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

Abbreviations B i First order model coefficients B ii Quadratic coefficients for the i-th variable B ij Interaction coefficients for the interaction of variables i and j CCFCD Centr

Extraction, Separation, and Bio-transformation of

Natural Plant Derived Compounds

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”

1 Gutachter: Prof Dr.-Ing G Brunner

2 Gutachter: Prof Dr.rer.nat A Liese

Prüfungsausschussvorsitzender: Prof Dr.rer.nat R Müller

Tag der mündlichen Prüfung: 10.12.2008

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

Abbreviations v

Summary vii

1 Introduction and Structure of The Work 1

2 Fundamentals and State of Knowledge 3

2.1 Supercritical extraction - Theoretical background 3

2.1.1 Supercritical fluid (SCF) 3

a) Definition of a supercritical fluid 3

b) Physico-chemical properties of the SCF 3

c) Solubility in SCF 5

2.1.2 Supercritical fluid extraction (SFE) 6

a) General description of SFE 6

b) Course of extraction for SFE 7

c) Supercritical fluid extraction modeling 8

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 SCCO 2 15

2.2.5 CO 2 -expanded organic solvent system 16

a) High pressure CO 2 - H 2 O - 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

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

3 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 SCCO 2 49

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 3 57

4 Sugar Fatty Acid Ester Synthesis in CO 2 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 4 73

5 MAG and DAG synthesis in CO 2 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-CO 2 system 75

5.1.3 Analytical method 76

a) Gas Chromatography (GC) 76

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 CO 2 -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 5 93

6 Conclusion and Outlook 95

Appendix 96

Bibliography 108

Abbreviations

B i First order model coefficients

B ii Quadratic coefficients for the i-th variable

B ij Interaction coefficients for the interaction of variables i and j

CCFCD Central composite face centered design

CER Constant extraction rate

DCER Diffusion controlled extraction rate

DS Degree of substitution

FER Falling extraction rate

PFAD Palm fatty acid distillates

RSM Response surface method

SCCO 2 Supercritical carbon dioxide

SCF Supercritical fluid

SFAE Sugar fatty acid esters

SFE Supercritical fluid extraction

Tc Critical temperature

Xi Independent variables

Y Value of the response

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 up-scaling 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 co-extracted 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

saturated 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 enzyme Novozyme 435 was also the best among the investigated 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

1 BIntroduction and Structure of The Work

Nowadays, industrial and research partners are looking for modern and improved 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 from the product Moreover, a supercritical fluid brings more benefits in mass transfer, phase distribution, etc

The oil palm fruit is a very good oil source; however after conventional palm oil extraction processing there is still a large amount of valuable minor compounds left behind Therefore, one purpose of this work is to evaluate the benefit of using supercritical fluid extraction to extract palm oil by better recovery of these minor compounds, compared to traditional screw pressing

Extracted palm oil comprises a high amount of palmitic acid, which is normally considered as a low value lipid-class in supercritical processing This fatty acid could be valorized by using enzymes to create more valuable compounds, e.g surfactants such as sugar fatty acid esters or mono- and di-glycerides The study investigates the use of high pressure CO2

for the enzymatic synthesis process, in order to accelerate the reaction or improve by-product separation

The study begins with a review on “Fundamentals and State of Knowledge” (Chapter 2)

to show how supercritical fluids became increasingly attractive as solvents in extraction and biotransformation A summary of palm oil and surfactant structure properties is presented Their processing is reviewed to demonstrate their application in food, cosmetic, or pharmaceutical industry

Chapter 3 presents the extraction of palm oil with SCCO2 and subcritical propane The oil and its valuable minor compounds are investigated in the extract and in the residue Modeling is performed for an up-scaling purpose

In Chapter 4, the esterification of palmitic acid with glucose to create sugar fatty acid ester is presented A new method for enzymatic reaction in CO2 expanded acetone is developed

A mechanism for the synthesis in this system is proposed

In Chapter 5, esterification of glycerol and palmitic acid for the synthesis of mono- and di-glycerides is reported A new apparatus is constructed to study the enzymatic synthesis in high pressure CO2-acetone with the ability to determine substrate loading The reaction is studied for total conversion and selectivity A response surface design is used to optimize the synthesis process

Finally, the conclusion and outlook is given in Chapter 6

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] As shown in Figure 2.1, the critical pressure is recorded

as the highest pressure at which a pure component liquid can be converted into a gas by an increase in temperature, while the critical temperature is the highest temperature at which a pure component gas can be converted into a liquid by an increase in pressure Critical temperatures and pressures of some substances can be found in Table 2.1

