ABSTRACT 
 
 
 
 
Title of Document: CHARACTERIZATION OF 
PHYSICOCHEMICAL PROPERTIES OF 
XANTHAN/CURDLAN HYDROGEL 
COMPLEX FOR APPLICATIONS IN FROZEN 
FOOD PRODUCTS 
  
 Patrick D. Williams, Doctor of Philosophy, 2011 
  
Directed By: Associate Professor, Y. Martin Lo, Department 
of Nutrition and Food Science 
 
 
Syneresis, expulsion of moisture from food, remains a major challenge in frozen 
products due to post-production temperature fluctuation during distribution, 
transportation, and storage. Curdlan, a FDA approved microbial polysaccharide, 
produces a hydrogel under certain conditions exhibiting good gel strength during 
freeze-thaw abuse, better than similar polysaccharides. However, curdlan alone 
cannot effectively suppress syneresis in aqueous solutions. The present study aims at 
developing an effective hydrogel complex containing curdlan and a secondary 
biopolymer to reduce or eliminate syneresis. Polysaccharides, ?-carrageenan, guar, 
locust bean, and xanthan gums were investigated. Xanthan gum, when added to 
curdlan, was found capable of eliminating syneresis for up to five freeze-thaw cycles 
(FTCs) while exhibiting stability in several rheological experiments. The xanthan 
curdlan hydrogel complex (XCHC) was further investigated to elucidate its structure 
 
  
in relation to its high elasticity and moisture holding capacity. A gel and indications 
of syneresis was clearly seen in magnetic resonance images of curdlan alone, whereas 
XCHC was homogeneous. The three dimensional network, indicated by frequency 
sweeps, of curdlan was responsible for its gel structure. Though not three 
dimensional, elasticity and angular frequency dependency for XCHC were 
significantly closer to curdlan than xanthan alone. Addition of xanthan to curdlan 
restricted spin-spin relaxation times of XCHC to intermediate and slower exchange 
regimes, promoting the polymer?s interaction with water while inhibiting 
intermolecular interactions found in curdlan. Furthermore, the effects of pH and NaCl 
concentration on the rheology of XCHC were examined. Though both pH and NaCl 
were found to reduce the hydrogel?s moduli, XCHC remained stable over the pH 
range 4 to 8 through FTCs and were not significantly different. Statistically, the 
modulus measurements of samples treated with 5-200 mM NaCl were slightly 
different and fluctuated throughout the FTCs. Nonetheless, the slope of the storage 
modulus over all NaCl-treated samples averaged ~9% different from untreated 
XCHC, indicative of similar gel behavior despite lower elasticity. Finally, the 
hydrogel was incorporated into a simulated pumpkin pie filling. Treating the filling 
with XCHC delayed moisture migration by one FTC, yielded a brighter color, 
reduced variability and minimized surface cracking that could lead to decreased 
quality and microbial growth.  
 
 
 
 
 
 
 
  
 
 
 
 
CHARACTERIZATION OF PHYSICOCHEMICAL PROPERTIES OF 
XANTHAN/CURDLAN HYDROGEL COMPLEX FOR APPLICATIONS IN 
FROZEN FOOD PRODUCTS 
 
 
 
By 
 
 
Patrick Daniel Williams 
 
 
 
 
 
Dissertation submitted to the Faculty of the Graduate School of the  
University of Maryland, College Park, in partial fulfillment 
of the requirements for the degree of 
Doctor of Philosophy 
2011 
 
 
 
 
 
 
 
 
 
 
Advisory Committee: 
Professor Y. Martin Lo, Chair 
Professor Thomas Castonguay 
Professor Mark Kantor 
Professor Srinivasa Raghavan 
Professor Qin Wang 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
? Copyright by 
Patrick Daniel Williams 
2011 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ii 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
To my wife and family. 
 
 
 
 iii 
 
Acknowledgements 
 I would like to thank my advisor, Dr. Y. Martin Lo, for his continual support, 
guidance and support throughout my graduate career. In addition, his thoughtfulness 
and care for his students, abundance in leadership, and just a pleasure to work under 
has made him an excellent role model for me. I am also very thankful for the advice 
and direction from my committee members, Dr. Srinivasa Raghavan, Dr. Qin Wang, 
Dr., Mark Kantor, and Dr. Thomas Castonguay and for their considerable support, 
guidance, and knowledge. I would also like to thank Drs. Kathryn and Mike 
McCarthy and Mecit Oztop at the University of California ? Davis, they played a 
pivotal role in moving my project forward. 
 I want to thank all my labmates for being patient with me, their unwavering 
support, and laughs; Dr. Pavan Kumar Soma [SPK], Peter Machado, Dr. Sanem 
Argin, Ansu Cherian, Dr. Meryl Lubran, Dr. Marla Luther, Daniel Reese, Avani 
Sanghvi, Karen Silagyi, Dr. Margaret Slavin, Ana Aguado, Yuting Zhou, Tong Liu, 
Michael Wiederoder, Melody Ge, Eric Thornber, I-Chang Yang. I want to especially 
thank my wife, Cintia, for her patience, encouragement, and support. Finally, I would 
like to thank my entire family for their unconditional love and support that they have 
given me my entire life. 
 
 
 
 
 
 iv 
 
Table of Contents 
 
 
Acknowledgements ...................................................................................................... iii 
List of Tables ................................................................................................................ vi 
List of Figures .............................................................................................................. ix 
Chapter 1: Introduction ................................................................................................. 1 
1.1 Introduction ......................................................................................................... 1 
1.2 Research Objectives ............................................................................................ 3 
Chapter 2: Literature Review ........................................................................................ 5 
2.1 Introduction ......................................................................................................... 5 
2.2 Seed Hydrocolloids ............................................................................................. 6 
2.2.1 Guar Gum ..................................................................................................... 6 
2.2.2 Locust Bean Gum ......................................................................................... 8 
2.3 Seaweed Extract Hydrocolloids ........................................................................ 10 
2.3.1 Alginate ...................................................................................................... 10 
2.3.2 Carrageenan ................................................................................................ 12 
2.4 Microbial Hydrocolloid ..................................................................................... 14 
2.4.1 Xanthan ...................................................................................................... 14 
2.5 Cellulose Derivative Hydrocolloids .................................................................. 16 
2.5.1 Sodium Carboxymethyl Cellulose ............................................................. 16 
2.5.2 Microcrystalline Cellulose ......................................................................... 17 
2.6 Curdlan .............................................................................................................. 18 
2.7 Characterization Tools ...................................................................................... 20 
Chapter 3: Texture stability of hydrogel complex containing curdlan gum over 
multiple freeze-thaw cycles ......................................................................................... 23 
3.1 Introduction ....................................................................................................... 23 
3.2 Materials and Methods ...................................................................................... 25 
3.2.1 Materials and Sample Preparation .............................................................. 25 
3.2.3 Syneresis and Textural Measurements ....................................................... 26 
3.3 Results and Discussion ...................................................................................... 27 
3.3.1 Syneresis..................................................................................................... 27 
3.3.2 Apparent Viscosity and Heat Stability ....................................................... 29 
3.3.3 Storage and Loss Moduli ............................................................................ 32 
3.3.4 Gel Strength and Adhesiveness .................................................................. 34 
3.4 Conclusion ......................................................................................................... 36 
Chapter 4:  Characterization of Water Distribution in Xanthan-Curdlan Hydrogel 
Complex Using Magnetic Resonance Imaging, Nuclear Magnetic Resonance 
Relaxometry, Rheology, and Scanning Electron Microscopy .................................... 37 
4.1 Introduction ....................................................................................................... 37 
4.2 Materials and Methods ...................................................................................... 40 
4.2.1 Sample Preparation .................................................................................... 40 
4.2.3 Rheology .................................................................................................... 41 
4.2.4 Scanning Electron Microscopy .................................................................. 42 
4.2.5 Statistical Analysis ..................................................................................... 42 
 
 v 
 
4.3 Results and Discussion ...................................................................................... 42 
4.3.1 Spin-lattice relaxation time, T1, measurements .......................................... 42 
4.3.2 Magnetic resonance imaging ...................................................................... 43 
4.3.3 Spin-spin relaxation time, T2, measurements ............................................. 46 
4.3.4 Rheology and Morphology......................................................................... 50 
4.4 Conclusion ......................................................................................................... 53 
4.5 Acknowledgement ............................................................................................. 55 
Chapter 5: Effect of pH and salt concentration on the xanthan curdlan hydrogel 
complex utilizing rheological techniques .................................................................... 56 
5.1 Introduction ....................................................................................................... 56 
5.2 Methods and Materials ...................................................................................... 58 
5.2.1 Sample Preparation .................................................................................... 58 
5.2.2 Rheological Measurements ........................................................................ 59 
5.2.3 Statistical analysis ...................................................................................... 59 
5.3 Results and Discussion ...................................................................................... 59 
5.3.1 Effect of pH ................................................................................................ 59 
5.3.2 Effect of Salt Concentration ....................................................................... 65 
5.4 Conclusion ......................................................................................................... 69 
Chapter 6:  Efficacy of the xanthan curdlan hydrogel complex in pumpkin pie ........ 70 
6.1 Introduction ....................................................................................................... 70 
6.2 Methods and Materials ...................................................................................... 71 
6.3 Results and Discussion ...................................................................................... 73 
6.3.1 Adhesiveness and Gel Strength .................................................................. 73 
6.3.2 Water Activity and Color ........................................................................... 74 
6.4 Conclusion ......................................................................................................... 77 
Chapter 7: Conclusions and Recommendations .......................................................... 78 
Appendix A: Supplemental Information ..................................................................... 80 
Appendix B: Supplemental Data ................................................................................. 88 
Appendix C: Statistical Analysis ................................................................................. 91 
References ................................................................................................................. 109 
 
 
 
 
 
 
 
 
 
 
 
 vi 
 
List of Tables 
 
Table 2.1. Hydrocolloids in use as stabilizers in frozen foods and desserts, bold 
indicates most often used (Towle 1996; Marshal et al. 2003). 
 
Table 2.2. Summary of carrageenan properties. 
 
Table 2.3. Investigative methodology of hydrocolloidal systems. 
 
Table 3.1.  Syneresis* of 2.0% aqueous curdlan solutions alone and with equal 
fractions of curdlan with xanthan (X/C), guar (G/C), ?-carrageenan (?-Ca/C), or 
locust bean gum (LBG/C). 
 
Table 3.2. Apparent viscosity modeling at 20 ?C* exhibiting the Cross model over a 
shear rate range from 0.15 to 100 s-1 and apparent viscosity at 80 ?C*, G7dfGb3cGb34, a shear rate 
of 2 s-1. 
 
Table 3.3.  Gel Strength and adhesiveness* of 2.0% aqueous solutions with equal 
fractions of curdlan with xanthan, guar, k-carrageenan, or locust bean gum over five 
freeze-thaw cycles (FTCs). 
 
Table 4.1. T1 relaxation times of 2.0% xanthan, curdlan and xanthan curdlan 
hydrogel complex (XCHC) subjected to freeze-thaw cycles (FTCs). 
 
Table 4.2. Coefficient of variation (COV) values given as percentages for signal 
intensities acquired by the spin-echo sequence (COV? one standard deviation) of 
2.0% xanthan, curdlan and xanthan curdlan hydrogel complex (XCHC) subjected to 
freeze-thaw cycles (FTCs). 
 
Table 4.3. Spin-spin relaxation time,T2 , and relative area of 2.0% xanthan, curdlan 
and xanthan curdlan hydrogel complex (XCHC) subjected to freeze-thaw cycles 
(FTCs). 
 
Table 4.4. Values of storage modulus (G? = a?b) and syneresis over five freeze-thaw 
cycles (FTCs) for 2.0% xanthan, xanthan curdlan hydrogel complex (XCHC), and 
curdlan. 
 
Table 5.1. Modeling of the storage modulus, G? = a?b, for pH treated xanthan 
curdlan hydrogel complex (XCHC) samples. Table 5.2. Adhesiveness and gel 
strength of the xanthan curdlan complex solutions with the pH adjusted from 2 to 8 
over five freeze thaw cycles (FTCs). 
 
Table 5.3. Modeling of the storage modulus, G? = a?b, for NaCl treated xanthan 
curdlan hydrogel complex (XCHC) samples. 
 
 vii 
 
Table 5.4. Adhesiveness and gel strength of the xanthan curdlan complex solutions 
with 5 to 200 mM NaCl over five freeze thaw cycles (FTCs). 
 
Table 6.1.  Adhesiveness and gel strength* of pumpkin pie filling and treated with the 
xanthan curdlan hydrogel complex (XCHC) over five freeze-thaw cycles (FTCs). 
 
Table 6.2. CIELAB values, L*, a*, and b*, for surface and filling of control and 
xanthan curdlan hydrogel complex (XCHC) treated pumpkin pies over five freeze-
 thaw cycles (FTCs). 
 
Table B.1. The pH of HCl or NaOH treated XCHC samples over five freeze-thaw 
cycles (FTCs).  
 
Table B.2. Syneresis of samples treated to achieve pH = 2. 
Table B.2. The pH of NaCl treated XCHC samples over five freeze-thaw cycles 
(FTCs).  
 
Table B.3. Pumpkin pie filling ingredients. 
Table C.1. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous curdlan gum solutions 
over 5 FTCs (n=3).  
 
Table C.2. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan gum solutions 
over 5 FTCs (n=3).  
 
Table C.3. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) solutions over 5 FTCs (n=3).  
 
Table C.4. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 2over 5 FTCs (n=3).  
 
Table C.5. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 3 over 5 FTCs (n=3).  
 
Table C.6. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 4 over 5 FTCs (n=3).  
 
Table C.7. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 5over 5 FTCs (n=3).  
 
Table C.8. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 6 over 5 FTCs (n=3).  
 
 
 viii 
 
Table C.9. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 7 over 5 FTCs (n=3).  
 
Table C.10. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 8 over 5 FTCs (n=3).  
 
Table C.11. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 5 mM NaCl over 5 FTCs (n=3).  
 
Table C.12. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 15 mM NaCl over 5 FTCs (n=3).  
 
Table C.13. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 20 mM NaCl over 5 FTCs (n=3).  
 
Table C.14. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 75 mM NaCl over 5 FTCs (n=3).  
 
Table C.15. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 100 mM NaCl over 5 FTCs (n=3).  
 
Table C.16. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 200 mM NaCl over 5 FTCs (n=3). 
  
 
 ix 
 
List of Figures 
 
 
Figure 2.1. Structure of guar gum. 
Figure 2.2. Structure of locust bean gum. 
Figure 2.3. Structure of alginate gum. 
Figure 2.4. Structures of kappa-, iota-, and lambda-carrageenan. 
Figure 2.5. Structure of xanthan gum. 
Figure 2.6. Sodium Carboxymethyl Cellulose. 
Figure 2.7. Structure of cellulose. 
Figure 2.8. Structure of curdlan. 
Figure 3.1. Frequency sweeps of 2.0% aqueous curdlan solutions alone and with 
equal fractions of curdlan with xanthan (X/C), guar (G/C), ?-carrageenan (?-Ca/C), or 
locust bean gum (LBG/C). Storage, G?, and loss modulus, G?,  prior to (a), and after 
the first (b), second (c), third (d), fourth (e), and fifth (f) freeze-thaw cycles. 
 
Figure 4.1. Representative MR images of curdlan prior to freezing (a) after one 
freeze thaw cycle (b) and after the fifth freeze thaw cycle (c). The scale on the right 
gives the relationship between signal intensity and grayscale for the three images. 
Axes in the images denote the voxel numbers. 
 
Figure 4.2. Frequency sweeps of 2.0% xanthan, curdlan, and xanthan curdlan 
hydrogel complex (XCHC). Storage, G?, and loss modulus, G?,  prior to (a), and after 
the first (b), second (c), third (d), fourth (e), and fifth (f) freeze-thaw cycles. 
 
Figure 4.3. Scanning electron microscope micrographs of the 2.0% curdlan (a), 2.0% 
xanthan (b), and 2.0% xanthan curdlan hydrogel complex (c) freeze dried samples. 
 
Figure 5.1. Frequency sweeps of 2.0% xanthan curdlan hydrogel complex (XCHC) 
with pH values of 2, 3, 4, 5, 6, 7, 8. Storage, G?(  101 - 102 102 - 103 103 - 104), and 
loss modulus, G? (  101 - 102  102 - 103  103 - 104),  prior to (a), and after the first (b), 
second (c), third (d), fourth (e), and fifth (f) freeze-thaw cycles. 
 
Figure 5.2.  Temperature ramps of the storage modulus, G?, of 2.0% xanthan curdlan 
hydrogel complex (XCHC) with pH values of 2, 3, 4, 5, 6, 7, 8. Heating ramp, G?  (
 101 - 102 102 - 103), and cooling ramp, G?( 101 - 102 102 - 103),  prior to (a), and 
after the first (b), second (c), third (d), fourth (e), and fifth (f) freeze-thaw cycles. 
 
 x 
 
Figure 5.3. Frequency sweeps of 2.0% xanthan curdlan hydrogel complex (XCHC) 
treated with 5, 15, 20, 75, 100, and 200 mM NaCl. Storage, G?(  101 - 102 102 - 103
 103 - 104), and loss modulus, G? (  101 - 102  102 - 103  103 - 104),  prior to (a), and after 
the first (b), second (c), third (d), fourth (e), and fifth (f) freeze-thaw cycles. 
 
Figure 5.4.  Temperature ramps of the storage modulus, G?, of 2.0% xanthan curdlan 
hydrogel complex (XCHC) treated with 5, 15, 20, 75,100, and 200 mM NaCl. 
Heating ramp, G? ( 101 - 102 102 - 103),  and cooling ramp, G?( 101 - 102 102 - 
103), prior to (a), and after the first (b), second (c), third (d), fourth (e), and fifth (f) 
freeze-thaw cycles. 
 
Figure 6.1. Water activity of the crust (square7) and filling (square7) of control samples over 
five freeze-thaw cycles (n=3). 
 
Figure 6.2. Water activity of the crust (square7) and filling (square7) of the xanthan curdlan 
hydrogel complex (XCHC) treated samples over five freeze-thaw cycles (n=3). 
 
Figure 6.3.  Images of the control pumpkin pie surface on the left and XCHC treated 
pie surface on the right. 
 
Figure A.1. Cross sectional view of cone and plate rheological apparatus. 
 
Figure A.2. Strain input (?) and stress output (?) as a function of time, where the 
difference in the stress and strain curves is the phase lag, ?. Where the strain 
amplitude is ?o, and the stress amplitude is ?o. 
 
Figure A.3. Typical frequency sweeps of a dilute solution made from 5% dextrin (a), 
concentrated solution made from 5% lambda carrageen (b), and a gel made from 1% 
agar (c) (Ross-Murphy, 1988). 
 
Figure A.4. Cross sectional view of concentric cylinder, or cup and bob, rheometer 
apparatus. 
 
Figure A.5. Typical texture analyzer output for the samples in the current work, 
where the maximum peak is the gel strength and the blue shaded area is the 
adhesiveness of the gel. A 0.5? dia. plastic probe was used with a lowering and 
raising rate of 0.5 mm/s 20 mm into the sample. 
 
Figure B.1.  Crust separation can be seen in the control (on the left) after three 
freeze-thaw cycles, whereas the crust of the XCHC treated pie filling (on the right) 
does not show separation. 
 
 1 
 
Chapter 1: Introduction 
1.1 Introduction 
According to a 2011 IBIS World industry report, frozen foods are expected to 
be a $28 billion industry by 2012 and during the economic downturn, revenue has 
increased by 3.1% and 2.0% in 2008 and 2009, respectively, due to consumer 
recognizing the affordability and durability of frozen foods (IBIS 2011). Mintel 
reports a growth of more than 16% in frozen desserts and novelties in the U.S. market 
from 2003 to 2008 (Mintel 2009) and it was also reported that the frozen food growth 
rate has risen from 3.1% to 5.8% in one year beginning in January of 2008 in the 
U.K. (TNS 2008). However, the frozen desserts and novelties market has been 
stagnant due to the lack of innovation and the economic downturn (Mintel 2009). It is 
evident that frozen foods and desserts are an important sector in the food industry, but 
needs innovation. Despite this, the demand for high quality products is always an 
importance aspect in the market, where the texture and stability of the products are 
undoubtedly of the utmost importance for existing as well as new frozen products to 
elevate their quality standards, gain consumer acceptance, and spur innovation.  
To date, various hydrocolloidal polysaccharides such as guar gum or locust 
bean gum have been employed as stabilizing, thickening, emulsifying, or suspending 
agents in the development of texture and/or sustaining texture in frozen specialty 
foods and desserts such as ice cream, sauces, and whip toppings. In most cases, 
individual hydrocolloids are capable of improving the quality of food products if the 
characteristic rheological attributes of the hydrocolloid were able to give the products 
 
 2 
 
the desired textural properties. However, increasing reports have shown that, when 
combining different gums, some synergistic effects can occur that increase the 
functionality and/or enhance viscous properties of the gum combination (Westra 
1989; Towle 1996; Imeson 1997; Wang et al 2002a,b; Richter et al. 2005; Higiro et 
al. 2006; Kim et al. 2006; Makri et al. 2006; Fernandez et al. 2007).  
Moreover, while the emulsifying, thickening, and suspending properties of 
hydrocolloids might work well in conventional foods, their applicability in frozen 
foods is often challenged, especially with loss of moisture in the form of dripping 
during freezing-thawing or temperature fluctuations during distribution/storage 
remains the most significant issue in retaining product quality and nutrients. A 
number of hydrocolloids, including carrageenan, xanthan gum, locust bean gum, guar 
gum, MCC, and CMC, are used as texture modifiers to protect the products from heat 
shock deteriorations and bodying agent (Towle 1996; Marshall et al 2003), help 
stabilize frozen foods (Sworn 2000), and reduce recrystallization (Goff 2006). 
Specifically, locust bean and guar gum are used to give and improve the texture of ice 
cream (Towle 1996; Marshall et al. 2003); MCC can be used to reduce ice crystals in 
frozen desserts (Marshall et al 2003) or a low-fat texture modifier (Champion et al. 
1982); and when used as an emulsifier, stabilizer or thickener, xanthan gum has the 
ability to keep its viscosity after being defrosted (Imeson 1997; Sworn 2000). 
Nevertheless, there are limited studies in the literature characterizing the textural 
effects of different gum combinations under freeze-thaw conditions.  
Xanthan in combination with curdlan has been demonstrated by our research 
group to reduce syneresis, water loss, to a point where it is not detected and yields 
 
 3 
 
stable rheological and physical properties over freeze-thaw cycles when compared to 
curdlan combined with guar, locust bean, or k-carrageenan gums. Therefore, the 
ultimate goal of the following research is to characterize the physicochemical 
properties of the xanthan/curdlan system and investigate its efficacy in pumpkin pie.  
1.2 Research Objectives 
The ultimate goal of the proposed research is to screen and identify possible 
hydrocolloids that could possibly reduce or eliminate syneresis, water loss, when 
added to curdlan, while maintaining elastic behavior and improving freeze-thaw 
stability and characterize the complex. Combining xanthan with curdlan proved the 
most viable candidate for further study, therefore possible reasons for the 
combinations ability to be resilient under temperature abuse using advanced 
techniques was explored. Characterization was achieved by utilizing rheological, 
microscopy, and resonance techniques over freeze-thaw abuse. Moreover, the effects 
of NaCl and pH on rheological properties of the complex were investigated for the 
optimal range in which the hydrogel is least affected. Finally, the efficacy of the 
system was applied to pumpkin pie, where the impact of the xanthan curdlan hydrogel 
on the moisture migration, observable physical changes, color, gel strength and 
adhesiveness of the pies was investigated. In order to achieve this goal, there were 
four specific objectives, where the subsequent chapters following the literature review 
are presented as separate manuscripts: 
 
 
 
 
 4 
 
Objective #1: Screen curdlan with xanthan, locust bean, guar, and ?-carrageenan to 
increase its freeze-thaw stability through their physical and rheological 
properties.  
 
