Extraction, separation, and bio transformation of natural plant derived compounds within supercritical CO2 environment

Extraction, Separation, and Bio-transformation of Natural Plant Derived Compounds Within Supercritical CO2 Environment B B Vom Promotionsausschuss der Technischen Universität Hamburg-Harburg zur Erlangung des akademischen Grades Doktor-Ingenieur genehmigte Dissertation von M.Sc Huan Phan Tai aus Vietnam 2008 “Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes” Gutachter: Prof Dr.-Ing G Brunner Gutachter: Prof Dr.rer.nat A Liese Prüfungsausschussvorsitzender: Prof Dr.rer.nat R Müller Tag der mündlichen Prüfung: 10.12.2008 ACKNOWLEDGEMENTS This research was made possible through a study grant awarded by the DAAD - German Academic Exchange Service First of all, I would like to express my deep gratitude and appreciation to my supervisor, Prof Dr Gerd Brunner, former Head of the Institute of Thermal and Separation Processes, Hamburg University of Technology I have learned much from his tutelage and am fortunate to have had such a dedicated supervisor I give special recognition to Prof Dr Andreas Liese for his co-evaluation of my dissertation My gratitude is also express to Prof Dr Rudolf Müller, who chaired the examination committee There are also many people whom I would like to be grateful to: Dr Carsten Zetzl and Stefanie Meyer-Storckmann for their many supports, especially in registration procedure, Marianne Kammlott for analysis , Ralf Henneberg and Thomas Weselmann for technical assistance I also thank my students, Sabine Mattheeßen and Uche Okekearu, for their laboratory help I will never forget Tim Rogalinski, Gustav Schrenk, Tobias Albrecht and Thomas Ingram, who share the same office with me, and the other colleagues: Kristin Rosenkranz, Leandro Danielski, Meng-Han Chuang, Alexandre Paiva, Daniela Doncheva and Wei Sing Long We really had a good time together in TUHH Finally, I would like to thank my family and Vietnamese friends for their supports during the time I have been in Germany Table of contents Table of Contents Abbreviations v Summary vii Introduction and Structure of The Work Fundamentals and State of Knowledge 2.1 Supercritical extraction - Theoretical background 2.1.1 Supercritical fluid (SCF) a) Definition of a supercritical fluid b) Physico-chemical properties of the SCF c) Solubility in SCF 2.1.2 Supercritical fluid extraction (SFE) a) General description of SFE b) Course of extraction for SFE c) Supercritical fluid extraction modeling d) SFE application in vegetable oil extraction 12 2.2 Biotransformation with lipase 13 2.2.1 Lipase 13 2.2.2 Progress curve and determination of reaction velocity 14 2.2.3 Lipase catalysis in a conventional solvent 15 2.2.4 Lipase catalysis in SCCO2 15 2.2.5 CO2-expanded organic solvent system 16 a) High pressure CO2 - H2O - organic solvent system 17 b) Solubility of polar compounds in a multi-phase system 18 2.3 Palm Fruits and Palm Oil Extraction 20 2.3.1 Palm fruits and their composition 20 a) Palm fruits 20 b) The composition of palm oil 21 2.3.2 Palm oil processing 23 2.3.3 SFE of palm oil and derivative products – State of the art 24 a) Fractionation and purification 24 b) Extraction 24 i Table of contents 2.4 Sugar Fatty Acid Esters 25 2.4.1 Sugar fatty acid esters as surfactants 25 2.4.2 Sugar Ester Synthesis 27 a) Chemical synthesis 27 b) Enzymatic synthesis 28 2.4.3 SCF for sugar ester synthesis – State of the art 31 2.5 Mono- and di-acylglycerols 32 2.5.1 Mono- (MAGs) and di-acylglycerols (DAGs) as surfactants 32 2.5.2 MAGs and DAGs synthesis 33 a) Chemical synthesis 33 b) Enzymatic synsthesis 33 2.5.3 SCF for MAGs and DAGs synthesis 36 2.6 Response Surface Method as Experimental Design and Regression Modeling 37 Supercritical Fluid Extraction of Palm Oil 40 3.1 Materials and Methods 40 3.1.1 Materials 40 3.1.2 Equipment and experimental procedure 40 3.1.3 Analytical method 41 a) High Performance Liquid Chromatography (HPLC) 41 b) Gas Chromatography (GC) 41 c) Soxhlet