One of the most commonly used supercritical fluids is supercritical carbon dioxide (SCCO2) Compared to other substances, CO2 has a moderate critical pressure (Pc = 7.38 MPa) and a critical temperature close to room temperature (Tc = 304.1°K) Propane is also an interesting fluid with a low critical pressure (Pc = 4.25 MPa) However, because critical temperature of propane is rather high (Tc = 369.8°K), this fluid is preferably used at subcritical conditions (at T < Tc)

Figure 2.1: Pressure-temperature diagram for a pure component (adapted from [1])

b) Physico-chemical properties of the SCF

There is only one phase existing at supercritical conditions It is neither a gas nor a liquid and it is best described as intermediate between these two extremes (Table 2.2) The solubility behavior approaches that of a liquid, while penetration into a solid matrix is facilitated by the gas-

Super-like viscosity and enhanced diffusion As a result, the extraction rate and phase separation can be significantly faster than for conventional liquid based extraction processes

Table 2.1: Critical parameters of selected supercritical fluids (after [1])

Viscosity (g/cm s)

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 solute in a SCF solvent are the exponential solubility enhancement with increasing pressure and the retrograde solubility behavior [4] As a result of marked changes in solvent density, solubility in general increases with increasing pressure Solubility of a solute in a SCF can be expressed by using Hildebrand-solubility parameters According to Elssier [5], the solvent power of a SCF can be correlated with the solubility parameter concept, where the solubility parameter of the fluid, 1, is given by:

L r

SF r c P

On the other hand, the solubility of solutes is defined by the solubility parameter (2), which is directly related to the energy contribution and group volumes [6]

i v

v

E

) (

) (

2

Where (E)i and ()i are the vaporization energy and the molar volume of the chemical group

i

The values of solubility parameters of common plant lipid compounds are presented in Table 2.3

Table 2.3: Solubility parameters for compounds found in seed oils (after [6, 7])

Lipid type Solubility parameter

(cal1/2/cm3/2)

Lipid type Solubility parameter

(cal1/2/cm3/2) Hydrocarbons

Diacylglycerols

Sterols

PC Monoacylglycerols

PI

9.45 9.52 9.77 10.2 12.05

The difference between the solubility parameter of the fluid and the solute (1 and 2) will determine the solubility of a solute in a SCF The smaller the difference, the higher is the solubility As a result, the solvating power of a SFC can be tuned by changing pressure and/or temperature to be equal to the lipid compounds By this way, one SFC can be used to extract various solutes from a feed matrix, which in conventional liquid extraction several different solvents are needed

2.1.2 Supercritical fluid extraction (SFE)

a) General description of SFE

In supercritical fluid extraction, one or more components can be separated from a mixture

by dissolving it in a supercritical solvent The product can be either the extract, the raffinate or both A typical supercritical fluid extraction system consists of a tank for the supercritical compound, a pump to pressurize the gas, an extraction vessel with a temperature control system,

a pressure reducing valve to maintain the high pressure in the extraction line and a vessel to collect the extract First, the raw material and supercritical fluid are brought to close contact in order to extract the soluble components from the starting material Extraction conditions, mainly

pressure and temperature, are adjusted to isolate components selectively The dissolved substances together with the fluid are then removed from the bulk material After that, the extracted components are completely separated from the fluid by means of changing pressure and/or temperature The supercritical fluid can be recompressed to the extraction condition, or just simply vented at ambient pressure (Figure 2.3) Recently, separation of extracted components from the SCF using membrane separation has been drawing attention in SFE fields [8, 9]

Figure 2.3: Typical supercritical fluid extraction (after [10])

b) Course of extraction for SFE

According to Brunner [1], the course of a solid extraction can be represented by the overall extraction curve (OEC), in which the amount of extract accumulating during the course of the extraction is plotted as a function of time or amount of solvent The schematical curve of an OEC is shown in Figure 2.4 The first part (I) of the curve is linear, corresponding to a constant extraction rate The gradient of this part may represent the equilibrium solubility of the extract in SCF However, the straight line of the OEC could correspond to a constant mass transfer resistance In the second part (II) extraction rate is declining and the graph approaches a limiting value where all the extractible substances are removed from the input material

Cooler condensor

Heater

Solvent recycle

Separator

Gas

EXTRACT FEED

T (°C)

T c

Extractor

Figure 2.4: Integral extraction curve Total amount of extract vs time of extraction (after [1])

extraction of theobromine from cocoa seed shells [1] or oil from rice bran [11] For a successful model, to calculate the course and result of an extraction, the following parameters have to be considered:

- equilibrium distribution between solid and supercritical fluid (adsorption isotherm)

- diffusion in the solid (effective diffusion coefficient or effective transport coefficient defined by the transport model)

- mass transfer from the surface of the solid to the bulk of the supercritical fluid phase

- axial dispersion (effective dispersion coefficient, related to inhomogeneities of the fixed bed, the solvent distribution and the influence of gravity)

