product.

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INTRODUCTION Diacylglycerols (DAGs) are widely applied as emulsifiers and surfactants in the food, pharmaceutical, cosmetic, chemical, and other industries.1,2 DAGs are formed when one fatty acid at the glycerol bone of triacylglycerols (TAGs) is released. They are divided into two isomers: 1,2-DAGs and 1,3-DAGs. Different from 1,3-DAGs, 1,2-DAGs are featured in many functions. 1,2DAGs are an intracellular second messenger mediating the migratory response of leukocytes, so as to accelerate the healing of wounds,3 and are also involved in ameliorating myocardial dysfunction in diabetic rats.4 Besides, pure 1,2-DAGs have great potential as intermediates for the synthesis of phospholipids, glycolipids, prodrugs, and structured TAGs and the preparation of numerous enzyme agonists and antagonists.5−7 To date, the reported methods for the purification of 1,2DAGs include column chromatography, molecular distillation, preparative high-performance liquid chromatography (HPLC), and liquid carbon dioxide extraction.8−11 In addition to molecular distillation, none of them is suitable for large-scale purification. Furthermore, purification by preparative HPLC and liquid carbon dioxide extraction needs high equipment input and maintenance costs. Liquid carbon dioxide extraction only separates 1,2-DAGs from fatty acid esters. For the separation of 1,2-DAGs from other impurities, such as monoacylglycerols (MAGs) and TAGs, the method is not feasible.9,12 Molecular distillation is suitable for large-scale purification, but the method usually needs a high temperature to separate 1,2-DAGs from fatty acid esters based on their differences in volatility. The ratio of 1,3-DAGs to 1,2-DAGs isomers is known to be 2:1 at equilibrium.9,13 Therefore, 1,2DAGs are not stable. Temperature is a key factor affecting acyl migration of 1,2-DAGs. Purification by molecular distillation at a high temperature will promote acyl migration of 1,2-DAGs toward 1,3-DAGs, causing the loss of regiopurity of 1,2-DAGs. © XXXX American Chemical Society

To avoid the disadvantages of the previous methods, a much simpler, effective, and scalable method was developed for the purification of 1,2-DAGs. First, 1,2-DAGs were synthesized by enzymatic ethanolysis of high oleic sunflower oil (HOSO). The crude product contained 1,2-DAGs, MAGs, glycerol, TAGs, and fatty acid ethyl esters. A two-step purification was designed to remove the impurities. The first step of purification aimed to remove fatty acid ethyl esters and TAGs by crystallization in a nonpolar solvent. The second step of purification aimed to further separate 1,2-DAGs from MAGs and glycerol in a polar solvent. The crystallization conditions were optimized to maximize the yield and purity of 1,2-DAGs. The main advantages of this method are that it is scalable and is conducted at a much low temperature, which is beneficial for the inhibition of acyl migration of 1,2-DAGs toward 1,3-DAGs.

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MATERIALS AND METHODS Materials. HOSO containing 83% oleic acid was purchased from Shanghai Liangyou Haishi Oils and Fats Industry Co. Ltd. (Shanghai, China). Novozym 435 (lipase B from Candida antarctica, immobilized on a macroporous acrylic resin) and Lipozyme 435 (a recombinant lipase from C. antarctica, expressed on Aspergillus niger and immobilized on Lewatit VP OC 1600) were obtained from Novozymes (Beijing, China). Diolein standard containing 85% 1,3-diolein and 15% 1,2diolein and dipalmitin were purchased from Sigma-Aldrich Chemical Co. (Shanghai, China). Hexane and isopropyl alcohol were of HPLC-grade and were purchased from Beijing J&K Scientific Co. Ltd. (Beijing, China). All other reagents including

Received: Revised: Accepted: Published: A

October 16, 2016 February 6, 2017 February 8, 2017 February 8, 2017 DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research Table 1. Experimental Design for the Optimization of 1,2-DAG Purification by a Two-Step Crystallizationa first step of purification

a

second step of purification

level

X1

X2 (w/v)

X3 (°C)

X4 (h)

X1

X2 (w/v)

X3 (°C)

X4 (h)

1 2 3 4

hexane isohexane isooctane petroleum ether

1:6 1:8 1:10 1:12

4 −20 −40

6 12 18 24

methanol acetonitrile ethanol isopropyl alcohol

1:10 1:12 1:15 1:18

4 −20 −40

2 4 5 8

X1 = type of solvent; X2 = ratio of product to solvent; X3 = temperature; X4 = time.

