RHEOLOGICAL STUDIES on the INTERACTION of XANTHAN and LOCUST BEAN GUM in AQUEOUS DISPERSIONS by SONIA DENISE GATCHAIR B.Sc, Spec
RHEOLOGICAL STUDIES ON THE INTERACTION OF
XANTHAN AND LOCUST BEAN GUM IN AQUEOUS DISPERSIONS
by
SONIA DENISE GATCHAIR
B.Sc, Special, University of the West Indies, 1979
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES
(Department of Food Science)
We accept this thesis as conforming
to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
March, 1985 21
© Sonia Gatchair, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of pnnn RHTT'NTH'R
The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3
Date WioL SLffj iqge,
DE-6 (3/81) - ii -
ABSTRACT
Aqueous dispersions of xanthan and locust bean gum, in combina• tion, show a synergistic increase in viscosity. At sufficiently high concentrations, firm gels are formed, although neither component forms true gels when alone. The actual molecular processes resulting in this phenomenon are still incompletely understood and Theological studies can provide some clues to the mechanism of the interaction. Rheological properties of the polysaccharide blend were therefore investigated.
Moisture, ash and inorganic elements and protein content as well as the intrinsic viscosity of the individual polysaccharides were determined. Dynamic viscoelastic properties of dispersions prepared from the polysaccharide blend were evaluated in four solvent treatments capable of disrupting weak intermolecular bonds. The effect of polysaccharide concentration, temperature, ionic strength, pH, ratio of mixing of the two gums and urea concentration on steady shear rheological properties were evaluated in a fractional factorial experiment. More detailed studies were carried out on the effects of temperature at two levels of concentration and ionic strength.
Solvent treatments significantly affected the viscoelastic properties of xanthan-1ocust bean gum solutions. At 20°C and under the conditions used, dipole interactions appeared to be the primary force responsible for stabilizing the xanthan-1ocust bean gum interacting system. Hydrogen bonding and hydrophobic interactions seemed to play less important roles. Under conditions of low ionic strength and increased temperature, the interactions were lost and polysaccharide solution behavior passed from that of a viscoelastic solid to that of a viscoelastic liquid. At 60°C and at high ionic strengths, hydrophobic interactions may become important in the stabilization of the three dimensional gel network.
Temperature effects on steady shear viscosity of xanthan-locust bean gum solutions were dependent on the concentration and ionic strength of the system. In general, steady flow properties were comparable to the reported behavior of xanthan solutions and so reflected the weakness of the interaction (dipole interactions) between the two polysaccharides. - iv -
TABLE OF CONTENTS
Page
ABSTRACT 1 i
LIST OF TABLES vi
LIST OF FIGURES vii
ACKNOWLEDGEMENTS ix
INTRODUCTION 1
LITERATURE REVIEW 3
A. General 3 B. Molecular Structure and Conformation of the Polysaccharides 5 C. Xanthan-Locust Bean Gum Interaction 9
D. Rheological Studies 13 E. Solvent Treatments 16
MATERIALS AND METHODS 18
A. Chemical Composition and Physical Properties 18
1. Moisture 18 2. Ash and Inorganic Components 18 3. Nitrogen Content 19 4. Intrinsic Viscosity 20 5. Determination of pH 22
B. Rheological Studies 22
1. Sample Preparation...... 22
a) Viscoelastic Studies 22 b) Steady Shear Flow Studies 23
2. Rheological Measurements 25
a) Viscoelastic Properties 25 b) Steady Shear Flow Properties 27
3. Statistical Analysis 29
a) Evaluation of Viscoelastic Properties 29 b) Steady Shear Studies 30 - v -
Page
RESULTS AND DISCUSSION 31
A. Composition of Materials 31
1. Moisture 31 2. Ash and Inorganic Components 31 3. Nitrogen Content 35 4. Other Components. 35 5. Intrinsic Viscosity 36
B. Rheological Studies 42
1. Viscoelastic Properties of the Polysaccharides.... 42
a) Xanthan 42 b) Locust Bean Gum 45 c) Xanthan-Locust Bean Gum Solutions 46 d) Frequency Effects on Entanglements and Cross-Linkages 48 e) Effect of Solvent Treatment and Temperature... 49
i) Effect of Urea 49 ii) Effect of Added Electrolytes 54 iii) Effect of Alkali 57 iv) Temperature Effects 59
f) Limitations 69
i) Failure to Meet Some Assumptions in Viscoelastic Theory 69 ii) Effect of Added Solutes 71 iii) Choice of Frequency 72 iv) Nature of the Crosslinkages 74
2. Steady Shear Flow Properties 76
a) General Considerations 76 b) Flow Properties Under Different Conditions.... 79
CONCLUSIONS 90
REFERENCES 92
APPENDICES 98 - vi -
LIST OF TABLES
Page
Table 1. Treatment combinations for xanthan-1ocust bean gum solutions for the fractional factorial experiment according to the scheme of 13 Taguchi L27 (3 ) 24
Table 2. Summary of variables and levels used in Taguchi's Fractional Factorial Experiment 28
Table 3. Summary of non-carbohydrate components for xanthan and locust bean gum powders 32
Table 4. Intrinsic viscosity and pH for xanthan and locust bean gum solutions 39
Table 5. Summary of flow parameters (Y = 0.2 - 175 s"1) obtained from Taguchi1s fractional factorial experiment 80 - vii
LIST OF FIGURES
Page
Figure 1. Pentasaccharide repeating unit of xanthan 5
Figure 2. Representative structure of locust bean gum 6
Figure 3. Representation of xanthan-1ocust bean gum interaction (from Dea et al., 1977) 10
Figure 4. Combined Huggins' (•) and Kraemer's (o) extrapolation to intrinsic viscosity for xanthan (0.1 M NaCl, 20°C) 37
Figure 5. Combined Huggins' (•) and Kraemer's (O) extrapolation to intrinsic viscosity for locust bean gum (0.1 M NaCl, 20°C) 38
Figure 6. Dynamic storage and loss moduli for xanthan, locust bean gum and xanthan-1ocust bean gum solutions (0.4% in water at 20°C) 43
Figure 7. Loss tangent for xanthan, locust bean gum and xanthan-1ocust bean gum solutions (0.4% in water at 20 °C) , 44
Figure 8. Dynamic storage (a) and loss (b) moduli for xanthan-locust bean gum solutions in water, 8 M urea, 0.6 M KC1 and 1 M K0H at 20°C 50
Figure 9. Dynamic storage (a) and loss (b) moduli for xanthan-locust bean gum solutions in water, 8 M urea, 0.6 M KC1, and 1 M K0H at 60°C 51
Figure 10. Loss tangent for xanthan-locust bean gum solutions in water, 8 M urea, 0.6 M KC1 and 1 M K0H at (a) 20°C and (b) 60°C 52
Figure 11. Effect of temperature on the storage modulus for xanthan-locust bean gum solutions in water 61
Figure 12. Effect of temperature on the loss modulus for xanthan-locust bean gum solutions in water 62
Figure 13. Effect of temperature on the loss tangent for xanthan-locust bean gum solutions in water 63
Figure 14. Effect of temperature on the storage modulus for xanthan-locust bean gum solutions in 0.6 M KC1... 64 vi i i -
Page
Figure 15. Effect of temperature on the loss modulus for xanthan-1ocust bean gum solutions in 0.6 M KC1... 65
Figure 16. Effect of temperature on the loss tangent for xanthan-locust bean gum solutions in 0.6 M KC1... 66
Figure 17. Rheograms for xanthan-locust bean gum solutions at 20°C plotted according to the power law flow model 77
Figure 18. Effect of temperature on flow behavior index of xanthan-locust bean solutions 82
Figure 19. Effect of temperature on the consistency coefficient of xanthan-locust bean gum solutions 83
Figure 20. Effect of temperature on the apparent viscosity (y = 50 s_1) of xanthan-locust bean gum solutions 84 - ix -
ACKNOWLEDGEMENTS
The author wishes to express her sincere appreciation to Dr.
Marvin A. Tung, Department of Food Science, for his advice and encouragement throughout the course of this work. Thanks are also extended to Dr. Donald E. Brooks of the Department of Pathology and Dr.
Shuryo Nakai and Dr. William D. Powrie of the Department of Food Science for their interest and review of this thesis.
The guidance of the Statistical Consultation Laboratory as well as the help and support from members of the Department of Food Science is also greatly appreciated. The assistance of Mr. A. Paulson, who provided the computer program for the calculation of viscoelastic parameters is also gratefully acknowledged.
The author also wishes to acknowledge the Canadian Commonwealth
Scholarship and Fellowship Committee and the Natural Sciences and
Engineering Research Council of Canada for provision of financial support for these studies. - 1 -
INTRODUCTION
Hydrophilic colloids are long chain polymers which dissolve or disperse in water to give a thickening or viscosity producing effect. A
few colloids, also have the ability to form firm, rigid networks which
trap or immobilize water in large proportions compared to their weight.
Rheological measurements provide an objective assessment of these
functional properties and in addition, can provide information on the
conformations and dimensions of these macromolecules in solution.
The ability of a colloid to form a three dimensional matrix or
gel is dependent on the development of suitable intermolecular
interactions. These materials cannot be adequately described in terms
of the classical models of an elastic solid or a Newtonian fluid.
Rheologically, they are termed viscoelastic, that is, materials
possessing both viscous and elastic properties.
Several techniques are available for characterizing viscoelastic
behavior. Dynamic oscillatory shear is non-disruptive and provides a measure of both the solid and liquid-like components of material
response to applied stress. It provides information on molecular movements at both long (low frequencies) and short (high frequencies)
times. These material properties are inherently dependent on the
strengths and lifetimes of bonds present in the system. Thus, under
suitable conditions, this technique has the potential for providing
information on the nature of bonds or forces involved in stabilizing an
interacting system. Knowledge of the forces involved and their relative - 2 -
importance can provide information on the actual interaction between atoms or groups of atoms on the macromolecules, thereby contributing to an understanding of the mechanism of gelation.
Steady shear flow is another technique used in characterizing rheological behavior. It can result in the disruption of weak inter-molecular interactions, but at low shear rates, it provides information on material behavior during pouring or stirring as well as for the objective assessment of organoleptic properties. Rheological properties, at higher shear rates can be used in predicting material behavior and energy requirements during processing operations such as extrusion, pumping or filling. The determination of these properties and factors affecting them, are therefore useful in the practical application of the colloidal system.
In this study, the principal forces responsible for stabilization of the xanthan-locust bean gum interacting system were determined.
Changes in dynamic viscoelastic properties in different solvents were monitored, in order to provide this information. In addition, the effect of several factors, on steady shear rheological properties at low shear rates, were evaluated. The conditions chosen, were such that the effects of the reported conformational change of xanthan on the interaction, could be evaluated. - 3 -
LITERATURE REVIEW
A. General
Hydrophilic colloids, hydrocolloids or gums are terms used to describe materials widely used in the food and other industries to produce suitable structural and textural characteristics in various products. In addition to their thickening, suspending and gelling properties, they are used as emulsion stabilizers, as protective coatings, as well as for a host of other functions (Glicksman, 1982).
Hydrocolloids are generally considered as food additives and, despite their important functional role, are used in only small quantities in foods. Thus in 1979, the estimated world use of hydro- colloids in food was just under 500 million pounds (Glicksman, 1982) with starch, gelatin and cellulose derivatives comprising the bulk of the material used.
Apart from gelatin, a proteinaceous hydrocol1oid, all commonly used gums or hydrocolloids are polysaccharide in nature. The functional properties of these polysaccharides are inherently dependent on their chemical structures, not only in the nature of component sugar residues but also in the relative orientation of the glycosidic bonds to and from each sugar ring (Aspinall, 1982). Structural types include polysacchar• ides having straight, unbranched polymer molecules formed from a single type of sugar unit, i.e., linear, homopolysaccharides such as cellulose; homopolysaccharides with branched structures, e.g., the amylopectin component of starch; and heterotypic polysaccharides which contain two or more different types of sugar units. These may be arranged in a - 4 -
relatively simple manner to give a fairly obvious repeating unit, as in agarose. The repeating unit may be masked, as in A-carrageenan, or may not be generated at all, e.g., the galactomannans (Rees, et al. 1982).
In alginates, homopolymeric sequences as well as heterotypic sequences may arise in the same molecule.
The sequence of covalently bound monomer units in the polysaccha•
ride chain forms the primary structure; secondary structure defines any geometrically regular arrangement in space that this sequence may adopt; and the tertiary structure defines the way in which these arrangements
pack together to form a higher level of organization (Rees and Welsh,
1977). For cellulose, a g-1+4 linked polysaccharide, the most likely
secondary structure is that of an extended ribbon (Rees et al. 1982).
Various members of the carrageenan family form single or double
helices. Secondary structures in polysaccharides are stabilized by a favorable energy balance resulting from interactions such as hydrogen bonding, dipolar and ionic interactions and solvent effects. The co-operative interaction between residues on long, regular sequences of the polysaccharide chain are required to bring about stabilization and outweigh the conformational entropy of the polysaccharide (Rees, 1972;
1977; Rees and Welsh, 1977). The ordered, secondary structures are more favored in the solid state but may persist in solution. In some
instances, more favorable solvent-polymer interactions and the natural flexibility of the molecule can lead to the development of a disordered
or random coil conformation in solution, e.g., the galactomannans, guar
and locust bean gum (Morris et al. 1981). The interaction of like and
unlike polysaccharide chains in solution may lead to the development of - 5 -
tertiary structures in solution. This results in solution properties which give rise to the functional characteristics of polysaccharides.
Chemical modification and derivatization serve to improve functional properties such as water solubility (cellulose to carboxymethyl cellulose) or acid resistance (alginate to propylene glycol alginate) (Glicksman, 1982).
B. Molecular Structure and Conformation of the Polysaccharides
Xanthan gum is the extracellular polysaccharide produced by the microorganism Xanthomonas campestris. It is a branched, acidic polysaccharide with a backbone consisting of g-1+4 linked glucose residues, as in cellulose, but having trisaccharide side chains of mannose-glucuronic acid-mannose residues on every other glucose residue to give the pentasaccharide repeating unit shown in Figure 1.
> 4)-g-D-Glcp-(l + 4)-3-D-Glcp-(l -»• 3 + 1 a-D-Manp-6-0Ac 2 + 1 3-D-GlcAp 4 + 1 3-D-Manp
4^ 6
C"H7 ^COOH
Figure 1. Pentasaccharide repeating unit of xanthan. - 6 -
Mannose residues closest to the main chain may be substituted with acetate residues (6-0-acetyl-D-mannose) while the terminal mannose
residue may be substituted with pyruvate to give a sugar ketal
(4,6-(l'-carboxyethylidene)-D-mannose) (Jansson et al. 1975; Melton et
al. 1976). The degree of pyruvate and acetate substitution varies
according to the bacterial strain, fermentation conditions and history of the gum and gives rise to differences in the solution properties of
xanthan (Sandford et al. 1977; Smith et al. 1981). Xanthan gum binds
cooperatively with polysaccharides having unbranched sequences of 3-1+4
linked D-glucose, D-mannose or D-xylose residues (Morris et al. 1977).