Equations 2.3 to 2.8 present the key equations of the model

Mass balance for the fluid phase is:

t

zcε

ε1z

(z)cε

uz

zcD

t

2 F 2 ax

F oG

ρ

cKzczck

C = mean concentration of extract components in the solid phase

cF = concentration of extract in the fluid

Dax = axial dispersion coefficient

uz = void volume linear velocity of supercritical solvent

cf* = equilibrium concentration of extract in the fluid phase

K = equilibrium distribution coefficient between liquid and solid phase

Des = effective diffusion coefficient in the solid phase

koG = overall mass transfer coefficient for the gas phase

z = coordinate in axial direction

ε= porosity of the fixed bed

t = time of extraction

a = specific surface of solid phase

ρs = density of the solid material

k1, k2 = coefficients of the sorption isotherm (Freundlich’s isotherm)

βF = mass transfer coefficient for the fluid phase

R =solid particle’s diameter

For the balance, in the fluid phase the following assumptions have been made:

- Gradients are neglected in radial direction

- Convection and axial dispersion cause dispersed plug flow

- The process is isothermal

- The loading of the supercritical fluid is relatively low

For the balance for the solid phase, some assumptions have been made:

- Transport can be considered one-dimensional

- Solid particles are uniform and the extract is evenly distributed over the solid

material

- Effective transport coefficient (effective diffusion coefficient) is used to display

the real transport phenomena, like membrane transition, pore diffusion, diffusion in the solid, etc

- Phase equilibrium is assumed at the interface solid/fluid

Sovova model:

Sovova [12] used a pseudo steady model for physical description of the extraction process The extraction process can be divided into 3 stages with different functions In the first stage, where the oil is easily accessible throughout the fixed bed, the extraction is carried on at a constant extraction rate (CER) When the easily accessible oil becomes exhausted at the fluid entrance, a transition period between the fast and slow extraction periods begins In this period, called the Falling Extraction Rate period (FER), the easily accessible oil is still extracted in one section of the fixed bed, while the extraction from the inside of the particles takes place in the other section

The boundary between the two sections described above, passes through the bed until it reaches its end After the boundary has reached the end of the fixed bed the Diffusion Controlled Extraction Rate period (DCER) comes with extraction rate controlled by diffusion of the oil from inside of the particles to outside

Key equations of the Sovova model are given in Equations 2.9 to 2.17

cer s

ya

y t Q

M qo

k

k r o

*

*

*

)60

*/(

*

*

*

Xk Xp

X

kya H S

tcer Mcer

Y

with

X s

Y kya

k m o

o

r

w

r o k

o k m

n

r k o

m

x x

x q q W x

x x W q

q

Z y x x

exp.ln

)/exp(

)(

ln1

./)(

/)(

exp1)/.exp(

1ln

1

k m

r o r

n m

w m

r

m r

q q x

x q q W y

x W W

y xo

q q q Z

z q

q y

q q Z

y q N

(2.17)

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 solute (kg/kg)

Mbed = Total mass of solid used for extraction (kg)

N = Mass of solute free solid phase (kg)

P = Mass of easily accessable solute (kg)

Mextr = Mass of extract (kg)

rho(g) = Density of solvent phase (kg/m3)

Mcer = Mass of extract till the end of constant extraction rate period (kg)

tcer = Time of extraction till the end of constant extraction rate period (min)

xo = Initial concentration of solid (kg/kg)

xp = Concentration of easily accessable solute (kg/kg)

xk = Mass of inaccessable solute inside the particles (kg/kg)

xp/xo = Ratio of easily accessable solute to the initial concentration of solute

ε = Porosity of the solid phase

S = X-sectional area of the extractor (m2)

d = Diameter of the extractor (m)

I = Length of the extractor (m)

kya = Solvent phase mass transfer coefficient

kxa = Solid phase mass transfer coefficient

yr = Solubility of the oil in the extraction solvent (kg/kg)

d) SFE application in vegetable oil extraction

SCCO2 and subcritical propane are common solvents used in edible oil extraction from plant materials Recently the number of studies on SFE of lipid from natural products has been growing: canola [13], soybean [14], millet bran [15], sunflower [16-18], hazelnut [19], olive [20] and rice bran [11, 21], coriander seed [22] Good reviews over SFE were given in [23, 24] From

a technical point of view, supercritical extraction could be used to produce valuable oil from these plant sources However, production cost is still high for a comparative market production Therefore, finding an optimum extraction method or a downstream production of more beneficial products is necessary Among solutions investigated in this work is adding value to lipids by enzymatic catalysis, especially with lipases within supercritical environment