solvents were of analytical-grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Synthesis of 1,2-DAGs by Enzymatic Ethanolysis. The reaction mixture containing HOSO and ethanol (1:50 molar ratio of HOSO to ethanol) was stirred at 50 °C for 15 min for emulsification. Ethanolysis was initiated at 50 °C by adding 6% Novozym 435 lipase (relative to the weight of the total reactants) as a catalyst and conducted in a three-necked roundbottom flask with condensation reflux. At the end of the 1 h reaction, the ethanolysis reaction was stopped. Subsequently, the lipase was removed by centrifugation at 4000 rpm for 10 min, and the solvent was removed by rotary evaporation at 35 °C. The resultant product consisted of 1,2-DAGs, MAGs, glycerol, fatty acid ethyl esters, and a small amount of TAGs. The crude product was used as a starting material for purification by crystallization. Purification of 1,2-DAGs by Crystallization. A two-step crystallization method was adopted to purify the crude product containing 1,2-DAGs. The optimum conditions were selected based on the purity and yield of 1,2-DAGs. When the purification conditions were optimized, one factor was changed at different levels, whereas the other factors were kept constant. After one of the factors was optimized, the optimal value of this factor was employed for the next factor optimizations. All crystallizations were run in duplicate unless otherwise specified. The results were expressed as the mean ± standard deviation. First Step of Purification To Remove Fatty Acid Ethyl Esters and TAGs. In the first step of purification, four parameters, including the solvent type, ratio of product to solvent (w/v), crystallization temperature, and time, were evaluated until optimization of the process was achieved. The design for the optimization experiments is outlined in Table 1 and Figure 1. The crude ethanolysis product (5 g) was mixed with a nonpolar solvent at 40 °C for 1 min until a clear phase formed. Subsequently, the solution was placed in a 100 mL roundbottom flask at a controlled temperature for the formation of crystals (the variation in the temperature during crystallization of the samples was about ±1 °C of the set values). Crystallization was stopped at the indicated time points for analysis of the yield and purity of 1,2-DAGs. As shown in Table 1, the selected nonpolar solvents for optimization of the solvent type included hexane, isohexane, isooctane, and petroleum ether. The ratio of crude product to solvent ranged from 1:6 to 1:12 (w/v). The crystallization temperature and time were in the ranges of +4 to −40 °C and 6−24 h, respectively. At the end of crystallization, the solid fraction containing 1,2DAGs, MAGs, and a small amount of glycerol was collected by filtration. Other materials, such as unreacted TAGs and fatty acid ethyl esters, were soluble in a nonpolar solvent and were discarded as the liquid fraction. The solvent in the solid and liquid fractions was evaporated under reduced pressure at 35 °C. Subsequently, the solvent-free solid fraction was stored at

Figure 1. General flowchart for the purification of 1,2-DAGs.

−20 °C and analyzed by HPLC, as described in the following section. Second Step of Purification To Remove Glycerol and MAGs. After the removal of TAGs and fatty acid ethyl esters in the first step, the semipurified product was mainly composed of MAGs, 1,2-DAGs, and glycerol. Therefore, further crystallization in a polar solvent is needed to remove MAGs and glycerol. In the second step of purification, the effects of four variables including the solvent type, ratio of product to solvent (w/v), crystallization temperature, and time on the purity and yield of 1,2-DAGs were investigated until optimization of the process was achieved. After the crystals (5 g) was completely dissolved in a solvent at 40 °C, the solution was placed in a 150 mL round-bottom flask at a controlled temperature for the formation of crystals (the variation in the temperature during crystallization of the samples was about ±1 °C of the set values). The crystallized samples were taken periodically from the freezer for analysis of the yield and purity of 1,2-DAGs. As shown in Table 1, the selected nonpolar solvents for optimization of the solvent type included methanol, acetonitrile, ethanol, and isopropyl alcohol. The optimum ratio of product to solvent was in the range of 1:10 to 1:18 (w/v). Additionally, the crystallization temperature ranged from +4 to −40 °C, and the crystallization time ranged from 2 to 8 h. B

DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research At the end of crystallization, the solid and liquid fractions were separated by filtration. The solid fraction containing 1,2DAGs was collected, whereas the liquid fraction containing MAGs and glycerol, which remained in a polar solvent, was discarded. The solvent in the solid and liquid fractions was evaporated under reduced pressure at 40 °C. Subsequently, the solvent-free solid fraction was stored at −20 °C and analyzed by HPLC, as described in the following section. HPLC−Evaporative Light-Scattering Detection (ELSD) Analysis. The crude and crystallization products were diluted to 0.6 mg/mL with a solvent mixture of hexane and isopropyl alcohol [1:1 (v/v) hexane/isopropyl alcohol] and subsequently quantified by HPLC−ELSD using a Waters 1525 liquid chromatographic system (Waters Corp., Milford, MA) equipped with a Sepax HP-Silica column (particle size of 5 μm, 4.6 mm × 250 mm, Sigma-Aldrich Corp., K.K., Tokyo, Japan), and eluted with a binary gradient of solvent A [1:99 (v/ v) isopropyl alcohol/hexane] and solvent B [1:1:0.01 (v/v/v) isopropyl alcohol/hexane/acetic acid] at 1 mL/min. The column temperature was kept at 35 °C, and the injection volume was 5 μL. Samples were analyzed based on the following gradient profiles: solvent A was decreased from 100 to 80% over 10 min, decreased further to 70% from 10 to 14 min, increased further to 100% from 14 to 20 min, and held at 100% for 5 min. The total run time was 25 min. A DAG mixture containing 85% 1,3-diolein and 15% 1,2diolein was used as the external standard, and the 1,2-DAG peak was identified by the retention time. The purity of the 1,2DAGs was calculated based on its mass relative to the total mass of a particular sample. Statistical Analysis. All data were analyzed by using oneway analysis of variance (ANOVA) of Origin 9.0 software (OriginLab, Northampton, MA). The differences among the means were compared at P = 0.05 using Tukey’s test.

of 1,2-DAGs from the impurities is mainly based on the differences in the polarity and solubility in different solvents. In the first step, nonpolar solvents were employed to remove the nonpolar impurities mainly including TAGs and fatty acid ethyl esters. The results are outlined in Figure 2a. The examined nonpolar solvents were hexane, isohexane, isooctane, and petroleum ether. The data showed that the purity of the 1,2-DAGs present in the solid fraction ranged from 78.1% to 84%. No significant differences were observed in the 1,2-DAG purity for all of the solvents. Different solvents gave similar results because fatty acid ethyl esters and TAGs had good solubility in all of the nonpolar solvents and could not be crystallized out from the solutions at −40 °C, whereas the 1,2DAGs were crystallized easily at such a temperature. Generally, fatty acid ethyl esters have pretty low melting points and are soluble in nonpolar solvents at a pretty low temperature.17 For example, ethyl oleate has a melting point of −32 °C. Therefore, all unsaturated fatty acid ethyl esters, independent of the chain length or number of double bonds, remained in the liquid fraction at the temperature investigated (−40 °C). Similarly, TAGs are not likely crystallized out from a nonpolar solvent either because of a very small amount of TAGs unreacted in the crude product. In terms of the yield of 1,2-DAGs, significant differences were found between hexane and petroleum ether. The 1,2-DAG yield obtained with hexane was significantly higher than that obtained with petroleum ether probably because petroleum ether could solubilize 1,2-DAGs better than hexane, resulting in decreased crystallization of 1,2-DAGs from the petroleum ether at −40 °C. For the rest of the solvent, no statistical differences were found in the yield. Taking both the purity and yield into account, hexane was selected as the optimum solvent for further crystallizations. Subsequently, the effects of the ratio of crude product to hexane on the purity and yield of 1,2-DAGs were investigated. Figure 2b reports the purity and yield of 1,2-DAGs in the solid fraction. When the ratio was changed from 1:6 to 1:12, the 1,2DAG purity increased from 70% to 84.1%. The purity was significantly lower at the 1:6 ratio than at the rest of the ratios. This result may be attributed to crystallization of impurities such as saturated fatty acid ethyl esters and MAGs at a low ratio of product to hexane. Unsaturated fatty acid ethyl esters and TAGs were not likely to crystallize out at −40 °C. For the ratio in the range of 1:8 to 1:12, there were no statistical differences observed in the 1,2-DAG purity. In contrast to the purity, the 1,2-DAG yield showed an inverse relationship with the ratio. The yields of the 1,2-DAGs ranging from 80.8% to 94.9% followed this order: 1:6 > 1:8 > 1:10 > 1:12. The yield obtained at the 1:12 ratio was significantly lower than that obtained at the rest of the ratios. Regarding the ratios ranging from 1:6 to 1:10, the differences were insignificant, although the 1,2-DAG yield decreased slightly with the ratio. When both the yield and purity were considered, the 1:10 ratio was an ideal value for further experiments. For the effect of the temperature investigated, the results are given in Figure 2c. The purity and yield of the 1,2-DAGs exhibited an inverse relationship with the temperature. When crystallizations were performed at −20 and 0 °C, respectively, no crystals were formed in the solutions. The results indicated that the 1,2-DAGs mainly composed of 1,2-diolein tended to be nonpolar compounds and were soluble in hexane at the temperatures investigated. In our preliminary study, pure saturated 1,2-DAG standards could be crystallized out from hexane with a ratio of 1:10 at −20 °C for 24 h. However,

