Intensified synthesis of structured triacylglycerols from fish, flaxseed and rice bran oil using supercritical CO2 or ultrasound

Chemical Engineering & Processing: Process Intensification 144 (2019) 107650

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Intensified synthesis of structured triacylglycerols from fish, flaxseed and rice bran oil using supercritical CO2 or ultrasound ⁎

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Snehal B. Morea, Parag R. Gogatea, , Jyotsna S. Waghmareb, , Satyanarayan N. Naikc a

Chemical Engineering Department, Oleochemicals and Surfactant Technology, Institute of Chemical Technology, Matunga, Mumbai 40019, India Department of Oils, Oleochemicals and Surfactant Technology, Institute of Chemical Technology, Matunga, Mumbai 40019, India c Department of Rural Development, Indian Institute of Technology, Delhi, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Acidolysis Enzymes Structured lipids Supercritical carbon dioxide Ultrasound Intensification

Intensified synthesis of structured triacylglycerols based on the reaction of different medium chain fatty acids (MCFA) with long chain oils (LCO) such as rice bran oil (RBO), fish oil (FO) and flaxseed oil (FlO) catalyzed by enzymes and using ultrasound (US) or supercritical carbon dioxide (SC-CO2) as the intensification approach has been studied. Combination of MCFA and LCO to give ST was focused to harness the advantages offered by MCFA and LCO. Effect of various reaction parameters such as temperature, time, molar ratio, type of catalyst and loading as well as the use of intensification methods based on US and SC-CO2 has been studied. It was observed that SC-CO2 based approach under conditions of 35 °C as the temperature, 100 bar as pressure, 3:1 as the reactant molar ratio and 6 h as the reaction time resulted in yield of ST as 84.4%. Almost similar yield of 84.5% was also obtained using US under conditions of optimum duty cycle of 6 s/4s (on/off) in 9.6 h which was higher compared to the yield obtained with conventional method (77.1% yield after 24 h). Overall an efficient and intensified method for synthesis of ST based on US or SC-CO2 has been demonstrated.

1. Introduction ST consisting of medium chain triacylglycerols (MCT) with poly unsaturated fatty acids (PUFA) or long chain fatty acids (LCFA) or the MCFA at specific positions has significant demand due to the various applications in the field of absorption studies and clinical nutrition [1]. ST consisting of MCT/MCFA or Long chain triacylglycerols (LCT)/ PUFA also shows vast applications as functional lipids and in nutraceuticals as well as medicinal applications [2]. The most common method for synthesis of ST is acidolysis of LCT and MCFA catalyzed using enzymes, most commonly using a 1,3 specific lipase as it shows higher activity toward MCFA as compared to the long chain fatty acids, especially PUFAs [1]. Omega-3 and omega-6 are PUFA, which are considered essential for various body functions. The important source of omega-6 is linoleic acid (18:2), which is an important constituent in different vegetable oils such as flaxseed, hempseed, grape seed, safflower and sunflower oil as well as fish oil. The important source of omega-3 is α-linolenic acid (ALA; 18:3), which is found in linseed, rapeseed, soya bean, walnuts, fish, chia seeds, flaxseeds [3,4]. The health benefits of PUFA has directed investigations into synthesis of fats and oils rich in PUFA and also created new opportunities for the industry to add significance to other forms of fats and oils by converting to ST [5].

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PUFA have fascinated attention because of its ability to participate in the functional and structural organization of cell membranes by regulating the fat metabolism, helping in reduction of blood cholesterol level and showing cardio protective effect [6]. Omega-6 fatty acids help in lowering the cholesterol in blood, whereas omega-3 fatty acid has anti thrombotic effect [7]. Omega-6 is also used for treatment of rheumatoid arthritis and symptoms of chronic diseases [8]. Kenny et al. [9] reported that use of omega-6 as a supplement along with the drugs for breast cancer patients was more effective in treating the disease. Omega-3 improves cardiac health by reducing triglycerides, blood pressure and also supports mental health of humans [10,11]. It also promotes bone health and prevents asthma [12,13]. Flaxseed is rich source of active substances such as omega-3 and omega-6 [14] and considered to be essential for all healthy people during different mental and physical activities. It is also considered particularly beneficial for the elderly, weakened children and postsurgical patients [15]. FlO is vegetable source of omega-3 and is relatively more stable to oxidation than FO [16]. Flaxseed shows protective effects against diabetic risk with postmenopausal women and prevents the formation of malignant tumors [17,18]. Flaxseed also offers protection against cardiac diseases and used for curing disorders associated with high cholesterol, atherosclerosis, and hypertension [19].

Corresponding authors. E-mail addresses: [email protected] (P.R. Gogate), [email protected] (J.S. Waghmare).

https://doi.org/10.1016/j.cep.2019.107650 Received 17 March 2019; Received in revised form 26 August 2019; Accepted 30 August 2019 Available online 31 August 2019 0255-2701/ © 2019 Elsevier B.V. All rights reserved.

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of our knowledge, such exhaustive study involving different starting long chain oils and medium chain fatty acids has not been reported in the open literature clearly establishing the originality and importance of work.

