Generation of flavor esters from coconut lipids by lipase mediated biocatalysis

GENERATION OF FLAVOR ESTERS FROM COCONUT LIPIDS BY LIPASE-MEDIATED BIOCATALYSIS SUN JINGCAN NATIONAL UNIVERSITY OF SINGAPORE 2013 GENERATION OF FLAVOR ESTERS FROM COCONUT LIPIDS BY LIPASE-MEDIATED BIOCATALYSIS SUN JINGCAN (M. Eng. Chinese Academy of Sciences; B. Eng. Beijing Technology and Business University) A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHIAE DOCTOR DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2013 Acknowledgements Foremost, I would like to express my sincere and deep gratitude to my supervisor Dr. Liu Shao Quan for his continuous support of my Ph.D study and research. I appreciate all his contributions of time, ideas, and encouragement to make my Ph.D. experience productive and stimulating. Without his understanding, inspiration and guidance, I could not have been able to complete this project. I would also like to thank my co-supervisor Dr. Yu Bin for his valuable instructions. His profound knowledge, abundant experience and analytical expertise are incredibly helpful in overcoming many of the difficulties which arose throughout my doctoral program. Besides my supervisors, I also wish to give my thanks to Prof. Zhou Weibiao who gave me insightful and constructive suggestions for my research. My sincere thanks also go to my lab mates, Lee Pin Rou, Cheong Mun Wai, Li Xiao, Chin Jin Hua, Lim Yunwei, Chan Li Jie, Chen Dai, Wilson Lee for their contributions and help in my work and life. In addition, I would also like to thank FST laboratory staff, Ms. Lee Chooi Lan, Ms. Lew Huey Lee, Ms. Jiang Xiao Hui and Mr. Abdul Rahman for their consistent technical assistance. I am also grateful to the National University of Singapore for granting the research scholarship. Last but not least, I would like to thank my family for their unconditional support and endless love which are the source of strength for me. I Table of Contents ACKNOWLEDGEMENTS . I LIST OF PUBLICATIONS AND MANUSCRIPTS . VI LIST OF TABLES VII LIST OF FIGURES VIII LIST OF SYMBOLS XIII SUMMARY CHAPTER INTRODUCTION AND LITERATURE REVIEW .3 1.1 BASIC KNOWLEDGE OF FLAVOR ESTERS .3 1.2 BIOTECHNOLOGICAL METHODS OF FLAVOR PRODUCTION 1.2.1 Plant cell and tissue cultures 1.2.2 Fermentation .5 1.2.3 Enzymatic biocatalysis 1.3 LIPASE-CATALYSED BIOCATALYSIS .7 1.3.1 Characteristics of lipases 1.3.2 Applications of lipases in industries 1.3.3 Synthesis of esters through esterification 10 1.3.4 Synthesis of esters through transesterification 11 1.3.5 Ester synthesis in solvent-free systems 11 1.3.6 Ester synthesis in organic solvents 12 1.3.7 Ester synthesis in ionic liquids 13 1.3.8 Ester synthesis in supercritical fluids 14 1.3.9 Ester synthesis in aqueous media 15 1.4 COCONUT LIPIDS FOR ESTER SYNTHESIS 16 1.5 FUSEL OIL AS AN ALCOHOL SOURCE FOR ESTER SYNTHESIS 17 1.6 OBJECTIVES 18 CHAPTER HS-SPME GC-MS/FID METHOD DEVELOPMENT FOR OIL SAMPLE ANALYSIS 20 2.1 INTRODUCTION .20 2.2 MATERIALS AND METHODS 22 2.2.1 Materials and reagents 22 2.2.2 Transesterification of coconut oil with fusel oil 22 2.2.3 Standard solution preparation .23 2.2.4 HS-SPME procedure and optimisation .23 2.2.5 GC-MS analysis 24 2.2.6 Method validation .25 2.3 RESULTS AND DISCUSSION .25 II 2.3.1 Transesterification of coconut oil 25 2.3.2 Matrix modification .28 2.3.3 HS-SPME parameters optimisation 31 2.3.4 Quantitative analysis .32 2.4 CONCLUSION .35 CHAPTER DETERMINATION OF FLAVOUR ESTERS IN ENZYMATICALLY TRANSFORMED COCONUT OIL .36 3.1 INTRODUCTION .36 3.2 MATERIALS AND METHODS 37 3.2.1 Materials and reagents 37 3.2.2 Transesterification in solvent-free system .37 3.2.3 Sample preparation and analysis 37 3.2.4 Calibration and validation 38 3.3 RESULTS AND DISCUSSION .39 3.3.1 Analysis of transesterified coconut oil 39 3.3.2 Calibration curves, LOD and LOQ .41 3.3.3 Reproducibility 44 3.3.4 Recovery test .44 3.3.5 Time-course of Lipozyme TL IM-catalysed transesterification of coconut oil 47 3.4 CONCLUSION .48 CHAPTER LIPASE-CATALYSED ESTER SYNTHESIS FROM COCONUT OIL IN