and docosahexaenoic acid (DHA) enriched fish oil triacylglycerols

Two-stage enzymatic preparation of eicosapentaenoic acid (EPA). 1 and docosahexaenoic acid (DHA) enriched fish oil. 2 triacylglycerols. 3. Running Hea...
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Two-Stage Enzymatic Preparation of Eicosapentaenoic Acid (EPA) And Docosahexaenoic Acid (DHA) Enriched Fish Oil Triacylglycerols Zhen Zhang,†,⊥ Fang Liu,‡,⊥ Xiang Ma,§ Huihua Huang,*,† and Yong Wang*,‡,∥ †

School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China Guangdong Saskatchewan Oil Seed Joint Laboratory, Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China § Research School of Chemistry, The Australian National University, Canberra 2601, Australia ∥ Guangdong Engineering Technology Research Center for Oils and Fats Biorefinery, Guangzhou 510632, China ‡

ABSTRACT: Fish oil products in the form of triacylglycerols generally have relatively low contents of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and so it is of potential research and industrial interest to enrich the related contents in commercial products. Thereby an economical and efficient two-stage preparation of EPA and DHA enriched fish oil triacylglycerols is proposed in this study. The first stage was the partial hydrolysis of fish oil by only 0.2 wt.‰ AY “Amano” 400SD which led to increases of EPA and DHA contents in acylglycerols from 19.30 and 13.09 wt % to 25.95 and 22.06 wt %, respectively. Subsequently, products of the first stage were subjected to transesterification with EPA and DHA enriched fatty acid ethyl esters (EDEE) as the second stage to afford EPA and DHA enriched fish oil triacylglycerols by using as low as 2 wt % Novozyme 435. EDEEs prepared from fish oil ethyl ester, and recycled DHA and EPA, respectively, were applied in this stage. Final products prepared with two different sources of EDEEs were composed of 97.62 and 95.92 wt % of triacylglycerols, respectively, with EPA and DHA contents of 28.20 and 21.41 wt % for the former and 25.61 and 17.40 wt % for the latter. Results not only demonstrate this two-stage process’s capability and industrial value for enriching EPA and DHA in fish oil products, but also offer new opportunities for the development of fortified fish oil products. KEYWORDS: enzymatic hydrolysis, enzymatic transesterification, urea complexation

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concentrates.9 This is not only because EPA and DHA present naturally as triacylglycerols but also due to various advantages. EPA and DHA can be administered for medical or dietetic purposes in different forms, including fish oil, free fatty acids, ethyl esters, or triacylglycerols. Free fatty acids and triacylglycerols have also been shown to be metabolized more rapidly and completely than ethyl esters. However, free fatty acids are oxidatively unstable.10 Therefore, triacylglycerols are considered the most desirable form.11 For example, triacylglycerols are more stable and readily utilized by humans. Triacylglycerols in fish oil were more rapidly recovered in lymph at an early stage after the administration than as free fatty acids and ethyl esters.12 Ikeda and co-workers13 also reported that the lowest recovery of cholesterol was observed when triacylglycerols in fish oil were given, because lymphatic recovery of EPA and DHA given as triacylglycerols was fast and more efficient than corresponding ethyl esters and free acids particularly shortly after administration. The limited related reports prompt us to explore the enrichment of fish oil products with EPA and DHA in the form of triacylglycerols. Wang et al.14 reported that immobilized MAS1 lipase displayed high catalytic efficiency in the production of n-3 polyunsaturated fatty acids enriched triacylglycerols. Signifi-

INTRODUCTION Polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are nutritionally essential for humans, and they are known for many beneficial properties, such as reducing triacylglycerols, anti-inflammation, anticancer, and slowing the progression of Alzheimer’s disease.1 DHA is the most abundant omega-3 fatty acid in central nervous system including the brain and retina, making it of great importance for infants and pregnant women.2 However, most fish oils have limited contents of EPA and DHA, with only about 18 and 12 wt %, respectively.3 Other saturated and low unsaturated fatty acids account for a large amount of the fatty acid compositions of fish oil triacylglycerols.3 These fatty acids are of relatively low nutritive values compared with EPA and DHA. Considering the increasing demand for high-end functional food, exploring the enrichment of EPA and DHA in fish oil products has attracted attention so as to enhance the beneficial effects of fish oils and increase profits for the fishery industry. Existing methods for enrichment of EPA and DHA generally consist of two steps. First, fish oil is subjected to hydrolysis or transesterification to afford EPA and DHA free fatty acids or corresponding esters. The first step’s products are then subjected to purification by low temperature solvent crystallization,4 supercritical carbon dioxide extraction,5 AgNO3 complexation,6 urea complexation,7 or molecular distillation.8 Currently, most commercial concentrates are omega-3 ethyl ester derivatives. However, ongoing research is attempting to obtain more natural and more digestible triacylglycerol © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

