Interesterification is the reaction between esters or TAG molecules. Not much research has been reported on MLCT production via interesterification reaction. For MLCT production, interesterification often involved the coconut oil or palm kernel oil or saturated TAG such as tricaprylin that will provide the MCFA. LCFA part in MLCT is contributed by the vegetable oil such as soybean oil, rapeseed oil (Fomuso and Akoh 1998; Lopez-Hernandez and others 2005; Adhikari and others 2011b). The progress of the interesterification reaction is measured by the changes in the TAG composition before and after the reaction (Fomuso and Akoh 1998; Lopez-Hernandez and others 2005).
For interesterification, substrate ratio is an important parameter that will affect the desirable yield. For example: substrate mole ratio of trilinolein to tricaproin from 1 : 1 to 1 : 4, the mole ratio of 1 : 2 gave the highest yield of 50.7% dicaproyllinolein (ECN 33) and 23.6% monocaproyldilinolein (ECN 45) (Fomuso and Akoh 1998).
The first commercially available MLCT sold in the market by Nisshin Ollio Ltd. Group, Ltd also uses the interesterification process for its production. This MLCT oil was produced via interesterification reaction involving coconut or palm kernel oil and edible vegetable oil, which is rapeseed, cottonseed and soybean oil. This produced a randomly structured MLCT with TAG composition of LLL (49.5% to 52.7%) LLM or LML (37.3% to 39.6 3%) LMM or MLM (8.6% to 9.3 4%) and MMM (0.1% to 0.2%)
Acidolysis involved the exchange reaction between acyl moiety of acylglycerol and a free carboxylic acid. For MLCT production via acidolysis reaction, the acylglycerol mostly came from native oils such as soybean oil, canola oil, lard, fish oil, sesame oil, borage oil, menhaden oil, and chicken fat (Lee and others 1999; Xu and others 2000; Kawashima and others 2001, 2002; Kim and Akoh 2006; Zhao and others 2007; Li and others 2008; Shuang and others 2009). These native oils were used in acidolysis as it contained essential fatty acids like, linoleic acid (LA), alpha- linoleic acid (ALA), eicosapentanoic acid (EPA), docosahexanoic acid (DHA), and y-linolenic acid (GLA) that impart good health to our body. However, tripalmitin, tristearin, and triolein also can be used as the acyglycerol for this reaction (Sellappan and Akoh 2000; Yankah and Akoh 2000). As for the free carboxylic acid, the most commonly used is the caprylic acid (CA). Little has been done on capric or lauric acid (Sellappan and Akoh 2000; Nunes and others 2011). Summary of research done on MLCT production via acidolysis is shown in Table 2.
Table 2–. Acidolysis reaction for MLCT production: source of long chain triacylglycerol, type of lipase used, reaction condition, study design, and the amount of CA incorporated in the TAG molecule.
|Study||Edible oil||Lipase producing microorganisms||Parameter||Study design||Incorporation of CA (mol%)|
|Temperature/°C||Enzyme load/%||Water content/%||Time/h||Edible oil/CA ratio|
|Kim and others (2010)||Borage oil||Pichia lynferdii NRRL Y-7223||10–20||4||–||24||1 : 4||Screw cap Erlenmeyer flask shaking at 300 rpm||45.70|
|Shuang and others (2009)||Soybean oil||RM IM||38.5||11.9||15.4||20.4||5.7||Shake flask at 200 rpm.||44.90|
|Li and others (2008)||Soybean oil||TLIM||40||16||4||20||1 : 3||Stirred at 150 rpm||27.01|
|Zhao and others (2007)||Lard||TLIM||55||5 to 10||10||24||1 : 2||Screw cap test tube, orbital shaking water bath at 150 rpm||50.14|
|Akoh and Moussata (2001)||Fish Oil and Canola Oil||RMIM||–||–||–||–||–||Lab scale packed bed reactor||29.50 for fish oil and 40.1 canola oil|
|Nunes and others (2011)||Olive oil||Rhizopus oryzae||40||5/10||–||24||1 : 2||Thermostated capped cylindrical glass vessel,400 rpm||34.80|
|Kawashima and others (2002)||Borage oil||Rhizopus oryzae.||30||5|| || ||1 : 2||Fixed packed bed reactor with 15 g.||55.60|
|Shuang and others (2009)||Menhaden oil||RM IM||65||290||0.15||3 to 33.5||4.5||Packed bed reactor||40.00|
Besides, choosing the right type of enzyme is important when running acidolysis reaction as different types of enzyme will affect the yield of MLCT. For acidolysis reaction to produce MLCT, 1,3 specific immobilized enzyme Rhizomucor meihei (RM IM) (Novozyme, Bagsvard, Denmark) lipase is commonly used compared to 1,3 specific enzyme TLIM though sometimes nonspecific enzymes such as Rhizopus oryzae and Rhizopus delemar lipase may be used. 1, 3 specific enzymes will make sure that the essential fatty acid is maintained in the sn2 position, creating the desired structured type of MLCT (Sellappan and Akoh 2000; Yankah and Akoh 2000; Foresti and Ferreira 2010; Nunes and others 2011). When RM IM enzyme is utilized for acidolysis reaction, it normally gives around 40 to 50 mol% of CA incorporation at reaction times of around 20 to 24 h (Akoh and Moussata 2001; Kim and Akoh 2006; Li and others 2008; Shuang and others 2009). As for Thermomyces languninosa (TLIM) (Novozyme, Bagsvard, Denmark) enzyme at the same duration time, 20 h, and only 27.01 mol% of CA can be incorporated into soybean oil. However, when organic solvent such has hexane and isooctane is added in the reaction, TLIM will give a 50.14% of CA incorporation (Zhao and others 2007). The enzyme load that was normally used is in between 5% and 10% (w/w). At least 24 h of residence time were required for a maximum CA incorporation.