SOLVENT-FREE SYSTEM 49 4.1 INTRODUCTION .49 4.2 MATERIALS AND METHODS 50 4.2.1 Materials and reagents 50 4.2.2 Solvent-free transesterification of coconut oil 50 4.2.3 Stability of Lipozyme TL IM 51 4.2.4 Sample analysis .52 4.3 RESULTS AND DISCUSSION .52 4.3.1 Effect of reactant molar ratio 53 4.3.2 Effect of enzyme loading .55 4.3.3 Effect of reaction temperature .55 4.3.4 Effect of shaking speed 56 4.3.5 Effect of reaction time .57 4.3.6 Operational stability of Lipozyme TL IM 58 4.3.7 Determination of key esters formed under optimised conditions 60 4.4 CONCLUSION .61 CHAPTER OPTIMIZATION OF ESTER SYNTHESIS FROM COCONUT OIL WITH RESPONSE SURFACE METHODOLOGY .63 III 5.1 INTRODUCTION .63 5.2 MATERIALS AND METHODS .64 5.2.1 Materials and reagents 64 5.2.2 Transesterification reaction assays and analysis 64 5.2.3 Experimental design and statistical analysis 65 5.3 RESULTS AND DISCUSSION 66 5.3.1 Model fitting 66 5.3.2 Effects of enzymatic synthesis parameters 69 5.3.3 Attaining optimum condition and verification .75 5.4 CONCLUSION .76 CHAPTER MECHANISM STUDY ON THE LIPASE-CATALYSED REACTIONS IN OIL SYSTEM .77 6.1 INTRODUCTION .77 6.2 MATERIALS AND METHODS 78 6.2.1. Materials and reagents .78 6.2.2. Lipase-catalysed synthetic reactions with ethanol .78 6.2.3. Lipase-catalysed synthetic reactions with fusel oil 79 6.2.4. Sample preparation and analysis .79 6.3 RESULTS AND DISCUSSION .79 6.3.1 Ester synthesis in coconut oil spiked with ethanol and acids 80 6.3.2 Ester synthesis in coconut oil spiked with fusel oil and acids .83 6.4 CONCLUSION .85 CHAPTER LIPASE-CATALYSED ESTER SYNTHESIS FROM COCONUT CREAM IN AQUEOUS SYSTEM 86 7.1 INTRODUCTION .86 7.2 MATERIALS AND METHODS 87 7.2.1 Materials and reagents 87 7.2.2 Biosynthesis of fatty acid esters 88 7.2.3 Sample preparation and analysis 88 7.2.4 The Taguchi methodology .89 7.2.5 Experimental design 90 7.3 RESULTS AND DISCUSSION .90 7.3.1 Experimental results and statistical analysis 90 7.3.2 Effects of parameters on biosynthesis of esters .93 7.3.3 Confirmation experiment 97 7.4 CONCLUSION .98 CHAPTER MECHANISM STUDY ON THE LIPASE-CATALYSED REACTIONS IN AQUEOUS SYSTEM .99 IV 8.1 INTRODUCTION .99 8.2 MATERIALS AND METHODS .100 8.2.1 Materials and reagents 100 8.2.2 Synthesis of esters in coconut cream with ethanol 101 8.2.3 Synthesis of esters in coconut cream with fusel oil .101 8.2.4. Synthesis of esters in buffer system 101 8.2.5 Sample preparation and analysis 102 8.3 RESULTS AND DISCUSSION .102 8.3.1 Ester synthesis in coconut cream spiked with ethanol 103 8.3.2 Ester synthesis in coconut cream spiked with fusel oil 106 8.3.3 Esterification catalysed by lipase in buffer system 110 8.4 CONCLUSION .112 CHAPTER SYNTHESIS OF FLAVOR ESTERS VIA SYNCHRONOUS FERMENTATION AND BIOCATALYSIS 113 9.1 INTRODUCTION .113 9.2 MATERIALS AND METHODS 114 9.2.1 Materials and reagents 114 9.2.2 Preparation of yeast pure culture and preculture 115 9.2.3 Ester synthesis through fermentation and biocatalysis . 115 9.2.4 Analysis of pH value, yeast enumeration and sugar concentrations . 116 9.2.5 Quantitative analysis of volatile compounds . 116 9.3 RESULTS AND DISCUSSION .117 9.3.1 Dynamic changes of yeast viable cell count and pH level 118 9.3.2 Sugar utilization during fermentation .120 9.3.3 Formation of ethyl esters and fatty acids after addition of Palatase 123 9.4 CONCLUSION .126 CHAPTER 10 GENERAL CONCLUSIONS AND FUTURE STUDY 127 10.1 GENERAL CONCLUSIONS .127 10.2 FUTURE STUDY 130 BIBLIOGRAPHY 132 V List of Publications and Manuscripts Publications and manuscripts derived from this thesis: 1. Sun, J., Yu, B., Curran, P., Liu, S.-Q., Quantitative analysis of volatiles in transesterified coconut oil by headspace-solid-phase microextraction-gas chromatography-mass spectrometry, Food Chemistry. 2011, 129, 1882-1888. 2. Sun, J., Chin, J. H., Yu, B., Curran, P., Liu, S.-Q., Determination of flavor esters in enzymatically transformed coconut oil, Journal of Food Biochemistry. 2012, DOI: 10.1111/j.1745-4514.2012.00660.x. 3. Sun, J., Yu, B., Curran, P., Liu, S.