September 13, 2017 November 28, 2017 December 12, 2017 December 12, 2017 DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. (a) Enzymatic refining approach of fish oil for the EPA and DHA enriched triacylglycerols. (b) Selective hydrolysis pathway of fish oil by 1,3-specific lipase for the enrichment of EPA and DHA acylglycerols.

was also investigated. Meanwhile, since EPA and DHA have limited sources, we made the most of fish oil to ensure thorough utilization of EPA and DHA. To achieve this goal, MD was applied to recover EPA and DHA from hydrolyzed products and EDEE was purified by a urea complexation approach. Overall, the positive outcome of this work demonstrates successful enrichment of EPA and DHA in fish oil products and suggests the economic value of this two-stage process. Our refining process could contribute to the development of value-added products from fish oil and holds good economic potential.

cantly higher triacylglycerol content (73.9%) was obtained with immobilized MAS1 lipase than those with Novozyme 435 (29.6%) and Lipozyme RM IM (10%). Moreno-Perez et al. also studied enzymatic transesterification of ethyl esters of DHA with glycerol and under optimal conditions, an 82% yield of triacylglycerol was obtained at 50 °C.9 In this research, a novel economical and efficient two-stage enzymatic refining process of fish oils is proposed to prepare EPA and DHA enriched triacylglycerols (EDT) (Figure 1a). The first stage is the partial hydrolysis of fish oil by enzyme with the intention of selectively hydrolyzing saturated fatty acid (SFA) ester. The resulting SFA is removed by molecular distillation (MD), with EPA and DHA enriched in acylglycerols (residual phase, RP). In the second stage, products of the last stage participate in the enzymatic transesterification with EPA and DHA enriched fatty acid ethyl esters (EDEE) to give EPA and DHA enriched fish oil triacylglycerols. EDEEs sourced from fish oil ethyl ester or recycled DHA and EPA in the distillate phase (DP) of MD were applied and investigated. Conditions of hydrolysis, molecular distillation, urea complexation, and transesterification have been investigated. Besides research interests, we also intend to propose an economic process of industrial value. The industrial use of enzymes has been hampered by their high production costs and thus, it is of significance to find the most efficient enzyme in the hydrolysis of fish oil and esterification of the second stage. Simultaneously, minimizing the enzyme usage to lower costs

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MATERIALS AND METHODS

Materials. Fish oil was purchased from NovoSana (Taicang) Co., Ltd., China (acid value = 0.19 mg KOH/g, peroxide value = 1.16 mequiv/kg, saponification value = 196.35 mg KOH/g). Commercially available lipases (E.C. 3.1.1.3) AY “Amano” 400SD (400 SD for short), MER “Amano” (MER), DF “Amano” 15 (DF), A “Amano” 12-K (12K) were purchased from Amano Enzyme China Lit. Lipozyme CALB (CALB), Novozym 435 (435), R. miehi lipase (RML), immobilized Lipozyme RML (RM IL), Lipozyme TL IM (TL IM), and phospholipase A1 (A1) were provided by Novozymes, Denmark. Boron trifluoride (BF3) solution in methanol (14%) was obtained from ANPEL Laboratory Technologies (Shanghai) Inc. Deionized water was used and all other reagents were of analytical grade. Preparation of DHA and EPA Enriched Fish Acylglycerols. (Selective hydrolysis of fish oil and molecular distillation). In a typical procedure, 3.0 g fish oil, 6.0 mL sodium phosphate buffer (0.1 M, pH B

DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry 7), and enzyme were added into a 25 mL conical flask. Nitrogen was filled into the flask, and the flask was sealed. The flask was mounted onto a constant temperature shaker, and the reaction was conducted at a stirring rate of 200 r/min. The reaction was quenched by adding 10 mL ethanol (95%). With the addition of phenolphthalein indicator (3 drops), KOH solution (1 M) was used to titrate the reaction mixture until a pink color was observed. Then the mixture was filtered, and the filtrate was transferred into a separation funnel, and 20 mL n-hexane was added to extract the acylglycerols. The n-hexane phase was collected and washed to remove soap with deionized water until the organic phase was clear. Then the n-hexane phase was collected and concentrated, affording raw acylglycerols enriched with EPA and DHA. The raw acylglycerols were stored at −16 °C for further usage. The efficiency of hydrolysis (EH) was calculated according to the following equation:

EH =

A p − Ac Sr − A r

× 100%

reaction, the reaction mixture comprising EDT were filtered, and the filtrate was analyzed by GC. Preparation of EDEE from the DP and Production of EDT. To facilitate further enrichment of EPA and DHA from the DP, the urea complexation was applied. Single factor experiment design was performed to optimize the urea complexation conditions, including mass ratio of urea to fatty acid (1:1, 1.5:1, 2:1, 2.5:1 and 3:1), complexation temperature (−15, − 10, − 5, 0, 5, 10, and 15 °C), complexation time (0.5, 1, 1.5, 2, 2.5, and 3 h), and mass ratio of ethanol to urea (2:1, 3:1, 4:1, 5:1, and 6:1). In a typical procedure, urea and ethanol (95%) were added into a round-bottom flask equipped with a magnetic stir bar and a condenser. The flask was stirred and incubated at 65 °C to completely dissolve the urea. Next, 10.0 g DP was added and the incubation was lasted for 1 h. After 1 h, the reaction mixture was cooled down for complexation. After complexation, the crystals formed were collected by filtration using a Buchner funnel, ethanol in crystals were removed by evaporation under vacuum, and fatty acids in crystals were extracted by 40 mL petroleum ether (60−90 °C). After the evaporation of petroleum ether under vacuum, EPA and DHA enriched fatty acids were obtained and analyzed by GC. EPA and DHA recovery rates (RED) in the final products of each trail were calculated and analyzed according to the following formula:

(1)

where Ar, Ac, and Ap and are the acid values of the reactant, control sample, and hydrolysis product, respectively, while Sr is the saponification value of the hydrolysis product. Different enzymatic hydrolysis reaction conditions including enzyme type (A1, CALB, RML, RM IM, TL IM, 435, 400SD, MER, DF, and 12-K), enzyme loading amount (0.1, 0.2, 0.3, 0.4, and 0.5 wt ‰), mass ratio of buffer to oil (0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, and 3:1), reaction temperature (25, 30, 35, 40, 45, and 50 °C), pH (5, 6, 7, 8, and 9) and reaction time (8, 12, 16, 20, and 24 h) were screened. A single factor experiment was conducted to find the optimal conditions. Free fatty acids in the hydrolysis product mixture were separated from the raw acylglycerols by MD. Conditions of molecular distillation were as follows: weight of raw acylglycerols 100.0 g, a feeding rate of 1.0 mL/min, a feed temperature of 90 °C, evaporator temperatures of 140−190 °C with an interval of 10 °C, an evaporator vacuum of 0.1 Pa, a roller speed of 300 r/min, and a condenser temperature of 60 °C. Both distillate and residual phases were collected for further usage, and their compositions of fatty acids were analyzed by gas chromatography (GC). Preparation of EDEE from Fish Oil Ethyl Esters (FOEE) and Production of EDT. The production of FOEE was performed under a 3:1 mass ratio of fish oil to ethanol, a 0.5 wt % loading amount of KOH (based on the substrates mass), a reaction temperature of 70 °C, and a reaction time of 1 h. Fish oil (30.0 g), ethanol (10.0 g) and KOH (0.2 g) were added to a 250 mL round-bottom flask. The mixture was then heated in a thermostatic oil bath (DFS101S-2L, Yuhua Instruments Equipment Co., Ltd., Gongyi, China) at 70 °C and stirred by a mechanical impeller with a plastic paddle (diameter = 2 cm). After 1 h, the stir was stopped, and the reaction mixture was cooled and settled into two layers. The upper phase consisting of FOEE was concentrated under reduced pressure, filtered, and subjected to the urea complexation. 10.0 g urea was dissolved in 20.0 g ethanol (95%) at 65 °C in a round-bottom flask followed by the addition of FOEE. A mechanical impeller with a plastic paddle rotating (200 r/min) was used to stir the reaction mixture. After 0.5 h, the mixture was settled for 2 h at 5 °C. The mixture was then concentrated under reduced pressure and filtered. The filtrate enriched with EDEE was collected and extracted by 20 mL petroleum ether (60−90 °C). After extraction, the petroleum ether phase was collected, water washed, and concentrated to give EDEE. The production of EDT was performed (conditions: molar ratio of RP hydroxyl group (calculated from the mass ratio of monoacylglycerols and diacylglycerols in acylglycerols) to EDEE of 1:1) with different lipases (RM IM, TL IM, and 435), loading amount of enzyme (1, 2, 3, 4, and 5 wt %, based on substrate mass), reaction temperature (30, 40, 50, 60, and 70 °C), reaction time (2, 4, 6, 8, 10, and 12 h), and vacuum pressure (20, 60, 100, 300, and 500 Pa). 7.18 g RP, 2.18 g EDEE, and lipase were added into a 50 mL round-bottom flask. The flask was heated in a thermostatic oil bath. A mechanical impeller was used to stir the reaction mixture with a plastic paddle at 1000 r/min. After

