Synthesis and Optimization Studies of Fructose Palmitate
by
Azalina Mohamed Nasir (0631110105)
A thesis submitted
In fulfillment the requirements for the degree of Master of Science (Bioprocess Engineering)
School of Bioprocess Engineering UNIVERSITI MALAYSIA PERLIS
2010
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AKNOWLEDGEMENTS
Alhamdulillah, all praises to Allah the Al-Mighty for His strengths and blessings towards the completion of this my master’s research project.
First and foremost, I am especially grateful to my supervisor, Associate Professor Dr. Mohamed Zulkali Mohamed Daud for his guidance and advice. His constructive comments and suggestions have assisted me in making this project a success. I would like also to express my genuine appreciation to my co-supervisor, Associate Professor Dr. Dachyar Arbain for his incessant support, guidance and constructive comments, during this period of study.
My deepest gratitude to my husband, Mr. Zulhillizan Othman and my son Amier Zuhairi whose love, understanding, prayers, patience and endless support have motivated me to work harder through the whole course of this study. I would also like to express my heartfelt gratitude to my mother Pn. Nurhapifahwati Ahmad and my family in law, for their continuous support and encouragement.
Special thanks to all the respective lecturers, staffs and PLVs, School of Bioprocess Engineering for their cooperation and support. Last but definitely not least, thanks to all my beloved friends, Alina, Najwa, Linda, Mai, Shira, Wani and Azira for their help, kindness, concern and moral support. Thanks for the friendship and memories, hope we all have a very bright future. I would also like to thank MOSTI for providing the Pascasiswazah financial support.
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TABLE OF CONTENTS
AKNOWLEDGEMENTS i
TABLE OF CONTENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF SYMBOLS xi
LIST OF ABBREVIATIONS xii
ABSTRAK xiii
ABSTRACT xiv
CHAPTER 1
1. INTRODUCTION
1.1. Introduction to Sugar Ester Surfactant 1.2. Synthesis of Sugar Ester
1.3. Problem Statement 1.4. Research Objectives
1 2 4 7
CHAPTER 2
2. LITERATURE REVIEW
2.1. Non Ionic Sugar Ester Surfactant 2.2. Chemical Synthesis of Sugar Ester 2.3. Enzymatic Synthesis of Sugar Ester
2.3.1. Introduction to Lipase
2.3.2. Lipase Catalyzed The Enzymatic Synthesis of Sugar ester 2.3.3. Immobilized Enzyme
2.4. Reaction System in Enzymatic Synthesis of Sugar Ester 2.4.1. Effect of Acyl Donor and Their Initial Concentration 2.4.2. Effect of Temperature
2.4.3. Effect of Solvent
2.5. Current Problem in Enzymatic Synthesis of Sugar Ester 2.5.1. Solubility of Sugar as Acyl Acceptor
9 13 15 17 18 22 23 23 26 28 32 32
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2.5.2. Effect of Water in Enzymatic Esterification 2.6. Introduction to Design of Experiment
2.6.1. Response Surface Methodology (RSM)
2.6.1.1. Choice of The Experimental Design Central 2.6.1.2. Central Composite Design (CCD)
2.6.2. Response surface methodology (RSM) of lipase catalyzed esterification of sugar ester
36 40 41 43 44
46
CHAPTER 3
3. MATERIALS AND METHODS
3.1. Materials, Chemicals and Equipments 3.1.1. Description of substrate
3.1.2. Description of enzyme 3.1.3. Analytical Reagents 3.2. Overall Experimental Flowchart
3.3. Preliminary study: Optimization of reactions condition for higher conversion
3.3.1. Effect of different solvent
3.3.2. Comparisons between supersaturated sugar solution under anhydrous condition, supersaturated sugar solution and control
3.3.2.1. Anhydrous Supersaturated Solution (ASS) System 3.3.2.2. Supersaturated Solution (SS) System
3.3.2.3. Non Supersaturated Solution System
3.3.3. Influence of stepwise addition of molecular sieve during the esterification
3.3.3.1. Effect of different reaction time on molecular sieve loading
3.3.3.2. Effect of molecular sieve loading during the esterification
3.4. Esterification: Process parameters studies 3.4.1. Effect of immobilized enzyme loading 3.4.2. Effect of fatty acid concentration