extraction 43 d) Spectrometer 43 e) Karl - Fischer titration 43 3.2 Results and Discussion 44 3.2.1 Characteristics of palm mesocarp 44 3.2.2 Effect of process parameters 44 a) Effect of pressure 44 b) Effect of temperature 45 c) Effect of flow rate 45 3.2.3 Extraction with different fluids 47 a) Total amount of extract 47 b) Solubility of palm oil in subcritical propane and SCCO2 49 ii Table of contents c) Co-extracted water 49 d) Extraction of tocochromanols and carotenoids 51 3.2.4 Mathematical modeling of the extraction 54 a) Sovova model 54 b) VT II model 55 3.3 Conclusion of Chapter 57 Sugar Fatty Acid Ester Synthesis in CO2 saturated acetone 58 4.1 Materials and Methods 58 4.1.1 Materials 58 4.1.2 Equipment and experimental procedure 58 4.1.3 Analytical method 59 4.2 Results and Discussion 60 4.2.1 Screening the reaction – First observation 60 4.2.2 Effect of acetone concentration 61 4.2.3 Effect of enzyme type 62 4.2.4 Effect of enzyme concentration 63 4.2.5 Temperature effect 64 4.2.6 Pressure effect 65 4.2.7 Molar ratio effect 66 4.2.8 Effect of adding water 67 4.2.9 Reaction mechanism 69 4.2.10 Reaction progress and reaction kinetics 71 4.2.11 Enzyme stability 73 4.3 Conclusion of Chapter 73 MAG and DAG synthesis in CO2 saturated acetone 74 5.1 Materials and methods 74 5.1.1 Materials 74 5.1.2 Equipment and experimental procedure 74 a) Reaction in acetone at atmospheric pressure 74 b) Reaction in the high pressure acetone-CO2 system 75 5.1.3 Analytical method 76 a) Gas Chromatography (GC) 76 iii Table of contents b) Calculation of conversion and glyceride yield 76 5.1.4 Response surface methodology 77 5.2 Results and discussion 78 5.2.1 Solubility behavior in CO2 -expanded acetone 78 a) Solubility of palmitic acid 78 b) Solubility of a mixture 80 c) Effect of process parameters on reactant concentration 80 5.2.2 Screening the MAG and DAG synthesis reaction 81 5.2.3 Reaction progress and reaction kinetics 82 5.2.4 Effect of enzyme type 83 5.2.5 Effect of substrate ratio 84 5.2.6 Effect of adding water 85 5.2.7 Response surface analysis of MAG and DAG synthesis 87 a) Effect of process variable 89 b) Optimum condition 91 5.2.8 Screening reactions with other types of fatty acids 92 5.2.9 Enzyme stability 92 5.3 Conclusion of Chapter 93 Conclusion and Outlook 95 Appendix 96 Bibliography 108 iv Abbreviations Abbreviations AP Palmitic acid B0 Intercept Bi First order model coefficients Bii Quadratic coefficients for the i-th variable Bij Interaction coefficients for the interaction of variables i and j CCFCD Central composite face centered design CER Constant extraction rate CPO Crude palm oil DAG Diacylglycerol DCER Diffusion controlled extraction rate DS Degree of substitution FER Falling extraction rate FFA Free fatty acid GC Gas chromatography Gly Glycerol HLB Hydrophilic-lipophilic balance HPLC High performance liquid chromatography IS Internal standard L1 Heavy phase L2 Light phase MAG Monoacylglycerol OEC Overall extraction curve P Pressure Pc Critical pressure PFAD Palm fatty acid distillates RSM Response surface method SCCO2 Supercritical carbon dioxide SCF Supercritical fluid SFAE Sugar fatty acid esters SFE Supercritical fluid extraction t Time T Temperature Tc Critical temperature v Abbreviations Xi Independent variables Y Value of the response vi Summary Summary The aim of this study included extracting the interesting compounds from plant material, studying the solubility behavior of compounds in different high- and low-value classes, and transforming a low-value compound into a higher valuable one by enzymatic reaction All of these tasks were conducted in the presence of a supercritical fluid, which has many advantages in both processing and environmental aspects Firstly, oil palm mesocarp was extracted by supercritical CO2 and subcritical propane at different pressures, temperatures and flow rates Total oil yield and co-extracted water were investigated in the course of extraction Tocochromanols and carotenoids, which are very important and