RED =

mpED mrED

× 100%

(2)

where mrED and mpED are the total mass of EPA and DHA in raw material and the complexation product. EDEE was also prepared from the obtained EPA and DHA enriched fatty acids under the fixed conditions: a mass ratio of ethanol to EPA and DHA enriched fatty acids of 10:3, a Novozyme 435 loading amount of 3 wt %, a temperature of 40 °C, and a reaction time of 12 h under nitrogen atmosphere. The obtained EDEE was transesterified with RP for producing EDT. 71.8 g RP, 10.2 g EDEE from the DP and 1.64 g Novozyme 435 were added into a round-bottom flask and mixed. The transesterification was conducted with constant magnetic stirring at 60 °C under vacuum (∼2000 Pa). After 6 h, the lipase was collected by filtration. The oil phase collected was the EDT which was analyzed by GC. Analytical Methods. Fatty acids in samples were converted into the corresponding fatty acid methyl esters (FAMEs) by transesterification using established methods.15 The analysis was performed on a Shimadzu GC-2010 Plus GC (Shimadzu Corporation, Japan) equipped with a Shimadzu AOC-20i auto injector, a flame ionization detector and a DB-Wax (10 m × 0.1 mm, 0.1 μm) capillary column. Responses of fatty acids in the detector were considered as identical so that the area normalization method was used to determine the mass percentage of each fatty acid. Acyglycerols products were analyzed by a GC system equipped with a capillary column (DB-1HT, 15 m × 0. 250 mm i.d., 0.1 μm in film thickness, Agilent Technologies Inc., Palo Alto, CA, U.S.A.). Yields of acylglycerols were expressed as the percent content of the corresponding peak area response compared with the total peak area using a hydrogen flame ionization detector (FID).16 Statistical Analysis. Analyses were done in triplicate, with data reported as the means ± standard deviations. One-way ANOVA was performed using SPSS 16 statistical software (SPSS Inc., Chicago, IL). Differences were considered to be significant at p ≤ 0.05, according to Duncan’s Multiple Range Test.

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RESULTS AND DISCUSSION Partial Hydrolysis of Fish Oil. Figure 1b shows the scheme of enzymatic hydrolysis of fish oil. Feasible conditions selection starts from the selection of enzyme types applying the following reaction conditions: enzyme loading amount (based on oil mass) 2 wt %, mass ratio of buffer to oil 2:1, reaction temperature 37 °C, pH 7, and reaction time 12 h. Aiming at higher EPA and DHA contents in acylglycerols after hydrolysis,

C

DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. (a) Effects of enzymes on the selective hydrolysis of fish oil; (b) effects of enzymes on the EPA and DHA contents in acyglycerols after hydrolysis; (c) effects of enzyme loading amount on the selective hydrolysis of fish oil and EPA and DHA contents in acyglycerols after hydrolysis; (d) effects of mass ratio of buffer to oil on the selective hydrolysis of fish oil and EPA and DHA contents in acyglycerols after hydrolysis; (e) effects of reaction temperature on the selective hydrolysis of fish oil and EPA and DHA contents in acyglycerols after hydrolysis; (f) effects of buffer pH on the selective hydrolysis of fish oil and EPA and DHA contents in acyglycerols after hydrolysis; (g) effects of reaction time on the selective hydrolysis of fish oil and EPA and DHA contents in acyglycerols after hydrolysis; (h) effects of molecular distillation temperature on the acid value and EPA and DHA content of RP; (i) and effects of molecular distillation temperature on the acid value and EPA and DHA content of DP. D

DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. (a) Effects of enzymes on the transesterification of EDEE and RP; (b) effects of 435 loading amount on the transesterification of EDEE and RP; (c) effects of reaction time on the transesterification of EDEE and RP; (d) effects of reaction temperature on the transesterification of EDEE and RP; and (e) effects of reaction vacuum on the transesterification of EDEE and RP.