Locust bean gum (from the legume, Ceratonia siliqua) is a
branched polysaccharide with a backbone of 3-1+4 linked D-mannose
residues. The mannose backbone is partially substituted at 0-6 with a-D
galactosyl units (McCleary et al. 1984). A representative structure is
shown in Figure 2.
a-D-Gal p a-D-Gal p 1 1 + + 6 6 4)-3-D-Manp- (1 4)-3-D-Manp-(l •»• 4)-3-D-Manp-(l 6 6 + + 1 1 a-D-Galp a-D-Galp
Figure 2. Representative structure of locust bean gum. - 7 -
Locust bean gum (LBG) is a member of the family of polysacchar• ides known as the galactomannans which differ in the degree and pattern of substitution of galactose residues along the mannose main chain. The galactomannans are able to interact with other polysaccharides apart from xanthan, with the degree of interaction being roughly inversely proportional to the degree of galactose substitution on the mannose backbone. In LBG, only about 30% of the D-mannosyl residues are substituted, with the result that 3-dimensional gel complexes can form with xanthan, agarose and carrageenan at suitable concentrations (Dea et al. 1972).
The molecular conformation of xanthan and consequent solution properties have generated considerable interest in recent years. In the native state, xanthan molecules exist in an ordered conformation which persists on dissolution at ambient temperatures. Under conditions of low ionic strength and increased temperature, the molecule undergoes an intramolecular conformational change to a more disordered form (Rees,
1972; Holzwarth, 1976; Morris et al. 1977). X-ray diffraction
(Moorhouse et al. 1977) and electron microscopic evidence (Holzwarth and
Prestridge, 1977) suggest that the ordered form of xanthan is a right handed helix. Morris et al. (1977) suggest that in the ordered conformation, the side chains are folded down and aligned with the main chain to give a rigid structure stabilized by intra-molecular non- covalent bonding.
Norton et al. (1980) monitored the salt (K+) induced disorder- order transition using stopped flow polarimetry. Results were in good agreement with a reaction of the form - 8 -
Coil -»• Helix [1]
where the first-order rate equation for the process is given by
£n[a0/(a0 - x)] = ^t [2]
In the equation, a0 is the total pentasaccharide residue concentration (based on a mean repeat unit of 1000 molecular weight) and x is the residue concentration in the helix form at time t.
The concentration independence of the transition process as well as the lack of increase in molecular weight as monitored by light scattering (Norton et al. 1984) suggest the ordered state is a single stranded helix. However, Holzwarth and Prestridge (1977) and Holzwarth
(1978) have suggested that the ordered state is multistranded with two or perhaps three strands based on electron microscopic evidence and hydrodynamic properties. Other workers (Morris, 1977; Whitcomb and
Macosko, 1978; Rinaudo and Milas, 1978; Jamieson et al. 1982) have suggested that xanthan exists in solution as a rigid, rodlike molecule with a length between 6000 and 10,000A. Based on the hydrodynamic behavior of xanthan in solution, Holzwarth (1981) pointed out that the conformation of xanthan could also be interpreted as a wormlike chain, and that this model was more consistent with the hydrodynamic properties. Norton et al. (1984), after analysis of equilibrium data for the coil to single-helix transition, concluded that ordered and disordered helical sequences co-existed within the same molecule giving an overall broken-rod structure. - 9 -
The conformational properties of LBG have generated no such interest. Based on computer model building studies and the work of various researchers on related polysaccharides, Rees (1972) and Dea et al. (1972) suggested that locust bean gum existed in a 2-fold, extended ribbonlike conformation in the solid state and adopted a random coil conformation in solution. The molecule is not totally rigid, but has severely restricted mobility about the glycosidic linkages.
C. Xanthan-Locust Bean Gum Interaction
According to Rees (1972), Morris (1973), and Dea and Morris
(1977), the interaction of xanthan with locust bean gum involves a co-operative association of the unsubstituted or lightly substituted regions of the LBG molecule with the cellulose-like backbone of xanthan
(Figure 3). Locust bean gum reverts to its ribbon-like conformation on interaction with xanthan. Gel formation occurs as a result of the association of unsubstituted regions of the galactomannan to form long, structurally regular junction zones. Regions containing galactose residues are unassociated and help in the solubilization of the gel network by extensive hydration. Both associating and non-associating molecular regions are essential for gelation, with too much association leading to precipitation and too little preventing the formation of a cohesive network.
The number of sugar residues involved in junction zone formation has not been determined as yet. Based on the solubilities of gal acto• mannan oligosaccharides, Rees et al. (1982) suggested a critical chain length for stable association of at least 10-15 residues. - 10 -
Mixed gel
Figure 3. Representation of xanthan-locust bean gum interaction (Dea et al. 1977).
g-1+4 or 1+3 linked linear polysaccharides of uniform structure can fit together to form ordered, crystalline arrays which are difficult to separate (Rees, 1972; Whistler, 1973). Branching or the presence of ionic charges reduces the possibility of inter-molecular association and increases the solubility of the polysaccharide. Thus mannan, a g-1+4 linked linear polysaccharide is insoluble, whereas the galactomannans, galactose substituted mannans, are soluble with solubility increasing as the proportion of galactose substituents increase.
The interaction of xanthan with galactomannans decrease as the galactose substituents on the mannan main chain increases. The most likely arrangement of galactose substituents which facilitates this - 11 -
interaction is one in which the galactose substituents are arranged in a block-like manner (Painter, 1982). From chemical degradation studies,
Baker and Whistler (1975) concluded that locust bean gum, on an average, consisted of regions in which there were twenty-five contiguous substituted mannose residues and eighty-five contiguous unsubstituted residues. Painter (1982) noted that other workers (e.g. Courtois and Le
Dizet, 1970), using enzymatic studies, also came to similar conclu• sions. However, Marchessault et al. (1979), using X-ray diffraction studies and Painter (1982), concluded that galactose substituents were arranged on the mannan main chain in a random manner. McCleary et al.
(1984), using enzymatic degradation studies and computer model building, concluded that the galactose residues were arranged in an irregular rather than random manner. From his studies, McCleary concluded that locust bean gum structure was typified by the presence of high propor• tions of substituted couplets but not triplets. However, this picture was not considered to alter the theory that regions in the galactomannan which form the "junction-zones" with xanthan are those unsubstituted or lightly substituted by D-galactose (McCleary, 1984). McCleary (1979),
Rees et al. (1982) and Painter (1982) pointed out that in the most stable conformation of the mannan chain, alternating sequences of galactose residues could all be on the same side of the mannose main chain. This would leave large areas of unsubstituted regions for inter• action with other molecules according to the proposed model. However, in a consideration of interactions between locust bean gum molecules,
Marchessault, et al. (1979), came to quite different conclusions on the nature of interaction. Since the mannan crystalline structure was - 12 -
absent in stretched and dried films of locust bean gum, he and co• workers considered it unlikely that the hydrated gels would contain crystalline regions organized in the same way as those in mannan poly• saccharide. They concluded that the D-galactose residues on one chain interact directly with the D-mannose residues of another. It is
possible that the interaction of xanthan with locust bean gum could occur by such a mechanism.
The stoichiometry of binding between the two polysaccharides has not been determined conclusively. However, Kovacs (1973) suggested a junction zone stoichiometry of 1:1 since maximum gel strengths occurred when the two polysaccharides were in nearly equal proportions. A
similar stoichiometry is also assumed in the molecular model presented by Dea and co-workers (1977). McCleary (1979) removed soluble poly•
saccharide from gelling mixtures of xanthan and locust bean gum in
ratios ranging from 5:1 to 1:5. Concentrations of polysaccharide in the
isolated gel residue were highest when the proportion of xanthan was greater than or equal to that of locust bean gum. Soluble poly• saccharide concentrations were greatest when locust bean gum was in the higher proportion. These observations suggested that a 1:1 stoichio• metry was quite likely.
Morris (1973) attributed the binding of the two polysaccharides to non-covalent interaction forces. Secondary, non-covalent forces which act between molecules and chemical groups giving rise to inter- and intramolecular interactions are primarily electrostatic in nature
(Richards, 1980). These forces arise in several ways and include the interaction of charged or polar groups (multipole interactions and Van - 13 -
der Waals forces), hydrogen bonding, hydrophobic interactions and
exclusion forces. Unlike most of the other interactions, hydrophobic
interactions arise from entropy related processes taking place in the
system rather than by Coulombic attractions.
Rees (1969; 1972) suggested that the most relevant interactions for polysaccharides were hydrogen bonding, dipole and ionic (monopole)
interactions. Contributions from hydrophobic and charge transfer inter•
actions which are important for proteins and nucleic acids probably have
little relevance. The secondary interactions are individually so weak that stability of the system is achieved only when a large number of them
are simultaneously favorable, that is, when they act co-operatively.
McCleary et al. (1982) showed that locust bean gum (0.1% w/v) in the presence and absence of xanthan (0.1% w/v) was hydrolyzed to the
same degree by an endo-hydrolase, B-D-mannanase. He concluded that only
a small proportion of the unsubstituted locust bean gum molecule was
involved in interaction with the xanthan gum at the "junction zones".
However, an alternative interpretation is that the observed results are due to the weakness of the interaction between the two polysaccharides.
D. Rheological Studies
"Rheological" measurements have been used extensively to demon•
strate the interaction of xanthan with galactomannans. Kovacs (1973)
presented information on gel strengths and 'elasticity' of xanthan-
locust bean gum gels as a function of gum ratio, total gum concentration
and pH. Gel strengths were measured with a Bloom Gelometer. For a 1%
gel, maximum gel strengths were obtained with xanthanrlocust bean gum - 14 -
ratios in the range of 6:4 and 4:6. Gel strengths increased with increasing polysaccharide concentration. Maximum gel strengths were obtained in the range of pH 6 to 8 with a rapid decline in gel strength being noted at both the alkaline and acid sides of the pH spectrum.
Elasticity, as determined from the force-deformation relationship produced by a Marine Colloids Gel Tester, was greatest when xanthan- locust bean gum were in the ratio of 9:1 and 1:9. The least elastic gels contained equal proportions of the two gums. Consistency of xanthan-locust bean gum solutions at different gum concentrations were obtained using a Brookfield Model LVT Viscometer (60 r.p.m.).
Consistency increased with increasing gum concentration and decreased with temperature.
Morris et al. (1980b) determined the gel strengths of xanthan- locust bean gum gels by compressing samples between parallel plates of an Instron Universal Materials Tester Model 1122 equipped with a 20 N load cell and using crosshead speeds of 10 mm/min. They were primarily concerned with demonstrating the effects of added guar gum on gel strengths (yield stresses). The presence of guar gum resulted in a lowering of yield stress and this was attributed to competitive inhibi• tion of the formation of network structure.
Although Dea et al. (1977) used oscillatory techniques to demonst• rate a marked enhancement in the dynamic rigidity and viscosity of xanthan-guar blends, similar results have not been presented for xanthan- locust bean gum blends. Morris et al. (1980b) also demonstrated the enhancement of viscosity of xanthan-guar blends. Viscosity measurements were obtained using coaxial cylinder fixtures with a shear rate of 10 s"1. - 15 -
McCleary (1979) used a Brabendor Amylograph with a 350 cmg cartridge at 80 r.p.m. and a Brookfield viscometer at . 20 r.p.m. to investigate the effects of enzymic treatment on the gelling interaction of xanthan and galactomannans in different proportions. Amylograph viscosities were highest when xanthan and locust bean gum were in equal proportions and decreased as the proportion of locust bean gum to xanthan decreased with the individual polysaccharide solutions showing the lowest amylograph viscosities. A rapid drop in amylograph viscosity was observed in the presence of the enzyme, 3-mannanase. An optimum was observed in Brookfield consistency when locust bean gum and xanthan were in a 1:2 ratio.
Consistency measurements and gel strengths have been used extensively to demonstrate the interaction of xanthan with locust bean gum. The techniques used, e.g., Marine Colloids Gel Tester, Bloom
Gelometer, have been mostly empirical and do not provide fundamental information. Single point viscosity measurements are also totally inadequate for characterizing flow behavior of these non-Newtonian dispersions. As a result, very little comparison can be made with the results of different workers, as these are only valid where the same instrument and testing conditions are used.
Mitchell (1976; 1980) reviewed rheological studies on gels formed by macromol ecular food additives. In particular agar, alginate, carrageenan, gelatin, pectin and microbial polysaccharide gels were considered. In addition to the empirical methods mentioned previously, creep and stress relaxation tests have been the most commonly used - 16 -
techniques in the study of polysaccharide gels. Dynamic shear methods have been applied to only a limited extent.
E. Solvent Treatments
Protein denaturation has been extensively studied with the hope of gaining insights on the structure, properties and functions of these important biological macromolecules. Haschemeyer and Haschemeyer (1973) and Lapanje (1978) have defined protein denaturation as a change in conformation or three dimensional structure, without any change in the primary valence bonds. The breakage of disulfide linkages is considered the exception to this general rule. One approach taken to understand the mechanism of denaturation, is the determination of the forces involved in holding the molecule in its compact, folded form. Denaturation agents such as heat, pH, urea, guanidinium salts, inorganic salts, organic solvents and detergents have been employed to this end.
The ability of urea to act as a denaturant has been explained both in terms of its ability to bind directly with proteins replacing existing hydrogen bonding interactions and, in terms of its ability to disrupt long range order characteristics of pure water. Changes in the structure of water leads to changes in the nature of polymer-water interactions. This, in turn, determines whether polymer-solvent or polymer-polymer interactions become more favorable. Salt effects have also been attributed in part, to differences in the solubility of the protein in salt solutions when compared with water, in addition to its effect on electrostatic interactions.
The use of these dissociating agents have been less extensively applied to polysaccharides. Dintzis et al. (1970), based on - 17 -
determinations of xanthan molecular weight, concluded that true macro- molecular solutions of xanthan were formed only after heating the poly• saccharide in 4 M urea. Frangou et al. (1982), Ross-Murphy et al. (1983) found that viscoelastic properties of xanthan (0.5%) passed from that of a viscoelastic solid to that of a viscoelastic liquid after being subjected to heat treatment in 4 M urea. Jamieson et al. (1982) and Southwick et al. (1983) concluded that xanthan existed in a random coil conformation after heat treatment in salt-free 4 M urea solutions. These effects were generally attributed to the disruption of inter- and intra-molecular hydrogen bonding in xanthan aggregates and molecules.
