Production of MAG via enzymatic glycerolysis

Production of MAG via enzymatic glycerolysis Norul Naziraa Ahmad Jamlus, Darfizzi Derawi, and Jumat Salimon Citation: AIP Conference Proceedings 1678, 050034 (2015); doi: 10.1063/1.4931313 View online: http://dx.doi.org/10.1063/1.4931313 View Table of Contents: http://scitation.aip.org/content/aip/proceeding/aipcp/1678?ver=pdfcov Published by the AIP Publishing Articles you may be interested in The utilization natural mineral in the process of palm oil glycerolysis AIP Conf. Proc. 1677, 100004 (2015); 10.1063/1.4930762 Production of enzymatic protein hydrolysates from freshwater catfish (Clarias batrachus) AIP Conf. Proc. 1614, 323 (2014); 10.1063/1.4895216 Application of consolidate enzymatic system of Fusarium and Saccharomyces to enhance the production of ethanol from spent grain J. Renewable Sustainable Energy 5, 053112 (2013); 10.1063/1.4821518 Optimization of enzymatic hydrolysis of prairie cordgrass for improved ethanol production J. Renewable Sustainable Energy 4, 033118 (2012); 10.1063/1.4729587 TWT, mags still kicking Phys. Today 30, 65 (1977); 10.1063/1.3037513

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Production of MAG via Enzymatic Glycerolysis Norul Naziraa Ahmad Jamlus, Darfizzi Derawi and Jumat Salimon School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia Abstract. Enzymatic glycerolysis of a medium chain methyl ester, methyl laurate was performed using lipase Candida antarctica (Novozyme 435) for 6 hours at 55 . The percentage of components mixture of product were determined by using gas chromatography technique. The enzymatic reaction was successfully produced monolaurin (45.9 %), dilaurin (47.1 %) and trilaurin (7.0 %) respectively. Thin layer chromatography (TLC) plate also showed a good separation of component spots. Fourier transformation infra-red (FTIR) spectrum showed the presence of ester carbonyl at wavenumber 1739.99 cm-1 and hydrogen bonded O-H at 3512.03 cm-1. The product is potentially to be used as emulsifier and additive in food industry, pharmaceutical, as well as antibacterial. Keywords: Methyl laurate, enzymatic glycerolysis, monolaurin, lipase candida antarctica (Novozyme 435)

INTRODUCTION Enzymatic reaction is a green approach to perform a chemical reaction. It requires moderate reaction condition with non-toxic chemicals. This green and moderate chemical reaction is favored in research to produce a human consumption product such as monoacylglycerols and diacylglycerols. Monolaurin (ML) is a medium chain monoacylglycerol with 12 carbons number. Conventionally, it has been used as an emulsifier in the food industries. It also has been reported as anti-bacterial, particularly against gram-positive bacteria [1] ML compounds can inhibit the bacterial cells from reproducing by dissolving and penetrating into the phospholipid layer of bacterial cell. Then, the cell membrane layer will be disintegrated. Thus, the bacteria cell replication process will be inhibited [2]. Naturally, monolaurin is an active ingredients in breast milk, which act as a booster to the infant's immune system [3]. There are some reports discussed about ML production. Previously, researchers are focusing on esterification of fatty acid to produce ML. Generally, ester compounds produced commercially using acid catalyst such as sulfuric acid and hydrochloric acid. Homogeneous catalysts will lead to pollution problem because toxic, corrosive and produces byproducts that makes it difficult to be separated in the reaction medium [5]. These pollution problem will increase the cost of production although it’s effectively as a catalyst reaction. Furthermore, the acid catalyst is needed in large quantities and the reactants used excessive need to ensure the reaction always forward so that can produce a lot of ester. There is also the esterification process using heterogeneous catalysts such as aluminium and zirconium. Heterogenous catalysts can react in continuous process, producing good quality product, reusable, environmentally friendly and more effective than acid catalysts and enzymes [6]. Reaction using metal oxide catalyst (Sn, Al2O3) was also carried out which produce high yield of reaction that can reached until 89% [7]. In addtion, there are new method to produce MAG that have high purity palm oil glycerolysis fully hydrogenated catalyzed by loaded with K2CO3(K2O3/HT). The reaction temperature is 200 , K2CO3/HTI was loading at 0.8% by weight( mass FHPO), the mass ratio FHPO to glycerol 5: 2 and the reaction time is 2 hours [8]. Malaysia is a major oleochemicals exporter. The main source of oleochemicals in Malaysia is palm oil. Glycerin or glycerol is a byproduct from the methyl esters (biodiesel) production. The bulky production of glycerol cause low and unstable market value. Chemists are actively conducting research in glycerol modification in order to increase its value. Glycerol can be reacted with methyl esters to produce medium chain monoacylglycerols and diacylglycerols. These products give higher value to the glycerol. Methyl laurate is a medium chain methyl ester. It can be used as a raw materials in enzymatic glycerolysis reaction to produce ML. There are limited sources discussed about monolaurin production from methyl laurate using enzymatic glycerolysis. In this paper, we report the production of monolaurin, dilaurin synthetically via enzymatic glycerolysis of methyl laurate. Thin layer chromatography (TLC), fourier transformation infra-red (FTIR) spectroscopy and gas chromatography (GC) were performed as characterization techniques. The 2015 UKM FST Postgraduate Colloquium AIP Conf. Proc. 1678, 050034-1–050034-7; doi: 10.1063/1.4931313 © 2015 AIP Publishing LLC 978-0-7354-1325-2/$30.00

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EXPERIMENTAL Raw Material Glycerol 99%, methyl laurate C13H26O2, lipase Candida antarctica (Novozyme 435), sodium chloride, sodium bicarbonate, chloroform, diethyl ether, ethyl-ethanol, sodium sulfate are purchased from Sigma Aldrich and distilled water supplied from the oleochemicals lab.