FO consists of eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) that are helpful for various body functions. EPA is an antagonist of arachidonic acid and as a substrate produces eicosanoids and leukotrienes. DHA is important for development of brain and retina especially in the infants [20,21]. FO shows benefits in ameliorating inflammatory disorders and also improves mental health including cognitive functions [22]. It is also reported that low intake of FO is associated with poor fetal development, and risk of the development of Alzheimer’s disease [23,24]. RBO contains mainly the unsaturated fatty acids accounting to about 75% content (specifically oleic acid as 38.4%, linoleic acid as 34.4%, and linolenic acid as 2.2%) and almost one third of saturated fatty acids (specifically palmiticas 21.5% and stearic as 2.9%) [25]. RBO due to the presence of elements such as oryzanol, tocopherols, tocotrienol and phytosterols, finds its applications in various nutraceutical and pharmaceutical industries [25]. Presence of tocopherols and tocotrienols improves oxidative stability by inhibiting oxidation of the RBO [25]. RBO improves the blood circulation, reduces the oxygen demand of human body, and have been also reported to reduce serum cholesterol [26]. Oryzanol present in RBO was reported to yield reduction in serum low-density lipoprotein (LDL) cholesterol in humans [27]. Rice bran also finds its potential use in reducing plasma cholesterol, Low density lipoprotein (LDL), LDL-C and LDL-C/HDL-C ratio as well as in reducing liver cholesterol levels [28,29]. There have been some studies reported for the synthesis of structured triacylglycerols based on RBO also demonstrating successful subsequent applications. Structured triacylglycerols prepared from RBO can be used in various food products. As a specific example, RBO rich ST was reported as value addition to RBO so as to encourage its utilization as dietary oil [30] with applications in sweet potato chips and energy bars. We now present an overview of studies related to synthesis of ST also focusing on the use of intensification approaches. Zhang et al. [31] reported synthesis of ST from ethyl ferulate (EF) with triglycerides in the presence of US pretreatment. Optimized reaction parameters giving conversion of 92.7% were established as 16% as enzyme loading, substrate ratio of 1:5 (oil/EF, mol/mol), 85 °C as the temperature, US treatment for 1 h, pulse mode of operation with duty cycle of 3 s/3 s (on/off), and 17.0 W/mL as the power density. Linyuan et al. [32] reported synthesis of structured lipid using immobilized lipase TL IM in solvent free system with medium chain fatty acids at sn-1,3 position and long chain fatty acids at sn-2 position. The optimized reaction parameters were established as ultrasonic irradiation time of 4 min, ultrasonic power of 100 W and duty cycle of 5 s on/ 10 s off. It was also reported that enzyme was reusable 10 times though the activity reduced to about 50% at the end of 10 cycles. Lui et al. [33] reported synthesis of structured triacylglycerols from tripalmitin (PPP) and oleic acid (OA) catalyzed by lipase and intensified using US. Optimized reaction parameters were established as irradiation time of 6 min at 50% power dissipation and duty cycle of 3 s on/9 s off, 1:8 as the optimum PPP/OA molar ratio, enzyme loading of 12% and temperature of 50 °C with an OPO yield of 51.8% in 4 h. Analysis of the literature revealed that focus of most of the earlier studies has been based on biodiesel production or dealing with limited starting raw materials in terms of a specific combination of acid and oil. The current work focuses on synthesis of structured triacylglycerols using a variety of MCFA and LCO such as rice bran oil, fish oil and flaxseed oil catalyzed by enzymes. ST containing MCT and FlO, FO and RBO will have characteristics of both MCT and essential fatty acids. The product adds value to normal oils based on processing to give ST and is commercially very attractive. The work also deals with using ultrasound (US) or supercritical carbon dioxide (SC-CO2) as a process intensification approach and developing understanding into the effect of different reaction parameters with an objective of developing lucrative process for commercial exploitation. Comparison with the conventional method of synthesis has also been presented to highlight the beneficial effects of using ultrasound or the SC-CO2 based approaches. To the best

2. Materials and methods 2.1. Materials Different MCFA as caprylic acid (CpA) (99.9% purity), lauric acid (LA) (99.9%) and capric acid (CaA) (99.9%) used in the work were obtained from Hi-Media, (India) Ltd. Mumbai. Lipase samples Novozyme 435 (N-435) and Lipozyme RM (LRM)) were given by Brenntag India Pvt. Ltd. Mumbai as gift samples. Nature make FO (99%) and FlO (99.9%) was obtained from Kamani Oil Industries Pvt. Ltd. Khopoli, and Pure RBO (100%) was obtained from KS Essentials. All the other chemicals required for analysis were procured from S.D Fine-Chem Pvt. Ltd., Mumbai, India. 2.2. Experimental setup 2.2.1. Experimental set up for conventional method of synthesis The conventional synthesis approach was based on the use of a two neck flask in which one port is used for adding sample and removal of samples for HPLC study whereas second port was connected to thermometer to monitor the temperature. The reaction mixture consisting of MCFA and FO/FlO /RBO with enzymes was introduced in the reactor placed on magnetic hot plate used for obtaining desired mixing and temperature. The magnetic stirrer was used to mix the reaction mixture. 2.2.2. Experimental set up for supercritical CO2 assisted synthesis SC-CO2 assisted synthesis was based on using a reactor of 600 mL capacity (jacketed stainless steel vessel: A2560HC13EE, Parr Instruments Co., USA). Parr 4848 controller was used to measure and maintain the temperature, pressure and rotational speed. The reaction substrates as 25 g of FO/FlO/RBO along with 100 g of CpA, CaA and LA at 3% enzyme loading were added to corresponding batches to investigate the effect of operating conditions. Periodically, samples were withdrawn from the sampling line and analyzed to monitor the conversion using high performance liquid chromatography (HPLC). The vessel was equipped with stirrer for mixing with pressure being maintained using CO2 and temperature using the external heating mantle. 2.2.3. Experimental set up for ultrasound assisted synthesis US assisted synthesis was based on using 100 mL beaker. Ultrasonic horn (M/s. Dakshin, India, 22 kHz, rated supplied power of 240 W and tip diameter of 20 mm) was directly immersed into the reaction mixture. The typical calorimetric efficiency was around 10.8% indicating that the calorimetric power dissipation was 25.9 W. Ultrasonic horn tip was placed at a fixed height of 2 mm below the liquid surface. The reaction mixture was also stirred with the help of magnetic stirrer to ensure proper mixing. Reaction parameters such US pulse cycle, and power were optimized so as to arrive at best conditions to get high yield. 2.2.4. Experimental methodology In conventional approach, the batch reactor equipped with stirrer was used with different experiments involving varying parameters such as time (range of 1–24 h), temperature (range of 30–60 °C), and molar ratio of the reactants (MCFA: FO/FlO/RBO as 1:1 to 7:1) to understand the effect on the yield of structured triacylglycerols. Two types of enzyme as N-435 and LRM at different loadings (1–5%) were used to analyze the effect of type and loading of enzyme on yield of the ST. In US assisted synthesis, in addition to the effect of molar ratio (MCFA: FO/FlO/ RBO ratio over the range of 1:1 to 6:1), time, temperature, type and amount of enzyme, parameters pertaining to US such 2