activity. 0.2 wt.‰ enzyme loading amount presented decent hydrolysis efficiency (42.54%), with 26.27 wt % of EPA and 20.04 wt % of DHA correspondingly after hydrolysis. When over 0.2 wt.‰ enzyme was used, the efficiency of hydrolysis and the content of DHA in acylglycerols after hydrolysis did not increase significantly while the content of EPA remained almost unchanged. This result is of great industrial value since the low enzyme loading amount suggests a practical and economical hydrolysis. Therefore, an enzyme loading amount of 0.2 wt.‰ was used in the rest of the condition selection experiments. Figure 2d displays collected data of mass ratio selection of buffer to oil. The effect of mass ratio of buffer to oil on EH and EPA-DHA contents was examined by varying ratios from 0.5:1 to 3:1 while other process parameters were kept constant at 400SD loading amount 0.2 wt.‰, reaction temperature 37 °C, pH 7, and reaction time 12 h. When the mass ratio of buffer solution to oil was 1:1, the corresponding efficiency of hydrolysis (45.84%), the content of EPA (26.15 wt %) and DHA (21.29 wt %) all reached their highest values. Consequently, the ratio of 1:1 was selected. The reaction

single factor experiment was conducted to select suitable hydrolysis conditions. When reaction conditions are selected, one factor is changed to different levels, whereas other factors are kept constant. After one of the factors is selected, the selected value is employed for the next factor selection. The effect of enzyme types on the fish oil partial hydrolysis is shown in Figure 2a and as demonstrated, AY “Amano” 400SD (400SD) from Candida rugosa exhibited the highest efficiency of hydrolysis with the highest percentages of EPA and DHA obtained (Figure 2b). This result corresponds to a previous study in which the high catalytic efficiency of 400SD could be explained by the specific hydrolytic ability of Candida rugose lipase. It has been reported to present higher hydrolyzing ability toward short chain fatty acids (C18 or below).17 Therefore, 400SD was selected for the hydrolysis of fish oil. The selection of 400SD loading amount is shown in Figure 2c. The loading amount ranged from 0.1 wt.‰ to 0.5 wt.‰, while other reaction process parameters were kept constant at mass ratio of buffer to oil 2:1, reaction temperature 37 °C, pH 7 and reaction time 12 h. It should be noted that a very low enzyme loading amount was found to exhibit high hydrolysis E

DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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lipases, and facilitate the transesterification of sn-1 and 3 free hydroxyl groups. In contrast, 435, as a nonselective lipase, can catalyze transesterification of hydroxyl groups at any position.17 Therefore, more substrates could be consumed during the transesterification with 435 than those with RM IM and TL IM, yielding the highest content of triacylglycerols with 435.23 Accordingly, 435 was selected for transesterification in subsequent studies. In order to investigate the effect of the enzyme loading amount on the transesterification, 435 loading amount 1, 2, 3, 4, and 5 wt % (substrates mass) were selected for the experiment under conditions of temperature 50 °C, vacuum pressure 1.3 kPa, and reaction time 6 h. The result is shown in Figure 3b. As described, when the addition of lipase increased from 1 wt % to 2 wt %, the content of triacylglycerols increased accordingly by 10.53 wt % to 87.31 wt %. Nonetheless, if over 2 wt % enzyme was applied, then the content of triacylglycerols did not change significantly, indicating an established transesterification equilibrium. This could be attributed to that 2 wt % enzyme may have provided sufficient active sites for all reactants and any additional enzyme usage would not enhance the transesterification significantly.24 Hence, taking the cost and catalytic efficiency into consideration, the loading amount of enzyme was set at 2 wt %. Under conditions of temperature 50 °C, 435 loading amount 2 wt %, and vacuum pressure 1.3 kPa, in order to investigate the effect of time on the transesterification, the content of triacylglycerols was analyzed with different reaction time and results are displayed in Figure 3c. Initially, there were 70.77 wt % triacylglycerols in RP. In the first 6 h, the content of triacylglycerols increased with reaction time, reaching 87.37 wt % at 6 h. However, enlonged reaction time posed insignificant effects on the content of triacylglycerols which underwent a mere increase of 2.17 wt % in the next 6 h. This could be explained by the established transesterification equilibrium at 6 h, and thus, 6 h was selected as the reaction time for subsequent experiments. Temperature affects enzyme catalyzed reactions because the activity and stability of the enzyme are closely related to temperature. Under conditions of 435 loading amount 2 wt %, vacuum pressure 1.3 kPa, and reaction time 6 h, temperatures of 30, 40, 50, 60, and 70 °C were selected to investigate the effect of temperature on the transesterification, and the result is depicted in Figure 3d. It could be found that under 60 °C the content of triacylglycerols increased in accordance with temperatures. The highest content of triacylglycerols was 91.00 wt % at 60 °C. If the temperature continued to increase, then the triacylglycerol content began to decrease. According to the Arrhenius law, raising the reaction temperature will result in an acceleration effect on reaction rate. Meanwhile, the viscosity could be reduced at high temperatures, allowing the improvement of substrate diffusion.25 But excessive temperatures are not favorable for stabilizing the enzyme’s structure which is the key to the stability and activity of enzyme.26 After considering catalytic efficiency, the selected reaction temperature was set at 60 °C. The effect of reaction vacuum pressure on the transesterification was also studied and the results are shown in Figure 3d. In the experiment, vacuum pressures of 20, 60, 100, 300, and 500 Pa were selected under the above selected conditions of 435 loading amount 2 wt %, reaction temperature 60 °C, and reaction time 6 h. It can be seen that compared with above results under 1.3 kPa (triacylglycerols in acylglycerols

