Relation between the effect of increasing reaction time, reaction temperature and ratio of substrate on incorporation of lauric acid into palm kernel oil

Lauric acid is a medium chain fatty acid that is composed of 12 C atoms, also found in palm kernel oil (PKO). PKO belongs to medium chain triglycerides (MCT) containing high levels of lauric acid which has health benefits. This research aims to find the relationship between the effect of increasing reaction time and reaction temperature and the mole ratio of substrate on the incorporation of lauric acid into palm kernel oil. From the research results it was revealed that increasing the reaction time up to 24 hours could increase the incorporation of lauric acid but decreased if the reaction time was increased to 40 hours. Furthermore, an increase in the reaction temperature from 44°C to 64°C increased the incorporation of lauric acid, if it was increased from 64°C to 84°C it resulted in a decrease in incorporation of lauric acid, if it was increased again to 104°C it resulted in incorporation of lauric acid -21.6%. In increasing the substrate ratio, the higher the substrate ratio (1:11), the higher the incorporation of lauric acid.


Introduction
Palm oil has a high saturated fat content, is semi-solid at room temperature, and contains various types of saturated fat, lauric acid (0.1%), myristic acid (1%), stearic acid (5%) and palmitic acid (44%) [1].In the human body, lauric acid is converted into monolaurin which contains a natural antibiotic that works by damaging the membrane covering germ cells, viruses, and microorganisms that are infested with fatty acids, that's why researchers pay special attention to the synthesis of lauric acid [2].
Palm kernel oil is an oil in which more than 50% of the fatty acid content is medium-chain fatty acids, namely C8 (3-5%), C10 (3-5%), and C12 (46-52%) so that it can be used as an MCT substrate.MCT products have the potential to be developed in Indonesia, taking into account that the raw material for palm kernel oil is abundant and the price is cheaper than coconut oil [3][4][5].MCT is not available naturally so it needs to be synthesized [6,7].MCT synthesis is enzymatically more advantageous than chemically such as environmentally friendly reaction conditions, proceeding at lower temperatures, little or no reaction by-products, ease of product recovery and in controlling the reaction [8,9].
Incorporation of medium chain fatty acids (capric acid) into pumpkin seed oil by solvent-free enzymatic acidolysis system was carried out under the best reaction conditions, namely at 45 ℃ , 1:2 substrate ratio (molar basis), and 5% enzyme weight [9][10][11].The results of previous studies revealed the success of incorporated lauric acid into Echium seed oil by enzymatic acidolysis of immobilized commercial lipase [12][13][14].This research aims to find the relationship between the effect of increasing reaction time and reaction temperature and the mole ratio of substrate on the incorporation of lauric acid from palm kernel oil.

Research design
This research was carried out from January 2022 to August 2022 at the Faculty of Mathematics and Natural Science of Universitas Sumatera Utara and the Indonesian Oil Palm Research Institute Laboratory.This research is classified as exploratory.Each reaction in this study was carried out with two repetitions.

Experimental methodology 2.2.1. Lipase-catalyzed acidolysis reaction.
Enzymatic acidolysis reaction was carried out referring to the modified method [15].A total of 10 g of the substrates (palm kernel oil and lauric acid with a molal ratio of 1:3, 1:7, and 1:11) was transferred into a 50 mL Erlenmeyer flask.Then the substrate mixture was added with non-specific lipase enzyme NS 400190 as much as 7% (of the total weight of the substrate).The reactions were carried out in a solvent-free system with different temperature treatments (44 -104 ℃ ) for 0 -48 hours on a heater shaker with a speed of 200 rpm.After the acidolysis reaction, the product mixture was separated from the enzyme using Whatman filter paper No. 4. Products were stored in a freezer (T, -4 ℃ ) for further analysis.Each reaction in this research was carried out with two replicates.The residual activity of the recovered enzyme was calculated to assess the operational stability of the enzyme.

Separation and analysis of triglycerides using TLC.
This procedure was done to the modified method [16].Oil triglycerides resulting from the acidolysis reaction were analyzed using Thin Layer Chromatography (TLC) silica gel 60 F 254 20 x 20 cm glass plate (Merck, Darmstadt, Germany), with a developer solution of hexane: diethyl ether : glacial acetic acid (80:20:1, v/v/v).Spots formed were visualized with iodine gas.Triglycerides were identified by comparing them with the standard.Spots that were triglycerides were crushed and dissolved in hexane.The hexane fraction was used for fatty acid composition analysis using GC.