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Fig. 1. HPLC Chromatogram of ST for representative case of FO and CpA.

2.3.3. Peroxide value Oxidation of oil causes oil to become rancid making it unpleasant for consumption with deleterious effect on health. Peroxide value test is used to determine amount of rancidity in given sample of oil. The test was performed as per the standard procedure of American Oil Chemical Society, also described in our earlier work in details [34].

as duty cycle was also varied over the range of 1 s/9 s work/pause cycle to 9 s/1 s work/pause cycle. Similarly in the case of SC-CO2 based approach, in addition to the various reaction parameters over the same range (except as specified otherwise in the discussion), effect of operating pressure was also studied over the range 80–120 bar. Samples were withdrawn at regular intervals of 1 h for HPLC analysis as well as for acid value testing for determination of free fatty acid. After the reaction was completed, enzymes were separated using vacuum filtration (Whatman filter paper no.1). The enzymes retained on the filter cake were washed with hexane to remove any residue and subsequently dried at 40 °C for 5 h. The recovered enzymes were also reused in the reaction. The obtained product as filtrate was washed with 1% NaOH to remove any free fatty acids and then passed through silica gel column to obtain the final product of desired purity.

3. Results and discussion Synthesis of ST was performed using medium chain acids such as CaA, CpA and LA reacted with FO, FlO and RBO. Effect of different reaction variables as temperature, time, molar ratio, type of enzyme and amount of enzymes on the yield of ST have been studied. It is important to note that the enzyme catalyzed reactions are also driven by mass transfer limitations and hence true equilibrium is not always expected to be reached under all the operating conditions. Ultrasonic irradiation causes cavitational effects, which improve the mixing between two phases due to the turbulence effects causing increased rates of transfer [35]. Similarly SC-CO2 is good substitute to organic solvents because of its exceptional properties, such as low viscosity and high diffusivity [36]. Use of SC-CO2 definitely gives much better mixing effects based on the high diffusivity, which enhances the reaction rate as well as the observed yields based on the elimination of the mass transfer limitations. SC-CO2 also causes activation of enzymes by causing movement of surface groups and creating active sites [37]. Considering this analysis, an indepth study into effects of using ultrasound or SC-CO2 on both the obtained yields and the rate of reaction is important, which has been indeed attempted in the current work establishing the importance. It is important to note that true equilibrium conditions may not be reached due to the mass transfer limitations and hence eliminating the mass transfer resistances using intensification approach can give enhanced yields trying to reach the value of true equilibrium.

2.3. Analytical techniques 2.3.1. HPLC analysis HPLC analysis was performed to determine the amount of product formed in the reaction. The HPLC instrument used for the study was Shimadzu HPLC system (HiQSil C18 column with dimensions as 4.6 mm ID and 250 mm height, RID 6A refractive index detector, CR 4A integrator and LC-AS Pump, ans CTO 10 column). Mobile phase used in the study was mixture of acetonitrile and acetic acid (94:6 v/v) at a flow rate of 1 mL/min. The sample volume used for the analysis was 2 μl. 2.3.2. Determination of acid value The amount of unreacted fatty acids in 1 g reaction mixture was determined by acid value test by neutralizing free fatty acids with potassium hydroxide. Acid value is calculated as per Standard procedure of American Oil Chemical Society Official Method Te 1a-64. One gram of sample is taken in conical flask to which 20 mL neutral alcohol is added and refluxed gently to ensure complete dissolution of sample. The resultant solution is now titrated against 0.01 N potassium hydroxide solution using phenolphthalein indicator and colour change at the end point was recorded as from colourless to pink. Acid value was calculated using the following formula.