48 49 50 51
52
53 53 54 54 55
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3.4.4. Effect of agitation speed
3.5. Interaction and optimization of sugar ester esterification 3.5.1. Response surface methodology (RSM)
3.5.2. Optimization of fatty acid conversion
3.6. Reusability of immobilized enzyme in batch system 3.7. Sampling procedure
3.8. Preparation of calibration curve
3.8.1. Preparation of palmitic acid standard 3.9. Analytical analysis
3.9.1. Determination of palmitic acid 3.10. Determination of fatty acid conversion 3.11. Determination of initial reaction rate
58 58 59 59 62 63 63
64
65 65 65
CHAPTER 4
4. RESULTS AND DISCUSSIONS
4.1. Fructose Palmitate Conversion From Experimental Data
4.2. Preliminary study: Optimization of reaction conditions for higher conversion of fructose palmitate
4.2.1. Effect of organic solvent to the conversion and sugar solubility
4.2.2. Esterification of fructose palmitate in different systems 4.2.3. Influence of stepwise addition of molecular sieve during
the esterification
4.2.3.1. Effect of different time added of molecular sieve during the esterification
4.2.3.2. Effect of molecular sieve loading on the esterification
4.3. Studies on process parameters effect on esterification of fructose palmitate to the initial reaction rate and fatty acid conversion 4.3.1. Effect of immobilized enzyme loading
4.3.2. Effect of temperature
4.3.3. Effect of fatty acid concentration
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69 71
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4.4. Process optimization of fructose palmitate esterification 4.4.1. Statistical analysis
4.4.2. Model analysis
4.4.2.1. Effect of enzyme loading and its interaction with other factors
4.4.2.2. Effect of fatty acid concentration and its interaction with other factors
4.4.2.3. Effect of reaction time 4.4.2.4. Effect of temperature 4.4.3. Optimization analysis
4.5. Effect of reusability study on the immobilized enzyme in batch system
86 88 90 96
97
101 104 105 106
CHAPTER 5
5. CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion
5.2 Recommendations
108
111 113
REFERENCES 114
APPENDIX APPENDIX A
APPENDIX B
Calibration curve of palmitic acid obtained from HPLC
Result from Polymath for fatty acid conversion at 50oC temperature
125
126
LIST OF PROCEEDING 127
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LIST OF TABLES
Table Page
2.1 Fields of application and production capacities for sugar
based surfactants 10
2.2 The characteristics of enzyme synthesized surfactants 16 2.3 Sugar esters conversion (%) of enzymatic synthesis using
different commercialized lipase 21
2.4 Types of acyl donor and their by-product. 24
2.5 Optimum temperature of various solvent and sugar used for
the production of sugar ester. 27
2.6 Glucose solubility in different solvent hydrophobicity 30 2.7 Solubility of fructose and glucose in acetone, tert-BuOH and
2M2B at 60oC (g/L). 32
3.1 Physical properties of substrates 48
3.2 Physical characteristic of the enzyme 49
3.3 List of chemical and chemical used 50
3.4 List of hydrophilic solvents used for screening of suitable
reaction system 52
3.5 Schedule of molecular sieves addition during the course of
the esterification reaction. 56
3.6 Range of variables for the CCD design. 60
3.7 Experimental design for optimization of sugar ester
esterification. 61
3.8 The goal and limits of the factors and response of the
optimized process. 62
4.1 List of solvent used for screening of suitable reaction system. 69 4.2 The initial reaction rate and fatty acid conversion at 24h of
reaction time. 73
4.3 Influence of speed through the initial reaction rate, fatty acid
conversion and equilibrium time. 87
4.4 Experimental design results for esterification of fructose
palmitate. 91
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4.5 Analysis of variance (ANOVA) for quadratic model. 92 4.6 Statistical parameters obtained from ANOVA. 94 4.7 Optimum condition found by Design Expert for esterification
