Intensified synthesis of structured lipids from oleic acid rich moringa oil in the presence of supercritical CO2

Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 86–95

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Intensified synthesis of structured lipids from oleic acid rich moringa oil in the presence of supercritical CO2 Snehal More a , Parag Gogate a,∗ , Jyotsna Waghmare b,∗ , Satyanarayan N. Naik c a

Chemical Engineering Department, 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, Hauz Khas, New Delhi 110016, India b

a r t i c l e

i n f o

a b s t r a c t

Article history:

Structured lipids have been synthesized using different medium chain fatty acids based on

Received 30 March 2018

the reaction with long chain triglyceride viz. moringa oil, in the presence of supercritical

Received in revised form 28 August

CO2 . Effect of different operating parameters related to supercritical CO2 approach on the

2018

product yields has been studied also establishing the effect of type of biocatalyst (using

Accepted 4 September 2018

two forms of commercial lipases as Novozym 435 and Lipozyme RM). It was observed that

Available online 8 September 2018

100 bar pressure at 50 ◦ C temperature with 300 rpm speed, molar ratio of 4:1 (medium chain

Keywords:

were the best conditions giving 63.2% yield which was much higher as compared to the

fatty acids: long chain triglycerides), reaction time as 5 h and Novozym 435 as the catalyst Medium chain triglycerides

conventional approach where only 28.2% yield was obtained. After acidolysis, the residual

Acidolysis

unreacted triglycerides were extracted using hexane and allowed to react again with free

Structured lipids

corresponding medium fatty acids under same reaction conditions. After complete reaction

Supercritical CO2

with medium fatty acids, product obtained was structured lipids with medium chain fatty

Moringa oil

acids at 1,3 position and long chain fatty acid at 2-position. The enzyme reusability studies confirmed that enzyme was efficient up to 15 cycles providing a cost effective proposition.

Lipase

Overall, it was established that structured lipids possessing significant nutritional benefits could be effectively synthesised using green intensified approach based on supercritical CO2 . © 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1.

Introduction

Structured lipids (SL) are reorganized form of lipids offering added nutritional benefits, and hence find applications in various food and nutraceutical products. SL can be produced either enzymatically or

tured lipids can be synthesized by interesterification, acidolysis, and alcoholysis methods (Kapoor and Gupta, 2012). In the present work, structured lipids have been synthesized by acidolysis of long chain triglyceride (LCT) with medium chain fatty acids (MCFA) catalyzed by lipase with intenification studies based on supercritical carbon dioxide.

chemically (Akoh, 2002). Chemical method is inexpensive compared to

SL are generally synthesized from Medium chain triacylglycerols

enzymes but lacks specificity. Enzyme catalysed synthesis of structured

(MCT) and long chain triglycerides. MCT are easily absorbed in the body

lipids is favourable over chemical method because of its specificity and green nature (Kim and Akoh, 2006). Biocatalysts offer high specific activity and a low impact on the environment (Basri et al., 1996). Struc-

cells and break down quickly which drives the important applications

∗

in the areas of treatment of malabsorption syndrome cases, infant care and also offer as a high energy nutrient source (Babayan, 1987). The ease of solubility and rapid metabolism of MCT provide health benefits when incorporated into SL (Jennings and Akoh, 2009). MCT are utilized quickly as energy source, not stored in adipose tissue as fat and are metabolized

Corresponding authors. E-mail addresses: [email protected] (P. Gogate), [email protected] (J. Waghmare). https://doi.org/10.1016/j.fbp.2018.09.004 0960-3085/© 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 86–95

through the portal system instead of the lymphatic system (Jennings and Akoh, 2000). Even though MCT is a fast energy source with wide variety of applications, it lacks presence of essential fatty acids. Considering this lacunae, combination of MCT with essential fatty acids can be a excellent way of obtaining very effective SL. One such source of fatty acids is moringa oil (MO), which is rich in oleic acid (OA) and posses various health benefits. Oleic acid reduces blood pressure, aids weight loss and protects cells from free radical damage (Teres et al., 2008). MO is extracted from the seeds of the tree. Moringa oleifera seeds contain a high proportion of oil that makes the plant as a major source of oil for edible as well as non-edible purposes. MO typically consists of 70–78% oleic acid similar to that of olive oil (Nestel et al., 1994). The high percentage of oleic acid in the oil makes it desirable in terms of nutrition, health friendly nature and the offered high stability makes it ideal for use as cooking and frying oil (Tsaknis et al., 1998). The high oleic acid content of the oil also confers it a functional food property as oils containing high oleic content have been shown to reduce risks of coronary heart disease. MO is highly resistant to autoxidation and can be used as a replacement of synthetic oxidants (Nadeem et al., 2014). MO also provides very good oxidative and thermal stability because of presence of high oleic acid and can be used as a substitute to commercial raw and refined edible oils. MO is hence considered as very stable and healthy substitute for commercial groundnut oil as a cooking and frying medium (Babatunde et al., 2014). M. oleifera is food crop typically with all the parts of the plant considered useful for human and animal consumption after some processing. In India, the annual production of

Fig. 1 – Schematic representation of the experimental setup. tigated at all though it offers significant benefits as discussed earlier. Also the comparison of different types of lipase enzymes is lacking in the literature for the synthesis of SL. Considering these aspects, the novelty of the work dealing with intensified synthesis of SL from MCT and moringa oil in the presence of different lipases is clearly established. The work also presents kinetic analysis which gives important design related information for possible commercial scale operations.