Acid value(mg KOH/g) =

3.1. HPLC analysis The formation of the desired products was confirmed using HPLC analysis. The HPLC chromatogram for the representative case of structured lipid obtained from FO and CpA is shown in Fig. 1. The peak of synthesized ST matched with the standard and was observed at retention time of 6.733 min. It was observed that only one prominent peak is obtained for the product and no separate peaks for any byproduct could be seen after the desired time for the reaction. The

56.1 × 0.01×CBR Weight of oil sample

56.1 is the molecular weight of the KOH (g/mol) and CBR is constant burette reading. 3

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approach and US assisted approach. The obtained results for the effect of temperature on the yield of ST based on the reaction of FO, FlO and RBO with CpA as the model case are represented in Fig. 2A, Fig. 2B and C respectively (the trends for reactions with CaA and LA are same though with different quantitative values as discussed later). It can be seen from the figure that temperature of 35 °C was sufficient for giving best yields of the product and comparatively much lower yields were obtained at 30 °C. With an increase in temperature initially, the yield has been observed to dominantly increase but beyond temperature of 40 °C, not much increase in the yield is observed. The observed trend can be attributed to the fact that even if the temperature of more than 40 °C drives the reaction faster chemically, at higher temperature enzyme activity is also negatively affected giving marginal changes in the final yield of ST. Considering the stability of enzyme, temperature between 30–40 °C is best for the synthesis. The maximum effects in terms of higher rates and yields using enzyme were found when temperature was maintained at 40 °C for RBO and at 35 °C for FO and FlO. FO and FlO are prone to degradation at higher temperature as well and hence considering the enzyme as well as FO and FlO stability, the best temperature conditions were fixed at 35 °C. As RBO has greater oxidative stability compared to FO and FlO, higher temperature of 40 °C was considered as best for the synthesis of RBO-ST. The best yields for ST containing FO and CpA were 77.1% using conventional, 83.2% using US and 83.4% using SC-CO2 based approaches. For the combination of FO and CaA yields were 77% using conventional, 83.1% using US and 83% using SC-CO2 assisted approaches whereas for the FO and LA combination, the yields were 76.8% using conventional, 83% using US and 83.1% using SC-CO2 based approaches. For the combination of FlO and CpA yields were 76.5% using conventional, 84.2% using US and 84.2% using SC-CO2 based approaches whereas ST involving FlO and CaA gave yields of 76.6% using conventional, 84.1% using US and 84% using SC-CO2 assisted approaches. ST based on FlO and LA showed yields of 76.7% using conventional, 84% using US and 84.3% using SCCO2 based approaches. ST involving RBO and CpA demonstrated 76.5% yields using conventional, 84.5% using US and 84.4% using SC-CO2 whereas that involving RBO and CaA showed yields of 76.4%, using conventional, 84.5% US and 84.3% using SC-CO2 based approaches. Finally the combination of RBO with LA showed best yields of 76.8% using conventional, 84.4% using US and 84.1% using SC-CO2 assisted approach at a constant temperature of 40 °C for all the combinations. Shimada et al. [39] also reported that lipase catalysed synthesis of structured triacylglycerols from linseed oil with CpA demonstrated best results of yield as 48.4% at optimum temperature of 30 °C with other reaction parameters as molar ratio of 2: 1 (CA:SO/LO) and reaction time of 48 h. Temperature of 40 °C was reported as optimum temperature for lipase catalysed synthesis of triacylglycerols from virgin olive oil with CaA, with other parameters optimized as molar ratio olive oil: free fatty acid as 1:2 and 24 h as the reaction time [40]. Nhivekar and Rathod [41] reported best yields of polyethylene glycol stearate as 84.34% at temperature of 60 °C with other reaction parameters being fixed as enzyme loading of 0.5 wt%, molar ratio of 1:1, ultrasonic frequency of 22 kHz and power dissipation of 50 W. Martin et al. [42] reported that US-assisted ester synthesis using commercial immobilized lipase B from Candida antarctica (N-435) showed best results of yield of 94% at temperature of 46 °C with other reaction parameters optimized as substrate molar ratio of 3.6:1 (butanol: acetic acid) and enzyme content of 7%. The obtained results in the present work and comparison with the literature have showed that the optimum temperature is clearly dependent on the type of oil and enzyme used for the synthesis highlighting the importance of the current work.

Fig. 2. Effect of temperature on yield of ST obtained using Conventional, Ultrasound and supercritical CO2 assisted synthesis approaches CpA with FO (Fig. 7.2.A), CpA with FlO (Fig. 7.2.B), CpA with RBO (Fig. 7.2.C), C-Conventional with reaction time of 24 h, U- Ultrasound with reaction time of 6 h, SCSupercritical CO2 method with reaction time of 6 h.

reaction parameters for obtaining ST used in the analysis were set as temperature of 30 °C, N-435 lipase at loading of 3% and molar ratio of 3:1. Lee et al. [38] also reported HPLC study for identification of ST obtained using lipase catalysed synthesis. It was reported that after acidolysis reaction, the 1 and 3 positions of FO were replaced by corresponding medium chain fatty acid. Thus the obtained ST was reported to consist of CpA at 1 and 3 position on backbone of FO. The HPLC study of all the ST was performed similarly and the data was used to interpret yield of ST as well as confirm the formation of the ST with incorporation at the specific positions for the different combinations applied in the work.