Objective #2: Utilize magnetic resonance imaging (MRI), nuclear magnetic resonance 
(NMR) relaxometry, scanning electron microscopy (SEM), and rheometry to 
investigate structure and water distribution of the most stable system, further 
characterizing the combination. 
 
Objective #3: Build a foundation for the combinations optimal range, by studying the 
effects of pH and sodium chloride as major food components. 
 
Objective #4: Explore efficacy of the complex in pumpkin pie, investigating quality 
aspects, including moisture migration, color, and texture. 
 
 
 
 
 
 
 5 
 
Chapter 2: Literature Review 
 
Adapted from: Soma PK, Williams PD, Lo YM. 2009. Front Chem Eng China 3(4): 
413-426. 
 
 
 
2.1 Introduction 
Hydrocolloids are polysaccharides and proteins (Ockerman, 1978; Williams 
and Phillips, 2000) with hydrophilic and colloidal properties (Graham 1977; Hoefler 
2004) that could be used to provide certain textural properties such as thickening or 
stabilizing. Fixed length polypeptide chains of 50 or more amino acid residues with 
possibly 20 different amino acids make up the protein structure, where as 
polysaccharides colloids vary in length and consist of heteropolysaccharides and 
homopolysaccharides (Graham 1977). Hydrocolloids stem from several sources, 
including, botanical, algal, microbial, and animal sources (Glicksman 1982; Williams 
and Phillips 2000; Hoefler 2004). They are used in sauces and dressings (Mandala et 
al. 2003; Sikora et al. 2008), as emulsion stabilizers (Garti et al. 2001; Dickenson 
2008; Mikkonen 2009), and for their freeze-thaw stability (Freeland 2002).  
In frozen foods, gums help maintain or control textural aspects including 
reducing ice re-crystallization, minimize water distribution, and viscosity 
enhancement (Goff 2006). Hydrocolloids that provide these functions in frozen foods 
and desserts are included in Table 2.1, where the bold hydrocolloids are most often 
used in the frozen food industry. Due to their frequent use in frozen foods and 
desserts, several of the gums in bold will be further examined as relevant 
hydrocolloids in frozen foods.  
 
 6 
 
  
Table 2.1. Hydrocolloids in use as stabilizers in frozen foods and desserts, bold indicates most often 
used (Towle 1996; Marshal et al. 2003). 
Sources  Hydrocolloids  
Proteins  Gelatin, Microparticulated Milk and Egg Proteins  
Plant Exudates  Arabic, Ghatti, Karaya, and Tragacanth  
Seed Gums  Locust Bean, Guar, Psyllium, Tara, Konjac Glucomannan, Starch and 
Modified Starches  
Microbial Gums  Xanthan, Gellan  
Seaweed Extracts  Agar, Alginates, Carrageenan  
Pectins  Low and High Methoxyl  
Cellulose  Microcrystalline Cellulose, Sodium Carboxymethylcellulose, Methyl and 
Methylethyl Celluloses, hydroxypropyl and Hydroxypropylmethyl Celluloses  
 
2.2 Seed Hydrocolloids 
2.2.1 Guar Gum 
Native to India and Pakistan, guar gum is obtained from the guar plant, 
Cyamposis tetragonolobus of the Leguminosae family (Glicksman 1986; Wielinga et 
al. 2000). Currently, the plant is commercially grown annually in Texas, Oklahoma, 
Arizona, and the southern hemisphere in response to the fluctuations in availability of 
obtaining guar bean gum from foreign sources (Glicksman 1986; Wielinga et al. 
2000). Made up of nearly all galactomannan, the nonionic structure of guar gum 
consists of a backbone with (1?4)-linked ?-D6 mannopyranosyl unit with a side 
chain unit consisting of (1?6)-linked ?-D-galactopyranosyl at a ratio of 1.8:1, 
respectively (Fig. 2.1). The large amount of galactose substitutions prevents strong 
cohesion of the main backbone and therefore extensive crystalline regions cannot be 
 
 7 
 
formed allowing hydration at and above room temperature (Wielinga et al. 2000).  
Hydration of guar gum can be correlated to the inter- and intramolecular hydrogen 
bonding through the unsubstituted regions of the backbone (Goycoolea et al. 1995; 
Sandolo et al. 2008) and thus makes guar gum soluble in cold, highly agitated water 
while exhibiting pseudoplastic behavior. It is used as a formulation aid, stabilizer, 
firming agent, and thickener (Towle 1996) with a classification of Generally 
Recognized As Safe (GRAS) by the U.S. Food and Drug Administration (FDA). 
 
Figure 2.1. Structure of guar gum. 
 
 Wielinga et al. (2000) first reported that guar gum can help enhance the 
freeze-thaw stability of solutions. It was found that guar gum can be stable over two 
freeze-thaw cycles in aqueous, NaCl, CaCl2, citric acid, acetic acid and milk serum 
solutions at a concentration of 1.0%. Another study found that when added to curdlan 
gum in the same amount totaling 2.0% (w/v), syneresis was undetectable over five 
freeze-thaw cycles (Williams et al. 2009). Galactomannans such as guar gum and 
locust bean gum are amongst the most frequently used ingredients for the stabilization 
of ice cream (Marshall et al. 2006). Among fresh and frozen vegetable puree, guar 
O
 H
 O
 HO
 H
 H
 H
 OH
 O O
 H
 O
 O
 OH
 OH
 HO
 H
 OH
 H
 HH
 H
 H
 H
 H
 HO
 OH
 OH
 O
 H
 O
 HO
 H
 H
 H
 OH
 O O
 H
 O
 O
 OH
 OH
 HO
 H
 OH
 H
 HH
 H
 H
 H
 H
 HO
 OH
 OH
 O
 
 8 
 
gum was found effective in reducing drip loss in potatoes, carrots, and turnips 
(Downey 2002). Moreover, when added to frozen dough, guar gum has shown 
improvements in bread texture, volume and crumb structure (Ribotta et al. 2004). 
2.2.2 Locust Bean Gum 
Locust bean gum (LBG), a.k.a. carob bean or St. John's gum, is obtained from 
the seeds of the carob tree, Ceratonia siliqua, grown in Mediterranean countries 
(Glicksman 1986; Wielinga et al. 2000). The galactomannan structure of LBG (Fig. 
2.2), similar to guar gum, consists of a backbone with (1?4)-linked ?-D-
 mannopyranosyl units and a side chain consisting of a single (1?6)-linked ?-D-
 galactopyranosyl unit with a ratio of 3.9:1. Contrary to the structure of guar gum, 
LBG side chains are very unevenly substituted with sections of the backbone 
concentrated with substitutions and sections with no substitutions. Slightly soluble in 
water at room temperature, LBG must be heated to approximately 60 - 85 ?C to 
achieve complete hydration. LBG solution exhibits pseudoplasticity (shear-thinning 
behavior), which shows reduced viscosity at increased shear rate, and can form a 
weak gel network with concentrations as low as 0.5% (Dea et al. 1977). Classified as 
GRAS by the FDA, LBG is used for its stabilizing, thickening, and fat-replacing 
properties.  
 
 9 
 
 
Figure 2.2. Structure of locust bean gum. 
 
 Unlike guar gum, there was little stability in a system containing LBG. For 
instance, LBG forms a gel after the first freeze-thaw cycle in the presence of NaCl, 
acetic acid, and milk serum, and syneresis occurs in both aqueous and CaCl2 solutions 
(Wielinga et al. 2000). This could be attributed to the unevenly substituted sections of 
the backbone that create junction zones and thus a gel or gel-like network. It has been 
shown that freeze-thaw cycles and rate of freezing and/or thawing affect its gel 
strength (Tanaka et al. 1998; Lozinsky et al. 2000, 2001; Zeira et al. 2004).  
Therefore, LBG alone could not provide the freeze-thaw stability over multiple 
cycles.  
 Several studies with LBG and xanthan gum have been performed because of 
their unique synergistic gelling behavior that is not formed with the other 
galactomannan, guar gum (Rocks 1971; Goycoolea et al. 1995; Wang et al. 2002b). 
Conformational studies via parameters such as intrinsic viscosity that depicts 
molecular occupancy in highly dilute solutions have been used to better understand 
this interaction. It was reported that the intrinsic viscosity exhibited lower values than 
O
 H
 OH
 HO
 H
 H
 H
 OH
 O O
 H
 O
 H
 H
 H
 H
 HO
 OH
 OH
 O
 H
 O
 HO
 H
 H
 H
 OH
 O O
 H
 O
 O
 OH
 OH
 HO
 H
 OH
 H
 HH
 H
 H
 H
 H
 HO
 OH
 OH
 O
 
 10 
 
weighted averages, suggesting that there is some flexibility in the structure of xanthan 
that may allow LBG to bond to the backbone (Wang et al. 2002b; Higiro et al. 2006). 
Even so, LBG is best known for its use in ice cream to prevent iciness during heat 
shock (Glicksman 1986; Towle 1996; Marshall et al. 2003) and many times combined 
with guar and carrageenan.  However, little to no studies have been conducted to 
examine any potential synergistic effects provided by LBG and xanthan under freeze-
 thaw abuse. On the other hand, frozen dough containing LBG showed better gluten 
quality through dough extensibility analysis and lower proof times compared to 
control samples and exhibited increased specific loaf volume along with improved 
external and internal bread characteristics compared to the control along with high 
moisture content (Sharadanant and Khan 2003a;2003b; Mandala et al. 2008). 
 
2.3 Seaweed Extract Hydrocolloids 
2.3.1 Alginate 
            The extraction and processing of brown algae from host of species including 
Laminaria hyperborean, Macrocystis pyrifera, Laminaria giditata, Ascophyllum 
nodosum, Laminaria japonica, Eclonia maxima and others can produce the 
intermediate product, alginic acid. After the process of neutralization with sodium 
carbonate or sodium hydroxide, alginic acid forms the more stable water-soluble 
product called sodium alginate (Onsoyen 1997; Draget 2000). The linear structure of 
alginate consists of either the homopolymeric blocks ?-D-mannuric acid (M) and ?-L-
 guluronic acid (G) or the heteropolymeric blocks of alternating M and G linked by 
(1?4)-glycosidic linkages, making it a polyanionic hydrocolloid (Fig. 2.3). The 
amount of homo- and heteropolymeric blocks present in alginate is dependent upon 
 
 11 
 
the sources previously mentioned. Thermo-irreversible gels can be formed by 
controlling the amount of calcium and acidity. On the other hand, low acid solutions 
below pH 4.0, form thermo-reversible gels when combined with high-methoxyl 
pectin.  There are a variety of different viscosity grades of alginates due to the amount 
of G-blocks available for Ca2+ to from an ?egg-box? structure at junction zones 
creating a strong gel. Due to its ability to form strong gels, they are used to control 
the shape of foods such as onion rings, pimiento and anchovy olive fillings, apple 
pieces for pie fillings, cocktail berries, meat chunks for pet food, shrimp-like fish 
products, and fish patties (Onsoyen 1997). 
 
Figure 2.3. Structure of alginate gum. 
 
 Although temperature does not hinder gelation of sodium alginate, it affects 
the final gel properties; however, once a gel it is heat and freeze-thaw stable 
(Onsoyen 1997; Draget 2000). Sodium alginate has been seen to improve frozen 
dough stability along with whey by increase the specific loaf volume (Shon et al. 
2009). Following each of seven freeze-thaw cycles, alginate increased dough 
development, water adsorption, and reduced syneresis, however, yielded different 
texture aspects as indicated by tensile strength and exhibiting a firmer dough when 
compared to control samples (Lee et al. 2008).   
O
 O
 H H
 O
 -OOC
 H
 H
 HO
 HO
 H
 O
 H
 OH H
 O
 OH
 H
 -OOC
 H
 H
 O
 H
 -OOC
 HO
 HO
 H
 H
 H
 H
 O
                   M                          G                             M 
 
 12 
 
2.3.2 Carrageenan 
Three varieties of carrageenan, kappa (?), lambda (?), and iota (?), can be 
extracted from the red seaweeds. They are primarily extracted from the Gigartina 
species and Chondrus crispus which produce kappa and lambda types, and Eucheuma 
cottonii and spinosum species which produce kappa and iota types, respectively 
(Imeson 2000; Hoefler 2004). The general structure of carrageenan contains repeating 
galactose units and 3,6-anhydrogalactose both with 15-40% (w/w) ester sulfate 
content and side chain consisting of alternating ?-(1?3)- and ?-(1?4)-glycosidic 
linkages. The three types of carrageenan do not exist singly, but as a combination of 
two types and available with one predominating type or molecules containing 
structural components of more than one type. Each type of carrageenan has a unique 
set of characteristics, including gel strength, viscosity, temperature stability, 
synergism, and solubility. The solubility of each type of carrageenan depends on the 
number of sulfate groups, which increases water solubility, compared to anhydro 
bridges, which is hydrophobic. Having the least water solubility, ?- carrageenan has 
one sulfate group for every two galactose units and one anhydro bridge, ?- 
carrageenan has two sulfate groups for every two galactose units along with one 
anhydro bridge, and with the highest solubility, ?-carrageenan has three sulfate 
groups for every two galactose units and no anhydro bridges (Imeson 2000; Hoefler 
2004).  
 
 
 
 13 
 
 
  
    
 
Figure 2.4. Structures of kappa-, lambda-, and iota-carrageenan. 
 
 There are distinct differences between each type of carrageenan, but all types 
are soluble at high temperatures and are stable above pH 4.5. Though the least 
soluble, ?-carrageenan provides the strongest, yet brittle, gel that allows for some 
syneresis. The ?-carrageenan type is more elastic and is freeze-thaw stable allowing 
for no syneresis and ?-carrageenan thickens without gelling. Moreover, due to 
syneresis, ?-carrageenan has poor freeze-thaw stability; however, by the right 
combination of ?- and ?-carrageenan intermediate freeze-thaw stability can be 
acquired along with a range of gel textures without syneresis (Imeson 2000). In ice 
cream and other frozen milk-based products, a small amount of ?-carrageenan (0.01-
 O
 -O3SO
 OH
 H
 O
 H
 H OH
 H
 O
 H
 O
 H
 O
 OH
 H
 H
 H
 H
 H2C
 O
 O
 O
 OH
 OH
 H
 OSO3-
 H
 OO
 H
 H
 H
 CH2OSO3-
 OH
 H
 O
 OSO3-
 H
 H
 H
 H
 O
 O
 -O3SO
 OH
 H
 OH
 H
 OO
 H
 H
 H
 H2C
 O
 H
 O
 OSO3-
 H
 H
 H
 H
 kappa-carrageenan 
lambda-carrageenan 
iota-carrageenan 
 
 14 
 
0.3%) is used to prevent phase separation of casein (Fox 1997; Marshall et al. 2003). 
?-Carrageenan alone and along with whey has exhibited higher specific volume in 
bread after the dough was frozen storage (Sharadanant and Khan 2003b; Shon et al. 
2009).   
 
Table 2.2. Summary of carrageenan properties. 
 Kappa Iota Lambda 
Sulfate Group  1 2 3 
Anhydro Bridge  1 1 0 
Texture  
Firm, brittle, syneresis, 
strong gels with K+ 
Elastic, cohesive, no 
syneresis , strong gels 
with Ca++ 
No gelling, thickening, 
provides ?body? 
 
 
2.4 Microbial Hydrocolloid 
2.4.1 Xanthan 
Produced by fermentation of Xanthomonas campestris, xanthan gum is an 
anionic microbial polysaccharide with a disaccharide backbone and a trisaccharide 
side chain. The backbone consists of two repeating (1?4)-?-D-glucose units, the 
same as cellulose, and located on C3 of every other glucose, ?-D-mannose, ?-D-
 glucuronic acid, and ?-D-mannose trisaccharide is attached. Various terminal 
mannose residues are pyruvated depending on the X. campestris strain.  
 
 
 15 
 
 
Figure 2.5. Structure of xanthan gum. 
 
Xanthan is soluble in water with the ability to hydrate in cold water, 
exhibiting pseudoplastic behavior. The viscosity of xanthan is stable over a range of 
pH values, salt concentrations, and temperatures (Moorhouse et al. 1977a; Whitcomb 
et al. 1978; Sworn, 2000). Thickening, stabilizing, and emulsifying are some of the 
well recognized characteristics of xanthan gum. As a thickening agent, low shear 
rates show stiff, aggregated, highly ordered molecules and, as shear is increased, the 
stiff molecules separate and align in the direction of shear force. As a consequence, 
pseudoplasticity is exhibited (Wilkes 1999). While 1.0% xanthan solution produces 
gel-like consistency, as little as 0.1% xanthan gum can increase viscosity and shows 
similar rheological properties. Giannouli and Morris (2003) suggested that xanthan 
can from stronger more cohesive networks, similar to how Ca2+ enhances xanthan?s 
weak gel network, when frozen and thawed. 
Because of its resilience to pH and temperature fluctuations, xanthan gum has 
been extensively studied for its own properties and synergistic effects with other 
gums, starches, and food ingredients. The interactions between xanthan and 
galactomannans have been examined to identify their synergy that alter viscous and 
O
 O
 H
 HO
 HO
 HO
 H
 H
 OHH
 H
 O
 HO
 H
 OH
 H
 H
 H
 H
 H
 O
 O
 H
 O
 O
 HO
 H
 H
 OHH
 H
 O
 HO
 H
 OH
 H
 H
 H
 H
 H
 OH
 O
 H
 H
 OH
 H
 OH
 H
 HO
 CH3
 O
 O
 O
 O
 O
 H
 H
 -O
 H
 H
 H
 HO
 O
 H
 O
 HO
 H
 H
 HO
 H
 H
 O
 -O
 O
 H3C
 n
 
 16 
 
gelation properties (Rocks 1971; Sworn 2000; Pai et al. 2002; Wang 2002(a,b); 
Richter et al. 2004; Higiro 2006). Synergy of xanthan has also been explored with 
xyloglucan (Kim et al., 2006), glucomannan (Paradossi et al. 2002), and its positive 
effects with arabic and LBG in emulsions (Makri 2006), as well as its effect on starch 
when combined with other gums (Chaisawang et al. 2006).  
Furthermore, the stability of frozen entrees and sauces was improved by 
xanthan gum with respect to syneresis and viscosity control over freeze-thaw cycles 
(Sworn 2000). The presence of xanthan gum in starch gels has significantly increased 
its freeze-thaw stability, according to Lo and Ramsden (2000), indicating an excellent 
compatibility with major food components. Xanthan gum has also had a positive 
impact with frozen dough by increasing the specific volume of the bread after one 
week of frozen dough storage and heating via a microwave (Mandala 2005), with 
better efficiency in combating crust deterioration than LBG and guar (Mandala et al. 
2008). Improved dough quality by the addition of xanthan could be due to the 
reduction of free water as indicated by a reduction in fusion enthalpy (Matuda 2008). 
2.5 Cellulose Derivative Hydrocolloids 
2.5.1 Sodium Carboxymethyl Cellulose 
Sodium carboxymethyl cellulose (CMC), or cellulose gum, is produced by 
treating cellulose with sodium hydroxide and reacted with sodium monochloroacetate 
and finally washed (Ganz 1977). CMC is water soluble with the ability to increase 
viscosity to 5 Pa s in a 1% aqueous solutions, which is considerably lower than that of 
xanthan gum. CMC solutions exhibit shear-thinning (Murray 2000) and sometimes 
Newtonian behavior or shear-thickening (Ya?ar et al. 2007). It is currently used as a 
 
 17 
 
thickener or bodying agent for instant products, sauces, dressings, and soft drinks; 
however, high viscosity forms of CMC can cause a ?gummy? mouth feel (Murray 
2000). Similar to carrageenan and locust bean gum, CMC is used to stabilize frozen 
products like ice cream by inhibiting ice crystal formation (Murray 2000; Marshall 
2003).  
 