temperature, buffer pH, and reaction time were screened in this manner, (Figure 2e, 2f,g). The resulting feasible conditions for the selective hydrolysis of fish oil were 400SD loading amount 0.2 wt.‰, mass ratio of buffer to oil 1:1, 35 °C, buffer pH 6, and hydrolysis time 10 h. By applying these conditions, EPA and DHA were enriched from 19.30 wt % to 25.89 and 13.09 wt % to 21.38 wt %, respectively, after hydrolysis. After hydrolysis, the effect of molecular distillation temperature on the separation efficiency of free fatty acid from acylglycerols was studied. First, hydrolyzed products were degassed and dewatered at the first stage of MD, followed by separation in the second stage to obtain acylglycerols and fatty acids.18 In order to find the optimum distillation temperature, acid values of both heavy and light phases were measured, and their compositions were analyzed by GC (Figure 2h,i). As the temperature increased, the fatty acid in hydrolyzed products escaped into the DP, and the acid value of RP decreased (Figure 2h). The acid value of RP product was 0.92 mg KOH/g at 160 °C, meeting the requirement of SC/T 3503−2000 (the aquatic industry standard of polyene fish oil products of China). When temperatures were over 160 °C, acid values of RP did not change significantly. In contrast, the acid value of DP was much higher (>200 mg KOH/g) (Figure 2i) but the acid value of DP decreased when distillation temperatures were over 160 °C. It could be due to that above 160 °C, monoacylglycerols, which may contain EPA and DHA, begin to transfer to the DP, resulting in the decreased content of monoacylglycerols in the DP. Meanwhile, because of the temperature increase, the molecular average free path and vapor pressure increase continuously, allowing EPA and DHA to escape to the DP more easily. Furthermore, too high temperature can lower the yield of acylglycerols and affect the quality of fish oil negatively since thermosensitive unsaturated fatty acids abound in it. After overall consideration, the choice of 160 °C was considered as the appropriate temperature for the separation of acylglycerols and free fatty acid by MD, and contents of EPA and DHA in RP reached 25.95 and 22.06 wt %, respectively, with 70.77 wt % triacylglycerols, 28.31 wt % diacylglycerols, 0.85 wt % monoacylglycerols, and 0.15 wt % free fatty acids. Production of EDT by the Transesterification of EDEE from FOEE and RP. Fish oil is thermosensitive because it is rich in unsaturated fatty acids. Therefore, lipase, which can catalyze transesterification at mild conditions with fewer byproducts and high catalytic efficiency, is a very desirable catalyst for fish oil biorefinery. Under established conditions of temperature 50 °C, vacuum pressure 1.3 kPa, and enzyme loading amount 5 wt %, the content of triacylglycerols in acylglycerols was determined after reacting for 6 h. In natural fish oil triacylglycerols, EPA is randomly distributed at any position of the triacylglycerol backbone while DHA prefers the sn-2 position.19 Hence, in hydrolyzed products which act as the substrate for the transesterification, different acylglycerols with one (or two) free hydroxyl group(s) at sn-1, 2, or 3 positions coexist. The effect of enzyme type on the transesterification is outlined in Figure 3a, and the results demonstrate capacities of RM IM, TL IM, and 435 to catalyze the transesterification of EDEE from FOEE and RP to give triacylglycerols.20−22 The highest content of triacylglycerols, 86.35 wt %, was recorded with 435, significantly higher than those with other two enzymes. The difference of catalytic capabilities among these enzymes may result from their transesterification selectivity. RM IM and TL IM are selective F

DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. (a) Effects of mass ratio of urea to fatty acids on the EPA and DHA recovery; (b) effects of urea complexation temperature on the EPA and DHA recovery; (c) effects of urea complexation time on the EPA and DHA recovery; and (d) effects of mass ratio of ethanol to urea on the EPA and DHA recovery.