2.2 Biotransformation with lipase

2.2.1 Lipase

Triacylglycerol lipases (E.C 3.1.1.3) have been used to catalyse the hydrolysis reaction of acylglyceride or some types of esters such as thiol-, polyol- or polyacid- esters [25] Lipases are also used as catalysts for synthesis of high value acylglycerides or esters Lipases can be produced from animal pancreas or stomach, but lipases from microorganisms are preferred because of higher purity and productivity Nowadays, lipases are broadly applied in pharmaceutical, cosmetic, leather, detergent, food, or biofuel industry

Lipases have the ability to catalyze a variety of reactions with lipids In general, lipase reactions can be classified into 2 categories: hydrolysis and synthesis [25]

Hydrolysis reaction:

RCOOR’ + H2O  RCOOH + R’OH

The hydrolysis reaction can take place with a specific carbon position (Rhizopus oryzae, Humicola lanuginosa, Rhizomucor meihe), a specific fatty acid (Geotrichum candidum, Brassica napus, Mucor miehei), or no specification (Candida antarctica, Candida rugosa, Pseudomonas cepacia)

Synthetic reaction:

These can be divided into 4 small groups:

 Esterification: RCOOH + R’OH  RCOOR’ + H2O

 Interesterification: RCOOR’ + R”COOR’’’  RCOOR’’’ + R”COOR’

 Alcoholysis: RCOOR’ + R”OH  RCOOR” + R’OH

 Acidolysis: RCOOR’ + R”COOH  R”COOR’ + RCOOH

2.2.2 Progress curve and determination of reaction velocity

To determine enzymatic reaction velocities, according to [26], it is necessary to generate a progress curve A first-order exponential decrease in substrate concentration describes the conversion of substrate (S)

S S

Reaction velocities can be generated from the progress curve The rate of the reaction (reaction velocity  ) corresponds to the initial slope of the progress curves

dt

dP dt

2.2.3 Lipase catalysis in a conventional solvent

For an enzymatic reaction, substrates are required to be contacted with the active sites of the enzyme Usually an organic solvent is used as a reaction medium The solvent should be miscible with substrates Another important factor is water content Water as a by-product of the reaction plays an important role in an enzymatic esterification [27] A mono hydration layer on the surface of the enzyme is required to maintain the flexibility of the protein structure for its catalytic activity However, a large amount water will shift the equilibrium of the reaction towards the hydrolysis side Therefore, there are many approaches to remove water from the reaction phase When the esterification is carried out in a free solvent system, vacuum evacuation can be used When organic solvents are used as reaction media, selective distillation must be applied Usually, a solvent with a higher boiling point than that of water is not accepted in food industry It is difficult to remove water out of a solvent with a lower boiling point In this case, water generated during the reaction must form an azeotrope with the selective solvent and can then be removed from the reaction medium by azeotropic distillation Another way to remove water is using adsorbents However, this method is not practical at industrial scale because of mass-transfer limitations Water removal methods from literature will be discussed in the following parts for the synthesis of sugar ester, mono- and di-glycerides A novel method to remove water is then presented: using SCCO2 as reaction medium

2.2.4 Lipase catalysis in SCCO 2

Enzymatic catalysis in supercritical fluids has gradually attracted attention because it can overcome many problems of traditional media: (i) solubility of substrates in the medium can be modified, (ii) products, by-products, and catalyst can be easier removed, (iii) there is no toxic solvent remaining in the final products However, enzymatic reaction at supercritical conditions still follows general rules of catalysis as in a traditional solvent Because enzymes are sensitive to temperature, pressure, nature of solvent, inhibitor, and water activity [28-33], knowledge of the activity of the enzyme at supercritical conditions must be obtained for a reaction for every new substance and enzyme

Concentration of substrate or enzyme directly influences the final conversion rate It is observed that most of research on enzymatic reactions in SCF is concerned with temperature and pressure of the fluids This can be explained by the effect of temperature and pressure on the solubility of the substrate in SCF [1]

Like major hydrolases, most of the lipases have no cofactors but an active site with specific acid amines such as imidazol of histidin or hydroxyl of serine As a result, lipases are inactivated at high temperatures due to both partial unfolding and covalent alterations in the primary structure [29]

In contrast, previous studies on enzymatic catalysis showed that there is no activity change for lipases when exposed to high pressure of CO2, but the pressure influences how far the enzyme can withstand temperature [30, 31]

Water has great effect on the enzymatic reaction in any supercritical medium because some water is required for an enzyme to retain catalytic activity, but excess water can lead to agglomeration of the enzyme or accelerate the backward hydrolysis [30, 33, 34]

On the other hand, the nature of the solvent has a strong effect on the enzymatic activity [35] Besides CO2, propane is a common solvent to replace an organic solvent in bio-catalysis [35, 36]

With the presence of some substances, called inhibitors, the reaction rate is reduced In supercritical reactions, modifiers like alcohols, which are used to enhance the solubility of reactants, commonly inhibit enzyme activity [34] Reaction products are also inhibitors Especially, in the case of esterification, the excess of water produced, decreases total conversion