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RESULTS AND DISCUSSION Enzymatic Synthesis of 1,2-DAGs. Enzymatic alcoholysis was carried out in ethanol without the addition of other solvents because a previous study reported by our team showed that the addition of a nonpolar solvent had an adverse effect on the yield of the target product.14 Polar reaction solvents, except methanol, could enhance the selectivity of lipase and selectively alcoholyze the fatty acids at the sn-1,3 positions of the TAGs to produce 1,2-DAGs without the formation of 1,3-DAGs as a byproduct.14−16 At the end of 1 h of reaction, the crude product contained 49.8% 1,2-DAGs, 40.3% fatty acid ethyl esters, 6.9% MAGs, and small amounts of unreacted TAGs and glycerol. The alcoholysis time is a key factor affecting the 1,2DAGs yield. When the time was prolonged from 1 to 2 h, only 19% 1,2-DAGs was formed in the crude product, suggesting further alcoholysis of 1,2-DAGs by Novozym 435 to form 2MAGs. 2-MAGs are extremely unstable and will be converted to glycerol and fatty acid ethyl esters after being migrated to 1MAGs in the presence of Novozym 435 as the catalyst. The crude product obtained was used as the starting material for the purification of 1,2-DAGs. First Step of Purification. The impurities in the crude product can be classified into two types, namely, nonpolar compounds, such as TAGs and fatty acid ethyl esters, and polar compounds, such as MAGs and glycerol. The polarity of the 1,2-DAGs is between them. Therefore, the impurities can be removed by crystallization in a suitable solvent. The separation C

DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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temperature of −40 °C, and a crystallization time of 24 h; (c) 1:10 ratio of crude product to hexane and a crystallization time of 24 h; (d) 1:10 ratio of crude product to hexane and a crystallization temperature of −40 °C.

different from the preliminary study, saturated 1,2-DAGs were not crystallized out in the current study probably because of the presence of unsaturated 1,2-DAGs and MAGs in the solutions as emulsifiers in the crude product. Crystallization at −40 °C provided the highest purity and yield. On the basis of the results given above, −40 °C was chosen as an optimum crystallization temperature. Last, the effects of the crystallization time on the purity and yield of the 1,2-DAGs were studied. The results in Figure 2d indicate that the crystallization time had no effect on the 1,2DAG purity. When the crystallization time was varied from 12 to 24 h, the purity remained constant at around 84%. Within the first 6 h, the purity increased slightly with time. The 1,2DAG purity obtained at 6 h was not significantly lower compared to that at other time points. A slight increase in the 1,2-DAGs within the first 6 h may be attributed to first crystallization of MAGs and then more and more DAGs, leading to a slight increase in the purity. Regarding the 1,2DAG yield, the results showed that the crystallization time had a significant effect on the yield. When the crystallization time ranged from 6 to 24 h, the yield ranged from 50.4% to 90.3%. However, the 1,2-DAG yield obtained at 18 h was not significantly higher than that at 24 h. There was no need to prolong the crystallization time further. Therefore, 18 h was the optimum crystallization time. After the first step of purification, the optimum crystallization conditions were obtained after optimization. Under these conditions (1:10 ratio of crude product to hexane at −40 °C for 18 h), the purity of the 1,2-DAGs reached 85.5% in a 90.3% yield. All fatty acid ethyl esters and TAGs were removed. The 1,2-DAGs with 85.5% purity were used as the starting materials for further purification at the second step. Second Step of Purification. 1,2-DAGs containing one −OH group tend to be nonpolar, whereas MAGs containing two −OH groups tend to be polar. Thus, they can be separated by crystallization or extraction in a polar solvent.18,19 When the product after the first step of purification is crystallized in a polar solvent, the 1,2-DAGs crystallize out from the solution as the solid fraction, whereas the polar impurities, such as MAGs and glycerol, remain in the liquid fraction. After filtration, pure 1,2-DAGs will be obtained with the removal of glycerol and MAGs. In the second step, the same parameters were evaluated for the removal of MAGs and glycerol. The results are presented in Figure 3a−d. First, the effects of the solvent type on the purity and yield of the 1,2-DAGs in the solid fraction were investigated. The type of solvent significantly affected the crystallization process. When methanol was used as the solvent, the solid fraction contained 99.6% 1,2-DAGs, which was significantly higher than those with the rest of the solvents. Compared to the starting material containing 85.5% 1,2-DAGs, the increase in the 1,2-DAG purity in acetonitrile was limited. Surprisingly, no crystals were formed at −20 °C with ethanol and isopropyl alcohol as the solvents and a 1:15 ratio of semipurified product to solvent. The yield ranged from 0 to 82.4%. Methanol also provided the best results compared to