-Q., Lipase-catalysed transesterification of coconut oil with fusel alcohols in a solvent-free system, Food Chemistry. 2012, 134, 89-94. 4. Sun, J., Chin, J. H., Zhou, W., Yu, B., Curran, P., Liu, S.-Q., Biocatalytic conversion of coconut oil to natural flavor esters optimized with response surface methodology, Journal of the American Oil Chemists' Society. 2012, 89, 1991-1998. 5. Sun, J., Yu, B., Curran, P., Liu, S.-Q., Optimisation of flavour ester biosynthesis in an aqueous system of coconut cream and fusel oil catalysed by lipase, Food Chemistry. 2012, 135, 2714-2720. 6. Sun, J., Yu, B., Curran, P., Liu, S.-Q., Lipase-catalysed ester synthesis in solvent-free oil system: is it esterification or transesterification? Food Chemistry. 2013, 141, 2828-2832. 7. Sun, J., Lim, Y., Liu, S.-Q., Biosynthesis of flavor esters in coconut cream through coupling fermentation and biocatalysis. European Journal of Lipid Science and Technology. 2013,115, 1107-1114. Other achievements (not part of this thesis): 8. Liu, S.-Q, Lee, H.Y., Yu, B., Curran, P., Sun, J., Bioproduction of natural isoamyl esters from coconut cream as catalysed by lipases. Journal of Food Research. 2013, 2(2), 157-166. 9. Koh M.K.P, Sun, J., Shao-Quan Liu. Optimization of L-methionine bioconversion to aroma-active methionol by Kluyveromyces lactis using the Taguchi method. Journal of Food Research. 2013, 2(4), 90-100. VI List of Tables Table 2.1 Composition of fusel oil used for the transesterification reaction .23 Table 2.2 Calibration and validation of the HS-SPME-GC-MS/FID quantification method .34 Table 3.1 Major volatile compounds detected in transesterified coconut oil 42 Table 3.2 Quantification method and validation characteristics 45 Table 3.3 Recovery test results of the developed quantification method .45 Table 4.1 Fatty acid composition of the coconut oil 51 Table 4.2 Major flavor compounds identified in transesterified coconut oil .61 Table 5.1 Factors and their levels for central composite design (CCD) a 66 Table 5.2 CCD experimental design and actual, predicted conversions 67 Table 5.3 Coefficients of the model and ANOVA results 68 Table 7.1 Parameters and their levels employed in the Taguchi design for optimisation of octanoic acid ester production. 88 Table 7.2 Experimental results for the yields of octanoic acid esters and corresponding S/N ratios .91 Table 7.3 Analysis of variance for the yield of octanoic acid esters 93 VII List of Figures Figure 1.1 3D Structure of lipase from Thermomyces laguginosus (48). Figure 2.1 Chromatogram (GC-MS) of the major volatile compounds identified in transesterified coconut oil. Major peaks: (1) isobutyl alcohol; (2) isoamyl acetate; (3) *(iso)amyl alcohol; (4) ethyl octanoate; (5) unknown; (6) propyl octanoate; (7) ethyl nonanoate (IS); (8) isobutyl octanoate; (9) methyl decanoate; (10) 2-undecanone; (11) butyl octanoate; (12) ethyl decanoate; (13) *(iso)amyl octanoate; (14) propyl decanoate; (15) isobutyl decanoate; (16) methyl dodecanoate; (17) ethyl dodecanoate; (18) isoamyl decanoate; (19) propyl dodecanoate; (20) isobutyl dodecanoate; (21) ethyl tetradecanoate; (22) octanoic acid; (23) *(iso)amyl dodecanoate. *Isoamyl and active amyl alcohols and corresponding esters are referred as (iso)amyl alcohols and esters. .26 Figure 2.2 Time-course production of octanoic acid esters during lipase-catalysed transesterification of coconut oil with fusel alcohols. 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Biocatalysis and Biotransformation 1990, 3, (4), 307-316. 160 [...]