Non-immobilised enzymes are often deactivated by SCCO2 while immobilised enzymes are better, depending on the type of the immobilising support A screening process of several commercial lipases to find a lipase with superior performance for the conversion of lipid moieties

in SCCO2 has been reported by Frykman [37]

Recently, progress of using SCCO2 as a medium for enzymatic reaction has well been studied and applied [38] Good reviews on enzymatic catalysis in SCF were presented [30, 39, 40]

2.2.5 CO 2 -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 from the 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

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 were reported [41, 43]:

- abilities to alter the physicochemical properties of the solvent such as viscosity, dielectric constants, solvent power

- comparable or better product selectivity than in neat organic solvent or SCCO2

- milder process pressure (tens of bars) compared to SCCO2 (hundreds of bars)

- enhanced reaction rates and turnover frequencies

- substantial replacement of organic solvents with dense-phase CO2

On this basis, CO2 expanded liquids have been proven to be good media for important chemical reactions [42-45]

a) High pressure CO 2 - H 2 O - organic solvent system

When water is present together with an organic solvent, phase equilibrium should be investigated Lim [46] reported the vapor-liquid equilibrium for the CO2-water-ethanol system for 40 and 70 °C and pressures up to 18.5 MPa over a wide range of ethanol concentration

Data on liquid-liquid-vapor equilibria and critical points in the ternary system carbon dioxide-water-acetone were reported at 20, 40, and 60 °C, for pressures between 1.8 and 9.5 Mpa [47] Figure 2.6 shows the critical point in the binary system Above this pressure, the liquid-vapor equilibrium region L2V detaches from the CO2-acetone side With further increasing pressure, the liquid-liquid-vapor equilibrium region L1L2V is obtained, in which L1 is the water rich phase and L2 is acetone rich phase The L2V region becomes smaller with increasing pressure Finally, a pressure is reached, in which liquid phase L2 and the vapor phase V become critical

In another work, vapor-liquid equilibrium data have been measured for the ternary carbon dioxide-isopropanol-water system and for the quaternary carbon dioxide-isopropanol-water-glucose system at 40 and 60 °C and at pressures between 13.8 and 30.0 MPa [48]

A general review about mixtures of water and a hydrophilic organic solvent (for example:

an alcohol, a ketone, or a carboxylic acid) pressurized with near critical carbon dioxide at temperatures near the critical temperature of carbon dioxide can be found in [49]

Figure 2.6: The phase diagram for CO2-water-acetone (after [47])

b) Solubility of polar compounds in a multi-phase system

Solubility of polar compounds in multi-phase systems was also reported Dohrn [50] studied a glucose-water-ethanol-CO2 system Vapor-liquid equilibrium data have been reported at temperatures from 50 to 70 °C and pressures up to 30MPa The study showed that there was a very small amount of glucose detected in the vapor phase It also showed that the solublity of glucose increases remarkably when ethanol is added compared to pure SCCO2

Pfohl et al [51] reported the partioning of five carbohydrates (xylose, fructose, glucose, saccharose, and maltose) in the vapor-liquid-liquid region of the 2-propanol-water-CO2 system The study presented the data of phase compositions from all three coexisting phases, determined

at pressures between 9 and 13 MPa and temperatures between 50 and 70 °C

In another work, Bünz et al [48] reported the vapor-liquid equilibrium data measured for the quaternary CO2-isopropanol-water-glucose system at 40 and 60 °C and at pressures between 13.8 and 30 MPa While pressure and vapor-phase density do not influence the solubility of glucose in the vapor phase significantly, the glucose concentration is strongly dependent on the vapor-phase concentration of isopropanol and water

The most interesting study related to this work is the phase equilibrium in the water-CO2-glucose system reported by Pfohl et al [52] Table 2.4 shows the phase composition

acetone-of all three coexisting phases at temperature acetone-of 40, 50 and 60 °C and pressures acetone-of 4, 6 and 8 MPa

The Soave-Redlich-Kwong equation of state (EOS) has been used to correlate the experimental data for this quaternary system

Table 2.4: Mole fractions from VLLE measurements in the CO2-acetone-water-glucose system (after [52])

