RHEOLOGlCAL PROPERTIES OF GELATlNlSTARCH
COMPOSITE GELS
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
MICHAEL D. H. ROGERS
In partial fulfilment of requirements
for the degree of
Master of Science
April, 2001
O Michael D. H. Rogers, 2001 National tibraty Bibliothèque nationale 1+1 ,,,da du Canada Acquisitions and Acquisitions et 8ibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON KiA ON4 Ottawa ON KiA ON4 Canada Canada
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RHEOLOGICAL PROPERTIES OF GELATIN/STARCH COMPOSITE GELS
Michael D. H. Rogers Advisors: University of Guelph, 2001 Dr. Y. Kakuda and Dr. I.J. Britt
Rheological properties of gelatin, gelatin/regular and gelatinlwaxy corn starch
composite gels were investigated using oscillatory shear, constant rate uniaxial and
dynamic compression methods to study the sirnilarities between the moduli to
interpref the protein-polysaccharide interactions within each system. Composite gelatinktarch gels were tested at a 5% (wfw) gelatin concentration and 0, 1, 3 and
5% (wfw) starch concentration. Scanning electron microscopy, volume fraction and particle sizing experiments further support the results of the rheological tests and help define the protein-polysaccharide interactions.
In gelatinfregular starch composite gels, Grand E increased linearly, thus the rigidity of the gels increased with increased volume fraction as a result of the synergistic effect of a secondary amylose gel. This indicated that regular corn starch behaved as an active filler particle. The results for Gr and E , for gelatin/waxy starch composite gels, dernonstrated a minimal change in rigidity over the concentration range of the waxy corn starch since little response was observed from either test method. Both rheological tests generated similar conclusions about the structure of gelatinkegular and gelatidwaxy corn starch composite gels. To the late Drs. Mawin A. Tung and Chester D. Myers 1 would Iike to express my gratitude to Dr. lan J. Britt and to the other mernbers of the Food Packaging Research Group for their support and encouragement throughout my research project. I would also Iike to thank the other members of rny cornmittee: Drs. Yukio Kakuda, Donald Mercer, and Allan Paulson for their valued input.
Furthermore, I want to thank Sylvia Yada and Susan Tosh for their advice, support and enlightening points of view and assistance durhg the writing of this thesis.
Finally, special thanks to my parents Sharon and John Rogers and to Patricia Bell who each supported and encouraged me through the course of my stud ies.
This research was made possible throug h research funding provided by the Natural Sciences and Engineering Research Council of Canada and the NSERClGeorge Weston Industrial Research Chair in Food Packaging Technology. TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... i .. TABLE OF CONTENTS...... 11 LIST OF TABLES ...... iv LIST OF FIGURES...... v
1. INTRODUCTION...... 1
2. LITERATURE REVIEW...... 3 2.1 Hydrocolloids in Food...... 3 2.11.1 Gelatin...... 5 2.1.2 Starch...... 7 2.1.3 Composite Polymer Gels...... 9 2.2 Gel Microstructure...... 14 2.2.1 Scanning Electron Microscopy ...... 14 2.2.2 Particle Sizing ...... 16 2.3 Rheology of Food Polymer Gels...... 16 2.3.1 Small Amplitude Oscillatory Shear Testing...... 18 2.3.2 Constant Rate Uniaxial Testing ...... 22 2.3.3 Dynamic Compression Testing ...... 25 2.3.3.1 Elastic Behaviour...... 26 2.3.3.2 Viscous Behaviour...... 27 2.3.3.3 Viscoelastic Behaviour...... 28 2.4 Gel-Sol Transition and Gelation ...... 29
3 . OBJECTIVES ...... 34 4 . MATERIALS AND METHODS...... 35 4.1 Gelatin and Starch Preparations ...... 35 4.2 CryoScanning Electron Microscopy ...... 36 4.3 Particle Size Determination...... 37 4.4 Mechanical Testing...... 37 4.4.1 Small Amplitude Oscillatory Shear Testing ...... 37 4.4.2 Constant Rate Uniaxial Testing...... 39 4.4.3 Dynamic Compression Testing...... 40 4.5 Volume Fraction Determination ...... 45 4.6 Gel-Sol Transition of GelatinIStarch Composite Gels ...... 46 4.6.1 Differential Scanning Calorimetry (DSC) ...... 46 4.6.2 Gr - G r f Crossover...... 47 RESULTS AND DISCUSSION...... 48 5.1 Gel Structure...... 48 5.2 Volume Fraction...... 51' 5.3 Mechanical Properties of GelatinlStarch Gels ...... 55 5.3.1 Small Amplitude Oscillatory Shear Testing...... 56 5.3.2 Constant Rate Uniaxial Testing...... 64 5.3.3 Comparison of Results ...... 66 5.3.4 Dynamic Compression Testing...... 71 5.4 Gel-Sol Transition ...... 72 CONCLUSIONS ...... 79 REFERENCES...... 82 APPENDIX...... 89
iii LIST OF TABLES
Table Page
5.1 A comparison of typical dynamic shear storage moduli ( Gr) obtained from small amplitude oscillatory rheometry (3 rads-', 22.5 OC)to theoretical shear moduli ( G ) for various gelatin and gelatin/starch composite gels ...... 67
5.2 A comparison of phase angles and maximum force values obtained from three ideal mechanical models (0.1 Hz, room temperature)...... 71
5.3 Gel-sol transition temperatures for both gelatin and gelatinfstarch composite gels obtained from the G' - G" cross-over through small amplitude oscillatory rheometry (3 radd, 3200 pNm, 0.5 Co-min-')...... 76 LIST OF FIGURES
Figures Page
Structural diagrams of the principal amino and irnino acids in gelatin...... 5
Glucose monomers connected by a-1,4 linkages to form a linear amylose polymer...... 0ww.....8
Glucose monomers Iinked by a-1,4 and a-1 ,6 linkages to form a branched chain-. arnyiopectin polymer. Branch points occur via a-i,6glucosidic bonds...... -8
Mechanical models illustrating: A. Elastic Bodies; B Viscous Bodies; and C. Viscoelastic Bodies (Kelvin Model)...... -26
The conformational changes involved in the network formation of random coi1 gelatin chains (adapted from Johnston-Banks, i990) ...... 31
Model apparatus used to represent a dashpot for experimental analysis of an ideai viscous rnaterial (Newtonian Fluid)...... 43
Model apparatus used to represent a dashpot and a spring in parallel (a Kelvin Body) for experimental analysis of an ideal viscoelastic material (Newtonian Fluid)...... d
Cryo-Scanning Electron Micrographs (10-1 5 kV, 5000 x magnification) of gelatin, starch, and gelatinlstarch composite gels (A) 5% (w/w) Gelatin, (B) 5% (wlw) Regular Corn Starch, (C) 5% (wlw) Waxy Corn Starch, (D) 5% (wlw) Gelatinl5% (wlw) Regular Corn Starch, E) 5% (w/w) Gelatin/5% (wlw) Waxy Corn Starch. All samples were frozen in ... Iiquid nitrogen slush...... -49
Standard curve of storage modulus, G' (3 rad s", 3200 pNm, 22.5"C) versus gelatin concentration (c) for determination of the gelatin fraction concentration in a gelatinlcorn starch aqueous dispersion (Standard Error= 0.034) ...... w.....52 Relationship between volume fraction data for regular corn starch in 5% gelatin composite gels (225°C) detemined by srnall amplitude oscillatory rheometry. (Standard Error = 0.035) ...... -...... *53
Relationship between volume fraction data for waxy corn starch in 5% gelatin composite gels (22.5"C) deterrnined by small amplitude oscillatory rheometry. (Standard Error = 0.029) ...... 53
Gel moduli for gelatinlregular corn starch composite gels obtained from small amplitude oscillatory testing ( G' , 6 cm diameter acrylic parallel plate 3 rad s-' , 22.5"C;Standard Error = 18.50) and large deformation mechanical testing (E, 20% Engineering strain, room temperature; Standard Error = 157.27) ...... =-....57
Gel moduli for gelatinlwaxy corn starch composite gels obtained from small amplitude oscillatory testing ( G' , 6 cm diameter acrylic parallel plate, 3 rad s-', 22.5"C,Standard Error = 11.60 ) and large deformation mechanical testing (E, 20% Engineering strain, room temperature, Standard Error = 845.69)...... 58
Dynamic storage moduli for 5% gelatin with gelatinized waxy corn starch, WCS (A), in comparison with the theoretical rnoduli, isostrain and isostress, from the moduli for starch and gelatin alone ...... 60
Dynamic storage moduli for 5% gelatin with gelatinized regular corn starch, RCS (A),in comparison with the theoretical rnoduli, isostrain and isostress, from the moduli for starch and gelatin alone...... -...... 61
A typical stress-strain curve illustrating the large deformation behaviour of gelatin and filled gelatin gels (3 mm-min-') at room temperature obtained for determination of elastic moduli...... 65
Correlation between the dynarnic shear storage modulus ( Gr) and Young's modulus (E) for gelatinkegular corn starch gels (Standard Error = 32.1 1)...... 68
Correlation between the dynamic shear storage modulus ( Gr) and Young's rnodulus (E) for gelatinlwaxy corn starch gels (Standard Error = 16-68)...... 70 5.12 Typical temperature profile cuwe for the heating of a gelatinkorn starch composite gel for measuring the gel-sol transition temperature (temperature ramp = 0.5 Co-min-')...... 73
5.13 A more detailed view of the G' - G" intercept region, from Figure 5.1 2, of a gelatinkorn starch gel (temperature ramp = 0.5 Camin-')...... 74
vii 1. INTRODUCTION
Protein and polysaccharide polymers, also known as hydrocolloids, provide a valuable tool for food developrnent due to their ability to gel and thus alter the mechanical and rheological properties of a food system. Gels have been well defined by Ferry (1970) who described thern as a system having no steady state flow and cross-Iinked in solution by junction zones.
The use of composite filled or rnixed polymers in food systems is a means of preparing gels that have specific mechanical or textural characteristics.
A composite gel systern may be defined as one in which particulate inclusions such as starch granules, solid particles, or fat droplets modify the properties of the biopolymeric gel matrix (Brownsey et al., 1986). There is a need to better understand their rheological, physical-chernical and textural characteristics in order to optirnize their use in food systems.
Previous studies have investigated the rheological properties of protein gels containing polysaccharides (Marrs, 1982; Papageorgiou and Kasapis, 1995; Michon et al., 1996). Those studies lead to a better understanding of the rheological properties and the chemical interactions observed between gelatin and specific polysaccharides by relating the structure of the protein-polysaccharide material using one rheological test method. Cornparisons among different rheological methods were not performed. It is of interest to examine the results of different mechanical properties, obtained through different methods, to determine if the resulting interpretation of the physico-chernical nature of gelatin/com starch gels yield the same conclusion.
Knowledge of the physico-chernical properties of gels is of significant value when formulating fabricated foods. Understanding these properties would allow for the development of a maximum number of product types for a given ingredient, preparation of products from any one of several raw materials, and application of new gelling and thickening materials to food systems (Morris, 1986).
Further, the study of composite gelatin gels containing pre-gelatinized corn starch will provide greater insight into changes in the mechanical properties of the composite gel rnatrix which occur as a result of combining the phase separated components. 2. LITERATURE REVIEW
2.1 Hydrocolloids in Food
Hydrocolloids can undergo geIation in food products. They have been
defined in several different ways. A hydrocolloid may be defined as a water-soluble
stabilizing material of high but ill-defined molecular weig hi(Dickinson and Stainsby,
1982). Blanshard (1982) characterized hydrocolloids as biopolymers that interact with waterto cause a rearrangement of the polymer and water molecules depending on temperature, and Morris (1984) defined a hydrocolloid gel as a substance capable of supporting its own weight against gravity. Some common examples of hydrocolloids are: starch, guar gum, gellan gum, carrageenan as well as the protein gelatin.