3.3. Effect of time on yield of ST 3.2. Effect of temperature The second parameter studied was the optimum time required for reaching the desired yields using conventional, US and supercritical CO2 approaches. The obtained results for the effect of time on the yield

The effect of temperature was studied over the range of 30–60 °C for all the three approaches as conventional, supercritical CO2 assisted 4

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both US and supercritical CO2 approach in 9.6 h and 6 h respectively whereas conventional approach required more than 24 h reaction time for yield of 77.1%. Similar trends for the effect of reaction time can be seen in literature. Enzymatic synthesis of ST using acidolysis of blubber oil with CaA showed best yield of 65% after 24 h of reaction under optimized conditions of oil/fatty acid mole ratio of 1:3, use of hexane, temperature of 45 °C and 10% as the Lipozyme-IM lipase loading [43]. Sun et al. [44] studied the synthesis of lipophilic structured lipids using soybean oil and reported high lipid yields of around 73.5% in 60 h of reaction under conditions of temperature of 85 °C, enzyme loading of 25% w/w and reactant molar ratio of 1:6. Chen et al. [45] studied enzymatic acidolysis for synthesis of caffeic acid phenethyl ester using US and reported that reaction time of 9.6 h gave highest yield of 93.08% for the substrate molar ratio of 1:71, enzyme loading of 2.9%, and ultrasonic power intensity of 2 W/cm2. One of the advantages of using intensification techniques demonstrated in the work and also confirmed using the reported literature data is the faster rate of reaction. It can be clearly seen from the results and comparison with literature that US reduced the reaction time from 24 h to 9.6 h whereas reaction time reduced from 24 h to 6 h using supercritical CO2 approach. Also higher amounts of ST were formed using US and supercritical CO2 approach compared to conventional method of synthesis. US treatment using probe intensifies the rate of reaction based on the enhanced mass transfer and easy access of the active sites to substrates, which favors the fast completion of reaction [46]. Waghmare and Rathod [47] also reported that US assisted synthesis of glycerol carbonate using enzymatic transesterification based on commercial immobilized lipase (N435) required lesser time as 4 h compared to the conventional method, which required 14 h under optimized reaction parameters as catalyst amount of 13% (w/w), power input of 100 W, duty cycle of 50% and temperature of 60 °C. Kim et al. [37] also reported that the synthesis of ST in the presence of supercritical CO2 was faster with lower reaction time of 12 h compared to 24 h required in the conventional approach. Thus, it can be concluded that reaction time is greatly reduced from 24 h to 6 h and 9.6 h using intensification techniques based on SC-CO2 and US respectively. 3.4. Effect of molar ratio The effect of molar ratio of medium chain fatty acids (MCFA) to FO/ FlO/RBO was studied over the range from 1:1 to 6:1(MCFA:FO/FLO/ RBO) and the obtained results for FO, FlO and RBO based on the reaction with CpA are represented in Fig. 4A, Fig. 4B and C respectively. It can be seen from the figure that as molar ratio increased, the yield of ST increased till an optimum. The increase in molar ratio increases free fatty acids available for the reaction, which in turn maximizes rate of reaction but only till an optimum value. Excess molar ratio provides with excess fatty acids beyond the optimum requirement which will not be utilized favorably and also creates problems for the removal of unreacted fatty acids. Considering this aspect, optimum molar ratio was fixed at 3:1 (MCFA: FO/FLO/RBO), which was found to same for all the combinations studied in the work and also the type of approaches. Silroy et al. [48] reported that for the lipase catalysed synthesis of RBO with CaA, 3:1 was the optimum molar ratio giving best results of yield as 30.8% with other reaction parameters as reaction time of 72 h, temperature of 45 °C and enzyme concentration of 10%. Similarly the lipase catalysed synthesis of structured triacylglycerols from corn oil (CO) showed best results of 21.5% as the yield at optimum molar ratio of 4:1 (CpA/corn oil) after 3.1 h of reaction [49]. Jennings et al. [50] also reported that molar ratio of 1:6 (FO/CaA) was optimum with best yield of structured triacylglycerols as 41.2% from the reaction of CaA with FO using immobilized lipase, IM60, obtained from Rhizomucor miehei under conditions of reaction time of 72 h, enzyme loading of 10%, temperature of 55 °C and speed of 200 rpm. It can be concluded from the present study as well as from the literature that optimum ratio

Fig. 3. Effect of time on yield of ST using conventional, Ultrasound and supercritical CO2 assisted approaches (A-FO with CpA: B- FlO with CpA: C: RBO with CpA).

of ST using FO, FlO and RBO based on the reaction with CpA are shown in Fig. 3A, Fig. 3B and C respectively. It can be seen that the reaction proceeds rapidly using both US as well as supercritical CO2 assisted methods compared to the conventional approach as represented in Fig. 3. It was also clearly observed that reaction time has an impact on the yield of the product. Typically, as the time increases, yield of the ST also increases till an optimum time and then the yield almost remains constant. In conventional synthesis approach, 24 h of reaction time was required for attaining the constant reaction yield whereas the time reduced to 9.6 h for the US and 6 h for the supercritical CO2 assisted approaches for all the ST. Best results of ST yield of 83.5% was found for the US assisted and supercritical CO2 approach for synthesis of ST from FO and CpA whereas the observed yield for the conventional approach was 76.8%. Higher obtained in the case of US and Sc-CO2 based approaches means that true equilibrium is not reached in the case of conventional approach. Similar trends for the yields were also obtained for other combination of FlO and RBO with CaA and LA. Highest yield among all ST was obtained with RBO and CpA as 84.5% using 5

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Fig. 5. Effect of different types of lipase and lipase loading on the yield of ST using the Conventional approach for reaction time of 24 h, Ultrasound with reaction time of 9.6 h, Supercritical CO2 method with reaction time of 6 h (A Novozyme-435 with FO/CpA; B - Lipozyme RM with FO/CpA).