0.014 0.383 0.066

0.003 0.107 0.885

0.983 0.510 0.028 6.11 40 V

L2

L1

- 1.00E-06 1.82E-02

0.014 0.183 0.019

0.003 0.031 0.934

0.983 0.786 0.029 4.1 50 V

L2

L1

- 9.20E-05 2.15E-02

0.022 0.442 0.083

0.008 0.187 0.870

0.970 0.371 0.025 6.1 50 V

L2

L1

- 5.2E-06 1.93E-02

0.020 0.311 0.032

0.005 0.051 0.924

0.975 0.638 0.025 4.16 60 V

L2

L1

- 3.0E-04 2.09E-02

0.030 0.450 0.094

0.008 0.253 0.856

0.962 0.297 0.029 6.07 60 V

L2

L1

- 2.30E-05 2.04E-02

0.030 0.350 0.059

0.006 0.041 0.944

0.963 0.788 0.025 8.23 60 V

L2

L1

- 1.00E-06 1.96E-02

0.031 0.171 0.011

0.006 0.041 0.944

0.963 0.788 0.025

2.3 Palm Fruits and Palm Oil Extraction

2.3.1 Palm fruits and their composition

a) Palm fruits

It is reported that the Oil Palm (Elaeis guineensis) originated in the tropical rain forest region of West Africa During the 14th to 17th centuries some palm fruits were taken to the Americas and from there to the Far East [53] Nowadays, the oil palm is grown as a plantation crop in most countries with high rainfall in tropical climates within 10° of the equator, because of its economic importance as an high-yielding source of edible and technical oil [54] Among the producers, Malaysia is the biggest one with production of 11.41 millions tons per year (contribution is 54% of total global palm oil)

According to Poku [53], the palm bears its fruit, ranging from 6 to 20 g, in bunches varying in weight from 10 to 40 kg The fruits (Figure 2.7) are made up of an outer skin (exocarp), a pulp (mesocarp) containing the palm oil in a fibrous matrix; a central nut consisting

of a shell (endocarp); and the kernel, which itself contains an oil, quite different to palm oil, resembling coconut oil

Figure 2.7: Palm fruits: a) Exocarp, b) Mesocarp, c) Endocarp, d) Kernel.1

b) The composition of palm oil

Triglycrides constitute the major component of palm oil (90%) [54] Monoglycerides and diglycrides follow with 5% The fatty acid composition of glycerides is presented in Table 2.5 C-

16 and C-18 are the main fatty acids It could be observed that palm oil has a balanced fatty acid composition in which the level of saturated and unsaturated fatty acids are almost equal (50% saturated, 40% monounsaturated and 10% polyunsaturated fatty acids) As a consequence of high polyunsaturated acid content palm oil is a good oil with the ability to reduce blood cholesterol and the risk of coronary heart disearse Besides, palm oil contains ca 3% of free fatty acids and 1% of other minor components The minor constitutents of palm oil include carotenoids, tocopherols, sterols, phosphatides, triterpenic, and aliphatic alcohols (Table 2.6) Among them, the most important are carotenoides and tocochromanols

Table 2.5: Fatty acid composition of Malaysian palm oil (after [55])

beta-800 ppm tocopherols and tocotrienols (the whole group called tocochromanols) (Figure 2.9) The major portion of total tocochromanols in palm oil is alpha-tocopherol, known as vitamin E, and gamma-tocotrienol These compounds are also antioxidants and provide some natural oxidative protection to the oil

Table 2.6: Minor components of crude palm oil (after [55])

Carotenoids 500-700

Sterols 326-527 Phospholipids 5-130

Figure 2.8: Strucstrures of carotenes

Figure 2.9: Tocopherols and tocotrienols

It is obvious that the combined effects of properties of carotenoids, tocochromanols and

the high portion of unsaturated acids give palm oil a higher oxidative stability compared to many

other edible oils However, these carotenoids are thermally destroyed during the deodorization

stage Commonly refined palm oil retains about 50% of the tocochromanols A lot of new and improved methods for palm oil production have been investigated and applied during the last decades

2.3.2 Palm oil processing

According to Shahidi [56], palm oil extraction includes following steps:

- Fruit Reception: Palm fruit should be handled with utmost care to minimize the damage to the fruit

- Sterilization: This step is carried out by exposing the fruits to a steam pressure of 3 kg/cm2(143°C) for approximately 60 min The objectives of sterilization are: prevention of further rise in the free fatty acid (FFA) of the oil due to enzymatic reaction (i), facilitation of mechanical stripping (ii), preparation of the pericarp for subsequent processing (iii), and preconditioning of the nuts to minimize kernel breakage (iv)

- Stripping: The objective of this step is the separation of the sterilized fruit from the bunch stalks

- Digestion: Sterilized fruits are reheated to a temperature between 95 and 100°C for approximately 20min The objective of this step is to loosen the pericarp from the nuts and to break the oil cells before passing to the oil extraction unit

- Oil Extraction: Commonly a continuous screw pressing system is applied The products from the press included a mixture of oil (66%), water (24%) and non-oily solid (10%), and a press cake containing fibers and nuts

- Clarification: The crude oil is separated from fibrous materials by screening and water by settling and centrifugating The oil is then processed in a vacuum dryer and finally a cooler before going to the storage tanks