Figure 2.6. Sodium Carboxymethyl Cellulose. 
 
Similar to LBG, CMC was found to reduce proof times and more resistant to 
extension in previously frozen dough and improve external and internal 
characteristics of bread, but also increased a desired yellow color in the crust 
compared to control samples (Sharadanant and Khan 2003a,b). 
2.5.2 Microcrystalline Cellulose 
 Microcrystalline cellulose (MCC) is a linear structure consisting of 
anhydroglucose units linked by a (1?4) ?-glycosidic bond. Acid hydrolysis is 
performed on to depolymerize wood cellulose under high shear conditions to liberate 
small crystalline structures. MCC is water-insoluble and thus needs sufficient shear or 
copolymer network to properly disperse in water (Iijima and Takeo 2000). 
Temperature has little to no effect on the functionality or apparent viscosity of the 
networks and remains stable during high temperature processing such as baking or 
 
 18 
 
retort. Not only can MCC be applied to high temperature processes, it can be used as 
a texture modifier for low-fat desserts (Marshall et al. 2003) and prevents growth of 
ice crystals in frozen foods during freeze-thaw cycles (Champion et al. 1982). 
Synergy is important to create and improve texture properties, however, it is more 
difficult to achieve with other material or gums due to its highly linear structure. In 
many cases, co-processing under specific conditions is required to form hydrogen 
bonds between MCC and the material (Glicksman 1986). 
 
Figure 2.7. Structure of cellulose. 
2.6 Curdlan 
Alcaligenes faecalis var. myxogenes, now classified as Agrobacterium biovar. 
1, produces the neutral, linear polysaccharide curdlan. Curdlan is the third microbial 
extracellular polysaccharide following xanthan gum and gellan gum to be approved 
for food uses in the United States (FDA 1996). Improved production continues to be 
explored (West 2009; Puliga et al., 2010; Seviour et al., 2011; Yu et al., 2011). 
Repeating units of (1?3)-?-glucan can form a low-set thermo-reversible gel when 
heated between ~55 ?C and 80 ?C or a high-set, triple helix, thermo-irreversible gel 
when heated above ~80 ?C and then cooled (Harada 1994; Hirashima et al. 1997; 
Nakao 1997; McIntosh et al. 2005; Gagnon et al. 2007). The gelation mechanism of 
the low- and high-set gels continues to be studied (Tada et al. 1999; Hatakeyama et 
O
 O
 H
 OH
 H
 OH
 H
 OHO
 HO
 H
 H
 H
 H
 H
 O
 H
 HO
 OH
 OH
 H
 O
 O
 H
 OH
 H
 OH
 H
 OHO
 H
 H
 H
 H
 H
 OH
 H
 HO
 OH
 OH
 H
 n
 
 19 
 
al. 2006; Gagnon et al. 2007). Curdlan has a lack of water solubility, alcohols, and 
most organic solvents but shows solubility in alkaline solutions (Pederson 1979; 
McIntosh 2005). It is known to be able to hold moisture in meat processing and flour 
products, stable over a pH range of 2-12 and freeze-thaw conditions, except as an 
aqueous solution there is syneresis (Nakao 1997).  
Composed of a three dimensional structure stabilized by crosslinks that 
connect junction zones between molecules (Harada et al. 1966; Saito et al. 1978; 
Marchessault and Deslandes 1978; Nakao et al. 1991; Jezequel 1998), curdlan gum 
was once the focus of extensive research efforts to expand its applications to improve 
the texture of various food products (Kanzawa et al. 1987; Nakao et al. 1991; 
Sanderson 1996; Wang et al., 2010) as well as the delivery of medicinal ingredients 
(Harada and Harada 1996; Na et al. 2000) and in combination with chitosan as an 
antibacterial gel (Sun et al., 2011). To date, unfortunately, despite curdlan?s ability to 
form viscous aqueous suspensions with shear-thinning flow behavior (Hirashima et 
al. 1997; Lopes da Silva et al. 1998; Funami et al. 1999), the applicability of curdlan 
in the U.S. market remains scarce and limited, due mainly to its less profound 
viscosity than xanthan gum in solution and inferior gel formation capacity when 
compared with gellan (Sadar 2004).   
Nevertheless, curdlan continues to be used as a texture modifier in Chinese 
and Japanese noodles and surimi-based products (Nakao et al. 1991; Nishinari 2000) 
as well as in processed meats such as pork, fried battered chicken, hamburger patties, 
duck and meatballs to yield juicier and softer products (Nakao, 1997; Hsu and Chung 
2000; Chen et al., 2010). Though insoluble in water (Pederson 1979), curdlan is 
 
 20 
 
known to form thermo-reversible or irreversible aqueous gels via heating (Maeda et 
al. 1967; McCleary 1979; Fulton and Atkins 1980; Harada et al. 1994) or addition of 
Ca2+ or Mg2+ (Kanzawa et al. 1987, 1989; Konno and Harada 1994). Furthermore, 
curdlan is also capable of forming tasteless, odorless, and colorless hydrogel 
complexes with other polysaccharides (Lo and Ramsden 2000; Lee et al. 2002). This 
unique gelling mechanism could be used to increase retention or absorption of 
moisture and other ingredients (Lo et al. 2003) while withstand the temperature 
extremes of freezing and retorting processes (Wielinga and Maehall 2000; McIntosh 
et al. 2005). However, the stability of such complexes when subjected to temperature 
abuse remains uncharacterized.   
 
Figure 2.8. Structure of curdlan. 
 
2.7 Characterization Tools 
Several tools, listed in Table 2.3, have been reported in the literature capable 
of characterizing different gums to aid in understanding any positive effects that 
could exist when more than one polymer are present in a system. Rheological 
properties, including viscosity and dynamic modulus (Appendix A), are often used to 
characterize the behavior of gum, starch, and gum/starch combinations (Whitcomb et 
al. 1978; Williams and Langdon 1996; Wang 2002(a,b); Giannouli et al. 2003; Ikeda 
O
 H
 H2C
 OH
 HO O
 H
 OH
 H
 H O
 H
 H2C
 OH
 HO O
 H
 OH
 H
 H O
 H
 H2C
 OH
 HO O
 H
 OH
 H
 H O
 H
 H2C
 OH
 HO O
 H
 OH
 H
 O
 H
 
 21 
 
et al. 2004; Higiro et al. 2006; Khouryieh et al. 2006; Kim et al. 2006; Nishinari 
2007; Pongsawatmanit et al. 2007; Choi et al. 2008). Other rheological properties 
such as gel strength and adhesiveness could also provide a broader view of the 
behavior and stability of the gum system. Utilizing a variety of rheological 
experiments assists in a well-formed characterization of the structural and textural 
information of hydrogels. 
In order to further depict structural characteristics and water-polymer 
interactions, methods such as microscopy and magnetic resonance are useful (Table 
2.3). Surface morphology can be investigated using scanning electron microscopy 
(SEM) to elucidate the network properties and microstructure of the combination. It 
has been successfully employed for investigating skim milk with xanthan and locust 
bean gum (Sanchez 2000), tapioca starch modified by guar and xanthan gum 
(Chaisawang 2006), effects of xanthan, locust bean gum, and guar on sucrose 
(Fern?ndez et al. 2007), chitosan and methylcellulose (Pinotti et al. 2007), and 
carrageenan/O. ficus indica (Medina-Torres et al. 2006).  
To delve further in to the nature and adjustments in the molecular structure of 
polysaccharides in water, nuclear magnetic resonance (NMR) relaxometry and 
magnetic resonance imaging (MRI) has been used for molecular analysis in 
carbohydrates (L?sse and Arnold, 1998; Vittadini et al., 2002), including xanthan and 
xyloglucan (Kim et al. 2006), xanthan and locust bean gum (Gil et al., 1996) and 
pectin molecules (Kerr and Wicker, 2000). In these studies the spin-spin relaxation 
time, T2, was monitored to identify inter- and intramolecular interactions, along with 
 
 22 
 
their interactions with water, indicating syneresis, water distribution, and structural 
behavior that can be related to rheology.   
 
Table 2.3. Investigative methodology of hydrocolloidal systems. 
 
Investigative Methodology Test Properties Reference 
Rheological 
 
Flow characteristics, 
elasticity classification 
Whitcomb 1978; Steffe 
1992; Williams and 
Langdon 1996; Tada et al. 
1999; Wang et al. 2002; 
Giannouli et al. 2003; 
Ikeda et al. 2004; Jin et al. 
2006; Khouryieh et al. 
2006; Kim et al. 2006;  
Differential Scanning 
Calorimetry 
Thermal analysis, 
gelation mechanisms 
explored 
Annable et al. 1994; 
Williams and Langdon 
1996; Ikeda et al. 2004; 
Hatakeyama et al. 2006; 
Jin et al. 2006; Kim et al. 
2006; 
Nuclear Magnetic Resonance Molecular structure and 
structural changes 
L?sse and Arnold, 1998; 
Vittadini, 2002; Kim et al. 
2006; 
Electron Spin Resonance Molecular structure and 
structural changes 
Shimada et al. 1993; 
Annable et al. 1994; 
Williams and Langdon 
1996 
Dynamic Light Scattering Particle Size Coviello and Burchard 
1992; Rodd et al. 2001; 
Richter et al. 2004,2005;  
Atomic Force Microscopy Surface morphology Ikeda et al. 2004; Jin et al. 
2006 
Scanning Electron 
Microscopy 
Surface morphology Kanzawa et al. 1989; 
Sanchez et al. 2000 
 
 
 
 
 
 
 
 23 
 
Chapter 3: Texture stability of hydrogel complex containing 
curdlan gum over multiple freeze-thaw cycles 
Williams et al., 2009. J Food Process Pres 33: 126?139. 
 
 
3.1 Introduction 
 
 A neutral, linear, polysaccharide produced by the microorganism Alcaligenes 
faecalis var. myxogenes, curdlan is the third microbial extracellular polysaccharide 
following xanthan gum and gellan gum to be approved for food use in the United 
States (FDA 1996). Composed of a three dimensional structure stabilized by 
crosslinks that connect junction zones between molecules (Harada et al. 1966; Saito 
et al. 1978; Marchessault and Deslandes 1978; Nakao et al. 1991; Jezequel 1998), 
curdlan gum was once the focus of extensive research efforts to expand its 
applications to improve the texture of various food products (Kanzawa et al. 1987; 
Nakao et al. 1991; Sanderson 1996), as well as the delivery of medicinal ingredients 
(Harada and Harada 1996; Na et al. 2000). To date, unfortunately, despite curdlan?s 
ability to form viscous aqueous suspensions with shear-thinning flow behavior 
(Hirashima et al. 1997; Lopes da Silva et al. 1998; Funami et al. 1999), the 
applicability of curdlan in the U.S. market remains scarce and limited, due mainly to 
its less profound viscosity than xanthan gum in solution and inferior gel formation 
capacity when compared with gellan (Sadar 2004).   
 Nevertheless, curdlan continues to be used as a texture modifier in Chinese 
and Japanese noodles and surimi-based products (Nakao et al. 1991; Nishinari 2000) 
 
 24 
 
as well as in processed meats such as pork, fried battered chicken, hamburger patties, 
and meatballs to yield juicier and softer products (Nakao, 1997; Hsu and Chung 
2000). Insoluble in water, alcohol, and most organic solvents (Pederson 1979), 
curdlan is known to form thermo-reversible or irreversible gels via heating (Maeda et 
al. 1967; McCleary 1979; Fulton and Atkins 1980; Harada et al. 1994) or addition of 
Ca2+ or Mg2+ (Kanzawa et al. 1987, 1989; Konno and Harada 1994). Furthermore, 
curdlan is also capable of forming tasteless, odorless, and colorless hydrogel 
complexes with other polysaccharides (Lo and Ramsden 2000; Lee et al. 2002). This 
unique gelling mechanism could be used to increase retention or absorption of 
moisture and other ingredients (Lo et al. 2003) while withstand the temperature 
extremes of freezing and retorting processes (Wielinga and Maehall 2000; McIntosh 
et al. 2005). However, the stability of such complexes when subjected to temperature 
abuse remains uncharacterized.   
 The present study investigates the feasibility of using curdlan gum to form 
stable hydrogel complexes that could improve texture stability over multiple freeze-
 thaw cycles (FTCs). Hydrogel complexes formed by curdlan and a secondary 
biopolymer, namely ?-carrageenan, xanthan, guar, and locust bean gum, were 
subjected to discrete temperature fluctuations. Five FTCs were conducted to resemble 
the possible temperature abuses of a frozen product (1) from processing plant to 
warehouse, (2) from warehouse to distributor, (3) from distributor to retail, (4) from 
retail to consumer, and (5) thawing and refreezing by consumer at home. The extent 
of syneresis, or water loss, and a spectrum of textural attributes of the gel complexes 
after each FTC were characterized and compared.   
 
 25 
 
3.2 Materials and Methods 
3.2.1 Materials and Sample Preparation 
 Odorless, fine, free-flowing white powder curdlan containing a minimum of 
90% ?-D-glucan and with a maximum of 10% water was used (Takeda Vitamin & 
Food USA, Orangeburg, NY). Samples of ?-carrageenan (Colloid F-390), xanthan 
(Ticaxan?), guar (Guar Gum 8/22A), and locust bean gum (Pre-Hydrated LBG) were 
kindly supplied by TIC Gums (Belcamp, MD). A predetermined amount of each of 
the biopolymer powders was weighed and dry blended at ambient temperature before 
introduced to deionized (DI) water under constant stirring to achieve an aqueous 
biopolymer solution containing 1.0% (w/v) curdlan and 1.0% (w/v) of ?-carrageenan, 
xanthan, guar, or locust bean gum.   
3.2.2 Hydrogel Complexes and Freeze-Thaw Cycles  
 Each of the biopolymer solutions continued to be magnetically stirred while 
gradually heated to reach 90?C on a heat plate. Once the temperature was met, the 
biopolymer solutions were removed from the heat and poured into 50 mL test tubes.  
The solutions were allowed to cool to room temperature to form the hydrogel 
complexes, which were covered and refrigerated for 24 h at 4?C before subjected to 
FTCs. Five FTCs were employed where each of the hydrogel complexes was stored at 
-16?C for 18 h, followed by thawing at 25 ?C for 6 h. The thawed samples were 
collected and tested for syneresis, viscosity, heat stability, storage and loss moduli, 
gel strength, and adhesiveness. 
 
 26 
 
3.2.3 Syneresis and Textural Measurements 
 Syneresis of the hydrogel complexes after each FTC was determined by 
centrifuging the samples at 2200 rpm (707 ? g) for 20 min in a Beckman Model TJ-6 
centrifuge (Beckman Coulter, Palo Alto, CA). The volume of exuded water was 
determined using a laboratory graduated cylinder (Fisher Scientific, Fairlawn, NJ). A 
TA.XT2i texture analyzer (Texture Technologies, Inc., Scarsdale, NY) was employed 
to determine the gel strength and adhesiveness profiles using a P/0.5R probe with a 5-
 kg load cell (Fiszman and Damasio 2000). Hydrogel complexes (50 mL) formed in 
cylindrical centrifuge tubes (1-in diameter; Fisher Scientific) were gently transferred 
onto the texture analyzer platform. For this analysis, when 5 g of force was detected 
by the probe, it continued to descend into the sample for 10 s before returning to the 
starting position. 
 General and dynamic rheological measurements of the hydrogel complexes 
were conducted using an AR2000 Rheometer (TA Instruments, New Castle DE) with 
a 60-mm aluminum truncated cone before and after each of the FTCs. The apparent 
viscosity of the hydrogel complexes was measured at 20?C under a shear rate of 0.15 
to 100 s-1. The heat stability of the complexes was assessed by ramping the 
temperature of the samples after each FTC from 20?C to 80?C and measuring their 
viscosity at a shear rate of 2 s-1. The storage (G?) and loss (G?) moduli of samples 
before and after each FTC were investigated using a frequency sweep over a range of 
angular frequency (6.283?150  rad/s) while controlling respectively the strain at 10-2, 
2.5 ? 10-3, 4 ? 10-3, and 10-2 for samples containing ?-carrageenan, xanthan, guar, and 
locust bean gum. 
 
 27 
 
3.3 Results and Discussion 
3.3.1 Syneresis 
 Syneresis, or loss of water, is an important parameter critical to the stability of 
a gel system (Xu et al. 1992; Hoover et al. 1997). Prior to freezing-thawing 
treatments, no syneresis was detected in the hydrogel complexes investigated (Table 
3.1), indicating good water holding capacity of the hydrogels upon formation. After 
the first FTC, apparent syneresis averaging 18% and 32% were detected in the 
curdlan complexes formed with ?-carrageenan (?-Ca/C) and locust beam gum 
(LBG/C), respectively. Loss of moisture in the gel complexes after additional FTCs in 
the curdlan/locust bean system (approaching 60% after the fifth cycle) was more 
profound than the curdlan/?-carrageenan complex where an average of 15-20% 
syneresis was observed after each FTC. No syneresis was detected in curdlan 
hydrogel complexes formed with guar gum (G/C) and xanthan gum (X/C) up to five 
FTCs, indicating strong and stable water holding capacity in these two hydrogel 
systems.   
 
Table 3.1.  Syneresis* of 2.0% aqueous curdlan solutions alone and with equal fractions of curdlan 
with xanthan (X/C), guar (G/C), ?-carrageenan (?-Ca/C), and locust bean gum (LBG/C). 
Syneresis (%) 
FTC Curdlan X/C G/C ?-Ca/C LBG/C 
0 46.7 ? 0.9 ND? ND ND ND 
1 54.1 ? 4.3 ND ND 18.56 ? 1.21 31.63 ? 3.84 
2 56.5 ? 4.5 ND ND 15.75 ? 1.46 43.95 ? 2.44 
3 58.1 ? 5.1 ND ND 17.74 ? 1.02 46.78 ? 2.57 
4 59.8 ? 3.9 ND ND 18.31 ? 0.60 57.01 ? 1.60 
5 61 ? 3.9 ND ND 15.55 ? 0.54 59.61 ? 2.28 
*Data were expressed as mean ? SD (n=3).  
?ND = Not Detected. 
 
 28 
 
 The ability of the hydrogel complexes to hold moisture is dependent upon the 
conformation of curdlan and the secondary biopolymer in the system, since curdlan is 
a linear polysaccharide that forms complex tertiary structures resulting from 
intramolecular and intermolecular hydrogen bonding (Nishinari 2000; Lo et al. 2003).  
Before FTCs, the heating (up to 90 ?C) involved in the hydrogel formation process 
could be responsible for the strong initial water-holding capacity, since temperature is 
known to influence the conformation of curdlan molecules. It has been shown that 
curdlan swells at around 55?C, resulting in partial rupture of intramolecular hydrogen 
bonding (Jezequal 1998). When the temperature is reduced, new hydrogen bonds then 
crosslink the single-helix curdlan micelles to form a low-set gel (Maeda et al. 1967; 
Kimura et al. 1973). A high-set, thermal-irreversible gel could be formed if the 
aqueous solution is heated to above 80?C (Pszczola 1997; Jezequal 1998). 
 Furthermore, the conformation of curdlan is also known to vary with the 
concentration of alkaline in a solution (Ogawa et al. 1972). At low NaOH 
concentrations, curdlan shows an ordered (helical) conformation. A significant 
change occurs once the concentration of NaOH is increased to between 0.19 and 0.24 
N. Curdlan becomes fully soluble with a random structure once the NaOH exceeds 
0.24 N. The random structure returns to an ordered state once the solution is 
neutralized. Therefore, the stability of the intramolecular and intermolecular 
hydrogen bonding was also dependent on the chemical compatibility of curdlan with 
the secondary biopolymer in the system. Although the ?-1,3 glucosidic linkages that 
connect curdlan?s repeating D-glucose subunits (Harada and Harada 1996; Jezequal 
1998; Funami et al. 1999; Imeson 2000) are also found in ?-carrageenan, the neutral 
 
 29 
 
nature of the D-glucose that makes up curdlan might have hindered the interactions 
between curdlan and ?-carrageenan, consequently losing its water-holding capacity, 
as evidenced by the 15-20% syneresis after freeze-thaw treatments (Table 3.1).   
 Locust bean gum is a neutral galactomannan with linear chains of 1,4-linked 
beta-D-mannan backbone with 1,6-linked alpha-D-galactose side units (Meer 1977; 
Wielinga and Maehall 2000). The galactose side units are the only portion responsible 
for hydration and hydrogen-bonding activity; however, at a 4:1 mannose:galactose 
ratio the galactose units could easily become unavailable if they are trapped 
intramolecularly (Dea et al. 1977). Contrarily, both xanthan and guar gum are highly 
substituted. Xanthan gum is an anionic linear hydrocolloid with a (1?4) linked ?-D-
 glucose backbone, and has a large side unit on every other glucose unit at location C-
 3 (Kovac and Kang 1977; Sworn 2000; Giannouli and Morris 2003). Guar gum, a 
neutral hydrocolloid with linear chains of D-mannopyranosyl units with D-
 galactopyranose substituents protruding by (1?6) linkages, contains approximately 
two mannose residues per galactose (Maier et al. 1992). The highly substituted nature 
of these two polymers allows for excellent hydration and hydrogen-bonding activity 
(Hoyt 1966; Argin-Soysal et al. 2009), which could attribute to the zero syneresis 
achieved when xanthan or guar was incorporated in the curdlan hydrogel complexes 
(Table 3.1).   
 3.3.2 Apparent Viscosity and Heat Stability 
Xanthan/curdlan and guar/curdlan hydrogel complexes conducted in the 
present study were found to exhibit pseudoplastic behavior, as indicated by the Cross 
model below, representing the curve better than the simpler power law model.  
 
 30 
 
                                           Gbb6 Gc0eGc2cGb3fGc0eGcaeGb35Gb3eGbdeGcb4 Gc0aG1236 Gcd8                                               [1] 
Where ? is the viscosity, G7dfGbb6 is the infinite shear viscosity, G7dfGb34 is the zero shear 
viscosity, kC is the consistency coefficient of the Cross model, m is the flow behavior 
index and G7dbG1236  is the shear rate. There was variation in the values over the FTCs, 
however, a trend can be elucidated from Table 3.2. As expected from gels, the 
viscosity of ?-Ca/C and LBG/C at zero shear viscosity was significantly higher than 
G/C and X/C complexes. The zero shear viscosity of X/C was greater than G/C, 
signifying a texture that is possibly more ?gel like.? Conversely, the G/C and X/C 
samples showed higher consistency coefficients than other complexes tested.   
 More consistent with gel behavior, the consistency coefficient was lower for 
?-Ca/C and LBG/C. Interestingly the latter also exhibited shear thickening behavior, 
where the flow behavior index is greater than 1. Complexes G/C and X/C had a rate 
index below 1, indicating shear thinning behavior. Finally, the quick ramp (30 
?C/min) in temperature yielded a significant increase in apparent viscosity at 80 ?C, 
G7dfGb3cGb34, and 2 s-1. As shown in Table 3.2, ?-Ca/C and LBG/C exhibited the least stability 
through FTCs, where LBG/C increased through FTCs until decreasing after the fifth 
FTC and ?-Ca/C decreased over the FTCs.  With the exception of the third FTCs, the 
heat ramping had the same affect throughout FTCs on G/C and X/C. The lack of heat 
stability between all samples may be due to some water evaporation though a solvent 
trap was utilized.   
 
  
31
  
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 32 
 
3.3.3 Storage and Loss Moduli 
 Frequency sweep testing was conducted within the linear viscoelastic range 
determined from strain sweep measurements. With the exception of locust bean gum, 
all hydrogel complexes exhibited a greater elastic (or solid-like) portion, as indicated 
by the storage modulus, G?, predominating the loss modulus (viscous portion), G?, 
throughout the five FTCs in Figure 3.1. The differences between G? and G? in the 
curdlan/guar and curdlan/xanthan combinations were significantly lower than the 
other two combinations, suggesting the formation of polymer entanglements when 
curdlan was mixed with guar or xanthan (Lo et al. 2003). This is in agreement with 
the low apparent viscosity of G/C and X/C at 20 ?C (Table 3.2). The values of G?, a 
measurement of energy stored during deformation and related to the solid-like portion 
of the system, were significantly higher when curdlan was mixed with ?-carrageenan 
or locust bean gum. The greatest G? values were observed with the locust bean/ 
curdlan combination, which also showed two distinct crossover points between G? 
and G? before the hydrogels were subjected to FTCs?first at angular frequency of 
0.15 rad/s and modulus of 2.21 Pa and second at 17.37 rad/s and 46.03 Pa.   
 The crossover points, the overlap frequency that represents the point at which 
the characteristic behavior of the gels shifted from elastic to viscous, in the 
curdlan/locust bean combination were indicative of its weak gel structure. The 
hydrogel complexes continued to behave like viscous solutions where the two 
polymers formed typical entanglement solutions between the two crossover points.  
Although the significance of the second crossover point at high angular frequency on 
the structure of curdlan/locust bean hydrogels remained unclear, this unique 
 
 33 
 
phenomenon could also attribute to the dramatic changes of viscosity and heat 
stability (Table 3.2) observed in the system after multiple FTCs. 
 
 
Figure 3.1. Frequency sweeps of 2.0% aqueous curdlan solutions alone and with equal fractions of 
curdlan with xanthan (X/C), guar (G/C), ?-carrageenan (?-Ca/C), or locust bean gum (LBG/C). 
Storage, G?, and loss modulus, G?,  prior to (a), and after the first (b), second (c), third (d), fourth (e), 
and fifth (f) freeze-thaw cycles. 
 
It has been postulated that, at higher frequencies, the polymer chain entanglements 
are retained throughout the short period of oscillation and they act as temporary 
cross-links, causing the material to exhibit pronounced elasticity (Dea 1979; Imeson 
2000; Lo et al. 2003). However, after freeze-thaw treatments, the curdlan/locust bean 
1
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ? (rad/s)
 a
 G'   G" ?-Ca/C     G'   G" X/C
 G'   G" LBG/C  G'   G" G/C
 1
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ? (rad/s)
 b
 G'   G" ?-Ca/C     G'   G" X/C
 G'   G" LBG/C  G'   G" G/C
 1
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ? (rad/s)
 c
 G'   G" ?-Ca/C     G'   G" X/C
 G'   G" LBG/C  G'   G" G/C
 1
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ? (rad/s)
 d
 G'   G" ?-Ca/C     G'   G" X/C
 G'   G" LBG/C  G'   G" G/C
 1
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ? (rad/s)
 e
 G'   G" ?-Ca/C     G'   G" X/C
 G'   G" LBG/C  G'   G" G/C
 1
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ? (rad/s)
 f
 G'   G" ?-Ca/C     G'   G" X/C
 G'   G" LBG/C  G'   G" G/C
 
 34 
 
combination showed distinct gel-like properties where G? predominated G? over the 
entire frequency range. 
3.3.4 Gel Strength and Adhesiveness 
 The gel strength data were in support of the moduli measurements where the 
curdlan/?-carrageenan complexes were found to have the highest gel strength prior to 
FTCs (Table 3.3). The gel strength of curdlan/?-carrageenan continued to decline 
after each FTC, suggesting the inability of the system to withstand temperature 
fluctuations. At the end of the fifth FTC, the gel structure was completely destructed.  
Conversely, the gel strength of curdlan hydrogels formed with xanthan or guar gum 
both stayed extremely low throughout the five FTCs, reflective of the pudding-like 
texture of both hydrogel complexes. The gel strength of curdlan/locust bean 
complexes was significantly increased after the first FTC, and remained relatively 
stable over the next three cycles before declining again after the fifth FTC. Such high 
gel strength even after multiple FTCs could be attributed to the considerable smooth 
mannose backbone regions that give locust bean gum the ability to form strong gels 
with other polymers such as xanthan, carrageenan, and agarose (Meer 1977; 
McCleary 1979; Launay and Cuvelier 1986).  
 Adhesiveness, the work required to separate a surface from food materials, is 
an important attribute that could have both positive and negative ramifications 
depending on the applications (Meiron and Saguy 2007). The adhesiveness of 
curdlan/locust bean hydrogels was the highest amongst all combinations prior to 
FTCs, followed by guar, ?-carrageenan, and xanthan (Table 3.3). Except for xanthan,  
 
 
 35 
 
Table 3.3.  Gel Strength and adhesiveness* of 2.0% aqueous solutions with equal fractions of curdlan 
with xanthan, guar, k-carrageenan, or locust bean gum over five freeze-thaw cycles (FTCs). 
Gel Strength and Adhesiveness of Curdlan Combinations 
FTC X/C G/C k-Ca/C LBG/C 
Gel Strength (N) 
0 0.075?0.001 0.089?0.013 0.205?0.019 0.079?0.002 
1 0.080?0.001 0.091?0.015 0.164?0.005 0.494?0.117 
2 0.082?0.002 0.077?0.001 0.156?0.020 0.301?0.025 
3 0.084?0.001 0.089?0.014 0.146?0.017 0.262?0.025 
4 0.086?0.001 0.075?0.001 0.114?0.004 0.268?0.009 
5 0.085?0.001 0.076?0.001 0.090?0.002 0.202?0.039 
Adhesiveness (|N s|) 
0 0.044?0.003 0.186?0.019 0.104?0.011 0.262?0.044 
1 0.048?0.012 0.097?0.001 0.019?0.002 0.004?0.006 
2 0.060?0.015 0.106?0.000 0.005?0.003 0.005?0.004 
3 0.060?0.005 0.165?0.044 0.001?0.014 0.004?0.004 
4 0.063?0.003 0.050?0.011 0.007?0.001 0.005?0.001 
5 0.060?0.000 0.047?0.002 0.007?0.001 0.005?0.001 
*Data were expressed as mean ? SD (n=3).  
 
 
the adhesiveness of the complexes quickly declined after the first FTC, with 
curdlan/locust bean being the most drastic. However, it was drastically reduced after 
the first FTC and the hydrogels remained almost non-adhesive after subsequent FTCs.  
Unlike locust bean gum or ?-carrageenan, guar gum in combination with curdlan 
formed hydrogels that showed an unusual spike in adhesiveness after the third FTC 
yet continued to decline in following cycles.   
 The tendency of a food to adhere and possibly interact with its packaging 
material could have significant ramifications that may affect its overall quality, 
appearance, and shelf life (Meiron and Saguy 2007). It could also affect the barrier 
characteristics of the package, which could have significant ramifications (e.g., 
flavor, color, and nutrients deterioration) (Hwang et al., 2000; Ayhan et al., 2001).  
 
 36 
 
With regard to product quality, especially concerning the product?s shelf life and 
shelf stability, it is highly desirable to employ a hydrogel complex that could remain 
stable over multiple FTCs. Therefore, the most consistent adhesiveness observed in 
the curdlan/xanthan hydrogel complexes up to five FTCs suggests that curdlan in 
combination with xanthan could serve as an excellent candidate to prevent syneresis 
in frozen products over temperature fluctuations, either intentionally or 
unintentionally, without altering the textural quality of the product.   
3.4 Conclusion 
 Over the five FTCs investigated, the curdlan/xanthan hydrogel complexes 
proved to be the most resilient, showing zero syneresis with superior consistency 
storage and loss moduli, gel strength, and adhesiveness compared to curdlan 
complexes formed with guar, ?-carrageenan, or locust bean gum. Guar and curdlan 
combination also showed good stability with no syneresis, but yielded lower zero 
shear viscosity, fluctuations in both the dynamic modulus and adhesiveness.  
Conversely, locust bean gum, when incorporated into curdlan hydrogels, was found 
the most unstable due mainly to the formation of a weak gel network that quickly 
broke down, leading to increased syneresis as FTCs progressed. The curdlan/?-
 carrageenan formulation gave high gel strength; however, besides noticeable 
syneresis (15-20%) the drastic changes in viscosity and adhesiveness after FTCs were 
undesirable for food applications. Further investigations are warranted in order to 
optimize the formulation that fulfills product-specific requirements. 
 
 
 
 37 
 
Chapter 4:  Characterization of Water Distribution in Xanthan-
 Curdlan Hydrogel Complex Using Magnetic Resonance 
Imaging, Nuclear Magnetic Resonance Relaxometry, Rheology, 
and Scanning Electron Microscopy 
 
Williams et al., 2011. J Food Sci: Food Engineering and Physical Sciences 
 
 
4.1 Introduction 
Syneresis, the spontaneous expulsion of liquid from a gel (Parker, 1984), 
remains a challenge in processed foods, especially frozen products, since product 
quality could be drastically reduced by uncontrolled moisture loss or migration, 
which consequently creates ice crystals that could lead to microbial growth and 
texture deterioration in food (Goff et al., 1993; Lund 2000). In our previous study 
(Williams et al., 2009), we successfully demonstrated that a hydrogel complex 
formed by xanthan and curdlan gum significantly reduced syneresis over five freeze-
 thaw cycles (FTCs) to an undetectable level, while retaining stable rheological and 
textural properties. The viscosity, heat stability, loss and storage moduli, gel strength, 
and adhesiveness remained relatively consistent throughout the FTCs investigated.  
However, the distribution of water and its interactions with the xanthan curdlan 
hydrogel complex (XCHC) remains uncharacterized.  
Xanthan gum, an anionic microbial polysaccharide widely used as a 
thickening, stabilizing, and emulsifying agent in the food, chemical, and 
pharmaceutical industries due to its desirable pseudoplastic behavior in water 
(Whitcomb and Macosko, 1978), is produced by Xanthomonas campestris 
fermentation and consists  of repeating (1?4)-?-D-glucose units in the backbone 
with a trisaccharide side chain, namely ?-D-mannose, ?-D-glucuronic acid, and ?-D-
 
 38 
 
mannose, located on C3 of every other glucose. Besides having excellent 
compatibility with major food components, xanthan, when combined with other gums 
or starches, showed synergistic effects in viscosity and moisture control (Lo and 
Ramsden 2000; Sworn 2000). Curdlan, on the other hand, is a neutral linear microbial 
polysaccharide made of (1?3)-?-glucan repeating units, making it insoluble in water, 
alcohols, and most organic solvents, except for alkaline solutions (Pederson, 1979; 
McIntosh et al., 2005). It can form a low-set thermo-reversible gel when heated 
between ~55?C and 80?C, or a high-set, triple helix, thermo-irreversible gel when 
heated above ~80?C and then cooled (Harada et al., 1994; Hirashima et al., 1997; 
Nakao, 1997; McIntosh et al., 2006; Gagnon and Lafleur, 2007). Capable of holding 
moisture in processed meat and flour products, curdlan is stable over a pH range of 2 
to 12 and freeze-thaw conditions, except as an aqueous solution wherein syneresis 
occurs (Nakao, 1997). The gelation mechanism of the low- and high-set gels 
continues to be of interest in the literature (Tada et al., 1999; Hatakeyama et al., 2006; 
Gagnon et al., 2007). Nevertheless, no information is yet available on the interaction 
of xanthan and curdlan undergone freezing-thawing treatments.  
Nuclear magnetic resonance (NMR) relaxometry and magnetic resonance 
imaging (MRI) techniques have allowed identification of water distribution and 
molecular interactions of gums without destruction of the sample. These non-invasive 
techniques have been used to study the gelation process of combined hydrocolloids 
and their interactions with water. For instance, NMR was employed to characterize 
molecular mobility for the combination of several polysaccharides, including xanthan 
gum (L?sse and Arnold, 1998; Vittadini et al., 2002), as well as structural interactions 
 
 39 
 
(Whitney et al., 1998; Lazaridou et al., 2000; Tojo and Prado, 2003; Kim et al., 
2006). With the assistance of NMR, the effect of water on pectin molecules was 
elucidated (Kerr and Wicker, 2000), whereas the molecular interactions between 
locust bean gum and cellulose was specified to involve mannosyl residues and not 
galactosyl residues as first thought (Newman and Hemmingson, 1998). Capable of 
measuring the proton density of water throughout a sample, MRI, when used in 
conjunction with NMR, could relate the homogeneity of a hydrogel to its textural 
behavior (Gil et al., 1996). Oztop and others (2010) recently quantified the effect of 
water uptake on whey protein hydrogels by using MRI and NMR relaxometry. They 
showed that the spin-spin relaxation time (T2) distributions were highly correlated 
with the water uptake and were able to explain the interactions of the gel with water 
at different pH values. Integration of MRI and NMR relaxometry with conventional 
rheological measurements widely used to investigate textural behavior of 
polysaccharides (Whitcomb and Macosko, 1978; Wang et al., 2002a,b; Giannouli and 
Morris, 2003; Ikeda et al., 2004; Higiro et al., 2006; Khouryieh et al., 2006; Kim et 
al., 2006; Nishinari 2007; Pongsawatmanit et al., 2007; Choi and Yoo, 2008), should 
provide a powerful approach in attaining a more complete understanding of how the 
water distribution is affected by the structure of polymers in a hydrogel complex. 
In the present study, the moisture distribution in xanthan, curdlan, and XCHC 
was investigated using MRI and NMR relaxometry, and the results were correlated 
with respective rheological measurements. The surface morphology, as well as the 
microstructure of the polysaccharides in question, was visualized by scanning 
electron microscopy (SEM).  
 
 40 
 
4.2 Materials and Methods 
 4.2.1 Sample Preparation 
 Xanthan and curdlan were provided by TIC Gums (Belcamp, Md., U.S.A.) 
and Takeda USA (Orangeburg, N.Y., U.S.A.), respectively. Each of the sample 
solutions containing xanthan or curdlan alone as well as equal fractions of both were 
prepared to reach a final concentration of 2% (w/v) in de-ionized water. The 
polysaccharides were gradually added to continuously stirred water until well 
hydrated, then heated to 90 ?C under reflux using a water bath. Samples were then 
allowed to cool to room temperature and finally placed in the appropriate testing 
vessel at 4 ?C for 24 hrs. To complete a freeze-thaw cycle (FTC), the samples were 
placed at -20 ?C for 18 hrs, followed by 6 hrs of thawing at room temperature. 
 4.2.2 Magnetic Resonance imaging and nuclear magnetic resonance 
 relaxometry 
MRI and NMR relaxometry experiments were carried out using a 1.03T NMR 
spectrometer with 43.8 MHz for 1H-resonance frequency (Aspect Imaging,  
Hevel Modi'in Industrial Area, Israel). A T1 weighted spin echo sequence was used 
for MR acquisition. The echo time (TE) was 7.6 ms and the repetition time (TR) was 
500 ms. Four saggital slices with a thickness of 3 mm were acquired.  The 3rd slice 
was used for data analysis. The field of view (FOV) was 50 mm by 50 mm which 
yields an in-plane spatial resolution of 391 ?m/pixel. All imaging experiments were 
performed with a time invariant reference vial of distilled water in the image. The 
image files were analyzed by using MATLAB? v. 7.8.0 (The MathWorks Inc., 
Natick, Mass., U.S.A.).  
 
 41 
 
 For the NMR relaxometry, the spin-spin relaxation (T2) measurements were 
performed on the same samples with the same MRI spectrometer. A Carr-Purcell-
 Meiboom-Gill (CPMG) pulse sequence was used with echo time (TE) of 1 ms, 16 
acquisition points, 12000 echoes for curdlan, 1024 echoes for xanthan, and 2048 
echoes for XCHC. T2 measurements were acquired four times for each sample.  
Multi-exponential decay fitting of the CPMG curves (T2 distributions) were 
performed using the Nonnegative-Least-Square (NNLS) macro of the software 
PROSPA (Magritek, New Zealand).  
 
The spin-lattice relaxation (T1) measurements were performed by using a 
saturation recovery pulse sequence with delay time ranging from 1 ms to 20000 ms, 
with 1024 acquisition points and 4 scans. The T1 values were analyzed by fitting the 
recovery data to a monoexponential fit by using MATLAB?. 
 4.2.3 Rheology 
 Frequency sweeps, i.e. angular frequency vs. storage and loss moduli, were 
performed with controlled strain AR2000 rheometer (TA Instruments, New Castle, 
Del., U.S.A.) prior to and after each FTC. A 60-mm aluminum truncated cone (1?) 
geometry with a gap of 30 ?m was implemented. The frequency sweep was 
performed from 100 to 0.1 rad/s at 20 ?C within the linear viscoelastic region, with % 
strain being 1.6, 0.16, and 0.02 for curdlan, xanthan, and the xanthan curdlan 
hydrogel complex, respectively. Three replications were conducted for each sample. 
  
 
 
 42 
 
 4.2.4 Scanning Electron Microscopy 
Samples undergoing SEM were first freeze dried after the 24 hr refrigeration 
period. SEM was performed using a SU-70 Ultra-High Resolution Analytical SEM 
(Hitachi, Tokyo, Japan). Freeze dried samples were gold sputtered for analysis by the 
microscope. 
 4.2.5 Statistical Analysis 
 For statistical analysis, Minitab? v. 15 (Minitab, Inc., State College, Penn., 
U.S.A.) was used. Analysis of variance was performed to evaluate the effect of gel 
type and processing (control and freeze-thaw cycle) on T1 and T2.  Tukey?s test at 5% 
significance level was used as the comparison test. For rheological samples, Tukey?s 
HSD test at 5% significance level was employed as the comparison test using SAS? 
v. 9.2 (SAS Institute Inc., Cary, N.C., U.S.A) (see Appendix C). 
4.3 Results and Discussion 
 4.3.1 Spin-lattice relaxation time, T1, measurements 
 The spin-lattice relaxation time, T1, a time constant characterizing the 
exchange of energy between the nuclear spins and their molecular surrounding, are 
shown in Table 4.1. As expected, the proton spin-lattice relaxation times for each type 
of curdlan and/or xanthan hydrogel were shorter than water (Table 4.1).  
Additionally, the values were significantly different from one another (p<0.05). The 
xanthan hydrogel exhibited the longest T1 and the curdlan hydrogel exhibited the 
shortest. The combination of xanthan and curdlan (XCHC) had spin-lattice relaxation 
 
 43 
 
times between those of the pure gums. There was no significant difference in T1 
values between different days, indicating that FTC did not have an effect on T1.   
 
Table 4.1. ? Spin-lattice relaxation timesa, T1, of 2.0% xanthan, curdlan and xanthan curdlan hydrogel 
complex (XCHC) subjected to freeze-thaw cycles (FTCs). 
 Spin-lattice relaxation times, T1 (ms) 
FTCs Xanthan XCHC Curdlan 
0 2067 ? 34 1934 ? 4 1537 ? 58 
1 2171 ? 110 1946 ? 11 1714 ? 42 
5 2210 ? 101 1918 ? 21 1671 ? 59 
aEach value is the mean ? SD, n = 3. 
 
T1 values were characteristic of each hydrogel and were used to optimize the image 
acquisition. The T1 spectra have been used to quantify the porous microstructure of 
hydrogels (Chiu et al., 1995). 
 Although T1 measurements were not used in this case to provide information 
about the interaction of water with the biopolymers and the water distribution in the 
hydrogels directly, differences in T1 values are reflected in MR signal intensities for 
the hydrogels. Since the MR images were T1 weighted, lower signal intensity was 
observed in the hydrogels with the longer T1 values, this is described in detail in the 
next section.  
 4.3.2 Magnetic resonance imaging 
 The MR signal intensity equation for a spin-echo sequence is given by 
(Bernstein et al., 2004):   
                                                             [1] 
 
 44 
 
where M0 is the initial magnetization, TR is the repetition time, and TE is the echo 
time; T1 and T2 are spin-lattice and spin-spin relaxation times, respectively. When the 
repetition time, TR, is set such that it is lower than T1, and TE is small compared to the 
T2 of the sample, the image becomes T1 weighted. The TR value used in this study was 
500 ms, which was lower than the T1 of all hydrogels (Table 4.1). Therefore, the low 
signal indicates long T1 component and a high signal indicates shorter T1.   
Representative images of curdlan hydrogel are given in Figure 4.1. The first 
image illustrates the sample prior to a FTC. The second and third images illustrate the 
sample after 1 and 5 FTCs respectively. Each image has a reference vial of water on 
the left. The reference water vial has lower signal intensity due to the longer T1 of 
water than that of the hydrogels. The curdlan hydrogel was less homogeneous than 
other hydrogels in this study, as seen in the images by regions of higher and lower 
signal intensity within the sample. These images are characteristic of samples that 
exhibit phase separation, e.g., syneresis. The mean signal intensity and the standard 
deviation of the signal intensities were calculated in the sample region for each 
image. The decrease in signal intensities for curdlan hydrogels was about 8% (Figure 
4.1). Other hydrogels also decreased in signal intensity after one FTC but did not 
change after multiple FTCs. 
 
 
 
 
 45 
 
 
Figure 4.1. Representative magnetic resonance images (MRI) of curdlan (top row) and the xanthan 
curdlan complex hydrogels (bottom row) prior to freezing (a, d) after one freeze thaw cycle (b, e) and 
after the fifth freeze thaw cycle (c, f). The scale on the right gives the relationship between signal 
intensity and grayscale for the images. Axes in the images denote the voxel numbers. 
 
Coefficient of variation (COV) values were calculated as the ratio of the 
standard deviation of the signal intensity in an image to the mean signal intensity 
(Table 4.2). Larger COV values were observed for curdlan hydrogels and increased 
with additional FTCs, while other hydrogels? COV values did not change 
significantly. The lower decrease in signal intensity and no change in COV values 
compared to the curdlan hydrogels could be explained by the stabilizing effect of 
xanthan. Syneresis was not visually observable in these gels and the signal intensity 
in the MR images was homogeneous indicating no syneresis. 
 
 
 
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
  
 
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
  
 
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
  
 
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
  
 
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
  
 
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
 0
 500
 1000
 1500
 2000
 2500
 3000
 3500a b c
 d e f
 
 46 
 
Table 4.2. Coefficient of variationa (COV) values given as percentages for signal intensities acquired 
by the spin-echo sequence in relation to magnetic resonance images of 2.0% xanthan, curdlan and 
xanthan curdlan hydrogel complex (XCHC) subjected to freeze-thaw cycles (FTCs). 
 Coefficient of Variation (%) 
FTCs Xanthan XCHC Curdlan 
0 5.37 ? 0.36 10.33 ? 0.90 12.37 ? 3.19 
1 6.56 ? 0.08 8.76 ? 0.63 13.60 ? 1.39 
5 6.56 ? 0.12 9.44 ? 0.54 14.48 ? 0.56 
aEach value is the mean ? SD, n = 3. 
 
 4.3.3 Spin-spin relaxation time, T2, measurements 
 T2 distributions of the hydrogel samples were obtained from the CPMG decay 
curves by using the Non-Negative Least Square Algorithm in PROPSA. T2 
characterizes the rate at which the Mxy component of the magnetization vector decays 
in the transverse plane and characterizes the interactions between neighboring nuclear 
spins. In gels, this relaxation rate is affected by the exchangeable hydroxyl protons 
resulting in a multi-exponential behavior that is quantified by a T2 distribution. The 
conformation and mobility of the biopolymer chains resulting from FTC is reflected 
in the T2 distributions. The T2 distributions for the hydrogel samples are given in 
Table 4.3. The distributions include the peak T2 values and the areas associated with 
the peak.   
 Initially, there were two proton pools (or compartments) for the curdlan: one 
associated with the gel structure and one associated with water not entrapped by the 
gel structure. With one FTC, a third compartment was observed. The T2 of all the 
proton pools increased with respect to FTC which was consistent with curdlan?s 
unstable behavior after a freeze-thaw treatment. The first two compartments 
associated with gel structures had different amounts of water. These were related to 
 
 47 
 
the exchangeable protons of the biopolymer, such as hydroxyl and hydroxymethyl 
groups (-OH, -CH2OH) corresponding to the intermolecular interaction of the 
biopolymer. The first proton pool was in an intermediate exchange regime whereas 
the second pool was in a slower exchange regime.  
The third compartment with the longest T2 was associated with the water not 
entrapped in the gel structure but the water moving between the gel particulates.  
These protons were also associated with protons that were not interacting strongly 
(Kerr and Wicker 2000) with the polymer molecules, which might be indicative of the 
hydrophobic effect that causes curdlan?s insolubility in water.   
All compartments experienced a change in the relative areas of the proton 
pools after the FTCs (Table 4.3). The first compartment?s contribution increased, 
whereas a decrease in the relative area of the second proton pool was observed. The 
increase in the area of the first compartment could be attributed to an increasing 
development of a three-dimensional network observed with high-set curdlan gels. The 
decrease in the area of the second compartment was compensated by the increase in 
the first area. The third compartment?s area also showed a 25% increase which was 
consistent with images and the syneresis occurring in the hydrogel.   
Due to its high water solubility, only one compartment with a T2 value of 186 
ms was observed for the xanthan hydrogels. Unlike curdlan, the T2 values in xanthan 
did not change nor did the relative proton pool areas change upon freeze-thaw. These 
results were also consistent with the imaging experiments. 
 Two compartments were observed with the XCHC. The relative areas of the 
proton pools were different compared to that of the curdlan only hydrogels, where the 
 
 48 
 
lower T2 value (~110 ms) was very small (4%) versus the relative area of the second 
pool at 342 ms. In XCHC, curdlan hydrogels may be suspended in the xanthan 
solution. In that regard the first peak could be attributed to the protons coming from 
the suspended curdlan hydrogels. There was no significant difference (p>0.05) in the 
T2 values or relative areas of the compartments due to FTCs (Table 4.3). A third 
compartment was not found with XCHC as it was in curdlan, indicating XCHC was 
more resistant to syneresis than curdlan. 
 In this study, T2 distributions were used to characterize changes in hydrogels 
that were subjected to severe treatments. T2 relaxation spectrums were sensitive to 
composition for all hydrogels. For the hydrogel that exhibited increasing syneresis 
after FTCs, the relaxation spectrums also changed and hence were sensitive to 
process induced structural changes. 
  
  
49
  
 
  
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 50 
 
 4.3.4 Rheology and Morphology 
 Frequency sweeps of curdlan samples, prior to and following the five FTCs, 
showed greater storage modulus, G?, than the loss modulus, G?, over the entire 
frequency range, indicating gel behavior (Figure 4.2). A fully cured three-
 dimensional gel network is indicated when G? predominates G? with their slopes 
being independent of the angular frequency.   
 
 
Figure 4.2. Frequency sweeps of 2.0% xanthan, curdlan, and xanthan curdlan hydrogel complex 
(XCHC). Storage, G?, and loss modulus, G?,  prior to (a), and after the first (b), second (c), third (d), 
fourth (e), and fifth (f) freeze-thaw cycles. 
 
 
10
 100
 1000
 10000
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 G
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 " 
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 a)
 Angular Frequency, ?, (rad/s)
 f
 circle7-G' Curdlan ?-G" Curdlan
 square7-G' XCHC   box1-G" XCHC
 triangleup-G'Xanthan triangleopenup-G" Xanthan
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ?, (rad/s)
 d
 circle7-G' Curdlan ?-G" Curdlan
 square7-G' XCHC   box1-G" XCHC
 triangleup-G'Xanthan triangleopenup-G" Xanthan
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ? (rad/s)
 b
 circle7-G' Curdlan ?-G" Curdlan
 square7-G' XCHC   box1-G" XCHC
 triangleup-G'Xanthan triangleopenup-G" Xanthan10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
  
Angular Frequency, ? (rad/s)
 a
 circle7-G' Curdlan ?-G" Curdlan
 square7-G' XCHC   box1-G" XCHC
 triangleup-G'Xanthan triangleopenup-G" Xanthan
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ?, (rad/s)
 c
 circle7-G' Curdlan ?-G"Curdlan
 square7-G' XCHC   box1-G" XCHC
 triangleup-G'Xanthan triangleopenup-G" Xanthan
 10
 100
 1000
 10000
 0.1 1 10 100
 G
 ', 
G
 " 
(P
 a)
 Angular Frequency, ?, (rad/s)
 e
 circle7-G' Curdlan ?-G" Curdlan
 square7-G' XCHC   box1-G" XCHC
 triangleup-G'Xanthan triangleopenup-G" Xanthan
 
 51 
 
 
 
The slope can be quantified by modeling the storage modulus with the power law,  
                                                                Ga G11f1 Gd4c G73dG7f1Gbd5                                                    [2] 
where a is the intercept, b is the slope and ? is the angular frequency. Curdlan 
exhibited a positive, but slight, dependency upon the angular frequency and therefore 
did not definitively exhibit a three-dimensional gel structure from rheological data.   
As seen in Table 4.4, the slope of curdlan prior to FTCs was 0.069, whereas the slope 
of 1% agar, a known fully cured gel, was 0.037 (Ross-Murphy, 1988; Steffe, 1996).   
 
Table 4.4. Linear regression of storage modulus (G? = a?b) and syneresis over five freeze-thaw cycles 
(FTCs) for 2.0% xanthan, xanthan curdlan hydrogel complex (XCHC), and curdlan. The intercept, a, 
and slope, b, of the storage modulus are presented. 
 Xanthan XCHC Curdlan 
FTC a (Pa sb) b Syneresis (%)  a (Pa sb) b Syneresis (%) a (Pa sb) b Syneresis? (%) 
0 120 0.211 - 1298 0.102 - 2280 0.069 46.7 ? 0.9 
1 128 0.198 - 1028 0.107 - 2570 0.065 54.1 ? 4.3 
2 170 0.190 - 1054 0.103 - 3515 0.062 56.5 ? 4.5 
3 177 0.183 - 1091 0.093 - 2738 0.066 58.0 ? 5.1 
4 221 0.169 - 1062 0.106 - 4190 0.064 59.8 ? 3.9 
5 208 0.173 - 954 0.111 - 3141 0.063 61.0 ? 3.9 
?Each value is the mean ? standard deviation, n = 3. 
 
The fluctuation of the dynamic modulus was due in part to the difficulty in handling 
the sample. The syneresis of curdlan caused the water-to-gel ratio to become 
inconsistent for each FTC when placed on the plate for rheological tests.  
Nonetheless, the SEM image of curdlan confirmed the three dimensional network 
(Figure 4.3), showing the concentrated polymers in a ?honeycomb? like orientation 
that could be responsible for curdlan?s elastic behavior, where free water could be 
 
 52 
 
moving between and around the gel matrix. This potentially trapped water could be 
represented by the longest T2 relaxation time, which is subsequently released over 
FTCs as seen in the MR images (Figure 4.1).  
 Frequency sweeps of xanthan showed G? predominating G? over the entire 
frequency range with a significant positive slope. The modulus values were at least 
one order of magnitude lower than that of curdlan solution, indicating low elasticity. 
Though there was no detectable syneresis in xanthan samples, the modulus for the 
second through fifth FTCs was noticeably higher than the modulus of previous 
cycles. Xanthan is known to exhibit ?gel-like? behavior, which is indicated by its 
frequency sweep; however, the SEM photos did not reveal a three-dimensional 
network.  SEM images in Figure 4.3 showed two-dimensional layers closely packed. 
The lack of a three-dimensional network in the SEM micrographs was expected due 
to xanthan?s dynamic modulus dependency upon angular frequency and low modulus 
values. 
  
 
Figure 4.3. Scanning electron microscope micrographs of the 2.0% curdlan (a), 2.0% xanthan (b), and 
2.0% xanthan curdlan hydrogel complex (c) freeze dried samples. 
 
a b c 
 
 53 
 
The storage modulus was greater than the loss modulus for XCHC over the 
entire frequency range with a lesser positive slope than that of xanthan but a greater 
slope when compared to curdlan. Furthermore, the elasticity of XCHC was 
significantly closer to curdlan than xanthan.  The elasticity did decrease after the first 
FTC remaining constant until a slight increase was observed after the fifth FTC. 
Table 4.4 shows a 33-56% difference in the slope of the storage modulus, b, of 
XCHC from curdlan, whereas xanthan exhibited 89-100% difference from curdlan. 
However, no syneresis was detected. While the rheological behavior of XCHC was 
found relatively close to curdlan than xanthan, the surface morphology exhibited 
layers. Unlike xanthan, these wavy layers were not closely packed, the layers bend or 
curve towards each other mimicking a three-dimensional structure, however there 
was no evidence of a connection between the layers. The structure could explain the 
positive slope found in the frequency sweeps of XCHC but relatively high elasticity 
when compared to the xanthan solution. Hence, the tightly packed layers may 
contribute to the frequency dependency of XCHC. 
4.4 Conclusion 
 A comprehensive view of curdlan, xanthan and XCHCs structural behavior in 
water was achieved by combining NMR relaxometry, MRI, rheology, and SEM. The 
fully cured gel of curdlan, interpreted from frequency sweeps, SEM, and MRI, 
yielded increasing amounts of syneresis under freeze-thaw abuse due to the weak 
interactions between the biopolymer and water as elucidated from NMR 
relaxometry. The addition of xanthan to curdlan  (XCHC) reduced syneresis to an 
undetectable level and exhibited elasticity closer to that of curdlan than xanthan ? 
 
 54 
 
while lacking a three dimensional network.  Moreover, XCHC remained unaffected 
after the first FTC, making XCHC an ideal candidate to simulate a stable gel without 
syneresis under freeze-thaw abuse.   
  
 
 55 
 
4.5 Acknowledgement 
We acknowledge the support of the Maryland NanoCenter and its NispLab. The 
NispLab is supported in part by the NSF as a MRSEC Shared Experimental Facility.   
 
 56 
 
Chapter 5: Effect of pH and salt concentration on the xanthan 
curdlan hydrogel complex utilizing rheological techniques 
 
5.1 Introduction 
 Curdlan is well known to be insoluble in water, but can produce an aqueous 
gel under the proper conditions. However, syneresis occurs and increases under 
freeze-thaw abuse. Syneresis remains a challenge in processed foods, especially 
frozen products, since product quality could be drastically reduced by uncontrolled 
moisture loss or migration, which consequently creates ice crystals that could lead to 
microbial growth and texture deterioration in food (Goff et al., 1993; Lund 2000). In 
our previous study (Williams et al., 2009), we successfully demonstrated that a 
hydrogel complex formed by xanthan, a negatively-charged hydrocolloid, and curdlan 
gum, a neutral, linear microbial polysaccharide, significantly reduced syneresis over 
five freeze-thaw cycles (FTCs) to an undetectable level, while retaining rheological 
and textural properties. Though the usage of xanthan and curdlan alone in food 
matrices has been studied, the fundamental effects of basic food components such as 
sodium chloride and pH has yet to be explored in regards to this combination. 
 The uncommon microbial gum produced by Agrobacterium biovar 1, curdlan, 
is one of three microbial polysaccharides approved by the FDA (21CFR 172.809). 
The linear (1?3) ?-glucan is insoluble in water, alcohols and most organic solvents, 
however, solubility has been shown in alkaline solutions (Pederson 1979; McIntosh 
2005). Curdlan has been used as a texture modifier for Chinese and Japanese noodles 
and processed meats for juicier texture (Nakao et al. 1991; Nakao, 1997; Hsu and 
Chung 2000; Nishinari 2000). Despite its insolubility in water, curdlan can produce 
 
 57 
 
both a low-set thermo-reversible and high-set thermo-irreversible gel in aqueous 
solutions (Harada 1994; Hirashima et al. 1997; Nakao 1997; McIntosh et al. 2005; 
Gagnon et al. 2007). The high-set thermo-irreversible aqueous gel is created when 
curdlan is heated above 80 ?C, where its gel strength is consistent after one freeze-
 thaw cycle, unlike agar, carrageenan, and konjac (Nakao 1997). Over the pH range of 
2-10, curdlan can gel in the presence of sugars, starches, salt and the incorporation of 
fats and oils (Nakao 1997; McIntosh et al., 2005). 
 Xanthan gum is another microbial polysaccharide approved by the FDA 
(21CFR 172.695) produced by Xanthomonas campestris. The structure consists of 
two repeating (1?4)-?-D-glucose units making up the backbone with a trisaccharide 
side chain consisting of ?-D-mannose, ?-D-glucuronic acid, and ?-D-mannose. It is 
known that xanthan gum is unaffected by salt concentrations above approximately  
10-2 M in the dilute and semi-dilute regime (Rochefort and Middleman 1987; Dolz et. 
al, 2002) and is stable over a pH range of 1 to 9 (Rao et al., 1998; Hoefler 2004).  
 In the present study, the combination of frequency sweeps, temperature 
ramps, gel strength, and adhesiveness will yield an optimal range of texture stability 
for the xanthan curdlan hydrogel complex (XCHC) over a range of pH values and 
sodium chloride concentrations. By investigating the effects of salt and pH under 
temperature abuse utilizing rheological properties, a foundation for further 
applications involving the combination can be established.  
 
 58 
 
5.2 Methods and Materials 
5.2.1 Sample Preparation 
 Odorless, fine, free-flowing white powder curdlan containing a minimum of 
90% ?-D-glucan and with a maximum of 10% water was used (Takeda Vitamin & 
Food USA, Orangeburg, NY). Xanthan (Ticaxan?) was kindly supplied by TIC Gums 
(Belcamp, MD). The biopolymer powders were weighed and dry blended at ambient 
temperature before introduced to deionized (DI) water under constant stirring to 
achieve an aqueous solution containing 1.0% (w/v) curdlan and 1.0% (w/v) xanthan 
gum.  
 The pH was adjusted after the gums were well hydrated. Values of pH were 
evaluated from 2 to 8 by 1. Solutions of 1.0N HCl and 1.0 N NaOH were added once 
the gum solution was well hydrated using a RW 20 Digital overhead stirrer (IKA, 
Germany) to achieve the desired pH values below six and six and above, respectively. 
Sodium chloride was blended and introduced to water along with the biopolymer 
powders until well hydrated using the overhead stirrer, yielding 5, 20, 75, 100, and 
200 mM of NaCl. Either the pH or NaCl solutions were then immersed in a water 
bath to reach a temperature of 90 ?C under reflux. The biopolymer solutions were 
removed from the heat and allowed to cool to room temperature and subsequently 
poured into 50 mL centrifuge tubes. The solutions were refrigerated for 24 h at 4 ?C 
before subjected to freeze-thaw cycles (FTCs). Five FTCs were employed where each 
solution was stored at -18?C for 18 h, followed by thawing at 25 ?C for 6 h. The 
thawed samples were collected for rheological experimentation. Each sample and 
their two replications were averaged and presented. 
 
 59 
 
5.2.2 Rheological Measurements 
 Frequency sweeps and temperature ramps were performed with controlled 
strain AR2000 Rheometer (TA Instruments, Delaware) prior to and after each FTC. A 
60-mm aluminum truncated cone (1?) geometry with a gap of 30 ?m was 
implemented. The frequency sweep was performed from 100 to 0.1 rad/s. 
Temperature ramping was performed at an angular frequency of 6.283 rad/s, first with 
a heat ramp step from 18 ?C to 80 ?C, immediately followed by a cooling step 
returning the sample to 18 ?C at a rate of 2 ?C /min. A  TA-XT2i texture analyzer 
(Texture Technologies, Inc., Scarsdale, NY) was used to measure the gel strength and 
adhesiveness with a 0.5? dia. acrylic probe attachment.  
5.2.3 Statistical analysis 
 For statistical analysis, SAS (v. 9.2) was used. Tukey?s HSD test at 5 % 
significance level was employed as the comparison test (see Appendix C).  
5.3 Results and Discussion 
5.3.1 Effect of pH 
 A fully cured three-dimensional network is indicated when the storage 
modulus, G?, predominates the loss modulus, G?, and both are independent of the 
angular frequency (Steffe 1996). Curdlan, 2% aqueous solution, closely resembles 
this behavior. Xanthan alone exhibits ?gel-like? behavior where G? predominates G? 
over the entire angular frequency range, however, the moduli are directly proportional 
to the angular frequency and exhibits low modulus values. The dynamic modulus of 
 
 60 
 
the untreated xanthan curdlan hydrogel complex (XCHC), or control, falls in between 
curdlan and xanthan solutions, with values closer to that of curdlan.  
 It was evident that adjusting the pH, above or below its original pH of 
approximately 5.83, causes a reduction in dynamic modulus values during frequency 
sweeps (Figure 5.1). However, the moduli of XCHC with pH of 2 began to increase 
after the second FTC and continued to increase through the fifth FTC above control 
values. Furthermore, its modulus was significantly larger than solutions with pH of 3 
through 8 over all FTCs. 
 
Figure 5.1. Frequency sweeps of 2.0% xanthan curdlan hydrogel complex (XCHC) with pH values of 
2, 3, 4, 5, 6, 7, and 8. Storage, G?(  101 - 102 102 - 103 103 - 104), and loss modulus, G? (  101 - 
102  102 - 103  103 - 104),  prior to (a), and after the first (b), second (c), third (d), fourth (e), and fifth 
(f) freeze-thaw cycles. 
G"
 Control
 2
 3
 4
 5
 6
 7
 8
 G
 ', 
G
 "(
 Pa
 )
 5 FTCs
 102
 10-1
 101
 100
 G'G"
 Control
 2
 3
 4
 5
 6
 7
 8
 G
 ', 
G
 "(
 Pa
 )
 4 FTCs
 102
 10-1
 101
 100
 G'
 G"
 Control
 2
 3
 4
 5
 6
 7
 8
 G
 ', 
G
 "(
 Pa
 )
 3 FTCs
 102
 10-1
 101
 100
 G'G"
 Control
 2
 3
 4
 5
 6
 7
 8
 G
 ', 
G
 "(
 Pa
 )
 2 FTCs
 102
 10-1
 101
 100
 G'
 G"
 Control
 2
 3
 4
 5
 6
 7
 8
 G
 ', 
G
 "(
 Pa
 )
 1 FTC
 102
 10-1
 101
 100
 G'G"
 Control
 2
 3
 4
 5
 6
 7
 8
 G
 ', 
G
 "(
 Pa
 )
 Prior to FTCs
 102
 10-1
 101
 100
 G'
 a
 c d
 f e
 b
 
 61 
 
XCHC with a pH of 3 was also larger than samples with pH values of 4 to 8 prior to 
FTCs, but consistent over the five FTCs. Subsequent FTCs showed modulus values 
for pH equal to 3 were higher than other treated samples near or above angular 
frequencies of 1 rad/s after the first FTC and above 10 rad/s for the following FTCs. 
Though there were fluctuations at high frequencies (above 10 rad/s) over the five 
FTCs for samples at pH = 4, the samples were not different from samples with pH 
values from 5 through 8. FTCs did not affect XCHC samples with pH values of 5 
through 8, nor were they significantly different from each other (Figure 5.1).  
The storage modulus of these samples held in the range of approximately 50-
 180 Pa over the frequency range. Whereas, the storage modulus values of the control 
was an order of magnitude larger ranging from 600 to 2100 Pa. The slope of the 
dynamic modulus over the frequency range indicates the possibility of gel or gel-like 
properties. The slope, b, of the storage modulus for the curdlan gel ranged from 0.062 
to 0.069 over five freeze-thaw cycles. Though storage modulus values were reduced 
due to pH adjustment, the difference in the slope of the storage modulus from curdlan 
was 44-84%, with slopes ranging from 0.109-0.152 in Table 5.1, and only averaged 
15% different from the control. This implied less dependency upon the angular 
frequency than xanthan alone, where an 89-100% difference from curdlan was 
exhibited. Electrostatic interaction between the adjusted pH and negatively charged 
xanthan may be the cause for the reduction in modulus by an order of magnitude. 
Nonetheless, XCHC frequency sweeps were relatively stable over a pH range of 4 to 
8 during freeze-thaw abuse.   
 
 62 
 
Table 5.1. Linear regression of the storage modulus, G? = a?b, for pH treated xanthan curdlan 
hydrogel complex (XCHC) samples. The intercept, a, and slope, b, of the storage modulus are 
presented. 
 
Freeze-Thaw Cycles 
0 1 2 3 4 5 
Modulus slope, b 
pH 
2 0.124?0.004 0.123?0.005 0.119?0.001 0.115?0.002 0.113?0.003 0.110?0.001 
3 0.116?0.005 0.11  ?0.003 0.124?0.003 0.121?0.002 0.121?0.004 0.121?0.003 
4 0.136?0.013 0.147?0.005 0.152?0.012 0.143?0.008 0.144?0.008 0.142?0.003 
5 0.141?0.007 0.134?0.006 0.138?0.009 0.128?0.007 0.136?0.010 0.132?0.005 
6 0.116?0.003 0.113?0.004 0.117?0.004 0.112 0.001 0.113?0.003 0.115?0.005 
7 0.114?0.001 0.109?0.003 0.111?0.004 0.116?0.001 0.113?0.001 0.112?0.002 
8 0.109?0.003 0.114?0.003 0.116?0.008 0.122?0.004 0.112?0.006 0.111?0.005 
Modulus intercept, a (Pa sb) 
pH 
2 506?67.3 525?87.1 584?144.5 650?23.2 1375?218.8 2211?750.9 
3 213?59.9 168?30 141?26.5 140?8.5 145?39.6 142?17.8 
4 130?62 74?3 74?19.7 99?20.1 78?21.9 93?14.4 
5 95?30 78?12 75?10.4 92?11.4 77?18.7 74?8.5 
6 98?21 86?4.9 79?7.6 92?3.3 88?6.1 85?6.2 
7 112?17.4 103?8.6 110?12.5 96?3.8 98 ?10.6 95?14.4 
8 116?8.8 96?11.8 83?18.2 73?7.7 95?19.4 84?10.6 
 
Similar to the frequency sweep results, the adhesiveness and gel strength of 
XCHC samples with pH values from 4 to 8 were the same and were not affected by 
freeze-thaw abuse (Table 5.2). Adhesiveness for samples with pH equal to 2 was 
highest and decreased until after the third FTC remaining the same until after the fifth 
freeze-thaw cycles. A 2.9% water loss, syneresis, was yielded after the second freeze-
 thaw cycle and noticeably increased to 24.6% after the third FTC (Table B.2). 
 
 
 
 
 
 63 
 
Table 5.2. Adhesiveness and gel strength of the xanthan curdlan complex solutions with the pH 
adjusted from 2 to 8 over five freeze thaw cycles (FTCs). 
 
This amount of syneresis coincides with reduction in adhesiveness and increased 
dynamic modulus. The gel strength for pH of 2 was also the highest of the treated 
samples, but decreasing slightly after the first FTC remaining stable during the 
following FTCs. Contradicting the behavior of pH = 2, a pH of 3 exhibited greater 
values of adhesiveness and gel strength and was not affected by freeze-thaw abuse. 
The increased modulus and gel strength  of XCHC at pH 2 and 3 could be due the fact 
that xanthan is negatively charged and electrostatic interactions caused by the 
introduction of positive charges created a stronger gel. 
In order to further investigate the stability of the pH adjusted samples, the 
ability to withstand temperature ramps at an angular frequency of 6.283 rad/s was 
explored. Generally, the storage modulus, presented in Figure 5.2, increased during 
 Freeze Thaw Cycles 
 0 1 2 3 4 5 
Adhesiveness (|N s|) 
pH       
2 0.940?0.043 0.579?0.059 0.252?0.058 0.131?0.080 0.081?0.025 0.074?0.012 
3 0.960?0.328 1.003?0.311 0.901?0.456 0.950?0.280 0.956?0.289 1.026?0.396 
4 0.438?0.101 0.438?0.210 0.479?0.209 0.426?0.167 0.414?0.081 0.454?0.178 
5 0.371?0.012 0.377?0.005 0.418?0.051 0.405?0.024 0.439?0.042 0.473?0.030 
6 0.329?0.053 0.341?0.019 0.356?0.019 0.378?0.022 0.408?0.029 0.440?0.087 
7 0.332?0.036 0.358?0.036 0.395?0.040 0.498?0.123 0.445?0.012 0.535?0.156 
8 0.348?0.020 0.361?0.032 0.391?0.038 0.407?0.024 0.444?0.059 0.467?0.047 
Gel Strength (N) 
pH       
2 0.106?0.004 0.094?0.003 0.085?0.006 0.099?0.015 0.094?0.013 0.113?0.022 
3 0.108?0.009 0.109?0.011 0.111?0.018 0.112?0.011 0.110?0.011 0.112?0.012 
4 0.086?0.005 0.085?0.008 0.085?0.006 0.084?0.006 0.085?0.006 0.088?0.010 
5 0.084?0.000 0.085?0.000 0.086?0.001 0.086?0.000 0.086?0.001 0.087?0.001 
6 0.079?0.001 0.083?0.000 0.084?0.001 0.085?0.000 0.086?0.001 0.087?0.003 
7 0.079?0.001 0.082?0.001 0.083?0.000 0.087?0.002 0.086?0.000 0.091?0.005 
8 0.083?0.001 0.086?0.000 0.088?0.001 0.089?0.001 0.091?0.001 0.092?0.002 
 
 64 
 
both the heating and cooling cycles, displaying hysteresis. The storage modulus was 
lower at pH = 5, 6, 7, and 8, where samples at pH = 5 yielded the most hysteresis. 
 
 
Figure 5.2.  Temperature ramps of the storage modulus, G?, of 2.0% xanthan curdlan hydrogel 
complex (XCHC) with pH values of 2, 3, 4, 5, 6, 7, and 8. Heating ramp, G?  ( 101 - 102  102 - 103), 
and cooling ramp, G?(  101 - 102  102 - 103),  prior to (a), and after the first (b), second (c), third (d), 
fourth (e), and fifth (f) freeze-thaw cycles. 
 
Conversely, there was minimal hysteresis with XCHC at pH = 2. Reduction in the 
sample pH (Table B.1) following refrigeration could be an indication of electrostatic 
interactions creating sensitivity to temperature allowing for fluctuations in elasticity 
during temperature ramps. However, this does not explain the reduction in modulus 
for the control and hysteresis with the control. Hysteresis under temperature ramps 
Control
 2
 3
 4
 5
 6
 7
 8S
 to
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 Pa
 )
 4 - FTCs
 20
 80
 70
 60
 50
 40
 30
 G' HeatingG' Cooling
 Control
 2
 3
 4
 5
 6
 7
 8
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 Pa
 )
 5 - FTCs
 20
 80
 70
 60
 50
 40
 30
 G' HeatingG' Cooling
 Control
 2
 3
 4
 5
 6
 7
 8St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 Pa
 ) 2 - FTCs
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 Control
 2
 3
 4
 5
 6
 7
 8S
 to
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 Pa
 ) 3 - FTCs
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 Control
 2
 3
 4
 5
 6
 7
 8
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 Pa
 )
 Prior to FTCs
 20
 80
 70
 60
 50
 40
 30
 G' HeatingG' Cooling
 Control
 2
 3
 4
 5
 6
 7
 8S
 to
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 Pa
 ) 1 - FTC
 G' Heating20
 80
 70
 60
 50
 40
 30
 G' Cooling
 a
 c
 b
 d
 fe
 
 65 
 
may be impart due to slow temperature ramping and the geometry gap of 30 ?m 
(Figure A.1) allowing the sample enough surface area permitting curdlan to form a 
network that was hindered by xanthan in solution.  
5.3.2 Effect of Salt Concentration 
Similar to the effects of pH, but to a lesser extent, sodium chloride reduced the 
dynamic moduli of the hydrogel complex prior to FTCs (Figure 5.3). Treatments of 5, 
20, 75,100, and 200 mM NaCl were different from each other and fluctuated 
throughout FTCs with no discerning patterns. However, this disparity regarding 
significant differences for pH and NaCl treated samples could be explained by the 
coefficient of variance (COV). The COV was 17 for NaCl treated samples, whereas 
COV was 57 for pH treated samples exhibiting more variability between samples. 
Furthermore, the slope of G? in Table 5.3 averaged 9 % difference from the control, 
yielding similar gel behavior at lower modulus values.  
 
 66 
 
 
Figure 5.3. Frequency sweeps of 2.0% xanthan curdlan hydrogel complex (XCHC) treated with 5, 15, 
20, 75, 100, and 200 mM NaCl. Storage, G?(  101 - 102 102 - 103 103 - 104), and loss modulus, 
G? (  101 - 102  102 - 103  103 - 104),  prior to (a), and after the first (b), second (c), third (d), fourth 
(e), and fifth (f) freeze-thaw cycles. 
 
Similar to dynamic modulus, variation was displayed with adhesiveness in 
Table 5.4. Analogous to dilute and semi-dilute xanthan solutions (Rochefort and 
Middleman 1987; Dolz et. al, 2002), the low sodium chloride concentrations affected 
adhesiveness. Samples treated with 5 and 15 mM NaCl were significantly lower than 
subsequent treatments and increased over FTCs. Increased molecular mobility of the 
system could be responsible for increased adhesiveness (Adhikari et al., 2001).
 G"
 Control
 5
 15
 20
 75
 100
 200
 G
 ', 
G
 ' (
 Pa
 )
 Prior to FTCs
 102
 10-1
 101
 100
 G'
 G"
 Control
 5
 15
 20
 75
 100
 200
 G
 ', 
G
 ' (
 Pa
 )
 2 FTCs
 102
 10-1
 101
 100
 G' G"
 Control
 5
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 102
 10-1
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 G'
 G"
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 102
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 102
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 5
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 68 
 
With decreased adhesiveness, higher gel strength was expected for 5, 15, and 20 mM 
NaCl compared to higher NaCl concentrations, however, both adhesiveness and gel 
strength were lower. This behavior could be explained by the fact that xanthan and 
curdlan are not oppositely charged and are unlikely to bond, thus being separate 
solutions while keeping its structure. 
 
 
Table 5.4. Adhesiveness and gel strength of the xanthan curdlan complex solutions with 5 to 200 mM 
NaCl over five freeze thaw cycles (FTCs). 
 
The changes in stability of sodium chloride treated samples during 
temperature ramping were similar to its behavior during frequency sweeps. 
Temperature ramps of the storage modulus in Figure 5.4 showed some hysteresis 
upon cooling and minor fluctuations throughout FTCs for all treated samples, 
exhibiting better heat stability than pH treated samples. 
 Freeze Thaw Cycles 
 0 1 2 3 4 5 
Adhesiveness (|N s|) 
5  0.563?0.006 0.681?0.058  0.755?0.022  0.763?0.023  0.921?0.123  0.916?0.029  
15  0.436?0.053  0.434?0.049  0.755?0.048  0.763?0.028  0.921?0.306  0.916?0.053  
20  1.285?0.026  1.263?0.183  1.401?0.093  1.508?0.101  1.571?0.095  1.556?0.046  
75  1.770?0.155  1.533?0.106  1.750?0.162  1.687?0.049  1.561?0.017  1.677?0.025  
100  1.588?0.046  1.520?0.070  1.449?0.034  1.554?0.171  1.507?0.224  1.585?0.060  
200  1.619?0.062  1.275?0.037  1.314?0.095  1.265?0.122  1.513?0.104  1.418?0.050  
Gel Strength (N) 
5  0.095?0.001  0.098?0.001  0.102?0.002  0.104?0.001  0.107?0.003  0.107?0.001  
15  0.104?0.004  0.107?0.005  0.110?0.007  0.110?0.004  0.112?0.002  0.108?0.002  
20  0.116?0.001  0.118?0.006  0.122?0.003  0.123?0.001  0.126?0.002  0.127?0.002  
75  0.136?0.004  0.132?0.004  0.133?0.004  0.130?0.003  0.128?0.002  0.132?0.002  
100  0.132?0.001  0.129?0.003  0.127?0.002  0.130?0.007  0.124?0.002  0.130?0.001  
200  0.132?0.001  0.124?0.001  0.123?0.002  0.121?0.003  0.127?0.004  0.124?0.006  
 
 69 
 
 
Figure 5.4.  Temperature ramps of the storage modulus, G?, of 2.0% xanthan curdlan hydrogel 
complex (XCHC) treated with 5, 15, 20, 75,100, and 200 mM NaCl. Heating ramp, G? ( 101 - 102 
102 - 103),  and cooling ramp, G?(  101 - 102  102 - 103), prior to (a), and after the first (b), second 
(c), third (d), fourth (e), and fifth (f) freeze-thaw cycles. 
5.4 Conclusion 
 Electrostatic interactions between xanthan and respective treatments may have 
resulted in reduced modulus values during frequency sweeps. Nonetheless, the effects 
of pH values of 4, 5, 6, 7, and 8 or 75,100, or 200 mM NaCl were similar to each 
other, where freeze-thaw cycles had little effect on the dynamic modulus, gel 
strength, and adhesiveness of the samples. Sodium chloride samples were more 
resistant to temperature ramping over the five FTCs. Despite the reduction in 
dynamic modulus, the aforementioned treatments show XCHC as a viable candidate 
in food matrices with like food components due its stability and strong gel behavior. 
Control
 5
 15
 20
 75
 100
 200
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 P
 a) 4 - FTCs
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 Control
 5
 15
 20
 75
 100
 200
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 P
 a) 5 - FTCs
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 Control
 5
 15
 20
 75
 100
 200
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 P
 a) 2 - FTCs
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 Control
 5
 15
 20
 75
 100
 200
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 P
 a) 3 - FTCs
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 Control
 5
 15
 20
 75
 100
 200
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 P
 a) Prior to FTCs
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 Control
 5
 15
 20
 75
 100
 200
 St
 o
 ra
 g
 e 
M
 o
 d
 u
 lu
 s,
  G
 ' (
 P
 a) 1 - FTC
 G' Heating
 20
 80
 70
 60
 50
 40
 30
 G' Cooling
 a
 c
 b
 d
 f e
 
 70 
 
Chapter 6:  Efficacy of the xanthan curdlan hydrogel 
complex in pumpkin pie 
 
 
6.1 Introduction 
 Previous studies have shown how the xanthan curdlan complex is a potential 
candidate for food matrices through its ability to maintain texture qualities throughout 
freeze-thaw abuse, while not allowing water to release in an aqueous solution. 
Furthermore, it has been shown that the complex continues to be resistant to water 
loss and stable after freeze-thaw abuse when adjusted to pH values from 4 to 8 and 
sodium chloride concentrations of 75 to 200 mM. However, its efficacy in a food 
product has yet to be investigated. Thus, pumpkin pie filling was found to be a 
possible food product to improve, where not only moisture migration from the filling 
to the crust can occur, but also there is the common problem of the filling cracking 
once backed which could potentially be a reservoir for bacteria (Kane et al., 1971).  
One of three microbial polysaccharides approved by the FDA (21CFR 
172.809), curdlan is produced fermentatively by Agrobacterium biovar 1. The linear 
(1?3) ?-glucan is insoluble in water, alcohols and most organic solvents, however, 
show solubility in alkaline solutions (Pederson 1979; McIntosh 2005). Curdlan has 
been used as a texture modifier for Chinese and Japanese noodles and processed 
meats for juicier texture (Nakao et al. 1991; Nakao, 1997; Hsu and Chung 2000; 
Nishinari 2000). Despite its insolubility in water, curdlan can produce both a low-set 
thermo-reversible and high-set thermo-irreversible gel in aqueous solutions (Harada 
1994; Hirashima et al. 1997; Nakao 1997; McIntosh et al. 2005; Gagnon et al. 2007). 
The high-set thermo-irreversible aqueous gel is created when curdlan heated above 80 
 
 71 
 
?C, where its gel strength is consistent after one freeze-thaw cycle, unlike agar, 
carrageenan, and konjac (Nakao 1997). Over the pH range of 2-10, curdlan can gel in 
the presence of sugars, starches, salt and the incorporation of fats and oils (Nakao 
1997; McIntosh et al., 2005). 
 Xanthan gum is another microbial polysaccharide approved by the FDA 
(21CFR 172.695) produced by Xanthomonas campestris. The structure consists of 
two repeating (1?4)-?-D-glucose units making up the backbone with a trisaccharide 
side chain consisting of ?-D-mannose, ?-D-glucuronic acid, and ?-D-mannose. It is 
known that at xanthan gum is unaffected by salt concentrations above approximately 
10-2 M in the dilute and semi-dilute regime (Rochefort and Middleman 1987; Dolz et. 
al, 2002) and is stable over a pH range of 1 to 9 (Rao et al., 1998; Hoefler 2004).  
By combining curdlan with xanthan, high elasticity is retained, resilience is 
increased and syneresis is prevented. Moreover, xanthan is known to be an excellent 
stabilizer of dairy products (Sworn 2000). Therefore, introducing the complex to 
pumpkin pie could better maintain its quality during freeze-thaw abuse.   
6.2 Methods and Materials 
 Odorless, fine, free-flowing white powder curdlan containing a minimum of 
90% ?-D-glucan and with a maximum of 10% water was used (Takeda Vitamin & 
Food USA, Orangeburg, N.Y.). Xanthan (Ticaxan?) was kindly supplied by TIC 
Gums (Belcamp, Md.). The biopolymer powders were weighed and dry blended 
before introduced to pumpkin filling.  
 The pumpkin filling procedure was adapted from Beranbaum (1998) 
substituting milk for a combination of water, milk solids non-fat, and heavy cream 
 
 72 
 
resulting in the composition of whole milk. Well hydrated, 2.0% aqueous solution of 
equal amounts of xanthan and curdlan (XCHC) was added in substation for two-thirds 
portion of the water added in the standard.  Pumpkin puree, brown sugar, spices, and 
salt (Table B.3) where mixed together. Over medium heat, the mixture was brought to 
a sputtering simmer (~90 ?C), stirring constantly. The heat was reduced to low, 
stirring constantly, for 9 minutes until thick and shiny. The puree mixture was 
scraped out of the pot and placed into a food processor and processed for 1 minute. 
The non-fat milk solids (4.84 g) were dissolved in 14.3 g of water then added to the 
pumpkin puree mixture along with 28.8 g water (control) or XCHC and cream, 
processing until incorporated. The eggs (50 g) were whisked to achieve the 
appropriate amount and added slowly, finally adding the vanilla extract. 
 The mixture, 90 g of pumpkin filling, was then poured into ~45 g pie shells 
that were pre-baked at 350 ?F for 24 minutes with a 40 g weight in 4-3/4" by 3/4" 
deep circular aluminum baking pans. Pies were baked for 35 min at 350 ?F and 
allowed to cool to room temperature, before refrigerating at 4 ?C for 24 hrs and 
subjecting them to five FTCs.  
 A TA-XT2i texture analyzer (Texture Technologies, Inc., Scarsdale, NY) was 
used to measure the gel strength and adhesiveness with a 0.5? dia. acrylic probe 
attachment. An IC-500 AW-LAB (Novasina, Switzerland) water activity meter was 
used to measure the water activity of both the crust and pumpkin pie filling prior to 
FTCs and then after each subsequent FTC. The ColorFlex colorimeter with CIELAB 
color space (HunterLab, Reston, Va.) was implemented to compare the color of the 
 
 73 
 
original pie filling recipe to the pie filling with the xanthan/curdlan complex before 
and after baking the filling. 
6.3 Results and Discussion 
 6.3.1 Adhesiveness and Gel Strength 
 Besides cracking of the filling, adhesiveness can often be a problem in 
manufacturing operations along with lack of adherence of pie filling to the crust 
(Kane et al., 1971; Adhikari et al., 2001). Therefore, the adhesiveness of the baked 
filling was investigated. Adhesiveness results in Table 6.1 showed that the xanthan 
curdlan hydrogel complex (XCHC) did not contribute additional adhesiveness to the 
filling, thus, no different than that of the control. Nevertheless, it is clear that there 
was less deviation between samples for XCHC treated filling. Similarly, the gel 
strength for the XCHC treated pie filling was not significantly different from the 
control. The XCHC may not contribute to either adhesiveness or gel strength due the 
relatively small amount added compared to other texture contributing components 
such as the pumpkin puree and coagulated milk proteins. 
 
Table 6.1.  Adhesiveness and gel strength* of pumpkin pie filling and treated with the xanthan curdlan 
hydrogel complex (XCHC) over five freeze-thaw cycles (FTCs). 
 
Adhesiveness (|N s|) Gel Strength (N) 
FTC Control XCHC Control XCHC 
0 25.982 ? 8.690 23.730 ? 1.956 2.325 ? 0.296 2.009 ? 0.21 
1 26.292 ? 7.641 24.047 ? 2.551 2.774 ? 0.544 2.200 ? 0.116 
2 28.328 ? 10.483 28.703 ? 7.268 2.554 ? 1.11 2.120 ? 0.677 
3 27.999 ? 4.053 27.689 ? 3.557 2.449 ? 0.37 2.130 ? 0.579 
4 18.597 ? 6.795 26.214 ? 3.016 2.143 ? 0.435 2.325 ? 0.384 
5 23.335 ? 4.058 22.561 ? 0.704 2.621 ? 0.427 1.958 ? 0.173 
*Values represent the means ? standard deviations (n = 3). 
 
 
 74 
 
 6.3.2 Water Activity and Color 
 Though subtle, the water activity, aw, of the pie filling was greater than the 
crust. The difference in water activity between the filling and crust for both the 
control and XCHC over FTCs, signifying moisture migration to the crust. 
Interestingly, the water activity of the pie filling remained the same over FTCs for 
both the control, Figure 6.1, and XCHC treated samples, Figure 6.2. This 
phenomenon could be attributed to the continual release of water throughout the 
filling resulting in separation, where an amount of water is transferred to the crust and 
an amount remains with the filling allowing its water activity to remain constant. 
XCHC treated samples proved better at delaying water migration to the crust until 
after the fourth FTC, whereas the water activity of the control crust increased after the 
third FTC. Furthermore, once the filling was removed, there was a noticeable 
difference in the surface attributes of the crusts for the control and XCHC treated pie 
(Figure B.1). Crust separation was exhibited in the control after three FTCs where it 
was not seen with the XCHC treated pie and could be attributed to an affinity of the 
crust to the water rather than a polysaccharide solution in the filling. 
 
 
 
 
 75 
 
 
 
Figure 6.1. Water activity of the crust (square7) and filling (square7) of control samples over five freeze-thaw 
cycles (n=3). 
 
 
 
Figure 6.2. Water activity of the crust (square7) and filling (square7) of the xanthan curdlan hydrogel complex 
(XCHC) treated samples over five freeze-thaw cycles (n=3). 
 
 The color of both the surface of the pie and the filling was measured. From 
Table 6.2, color values a* and b* were greater for the XCHC treated samples than the 
control, yielding more red and yellow hues, respectively, translating into a brighter 
pie surface seen on the right in Figure 6.3. A sensory evaluation regarding appearance 
0.900
 0.910
 0.920
 0.930
 0.940
 0.950
 0.960
 0.970
 0.980
 0.990
 1.000
 0 1 2 3 4 5
 W
 at
 er
  A
 ct
 iv
 it
 y,
  a
 w
 Freeze Thaw Cycles
 Crust Control Filling
 0.900
 0.910
 0.920
 0.930
 0.940
 0.950
 0.960
 0.970
 0.980
 0.990
 1.000
 0 1 2 3 4 5
 W
 at
 er
  A
 ct
 iv
 it
 y,
  a
 w
 Freeze Thaw Cycles
 Crust XCHC Filling
 
 76 
 
could yield positive results for the treated pie. Generally, FTCs did not affect the 
surface color of either pie. Inspecting the surfaces of the pies in Figure 6.3, cracking 
and wrinkles are noticeable on the control pie due to upward expansion of the filling 
during baking and subsequent contraction upon cooling. However, a smoother surface 
was found with the XCHC treated pie. The limited amount of cracks and wrinkles 
could be due to the addition of the complex, where its elastic properties allowed the 
surface to stretch and contract, maintaining its structure. 
 
Table 6.2. CIELAB values, L*, a*, and b*, for surface and filling of control and xanthan curdlan 
hydrogel complex (XCHC) treated pumpkin pies over five freeze-thaw cycles (FTCs). 
 
Surface Filling 
FTC L* a* b* L* a* b* 
Control 
0 40.78 ? 2.86 15.95 ? 0.67 38.41 ? 5.88 43.55 ? 0.92 14.67 ? 0.45 42.61 ? 0.24 
1 42.28 ? 2.87 16.46 ? 0.17 38.83 ? 6.01 42.85 ? 1.83 15.27 ? 0.48 42.58 ? 0.87 
2 43.70 ? 2.74 15.70 ? 0.33 40.36 ? 6.71 44.48 ? 2.05 14.49 ? 0.51 42.79 ? 1.69 
3 43.66 ? 1.64 16.06 ? 0.63 40.39 ? 4.42 43.18 ? 2.08 14.70 ? 0.56 44.50 ? 2.07 
4 43.87 ? 1.64 15.96 ? 1.14 40.79 ? 5.70 43.66 ? 1.77 14.41 ? 0.19 43.46 ? 1.03 
5 43.66 ? 2.11 16.09 ? 0.80 40.66 ? 6.76 43.24 ? 0.59 14.60 ? 0.17 43.32 ? 0.45 
XCHC 
0 42.41 ? 0.30 18.14 ? 0.99 46.46 ? 1.61 42.67 ? 1.34 14.84 ? 0.68 43.46 ? 0.55 
1 42.67 ? 0.13 18.03 ? 0.36 45.05 ? 0.92 43.83 ? 1.74 14.76 ? 0.59 43.56 ? 0.58 
2 43.01 ? 0.82 17.88 ? 0.34 43.90 ? 0.94 44.41 ? 1.94 14.22 ? 0.31 43.52 ? 0.16 
3 43.64 ? 0.88 18.08 ? 0.59 47.25 ? 0.57 42.38 ? 0.87 14.71 ? 0.30 43.01 ? 0.28 
4 43.57 ? 0.29 17.99 ? 0.23 45.79 ? 1.34 45.13 ? 0.82 13.91 ? 0.01 43.19 ? 0.17 
5 44.37 ? 0.67 17.72 ? 0.19 47.53 ? 0.40 43.45 ? 1.95 14.45 ? 0.34 42.94 ? 1.16 
 
Though the surface was brighter, the color of pie filling under the surface of 
XCHC treated sample was indistinguishable from the control (Table 6.2).  This 
disparity between the surface and filling could explain the brighter attribute of the 
XCHC samples.  The hydrogel complex could be hindering Maillard browning 
 
 
 
reactions between lactose and milk proteins and/or caramelizati
 brown sugar.
  
Figure 
6.
  
pumpkin pie were indi
 moisture migration
 Furthermore, the 
harbor ba
 further studies with an increased amount of XCHC could 
migration from the filling to the crust throughout all freeze
 warranted.  
 
 
4 Conclusion
 6.
 Gel strength, adhesiveness, and pumpkin pie filling color of the XCHC tr
 3.  
cteria.
  
Images of the control pumpkin pie surface on the left and XCHC treated pie surface on the 
 
 
 
surface was b
 XCHC was found to improve certain aspects of pumpkin pie, thus 
 by an additional
 stinguishable from the control.
  
righter, exhibiting
                   
 freeze
 77
 right.
  
 
 
 
-thaw cycle
  less cracks and wrinkles 
 However, XCHC limited 
 compared to the control
 on of sucrose from the 
further 
-thaw cycles are 
 
 78 
 
Chapter 7: Conclusions and Recommendations 
 
Curdlan has been shown to yield a gel under specific conditions and retain its 
gel strength after a freeze-thaw cycle, however, syneresis occurs?an undesirable trait 
for frozen foods. By combining curdlan with xanthan, syneresis was eliminated while 
maintaining good texture properties and excellent stability throughout five freeze-
 thaw cycles. Coupling rheology with non-invasive techniques such as relaxometry, 
imaging, and microscopy provided clear understanding of the combinations? behavior 
that leads to its reliance under freeze-thaw abuse. Through NMR relaxometry it was 
confirmed that water was interacting relatively strongly with the polysaccharides.  
Wavy layers seen in microscopy explained the ability of the hydrogel complex to 
achieve higher elasticity than xanthan alone.   
Furthermore, the combination was found to be stable when pH values were 
altered from 4 to 8 and sodium chloride concentrations from 75 to 200 mM, 
demonstrating the potential to impart its resilience on a range of frozen food products.  
Due its elasticity and consistent behavior, introduction of the xanthan curdlan 
hydrogel complex to pumpkin pie has resolved the issue of surface cracking and 
wrinkling that may harbor bacteria. Moreover, addition of the complex produced a 
brighter pie surface that could be appealing in sensory evaluations. Finally, products 
such as ice cream, dough, sauces, and other types of pies could benefit from the 
texture enhancing and stabilizing ability of the xanthan curdlan hydrogel complex. 
 
 
 
 79 
 
To narrow an optimum range of usage for this complex, it is recommended 
that further studies on the effects of major food components such as whey and casein 
protein, divalent ions, higher sodium chloride concentrations and pH values should be 
investigated.  
  
 
 
 
A
  
A.1 
 
 
manner which materials respond to stress and strain
 properties are an integral part in understanding the effects of food po
 a food system.
 related to the 
ensure consistency of some stage of food production involving gum
 importa
 oscillatory rheology,
 geometry mus
 a parallel plate 
plate ap
 placed on the plate wh
 fixed gap specific to that
  
 
ppendix A
 Rheo
 Rheology is the science of the deformation and flow of matter referring to the 
meter Operation and Understanding
 ntly, rheology can indicate 
paratus
 Solvent trap
 :
  The ability to thicken or modify the texture of a fo
 viscosity of the food system and thus rheo
 t be d
  was used
 Figure 
 Supplemental Information
 can be utilized
  g
 etermined. Curdlan is known to form a 
 
el behavior can be determined. 
 
ere a particular geometry is positioned
  geometry.
 A.1
 for the experiments in this work (Figure 
. Cross 
Solvent in 
solvent well
 . However, to reduce slippage a 
sectional view of c
  
structural behavior. Through small angle 
 
80
  
 
 
one and plate
 Truncated cone geometry
  
 
First
 (Seffe
 logy can serve as 
high
  
 rheological
 Sample
 , the proper rheological 
atop the sample cr
  199
 -set 
truncated 
A.1
 6
 lysaccharides in 
od system can be 
gel and therefore 
 apparatus.
  
). 
). The sample 
 
Rheolo
  
s. 
cone and 
 
Geometry 
gical 
too
 M
 eating 
Gap
 l in 
ore 
is 
 
 
 81 
 
 
The geometry, here a truncated cone, oscillates about an angle specified for that 
geometry and applies a strain, in turn, yielding a stress, Figure A.2. The difference in 
the stress and strain curves at their amplitudes, ?o and ?o, respectively, yields the 
phase lag (?).   
 
 
Figure A.2. Strain input (?) and stress output (?) as a function of time, where the difference in the 
stress and strain curves is the phase lag, ?. Where the strain amplitude is ?o, and the stress amplitude is 
?o. 
 
These three parameters are used to acquire the dynamic moduli, storage (G?) and loss 
(G?) modulus, and assists in determining the structural behavior of the sample in the 
following manner:  
 G729 ? Gd4c Gd6cG7eaGbe2G7dbGbe2 Gd70 G85G91G95G123aG7dc G123b [1] 
 G729G336 Gd4c Gd6cG7eaGbe2G7dbGbe2 Gd70 G95G8bG90G123aG7dc G123b [2] 
 
 The storage modulus, G?, represents the elastic or solid like behavior and the 
loss modulus, G?, represents the viscous or more liquid like behavior. Viscoelastic 
materials consist of both viscous and elastic behavior. In order to yield meaningful 
information, the amount of strain applied to the sample should be determined by a 
strain sweep and must be within the linear viscoelastic range (LNR). The sweep is 
 
 82 
 
increased from 0.01% strain to 50% strain, depending on the geometry, where the 
dynamic modulus exhibits independence of strain at low strains and abruptly declines 
quickly at some % strain. The percent strain approximately 75% of the strain value 
immediately prior to the modulus decline is used.  
 Once the % strain is found, oscillatory experiments, including a frequency 
sweep and temperature ramps, can be performed. Depending upon the response of the 
moduli, texture properties can be interpreted. A frequency sweep entails decreasing 
the angular frequency and measuring the corresponding moduli. Viscous behavior is 
exhibited when G? values are greater than G? values with an increasing slope over the 
entire frequency range, Figure A.3a. Figure A.3b shows elastic behavior, exhibiting 
greater values of G? than G? with angular frequency independence indicating a fully 
cured three-dimensional network. When G? is predominating G? the entire angular 
frequency range with positive slopes, a ?gel-like? behavior is exhibited.   
 
 
 
 
 
 
 83 
 
 
 
 
Figure A.3. Typical frequency sweeps of a dilute solution made from 5% dextrin (a), 
concentrated solution made from 5% lambda carrageen (b), and a gel made from 1% agar (c) (Ross-
 Murphy, 1988). 
 
b 
c 
a 
 
 
 
 
 
modulus values (
 rheomet
 configuration)
 apparatus ensures less slippage of the geometry
 between the cup and bob
 behavior of a less viscous substance such as xanthan gum.
  
 
 
help
 simply involves increasing 
angula
 solvent trap shown in Figure 
above the geometry and enclosed to ensure the solve
 temperature ramp
  
 
Xanthan, however, exhibited weak ?gel
 As previously mentioned, a temperature
 understand the samples behavior during temperature changes. This experiment 
r frequency
 er 
Figure 
apparatus called a concentric cylinder where the bob oscillates
  producing strains as the truncated cone does
 A.4
 highest values 
. Cross sectional
 , 
ing
 measuring the storage and loss moduli. In this experiment, the 
. 
, thus providing a more accurate depiction of the textural 
 view of 
temperature
 A.
 be
 1 is vital. The solvent is placed in a well directly 
low 100Pa) 
concentric
 84
  at some given rate and at some
 Cup
  
 
 
 cylinder
  
-like? behavior with low storage 
and 
ramp could also be performed and 
could utilize
  an
 , or cup and bob, rheom
 d minimal heat distribution 
 
 
nt does not evaporate during
 Bob
 Sample
  
, Figure 
 
 a more sensitive 
A.
 eter
  
4
  apparatus.
  specific 
(Searle 
. This 
 
 
 
 
 
A.2 
 
 
the sample and measuring the force acting on the probe. In the current dissertation, 
the 0.5? dia. probe was lowered 20mm into the sample at a rate o
 yielded at 20mm, this was also the maximum, was considered the gel strength of the 
gums. The probe was then raised at the same rate until it was out of the solution. As 
the probe was being raised, the force was negative. The absolute v
 above this negative curve was considered the adhesiveness. Figure 
example of
 Figure 
Texture Analyzer
 peak is the gel strength and the blue shaded area is the adhesiveness of the gel. 
The 
A.5.
 probe was used with a lowering and raising rate of 0.5 mm/s 20 mm into the sample.
 texture 
 a 
 Typical texture analyzer output for the samples in the current work, where the maximum 
typical gel strength and adhesiveness output.
  
analyzer performs experiments by lowering and raising probe into 
Gel Strength
 85
  
 
 
 
f 0.5mm/s. The force 
alue of the area 
A 0.5? dia. plastic 
Adhesiveness
 A.5 shows an 
 
 
 
 86 
 
A.3 Ratio Justification 
 Preliminary studies of various ratios of xanthan to curdlan were performed. 
Lower modulus values were seen with increased xanthan (while holding the total gum 
percent at 2%), but lower slopes compared with when more curdlan was added (Table 
A.1). However, sedimentation of curdlan was observed upon visual inspection, where 
the initial sample preparation was used (Section 3.2.1) with the samples not under 
reflux. Under reflux, a stronger gel was produced for curdlan alone, it was thought 
that adding less than an equal amount of xanthan would eliminate syneresis similar to 
the 1:1 ratio used throughout the studies, and keep the same rheological properties as 
curdlan. However, a 1:4 ratio of xanthan to curdlan showed gel particulates in 
magnetic resonance images in Figure A.6, and a third proton pool in nuclear magnetic 
resonance relaxometry (Table A.2.), indicating syneresis that was seen during visual 
observations. Therefore, a ratio of 1:1 underwent further studies. 
 
Table A.1. Comparison of the intercept, a, and slope, b, of the storage modulus (G? = a?b) over 
various ratios of xanthan to curdlan (X:C). 
X:C a (Pa sb) b 
1:3 322 0.1465 
1:1 56 0.1185 
2:1 55 0.1146 
5:1 31 0.1539 
 
 
 
 
  
 
 87 
 
0
 500
 1000
 1500
 2000
 2500
 3000
 3500
  
Figure A.6. Representative magnetic resonance images (MRI) of 4:1 xanthan to curdlan prior to 
freezing (a) after one freeze thaw cycle (b) and after the fifth freeze thaw cycle (c). The scale on the 
right gives the relationship between signal intensity and grayscale for the images. Axes in the images 
denote the voxel numbers. 
 
Table A.2. Spin-spin relaxation timea,T2 , and relative area of 2.0% xanthan curdlan hydrogel complex 
(XCHC) with a ration of 1:4 xanthan to curdlan subjected to five freeze-thaw cycles (FTCs) from 
nuclear magnetic resonance relaxometry. 
 Xanthan:Curdlan 1:4 
 1st Proton Pool 2nd Proton Pool 3rd Proton Pool 
FTC T2 (ms) 
Relative 
Area (%) T2 (ms) 
Relative 
Area (%) T2 (ms) 
Relative 
Area (%) 
0 - - 248?14.68 21.5?3.58 849?0.00 77.35?3.20 
1 120?17.47 6.33?1.71 328?56.29 15.96?1.73 881?36.89 78.33?2.18 
5 28?2.99 1.36?0.08 272?18.56 17.52?4.87 865?31.95 80.64?4.34 
aEach value is the mean ? SD, n=3. 
 
 
 
 
 
 
 
  
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
 20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
  
 
20 40 60 80 100 120
 20
 40
 60
 80
 100
 120
 a b c 
 
 88 
 
Appendix B: Supplemental Data 
 
 
Table B.1. The measured pH of HCl or NaOH treated XCHC samples over five freeze-thaw 
cycles. 
Freeze Thaw Cycles 
pH 0? 1 2 3 4 5 
2 1.95?0.03 1.90?0.01 1.92?0.02 1.90?0.03 1.90?0.02 1.90?0.03 
3 2.88?0.05 2.78?0.03 2.84?0.06 2.75?0.09 2.79?0.03 2.80?0.16 
4 3.79?0.21 3.84?0.23 3.83?0.23 3.83?0.23 3.82?0.23 3.80?0.20 
5 4.81?0.14 4.74?0.13 4.77?0.12 4.77?0.18 4.79?0.19 4.79?0.19 
6 5.63?0.16 5.65?0.10 5.67?0.08 5.55?0.21 5.63?0.07 5.50?0.12 
7 6.28?0.18 6.23?0.23 6.28?0.14 6.32?0.18 6.30?0.26 6.16?0.38 
8 6.65?0.09 6.61?0.08 6.60?0.10 6.57?0.11 6.64?0.08 6.59?0.21 
?Values of pH do not match prior to FTCs due to the 24 hr refrigeration period allowing pH 
to drop.  Each sample was initially adjusted to the corresponding pH. 
 
Table B.2. Syneresis of samples treated to achieve pH = 2. 
 
Syneresis (%) 
FTCs 
 
0 - 
1 - 
2 2.92 
3 24.64 
4 32.50 
5 41.19 
 
Table B.2. The measured pH of NaCl treated XCHC samples over five freeze-thaw cycles. 
 