Enzymatic Glycerolysis of Methyl laurate Reaction ratio between glycerol and methyl laurate is 1: 1 (mol/mol). About 0.5 g of lipase candida antarctica (Novozyme 435) (based on 500 unit/g glycerol) is used as a catalyst in the reaction. Mixtures are heating at 60oC for 6 hours under reflux. After the reaction ended, the sample was post-treated with sodium bicarbonate solution, 40 ml of sodium chloride, 60 mL of distilled water, 40 mL of chloroform 40 mL of ethanol. Unreacted glycerol and water as by-product were dissolved in ethanol, while the desired product was dissolved by chloroform. Then, the product is characterized to confirm its components.

Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography (GC) Analysis A total of 100 mL of TLC mobile phase was provided by the ratio of solvent toluence: chloroform: acetone is 70:20:10 (v / v) [8]. FTIR Spectrometer Perkin-Elmer model Spectrum BX with NaCl window method is used to analyze the present of significant functional groups. GC was performed by using column DB-ht (30m x 0.25mmx 0.10 ), temperature is 100(0) -380(5) rate 5/min, injector 250 and detector 400 . The sample was prepared by adding 0.03 ml with 1.0 ml ethyl acetate.

RESULT AND DISCUSSION The samples were analysis by Gas Chromatography (GC), Thin Layer Chromatography (TLC) and Fourier Transform Infrared Spectroscopy (FTIR).

Lipase Candida antarctica (Novozymes 435) The reaction conditions are optimum and effective because it occurs at low temperature. At low temperature the sample 1-MAG more easily formed as a precipitate and can be separated from the reaction medium through the screening process. Meanwhile, it can prevent changes in discoloration and unsaturated fatty acids. Application to reaction catalysis, lipase is limited to aqueous systems because it will affect the weak activity when the presence of organic solvents. Therefore, this reaction is carried out in medium without solvent [9]. Lipase candida antarctica (novozymes 435) can be used ten times without loss of significant activity[10].


OH O

Lipase candida antarctica

+

OH

OCH3

OH

Glycerol

Methyl laurate

(Novozyme 435)

O

O OH OH

Monolaurin (ML) +

O

O O

O

Dilaurin (DL)

OH

+ O

O O

+

O

3H2O

O

O

Trilaurin (TL) FIGURE 1 Reaction between methyl laurate and glycerol.


Gas chromatographic analysis FIGURE 2 and 3 show result gas chromatographic of reaction. The peaks of ML, DL and TL clearly detected at tR(s)= 15.5, 30.6 and 40.3 min., respectively. Results were identified by direct comparison with standard mixture. Composition of monolaurin, dilaurin and trilaurin were related to the total area of the chromatogram. The result show in TABLE (1).

Biodiesel

Dilaurin Monolaurin

Trilaurin

FIGURE 2 Chromatogram of reaction sample

Monolaurin Standard

FIGURE 3 Chromatogram of monolaurin standard


Table (1) Composition of product components [11]

Component Composition (%)

Monolaurin 45.9

Dilaurin 47.1

Trilaurin 7.0

Thin layer chromatography

Biodiesel

Biodiesel

TL TL Fatty acid

Fatty acid

1,3- DL 1,3-DL

1,2- DL

1,2-DL

ML

ML

Glycerol (a)

Methyl laurate

Sample

(b)

Figure 4 a) TLC of reference lipids and biodiesel samples [12]. b) TLC of sample reaction methyl laurate and glycerol.

TLC plate is stationary phase, which adsorbs molecules by its polarization. Separation occurs as a component of the reaction products have different affinity degrees the stationary phase. Therefore, the adsorption depends on the interaction of certain components with the stationary phase [12]. Sample response dripped on TLC plates is called the substrate. More polar substrates will adsorb first. This is because the interaction of the substrate with the stationary phase is very strong. Products of the reaction are ML, DL and TL . ML has two OH groups and more polar than the DL and TL. It moves much more slowly during the elution process and form hydrogen bonds with the stationary phase. Therefore, ML was eluted first compared to DL and TL.While, DL was eluted first compared TL because DL have one OH group (FIGURE 4). This causes the interaction between DL with the stationary phase is stronger than TL [13]. The analysis showed that the desired compounds of ML produced during the reaction is carried out. However, DL and TL also produced during the reaction.

FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR) FTIR analysis for 6 hours reaction time were carried out. In these analysis, the functional group should exist are ester, 1735 cm-1- 1746 cm-1 and hydroxyl, 3200 cm-1 – 3600 cm-1. FIGURE 5 displays a peak at a frequency of 1739.99 cm-1 and are identified as C=O ester bond. However, the peaks are not really sharp. This was because during the process of neutralization reaction products using NaHCO 3, lauric acid is likely not neutralize properly. The OH stretch occurs at 3512.03 cm-1. This mean that the sample is neat liquid.


Figure 5 The infrared spectrum of monolaurin

CONCLUSION As a conclusion, ML was successfully produced with 8.8 % weight of yield. About 3.6 % percentage peak area of ML was analyzed by using gas chromatography (GC). Besides that, monolaurin clearly seen at TLC plate using toluence: choloroform: acetone (70:20:10) solvent. ML is potentially to be used as antibacterial emulsifier.

ACKNOWLEDGEMENTS The authors would like to acknowledge the Universiti Kebangsaan Malaysia for the research facilities and research grant no. GGPM-2014-033, also we would like to thank to the Ministry of Education for the research grant no. FRGS/2/2014/ST01/UKM/01/2 and My Brain 15 scholarship.

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