M. oleifera fruits is 1.1–1.3 million tonnes, which can yield about 9500 kl of oil. Thus, it can be said that MO is also available in good amount

2.

Materials and methods

(though the cost is higher as compared to other oils like groundnut, sunflower or palm; off course the quality attributes can compensate the cost) and hence it can prove as a potential source of value addition especially if used in small proportions in SL giving significant quality

2.1.

Materials

attributes (Mathur, 2014). MO contains all the main fatty acids similar to that of olive oil, and therefore, can be used as a possible substitute to the expensive olive oil (Abdulkarim et al., 2005), which is commonly used currently. The properties of MO can be highly advantageous particularly with the current trend of replacing polyunsaturated vegetable oils with the better substitutes in the production of commercial products with value addition (Corbett, 2003). Considering this analysis, MO was selected as rich source of oleic acid in the present work aimed to utilize MCT and oleic acid rich MO in synthesis of SL that can offer combined health and nutritional benefits. There has been an increased interest in the biosynthesis of SL especially the form containing long chain fatty acids, at the sn-2 position, as well as MCFA at the sn-1,3 positions, often described as MLM-SL (Ingle et al., 1999). Additional health benefits such as improved nitrogen balance and reduction in cancer risk are shown by these types of SL (Akoh and Kim, 2008). Use of SLs in diet also causes significant reduction in accumulated body fat and serum cholesterol (Kasai et al., 2003). Thus, structured lipids synthesized using medium chain fatty acids and essential fatty acids offer increased health benefits and hence the importance of the present work is established. Conventional method of synthesis of SL includes use of organic solvents like hexane (Jennings and Akoh, 2000) typically requiring reaction time of more than 24 h. Although organic solvents in the presence of enzymes show good results in terms of yield compared to solvent

The different medium chain fatty acids used such as caprylic acid (99.9%), capric acid (99.9%) and lauric acid (99.9%) were procured from Hi-Media, (India) Ltd. Mumbai. Samples of enzymes were obtained from Brenntag India Pvt., Ltd. Mumbai as gift samples. Moringa oil was obtained from Earth Expo Pvt., Ltd. (India), Gujarat. Sodium thiosulphate, potassium iodide, potassium hydroxide and starch were procured from S.D FineChem Pvt., Ltd., Mumbai, India.

2.2.

Reaction scheme

The reaction considered for the present study is an acidolysis reaction between medium chain fatty acids (caprylic acidCpA, capric acid-CA and lauric acid-LA) with moringa oil in the presence of enzymes (Novozym 435 (N435) and Lipozyme RM (LRM)) as the catalyst. Supercritical carbon dioxide is applied as green solvent and as process intensification approach. The reaction scheme can be represented as follows: Medium chain fatty acids +Moringa Oil

Bio -catalyst

→

Supercritical CO2

Structured Lipid

free conditions but the adverse effect on environment confine the use of such SL in food and health related applications. Use of supercritical CO2 (SC-CO2 ) to replace the organic solvents is a green approach for synthesis of structured lipids. SC-CO2 mediated enzymatic reaction offers an intensification approach because of advantages of high diffusivity, low surface tension, and low viscosity that induces accelerated mass transfer (Habulin et al., 1999; Oliveira and Oliveira, 2000). A review of literature revealed that there are some reports dealing with use of SC-CO2 in synthesis of structural lipids. Kim et al. (2004) investigated enzymatic alcoholysis of Palm Kernel Oil in SC-CO2 and reported 26.4% as the obtained yield after 4 h of reaction. Sellappan and Akoh (2001) studied synthesis of SL from trilinolein and caprylic acid catalyzed using lipase Lipozyme IM. Analysis of the literature also revealed that the use of MCT and moringa oil combination has not been inves-

2.3.

Experimental setup

The reactor used in the work is a jacketed stainless steel vessel with 600 ml capacity (A2560HC13EE, Parr Instruments Co., USA) as depicted in Fig. 1. The temperature of reaction vessel, pressure and rotating speed were controlled using Parr 4848 controller with the values being shown on digital display. The sampling line for the liquid phase was located at the top of the reactor and the withdrawn samples were used for HPLC analysis. The desired pressure was maintained by pumping CO2 into the reactor and temperature was achieved by heat-

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Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 86–95

ing the autoclave using external heating mantle. The reaction mixture was continuously stirred using magnetic stirrer. In conventional method of synthesis (without the use of supercritical CO2 ), the reaction mixture was taken in 100 ml capacity two neck round bottom flask equipped with heating mantle to maintain the desired temperature and magnetic stirring for uniform mixing. Thermometer was used in one opening for temperature measurements whereas through the second opening, substrates and catalyst were added at the start of experiment and then during the actual experiment, samples withdrawn for analysis from the same opening.

2.4.