valuable minor compounds in palm oil, were evaluated not only in the extraction oil, but also in the residual fiber Additionally, mathematical modeling was performed for upscaling the process The result showed that oil yield up to 90% was obtained after 120 minutes Using supercritical CO2 or subcritical propane, tocochromanols and carotenoids can be coextracted with a concentration in the same range of normal commercial processing of palm oil Moreover, the recovery factors of these compounds were much higher in case of extraction with supercritical fluids than those with traditional screw pressing Among the investigated methods, recovery of tocochromanols by propane extraction was better than by CO2 extraction, while recovery of carotenoids was nearly the same However, extractions with CO2 gave a better total oil yield after 45 minutes than those with propane Palm oil has a large amount of palmitic acid in its free fatty acid content, which had been successfully separated by countercurrent gas extraction at the VTII department, TUHH Therefore, the second task of this study was about downstream processing of this fatty acid Esterification of palmitic acid and glucose by different types of enzymes was performed in CO2 expanded acetone The study included investigating key process parameters such as pressure, temperature, substrate and amount of enzyme Novozyme 435, a lipase, was selected as the best enzyme An amount of acetone up to 3% (Vacetone/Vreactor) is required to ensure that the reaction takes place in an expanded liquid phase, where the mass transfer is improved and reaction rate is accelerated A good esterification performance could be found with 30% wt enzyme related to the amount of dissolved fatty acid at an optimum temperature of 50°C and a pressure of 65 bar Additionally, a new mechanism for removal of water as a by-product of the reaction is discussed, which is due to the multi-phase distribution of acetone-CO2-water-glucose system Acetone vii Appendix Appendix C4: Extraction of palm mesocarp with SCCO2 at 35 (kg/h)/kg Pressure 400 bar 300 bar Temperature Extraction time (min) 10 20 30 45 60 90 120 Extracted oil (g) Total yield (%) 65°C Oil yield (%) Water (g) 0.89 14 0.01 2.29 16 35 0.03 3.24 22 50 0.06 4.23 29 65 0.1 4.85 33 74 0.15 5.63 39 86 0.23 5.98 41 92 0.24 Extracted oil (g) Total yield (%) 55°C Oil yield (%) Water (g) 1.03 16 0.01 2.24 15 34 0.03 3.16 22 48 0.05 3.95 27 61 0.07 4.42 30 68 0.11 4.91 34 75 0.14 5.11 35 78 0.14 Extracted oil (g) Total yield (%) 45°C Oil yield (%) Water (g) 0.7 11 0.01 1.88 13 29 0.03 2.78 19 43 0.04 3.37 23 52 0.07 3.87 27 59 0.1 4.57 32 70 0.16 5.01 35 77 0.21 Extracted oil (g) Total yield (%) 65°C Oil yield (%) Water (g) 0.88 13 0.01 2.12 15 32 0.02 21 46 0.04 3.82 26 59 0.07 4.45 31 68 0.11 5.05 35 77 0.17 5.24 36 80 0.17 Extracted oil (g) Total yield (%) 55°C Oil yield (%) Water (g) 0.86 13 0.05 1.95 13 30 0.07 2.68 18 41 0.08 3.45 24 53 0.11 3.95 27 61 0.14 4.69 32 72 0.17 4.97 34 76 0.19 Extracted oil (g) Total yield (%) 45°C Oil yield (%) Water (g) 0.46 1.51 10 23 0.02 2.36 16 36 0.03 3.1 21 48 0.04 3.64 25 56 0.07 4.16 29 64 0.1 4.45 31 68 0.11 102 Appendix Appendix C5: Screening synthesis of sugar ester (40 mL acetone, 2h, mg palmitic acid, mg glucose, Novozyme 435 ) Condition Conversion (%) 50°C, 100 bar, 60mg enzyme 50°C, 100 bar, 30mg enzyme 40°C, 100 bar, 30 mg enzyme 40°C, bar, 30 mg enzyme 34 44 37 Appendix C6: Effect of acetone concentration on the initial reaction rate (P=65bar, T=50°C, 50mg palmitic acid, 35mg glucose, 15mg Novozyme 435) Acetone concentration [Vacetone/Vreactor] Initial rate [μmol/(genzyme.h)] 402 905 705 592 239 Appendix C7: Screening of different lipases for sugar ester synthesis (P=65bar, 50mg palmitic acid, 35mg glucose, 15mg enzyme, 20mL acetone ) Enzymes Initial rate [umol/h/g enzyme] Temperature [°C] Novozyme 435 436 751 788 40 50 60 Lipozyme RM IM 155 272 40 50 60 Lipozyme TL IM 65 0 40 50 60 103 Appendix Appendix C8: Effect of Novozyme 435 concentration on the