Figure 2. Optimization of the crystallization conditions of 1,2-DAGs in the first step. Crystallization conditions: (a) 1:10 ratio of crude product to solvent, a crystallization temperature of −40 °C, and a crystallization time of 24 h; (b) hexane as the solvent, a crystallization D

DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research Figure 3. continued

crystallization temperature of −20 °C, and a crystallization time of 6 h; (c) 1:12 ratio of semipurified product to methanol and a crystallization time of 6 h; (d) 1:12 ratio of semipurified product to methanol and a crystallization temperature of −20 °C.

other solvents. Crystallization in ethanol and isopropyl alcohol was not satisfactory, although ethanol as a crystallization solvent is preferred because it is safer and greener. However, the purification of 1,2-DAGs in ethanol is feasible by either increasing the ratio and crystallization time or decreasing the crystallization temperature to −40 °C. On the basis of the purity and yield of 1,2-DAGs in the solid fraction, methanol was selected as a solvent for further experiments. Subsequently, the effects of the ratio of semipurified product to methanol were evaluated because the solvent quantity affects the solubilization of the product and thus influences the crystallization of the product. Figure 3b gives the changes of the purity and yield of 1,2-DAGs with the ratio of product to methanol. The results showed that the purity was not affected by the ratio. There was a slight increase in the purity observed when the ratio was varied from 1:10 to 1:12, but no significant differences were seen. When the ratio was in the range of 1:12 to 1:18, the purity of the 1,2-DAGs remained constant. In contrast to the purity, the 1,2-DAG yield decreased with the ratio. The yield was significantly higher at the 1:10 ratio than at the 1:18 ratio. No statistical differences were found among the rest of the ratios, even though the yield obtained at the 1:12 ratio tended to be higher than that obtained at 1:18 (P = 0.07). Increasing the ratio caused a decrease in the yield mainly because a high solvent addition quantity was beneficial for solubilization of the product, making crystallization of the 1,2DAGs from the solution more difficult. On the basis of the results given in Figure 3b, the 1:12 ratio was used as an optimum value for the next purification. The effects of the crystallization temperature were investigated because it affected the crystallization rate, time, and crystal quantity. Generally, the temperature has a negative correlation with the yield, whereas the relationship between the temperature and purity should be positive. As shown in Figure 3c, the purity increased slightly when the crystallization temperature was increased from −40 to −20 °C. This result can be explained by the fact that the impurities remained in the liquid fraction when crystallization was conducted at −20 °C, whereas they were crystallized out as the solid fraction at −40 °C. When the temperature was elevated further from −20 to +4 °C, no crystals formed as the solid fraction and the product remained a liquid in the solution. Regarding the effect of the temperature on the 1,2-DAG yield, the results showed an inverse correlation between them. There were no significant differences in the yield between −20 and −40 °C. However, when crystallization was performed at 0 °C, both the purity and yield dropped to 0. Therefore, the optimum temperature was −20 °C for further experiments. Last, the effects of the crystallization time were examined. As shown in Figure 3d, with an increase in the crystallization time, the 1,2-DAG purity did not changed significantly and 100% purity was achieved at 2 and 4 h time points. Different from the changes of the purity, the yield ranging from 64.6% to 86.3% increased with time. This result was attributed to a long time causing crystallization of the 1,2-DAGs, thereby increasing the yield. However, the impurities always remained a liquid in the