Experimental methodology

Effect of different reaction parameters such as time, temperature, pressure, enzyme loading, stirring speed and molar ratio on the progress of reaction has been investigated to establish the best operating conditions which can give maximum yield of structured lipid. Two different enzymes namely Novozym 435 and Lipozyme RM were used in the study to establish the effect of type of enzymes on the yield of structured lipid. The time of reaction was varied from 1 to 10 h and the yield obtained in the presence of SC-CO2 was compared with the conventional synthesis approach (without SC-CO2 ). The molar ratio of medium chain fatty acid (MCFA) viz. caprylic acid, capric acid and lauric acid to the moringa oil (MO) was varied over the range of 1:1–7:1 (MCFA:MO) whereas the temperature was varied over the range of 30–60 ◦ C. The operating pressure was varied over the range 80–120 bar whereas rotation speed was varied from 100 to 400 RPM to understand the effect on the progress of the reaction. During the reaction, aliquots of samples were taken at regular intervals of 1 h and HPLC analysis was performed to know the progress of the reaction. After withdrawing the samples, the mixture was cooled to ambient conditions, filtered using whatmann filter paper no.1 to remove the enzyme and then analysed using HPLC. After completion of reaction, the reaction mixture was allowed to attain room temperature and then vacuum was released. Enzymes in the reaction mixture were removed by filtering the mixture through Whatman filter paper no.1 in vacuum filtration followed by washing the enzyme with hexane to remove any residue and drying it in oven at 40 ◦ C for 6 h. The recovered enyme was again used in subsequent cycles to check the reusability. Free fatty acids were removed by washing the reaction medium (filtrate obtained in the earlier step) using 1% NaOH. Pure product was subsequently obtained by passing reaction mixture through silica gel column. The experiments were repeated at least 3 times to check the reproducibility of the obtained results. The data reported in the figures and used in the discussion is the average of three readings and the typical experimental errors were found to be in the range of ±2% of the reported value.

2.5.

Estimation of kinetic rate constant

The enzyme-catalysed acidolysis of caprylic acid (for that matter any fatty acid used in the work) with moringa oil progresses with the attachment of fatty acids of moringa oil at syn-2 position and caprylic acid at syn-1,3 position of triglyceride. The acidolysis reaction due to the presence of excess caprylic acid can be considered as an irreversible pseudo second-order reaction. Pseudo second order kinetic model was considered for studying the kinetics of acidolysis reaction for synthesis

of structured lipid. The rate equation for the reaction can be expressed as follows: −dCMO /dT = rMO = K × C2 MO

(1)

where CMO is the moringa oil molar concentration in mol/l, t is the reaction time in min, rMO is the reaction rate in mol/(l min) and k is the reaction rate constant in l/(mol min). Integration of Eq. (1) gives the following equation: 1/CMO = k. t + 1/CMO0

(2)

Rearranging Eq. (2) in terms of the conversion gives the following equation: X/(1 − X) = CMO0 .k.t

(3)

where CMO0 is molar concentration of moringa oil at time t = zero in mol/l and X is moringa oil conversion at any time t. Based on information of structured lipid yield and reaction stoichiometry, the amount of residual moringa oil during acidolysis reaction can be obtained to calculate the conversion. A plot of X/(1 − X) versus t will be a straight line if the model is valid, and k can be obtained from the slope of this line. Arrhenius equation gives the relationship between specific reaction rate constant (k), absolute temperature (T) and energy of activation (Ea) and can be given as follows: K = A exp [−Ea/RT]

(4)

In Eq. (4) R and A represent universal gas constant (J/(mol K)) and frequency factor respectively. Rearranging the equation, we get Eq. (5), ln(k) = [−Ea/RT] + ln (A)

(5)

Slope (−Ea/R) was calculated from the plot of ln(k) vs. 1/T and knowing R, activation energy was determined.

2.6.

Analytical techniques

2.6.1.

HPLC analysis

Product formation was analysed using the Shimadzu HPLC system consisting of LC-AS Pump, CTO 10 column, RID 6A refractive index detector and CR 4A integrator. The column used for analysis of triglycerides was HiQSil C18 having dimensions as 4.6 mm ID and 250 mm length. Mixture of acetonitrile and acetic acid (94:6 v/v) was used as mobile phase with flow rate of 1 ml/min. The sample was dissolved in the mobile phase before the analysis and injection volume into equipment was 2 ␮l.

2.6.2.

Determination of acid value

Acid value test is used to calculate amount of free fatty acids present in reaction mixture. It is calculated as milligrams of potassium hydroxide required to neutralize the free acids in one gram of sample. Standard procedure available as per the reports of American Oil Chemical Society Official Method Te 1a-64 was used for performing the acid value test. 1 g of sample was weighed accurately and transferred in 250 ml conical flask followed by addition of 20 ml neutral alcohol. The mixture was refluxed gently until the sample was completely dissolved. The solution was titrated against 0.01 N potassium hydroxide solution. End point was colourless to pink. The amount of KOH

Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 86–95

70 60

Yield of product (%)

50 40 30

CpA+MO+N435 CA+MO+N435 LA+MO+N435 CpA+MO+LRM CA+MO+LRM LA+MO+LRM Convenonal Synthesis

20 10 0

1

2

3

4

5

6

7

8

9

10

Time (h)

Fig. 2 – Comparison of SC-CO2 approach with conventional route of synthesis (CpA—caprylic acid, CA—capric acid, LA—lauric acid, MO—moringa oil, N435—Novozym 435, LRM—Lipozyme RM). required was measured and acid value was calculated using the following formula. Acid value (mg KOH/g) =

56.1 × 0.01 × C.B.R Weight of oil sample

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

2.6.3.