process. 107
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LIST OF FIGURES
Figure Page
2.1 Structure of sugar ester surfactant. 11
2.2 Structure of fructose furanose ester. R1and R2 are fatty acid
carbon chains. 12
2.3 Lipase catalyzed esterification synthesis of sugar fatty acid
ester of carboxylic acid. 19
2.4 Lipase catalyzed transesterification synthesis of sugar fatty
acid ester of ester. 19
2.5 Lipase catalyzed transesterification synthesis of sugar fatty
acid ester by enol ester. 20
2.6 Molecular sieve structure of tetrahedras alumino silicate
structure (AlO4) 40
2.7 Central composite design for evaluating (A) 2 factors and
(B) 3 factors 45
3.1 Illustrates the flowchart of the overall study 51 4.1 Comparison HPLC chromatogram of fructose palmitate
obtained from the study (a) and Ferre et al.,(2005) (b). 67 4.2 Plot of area HPLC of mono-ester versus fatty acid
conversion. 68
4.3 Enzymatic synthesis of fructose palmitate under different
systems during 10hrs of reaction. 72
4.4 Fructose palmitate synthesis by influence of molecular sieve
added during the esterification reaction. 75 4.5 Effect of differences molecular sieves loading. 77
4.6 Effect of immobilized enzyme loading. 80
4.7 Effect of temperatures. 82
4.8 Effect of fatty acid concentration. 84
4.9 Effect of various agitation speeds. 86
4.10 Normal plot of residuals for fatty acid conversion of fructose
palmitate esterification. 95
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4.11 Residual versus predicted plot for fatty acid conversion of
fructose palmitate esterification. 96
4.12 One factor plot for enzyme loading. F.A conversion as function of enzyme loading at intermediate of fatty acid concentration (0.75M), reaction time (8.0hrs) and
temperature (53.5oC). 97
4.13 (A) Interaction plot for enzyme loading and fatty acid concentration when the fatty acid concentration at their low ŶDQGKLJKŸOHYHOV%5HVSRQVHVXUIDFHSORWIRU)$
conversion at 8.0hrs of reaction time and 53.5oC of
temperature. 98
4.14 (A) Interaction plot for enzyme loading and temperature ZKHQ WKH WHPSHUDWXUH DW WKHLU ORZ Ŷ DQG KLJK Ÿ OHYHOV (B) Response surface plot for F.A conversion at 8.0hrs of
reaction time and 0.75M of fatty acid concentration. 100 4.15 One factor plot for fatty acid concentration. F.A conversion
as function of fatty acid concentration at intermediate of enzyme loading (11.0 % w/w substrates), reaction time
(8.0hrs) and temperature (53.5oC). 101
4.16 (A) Interaction plot for fatty acid concentration and reaction WLPH ZKHQ WKH UHDFWLRQ WLPH DW WKHLU ORZ Ŷ DQG KLJK Ÿ levels. (B) Response surface plot for F.A conversion at
53.5oC of temperature and 10.68% enzyme loading. 102 4.17 One factor plot for reaction time. F.A conversion as function
of reaction time at intermediate of enzyme loading (11.0 % w/w substrates), fatty acid concentration (0.75M) and
temperature (53.5oC). 104
4.18 One factor plot for temperature. F.A conversion as function of temperature at intermediate of enzyme loading (11.0 % w/w substrates), fatty acid concentration (0.75M) and
reaction time (8.0hrs). 105
4.19 Response surface plot for optimum F.A conversion from
experiment observation. 107
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4.20 Reusability of immobilized enzyme in batch system every
10hrs of reaction. 109
A.1 Calibration curve of palmitic acid obtained from HPLC
analysis. 125
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LIST OF SYMBOLS
Symbol Unit
Į Alpha (axial distance from center point which makes the design rotatable)
ȕ0 regression coefficients for the intercept coefficient ȕi regression coefficients for the linear coefficient ȕii regression coefficients for the quadratic coefficient ȕij regression coefficients for the interaction coefficient
ı Standard deviation
ȤiȤj coded independent variables
İ residual associated to the experiments
3A 3 Ångstrom
4A 4 Ångstrom
2k Two level full factorial design
A immobilized enzyme loading %(w/w subs.)