Attempts have been made to study changes in the viscoelastic properties of hyaluronic acid, a glycosaminoglycan, under different condi• tions of pH and ionic strength (Gibbs et al. 1968; Welsh et al. 1980;
Morris, et al. 1980a). Lowering pH and increasing ionic strength enhanced dynamic network properties and were attributed to increased intermolecular associations by suppression of electrostatic repulsions.
Solvent treatment and levels used in this study were adapted primarily from the work of Matsumoto (1979). He studied chemical changes taking place in fish protein during frozen storage after being subjected to various solvent treatments. The solvent treatments used, included water (to test for non-specific association forces), 0.6 M KC1 (to test for ionic bonds), 8 M urea (to test for hydrogen bonds and non-polar bonds) and 1 M KOH (to test for ionic bonds and others). In the study of
Matsumoto, differences in the degree of cross-linking between the protein molecules were monitored by measuring changes in the solubility of the protein. - 18 -
MATERIALS AND METHODS
A. Chemical Composition and Physical Properties
In order to draw conclusions on events taking place at the molecular level from the gross behavior of the system, pure materials or materials of a reasonably well defined composition should be used. The latter approach was used in this study. Commercially available samples of xanthan (Keltrol, Kelco Division of Merck Inc., San Diego, CA) and locust bean gum (Tic Gums Inc., New York, NY) were analyzed for moisture, ash, principal inorganic components and nitrogen content.
Intrinsic viscosity and pH of the samples were also determined.
1. Moisture
The AOAC vacuum oven method (AOAC, 1980, Section 14.002) was used to determine the moisture contents of the polysaccharides. Samples
(2 g) of each gum were accurately weighed into aluminum dishes (65 mm diameter x 15 mm deep) which had previously been dried, cooled in a desiccator and weighed. Samples were dried in the oven at 100°C for
3 h, cooled in a desiccator and weighed quickly to minimize moisture pick-up. Drying and weighing were repeated for additional 1 h periods until very little difference in weight existed between successive weighings. Four determinations were carried out for each gum.
2. Ash and Inorganic Components
Ash content of the polysaccharides was obtained using the dry ashing method of the AOAC (AOAC, 1980, Section 14.006). Crucibles were - 19 -
acid washed, then heated in a muffle furnace at ca. 550°C overnight, cooled in a desiccator, and weighed. Samples (1 g) of polysaccharide were accurately weighed into the crucibles and ashed at 550°C, cooled in a desiccator and reweighed. This process was repeated until constant weight was obtained, which was approximately 36 h. Four determinations were carried out for each polysaccharide. The residual ash was diluted
to 100 mL with 0.5% HN03 and analyzed for a range of inorganic components using an inductively coupled plasma spectrometer, Jarrel-Ash
Plasma Atomcomp (Fisher Scientific Co., Waltham, MA). Further dilutions of the above solutions (100 x for xanthan and 10 x for locust bean gum) were made with 0.5% HNO3 for determinations of potassium and sodium using an atomic absorption spectrophotometer, Perkin Elmer 306
(Perkin Elmer Corp., Norwalk, CT).
3. Nitrogen Content
Nitrogen determination was carried out using the micro Kjeldahl technique of Concon and Soltess (1973). Using a Mettler analytical balance, 0.15 g of the polysaccharides were weighed into clean, dry
Kjeldahl flasks which had been acid washed. A catalyst, 2.3 g K2S0lt -
HgO mixture (190:4 w/w) was added to the sample and mixed. This was followed by addition of 2.3 mL concentrated H^SO^. The samples were digested by heating with periodic addition of H2O2 (30%) until all traces of organic material had been digested and the solution in the flask was clear and colorless. When cooled slightly, the digest was diluted with 10 mL distilled, deionized water and then transferred quantitatively to a 25 mL volumetric flask and made up to volume. An - 20 -
aliquot of this diluted solution was analyzed for nitrogen content using a Technicon Autoanalyzer II (Technicon Industrial Systems, Tarrytown,
NY). The nitrogen values were converted % N values based on dry weight
of polysaccharide used. Five estimates of nitrogen content were
obtained for each sample.
4. Intrinsic Viscosity
Stock solutions of xanthan and locust bean gum (ca. 0.1%) were
prepared by mixing the individual gums, (2 min, Polytron Homogenizer,
Model PT10/35, Brinkmann Instruments Inc., Westbury, NY) into 0.1 M
sodium chloride solution containing 0.02% sodium azide. Locust bean gum
solutions were then heated with stirring for 10 min at 80°C, in order to dissolve the gum (Doublier and Launay, 1981).
Locust bean gum typically consists of three fractions of differ•
ing solubilities, a cold water soluble fraction, a hot water soluble
fraction and an insoluble residue. These fractions correspond to
different degrees of galactose substitution on the main chain with
solubility increasing as the number of galactose substituents increase.
Strong interchain association between unsubstituted or lightly substit•
uted regions of the locust bean gum molecules restrict hydration and decrease solubility (Whistler, 1973; Morris et al. 1977). The method
used was considered most likely to maximize dissolution of the gum as well as minimize oxidative or hydrolytic reactions. Some methods used
by other workers e.g., stirring for 90 min at 70°C (Sharman et al.
1978), or for 30 min at 80°C (Dea and Morrison, 1975) were considered
unsuitable for these reasons. - 21 -
The polysaccharide solutions were centrifuged at 10,400 xg for 15 min to remove air and undissolved residue. In general, no sedimentation was observed in xanthan solutions after this centrifugation process.
The stock solutions were diluted with 0.1 M sodium chloride to give concentrations in the range 0.004 to 0.075% for xanthan and
0.005 to 0.1% for locust bean gum. Viscosities of these solutions as well as 0.1 M NaCl solution were obtained with a No. 100 (Z13) Cannon
Fenske, calibrated capillary viscometer. Additional evaluations were carried out using a No. 200 calibrated Cannon-Fenske capillary visco• meter to check for Newtonian flow behavior. Calibration of the viscometers was checked with standard oils by solving for the calibra• tion constant, ki in the equation
p=P=klt+^I C3]
where u is the kinematic viscosity in centistokes.
The constant ki was in good agreement with the calibration constant supplied by the manufacturer. The kinetic energy correction
2 term, k2/t formed only a small fraction (<0.06%) of the kit term and was considered negligible (Van Wazer et al. 1963). Concentrations of the stock solution were obtained by drying and weighing after dialyzing
(mol wt cut-off of 12,000 - 14,000) against distilled water. Kraemer's
(Eq. 4) and Huggins (Eq. 5) methods were used to model the concentration functions of viscosity after converting to relative viscosities (nrel) and specific viscosities (nsp)« - 22 -
2 (An npel)/c = [n] + k' Cn] c [4]
2 nsp/c = [n] + k" [n] c [5]
The mean of the intercept from the graph of {in nre-|)/c against c and
3 1 nSp/c against c gave the intrinsic viscosity IT\1 in m kg" .
5. Determination of pH
Duplicate determinations of pH were obtained for 0.1% and 0.4% solutions of the polysaccharides in distilled, deionized water.
Measurments were taken at 20°C using a Fisher Accumet Model 230 pH/Ion
Meter (Fisher Scientific Company, Pittsburgh, PA).
B. Rheological Studies
1. Sample Preparation
a) Viscoelastic Studies
Dispersions (ca. 0.4%) were prepared in a manner similar to those used for intrinsic viscosity determinations. In this case, solutions were prepared in distilled water, 8 M urea, 0.6 M potassium chloride and for xanthan only, 1 M potassium hydroxide. Solutions of locust bean gum in potassium hydroxide (for reasons discussed later), were prepared by first dispersing twice the required concentration of gum in distilled water, heating to dissolve as before, then diluting with 2 M potassium hydroxide to the required concentration of polysaccharide and potassium hydroxide. Xanthan-locust bean gum blends - 23 -
were made by mixing equal weights of the prepared solutions at 80°C, then held at this temperature for 10 min to remove air bubbles. Pulling a small vacuum on the samples also facilitated the removal of air bubbles. After each rheological test, polysaccharide concentrations in the blends and individual solutions were obtained after dialyzing against distilled water, then drying and weighing.
Concentrations of ca. 0.4% total polysaccharide by weight were chosen for this experiment because: (1) they showed linear viscoelastic behavior over the accessible frequency range; (2) they were relatively dilute, thereby minimizing the possibility of entanglements and (3) they were easy to handle.
b) Steady Shear Flow Studies
Solutions of xanthan and locust bean gum at concentrations 0.20,
0.10 and 0.05% were prepared in a manner similar to those used for dynamic shear studies. Samples to be evaluated in the fractional factorial experiment had combinations of ionic strengths and urea concentrations as shown in Table 1 for each experimental unit. The solutions were adjusted to the desired pH (Table 1) with either 1 M HC1 or 1 M NaOH just before blending. The pH of the samples did not change appreciably after blending. Samples for the more detailed evaluation of temperature effects on viscosity were prepared in either distilled, deionized water or 0.1 M NaCl at their natural pH. Xanthan-locust bean gum blends were prepared by mixing solutions of equal concentration by weight, in the desired ratio. - 24 -
Table 1. Treatment combinations for xanthan-locust bean gum solutions for
the fractional1|ac^orial experiment according to the scheme of
Taguchi L27 (3 ).
Factors
Concentration Temperature Ionic Strength pH Gum Ratio2 Urea (% w/w) (°C) (M added NaCl) Concentration (M)
1 0.20 25 0 6 1 4 0 2 0.20 25 0.05 3 1 1 2 3 0.20 25 0.10 9 4 1 4 4 0.20 35 0 3 1 :1 4 5 0.20 35 0.05 9 4 1 0 6 0.20 35 0.10 6 1 :4 2 7 0.20 55 0 9 4 1 2 8 0.20 55 0.05 6 1 4 4 9 0.20 55 0.10 3 1 1 0 10 0.10 25 0 3 4 :1 4 11 0.10 25 0.05 9 1 4 0 12 0.10 25 0.10 6 1 :1 2 13 0.10 35 0 9 1 •4 2 14 0.10 35 0.05 6 1 1 4 15 0.10 35 0.10 3 4 1 0 16 0.10 55 0 6 1 1 0 17 0.10 55 0.05 3 4 1 2 18 0.10 55 0.10 9 1 4 4 19 0.05 25 0 9 1 1 2 20 0.05 25 0.05 6 4 1 4 21 0.05 25 0.10 3 1 4 0 22 0.05 35 0 6 4 1 0 23 0.05 35 0.05 3 1 4 2 24 0.05 35 0.10 9 1 1 4 25 0.05 55 0 3 1 4 4 26 0.05 55 0.05 9 1 .1 0 27 0.05 55 0.10 6 4 1 2
From Taguchi's L27 (3 ) fractional factorial design (Taguchi, 1957).
Ratio, xanthan: locust bean gum. - 25 -
2. Rheological Measurements
a) Viscoelastic Properties
Dynamic viscoelastic properties of solutions of xanthan, locust bean gum and blends of the two in water were obtained at 20°C using a
Weissenberg Rheogoniometer, Model R19 (Sangamo-Schlumberger Ltd., Bognor
Regis, England). Viscoelastic properties of the polysaccharide blends in the different solvents were obtained at 20 and 60°C in a similar manner. Samples were evaluated over the frequency range 0.6 to 60 s"1.
Data were collected at nine equally spaced, logarithmic intervals of frequency for the individual gum solutions in water and in preliminary evaluations of the blend. For the evaluation of the effect of the different solvents on viscoelastic properties of the blend, data were collected at only five equally spaced, logarithmic intervals.
Viscoelastic parameters were also evaluated at 5°C then at 10 C° intervals over the temperature range 0 to 60°C for blends prepared in distilled water and 0.6 M KC1.
Parallel plate fixtures, 7.5 cm in diameter, were used with a gap thickness of 500 pm. Parallel plate fixtures are generally advantageous for oscillatory testing when compared to other geometries such as cone and plate or co-axial cylinders (Walters, 1975). The required gap thickness can be set and varied with less difficulty than with the other geometries and if inertial effects are significant, these can be treated more simply mathematically. The upper platen was supported by a #7 torsion bar (9.4 Pa cm3/pm). A Haake PG2 circulating bath (Haake Inc.,
Saddle Brook, NJ) was used to maintain a flow of fluid through the lower - 26 -
platen (a modified Ferranti-Shirley fixture) in order to produce the desired temperatures.
Preliminary experiments indicated that aqueous solutions of the polysaccharides exhibited linear viscoelasticity in the strain range of
0.04 to 0.23 at 20°C, using a frequency of 1.9 s"1 and 0.04 to 0.19 at
60°C. Therefore, all the experimental runs were carried out with a maximum strain of 0.19.
The dynamic storage modulus (see Appendix I) was a slowly increasing function of time, appearing to level off after 5 h at 20°C.
At 60°C, erratic behavior was observed after 3 h. This possibly resulted from drying out of the sample at the edge. Therefore, it was decided to evaluate the samples after aging for 1 h on the rheogoniometer. This was because the rate of change of the viscoelastic parameters was relatively slow at this time and consistent reproducible results could be obtained. Silicone oil was placed around the sample edge during the aging period. Excess oil was removed at the time of measurement to leave only a thin film. A Tronotec Model 703A digital phase meter (Tronotec Inc., Franklin, NJ) was used to monitor the amplitudes of the input (strain) and output (stress) voltage signals and phase difference between the two signals.
From these data, values of the storage modulus (G1), a measure of the energy stored elastically during a cycle of sinusoidal deformation; the loss modulus (G"), a measure of the energy dissipated as heat and the loss tangent (tan 6), the ratio of energy lost to energy stored, were calculated using a program written for an Apple II microcomputer. - 27 -
b) Steady Shear Flow Properties
Steady shear flow studies were carried out using Brookfield
Synchro-lectric Viscometers, Models RVT and LVT (Brookfield Engineering
Lab. Inc., Stoughton, MA) with co-axial cylinder fixtures. Samples were sheared from the lowest to the highest rotational speeds with shearing continued at the highest speed until a nearly constant value was
attained. Scale readings were then recorded using decreasing steps of
rotational speed. The shear rate range covered varied with the geometry of the fixtures and model of the viscometer used and was in the range
0.2 to 175 s"1. Four subsamples of each treatment combination were
evaluated for the fractional factorial experiment.
Samples corresponding to the upper and lower levels of concentra• tion and ionic strength (Table 2) were evaluated over the temperature
range 10 to 60°C at 10 C° intervals. Temperature control was achieved
using a Lauda-Brinkmann Model RM3 circulating bath (Brinkmann
Instruments Inc., Westbury, NY) which pumped fluid through a jacket
surrounding the outer cylinder of the viscometer. No data were obtained
at 10°C for xanthan-locust bean gum blends at 0.2% since firm gels were formed under these conditions. Equal proportions of the two gums at their natural pH were used.
The viscometers were calibrated with viscosity standard oils, S6
(0.7775 poise) and S60 (1.044 poise), (Cannon Instrument Co. State
College, PA). Calibration was checked at both 25 and 40°C. The same
calibration constant could be used to convert scale readings to shear
stress values at all the rotational speeds for the RVT viscometer. - 28 -
However, a different calibration constant was required for each rota- tional speed of the LVT viscometer. The calibration constants at the two temperatures were essentially the same.
Table 2. Summary of variables and levels used in Taguchi's Fractional Factorial Experiment
Level
Variable 1 2 3
Concentration (% w/w) 0.20 0.10 0.05 Temperature (°C) 25 35 55 Ionic Strength (M added NaCl) 0 0.05 0.10 pH 6 3 9 Gum Ratio (X:LBG) 1:4 1:1 4:1 Urea (M) 0 2 4
Shear stress (a, Pa), shear rate (Y, S"1) and apparent viscosity
(n, Pa s) were calculated using a computer program written for this purpose together with the facilities of the statistical package MIDAS
(Fox and Guire, 1976). Least squares regression was used to examine the fit of the power law model (Eq. 6) to the transformed data.
log n = log m + (n - 1) log y [6] where m is the consistency coefficient (Pa sn) and n is the flow behavior index. The Kreiger-Maron correction for non-Newtonian behavior
(Haugen and Tung, 1976), was applied to recalculate each shear rate value using values of the flow behavior index evaluated from the data. - 29 -
The coefficients of determination, r2, were calculated as a measure of goodness of fit of the model. Values of apparent viscosity at a shear rate of 50 s"1 were calculated from the power law parameters.
3. Statistical Analysis
a) Evaluation of Viscoelastic Properties
The effects of the four solvent treatments on the viscoelastic properties of xanthan-locust bean gum solutions at 20 and 60°C, were evaluated using a split-plot experimental design. The two temperature levels were considered as the main plots over which the four treatments were randomized. There were three replications of each treatment within a plot, and the entire experiment was rerun three times. This gave a total of nine observations for each treatment-temperature combination at the five levels of frequency examined. Analyses of variance were performed on the storage and loss moduli and the loss tangent using the statistical package UBC Genlin (Greig and Bjerring, 1980), available on the UBC computer. The analyses were performed separately for each frequency.
The logarithmically transformed data were used for the analysis as the normality assumption was well met when the data were in this form. The variability between the standard deviations of the means for the different treatment combinations was fairly large and this made it unclear as to whether the assumption of homogeneity of variance was fully met. However, because the analyses were performed on a fully balanced design, the results of the analyses of variance could still be interpreted with confidence. - 30 -
Means for the various treatments were not compared after the analysis of variance because of difficulties in estimating appropriate standard errors and the associated degrees of freedom. These difficulties arose because of the presence of missing data as well as significant interaction terms involving replications.
b) Steady Shear Studies
The effects of total gum concentration, temperature, ionic strength, pH, gum ratio and urea on steady shear flow behavior were evaluated. The levels of the factors used are summarized in Table 2.
Analyses of variance were carried out on the data obtained for the consistency coefficient, flow behavior index and the apparent viscosity at a shear rate of 50 s-1. The analyses was performed for a 3-level
13
fractional factorial experiment according the scheme (L27 (3 )) outlined by Taguchi (1957). Computations were carried out using a program written for the Munroe Model 1880 calculator (Litton Business
Systems Inc., Orange, NJ). Temperature effects on steady shear flow properties of xanthan-locust bean gum solution in equal proportions and at two levels of concentration and ionic strength were evaluated by conducting the experiment in a split-plot design. Again the temperature levels constituted the whole plots over which all combinations of the two factors were randomized. Analyses of variance were performed on the data for the consistency coefficient, the flow behavior index and the apparent viscosity at a shear rate of 50 s_1 using the statistical package UBC Genlin (Greig and Bjerring, 1980). Data for the consistency coefficient and the apparent viscosity at 50 s-1 were transformed logarithmically before the analyses were carried out. - 31 -
RESULTS AND DISCUSSION
A. Composition of Materials
Mean values for moisture, ash, major metallic elements and nitrogen contents for xanthan and locust bean gum samples are shown with their standard errors in Table 3.
1. Moisture
The moisture contents of both xanthan and locust bean gum were approximately 11%. Typically, polysaccharides contain 8-10% water as water of hydration at normal humidities (Glicksman, 1982). In an amorphous state, polysaccharides have numerous unsatisfied hydrogen bonding positions which tend to hydrate easily. Thus, completely dry polysaccharides have a strong affinity for water. It is unlikely that all the water of hydration was removed using the oven-drying technique.
2. Ash and Inorganic Components
The ash content for xanthan polysaccharide was considerably greater than that of locust bean gum (Table 3). This may be a con• sequence of the polyelectrolyte nature of xanthan. The predominance of potassium ions over sodium as the chief counterion present, is expected for biological systems. However, the addition of sodium chloride during commercial processing of some food products usually results in the reverse situation in processed foods. Thus, the higher concentration of - 32 -
Table 3. Summary of non-carbohydrate components for xanthan and locust bean gum powders based on dry weights of the polysaccharides.
Xanthan Locust Bean Gum
Moisture (%) 11.5 11.0 (0.23)1 (0.05)
Ash (%) 10.6 1.4 (0.23) (0.16) K (%) 3.5 0.36
Na (%) 1.6 0.13
Ca (%) 0.090 0.105
Mg (%) 0.104 0.048
Al (ppm) 70 50
Fe (ppm) 20 10
Cu (ppm) 10 10
Nitrogen (%) 0.37 0.84 (0.073) (0.055)
1Data in parentheses are standard errors. - 33 -
potassium was not unexpected for xanthan as large quantities of sodium chloride were not added during the processing of xanthan (Pettitt,
1982).
Combining the molar contributions of sodium and potassium ions for xanthan, a total of 1.55 x 10"3 moles per gram of polysaccharide was calculated from the percentages obtained in the elemental analysis. The amount or distribution of pyruvate residues in the sample used is not known. However, assuming pentasaccharide repeating units containing acetate substitutents, the number of moles of available sites for neutralization can range from 1.11 x 10"3 moles per gram polysaccharide where no pyruvate substitutents are present to 1.98 x 10"3 moles/gram for a pyruvate residue in every repeat unit (Sandford et al. 1977).
Although it is not completely valid to relate the ion content obtained in the elemental analysis to the degree of neutralization of carboxylic acid substituents, the results do suggest that a large excess of ions is not present. It also suggests that either the carboxylic acid substit• utents are incompletely neutralized or the sample corresponds to one with a low degree of pyruvic acid substitution. For comparable values of sodium and potassium concentration, Sandford et al. (1977) and
Rinaudo and Mil as (1978) suggested a pyruvate substituent on every fourth repeating unit.
The combined molar contribution of calcium ions for xanthan- locust bean gum blends in equal proportions is about 2.4 x 10"3 moles per gram of polysaccharide. Holzwarth (1976) found that Ca2+ was more effective at lower concentrations than Na+, in stabilizing the xanthan conformation. At concentrations as low as 0.0005 M CaCl2, the - 34 -
conformation of purified xanthan (0.6 mg mL"1) appeared to be stabilized at temperatures of 80°C as judged by optical rotation changes. Rinaudo and Milas (1978) also found that xanthan exhibited greater selectivity towards divalent counterions, especially calcium, when compared to monovalent counterions.
The effects of divalent ions on the xanthan-locust bean gum
interaction have not been studied in detail. It is possible that these
ions might have some effect on the interacting system. Gelation of
polyuronates (polyanions) e.g., alginate and pectate is induced by the
presence of Ca2+. However, it is considered that cation binding by
carboxyl and glycosidic oxygens of two polymer chains giving rise to the
"egg box" model of junction zones, is responsible for the formation of
the gel network in these polysaccharides (Rees et al. 1982).
The counterions present in a polyelectrolyte solution can make a
significant contribution to the ionic strength of the solution. Ionic
strength increases with increasing polymer concentration and decreases with decreasing concentration. If these effects are not taken into
consideration and corrected for, misleading conclusions can be drawn
about the solution properties of the polymer. This is important in the
determination of properties such as intrinsic viscosity and for xanthan,
the transition temperature of the order-disorder transition process.
Thus, the transition temperature for xanthan is independent of
concentration once the contribution of the polymer's own counterions are
taken into account (Milas and Rinaudo, 1979). In this study, solutions
of xanthan with no external salt added were considered to have low ionic - 35 -
strengths. In dynamic shear studies, the same polysaccharide concentra• tion was used throughout so all solutions would be expected to have
roughly the same contributions to solution ionic strength. In steady
shear studies, solution concentration was varied over the narrow range of approximately 0.05 - 0.2%. However, the contribution of the polymer
counterions to the ionic strength of the solution may still influence
solution behavior of the polysaccharides.
3. Nitrogen Content
The nitrogen contents of xanthan and locust bean gum shown in
Table 3 can be converted into crude estimates of protein content (% N x
6.25). This gives values of 2.28% and 5.26% respectively. Nitrogen or
protein content of the locust bean gum sample was more than twice that
of xanthan. Locust bean gum is derived from a legume and would be
expected to have a higher proportion of protein associated with it than the bacterial extracellular polysaccharide. Complete removal of protein
using similar isolation procedures would therefore be somewhat more difficult for locust bean gum than for xanthan. The differences in
protein content probably arise from differences in the quality standards
accepted by the different manufacturers. The effect of protein content
on the interacting system is not known specifically.
4. Other Components
The pure polysaccharides in question are considered to comprise the major portion of the remainder of the samples analyzed. However, it - 36 -
is possible that small quantities of mono- or disaccharides from hydrolytic reactions as well as residual lipids might be present.
5. Intrinsic Viscosity
Kraemer's and Huggins' equations provided slightly different estimates of intrinsic viscosity for both xanthan and locust bean gum as shown in Figure 4 and Figure 5, respectively. For xanthan gum, these were 2.07 m3 kg"1 using Huggins' method and 2.16 m3 kg"1 using Kraemer's method while the corresponding values for locust bean gum were 1.43 and
1.52 nr kg". The mean of these two values is shown in Table 4, as a combination of these two methods may provide a more accurate estimate of intrinsic viscosity than either alone (Morris and Ross-Murphy, 1981).
Intrinsic visosity is a measure of the contribution of individual polymer molecules to overall solution viscosity. It is defined as
n n [n] = lim ~ s m c+o n c L J s
where c is the concentration of polymer and ns, the viscosity of the solvent. It is obtained at the limit of infinite dilution where inter- molecular effects would not be present. The intrinsic viscosity is dependent on the molecular weight, volume and shape of the macromole- cular particle and can be used to provide estimates of these where sufficient information is available.
Additional complications arise for polyelectrolytes because coil dimensions vary with changes in ionic strength. At low ionic strengths, - 37 -
Figure 4. Combined Huggins' (•) and Kraemer's (O) extrapolation to intrinsic viscosity for xanthan (0.1 M NaCl, 20°C). - 38 -
Figure 5. Combined Huggins' (•) and Kraemer's (O) extrapolation to intrinsic viscosity for locust bean gum (0.1 M NaCl, 20°C). - 39 -
Table 4. Intrinsic viscosity and pH for xanthan and locust bean gum solutions.
Xanthan Locust Bean Gum
Intrinsic viscosity 2.12 1.47 (m3 kg-1 0.1 M NaCl 20°C)
pH (0.1% solution, H20, 20°C) 6.0 6.1 (0.27)1 (0.10)
^ata in parentheses are standard errors. - 40 -
the molecule adopts an expanded conformation which collapses to a more compact form as the ionic strength is increased and intramolecular repulsions are reduced. For polyelectrolytes, ionic strength is reduced as the concentration of polymer in solution is decreased. In order to compensate for this effect, intrinsic viscosity was determined in 0.10 M
NaCl. Rinaudo and Milas (1978) have reported that the conformational properties of xanthan change very little above ionic strengths of
10'2 M.
For Newtonian fluids, the intrinsic viscosity is independent of shear rate. However if there is non-Newtonian flow behavior, data for intrinsic viscosities obtained at finite shear rates should be extrapol• ated to conditions corresponding to zero shear. Low shear rates are generated in capillary viscometers with no externally applied pressure.
It is anticipated that the choice of capillary viscometers will provide sufficiently low shear rates so that viscosities areessentially independent of shear rate.
The Huggins plot for xanthan data shows slight curvilinearity, becoming more pronounced at higher concentrations. This is probably due to the presence of intermolecular interactions; thus for xanthan, the highest concentration was not included among the values used for estima• tion of the intrinsic viscosity. This effect was less pronounced in locust bean solutions. A range of values for the intrinsic viscosity of xanthan obtained under a variety of conditions have been reported in the literature. Morris and Ross-Murphy (1981) reported an intrinsic visco• sity of 1.67 m3 kg-1 for xanthan gum in the potassium salt form at 0.10 - 41 -
ionic strength. In contrast, Holzwarth (1981) reported a value of 8.00 m3 kg"1 for unpurified xanthan in 0.80 M NaCl.
A range of values has also been reported for the intrinsic viscosity of locust bean gum solutions. Sharman et al. (1978) reported
a value of 2.71 m3 kg"1 for locust bean gum at a shear rate of 1 s"1.
However, this value should not be considered reliable as it was obtained by extrapolating from a rather high concentration of 0.10% where both molecular interactions and non-Newtonian flow behavior are likely to be
present. Doublier and Launay (1981) reported values ranging from 0.77 to 1.00 m kg" for different samples of locust bean gum. In comparison with literature reports, the value of 1.47 m3 kg"1 obtained in this
experiment seems to be a reasonable estimate of intrinsic viscosity.
The intrinsic viscosity of locust bean gum is likely to be lower
in sodium chloride solution than in water since sodium chloride is a
poorer solvent. Molecular expansion, coil dimensions and solution
hiscosity would be expected to be lower in the salt solution.
Viscosities obtained using the No. 200 Cannon-Fenske capillary
viscometer were somewhat higher than those obtained with the No. 100 for
all locust bean gum solutions, except the highest concentration.
However for xanthan solutions, this trend was seen only below concentra• tions of approximately 0.02%. Intrinsic viscosities obtained using the
No. 200 capillary viscometer were somewhat lower than those using the
No. 100. Data obtained using the No. 100 capillary were considered more
accurate as this gave longer efflux times and showed greater
sensitivity. - 42 -
B. Rheological Studies
1. Viscoelastic Properties of the Polysaccharides
In Figure 6, the dynamic storage modulus (G1) and the loss modulus (G") are shown as functions of frequency for xanthan, locust bean gum and blends of the two. The corresponding plot for the loss tangent is shown in Figure 7.
a) Xanthan
The curves for the storage and loss moduli of xanthan gum dispersions could be divided into two distinct regions. At low frequencies, the curves of the storage and loss moduli indicated the development of a plateau region, in which the moduli changed slowly with changes in frequency or time. In contrast with locust bean gum
solutions, the values of the storage moduli were larger than those of the loss moduli. The loss tangent changed very little with increasing frequency and had an average value of about 0.4. At the highest frequencies, the moduli increased sharply with increasing frequency.
The solution behavior of xanthan at 0.4% total polysaccharide concentration, is typical of polymer systems in which molecular inter• actions or entanglements are present. Interactions are increased by
increasing polymer concentration or molecular weight with the result that the plateau region in the G', G"-frequency curves becomes more
pronounced and extends over a wider range of frequency (Ferry, 1980).
Entanglements arise from physical interlocking or coupling of
segments of molecules. At. short time periods, entangled molecules
appear to be cross-linked. However, at long time periods, entanglements - 43 -
Figure 6. Dynamic storage and loss moduli for xanthan, locust bean gum and xanthan-locust bean gum solutions (0.4% in water at 20°C). - 44 -
oo
CM r-j , 1 1 1 1 -0.5 0.0 0.5 1.0 1.5 2.0 FREQUENCY (LOG SCALE)
Figure 7. Loss tangent for xanthan, locust bean gum and xanthan-locust bean gum solutions (0.4% in water at 20°C). - 45 -
differ from chemical cross-links in that the loops can untie themselves because of thermal motions and the interactions are lost (Bird et al.,
1977). Coupling is usually topological rather than due to intermole- cular forces (Ferry, 1980). Cross-linking implies somewhat more perman• ent chemical interactions, and in rubber and polymer rheology, usually involves covalent linkages. The presence of entanglements or interac• tions results in an increase' in the solid-like or elastic properties of the solutions.
b) Locust Bean Gum
The storage and loss moduli for locust bean gum solutions increased rapidly with increasing frequency, when curves were compared with those of xanthan and the polysaccharide blend. The values of the loss moduli were larger than the storage moduli, at all frequencies studied, with values of the loss tangent being close to 3. The value of the loss tangent was comparable to that of amorphous polymers (Ferry,
1980). This behavior can be correlated with the expected solution conformation of locust bean gum. The most likely conformation of locust bean gum molecules in solution is that of a random coil (Rees, 1972).
At 0.4% total polysaccharide concentration, a greater proportion of energy was dissipated as heat during the deformation process. This form of viscoelastic behavior is typical of polymer solutions in which little or no interactions occur (Ferry, 1980).
Differences in the viscoelastic properties of xanthan and.locust bean gum at the same solution concentration, can be correlated with differences in their solution conformations. Xanthan exists in solution - 46 -
as an extended, helically coiled molecule with a rod-like configuration
(Morris et al. 1977; Whitcomb and Macosko, 1978; Rinaudo and Milas,
1978). There is a strong tendency for the molecules to align and form aggregates in solution (Dintzis et al. 1970; Morris et al. 1977;
Southwick et al. 1979; 1980). This results in the development of solu• tions with considerable structure and rigidity (Morris et al. 1977). In addition, entanglement phenomena are considerably more pronounced in solutions of rigid, rod-like molecules than in solutions of amorphous polymers of the same molecular weight. It could be argued, that differ• ences in the solution behavior of xanthan and locust bean gum are due to differences in the molecular weight of the two polymers. However, the reported literature values for the molecular weight of unaggregated xanthan (MW * 2 x 106, Dintzis et al. 1970; Rinaudo and Milas, 1978) and locust bean gum (MW * 1.4 x 106, Doublier and Launay, 1981) are comparable. The molecular weights of the samples used were not estima• ted, however, and some differences may exist between molecular weights of polysaccharides from different sources.
c) Xanthan - Locust Bean Gum Solutions
The viscoelastic properties of xanthan-locust bean gum solutions were similar, in some respects, to those of xanthan solutions. However, the plateau region was considerably more pronounced, with an essentially constant value over the frequency range of 0.6 to 19 s"1. The storage moduli for the blend were higher and the loss moduli were lower than those for xanthan gum solutions alone, with the result that the values of the loss tangent were as much as ten times lower than those for - 47 -
xanthan solutions. The values of the loss tangent were very small, with an average value of around 0.03. This is close to the value of 0.01 quoted by Ferry (1980), for lightly cross-linked polymers.
Xanthan-locust bean gum solutions showed considerably more elasticity with less energy being dissipated as heat, when compared with xanthan solutions of the same concentration. This suggests the presence of .more numerous or stronger interactions in the blend. When compared with the systems described by Ferry (1980), the viscoelastic behavior of xanthan-locust bean gum solutions corresponded to that of either high molecular weight entangled systems or to lightly cross-linked polymeric networks. Since polysaccharide concentrations and molecular weight were essentially the same as in the individual solutions, it is unlikely that entanglements would have developed sufficiently to bring about the changes seen. The enhancement in rigidity and reduction in the viscous properties is therefore attributed to the development of specific cross- linkages between xanthan and locust bean gum (Dea et al. 1977).
Cross-links in xanthan-locust bean gum systems are not considered to specifically involve covalent bond formation or to be of the tetra- functional type used in rubber elastic theory (Treloar, 1975). The
"cross-links" resulting in polysaccharide gel formation have been attributed to non-bonded interactions between two or more chain segments leading to localized regions of order. Several sugar residues are involved giving rise to junction zones. A three dimensional network is built up when the ordered sequences of sugar residues are interrupted and a single polysaccharide chain interacts with several others (Rees,
1972; Rees and Welsh, 1977). - 48 -
d) Frequency Effects on Entanglements and Cross-Linkages
In cross-linked systems, it is assumed that both entanglements and cross-links contribute additively to elastic moduli of the network.
At high frequencies (short times), entanglements make a substantial
contribution, whereas cross-links may not add much. In the transition
zone from rubberlike to glasslike behavior (high frequencies),
configurational changes which occur between entanglements are of greater
significance and these give rise to short relaxation times. At low
frequencies, configurational changes which take place beyond
entanglements are of greater significance and entanglements do not
contribute much to the moduli. These changes are characterized by
longer relaxation times. For uncross-1inked polymeric systems, the
value of the moduli fall sharply with decreasing frequency giving rise
to the terminal zone. In this region, energy stored becomes negligible
in comparison to that dissipated as heat and the phase angle between
stress and strain approaches 90°. In contrast, the moduli for
cross-linked systems remain relatively unchanged. Thus, the greatest
differences between cross-linked and uncross-1inked systems are seen at
low frequencies (Ferry, 1980).
The terminal zone was not observed in the curves of the moduli for xanthan-locust bean gum blends, and this lends additional support to
the conclusion that the viscoelastic behavior of the polysaccharide
blends corresponds to that of a lightly cross-linked network. However,
it is possible that experiments were not carried out at low enough
frequencies to allow the observation of the terminal zone.
A pronounced minimum was observed in the curve of the loss moduli - 49 -
at 12 s-1. This was also reflected in the curve for the loss tangent.
The minimum value appeared in all three replications carried out at that frequency and so was not considered a spurious occurrence. A minimum is expected in the G" - frequency curve of entangled polymeric systems
(Ferry, 1980). However, this minimum is expected to encompass most of the plateau region and not just a single point in frequency. The minimum in the curve was probably due to a specific relaxation process which arose from movements of the polysaccharide chain.
e) Effect of Solvent Treatment and Temperature
The storage and loss moduli evaluated at 20 and 60°C for xanthan- locust bean gum blends in KOH, KC1 and urea are shown in Figure 8 and
Figure 9 as functions of frequency along with the curves for blends in water. Corresponding values for the loss tangent are shown in Figure
10. The F-ratios from the analysis of variance for the storage and loss moduli and the loss tangent are summarized in Tables Al, A2 and A3 of
Appendix II, respectively. Both temperature and solvent treatments were significant (p < 0.01) sources of variation. The significant (p < 0.01) temperature by solvent interaction indicated treatment effects were not the same at the two temperatures.
i) Effect of Urea
At 20°C, the storage and loss moduli for urea treated samples changed very little with increasing frequency over the frequency range used (0.6 to 19 s"1). However, at the highest frequency, the moduli increased sharply. This was comparable to the behavior of aqueous Figure 8. Dynamic storage (a) and loss (b) moduli for xanthan-locust bean gum solutions in water, 8 M urea, 0.6 M KC1, and 1 M KOH at 20°C. Figure 9. Dynamic storage (a) and loss (b) moduli for xanthan-locust bean gum solutions in water, 8 M urea, 0.6 M KC1, and 1 M KOH at 60°C. - 52 -
d dq
Fiqure 10. Loss tangent for xanthan-locust bean gum solutions in water, 8 M urea, 0.6 M KC1 and 1 M KOH at (a) 20°C and (b) 60°C. - 53 -
solutions. Values of the storage moduli for blends in urea were slightly lower than those of aqueous solutions. At 20°C, both the loss moduli and the loss tangent for urea treated blends were larger than those of aqueous solutions over the entire frequency range studied.
This indicates that more energy is being dissipated as heat for blends made in urea solutions when compared with solutions in water.
The results suggest that the interaction of xanthan with locust bean gum is not greatly disturbed by the presence of 8 M urea at 20°C, although some disruption in the system does take place. In urea solu• tions, intermolecul ar hydrogen bonds become disrupted as hydrogen bonds between urea and the polymer molecules are formed in preference
(Kauzmann, 1959; Lapanje, 1978). The end result would be that aggrega• tion of polymer molecules could be inhibited. These results suggest that hydrogen bonding plays only a minor role in the xanthan-locust bean gum interaction. Polysaccharide molecules form strongly hydrogen bonded structures in the solid state. However, in aqueous solutions, hydrogen bonds are considerably weaker, unless the shapes of the molecules are such that they can approach each other very closely with hydroxyl groups suitably orientated for hydrogen bond formation. Hydrogen bonds are strongest when the participating atoms are colinear, with bond strengths decreasing somewhat as the angle between the two participating groups increase (Richards, 1980). Therefore, a possible reason for the lack of importance of hydrogen bonding in this system could be unfavorable orientation of hydrogen bonding atoms in a large number of cases. - 54 -
Urea may also have a small direct effect in weakening hydrophobic bonds (Kauzmann, 1959; Lapanje, 1978). Hydrophobic sites on poly• saccharides develop on the interior of coiled helices (Kauzmann, 1959;
Rees, 1969). Interlocking of two helically coiled polysaccharides could give rise to hydrophobic interactions. From the results, it would appear that hydrophobic interactions would be minimal and this is consistent with proposed solution conformations of the interacting systems. Although xanthan exists in solution as a helically coiled molecule (Holzwarth and Prestridge, 1977; Moorhouse et al. 1977; Morris et al. 1977), the exact conformation of locust bean gum in solution is not known with certainty. It has been suggested that locust bean gum exists in solution as a random coil molecule, which reverts to the extended ribbon conformation found in the solid state, on interaction with xanthan (Dea et al. 1972, 1977). In addition, neither poly• saccharide carries a large proportion of non-polar side groups, e.g., alkyl substituents, so the possibilities for hydrophobic interactions are limited.
ii) Effect of Added Electrolytes
For both KOH and KC1 treated samples, the storage and loss moduli were almost linearly increasing functions of frequency. The storage moduli increased much more rapidly than the loss moduli, with the loss moduli being greater than the storage moduli at lower frequencies in both cases. At intermediate frequencies, the storage and loss moduli cross over, so that at the highest frequencies, the storage moduli becomes larger. The changes in the storage and loss moduli can be - 55 -
interpreted as a change from viscous or liquid-like behavior at low
frequencies (long times) to a more elastic or solid-like behavior at
higher frequencies (short times).
When blends made in KC1 and water are compared, the storage moduli were lower at the two lowest frequencies, with little difference
at higher frequencies. As with the urea treated samples, at 20°C values
for both the loss moduli and the loss tangent were larger than similar
values for samples in water, at all frequencies studied. Thus energy
dissipated as heat during cyclic deformation was greater for xanthan-
locust bean gum blends made in KC1 than for those made in water. The
greatest differences in viscoelastic behavior for cross-linked systems
occur at low frequencies where changes in the strength or degree of
cross-linking are apparent. The similarities in the storage moduli at
high frequencies, suggest that entanglements contribute similarly to the
values of the moduli for water, urea and KC1 treated samples.
In solutions of high ionic strength e.g., 0.6 M KC1 or 1.0 M KOH,
charges on polar groups or atoms become surrounded by a stabilizing
cloud of counterions. This minimizes ionic or electrostatic interac•
tions between oppositely charged groups (Kauzmann, 1959; Richards,
1980). The formation of induced dipoles leading to the development of
Van der Waals forces (London dispersion forces) and other dipolar inter•
actions between polysaccharide chains are also reduced in solutions of
high ionic strength. From a comparison of viscoelastic properties of
xanthan-locust bean gum blends in the different solvents, it would
appear that dipole interactions of a rather general nature are likely to
play a major role in stabilizing the gel structure. Dipole interactions - 56 -
are probably of greater importance than the more specific hydrogen bonded interactions which arise from similar fundamental causes. The most likely interactions probably arise from monopole (e.g., charged carboxyl group)-dipole as well as permanent and induced dipole-dipole interactions. Monopole-dipole interactions are considerably stronger than dipole-dipole interactions (Richards, 1980), however the latter are likely to occur more frequently. These interactions are relatively weak and are easily disrupted. The system probably owes its stability to the fact that a very large number of these interactions are formed co-operatively (Rees and Welsh, 1977).
Steric fit is an important contributor to the stability of carrageenan and pectin gels. Bulky substituents can diminish gelling ability drastically (Rees, 1969). It is possible that xanthan and locust bean molecules can approach each other more closely than can two molecules of the same type (Morris, 1973). This would enhance the degree of interaction between the two, to a level greater than that seen in like molecules.
The results obtained here for the effects of added electrolyte on xanthan-locust bean gum gels are opposite to that reported by Symes
(1980). In an empirical evaluation of gel strengths at 1% total poly• saccharide concentration, he found that gel strengths increased with increasing salt concentration when the salt concentration was in the range 0 to 4%. The proportion of added electrolyte to total poly• saccharide in this study is much higher than that used by Symes. It is possible that differences in the conclusion reached are due to differences in the proportion of salt to total polysaccharide. Jeanes - 57 -
et al. (1961), Symes (1980) and Southwick et al. (1983) have noted that the effects of salt on the viscosity of xanthan solutions depend not only on salt or polysaccharide concentrations, but also on the pH or degree of ionization of the polyanion.
iii) Effect of Alkali
At 20°C the storage moduli for blends made in potassium hydroxide were lower than those of blends in water, at all frequencies tested.
Values of both the loss moduli and the loss tangent were also larger than those of water at all frequencies tested.
Potassium hydroxide serves a similar purpose in the system as
KC1, in that, it brings about the disruption of polar interactions, including hydrogen bonds (Whistler, 1973). The close similarity of the
G' ,G"-frequency curves of KOH and KC1 is consistent with this and conclusions similar to those drawn for KC1 on the nature of cross-links in the system can also be drawn here.
However, the high pH of KOH, results in greater disruption of the interacting system, as this solvent is capable of disrupting covalent bonds. The values of the storage moduli for blends in KOH were lower than those in water and the other solvent treatments even at the highest frequencies. This suggests that the entanglement density in KOH solu• tions may have decreased somewhat. The number or density of entangle• ments varies with concentration or molecular weight of the polymer
(Ferry, 1980). The recovery of non-dialyzable solids from KOH treated blends were, on the average, somewhat higher than those of the other - 58 -
solvent systems except urea. This is probably due to the greater
solubility of polysaccharides in alkali (Whistler, 1973). Therefore, lower concentrations of polysaccharides in the system are unlikely to be the cause of the difference.
It is possible that base catalyzed hydrolysis of the glycosidic
linkages could have brought about reduction in the molecular weight of the polysaccharides. The viscosity of xanthan solutions is fairly
stable up to pH 11 (Jeanes et al. 1961; Rocks, 1971) but its stability above this pH is not known specifically. Galactomannans on the other
hand are rapidly degraded above pH 10.5, especially if heated (Dea and
Morrison, 1975). The glycosidic linkage is generally stable under
alkaline conditions. However, under certain conditions, e.g., in the
presence of strongly electron withdrawing functional groups, a base
catalyzed elimination reaction can be initiated (Aspinall, 1982). The degradation reaction begins at the reducing end of the molecule, and
proceeds with successive sugar residues being stripped from the molecule
in a stepwise manner. This type of reaction is particularly susceptible to competing reactions and a base stable terminal sugar unit can be
produced by side reactions. No further degradation will proceed if this occurs. Isosaccharinic acids (M.W. 192) are produced from the
hydrolysis reaction and these are expected to be lost on dialysis.
However, the solids recovered after dialysis, for the KOH treated samples, were on the average somewhat larger than those prepared in water or KC1, not smaller as would be expected if hydrolysis had proceeded to a significant extent. However, this is not consistent with the results of viscoelastic studies. It is possible that, hydrolysis - 59 -
reactions resulting in smaller molecular weight molecules, with fewer entanglements, have taken place by some other mechanism. Rees (1969), suggested that strong alkali can cause ionization of hydroxyl groups.
Association and interaction of polysaccharide chains becomes reduced by the repulsion of similarly charged groups. Such a mechanism could be responsible for the reduction in the values of the viscoelastic para• meters of xanthan-locust bean gum blends. Alternatively, potassium hydroxide may have brought about the disruption of fairly strong electrostatic or ionic type linkages not disrupted by KC1.
iv) Temperature Effects
The changes in the viscoelastic parameters were somewhat different at 60°C when compared with those obtained at 20°. The values of the storage and loss moduli increased almost linearly with increasing frequency and were smaller than corresponding values at 20°C. Except for samples prepared in KC1, values of the loss moduli were generally larger than the storage modulus. Values of the storage and the loss moduli for water and urea treatments became very similar. The loss tangent of samples in water, urea and KOH were also very close to each other but these were quite different from the values of KC1 treated
samples at corresponding frequencies.
When the effect of temperature on the viscoelastic properties of xanthan-locust bean gum blends in water and 0,6 M KC1 were examined in greater detail over the temperature range 5 to 60°C, the storage moduli and loss tangent for blends in water were most sensitive to changing
temperature. Over the temperature range 5 to 20°C, the storage moduli - 60 -
for samples in water changed very little, but at higher temperatures, the moduli decreased gradually and the G'- frequency curves changed shape (Figure 11). There appeared to be very little difference in values of the loss moduli (Figure 12), while the loss tangent increased with temperature (Figure 13). Viscoelastic behavior passed from that of a viscoelastic solid to that of a viscoelastic liquid. The storage moduli for blends in KC1 changed very little over the temperature range
5 to 50°C, then at 60°C, a plateau region developed in the G'-frequency curve (Figure 14). The storage moduli also became larger than the loss moduli (Figure 15) at all frequencies evaluated. Values of the loss tangent for KC1 treated samples appeared to change little over the
temperature range 5 to 50°C (Figure 16). At 60°C, values of the loss tangent became lower than corresponding values at 20°C while the reverse was true for samples prepared in the other solvents. These changes in viscoelastic behavior indicate an increase in the elastic or solid-like
characteristics of blends in KC1 at 60°C.
Changes in the viscoelastic properties of xanthan-locust bean
gum solutions can be correlated with the solution properties of xanthan
under different conditions of temperature and ionic strength. At
elevated temperatures, under conditions of low ionic strength, the
ordered conformation of xanthan is disrupted and a random coil conforma•
tion is adopted (Morris et al. 1977). The ordered conformation is
required for the interaction of xanthan with locust bean gum (Dea
et al. 1977), and the interaction is lost when the transition takes
pi ace. - 61 -
m CN CM
Figure 11. Effect of temperature on the storage modulus for xanthan-locust bean gum solutions in water. - 62 -
Figure 12. Effect of temperature on the loss modulus for xanthan-locust bean gum solutions in water. - 63 -
Figure 13. Effect of temperature on the loss tangent for xanthan-locust bean gum solutions in water. - 64 -
Figure 14. Effect of temperature on the storage modulus for xanthan-locust bean gum solutions in 0.6 M KC1. - 65 -
o
°C • 5 o 10 A 20 + 30 X 40 0 50 V 60 in
9-| ! 1 1 1 — -0.5 0.0 0.5 1.0 1.5 2.0 FREQUENCY (LOG SCALE)
Figure 15. Effect of temperature on the loss modulus for xanthan-locust bean gum solutions in 0.6 M KC1. - 66 -
Figure 16. Effect of temperature on the loss tangent for xanthan-locust bean gum solutions in 0.6 M KC1. - 67 -
Xanthan-locust bean gels are thermoreversible systems. Dea et al. (1977) reported that the gels have sharp melting and setting points which increase with increasing polysaccharide content, but show little dependence on the relative proportions of the two polymers. The effect of ionic strength on the gel melting and setting temperatures is not
known. It is possible that melting of xanthan-locust bean gum gels occurs at the same time as the conformational change of xanthan.
Snoeren and Payens (1976) studied gelation and the order-disorder transition in intensively decalcified K-carrageenan systems using light
scattering and optical rotation techniques, in an attempt to correlate the gel point with the reported double helix parameters of the poly•
saccharide. Both the gel point and the helix transition temperature,
increased linearly with the logarithm of the salt concentration. Since
both sets of experimental data coincided, they concluded that gel melting and the conformational change of K-carrageenan occurred
simultaneously. The midpoint of the transition temperature of xanthan
(Holzwarth, 1976; Milas and Rinaudo, 1979) and its reciprocal (Norton
et al., 1984) have also been shown to vary linearly with the logarithm
of cation concentration. However, no correlation has yet been made with
gel melting temperatures of xanthan-locust bean gum systems.
The ordered conformation of xanthan is stabilized to temperatures
in excess of 100°C at high ionic strengths (> 0.1 M KC1) (Morris et al.
1977). It would appear then, that the forces of interaction between
these two polysaccharides are still present under these conditions. The
dominant forces resulting in a gel structure at elevated temperatures,
are probably not the same as at 20°C. High ionic strength - 68 -
appears to enhance the interaction of xanthan with locust bean gum at
60°C. These results suggest that hydrophobic or non-polar interactions may now be more important. Hydrophobic interactions are more stable at elevated temperatures because the transfer of non-polar groups from water to a non-polar environment is endothermic (Kauzmann, 1959).
Entropy contributions to the overall free energy change of the system as a result of hydrophobic interactions, become more negative with increasing temperature, thus leading to greater stability of the interaction (Ben-Nairn, 1980). In addition, electrolytes tend to strengthen non-polar bonds because they decrease solubility of non-polar materials (Kauzmann, 1959).
Temperature also has a direct effect on the values of the visco• elastic parameters. With increasing temperature, the free volume of the solution is increased with a corresponding reduction in the fractional resistance to movement between different parts of the molecule (Ferry,
1980). Thus, the plateau region for blends in KC1 at 60°C occurs at lower values than the plateau region in water at 20°C. All other factors remaining constant, the evaluation of viscoelastic parameters at elevated temperatures corresponds to an evaluation at lower frequencies.
By applying the method of reduced variables to the viscoelastic parameters obtained at different temperatures, the frequency range over which the parameters are compared, can be extended. In order to use this procedure, the shapes or forms of the curves at different temperat• ures should match sufficiently to provide adequate regions of overlap.
No other changes in the system, apart from temperature effects, should affect the viscoelastic parameters. This is clearly not the case for - 69 -
the xanthan-locust bean gum system, where the conformational change under conditions of low ionic strength has affected the viscoelastic properties. The method is also not applicable to solutions at high
ionic strength since only a limited temperature range is available for evaluation of aqueous solutions. Changes in viscoelastic properties were not appreciable under these conditions.
f) Limitations
i) Failure to Meet Some Assumptions in Viscoelastic Theory
The results obtained here are based on a qualitative comparison of the viscoelastic behavior with the viscoelastic behavior of different polymeric systems described by Ferry (1980). It is difficult to apply established mathematical models to the xanthan-locust bean gum blends, as in many cases the assumptions inherent in the development of the theory are not met.
The systems to which xanthan-locust bean gum blends most closely corresponds, are either concentrated polymer solutions or lightly cross- linked polymeric networks. The modified Rouse Theory for concentrated
solutions for example, relates the values of the moduli with the frequency and relaxation times of the system. However, this theory holds for polymers with molecular weight less than 200,000. The molecu•
lar weight of xanthan and locust bean gum are both approximately to the
order of 2 x 106 and samples are generally polydisperse. Both these factors complicate the application of the theory. In addition, xanthan exists in solution as a rather rigid, helical rod. The domain of rod•
like molecules is greater than flexible molecules of similar molecular - 70 -
weight, thus these molecules interact and show concentrated solution behavior at concentrations much lower than their flexible counterparts
(Ferry, 1980). Ferry, in an examination of the viscoelastic properties of rod-like DNA (MW 5.8 x 106), found a plateau region extending over several decades of frequency at a concentration of 0.012 gmL-1.
However, attempts by the author to estimate the average molecular weight of polymer between coupling points, lead to a meaninglessly small value.
For cross-linked polymers, the relationship which stems from theory of rubber elasticity gives the equilibrium value of the shear
modulus (Ge) as
Ge = cRT/Mc [8]
where c is the concentration of the polymer solution, R, the universal
gas constant, T, the absolute temperature and Mc, the average molecular weight between coupling points. The above relationship for rubber-like polymers (cross-linked networks), stems from entropic considerations and assumes (among other things), a Gaussian distribution of the end-to-end vectors of the polymer chains. Although Holzwarth
(1981) and Norton et al. (1984) have implied that there exists a certain amount of flexibility in xanthan molecules it is unlikely that this assumption will hold for xanthan-locust bean gum molecules. This is because the molecules are considered to be in an extended configuration in the gel state (Mitchell, 1980). Rubber elastic theory also predicts an increase in the elastic modulus with temperature provided no cross• links are broken. This relationship does not hold for xanthan-locust - 71 -
bean gum blends in water or 0.6 M KC1 over the temperature range examined and attempts to apply this relationship to xanthan-locust bean
gum systems in water where Ge averaged 22 Pa, also lead to a small value for MQ.
To date, studies on the viscoelastic properties of aqueous biological systems have not been as extensive as plastic polymer systems. As a result, ability to treat the results from these systems mathematically is very limited. More systematic studies are required to develop suitable theories applicable to biological systems.
ii) Effect of Added Solutes
It is possible that the presence of added solutes may bring about changes in the shape of the viscoelastic curves without any changes having been made in the degree of cross-linking. Solutions in 8 M urea, with the greatest percentage of added solutes at 48% w/v, had the least perturbation of the three systems compared with water. Frangou et al.
(1982), in studies of xanthan viscoelastic properties (0.5%; 0.02 M KC1) over a similar frequency range, found that material behavior passed from that of a viscoelastic solid in 0.02 M urea (G' > G") to that of a viscoelastic liquid in 4 M urea. The values for the storage and loss moduli in 0.02 M urea increased much more slowly with increasing frequency than values in 4 M urea. These authors attributed the changes occurring in xanthan solutions, to the disruption of hydrogen bonds in xanthan aggregates, by 4 M urea. However, an even higher concentration of urea was used in the current study with considerably less disruption of the xanthan-locust bean gum interaction. Thus, earlier conclusions - 72 -
that hydrogen bonding plays a minor role in the xanthan-locust bean gum interactions still hold. Alternatively, the high concentration of solids and the resulting increase in frictional resistance between moving parts of the polysaccharide chain, could lead to an enhancement of both moduli which offsets any decrease brought about by rupture of hydrogen bonds.
Studies on the viscoelastic properties of xanthan in 0.1 M NaCl
show an increase in the storage moduli of xanthan and a decrease in the loss moduli when compared with water (Lim et al. 1984). These results have been attributed to an increase in aggregation of xanthan because
0.1 M NaCl is a poorer solvent than water. Southwick et al. (1983) suggested that at high ionic strengths, negative charges on xanthan were shielded, resulting in a decrease in interchain repulsion and an
increase in aggregation. However, these results are opposite to that obtained in this study using 0.6 M KC1 and this supports the conclusion that molecular interaction has been reduced in xanthan-locust bean gum blends, in this solvent. The results also suggest that dissolved
solutes do not directly affect viscoelastic properties. More studies along these lines are required to determine conclusively the effect of different solvents and solute concentrations on viscoelastic properties of biopolymers.
iii) Choice of Frequency
The frequency range used in this experiment, 0.6 to 60 s-1, approximately covers the range in which many changes may occur in the
xanthan-locust bean gum system. For materials whose rheological - 73 -
behavior approaches that of a viscoelastic liquid, it becomes progres•
sively more difficult to obtain data at low frequencies using
oscillatory shear techniques. Thus, the lower limit used in this
experiment, corresponded to the lowest frequency level at which reliable
data could be collected for samples evaluated at 60°C. The upper limit
approximated the upper limit of the instrument used. As a result the
experimentally accessible frequency range spanned only two logarithmic
intervals. In the systems discussed by Ferry (1980), at least ten to
fifteen logarithmic intervals of frequency were used to characterize the
different systems. The use of two logarithmic intervals would not be
sufficient to accurately characterize the viscoelastic behavior. It has
already been mentioned, that the absence of the terminal zone in
xanthan-locust bean gum blends, could be due to the fact that studies were not carried out at sufficiently low frequencies. In entangled
systems, the terminal zone is pushed to lower frequencies (Ferry, 1980)
and this could possibly be the case operating here. Investigation of
the viscoelastic properties of xanthan-locust bean gum blends at lower
frequencies may help to clarify this point.
In order to increase the experimentally accessible frequency
range or time period of evaluation, the time-temperature superposition
principle is usually applied. As discussed before, this method does not
appear to be applicable to xanthan-locust bean gum gels. Results from
creep or stress relaxation experiments which provide information on molecular movements requiring long times, can also be used to extend the
frequency or time range over which viscoelastic characterization is
carried out. These results are combined with those obtained from - 74 -
dynamic experiments, after conversion to common units. It may be possible to apply either of these two techniques to xanthan-locust bean gum gels at higher concentrations as firm, self-supporting gels are required. At 0.4% total polysaccharide concentration, weak gels are formed and creep or stress relaxation tests cannot be used.
iv) Nature of the Cross-linkages
Evaluation of the viscoelastic properties of the blends was carried out approximately one hour after the preparation of the gel.
Because of limitations discussed earlier, tests were carried out before the system had come to equilibrium. It is possible that early bonds formed were not the same as those which would predominate in older gels. Ferry (1980) and Treloar (1975) suggested that initially weak bonds may dissociate as the system ages and be replaced by stronger bonds which are formed more slowly. Relative strengths of bonds may also change and as the bonds age, they become progressively more difficult to break. Therefore hydrogen bonds or hydrophobic inter• actions may increase in importance in older gels. The relative import• ance of different bonds may also change with increasing concentration.
Increasing concentration can lead to the development of more favorable sites for cross-linking. Thus, cross-linking density is not only a function of time or temperature but also of concentration (Ferry, 1980).
The experiment provides no information on the stoichiometry of binding or the number of sugar residues involved in junction zone formation. Although a 1:1 stoichiometry is the simplest and most likely relationship, other stoichiometrics cannot be ruled out. It is also - 75 -
possible that there is no exact stoichiometry of association or even binding of the polysaccharides.
Carroll et al. (1984) have examined the interaction of ic-carrageenan and locust bean gum using X-ray fiber diffraction. Pure
K-carrageenan has a minimum gelling concentration of 1.5%. However, on the addition of locust bean gum, gelation occurs at total polymer concentrations as low as 0.5%. The currently-accepted model for this synergism is the same as that proposed for xanthan-locust bean gum systems, as shown in Figure 3. In their study, Carroll et al. (1984) found that the X-ray fiber diffraction patterns of the carrageenan- locust bean gum systems were identical to those of pure carrageenan.
Measured unit cell dimensions were essentially unchanged. They concluded that molecules of locust bean gum had not been incorporated
into the carrageenan gel network. Since there was no superimposition of the locust bean gum diffraction pattern onto that of carrageenan, they suggested that a specific molecular interaction involving parallel alignment of carrageenan and locust bean gum molecules was unlikely. A model involving the linking of relatively large carrageenan crystallites by attachment of a few locust bean gum molecules to the surface of the crystallites was not considered unreasonable.
Such a model is also quite feasible for xanthan-locust bean gum systems. The tendency for xanthan molecules to aggregate in solution has been repeatedly documented (Dintzis et al. 1970; Morris et al. 1977;
Southwick et al. 1979; 1980; Norton et al. 1984). Locust bean gum molecules might serve as "bridging" molecules between xanthan aggregates
resulting in the formation of a less open three-dimensional network. - 76 -
The flexibility of locust bean gum molecules may facilitate its ability to act as a "cross-linking" agent. Weak dipole interactions and hydrogen bonding may be responsible for holding xanthan aggregates as well as bridging locust bean gum molecules together.
2. Steady Shear Flow Properties
a) General Considerations
Typical flow behavior rheograms are shown in Figure 17, for xanthan-locust bean gum solutions at 0.2% and 0.05% total polysacchar• ide concentration. Rheological behavior corresponded to that of non-
Newtonian pseudoplastic fluids as indicated by the negative slopes of the rheograms. Samples exhibited a limited amount of time dependence as rheograms taken first with increasing, then decreasing values of shear rate did not follow the same path. Rheological data obtained using decreasing values of shear rate were collected after the samples had been sheared to a nearly constant shear stress value at the maximum
shear rate, Ymax. This was considered an adequate method for producing a well defined condition at the start of shear (Mewis, 1979). All further analyses were carried out using data obtained in this manner.
The apparent viscosity, n at any shear rate, y was then a function of the maximum shear rate used, the shear rate of measurement and the time, t during which shear was applied. That is,
n = f(Y > y, t) v max' * - 77 -
• 0.2 °to, HaO O 0.05O/O, H2O A 02%, 0.1 M NaCl + 0.05%, 0.1 M NaCl
T 1 1 I I I I I 1 1 1 1 I I I I I T 1 1 I I I I -1 2 10 10° 101 SHEAR RATE ( sr1 ) 10
Figure 17. Rheograms for xanthan-locust bean gum solutions at 20°C plotted according to the power law flow model. - 78 -
In more complicated relationships, the term Ymax may be replaced by a
structural parameter, X which more completely describes the structural
history of the material.
Pseudoplastic and time dependent effects arise from processes taking place at the molecular level. Intermolecular forces of attrac• tion result in the association of particles in solution leading to increased resistance to flow and higher apparent viscosities. If these forces are weak, hydrodynamic forces arising from steady shear are sufficient to disrupt the associated structural units. Smaller size
units give reduced resistance to flow and lower apparent viscosities.
Alignment of molecules along the shear planes, especially if long and
rigid, gives rise to the phenomenon of shear thinning or pseudoplasti- city. If the rate of recovery of structure is slow, apparent viscosit•
ies from successive shearing regimes are different and time dependent flow behavior is observed. In the previous section, it was shown that the forces of interaction between xanthan and locust bean gum were
predominantly weak, dipole interactions. These are easily disrupted by
shearing forces. The interactions are not recovered instantaneously, giving rise to time dependent flow behavior. It is possible that complete recovery of structure to the original level does not occur
(rheodestruction).
The presence of a yield stress is also a common characteristic of materials of this nature. However, the time dependent properties or yield stresses of xanthan-locust bean gum blends were not examined specifically in this study. - 79 -
In isolated cases, scatter plots indicated the development of a
low shear Newtonian region. Since only a limited number of data points were available in this region and the power law model provided an
adequate fit to the data (r values of Table 5), no other rheological
model was fitted to the data.
b) Flow Properties Under Different Conditions
The power law parameters n and m, and the viscosity at 50 s_1
obtained' in Taguchi's fractional factorial are also shown in Table 5.
Results of the analysis of variance for these variables are shown in
Tables A4, A5 and A6 of Appendix II. The analyses were performed in two
steps, with insignificant terms in an initial analyses being combined
with the error term to give the final analyses shown here.
The pseudoplasticity of xanthan-locust bean gum solutions
varied considerably (n values ranged from 0.25 to 0.68). All the
variables examined except concentration, temperature and the correspond•
ing interaction term brought about significant changes in the flow
behavior index (p < 0.05). However, the second . experiment in which
temperature, concentration and ionic strength effects were examined in
greater detail, indicated that these factors had significant effects
(p < 0.01) on the flow behavior index (Table A7 of Appendix II).
Differences in the results obtained from the two experiments are
probably due to differences in the statistical precision of the two
analyses. There were only two degrees of freedom associated the error
term in the fractional factorial experiment while there were forty
degrees of freedom associated with error in the second analysis. It is - 80 -
Table 5. Summary of flow parameters (Y = 0.2 - 175 s" ) obtained from Taguchi's fractional factorial experiment.
1 2 Expt. m n1 n(at Y = 50 s_1) r No. mPa sn mPa s
1 200.0 0.493 21.4 0.977 2 672.0 0.480 87.9 0.957 3 1414.0 0.381 125.6 0.973 4 97.9 0.642 24.2 0.973 5 1188.0 0.380 105.0 0.854 6 187.0 0.558 33.0 0.602 7 288.0 0.416 29.3 0.990 8 58.2 0.667 15.8 0.980 9 53.5 0.628 12.5 0.932 10 61.1 0.683 17.7 0.945 11 19.5 0.666 5.3 0.897 12 137.0 0.460 16.5 0.968 13 87.1 0.592 17.7 0.660 14 200.4 0.594 41.0 0.828 15 213.9 0.448 24.7 0.952 16 332.0 0.278 19.7 0.983 17 28.6 0.649 7.3 0.876 18 18.5 0.610 4.0 0.917 19 49.1 0.538 8.1 0.958 20 95.3 0.482 12.6 0.985 21 10.0 0.609 2.2 0.856 22 546.0 0.246 28.5 0.979 23 7.6 0.730 2.7 0.728 24 46.2 0.633 11.0 0.887 25 12.9 0.484 1.7 0.947 26 16.0 0.636 3.8 0.863 27 16.4 0.651 4.2 0.907
Obtained from pooled regression of 4 sub-samples.
Calculated from regression parameters. - 81 -
therefore much easier to declare significant differences in the second experiment. The more detailed analysis also showed that the combined effects of temperature, concentration and ionic strength were signific•
ant in affecting the flow behavior index (p < 0.01). Changes in the flow behavior index with temperature are shown for xanthan-locust bean gum blends at two different levels of concentration and ionic strength
in Figure 18. For blends not stabilized by added electrolyte, the rate of structure breakdown increased with increasing temperature (decreasing n values), up to a 40°C for solutions at the lower concentration and to
50°C for solutions at the higher concentration. For blends at low
concentration in 0.1 M NaCl, the rate of structure breakdown decreased with increasing temperature while at the higher concentration, values of the flow behavior index changed in a less consistent manner. These trends were also indicated by the fractional factorial experiment.
The effect of temperature on the consistency coefficient
(numerically equal to the apparent viscosity at 1 s-1), and the apparent
viscosity at 50 s"1 also varied with polysaccharide concentration and
ionic strength (Tables A8 and A9 of Appendix II). For blends in water, there was an increase in apparent viscosity with temperature up to 40°C followed by the normal decrease with increasing temperature. These effects were more pronounced for the solutions at lower concentrations
and for the consistency coefficient (Figures 19 and 20). For blends in
0.1 M NaCl, the apparent viscosity remained constant up to temperatures
of 40°C, before the usual decrease with temperature was observed. The values of the consistency coefficient for solutions of 0.05% poly•
saccharide concentration in 0.1 M NaCl, decreased steadily with - 82 -
; i 1 1 1 1 1— 0.0 10.0 20.0 30.0 40.0 50.0 60.0 TEMPERATURE (°C)
Figure 18. Effect of temperature on flow behavior index of xanthan-locust bean solutions. - 83 -
c
2 UJ O
Ul o o — o >- o 2
00
10 z o o
oJ
• 0.2 °to, HaO o 0.050/0, H2O
A 02%, 0.1 M NaCl + 005O/o, 0.1 M NaCl
1 1 1 1 0.0 10.0 20.0 30.0 40.0 50.0 60.0 TEMPERATURE (°C)
Figure 19. Effect of temperature on the consistency coefficient of xanthan-locust bean gum solutions. - 84 -
• 0.2 °to, HjO O 0.05 o/b, HaO A 0.2%, 0.1 M NaCl + 005%, O.lMNaCI
O a.
CO o 00 2-
2 UJ or < <
O-
1 1 ! I 1 1 0.0 10.0 20.0 30.0 40.0 50.0 60.0 TEMPERATURE (°C)
Figure 20. Effect of temperature on the apparent viscosity (Y = 50 s"1) of xanthan-locust bean gum solutions. - 85 -
temperature. Apparent viscosities of solution in 0.1 M NaCl were considerably lower than solutions of the same concentration in water.
The rheological behavior of xanthan-locust bean gum solutions under different conditions of temperature and ionic strength was very similar to the reported behavior of xanthan solutions. Jeanes et al.
(1961) found that below concentrations of 0.1%, apparent viscosities of xanthan solutions decreased on addition of salt whereas at higher concentrations, apparent viscosities increased on the addition of salt.
The decrease in viscosity of xanthan solutions in the presence of salts follows normal polyelectrolyte behavior in which, increased ionic strength results in charge screening and a more compact coil form. In the previous section, it was shown that high ionic strengths also serve to disrupt the interaction between xanthan and locust bean gum. Thus, under the conditions used here, both the above mentioned effects may be in operation, leading to lower apparent viscosities of xanthan-locust bean gum solutions in the presence of external salt.
The reported solution behavior of xanthan polysaccharide in the presence of salt is complicated, with the observed effects being dependent on polysaccharide concentration, the concentration of salt used and the pH or degree of ionization of the polyanion. The number of ionizable groups present, in turn, depends on the degree of pyruvate substitution on the polymer molecule. In addition to effects of salt on xanthan solutions at low concentrations (<0.1% polysaccharide concentra• tion), Jeanes et al. (1961) and Smith et al. (1981) also noted that above concentrations above 0.4% polysaccharide, viscosity increased to a maximum with increasing concentrations of salt. Fluctuating behavior - 86 -
was observed at intermediate concentrations. Symes (1980) suggested that the changes in solution viscosity upon addition of salt to purified xanthan at high concentration, depended on the degree of pyruvate
substitution or ionization of the polyanion. When the carboxylate groups are fully protonated, interchain repulsion is minimized and attractive forces may even be accentuated by virtue of hydrogen bonding. An increase in ionic strength resulted in a decrease in viscosity whereas the reverse was observed for solutions of the
polysaccharide in the salt form.
In the K+ salt form, if the degree of pyruvate substitution exceeded 0.31, viscosity increased with increasing ionic strength (Smith et al. 1981). The increase was attributed to an increase in macro- molecular associations, as a result of interactions of pyruvate methyl groups which are suitably situated near the periphery of the helical
conformation. Southwick et al. (1983), based on the diffusion coeffi• cients, viscosity and flow of solutions through millipore filters,
suggested that the hydrodynamic size of xanthan was increased by
addition of electrolyte above a critical concentration (ca. 0.02M
NaCl). However, concentrations of polysaccharide used in his study
appeared to be less than the critical concentration suggested by Jeanes et al. (1961). Rinaudo and Mil as (1978) suggested that the hydrodynamic
size of xanthan molecules was essentially constant above ionic strengths of 10"2M. Additional studies are required over a wide range of salt and
polysaccharide concentration to determine whether xanthan-locust bean gum blends show similar complicated behavior.
The insensitivity of xanthan solutions to temperature in the presence of salts has been frequently documented (Whitcomb and Macosko, - 87 -
1978; Anon. 1978). Jeanes et al. (1961) and Dea et al. (1977) showed that in the absence of salt, xanthan solutions showed an anomalous increase in viscosity with increasing temperature. The increase in viscosity was attributed to an increase in the hydrodynamic volume of xanthan as the molecule changed conformation. Normal viscosity decrease with temperature was observed when the conformational change was complete and only the random coil form was present (Morris et al.
1977). The less pronounced increase in viscosity for blends at 0.2% polysaccharide concentration, may possibly be due to higher ionic strengths of these solutions, as a result of counterion contributions.
Launay et al. (1984) suggested that it was not justified to associate the increase in apparent viscosity with the onset of helix melting. This was because the temperature, at which the maximum appeared, varied with the shear rate used to evaluate the viscosity. In addition, the transition temperature obtained viscometrically, was often much lower than the melting temperature determined by other experimental methods. However, an alternative rationalization for the increase in viscosity of xanthan solutions, under conditions of low ionic strength and increasing temperature is not immediately obvious.
The ratio in which the two polysaccharides were mixed was also a significant factor (p < 0.05) affecting the flow behavior index and the apparent viscosity (Tables A4 and A6 of Appendix II). Both pseudo- plasticity and apparent viscosity increased when the proportion of xanthan increased. In preliminary experiments, apparent viscosities (up to shear rates of 50 s'1 of xanthan solutions were higher than those of locust bean gum at equivalent concentrations. Above this shear rate the - 88 -
reverse became true, because of the greater pseudoplastic nature of xanthan solutions. Thus it is possible that solution properties of blends simply reflect the solution properties of xanthan polysaccha• ride. Increased • proportions of xanthan increased the pseudoplastic character of the system, as xanthan molecules have a greater tendency to align in solution (Morris et al. 1977; Southwick et al. 1980). At low shear rates, solutions with a higher proportion of xanthan had higher apparent viscosities. At higher shear rates, increasing the proportion of xanthan may result in solutions of progressively lower apparent viscosities. Kovacs (1973) suggested the optimal gum ratio for gel strengths reflected the stoichiometry of binding of the two polysaccha• rides. However, no optimum was observed under the experimental conditions used here. This suggests that no exact stoichiometry of binding exists between the two polysaccharides under these conditions.
The flow behavior index and viscosities of xanthan-locust bean gum solutions were also influenced by the pH of the solution. Over the pH range used, viscosity tended to increase with increasing pH possibly due to greater solubilization of the polysaccharide. It is expected that viscosity would fall somewhat if more extreme pH conditions were used.
The viscoelastic properties of xanthan-locust bean gum solutions were considerably affected by 8 M urea. However, under the conditions used, 4M urea was not a significant factor (p > 0.05) affecting steady shear flow viscosity of xanthan-locust bean gum solutions. It is possible that either the concentration of urea or the conditions used were not sufficient to bring about a marked change in viscosity. - 89 -
Alternatively, a balance may have been achieved in two opposing forces:
(1) increased viscosity due to the greater ability of urea to solubilize the polysaccharides, and (2) decreased viscosity as result of disruption of hydrogen bonds in the interacting system. - 90 -
CONCLUSIONS
The viscoelastic properties of solutions of xanthan, locust bean gum and blends of the two indicate that various levels of association exist between the polysaccarides in solution. The associations between locust bean gum molecules at 0.4% concentration are low in number and are very weak. The intermolecular associations between xanthan molecules, at the same concentration, are considerably greater, with the result that xanthan solution behavior is typical of highly entangled systems. Strong intermolecular associations of greater magnitude than those in xanthan exist between molecules of xanthan and locust bean gum. These associations are considered more specific than simple entanglements and give the system viscoelastic properties typical of a lightly cross-linked network.
Solvent treatments, aqueous solutions of 8 M urea, 0.6 M KC1 and
1 M KOH reduce the interactions between the two polysaccharides to varying extents. The results suggest that early in the formation of the gelling system, at 0.4% concentration, dipole interactions are the principal forces responsible for stabilizing the network. Hydrogen bonding and hydrophobic interactions may play only limited roles.
Under conditions of low ionic strengths, xanthan-locust bean gum solutions lose their solid-like behavior with increasing temperature and eventually behave as viscoelastic liquids at 60°C. The combined results of dynamic and steady shear experiments suggest that gel melting and the order-disorder transition of xanthan occur simultaneously. When the ordered conformation is stabilized by high ionic strengths, the inter- - 91 -
acting system maintains its elasticity and even appears to become more
stable at the temperature of 60° C. It is possible that hydrophobic
interactions play a dominant role in the formation of the three dimensional network structure under these conditions.
In steady shear flow studies an anomalous increase in viscosity
is observed in xanthan-locust bean gum solutions under conditions of low
ionic strength. This is similar to reported steady shear rheological
behavior of xanthan. This behavior has been attributed to the conforma•
tional change of xanthan under conditions of low ionic strength and ele•
vated temperatures. Thus, as observed with optical rotation studies of
xanthan-guar and xanthan-tara solutions (Dea et al. 1977), the order- disorder conformational transition of xanthan is still in evidence for
xanthan-locust bean gum blends, as shown viscometrically, in this study.
The viscosity of xanthan-locust bean gum solutions increased with
increasing polysaccharide concentration. Temperature effects were
strongly dependent on the ionic strength of the solution. Viscosity appeared to increase as the pH of the solutions and the proportion of
xanthan increased. Ionic strength, pH and gum ratio all affected the flow behavior of xanthan-locust bean gum solutions.
•Results of viscoelastic studies indicate that, under the conditions used, dipole interactions may be responsible for
stabilization of the interacting system. These forces are very weak and this is consistent with the observation that both dynamic and steady
shear flow properties of xanthan-locust bean gum solutions appear to be dominated by the solution properties of xanthan. - 92 -
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APPENDIX I. Basic viscoelastic theory.
In small amplitude oscillatory shear, the material is subjected to a sinusoidally varying strain. The strain, as a function of time can be represented by
Y = Y0 cos u>t [Al]
1S tne where Y0 strain amplitude (Whorlow, 1980). If the strain amplitude is sufficiently small the resultant stress can be represented by
a = aQ cos (cot + 6) [A2]
where o0 is the amplitude of the shear stress and 6 is the phase shift
of the stress relative to the strain. The dependence of a/y0 on time
tnus is independent of Y0 Equation A2 can be rewritten
^ = ^ cos (u>t + 6) [A3] 0 To
1s If Y0 sufficiently small, the amplitude ratio (a0/y0) and the phase shift 6 are independent of amplitude. This means that the results of small amplitude oscillatory shear tests can be completely described - 99 -
by plots of (O0/YO) and 6 as functions of frequency (Dealy, 1982).
However it is customary to write equation A2 in the form
a = Yq (G' cos wt - G" sin wt) [A4)
The storage modulus
a cos 6 G' =-2— [A5] To
is a ratio of the stress in phase with the strain to the strain. It is a measure of energy stored elastically during a cycle of deformation.
The loss modulus
a sin 6 G" = -°— [A6] To
gives the ratio of the stress 90° out of phase with the strain to the strain. It is a measure of the energy dissipated as heat. If the stress and strain are in phase (G" = 0), material behavior is that of an ideal elastic (Hookean) material. If G' = 0 but G" * 0 the stress and strain are 90° out of phase and one finds that all of the energy introduced into the system is dissipated as heat. This is the viscous portion of the material response. - 100 -
The ratio of the two moduli, the loss tangent
tan 6 = ^ [A7]
is the ratio of energy dissipated, to the energy stored.
If use is made of the relationship
cos tot + isin oot = ela)t
then Equation Al and Equation A2 can be rewritten
iwt Y - R (Y0 e ) CA8]
a = R (a ei^6,t+6)) = R (a e16 eia)t) [A9] \ Q o
l5 It is convenient to refer to o0e as the complex amplitude of a * . The complex modulus G is defined as
G* = complex stress amplitude ~ complex strain amplitude
a e16 -°- = G' + 1G" [A10] o
The shear rate is also periodic and is given by - 101 -
Y = -UY_ sin wt = -y sin wt [All]
so that Equation A4 may be written in yet another form
a = y (- n' sin wt + TI" cos wt) CA12]
where = (°0/v0) sin 6 [A13]
II a = G'/w = ( 0/"Y0) COS 6 CA14]
The function n1 is the dynamic viscosity and is the ratio of the stress in phase with the rate of strain to rate of strain and n" is the stress 90° out of phase with the rate of strain divided by the rate of strain. The in-phase or real component n' for a viscoelastic liquid
approaches steady flow viscosity n0 as the frequency w approaches zero
(Ferry, 1980).
The phase relationships can also be expressed in terms of a complex viscosity n*, where
* , II [A15] n = n -in - 102 -
APPENDIX II. Analyses of variance for rheological parameters.
Table Al. Summary of F-ratios obtained from split-plot experimental design for dynamic storage modulus.
Frequency s' Source : 0.60 1.90 6.0 19.0 60.0
Run 0.48 NS 0.02 NS 0.86 NS 3.31* 2.27 NS
Temperature 167.15** 1159.** 191.6** 318.44** 292.26**
Run x Temp 1.39 NS 0.41 NS 0.50 NS 3.99* 4.18*
Sol vent 16.71** 23.92** 87.05** 71.13** 81.43**
Sol x Run 1.34 NS 1.97 NS 1.05 NS 1.08 NS 0.98 NS
Sol x Temp 68.06** 21.02** 14.94** 6.16* 6.82*
Sol x Run x Temp 0.67 NS 1.74 NS 13.06* 3.94** 5.12**
NS = Not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at p < 0.01. - 103 -
Table A2. Summary of F-ratios obtained from split-plot experimental design for the loss modulus.
Frequency s"1
0.60 1.90 6.0 19.0 60.0
Run 0.89 NS 0.24 NS 2.65 NS 6.71** 2.87 NS
Temperature 42.48* 225.62** 362.03** 43.89** 60.51*
Run x Temp 5.07* 1.16 NS 1.26 NS 6.45** 3.69*
Sol vent 16.16** 108.09** 126.33** 80.14** 20.80**
Sol x Run 2.63 NS 0.46 NS 0.66 NS 1.29 NS 3.28**
Sol x Temp 3.96 NS 12.01** 16.42** 21.61** 21.97**
Sol x Run x Temp 2.50* 1.70 NS 13.02* 3.28** 4.17**
NS = Not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at p < 0.01. - 104 -
Table A3. Summary of F-ratios obtained from split-plot experimental design for the loss tangent.
Frequency s" 1 Source 0.60 1.90 6.0 19.0 60.0
Run 1.42 NS 0.27 NS 0.42 NS 6.71** 2.88 NS
Temperature 44.19* 732.33** 1767.4** 43.89** 60.51*
Run x Temp 0.61* 0.11 NS 0.14 NS 6.44** 3.69*
Sol vent 36.17** 29.55** 102.46** 80.14** 20.80**
Sol x Run 1.09 NS 1.54 NS 1.05 NS 1.29 NS 3.27**
Sol x Temp 112.06** 55.87** 244.47** 21.61** 21.97**
Sol x Run x Temp 0.75 NS 1.11 NS 0.59 NS 3.28** 4.17**
NS = Not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at p < 0.01. - 105 -
Table A4. Analysis of variance for flow behavior index n, from
Taguchi's Fractional Factorial Experiment L27 (3 ).
df1 MS F-ratio
Ionic Strength 2 0.02390 9.51* pH 2 0.02370 9.43*
Gum Ratio 2 0.03200 12.72*
Urea 2 0.02072 8.24*
Cone x Temp. 4 0.00906 3.60NS
Cone x I.S. 4 0.01621 6.45*
Temp x I.S. 4 0.02396 9.53**
Error1 6 0.02515
Total 26
ldf for concentration and temperature pooled with error terms as they were not significant in original analysis of variance.
NS = not significant (P > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at p < 0.01. - 106 -
Table A5. Analysis of variance for consistency coefficient m, from
Taguchi's Fractional Factorial Experiment L27 (3 ).
df1 MS F-ratio
Concentration 2 1.8850 36.53**
Temperature 2 0.6860 13.29** pH 2 0.4616 8.94*
Gum Ratio 2 1.1102 21.51**
Cone x Temp. 4 0.1556 3.02NS
Cone x I.S. 4 0.1610 3.12NS
Temp x I.S. 4 0.1307 2.53NS
Error1 6 0.0516
Total 26
df for ionic strength and urea pooled with error terms as they were not significant in original analysis of variance.
NS = not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at p < 0.01. - 107 -
Table A6. Analysis of variance for viscosity obtained at a shear rate of 50 s from Taguchi's Fractional Factorial Experiment 13 L27 O ).
df1 MS F-ratio
Concentration 2 1.4944 121.72**
Temperature 2 0.4954 40.35** pH 2 0.1807 14.71**
Gum Ratio 2 0.5880 47.89**
Cone x Temp. 4 0.0853 6.95*
Cone x I.S. 4 0.0491 4.00NS
Temp x I.S. 4 0.0117 0.95NS
Error1 6 0.0123
Total 26
1df for ionic strength and urea pooled with error terms as they were not significant in original analysis of variance.
NS = not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at p < 0.01. - 108 -
Table A7. Analysis of variance for flow behavior index from steady shear-temperature studies.
Source df Mean Square F-ratio
Temperature 4 0.01652 8.90**
Ionic Strength 1 0.22861 123.13**
Concentration 1 0.04772 25.70**
I.S. x Cone. 1 0.04987 26.86**'
Temp x I.S. 4 0.05866 31.59**
Temp x Cone 4 0.04924 265.24**
Temp x Cone x I.S. 4 0.00911 4.90**
Residual 40 0.00187
Total 59
NS = not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
= significant at p < 0.01. - 109 -
Table A8. Analysis of variance for consistency coefficient from steady shear-temperature studies.
Source df Mean Square F-ratio
Temperature 4 1.5823 96.97**
Ionic Strength 1 11.203 686.54**
Concentration 1 25.127 1,539.83**
I.S. x Cone. 1 0.1108 6.79**
Temp x I.S. 4 0.2376 14.51**
Temp x Cone 4 0.0118 0.72 NS
Temp x Cone x I.S. 4 0.0578 3.54*
Residual 40 0.0163
Total 59
NS = not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at P < 0.01. - 110 -
Table A9. Analysis of variance for viscosity at 50 s"1 from steady shear-temperature studies.
Source df Mean Square F-ratio
Temperature 4 1.3247 80.39**
Ionic Strength 1 6.4248 389.90**
Concentration 1 21.544 1,307.44**
I.S. x Cone. 1 0.5074 30.79**
Temp x I.S. 4 0.3324 20.17**
Temp x Cone 4 0.1568 9.51**
Temp x Cone x I.S. 4 0.1413 8.58*
Residual 40 0.0165
Total 59
NS = not significant (p > 0.05);
* = significant at 0.01 < p < 0.05;
** = significant at p < 0.01.