represented in the figures, though with different actual value). It can be clearly seen from the figures that using N-435 as the catalyst is more beneficial as compared to LRM. Lipases typically show different activity in different reactions. The activity of Novozyme 435 for transesterification type reactions is higher attributed to the presence of specific active sites required for the reaction. There have been many such instances reporting the use of this type of lipase for carrying out reactions with higher activity as compared to the different forms of lipase as Lipozyme TL-IM, Lipozyme RM-IM, CALB etc. [51] confirming the obtained results in the present work. From the results presented in Fig. 4 Fig. 5, it can be also seen that an increase in the loading of enzyme increases the yield of ST significantly till an optimum loading established as 3% which was the best to give high yield of ST in a cost effective manner. It was also demonstrated that only marginal increase in yield of ST was obtained at higher loading beyond 3%. Credence to the existene of optimum loading can be obtained based on comparison with the literature. Zhou et al. [52] reported that lipase-catalyzed synthesis of structured triacylglycerols from soybean oil with methyl caprylate using enzyme, Lipozyme rhizomucor miehei in immobilized form (LRM) resulted in highest yield of 59.45 mol% at optimum enzyme loading of 16% with the other reaction parameters being temperature of 42 °C, substrate ratio of 8∶1 and reaction time of 16 h. Similarly 10% enzyme loading was reported as the best for synthesis of ST from blubbler oil and CaA using lipase at conditions of reactant molar ratio of 1:3 and temperature of 45 °C with reaction time of 24 h [52]. In another work, 1 g enzyme loading of N-

Fig. 4. Effect of molar ratio on yield of the ST using the Conventional approach for reaction time of 24 h, Ultrasound with reaction time of 9.6 h, Supercritical CO2 method with reaction time of 6 h (A-FO with CpA: B- FlO with CpA: C: RBO with CpA).

(specific to the combination of reactants) is required for obtaining best results for synthesis of ST and in the present work, best results were obtained at molar ratio of 3:1 (MCFA to oil), which was considered for the further studies. 3.5. Effect of type and amount of Lipase Effect of using two types of lipase namely N-435 and LRM and the loading of enzyme (over the range of 1 to 5%) on the yield of ST using different approaches as conventional, US and supercritical CO2 assisted has been studied. The obtained results for the effect of enzyme loading and type of enzyme on the yield of ST obtained from the specific combination of FO with CpA using N-435 as the catalyst has been represented in Fig. 5A whereas for the case of Lipozyme RM as the catalyst, the data has been shown in Fig. 5B for the same combination (trends for all the other combinations of oil and acid are similar to that 6

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435 was reported sufficient for synthesis of ST by esterification of 2MAGs with CpA catalyzed by 1,3 specific lipase with highest yield of 65% [53]. Similarly, with a study of two lipases namely LRM and N-435 used for synthesis of ST from Triacetin and camellia oil, it was demonstrated that N-435 was more suitable for the reaction system giving 98.4% yield which is higher compared to LRM [40]. Another study into enzymatic synthesis of cetyl Octanoate structured lipid using two commercial immobilized lipases, i.e., LRM (Rhizomucor miehei) and N435 (Candida antarctica) revealed that using N-435 results in best yield of 98% proving to be a more efficient biocatalyst than LRM which gave 94% yield under optimized reaction parameters as time of 5 h, reaction temperature of 45 °C, substrate molar ratio of 3:1 and enzyme loading of 30% [54]. Lee et al. [55] also studied synthesis of structured triacylglycerols using two immobilized lipases, LRM and N-435 demonstrating higher activity of N-435 with best yields of 57.7% obtained at enzyme loading of 4%. The enzyme reusability was also studied in the same work using same enzymes repetitively for synthesis of ST. It was reported that enzymes were stable up to 15 cycles in conventional approach whereas stability marginally decreased to 14 cycles in the case of US and supercritical CO2 approach. Pressure and cavitation effects to some extent may have ceased the enzyme stability to a marginal extent, and it was concluded that use of US and supercritical CO2 did not demonstrate any significant harmful effect on the enzymes especially considering the obtained benefits. Another study also established that the enzyme was reusable over four successive cycles beyond which decline in rate of reaction was reported [56], indicating that the reusability and the observed activity is dependent on the specific system. Overall the obtained results in the present work and also overview of the literature confirmed that N-435 lipase is more specific for synthesis of ST with higher demonstrated activity. It is also clearly established that an optimum loading specific to the system is required for achieving a cost effective operation.

Fig. 7. Effect of duty cycle on the yield in Ultrasound assisted synthesis approach (reaction time of 9.6 h).

that 150 bar was the optimum pressure for the enzyme catalysed synthesis of ST at operating temperature of 40 °C. Yan et al. [59] reported that 19.6 MPa was optimum pressure for increased activity of lipase in the presence of SC-CO2 at 35 °C temperature and reaction time of 1 h. Generally it is reported that higher reaction yield could be obtained at optimum conditions of the pressure, which is specific to the system in question. With increase in pressure, typically the solubility of the substrate in SC-CO2 increases giving favorable effects for reaction. However beyond the optimum pressure, favorable effects of solubility may not be seen on the reaction rates due to other simultaneously acting effects. For example, Inactivation of enzymes take place at higher pressure typically above 103 bars pressure [60]. Silveira et al. [61] also reported that the substrate-binding region of the lipase is typically exposed to carbon dioxide favorably during the supercritical CO2 treatment only under optimum conditions of pressure, which facilitates diffusion of large fatty acids into active sites, without affecting functional structure of the enzyme. The optimized pressure established in the present case was 100 bars, which was observed to enhance rate of reaction without affecting the enzyme activity and hence used in subsequent set of experiments involving Sc CO2 based approach.