Peroxide value

Rancidity of the sample was measured using the peroxide value test. The test was performed as per the standard procedure available from the reports of American Oil Chemical Society. 5 g oil sample was taken and 30 ml (3:2 proportion by vol) acetic acid: chloroform solution was then added along with 0.5 ml saturated KI and kept in dark for 1 min. After liberation of iodine, 30 ml distilled water was added. Blank was prepared similarly, using all the components with same amount except the sample of oil. The mixture was titrated against 0.01 N Sodium thiosulphate using starch indicator. Peroxide value was calculated according to the following equation: Peroxide value = (sample − blank) × 1000 × Normality/wt. of sample

3.

Results and discussion

3.1. Comparison of conventional and supercritical CO2 based approach of SL synthesis The yield of SL obtained using SC-CO2 approach has been compared with conventional approach to establish the process intensification benefits and obtained data for the two approaches have been shown in Fig. 2. It was observed that in SC-CO2 assisted synthesis, the SL were formed to higher extent compared to conventional method of synthesis without any solvent. After 1 h reaction time, 43.2% product was obtained using SC-CO2 but only 12.3% was obtained using conventional method. Marked difference between two methods was observed with increasing time of reaction. The increase in yield of SL using SC-CO2 can be attributed to the fact that, lipase activity increases in the presence of SC-CO2 thus enhancing rate of reaction. The overall formation of SL in the presence of SC-CO2 was higher as compared to the con-

89

ventional method in solvent free conditions. In conventional method, the amount of SL formed was found to be 28.2% after 6 h, which for the SC-CO2 approach was 63.2% clearly establishing the efficiency of SC-CO2 to enhance the SL formation. In the case of conventional approach, continuing the reaction even for 24 h resulted in only 42% yield. Supercritical fluids as solvents have been reported to play vital role in enhancing the activity of enzymes (Wimmer and Zarevucka, 2010). The use of supercritical conditions also increases the solubility of substrates by altering the solvent strengths increasing the yield of the product (Wimmer and Zarevucka, 2010; Zhao et al., 2007). The application of SC-CO2 as a solvent in enzymecatalysed reactions favours higher diffusivity, which results in enhanced mass transfer rates and hence the higher reaction rates as compared to conventional method (Oliveira and Oliveira, 2000). Credence to these mechanisms can also be found in the literature. Giessauf and Gamse (2000) reported that the lipase activity increases several fold in the presence of SC-CO2 at pressure of 150 bar and temperature of 75 ◦ C. Similar results in terms of increase in the relative product yields in the presence of SC-CO2 as compared to the conventional method were reported by Hlavsová et al. (2008), who attributed the trends to low viscosities and high diffusivities increasing the rates of mass transfer of the substrates to the enzyme ultimately enhancing rate of reaction.

3.2.

Effect of time on yield of SL

The variation of yield with time of reaction was studied to establish the time required for obtaining maximum yield of SL in optimum time in the presence of SC-CO2 . The experiments were performed over reaction time of 10 h using conditions of 100 bar as the pressure, 50 ◦ C as the temperature, 300 rpm as the speed and molar ratio of 4:1 (MCFA: MO) for SC-CO2 assisted synthesis. The obtained data has been shown in Fig. 3A. It can be seen from the figure that as reaction time increases the amount of product formed typically increases. It was established that the yield increases rapidly over the time of 1 h to 5 h for both enzymes namely Novozym 435 and lipozyme RM with the obtained yields for caprylic acid (CpA), capric acid (CA) and lauric acid (LA) as 63.2%, 61.2%, 63.1% (in the case of Novozym 435) and 60.4%, 63%, 60.1% (in the case of Lipozyme RM) respectively. Beyond 5 h as the reaction time, the product yield remained almost constant, which can be attributed to the fact that a thermodynamic equilibrium is reached and hence product yield remains constant. Kim et al. (2004) reported similar results of optimum reaction time of 6 h for the synthesis of SL from corn oil and caprylic acid in the presence of SC-CO2 . More et al. (2018) reported similar results for esterification reaction of caprylic acid in presence of SCCO2 with optimum reaction time of 6 h. Zhanga et al. (2000) also reported that lipase catalysed interesterification for the production of margarine fats SL required optimum reaction time of 6 h below which the time greatly influenced interesterification. Considering the obtained results in the present work, optimum time was fixed at 5 h for further study of effect of operating parameters.