In the formation of a gel, hydrocolloid polymer molecules become associated with each other forming areas, known as junction zones, to form a three- dimensional network (Rees, 1969). A hydrocolloid gel rnay take the form of an aggregated dispersion or a polymeric network (Dickinson and Stainsby, 1982). An aggregated dispersion is an ordered, tightly packed array of droplets (Dickinson and
Stainsby, 1982). Hydrocolloid gels formed as poiymeric networks consist of droplets more loosely packed but cross-linked by polymer molecules. Polymeric networks generally have a lower gel strength than aggregated dispersions
(Dickinson and Stainsby, 1982).
Due to their ability to form a gel, polysaccharides and proteins are the two most important hydrocolloid biopolymers used to modiw food texture (Smewing,
1999). Close examination of three-dimensional polysaccharide and protein structures, together with a knowledge of their interaction with water and other food ingredients is necessary to understand their properties with respect to food applications (Cesaro, 1994).
A "solvent effect" describes the solute-solvent interactions of a system.
Cesaro (1994) described a "good solvent'' as one in which solute-solvent and solvent-solvent interactions are dominant as cornpared to a poor solvent where the solute-solute interactions dominate. Understanding the effects of a solvent on hydrocolloids provides a means for understanding the conditions by which a hydrocolloid forms a gel network.
There are four general types of water-solute interactions that may be involved in the formation of a hydrocolloid gel: hydrogen bonding, ionic bonding, hydrophobic association, and van der Waals forces (Busk, 1984; Fennema, 1996).
Hydrogen bonding occurs when hydrogen atoms interact with a highly electronegative atom or region in a compound, such as a food polymer, thereby forming a dipole. This type of bonding is readily seen in a solvent such as water where oxygen atoms bond with hydrogen. lonic bonding involves interaction between an electric dipole and a charged ion. Non-polar molecules placed in a polar environment such as water. may undergo an interaction known as hydrophobic association which involves two non-polar groups separated by a polar aqueous environment. The polar aqueous environment will promote association of
4 the non-polar groups thus decreasing the prolar-non-polar interfacial area.
Therrnodynamically unstable situations, such as theassociation of non-polar protein
groups with water, require hydrophobie association so as to create a
thermodynamically stable state. Finally, vam der Waals forces describe
intermolecular attraction among atoms due to dipoles that result from electron
movement (Busk, 1984)-
2.1 .l Gelatin
Gelatin is a protein, derived from collagen, the primary component of the white fibrous connective tissue in vertebrates. Gelatin consists predominantly of glycine (33%); proline and hydroxyproline imino acids (20%); and alanine (11%)
(te Nijenhuis, 1997). Figure 2.1 shows the amina and imino acids characteristic to gelatin. lmino acids feature a pyrrolidine ring which distinguishes them from arnino acids,
g Iycine. proline hydroxy*proIine alanine
Figure 2.1 : Structural diagrams of the principaal amino and imino acids in gelatin
Commercially available sources of collagem are manufactured from pigskin, cattle skin and cattle bone and are available in large quantities, at reasonable cost
(JOhnston-Ban ks, 1990). The production of gelatin involves the destruction of the secondary structure of collagen by either an acid pre-treatment producing a type A gelatin or an alkaline pre-treatment producing a type B gelatin (te Nijenhuis, 1997).
Type A gelatin is treated with sulfuric or hydrochloric acid and pretreatment can be achieved in one day; whereas, type 6 gelatin is treated with lime or caustic soda and can require 2 to 8 weeks for complete pretreatment. In both cases, pretreatment increases the solubility of the collagen making it suitable for extraction
(Johnston-Banks, 1990). The soIuble material is subsequently washed, extracted, purified, concentrated and then dried to form a stable powder.
Gelatin is more insoluble in cold water but, can be readily hydrated in warm water (60 - 90°C) to form a gel network upon cooIing. Gelatin can, however, initially be swelled in cold water, then warmed to 60°C to enhance the rate of hydration.
Hot water dissolution involves hydrating the gelatin powder by adding it to water and heating it to 90°C. Using either of these methods results in intermolecular bonding and thus gel formation upon cooling. The resulting gelatin gels are clear and elastic
(Smewing, 1999). Elastic materials restore their shape after the removal of a deforming stress. Gelatin gels are also therrnoreversible and thus will melt when heated.
Gel rigidity is a measure of strength which depends on factors such as time, temperature and pH. It can be defined as the maximum applied stress before a gel fractures (Dobraszczyk and Vincent, 1999). The rigidity of gelatin gels increases with time as the polymer matures and this process is independent of pH between
6 4 and 10 (Stainsby, 1977). Gelatin rigidity reaches an equilibriurn stage after approximately 18 hours of setting time (Johnston-Banks, i990). A Bloom value is a correlative measure of the rigidity of gelatin gels at a temperature of 10°C and a gelatin concentration of 6.66% (wfw).
The principal disadvantage of gelatin over other hydrocolloids is that the peptide bonds are heat labile (Ledward, 1986). As a result, thermal processing tends to cause signjficant loss of gelling ability. Gelatin, however, could have advantages over polysaccharide gelling agents if the thermal characteristics of gelatin could be controlled. Ledward (1986) indicated that the gelling ability of gelatin is often enhanced by the addition of components that could introduce additional cross-links between gelatin chains. Kasapis (1999) demonstrated in his study of microcrystalline cellulose (MCC) and gelatin that the addition of MCC, ranging in concentration from 0.025% to 1.6%, altered the network strength and produced a composite gel with a dynamic shear storage moduius approximately 8.5 times that of gelatin alone.
2.1.2 Starch
Corn starch granules consist of both amylose and amylopectin fractions, and these granules swell to several times their original size at their gelation temperature
(Ring, 1985). Amylose (Figure 2.2) is a linear polymer, whereas amylopectin
(Figure 2.3) is a highly branched polymer (Smith, 1999). Figure 2.2: Glucose monomers connected by a-1,4 linkages to form a linear amylose polymer-
Figure 2.3: Glucose rnonomers connected by a-1.4 and a-1,6 linkages to form a branched chain amylopectin polymer. Branch points occur via a-1.6 glucosidic bonds. When starch granules are added to water and heated beyond the gelatinization temperature. the granules swell. causing amylose and amylopectin to be released and become solubilized (Nguyen et al., 1998). If the concentration is sufficient, the result of this treatrnent would be a viscous paste that can be transformed into a gel if sufficiently cooled. Starch gels are generally opaque and possess a short texture (Smewing, 1999). Gels with a short texture are observed to have little stringiness- The rheoIogica1 properties of starch are an important consideration when evaluating the use of starch as a thickening agent.
Amylose solubilized during the gelation process can form a gel matrix after the retrogradation of amylose (Ring, 1985; Nguyen et ai., 1998). Retrogradation is the tendency of starch to become increasingly insoluble when cooled. Amylose is more susceptible to retrogradation than amylopectin. Gels containing amylose and starch granules are approximately three times stronger than amylose gels that lack starch granules (Ring. 1985). The amylopectin component of the gelatinized starch granules thus fills an amylose gel interpenetrating matrix (Morris, 1986).
Amylopectin gel stiffness has also been dernonstrated to be closely related to amylopectin chah association (Inaba et al., 994).
2.1.3 Composite Polymer Gels
A filled gel is a type of composite polyrner gel where one ccmponent can form a continuous network through the entire volume of the system. Single-phase filled gels contain a filler in a molecularly dispersed state whereas two-phase filled gels possess dispersed particles of liquid or gel (Tolstoguzov, 1986). The dispersed phase can be made up of a variety of filler particles. Changes in the type and concentration of filler particle may influence the texture of the overall system by altering such properties as the brittleness, rigidity or elasticity of the food systern.
When a gel is filled with rigid spherical particles the gel can be reinforced but the arnount of reinforcement depends on the volume fraction that the particles occupy and size of the particles (Ring, 1985).
Filler particles can be important as a food additive because of their ability to alter the mechanical properties of a biopolymer gel. For example, the gel rigidity rnay be altered by the adhesion of Sephadex to a gelatin biopolymeric matrix
(Brownsey et al., 1986). The rigidity of the Sephadex beads increases as the amount of cross-linking between Sephadex beads and gelatin increases. They also reported that alteration of mechanical properties was due to the affinity of the filler particle for the continuous matrix. In confectionery prcducts, there have been attempts to use starch as a gelatin repiacernent to reduce ingredient costs. While starch may not cornptetely replace gelatin, starch granules have partially supplemented gelatin without large variations in the textural and mechanical properties of the system (Johnston-Banks, 1990).
Early work with gelatin composite gels containing both rigid and deformable filler particles demonstrated that the rigidity of the system depended on the rigidity of the continuous matrix, the volume fraction of the dispersed phase, and the overall deforrnability of the dispersed phase (Ring and Stainsby, 1 982). Tolstoguzov (1986) found that the gel rigidity of filled, single phase, gelatin gels increased as the concentration of the agarose filler particle increased. In contrast, filled, two-phase, gelatin gels demonstrated a drop in gel rigidity when the fiiler particle concentration increased. Decreased rigidity in the filled gelatin gels was beIieved to be due to increasing structural imperfections caused by the rising volume fraction of the dispersed phase (Tolstoguzov,1 986).
The dynamic shear storage rnodulus of a composite gel may be predicted by assuming that strain is unifom (isostrain) or that stress is uniform (isostress)
(Clark,l987). These cases represent the upper and lower bounds of the shear storage modulus, respectively and are described by the following equations:
(Isostrain) G: = $?jXGi+ $dYG;. (1)
(Isostress)
where G: , G:, and G; are the shear storage moduli of the composite gel and the x and y components of the composite gel, respectively, and, g$ and @y are the volume fractions of the x and y components, respectively.
Davies (1971) developed a relationship to describe the dynamic storage moduli of a bicontinuous composite polymer, formed from agarose and waxy corn starch, and found that an important factor for determining the moduli of a bicontinuous network was to obtain a reliable rnodulus of the starch phase. The modulus should be that of the network formed between the granules, which can
be achieved if the starch concentration is known. Mohammed et al- (1998) used this principle to find the effective concentration of waxy corn starch by determining the volume that one gram of starch occupies. Abdulmola et al.
(1996) found that the effective concentration of the starch is the concentration where the total volume of the starch granules would equal the volume of the system. The effective volume of both systems can be determined from the slope of a plot of volume fraction versus starch concentration. Davies (1971) developed a relationship for bicontinuous networks:
where the nomenclature is the same as in equations (1) and (2). Piculell et ai.
(1992) examined iota and kappa carrageenan mixtures and determined theoretical moduli (GL) through the isostrain (equation 1) and the isostress
(equation 2) models. The isostrain model produced moduli higher than the experimental results and the isostress model produced moduli lower than the experimental results, but he found that Davies' (1971) relationship fit the experimental data well.
One rnethod of determining the volume fraction of a dispersed phase within a continuous phase has been reported by Abdulmola et al. (1996) where the continuous gelatin phase was isolated from a modified waxy starch phase by centrifugation. The ratio of the concentrations of the gelatin supernatant to that of
12 the gelatin originally added to the composite gel can then be deterrnined, The reciprocal of this ratio provides a measure of the fraction of volume occupied by continuous phase (gelatin fraction, p,) in the composite system since concentration is inversely proportional to volume. Thus, the volume fraction occupied by the filler particle (swollen starch granules g>,) is equal to one minus the volume fraction of the continuous phase ( p, = 1- p, ). In both the work by
Abdulrnola et al. (1996), and more recently by Gilsenan and Ross-Murphy (2000), the density of the gelatin gel was assumed to be near that of water (1000 kg-mJ).
Another method for determining the volume fraction of a dispersed phase within a starch system was suggested by Evans and Haisman (1979).
Suspensions of starch were prepared in a dilute solution of blue dextran, and the gelatinized starch granules excluded the blue dextran dye due to the high molecular weight of the dye. The volume occupied by the granules was determined by first rneasuring the absorbance (630 nm) of the initial solutions and of the supernatant after settling in order to determine the concentration of the blue dextran. The dry weight combined with the added weight of the blue dextran was measured to determine the starch concentration. They used the following equation to determine the weight of the water absorbed by the starch:
where Ws is the weight of the water associated with the starch and excluded by blue dextran. W, is the weight of the water used to prepare the sarnple, C, and
C,are the concentration of the blue dextran before and after the experiment,
respectively.
2.2 Gel Microstructure
The study of gel microstructure is important so that researchers may gain a better understanding of the interactions between the biopolymer and the solvent or
between two or more polymers in the structure. Furthermore, gel microstructure studies provide a means of interpreting the effects that gelling conditions have on the gelled sample (Clark, 1995). Knowledge of structural information can assist in predicting the mechanical and textural properties of the gel. Severat analytical tools such as microscopical techniques and particle sizing are available for studying the microstructure of a gel.
2.2.1 Scanning Electron Microscopy
Kessel and Shih (1974) described the function of scanning electron microscopy (SEM) as "a closed-circuit video display showing the sample of interest as a moving spot of electrons." Electrons are emitted frorn a "gun" by heating a tungsten filament at variable voltage. Below the gun are three condenser
lenses which acceierate the electron bearn and focus it to a fine point. Scanning coils deflect the electrons and move :he beam over a specimen in a square raster or grid (Kessel and Shih, 1974). This bearn is synchronized with the electron beam from a cathode ray tube. The specimen surface appears on a video screen as lig ht and dark images that represent the three-dimensional topography of the specimen. Gel samples for SEM are generally prepared using freezing in order to preserve the structure of the specirnen (Kessel and Shih, 19~74).Cryogens used in
this application should have a low melting point, a high boilimg point, a high thermal conductivity and a high heat capacity (Echlin, 1992). One difficulty with cryo-SEM is that samples are often large and have poor heat transfer characteristics (Smith, 2000). As a result, production of ice crystals below the resolution of the SEM is difficult and, therefore, the image is distorted (Smith, 2000). Furthermore, freezing may result in the formation of a gas between the warm object and the coolant
known as the "Leidenfrost" phenomenon (Robards and Sleytr, 1985). This gas barrier interferes with heat transfer slowing the cooling rate cxf the specimen. Since the outside of a sample freezes first, ice crystals near the surface form more quickly and are smaller than those formed slowly near the centre oif a specimen. Two common cryogens are liquid nitrogen slush andl propane, with melting points of -21 0°C and -187"C, respectively. The rate of cool ing in propane is much greater than in liquid nitrogen slush. The boiling point ofif Iiquid nitrogen slush (-1 96°C) is much lower than propane (-42°C) and therefore Iiquid nitrogen slush is more susceptible to the Leidenfrost phenomenon (Echlin, 1992). This causes a reduction in the cooling rate of liquid nitrogen siush compared to propane. As a result, ice crystals formed by propane are smaller which results in less disruption of the gel structure by lirniting artifacts related to the freezing process (Smith, 2000). SEM is one of the primary methods of obtaining microstructural data in food systerns (Abeysekera and Robards, 1995). Abeysekera and Robards (1 995) found that macromolecular aggregates developed by phase sepai ration could be viewed effectively using SEM. The authors observed that the study of phase separation between gelatin and modified corn starch mixtures could also be obtained from confocal laser scanning microscopy or simple light microscopy. ln general, though, it was noted that the depth of field and three-dimensiomal topographie detail
15 obtained by SEM could not be achieved using a Iight microscope.
2.2.2 Particle Sizing The size of a particle, such as a starch granule, is important since particle size can affect the mechanical and textural properties of the food system. Particle sizing can be accomplished by a number of methods. Microscopy and low angle laser light scattering are two of the most common methods. Microscopy is a comrnon and effective method for particle sizing but is considered to be laborious (Tung and Jones, 1981). Also, particle sizing can be difficult at sizes less than i Fm and the background may be obscured by smaller particles which hide iarger particles (Dickinson and Stainsby, 1988). Low angle laser light scattering is used to measure particle sizes of dry powders and emulsions. It provides repeatable volume distributions at high resolution as shown by Tecante and Doublier (1999). A common method for determining the average size of the particles under investigation is by calculating the volume moment mean or D[4,3] (Tecante and Doublier, 1999) which is calculated as the sum of each particle length raised to the fourth power divided by the sum of each particle length raised to the third power (D[4,3] = (a4+b4+c4+...) /(a3 +b3+c3+...)). This method has been used to quantify particle size distributions of waxy corn starch dispersions and gellan beads (Tecante and Doublier, 1999; Jampen, 1998)
2.3 Rheology of Food Polymer Gels
Rheology is defined as "the science concerned with the deformation and flow of matter" (Tung and Paulson, 1995). Rheological behavior is dependent on macroscopic properties that define the conformational order or disorder of the polyrner network arising frorn interactions arnong the components of the system
(Cesaro, 1984). In general, knowledge of the particle-rnatrix interactions in a food system allows for a better understanding of the texturai and rheological properties of structured foods (Jampen, 1998)- Rao (1986) classified structured foods as dispersions that, in many cases, are predominately water. ln orderto characterize the rheological properties offood products, a nurnber of rnethodologies have been developed to apply stresses or strains to a material in a known geometry and to observe the resulting response of the material. This field of study, known as rheometry, has been shown to be useful in quantifying the mechanical behavior of structured food systerns (Dobraszczyk and Vincent, 1999). Obtaining modulus values for food materials can help relate the stress to the resulting strain or vice versa. Moduli are often rneasured either in shear or uniaxial compression. Simple and dynamic moduli obtained by either shear or compression can, in theory, be related to the Poisson ratio which is the ratio of Iateral strain to axial strain in compression. This should allow for an interchangeable use of either dynamic or simple moduli in developing a conclusion for the effects of specific biopolymer interactions on the rnechanical properties of the system. The relationships can be written as follows (Ferry, 1970):
(Simple)
(Dynamic) Er = 2G'(l+ u) (6) where E is the apparent Young's Modulus, G is the shear rnodulus, E' is the dynamic compression modulus, G' is the dynamic shear storage modulus, and o is the Poisson ratio.
For an incompressible fiuid the Poisson ratio is 0.5 and thus: E=3G (7)
E' = 3G' (8)
The study of the mechanical properties of foods is important so that an
interpretation can be made regarding the quality characteristics of food materials with respect to handling properties and sensory acceptance, as well as provide information regarding the structure of the product (van Vliet, 1995). When relating the structure of a gel to its mechanical properties, small deforrnation testing iç the dominant mode of analysis (van Vliet, 1995). Small deforrnation testing does not affect the structure of the rnaterial if experiments are conducted within the linear viscoelastic region (LVR). Often though, food product development requires testing of mechanical properties related to eating characteristics, handling, fracturability and shaping (van Vliet, 1995). In these cases, large deformation testing is used. van Vliet (1995) indicated that it is difficult to obtain a simple relation between the fundamental mechanical properties obtained frorn small deformation studies and the empirical properties obtained from large deformation studies. For example, the moduli of low fat dough is lower than high fat dough at small deformations. However, at large deformations, the reverse effect on the rnoduli of the dough is observed (van Vliet, 1995).
2.3.1 Small Amplitude Oscillatory Shear Testing Oscillatory testing involveç the application of a sinusoidal stress or strain to a sample and observing the resulting strain or stress. Such tests rnay be conducted in uniaxial tension or compression, bulk compression or shear. Oscillatory shear is the most common rnethod used in dynarnic testing of food materials (Steffe, 1996). A large variety of fixture geometries and sizes are available for smalf amplitude oscillatory shear testing. Comrnon geometries include: parallel plate, cone and plate and concentric cylinder. The principaldifference between the parallel plate fixture and the cone and plate fixture is that at smali angles the shear strain rate is uniform across the gap with the cone and plate geornetry; whereas, parallel plates exhibit a variation of stress, strain and strain rate as the distance from the centre of the plate changes (Steffe, 1996). The parallel plate fixture can, however, accommodate large particle sizes (Van Wazer et al.,1966) whereas cone and plate geornetry cannot accommodate such samples because of the small cone angle (Steffe, 1996). Oscillatory instruments are designed to perform dynamic tests in either a controlled strain mode (measured stress) or controlled stress mode (measured strain) (Steffe, 1996). Oscillatory testing is used to measure the elastic, viscous or viscoelastic response of a system subjected to an applied stress or strain. Deformable materials that exhibit only partial recovery after removing a deforming stress are known as viscoelastic materials (Tung and Paulson, 1995). AI1 gels are considered viscoelastic materials (Mitchell, 1976). Measuring the dynamic response of viscoelastic materials can provide information about molecular structure and mechanical behavior (Tung and Paulson, 1995). Prior to measuring the dynamic viscoelastic response of a material, it is necessary to ensure that applied stress, strain or strain rate are within the linear viscoelastic range (LVR) of the material. Within the LVR, rheological properties are not stress or strain dependent (Steffe, 1996). The LVR is comrnonly measured using a torque rarnp at constant frequency and ternperature.
Considering Hookean elasticity (ideal elastic solid), the shear stress (a)can
be related to the shear strain (y ) by the modulus value (k) through the following
eq uation:
where the rnodulus value can be measured in either shear or compression modes. Dynamic oscillatory testing subjects a sample to a small sinusoidal strain.
This sinusoidal strain (y ) may be expressed as a function of time ( t ):
where y, is the maximum strain imposed on the systern and o is the oscillatory frequency (rad-s"). In the case of a Hookean solid, the shear stress is directly proportional to the shear strain. Therefore, substituting equation (10) into equation (9) we derive the equation:
In the case of Newtonian fluids however, the shear stress is proportional to shear strain rate (y) not shear strain. This is described by the relationship: where 7 is the viscosity. From equation 10. and, thus Since a viscoelastic material consists of both a viscous and an elastic cornponent, combining equations (1 1) and (14) provides a stress relationship for a viscoelastic material: whereGf , the dynamic shear storage modulus, is a measure of the energy recovered per cycle of sinusoidal deformation (Tung and Paulson, 1995) and the dynamic shear loss modulus, G" , is the arnount of energy not recovered during a cycle of sinusoidal deformation relating to viscous Row (Tung and Paulson, 1995). The dynamic response of a material for an elastic, viscous or viscoelastic material can be described for oscillatory shear by relating the response to the phase angle, 6 , where: The tangent of the phase angle compares the energy lost to the energy stored during a sinusoidal deformation cycle (Steffe, 1996). As 6 approaches O", Guapproaches O and the material behaves as an elastic solid. When the phase angle is infinitesimal or undefined, G' is very small, 6 + 90" ,and the material behaves as a viscous liquid. If, however, the phase angle is between 0" and 90" then the system has both elastic and viscous behaviors and is classified as 2.3.2 Constant Rate Uniaxial Testing Evaluation of the mechanical properties of food materials is often based on ernpirical testing. Empirical tests measure poorly-defined parameters that can be related to textural quality (Bourne, 1982); however, they do not provide a fundamental understanding of the material being tested. Large deforrnations are often required to imitate the process of mastication of food materials. Such large deformations may be accomplished by squeezing a food material between two lubricated parallel plates and in that case, a cylindrical sample is cornpressed in the axial direction and expands in the radial direction (biaxial extension). Thus the compression of a material between two plates involves a change in height (axial strain) and a change in area (lateral strain) (Steffe, 1996). The ratio of lateral strain to axial strain is known as Poisson's ratio ( v ) and is described for cylindrical samples as: lateral strain 8r / ro v= - axial strain 6h / h, where 6r is the change in sample radius, r, is the initial sample radius, 6h is the change in sample height, and ho is the initial sarnple heig ht. P O i s s O n ' s rati O ranges in value from O to 0.5 where O represents a rigid material and 0.5 represents a liauid-like material (Steffe, 1996). The effects of specimen dimensions (Le.,height to diameter ratio or aspect ratio) are often consiasred when evaluating food materials At a known deformation, the force on the specimen is directly proportionalto the cross-sectional area and inversely proportional to the specirnen length (Peleg, 1977): where F is the applied force, A is the cross-sectional area, and h is the height of the specimen. Uniaxial compression is useful for studying the compression characteristics of Hookean solids. For small deformations, normal engineering stresses (o, ) and strains (E ) can be calculated as follows (Steffe, 1996): where F is the applied force and A is the initial cross-sectional area of the sample, and: where6h is the change in sarnple height and ho is the initial sample height. 23 When deformations are large, however, it is necessary to calculate the true stress (a ) and true strain ( gh) as described in the following relationships (Tang et and Knowing true stress and strain values allows one to calculate the apparent Young's modulus (E), also known as the rnodulus of elasticity. The apparent Young's modulus depends on the crosshead speed of the mechanical testing apparatus and the aspect ratio of the sample. A greater crosshead speed results in a greater apparent Young's modulus. An aspect ratio of less than or equal to unity for uniaxiai compression testing appears to be the most common choice among researchers (Christianson et al., 1985; Tang et al.,? 996). The dope of the tangent line through the steepest portion of the stress-strain curve provides an estimate of the apparent Young's rnodulus. For any point along the linear portion of the stress-strain curve it is calculated as: 2.3.3 Dynamic Compression Testing The theory behind dynamic compression testing is sirnilar to that of small deformation oscillatory testing. Using a servohydraulic actuator and the appropriate control and data collection software, wavefoms can be established at a desired frequency and amplitude. A cornparison of the dynamic storage moduli obtained from both sinusoidal compression ( Er) and srnall amplitude shear ( Gr) oscillatory tests could prove interesting if there is strong correlation between the results. Sale et al. (1984) developed a method for measuring the rheological properties of semi-solid foods using sinusoidal compression. Based on studies for the chewing of meat, the mechanical testing apparatus that was employed allowed for various rates of deformation and a variable compression stroke length. Despite the development of this novel test, the dynamic compression of food specirnens has not been widely applied in food science. An examination of theoretical elastic and viscous bodies will aid in deveIoping a methodology that can be applied to the study of the mechanical properties of food composite polymers and can serve as reference points for materials possessing a more complex dynamic behavior. Ultimately, the theory described here can be used to form a basis of understanding viscoelastic materials. Elastic, viscous and viscoeIastic bodies can be depicted, as seen in Figure 2.4, as springs, dashpots and springfdashpot combinations, respectively (Mohsenin, 1970). Figure 2.4: Mechanical models illustrating: A. Elastic bodies; B. Viscous Bodies; and C. Viscoelastic bodies (Kelvin model). 2.3.3.1 Elastic Behaviour Robert Hooke developed the theory of Hookean eIasticity to describe the behaviour of ideal elastic bodies (Tung and Paulson, 1995). An ideal elastic body will change its shape in proportion to the magnitude of the stress applied to the system. Hooke's Law is given as: F= ky (24) where F is the applied force, y is the deformation and k is a constant. A common example of an ideal elastic body is a spring and that serves as a model system for elastic behaviour. These materials are linearly elastic (Steffe. 1996) and thus k is independent of the strain or the rate at which the force is applied. Application of a sinusoidal waveform can produce both data for stress and strain. Testing of an elastic body would illustrate that there is a zero degree phase shift between the stress curve and the strain curve, over time. Knowing this, the dynamic storage modulus in compression can be calculated as follows (Ferry, IWO): where O,is the maximum applied stress, is the maximum strain, and 6 is the phase angle which in this case would be equal to zero. WhenG equals zero, then cos(@ will equal one and thus, the dynamic storage modulus will be equal to Young's modulus. It is important to note that equation (25) can also define the shear storage modulus ( G' ) since both are dynarnic quantities. 2.3.3.2 Viscous Behaviour Viscous behaviour lies ai the other end of the spectrum from elastic behaviour. A viscous material will continue to flow and deform so long as the a stress is applied and will not recover once the stress is removed (Tung and Paulson, 1995). An ideal viscous liquid (Newtonian Iiquid) is the simplest case when considering the measurernent of viscosity (de Man, 1976). de Man (1976 ) considered a plate moving over a Newtonian fluid with a velocity v, as a result of a shear stress a . The plate will produce a shear strain rate (y). Viscosity ( 7 ) is the proportionality constant between o and y and: O=?y Now considering a viscous Newtonian fluid such as corn syrup, a mode1 system can be developed so that the system behaves Iike a d ashpot. For a viscous body, the phase angle (6)is equal to 90 degrees indicating that the curves for load and displacement are 90 degrees out of phase. The viscous cornponent, the dynamic loss rnodulus in compression (Eu) is defined as follows (Ferry, 1970): where o,is the maximum applied stress, E, is the maximum strain, and 6 is the phase angle, which in this case would equal90 O. When 6 equals 90" then çin(6) wilt equal one and thus the dynamic loss modulus is also equal to the proportionality constant (7 ) where 7 is the viscosity of the material. 2.3.3.3 Viscoelastic Behaviour Materiafs that possess elastic and viscous properties and undergo partial elastic recovery upon removal of a deforrning force are known as viscoelastic materials (Tung and Paulson, 1995). Information of about the molecular and macroscopic behaviour of a viscoelastic systern can be obtained from a study of the dynamic mechanical properties of the system (Tung and Pa ulson, 1995). The Kelvin mode1 (Figure 2.4~)describes a spring and a dashpot in parallel. In order to derive solutions for the elastic and the viscous cornponents the following two parameters must be known. First, the stress on the system is divided between the spring and the dashpot (equation (28)) and second, the total strain is equal to the strain on the dashpot or the spring (equation (29)). Now knowing that for any tirne (t) in dynamic testing we can define the stress at time, t as the following : a, = oosin(@ t + 6)= Ekosin(@ t)+ IJ w COS(@t) (31 By solving equation 31 at two different strains two equations and two unknowns are acquired. Solving these two equations leads to a solution for E' and q~which are the dynamic compression modulus and the viscosity respectively. 2.4 Gel-Sol Transition and Gelation The procesç of gelation involves transforming a Iiquid solution of macro- molecules or particles into an well-shaped elastic solid (Djabourov ef al., 1988a). The mechanism of gelation is considerably more complicated for a food polymer than for a synthetic polymer, because of the involvement of factors such as coi1 - helix transitions, disulfide bonds and hydrogen bonds (Kumagai et al., 1993). The temperature for a biopolymer to melt (Tm)may be defined as the temperature where the dynamic shear storage modulus ( Gr) equals the shear loss modulus (G" ) (the gel-sol transition) (Djabourov et al., 1988a). This can be easily measured using a controlled stress rheometer in oscillatory mode. It is important to recognize that 29 viscoelastic moduli are frequency dependent and this dependence must be recognized when measuring the rnelting or gelling points (Djabourov et al., 1988a). Interpretation of gel-sol transition data in'the form of a plot of rnodulus versus temperature can help to define the phases within a system: continuous, discontinuous or bicontinuous. Understanding the arrangement of polymer phases in a gel systern can lead to theories of therrnodynamic incornpatibility in the systern and thus a better understanding of the nature of the mechanical properties of the gel. An alternative method for examining gel-sol transition points is by the use of differential scanning calorimetry (DSC). DSC allows for examination of molecular and intermolecular transformations when water is present in the gel (Davis and Gordon, 1984). Water, or another aqueous solvent, is required for gelation and is, in part, responsible for the textural, rheological and stability properties of a gel. DSC results indicate either an endothermic or an exothermic peak temperature. These peaks represent the melting temperature and setting temperatures of a gel, respectively (Miyoshi et al., 1994). When measuring melting point temperatures of protein gels using DSC, a minimum protein concentration must be used in order to obtain a reliable signal- In the cases of low protein concentrations (s0.05%), a rnicrocalorimetric technique may be useful (Marcone, personal communication). Microcalorimetry was not pursued in this research since the concentration of gelatin was sufficiently high to rnake cleaning of the cell impractical. RANDOM COlL TRIPLE HELlX THREE-CHAIN JUNCTION ZONE TWO-CHAIN JUNCTION ZONE GEL NETWORK Figure 2.5: The conformationai changes involved in the network formation of random coi1 gelatin chains (adapted from Johnston-Banks, 1990) In the case of gelatin, gelation results from a coi1 - helix transition that leads to chain association, ând thus a three-dimensional structure (see Figure 2.5) (Djabourov et al.,l988b). The rate of this transition increases as the temperature decreases (Djabourov et al., 1988a). Gelation of gelatin involves two principal mechanisms. First, the junctions within the molecular network become more ordered and rigid, and second, the network thickens. When gelatin sets, the gel network undergoes a partial return to a collagen structure (Stainsby, 1977). The dominant factors which affect the melting point of a gelatin gel are viscosity and gelatin concentration (Johnston-Banks, 1990). The rnelting of a gelatin gel initially involves the areas of amino acid sequence with the Iowest content of imino rich glycine-proline-hydroxyproline triplet residues, which are the first residues to become disordered. The glycine-proline-hydroxyproline triplet content is proportional to the strength of the gel (Johnston-Banks, 1990). Stronger links remain intact until the temperature reaches the melting point of the gel while some chemical links remain in the viscous phase a few degrees above the melting point (Stainsby, 1977). Johnston-Banks (1990) reported that the melting point of a gelatin gel increases dramatically as the gelatin concentration approaches 10%. Also the melting point of 1-5% gelatin is essentially independent of pH in the range of 2-1 1 (Stainsby, 1977), indicating that neither the ionizable carboxyl nor the amino groups in the protein affect gel stability. Another important consideration to include when discussing the gel-sol 32 transition is the ternperature history of the gelatin. During the aging of a gel at a given temperature there is growth of nuclei causing the crosslinking oftriple helices (te Nijeenhuis, 1997) and therefore, an increase in the storage modulus. As reported by te Nijenhuis (1997) many researchers believe that the aging process is independent of ternperature and it has been noted that the effects of excess aging, caused by lowering the temperature, decrease as the concentration of the gelatin increases. In any case, the effects of excess ageing is eliminated when the ternperature is increased (te Nijenhuis, 1997). "Percolation theory" is a method used for examining chemical gelation. Percolation theory predicts the probability that a given number of chernical bonds will react and describes the increasing interconnections in a random medium (Djabourov et al., l988a). Where P is the probability of bond formation and P, is the percolation threshold, the gel point of the system is reached as 1 P - P,I - O (Stauffer, 1985). This theory predicts that below the gel point the static viscosity diverges whereas above the gel point the modulus increases with a power law relationship. In the case of composite gels, a percolated system would be one which describes the microstructure when both polymers are continuous (Brown et al., 1995). Study of the mechanical properties of proteinlcarbohydrate composite systerns is important so that the particle-matrix interactions can be better understood. Gelatin and gelatinized corn starch were chosen as mode1 systerns for protein-polysaccharide interactions in food systerns with waxy and regular corn starches used as filler particles. Waxy and regular corn starch have different amyIopectin contents. Regular corn starch, unlike waxy corn starch, possesses considerable amylose which, during gelatinization, rnay be exuded from the granule to form an amylose gel. The objective of this research was to investigate gelatinfcorn starch composite gels having different functional properties in order to investigate the effects of protein-polysaccharide interactions on the gel moduli obtained experirnentally using three different test methods: srnall amplitude oscillatory rheometry, uniaxial compression and dynamic compression testing. These measurernents allowed for testing of the hypothesis that in a protein- polysaccharide gel, dynamic or constant rate uniaxial testing, either in shear or compression, wilt allow for the sarne interpretation of the protein-polysaccharide interactions within a system. To better comprehend the data obtained from the mechanical tests, cryo- SEM was used to provide a visual representation of the gel microstructure. As well, the gel-sol transition temperature was measured to determine whether the composite polymers exist as a bicontinuous network or if one of the polymers is discontinuous and the system exhibits phase separation. 34 4. MATERIALS AND METHODS 4.1 Gelatin and Starch Preparations Type A pigskin gelatin polymer (250 Bloorn, 40 mesh) was obtained from Cangel Inc. (Toronto, ON). Waxy corn starch (S-9679, = 100% arnylopectin, trace amylose) and regular corn starch (S-4126, 73% amylopectin, 27% amylose) were obtained from Sigma Chemical Inc. (St. Louis, MO). Aqueous preparations of gelatin and starch were made on a weight to weight basis to form dispersions and gels of various concentratioriis as required for subsequent studies. For gelatin solutions, gelatin powder was added to hot (95°C) distiiled water while stirring constantly using a rnagnetic stir bar. Starch mixtures were prepared by adding waxy or regular corn starch to distilled water at room temperature. The resultant opaque starchlwater dispersions were then heated to 95°C with continual mixing. Starch gelatinization was considered complete when the system thickened and becarne translucent. To form 5% gelatin or 5% starch gels, the corresponding aqueous preparations were poured into glass vials (21 x 70 mm, borosilicate glass) and allowed to set, overnight, in a refrigerator at 5°C. To form various gelatinktarch composite gels, a 10% (w/w) gelatin solution was added to an equal arnount of 2,6,or 10% starch dispersion once gelatinization was complete. The polymer mixtures were stirred for 15 minutes using a magnetic stir bar, before being allowed to set as described above. Composite gels were prepared by this method so that the final concentration of the continuous gelatin phase was held constant at 5% (w/w) and the fina! concentrations of the dispersed starch phase were 1,3 or 5% (w/w) waxy or regular corn starch. 4.2 Cryo-Scanning Electron Microscopy The microstructure of 5% gelatin, 5% gelatin/5% regular corn starch, 5% gelatin/5% waxy corn starch, 5% regular corn starch and 5% waxy corn starch gels were examined using cryo-scanning electron microscopy (cryo-SEM). An Hitachi S-570 SEM (Hitachi Ltd., Tokyo, Japan) was used for al1 tests. Gels were prepared as described in Section 4.1. Each sample was carefully cut into small pieces, approximately 1 mm x 1 mm x 2 mm, and placed into a copper mounting block so that the sample protruded out of the sarnple mounting hole for freezing in liquid nitrogen slush. Propane was also used as a cryogen in an attempt to reduce ice crystal size. SrnaII divers (1 mm x i mm x 2 mm) were cut and placed in a rivet. The propane was pre-cooled in Iiquid nitrogen (-187°C) that was added to a Dewar fiask surrounding the propane chamber. Cryo-gel (Tissue Tech) was used to hold the sampie in place during fracturing. Samples frozen in liquid nitrogen slush were surrounded by an argon atrnosphere to avoid condensation. Under vacuum and freezing conditions (=A 50 Pa and -125°C) the gels, frozen in either cryogen, were fractured to 36 expose the surface structure. Fractured gels were then sublimated, either on the SEM stage or in the cryo unit, at elevated temperatures (-80°C)for 30 to 45 min. The fractured surfaces of each gel were then sputter coated with a thin layer of gold (=30 nm) and viewed on the SEM with an accelerating beam voltage of 5- 10 kV ai temperatures ranging from -1 25 to -1 60 OC. 4.3 Particle Size Determination Both waxy and regular corn siarch granule particle distributions were studied using a low angle laser light scattering method on a Malvern Mastersizer X (Malvern Co., Malvern, UK). Dispersions (2% wlw) of waxy and native corn starch in distilled water were prepared in glass vials. The vials were shaken to ensure that the starch granules were well dispersed in the distilled water. Then each sample was added drop-wise until the appropriate particle concentration was achieved in the Mastersizer blender as determined by the computer software. Samples were tested in duplicate for both the waxy and native corn starches. 4.4 Mechanical Testing 4.4.1 Small Amplitude Oscillatory Shear Testing Small amplitude oscillatory measurements on 5% gelatin and gelatinlstarch composite gels, consisting of 5% gelatinhegular corn starch (1, 3, and 5% wlw) and 5% gelatinlwaxy corn starch (1, 3, and 5% w/w), were carried out using a 37 Carri-Med CSLZ 500 controlled stress rheometer (TA Instruments, New Castle, DE) equipped with a 6 cm acrylic flat plate upper-fixture- Gelatin and gelatinfstarch gel samples in glass wials were melted at 60°C using a temperature controlled circulating water bath. SrnaII samples (~0.5rnL) of the melted gels were applied to the lower platen usirng a syringe. A pneumatic ram raised the lower platen until the sample made coniitact with the upper-fixture. The gap between the lower platen and the Mure was; adjusted to 60 Hm which defined the initial sample thickness. A light coating of paraffin oil was applied to the rim of the upper fixture around the circumferencee of the sample to protect against evaporation. The linear viscoelastic range (LVR) for each : sample composition was determined at an oscillatory frequency of 3 rads-' ovarr a torque sweep of 100 -10,000 pN-m. The specified oscillatory frequency was used as it was at the middle of the desired frequency range (1.0 to 10.0 rard-s-') . Temperature was controlled at 22.5 f 0.1 OC using a Peltier plate mounted in the lower platen and samples were equilibrated for 60 min in the instrumemt before testing. Due to the compliant nature of the samples a large LVR was olbserved. Viscoelastic properties of the gels were deterrmined from a frequency sweep and calculated using the rheometer software. Frequency sweeps were performed over a range of 1.0 to 10.0 rads-' at an coscillatory torque of 3200 pN-m. These conditions were within the LVR for aall samples. Al1 samples were tested in triplicate- 4.4.2 Constant Rate Uniaxial Testing An lnstron Model 1122 mechanical testing machine was used in conjunction with the Merlin Series IX computer software (Instron Corporation, Canton, MA) to study the elastic moduli of the composite polymer gels. Samples were prepared as above except that the composite aqueous dispersions were poured into stainless steel cylindrical molds with a 21 mm interna1 diameter. The inner surfaces of the steel moIds were lubricated with a vegetable oil spray (PAM", International Home Foods (Canada), Inc.) to allow easy removal of a gel from the rnold. In the absence of lubrication, gels would fracture in the mold as pressure was applied to remove them. Sarnples were placed in a refrigerator at 5°C and allowed to set overnight. After the samples were removed from the mold, they were cut into 20 mm lengths using a holder and a wire sectilorneter. Samples were then equilibrated to roorn temperature. Samples were compressed uniaxially between parallel Teflon (10.5 cm diameter) platens. Vegetable oil spray was applied to the surface of the upper and lower platens prior to testing to reduce surface friction. This rninimized "barreling" of the samples during compression. The crosshead speed was set to a constant deformation rate of 3 mmlmin and a calibrated 2.0 kg load cell was used to measure the applied force on the sample. The samples were placed on 39 the lower platen and compressed to a height of 10 mm (50% deformation). From the force-deformation data, transformations were perforrned to obtain values of true stress and engineering strain. Engineering strain was used instead of true strain since this definition of strain provides a more linear correlation between stress and strain as was described by Bagley et al. (1984). The apparent Young's Modulus was determined for each sample by performing Iinear regression analysis over an engineering strain range of O to 20%. AI! samples were tested in triplkate- 4.4.3 Dynamic Compression Testing An lnstron Modef 8871 servohydraulic testing system equipped with Fast TrackTM 8800 software (Instron Corporation, Canton, MA) was used for al1 dynamic compression tests. In addition, Wavernakerm editor and WavemakerTM Runtime (Instron Corporation, Canton, MA) were used to develop and control the sine wave motion of the actuator. The principal reason for developing a methodology for sinusoidal deformation of soft materials instead of measuring the moduli for the gelatinicorn starch gels was due to the sensitivity limits of the equiprnent. Observations showed that the inertia effects of the instrument were caused by the movement of the actuator. This resulted in a noise output being added to the signal of the force sensor. Similar observations were seen in the work of Sale et al. (1984). A methodology was developed for measuring the dynamic compression moduli in simple elastic. viscous and viscoelastic systems. Mode1 systerns were used to obtain data for each of the cases. The simple elastic case required the use of a spring; the viscous case used a cup containing corn syrup with a plunger (a dashpot) that moved against the corn syrup in a sinusoidal fashion (see Figure 4.1); and the viscoelastic case used a Kelvin model of the spring and dashpot, then a gelatin gel (5% w/w). The difference between the phase angle of the stress and strain curve indicates the nature of the material: elastic, viscous or viscoelastic. Measurernent of the ideal elastic storage modulus involved placing a spring between the Ioad cell and the frarne. The spring (16 x 40 mm) was compressed by an initial amount which was greater than the amplitude used in the test. The deformation properties of the spring were measured at a frequency of 0.1 Hz which allowed elastic recovery of the sample. The ideal loss modulus was determined by using a Newtonian fluid of high viscosity (corn syrup), and a plunger and a cup (see Figure 4.1) combined to form a model dashpot. The diameter of the plunger was 94 mm and the annular gap was 3 mm. The plunger was moved, up and down. through the corn syrup in a sinusoidal fashion. The loss modulus was determined at a frequency of 0.1 Hz. To measure an ideal viscoelastic system, a combination of the spring, described above. and the dashpot was tested as a Kelvin body (Figure 4.2). The spring was positioned, upright. on the bottom of the cup and corn syrup was added to the cup to cover the spring. The plunger was Iowered to pre-strain the spring. The plunger was moved, sinusoidally, against the spring and the corn syrup in a sinusoida1 motion at a frequency of 0.1 Hz. To measure the phase angle shift of the Ioad or position curves, Table Curve 2D cornputer software (Version 4, AISN Software Inc.) was applied to fit the resulting sine function in the form: y = a + b sin(2/rt/T+ 6) where y is the stress or strain applied in compression, L is the time at which the stress or strain was applied, S is the phase shift (horizontal) of the cuwe, n is the vertical shift (assumed to be zero), b is the amplitude of the sine wave and T is the period. The maximum load was determined from the peak amplitude on the load curve. This force value was used to compare the measured viscoelastic response to a calculated viscoelastic response. Plunger C~P Annular Gap Figure 4.q : Model apparatus used to represent a dashpot for experimental analysis of an ideal viscous material (Newtonian Fluid). Plunger Viscous Matenal Spring Figure 4.2: Mode1 apparatus used to represent a dashpot and a spring in parallel (a Kelvin Body) for experimental analysis of an ideal viscoelastic material (Newtonian Fluid) . 4.5 Volume Fraction Determination Six different composite polymer aqueous dispersions, 5% gelatinll , 3 and 5% regular corn starch and 5% gelatinfl, 3 and 5% waxy corn starch, were prepared and cooled to approximately 40°C. An aliquot of each polymer dispersion was centrifuged in a 50 rnL polycarbonate centrifuge tube (Nalgene Brand Products, Rochester, NY). Gelatinfwaxy corn starch dispersions were centrifuged using an ultracentrifugewith a 70.1Ti rotor (Beckman L8-70M, Beckrnan Instruments, Inc., Palo Alto, CA) at 246,960 x g for 60 min, gelatidregular corn starch dispersions were centrifuged (IEC 21000, International Equipment Company, Needham Heights, MA) at 22,500 x g for 75 min. The waxy corn starch dispersions required a higher g force since neither the g forces available in the IEC 21000 series centrifuge nor vacuum filtration using a Whatman 1 or a Whatrnan 2 filter paper were able to separate the starch phase from the gelatin phase. The clear gelatin supernatants present after centrifugation were then extracted, using a Pasteur pipette, and stored at 5°C overnight. Small amplitude oscillatory rheometry was used to measure shear storage moduli of the gelatin fractions. Shear storage moduli for standard gelatin solutions of 2, 4, 5 and 8% w/w concentration were measured using the same method. The G' data obtained from the standard solutions were used to construct a standard curve of shear storage moduli versus gelatin concentration plotted on logarithmic axes. The standard curve was then used to obtain the concentration of the gelatin fractions obtained from centrifugation. The ratio of the gelatin concentration in the fraction to the gelatin concentration in the initial solution was calculated for each waxy and regular corn starch concentration (0, 1, 3 and 5% w/w). The inverse of this ratio provided a measure of the volume fraction of the gelatin phase since concentration is inversely proportional to volume. Plots of volume fraction versus starch concentration were prepared for the regular and waxy corn starches. The swelling volume for each starch was calculated from the slope of those curves using the methods proposed by Mohammed (1998). 4.6 Gel-Sol Transition of GelatinIStarch Composite Gels Study of the gel-sol transition point was used to determine whether samples were bicontinuous or if the samples had phase separated components. This was determined by identifying which of the component(s) dominate during melting of each gel and was thus regarded as the continuous phase(s). 4.6.1 Differential Scanning Calorimetry (DSC) Experiments were conducted to determine melting point temperatures. Attempts were made to measure the melting point temperatures for ail gels by DSC in order to compare with results obtained for the gel-sol transition temperature obtained through small deformation oscillatory temperature sweeps. 46 A mode1 TA291 0 Differential Scanning Calorimeter (T.A. Instruments, New Castle, DE) was used for these measurements. Temperature and cell constant calibration tes& were carried out using an indium standard (melting point = 156.6"C). Approxirnately 15 mg of 5% aqueous gelatin dispersion or gelatidstarch composite aqueous dispersion were placed in DSC pans using a Pasteur pipette. A lid was placed on each pan and then hermetically sealed. The sample pan was placed in the DSC chamber and allowed to equilibrate to 10°C for one hour. Following equilibration, each sample was scanned at 1 Co-min-' frorn 10 to 35°C to obtain a DSC melting endotherm for each of the aqueous colloid dispersions. 4.6.2 Gr- Gu Crossover This method for determining the continuous phase(@ of the composite gels is similar to that described earlier (Section 4.4.1) but with the following changes to the testing conditions. lnstead of a frequency ramp, a temperature ramp was employed in order to find the gel-sol transition temperature. Samples were allowed to equilibrate on the instrument for 60 min at 10°C. Following equilibration, the temperature was ramped at a rate of 0.5 Co-min-' from 10 to 35°C. The frequency was constant at 3 rad s-' and the oscillatory torque was set at 3200 pN-rn, which was within the linear viscoelastic region for each composite gel. Data were gathered for both shear storage and shear loss moduli. 5. RESULTS AND DISCUSSION 5.1 Gel Structure During the gelatinization of regular corn starch, solubilized amylose leaches out and undergoes retrogradation during cooling (Keetels et al., 1995). Retrogradation is the formation of an insoluble aggregate and in the case of regular corn starch, the amylose forms an insoluble aggregate leading to gel formation (Nyugen et ai., 1998). In contrast, waxy starch granules are not susceptible to retrogradation due to the highly branched structure of the arnylopectin polymer. The formation of a gel rnay result from the close association of the arnylopectin branch chahs (Nyugen et al., 1998). Figure 5.1 is a series of cryo-SEM micrographs which illustrate the structural differences between gelatin gels and gelatinlstarch composite gels. The 5% gelatin gel (Figure 5.1A) exhibits a "honeycomb" structure with large void spaces. The gels of 5% regular and waxy corn starches (Figures 5.1 B and 5.1C,respectively) appear to have a network that is less defined than that of the gelatin gel, but void spaces are clearly visible, The composite gelatinkegular corn starch gel (Figure 5.1 D) is shown to have a very densely packed network where the rnost densely packed areas rnay be dominated by starch while the less densely packed area has a honeycomb pattern similar to the gelatin gel, but with smaller fibre lengths. In this case, void spaces that can be occupied by Photo Width = 18-67 Mm Figure 5.1: Cryo-scanning electron micrographs (10-1 5 kV, 5000 x magnification) of gelatin, starch and gelatinlstarch composite gels (A) 5% (w/w) Gelatin, (8) 5% (w/w) Regular Corn Starch, (C) 5% (wlw) Waxy Corn Starch, (D) 5% (wlw) GelatinlS% (wlw) Regular Corn Starch, (E) 5% (w/w) Gelatinl5% (wlw) Waxy Corn Starch. All samples were . frozen in liquid nitrogen slush. water appear to be smaller as compared to gelatin or starch alone. Figure 5.1 E shows that the composite gel made with gelatin and waxy corn starch has a network similar to that of gelatin (Figure 5.1A) but the void spaces in the network are visibly smaller than in the gelatin sample. Exuded amylose from gelatinized regular corn starch can extend throughout the gel network, entrapping the aqueous continuous phase and forming an amylose gel (Nguyen et al., 1998). Conversely, waxy corn starch consists primarily of amyiopectin with only trace amounts of amylose present. Nguyen et al. (A998) proposed that waxy corn starch does not form a gel upon cooling and that the gel-like structures observed are due to swelling and close packing of amylopectin. Thus, in gelatinlwaxy corn starch gels, the gelatinized starch granules would act as filler particles occupying the void space in the gelatin network. In contrast, the structure of the gelatinlregular starch gels shows what may be separate domains dorninated by either gelatin or starch that have both gelled. This may explain the compaction of the polyrner network seen in Figure 5.1 D. The size of the ice crystals present after freezing is mainly a function of the freezing rate. The honeycomb shaped structure. particularly evident in gelatin gels frozen in Iiquid nitrogen slush, was likely due to ice crystal formation. Freezing of the gel samples in propane was performed to increase the freezing rate, rnove water closer to the vitreous state and reduce ice crystal size. The use of a propane cyrogen was observed to reduce the wall thickness but the 50 overall topography for each gelatin and gelatinlstarch composite gel remained essentially unchanged. The vitreous state of water requires a rate of freezing greater than or equal to 1 x IO5 Kelvin-s" (Djabourov, 1988b). Vitrification is a process whereby low viscosity water becomes amorphous high viscosity water, often termed a glass. It should be noted that cryo-SEM images for each specimen were studied at areas near their surface so that ice crystal size would be minimal. 5.2 Volume Fraction Linear relationships between the volume fraction of the two types of corn starch and their respective concentrations in gelatinktarch composite gels are consistent with the results reported by AbduImola et al. (1996). As seen in Figure 5.2, dynamic storage modulus (G' ) values increased linearly with gelatin concentration in aqueous solutions when plotted on log-log coordinates. This provided a standard curve to determine the concentration of the gelatin fractions obtained after centrifugation of the composite gelatinktarch dispersions polymers. The gelatin fraction obtained after centrifugation was assumed to have undergone complete phase separation. This was supported by Abdulmola et al. (1 996) with their work on gelatin and starch, and by Kasapis (1 999) with his work on gelatin and microcrystalline cellulose. Both studies indicated that the gelatin molecules would be confined to the supernatant, obtained from centrifugation, as it was unlikely that gelatinized starch or cellulose particles would be penetrated by the gelatin. The relationship between volume fraction and the concentration of regular and waxy corn starches in 5% gelatin are presented in Figures 5.3 and 5.4, respectively. In both cases, volume fraction increased linearly with concentration with a dope of 0.068 mL-g-lfor regular and waxy starch. Thus 1 g 10 Gelatin Concentration, c (% w/w) Figure 5.2: Standard curve of storage modulus, G' (3 rads-', 3200 pNm, 225°C) versus gelatin concentration (c) for determination of the gelatin fraction concentration in a gelatinfcorn starch aqueous dispersion (Standard Error = 0.034). O 1 2 3 4 5 6 Regular Corn Starch Concentration, c (% whv) Figure 5.3: Relationship between volume fraction and concentration of regular corn starch in 5% gelatin composite gels (22.5"C)determined by small amplitude oscillatory rheometry. (Standard Error = 0.035). 7 O 1 2 3 4 5 6 Waxy Corn Starch Concentration, c (% wlw) Figure 5.4: Relationship between volume fraction and concentration of waxy corn starch in 5% gelatin composite gels (22.5"C)determined by small amplitude oscillatory rheometry. (Standard Error = 0.029) of gelatinized regular corn starch or gelatinized waxy corn starch occupies a volume of 6.80 mL in a composite 5% gelatin/5% starch dispersion. Abdulmola et al. (1996) observed a swelling volume for waxy corn starch of 9.65 mUg. The difference between the swelling volumes observed in this study and the swelling volumes determined by Abdulrnola et al- (1996) may be attributed to the concentration of gelatin used in the experiments. Abdulmola et al. (1996) used a maximum of 1.5% gelatin in their gelatinfstarch composites; whereas, the composite gels in this study used 5% gelatin. More water would have been available for the additional swelling of the starch after the starch is added to the gelatin solution at the lower concentration. In this study, preparation of gelatin fractions from gelatin/waxy corn starch aqueous dispersions required ultracentrifugation (246,960 x g) in order to sediment the gelatinized starch. However, Abdulrnola et al. (1996) reported the sedirnentation of waxy corn starch from gelatin at 4,000 x g, suggesting that perhaps the higher concentration of gelatin used in this work suspended the starch more effectively than the lower concentration used by Abdulmola et al. (1996). A clear gelatin fraction was obtained from getatin/regular corn starch samples at 22,500 x g, whereas ultracentrifugation led to a cloudy, nearly opaque gelatin fraction when the sample was cooled. Ultracentrifugation is designed to operate under vacuum and attain high centrifugal forces required to sediment small molecules. It is hypothesized that when the greater centrifugal forces were applied to this aqueous dispersion, the amylose and absorbed water were exuded into the surrounding medium resulting in a cloudy supernatant- Results of particle sizing indicated that the diameter of the ungelatinized waxy corn starch granules was smaller than the diameter of the regular corn starch granules,l4.55 I 0.12 pm and 15.38 * 0.13 Pm, respectively. Past research (Nguyen et al., 1998) indicated that waxy corn starch granules had a larger diameter and had a greater swelling ability than regular corn starch granules. The swelling volume of the waxy cornstarch was determined to be equal to the regular corn starch. This would appear to indicate that differences in mechanicat properties could not be accounted for by swelling volume variations. 5.3 Mechanica1 Properties of GelatinlStarch Gels There has been Iittle experimental research reported on the characteriration of the mechanical behaviour of composite gels in food systems. In this study, the rigidity of composite gels containing 5% gelatin and either waxy or regular corn starch at concentrations of O, 1, 3 and 5% w/w was measured using small and large deformation testing methods. Small amplitude oscillatory testing was performed using a controlled stress rheometer, whereas uniaxial compression was completed using a mechanical testing machine. As well, a methodology was investigated for using dynamic uniaxial compression testing to provide information on the mechanical properties of gels. 5.3.1 Small Amplitude Oscillatory Shear Testing Gr values for regular corn starch and waxy corn starch samples were compared at an oscillatory frequency of 3 rads' for torques between 650 and 3900 pN-m- Different torque ranges were used to stay in the linear viscoelastic range for different sample compositions. Results (Figure 5.5) indicated a linear increase in rigidity with respect to the starch volume fraction for al1 regular corn starch volume fractions tested. Thus, there was an overall increase in composite gel rigidity as the volume fraction of gelatinized corn regular starch increased. Composite gelatin gels containing waxy corn starch demonstrated Iittle change in rigidity over the volume fraction range of waxy corn starch. Figure 5.6 indicates that the rigidity of these gels was independent of concentration. 8000 7000 1- 5000 1- w 4000 :- iooo :- G' = 1096.OVF + 73-87 ?=0.99, n=4 G' A * O O 0.05 O. 1 0.15 0.2 0.25 0.3 0.35 0-4 Volume Fraction of Regular Corn Starch Figure 5.5: Gel moduli for gelatin/regular corn starch composite gels obtained from small amplitude oscillatory testing (Gr, 6 cm diameter acrylic parallel plate, 3 rads-', 225°C;Standard Error = 18.50) and large deformation mechanical testing (E, 20% engineering strîin, room temperature; Standard Error = 157.27). O 0-05 0.1 0.13 0.2 0.25 0.3 0.35 0.4 Volume Fractiion for Waxy Corn Starch Figure 5.6: Gel moduli for gelattinlwaxy corn starch composite gels obtained from small amplitude oscillatory testing ( G' , 6 cm diameter acrylic parallel plate 3 rad~~-'.2Z.5~C,Standard Error = 11.60) and large deformation mechanical testing ! ( E . 20% engineering strain, room temperature, Standard Error = 8145.69). Blending law analysis, as reported by Abdulmola et al- (1996) and Mohammed et aL (1998), can be an important tool for understanding the behaviour of phases in a gelatinlstarch composite gel. Figures 5.7 and 5.8 illustrate the measured moduli of gelatinlwaxy starch and gelatin/regular starch composite gels, respectively. Furthermore, these figures show the calculated values for the isostrain, isostress and bicontinuous network rnodels. In the case of gelatin/waxy corn starch gels the gelatin is more rigid than the starch filler systern thus, the modulus would be expected to be closer to the upper- bound determined by the isostrain rnodel (see equation (1)) (Abdulmola et al., 1996). Figure 5.7 shows that the gelatidwaxy corn starch composite has moduli near the isostrain moduli and the gelatin phase mod uli, thus gelatin dominates the modulus of the system. However, if the gelatin is less rigid than the gelatinized starch filler, as in the case of gelatinlregular starch gels, the gel modulus is expected to lie closer to the isostress rnodel (see equation (2)) if the gelatin dominates the rheology of the systern. The isostress model predicts the lower-bound of gel modulus and Figure 5.8 shows that the rnoduli for the gelatinlregular starch gels closely agree with calculated values from the isostress model. Exarnination of the blending law analysis for gelâtin starch composite gels (Abdulmola et al., 1996) and bicontinuous network theory (Davies, 1971; Mohammed et al., 1998) was performed once the effective concentrations of both the waxy and regular corn starches were deterrnined. The swelling ; . Gelatin Phase (WCS) - -&-. WCS Composite WCS Atone - - - - - lsostress (WCS) -Isostrâin (WCS) _------Bicontinuous (WCS) ; O 1 2 3 4 5 6 Waxy Corn Starch Concentration (% whiv) Figure 5.7: Dynamic storage moduli for 5% gelatin with gelatinized waxy corn starch, WCS (A),in comparison with the theoretical moduli, isostrain and isostress, from the moduli for starch and gelatin alone. i -, , -, Gelatin Phase (RCS) RCS Alone I ...... lsostress (RCSJ - - ,, , RCS Composite -Isostrain (RCS) - - _ - ., , Bicontinuous (RCS) 1 O 1 2 3 4 5 6 Regular Corn Starch Concentration (% wlw) Figure 5.8: Dynamic storage moduli for 5% gelatin with gelatinized regular corn starch, RCS (A),in cornparison with the theoretical moduli, isostrain and isostress, from the moduli for starch and gelatin alone. volumes for each starch indicated an effective concentration of -44.70% (wlw). G' at the effective concentration of waxy corn starch was 6.7 Pa (3 rads1,225°C). whereas the modulus for regular corn starch was 1230 Pa (3 rad-s-', 22.5"C). Both gelatinlwaxy corn starch (Figure 5.7) and gelatinlregular corn starch (Figure 5.8) results indicated that the gelatin biopolymer dominated the rheology of the system up to the 1% starch concentration level. Above 7% (VF=0.07) regular corn starch level, the mechanical properties of the gel continued to be dominated by the gelatin but the starch played a role in increased rigidity. Also, above the 1% (VF=0.07) waxy corn starch concentration level, the mechanical properties of the gels continued to be dominated by gelatin but the waxy corn starch had a Iimited effect on the rigidity of the system. The regression analysis for the Grdata gathered over the series of waxy corn starch volume fractions (Figure 5.6) showed that the rigidity of the of system remained essentially unchanged by the addition of waxy corn starch. Study of the volume fraction occupied by the reguiar and waxy corn starches in gelatin indicated that after the addition of the starch to the gelatin, the starch continued to absorb the free water avaifable to the gelatin. This caused the gelatin concentration to increase as the volume fraction of the starch increased. The negative effect on the rigidity of the gelatin/waxy corn starch gel with increased volume fraction of waxy corn starch may be due to the competing effects of increasing gelatin concentration and strength as a result of the starch taking up more of the available water and the interruption of the gelatin continuous matrix. This may have inhibited the development of rigidity in the composite gel, As shown in Figures 5-7 and 5.8, the bicontinuous network rnodel underestimated the composite rnoduli for both gelatinlregular starch gels and gelatin/waxy starch gels. The correlation coefficients (pe0.05) were -0.64 and -0.013 for the regular and waxy corn starches, respectively. The poor fit of the bicontinuous network rnodel to the data suggests that one of the two phases is discontinuous within the system. In the case of gelatinlwaxy starch gels (Figure 5.7) the rnoduli, determined by oscillatory testing are between the calculated values of the isostrain (maximum rnoduli) and bicontinuous models. The moduli of the gelatidwaxy starch gels were dorninated by the gelatin rnatrix since these moduli were closer to the gelatin phase and did not fit the bicontinuous model. This result is similar to that obtained by Abdulmola et al. (1996) in that gelatin dominated the modulus of both the gelatinlwaxy corn starch and gelatinlcross- Iinked waxy corn starch gels. Gelatin/regular starch gels show moduli near the calculated isostress values (minimum moduli) and the rneasured moduli for the gelatin phase (Figure 5.8). As a result, this suggests the gelatin is the dominant polymer in a gelatinlregular starch system. This supports the theory of phase separation within each of the composite gels developed later in the discussion of the gel-sol transition. 5.3.2 Constant Rate Uniaxial Testing Figure 5.9 is a typical stress-strain curve obtained for uniaxial compression of gelatin and gelatinfstarch composite gels. From the stress-strain data obtained from the gelatinfstarch composite gels, the apparent elastic moduli were measured in the linear part of the stress-strain cuwe within the Iinear viscoelastic Iimits of the gels. Reported values of the apparent elastic modulus were measured at 20% strain for both gelatin/regular corn starch and gelatinfwaxy corn starch gels. Young's moduli for gelatinfregular corn starch gels demonstrated a Iinear dependence on starch concentration (Figure 5.5). The linearity of this relationship indicates that as the volume fraction of starch increases so does the strength of the gel. Uniaxial compression showed that over al1 volume fractions of waxy corn starch Young's rnodulus rernained essentially unchanged (Figure 5.6). As the concentration of starch within the gelatin matrix increased, the concentration of the gelatin also increased. This was determined from the volume fraction studies for both regular and waxy corn starch in gelatin. Despite the increase in starch, the void volume in the gelatin matrix was principaliy filled by water and the overall structure (Figure 5.1 E) was similar to the overall structure of 5% gelatin (Figure 5.1A). It seems that the addition of waxy corn starch negated any effect on rigidity due to the increase in gelatin concentration. Engineering Strain Figure 5.9: A typical stress-strain curve illustrating the large deformation behaviour of gelatin and filled gelatin gels (3 mm-min-') at room temperature obtained for determination of elastic moduli. 5.3.3 Comparison of Results Since Young's Modulus cannot be directly compared to the shear storage rnodulus it was necessary to convert the dynarnic shear storage rnoduli, obtained from small amplitude oscillatory rheometry, to shear moduli ( G ). Huseby and Blyler (1967) proposed a relationship which allowed for the quantity G to be written in terms of the dynamic quantity G' . It was found that: (33) where o = j . Thus over a given frequency range, G will equa1 O r exceed G' depending on the value of 6 (Huseby and Blyler, 1967). Table 5.1 shows the values of G . These were calculated by substituting the experimentally determined G' and tan& data, measured from smali amplitude oscillatory testing at 3200 pNm, 3 rads-' and 22.5"C,into equation (33). The calculated value of G differed only slig htly from the measured Gf since 6 remained very close to zero. The close correspondence between calculated G and measured G' values (Table 5.1) indicates that the same trends seen for the G' moduli over increasing regular and waxy starch volume fractions will also occur for the G moduli. A comparison between G' and E' or G and E can be established by knowing the Poisson ratio (u ) and the relationships described in equations (5) and (6). This can provide a means for measuring the correlation of the results obtained from different mechanical testing methods. E'is the dynamic compression modulus obtained from dynamic uniaxial compression. Table 5.1: A cornparison of typical dynamic shear storage moduli ( G' ) obtained from small amplitude oscillatory rheometry (3 rads', 22.5 OC) to theoretical shear moduli ( G ) for various gelatin and gelatidstarch composite gels. Sample T~W) 1 G' (~a) 1 G (~a) 5% Gelatin 1 0.03288 79-6 79 -7 5% Gelatin/l % Regular Corn Starch 0.02390 104.6 104.6 1 5% Gelatinl3% Regular Corn Starch 1 0.02559 / 299.4 1 299.5 1 5% Gelatin/5% Regular Corn Starch 1 0.0371 0 1 467.5 1 467.8 1 5% Gelatin/l% Waxy Corn Starch 1 0.02986 1 128.4 1 128.4 1 5% GelatinM% Waxy Corn Starch 1 0.05654 1 116.8 / 117.0 1 5% Gelatin/5% Waxy Corn Starch 1 0.1406 / 72.5 1 73.2 There was a Iinear correlation between the dynamic shear storage modulus (G' ) and Young's Modulus (E) for gelatinlregular starch composite gels (Figure 5.10). The linear correlation coefficient was found to be 0.97 (p<0.05). This corresponds well to the work of Tung et ai. (1991) who, as part of their experimental work, compared the moduli of deformability to the storage moduli of surimi gel samples. The linear increase in gelatinJstarch composite gel rigidity with increased regular starch volume fraction, indicates that the regular corn starch behaves as an active filler phase. The addition of regular corn starch resulted in the Figure 5.10: Correlation between the dynamic shear storage modulus ( G' ) and Young's Modulus (E) for gelatinlregular corn starch gels (Standard Error = 32.1 1). increased rigidity of the composite gel beyond the more compliant gelatin gel as the Iikely result of the synergistic effect with a secondary amylose gel. Differences between the mechanical properties of the gelatinlwaxy starch gels measured by oscillatory testing and uniaxial testing were observed (Figure 5.6). A cornparison of these two methods showed that those gels having waxy starch concentrations between O and 5% (VF=0.34) demonstrated trends in the dynamic storage moduli and Young's moduli that did not correlate (Figure 5.11). No change in response was observed for the gelatin/waxy corn starch gels. The moduli remained unchanged and thus a lack of correlation between the oscillatory testing data and the uniaxial testing is consistent with the observations for both tests. These tests involved strains that were kept within the Iinear viscoelastic region- As a result, the structure of the sarnple shouId be maintained throughout the course of the experiment since the foming structures undergo minimal disturbance and they are not destroyed during the course of the experirnent (Beveridge and Timbers, 1985). Similar to the trend in the storage moduli obtained in the present study from the oscillatory testing of gelatin/waxy starch composite gels, Alloncle and Doublier (1991) showed results with locust bean gum and corn starch while Mohammed et ai. (1998) found similar results with agarose and waxy corn starch. The response in observed moduli measured by Mohammed et al. (1998), similar to the results presented here, were not expected since the data fit different models as the concentration of the starch increased. The negligible 69 change of composite moduli could not be explained in ternis of isostrain or isostress blending laws (Mohammed et al., 1998). The negative effect on the moduli for the gelatidwaxy starch gels indicates that the waxy starch behaves Iike a deformable filler particle. Figure 5.1 1: Correlation between the dynamic shear storage modulus ( G' ) and Young's Modulus (E) for gelatinfwaxy corn starch gels (Standard Error = 16.68). 5.3.4 Dynamic Compression Testing The development of a methodology for dynamic compression testing required that ideal elastic and viscous samples be tested to help interpret the viscoelastic element. The rheological components for the three test standards are provided in Table 5.2. Dynamic uniaxial compression of three rnodel systems yielded phase angle (6) responses consistent with those for ideal elastic, viscous, and viscoelastic systems: O", 90" and 45"respectively. Table 5.2: A comparison of phase angles and maximum force values obtained from three ideal mechanical models (0.1 Hz, room temperature). Spring (Elastic) 1 0.15 7.37 Dashpot (Viscous) 88.53 8.06 Kelvin Body (Viscoelastic) / 44.09 1 9.34 E' and E" are equal to the force as a function of cross-sectional area (F(A))through stress calculations. In these stress calculations, the cross- sectional area (A) is undefined for the mode1 systems. As a result, a unit measure was assumed for both the cross-sectional area and the maximum strain (E~) To calculate Er and E" equations (25) and (27) would be used but, since A equals one and go is a scaling factor the systems are compared by examining the maximum force values ( Fm, ). The following relationship was used to calculate the theoretical Fm, value for the viscoelastic body using the measured Fm, values for the elastic and viscous bodies: The calculated maximum force value was 10.9 N. This is comparable to the measured maximum force value for the viscoelastic body (Table 5.2). The results indicate that the dynamic compression testing equipment used in this study was able to provide an accurate measure of the moduli for each type of system. Although results were obtained for a 5% gelatin gel, the validity of the information was in doubt since there was considerable electricat noise affecting the load data and a lack of sensitivity in the load cell. A 5 kN load ceIl was used in this study. If a load ce11 with increased sensitivity was used, the findings for the mode1 systems suggests that representative data could be obtained for weak systems such as gelatin and gelatinktarch composite gels. However, due to time constraints on this research a load cell could not be acquired in an acceptable amount of time. 5.4 Gel - Sol Transition Figure 5.12 shows a typical heating profile for gelatin and gelatinktarch composite gels. Measurement of the gel-sol transition temperature required knowledge of the location of the G'- G" intercept. Figure 5.13 is a typical illustration of the region where the dynamic shear storage modulus (G' ) 72 15 20 25 30 35 40 Temperature (OC) Figure 5.1 2: Typical temperature profile curve for the heating of a gelatinlcorn starch composite gel for measuring the gel-sol transition temperature (temperature ramp = 0.5 Co-min") 29 31 Temperature (OC) Figure 5.13: A more detailed view of the G' - G" intercept region, from Figure 5.12, of a gelatinlcorn starch gel (temperature ramp = 0.5 Co-min-') intercepts the shear loss modulus ( G" ) for gelatinlregular starch and gelatinlwaxy starch geis. The temperature at the intercept point is where the polymers underwent a transition frorn an elastic material (a gel) to a viscous material (a solution), referred to as the gel-sol transition. The G" curve was assumed to be Iinear with a slope near zero at G" approaching O Pa at temperatures above that at the intercept point. To find the equation of the intercepting Gy heating cuwe, a Iinear portion of the curve was selected and Iinear regression analysis was performed on the selected points. Frorn resulting slope and intercept a Iinear equation was derived ( Gr = rnT + b) and the gel-sol transition ternperature was calculated for Gr = G" = O for each G' curve. Table 5.3 shows the gel-sol transition temperature data obtained for gelatinlregular corn starch and gelatinlwaxy corn starch composite polymer gels at four different starch concentrations (0, 1, 3 and 5%). Statisticai analysis of the gel-sol transition ternperature data, using Dunnet's test (Kuehl, 1994) (see Appendix 1) to compare al1 treatments to the 5% gelatin control, indicated no significant differences between melting temperatures of gelatin gel and the composite polymer gels with increasing fraction of regular or waxy corn starch in the dispersed phase (p4.01). At the melting point, gelatin undergoes a helix-coi1 transition and thus the gel network collapses. The observed helix-coi1 transition temperature for gelatin (Table 5.3) corresponded well to the values found in the literature. Djabourov et Table 5.3: Gel-sol transition temperatures for both gelatin and gelatinktarch composite gels obtained from the G' - G" cross-over through small amplitude oscillatory rheornetry (3 rads-', 3200 pNm, 0.5 Gomin"). 1 1 Gel-Sol Transition Temperature (OC) Gel Sample Rep 1 Rep 2 Rep 3 Mean* 2 l Std. Dev. L 1 I 1 5% Gelatin 1 27.1 1 27.3 1 27.4 I 5% Gelatinl 27.4 27.6 27.3 1% Regular Corn Starch 5% Gelatin/ 27.9 27.9 27.7 3% Regular Corn Starch 5% Gelatinl 28.6 28.0 28.8 5% Regular Corn Starch 5% Gelatinl 27.6 27.3 1% Waxy Corn Starch 5% Gelath/ 1 27.5 I3% Waxy Corn Starch i 27-6 I 5% Gelatinl / 27.8 1 28.7 1 28.4 5% Waxy Corn Starch F * Mean values were not significantly different (p<0.05) al. (1988a) reported that the transition temperature range for gelatin was 27.4 - 27.5" C using both rheological and polarimetric methods. The temperature profiles of the composite gels closely resembled the temperature profiles of the gelatin gek Melting of the gelatinlstarch gels would involve another domain occurring if the composite gelatinlstarch systern were bi- continuous. The second domain would represent the melting of the starch phase. The lack of a shoulder on any of the temperature profiles appears to indicate that the system was made up of one continuous phase, in this case gelatin. The presence of one continuous phase in the gelatinktarch composite gels demonstrates that the gel contains a dispersed filler phase (Brown ef al, 1995). The presence of a dispersed phase in a continuous matrix indicates that the systern is thermodynamically unstable. As a result, phase separation disrupts the matrix. Further examination of the gel-sol transition temperature for gelatinlcorn starch composite gels involved Differential Scanning Calorimetry. DSC testing met with little success. Scanning rates of 1 and 5 CO/min were attempted. The lower scanning rate generated noise as the therrnogram resembled a "saw-tooth" and the higher scanning rate did not pick-up a signal. The lack of a clear signal was attributed to an insufficient protein concentration where the recommended protein concentration for DSC is 15% (Maurice and Page, 1983). The prospect of using a microcalorimetric method to study the gel-sol 77 transition was also explored but the gelatin concentration (5% w/w) of the gels was approximately one hundred times too great. The excessive concentratiron of the gelatin polymer would not permit effective cleaning of the test charnber af the microca lorimeter. 6. CONCLUSIONS The mechanical properties of gelatin/regular corn starch and gelatidwaxy cornstarch gels were studied to compare the results of protein-polysaccharide interactions in the two systems, using both dynamic and uniaxial rheological methods. Cryo-SEM of composite gels revealed considerable variation in the topography of the geIatin/regular starch (Figure 5.1 D) and gelatinfwaxy starch gels (Figure 5.1E). These variations rnay explain potential phase separation in the gelatinfstarch composite gels and ultimately aid in understanding the differences in their mechanical properties. In order to study phase separation in more detail, it would be necessary to stain the sarnple for protein and starch to identify their locations in the gel structure. That phase separation occurred within the gelatinlstarch systerns was supported by small amplitude oscillatory rheometry where gelatin appeared to dominate the mechanical properties of the gel. As well, those data failed to correlate with the bicontinuous network mode1 indicating that the composite gel was composed of a gelatin continuous matrk with a diçpersed starch phase. The rigidity of the gelatinlregular corn starch gels increased linearly. This indicated a synergistic interaction between the protein and polysaccharide cornponents as a result of a secondary amylose gel being formed around the gelatin network. The regular corn starch thereby behaved like a rigid filler particle. Gelatin/waxy starch gels demonstrated no change in gel rigidity over 79 the waxy corn starch concentration range. This demonstrated that no response was obtained as the concentration of waxy corn starch increased. Perhaps, given more sensitive equipment, slight contributions to composite gel rigidity by the waxy corn starch might become more discernable. Large deformation uniaxial compression data for gelatinlregular starch gels correlated well with the respective data from small amplitude oscillatory rheometry. This supported the trend for increased rigidity of gelatinlregular starch gels with increasing starch and the development of the secondary amylose gel forming a further supporting matrix around the gelatin matrix. Uniaxial compression of gelatinlwaxy corn starch gels, again, showed little change in the moduli of the systern as the starch concentration increased. This may be due to an interrupting effect of the waxy corn starch on the gelatin matrix. These data did not correlate with the results from small amplitude oscillatory rheometry. This was to be expected since the contribution of the waxy corn starch could not be readily determined to be responsible for any variation in the moduli. Experimentation with dynamic uniaxial compression demonstrated the ability to rneasure elastic, viscous and viscoelastic bodies. Better data would likely be the result if a more sensitive load cell were available with the present equipment. A major advantage of this method over oscillatory rheometry is the ease of sample preparation and sample loading ont0 the equiprnent platen. Finally, the Gr- G" crossover results from the investigation of the gel-sol 80 transition of gelatin and gelatinlstarch composite gels supported the theory that phase separation occurred in the protein-polysaccharide gels. The Gr- temperature profile, for gelatin/regular starch and gelatin/waxy starch gels, illustrated results consistent with the gel-sol trans ition of gelatin alone. This indicated that gelatin behaved as the continuous phase in each composite gel. In future studies, it would be interesting to investigate the mechanical properties of protein-polysaccharide gels using dynamic uniaxial compression. The sample size flexibility of the dynamic servohydraulic mechanical testing equipment could provide a means of rneasuring dynamic quantities for in-situ food samples. Obtaining further information about the mechanical and structural properties of food systems, as well as rnethods for obtaining this information is important to the developrnent of new food products. The gelatinlstarch composite gels have provided an interesting phase-separated protein-polysaccharide system for cornparing the mechanical properties of each gel rneasured by dynamic and uniaxial tests. 7- REFERENCES Abdulmola, N.A,, Hember, M.W.N., Richardson, R.K. and Morris, E.R. 1996. Application of polymer blending laws to starch-gelatin composites. Carbohydr. Polym. 31: 53-64. 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In Proceedings of the Fifieenth Annual Conference of the Tropical and Subtropical Fishenés Technological Society of the Amencas, ed. S.W. Otwell- University of Florida, Gainsville, FL. van Vliet, T. 1995. Mechanical Properties of Concentrcited Food Gels- ln Food Macrornolecules and Colloids, ed. E. Dickinson and D. Lorient. Royal Society of Chemistry, Cambridge, UK, pp. 447-461. Van Wazer, J.R., Lyons, J.W., Kim, K.Y. and Colwell, R.E. 1966. Viscosity and Flow Measurement: A Laboratory Handbook of Rheology. Interscience Publishers, New York, NY. Appendix 1 Cornparison of the Gel-Sol Transition Temperature of 5% GelatinIStarch (1, 3, and 5% Regular and Waxy Starch) Composite gels to 5% Gelatin Gel. The Dunnett Test Test for significant difference in the gel-sol transition temperatures at the G' - Gr' cross-over. - the Dunnett Criterion is, where da,,,,is obtained from the table for 6 treatments. 14 degrees of freedorn for error and a 0.05 level of significance. s2(Mean Squared Error), obtained from ANOVA is 0.65 and the number of replications (r) is 3. Sample Average Gel-Sol Transition Temperature Gelatin 1 5% Gelatinll% Regular Corn Starch 1 27.4 1 O. 1 1 5% Gelatin/3% Regular Corn Starch 27.8 0.5 5% Gelatin/5% Regular Corn Starch 28.5 1-2 5% Gelatinll % Waxy Corn Starch 27.4 O. 1 5% Geiatin/3% Waxy Corn Starch 27.6 0.3 5% Gelatin/S% Waxy Corn Starch 28.3 1-0 Cornparison of: 5% Gelatin and 5% Gelatin/l% Regular Corn Starch 0-1 < 1.92 =. NOT significant 5% Gelatin and 5% Gelatin/3% Regular Corn Starch 0.5 c 1-92 .-.NOT significant 5% Gelatin and 5% GeIatin/5% Regular Corn Starch 1.2 c 1.92 =. NOT significant 5% Gelatin and 5% Gelatin/l% Waxy Corn Starch 0.1 c 1.92 =. NOT significant 5% Gelatin and 5% Gelatin/3% Waxy Corn Starch 0.3 < A -92 :-NOT significant 5% Gelatin and 5% Gelatin/5% Waxy Corn Starch 0.5 < 1.92 .-. NOT significant Therefore there was no significant difference between the gel-sol transition temperatures of the gelatidstarch composite gels and gelatin alone.