3.6. Effect of pressure in supercritical CO2 assisted synthesis The effect of pressure was studied over the range of 80 bar to 130 bar as this is an important parameter in the supercritical CO2 assisted approach. The obtained results have been represented in Fig. 6 for the specific case of synthesis of ST from combination of FO, FlO and RBO with CpA. It can be seen from the figure that best yield of ST was obtained at 100 bar pressure and as the pressure increased beyond 100 bar, yield almost remained constant for all the combinations of ST and hence 100 bar pressure was considered as optimized pressure for obtaining best yield of ST. More et al. [57] also reported that 100 bar was the optimum pressure established in the case of supercritical carbon dioxide pretreatment applied for intensifying the enzymatic synthesis of medium chain triacylglycerol. Hlavsova et al. [58] reported

3.7. Effect of duty cycle Duty cycle is an important parameter in US assisted synthesis as this directly affects the cost of treatment and also the durability of the equipment. The duty cycle was varied over the range of 1 s/9 s (on/off) to 9 s/1 s (on/off) and the obtained results are represented in Fig. 7 with duty cycle being represented in the form of percentage on time (10% on time corresponds to 1 s (on)/ 9 s (off) in the cycle). It can be seen from the figure (data shown for specific combination with CpA only for clarity and for other acids as well similar trends are observed) that the duty cycle of 6 s/4s (on/off) showed highest yield of 84.5% and this optimum value was the same for all the combinations of oil and acid. Thus, it can be concluded that duty cycle of 6 s/4s (on/off) exhibits best intensification activity. Typically an increase in duty cycle increases the on time of ultrasound meaning the exposure to cavitating conditions is higher. Due to higher exposure to ultrasound at faster frequency, the reaction is benefitted at higher duty cycles till the optimum. It was indeed observed that increasing time of exposure to cavitation till the optimum and decreasing off time makes reaction proceed faster. Longer pause time can weaken the ultrasound induced cavitation effects and lower the rate of reaction [29]. It is also important to note that long exposure of cavitation can also affect the enzyme activity, which can affect the reusability of the enzyme and also the activity in the reaction. Considering both the aspects, the duty cycle of 6 s/4s (work/pause) was established as the optimized condition in the present work. Khan et al.

Fig. 6. Effect of pressure on the yield in the case of supercritical CO2 assisted approach (reaction time of 6 h). 7

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Fig. 9. Effect of storage time on acid value (A-FO with CpA: B- FlO with CpA: C: RBO with CpA).

Fig. 8. Effect of storage time on peroxide value (A-FO with CpA: B- FlO with CpA: C: RBO with CpA).

with triacetin using hexane as a solvent and reported that 7 s/3 s (on/ off) duty cycle showed highest yield of 83% under conditions of catalyst as LRM, molar ratio of 1:1 (hexanol to triacetin), 4% as the enzyme loading (w/v), 60 W as the power, 50 °C as the temperature and reaction time of 4 h. Thus, it can be established that use of US under optimum duty cycle (dependent on the specific system) is required to give best efficacy for the enhanced rate of reaction and high yield.

[62] studied synthesis of ester n-butyl palmitate using US in solvent free approach and reported that 7 s/3 s (on/off) duty cycle exhibited highest yield of about 96.6% under optimized conditions of 50 min as reaction time, 1:1 as the molar ratio of palmitic acid to n-butanol, 70 °C as the temperature, 4% w/w of enzyme loading and 25 kHz as the frequency. Zhu et al. [63] also reported that US assisted synthesis of feruloylated structured triacylglycerols (FSLs) by enzymatic transesterification of ethyl ferulate (EF) with triglycerides showed best results at duty cycle of 3 s/3 s (on/off) with highest yield of 92.7% and the other optimum reaction parameters were treatment time as 1 h, 16% as the enzyme loading, substrate ratio of 1:5 (oil/EF, mol/mol) and temperature of 85 °C. Similar results for the effect of duty cycle were reported by Lui et al. [64] in the US assisted synthesis of ST from tripalmitin (PPP) and oleic acid (OA) with best results of yield as 51.8% being obtained at duty cycle of 3 s/9 s (work/pause) at 50 °C temperature in 4 h. Deshmukh et al. [65] studied lipase catalysed transesterification of hexanol

3.8. Effect of storage time on peroxide value of the synthesized ST The peroxide value test is used to study the oxidative stability of synthesized ST. The peroxide value test was performed after every 7 days over a total period of 28 days. The obtained values for the peroxide value in the case of ST obtained from FO, FlO and RBO using CpA have been represented in Fig. 8A, Fig. 8B and C respectively (trends for CaA and LA were also similar to that represented for the specific case of CpA). It was observed that the peroxide value of the freshly synthesized 8

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Table 1 Effect of cycles of usage of enzymes on the obtained yields (expressed as % for different combinations). Cycle number

FO + CaA (U)

FlO + CaA (U)

RBO + CaA (U)

FO + CaA (SC)

FlO + CaA (SC)

RBO + CaA (SC)

1 3 5 7 9 11 13 15

83.6 83.7 83.8 83 80.3 76 71.1 65.3

84.3 84.4 84.5 84.1 80.4 76.9 70.1 65.6

84.7 84.7 84.8 84.4 81.5 79.8 73.4 68.7

83.6 83.5 83.1 82.9 80.1 75.8 70.3 64.1

84.3 84.2 84.1 81.3 79.4 74.3 69.4 62.4

84.7 84.6 84.2 82.4 81.6 78.6 72.6 66.7

CO2 is seen till about 15 cycles. Similar results were also reported by Keng et al. [69] with lipase stability upto 16 cycles. It was observed that the yield of the product only decreased gradually and no sharp decrease in yield was observed even after 15 successive cycles. Thus, it can be said that lipase N-435 can be efficiently used over 15 cycles, though with a marginal reduction in the activity.