3.3. Effect of temperature on yield of SL in presence of supercritical CO2 The temperature range used in the current study was from 30 ◦ C (subcritical region) to 60 ◦ C (supercritical region). Temperature was not increased further beyond 60 ◦ C as lipase

90

Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 86–95

2

60

1.8

50

1.6

30 20

0.4

4

5

6

7

8

45 50 55

0.8

0

3

40

1

0.6

2

35

1.2

10 1

30

1.4

CpA+MO+N435 CA+MO+N435 LA+MO+N435 CpA+MO+LRM CA+MO+LRM

40

X/1-X

Yield of product (%)

70

9

10

Linear (30) Linear (35) Linear (40)

0.2

Time (h)

0

(A)

Linear (45) 0

1

2

70 60

4

5

Linear (50)

6

Linear (55)

Fig. 4 – Kinetic study for calculating rate constant.

50 Yield of product (%)

3 Time (h)

CpA+MO+N435 CA+MO+N435 LA+MO+N435 CpA+MO+LRM CA+MO+LRM LA+MO+LRM

40 30 20 10 0

30

35

40

45 50 Temperature (C)

0 0.003

0.00305

0.0031

0.00315

0.0032

0.00325

0.0033

0.00335

-0.5

55

-1

60

-1.5

(B) 70

-2

60

Yield of product (%)

50 40

CpA+MO+N435 CA+MO+N435 LA+MO+N435 CpA+MO+LRM CA+MO+LRM

30 20 10 0

80

90

100

110 120 Pressure (Bars)

(C)

Fig. 3 – Effect of operating parameters on the yield of SL (A) time, (B) temperature, (C) pressure. activity is typically observed to be optimum over the temperature range of 30–60 ◦ C. The other operating conditions for the study involving effect of temperature were 100 bar as the pressure, 300 rpm as the speed, molar ratio of 4:1 (MCFA:MO) and reaction time as 5 h. It was observed that the temperature has very promising effect on yield of the product till an optimum as per the results shown in Fig. 3B. With an increase in the temperature, the lipase activity also increases resulting in higher yield of the structured lipid. However, protein in the enzymes can be denatured at higher temperature and pressure affecting its activity, due to which beyond 50 ◦ C, no increase in the yield is observed. Lipases have been reported to be are more active at higher temperatures till an optimum (Hasnisa and Jumat, 2012) with the most suitable temperature range for enzymatic acidolysis as 50–55 ◦ C (Zhao et al., 2007). Cao et al. (2013) studied synthesis of structured lipids from camellia oil and triacetin and reported that at temperature of 50 ◦ C, maximum yield was obtained as 52.1% without any adverse effect on enzyme activity. Similar results were reported by Zou et al. (2016) with 48.2% yield obtained in synthesis of SL from catfish oil and sunflower oil at 50 ◦ C. Huang and Akoh (1996) reported that temperature of 45 ◦ C was the optimum for synthesis of SL from caprylic acid and triolein. Kim et al. (2004) also reported highest yield of SL from corn oil and caprylic acid at 55 ◦ C. It is also important to note that in some specific cases, the temperature stability of enzymes and hence the optimum

-2.5

y = -5915.3x + 17.167 R² = 0.9694

Fig. 5 – Arrhenius plot for estimation of activation energy. temperature can be still higher. For example, Overmeyer et al. (1999) reported that enzymes remain active above 100 ◦ C in the presence of supercritical CO2 as reaction medium. Thus, it can be said that the optimum temperature where the protein structure may largely be retained is dependent on the system and specific type of enzyme (Mozhaev et al., 1996). Considering the results obtained in present work, temperature was fixed at 50 ◦ C for subsequent studies related to understanding the effects of other parameters.

3.4.

Kinetic analysis and Arrhenius plot

Kinetic analysis of reaction was performed over temperature range of 30–60 ◦ C to find the rate constants and then the activation energy. The molar ratio (MO:CA) was maintained constant and the enzyme used was Novozym 435 at pressure of 300 bar and 300 rpm as speed of rotation. The kinetic plots of X/(1 − X) vs time at varying temperatures are given in Fig. 4. The enzyme catalysed reaction is greatly influenced by the temperature. Elisa et al. (2011) reported kinetic study for synthesis of structured lipid from soyabean oil with sardine oil and fitting of the Arrhenius plot with R2 value as 0.95. Wojdyla et al. (2014) reported that activation energy for synthesis of structured lipid from milk fat and rapeseed oil was 91.97 kJ/mol. As per the values of kinetic constants given in Table 1, the obtained results established that the rate constant increased from 0.09 to 0.3 l/(mol min) over the temperature range of 30–55 ◦ C respectively. It was also observed that although rate constant increased after 50 ◦ C, the yield of SL does not increase significantly and considering enzyme economy, 50 ◦ C temperature has been considered as optimum. The arrhenius plot of ln(K) vs T represented in Fig. 5 was a straight line and slope enabled to calculate activation energy. The obtained value was 49.18 kJ/mol and the pre-exponential

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Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 86–95

Table 1 – Obtained values of rate constant and activation energy. Temperature (◦ C)

Rate constant (k)

Correlation factor R2

Frequency factor (K0 )

Activation energy(kJ/mol)

30 35 40 45 50 55

0.098 0.129 0.159 0.249 0.369 0.379

0.840 0.882 0.880 0.922 0.855 0.835

1.51

49.18

factor was found to be 1.51 l/(mol min) as per the data shown in Table 1.

