Design, synthesis and characterization of fatty acid ...
DESIGN, SYNTHESIS AND CHARACTERIZATION OF FATTY ACID ESTERS IN FOOD FLAVOURS AND NUTRITIONAL SUPPLEMENTS
LI CHENG
School of Physical and Mathematical Sciences
A thesis submitted to the Nanyang Technological University
in fulfillment of the requirement for the degree of
Doctor of Philosophy
2014
Acknowledgements
Looking back, I am surprised and meanwhile very appreciative for all I have received throughout these years. It has certainly shaped me as a person and has led me where I am now. All these years of Ph.D studies are full of such gifts.
First of all, I would like to express my deepest respect and sincere appreciation to my supervisor Prof. Li Tianhu and NUS Prof. Huang Dejian, Prof. Liu Shaoquan for their unfailing guidance, edification and support throughout my Ph.D project. They have helped me to undertand life and science in their full depth. They also taught me how to appreciate the good scientific work that helps other researchers to build on it. I am so grateful for their invaluable advice, support and friendship.
I also thank my colleagues, especially Ms. Sun Jingcan from National University of Singapore, for her fruitful collaborations and giving me many helpful suggestions for my experiments and thank all other lab colleagues (Tan Hong Kee, Zhang Hao, Li Yiqin Jasmine, Li Dawei, Yang Zhaoqi, Lei Qiong and Ba Sai) for their scientific help during my graduate study. As a source of collaboration and freidenships, I am so honored to work in the research group.
Last, but by no means the least, I thank my family for their love and encouragement. Special thanks to my husband, Justin Yeo, for his unequivocal support and patience at all times. Without him I would not focus on my Ph.D study, and it would have been certainly much harder to finish a Ph.D. Thank him for being with me all the time.
Li Cheng
2014
Table of Contents
Acknowledgement...............................................................................................................I
Abstract............................................................................................................................XII
List of Tables..................................................................................................................XVI
List of Figures...............................................................................................................XVII
List of Schemes.............................................................................................................XXII
List of Symbols and Abbreviations...........................................................................XXIII
Chapter 1— Introduction
1.1 Methionol...................................................................................................................1
1.1.1 Volatile Sulphur Flavour Compounds and Their Characteristics........................1
1.1.2 The Characteristics and Uses of Methionol.........................................................3
1.1.3 Sources and Synthesis Method of Methionol......................................................3
1.1.4 Methionol Derived Flavours................................................................................4
1.2 2-Phenethyl Alcohol...................................................................................................5
1.2.1 The Characteristics and Uses of 2-Phenethyl Alcohol........................................6
1.2.2 Natural Sources and Synthesis Method of 2-Phenethyl Alcohol........................6
1.2.3 2-Phenethyl Alcohol Derived Flavours...............................................................9
1.3 Fatty Acids as the Flavour Compounds in Food Science.........................................10
1.3.1 Common Fatty Acid Flavours...........................................................................10
1.3.2 Pathways to Generate Acid Flavours................................................................11
1.4 Natural Oil as the Precursors in the Synthesis of Flavouring Products....................12
1.5 Octacosanol..............................................................................................................14
1.5.1 Introduction to Octacosanol..............................................................................14
1.5.2 Physiological Function......................................................................................15
1.6 Flavour Synthesis Method........................................................................................18
1.6.1 Traditional Flavour Synthesis Method..............................................................19
1.6.2 Esters Synthesis Method....................................................................................21
1.6.2.1 Characteristics of Esters as Flavours..........................................................21
1.6.2.2 Chemical and Bioconversion Synthesis Method…....................................22
1.7 Analytical Method....................................................................................................27
1.7.1 Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID)...................................27
1.7.2 Solid-Phase Microextraction (SPME)...............................................................30
1.7.2.1 Introduction to SPME.................................................................................30
1.7.2.2 The Limitation of SPME............................................................................31
1.8 Optimization by Using Response Surface Methodology (RSM).............................33
1.8.1 Introduction to RSM..........................................................................................33
1.8.2 Application of RSM in Food Analysis...............................................................34
Chapter 2 — Design, Synthesis and Study Sensory Attributes of Methionol Derived Fatty Acid Esters
2.1 Introduction..............................................................................................................36
2.2 Materials and Methods.............................................................................................39
2.2.1 Materials and Reagents......................................................................................39
2.2.2 Analytical Method.............................................................................................40
2.2.3 Esterification of Methionol with Fatty Acid through Chemical Method..........41
2.2.4 Esterification of Methionol with Fatty Acid through Enzymatic Method.........41
2.2.4.1. 1H NMR and HREI Mass Spectra Results..........................................42
2.2.5 Transesterification of Butter Oil with Methionol by Enzymatic Method..........43
2.2.6 Standard Solutions for Calibration Curves........................................................44
2.2.7 Qualitative and Quantitative Analysis of Methionyl Esters..............................45
2.2.8 Stability Evaluation of Fatty Acid Methionyl Esters in Acidic Media and Heat Treatment.........................................................................................................45
2.2.8.1 Acid Stability Test..................................................................................45
2.2.8.2 Thermal Stability Test............................................................................46
2.2.9 Odour Descriptions and Threshold Testing of Methionyl Esters......................46
2.3 Results and Discussion.............................................................................................48
2.3.1 Qualitative and Quantitative Analysis of Methionyl Esters..............................48
2.3.2 Acid and Thermal Stability Assay of Methionyl Esters....................................53
2.3.3 Odour Descriptions and Threshold Testing of Methionyl Esters......................53
2.4 Conclusion................................................................................................................54
Chapter 3 — Design, Synthesis and Study Sensory Attributes and Improved Yield of 2-Phenethyl Alcohol Derived Fatty Acid Esters………………….…………………....56
3.1 Introduction................................................................................................................56
3.2 Materials and Methods...............................................................................................58
3.2.1 Materials and Reagents......................................................................................58
3.2.2 Analytical Method.............................................................................................59
3.2.3 Esterification of 2-Phenethyl Alcohol with Fatty Acid through Chemical Method..............................................................................................................60
3.2.4 Esterification of 2-Phenethyl Alcohol with Fatty Acid through Enzymatic Method..............................................................................................................60
3.2.4.1 1H NMR and HR-APCI Mass Spectra Results.....................................61
3.2.5 Transesterification of Butter Oil with 2-Phenethyl Alcohol Catalyzed by Lipase........................................................................................................63
3.2.6 Standard Solutions for Calibration Curves........................................................64
3.2.7 Qualitative and Quantitative Analysis of 2-Phenethyl Esters............................65
3.2.8 Stability Evaluation of 2-Phenethyl Esters in Acidic Media and Heat Treatment.........................................................................................................66
3.2.8.1 Acid Stability Test..................................................................................66
3.2.8.2 Thermal Stability Test............................................................................66
3.2.9 Sensory Evaluation............................................................................................67
3.3 Results and Discussion.............................................................................................68
3.3.1 Qualitative and Quantitative Evaluation of Two Methods in Preparing 2-Phenethyl Esters..........................................................................................68
3.3.2 Acid and Thermal Stability Assay of 2-Phenethyl Alcohol Derived Fatty Acid Esters................................................................................................................72
3.3.3 Flavour Attributes of 2-Phenethyl Esters..........................................................73
3.3.4 Effect of Reaction Parameters on the Synthesis of 2-Phenethyl Esters during Transesterification of Butter Oil with 2-Phenethyl Alcohol.............................74
3.3.4.1 Effect of Reactant Molar Ratio...................................................................74
3.3.4.2 Effect of Enzyme Loading..........................................................................76
3.3.4.3 Effect of Shaking Speed.............................................................................78
3.3.4.4 Effect of Incubation Temperature..............................................................79
3.3.4.5 Effect of Reaction Time.............................................................................81
3.3.4.6 Determination of Conversion of 2-Phenethyl Alcohol and Key Esters Formed under Optimized Conditions.......................................................82
3.4 Conclusion................................................................................................................83
Chapter 4 — Biocatalytic Conversion of Butter Oil to Natural Flavour Esters Optimized with Response Surface Methodology...........................................................85
4.1 Introduction..............................................................................................................85
4.2 Materials and Methods.............................................................................................87
4.2.1 Materials............................................................................................................87
4.2.2 Transesterification Reaction Assays and Analysis............................................88
4.2.3 Experimental Design and Statistical Analysis...................................................90
4.3 Results and Discussion.............................................................................................91
4.3.1 Transesterification of Butter Oil with 2-Phenethyl Alcohol..............................91
4.3.2 Model Fitting.....................................................................................................92
4.3.3 Effects of Enzymatic Synthesis Parameters......................................................96
4.3.4 Attaining Optimum Condition and Verification..............................................103
4.4 Conclusion..............................................................................................................104
Chapter 5 — Design and Synthesis Octacosanol Derived Fatty Acid Esters to Improve the Solubility of Octacosanol.........................................................................106
5.1 Introduction............................................................................................................106
5.2 Materials and Methods...........................................................................................110
5.2.1 Materials..........................................................................................................110
5.2.2 Esterification of Octacosanol with Fatty Acid through Chemical Method..........................................................................................................111
5.2.3 Esterification of Octacosanol with Fatty Acid through Enzymatic Method..........................................................................................................111
5.2.3.1 1H NMR and HREI Mass Spectra Results.......................................112
5.2.4 Solubility Test of Octacosyl Esters in Edible Oil............................................113
5.2.5 Stability Evaluation of Octacosyl Esters in Acidic Media and Heat Treatment......................................................................................................113
5.2.5.1 Acid Stability Test..............................................................................113
5.2.5.2 Thermal Stability Test.........................................................................114
5.2.6 Hydrolysis of Octacosyl Esters by Lipase.......................................................114
5.3 Results and Discussion...........................................................................................115
5.3.1 Qualitative and Quantitative Analysis of Two Methods to Synthesis Octacosyl Esters..............................................................................................................115
5.3.2 Stability Evaluation of Octacosyl Esters in Acidic Media and Heat Treatment......................................................................................................117
5.3.3 Solubility Test of Octacosyl Esters in Edible Oil............................................118
5.3.4 Hydrolysis of Octacosyl Esters by Lipase.......................................................119
5.4 Conclusion..............................................................................................................124
Chapter 6 — Future Perspectives.................................................................................126
Reference.........................................................................................................................132
List of Publications and Conference.............................................................................144
Abstract
Flavour esters are the most important and versatile components of flavoring compounds and fragrances. These esters have been widely used in the food, beverage, fine cosmetic, non-cosmetic and pharmaceutical industries.1 Methionol is an alcohol with low odour threshold ranging from about 1 to 3 ppm and imparts powerful sensory notes described as meaty, boiled potato-like, vegetable-like, savory or toasted cheese-like.2 2-Phenethyl alcohol is a colourless liquid with a pleasant floral odour with a mild, warm and rose-honey-like note of moderate to poor tenacity. It has been wildly found in a variety of essential oils such as rose, hyacinth, aleppo pine, orangeblossom, ylangylang, allium, anise, carnation, geranium, neroli and champaca.3 This material is therefore used as a fragrance ingredient in many compounds, particularly when rose odour is desired. Novel methionol derived fatty acid esters of up to 14 carbon fatty acid and 2-phenethyl alcohol derived fatty acid esters of up to 18 carbon fatty acid were synthesized by both chemical and lipase catalysed esterification between fatty acids and methionol or 2-phenethyl alcohol, respectively. The high acid- and heat-stability of methionyl esters and 2-phenethyl esters meet the requirement of food matrix. Most importantly, the sensory test showed that fatty acid carbon chain length had important effects on the flavour attributes of methionyl esters and 2-phenethyl esters.
Through Lipozyme TL IM-mediated tranesterification in solvent-free system, valuable methionol or 2-phenethyl alcohol derived esters were synthesized by using the readily available natural materials butter oil as the fatty acid source. The conversion of methionol or 2-phenethyl alcohol and yield of each methionyl ester or 2-phenethyl ester were also obtained by Gas Chromatography-Mass Spectrometry-Flame Ionization Detector (GC-MS-FID). The process parameters (reaction temperature, substrates molar ratio, enzyme loading, shaking speed and reaction time) were studied to achieve the highest conversion of 2-phenethyl alcohol to 2-phenethyl esters. Maximum conversion of 2-phenethyl alcohol (80.0 %) were achieved after 20 hours of reaction at 40 oC, shaking speed of 100 rpm, an enzyme loading of 20 % (w/w) of reaction mixture and molar ratio of 3.0:1 (mmol/mmol of 2-phenethyl alcohol to butter oil). This process was further optimized using response surface methodology (RSM) based on a three level- three-factor box-behnken design to investigate the effects of single and multiple interactive factors on the conversion. The optimum molar conversion (86.5 ± 2.6 %) were achieved for 2-phenethyl esters by using molar ratio of 4.0:1 (2-phenethyl alcohol/butter oil), 20 % enzyme loading of all the reactant at 137 rpm and 41 oC for 8 hours.
The beneficial effects of both methods were compared qualitatively and quantitatively by GC-MS/GC-FID results. Compared by chemical method, the enzymatic ones seem a superior strategy due to the advantage of enzymatic reactions, such as high substrate specificity, high reaction specificity, purer products, reduction of waste product formation and the milder reaction conditions. Moreover, the transesterification of butter oil with methionol or 2-phenethyl alcohol reacted in solvent-free system have more advantage. This is because butter oil was applied as the inexpensive, readily available, and renewable natural precursors to synthesize flavour esters. Also, the solvent-free media was considered as safe in the food industry. Consequently, with the increase in consumer demand for natural flavouring materials, the use of specific enzymes in biosynthetic processes presents great potential to meet this demand.
Nutritional supplements or nutraceuticals have aroused increasingly interest among the public. As one of such supplements, octacosanol has been the subject of numerous studies. Octacosanol is a water-insoluble and stable long chain fatty alcohol. In a long time study, it has been shown to have some health benefits such as cholesterol lowering, antiaggregatory effects, athletic performance improvment and cytoprotective effect.4-9 Thus, the high stability and encouragingly physiological function of octacosanol makes it a potential candidate for a supplement in food, beverage, pharmaceutical and cosmetics industries. In addition, the physicochemical and physiological properties of octacosanol are effective due to its high melting point; however, the poor solubility of octacosanol in oil becomes its largest obstacle for their application as nutritional supplements in food, beverage, pharmaceutical and cosmetic products. Consequently, in order to improve the solubility, new octacosanol derived esters were synthesized by both chemical-catalyzed and enzyme-catalyzed esterification of fatty acids with octacosanol. Besides, two synthesis methods were compared qualitatively and quantitatively. In our study, enzymatic method shows more advantages over chemical method, especially for the higher yield (>80 %) of enzymatic mehtod. Most importantly, the solubility of the esterified octacosanol were improved significantly which was up to 6 times that of octacosanol and our further study showed that different length of fatty acid carbon chain had effects on the solubility attributes of these esters. In addition, the hydrolysis test by lipase can prove that octacosanol derived esters can be digested by the human body.
The increasing consumer demand for flavourful food and nutritional supplements supply has led to an increasing demand for food grade starting materials that may be considered natural. The use of specific enzymes in biosynthetic processes presents great potential to meet this demand. Due to the advantage of enzyme-catalyzed esterification or tranesterification, the esters in food could be synthesized through this strategy in the food industry.
List of Tables
Table 1.1 Structures of VSFCs. 2
Table 2.1 Comparison of yield of methionol derived fatty acid esters synthesized by chemical and enzymatic methods. 49
Table 2.2 The yield of methionol derived fatty acid esters synthesized by lipase-catalyzed transesterification of butter oil and methionola. 52
Table 2.3 Comparison of sensory description and thresholds of methionyl esters. 54
Table 3.1 Comparison of yield of 2-phenethyl alcohol derived fatty acid esters synthesized by chemical and enzymatic methods. 70
Table 3.2 Comparison of sensory descriptions of 2-phenethyl esters. 74
Table 4.1 Factors and their levels for Box-Behnken Design. 91
Table 4.2 Box-Behnken experimental design and actual, predicted conversions for 2-phenethyl alcohol to 2-phenethyl esters. 92
Table 4.3 Coefficients of the model and analysis of variance (ANOVA). 94
Table 5.1 Comparison of yield of octacosanol derived fatty acid esters synthesized by chemical and enzymatic methods. 117
Table 5.2 Solubility of designed octacosyl esters in soy bean oil (mg mL-1) at room temperature. 118
Table 6.1 Alcohols employed in the further study and their sensory descriptions. 128
Appendix Table Ethics, gender and age of the sensory panelists. 131
List of Figures
Figure 2.1 Chromatogram (GC-MS) of major volatile compounds identified in butter oil transesterified with methionol. Major peaks identified: peak 1, methionol; peak 2, 3-methylthio-propyl butanoate; peak 3, 3-methylthio-propyl hexanoate; peak 4, 3-methylthio-propyl octanoate; peak 5, methyl pentadecanoate; peak 6, 3-methylthio-propyl decanoate; peak 7, 3-methylthio-propyl dodecanoate; peak 8, 3-methylthio-propyl tetradecanoate; peak 9, 3-methylthio-propyl hexadecanoate; peak 10, 3-methylthio-propyl oleate; peak 11, 3-methylthio-propyl octadecanoate.
51
Figure 2.2 Time-course production of methionyl esters during lipase-catalyzed transesterification of butter oil with methionol. A reaction mixture containing 4.0 g of butter oil, 1.5 g methionol and 0.28 g of Lipozyme TL IM was incubated at 40 oC, 130 rpm. All the reactions were conducted in triplicate and responses presented from GC-MS-FID were the average of three determined values. 52
Figure 3.1 Chromatogram (GC-MS) of 2-phenethyl alcohol and 2-phenethyl esters of C4-18 fatty acids. Major peaks identified: peak 1, 2-phenethyl alcohol; peak 2, 2-phenethyl butanoate; peak 3, 2-phenethyl hextanoate; peak 4, methyl pentadecanoate; peak 5, 2-phenethyl octanoate; peak 6, 2-phenethyl decanoate; peak 7, 2-phenethyl dodecanoate; peak 8, 2-phenethyl tetradecanoate; peak 9, 2-phenethyl hexadecanoate; peak 10, 2-phenethyl octadecanoate. 65
Figure 3.2 Chromatogram (GC-MS) of the major volatile compounds identified in transesterified butter oil with 2-phenethyl alcohol. Major peaks identified: peak 1, 2-phenethyl alcohol; peak 2, 2-phenethyl butanoate; peak 3, 2-phenethyl hextanoate; peak 4, methyl pentadecanoate; peak 5, 2-phenethyl octanoate; peak 6, 2-phenethyl decanoate; peak 7, 2-phenethyl dodecanoate; peak 8, 2-phenethyl tetradecanoate; peak 9, 2-phenethyl hexadecanoate; peak 10, 2-phenethyl oleate; peak 11, 2-phenethyl octadecanoate. 71
Figure 3.3 Time-course production of 2-phenethyl esters during lipase-catalyzed transesterification of butter oil with 2-phenethyl alcohol. A reaction mixture containing 4.0 g of butter oil, 1.686 g of 2-phenethyl alcohol and 0.284 g of Lipozyme TL IM was incubated at 40 oC and 150 rpm shaking speed. All the reactions were conducted in triplicate and responses presented from GC-MS-FID were the average of three determined values.
72
Figure 3.4 Effect of molar ratio of 2-phenethyl alcohol to oil on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: 10 % wt/wt enzyme loading, 40 oC, 8 hours, 150 rpm; p < 0.05) Mean values labeled with same letters in the same figure are not significantly different according to LSD test.
76
Figure 3.5 Effect of enzyme loading on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 40 oC, 8 hours, 150 rpm; p > 0.05) Mean values labeled with same letters in the same figure are not significantly different according to LSD test. 78
Figure 3.6 Effect of shaking speed on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 20 % wt/wt enzyme loading, 40 oC, 8 hours; p < 0.05) Mean values labeled with same letters in the same figure are not significantly different according to LSD test. 79
Figure 3.7 Effect of reaction temperature on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 20 % wt/wt enzyme loading, 8 hours, 100 rpm; p < 0.05); Mean values labeled with same letters in the same figure are not significantly different according to LSD test.
81
Figure 3.8 Effect of reaction time on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 20 % wt/wt enzyme loading, 40 oC, 100 rpm; p < 0.05); Mean values labeled with same letters in the same figure are not significantly different according to LSD test. 82
Figure 4.1 The plot of predicted versus actual distribution. 96
Figure 4.2 The contour plot of the effects of molar ratio and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol. 98
Figure 4.3 The 3D plot of the effects of molar ratio and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol. 98
Figure 4.4 The contour plot of the effects of molar ratio and reaction temperature and their interactive effects on the conversion of 2-phenethyl alcohol. 100
Figure 4.5 The 3D plot of the effects of molar ratio and reaction temperature and their interactive effects on the conversion of 2-phenethyl alcohol. 100
Figure 4.6 The contour plot of the effects of reaction temperature and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol. 102
Figure 4.7 The 3D plot of the effects of reaction temperture and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol. 103
Figure 5.1 Octacosyl acetate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC. Peak 1, 2 and 3, unknown impurities. 120
Figure 5.2 Octacosyl acetate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak 1 and 2, unknown impurities; peak 3, acetic acid. 120
Figure 5.3 Octacosyl butyrate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC. Peak 1 and 2, unknown impurities. 121
Figure 5.4 Octacosyl butyrate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak 1, 2 and 3, unknown impurities; peak 4, butyric acid. 121
Figure 5.5 Octacosyl hexanoate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC. Peak 1 and 2, unknown impurities. 122
Figure 5.6 Octacosyl hexanoate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak 1, 2 and 3, unknown impurities; peak 4, hexanoic acid.
122
Figure 5.7 Octacosyl octanoate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC. 123
Figure 5.8 Octacosyl octanoate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak 1, octanoic acid. 123
List of Schemes
Scheme 1.1 The Grignard reaction between phenylmagnesium bromide and ethylene oxide. 7
Scheme 1.2 The Friedel-Crafts reaction between benzene and ethylene oxide. 7
Scheme 1.3 The Steglich esterification. 23
Scheme 1.4 The reaction mechanism of Steglich esterification. 23
List of Symbols and Abbreviations
|GC-MS |gas chromatography-mass spectrometry |
|GC-FID |gas chromatography-flame ionization detector |
|VSFCs |volatile sulphur flavour compounds |
|DMS |dimethyl sulphide |
|DMDS |dimethyl disulphide |
|DMTS |dimethyl trisulfide |
|VHS |vacuum headspace |
|AAAs |aryl alkyl alcohols |
|2-PEAc |phenylacetate |
|AAASAE |aryl alkyl alcohol simple acid esters |
|2-PE |2-phenoxyethanol |
|CAPE |caffeic acid phenethyl ester |
|SUS |saturated-unsaturated-saturated |
|CBE |cocoa butter equivalent |
|LDL |low-density lipoprotein |
|HDL |high-density lipoprotein |
|CHD |congenital heart disease |
|AOM |alcohol or mercaptan |
|DMAP |4-dimethylaminopyridine |
|DCC |N’, N’-dimethylcyclohexylcarbodiimide |
|FID |flame ionization detector |
|TCD |thermal conductivity detector |
|ECD |electron capture detector |
|PID |photoionization detector |
|AED |atomic emission detector |
|SIM |selected ion mode |
|MID |multiple ion detection |
|LR |low resolution |
|HR |high resolution |
|m/z |mass to charge |
|SPME |solid-phase microextraction |
|HS |headspace |
|DI |direct immersion |
|HPLC |high-performance liquid chromatography |
|MS |mass spectrometry |
|MSD |mass selective detector |
|DCM |dichloromethane |
|IR |infrared |
|1H NMR |hydrogen-1nuclear magnetic resonance |
|LCMS |liquid chromatography mass spectrometry |
|TLC |thin layer chromatography |
|HR-EI |high resolution electron impact |
|HR-APCI |high resolution atmospheric pressure chemical ionization |
|RSM |response surface methodology |
|3D |three dimensionoal |
|CAR/PDMS |carboxen/polydimethylsiloxane |
Chapter 1
Introduction
1. Methionol
1.1.1 Volatile Sulphur Flavour Compounds and Their Characteristics
Volatile sulphur flavour compounds (VSFCs) contribute a wide array of distinct and characteristic flavours and occur in many foods and beverages. Because the sensory thresholds and concentrations of VSFCs in food system are very low, so they are very important to the flavour and off-flavour characteristics of foods. The compound including methanethiol, dimethyl disulphide (DMDS), dimethyl trisulfide (DMTS), 3-(methylthio)propanal (methional), dimethyl sulphide (DMS), 3-methylthio-1-propanol (methionol) and S-methylthioesters of short chain fatty acids (acetate, propionate and butanoate) are identified as the common VSFCs.10 These compounds have been found in natural food such as vegetables (leeks, onions, garlic, broccoli, and cabbage), fruits and some food products (beer and bread).11-13 A wide variety of VSFCs have also contributed the integral flavour in fermented food products, such as ripened cheeses.14 The flavouring methionol is a potent VSFCs which was found as a main component in beer and other beverage.15 (See structures of VSFCs in Table 1.1)
Table 1.1 Structures of VSFCs
|VSFCs |Structures of VSFCs |
|methanethiol |H3C–SH |
|dimethyl sulphide |H3C–S–CH3 |
|dimethyl disulphide |[pic] |
|dimethyl trisulfide |[pic] |
|3-(methylthio)propanal |[pic] |
|3-methylthio-1-propanol |[pic] |
|S-methyl thioacetate |[pic] |
|S-methyl thiopropionate |[pic] |
|S-methyl thiobutanoate |[pic] |
Compared with many synthetic flavours, such as alcohols, aldehydes, ketones, esters and acetals, VSFC flavours show some distinct characteristics. The main characteristics are as follows:
A) Pure sulfur-containing flavours often present an unacceptable stinking odour, while their high diluted solutions have an attractive aroma reminiscent of food, especially in cooked dishes (meat aroma, roasted meat aroma, sea food aroma, coffee aroma, chive aroma, garlic aroma, leek aroma, cabbage aroma, and some tropical fruit aroma).12 The odour characteristics of sulfur-containing flavours decide their application, which is usually limited to food flavourings, particularly savory flavourings and tropical fruit flavourings.
B) Sulfur-containing flavours have low thresholds and high intensity of aroma. In general, they are a class of flavours having the lowest thresholds and strongest aroma intensity among all flavours. For example, the threshold of methionol in water (1-3 ppm) is only one 29th of that of maltol (29 ppm).16 The other features of VSFC correlated closely with their low thresholds and high intensity aroma including low use levels, small batch production and high prices.
1.1.2 The Characteristics and Uses of Methionol
Methionol is of particular interest among VSFCs with low sensory threshold and powerful odour. The range of its sensory threshold is from 1 to 3 ppm and its distinct odour is described as soup-tasty, meaty, vegetable-like, boiled potato-like or toasted cheese-like. Even though methionol has been regarded as an off-flavour contributor in beer and wine,2,10,17 it is still very important to the overall aroma profiles in cheeses. Because methionol contributes very essential sensory notes to the overall flavour in premium quality cheddar and camembert.10,18
3. Sources and Synthesis Method of Methionol
The common way to produce methionol is fermentation. Recently, a study investigated that methionol can be produced from yeast fermentation by the use of coconut cream as the growth medium and the supplementation with L-methionine.17 Due to the high fat content and low cost of coconut cream, it served as a secific flavoring retainer for the sulfur containing compounds. In addition, the lack of information on the constitution of coconut cream caused an obstacle on the analysis of the produced compounds. Another study reported that flavourful alcohols and esters were generated by the bioconversion process of amino acids by Erwinia carotovora subsp. Atroseptica.19 In this study, methionol was also generated by a fermentation method.
However, there is generally a challenge to produce sufficient amounts of methionol due to very low yield and formation of variable byproducts during the fermentation process. In addition, the instability of these natural sources makes the control of production, consistency and predictability difficult. Nonetheless, a relatively abundant amount of methionol can still be obtained by natural production process. A patent has developed a fermentation to yield the production of methionol up to 0.4 g L−1 from methionine in dairy media.17 Etschmann and others 20 have produced up to 3.5 g L−1 of methionol through generically-engineered yeast S. cerevisiae bioprocess supplemented with methionine. However, gene technology has been considered as a concern for many scientific fields, the production of methionol naturally is still pertinent.
1.1.4 Methionol Derived Flavours
Among the methionol derived compounds, methyl and ethyl 3-methylthio-propanoate dominated in the vacuum headspace method (VHS) extract of yellow passion fruits.21 Both compounds have previously been found in pineapple juice.22
Some other methionol derived flavours have also been reported. 3-Methylthio-propyl acetate (methionyl acetate) imparts herbaceous character and a typical vegetable-like odour impressions and has been described in the literature in numerous flavor systems.22 In addition, methionyl butanoate (sulfury, cheese and mushroom-like) and methionyl hexanoate (tropical fruit, methional and canned pineapple-like), which have threshold concentrations of 10-20 ppb and 500 ppb, respectively, were also identified in the VHS extract obtained from the juice of the yellow variety. Werkhoff and others 21 revealed 3-(methylthio) esters of propanoic acid impart in the yellow passion fruit variety. The aroma properties of the 3-(methylthio) esters of propanoic acid are not very interesting with the exception of the hexyl derivative. In general, the 3-(methylthio) propanoic acid esters have a sulfury, vegetable like odour, and only hexyl 3-(methylthio) propanoate with its fruity and geranium-like odour may contribute to the overall olfactory impression of the passion fruits.
Generally, the research results indicated that methionol derived flavour compounds have a basic meaty flavour. This presumption can be used to direct the development and formation of newly meaty flavours and avoid aimlessness in screening flavours. So their distinct characteristics are helpful in promoting research and development of meaty flavouring.
1.2 2-Phenethyl Alcohol
1.2.1 The Characteristics and Uses of 2-Phenethyl Alcohol
2-Phenethyl alcohol or 2-phenylethanol belongs to the fragrance structural group Aryl Alkyl Alcohols (AAAs) with the formula C6H5CH2CH2OH. Commonly, the structural elements for the AAAs are the alcohol group that includes primary, secondary and tertiary alkyl alcohols and an aryl group to which the alkyl alcohols covalently bonded. Because in AAA fragrance ingredients, the aryl group may be either a substituted or unsubstituted benzene ring, so the AAAs can be represented as an Ar-C-(R1)(R2)OH or Ar-Alkyl-C-(R1)(R2)OH group.
The aroma properties of 2-phenethyl alcohol is described as floral, mild and rose-honey notes.23 It is therefore contributed as a common fragrance component in flavours and perfumery, particularly when the smell of rose is desired. It may be found in fragrances used in decorative cosmetics, perfumery, shampoos, preservative in soaps and other toiletries as well as in non-cosmetic products such as household cleaners and detergents. Particularly, it is applied as an additive in cigarettes due to its stability in basic conditions and its antimicrobial properties. The main use of 2-phenethyl alcohol is as a flavour component in the modification of certain flavourings. As a precursor of the synthesis of high-value flavouring or pharmaceutical compounds such as 2-phenethyl esters, 2- phenethyl alcohol has also been applied.24 It has been reported that the worldwide consuming volume for 2-phenethyl alcohol as a fragrance ingredient in consumer products market is more than 1000 metric tons per year.24
1.2.2 Natural Source and Synthesis Method of 2-Phenethyl Alcohol
The natural resource of phenethyl alcohol has been observed in a variety of essential oils, including rose, carnation, hyacinth, aleppo pine, orange blossom, ylang-ylang, geranium, neroli and champaca, particularly with the highest quantities found in anise and species of allium plants.3,25 It is miscible with ethanol and ether, but with low solubility in water (2 mL/100 mL H2O).
2-Phenethyl alcohol can be prepared by a variety of chemical method, such as by a Grignard reaction (Scheme 1.1) and a Friedel-Crafs reaction (Scheme 1.2).
C6H5MgBr + CH2CH2O → C6H5CH2CH2OMgBr
C6H5CH2CH2OMgBr + H+ → C6H5CH2CH2OH
Scheme 1.1 The Grignard reaction between phenylmagnesium bromide and ethylene oxide.
C6H6 + CH2CH2O (+ AlCl3) → C6H5CH2CH2OH (+AlCl3)
Scheme 1.2 The Friedel-Crafts reaction between benzene and ethylene oxide.
Chemical synthesis is often defined as the environmentally unfriendly process due to their vigorous reaction condition (high temperature and high pressure) and toxic catalyst (strong acid or alkali), the chemical wastes and the unwanted byproducts which may reduce efficiency as well as increase downstream costs. Moreover, US and European legislations has determined that the uses of chemically synthesized flavour compounds are restricted in food, beverages, pharmaceutical and cosmetics industry.26 So the extraction method from natural materials such as the essential rose oils to synthesize 2-phenethyl alcohol is considered as safe to be food grade product. However, the low concentration of 2-phenethyl alcohol in natural materials led the extraction process complicated and costly. The productivity is also influenced by the available natural source. Thus, the significantly expensive extraction method to produce 2-phenethyl alcohol is not capable to meet the large market demands.27
Recently, according to the US Food and Drug Administration and European legislations, the products generated from natural origin through bioconversion can be considered as natural.28 Whole-cell microbial transformation is a promising natural method for the modification of substrates to other valuable compounds owing to its several advantages such as food grade reaction conditions, high reaction selectivity and pure produced compounds.26 So the low-cost production of 2-phenethyl alcohol by microbial fermentation or enzymatic transformation can be considered as natural. Based on Ehrlich pathway, several biotransformation processes has been applied to produce 2-phenethyl alcohol.29 Moreover, phenylaldehyde and phenylacetate (2-PEAc) were further transformed from 2-phenethyl alcohol. Beside Ehrilich pathway, a relative aboundant amount of 2-phenethyl alcohol could be generated from fermentation carried out by Saccharomyces cerevisiae and supplemented with grape must in semi-aerobic conditions, mainly during cellular growth.30 Another study indicated that 2-phenethyl alcohol can also be obtained by immobilized yeast Saccharomyces cerevisiae biotransformation from L-phenylalanine.31
1.2.3 2-Phenethyl Alcohol Derived Flavours
As mentioned above, a large scale of 2-phenethyl alcohol has been used for the synthesis of its esters, especially 2-phenethyl acetate. So, our emphasis is placed on the production of high value aromatic esters starting from 2-phenethyl alcohol.
The common used 2-phenethyl esters belong to the members of the fragrance structural group aryl alkyl alcohol simple acid esters (AAASAE), such as 2-phenethyl acetate, 2-phenethyl butyrate, 2-phenethyl isobutyrate and 2-phenethyl propionate.
The AAASAE are obtained by reacting an aryl alkyl alcohol with a simple carboxylic acid (a chain of 1-4 carbons) to generate acetate, butyrate, propionate, isobutyrate and carbonate esters. These esters may be found in fragrances used in decorative cosmetics, fine fragrances, shampoos, toilet soaps, other toiletries and non-cosmetics. Besides these AAASAE fragrance derived from 2-phenethyl alcohol, some other valuable phenethyl esters such as 2-phenethyl hexnotate and 2-phenethyl octanoate are also important. According to the notes reports of Flavour and Extract Manufacturers' Association, the sensory notes of these 2-phenethyl esters are unique, such as 2-phenethyl acetate (very sweet, rosy, fruity and honey-like), 2-phenethyl butyrate (warm, floral and fruity), 2-phenethyl hexnotate (fruity, green, rosy, fresh pineapple and banana-like) and 2-phenethyl octanoate (mild, fruity and wine-like ).32
Besides 2-phenethyl esters, there are some other 2-phenethyl alcohol derived compounds, such as valuable metabolites phenylacetaldehyde and 2-PEAc. Through the oxidation of 2-phenoxyethanol (2-PE) with acetic acid bacteria, phenylacetaldehyde can be prepared. Then starting from aliphatic hydrocarbons, aldehydes were further accumulated by two Acetobacter sp. strains (ALEF and ALEG).33
Another interesting 2-phenethyl alcohol derived compound is caffeic acid phenethyl ester (CAPE). It is a flavonoid like compound of antioxidant, anti-inflammatory, anti-carcinogenic and neuroprotective effects.34-42 Commonly, CAPE could be synthesized by immobilized lipase (Novozym® 435) catalyzed reaction from caffeic acid and 2-phenethyl alcohol in an isooctane system.43 Also, the chemical method has been applied to synthesize caffeic acid esters.44
1.3 Fatty Acids as Flavour Compounds in Food Science
1.3.1 Introduction to Fatty Acid Flavours
The sensory notes of short chain fatty acids are described as sharp unpleasant, strongly pungent and irritating odours in high concentration. As increasing fatty acid molecular weight, the odours are replaced by rancid, buttery and cheesy. Fatty acids containing above 14 carbon atoms possess only slight tallow-like odours.45 The fatty acids mainly in food aroma include acetate, butyrate, caproate, decanoate, isobutyrate, 2-methylbutyric acid, 3-methylbutyric acid, octanoate, phenylacetate, propionate and valerate.46 Except for the previous mentioned characteristics, each fatty acid contributes to complex aromas and accentuates unique aroma characteristic, like C3 to C6 fatty acids confer fruity character, while fatty acids containing 4, 6 to 10 carbons impart cheese-associated impression.
Aromatic acids are faintly balsamic with light, spicy and floral notes, even though a hydroxyl group tends to suppress odour. Di- and tricarboxylic acids are all odourless.46 Starting from fatty acids, many valuable flavours and fragrances with green, meaty and mushroom-associated notes can be produced through microorganisms, such as specific lactones and methylketones. Methylketones derived from medium chain fatty acids accentuate strong cheesy flavours, such as Roquefort, Camembert and Stilton. Jasmonic acid is formed by the fungal plant pathogen Botryodiplodia theobromae using α-linolenic acid as the substrate. And methyl (+)-7-isojasmonic acid with a sweet floral, jasmine like odour can be further obtained by lipase catalyzed esterification of jasmonic acid.47 Through lipoxygenase action, linoleic acid (C18:2) can also be converted to 1-octene-3-ol of which confers typical mushroom aroma.47
1.3.2 Pathways to Generate Acid Flavours
The common way to generate acids is microbial biogenesis. Lactic acid and the C1 to C5 aliphatic acids are believed to be formed primarily as the end products of fermentations. Unsaturated fatty acids are precursors of longer C6 to C18 saturated acids via oxygen dependent desaturase catalysis reaction. Isoacids arise from amino acid biosynthesis route, and other free fatty acids are derived from microbial lipase-mediated degradation of glycerides to respective fatty acids and glycerol.45 For example, acetic acid was produced by various species of Acetobacter. Butyric acid was generated by anaerobic fermentation carried out by various Clostridium and Butyribacterium species. Butyric acid as a short chain fatty acid has some important physiological functions. For example, butyric acid is the flavour contributors in tropic fruits and dairy products, being used to provide butter-like notes to flavours and may be used as the precursor for the production of ethyl, butyl, isobutyl and amyl butyrate.48 It is also an important flavour compound in beer. However, if the concentration of butyric acid is above its beer flavour threshold, it is a off-flavour. And further its abnormal concentration in beer can arise infections by anaerobic spore-forming bacteria of the genus Clostridium.49
1.4 Natural Oil as the Precursors in the Synthesis of Flavouring Products
Through biotechnological processes, plant cell and tissue cultures, microorganisms and enzymes can be used as possible alternatives for the production of natural flavouring compounds.50 Due to the advances in microbial fermentation and enzyme technology, individual flavour compounds or complex flavour mixtures are increasingly becoming targets for production on an industrial scale.51 Particularly, without prior isolation of free fatty acids, the natural oils have been widely used as the precursors in directly converting lipids and alcohols to desirable flavour esters through lipase-catalyzed transesterification.52,53 The commonly used natural oils include castor oil, coconut cream, butter oil, coconut oil and cocoa butter.
For example, castor oil, which contains 80 % of esterified ricinoleic acid in its constitution, was used as a precursor for the bioconversion process of ricinoleic acid into γ-decalactone by the yeast Yarrowia lipolytic.54 Coconut cream is also a natural source which was used to generate natural flavour such as methionol. For example, the flavour compound methionol has been produced from yeast fermentation by methionine metabolism in coconut cream.55
Butter oil is abundantly and natural available agricultural product. Additionally, it is composed of various C4 to C18 fatty acids,56 which makes it become a potential lipid substrate for producing valuable fatty acid esters together with proper alcohol substrates. Another natural available acid source is coconut oil which is a low-cost oil in food industry and consisting of various octanoic acid.57 Thus, it would be of big value to convert butter oil or coconut oil these natural acid sources to produce flavour-active compounds. To apply these natural materials as the procecurs to produce target flavour compounds, biocatalysis is a promising alternative among the bioconversion methods.58 By comparing with traditional methods such as chemical synthesis, extraction from natural source and yeast fermentation, this method allows production of natural flavour esters with less unwanted byproduct and higher yields due to its reaction specificity. Lipases are one group of important biocatalysts that have been frequently utilized to synthesis fatty acid esters through enzyme-catalyzed esterification or transesterification reaction.56,59
Cocoa butter is a high price raw material in the chocolate and related confectionery industries with a saturated-unsaturated-saturated (SUS) triacylglycerols structure. Because the triacylglycrol composition of the low costly cocoa butter equivalent (CBE) is similar with that of cocoa butter, CBE can be applied to replace cocoa butter in food industry. Thus, the preparation methods of CBE have been the subjects of numerous studies. Through the chemical interesterification of cocoa butter, the major triacylglycerol components of cocoa butter have been obtained.60 However, much more attention has been received on the preparation of CBE through 1,3-specific lipase-catalyzed interesterification, because the biocatalysis offer certain advantages over the chemical method, such as lower energy consumption, less by-product, high substrate and reaction specificity.61
1.5 Octacosanol
1.5.1 Introduction to Octacosanol
Nutritional supplements have aroused increasingly interest among the public due to their benefits to the human health. Numerous studies have been done on one of such supplements, octacosanol.4-9 Octacosanol, which is a high molecular weight long chain fatty alcohol, with the formula CH3(CH2)26CH2O14 , is the main component of a natural product wax extracted from plants. This wax commonly exists in whole grains, nuts, vegetable oils, sugar canes, fruit, leaves and surface of plants and whole seeds. Octacosanol must be ingested as a supplement due to its very small ingested amounts in the diet. Most studies have been used policosanol as the study subject. Because octacosanol is the main component of the policosanol which contain a natural mixture of primary alcohols.4,5
Octacosanol is insoluble in water, soluble in the aliphatic and aromatic compound at 70-80 °C. If octacosanol is first dissolved in hot oil, its bioavailability and efficacy can be increased. When octacosanol is placed in hot virgin coconut oil at a temperature slightly higher than its melting point (83 °C), it will dissolve quickly and completely into the oil after a minute or so of stirring. Once cooling down the virgin coconut oil, octacosanol solution will form a semi-solid paste with high bioavailability and efficacy.62
According to the acute and chronic toxicity, carcinogenicity and mutagenicity studies of policosanol, its safety is extremely high. Administration test to rats at 50-500 mg/kg/day for 12 months revealed no treatment-related toxicity of policosanol.63 Furthermore, according to in vivo and in vitro toxicity studies, policosanol showed no genotoxic effects.64 Since policosanol has been studied in long term clinical studies, it could be considered as well tolerated and safe.
1.5.2 Physiological Function
A) Lowering plasma cholesterol in humans
In a study published in 1995, rats fed a high fat diet were given octacosanol supplements for a period of time.4 After this period, rats with the high fat diet and supplemented with octacosanol had a significant decrease on cholesterol levels. In 1995, a report outlined a study involved healthy volunteers with normal cholesterol levels given two divided doses (10 or 20 mg of policosanol) or placebo for four weeks. The results showed that supplementation of subjects with policosanol led to a significant effect on lowing serum cholesterol levels. The decreases in low-density lipoprotein (LDL) levels and increases in high-density lipoprotein (HDL) levels were observed when the subjects were given the higher dose of policosanol. On the contrast, an increase in serum cholesterol and LDL levels were observed on the placebo. The results also showed good tolerance in all subjects given the policosanol in this study.6 Statin is often introduced to lower the cholesterol level. The available statin includes atorvastatin, fluvastatin, pravastatin and simvastatin. However, the side effects of statin restrict its uses in lowing cholesterol level such as muscle effects, altered liver function and hepatitis.65 Policosanol therefore could be a useful alternative to cholesterol lowing drugs. Compared by pravastatin which belongs to the group of statin, it has been reported that policosanol was more effective in lowering LDL levels, the ratios of LDL to HDL and total ratios of cholesterol to HDL. However, pravastatin induced a moderate, significant rise in serum transaminase which will reflect possible risk of hepatotoxicity, and an increase in serum creatine phosphokinase which will lead to myositis. Overall, due to the well tolerant and safety of policosanol, it is a more appropriate choice than pravastatin in the treatment of elderly patients with type II hypercholesterolemia and high coronary risk.66 In another study, the cholesterol lowing effects of policosanol and those drugs belong to fluvastatin group were compared. The results showed that a series of side effects was introduced by fluvastatin compared by policosanol.67 A study using lovastatin as the contrast subject with policosanol also found the similar results.68
From these studies, the well tolerant and safe octacosanol has a promising lowering cholesterol function. But long term trials involving more are required to test its cholesterol lowering effect and safety. Due to its fewer side effects and more significant cholesterol lowing effect than statins, the natural available and low costly octacosanol would be widely used in the treatment of patients.
B) Antiaggregatory effects
The antiaggregatory effect of policosanol was observed when the experiment subjects fed with 5 to 20 mg kg-1 doses of policosanol in one placebo-controlled study.69 Another study found that significant reduction in platelet aggregation can only be obtained when a sufficient dose must be administrated, because low dose of policosanol has little effect on platelet aggregation.7
The effects of policosanol and aspirin on platelet aggregation were investigated in a randomized, double-blind, placebo controlled study by using healthy volunteers as the experiment subjects. The results indicated that both policosanol and aspirin administered daily had a significant inhibition of platelet aggregation. However, aspirin caused an increasing chance of side effects such as gastric irritation. Moreover, the results indicated that the sufficient dose of policosanol daily was much lower than that of aspirin, but no side effects were noted.8
C) Athletic performance
The athletic performance of octacosanol such as the effects for grip, chest strength, stamina, cardiovascular function and reaction time has been investigated for a long time. Octacosanol was introduced as a performance improving agent in a placebo controlled study which showed its significantly effect on grip strength and reaction time in response to a visual stimulus. Another study aimed to monitor exercise tolerance of 45 patients with congenital heart disease (CHD) and myocardial ischemia who run on a treadmill with electrocardiography. The results showed that a significant decreasing effect on cardiac events after 20 mo was observed on the subjects administrated with policosanol.5
D) Cytoprotective mechanisms
Numerous studies have been investigated on the antiulcer activity of octacosanol. One study involved rats administered orally with D-002 as the experiment subject found anti-inflammatory effect. D-002 is a mixture of higher primary alcohols of wax, including triacontanol, octacosanol, dotriacontanol, hexacosanol and of which the concentration of major component octacosanol is 17.49 %. The most effective doses of orally administration of octacosanol were determined to be 25 and 50 mg kg-1. Aspirin has the similar cytoprotection effect with octacosanol. However, due to the side effect including gastric irritation caused by aspirin, policosanol is a favorable alternative in patients in whom aspirin is contraindicated.9
E) Supplement in health food for astronauts
In on study, tail-suspended rats fed with octacosanol were studied as the subjects to observe the physiological function of octacosanol in food and further to investigate whether it is possible to be added to natural food to benefit the health of astronauts. The results showed that the weight of thymus was elevated after administration of octacosanol daily. At the same time, the biomechanical properties of the femur and the membrane fluidity of red blood cell were also enhanced. It suggested that octacosanol can counteract some effects of simulated weightlessness on rats. Therefore, octacosanol may be applied as nutritional supplements in health food for cosmonauts due to its counteraction of simulated weightlessness.70
1.6 Flavour Synthesis Method
Flavours classified as natural by the European and U.S. food agencies have been preferred by consumers because they consider artificial ingredients to be unhealthy. Even though the chemical synthesis uses the natural substances to produce flavouring, the resulting flavours cannot be considered as natural. In addition, disadvantage of chemical methods such as lacking substrate specificity may result in low product purity and yield. So the further isolation step of the target isomer from the generated mixtures will be costly if the isolation is impractical. That is why flavour companies prefer to produce flavours through bioconversion pathway.45,50 For a long time, flavour compounds ranging from highly complex mixtures to individual compound have been extracted from raw materials such as plants and fruits. However, this process has a variety of problems, such as low extraction yield of the target flavour, seasonal variation of the substances and the effect from the changing of the weather, which can significantly affect quality of the flavours.71
Through biotechnological approaches, plant cell and tissue cultures, microorganisms, and enzymes can be employed as possible alternatives for the production of natural flavouring compounds.50 With the development of microbial fermentation and enzyme-catalyst technology, single flavour compound or complex mixed compounds are increasingly becoming target products in food industry.51 Biotechnological processes can be classified into two groups: microbiological process and enzymatic process.45 They can also be divided into two major classes: microbial fermentation which apply metabolizing cells and biotransformation or bioconversion which use suitable precursors to react with microorganisms or enzymes.45,51
In the present section, the biotransformation or bioconversion process will be the focus.
1.6.1 Traditional Flavour Synthesis Method
A) Microbial fermentation
Fermentation process which is well known as de novo synthesis, implicates the production of flavour compounds using simple culture media, without the addition of any special carbon source. This method uses the entire metabolic arsenal from the microorganism and generally produces the flavour mixture containing several compounds.72 So the microbial fermentation is an immense method to synthesize various flavour compounds which can be found in microbial culture media or their headspaces. However, the low concentrations of detected flavouring from microorganism can not satisfy industry scales.
B) Biotransformation or bioconversion
Biotransformation of a compound into the target product proceeds in living plant cells, enzymes or microorganisms. Also, biotransformation is considered as a single step reaction which catalyzes the conversion of the substrate. Anyway, bioconversion or biotransformation is defined as the synthesis of one or more flavour compounds with the existence of precursors in the culture media. And under the catalization by constitutive or inducible enzymes, this one step reaction can generate a desired product.73
The biotransformation has been used as a superior strategy to convert a structurally related precursor to a determinate flavour compound, because this method can enhance the accumulation of the product. The natural available and sufficient amounts of precursor are the prerequisite for this economic feasible strategy. So the highly valued flavouring can be converted by relative low costly, readily available and renewable natural precursors including fatty acids, amino acids or natural oils.
The use of enzymes for single flavour compound synthesis is also of great importance due to the characteristic of enzymatic reactions such as high substrate and reaction specificity, mild reaction conditions, high yield of the target product and reduced by-product. Biotechniques such as the development of enzyme immobilization allowed the stability of microbial enzymatic conversion to be enhanced. So, biocatalytic conversion has been applied widely on aroma production. The enzymes can be used directly as food additives and to provide aromas of the product in order to avoid undesirable aromas generated by some compounds.74
1.6.2 Esters Synthesis Method
1.6.2.1 Characteristics of Esters as Flavours
Esters are important flavour compounds because of their abundant and accessible amounts, their wildly occurrence in natural sources, their various odour characteristics and widely uses in flavour components. They present in natural with fairly low concentrations ranging from 1 to 100 ppm.50 They are recognized as important flavour compounds in fruit-flavoured products (beverages, candies, jellies and jams), baked goods, wines and dairy products (cultured butter, sour cream, yogurt and cheese). For example, the most commonly used acetate esters, such as ethyl acetate, hexyl acetate, isoamyl acetate and 2-phenylethyl acetate, are employed in wine and other grape-derived alcoholic beverages. The yeasts, molds and bacteria fermentation has been used to produce the flavour esters.75
The odour of esters varies with the acid and alcohol sources from which the esters derived. Moreover, with the increment of the molecular weight of esters, their odour intensity decreased.76 Thus, in order to prolong the duration of the odour of flavour esters, strong odours and high molecular weight are the prerequisite of these esters. Because sulfur containing flavour compounds always depict very low sensory threshold and distinct odour, so the sulfur-containing esters can be synthesized to produce flavourings with strong odour characteristics and high molecular weight.
1.6.2.2 Chemical and Bioconversion Synthesis Method
Esters containing sulfur can be divided into two groups according to where the sulfur atom belongs to. If the sulfur atom comes from the acid, the esters belong to the first group, such as CH3SCH2CH2COOCH3.The other group are the esters of which the sulfur atom comes from an alcohol or mercaptan (AOM). The facile and efficient chemical method has been used to synthesize the first sulfur-containing ester group such as thia-Michael addition reaction.77,78 The second group can be generated by both chemical and enzymatic method.
Chemical Method: There are three chemical methods to synthesize the second group ester. Either acidic or basic conditions or application of the heat have been reported as the requirement for the ester synthesis methods. The first chemical method is the catalyzed esterification between AOM and an organic acid in the basic condition using 4-dimethylaminopyridine (DMAP) as the catalyst 79 or the acidic condition using solid superacids 80,81 or p-toluenesulfonic acid 82 as the catalyst. As a superb nucleophilic catalyst, DMAP has been widely used to catalyze many organic reactions such as DMAP catalyzed regioselective acylation of 6-O-protected octyl β-d-glucopyranosides and the synthesis of electrophilic alkenes.83 Moreover, DMAP is not a toxic metal catalyst. At the same time, in order to remove water from the reactant and drive the reaction to completion, a coupling agent such as dimethylcyclohexylcarbodiimide (DCC) is needed. So the esterification with DCC as a coupling regent and DMAP as a catalyst is called Steglich esterification described by Wolfgang Steglich in 1978.84 The scheme and reaction mechanism of Steglich esterification are shown in scheme 1.3 and scheme 1.4. The second method to prepare the esters is to react AOM with an anhydride.85 The third method to yield the corresponding esters is the treatment of AOM with an acyl chloride.86 However, the side product such as hydrogen chloride must be eliminated by pyridine addition in this method.
[pic]
Scheme 1.3 The Steglich esterification.
[pic]
[pic]
Scheme 1.4 The reaction mechanism of Steglich esterification
Bioconversion methods are divided into fermentation and enzymatic methods.
Fermentation method: Esters could be generated by the fermentation process supplemented with specific amino acids in a fermentation broth such as the formation of acetate esters from the abundant acetic acid. For example, through yeast fermentation, the intermediate branched alcohols including isobutanol, 3-methylbutanol and 2-methylbutanol were formed from the amino acids including valine, leucine and isoleucine, respectively. Then the added intermediate fruity alcohols (isobutanol, 3-methylbutanol and 2-methylbutanol) were metabolized into the corresponding volatile branched acetates through alcohol acetyltransferases biochemically (isobutylacetate, 3-methylbutylacetate and 2-methylbutylacetate).47
Bacteria and filamentous fungi were capable to synthesize esters through direct esterification reaction of alcohols and acids. The biosynthesis of flavour esters in whole cell media was first found in dairy products such as the cheese production. For example, abundant ethyl or methyl esters in cheese are generally associated with fruity flavours, while thioesters bring about cabbage or sulfur-like notes. It has been reported that the esterification predominately carried out by several lactic acid bacteria and Pseudomonas can form ethyl esters (ethyl butyrate and ethyl hexanoate) and thioesters.45,46 A unique esterase from Lactococcus lactis has been reported to be partially capable for the synthesis of esterified aroma compounds.46
Enzymatic method: Enzymes have shown potential application in the formation of optically pure aliphatic and aromatic esters and lactones through stereo- and regiospecific hydrolyses and transesterification reaction.73 Aside from the highly substrate and reaction specificity, the other benefits of enzyme-catalyzed reaction to synthesize highly conjugated fatty acid esters enable the lipases to work under mild reaction condition and to reduce the waste chemicals.87
A) Enzymatic reaction in organic solvent media
In the enzymatic reaction, the employment of organic solvent media can increase the solubility of hydrophobic substrates when the reactants do not mix well with each other. It also makes the separation of enzyme from product easily after the reaction. However, some disadvantage from organic solvent will reduce the activity and stability of the reaction. So, immobilized enzyme has been employed to solve this problem.
The first application of immobilization of enzyme aimed only to reuse the enzyme for saving the cost. With the development of biotechnology, the enzyme immobilization offer other benefits such as easy separation of immobilized enzyme from the reaction mixture or easy retaining in bath reactors. The most important advantage is the improvement of enzyme activity and stability against extreme conditions such as extreme temperature, pH and adverse effect from the organic solvent medium.87 For instance, the enzymatic synthesis of water-insoluble compounds always reacts in organic solvent media, so the prerequisite of this reaction is the activity and stability of enzyme in organic solvent. Even though the stability of some natural enzymes in the organic solvents medium are able to against extreme conditions,88 however, the stabilizing methods for the enzyme in organic solvents are still required. For example, by treatment of phenolic acid and its derivatives with aliphatic alcohol in an organic media, the immobilized enzyme was used as the catalyst in the esterification reaction to synthesize the related esters.89 The immobilized enzyme-catalyzed esterification between cinnamic acid and oleyl alcohol in organic solvent media has also been reported.90
B) Enzymatic reaction in solvent free system
The solvent free system is considered safe in food industry. With the growing consumer demand for nutritious and flavourful food, the natural flavouring materials are becoming more and more popular among the public. The use of microbial cells or specific enzymes presents great potential to meet this demand. Biosynthetic processes may not yet be industrially worthy, but biotransformation of low costly precursors to highly valued compounds is a good alternative to overcome this obstacle. A good example is the direct enzymatic biotransformation of lactic acid and fatty acids. In this Candida antarctica lipase catalyzed esterification, a four fold excess of caprylic acid can be converted to a very pure ester of the L-enantiomer.91 In another study, the researchers used another enzyme Novozym 435 to synthesis hightly conjugated 1,3-dicylglycerol by the solvent-free esterification between monoolein and oleic acid.92 The most commonly used Lipozyme TL IM was also used as the catalyst in a solvent-free transesterification reaction. In this study, starting from coconut oil and fusel alcohols, a series of flavouring esters were generated.93
1.7 Analytical Method
1.7.1 Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID)
The choice of volatile isolation technique must consider some important properties and characteristics of the volatiles to be isolated. In addition, different analytical techniques should also be employed to analyze the obtained extracts according to their different physical and biochemical characteristics. The development of GC in aroma research area dates back to the mid-1960. In 1963, the number of the unknown aromatic compounds of foodstuffs was only about, while up to today, 7000 compounds has been identified. The excellent separating ability and extreme sensitivity on the volatile compounds of GC make it suitable to isolate volatile compounds.94,95 The main procedure of GC analysis is described as follow. As the injection of the extract into a mobile phase happens, the separation of the compounds by the absorption of the stationary phase occurs at the same time. There are some essential components which contribute to the overall efficiency of GC, such as insert gas, an inlet, a column, an oven, a detector and a data system. The mobile phase is a source gas. The function of an inlet is to deliver the sample to a column. The column contains a stationary phase which separates the volatile compounds by selectively attraction of the compounds. The thermostat for the column is an oven. The different chemicals in the column effluent are registered by a detector. And the chromatogram is recorded and displayed by a data.96 In order to protect the columns from the damage caused by the impurities when operated over 100 °C, the requirement of the carrier gas is its purity from moisture and oxygen. The commonly used pure gases include nitrogen, carbon dioxide, helium and argon. The suite of insert gas is associated to the type of the used detector. There are many choices of gas chromatographic detectors such as: flame ionization detector (FID), thermal conductivity detector (TCD), electron capture detector (ECD), thermoionic detector, photoionization detector (PID), flame photometric detector, chemiluminescence detector, and some more unusual and expensive detectors like atomic emission detector (AED). Among so many detectors, the most popular detector FID has been widely used in flavour and fragrance analysis. FID is very sensitive and universal applicable to separate the flavour compounds. Sometimes, the detection of different chemicals in complex mixtures needs to be carried out by selective detectors separately or in conjunction with the normal one. For example, the nitrogen-phosphorus detector is very sensitive to detect nitrogen and the flame photometric detector is applicable to detect sulfur compounds, respectively. The mandatory requirement of high-resolution columns (typically 25 m or longer) is the use of relatively nonpolar phases. The difficulty exists in high resolution columns is the separation of the more polar phases.97
The principle of GC is that volatile compounds which are tending to bind the stationary phase pass over a stationary phase. So the volatile compounds will pass slow compared to a gas that passes over the same surface, because the gas has no tendency to bind. Under GC conditions, the retention time is defined as the time that takes for a compound to pass through the column. While, in GC analysis, the absolute retention time is not reliable, and the compound is often characterized and identified by its relative retention time. Relative retention time is determined by relating the retention time of an unknown compound to that of a standard compound or a series of standard compounds. So by comparing the experimental relative retention time with the programmed temperature retention indices for the commonly used compounds, the characterization of unknown flavouring compounds can be obtained.98
However, the characterization of an unknown compound can be completely obtained by GC due to the same retention time of different substances. So the conjunction of GC and mass spectrometry (MS) can define the eluted compound. MS has the ability to provide mass spectra information about the unknown substances. So by the combination of these two analytical techniques, many pairs of compounds with similar retention times or with similar mass spectra can be differentiated.
By comparing with other techniques, GC-MS can resolve many problems which make the combination extremely valuable, such as the quantification of the compounds and chromatographic problems.99 In such cases, there are three operation modes of MS, such as selected ion mode (SIM), multiple ion detection (MID) mode and full scan mode. According to the sensitivity of mass spectrometers, its resolution can be low or high. The molecular weight of a compound can be determined by low resolution; conversely, the high resolution provides the information of elemental composition.
As a mass selective GC detector, MS has been wildly used to identify the unknown compounds in the flavouring area. By the coupling of GC and MS, the analysis of complex aroma of wine,100 fruits 101-103 and vegetables 104 have been reported. As an identification tool, MS serve to make the identification simply by comparing the experimental results with efficient MS libraries and searching algorithms. However, in order to avoid the incorrect identifications, some other data of the unknown compounds including Infrared (IR) or Nuclear Magnetic Resonance (NMR) are essential to support GC-MS identifications.
For the quantification analysis of the volatile compounds, GC-MS can determine the positional and geometric isomers of fatty acid methyl esters by SIM mode. On the other hand, GC-FID is the most common method to determine the fatty acids in biological samples.
1.7.2 Solid-Phase Microextraction (SPME)
1.7.2.1 Introduction to SPME
SPME was first developed to extract volatile organic compounds in water105 and further has been applied in the last 10 years to isolate aroma compounds from food.106-109 It was widely applied in numerous flavour study, for instance, strawberry,110 apple,111 orange juice,112 kiwifruits,113 pear, peach and apricot114.
SPME partition process occurs between a liquid or a gaseous phase and a thin solid phase adsorbent. And there are several choices of the equilibrium partition process according to the polarity and film thickness of the adsorbent. A syringe which generally serves as a direct injector associated with the adsorbents coated fibers.106 When an SPME coating is exposed in the headspace of an air tightly sealed sample bottle, an equilibrium partition process occurs between the sample and the SPME coating.
SPME fibers can be divided into two types according the principle of extract analytes. The first type is headspace (HS) SPME, of which the fiber is exposed in the vapor phase above a gaseous, liquid, or solid sample. The second type is direct immersion (DI) SPME, of which the fiber is directly immersed into the liquid samples. In order to increase the rate of equilibration, the sample is often agitated by a stirring bar during a suitable extraction time. After the end of the extraction, the fiber will be withdrawn into a needle which will be further removed from the septum. Then the following step is to insert the needle into the injection port of GC or the desorption chamber of the SPME-high-performance liquid chromatography (HPLC) interface. Commonly, HS- and DI-SPME techniques are combined with GC, GC-MS, HPLC or HPLC-MS system to analyze the extractors. The finial step is to load the analyte from the fiber into the analytical techniques in order to transfer the analyte directly to the column for analysis.106
1.7.2.2 The Limitation of SPME
SPME has been used widely due to its many advantages, such as automated and readily uses, high sensitivity, no added solvent and small sample loading volume.106,115,116 But the mechanisms of SPME determine a certain degree of selectivity of this technique. Many factors will affect the extraction of the analyte from the vapor phase of the sample or directly from the sample, including polarity, sample volume, temperature, volatility, partition coefficients and the nature of the adsorbent-coating material.117
The analyte recovery from HS-SPME fiber is affected by two different equilibriums. The first equilibrium which is measured by distribution coefficient K2 and is responsible for the HS composition is called the matrix/HS equilibrium. By measuring the distribution coefficient K1, the second one called the HS/polymeric fiber coating equilibrium is obtained. Some factors will determine the equilibrium partition of the analytical compounds between the HS-SPME coating and its sample bottle, such as the heating time, temperature, volume and concentration of the sample in the bottle.
HS mode of SPME is more wildly used compared by DI mode. This is because the direct immersion of the fiber in highly complex matrices such as the organic solvent could damage the fiber. For example, the sensitivity of these two modes was compared under the vacuum extraction of Camembert cheese volatiles from cryo-trapping of the aqueous phase. The results showed that DI-SPME is less sensitive than HS-SPME.118
During the absorption of analyte into the syringe needle, the available amount of the sorbent is always small, which means the extraction yield of the analyte is low. So, quantitative analysis has not been successful due to the challenges involved with incomplete adsorption.119 Aside of this disadvantage, some artifact formations have also been noticed. Due to the formation of maillard products during the desorption step, artifact was noticed in the flavour analysis of strawberry and apple fruits.120 If the fiber was rinsed with water before the thermal desorption, a significant reduction in artifact formation was noticed. However, the formation of artifacts is often unavoidable. For instance, it has been reported that the extraction of volatile sulfur compounds will form intrinsic artifact during SPME process.121 Recently, new internally cooled fibers have been described for the analysis of five tropical fruits to overcome this defect.122
1.8 Optimization by Using Response Surface Methodology (RSM)
1.8.1 Introduction to RSM
The optimization method is defined to improve the performance of the systems and to increase the yield of the product without increasing the cost. So in order to apply a process to obtain the best experimental response, the optimization method has been commonly used, especially in analytical chemistry.123
Traditionally, one-variable-at-a-time optimization technique has been carried out to monitor the influence of one factor at a time on a possible response. This optimization method changes only one parameter and keeps the others at a constant level at one time. By using this optimization method, the interactive effects among the studied parameters can not be analyzed. Consequently, the complete information of effect of the variables on the process can not be obtained.124 Another disadvantage of the one-factor optimization is the numerous experiments needed to conduct on the process. These time and reagents consuming experiments will increase the economic cost of the research. Conversely, the advantages of multivariate statistic techniques make them have been widely utilized on the optimization of analytical procedures. The most commonly used multivariate statistic techniques is RSM optimization method.
RSM is a multivariate mathematical and statistic techniques which apply a fit model to describe the behavior of the set data to make the statistical previsions. The construction of the fit model is based on the fitness of polynomial equation to the experimental data. If a response or a series of response of the process are affected by several variables, RSM can be well applied to analyze the interactive effects and complete effect of the studied variables. Simultaneously, the best system performance can be attained by the optimization of the levels of the studied variables.
RSM methodology includes several experimental design matrices which will decide the experiments should be carried out in the experimental region. For the data set which presents linear, the experimental designs for first-order models (e.g., factorial designs) should be used. However, for the data set which presents curvature functions, experimental designs for quadratic response surfaces should be used to approximate a response function to experimental data. The commonly used quadratic experimental designs include three level factorial, Box-Behnken, central composite and Doehlert designs.125
1.8.2 Application of RSM in Food Analysis
RSM has been wildly applied in the optimization studies in recent years. These optimization studies aim to design, develop and form new products, as well as to improve the existing product design. Through analyzing the interact effect on the independent variables, RSM methodology generates a mathematical and statistic fit model to describe the chemical or biochemical processes.126,127 The optimization of biochemical process has been performed by this methodology, such as hydrolysis of enzyme-mediated lignocellulose,128 enzymatic synthesis of fatty acid esters such as palm-based wax ester,129 lipase-catalyzed transesterification of soybean oil and methanol,130 extraction of phenolic compounds from wheat,131 extraction of germinant pumpkin seeds protein,132 optimized biotransformation of 2-phenylethanol to phenylacetaldehyde,133 anaerobic digested sludge conversion of starch to hydrogen,134 the application of pectinase to pretreat mosambi juice.135 Another application of RSM is to determine kinetic constants and investigate enzyme stability. It has been reported that RSM can evaluate kinetic constants for an alkaline protease from Bacillus mojavensis.136 The enzyme kinetics of alcohol dehydrogenase was also determined by this methodology.137 Overall, it is clearly that RSM has been widely applicable for numerous of purposes in food analysis.
Chapter 2
Design, Synthesis and Study Sensory Attributes of Methionol Derived Fatty Acid Esters
2.1 Introduction
Esters are important flavour compounds because of their occurrences in a wide range of natural sources, various odour characteristics and their wide range of uses in flavourings. They are naturally present in fruits in low concentrations, mostly between 1 and 100 ppm and are used in fruit-flavoured products, baked goods, and dairy products.50
The sensory notes of esters are related to the organic acid and alcohol moieties from which they are derived. Moreover, the odour intensity of esters decreases with the increase of molecular weight138; however, the flavour industry needs esters with strong odours and high molecular weight to prolong the duration of the odour of flavourings. Esters containing sulfur can be synthesized to yield flavour substances with strong odour and high molecular weight, as flavour compounds containing sulfur exhibit very low sensory threshold levels and very good odour characteristics.17 The sulfur-containing esters can be divided into two classes; one in which the sulfur atom belongs to the organic acid, such as methyl 3-(methylthio) propionate, and the other one in which the sulfur atom is from an AOM, such as methionol.80
Methionol is an alcohol with low odour threshold ranging from about 1 to 3 ppm and imparts a powerful odour described as soup-like, meaty, boiled potato-like, vegetable-like, savoury or toasted cheese-like. Although methionol has been regarded as an off-flavour compound in beer and wine,2 it is considered as an important constituent of the overall aroma profile in cheeses, particularly premium quality cheddar and camembert.10
Among the methionol related esters, methyl and ethyl 3-(methylthio) propanoate dominated in the vacuum headspace extract (VHS) of yellow passion fruits. Both compounds have previously been found in pineapple juice.22 3-Methylthio-propyl acetate (methionyl acetate) possesses herbaceous odour impressions and a typical vegetable-like character and has been described in numerous flavour systems.22 In addition, methionyl butanoate (sulphury, cheese like, mushroom-like) and methionyl hexanoate (tropical fruit note, methional-like and canned pineapple-like), which have threshold concentrations of 10-20 ppb and 500 ppb respectively, were also identified in the VHS extract obtained from the juice of the yellow variety of passion fruits.22 Werkhoff and others21 revealed the presence of 3-(methylthio) esters of propanoic acid in the yellow passion fruit variety. The aroma properties of the 3-(methylthio) esters of propanoic acid are not very interesting with the exception of the hexyl derivative. In general, the 3-(methylthio) propanoic acid esters have a sulfury, vegetable-like odour, and only hexyl 3-(methylthio) propanoate with its fruity and geranium-like odour note may contribute to the overall olfactory impression of the passion fruits. Overall, the research results indicated that methionol derived organic compounds usually have a basic meat flavour. This presumption can be used to direct the development of new meaty aromas, and avoid aimlessness in screening aromas. It is also helpful in promoting research and development of meaty flavour.
There are two methods to synthesize esters by esterification. Fatty acids or butter oil and methionol were used as the starting materials in both methods in this work. In the chemical method, DMAP was applied as the chemical catalyst and DCC was used as an esterification agent, removing water and driving the reaction to completion.139 The advantage of the DMAP/DCC system is that it does not require a toxic metal catalyst. The second method is the enzymatic method, in which Lipase TL IM was employed as the biocatalyst.53,140,141
The biocatalytic conversion of a structurally related precursor molecule is often a superior strategy, which allows the accumulation of a desired flavour product to be significantly enhanced. As a prerequisite for this strategy, the precursor must be present in nature, and its isolation in sufficient amounts from the natural source must be easily feasible in an economically viable fashion.142 Inexpensive, readily available, and renewable natural precursors, such as fatty acids which were used as the starting materials in our study, can be converted to more highly valued flavours.143
The use of enzymes for flavour compounds synthesis is also of great importance due to the characteristic of enzymatic reactions such as high substrate specificity, high reaction specificity, mild reaction conditions, and reduction of waste product formation. Especially the transesterification of butter oil with methionol reacted in solvent-free system. In the food industry, the solvent-free system is preferred because of the safety concern when solvent is needed.144 The increase in consumer demand for nutritious and flavourful food supply has led to an increased demand for flavouring materials that may be considered natural. The use of specific enzymes in biosynthetic processes presents great potential to meet this demand.
The objectives of the present study were to synthesize new methionol derived flavours by chemical-catalyzed and enzyme-catalyzed esterification of fatty acid with methionol for the first time, since there is no report on the enzymatic esterification of fatty acid with methionol. Moreover, the two methods were compared qualitatively and quantitatively. The sensory description and threshold of each ester were also understood. The sensory test of the new synthesized methionol esters showed that fatty acid carbon chain length have effect on the flavour attributes of methionol esters. In addition, we aimed to convert butter oil and methionol to valuable methionol derived esters through Lipozyme TL IM-mediated solvent-free transesterification and to understand the yield of the formed valuable esters.
2.2 Materials and Methods
2.2.1 Materials and Reagents
Hydrogen chloride solution (2 M) in diethyl ether, DMAP, DCC, methionol was purchased from Sigma Aldrich Chemical Company (Singapore) and used directly. Fatty acids (C4, C6, C8, C10, C12 and C14) were obtained from Firmenich Asia Private Ltd (Singapore). The solvents were all of analytical grade and used as received. Butter oil containing no free fatty acids (analysed by the SPME-GC-MS method) was supplied by Firmenich Asia Private Ltd (Singapore). The major fatty acid composition of butter oil by weight is as follows: butyric (C4) 10 %; caproic (C6) 5 %, caprylic (C8) 2.6 %; capric (C10) 5 %; lauric (C12) 5 %; myristic (C14) 12 %; palmitic (C16) 27 %; stearic (C18) 10 %; and oleic (C18:1) 23 %. 56 The molecular weight of butter oil used was estimated to be 863 g mol-1 according to its fatty acid composition and confirmed further with its average saponification value.145 Lipozyme TL IM is a lipase immobilized on silica gel from Novozymes (Bagsvaerd, Danmark); it is a food-grade lipase from Thermomyces lanuginosus with sn-1, 3-specific selectivity. Internal standard, methyl pentadecanoate (> 98 %) used for determining the composition of fatty acid esters was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan).
2.2.2 Analytical Method
Gas chromatography was performed with a GC-MS-QP2010 system (Shimadzu, Japan) equipped with a DB-5ms capillary column (30 m × 0.25 mm × 0.25 µm, Supelco, Woodbridge, USA), a 5975 inert mass selective detector (MSD) and FID. The injector and detector temperatures were set at 230 oC. The initial oven temperature was set at 50 oC for 5 min, ramped to 230 oC at 5 oC min-1, and then held for 30 min. Injection of 1.0 µL was performed and helium was used as the carrier gas with a flow rate of 1.2 mL min-1. Split mode was applied with a split ratio of 10:1. Proton NMR spectra were recorded on a 300 MHz spectrophotometer with chemical shift values reported in δ units (part per million) relative to tetramethylsilane. Thin layer chromatography (TLC) was done on precoated 0.2 mm Merck silica gel 60F254 plates. High-resolution mass spectra were obtained using micrOTOF chromatography-mass spectrometry (LCMS) (Bruker Biospin Pte Ltd, Germany).
2.2.3 Esterification of Methionol with Fatty Acid through Chemical Method
Fatty acids (C4, C6, C8, C10, C12 and C14 fatty acids) (each was 3.0 mmol), DCC (619.0 mg, 3.0 mmol), and methionol (265.5 mg, 2.5 mmol) were dissolved in dichloromethane (DCM) (20 mL). To the solution, catalytic amount DMAP (55.0mg, 0.45 mmol) were added and stirred at room temperature for 24 hours. N, N-dicyclohexylurea solid formed was filtered off and the filtrate was transferred to a 50.00-mL volumetric flask and topped by adding DCM. The resulting solution (100 μL) of was taken and spiked with 75 μL of internal standard solution of methyl pentadecanoate (2.2 mg mL-1) and diluted to 1.0 mL with DCM. The resulting solution was stored at room temperature for GC-FID analysis. The remaining filtrate was washed with hydrochloric acid (1 M, 3 × 20 mL), saturated sodium bicarbonate (3 × 20 mL) again with water (3 × 20 mL) and dried over anhydrous sodium sulphate. The solvent was removed under the reduced pressure to give the esters, which were chromatographed over silica gel column using n-hexane-ethyl acetate (25:1, v/v) mixture as an eluent. All these newly synthesized compounds were characterized by 1H NMR, and high resolution electron impact (HREI) mass spectra. The esterification reaction was carried out in triplicate and the mean and standard deviation yield of each methionol derived fatty acid ester was determined.
2.2.4 Esterification of Methionol with Fatty Acid through Enzymatic Method
Methionol (265.5 mg, 2.5 mmol), fatty acid (3.0 mmol) , Lipozyme TL IM (5 % (w/w) of all the reactants) and n-hexane (20 mL) were added into a 100-mL screw-capped glass bottle, followed by incubation at 40 oC in water bath at 150 rpm shaking speeds for 24 hours. The reaction was stopped by separating the enzyme from the reaction solutions through centrifugation. The supernatant was transferred to a 50.00-mL volumetric flask and topped with n-hexane. The solution (100 μL) was pipetted into a GC vial and spiked with internal standard methyl pentadecanoate solution (2.2 mg mL-1, 75 μL) and diluted to 1.0 mL with n-hexane for GC-FID analysis. The solvent of the remaining solution was removed under the reduced pressure to give the crude esters, which were chromatographed over a column of silica gel using n-hexane-ethyl acetate (25:1, v/v) as an eluent to obtain pure esters what were confirmed by 1H NMR and HREI mass spectra. The esterification reaction was carried out in triplicate and the mean and standard deviation yield of each methionol derived fatty acid ester was determined.
2.2.4.1. 1H NMR and HREI Mass Spectra Results
3-Methylthio-propyl butanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1/v:v), 1H NMR (300 MHz, CDCl3) δ 4.13 (t, J = 6.0 Hz, 2H, CH2O), 2.52 (t, J = 6.0 Hz, 2H, CH2S), 2.25 (t, J = 7.5 Hz, 2H), 2.00 (s, 3H, CH3S), 1.91-1.84 (m, 2H), 1.68-1.55 (m, 2H), 0.91 (t, J = 7.5 Hz, 3H, CH3CH2). HREI mass spectrum (CI) calcd for C8H16O2S m/z 176.0871, found 176.0871.
3-Methylthio-propyl hextanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1/v:v), 1H NMR (300 MHz, CDCl3) δ 4.14 (t, J = 6.0 Hz, 2H, CH2O), 2.53 (t, J = 6.0 Hz, 2H, CH2S), 2.27 (t, J= 7.5 Hz, 2H, CH2CO), 2.01 (s, 3H, CH3S), 1.92-1.85 (m, 2H), 1.62-1.57 (m, 2H), 1.30-1.27 (m, 4H), 0.87 (t, J = 7.5 Hz, 3H, CH3CH2). HREI mass spectrum (CI) calcd for C10H20O2S m/z 204.1184, found 204.1182.
3-Methylthio-propyl octanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1/v:v), 1H NMR (300 MHz, CDCl3) δ 4.14 (t, J = 6.0 Hz, 2H, CH2O), 2.54 (t, J = 6.0 Hz, 2H, CH2S), 2.25 (t, J = 7.5Hz, 2H, CH2CO), 1.92 (s, 3H, CH3S), 1.90-1.88 (m, 2H), 1.62-1.57(m, 2H), 1.28-1.26 (m, 8H), 0.86 (t, J = 7.5Hz, 3H, CH3CH2). HREI mass spectrum (CI) calcd for C12H24O2S m/z 232.1497, found 232.1490.
3-Methylthio-propyl decanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1/v:v), 1H NMR (300 MHz, CDCl3) δ 4.16 (t, J = 6.3 Hz, 2H, CH2O), 2.55 (t, J = 6.0 Hz, 2H, CH2S), 2.29 (t, J = 7.5Hz, 2H, CH2CO), 2.01 (s, 3H, SCH3), 1.91-1.87 (m, 2H), 1.63-1.60 (m, 2H), 1.26-1.24 (m, 12H), 0.87 (t, J = 7.5 Hz, 3H, CH3CH2). HREI mass spectrum (CI) calcd for C14H28O2S m/z 260.1810, found 260.1820.
3-Methylthio-propyl dodecanoate, liquid, Rf = 0.5 (n-hexane:EA=16:1/v:v), 1H NMR (300 MHz, CDCl3) δ 4.16 (t, J= 6.0 Hz, 2H, CH2O), 2.55 (t, J = 6.0 Hz, 2H, CH2S), 2.29 (t, J = 7.5 Hz, 2H, CH2CO), 2.00 (s, 3H, CH3S), 1.93-1.87 (m, 2H), 1.62-1.56 (m, 2H), 1.26-1.24 (m, 16H), 0.87 (t, J= 7.5 Hz, 3H, CH3CH2). HREI mass spectrum (CI) calcd for C16H32O2S m/z 288.2123, found 288.2135.
3-Methylthio-propyl tetradecanoate, liquid, Rf = 0.5 (n-hexane:EA = 16:1/ v:v), 1H NMR (300 MHz, CDCl3) δ 4.16 (t, J = 6.0 Hz, 2H, CH2O), 2.55 (t, J = 6.0 Hz, 2H, CH2S), 2.29 (t, J = 7.5 Hz, 2H, CH2CO), 2.10 (s, 3H, CH3S), 1.94-1.90 (m, 2H), 1.61-1.58 (m, 2H), 1.27-1.24 (m, 20H), 0.88 (t, J = 7.5 Hz, 3H, CH3CH2). HREI mass spectrum (CI) calcd for C18H36O2S m/z 316.2436, found 316.2446.
2.2.5 Transesterification of Butter Oil with Methionol by Enzymatic Method
Methionol was employed as the alcohol substrate for the transesterification reaction. Butter oil (4.000 g, 4.6 mmol), methionol (1.4653 g, 13.8 mmol) and Lipozyme TL IM (0.2750 g, 5% (w/w) of reactants) were added into a 100-mL screw-capped glass bottle, followed by incubation at 40 ºC in water bath and kept stirring at 130 rpm and samples were collected at different times for GC-FID analysis. The reaction was stopped by separating the enzyme from the reaction solutions through centrifugation. Samples (20 μL) obtained was spiked with internal standard solution (methyl pentadecanoate, 75 μL, 2.2 mg mL-1) and diluted to 1.0 mL with n-hexane. The resulting solution was stored in a fridge at 0 oC overnight to precipitate undesirable products such as glycerol, mono-, di- and triacylglycerols. The top layer was collected for GC-MS and GC-FID analysis. All the reactions were conducted in triplicate and responses from GC-MS-FID were the average of three determined values.
2.2.6 Standard Solutions for Calibration Curves
The purified esters were used as standards. A stock solution was prepared by dissolving six esters with 2000 ppm each in n-hexane (10 mL). These esters included 3-methylthio-propyl butanoate, 3-methylthio-propyl hexanoate, 3-methylthio-propyl octanoate, 3-methylthio-propyl decanoate, 3-methylthio-propyl dodecanoate, and 3-methylthio-propyl tetradecanoate. The internal standard stock solution (220 ppm) was prepared by accurately measuring methyl pentadecanoate (2.2 mg) and dissolving in n-hexane (10.00 mL). The internal standard solution (75 μL) was added into each sample vial for analysis with a final concentration of 165 ppm. The final concentrations of the mix ester standards are 150, 200, 400, 600, 800, and 1000 ppm. These standard solutions were applied in GC-FID test to calculate standard curve. The calibration curves were carried out in triplicate and the mean value and standard deviation were determined.
2.2.7 Qualitative and Quantitative Analysis of Methionyl Esters
Methionol derived fatty acid esters (3-methylthio-propyl butanoate, hextanoate, octanoate, decanoate, dodecanoate and tetradecanoate) were analyzed by GC-MS and GC-FID. The synthesized compounds found in esterified samples or transesterified oil samples were identified through mass spectral analysis and comparing their mass spectra with Wiley database. All these newly synthesized compounds were characterized by 1H NMR and HREI mass spectral data. While it is not possible to identify absolutely the esters with carbon-chain length longer than those were synthesized, because information on methionyl esters of fatty acids would not be in the MS database, standards of most of these esters are not commercially available, that is why C4 to C14 methionyl esters were synthesized in our study. Accordingly, those unidentified esters can be deduced by eliminating those identifiable, such as the synthesized esters of fatty acids and methionol.
2.2.8 Stability Evaluation of Fatty Acid Methionyl Esters in Acidic Media and Heat Treatment
2.2.8.1 Acid Stability Test
Esters (10 mg), hydrochloride in diethyl ether (9 mL, with concentration of 10, 0.1, and 0.001 mM respectively) and deionised water (1mL) were added into 100-mL screw-capped glass bottles and kept at room temperature for 24 hours. The top layer of the mixtures (100 μL) were mixed with with internal standard solution (75 μL, methyl pentadecanoate, and 2.2 mg mL-1) and diluted to 1.0 mL with n-hexane. The resulting solutions were analyzed by GC-MS and GC-FID. In order to dissolve the esters into solution completely, diethyl ether was selected as an ideal medium. Moreover, due to a small amount of water in HCl in diethyl ether, the acid stability test of esters was conducted in aquesous condition. Consequently, if the esters were decomposed by acid, carboxylic acid and methionol can be detected by GC-MS. The acid stability test was carried out in triplicate.
2.2.8.2 Thermal Stability Test
Ester (10 mg) and toluene (10 mL) were added into a 50-mL three necked-flask and stirred at 200 rpm at stipulated temperature for 2 hours at ambient atmosphere or under N2 protection, respectively. The temperatures tested were at 75, 80, 85, 90, 95, 100 and 105 oC. Samples were collected every two hours for GC-MS and GC-FID analysis. One hundred μL of reaction samples obtained were spiked with 75 μL of internal standard solution (methyl pentadecanoate, 2.2 mg mL-1) and diluted to 1.0 mL with toluene. The resultant solution was stored at room temperature until GC-MS and FID analysis. The thermal stability tests were carried out in triplicate.
2.2.9 Odour Descriptions and Threshold Testing of Methionyl Esters
In the odour description and threshold tests, the flavour attributes of all the six compounds were performed by a sensory panel consisting of five well-trained flavourists (four females and one male) from Firmenich Asia Private Ltd (Singapore) who are experienced in aroma evaluation. Before sensory analysis all the ester samples were diluted by 5 % ethanol in deionised water and kept at a room temperature for 24 hours. The blank samples containing 5 % ethanol in deionised water were also provided for each tested compound.
For odour description test, each test tube was coded and presented to the panels with the blank in a randomized order and done in triplicate. The panels individually evaluated the odour by sniffing the sample at room temperature. For the description of the perceived odour, the use of multiple odour attributes was allowed. The odour qualities assigned were collected, and the number of the same answers was listed. However, there was no overall agreement on one odour quality for all compounds, but multiple attributes were given for some odourants instead. After sniffing the samples, the assessors finally agreed upon the odour description of each compound on the basis of an intense discussion. Following this protocol, the odour qualities of the resting methionyl esters were determined in triplicate.
The detection thresholds of methionyl esters in water were determined using the forced choice ascending concentration series method of limits.146 Compounds were diluted in absolute ethanol (for the water threshold) before addition to the deionised water. The compound concentrations were serially diluted for five concentration series for the threshold test. Blank samples in each set were adjusted with the same concentration of ethanol to eliminate any bias due to the solvent used. Each 20-mL volume screw capped test tube was filled up to 8 mL and was allowed to equilibrate for 24 hours before testing. Each concentration in the series was presented to the panellists with the blank in a randomised order. The threshold of each compound was calculated as the geometric mean of the estimate thresholds of individual flavourist.
2.3 Results and Discussion
2.3.1 Qualitative and Quantitative Analysis of Methionyl Esters
Fatty acid esters can be synthesized with high yields via many chemical methods with low costs. However, for food grade flavouring materials, the residues of chemical reagents used in preparing the esters may disqualify the food grade status of the final products if purification is not done thoroughly. To eliminate such a concern, food grade lipases may be a good choice as the formed final products do not need to undergo purification steps and can be directly used as flavouring materials. In addition, immobilized lipases are especially suitable for food industry applications due to several advantages such as lack of contamination of products with enzymes, enzyme reuseability, and easy separation of immobilized enzymes from the reaction mixture or easy retention of enzymes in batch reactors. The most important advantage is the improvement of enzyme activity and stability against extreme conditions such as extreme temperatures, pH and adverse effects from the organic solvent medium.87 These advantages will overcome the drawback of costly enzymes needed if the conversion of the enzymatic reaction is satisfactory.
We compared both chemical and enzymatic methods for generating the methionyl esters and determined the conversion of the reaction using GC-FID (Table 2.1). From the data, it can be concluded that the conversion of the enzymatic reaction is comparable or even better than that of chemical methods. Moreover, there is a significant difference in the synthesis of each methionyl ester by two methods according to the ANOVA test result (p < 0.05). For the chemical method, purification steps are essential and this will reduce the yield due to product loss in the steps. In addition, chemical synthesis has other limitations such as the need to remove impurities and by-products, vigorous reaction conditions and being prone to form undesired by-products.
Table 2.1 Comparison of yield of methionol derived fatty acid esters synthesized by chemical and enzymatic methods.
|Methionol derived fatty acid estsers |Yield (%) |
| |Chemical method |Enzymatic methoda |
|3-Methylthiopropyl butanoate |67.2 ± 3.0 |90.6 ± 3.4 |
|3-Methylthiopropyl hexanoate |90.4 ± 4.1 |84.7 ± 3.0 |
|3-Methylthiopropyl octanoate |87.2 ± 4.2 |77.7 ± 3.0 |
|3-Methylthio-propyl decanoate |82.6 ± 3.5 |82.0 ± 3.2 |
|3-Methylthio-propyl dodecanoate |78.2 ± 3.5 |86.5 ± 2.0 |
|3-Methylthio-propyl tetradecanoate |86.0 ± 3.1 |84.4 ± 4.3 |
aIn the enzymatic method, Lipase TL IM was used as the catalyst.
In comparison, the enzymatic synthesis has great advantages, such as high substrate specificity, high reaction specificity, mild reaction conditions, and reduction of waste product formation. Therefore, an enzymatic procedure is far superior to the chemical method especially in food processing. In the food industry, the use of environmentally friendly lipases can alleviate complex downstream processes and thus leading to reduction of bulk chemical routes in overall operations.
To illustrate the utility of enzymatic reactions in food systems, we applied the same enzyme to convert butter oil and methionol to methionyl esters via transesterification reaction. The major volatile compounds identified in the transesterified butter oil were shown in the GC-MS chromatogram (Figure 2.1). The newly synthesized compounds were characterized by 1H NMR and high resolution mass spectra, which shown correct molecular formula. In addition to the known compounds we have synthesized (peak 2-4 and 6-8), there are three more peaks with higher retention time (peaks 9, 10, and 11). From the MS data, we can deduce their potential structures as methionyl esters of fatty acids with longer chain lengths. Peak 9 shows m/z of 344 (M+) for palmitic methionyl ester. In addition, characteristic fragments were detected for CH3SCH2CH=CH2 (m/z 88), methionol cation (m/z 106), and 239 ([C15H31CO]+). Peak 10 is methionyl ester of oleic acid with characteristic peaks at 265 ([C17H33CO]+), 106, and 88. Peak 11 is methionyl ester of stearic acid as it gave rise to m/z of 372 (M+), 267 ([C17H35CO]+), 106 and 88. These compounds are expected as the butter oil contains respective fatty acids (palmitic (27 %), oleic (23 %), and stearic (10 %)). These high molecular weight esters are not of interest as flavoring compounds; therefore, we did not attempt to prepare it from the respective acids and the methionol. The overall product profile shown in Figure 2.1 is consistent with the chemical compositions of the butter oil. Through the solvent-free transesterification, about 13.2 mg of 3-methylthiopropyl butanoate, 10.6 mg of 3-methylthiopropyl hextanoate, 6.9 mg of 3-methylthiopropyl octanoate, 10.4 mg of 3-methylthio-propyl decanoate, 9.4 mg of 3-methylthio-propyl dodecanoate and 20.6 mg of 3-methylthio-propyl tetradecanoate were produced per gram of butter oil. The conversion of methionol obtained was satisfactory at 81.3 % (Table 2.2 and Figure 2.2).
Enzymatic transesterification of butter oil with an alcohol is generally deemed as a direct alcoholysis reaction and/or a two-step process of simultaneous hydrolysis and esterification.147 Through the GC-MS results of the identification of unreacted butter oil, no peak was detected, suggesting that there were no free fatty acids in the starting butter oil. Therefore, the initial acidity of the butter oil was likely “neutral”. Since methionol contains a certain amount of water, hydrolysis and esterification may also occur during the solvent-free transeterification, but a further study is needed to verify the mechanism. In the reaction between butter oil and methionol, the conversion of methionol could not reach 100 %. Our results show that the reaction reached equilibrium with a conversion of 81.3 % after 48 hours.
[pic]
Figure 2.1 Chromatogram (GC-MS) of major volatile compounds identified in butter oil transesterified with methionol. Major peaks identified: peak 1, methionol; peak 2, 3-methylthio-propyl butanoate; peak 3, 3-methylthio-propyl hexanoate; peak 4, 3-methylthio-propyl octanoate; peak 5, methyl pentadecanoate; peak 6, 3-methylthio-propyl decanoate; peak 7, 3-methylthio-propyl dodecanoate; peak 8, 3-methylthio-propyl tetradecanoate; peak 9, 3-methylthio-propyl hexadecanoate; peak 10, 3-methylthio-propyl oleate; peak 11, 3-methylthio-propyl octadecanoate.
Table 2.2 The yield of methionol derived fatty acid esters synthesized by lipase-catalyzed transesterification of butter oil and methionola.
|Methionol derived fatty acid estsers |Yield (mg) per gram of butter oil |
|3-methylthiopropyl butanoate |13.2 ± 0.6 |
|3-methylthiopropyl hextanoate |10.6 ± 0.5 |
|3-methylthiopropyl octanoate |6.9 ± 0.3 |
|3-methylthio-propyl decanoate |10.4 ± 0.4 |
|3-methylthio-propyl dodecanoate |9.4 ± 0.4 |
|3-methylthio-propyl tetradecanoate |20.6 ± 0.8 |
aIn this reaction, Lipase TL IM was applied as the catalyst and the reaction was incubated for 48 hours.
[pic]
Figure 2.2 Time-course production of methionyl esters of fatty acids during lipase-catalysed transesterification of butter oil with methionol. A reaction mixture containing 4.0 g of butter oil, 1.5 g of methionol and 0.28 g of Lipozyme TL IM was incubated at 40 oC, 130 rpm. All the reactions were conducted in triplicate and responses presented from GC-MS-FID were the average of three determined values
2.3.2 Acid and Thermal Stability Assay of Methionyl Esters
One potential concern of flavour active esters is their stability towards weakly acidic food matrix and against heat during food thermal processing. We treated the esters with different concentrations of hydrochloric acids (10, 0.1, 0.001 mM) for 24 hours and analyzed the by GC. No new peaks were detected compared to untreated samples. Therefore, we concluded that the esters can resist acid environment fairly well. Moreover, heating these esters in toluene at 105 ºC for 2 hours did not result in any noticeable decomposition from the GC analysis. Therefore, the esters are acid and thermally stable. It should be pointed out that, in food matrix are typically complicated as it contains many types of molecules including proteins, lipids, and carbohydrates. The stability of the esters in real food matrix would need to be evaluated in real application of them as flavouring agents.
2.3.3 Odour Descriptions and Threshold Testing of Methionyl Esters
The sensory descriptions of C4 and C6 methionyl esters have been reported by Nijssen and others.22 The sensory tests of the other four methionyl fatty acid esters (C8, C10, C12, and C14) were needed to be understood. From our sensory test results, these methionyl fatty acid esters imparted their unique flavour notes.
The results of the detection thresholds of methionyl esters showed that the increased threshold of each methionyl ester was associated with the increased molecular weight. Hence, the long-chain fatty acid methionyl esters with high thresholds can prolong the duration of their flavouring. The aromatic characterization of the six methionyl esters and their thresholds were compared in Table 2.3.
Table 2.3 Comparison of sensory description and thresholds of methionyl esters.
|Compounds |Odour Assessment |Threshold |
|3-methylthio-propyl butanoate |Sulfury, cheese, mushroom-like |10-20 ppb |
|3-methylthio-propyl hextanoate |Tropical fruit-like, methional-like, |500 ppb |
| |canned pineapple-like | |
|3-methylthio-propyl octanoate |Green, fruity, slightly metallic, sulfury |1 ppm |
|3-methylthio-propyl decanoate |Canned pineapple-like, potato-like, |50 ppm |
| |metallic, methional-like, soft, | |
| |passion furit-like | |
|3-methylthio-propyl dodecanoate |Metallic, ripe papaya, pineapple-like |50 ppm |
|3-methylthio-propyl tetradecanoate |Fermented, juicy, pineapple-like, |50 ppm |
| |metallic, green, seedy, heavy, honey dew | |
2.4 Conclusion
Both chemical-catalyzed and enzyme-catalyzed esterification of fatty acid with methionol can effectively generated methionol derived flavours. In addition, these two methods were compared qualitatively and quantitatively by GC-MS-FID results. The sensory test of the new synthesized methionyl esters showed that fatty acid carbon chain length have effect on the flavour attributes of each methionyl esters. That is to say, all methionyl esters with different fatty acid carbon chain length have their unique sensory notes. Moreover, through Lipozyme TL IM-mediated transesterification, 3-methylthiopropyl esters were generated by using the low cost natural materials butter oil as the acid sources. The high acid and heat stability of our synthesized methionyl esters meet the food scale. Especially, the long fatty acid methionyl esters with high threshold can prolong the duration of their flavouring in food processing. Comparing by the chemical method, enzymatic synthesis has great advantage, especially the higher yield. So the valuable information on the Lipozyme TL IM-mediated transesterification of butter oil with methionol was provided for industry scale production. The transesterification of butter oil also provides a reference for the transeterification reaction not only for methionol derived esters but also for some other chemical derived esters such as 2-phenethyl alcohol derived esters. (Chapter 3 and 4)
Chapter 3
Design, Synthesis and Study Sensory Attributes and Improved Yield of 2-Phenethyl Alcohol Derived Fatty Acid Esters
3.1 Introduction
Flavour esters are important and versatile components of flavors and fragrances. These esters have been widely used in the food, beverage, fine cosmetic, and pharmaceutical industries.1 2-Phenethyl alcohol is a colourless alcohol with a pleasant floral, mild, warm and honey-like ordour of moderate to poor tenacity. It has been found in a variety of essential oils such as rose, carnation, hyacinth, anise, aleppo pine, orange, blossom, ylang ylang, geranium, neroli and champaca.3 2-Phenethyl alcohol is therefore used as an ingredient in many flavour and fragrance formulations with rose ordour.148
2-Phenethyl esters derived from 2-phenethyl alcohol are members of the aroma-active structural group aryl alkyl alcohol simple acid esters (AAASAE), such as 2-phenethyl acetate, 2-phenethyl butyrate, 2-phenethyl isobutyrate and 2-phenethyl propionate. The AAASAE aroma ingredients are commonly prepared by chemical reactions between an aryl alkyl alcohol and a simple carboxylic acid with a chain of 1-4 carbons to generate carbonate esters. These 2-phenethyl esters may be used as flavours and fragrances in cosmetics and non-cosmetic industries. The previous research indicated that 2-phenethyl alcohol derived compounds usually have a basic rose-like flavour.23 This presumption can be used to direct the development of new floral and rose-like aromas and avoid aimlessness in screening aromas.
Traditionally, these 2-phenethyl esters have been either isolated from natural sources or produced by chemical synthesis.45 But the extraction or synthetic yield is low and the cost is high. Thus, it is important to develop an effective or efficient method to generate 2-phenethyl esters. Herein, we reported both chemical and enzymatic methods to synthesize 2-phenethyl esters by esterification. Fatty acids or butter oil and 2-phenethyl alcohol were used as the starting materials. In the chemical synthesis, Steglich esterification described by Wolfgang Steglich in 1978 was used to produce 2-phenethyl esters.84 In this method, DMAP was applied as the chemical catalyst and DCC as the esterification agent which could remove water and drive the reaction to completion. The second esterification reaction is enzyme-mediated one, in which Lipozyme TL IM was employed as the biocatalyst.53,140,141
Increasing attention has been paid to the enzymatic production of flavor esters due to the advantage of enzymatic reactions, such as high substrate and reaction specificity, purer target products, reduction of waste product formation and milder reaction conditions. In addition, the products generated from enzyme-catalyzed reactions which use natural materials are usually deemed as natural.149 As a requirement, the precursor material must exist in nature and the cost of its isolation from sufficient amounts of the natural source should be reasonable.142 Inexpensive, readily available and renewable natural materials such as fatty acids can be converted into more highly valued flavours in an feasible and economically visible way.143 Another example of natural materials is butter oil, which can be transesterified with 2-phenethyl alcohol by lipases in a solvent-free system. In the food industry, the solvent-free system is considered as safe and natural compared with the solvent system.93
The objectives of the present study were to synthesize 2-phenethyl alcohol derived flavour esters by both chemical and enzyme-catalyzed esterification of fatty acids with 2-phenethyl alcohol. Moreover, we aimed to convert butter oil and 2-phenethyl alcohol into valuable 2-phenethyl alcohol derived esters through Lipozyme TL IM-mediated solvent-free transesterification and to understand the influence of some synthetic parameters (temperature, substrate molar ratio, enzyme loading, shaking speed and time) in order to achieve a higher conversion of 2-phenethyl alcohol to esters.
3.1 Materials and Methods
3.2.1 Materials and Reagents
Hydrogen chloride solution (2 M) in diethyl ether, DMAP and DCC were purchased from Sigma-Aldrich Chemical Company (Singapore). 2-Phenethyl alcohol was bought from Merck Schuchardt OHG (Hohenbrunn, Germany). Fatty acids (C4-18) were obtained from Firmenich Asia Private Ltd (Singapore). The solvents were all analytical grade. Butter oil which contained no free fatty acids (analysed by the SPME-GC-MS method) was supplied by Firmenich Asia Private Ltd (Singapore). The major fatty acid composition of butter oil by weight is as follows: butyric (C4) 10 %; caproic (C6) 5 %, caprylic (C8) 2.6 %; capric (C10) 5 %; lauric (C12) 5 %; myristic (C14) 12 %; palmitic (C16) 27 %; stearic (C18) 10 %; and oleic (C18:1) 23 %.56 So the calculation of molecular weight (863 g mol-1) of butter oil was done by the specific fatty acid composition and average saponification value.145 Lipozyme TL IM was obtained from Novozymes (Bagsværd, Denmark); it is a food-grade silica-granulated lipase from Thermomyces lanuginosus with sn-1, 3-specific selectivity. Internal standard of methyl pentadecanoate (>98 %) used for determining the composition and yield of fatty acid esters was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan).
3.2.2 Analytical Method
2-Phenethyl alcohol derived fatty acid esters (2-phenethyl esters of C4-18 fatty acids) were analyzed by GC-MS and GC-FID. Sample analysis was performed by a GC-MS-QP2010 system (Shimadzu, Japan) equipped with a DB-5ms capillary column (30 m × 0.25 mm × 0.25 µm, Supelco, Woodbridge, USA), a 5975 MSD and FID. The injector and detector temperatures were set at 230 oC. The initial oven temperature was set at 50 oC for 5 min, ramped to 230 oC at 5 oC min-1, and then held for 30 min. Injection of 1.0 µL was performed and helium was used as the carrier gas with a flow rate of 1.2 mL min-1. Split mode was applied with a split ratio of 10:1. Proton NMR spectra were recorded on a 300 MHz spectrophotometer with chemical shift values reported in δ units (part per million) relative to tetramethylsilane. TLC was done on precoated 0.2 mm Merck silica gel 60F254 plates. High-resolution atmospheric pressure chemical ionisation (HR-APCI) mass spectra were obtained using micrOTOF LCMS (Bruker Biospin Pte Ltd, Bremen, Germany). The synthesized compounds found in esterified samples or transesterified oil samples were identified through mass spectral analysis and comparing their mass spectra with WILEY database. All these newly formed compounds were characterized by 1H NMR and HR-APCI mass spectral data.
3.2.3 Esterification of 2-Phenethyl Alcohol with Fatty Acid through Chemical Method
Fatty acids (C4-18) (3.0 mmol each), DCC (3.0 mmol), and 2-phenethyl alcohol (2.5 mmol) were dissolved in DCM (20 mL). A catalytic amount of DMAP (0.45 mmol) was added to the solution, followed by stirring at room temperature for 24 hours. N, N-dicyclohexylurea solid formed was filtered off and the filtrate was transferred to a 50.00-mL volumetric flask and topped up by adding DCM and mixed thoroughly. The resulting solution (100 μL) was taken and spiked with internal standard methyl pentadecanoate (2.0 mg mL-1, 75 μL) and diluted to 1.0 mL with DCM. The resultant solution was stored at room temperature for analysis. The remaining filtrate was washed consecutively with hydrochloric acid (1 M, 3 × 20 mL), saturated sodium bicarbonate (3 × 20 mL), and water (3 × 20 mL), and then dried over anhydrous sodium sulfate. Solvent was removed under the reduced pressure to give the esters, which were chromatographed over silica gel column using a hexane-ethyl acetate (25:1, v/v) mixture as an eluent to obtain pure esters. All newly synthesized compounds were characterized by 1H NMR and HR-APCI mass spectra. The esterification reaction was carried out in triplicate and the mean and standard deviation yield of each 2-phenethyl alcohol derived fatty acid ester was determined.
3.2.4 Esterification of 2-Phenethyl Alcohol with Fatty Acid through Enzymatic Method
2-Phenethyl alcohol (2.5 mmol), fatty acids (C4-18) (3.0 mmol each), Lipozyme TL IM (5 % (w/w) of all the reactants) and hexane (20 mL) were added into a 100-mL screw-capped glass bottle, followed by incubation at 40 oC in water bath at 150 rpm shaking speeds for 24 hours. By the filtration of the enzyme from reaction solution, the reaction was stopped. The supernatant was transferred to a 50.00-mL volumetric flask and topped up with hexane. The solution (100 μL) was pipetted into a GC vial and spiked with internal standard of methyl pentadecanoate solution (2.0 mg mL-1, 75 μL) and diluted to 1.0 mL with hexane. The solvent of the remaining solution was removed under the reduced pressure to give the esters, which were chromatographed over a column of silica gel using hexane-ethyl acetate (25:1, v/v) as an eluent to obtain pure esters what were confirmed by 1H NMR and HR-APCI mass spectra. The esterification reaction was carried out in triplicate and the mean and standard deviation yield of each 2-phenethyl alcohol derived fatty acid ester was determined.
3.2.4.1 1H NMR and HR-APCI Mass Spectra Results
2-Phenethyl butanoate, liquid, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, Aceton-d6) δ 7.32-7.25 (m, 5H, C6H5), 4.26 (t, J = 7.5 Hz, 2H, CH2O), 2.93 (t, J = 7.5 Hz, 2H, CH2Ar), 2.24 (t, J = 6 Hz, 2H, CH2CO), 1.61-1.51 (m, 2H, CH2CH3), 0.88 (t, J = 6.0 Hz, 3H, CH3). HR-APCI-MS calcd for C12H16O2 m/z 192.1150, found 192.1154.
2-Phenethyl hextanoate, liquid, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, Acetone-d6) δ 7.30-7.21 (m, 5H, C6H5), 4.26 (t, J = 7.5 Hz, 2H, CH2O), 2.93 (t, J = 7.5Hz, 2H, CH2Ar), 2.26 (t, J = 7.5 Hz, 2H, CH2CO), 1.58-1.53 (m, 2H, CH2CH2CO), 1.29-1.23 (m, 4H, CH2CH2CH3), 0.87 (t, J = 6.0 Hz, 3H, CH3). HR-APCI-MS calcd for C14H20O2 m/z 220.1463, found 220.1458.
2-Phenethyl octanoate, liquid, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, acetone-d6) δ 7.33-7.19 (m, 5H, C6H5), 4.25 (t, J = 6.0 Hz, 2H, CH2O), 2.93 (t, J = 7.5 Hz, 2H, CH2Ar), 2.26 (t, J = 6.0 Hz, 2H, CH2CO), 1.57-1.53 (m, 2H, CH2CH2CO), 1.28-1.22 (m, 8H, (CH2)4CH3), 0.88 (t, J = 6.0 Hz, 3H, CH3). HR-APCI-MS calcd for C16H24O2 m/z 248.1776, found 248.1777.
2-Phenethyl decanoate, white crystals, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, acetone-d6) δ 7.33-7.19 (m, 5H, C6H5), 4.26 (t, J = 7.5 Hz, 2H, CH2O), 2.93 (t, J = 7.5 Hz, 2H, CH2Ar), 2.26 (t, J = 7.5 Hz, 2H, CH2CO), 1.58-1.53 (m, 2H, CH2CH2CO), 1.28-1.22 (m, 12H, (CH2)6CH3), 0.88 (t, J = 6.0 Hz, 3H, CH3). HR-APCI-MS calcd for C18H28O2 m/z 276.2089, found 276.2079.
2-Phenethyl dodecanoate, solid, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, Aceton-d6) δ 7.33-7.18 (m, 5H, C6H5), 4.26 (t, J = 7.5 Hz, 2H, CH2O), 2.93 (t, J = 7.5 Hz, 2H, CH2Ar), 2.26 (t, J = 6.0 Hz, 2H, CH2CO), 1.58-1.53 (m, 2H, CH2CH2CO), 1.28-1.22 (m, 16H, (CH2)8CH3), 0.88 (t, J = 6.0 Hz, 3H, CH3). HR-APCI-MS calcd for C20H32O2 m/z 304.2402, found 304.2393.
2-Phenethyl tetradecanoate, solid, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, acetone-d6) δ 7.33-7.18 (m, 5H, C6H5), 4.26 (t, J = 7.5 Hz, 2H, CH2O), 2.93 (t, J = 7.5 Hz, 2H, CH2Ar), 2.26 (t, J = 7.5 Hz, 2H, CH2CO), 1.58-1.53 (m, 2H, CH2CH2CO), 1.28-1.22 (m, 20H, (CH2)10CH3), 0.88 (t, J = 6.0 Hz, 3H, CH3). HR-APCI-MS calcd for C22H36O2 m/z 332.2715, found 332.2702.
2-Phenethyl hexadecanoate, solid, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, Aceton-d6) δ 7.33-7.19 (m, 5H, C6H5), 4.26 (t, J = 7.5 Hz, 2H, CH2O), 2.93 (t, J = 7.5 Hz, 2H, CH2Ar), 2.26 (t, J =6.0 Hz, 2H, CH2CO),1.57-1.53 (m, 2H, CH2CH2CO), 1.29-1.23 (m, 24H, (CH2)12CH3), 0.88 (t, J = 7.5 Hz, 3H, CH3). HR-APCI-MS calcd for C24H40O2 m/z 360.3028, found 360.3012.
2-Phenethyl octadecanoate, solid, Rf = 0.5 (hexane:EA = 14:1/v:v), 1H NMR (300 MHz, acetone-d6) δ 7.33-7.18 (m, 5H, C6H5), 4.26 (t, J = 7.5 Hz, 2H, CH2O), 2.93 (t, J = 7.5 Hz, 2H, CH2Ar), 2.26 (t, J = 7.5 Hz, 2H, CH2CO),1.58-1.53 (m, 2H, CH2CH2CO), 1.28-1.22 (m, 28H, (CH2)14CH3), 0.88 (t, J = 6.0 Hz, 3H, CH3). HR-APCI-MS calcd for C26H44O2 m/z 388.3341, found 388.3357.
3.2.5 Transesterification of Butter Oil with 2-Phenethyl Alcohol Catalyzed by Lipase
Butter oil (4.6 mmol), 2-phenethyl alcohol (13.8 mmol) and Lipozyme TL IM (5 % (w/w) of all the reactant) were added into a 100-mL screw-capped glass bottle, followed by incubation at 40 ºC in water bath with stirring at 150 rpm for 24 hours. Samples were collected at different times and analyzed to monitor the reaction progress. The reaction was stopped by separating the enzyme from the reaction solutions through centrifugation. Twenty microliters of reaction samples obtained was spiked with 75 μL of internal standard solution (methyl pentadecanoate, 2.0 mg mL-1) and diluted to 1.0 mL with hexane for GC analysis. The resultant solution was stored in a fridge at 0 oC overnight to precipitate undesirable products such as glycerol, mono-, di- and triacylglycerols. The top layer was collected for analysis. The transesterification reaction was carried out in triplicate and the mean and standard deviation yield of each 2-phenethyl alcohol derived fatty acid ester was determined.
3.2.6 Standard Solutions for Calibration Curves
The purified esters confirmed by 1H NMR and HR-APCI mass were used as standards. A stock solution was prepared by dissolving eight esters with 2000 ppm each in hexane (10 mL). These esters included 2-phenethyl butanoate, 2-phenethyl hexanoate, 2-phenethyl octanoate, 2-phenethyl decanoate, 2-phenethyl dodecanoate, 2-phenethyl tetradecanoate, 2-phenethyl hexadecanoate and 2-phenethyl octadecanoate. The internal standard stock solution (200 ppm) was prepared by accurately weighing methyl pentadecanoate (20.0 mg) and dissolving into hexane (10 mL). Seventy five microlitres of the internal standard solution were added into each sample vial. The final concentrations of the mixed ester standards are 150, 200, 400, 600, 800, and 1000 ppm respectively. The standard solutions were applied to calculate standard curves in GC-MS and GC-FID analyses. The calibration curves were carried out in triplicate and the mean value and standard deviation were determined. 2-Phenethyl alcohol and 2-phenethyl esters of C4-18 fatty acids standards had different retention times on GC-MS chromatogram (Figure 3.1).
[pic]
Figure 3.1 Chromatogram (GC-MS) of 2-phenethyl alcohol and 2-phenethyl esters of C4-18 fatty acids. Major peaks identified: peak 1, 2-phenethyl alcohol; peak 2, 2-phenethyl butanoate; peak 3, 2-phenethyl hextanoate; peak 4, methyl pentadecanoate; peak 5, 2- phenethyl octanoate; peak 6, 2-phenethyl decanoate; peak 7, 2-phenethyl dodecanoate; peak 8, 2-phenethyl tetradecanoate; peak 9, 2-phenethyl hexadecanoate; peak 10, 2-phenethyl octadecanoate.
3.2.7 Qualitative and Quantitative Analysis of 2-Phenethyl Esters
2-Phenethyl alcohol derived fatty acid esters (2-phenethyl butanoate, hextanoate, octanoate, decanoate, dodecanoate, tetradecanoate, hexadecanoate and octadecanoate) were analyzed by GC-MS and GC-FID.
The synthetized compounds found in esterified samples or transesterified oil samples were identified through mass spectral analysis and comparing their mass spectra with WILEY database. All these newly formed compounds were characterized by 1H NMR and HR-APCI mass spectral data.
However, it is not possible to identify absolutely the esters with carbon-chain length longer than those that were synthesized. This is because information on 2-phenethyl esters of fatty acids would not be in the MS database, and in addition, standards of most of these esters are not commercially available, that is why C4 to 18 2-phenethyl esters were synthesized in our study. Accordingly, those unidentified esters can be deduced by eliminating those identifiable, such as the synthesized esters of fatty acids and 2-phenethyl alcohol.
3.2.8 Stability Evaluation of 2-Phenethyl Esters in Acidic Media and Heat Treatment
3.2.8.1 Acid Stability Test
Esters (10 mg), hydrochloride in diethyl ether (9 mL, with concentration of 10, 0.1, and 0.001 mM respectively) and deionised water (1 mL) were added into 100-mL screw-capped glass bottles and kept at room temperature for 24 hours. The top layer of the mixtures (100 μL) were mixed with with internal standard solution (75 μL, methyl pentadecanoate, and 2.0 mg mL-1) and diluted to 1.0 mL with n-hexane. The resulting solutions were analyzed by GC-MS and GC-FID. In order to dissolve the esters into solution completely, diethyl ether was selected as an ideal medium. Moreover, due to a small amount of water in HCl in diethyl ether, the acid stability test of esters was conducted in aquesous condition. Consequently, if the esters were decomposed by acid, carboxylic acid and 2-phenethyl alcohol can be detected by GC-MS. The acid stability test was carried out in triplicate.
3.2.8.2 Thermal Stability Test
Ester (10 mg) and toluene (10 mL) were added into a 50-mL three necked-flask and kept stirring at 200 rpm at stipulated temperatures for 2 hours at ambient atmosphere or under N2 protection, respectively. The temperatures tested were at 75, 80, 85, 90, 95, 100 and 105 oC. Samples were collected every two hours. One hundred μL of reaction samples obtained were spiked with 75 μL of internal standard solution (methyl pentadecanoate, 2.0 mg mL-1) and diluted to 1.0 mL with toluene. The resulting solution was stored at room temperature before analysis. The thermal stability tests were carried out in triplicate.
3.2.9 Sensory Evaluation
Sensory experiments were performed at room temperature in a sensory room with single cabins. The sensorial evaluation of phenethyl esters was done by a panel of five well-trained flavourists (four females and one male) from Firmenich Asia (Singapore) who are experienced in aroma evaluation. The compound concentrations were diluted to 20000 ppm with deionised water. The blank samples containing deionised water were also provided for each tested compound. Each 20-mL screw capped test tube was filled up to 8 mL sample and was allowed to equilibrate for 24 hours before testing. Each test tube was coded and presented to the panels with the blank in a randomized order and done in triplicate. The panels individually evaluated the odour by sniffing the sample at room temperature. For the description of the perceived odour, the use of multiple odour attributes was allowed. The odour qualities assigned were collected, and the number of the same answers was listed. However, there was no overall agreement on one odour quality for all compounds, but multiple attributes were given for some odourants instead. Taking 2-phenethyl decanoate as an example, the majority of the panellists clearly described the solution of 2-phenethyl decanoate as animallic, mild, sweet, green, floral (Table 3.2). A total number of five odour descriptors were attributed to 2-phenethyl decanoate. After sniffing the samples, the assessors finally agreed upon the odour description of each compound on the basis of an intense discussion. Following this protocol, the odour qualities of the resting 2-phenethyl esters were determined in triplicate.
3.3 Results and Discussion
3.3.1 Qualitative and Quantitative Evaluation of Two Methods in Preparing 2-Phenethyl Esters
Traditionally, fatty acid esters are synthesized via chemical method with high yields and low costs. However, for the food grade flavouring materials, the residues of chemical reagents used in preparing the esters may disqualify the food grade status of the target products if purification is not done thoroughly. To eliminate such a concern, the food grade lipase-catalyzed conversion may be a good choice as the final products formed do not need to undergo purification steps and can be directly used as flavouring materials. This will also allow the accumulation of a desired flavouring to be significantly enhanced.
We compared both methods for generating the 2-phenethyl esters and determined the conversion of the reactions (Table 3.1). According to the ANOVA test result (p < 0.05), there was a significant difference in the two methods to synthesize each 2-phenethyl ester. The conversion the enzymatic reaction was comparable or even better than that of the chemical method, especially for the higher conversion of 2-phenethyl alcohol. This may be explained by product loss in the purification steps in the chemical processing which may reduce the yields of the desired compound. Chemical synthesis has other limitations such as the need to remove impurities and by-products from the final product, and harsh reaction conditions.
The enzymatic synthesis has greater advantages, such as high substrate specificity, high reaction specificity, mild reaction conditions and reduction of waste product formation. Therefore, the enzymatic procedure was superior to the chemical method. The use of lipases can alleviate complex downstream processes and thus, leading to reduction of bulk chemical routes in overall operation costs.
To illustrate the utility of enzymatic methods in food systems, we used the same enzyme to convert butter oil and 2-phenethyl alcohol into 2-phenethyl esters via transesterification. The major volatile compounds identified in the transesterified butter oil were shown in the GC-MS chromatogram (Figure 3.2). The newly synthesised compounds were characterised by 1H NMR and HR-APCI mass spectrometry. In addition to the known synthesized compounds (peaks 2-3, 5-9 and 11), there is another peak with higher retention time (peak 10). From the MS data, we can deduce its structures as 2-phenethyl ester of fatty acid with long chain length. Peak 10 is 2-phenethyl ester of oleic acid, with characteristic peaks at m/z 265 ([C17H33CO]+), 104 ([C6H5CH2CH]+) and 91 (C6H5CH2). This compound is expected, as the butter oil contains 23% oleic acid. This high molecular weight ester is not of interest as the flavouring compound; therefore, we did not attempt to prepare it from oleic acid and 2-phenethyl alcohol. The overall product profile shown in Figure 3.2 is consistent with the chemical compositions of the butter oil. Through the solvent-free transesterification, GC-FID results showed that about 74.5 mg of 2-phenethyl butanoate, 33.3 mg of 2-phenethyl hextanoate, 20.0 mg of 2-phenethyl octanoate, 37.8 mg of 2-phenethyl decanoate, 43.8 mg of 2-phenethyl dodecanoate, 124.1 mg of 2-phenethyl tetradecanoate, 343.0 mg of 2-phenethyl hexadecanoate and 102.2 mg of 2-phenethyl octadecanoate were produced per gram of butter oil. The conversion of 2-phenethyl alcohol obtained was 75.0 %. (Figure 3.3)
Enzymatic transesterification of butter oil with 2-phenethyl alcohol is deemed as a direct alcoholysis reaction and/or a two-step process of simultaneous hydrolysis and esterification. Since 2-phenethyl alcohol and butter oil contained a certain amount of water, hydrolysis and esterification may have also occurred during the solvent-free transeterification. And further information about the starting maerials is needed to known to verify the mechanism. So the conversion of 2-phenethyl alcohol to 2-phenethyl esters cannot reach 100 % in our study. Our results showed that the reaction reached equivalent after 48 hours.
Table 3.1 Comparison of yield of 2-phenethyl alcohol derived fatty acid esters synthesized by chemical and enzymatic esterification methods.
| |Conversion (%) |
|2-Phenethyl esters | |
| |Chemical method |Enzymatic methoda |
|2-Phenethyl butanoate |47.3 ± 1.8 |50.8 ± 1.9 |
|2-Phenethyl hextanoate |46.8 ± 1.7 |66.6 ± 2.7 |
|2-Phenethyl octanoate |54.3 ± 2.2 |88.4 ± 3.7 |
|2-Phenethyl decanoate |50.1 ± 2.0 |82.3 ± 3.0 |
|2-Phenethyl dodecanoate |53.8 ± 2.0 |86.5 ± 3.4 |
|2-Phenethyl tetradecanoate |53.2 ± 1.9 |85.2 ± 3.6 |
|2-Phenethyl hexadecanoate |62.7 ± 2.4 |83.2 ± 2.9 |
|2-Phenethyl octadecanoate |65.6 ± 2.7 |84.2 ± 3.4 |
aIn the enzymatic method, Lipase TL IM was used as the catalyst.
Figure 3.2 Chromatogram (GC-MS) of the major volatile compounds identified in transesterified butter oil with 2-phenethyl alcohol. Major peaks identified: peak 1, 2-phenethyl alcohol; peak 2, 2-phenethyl butanoate; peak 3, 2-phenethyl hextanoate; peak 4, methyl pentadecanoate; peak 5, 2-phenethyl octanoate; peak 6, 2-phenethyl decanoate; peak 7, 2-phenethyl dodecanoate; peak 8, 2-phenethyl tetradecanoate; peak 9, 2-phenethyl hexadecanoate; peak 10, 2-phenethyl oleate; peak 11, 2-phenethyl octadecanoate.
[pic]
Figure 3.3 Time-course production of 2-phenethyl esters during lipase-catalyzed transesterification of butter oil with 2-phenethyl alcohol. A reaction mixture containing 4.0 g of butter oil, 1.686 g of 2-phenethyl alcohol and 0.284 g of Lipozyme TL IM was incubated at 40 oC and 150 rpm shaking speed. All the reactions were conducted in triplicate and responses presented from GC-MS-FID were the average of three determined values.
3.3.2 Acid and Thermal Stability Assay of 2-Phenethyl Alcohol Derived Fatty Acid Esters
The pH and thermal stabilities of flavour esters are of great interest during food processing. C4-18 2-phenethyl esters were treated with different concentrations of HCl (10, 0.1, 0.001 mM) for 24 hours. According to the GC-FID and GC-MS results, no new peaks were detected compared to untreated samples, indicating that the esters could resist the acid environment well. Moreover, heating these esters in toluene at 105 ºC for 2 hours did not result in any noticeable decomposition, demonstrating that the esters were thermally stable. The stability of the esters would need to be evaluated in real food matrices.
3.3.3 Flavour Attributes of 2-Phenethyl Esters
The aromatic characterization of the eight 2-phenethyl esters synthesized in this study was compared in Table 3.2. The sensory descriptions of C4-8 2-phenethyl esters have been reported by Belsito, D.150 In our study, the flavour attributes of the five compounds were assessed by a trained sensory panel of five flavourists from Firmenich Asia Private Ltd (Singapore). Before and during sensory analysis, all the ester samples were kept at room temperature. From the sensory test results, these 2-phenethly alcohol derived esters imparted their unique flavour notes in comparison with that of 2-phenethyl alcohol.
Table 3.2 Comparison of sensory descriptions of 2-phenethyl esters.
|Compounds |Odour assessed in this study |Odour described in the literature |
|2-phenethyl butanoate |Floral, fruity |Warm, floral and fruity |
|2-phenethyl hextanoate |Frutiy, rosy, pineapple-like |Fruity-green, rosy, fresh |
| | |pineapple-like, |
| | |banana-like |
|2-phenethyl octanoate |Warm, fruity, wine-like |Mild, fruity, wine-like |
|2-phenethyl decanoate |Animallic, mild, sweet, green, floral |- |
|2-phenethyl dodecanoate |Floral, honey, mild, green, 2-phenylethyl |- |
| |alcohol-like | |
|2-phenethyl tetradecanoate |Green, 2-phenylethyl alcohol-like, honey, |- |
| |floral, slightly wine-like, pleasant, smooth, | |
| |sweet, chocolaty | |
|2-phenethyl hexadecanoate |2-Phenylethyl alcohol-like, honey-like, sweet, |- |
| |mild, chocolaty, floral, rosy | |
|2-phenethyl octadecanoate |Rubbery, plastic, honey, latex, burnt, heavy |- |
3.3.4 Effect of Reaction Parameters on the Synthesis of 2-Phenethyl Esters during Transesterification of Butter Oil with 2-Phenethyl Alcohol
3.3.4.1 Effect of Reactant Molar Ratio
The mechanism of lipase-catalyzed transesterification of palm oil has been indicated to be Ping-Pong Bi Bi type with an adverse effect caused by alcohol.147 Polar substrates are proposed to decrease the activity of enzymes which was explained by destroying the water micro-layer which can protect the enzyme active sites.151 Accordingly, abundant amount of alcohol is prone to inhibiting the reaction through inactivating the enzyme active sites, especially when the alcohol is insoluble in an oil phase.152 Hence, in order to generate the maximum amount of target esters, it is essential to avoid the inhibitory effects caused by the short-chain alcohols in the transesterification reaction.
An immobilized lipase Lipozyme TL IM was applied as the biocatalyst in the transesterification. Due to the polarity of 2-phenethyl alcohol,153 its excessive amount in the reactant will have inhibitory effect on the conversion. So the effect of reactant molar ratio on transesterification of butter oil with 2-phenethyl alcohol was investigated in the range of 1.0-5.0:1 (mmol/mmol of 2-phenethyl alcohol to butter oil). Figure 3.4 shows the effect of molar ratio on the conversion of 2-phenethyl alcohol to 2-phenethyl esters. The conversion of 2-phenethyl alcohol increased as the increment of the molar ratio from 1.0 to 3.0:1, and the highest conversion rate was achieved at a molar ratio of 3.0:1, which was selected as the optimum molar ratio for the subsequent studies. A slight decrease of the conversion was observed as the molar ratio was further increased from 3.0 to 5.0:1. This was because the reaction equilibrium would be equal upon the molar ratio being reached 5.0:1. Hence, reaction equilibrium may go to the reverse direction which leads to the decreased conversion of esters. In order to save the cost of 2-phenethyl alcohol, a slight change of the increasing conversion seems not significant. A molar ratio of 3.0:1 was selected as the optimized condition in the following tests.
[pic]
Figure 3.4 Effect of molar ratio of 2-phenethyl alcohol to oil on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: 10 % wt/wt enzyme loading, 40 oC, 8 hours, 150 rpm; p < 0.05) Mean values labeled with same letters in the same figure are not significantly different according to LSD test.
3.3.4.2 Effect of Enzyme Loading
Although a high enzyme loading may contribute to a high yield of fatty acid esters, excessive enzyme particles in a solvent-free reaction medium may decrease the mass transfer efficiency. To obtain high transesterification and economical feasibility, optimum enzyme loading was investigated. The effect of enzyme loading on the conversion of 2-phenethyl alcohol was studied in the range of 5-25 % (w/w, based on all the reactant weight) and using the optimized molar ratio (3.0:1) obtained from the previous result.
A positive effect of enzyme loading (5-20 %) on the conversion was demonstrated by the increased conversion rate of 2-phenethyl alcohol (p > 0.05). However, a further increased enzyme loading from 20 to 25 % (w/w of all the reactants) caused a slight reduction of conversion. The explanation is that the higher loading of lipase, especially in a solvent-free system, the greater viscosity of the reaction medium. Therefore, an excessive enzyme loading will lead to the lack of the substrates to the active sites of the excess enzyme molecules.154,155 It means the catalytic activity of the immobilized lipase may be affected by the effective interfacial area among the substrates and the enzyme particles.156 Consequently, as the substrates could not get access to the active sites of the excess enzymes, the effective interfacial area was reduced and further, the transesterification conversion failed to increase even though more immobilized enzyme was added. Another explanation is the difficulty in maintaining uniform suspension of the activity at higher enzyme concentrations. That is to say, the performance of enzyme will be also affected due to the high enzyme concentration. To obtain a high conversion of 2-phenethyl alcohol, 20 % (w/w) of all the reactants was selected as the optimum enzyme loading applied to the subsequent transesterification of butter oil with 2-phenethyl alcohol.
[pic]
Figure 3.5 Effect of enzyme loading on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 40 oC, 8 hours, 150 rpm; p > 0.05) Mean values labeled with same letters in the same figure are not significantly different according to LSD test.
3.3.4.3 Effect of Shaking Speed
In enzyme-catalyzed transesterification reactions, reactants have to get access to the external surface of enzyme particles from the liquid phase, and further activate the catalysts.157 The generated products need to diffuse out from the enzyme particles to the bulk liquid to alleviate product inhibition. Proper agitation at an optimum speed plays an essential role in enhancing external mass transfer. To observe the effect of mass transfer limitations, experiments were carried out at various agitation speeds ranging from 50 to 250 rpm. It was found (Figure 3.6) that the conversion of 2-phenethyl alcohol significantly increased as the shaking speed increased from 50 to 100 rpm. It was also observed that only a slight increment of in terms of the conversion when the shaking speed further increased to 250 rpm. This demonstrated that the mass transfer resistance had reached a minimum level. With the increment of shaking speed, the decreased film thickness around the solid lipase particles led to the decrease of the mass transfer resistance. The ideal optimized conversion of 2-phenethyl alcohol was at 100 rpm.
[pic]
Figure 3.6 Effect of shaking speed on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 20 % wt/wt enzyme loading, 40 oC, 8 hours; p < 0.05) Mean values labeled with same letters in the same figure are not significantly different according to LSD test.
3.3.4.4 Effect of Incubation Temperature
Reaction temperature has a significant influence on both the initial rate of the reaction and stability of the enzyme in the enzyme-catalyzed reaction. Usually, the initial rate of the reaction increases as the temperature rises, while the stability of enzymes declines.155 The enzymatic reaction rate increase with the increasing temperature can be explained by the transition state theory.158 Meanwhile, the enzyme might be inactivated when the temperature reaches to a certain degree.
To investigate the effect of reaction temperature on the conversion of 2-phenethyl alcohol to 2-phenethyl esters, the transesterification reaction was performed at temperatures varying from 20 to 60 oC. Figure 3.7 shows a significant difference in the conversion of 2-phenethyl alcohol within the studied temperature range according to the ANOVA test result (p < 0.05). The conversion rate increased gradually as the incubation temperature increased from 20 to 40 oC. This is because the melting point of butter oil is around 35 oC, which means the oil phase and the alcohol phase cannot be mixed well below 35 oC. By increasing the incubation temperature, all the reactants will be mixed well and the mass transfer will be enhanced.
A slight reduction of conversion was obtained when incubation temperature increased from 40 to 60 oC. This indicated that the enzyme may suffer from thermal denaturation due to the long reaction time at high temperatures. It was suggested that almost all the nonthermophilic enzymes will be inactive when the temperatures arises above 45 oC.159 In our experiment, the reaction may reach the fastest reaction rate around 40 oC, which could be one reason for the insignificant difference of the conversion rate observed from 40 to 60 oC. Therefore, an optimum reaction temperature should consider both the enzyme stability and the reaction rate of transesterification. To protect the enzyme from thermal deactivation and to obtain high conversion, 40 oC was selected as the reaction temperature for the subsequent experiment.
[pic]
Figure 3.7 Effect of reaction temperature on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 20 % wt/wt enzyme loading, 8 hours, 100 rpm; p < 0.05); Mean values labeled with same letters in the same figure are not significantly different according to LSD test.
3.3.4.5. Effect of Reaction Time
Under the above-optimized parameters, the time-course reaction was studied within a 20 hours period. Figure 3.8 shows that the conversion of phenethyl alcohol in the reaction solutions increased gradually with the increment of reaction time from 4 to 8 hours. Only a slight increment of conversion was observed as the reaction time was further increased from 8 to 20 hours. This is because the reaction rate was going slower as the reaction time increased. The reaction equilibrium was going to be equal around 8 hours. So in order to save reaction time and reduce energy consumption, 8 hours was selected as the optimum condition.
[pic]
Figure 3.8 Effect of reaction time on the synthesis of 2-phenethyl esters during transesterification of butter oil with 2-phenethyl alcohol. (Reaction conditions: molar ratio of 3.0:1, 20 % wt/wt enzyme loading, 40 oC, 100 rpm; p < 0.05); Mean values labeled with same letters in the same figure are not significantly different according to LSD test.
3.3.4.6 Determination of Conversion of 2-Phenethyl Alcohol and Key Esters Formed under Optimized Conditions
2-Phenethyl butanoate, 2-phenethyl hexanoate, 2-phenethyl octanoate, 2-phenethyl decanoate, 2-phenethyl dodecanoate, 2-phenethyl tetradecanoate, 2-phenethyl hexadecanoate and 2-phenethyl octadecanoate were quantified using methyl pentadecanoate as the internal standard. The results show that about 78.6 mg of 2-phenethyl butanoate, 35.0 mg of 2-phenethyl hexanoate, 20.5 mg of 2-phenethyl octanoate, 42.1 mg of 2-phenethyl decanoate, 44.9 mg of 2-phenethyl dodecanoate, 130.1 mg of 2-phenethyl tetradecanoate, 362.0 mg of 2-phenethyl hexadecanoate and 103.6 mg of 2-phenethyl octadecanoate were produced per gram butter oil under the optimized conditions. The total yield of C4-18 2-phenethyl estsers was about 816.8 mg per gram butter oil. The optimum reaction conditions led to an improved 80.0 % conversion of 2-phenethyl alcohol. Besides 2-phenethyl esters, other fatty acid esters were also formed such as long chain fatty acid esters. To separate each ester from the resultant reaction mixture, methods are available that include distillation based on different boiling points and membrane separation such as membrane dialysis and pervaporation.160
3.4 Conclusion
Both chemical and enzyme-catalyzed esterification of fatty acids with 2-phenethyl alcohol can effectively generate 2-phenethyl alcohol derived esters. Fatty acid carbon chain length had an effect on the flavour attributes of 2-phenethyl esters. Moreover, through Lipozyme TL IM-mediated tranesterification, valuable 2-phenethyl alcohol derived esters were synthesized from natural butter oil and 2-phenethyl alcohol. A better understanding of the influence of the process parameters (including temperature, substrate molar ratio, enzyme loading, shaking speed and time) on the transesterification reaction was achieved to reach a higher conversion of 2-phenethyl alcohol to the esters. The optimum condition may also provide a reference for the transeterification reaction for not only 2-phenethyl esters but also other chemically derived esters. The transesterified butter oil may be directly applied as a flavouring ingredient or further processed to obtain pure aroma chemicals. This novel flavour ingredient can have applications not only in flavour and fragrance formulations (especially upon extraction and separation), but also directly in food products, such as cheese.
Chapter 4
Biocatalytic Conversion of Butter Oil to Natural Flavour Esters Optimized with Response Surface Methodology
4.1 Introduction
Esters of short chain fatty acids and alcohols are known as flavour and fragrance compounds used in the food, beverage, cosmetic and pharmaceutical industries.161 Fats and oils are important inexpensive, readily available and renewable natural resources in the industry for the production of chemically pure compounds and biodiesels.162 In addition, fats and oils can also be applied in the food industry as starting materials for the synthesis of valuable fatty acid esters. To convert natural fats and oils to esters, enzyme-catalyzed bioconversion is a promising approach which normally uses lipases as the biocatalysts to obtain higher selectivity, specificity and predictability.163
Lipases play an important role in catalyzing esterification and transesterification reactions to synthesize various esters.164 Many studies demonstrated that esterification of fatty acids with alcohols is the most popular method for the synthesis of fatty acid esters.165 Aside from esterification reaction which need prior isolation of free fatty acids, transesterification allows the synthesis of various valuable fatty acid esters directly from fats or oils and alcohols, which makes the process more effective or efficient.140
The substrate investigated here was low-cost and sustainable butter oil. It contains considerable amount of fatty acids from C4 to C18 carbon which makes it an attractive alternative resource containing expensive dairy fats for the synthesis of fatty acid esters.166 It has been reported that a variety of lipases were used as the biocatalyst to synthesize flavour esters.167-169 However, in these studies, environmental unfriendly chemical acids were used and thus, the esters generated cannot be deemed as natural. Relatively little research has been done on the utilization of butter oil in the production of natural flavour esters by enzyme-mediated transesterification.170,171 Therefore, transestrification of butter oil to valuable fatty acid esters using immobilized lipase was studied here. In this biocatalytic process, 2-phenethyl alcohol was selected as the alcohol substrate.
The lipase-catalyzed transesterification reaction can be performed in both organic solvents and solvent-free medium. The solvent-free transesterification is favorable because it is safer, less toxic and more volumetric productive compared to that in organic solvents.140 However, in the case of conducting lipase catalytic transesterification in a solvent-free system, the increase in the viscosity of the reaction media could lead to lower mass transfer efficiency172 and lipase may be inactive due to the existence of polar alcohols.167 Hence, in order to obtain high transesterification yield, there is an interest in investigating the effects of independent reaction parameters such as alcohol concentration, agitation rate and incubation temperature and their mutual effects through a proper experimental design. Traditional optimization methods such as single-factor design only allow changing one factor at a time and other factors are fixed at constant levels. This method is laborious and time-consuming, since it requires a large number of designed experiments due to multiple factors being involved in our previous study. In addition, only single-dimensional information can be provided based on the one factor design results, interactive effects between studied parameters can not be compared.124 Response surface methodology (RSM) is a proven effective experimental design method and has been wildly applied in food area recently.128 This design method is able to generate sufficient information on statistically acceptable results with a reduced number of experiments.123 Owing to its advantages in studying multiple variables and providing important interactive effects between factors, RSM has been frequently applied in the optimization of lipase mediated biocatalytic processes.173
Therefore, the objective of this study was to convert butter oil and 2-phenethyl alcohol to valuable natural 2-phenethyl alcohol derived fatty acid esters through Lipozyme TL IM-mediated transesterification. The interactive effects of the independent reaction parameters including reactant molar ratio, incubation temperature and shaking speed on conversion were also studied with RSM. The independent reaction parameters for the RSM study were selected based on the comparison of p values obtained from our previous single factor optimized study in Chapter 3. Consequently, the optimized transesterification conversion was achieved by the RSM model and confirmed by our experimental results.
4.2 Materials and Methods
4.2.1 Materials
Butter oil (major fatty acid composition by weight: butyric (C4) 10 %; caproic (C6) 5 %, caprylic (C8) 2.6 %; capric (C10) 5 %; lauric (C12) 5 %; myristic (C14) 12 %; palmitic (C16) 27 %; stearic (C18) 10 %; and oleic (C18:1) 23 %.56) was provided by Firmenich Asia Pte. Ltd. (Singapore). 2-Phenethyl alcohol obtained from Firmenich Asia Pte. Ltd. (Singapore) was used as the alcohol substrate for butter oil transesterification. Lipozyme TL IM, a sn-1, 3 specific lipase from Thermomyces lanuginosus granulated onto silica, was supplied by Novozymes A/S (Bagsværd, Denmark). Hexane was of analytical grade. Standards such as (2-phenethyl butanoate, 2-phenethyl hextanoate, 2-phenethyl octanoate, 2-phenethyl decanoate, 2-phenethyl dodecanoate, 2-phenethyl tetradecanoate, 2-phenethyl hexadecanoate and 2-phenethyl octadecanoate) were the samples as used in Chapter 3. Internal standard of methyl pentadecanoate (> 98 %) used for determining the composition of phenethyl alcohol derived esters was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). The molecular weight of butter oil used was estimated to be 863 g mol-1 according to its fatty acid composition and confirmed further with its average saponification value.145,174
4.2.2 Transesterification Reaction Assays and Analysis
Lipozyme TL IM was used as the lipase in the transesterification of butter oil with 2-phenethyl alcohol. The reaction was performed in 50-mL screw-cap glass bottles with a defined amount of enzyme (20 %, w/w of all the reactant) and a mixture of 2-phenethyl alcohol in the range of molar ratios (2.0-4.0, alcohol/oil) and butter oil (4.0 g). The reactants were incubated in a linear shaking water bath (speed range 20-200 rpm) at different shaking speeds (50, 100 and 150 rpm) at different incubation temperature (30, 40 and 50 oC) for 8 hours.
The reaction was stopped by separating the immobilized enzyme from the reaction solutions through centrifugation. Twenty microliters of reactant samples obtained was spiked with 100 µL of internal standard solution (2.0 mg mL-1) and diluted to 1.0 mL with hexane. The resultant solution was stored in a fridge at 0 oC overnight to precipitate undesirable products such as glycerol, mono-, di- and triacylglycerols. The top layer was collected for the analysis. This sample pretreatment method allowed the removal of heavy compounds to protect the GC column from contamination.
C4-18 2-phenethyl esters were analyzed by Gas chromatography with a GC-MS-QP2010 system (Shimadzu, Japan) equipped with a DB-5ms capillary column (30 m × 0.25 mm × 0.25 µm, Supelco, Woodbridge, USA), a 5975 inert MSD and FID. The injector and detector temperatures were set at 230 oC. The initial oven temperature was set at 50 oC for 5 min, ramped to 230 oC at 5 oC min-1, and then held for 30 min. Injection of 1.0 µL was performed and helium was used as the carrier gas with a flow rate of 1.2 mL min-1. Split mode was applied with a split ratio of 10:1. Proton NMR spectra were recorded on a 300 MHz spectrophotometer with chemical shift values reported in δ units (part per million) relative to tetramethylsilane. TLC was done on precoated 0.2 mm Merck silica gel 60F254 plates. High-resolution mass spectra were obtained using micrOTOF LCMS (Bruker Biospin Pte Ltd, Germany).
The compounds found in transesterified oil samples were identified through mass spectral analysis and comparing their mass spectra with WILEY database. Quantification of major phenethyl esters was achieved by establishing calibration curves through plotting FID peak area ratios of ester standards to that of the internal standard against the concentrations of each ester standards. The conversion (molar conversion) was determined in terms of millimoles of 2-phenethyl esters to millimoles of 2-phenethyl alcohol present in the butter oil multiplied by 100 %.
4.2.3 Experimental Design and Statistical Analysis
The results of single factor design in our previous study suggested that the important factors affecting the transesterification of butter oil and 2-phenethyl alcohol were molar ratio (mmol/mmol) of alcohol to oil, incubation temperature (oC) and shaking speed (rpm). To further improve the bioconversion efficiency of Lipozyme TL IM mediated transesterification, a three-level-three-factor box-behnken design was employed in this study to evaluate the effects of above three factors on the conversion of 2-phenethyl alcohol. The 17 different experiments required were conducted in a random order by considering 7 factorial points, 5 axial points and 5 center points. The variables investigated and their levels selected for the study of conversion of 2-phenethyl alcohol were described in Table 4.1. The independent factors (A, B, C), levels studied and experimental design in terms of actual values were shown in Table 4.2. The results from experiments carried out for model development were analyzed with the Design-Expert 8.0.7.1 (Stat-Ease, USA) software to conduct the response surface regression procedure to fit a second-order polynomial model Eq. (1):
4 4 3 4
Y=b0+ ∑ bixi+∑biixi2 ∑ ∑ bijxixj
i=1 i=1 i=1 j=i+1
where Y is the response (%, molar conversion); b0, bi, bii, bij , constant coefficients (b0, constant; bi, linear effect term coefficients; bii, quadratic term coefficients; bij, cross term coefficients), xi , xj ,coded independent variables.
Table 4.1 Factors and their levels for Box-Behnken Design.
|Factors |Symbol |Coded factor levels |
| | |-1 |0 |1 |
|Molar ratio (mmol/mmol) of alcohol/oil |A |2 |3 |4 |
|Shaking speed (rpm) |B |50 |100 |150 |
|Incubation temperature (oC ) |C |30 |40 |50 |
4.3 Results and Discussion
4.3.1 Transesterification of Butter Oil with 2-Phenethyl alcohol
After Lipozyme TL IM-catalyzed transesterification, butter oil and 2-phenethyl alcohol were converted into various fatty acid esters. The major volatile compounds identified in the transesterified butter oil were shown in the GC-MS chromatogram (Figure 3.2). Besides C4-18 fatty acid esters and unreacted 2-phenethyl alcohol (peak 1 in chapter 3 Figure 3.2), 2-phenethyl alcohol esterified with a longer fatty acid (peak 10 in chapter 3 Figure 3.2) was also formed. Peak 12 is considered to be 2-phenethyl ester of oleic acid. This study only focused on the synthesis of C4-18 2-phenethyl esters because they impart more desirable fruity flavour notes than other longer chain fatty acid esters according to our previous study (Chapter 3). Moreover, the short and middle chain fatty acid esters have low threshed which could be widely used in food, beverage and cosmetic industry.
The mixture compounds of 2-phenethyl esters obtained may be used as aroma ingredients in many applications such as in food, beverage and cosmetic industry. These flavour esters may be extracted from the reaction mixture by using distillation and membrane separation such as pervaporation.160 Thus, they may be applied as individual compounds after separation, purification and concentration.
4.3.2 Model Fitting
The experimental and predicted conversions obtained from the 17 runs for the statistical design were shown in Table 4.2.
Table 4.2 Box-Behnken experimental design and actual, predicted conversions for 2-phenethyl alcohol to 2-phenethyl esters.
|Run |Factor A |Factor B |Factor C |Conversions |
| | | | | |
| |Molar ratio of |Shaking speed (rpm) |Incubation temperature |Actural Predicted |
| |alcohol/oil (mmol/mmol) | |(oC ) | |
|1 |2.0 |50 |40 |66.80 |66.30 |
|2 |4.0 |50 |40 |76.68 |76.32 |
|3 |2.0 |150 |40 |79.00 |79.36 |
|4 |4.0 |150 |40 |87.00 |87.50 |
|5 |2.0 |100 |30 |62.34 |62.01 |
|6 |4.0 |100 |30 |75.64 |75.17 |
|7 |2.0 |100 |50 |73.40 |73.87 |
|8 |4.0 |100 |50 |78.54 |78.87 |
|9 |3.0 |50 |30 |59.65 |60.48 |
|10 |3.0 |150 |30 |69.84 |69.80 |
|11 |3.0 |50 |50 |65.43 |76.28 |
|12 |3.0 |150 |50 |81.21 |80.38 |
|13 |3.0 |100 |40 |80.46 |80.64 |
|14 |3.0 |100 |40 |82.98 |80.64 |
|15 |3.0 |100 |40 |80.84 |80.64 |
|16 |3.0 |100 |40 |82.35 |80.64 |
|17 |3.0 |100 |40 |81.90 |80.64 |
Regression analysis was performed using second order regression Eq. (1). The coefficients Eq. (2) was obtained using Design-Expert 8.0.7.1 software which was given below:
Conversion (%) = 80.64+4.54*A+6.06*B+3.89*C-0.47*A*B-2.04*A*C+1.40*B*C+ 0.088*A^2-3.36*B^2-8.25*C^2
The statistical analysis of the developed second-order regression model was shown in Table 4.3. Indicated by analysis of variance (ANOVA) results, the second-order polynomial model [Eq. (2)] was statistically significant (p value < 0.0001). The result of lack of fit (p value > 0.05) indicated that the model was adequate to represent the experimental data. The coefficient of determination (R2) of the developed model was 0.9890, which demonstrated that the model could adequately indicate the actual relationships between the responses (conversion, %) and the three variables.
Table 4.3 Coefficients of the model and analysis of variance (ANOVA)
|Source |Sum of square |df |Mean square |F value |Probability>F |
|Model |953.73 |9 |105.97 |70.19 |< 0.0001 |
|Residual |10.57 |7 |1.51 | | |
|Lack of fit |2.80 |3 |0.93 |0.48 |0.7134 |
|Pure error |7.77 |4 |1.94 | | |
|Total |964.30 |16 | | | |
|R2 |0.9890 | | | | |
|Coefficients |Coefficient estimate |Standard error |F value |p value |
|Intercept |80.64 |0.55 |70.19 |< 0.0001 |
|A |4.54 |0.43 |109.22 |< 0.0001 |
|B |6.06 |0.43 |194.68 |< 0.0001 |
|C |3.89 |0.43 |80.13 |< 0.0001 |
|AB |-0.47 |0.61 |0.59 |0.4693 |
|AC |-2.04 |0.61 |11.03 |0.0128 |
|BC |1.40 |0.61 |5.17 |0.0571 |
|A2 |0.088 |0.60 |0.022 |0.8871 |
|B2 |-3.36 |0.60 |31.47 |0.0008 |
|C2 |-8.25 |0.60 |189.79 |< 0.0001 |
The F test results presented in Table 4.3 indicated that the investigated independent factors including molar ratio (A), shaking speed (B) and incubation temperature (C) were all statistically significant in the polynomial model. The coefficient values (Table 4.3) depicted that for the linear effect, the selected variables are all determining factors for the conversion of phenethyl alcohol due to the high coefficients of 4.54 (A), 6.06 (B) and 3.89 (C), respectively. Overall, by comparing the coefficients of A, B, C, A2, B2 and C2, the individual effects from the quadratic terms shaking speed, B2, and incubation temperature, C2, were both the important variables for the polynomial model of 2-phenethyl alcohol conversion as shown in Eq. (2). Moreover, the plots of normal residues, outlier T plot and predicted versus actual distribution did not show significant violations on the model assumptions as shown in Figure 4.1. Hence, the predictive model for the enzymatic synthesis of 2-phenethyl esters was adequate for the formulation, optimization and process simulation. From Table 4.3 it can be seen that the cross terms (AB) and A2 were not significant terms in the developed model, which indicates that the interactions between the molar ratio and shaking speed and second-order effect of shaking speed were not statistically significant in the studied ranges. Thus, the simplified model was determined as Eq. (3):
Conversion (%) = 80.68+4.54*A+6.06*B+3.89*C-2.04*A*C+1.40*B*C-3.35*B^2-
8.24*C^2
The R2 of the simplified model was 0.9881 and it was still considered adequate for determining the relationships of selected variables between the responses according to the ANOVA result obtained. Although the model shown in Eq. (3) was simpler, to obtain more desirable prediction results, Eq. (2) was used for the subsequent analysis. This was because the model presented by Eq. (2) was capable of offering a more adequate prediction since effects of more variable items were considered.
[pic]
Figure 4.1 The plot of predicted versus actual distribution.
4.3.3 Effects of Enzymatic Synthesis Parameters
The relationships between tested factors and response can be better understood by examining the series of contour and three-dimensionoal (3D) plots.
The two-dimensional contour plots and 3D plots generated based on the developed model [Eq. (2)] were used to evaluate the main and mutual effects of the independent factors on the conversion of 2-phenethyl alcohol. The interactive effect between molar ratio and shaking speed was shown in Figure 4.2 and 4.3. Enzymatic transesterification of butter oil with a 2.0-4.0:1 molar ratios of alcohol to oil and 50-150 of shaking speed could produce the maximum conversion of 2-phenethyl alcohol. The conversion of 2-phenethyl alcohol increased linearly with the rise of the shaking speed from 50 to 150 rpm. While, no significant effect was observed as molar ratio increased from 2.0 to 3.0 and the rate of conversion was slightly reduced as the molar ratio was further increased to 4.0 (Figure 4.2 and 4.3). This could be explained by the p value of the intercept of AB which is less than 0.05. So the no significant interactive effect between these two factors could be indicated by the two-dimensional contour plots and 3D plots. Moreover, the rate of conversion was observed to be slightly reduced in the range of 3.0 to 4.0 of molar ratio. This could be caused by the effect from the reaction equilibrium which was going to be equal from 3.0 to 4.0 of molar ratio.
Similarly, the increase in shaking speed from 50 to 150 led to a linearly increase in the conversion. In general, agitation showed positive effects on the conversion of butter oil to 2-phenethyl esters in the solvent-free system. With an abundant amount of alcohols, increasing the agitation speed effectively enhanced the conversion. On the other hand, the deactivation effect caused by a high concentration of alcohols in the solvent-free transesterification system could be relieved by agitation due to improved mass transfer between the oil phase and the enzyme support particles. This may be because with a higher shaking speed the alcohols were used up much faster than their being able to exert inhibitory effects on the lipase. The positive effects of agitation were also reported in a study on immobilized lipase-catalyst transesterification of vegetable oils.175 However, much higher shaking speed will cause the enzyme be thrown out of the reaction liquid and adhered to the reaction bottle, thereby the effective catalyst loading will be reduced,176 but this concern was not observed in our study.
[pic]
Figure 4.2 The contour plot of the effects of molar ratio and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol.
[pic]
Figure 4.3 The 3D plot of the effects of molar ratio and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol.
Figure 4.4 and 4.5 show the effects of molar ratio and incubation temperature and their interactive effects on the conversion of 2-phenethyl alcohol. The maximum conversion was achieved at an incubation temperature of 30-50 oC and a molar ratio of alcohol to oil that was in the range of 2.0-4.0:1. Incubation temperature showed positive effects on the conversion of 2-phenethyl alcohol to 2-phenethyl esters in the solvent free system. With an abundant amount of 2-phenethyl alcohol, increasing the incubation temperature effectively enhanced the conversion at the range from 30 to 42 oC and deducted the conversion from 42 to 50 oC. As the molar ratio increased from 2.0 to 4.0, the rate of the increment of conversion decreased gradually. This may due to the high concentration of 2-phenethyl alcohol which was responsible for the reduced catalytic activity of the lipase. Hence, as the molar ratio increasing, the reaction equilibrium may reach to be equal.
[pic]
Figure 4.4 The contour plot of the effects of molar ratio and reaction temperature and their interactive effects on the conversion of 2-phenethyl alcohol.
[pic]
Figure 4.5 The 3D plot of the effects of molar ratio and reaction temperature and their interactive effects on the conversion of 2-phenethyl alcohol.
Figure 4.6 and 4.7 illustrate the effects and mutual effects of incubation temperature and shaking speed on the conversion of 2-phenethyl alcohol for the enzymatic synthesis of 2-phenethyl esters. The best result predicted was around 42 oC incubation temperature and 90 rpm shaking speed. As the shaking speed increased, the conversion increased almost in a linearly way. A higher conversion was achieved at a higher shaking speed (150 rpm onwards) which was required to disperse the enzyme particles in the viscous oil system especially as high amount of immobilized enzyme was applied. This indicates that in the solvent-free system, agitation allows the immobilized enzyme to mix effectively with substrates in the viscous oil system to facilitate the transesterification reaction.176 Therefore, the external mass transfer and diffusion limitations caused by high enzyme loadings could be overcome by applying a proper agitation at an optimum shaking speed.
Similarly, the conversion of phenethyl alcohol increased linearly with the rise of the incubation from 30 to 42 oC and leveled off with a further increase. The drop of conversion at high molar ratios was indicated by the convex curvature in the 3D plot. Because the reaction rate may be the fastest one around 42 oC, the insignificant difference of conversion was observed from 42 to 50 oC. Generally, high temperature led to high reaction velocity during short time operation, however, during long term application, enzyme activity will be reduced which will have adverse effect on the operational stability. Therefore, an optimum incubation temperature should be considered both the enzyme stability and the rate of reaction. In order to avoid the enzyme from thermal deactivation and to obtain high yield especially during batch reactions, 42 oC was determined as the ideal reaction temperature.
[pic]
Figure 4.6 The contour plot of the effects of reaction temperature and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol.
[pic]
Figure 4.7 The 3D plot of the effects of reaction temperture and shaking speed and their interactive effects on the conversion of 2-phenethyl alcohol.
4.3.4 Attaining Optimum Condition and Verification
The maximum point determined according to the second order regression model was outside the studied range, to achieve which, a higher shaking speed than the studied upper limit should be applied. To evaluate the developed model and obtain optimal reaction conditions within the experimental range investigated, the optimization function of the Design-Expert software was applied. The optimized conditions for the Lipozyme TL IM-catalyzed transesterification of butter oil with 2-phenethyl alcohol were determined as a compromise between economic feasibility and high conversion, and they were as follows: a 4.0:1 molar ratio of 2-phenethyl alcohol to butter oil, a shaking speed of 137 rpm and an incubation temperature 41 oC. Under the optimized reaction conditions a conversion of 87.7 % was predicted by the developed polynomial model. To validate the predicted model, the transesterification experiments were carried out at for 20 hours with 20 % (w/w of all the reactant) enzyme loading over three different days under the optimized conditions. The conversions obtained in 3 days were 85.1, 84.3 and 89.5 %, respectively, and all the individual values fell into the 95 % prediction intervals of 83.3-92.1 %. The average conversion of 86.5±2.6 % was within 95 % confidence intervals of 82.2-90.8 % and close to the predicted value of 87.7 %. The deviation between the predicted and actual conversion was 1.2 % and thus confirming the goodness of fit of the model to the experimental data, validating it. And about 86.5 mg of 2-phenethyl butanoate, 38.7 mg of 2-phenethyl hextanoate, 23.2 mg of 2-phenethyl octanoate, 43.9 mg of 2-phenethyl decanoate, 50.9 mg of 2-phenethyl dodecanoate, 144.1 mg of 2-phenethyl tetradecanoate, 396.1 mg of 2-phenethyl hexadecanoate and 113.2 mg of 2-phenethyl octadecanoate were produced per gram of butter oil. In theory, about 87.7 % of 2-phenethyl alcohol could be converted to the corresponding esters since the enzyme applied is sn-1, 3 specific lipase. On the other hand, 2-phenethyl alcohol used in this study contained a certain amount of water, which could also affect the transesterification yield and enzyme activity. Thus, enhancing the migration of fatty acids and controlling the water content in the reaction system merit further research.
4.4 Conclusion
The Lipozyme TLIM-catalyzed transesterification in the solvent-free system can effectively convert relatively low cost natural materials, butter oil and phenethyl alcohol to valuable fatty acid esters. A better understanding of the relationship between variables and responses was achieved, and the optimum reaction conditions for the synthesis of phenethyl esters were also determined in this study with RSM. The results obtained indicate that in a solvent-free system, besides reactant molar ratio and reaction temperature, agitation also plays an important role in improving transesterification yield through reducing mass transfer limitations. Valuable information on the Lipozyme TL IM-mediated transesterification of buter oil was also provided which is useful for large-scale production of not only 2-phenethyl esters but also other important chemicals.
Chapter 5
Design and Synthesis Octacosanol Derived Fatty Acid Esters to Improve the Solubility of Octacosanol
5.1 Introduction
It is by now well known that very long chain fatty alcohols from dietary waxes, especially octacosanol are beneficial to human health. Octacosanol is a water-insoluble and stable compound. In a long time study, it has been shown some health benefits such as cholesterol lowering, antiaggregatory effects, athletic performance improving and cytoprotective effect.4-9 Recently, octacosanol has been aroused great interest in enhancing the sexual behaviours.177 Moreover, recent patent has demonstrated that cosmetics containing octacosanol could promote skin circulation, liven up the blood cells, diminish inflammation and prevent and cure the some skin diseases (such as beriberi, eczema, acne and itch).178 Thus, the high stability, encouragingly biological activities and physiological function of octacosanol make it become a potential candidate for a supplement in food, beverage, medicine and cosmetics industries.
High solubility, an absolute requirement for octacosanol, plays an important role in its effective functional performance. However, the physicochemical and physiological properties of octacosanol are effective due to its high melting point and low solubility in oil. Thus, the poor solubility of octacosanol becomes its largest obstacle for their application in food and pharmaceutical industries.
There are a number of octacosanol derived esters designed in our study, including octacosyl acetate, butyrate, hexanoate and octanoate. Each of these esters is a molecular chain composed of carbon, hydrogen and oxygen atoms. The main difference between the designed octacosyl esters is the number of carbon atoms of the fatty acid which make up the chain of the esters. For example, octacosyl acetate is composed of two carbon atoms from acetic acid, whereas the octacosyl butyrate is composed of four carbon atoms from butyric acid. Esterified octacosanol is generated in order to improve the solubility performance of octacosanol in oil, which in turn slows the release of the octacosanol from the site where it enters the body. Octacosanol has poor solubility in either oil or water. Once an ester group is added to octacosanol, it becomes even less soluble in water and more soluble in oil. Additionally, the more carbon atoms there are in an ester, the more soluble the ester is in oil.179 For example, the ester with two carbon atoms in the ester group is less soluble in oil than the ester with four carbon atoms in the ester group. Thus, the more carbon atoms the ester group have, the more soluble in oil. The term for this ratio between oil and water solubility is called the partition coefficient-the higher the solubility in oil, the higher the partition coefficient.180
The partition coefficient of the ester is important because it effects how long the compound itself stays in the system. If the octacosanol transfers too quickly to the digestion system, the result is that a large amount of octacosanol can not be digested by the human body. However, octacosyl esters have a high partition coefficient. When it is oral taken into the human body, the ester remains in its esterified form. From there, it will slowly enter the circulation as it is picked up in small quantities by the body. Once the esterified octacosanol is brought into the digestion system, esterase enzymes will cleave off the ester chain in a hydrolization process, thus leaving the octacosanol in its free form to perform its various actions and effects.
Consequently, it is important to develop an effective or efficient method to generate octacosanol derived esters. Among the available methods for modifying octacosanol, both chemical and enzymatic methods were used to synthesize esters by esterification in our study. C2 to C8 fatty acids and octacosanol were used as the starting materials in both methods. In the chemical esterification, DMAP was applied as the chemical catalyst and DCC as an esterification agent, removing water and driving the reaction to completion.139 The second method was the enzyme-mediated esterification which applied an immobilized lipase (Lipozyme TL IM) as biocatalyst.53,140,141
The lipase-catalyzed reactions are superior to conventional chemical methods owing to its mild reaction conditions, high catalytic efficiency and high conversion from the starting materials to the target products. Lipase-catalyzed esterification is one of the most common used methods for modification of natural materials such as fatty acid or vitamin C. Several industrial application of lipase-mediated esterification have been reported, such as the esterification of oleic acid and octanol by immobilized lipase,181 the production of 1-monoglyceride from lipase-catalyzed esterification of glycerol and fatty acid in reverse micelles.182 The increase in consumer demand for nutritious food supply has led to an increased application of natural compounds as the starting materials. Inexpensive, readily available and renewable natural precursors are demanded for nutritious food supply, such as fatty acids which were used as the starting materials to be converted to more highly valued esters in our study. Moreover, the short chain fatty acids used are safety to be oral intake even the products are hydrolyzed by the enzyme in human body.
The objectives of the present study were to synthetize newly octacosanol derived esters by both chemical- catalyzed and enzyme-catalyzed esterification of fatty acid with octacosanol for the first time, since there is no report on the esterification of fatty acid with octacosanol before. Moreover, the two methods were compared qualitatively and quantitatively. Most importantly, the solubility test in edible oil of our designed octacosyl esters showed that fatty acid carbon chain length have effect on the solubility attributes of these esters. The increasing number of carbon atoms of short chain fatty acid in the esterified octacosanol has the positive effect on the increment of the solubility in oil. Moreover, our study showed that octacosyl esters can be hydrolysis by lipase at 37 oC.
Therefore, our esterified modification of octacosanol can be established as a model to enhance the solubility of very long chain fatty alcohols such as heptacosanol, triacontanol, tetratriacontanol. Our present work might provide a substantial basis for the further research on modifying very long chain fatty alcohols by esterification with short chain fatty acid, monocarboxylic acid, dicarboxylic acid or vitamin C and further to improve the solubility. In food industry, our synthetized octacosyl esters could be employed as a nutrition supplement in edible oil to improve the nutrition component of oil.
5.2 Materials and Methods
5.2.1 Materials
Octacosanol (90 %) was purchased from Huzhou sifeng Biochem Co., ltd. Hydrogen chloride solution (2 M) in diethyl ether, DMAP, DCC were purchased from Sigma Aldrich Chemical Company (Singapore) and used directly. The solvents were all of analytical grade and used as received. Lipozyme TL IM was obtained from Novozymes (Bagsværd, Denmark), it is a food-grade silica-granulated lipase from Thermomyces lanuginosus with sn-1, 3-specific selectivity. Palatase 20000L was also obtained from Novozymes (Bagsværd, Denmark), it is a food-grade purified 1, 3-specific lipase from Rhizomucor miehei. Soy bean oil is food-grade edible oil.
Gas chromatography was performed with a GC-MS-QP2010 system (Shimadzu, Japan) equipped with a DB-5ms capillary column (30 m × 0.25 mm × 0.25 µm, Supelco, Woodbridge, USA), a 5975 inert MSD and FID. The injector and detector temperatures were set at 230 oC. The initial oven temperature was set at 50 oC for 5 min, ramped to 230 oC at 5 oC min-1 and then held for 30 min. Injection of 1.0 µL was performed and helium was used as the carrier gas with a flow rate of 1.2 mL min-1. Split mode was applied with a split ratio of 10:1. Proton NMR spectra were recorded on a 300 MHz spectrophotometer with chemical shift values reported in δ units (part per million) relative to tetramethylsilane. TLC was done on precoated 0.2 mm Merck silica gel 60F254 plates. High-resolution mass spectra were obtained using micrOTOF LCMS (Bruker Biospin Pte Ltd, Germany).
5.2.2 Esterification of Octacosanol with Fatty Acid through Chemical Method
Fatty acids (C2, C4, C6, C8 fatty acids) (each is 3.0 mmol), DCC (619.0 mg, 3.0 mmol), and octacosanol (1026.5 mg, 2.5 mmol) were dissolved in DCM (50 mL). To the solution, catalytic amount DMAP (55.0 mg, 0.45 mmol) were added and stirred at 55 oC for 24 hours. N, N-dicyclohexylurea solid formed was filtered off. The remaining filtrate was washed with hydrochloric acid (1 M, 3 × 50 mL), saturated sodium bicarbonate (3 × 50 mL) again with water (3 × 50 mL) and dried over anhydrous sodium sulphate. Solvent was removed under the reduced pressure to give the esters, which were chromatographed over silica gel column using n-hexane-ethyl acetate (30:1, v/v) mixture as an eluent. All these newly synthetic compounds were characterized by 1H NMR and HREI mass spectra. The esterification reaction was carried out in triplicate and the mean and standard deviation yield of each octacosanol derived fatty acid ester was determined.
5.2.3 Esterification of Octacosanol with Fatty Acid through Enzymatic Method
Octacosanol (1026.5 mg, 2.5 mmol), fatty acids (C2, C4, C6, C8 fatty acids) (each is 3.0 mmol) , Lipozyme TL IM (5 % (w/w) of reactants) and n-hexane (50 mL) were added into a 100-ml screw-capped glass bottle, followed by incubation at 55 oC in water bath at 150 rpm shaking speeds for 24 hours. The reaction was stopped by separating the enzyme from the reaction solutions through centrifugation. The solvent of the remaining solution was removed under the reduced pressure to give the crude esters, which were chromatographed over a column of silica gel using hexane-ethyl acetate (30:1, v/v) as an eluent to obtain pure esters what were confirmed by 1H NMR and HREI mass spectra. The esterification reaction was carried out in triplicate and the mean and standard deviation yield of each octacosanol derived fatty acid ester was determined.
5.2.3.1 1H NMR and HREI Mass Spectra Results
Octacosyl acetate, solid, Rf = 0.5 (n-hexane:EA = 25:1/v:v), m.p. 63-65 oC, 1H NMR (300 MHz, CDCl3) δ 4.07, 4.05, 4.02 (t, J = 6.9 Hz, 2H), 2.04 (s, 3H), 1.69-1.62 (m, 2H), 1.28-1.25 (m, 50H), 0.90, 0.88, 0.86 (t, J = 6.0 Hz, 3H). HREI mass spectrum (CI) calcd for C30H60O 2 m/z 453.4666, found 453.4682.
Octacosyl butyrate, solid, Rf = 0.5 (n-hexane:EA = 25:1/v:v), m.p. 62-64 oC, 1H NMR (300 MHz, CDCl3) δ 4.08, 4.06, 4.04 (t, J = 6.6 Hz, 2H), 2.30, 2.28, 2.25 (t, J = 7.5 Hz, 2H), 1.69-1.61 (m, 2H), 1.26-1.25 (m, 52H), 0.95-0.86 (m, 6H). HREI mass spectrum (CI) calcd for C32H64O2 m/z 480.4979, found 481.4997.
Octacosyl hexanoate, solid, Rf = 0.5 (n-hexane:EA = 25:1/v:v), m.p. 62-64 oC, 1H NMR (300 MHz, CDCl3) δ 4.08, 4.05, 4.03 (t, J = 6.6 Hz, 2H), 2.31, 2.29, 2.26 (t, J = 7.5 Hz, 2H), 1.62-1.59 (m, 2H), 1.30-1.25 (m, 56H), 0.92-0.85 (m, 6H). HREI mass spectrum (CI) calcd for C34H68O2 m/z 509.5292, found 509.5307.
Octacosyl octanoate, solid, Rf = 0.5 (n-hexane:EA = 25:1/v:v), m.p. 61-63 oC, 1H NMR (300 MHz, CDCl3) δ 4.08, 4.05, 4.03 (t, J = 6.6 Hz, 2H), 2.31, 2.29, 2.26 (t, J = 7.5 Hz, 2H), 1.62-1.59 (m, 2H), 1.30-1.25 (m, 60H), 0.92-0.85 (m, 6H). HREI mass spectrum (CI) calcd for C36H72O2 m/z 537.5605, found 537.5580.
5.2.4 Solubility Test of Octacosyl Esters in Edible Oil
Solubility of esters was determined in soy bean oil according to the following method. A small quantity of the purified esters was added to a 4 ml glass vial containing 1 ml soy bean oil and shaken. This procedure was continued until some solids were present in the solvent. A 1.5 x 8 mm Teflon-coated stirring bar was added and the vial sealed with a Teflon-lined screwcap. The vial was placed on a magnetic stirrer in a temperature-controlled room. Then to heat the glass vial until solid were not observed. After that, to cool down the vial to room temperature and more esters were added if solid were not observed. Saturation was assumed when solid were observed in the solvent at room temperature, then the vial was stirred for an additional 24 hours. Three determinations were made for each ester in soybean oil.
5.2.5 Stability Evaluation of Octacosyl Esters in Acidic Media and Heat Treatment
5.2.5.1 Acid Stability Test
Esters (10 mg), hydrochloride in diethyl ether (9 mL, with concentration of 10, 0.1, and 0.001 mM respectively) and deionised water (1mL) were added into 100-mL screw-capped glass bottles and kept at room temperature for 24 hours. The top layer of the mixtures (100 μL) were mixed with with internal standard solution (75μL, methyl pentadecanoate, and 2.0 mg mL-1) and diluted to 1.0 mL with n-hexane. The resulting solutions were analyzed by GC-MS and GC-FID. In order to dissolve the esters into solution completely, diethylether was selected as an ideal medium. Moreover, due to a small amount of water in HCl in diethyleter, the acid stability test of esters was conducted in aquesous condition. Consequently, if the esters were decomposed by acid, carboxylic acid and octacosonal can be detected by GC-MS-FID. The acid stability test was carried out in triplicate.
5.2.5.2 Thermal Stability Test
Ester (10 mg) and toluene (10 mL) were added into a 50-mL three necked-flask and stirred at 200 rpm at stipulated temperature for 2 hours at ambient atmosphere or under N2 protection, respectively. The temperatures tested were at 75, 80, 85, 90, 95, 100 and 105 ºC. Samples were collected every two hours for GC-MS analysis. 100 µL of reaction samples obtained were spiked with 75 µL of internal standard solution (methyl pentadecanoate, 2.0 mg mL-1) and diluted to 1.0 mL with toluene. The resultant solution was stored at room temperature until GC-MS-FID analysis. The thermal stability tests were carried out in triplicate.
5.2.6 Hydrolysis of Octacosyl Esters by Lipase
Each octacosyl ester (each is 0.022 mmol) was dispersed in 10 ml hexane in a 50-ml blue screwed bottle. Palatase 20000 L (10 % (w/w) of octacosyl ester) and deionised water (0.1 mg, 0.1 mmol) were added into the solution followed by incubation at 37 °C in a water bath and kept be shaking at 100 rpm for 24 hours. The incubation was terminated by placing the samples on dry ice or in a -80 ºC freezer.
The hydrolysis rate was determined by measuring released free fatty acids by SPME. An integrated automatic HS-SPME sampling system was used for extracting target volatile compounds from the headspace above an incubation sample. One millilitre of incubation sample was added into a SPME vial and incubated with a 85 µm carboxen/polydimethylsiloxane (CAR/PDMS) fibre (Supelco, Bellefonte, PA, USA) at 60ºC (250 rpm, 30 min) for adsorption. The fibre was desorbed at the Agilent (Palo Alto, CA, USA) 6890 N network GC system equipped with DB-FFAP capillary column (60 m × 0.25 mm × 0.25 µm, Agilent, Woodbridge, USA), 5975 inert MSD and FID. The whole HS-SPME-GC-MS/FID system was programmed and controlled by Agilent E.02.00 version MSD ChemStation software. Purified helium was used as the carrier gas with a flow rate of 1.2 mL min-1. Initial oven temperature was set at 50 ºC for 5 min, then ramped to 230 ºC at 5 ºC min-1, and kept at the highest temperature for 40 min. The split mode was splitless. Both injector and detector temperatures were 250 ºC. The free fatty acids released in the incubation sample in were identified through comparing mass spectra with those in the WILEY database and linear retention indices (LRIs) which were calculated based on the retention times of C8-C40 n-alkane hydrocarbon standards (Fluka, Buchs, Switzerland).
The FID peak area of the free fatty acids was used to monitor the hydrolysis of esters during the lipase catalyst hydrolysis reactions. Different reaction times led to the release of different amounts of the fatty acids from the esters. The hydrolysis tests were carried out in triplicate.
5.3 Results and Discussion
5.3.1 Qualitative and Quantitative Analysis of Two Methods to Synthetize Octacosyl Esters
Octacosyl esters can be synthesized with high yield via many chemical methods with low cost. However, for food grade flavouring materials, the residues of chemical reagents used in preparing the esters may disqualify food grade status of the final products if purification is not done thoroughly. To eliminate such a concern, food grade lipase may be a good choice as the formed final products do not need to undergo purification step and can be directly used as flavouring materials. This will overcome the drawback of costly enzyme needed if the conversion of the enzymatic reaction is satisfactory. We compared both methods for generating the octacosyl esters by the difference of the yield (Table 5.1). From the data, there was a significant difference in these two synthetical methods according to the ANOVA test result (p < 0.05). So it can be concluded that the conversion of the enzymatic reaction is better than that of chemical methods, especially the higher yield of the enzymatic method. For chemical method, purification step is essential and it will reduce the yield due to product loss in the step. In addition, chemical synthesis has other limitations such as bulk routes to removal impurities, vigorous reaction condition and prone to form undesired by-products. In comparison, the enzymatic synthesis has great advantage, such as high substrate specificity, high reaction specificity, mild reaction conditions and reduction of waste product formation. Therefore, enzymatic procedure was far superior to the chemical method especially in food processing. In industry, the use of environmentally friendly lipases can alleviates complex downstream processes and thus, leads to reduction of bulk chemical routes in overall operation costs.
Table 5.1 Comparison of yield of octacosanol derived fatty acid esters synthesized by chemical and enzymatic methods.
|Octacosyl esters |Yield (%) |
| |Chemical Method Enzymatic Methoda |
|Octacosyl acetate |70.0 ± 3.5 |82.3 ± 4.1 |
|Octacosyl butyrate |72.0 ± 3.4 |87.4 ± 3.6 |
|Octacosyl hexanoate |75.6 ± 2.0 |86.1 ± 2.9 |
|Octacosyl octanoate |74.3 ± 2.3 |81.0 ± 3.8 |
aIn the enzymatic method, Lipase TL IM was used as the catalyst.
5.3.2 Stability Evaluation of Octacosyl Esters in Acidic Media and Heat Treatment
One potential concern of esters is their stability towards weakly acidic food matrix and against heat during food thermal processing. We treated the esters with different concentration of hydrochloric acids (10, 0.1, 0.001 mM) for 24 hours and analyzed the stability by GC-MS-FID. No new peaks were detected compared to untreated samples. Therefore, we concluded that the esters can resist acid environment fairly well. Moreover, heating these esters in toluene at 105 ºC for 2 hours did not result in any noticeable decomposition from the GC-MS-FID analysis. Therefore, octacosyl esters are acid and thermally stable. It should be pointed out that, in food matrix are typically complicated as it contains many types of molecules including proteins, lipids, and carbohydrates. The stability of the esters in real food matrix would need to be evaluated in real application of them as nutrition supplement.
5.3.3 Solubility Test of Octacosyl Esters in Edible Oil
By using the previous mentioned method, the solubility of octacosanol and its derived esters are shown in Table 5.2. From the results shown in Table 5.2, it can be concluded that the solubility of octacosyl esters was improved up to 6 times that of octacosanol. By comparing from octacosyl acetate to octanoate, the more carbon atoms there were in the ester, the more soluble the ester was in oil. While, by increasing the number of carbon atoms of the fatty acid, the effect on increasing the solubility of esters seems not significant. Thus, in order to continue enhancing the solubility of the esterified octacosanol, it was considered not essential to continue to increase the number of carbon atoms of the fatty acid. Octacosyl octanoate was the optimum compound which owned the highest solubility in edible oil among the four designed esters.
Table 5.2 Solubility of designed octacosyl esters in soy bean oil (mg mL-1) at room temperature.
|Solubility in soy bean oil (mg mL-1) |
|at room temperature |
|Octacosanol |1.72 ± 0.06 |
|Octacosyl acetate |5.88 ± 0.25 |
|Octacosyl butyrate |8.89 ± 0.39 |
|Octacosyl hexanoate |10.32 ± 0.35 |
|Octacosyl octanoate |11.65± 0.41 |
Consequently, our synthetic octacosyl esters could be employed as a nutrition supplement in edible oil in food industry. Furthermore, the esterified modification of octacosanol can be established as a model to enhance the solubility of very long chain fatty alcohols such as heptacosanol, triacontanol and tetratriacontanol. That is to say our present work might provide a substantial basis for the further research on esterified modification on very long chain fatty alcohols. In addition, more work can be done to compare the effect on the solubility of esterified octacosanol which a variety of acids such as short chain fatty acid, monocarboxylic acid, dicarboxylic acid or vitamin C.
5.3.4 Hydrolysis of Octacosyl Esters by Lipase
The hydrolysis result obtained from SPME showed that octacosyl acetate, butyrate, hexanoate and octanoate can be hydrolyzed by lipase at 37ºC. From Figure 5.1 and 5.2, after 24 hours incubation of octacosyl acetate, the ester was hydrolyzed completely by Palatase 20000 L. The similar results shown in Figure 5.3, 5.4 5.5, 5.6, 5.7 and 5.8 also indicated that octacosyl butyrate, hexanoate and octanoate could be hydrolyzed by lipase in the same condition. Meantime, it can be observed that some impurities peaks such as peak 1, 2, 3 in Figure 5.1 which may come from the impurities from the incubation solution (hexane) or the fibre of SPME. Because between each run of SPME, the fibre can not be washed thoroughly, so this will lead to the contamination of the running result. However, due to very small abundance of the impurity peak, this effect can be neglected.
Figure 5.1 Octacosyl acetate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC. Peak 1, 2 and 3, unknown impurities.
Figure 5.2 Octacosyl acetate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak 1 and 2, unknown impurities; peak 3, acetic acid.
Figure 5.3 Octacosyl butyrate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC. Peak 1 and 2, unknown impurities.
Figure 5.4 Octacosyl butyrate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak1, 2 and 3, unknown impurities; peak 4, butyric acid.
Figure 5.5 Octacosyl hexanoate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC. Peak 1 and 2, unknown impurities.
Figure 5.6 Octacosyl hexanoate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak 1, 2 and 3, unknown impurities; peak 4, hexanoic acid.
Figure 5.7 Octacosyl octanoate was hydrolyzed by Palatase 20000 L for 0 hour at 37 ºC.
Figure 5.8 Octacosyl octanoate was hydrolyzed by Palatase 20000 L for 24 hours at 37 ºC. Peak 1, octanoic acid.
However, due to the limitation of SPME, the hydrolysis rate of esters can not be calculated accurately. The main limitation of SPME is the relatively low extraction yield due to the relatively small amount of sorbent available on the syringe needle which means quantitative analysis has not been successful due to the challenges involved with incomplete adsorption.119 Aside of this disadvantage, some artifact formations have also been noticed. Due to the formation of maillard products during the desorption step, artifact was noticed in the flavour analysis of strawberry and apple fruits.120 A significant reduction in artifact formation was obtained by rinsing the fiber with water prior to thermal desorption. However, the formation of artifacts is often unavoidable, and intrinsic artifact formation during the analysis of volatile sulfur compounds 121 in air has been reported. Thus, in the hydrolysis of octacosyl esters study, we could conclude that all these esters can be hydrolyzed by enzyme through 24 hours hydrolization process at 37 ºC.
5.4 Conclusion
Four new octacosanol derived esters were designed and generated by both chemical and enzymatic esterification of fatty acid with octacosanol in our study. In addition, the qualitative and quantitative comparison of the two methods has been made according to the reaction results. By esterification reaction, the solubility of esterified esters was improved significiantly, which is up to 6 times of the solubility of octacosanol. The increasing number of carbon atoms of short chain fatty acid in the esterified octacosanol has the positive effect on the increment of the solubility in oil. However, with the increment of fatty acid carbon chain length, the effect on increasing the solubility of esters seems not significant. Thus, octacosyl octanoate was the optimum compound which owned the highest solubility in edible oil among the four designed esters. And there is no need to continue to react the octacosanol with longer chain fatty acids to improve the solubility. Furthermore, in order to make our designed esters to reach food scale, hydrolysis test was done to prove that octacosyl esters can be hydrolyzed by lipase at 37 oC.
Consequently, four esterified modification of octacosanol in our study can be established as a model to enhance the solubility of very long chain fatty alcohols such as heptacosanol, triacontanol, tetratriacontanol. And our present work may provide a substantial basis for the further research on modifying very long chain fatty alcohols by esterification with short chain fatty acid, monocarboxylic acid, dicarboxylic acid or vitamin C to improve the solubility.
Chapter 6
Future Perspectives
The rapid growth of the flavour and fragrance industry has fueled demand for new generation flavours from synthetic or natural sources. Esters are common flavouring agents in fruit-flavoured products such as beverages, candies, jellies, and jams, baked goods and wines. They are also found in dairy products like cultured butter, sour cream, yogurt, and cheese. Ester extraction from natural sources has therefore become particularly appealing.
However, ester isolation from these sources is often expensive and time consuming. Comparing the chemical method, enzymatic synthesis has better advantage in terms of higher yield. Our information on the Lipozyme TL IM-mediated esterification or transesterificaton was provided as a reference for industry-wide production. Moreover, the general methodology described in our previous research can be applied to generate other chemical derived esters such as 3-methyl-2-buten-1-ol derived esters (see structure in Table 6.1). In addition, a series of esters with unique sensory description can be synthesized. Moreover, the flavour of the dairy product can be improved through the esterified composition of fatty acids at the sn-1 and sn-3 positions of triacylglycerols, such as milk fat.
The high acid and thermal stability of our synthesized methionyl and 2-phenethyl esters meet the food grid quality requirement. Especially, the long chain fatty acid methionyl and 2-phenethyl esters with high threshold, can prolong the duration of their flavouring in food processing. In order to apply these compounds as flavouring substances in the complicated real food matrix, further risk assessment work is needed per requirements from the Hazard Analysis & Critical Control Points (HACCP). These risk assessments include digestion tests such as the hydrolysis of synthesized compounds by human pancreatic lipase, toleration and safety in human body.
In the current trend of sustainable and green movement of food ingredients, simple and cost effective approaches to synthesize flavours are needed from a large scale perspective. The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst. As such, the prevention of waste can be achieved due to the reusability of immoblised enzyme (e.g. Lipozyme TL IM). Moreover, the reagents such as acids and bases can be filtered off, and can be regenerated (if needed) and reused in a subsequent run. Also, in the wide scale production of flavouring products, immobilized enzyme and reagents can be kept stationary while substrates are continuously added and pass through to yield a product that is continuously removed (for example by distillation). Furthermore, the results from chapter 4 provide an optimization methodology (RSM) to improve the conversion.
Lastly, our esterified modification of octacosanol can be established as a model to enhance the solubility of very long chain fatty alcohols such as heptacosanol, triacontanol and tetratriacontanol. Our present work will provide a substantial basis for the further research on modifying very long chain fatty alcohols by esterification with short chain fatty acid, monocarboxylic acid, dicarboxylic acid or vitamin C to improve the solubility.
Table 6.1 Alcohols employed in the further study and their sensory descriptions.a
|Alcohol |Chemical Structure |Sensory Description |
|3-Methyl-2-buten-1-ol |[pic] |Fresh, fruity, green, lavender |
|Geraniol |[pic] |Fresh, fruity, green, lavender |
|3-Heptanol |[pic] |Sweet, floral, rose, fruity |
|1-Carveol |[pic] |Herbaceous |
|1R-Myrtenol |[pic] |Caraway, spearmint |
|3-Methyl-1-pentanol |[pic] |Camphoraceous, minty |
|Farnesol |[pic] |Pungent ,wine-like, cocoa |
|Benzyl alcohol |[pic] |Delicate ,floral, oily |
|1-Propanol Alcohol |[pic] |Sharp, burning taste |
|3-Hexanol |[pic] |sweet |
|Trans-2-Hexen-1-ol |[pic] |Alcoholic ,ethereal, medicinal |
|Dl-Menthol |[pic] |Leafy, green, wine-like, fruity |
|Fenchyl alcohol |[pic] |Minty, woody |
|Para-anisyl alcohol |[pic] |Lemon |
|Vanillyl alcohol |[pic] |Floral, mild, sweet |
|Cinnamyl alcohol |[pic] |Balsamic, sweet |
|Cis-3-Hexen-1-ol |[pic] |Sweet, balsamic, hyacinth |
|Nonyl alcohol |[pic] |Fresh, green grass |
|3,7-Dimethyl-1-octanol |[pic] |Rose, citrus |
|α-Methylbenzyl alcohol |[pic] |Sweet, rose |
|3-Phenyl-1-propanol |[pic] |Mild hyacinth |
|2-Ethyl-1-hexanol |[pic] |Sweet, balsamic, floral |
|1-Octanol |[pic] |Mild, oily, sweet, slight rose |
|S-Perillyl alcohol |[pic] |Sharp, fatty, waxy, citrus |
|Amyl alcohol |[pic] |Green, pungent, fatty |
|2-Octanol |[pic] |Strong, somewhat sweet, balsamic |
|1-Penten-3-ol |[pic] |Fatty, oily, earthy |
|Furfuryl alcohol |[pic] |Butter, mild green |
aAll alcohols have an associated FEMA number (Flavour and Extract Manufacturers’ Association of the United States) and are commercially available in food-grade.183
Appendix Table Ethics, gender and age of the sensory panelists.
|Initials |Ethics |Gender |Age |
|AL |Chinese |Female |33 |
|JY |Japanese |Female |35 |
|YO |Chinese |Female |26 |
|QO |American |Male |45 |
|JO |Chinese |Female |28 |
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List of Publications and Conference
Peer-viewed Papers and Conference:
1. Li, C.; Sun, J. C.; Fu, C. L.; Liu, S-Q; Huang, D. J.; Li, T. H., The Effect of Fatty Acid Carbon Chain Length on the Flavour Attributes of Methionol Esters. Food Chem 2014, 145, 796-801.
2. Li, C.; Sun, J. C.; Liu, S-Q; Huang, D. J.; Li, T. H., Chemical and enzymatic synthesis of a library of 2-phenethyl esters and their sensory attributes. Food Chem 2014, 154, 205-210.
3. Li, C.; Sun, J. C.; Liu, S-Q*; Huang, D. J.; Li, T. H., Biocatalytic Conversion of Butter Oil to Natural Flavour Esters Optimized with Response Surface Methodology. Submitted.
4. Li, C.; Huang, D. J. *; Li, T. H., Design and Synthesis Octacosanol Derived Fatty Acid Esters to Improve the Solubility of Octacosanol. Submitted.
5. Li, C. *; Chin, K. F.; Li, T. H.; Zeng, H. Q.,To Study the Influential Factors on the Structure of Bulged DNA by Electrophoresis Method. Submitted.
6. Li, D. W.; Yang, Z. Q.; Zhao, G. J.; Long, Y.; Lv, B.; Li, C.; Hiew, S.; Ng, M. T. T.; Guo, J. J.; Tan, H.; Zhang, H.; Li, T. H. *, Manipulating DNA writhe through varying DNA sequences. Chem Commun 2011, 47 (26), 7479-7481.
7. Li, C.; Li, T. H. *, Study on chemical and enzymatic synthesis, sensory attributes and optimized conversion of a library of 2-phenethyl esters. The SIFST student symposium 2013 organized by Singapore Institute of Food Science&Technology. In conjunction with 13th ASEAN Food Conference, 8th June 2013. (Oral presentation)
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Time (min)
0
-3
10
20
30
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50
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70
mVolts
1/5.56
2/14.07
3/19.05
4/23.63
5/26.13
6/27.83
7/31.67
8/35.22
9/39.18
10/44.53
11/45.54
10
20
30
40
50
Time (min)
0
50
100
150
200
1/9.071
2/17.583
3/22.298
4/26.181
5/26.635
6/30.602
7/34.259
8/37.896
9/43.514
10/53.237
[pic]
250
mVolts
10
20
30
40
50
Time (Min)
0
25
50
75
125
mVolts
1/9.005
2/17.517
3/22.213
4/26.160
5/26.545
6/30.530
7/34.192
8/37.863
9/43.621
10/51.612
11/53.135
100
15.00
16.00
17.00
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Abundance
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23.00
16.00
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1000000
1200000
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2600000
Time
(min)
Abundance
1
2
3
2800000
17.00
18.00
19.00
20.00
21.00
22.00
23.00
24.00
25.00
26.00
27.00
28.00
200000
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600000
800000
1000000
1200000
1400000
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2800000
Time (min)
Abundance
1
2
18.00
19.00
20.00
21.00
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
200000
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600000
800000
1000000
1200000
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Time (min)
Abundance
1
2
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4
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
200000
400000
600000
800000
1000000
1200000
1400000
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1800000
2000000
2200000
2400000
2600000
2800000
Time (min)
Abundance
1
2
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
31.00
32.00
33.00
34.00
200000
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600000
800000
1000000
1200000
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2800000
Time (min)
Abundance
1
2
3
4
31.00
31.50
32.00
32.50
33.00
33.50
34.00
34.50
35.00
35.50
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
Time (min)
Abundance
36.00
31.00
31.50
32.00
32.50
33.00
33.50
3
4.00
34.50
35.00
35.50
36.00
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
Time (min)
Abundance
1
37.00
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