- Oil storage: Storage tanks should be internally coated with epoxy material to prevent iron pickup The temperature is maintained between 32 and 40°C during storage and transit The unloading or loading temperature is between 50 and 55°C The crude palm oil will be refined later by chemical or physical methods, which is well described by O'Brien [57]

Up to now, recovery of oil from the mesocarp by using a screw pressing system is the most commonly used method However, a significant quantity of carotenoids (4000-6000 ppm) and tocochromanols (2400-3500 ppm) remain in the residual oil (5-6% on dry basis) in the palm press fibers [58]

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 been conducted [63-67] Besides, the waste from palm oil processes like pressed palm fiber [68-71] were also investigated

a) 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] The refined oil can have less than 0.1% free fatty acids, higher carotene content, and low amount of diglycerides It was proven that a co-solvent can improve the refining process of palm oil

Carotenoids and tocohromanols can be purified from crude palm oil, processed with SCCO2 A well developed process had been proposed at VTII department, TUHH More details can be found in the literature reported by Chuang and Brunner [62] or Gast et al [72]

b) Extraction

SCCO2 has been used as a solvent to extract oil from the pulp or the pressed residue The main focus is the yield of extract material Pressure, temperature and flow rate of CO2 are commonly investigated parameters The extraction performance of an investigated system is usually evaluated by overall extraction curves

Bednarski [63] extracted palm mesocarp by SCCO2 at 30 MPa + 40°C, 40 MPa + 60°C and 50 MPa + 80°C The total extraction curves were studied, and carotene and tocopherols were also investigated However, the residual oil and its minor concentration in the residual fiber after the SCCO2 extraction were not reported

Suitable pre-treatment methods for raw material have been also studied Bisunadan [65] reported extraction of oil (triglycerides) from sonicated-dried, cooked-wet, sonicated-wet, cooked-dried, wet and dried palm fruits at pressures of 30, 40 and 50 MPa and temperatures of

40, 60 and 80°C Lau et al [64] also extracted oil from dried mesocarp The applied conditions

were 40 to 80°C and 14 to 30 MPa The oil obtained by SFE meets overall quality equivalent to those obtained by commercial processing of crude palm oil

Birtigh et al [70] also extracted carotene from the residue from the mechanical processing

of palm oil It was found that the extracted amount of carotenoids and tocopherols from the residue were not high enough to apply for an economic industrial size extraction

In a separate work, Luiz et al [69] studied the extraction of oil from pressed palm oil fibers using SCCO2 at 200, 250 and 300 bar and temperatures of 45 and 55°C It was shown that the oil extraction rate, as well as the total amount of carotene, increased largely from 200 to 250 bar, but not from 250 to 300 bar The extracted oil had a larger amount of free fatty acids than those found in commercial oils

In general, extraction of palm oil from palm fruit and pressed fiber using SFE has been explored However a full investigation about total oil yield, co-extracted water, carotenoids and tocochromanols in the extracted and the residue oil in a single run has not been reported

2.4 Sugar fatty acid esters

2.4.1 Sugar fatty acid esters as surfactants

Sugar fatty acid esters (SFAE) are non-ionic surfactants consisting of a sugar as hydrophilic group and a fatty acid as lipophilic group (Figure 1) They can be different in the degree of substitution (DS), defined as the number of hydroxyl groups esterified with fatty acids Sugar esters with DS 1 to 3 are hydrophilic, absorbable, and digestible Those with DS 4 to 8 (called sugar polyesters) are lipophilic, nondigestible, or partially digestible In industry, surfactant power of sugar ester is also classified by using hydrophilic-lipophilic balance (HLB) value, which is defined by Griffin’s method [73]

where M and Mh is the Hmolecular mass of the hydrophilic portion and the whole molecule respectively

M M

Glucose fatty acid ester Sucrose fatty acid ester

Figure 2.10: Structures of the carbohydrate fatty acid esters

Because SFAE have a wide range of HLB value, they can be used as both water in oil (W/O) or oil in water (O/W) emulsifiers SFAE are commonly used as food emulsifier because they are tasteless, odorless and nontoxic On the other hand, with the property of being non irritant to the eyes and skin, they have been employed widely as surfactants in food, detergent, and pharmaceutical applications [74] Their antimicrobial, antitumoral and insecticidal properties have been reported and might open new markets [75] Moreover, they are biodegradable With their wide application, sugar esters are produced in a huge amount For example, sucrose esters are produced at 4000 tons/year [76]

Carbohydrates:

Carbohydrate is the hydrophilic group of SFAE It is one of the most abundant biological molecules, and plays important role from storage and transport of energy (starch, glycogen) to structure of living cell (cellulose in plants, chitin in animals)