AB interaction between immobilized enzyme loading and substrate molar ratio (sugar to fatty acid)
AD interaction between immobilized enzyme loading and reaction time
B substrate molar ratio (sugar to fatty acid) M BC interaction between substrate molar ratio (sugar to fatty
acid) and reaction temperature
C reaction temperature oC
D reaction time h
F.A Fatty acid
k number of variable
N number of measurement
n Sample size
rA,obs initial mass transfer rate of substrate mg/ml of fatty
acid.min rpm Rotation per minute
T Temperature K
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LIST OF ABBREVIATION
2M2B 2-methyl-2-butanol
ANOVA Analysis of variance
AlO4 tetrahedras sodium alumino silicate
CCL Candida rugosaimmobilized on acrylic resin
CCD Central Composite design
CCRD Central composite rotatable design
DoE Design of Experiment
DMF dimethylformamide
DMP dimethylpyrrolidone
DMSO dimethylsulfoxide
K2CO3 Potassium carbonate
log P partition coefficient of solvent between water and octanol in two-phase system
Lipolase 100L Humicola Lanuginosaimmobilized on celite Lipolase 100T Humicola Lanuginosaimmobilized on acrylic resin
MEK methyl ethyl ketone
MMIM Mucor mieheilipase immobilized on a macroporous anion- exchanger resin of phenolic type
N435 Candida antarcticaB immobilized on acrylic resin PCL Penicillium chrysogenumimmobilized on celite
PCP Pseudomonas cepaciaimmobilized on toyonite -200-P RMIM Rhizomucor mieheiimmobilized on anion-exchange resin
RSM Response surface methodology
SP382 Candida antarcticaB immobilized on acrylic resin tert-BuOH 2-methyl-2-propanol
TLIM Thermomyces Larnuginosais silica granulated lipase
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xiii ABSTRAK
SINTESIS DAN KAJIAN PENGOPTIMUMAN FRUKTOS PALMITAT
Fruktos palmitat (gula ester) jika dibandingkan dengan surfaktan tanpa ionik yang lain, adalah antara yang terbaru dalam kelasnya. Kebolehannya sebagai surfaktan kebolehbiodegradasi, ketoksikan yang rendah dan keberkesanannya pada suhu, pH serta kemasinan yang melampau telah meningkatkan kegunaannya dalam pelbagai bidang.
Penghasilan gula ester secara skala besar telah didominasi oleh proses kimia konvensional sejak sekian lama. Walau bagaimanapun, proses kimia konvensional ini meninggalkan impak kesan yang buruk kepada manusia dan alam sekitar. Berbanding dengan proses sentesis secara enzim, proses ini menawarkan kaedah lain yang lebih selamat dan mudah. Dalam kajian ini, penghasilan gular ester secara kaedah enzim telah dibangunkan yang mana ia telah mengurangkan kebanyakan kelemahan dari proses kimia konvensional. Gula yang tidak dilindung dan asid lemak yang tidak diaktifkan digunakan secara terus sebagai bahan pemula. Kombinasi antara larutan gula terlampau tepu dibawah keadaan terhidrat dan penambahan molekul penapis sebagai penyerap air secara berskala sewaktu tindakbalas berlaku adalah kaedah paling sesuai untuk meningkatkan kadar tindakbalas dan penukaran asid lemak. Melalui kaedah ini, pengaruh beberapa parameter telah dikaji sebagai asas untuk proses pengoptimuman.
Keputusan dari kajian asas ini digunakan untuk pengoptimuman dan analisis esterifikasi fruktos palmitat (gula ester) menggunakan metadologi permukaan sambutan (RSM) berdasarkan reka bentuk komposit berpusat (CCD). Sejumlah 98.58 ± 0.52% penukaran asid lemak yang optimum telah diperolehi dengan menggunakan sebanyak 11.92%
(berat/berat bahan pemula) kuantiti enzim tersekatgerak, 0.50 M kepekatan asid palmitik dalam 10 jam masa tindak balas pada suhu 53.67oC. Kebolehgunaan semula enzim tersekatgerak menunjukkan hasil penukaran asid lemak yang bagus, dimana lebih 88% penukaran asid lemak dapat diperolehi selepas 10 kali kitaran tindak balas tanpa perlu melakukan penambahan rawatan terhadap enzim tersekatgerak tersebut.
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xiv ABSTRACT
SYNTHESIS AND OPTIMIZATION STUDIES OF FRUCTOSE PALMITATE
Fructose palmitate (sugar ester) is a relatively new class of nonionic surfactants.
Their excellent biodegradability and low toxicity surfactant as well as effectiveness at extreme temperature, pH and salinity show their increasing importance in numerous areas of application. For a long time, large scale production of sugar ester was dominated by conventional chemical processing. However, the conventional chemical process leaves out bad impact to the human and environment. Compared to the enzymatic synthesis, this process offers a safer and easier alternative. In the present work, sugar ester production was developed by a novel and effective enzymatic method which can reduce the advantages of conventional chemical process. Direct unprotected sugar and non activated fatty acid were used as a starting material. Combination of supersaturated sugar solution under anhydrous condition and stepwise addition of molecular sieve as water absorbent agent during the reaction were found to be a suitable method in increasing the reaction rate and fatty acid conversion. In this method, influences of several parameters were investigated as a screening to the optimization process. Results from screening were used to optimize and analyze fructose palmitate (sugar ester) esterification using a response surface methodology (RSM) based on central composite design (CCD). The 98.58 ± 0.52% of optimum fatty acid conversion was determined by 11.92% (w/w of substrates) immobilized enzyme loading, 0.50M fatty acid concentration, 10.0h reaction time and 53.67oC of reaction temperature. The reusability of the immobilized enzyme was shown good conversion, were greater than 88% of fatty acid conversion after 10th reaction cycles without additional treatment of the immobilized enzyme.
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1 CHAPTER I INTRODUCTION
1.1. Introduction to sugar ester surfactant
Surfactant or surface active agents are amphiphilic organic compounds. For decades, surfactant such as a general cleaning agent has been used in daily applications. However, progress in the area of surfactant has ultimately widened its possibilities. The product, which proved to be only marginally useful as detergents, showed good emulsifying, wetting solubilizing and foaming characteristics. The characteristics and applications of the surfactant differed by the charged groups in its heads. There were ionic and non-ionic surfactants. Among all surfactants, nonionic surfactants were the fastest in terms of growth with about 45% share of the overall industrial production. This was due to their increased use in the mentioned field (Patel, 2004).
In the past decade, glucoside (sugar based) head groups of nonionic surfactants has been introduced in the market. Sugar esters were one of the glucoside nonionic surfactant, which contained both hydrophobic tail groups (fatty acids) and hydrophilic head groups (sugars). The sugar base of nonionic surfactants structure also mimicked the glycolipids type of biosurfactant (Desai & Banat, 1997). Due to the close structure similarity, these sugar esters has the same characteristics as the biosurfactant, such as its biodegradability, biocompatibility, digestibility and low toxicity, which make them suitable for use in pharmaceuticals, cosmetics and food stuffs production (Deleu &
Paquot, 2004; Ferrer et al., 2005; Holmberg, 2001; Karmee, 2008; Kosaric, 1992).
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These various applications of the sugar ester surfactants brought about large scale production by most chemical company.
In 1996, over 5 billion pounds of surfactant were produced. In the Asia-Pasific region, the total surfactant consumption grew at an annual rate of 3.9% with a projection of 5.8 million tons in 2010. From the global perspective, the consumption and proportion of surfactants exhibited a different pattern for the North American and Western European region compared with the Asia-Pasific region. However, in the past decades, a new biodegradable surfactant which was known as sugar-based surfactant, has gained significant interests and increased market shares (John, 2001).
1.2. Synthesis of sugar ester
The synthetic sugar ester surfactants was usually produced by a chemical processing method (traditional method) using a base or acid catalysts and performed by toxic solvent such as dimethylformamide (DMF), dimethylsulfoxide (DMSO) or dimethylpyrrolidone (DMP) as the mutual solvent for solubilizing sugar. Potassium carbonate, lithium and sodium soup were also used as the solubilizing agent. This method utilized high temperature and pressure (Adamopoulos, 2006; Yan, 2001). This traditional method often led to major disadvantages such as high energy consumption, degradation of reactants, coloring of products and low selectivity. These disadvantages could cause difficulty in the production separation process. Moreover, certain processes of chemical synthesization of sugar ester were found to be toxic and not readily biodegradable. Thus their application in the cosmetic, food and pharmaceuticals
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industry was limited (Chang & Shaw, 2009; Karmee, 2008; Sabeder et al., 2006;
Soultani et al., 2001; Tarahomjoo & Alemzadeh, 2003; Yoo et al., 2007).
The use of enzymatic synthesis has been developed as an alternative route to the conventional chemical process. In enzymatic synthesis, a sugar ester was produced using a lipase enzyme as a biocatalyst. The enzymatic synthesis provided work under mild condition, easy recovery, reusability of the catalyst. Moreover, the level of contaminations in both the final products and the environment was also low (Deleu &
Paquot, 2004; Desai & Banat, 1997). The enzymatic synthesis gave out regeoselectivity, fewer isomers and side-products (Adamopoulos, 2006; Chua, 2005; Coulon et al., 1999). According to Desai et al., (1997) and Deleu et al., (2004), surfactant obtained through enzymatic synthesis was also known as natural surfactant and it was supported by the Food and Drug Administration (Chua, 2005; Deleu & Paquot, 2004; Desai &
Banat, 1997).
An enzymatic synthesis of sugar esters was a reaction between hydroxyl groups of sugar and carboxylic groups of fatty acid. Both sugar and fatty acid was joined by the ester bond to form sugar fatty acid esters or sugar esters. The enzymatic synthesis was carried out by lipase enzyme under nonaqueous conditions. In nonaqueous conditions, lipases were able to catalyze the reverse reaction to form ester bond and this process was known as esterification. There was a considerable number of studies on the lipase-catalyzed production of sugar fatty acid esters in organic solvent (Adachi
& Kobayashi, 2005; Kobayashi & Adachi, 2004), ionic liquids (Ganske &
Bornscheuer, 2005b; Lee et al., 2008), supercritical CO2system (Habulin et al., 2008;
Tai & Brunner, 2009) and in solvent free system (Xu et al., 2003) under nonaqueous condition.
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The rapid advances in the enzymatic synthesis of sugar esters has led to a considerable interest being generated in the development of this method for the manufacturing of surfactants and other value-added compounds on an industrial scale (Bommarius & Riebel, 2004; Chang & Shaw, 2009; Karmee, 2008; Zhang, 1999).
However, a limited source of enzyme and the low rate of production has made enzymatic synthesis quite unfavorable amongst large industry.
The problem posed by the enzyme catalyzed process is the high cost of the lipase used as the catalyst due to its limited availability. However, the high operational stability of an immobilized enzyme made recycling possible in a batch and continuous system. These has been reported by many researchers (Halim, 2008; Kim et al., 2004;
Mat et al., 2005).
Another problem in enzymatic approach for sugar ester production was low production caused by the negative effects of low sugar dissolution in the reaction medium and the present of water as a by-product. The water could lead to ester hydrolysis that is the reverse reaction of esterification. Therefore, the water should be removed while the reaction was in progress in order to increase the yield (Hari &
Divakar et al., 2001; Sekeroglu et al., 2004; Yu et al., 2008). Several methods of water removal during reaction has been reported namely the vacuum pressure (Dang, 2004;
Zhang, 1999), azeotropic distillation (Yan et al., 1999; Yan, Bornscheuer, & Schmid, 2001) and membrane pervaporation (Bufi-Bak et al., 2002; Sakaki et al., 2006; Yan,
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Bornscheuer, Stadler et al., 2001). However, the possibility of solvent loss, clogging and mass transfer restriction accuring during the evaporation process was high. This may increase the production cost in a large scale production set up. Many researchers used desiccants or drying agent such as molecular sieve, silica gel and anhydrous salt to remove the water content (Cauglia & Canepa, 2008; Chaiyaso et al., 2006; Sabeder et al., 2006; Yoo et al., 2007; X. Yu et al., 2004). The molecular sieve was commonly used as an absorbent but it has limited water absorbing capacity (Chua, 2005; Yan, 2001).
Thus, a stepwise addition of molecular sieve was proposed.
Most esterification of sugar ester was carried out in intermediate-polarity organic solvents. These solvent mediums played an important role in dissolving the two different substrates (sugars and fatty acids) so that esterification can occur. High yield was achieved in a solvent system compared to a solvent-free condition. Nevertheless, the production was still low. In this case, a low solubility of the sugar in the organic solvent system contributed to the low level of sugar ester conversion (Adachi &
Kobayashi, 2005; Kobayashi & Adachi, 2004). Several methods have been developed in order to solve this problem. They included the use of a highly polar organic solvents (DMSO, DMF or Pyridine) and a mixture of highly polar solvent with an intermediate- polarity solvent (Chang & Shaw, 2009; Karmee, 2008; Kennedy et al., 2006).