91.00 wt %), the content of triacylglycerols increased with the lowering vacuum pressure. When setting the vacuum at 20 Pa, the triacylglycerol content could reach 96.93 wt %. Under high vacuum, the boiling point of ethanol is decreased to around 40 °C which means ethanol is easily removed. As a consequence,

the reaction equilibrium shifts toward triacylglycerol production. Under the distillation temperature 160 °C, the transesterification product was subjected to MD to afford EDT which was generated under conditions of free hydroxyl ratio of EDEE from FOEE to RP 1:1, 435 loading amount 2 wt %, G

DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry vacuum pressure 20 Pa, reaction temperature 60 °C, stir speed 1000 r/min, and reaction time 6 h. The heavy phase product (EDT) contained 97.62 wt % triacylglycerols and 2.38 wt % diacyglycerols, which shows that the efficient MD separation of acylglycerols and ethyl esters, and there were nearly no free fatty acids in EDT products. Meanwhile, contents of EPA and DHA increased from 19.30 and 13.09 wt % to 28.20 and 21.41 wt %, respectively. Preparation of the EDEE from the DP. Urea can wrap saturated and low unsaturated fatty acids to form dense hexahedral complexation crystals at low temperatures. Fatty acids can be separated by urea complexation according to the difference of carbon−carbon double bond number.27 Unlike saturated and low unsaturated fatty acids whose conformations are much more flexible, conformations of EPA and DHA are very rigid since EPA and DHA contain 5 and 6 cis-carbon− carbon double bonds, respectively. Given the structural difference, urea complexation could be used to recycle EPA and DHA from the DP. Recycled fatty acids can be concentrated and used in EDT production after the ethyl esterification. Under the conditions of crystallization temperature of 5 °C, mass ratio of ethanol to urea of 3:1 and complexation time of 1 h, the urea and fatty acid mass ratios of 1:1, 1.5:1, 2:1, 2.5:1 and 3:1 were selected to investigate the effect of urea usage on contents and recovery rates of EPA and DHA. As shown in Figure 4a, contents of EPA and DHA in fatty acid increased correspondingly with the increase of urea ratio. When the ratio reached 2.5:1, contents of EPA and DHA were 44.12 and 9.27 wt % with recovery rates of 82.05% and 88.44%, respectively. If the ratio exceeded 2.5:1, then contents of EPA and DHA changed insignificantly. However, recovery rates of both EPA and DHA were greatly reduced. This could be attributed to an excess of urea. Theoretically, as the addition of urea increases, the saturated and low unsaturated fatty acids could be wrapped by the complexation network as much as possible and crystallize, and contents of EPA and DHA in the filtrate increase. Nonetheless, with excessive urea, portions of EPA and DHA with urea could gradually crystallize in the form of clathrates, leading to decreased recovery rates of EPA and DHA. However, excessive urea could result in more loss of filtrate containing fatty acids after complexation.28 Therefore, the urea and fatty acid mass ratio was set 2.5:1. To explore the effect of temperature on fatty acids contents and recovery rates of EPA and DHA, conditions were set as mass ratio of urea to fatty acid 2.5:1, mass ratio of ethanol to urea of 3:1, and complexation time 1 h. Results are shown in Figure 4b. Contents of EPA and DHA in the filtrate changed insignificantly under 5 °C, stabilizing at 45.05 and 7.58 wt %, respectively, whereas these contents began to decrease above 5 °C. Meanwhile, increased temperatures contributed to the recovery of EPA and DHA whose recovery rates experienced significant increases of 65.66 wt % to 93.63 and 67.77 wt % to 90.13 wt %, respectively, from −15 to 15 °C. Theoretically, urea complexation is an exothermic reaction, and low temperature favors the generation of complexation compound.27 But when the temperature is too low, the viscosity of the mixture is increased and this does not allow for the sufficient urea complexation of saturated and low unsaturated fatty acid. These in turn, on the one hand, lower the contents of EPA and DHA in the filtrate. On the other hand, the filtration becomes more difficult and lowers recovery rates of EPA and DHA.