3.6.

Effect of speed of rotation on yield of SL

The effect of rotation speed on the yield of SL was investigated using different values of speed as 100, 150, 200, 250, 300, 350 and 400 RPM at temperature of 50 ◦ C, molar ratio of 4:1 (MCFA:MO) and reaction time as 5 h. It was established from the results shown in Fig. 6 that yield of SL gradually increases as the speed increases. The maximum yield of SL as 63.2% was obtained at speed of 300 rpm beyond which further increase in speed of rotation did not cause marked increase in yield. Hence 300 rpm as the speed of rotation was established as optimum and kept constant for further studies. Adschiri et al. (1992) reported that lipase catalysed interesterification of triglyceride in the presence of SC-CO2 showed existence of optimum speed of rotation as 150 rpm. Lee and Akoh (1998) reported that low yield of product was obtained at lower speed of 200 rpm and the yield was higher at higher speed as 640 rpm, though no optimum was reported. Jennings and Akoh (2009) reported that optimum speed for synthesis of structured lipids

50 Yield of product (%)

The effect of pressure on yield of SL was studied using different values of pressure as 80, 90, 100, 110 and 120 bar at temperature of 50 ◦ C, speed as 300 rpm, molar ratio of 4:1 (MCFA:MO) and reaction time as 5 h. The obtained results are shown in Fig. 3C where it can be seen that as pressure increases from 80 to 100 bar, yield of SL increased significantly whereas a marginal increase in yield of SL was observed beyond 100 bar. As pressure is increased, solubility of substrate in SC-CO2 increases dominantly till an pressure of 100 bar which in turn shows positive effect on yield of SL. Adschiri et al. (1992) also reported that 100 bar pressure was best for interesterification of caprylic acid with methyl oleate. Similar results were reported by Shishikura et al. (1994) where 100 bar pressure showed good incorporation of medium chain triglycerides in the presence of supercritical carbon dioxide. Kim et al. (2004) reported that there was no significant increase in caprylic acid incorporation at pressures above 240 bar for 6 h of reaction time establishing 240 bar as the optimum. Nakaya et al. (1998) reported a continuous increase in the yield of transesterification of triolein and stearic acid catalysed by Lipozyme IM in the presence of SC-CO2 when the pressure was increased stepwise from 50 to 200 bar. The obtained results in the present work and the comparison with the literature establishes that the optimum operating pressure is specific to the system in terms of the type of reaction and enzyme used thus establishing the importance of the current work. Based on the obtained results in the present work, optimum pressure selected for the further set of experiments was 100 bar.

60

CpA+MO+N435 CA+MO+N435 LA+MO+N435 CpA+MO+LRM CA+MO+LRM LA+MO+LRM

40 30 20 10 0

100

150

200

250

300

350

400

Speed of rotaon (RPM)

Fig. 6 – Effect of speed of rotation on the yield of SL at pressure of 100 bar, temperature of 50 ◦ C, molar ratio of 4:1 (MCFA:LCT) and reaction time as 5 h. 70 60 50 Yield of product (%)

3.5. Effect of pressure on yield of SL in the presence of supercritical CO2

70

CpA+MO+N435

40

CA+MO+N435 LA+MO+N435

30

CpA+MO+LRM

20

CA+MO+LRM LA+MO+LRM

10 0

01:01

02:01

03:01

04:01

05:01

06:01

07:01

Molar Rao (MCFA:MO)

Fig. 7 – Effect of molar ratio on yield of SL at pressure of 100 bar, temperature of 50 ◦ C, speed of 300 rpm and reaction time as 5 h. from rice bran oil and caprylic acid was 200 rpm. It can be thus said that the optimum speed is also dependent on the specific reaction and the type of catalyst needing the detailed investigated into the effect of speed of rotation.

3.7.

Effect of molar ratio on yield of SL

The molar ratio is also one of the important parameters affecting the yield of SL. The molar ratio of medium chain fatty acids viz caprylic acid, capric acid and lauric acid to moringa oil (in different sets of experiments) was varied considering different values as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6 and 1:7 (MO:MCFA) at pressure of 100 bar, temperature of 50 ◦ C, speed of 300 rpm and reaction time as 5 h. The obtained results depicted in Fig. 7 establish that as the molar ratio increases till an optimum of 4:1 (MCFA:MO), the yield of SL increases dominantly attributed to the fact that the amount of medium chain fatty acids available increases. Using excess fatty acids drives the reaction in forward direction and hence higher yield is observed. Beyond optimum of 4:1, there was only marginal change in the yield. Rupani et al. (2014) also reported that trans-esterification of

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70

64

60

63 62 Yield of product (%)

Yield of product (%)

50 CpA+MO+N435 CA+MO+N435 LA+MO+N435 CpA+MO+LRM CA+MO+LRM LA+MO+LRM

40 30 20

60 59 58

10

57

0

56

0.5

1

1.5 2 2.5 Enzyme Load (%w/w)