Besides 1-step acidolysis reaction discussed previously, there is a 2-step enzymatic process for MLCT production. For this method, the 1st step involved the production of TriPUFA from EPA, DHA, and AA via esterification with glycerol at 3 : 1 mol/mol using Candida antratica (Novozyme, Bagsvard, Denmark) enzyme for 24 h. The TriPUFA of the following (1) 89% of y-linolenic acid (GLA), (2) 89% of archidonic acid (AA), (3) 88% of ecosapentaenoic acid (EPA), and (4) 83% of docosahexanoic acid (DHA) then undergone acidolysis with caprylic acid via Rhizopus delemar lipase (Ta-lipase Tanabe Seiyala Co. Ltd., Osaka, Japan) for 48 h producing acylglycerol containing CA. This 24-h acidolysis step was carried out consecutively for 3 cycles to increase the incorporation of CA. The repeated acidolysis reaction managed to increase the amount of CA incorporated in each of the TriPUFA with TriE 66%, TriA 63.8%, TriG 52.6%, and TriD 31.1%. MLM type structured lipid also increased with the 3 repeating acidolysis steps (diCA 86.5%, dice 85.7%, diCD 62.6%, and diCE32.3%). This process gives the highest yield of MLCT among all the other acidolysis methods carried out by other researchers. In this study, the enzyme can be reused up to 10 cycles in esterification step and 20 cycles in acidolysis step, respectively, reducing the cost of operation (Kawashima and others 2001). However, the concern about this method is that it is time consuming compared to the normal 1-step acidolysis method in which the first esterification step needed 24 h and the 3 consecutive acidolysis steps each required 48 h to be carried out.
Substrate mole ratio of fatty acid/glycerol also significantly affects the CA incorporation. Increase in the amount of fatty acid will lead to an increase in the CA incorporation (Li and others 2008). However, too much of the fatty acids will make the medium too acidic, thus inactivating the enzyme. Furthermore, the use of higher amounts of fatty acids will also incur higher cost of production. Thus, it is important get the optimum substrate mole ratio for acidolysis reaction to obtain a high yield of MLCT.
Temperature is a crucial parameter as it will affect the activity of enzyme, solubility and viscosity of the substrate. Temperature for acidolysis reaction normally falls in the range of 40 to 65 °C. However, recently, novel cold active lipase from Pichia lynferdii NRRL Y-7223, (culture collection of National Centre for Agricultural Utilization Research, Peoria IL, USA) was found to have comparable activity with RMIM at 20 °C. This Pichia lynferdii NRRL Y-7223 cold lipase gives 47.5% of CA incorporation on the borage oil at 20 °C, which is comparable to the RMIM with 45.7% incorporation at 40 °C (Kim and others 2010). Lower temperature for acidolysis reaction is preferable especially for structured lipid that contained PUFA such as fish oil, borage oil, GLA from evening primrose oil, which is susceptible to oxidation. Hence, it is necessary to maintain the temperature as low as possible during storage or reaction. Besides, lower temperature uses less energy consumption. However, this is not suitable to be applied to TAG production that has higher melting points such as palm kernel olein as this TAG will crystallize during the reaction.
The drawback of this acidolysis reaction is that a high ratio of fatty acids especially short chain and MCFA, which are easily soluble will create acidic condition in the reaction. This will inactivate the enzyme and restrict its reaction, leading to a low yield of MLCT.
Esterification is another alternative in the synthesis of MLCT. However, not much research has been done on the esterification reaction of MLCT production due to the high cost of fatty acids and glycerol. In esterification process, the desired fatty acid (such as oleic acid, stearic acid, capric acid, and so on) is made to react with glycerol in the presence of the enzyme lipase. To drive the reaction forward towards synthesis, vacuum pump or nitrogen gas purging is used to remove the water formed during the esterification reaction. Esterification process is a much more preferable method to produce a higher concentration of MLCT as it gives a high purity of MLCT and few unnecessary TAG, FFA, and MAG as compared to acidolysis. However, the drawback from this method is that it does not contain any natural antioxidants. Thus, natural antioxidants such as rosemary extract, sage extract, or chemical antioxidant such as tert–butyl hydroquinone (TBHQ), butylated hydroxyanisole (BHA) has to be added to increase the oxidative stability of this oil (Koh and others 2009).
Koh and others (2010) and Arifin and others (2011a, 2012) managed to produce MLCT oil via esterification reaction. Both studies use the same RMIM enzyme and same MCFA (capric acid) but different LCFA, with the former using oleic acid and the latter using stearic acid as their substrate. Both studies optimized the conditions for MLCT production using response surface methodology. Koh and others (2010) managed to get 58 wt% of MLCT oil under optimized conditions of 13.6 to 14 h reaction time, 7.9% to 8% for enzyme load, and 3 : 1 for fatty acid/glycerol molar ratio. This is comparable to that reported by (Arifin and others 2011a, 2012) who obtained 59.76 wt% of MLCT oil with 10% enzyme load, 70 °C reaction temperature, 14 h reaction time, and 3.5 : 1 substrate mole ratio.