... Time-course formation of octanoic acid esters during lipase- catalysed transesterification of coconut oil with fusel oil 47 Figure 4.1 Effect of reaction parameters on the synthesis of octanoic acid esters during transesterification of coconut oil with fusel alcohols A: Effect of molar ratio of alcohol to oil; B: Effect of enzyme loading; C: Effect of reaction temperature; D: Effect of shaking speed Mean... Lipase- catalysed transesterification could directly convert low-cost natural lipids into value-added flavor esters This research explored the potential of exploiting coconut lipids for the production of flavor esters, especially octanoic acid esters in both solvent-free and aqueous systems consisting of coconut oil and alcohols or coconut cream and alcohols This project investigated the synthesis of. .. synthesis of natural flavor esters Coconut cream is the thick and dense cream emulsion obtained from coconut milk Coconut cream can be further used for the production of coconut oil via dry or wet processes (125) It contains about 25% of fat and 70% of water Besides being used for cooking, coconut cream has been applied as the substrate for the production of natural aroma-active 2-phenylethyl esters via lipase- mediated. .. a novel application of coconut lipids for the synthesis of flavor esters with lipases as the biocatalysts The specific objectives of this research were to: 1 Develop sample preparation and analytical methods for the determination of flavor esters in oil samples (Chapter 2 and 3; Study described in Chapter 3 assisted by Chin Jin Hua as part of his Hons project) 2 Synthesise flavor esters in a solvent-free... occurring esters include sugar esters, fats and oils (esters of glycerol), free fatty acid esters, phosphoesters (backbone of DNA), nitrate esters, polyesters (plastic) Among these esters, only esters with low-molecular weight and low-boiling points play a significant role in the flavor and fragrance industry since they impart pleasant flavor/ aroma notes The structures of esters essentially determine... research gaps in the field of flavor ester synthesis, which are summarised below: 1 Although the synthesis of flavor esters via lipase- catalysed esterification of free fatty acids or transesterification of simple esters has been investigated, lipase- catalysed transesterification of vegetable oils for flavor synthesis is rarely reported; 2 Coconut oil has been used for the production of biodiesel, but research... the following discussion will only focus on lipase- mediated biocatalysis for ester synthesis 1.3 Lipase- catalysed biocatalysis 1.3.1 Characteristics of lipases Lipases are universal enzymes that can be obtained from animals, plants and microorganisms (37) One of the important characteristics of lipases is the interfacial activation property The activity of lipases is greatly enhanced at interfaces,... decreased, and flavor is enhanced (68, 69) In the flavor and perfume industries, lipases also play significant roles in synthesizing valuable flavor compounds, especially flavor esters Lipases have a high substrate selectivity and specificity, which makes it an effective alternative to chemical catalysts for the synthesis of flavor esters 1.3.3 Synthesis of esters through esterification Lipases are able... catalyse three different types of reactions to synthesise flavor esters, namely esterification, alcoholysis of esters with alcohols, and transesterification of esters with other types of esters (70) Among these three reactions, esterification is the most frequently employed one by researchers for ester synthesis Isoamyl acetate, one important flavor ester imparting banana flavor note, has been successfully... predication of the yield of esters Under optimised conditions, a yield of 7.3% (based oil weight) of octanoic acid esters was obtained To ascertain the reaction mechanism of lipase- catalysed transesterification, ester synthesis was carried out in a solvent-free system of coconut oil and ethanol or fusel alcohols The dynamic changes of free octanoic and decanoic acids indicate that ester synthesis catalysed by . GENERATION OF FLAVOR ESTERS FROM COCONUT LIPIDS BY LIPASE- MEDIATED BIOCATALYSIS SUN JINGCAN NATIONAL UNIVERSITY OF SINGAPORE. SINGAPORE 2013 GENERATION OF FLAVOR ESTERS FROM COCONUT LIPIDS BY LIPASE- MEDIATED BIOCATALYSIS SUN JINGCAN (M. Eng. Chinese Academy of Sciences; B. Eng. Beijing. occurring esters include sugar esters, fats and oils (esters of glycerol), free fatty acid esters, phosphoesters (backbone of DNA), nitrate esters, polyesters (plastic). Among these esters, only esters