3

3.5

Fig. 8 – Effect of type and amount of enzyme on yield of SL at pressure of 100 bar, temperature of 50 ◦ C, speed of 300 rpm, molar ratio of 4:1 (MCFA:MO) and reaction time as 5 h. alpha linolenic acid with olive oil showed significant changes in yield of SL till an optimum molar ratio 4:1. Similar results were also reported by Shieh et al. (1995) for synthesis of SL from triolein and capric acid with optimum molar ratio of 4:1 giving maximum yield (51%). Shimada et al. (1996) reported that molar ratio of 2:1 was the best for the synthesis of structured lipid from caprylic acid and sunflower oil giving maximum yield of 40%. It is important to note that, use of excess fatty acids increases the load on subsequent steps of purification including the removal of free fatty acids from the reaction mixture and hence overall process may not be economical at very high ratios necessitating the use of optimum molar ratio, which seems to be specific to the actual reaction. Base on the analysis and the obtained results in the present work, it was established that the optimum molar ratio was 1:4 (MO:MCFA), which was used for the subsequent studies.

3.8.

61

Effect of type and amount of enzyme on yield of SL

Two enzymes as Novozym 435 and Lipozyme RM were used in the present study to establish the effect of type of enzyme and also the amount of lipase was varied to ascertain effect on yield of SL in presence of SC-CO2 .The activity of enzymes is affected by temperature and hence the temperature of 50 ◦ C (established as optimum in the earlier set of experiments) was maintained constant for the study with other optimum conditions being molar ratio of MO:MCFA as 1:4, pressure as 100 bar, speed of rotation as 300 rpm and 5 h as the reaction time. The obtained results for both types of enzymes have been shown in Fig. 8. It can be seen from the figure that Novozym 435 enzyme shows slightly higher conversions as compared to Lipozyme RM. Caprylic acid reaction with MO showed 63.2% yield with Novozym 435 but only 61.2% yield was obtained with Lipozyme RM. The enzyme loading was also varied with different values as 1, 1.5, 2, 2.5, 3, 3.5 and 4% and the results are shown in Fig. 8. It was observed that lower enzyme loading show lower yield while higher loading of enzyme till an optimum of 3% gives dominant changes of higher yield. Beyond 3% enzyme loading, the yield of SL remained almost constant and using higher catalyst did not give any additional benefits. Hence, considering the high cost of enzymes, the optimum enzyme loading was fixed as 3%. Lee and Akoh (1998) reported that 2% enzyme loading showed maximum benefits in terms of yield in enzymatic synthesis of structured lipids from peanut oil and caprylic

2

4

CpA+MO+N435

CA+MO+N435

LA+MO+N435

CpA+MO+LRM

CA+MO+LRM

LA+MO+LRM

6

8

10

12

14

16

Enzyme Load (%w/w)

Fig. 9 – Reusability study of catalyst. acid. Zhanga et al. (2000) reported that interesterification of palm stearin and coconut oil showed best results at lipase concentration of 6%. Fomuso and Akoh (1997) reported that in the case of lipase catalysed acidolysis to produce reduced calorie SL, Lipozyme RM showed maximum yields at best enzyme loading of 10%. It can be thus established that the optimum enzyme loading is indeed dependent on the specific reaction and considering the present results, enzyme loading as 3% was selected as the best operating condition.

3.9.

Reusability of catalyst

The study of enzyme reusability becomes important considering the high cost of enzyme. In present study, the efficiency of enzyme to give high yield of SL in multiple cycles was studied. The used enzyme was washed, dried and again used for next set of reaction. The obtained results for the yield of product after repeated use of enzymes are shown in Fig. 9. It was observed that good yield of SL was obtained even after repeated use of enzymes upto 15 cycles. Impurity free substrates and washing with hexane every time after reaction completion were the main factors responsible for consistent activity of the enzymes. The results suggest that use of supercritical CO2 does not demonstrate any harmful effect on the enzymes and hence the recovered enzyme can be used upto 15 cycles. Lerin et al. (2011) reported good lipase activity and product yield even after 10 successive cycles. More et al. (2017) reported that use of Novozym 435 and lipozyme RM upto 10 cycles does not significantly affect activity of lipase for the esterification reaction. Keng et al. (2008) also reported the use of enzymes for esterification reaction for about 16 cycles without any changes in the activity.

3.10.

HPLC determination

The formation of SL containing MLM structure was confirmed using high performance liquid chromatography analysis. The HPLC Chromatogram of structured lipid obtained from caprylic acid (Dicapryloolein) is shown in Fig. 10. The peak of synthesized SL viz. Dicapryloolein was observed at retention time of 5.8 min, which matched well with the standards. For the confirmation study, acidolysis reaction using 1,3-specific enzyme was performed at molar ratio of 4:1 (acid to oil) and temperature of 50 ◦ C. During the same run, the fatty acid composition and acid value of moringa oil and structured lipids were analysed and the obtained results have been given in Table 2. It was observed that the fatty acid composition of moringa oil is significantly enhanced with MCFA. Originally,

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Table 2 – Comparison of physical characteristics of moringa oil and synthesized structured lipids (Dicapryloolein, Dicapriloolein and Lauriloolein SL are mentioned in the table as SL(MO + CpA), SL(MO + CA) and SL(MO + LA) respectively).