conversion of palmitic acid (P=65bar, T=50°C, 2h, 50mg palmitic acid, 35mg glucose, 20mL acetone) Enzyme concentration related to palmitic acid [%] 10 20 30 40 50 Conversion[%] 12 8 Appendix C9: Temperature effect on the conversion of palmitic acid (P=65bar, 50mg palmitic acid, 35mg glucose, 15mg Novozyme 435, 20mL acetone) Temperature 40°C 50°C 60°C Conversion[%] 0 60 3.32 4.16 5.09 120 6.86 11.54 11.6 180 18.1 19.95 20.25 240 20.51 22.91 23.3 Appendix C10: Effect of pressure on the initial reaction rate (50mg palmitic acid, 35mg glucose, 15mg Novozyme 435, 20mL acetone) Initial rate [umol/h/g enzyme] Temperature 40°C 50°C 60°C bar 152 204 65 bar 786 855 951 85 bar 887 954 1017 105 bar 925 974 1058 Appendix C11: Effect of substrate molar ratio on the initial reaction rate (P=65bar, T=50°C, 20mL acetone, 30% Novozyme 435) Molar ratio (Glucose: Palmitica acid) 1:1 1:2 1:4 1:6 1:8 Initial rate [μmol/(genzyme.h)] 788 976 616 560 471 104 Appendix Appendix C12: Effect of additional water on the initial reaction rate (P=65bar, T=50°C, 50mg Palmitic acid, 35mg glucose, 15mg Novozyme 435, 20mL acetone) Added water [Vwater/Vacetone, %] 0.5 Initial rate [μmol/(genzyme.h)] 751 937 599 472 267 169 Appendix C13: Reaction progress curve at different temperatures (P=65bar, 100mg palmitic acid, 35mg glucose, 30mg Novozyme 435, 20mL acetone) Temperature 40°C 50°C Reacted P.A (μmol/genzyme) hrs hrs hrs hrs hrs hrs 18 hrs 0 3031 622 36635 931 4388 1475 4957 2074 5236 2487 5704 Appendix C14: Reaction progress curve at different processing conditions Reacted palmitic acid (Mmol/genzyme) 85 bar, 50°C, 25% enzyme; 0.5 hrs hrs hrs hrs 23 hrs 24 hrs 5.4 6.29 8.97 14.09 20.72 24.93 Reacted palmitic acid (Mmol/genzyme) 85 bar, 55 °C, 23% enzyme, hrs hrs hrs hrs 23 hrs 24 hrs 4.43 7.56 9.89 11.24 19.32 19.74 105 Appendix Appendix C15: Screening of different lipases for MAG and DAG synthesis (P=75bar, T=50°C, 40 ml acetone, 300 mg palmitic acid, 300 mg glycerol, 45 mg enzyme) Time (min) Novozyme 435 RM IM TL IM Lipomod 34P 60 180 300 30.4 40.8 59.3 21.7 27.7 26.0 10.8 16.6 19.9 10.3 8.9 14.8 Appendix C16: Conversion of palmitic acid to MAG and DAG at different substrate ratios (P=75bar, T=50°C, 5h, 40 ml acetone, 300 mg palmitic acid, 15% Novozyme 435) Reacted palmitic acid (%) to Molar ratio ( palmitic acid : glycerol ) MAG DAG 1:1 1:3 1:6 45.33449 50.96496 51.51022 6.500243232 8.933811438 15.89745377 Appendix C17: Conversion of palmitic acid to MAG and DAG at different added amounts of water (P=75bar, T=50°C, 5h, 40 ml acetone, substrate ratio of 1:3 with 300 mg palmitic acid, 15% Novozyme 435) Reacted palmitic acid (%) to Added water [Vwater/Vacetone , %] MAG DAG 51.0 13.2 4.3 1.8 1.6 8.9 5.9 27.0 40.2 15.6 106 Appendix Appendix C18: Esterification of glycerol with a mixture of fatty acids (40 ml acetone, substrate ratio of 1:3 with 300 mg of FFA mixuture) Conversion (%) 75 bar, 50°C, 15% enzyme 85 bar, 55°C, 20% enzyme 85 bar, 50°C, 25% enzyme 60 180 300 17.1 18.7 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al., Enzymic transesterification in near-critical carbon dioxide: Effect of pressure, Hildebrand solubility parameter and water content Enzyme and Microbial Technology, 1992 14(8): p 649-655 115 CURRICULUM VITAE Personal Data: Name: Huan Phan Tai Date of birth: 07th August, 1978 Place of birth: Nha Trang, Vietnam Education: - 1996 - 2001: Engineer in Chemical Engineering & Food Technology, University of Technology, The National University - Ho Chi Minh City, Vietnam - 2002 - 2003: Asian-European Master's Degree of Science in Food Science and Technology Specialized in Agri-Food Industries Studies, ENSIA-SIARC, Montpellier, France Occupation: - From 2001: Lecturer, Faculty of Food Science & Technology, Nong Lam University, Ho Chi Minh City, Viet Nam - 2005 - 2008: Research assistant, Institute of Thermal and Separation Processes, Hamburg University of Technology, Germany [...]