The basic carbohydrate units are monosacharides Among them, glucose is an important monomer which is commonly available in the form of a white substance or a solid crystal Glucose can exist in 2 forms (Figure 2.11): an open chain (acyclic) and a ring form (cyclic) The latter is a result of intermolecular reaction between the aldehyde carbon atom and the C-5 hydroxyl group to form an intramolecular hemiacetal The regioselectivity of the sugar has a function to control the degree of substitution when esterification of sugars and fatty acids is performed

a) The Chain form of Glucose b) The Ring form of Glucose

Figure 2.11: Structure of glucose

Fatty acid:

Fatty acid belongs to the hydrophobic group of SFAE In general, a fatty acid consists of

a straight chain of an even number of carbon atoms, ending with a carboxyl group (-COOH) Fatty acids can be divided into saturated and unsaturated fatty acids A saturated fatty acid does not contain any double bond along its carbon chain When there is one or more double bonds in the fatty acid, it becomes mono- or polyunsaturated

As shown in Figure 2.12, palmitic acid (C-16) is one of the most common saturated fatty acids found in plants and present in every commercially processed food fat Fat or oil product with a certain palmitic acid content will crystallize in the beta-prime (β′) form, which is desirable for plasticity, smooth texture, aeration and creaming properties [57] Most chemically reactive are the fatty acids with several double bonds Linoleic (C-18:2) and linolenic (C-18:3), for example, are the notable polyunsaturated fatty acids or essential fatty acids

Figure 2.12 : Palmitic acid

2.4.2 Sugar Ester Synthesis

a) Chemical synthesis

For synthesis of SFAE chemical processes and enzymatic methods can be used Chemical processes are mainly performed at high temperatures in the presence of alkaline catalysts The Snell process is one of the most well known methods In this method sucrose fatty acid esters are synthesized by transesterification of sucrose with fatty acid methyl esters in the presence of potassium carbonate and sufficient solvent such as dimethylformamide (DMF) or dimethylsulfoxide (DMSO) The process is conducted in 9-12h at 90- 95°C under 80-100 mmHg

pressure The product of this process is not allowed to be used in food industry because of the solvent toxicity However the product can be approved, if the solvents are replaced by propylene glycol, water, or vegetable oil [77] Besides, the chemical process usually needs a high energy input

Acros et al [78] used acetone as solvent to produce emulsifier from glucose and fatty

acids, using an immobilized Candida antartica They found that for saturated fatty acids longer

than lauric acid, continuous precipitation of the monoester, as it is formed, permits a high conversion of glucose to the monoester (Figure 2.13) However, it required a long time (within 2-

3 days) to achieve a complete reaction

Yoo et al [79] performed enzymatic synthesis of SFAE in acetone and tert-butanol using an

immobilized Candida antartica lipase, and various sugars and oleic acid as substrates For a high

conversion and minimization of the residual fatty acid concentration, the optimal sugar-to-fatty acid ratio was in the range from 2:1 to 3:1 A molecular sieve column outside the reactor was used to remove the water formed during the reaction The highest yield (98 %) was obtained when xylitol and tert-butanol were used as the sugar and solvent

Chaiyaso et al [80] reported esterification of palm fatty acid distillates (PFAD) with

glucose and fructose by immobilized Candida antarctica lipase The ester could be obtained

with 76% yield from glucose and PFAD after reaction for 74 h with 150 U/ml immobilized lipase

at 40°C in acetone

Figure 13: Enzymatic synthesis of glucose laurate at different temperature (after [78]).2

Ferrera et al [81] used fatty acid vinyl esters as acyl donor for the synthesis of sugar esters The transesterification took place in 2-methyl-2-butanol:dimethylsulfoxide mixtures with the

lipases from Thermomyces lanuginosus and Candida antarctica It was found that both lipases

retained their initial activity and were similarly effective for the regioselective synthesis

Enzymatic synthesis of fructose esters was studied under reduced pressure by Coulon [82]

Immobilized Candida antarctica lipase was used as biocatalyst to test with oleic acid, oleic acid

methyl ester and rapeseed oil It was found that reduced pressure (200 mbar) allowed a high conversion yield and product concentration to be obtained, whatever the nature of the acyl donor tested It was explained that removal of by-product (water or methanol) shifted the equilibrium of the reaction toward sugar ester synthesis More than 90% of fructose was acylated compared to 50% under atmospheric pressure

It is known that many polar solvents such as tert-butanol, dimethylsulfoxide, are not accepted in food industry Because of the mass transfer limit, a rather long time for the reaction is required Like other esterification reactions, the presence of water in the final product inhibits the reaction rate Therefore, many methods were proposed to remove water during the reaction: addition of molecular sieve, vacumm distillation, or membrane gas permeation Esterification of

2 Conditions: 60mg glucose, 200mg fatty acid, 2 mL acetone, 100 mg Novozyme 435, and 200 mg molecular sieves