Figure 3. Optimization of the crystallization conditions of 1,2-DAGs in the second step. Crystallization conditions: (a) 1:15 ratio of semipurified product to solvent, a crystallization temperature of −20 °C, and a crystallization time of 6 h; (b) methanol as the solvent, a E

DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 4. HPLC chromatograms of the synthetic product before (a) and after crystallization in hexane (b) and methanol (c): FAEEs, fatty acid ethyl esters; TAGs, triacylglycerols; MAGs, monoacylglycerols. F

DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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solutions, regardless of the changes in time. That is why the 1,2DAG purity remained constant, whereas the yield increased with time. Because the purity and yield of the 1,2-DAGs obtained at 8 h were not significantly higher than those at 6 h, a crystallization time of 6 h was used as the optimum value. Under these conditions, 99.3% 1,2-DAGs was obtained in a 85.6% yield. To the best of our knowledge, no publication in the literature reports the purification of 1,2-DAGs by a low-temperature crystallization. Although various methods are available for purification of the target products, low-temperature crystallization is the preferred method because only a few are suitable for large-scale production.17,20 Generally, low-temperature crystallization is used for the purification of free fatty acids or fatty acid ethyl esters.20,21 We found that the method was also suitable for purification of the partial acylglycerols. For purification of the 1,2-DAGs, the temperature is of vital importance because unstable 1,2-DAGs undergo spontaneous acyl migration to form stable 1,3-DAGs at a relatively high temperature.22 Therefore, the enzymatic synthesis of partial acylglycerols, especially 2-MAGs and 1,2-DAGs, is usually conducted at a relatively low temperature in the range of 35−40 °C,7,23 and they are normally stored at a pretty low temperature below −20 °C. Thus, purification of the 1,2-DAGs also needs to be carried out at a low temperature. Our study exhibited that there was no 1,3-DAG formation during crystallization, suggesting that acyl migration of 1,2-DAGs toward 1,3-DAGs was inhibited under the conditions investigated (Figure 4). Therefore, the main advantage of this method is to achieve inhibition of acyl migration and scale-up production of 1,2DAGs.

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CONCLUSIONS

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AUTHOR INFORMATION

REFERENCES

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After a two-step purification, the nonpolar and polar impurities are fully removed. The purity of the 1,2-DAGs reaches almost 100% with a 77.3% yield. An effective and much simpler method is developed for 1,2-DAG purification in the current study. Low-temperature crystallization results in the production of pure 1,2-DAGs without the formation of 1,3-DAGs. The inhibition of acyl migration of 1,2-DAGs is very important because the separation of 1,2-DAGs from 1,3-DAGs is difficult. The purified 1,2-DAGs are useful in a broad range of cosmeceutical and pharmaceutical applications.

Corresponding Author

*E-mail: [email protected] (X.W.). Tel: +86-51085876799. Fax: +86-510-85876799. ORCID

Xiaosan Wang: 0000-0002-3731-6557 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

This work was financially supported by The Natural Science Foundation of Jiangsu Province (Grant BK20150137) and Program of Science and Technology Department of Jiangsu Province (Grant BY2016022-33). G

DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research (21) Mu, H.; Zhang, H.; Li, Y.; Zhang, Y.; Wang, X.; Jin, Q.; Wang, X. Enrichment of DPAn-6 and DHA from Schizochytrium sp. oil by low-temperature solvent crystallization. Ind. Eng. Chem. Res. 2016, 55, 737−746. (22) Andrews, P. C.; Fraser, B. H.; Junk, P. C.; Massi, M.; Perlmutter, P.; Thienthong, N.; Wijesundera, C. Large-scale synthesis of both symmetrical and unsymmetrical triacylglycerols containing docosahexaenoic acid. Tetrahedron 2008, 64, 9197−9202. (23) Hita, E.; Robles, A.; Camacho, B.; Ramírez, A.; Esteban, L.; Jiménez, M.; Muñío, M. M.; González, P. A.; Molina, E. Production of structured triacylglycerols (STAG) rich in docosahexaenoic acid (DHA) in position 2 by acidolysis of tuna oil catalyzed by lipases. Process Biochem. 2007, 42, 415−422.

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DOI: 10.1021/acs.iecr.6b03997 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX