PHASE BEHAVIOUR AND MODELING OF LOW AND HIGH-SOLID BIOPOLYMER MIXTURES: A TREATISE PREETI SHRINIVAS (B.Tech, U.I.C.T.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS_______________________________ __ Successful completion of this thesis would not have been possible without the research scholarship offered to me by NUS through the Food Science and Technology Programme. I therefore take this opportunity to express heartfelt gratitude and appreciation to have had this privilege. I am greatly indebted my guide and mentor, Prof. Stefan Kasapis whose constant and immense guidance, support and encouragement has been pivotal. I consider myself extremely fortunate to have had you as my supervisor and thank you for helping me endure patiently and sail through a difficult yet worthwhile and memorable journey! I am extremely grateful to my current supervisor Prof. Liu Shao Quan for taking me on as his student and assisting me through the final year. I would also like to extend my gratitude to the other faculty members and staff of the Food Science department for their valuable inputs and suggestions. In particular, I am grateful to Ms. Lee Chooi Lan, Ms. Huey Lee and Rahman. I would also like to take this opportunity to thank Mr. William Lee and Mr. Derrick for their technical assistance, Mr. Abel Gaspar Rosas for his constant encouragement and the staff at the Dept. of Chemistry, Dept. of Biological Sciences and IMRE, for permitting usage of their facilities. I owe a big thank you to Limei, Cynthia and Denyse for whole heartedly participating in this project, as also to my friends and fellow students at FST. ii I would next like to thank all those people whom I love immensely but haven’t expressed it often enoughMom and Dad, the very reason I have reached this far in life! Sujit, Sunil, Praveen, Rashmi and Archana- my anchors and pillars of strength. Jatin- through your encouragement I began this journey and in many ways you are the reason for its completion. My grandparents, uncles, aunts, cousins and well wishers whom I have not mentioned by name here. Dr. Ramamoorthy, Dr. Rao, Archana S. for being there when I needed you the most. My friends- old and new, Jiang Bin, Lilia, Shen Siung, Jorry, Mya, Neha, Sumantra, Tanmay and all those who have made Singapore home for me. Jayanth- for being extremely kind, understanding and supportive. Dinesh- for blessing me and being with me. Sadhguru- whom I deeply revere. And finally, God- for showing me who He really is. iii TABLE OF CONTENTS__ _____________________________ ACKNOWLEDGEMENTS Page ii SUMMARY vii LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xiii LIST OF PRESENTATIONS AND PUBLICATIONS xiv PREFACE xv PART I MECHANICAL PROPERTIES AND PHASE MODEL INTERPRETATION OF A COMPOSITE SYSTEM COMPRISING GELATIN, AGAROSE AND A LIPID PHASE CHAPTER 1: INTRODUCTION 1.1 Gelatin 1.2 Agarose 1.3 Biopolymer Mixtures 1.4 Phase Separation in Biopolymer Mixtures 1.5 Polymer Blending Laws of Takayanagi 11 1.6 Davies Law of Bicontinuity 15 iv CHAPTER 2: THEORY 2.1 Rheology 18 2.2 Differential Scanning Calorimetry 21 2.3 Scanning Electron Microscopy 22 CHAPTER 3: EXPERIMENTAL SECTION 3.1 Materials 23 3.2 Methods 25 CHAPTER 4: RESULTS AND DISCUSSION 4.1 Experimental Observations on Single Preparations of Gelatin, 28 Agarose and Lipids used 4.2 Experimental Observations on Mixed Systems of Gelatin, Agarose 36 and a Lipid Phase 4.3 Quantitative Analysis of Mechanical Functions in Support of the 48 Phase Topology of the Agarose/Gelatin/Lipid Mixture CHAPTER 5: CONCLUSIONS 58 CHAPTER 6: SUGGESTIONS FOR FUTURE WORK 59 REFERENCES 60 v PART II EFFECT OF CO-SOLUTE (GLUCOSE SYRUP) ON THE STRUCTURAL BEHAVIOUR OF AMYLOSE GELS CHAPTER 1: INTRODUCTION 68 1.1 Amylose 69 1.2 Glass Transition in High Solid Systems 71 CHAPTER 2: EXPERIMENTAL SECTION 2.1 Materials 78 2.2 Methods 79 CHAPTER 3: RESULTS AND DISCUSSION 3.1 Qualitative Aspects of the Effect of Increasing Levels of Co-Solute 82 on the Structural Properties of Amylose 3.2 Amylose Diverging from the Paradigm of Coil-to-Helix 86 Polysaccharides in a High Solids Environment 3.3 Utilization of the Method of Reduced Variables to Quantify the 94 Viscoelasticity of Amylose-Sugar Mixtures during Vitrification CHAPTER 4: CONCLUSIONS & FUTURE TRENDS 103 REFERENCES 105 vi SUMMARY: The first part of this thesis attempts at examining the structural properties of binary and tertiary mixtures made of the cold-setting biopolymers agarose and gelatin, and a lipid phase with solid or liquid-like viscoelasticity. The working protocol included the techniques of small-deformation dynamic oscillation on shear, modulated differential scanning calorimetry and scanning electron microscopy, and theoretical modeling that adapted ideas of relating morphology to elastic modulus of synthetic polyblends and block polymers. The experimental setting was designed to encourage extensive phase separation in the binary gel of agarose and gelatin whose mechanical properties were rationalized on the basis of a bicontinuous blending-law. The presence of two continuous phases allowed the slower-gelling component (gelatin) to exhibit favourable relative affinity for solvent with increasing concentrations of the protein in the system. This is an unexpected outcome that contradicts the central finding of a single value of the “p-factor” observed in the distribution of solvent between the continuous matrix and discontinuous inclusions of de-swelled binary gels reported earlier in the literature. Incorporation of a lipid phase of effectively zero elastic modulus or in excess of 108 Pa in the composite aqueous gel weakens or reinforces the matrix accordingly. The elastic moduli and morphology of the tertiary blend were related to changing the relative phase volumes of components using analytical expressions of isotropically dispersed soft or rigid filler particles in a polymeric matrix. The second half of the thesis presents data concerning the structural behavior of amylose in the presence of glucose syrup and a possible interpretation of the same. Observations were obtained once again by the aforementioned experimental methods of vii small-deformation dynamic oscillation on shear, modulated differential scanning calorimetry and scanning electron microscopy. In contrast to industrial polysaccharides that undergo readily a coil-to-helix transition (e.g., agarose, deacylated gellan and κcarrageenan), amylose holds its structural characteristics unaltered at low and intermediate levels of glucose syrup. This is followed by an early phase inversion from polysaccharide to co-solute dominated system at levels of solids above 70.0%, whereas industrial polysaccharides can dictate kinetics of vitrification at levels of solids as high as 90.0% in the formulation. Additional viscoelastic “anomalies” include a clear breakdown of thermorheological simplicity with data exhibiting two tan δ peaks in the passage from the softening dispersion to the glassy state. Besides phenomenological evidence, mechanistic modeling using the combined framework of the free volume / reaction rate theories argue for two distinct glass transition temperatures in the mixture. It is proposed that the amylose / glucose syrup / water system does not reach a state of molecular mixing, with the morphological features being those of a micro phase-separated material. viii LIST OF TABLES PART I Table 3.1 Composition of Soybean Oil 24 Table 3.2 Composition of Hydrogentated Vegetable Fat 24 ix LIST OF FIGURES PART I Figure 1.1 Structure of agarose Figure 1.2 Schematic representations of ideal rubber, gelatin and agarose Figure 1.3 Changes in calculated modulus as a function of SX Figure 4.1 Storage and loss modulus variation as a function of temperature and time of observation for gelatin and agarose on cooling to 0oC 29-30 Figure 4.2 Storage and loss modulus variation as a function of temperature and time of observation for gelatin and agarose on cooling to 25oC 31 Figure 4.3 Calibration curves of storage modulus as a function of polymer concentration for agarose and gelatin at and 25°C 33 Figure 4.4 Storage and loss modulus variation as a function of temperature and time of observation for Dalda Vanaspati lipid 35 Figure 4.5 Complex viscosity variation as a function of shear rate for soybean oil at 25oC 36 Figure 4.6 Heating profiles of storage and loss modulus for mixtures of gelatin, agarose and a lipid 38 Figure 4.7 DSC exotherms for single, binary and tertiary mixtures 40 Figure 4.8 Scanning electron microscopy images of binary and tertiary mixtures 42 Figure 4.9 Master curves of experimental storage modulus data obtained at 0oC and 25°C for single and binary mixtures 44 Figure 4.10 Experimental storage modulus data obtained as a function of lipid concentration in tertiary mixtures at 0oC and 25°C 47 Figure 4.11 a) Modeling the phase topology of the agarose/gelatin gel at 25°C using the isostrain and isostress blending laws for a binary sample comprising 1% agarose plus 10% gelatin 51 15 x 10.0 9.0 Log (G' / Pa) 8.0 7.0 6.0 5.0 4.0 3.0 2.0 -1.0 a -0.5 0.0 0.5 1.0 1.5 2.0 Log (frequency / rad s-1) 10.0 9.0 Log (G" / Pa) 8.0 7.0 6.0 5.0 4.0 3.0 b 2.0 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 Log (frequency / rad s-1) Fig. 3.8. Frequency variation of a) G' and b) G" for 2.0% amylose plus 70.0% glucose syrup. Bottom curve is taken at 0°C ( ); other curves successively upwards – 8°C ( ), 16°C ( ), - 20°C ( ), - 24°C ( ), - 28°C ( ), - 32°C ( ), - 36°C ( ) and – 40°C (―). Data at – 4°C, - 12°C, - 44°C, - 48°C, - 52°C, - 56°C and – 60°C have not been plotted to avoid clutter. 97 10 Log (G'p, G"p / Pa) G"p G'p -3 -1 11 -1 Log (ωa T / rad s ) Fig. 3.9. Master curve of reduced shear moduli (G'p and G"p) for the sample of 2.0% amylose plus 70.0% glucose syrup as a function of reduced frequency of oscillation (ωaT) based on the frequency sweeps of Figure 3.8 (for plotting purposes the reference temperature of - 4°C was used). Characterization of molecular mechanisms focused on three distinct domains in Figure 3.7 and the isothermal viscoelastic data at different temperatures in Figures 3.8a and 3.8b were utilized to construct composite curves. Data were processed choosing arbitrarily a point as the reference temperature (To) within each domain, and shifting the remaining spectra along the log frequency axis until a uniform curve was obtained. Thus, 98 temperature essentially plays the role of changing the time or frequency scale and assumes that a change in temperature within each domain shifts the time or frequency scale of all molecular mechanisms by the same amount. The reduced composite curves put together in the three temperature domains were further displaced horizontally for the single purpose of the pictorial representation in Figure 3.9. This depicts a master curve of viscoelasticity for an ultra-extensive range of oscillatory frequencies of about ten orders of magnitude. There is also a combined increase in the values of reduced shear modulus (G'p and G"p) of eight decades from 102 to ≈ 1010 Pa. The method of reduced variables employed in the construction of composite curves that correspond to the distinct temperature ranges in Figure 3.7 yields three sets of shift factors (aT) that can be treated with the popular concept of free volume in the form of the Williams, Landel and Ferry (WLF) equation:53 log aT = - (B/2.303fo)(T - To) (fo/α f ) + T - To (1) where, fo is the fractional increase in free volume at To, αf is the thermal expansion coefficient, and the value of B is set to be about one. The literature also defines the WLF parameters C1o and C 2o as the ratios of B/2.303fo and fo/αf, respectively. The WLF equation holds for any temperature within the glass transition region including Tg, a result which leads to the following relation:12 C1o = C1g C g2 /(C g2 + To - Tg) C o2 = C g2 + To - Tg (2) Thus the fractional free volume (fg) and the thermal expansion coefficient (αf) at Tg or any other temperature within the glass transition region can be calculated. 99 An alternative approach to the theory of free volume is the predictions of the reaction rate theory that can be formulated in terms of the modified Arrhenius equation:54 log aT = Εa 2.303R (1− Τ ) To (3) where, R is the gas constant. This mathematical expression follows the progress in shift factors by integrating two sets of temperature data. It returns a constant activation energy (Ea), which argues that relaxation processes in the glassy state are heavily controlled by specific chemical features. Figure 3.10 reproduces the outcome of the application of this school of thought (equations to 3) to the viscoelastic functions of the amylose / glucose syrup mixture. Three distinct patterns of structural relaxation have been featuring prominently, as documented in the factor aT for the horizontal superposition of mechanical spectra in Figures 3.8a and 3.8b. The high temperature fit has been taken by considering a reference temperature of – 4.0°C and the WLF equation. This exponential function of the reciprocal fractional free volume yields fg = 0.048, αf = 9.7 x 10-4 deg and Tg1 = - 14.0°C. Molecular dynamics are characterized by distinct kinetic rates in the second part of the experimental temperature regime, which, however, follow well the predictions of the combined WLF/free volume scheme; To = - 24.0°C, fg = 0.035, αf = 7.1 x 10-4 deg and Tg2 = - 32.0°C. It appears that in both cases, free volume is the molecular mechanism dictating diffusional mobility in the system. It should be noted that the lower temperature transition (Tg2) has a smaller value of fractional free volume (fg) and forms the more dense glass. In conjunction with data in Figure 3.3, which argue against a molecular state 100 of mixing between amylose and glucose syrup sequences, the two glass transition temperatures should be attributed to the morphology of a micro phase-separated material. The implication is that the polymer vitrifies first followed by the degree of “order” frozen into the glass structure formed by the co-solute. Arrhenius fit glucose syrup WLF fit Log a T amylose WLF fit glucose syrup T g amylose T g -1 -55 -45 -35 -25 -15 -5 Temperature (°C) Fig. 3.10. Temperature variation of the factor aT within the glass transition region of amylose ( ), the glass transition region of glucose syrup ( ) and the glassy state ( ) of 2.0% amylose plus 70.0% glucose syrup, with the solid lines reflecting the WLF and modified Arrhenius fits of the shift factors throughout the vitrification regime (dashed lines pinpoint the Tg predictions for amylose and glucose syrup). 101 At even lower temperatures, there is a clear change in the pattern of shift-factor development towards a linear behaviour that cannot be followed by the WLF equation. In here, the contribution of the mechanism of free volume is minimal and instead the modified Arrhenius equation offers a viable alternative to describing molecular dynamics. The latter yields a fixed value of the activation energy (650 kJ/mol) required to overcome an energetic barrier for local rearrangements to occur from one state to another. Such formalism assumes that the configurational entropy is essentially constant within the glassy state.55 The present value of activation energy is higher than estimates obtained by fitting the shift factor of amorphous synthetic polymers, which have been found to be in the order of 200 kJ/mol, for example, in the vitrification of poly(ethylene terephthalate).56 This discrepancy should be attributed to the high density of the bio-glass owing to the stiffness of the polysaccharide chain and its ability to form compact double helices. 102 CHAPTER 4: CONCLUSIONS & FUTURE TRENDS Amylose gels are rather different than those obtained from other polysaccharides. Industrial polysaccharides (e.g., agarose, deacylated gellan and κ-carrageenan), undergo readily a coil-to-helix transition as a result of which a drastic drop in their mechanical strength is observed in the presence of intermediate levels of co-solute. This has been interpreted as a transformation from a highly enthalpic, aggregated structure to a network of reduced cross-linking. Amylose, on the other hand holds its structural characteristics unaltered at intermediate levels of glucose syrup following which is observed an early phase inversion from polysaccharide to co-solute dominated system at levels of solids above 70.0%. In the case of industrial polysaccharides however, kinetics of vitrification are dictated upto levels of solids as high as 90.0% in the formulation. Additionally, viscoelastic “anomalies” in the form of occurrence of two tan δ peaks in the passage from the softening dispersion to the glassy state indicate a clear breakdown of thermorheological simplicity. Further, mechanistic modeling argues for two distinct glass transition temperatures in the mixture. It is therefore proposed that the amylose / glucose syrup / water system does not reach a state of molecular mixing, with the morphological features being those of a micro phase-separated material. Gelatin replacement in foodstuffs has always been the key incentive to understand the behaviour of polysaccharides in high sugar environments. With the use of gelatin increasingly falling out of fashion with consumers and producers alike, polysaccharides are invariably looked upon as alternatives. The behavioral response of gelatin in a high solids environment has been shown to be rather different from that of polysaccharides, 103 with sugar promoting chain association rather than inhibiting it. This rather unique behavioral response of gelatin in a high solids environment as compared to that of other polysaccharides has been speculated as one of the reasons behind difficulties encountered in its successful commercial replacement. Studying the behavioral pattern of amylose in further detail based on evidence from this piece of work could provide a much needed breakthrough as it appears to exhibit properties intermediary to those revealed by gelatin and other polysaccharides. Findings would prove vital for applications in the confectionery industry, and others such as flavour encapsulation, preservation/innovation of glassy or rubbery foodstuffs, and preservation of bioactive molecules in glassy carbohydrate matrices. In addition to the experimental techniques used in the current study, X-ray diffraction studies could help ascertain the presence or absence and thereby the role of crystallinity within amylose-glucose gels. In conclusion, it is hoped that the working combination of phenomenological and fundamental tools offers insights into the structural properties and phase topology of the amylose / co-solute mixture. 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(2004). Departure from the Vogel behaviour in the glass transition—thermally stimulated recovery, creep and dynamic mechanical analysis studies. Polymer, 45, 10071017. 56. Alves, N.M.; Mano, J.F.; Gomez Ribelles, J.L. (2002). Molecular mobility in polymers studied with thermally stimulated recovery. II. Study of the glass 111 transition of a semicrystalline PET and comparison with DSC and DMA results. Polymer, 43, 3627-3633. 112 [...]... Singapore, (June ’08) Awarded Best Poster 4 Shrinivas P., Kasapis S and Tongdang T (2009) “Morphology and Mechanical Properties of Bicontinuous Gels of Agarose and Gelatin and the Effect of Added Lipid Phase Langmuir, 25 (15), 8763-8773 5 Shrinivas P and Kasapis S (2010) “Unexpected Phase Behaviour of Amylose in a High Solids Environment” Biomacromolecules, 11 (2), 421-429 6 Kasapis S and Shrinivas... Gelatin, a protein; agarose and amylose, both of which are polysaccharides In addition, the properties of and roles played by sugars and lipids in composite biopolymer systems will be discussed The first part of this thesis will deal with a low solids biopolymer system comprising primarily of gelatin, agarose and a lipid An attempt is made thereof to address issues stemming from the phenomena of phase. .. AGAROSE AND A LIPID PHASE CHAPTER 1: INTRODUCTION The phenomenon of gel formation by biopolymers such as proteins and polysaccharides is widely known and has been a subject of interest to many academicians and scientists in the last few decades Structural manipulation of products using gelling biopolymers to obtain varieties of textures and profiles is commonly practiced in the food, beverage and pharmaceutical... (gelatin in this case), occurs at a specific mixture composition.5 1.4 Phase Separation in biopolymer mixtures The phenomenon of phase separation as mentioned earlier is a common feature in biopolymer mixtures It remains to be one of the basic tools of achieving the required structural properties in a variety of industrial products In terms of mechanistic understanding of phase behaviour, an early advance... hysterisis, i.e a difference/lag between gelling and melting temperatures, another important feature of agarose gels The gel hysteresis of agar greatly exceeds that of other gelling agents and is the basis for many of its applications in food and biotechnology In the food industry agarose is useful in applications such as low calorie foods It is also used as a gelling agent Agarose gels are also used for... excessive amounts of one polymer) ‘freezes’ the system and prevents diffusion of water between the two phases.20 The antagonistic effect operating between ordering-aggregation leading to gelation that arrests phase separation and the thermodynamic drive to extensive phase separation can be manipulated by the cooling regime Slow-cooled materials exhibit early phase separation and gel reinforcement in the way... ingredient as it can be used as a gelling agent, whipping agent, stabilizer, emulsifier, thickener, adhesive, binder or fining agent 5 1.2 Agarose: Fig 1.1 Structure of Agarose (Source: Ref 5) Agarose, the gelling component of Agar, is a neutral polysaccharide obtained from a family of red seaweeds (Rhodophyceae) As opposed to agaropectin, the charged polysaccharide fraction of agar, the sulphur content of agarose... Shrinivas P., Chong L-M., Tongdang T and Kasapis S “Structural Properties and Phase Model Interpretation of the Tertiary System Comprising Gelatin, Agarose and Lipids Part I: Inclusion of the Oil Phase Poster presentation at the 8th International Hydrocolloids Conference held in Trondheim, Norway, (June ’06) 2 Shrinivas P., Tongdang T and Kasapis S “Structural Properties and Phase Model Interpretation of. .. ordering and gelation are parameters that have a direct influence on the microstructure and rheology of biopolymer composites as well as the likelihood of phase separation.6 Numerous mixed systems of proteins and polysaccharides have been examined for their rheological properties and applications to food products Pioneering research to this effect was carried out using a gelatin-agarose model composite As... elongated, or irregularly shaped particles formed at high shear stresses, an outcome that may find application in the diffusional mobility of bioactive compounds within a polymeric matrix.23 1.5 Polymer Blending Laws of Takayanagi11,15-19 A combination of two simple viscoelastic models (parallel and series) in proportion to their phase volumes helps prediction of overall viscoelastic properties Takayanagi, . polymer concentration for agarose and gelatin at 0 and 25°C 33 Figure 4.4 Storage and loss modulus variation as a function of temperature and time of observation for Dalda Vanaspati lipid . vii SUMMARY: The first part of this thesis attempts at examining the structural properties of binary and tertiary mixtures made of the cold-setting biopolymers agarose and gelatin, and a lipid phase. Mixed Systems of Gelatin, Agarose 36 and a Lipid Phase 4.3 Quantitative Analysis of Mechanical Functions in Support of the 48 Phase Topology of the Agarose/Gelatin/Lipid Mixture CHAPTER 5: CONCLUSIONS