The effect of complexation time on contents and recovery rates of EPA and DHA is depicted in Figure 4c. Fixed conditions were mass ratio of urea to fatty acid ratio 2.5:1, mass ratio of ethanol to urea 3:1 and complexation temperature 5 °C. Contents of EPA and DHA gradually increased with the extension of the complexation time. After 1.5 h complexation, contents of EPA and DHA were 47.81 and 11.91 wt %, respectively. However, as the complexation time further extended, contents of EPA and DHA did not change significantly. Interestingly, when it comes to recovery rates of EPA and DHA, varying trends were recorded. Although the recovery rate of EPA decreased inversely with the complexation time, the recovery rate of DHA was insignificantly changed. This may be derived from the relative rigid structure of DHA which has one more cis-carbon−carbon double bond than EPA. After taking all these factors into account, 1.5 h was selected as the complexation time. Figure 4d shows the effect of various mass ratios of ethanol to urea on contents and recovery rates of EPA and DHA under selected conditions including mass ratio of urea to fatty acid 2.5:1, complexation temperature 5 °C, and complexation time 1.5 h. As a solvent in urea complexation, ethanol has good solubility of urea, and it does not form the clathrate with urea. When the addition of ethanol is not enough, urea cannot be fully dissolved, and there would be an insufficient complexation. Those undesired fatty acids would not be remove thoroughly and in turn, EPA and DHA contents and corresponding recovery rates become lower. With the increasing ethanol/urea ratio from 2:1 to 3:1, contents of EPA and DHA increased significantly from 41.59 and 7.88 wt % to 47.81 and 11.91 wt %, respectively. In contrast, it could be seen that with additional ethanol usage their contents began to decrease. This could be explained by the no saturated urea solution formed with excessive ethanol, and this does not allow for an efficient urea complexation process. A similar trend was recorded with the recovery rate of DHA while the recovery rate of EPA increased with the mass ratio of ethanol to urea. Therefore, the mass ratio of ethanol to urea was set at 3:1. The DP is mainly composed of saturated and shorter carbon chain fatty acids (data not shown). Among them, the content of palmitic acid was the highest, up to 26.40 wt %, followed by palmitoleic acid with a content of 16.45 wt %. Contents of myristic acid and stearic acid were 9.48 and 17.00 wt %, and EPA and DHA were 12.44 and 4.10 wt %, respectively. After urea complexation under the selected conditions, palmitic acid and myristic acid in the reaction products (DP after urea complexation) were largely removed (1.64 and 0.70 wt % respectively). Noticeably, EPA and DHA contents reached 47.83 and 11.99 wt %. Since EPA and DHA only exist in marine products, it is remarkable to collect and recycle them from byproducts. EPA and DHA enriched fatty acids from DP were converted into the corresponding EDEE which was subjected to the transesterification with RP to produce EDT. The reaction was performed under the selected conditions, including a free hydroxyl ratio of EDEE to RP 1:1, 435 loading amount 2 wt %, vacuum pressure 20 Pa, reaction temperature 60 °C and reaction time 6 h. The content of triglyceride in glyceride was 95.9 wt % in the reaction system, and EPA and DHA contents were 25.61 and 17.40 wt %, respectively. This work introduces a novel and economic biorefinery process of fish oil for producing EDT. Lipase AY “Amano” 400SD demonstrated potent selectivity in the hydrolysis of fish H

DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry oil. Free fatty acids were separated by MD at 160 °C. The RP obtained was mainly acylglycerols in which abounded EPA and DHA, with contents of mono-, di-, and triacylglycerols at 0.85, 28.31, and 70.77 wt %. Compared with raw fish oil, EPA and DHA contents were increased significantly from 19.30 wt % to 25.95 wt %, and 13.09 wt % to 22.06 wt %, respectively. For the further enrichment, EDEEs sourced from FOEE and DHA and EPA in the DP were applied in the second stage’s enzymatic transesterification. EDT of 97.62 wt % triacylglycerols with 28.20 wt % EPA and 21.41 wt % DHA was obtained from the transesterification of EDEE from FOEE and RP. The other transesterification between EDEE from the DP and RP afforded EDT products composed of 95.92 wt % triacylglycerols with 25.61 wt % EPA and 17.40 wt % DHA. Also, it must be emphasized that in our study, very low enzyme loading amounts were applied in each stage, with only 0.2 wt.‰ AY “Amano” 400SD and 2 wt % Novozyme 435 used, suggesting the high industrial value of our biorefinery process. The demonstrated efficiency of DHA and EPA enrichment approaches in this study could open up new possibilities for developing DHA and EPA fortified functional food.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.W.). *E-mail: [email protected] (H.H.). ORCID

Yong Wang: 0000-0001-7547-1542 Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The National Key Research and Development Program of China under grant 2017YFD0400200, the financial support from the National Natural Science Foundation of China under grants 31371785, 31671781, 31701525, and 31601503, the Department of Science and Technology of Guangdong Province under grants 2014A010107014, 2017B090907018, 2016YT03H132, and 2013B090800009, and the Bureau of Science and Information of Qingyuan under grant 2016D008 are gratefully acknowledged.

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ABBREVIATIONS USED: EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; EDEE, EPA and DHA enriched fatty acid ethyl esters; EDT, EPA and DHA enriched triacylglycerols; MD, molecular distillation; RP, residual phase; DP, distillate phase; EH, efficiency of hydrolysis; GC, gas chromatography; FOEE, fish oil ethyl esters; FAME, fatty acid methyl esters

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REFERENCES

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DOI: 10.1021/acs.jafc.7b04101 J. Agric. Food Chem. XXXX, XXX, XXX−XXX