Fatty acid composition analysis by GC.
The fatty acid composition was analyzed based on MPOB [17].The fatty acid composition was analyzed by GC 2 (Series 2010 Plus, Shimadzu) capillary column with flame ionization detector (FID) under operating conditions: Crossbond capillary column (Carbowax Polyethylene glycol) of 25 m length, 0.25 mm diameter, 0.25 µm film thickness.Column temperature from 120℃ for 7 minutes and then increased to 240℃ with a temperature run of 3℃/minute and for 26 minutes the temperature was constant at 240℃.Detector temperature 240℃, injection temperature 240℃ with helium carrier gas, gas flow rate 0.8 mL/min, make up gas Nitrogen with gas flow rate 30 mL/min, split injection mode with ratio 1:100.Fatty acid composition was identified by comparing the retention time of the sample peak with the standard solution.

Effect of increasing reaction time on incorporation of lauric acid into palm kernel oil
From the results of the study it was revealed that the longer the reaction time (hours) led to an increase in lauric acid incorporation, shown in Table 1 and for more details can be seen in Figure 1.
From Figure 1 it can be seen that there is a positive relationship between reaction time and lauric acid incorporation into triglycerides but if the reaction time is increased again until 40 hours it causes lauric acid incorporation to begin to decrease.These results indicate that the reaction time influences the concentration of lauric acid incorporation.This is in accordance with previous research conducted by [18] which stated that there are the best conditions for optimal synthesis of lauric acid [18,19].Based on this result, the reaction time of 24 hours showed the highest lauric acid incorporation concentration and chosen as the reaction time for further research.

Effect of increasing temperature reaction on incorporation of lauric acid into palm kernel oil
An increase in reaction temperature causes an increase in lauric acid incorporation, shown in Table 2 and for more details can be seen in Figure 2.

Table 2. Effect of increasing reaction temperature (℃) on lauric acid incorporation (%)
Increase in reaction temperature (℃) Lauric acid incorporation (%) Figure 2. The relationship between the increase in reaction temperature (℃) and the incorporation of lauric acid (%).
From Figure 2 it can be seen that there is a negative linear relationship between increasing reaction temperature and lauric acid incorporation, which means that an increasing reaction temperature of 20℃ will decrease the incorporation of lauric acid -13.85% (based on linear regression).The reaction temperature 44℃-64℃ shows the highest incorporation of lauric acid by 6,1% compared to temperatures of 64℃-84℃ and 84℃-104℃ (2,9 and -21,6%).The higher the reaction temperature until 64℃, the solubility of the reactants increases and decreases the viscosity of the solution and the enzyme performance is also more active [19,20].On the other hand, the extremely high temperatures (104℃) denature proteins of the enzyme, which disable the enzyme activity thereby decreasing lauric acid incorporation -21,6%.

Effect of increasing the molar ratio of substrate on incorporation of lauric acid into palm kernel oil
An increase in the substrate molar ratio causes an increase in the incorporation of lauric acid, shown in Table 3 and for more details can be seen in Figure 3.As shown in Figure 3, the substrate ratios have a great influence on the increase rate of incorporation lauric acid content.When molar ratio was increased from 1:3 to 1:11, incorporation of lauric acid increased from 71,3% to 80,0% respectivel, similar trends had been reported by Li et al [21].From Figure 3 it can be seen that there is a positive relationship between increasing the substrate ratio (mole basis) and lauric acid incorporation.The results indicated that substrate ratio had an effect on the yield of incorporation of lauric acid into PKO at all the time points studied.The yield of incorporation lauric acid was in the range of 71,3-80,0%.

Conclusion
From the research results, it can be concluded that the ideal conditions are as follows: an increase in reaction time up to 24 hours can increase the incorporation of lauric acid from palm kernel oil by 22.1% but decreases to 21.5% if the reaction time is increased to 40 hours.Furthermore, an increase in the reaction temperature from 44℃-64℃ increased the incorporation of lauric acid, if it was increased from 64℃-84℃ it resulted in a decrease in incorporation of lauric acid, if it was increased again to 104 ℃ it resulted in a decrease in incorporation of lauric acid -21.6%.In increasing the substrate ratio, the higher the substrate ratio (1:11), the higher the incorporation of lauric acid.

Table 3 .
Effect of increasing the molar ratio of substrate on lauric acid incorporation (%) Relation between increase in substrate ratio (molar basis) and lauric acid incorporation (%)