ST was around 3 Meq/kg. The storage from 7 to 28 days resulted in a marginal increase in the peroxide value (still below the value of 10 Meq/kg considered to be detrimental) as the ST starts oxidizing during storage. It was also observed that peroxide value of ST obtained from FO and FlO was high compared to RBO. Similar trends for increase in the peroxide value can be seen in the literature. Kimoto et al. [66] studied oxidative stability of ST (Marine Oil Triacylglycerols) and reported that peroxide value after 28 days was higher than the fresh value confirming that synthesized ST was susceptible to oxidation. Maduko et al. [67] performed the oxidative study of ST to be applied for infant milk formulation of tripalmitin with vegetable oil blends including FO and it was reported that coconut oil had the highest oxidative stability due to high content of saturated fatty acids whereas FO had the lowest resistance to oxidative deterioration. It was observed in the present work that peroxide value increased gradually till 28 days of storage. Maximum peroxide value obtained after 28 days was about 5.1Meq/kg for RBO and 6.4 Meq/kg for FO clearly confirming that the change is dependent on the type of oil used. Overall, it can be concluded that the ST showed good oxidative stability over 28 days as the period of storage without developing rancid flavor as the critical value for oil to turn rancid is above 10 Meq/kg.

4. Conclusions The present work focused on synthesis of structured triacylglycerols with process intensification studies involving US and supercritical CO2 and comparison with conventional method. Two enzymes as N-435 and LRM were used for synthesis and it was concluded that activity of N-435 was higher compared to other enzyme with reusability up to 15 cycles, though with a marginal lowering of the yields. The rate of reaction for synthesis of ST was established to be enhanced using US and supercritical CO2. Highest yield of 84.5% was obtained using US under condition of optimum duty cycle as 6 s/4s (on/off) whereas with supercritical CO2 assisted synthesis, best results of yield as 84.4% were obtained at 100 bar pressure; both higher as compared with conventional method (77.1% yield obtained in 24 h as reaction time). Another distinct advantage of using ultrasound and supercritical CO2 was significant reduction in the required reaction time to 9.6 h and 6 h respectively from 24 h required for conventional processing. The FFA values were low (less than 1) in all samples throughout 28 days of storage study indicating that no significant difference in the FFA content is obtained. The peroxide value measurements also demonstrated similar beneficial results during the storage. Overall, the present work demonstrated the process intensification benefits in terms of higher yields and much lower reaction times with good oxidation stability for obtained ST over 28 days.

3.9. Effect of storage time on acid value The amount of free fatty acids present in ST during the storage was also analyzed using the acid value test. The acid value test was performed after every 7 days over a storage period of 28 days and the obtained results for changes in acid value of ST obtained from FO, FlO and RBO using CpA are represented in Fig. 9A, Fig. 9B and C respectively (trends for CaA and LA were also similar to that represented for the specific case of CpA). It can be seen from the figure that acid value increased from 0.2 at 0 day to 0.5 after 28 days. The acid value increase was only marginal and within the recommended range of specifications. The oil is considered non potable if acid value is more than 1 which was not seen in the current work. Similar trends for stability of the synthesized ST have been reported in the literature. Martin et al. [68] applied the acid value test to study oxidative stability of ST synthesized enzymatically using fish and canola oil and it was reported that acid value of about 0.4 was seen after storage of 28 days, a similar increase to that obtained in the current work.

Acknowledgments One of the authors, SBM, is grateful to Dr. Babasaheb Ambedkar National Research and Training Institute (BARTI), Pune, India for financial support for the Ph.D. fellowship. References [1] M. Christensen, T. Redgrave, Intestinal absorption and lymphatic transport (EPA and DHA), Am. J. Nutr. 61 (1995) 56–61. [2] B. Haumann, Structured triacylglycerols allow fat tailoring, Inform. 8 (1997) 1004–1101. [3] R. Holman, S. Johnson, P. Ogburn, Deficiency of essential fatty acids and membrane fluidity during pregnancy and lactation, Proc. Natl. Acad. Sci. 88 (1991) 4832–4835. [4] A. Leaf, Y. Xiao, J. Kang, Interactions of n-3 fatty acids with ion channels excitable Tissues Prostaglandins Leukot, Essent Fatty Acids 67 (2002) 113–120. [5] J. Opstvedt, N. Urdahl, J. Pettersen, FO—an old fat source with new possibilities, in edible fats and oils processing, AOCS 9 (1990) 250–259. [6] D. Filippis, L. Sperling, Understanding omega-3′s, Am. Heart J. 151 (2006) 564–570. [7] A. Simopoulos, The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases, Exp. Biol. Med. 233 (2008) 674–688. [8] R. Zurier, M. Laposata, gamma-Linolenic acid treatment of rheumatoid arthritis. A randomized, placebo-controlled trial, Arthritis Rheum. 39 (1996) 1808–1817.

3.10. Reusability of catalyst Reusability study of immobilized lipase was also performed as this becomes very important for commercial applications especially considering the high costs of the enzyme. After the experiment, the lipase was removed by filtration and washed with hexane to remove any adherent impurity. After washing, the lipase was dried in oven for 15 min at 30 °C and then the lipase was reused for successive reaction. The study was focused on using N-435 lipase for both intensification techniques and observed results for the reusability are represented in Table 1. The obtained results suggest that only a marginal decrease in activity of enzymes in both the intensification techniques US and SC9

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