Unfortunately the method that has been proposed to increase solubility of the sugar has also created another problem with regards to the final products and the enzyme activity. A mixture of toxic solvent should be removed so that it will not contaminate the product. To solve the negative effect of dangerous solvent, moderate polar organic solvent such as tertiary alcohol has been proposed. Many researchers has
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shown that the use of tertiary alcohol which is a non-toxic solvent, has shown a high solubility rate of sugar as well as good stability of lipase enzyme (Cauglia & Canepa, 2008; Kim et al., 2004; Yoo et al., 2007).
Many sugars solubility rate was note to be very poor in the tertiary alcohol.
Therefore, sugar dissolution rate may limit the ester synthesis. Several researchers used various methods to increase the sugar dissolution rate such as the feed batch addition of solid sugar during esterification (Dang et al., 2005), sugar derivatives (protected sugar) or alkyl glygosides (Ganske & Bornscheuer, 2005b) and amorphous sugar (Dang, 2004). These methods not only require additional steps but can also affect the sugar structure. Thus, the uses of these methods were not a suitable approach for the production of sugar ester on a large scale because it incurred an increase in production costs. Thus, a supersaturated sugar solution has been proposed by several researchers (Cauglia & Canepa, 2008; Flores & Halling, 2002; Flores et al., 2002). In the supersaturated sugar solution, more solid sugar was dissolved compared to a cool solution.
In the present study, the esterification of fructose palmitate using Novozyme 435 as the catalyst in a tert-BuOH system was investigated. In this study, an experiment were supersaturated sugar solution under an anhydrous condition was performed. In this system, one step esterification was conducted in a batch process to get the optimum condition for the production of nonionic fructose palmitate surfactant. High yields and product could be obtained, since the problem of low sugar dissolution and water by- product can be solved using the supersaturated sugar solution under an anhydrous condition technique and stepwise addition of molecular sieve during the reaction. More
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sugar was dissolved by supersaturated sugar solution under the anhydrous environment.
The anhydrous environment was triggered by molecular sieves. Meanwhile, the water by-product was adsorbed by the feed batch addition of molecular sieves during the esterification process. Then, a statistical analysis software which was known as Design of Experiment (DoE) has been applied to the factor affecting the fatty acid conversion in supersaturated sugar solution under anhydrous condition technique, for the optimization and analysis of the parameters.
1.4. Research Objectives
The purpose of this research was to develop a novel and alternative method for sugar ester surfactant production using an enzymatic approach, which was considered safe to be used in food, pharmaceutical and cosmetic industries. In this method, fructose and palmitic acid would be used as reactants to produce a fructose palmitate as sugar ester in enzymatic esterification. The development of the enzymatic synthesis involved the improvement of reaction rate, fatty acid conversion and immobilized enzyme stability via batch system. The present research has the following objective:
General objective:
To study the effect of supersaturated sugar solution under anhydrous condition and feed batch addition of molecular sieve to the esterification of fructose palmitate (sugar ester) in an immobilized enzyme batch flask system.
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8 Specific objectives:
1) To study the effects of process parameters on esterification of fructose palmitate (sugar ester) in an immobilized enzyme batch flask system based on fatty acid conversion and initial reaction rate.
2) To identify the optimum condition for sugar ester surfactant synthesis in a batch flask system using Design of Experiment (DoE).
3) To study the effect of immobilized enzyme recycling to the fructose palmitate (sugar ester) surfactant enzymatic synthesis.
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9 CHAPTER II
LITERATURE REVIEW
2.1. Non ionic sugar ester surfactant
Nonionic surfactants are found today in a large variety of domestic and industrial products. They can be found in powdered forms or liquid formulations.
However, the market is dominated by ethoxylates, alkyl benzene sulfonates, alcohol ether sulphates, and alcohol sulphates surfactant products. These traditional surfactants exhibit a low rate of biodegradation and a high potential of aquatic toxicity (Deleu &
Paquot, 2004). For these reasons, new green surfactants are promising even if their performances could be slightly inferior or their price more expensive. Among these surfactants, sugar based surfactant such as alkylpolyglycosides (APG) are the most successful at this time (Table 2.1). In the past decade, sugar ester which was one of the alkylpolyglycosides group of surfactants has been introduced in the market due to their low toxicity, which make them more suitable for pharmaceuticals, cosmetics and food products application (Holmberg, 2001; Maag, 1984).