Acid value (mg KOH) Peroxide value (mEq/kg) Type of fatty acids Caprylic acid (C8:0) Capric acid (C10:0) Lauric acid (12:0) Myristic acid (C14:0) Palmitic (C16:0) Palmitoleic (C16:1) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2) Linolenic (C18:3) Arachidic (C20:0) Gadoleic (C20:1) Behenic (C22:0) Arachidic (C24:0)

Moringa oil

SL(MO + CpA)

SL(MO + CA)

SL(MO + LA)

0.40 1.62

0.32 3.55

0.32 3.56

0.33 3.56

ND ND ND 0.2 6.8 2.9 6.5 70 0.9 ND 4.2 1.4 5.8 1.3

63.2 ND ND 0.1 2.2 1.5 2.2 24.3 0.5 ND 2 1.2 2.1 0.7

ND 63.1 ND 0.1 2.1 1.5 2.2 24.4 0.6 ND 2.1 1.2 2.1 0.6

ND ND 63.1 0.1 2.2 1.5 2.1 24.4 0.6 ND 2 1.2 2.2 0.6

Table 3 – Results for changes in peroxide values at different time intervals.

Fig. 10 – HPLC Chromatogram of structured lipid (Dicapryloolein). oleic acid was predominant with about 70% composition in the moringa oil and no presence of the MCFA (caprylic, capric or lauric acids; as per data in Table 2 second column). However after acidolysis, the major fatty acid present was corresponding medium chain fatty acids (Table 2, SL as Dicapryloolein consisting of caprylic acid and MO has been given in third column whereas SLs as Dicapriloolein and Lauriloolein obtained from capric acid and lauric acid have been given in fourth and fifth column respectively) and oleic acid. After 5 h of reaction SL yield was 63.2% and 61.2% using Novozym 435 and Lipozyme RM respectively as per the data obtained from HPLC. Compared to conventional method, the yield of SL was very high confirming that the substrates readily dissolve in SC-CO2 , which in turn increases contact between substrate and lipase (Kima et al., 2002).

Time (days)

Peroxide value (Novozym 435)

Peroxide value (Lipozyme RM)

0 7 14 21 28 35 42 49 52 63

3.55 3.57 3.60 3.66 3.69 3.73 3.80 3.90 4.04 4.08

3.55 3.58 3.62 3.68 3.70 3.75 3.82 3.91 4.07 4.11

of SL produced from fish and canola oils was below 5 mEq/kg over the shelf life study period indicating good stability of synthesized SL. Thus it is important to establish the stability of the obtained SL and hence the peroxide value was tested over period of 63 days with the value being calculated at different storage periods of 0, 7, 14, 21, 28, 35, 42, 49, 56 and 63 days. The obtained results are given in Table 3. The initial peroxide value was 3.55. The results suggested that the peroxide value does not increase rapidly and remained almost constant for 63 days with the final value being only 4.11. Thus, it can be concluded that SL obtained using the present approach can remain stable for more than 2 months without becoming rancid.

4. 3.11.

Conclusions

Peroxide value

Rancid oil or lipids in general consists of many free fatty acids and is not suitable for human consumption and hence study of oxidative stability of synthesized SL becomes important. Peroxide value tests are used to calculate oxidative stability of the sample. Coulter and Jenness (1954) reported that peroxide value appears to be very satisfactory approach for determination of oxidation stability. Osborn and Akoh (2004) reported SL synthesized from canola oil and caprylic acid showed good oxidative stability upto 15 days. Senanayake and Shahidi (2002) reported that peroxide value of structured lipids synthesized from Borage and Evening Primrose Oil only marginally increased from 2.2 to 2.7 meq/kg after 4 days storage. Akoh and Moussata (2001) reported that peroxide value

The acidolysis of medium chain fatty acids such as caprylic acid, capric acid and lauric acid with moringa oil in presence of SC-CO2 demonstrated intensified approach with much higher yield of SL (63.1%) compared to conventional synthesis approach (42%). The required time for obtaining maximum yield also reduced from 24 h in conventional synthesis to 5 h in the approach based on SC-CO2 . The optimized reaction parameters for the synthesis of SL using the SC-CO2 approach established in the present work were reaction time of 5 h with temperature of 50 ◦ C, pressure as 100 bar, speed of rotation as 300 rpm and 3% enzyme loading. Kinetic studies for the reaction established that second order kinetic model fitted well. The activation energy was found to be 49.18 kJ/mol and the pre-exponential factor was found to be 1.51 l/(mol min). The

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structured lipid demonstrated properties of both the long and medium chain fatty acids. The stability studies performed in terms of the peroxide value confirmed the stable nature of the obtained SL. Overall the work demonstrated an intensified approach for synthesis of SL from moringa oil giving value addition in terms of possible nutraceutical application.

Acknowledgement One of the authors, SBM, is grateful to Dr. Babasaheb Ambedkar National Research and Training Institute (BARTI) for financial support for the Ph.D. fellowship.

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