... traditional concept of a ‘co-solvent’ for a CO2 based system CO2 expanded organic solvent medium starts with the organic solvent and increases its volume by the addition of CO2, whereas 16 Fundamentals and state of knowledge relatively small amounts of ‘co-solvent’ have traditionally been added to dense CO2 phases to improve solubilities of certain compounds [42] Many advandtages of CO2- expanded liquids... (2.17) 11 Fundamentals and state of knowledge Where: Q1 = Mass flow rate of solvent (kg/s) qo = Mass flow rate of solvent related to N (1/s) q = Specific amount of solvent = Mass of solvent/ Mass of solute free solid (kg/kg) q = qo x t qm = Mass specific mass of solvent at the start of extraction from inside of the solid (kg/kg) qn = Specific mass of solvent till the end of extraction of easily accessible... Gases SC -CO2 Liquids Physical-chemical properties of the SCF are easily tuned by changing pressure and/ or temperature Figure 2.2 shows the density of CO2 as a function of pressure and temperature 4 Fundamentals and state of knowledge Figure 2.2: Density–pressure projection of the phase diagram for pure CO2 (after [3]) c) Solubility in SCF Among the features characteristic of the solubility behavior of a... has well been studied and applied [38] Good reviews on enzymatic catalysis in SCF were presented [30, 39, 40] 2.2.5 CO2- expanded organic solvent system CO2- expanded liquids are formed by dissolution of CO2 in organic liquids They are intermediate in properties between a normal liquid and a supercritical fluid, both in solvating power and in transport properties [41] The CO2- expanded phases can be distinguished... fibers [58] 23 Fundamentals and state of knowledge 2.3.3 SFE of palm oil and derivative products – State of the art Supercritical fluid technology has been proven to be a modern technique for edible oil processing Recently, supercritical CO2 has been applied in purification and fractionation of crude palm oil [59-62] Extraction of palm oil direct from palm pulp or kernel under supercritical condition has... Fractionation and purification Markom et al [60] fractionated crude palm oil using SCCO2 at temperatures of 40, 50 and 60°C and pressures of 110, 140 and 200 bar It was found that the concentration of carotenoids increased as a function of amount CO2 Adding polar cosolvents such as ethanol did not affect the extraction of carotenoids from crude palm oil Crude palm oil can be refined by continuous SCCO2 [61]... technologies for less impact on environment In the fat and oil industry, extraction, fractionation and modification techniques require a large amount of organic solvents, which may be harmful to environment Therefore, supercritical fluids, especially supercritical carbon dioxide (SCCO2), became interesting solvents, thanks to the advantage of being non-flammable, non-toxic, cost effective, and easily be separated... optimize the synthesis process Finally, the conclusion and outlook is given in Chapter 6 2 Fundamentals and state of knowledge 2 Fundamentals and State of Knowledge 2.1 Supercritical extraction - Theoretical background 2.1.1 Supercritical fluid a) Definition of a supercritical fluid Any pure compound can become a supercritical fluid (SCF) when its temperature and pressure are above the critical values [1]... with CO2 proved to be a good medium for esterification of fatty acids and polar compounds Finally, glycerol as another type of polar compound was selected to demonstrate the advantages of esterification in such a CO2- acetone sytem Response surface method was selected as the experimental design for esterification of palmitic acid and glycerol at the condition ranging from 65-85bar, 40-60°C and 5-25% of. .. enzymes The kinetics and selectivity of this enzymatic reaction was investigated Optimum condition was found at 85bar, 50°C and 25% of enzyme related to the amount of dissolved fatty acid Water could be removed by phase distribution viii Introduction and structure of the work 1 Introduction and Structure of The Work B Nowadays, industrial and research partners are looking for modern and improved technologies