Freeze Thaw Cycles 
NaCl (mM) 0 1 2 3 4 5 
5 5.51?0.02 5.51?0.03 5.49?0.01 5.50?0.03 5.50?0.01 5.51?0.00 
15 5.26?0.02 5.27?0.03 5.28?0.03 5.31?0.03 5.24?0.03 5.23?0.03 
20 5.25?0.01 5.38?0.02 5.33?0.02 5.34?0.05 5.30?0.07 5.31?0.03 
75 5.17?0.01 5.16?0.02 5.13?0.03 5.15?0.06 5.12?0.03 5.20?0.05 
100 5.13?0.02 5.10?0.02 5.12?0.02 5.12?0.02 5.08?0.01 5.09?0.02 
200 4.89?0.08 4.91?0.04 4.98?0.06 4.99?0.02 4.98?0.01 4.95?0.01 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure B.1. 
whereas the crust of the XCHC treated pie filling (on the right) does not show separation
  Crust separation can be seen in the control (on the left) after three freeze
 Table 
 Ingredie
 Unsweetened pumpkin puree
 Light brown sugar
 Ground ginger
 Ground cinnamon
 Salt
 Water
 Milk solids non
 Heavy cream
 Fresh eggs 
Vanilla extract
  
Control
 B.3.
  
 Pumpkin pie filling ingredients
 nt
  
 
 
 
freeze thaw cycles
  
-fat
  
     
89
  
 
 
               
 
 
. 
Weight
 141.7
 54.3
 0.333 oz
 0.25 oz
 0.083 oz
 28.8
 4.84
 56
 50
 0.083 oz
  g
  g
  
 g
  g 
 g 
 g 
 
 
 
 
 
 
 
  
-thaw cycles, 
XCHC
  after five 
 
 
 
 90 
 
Table B.4. Water activity of the crust and pumpkin pie filling for untreated pumpkin pie (control) and 
xanthan curdlan hydrogel complex (XCHC) treated pie fillings. 
 
Water Activity 
 
Control 
FTC Crust Filling 
0 0.934?0.019 0.989?0.008 
1 0.945?0.007 0.973?0.007 
2 0.958?0.014 0.977?0.004 
3 0.966?0.002 0.975?0.006 
4 0.966?0.007 0.982?0.000 
5 0.969?0.008 0.986?0.008 
 
XCHC 
FTC Crust Filling 
0 0.951?0.007 0.989?0.001 
1 0.948?0.008 0.985?0.005 
2 0.953?0.016 0.985?0.004 
3 0.958?0.017 0.985?0.003 
4 0.985?0.012 0.992?0.005 
5 0.975?0.015 0.986?0.012 
 
 91 
 
Appendix C: Statistical Analysis 
 
 Tukey?s HSD (Honestly Significant Difference) was employed for statistical 
analysis in order to find significant differences over FTCs.  Tukey?s HSD requires the 
calculation of the test statistic T, utilizing the equation below: 
                                                               G736 Gd4c G74dGc08 Gda7Gbe6Gc2eGbe5 G3                                                   [1] 
Where q? is the value obtained from F distribution tables depending on the number of 
samples with ? = 0.01, s2 is the means squared error from analysis of variance, and r 
is the number of replications. The number of replications was the same for all tests at 
3.  To ascertain whether or not there is a significant difference, the difference between 
two means must be greater than T.  Here, the means of the storage modulus at each 
angular frequency was obtained, thus the following equation was used 
                                          G756G735G726Gbeb Gd46 G752G735G726Gbec Gd4c G736 Gd46 G123aG201G756G729G522Gd25 Gbeb Gd46 G752G729G522Gd25 Gbec G201G123b                              [2] 
where SD  is significant difference between the mean of the storage modulus, G729G522Gd25 , at 
freeze-thaw cycle x or  y (x ? y), where z and v (z ? v)  corresponds to pH or NaCl 
concentrations when applicable.  The shaded values in the following table indicate 
there was a significant difference between the means at its respective angular 
frequency, ?.  The following commands were used to yield necessary information to 
perform Tukey?s HSD using SAS v. 9.2. 
 
 
 
 
 
 92 
 
data Xanthan; 
 input FTC $ w @; 
 do i=1 to 3; 
  input G @; 
  output; 
 end; 
 cards; 
run; 
 
proc GLM data=Xanthan; 
 class FTC w;  
 model G= FTC w FTC*w; 
 means FTC FTC*w/tukey; 
run; 
 
where FTC is freeze-thaw cycle, w is the angular frequency, and G is the storage 
modulus. 
 
 
  
 
 93 
 
Table C.1. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous curdlan gum 
solutions over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for Curdlan 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 1990.0 2304.7 3274.7 2503.0 3846.0 2918.0 
0.03 1983.7 2290.0 3243.3 2483.0 3801.3 2892.7 
0.03 1979.0 2279.7 3214.0 2466.0 3766.7 2868.3 
0.04 1974.7 2269.7 3188.0 2450.3 3732.3 2847.0 
0.05 1973.3 2262.3 3166.3 2437.7 3710.7 2828.0 
0.06 1974.7 2258.0 3149.7 2425.7 3695.3 2814.7 
0.08 1978.3 2258.7 3139.0 2421.0 3688.3 2804.7 
0.10 1986.3 2263.0 3135.3 2422.0 3688.0 2800.3 
0.13 1997.3 2272.7 3138.0 2428.3 3703.7 2803.3 
0.16 2012.3 2286.3 3147.0 2439.7 3724.7 2814.7 
0.20 2031.0 2305.0 3164.3 2456.7 3756.3 2833.3 
0.25 2053.3 2329.3 3191.7 2479.3 3793.3 2856.7 
0.32 2080.3 2358.0 3228.0 2507.3 3839.0 2886.0 
0.40 2112.7 2392.0 3269.3 2541.3 3890.7 2920.0 
0.50 2147.0 2428.3 3315.7 2577.7 3952.7 2961.7 
0.63 2185.0 2468.3 3368.7 2620.3 4019.0 3007.7 
0.79 2224.3 2511.0 3425.3 2664.0 4089.7 3059.0 
1.00 2266.3 2556.0 3484.3 2712.7 4161.7 3112.3 
1.26 2308.7 2601.3 3545.3 2761.0 4237.0 3166.3 
1.59 2351.0 2646.7 3606.7 2809.7 4310.3 3219.7 
2.00 2394.3 2692.3 3667.7 2859.3 4384.7 3275.3 
2.51 2437.7 2738.3 3730.7 2909.7 4460.7 3331.0 
3.16 2483.0 2785.7 3793.7 2960.7 4538.0 3389.0 
3.98 2527.0 2831.3 3856.3 3011.3 4613.0 3444.3 
5.01 2570.7 2877.3 3918.0 3061.7 4689.0 3500.3 
6.31 2613.7 2922.0 3979.0 3111.7 4764.7 3555.0 
7.94 2656.7 2966.0 4039.3 3161.7 4839.7 3610.3 
10.00 2699.3 3009.3 4099.0 3210.7 4914.3 3663.3 
12.59 2741.0 3052.3 4157.7 3260.7 4988.3 3716.0 
15.85 2783.0 3094.7 4215.3 3310.0 5061.7 3767.3 
19.95 2824.7 3135.7 4271.7 3359.0 5134.0 3817.7 
25.12 2865.7 3175.7 4326.0 3407.7 5206.0 3867.0 
31.62 2905.7 3215.0 4379.7 3455.3 5276.3 3914.7 
39.81 2944.7 3252.7 4430.3 3502.7 5345.3 3959.7 
50.12 2983.0 3288.7 4479.7 3549.3 5413.7 4003.0 
63.10 3019.3 3322.0 4525.3 3594.7 5480.0 4042.7 
79.43 3055.7 3353.7 4568.3 3638.7 5543.3 4079.3 
100.00 3096.0 3389.7 4619.7 3691.3 5614.3 4123.0 
 
  
 
 94 
 
 
Table C.2. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan gum 
solutions over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for Xanthan 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 60.7 70.5 96.2 99.9 128.9 122.3 
0.03 62.0 71.4 97.4 100.9 130.9 123.8 
0.03 63.6 72.8 99.3 102.9 133.6 126.1 
0.04 65.6 74.6 101.9 105.3 136.8 128.9 
0.05 67.9 76.7 104.7 108.4 140.7 132.3 
0.06 70.4 79.0 108.1 111.9 144.8 136.2 
0.08 73.3 81.9 111.8 115.8 149.3 140.5 
0.10 76.3 84.9 115.9 119.9 154.2 144.9 
0.13 79.6 88.2 120.2 124.3 159.4 149.8 
0.16 83.2 91.8 124.8 129.0 164.9 155.1 
0.20 87.0 95.6 129.6 133.9 170.7 160.6 
0.25 91.2 99.6 134.6 139.2 176.9 166.5 
0.32 95.4 103.7 140.0 144.8 183.3 172.7 
0.40 100.0 108.2 145.3 150.5 190.0 179.2 
0.50 104.8 112.8 151.1 156.7 197.2 186.0 
0.63 109.7 117.7 157.1 163.0 204.6 193.1 
0.79 115.1 122.8 163.4 169.6 212.3 200.5 
1.00 120.6 128.2 169.8 176.4 220.4 208.1 
1.26 126.3 133.7 176.4 183.5 228.5 215.9 
1.59 132.2 139.4 183.3 190.8 237.1 224.1 
2.00 138.4 145.4 190.4 198.3 245.8 232.4 
2.51 144.8 151.6 197.6 206.2 254.8 241.0 
3.16 151.5 158.0 205.3 214.2 264.1 250.0 
3.98 158.3 164.6 213.1 222.5 273.7 259.2 
5.01 165.5 171.5 221.3 231.2 283.7 268.8 
6.31 173.1 178.8 229.9 240.3 294.1 278.7 
7.94 181.0 186.3 238.8 249.6 304.9 289.0 
10.00 189.4 194.3 248.1 259.7 316.2 299.9 
12.59 198.4 202.7 258.2 270.3 328.2 311.3 
15.85 207.8 211.8 268.9 281.4 341.0 323.6 
19.95 218.3 221.7 280.5 293.6 354.7 336.8 
25.12 229.8 232.6 293.2 307.0 369.7 351.3 
31.62 242.8 244.7 307.6 322.1 386.3 367.2 
39.81 257.8 258.7 324.0 339.3 405.3 385.5 
50.12 275.6 275.4 343.5 359.4 427.2 406.8 
63.10 297.5 295.6 367.2 383.9 453.5 432.5 
79.43 325.2 321.1 396.9 414.7 486.0 464.5 
100.00 361.5 354.5 435.6 454.5 527.9 505.7 
 
 
 
 
 95 
 
Table C.3. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 849.0 643.5 674.2 627.9 710.0 602.3 
0.03 852.3 651.2 698.4 648.7 720.5 611.0 
0.03 902.2 689.7 714.2 735.8 715.9 626.8 
0.04 920.2 706.0 733.2 822.3 730.3 645.7 
0.05 951.9 722.6 749.0 837.7 747.9 658.6 
0.06 975.7 745.1 766.3 1119.9 772.1 676.3 
0.08 996.2 761.7 787.3 1032.1 794.2 700.4 
0.10 1027.2 782.5 812.1 1096.3 814.3 724.6 
0.13 1050.7 804.0 835.5 915.4 838.9 743.3 
0.16 1071.0 831.5 859.0 927.8 862.4 766.5 
0.20 1095.7 857.8 884.6 919.5 888.8 792.7 
0.25 1116.3 881.7 909.9 941.2 913.5 815.6 
0.32 1142.7 913.0 936.6 949.2 939.8 840.6 
0.40 1169.7 936.3 961.7 976.7 965.4 864.9 
0.50 1203.3 959.8 986.7 1014.0 990.7 889.5 
0.63 1235.0 990.8 1013.5 1021.6 1019.3 912.2 
0.79 1269.3 1014.9 1042.5 1049.6 1046.3 937.6 
1.00 1305.3 1042.7 1067.9 1073.4 1071.3 964.7 
1.26 1338.0 1070.4 1094.7 1094.8 1100.7 990.0 
1.59 1371.0 1097.3 1121.7 1129.0 1128.7 1015.3 
2.00 1405.0 1124.2 1149.7 1160.1 1157.0 1042.4 
2.51 1439.0 1150.7 1176.3 1177.7 1185.7 1069.7 
3.16 1474.0 1179.3 1203.0 1206.3 1214.0 1096.0 
3.98 1509.7 1206.7 1230.0 1227.3 1243.0 1122.3 
5.01 1544.7 1232.0 1257.3 1258.3 1270.7 1150.3 
6.31 1580.7 1259.3 1284.3 1286.7 1299.3 1177.3 
7.94 1614.3 1285.0 1311.0 1311.3 1329.0 1205.7 
10.00 1648.0 1318.3 1336.3 1334.0 1360.3 1232.0 
12.59 1678.0 1347.0 1366.3 1366.3 1389.0 1261.3 
15.85 1721.3 1375.0 1395.7 1397.0 1420.0 1290.0 
19.95 1754.0 1406.3 1424.3 1428.7 1452.0 1321.0 
25.12 1794.3 1437.3 1454.3 1457.0 1483.7 1351.3 
31.62 1837.7 1470.3 1487.3 1492.7 1519.3 1387.0 
39.81 1876.7 1504.0 1521.3 1542.0 1555.7 1422.3 
50.12 1923.3 1540.7 1557.0 1575.7 1594.7 1462.7 
63.10 1972.0 1582.0 1597.7 1606.7 1638.3 1505.3 
79.43 2027.0 1630.0 1643.0 1669.7 1686.3 1554.7 
100.00 2088.7 1685.3 1697.7 454.5 1744.7 1613.0 
 
  
 
 96 
 
Table C.4. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 2 over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC at pH = 2 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 323.9 359.4 392.9 435.0 962.8 1566.7 
0.03 331.5 363.9 399.2 440.9 968.0 1578.7 
0.03 341.9 368.9 406.0 448.7 974.7 1592.3 
0.04 351.9 374.7 411.7 458.6 983.2 1610.0 
0.05 361.0 380.1 417.8 467.5 995.0 1629.0 
0.06 368.8 386.5 425.8 476.7 1010.0 1651.0 
0.08 376.7 393.2 433.9 486.0 1027.7 1675.3 
0.10 384.4 400.4 442.8 496.0 1046.6 1703.0 
0.13 393.0 408.1 452.7 506.8 1068.7 1736.7 
0.16 402.7 417.2 463.9 518.3 1093.0 1774.0 
0.20 412.9 427.2 475.2 530.5 1120.7 1815.7 
0.25 423.6 437.9 487.9 545.0 1150.4 1862.0 
0.32 433.6 449.6 501.6 560.2 1183.9 1912.7 
0.40 446.5 462.3 516.5 576.6 1220.9 1968.7 
0.50 459.1 475.9 532.6 594.0 1258.3 2025.7 
0.63 472.9 490.5 549.8 612.4 1299.3 2088.0 
0.79 487.5 506.4 567.8 631.7 1342.0 2154.0 
1.00 502.7 522.8 586.7 652.0 1386.3 2222.0 
1.26 518.7 539.8 606.1 672.9 1430.3 2291.0 
1.59 535.1 557.2 625.2 694.2 1475.7 2361.0 
2.00 552.2 575.3 644.4 715.2 1520.3 2431.7 
2.51 570.0 593.5 664.6 736.3 1565.3 2502.7 
3.16 588.3 612.3 684.4 757.6 1610.7 2574.0 
3.98 606.6 630.6 704.5 778.6 1654.0 2643.7 
5.01 625.2 649.8 724.2 799.6 1697.7 2713.7 
6.31 643.9 668.4 743.8 820.4 1741.3 2782.7 
7.94 662.9 687.3 763.3 840.9 1783.7 2850.0 
10.00 681.9 705.9 782.5 861.3 1825.3 2916.7 
12.59 700.9 724.7 801.6 881.8 1866.0 2981.7 
15.85 720.0 743.4 820.7 902.1 1906.3 3045.7 
19.95 739.1 762.0 840.0 922.5 1945.7 3107.7 
25.12 758.5 780.5 859.0 942.7 1984.3 3168.7 
31.62 777.8 799.7 877.6 962.8 2022.0 3227.0 
39.81 797.3 818.3 896.5 982.8 2059.0 3284.3 
50.12 816.8 837.1 915.7 1002.7 2095.3 3340.0 
63.10 836.5 855.9 934.8 1022.5 2131.3 3393.0 
79.43 856.8 875.3 953.8 1042.3 2167.0 3446.3 
100.00 878.4 896.0 976.5 1064.7 2209.7 3504.0 
 
  
 
 97 
 
Table C.5. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 3 over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC at pH = 3 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 134.2 106.0 87.8 92.0 91.5 91.8 
0.03 136.4 108.3 89.5 91.7 93.0 92.0 
0.03 139.1 110.3 90.4 92.5 95.1 93.2 
0.04 142.4 112.6 93.4 93.8 97.0 94.7 
0.05 145.6 114.3 95.5 95.8 99.3 96.9 
0.06 149.3 118.2 97.7 98.0 101.7 99.2 
0.08 153.5 121.5 99.8 100.7 104.4 102.0 
0.10 157.9 124.8 101.5 103.5 107.4 104.7 
0.13 162.8 128.3 106.4 106.5 110.5 107.8 
0.16 167.8 132.3 109.6 109.8 113.7 111.2 
0.20 173.0 136.3 112.6 113.5 117.5 114.7 
0.25 179.0 140.6 117.5 117.3 121.3 118.5 
0.32 184.8 145.2 121.4 121.2 125.3 122.5 
0.40 190.8 149.9 125.7 125.2 129.3 126.5 
0.50 196.7 154.8 130.0 129.4 133.7 130.7 
0.63 203.1 159.7 134.3 133.7 138.2 135.0 
0.79 209.2 164.9 138.6 138.0 142.6 139.5 
1.00 215.8 170.0 143.3 142.4 147.1 144.0 
1.26 222.2 175.3 147.8 146.9 151.8 148.5 
1.59 228.8 180.5 152.3 151.3 156.3 152.9 
2.00 235.3 185.9 156.6 155.7 160.8 157.6 
2.51 242.0 191.2 161.5 160.6 165.8 162.2 
3.16 248.6 196.6 165.7 165.3 170.5 166.8 
3.98 255.2 202.0 170.1 169.8 175.2 171.4 
5.01 261.8 207.4 176.4 174.5 180.0 175.9 
6.31 268.5 212.7 181.1 179.0 184.6 180.8 
7.94 275.2 218.1 185.8 183.7 189.4 185.5 
10.00 281.9 223.5 190.6 188.4 194.1 190.2 
12.59 288.7 228.9 195.3 193.0 198.8 194.8 
15.85 295.4 234.3 200.3 197.8 203.2 199.6 
19.95 302.1 239.8 205.2 202.5 208.9 204.4 
25.12 309.0 245.3 210.1 207.3 213.8 209.3 
31.62 315.9 250.8 215.0 212.0 218.7 214.1 
39.81 322.7 256.3 219.8 216.8 223.6 218.8 
50.12 329.6 261.7 224.8 221.6 228.5 224.0 
63.10 336.4 267.1 229.7 226.4 233.5 228.9 
79.43 343.4 272.5 234.7 231.2 238.3 233.9 
100.00 351.0 278.4 240.5 236.4 243.4 239.1 
 
  
 
 98 
 
Table C.6. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 4 over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC at pH = 4 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 86.1 50.6 46.8 69.8 65.3 63.2 
0.03 89.9 49.7 46.8 67.9 60.7 62.4 
0.03 90.0 49.6 47.3 66.9 57.3 62.4 
0.04 90.6 50.0 48.1 66.6 55.3 62.5 
0.05 91.5 50.5 48.9 67.2 55.1 63.3 
0.06 92.8 51.2 50.1 68.1 55.4 64.1 
0.08 94.3 52.3 51.4 69.6 56.2 65.9 
0.10 96.0 53.5 52.8 71.2 57.2 67.4 
0.13 98.1 54.9 54.3 73.1 58.4 69.0 
0.16 100.8 56.5 56.0 75.3 60.0 71.2 
0.20 103.6 58.1 57.9 77.6 61.8 73.5 
0.25 106.8 60.1 59.8 80.2 63.7 75.8 
0.32 110.2 62.3 62.0 82.9 65.8 78.4 
0.40 113.7 64.5 64.2 85.9 68.0 81.0 
0.50 117.5 66.8 66.7 89.1 70.5 84.0 
0.63 121.6 69.3 69.2 92.2 73.0 86.9 
0.79 125.7 71.8 71.7 95.5 75.7 90.1 
1.00 130.0 74.5 74.5 99.0 78.4 93.4 
1.26 134.3 77.3 77.3 102.7 81.3 96.8 
1.59 138.7 80.0 80.2 106.4 84.1 100.2 
2.00 143.2 83.1 83.2 110.1 87.1 103.7 
2.51 147.8 86.1 86.3 113.9 90.2 107.3 
3.16 152.6 89.2 89.4 117.9 93.4 111.1 
3.98 157.4 92.3 92.5 122.0 96.6 114.9 
5.01 162.3 95.5 95.8 126.0 99.9 118.8 
6.31 167.3 98.8 99.2 130.2 103.3 122.6 
7.94 172.3 102.2 102.7 134.4 106.7 126.6 
10.00 177.4 105.5 106.2 138.8 110.2 130.7 
12.59 182.6 109.1 109.7 143.1 113.7 134.8 
15.85 187.8 112.6 113.4 147.5 117.3 139.1 
19.95 193.1 116.2 117.1 152.0 121.0 143.3 
25.12 198.5 119.9 120.8 156.6 124.7 147.6 
31.62 204.0 123.7 124.8 161.3 128.5 152.1 
39.81 209.6 127.5 128.7 165.9 132.4 156.6 
50.12 215.3 131.3 132.7 170.6 136.3 161.2 
63.10 221.3 135.2 136.8 175.4 140.3 165.7 
79.43 227.3 139.1 141.1 180.2 144.2 170.4 
100.00 233.8 143.2 145.6 185.1 148.4 175.3 
 
  
 
 99 
 
Table C.7. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 5 over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC at pH = 5 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 51.7 51.7 47.4 61.2 50.1 50.4 
0.03 54.0 52.0 48.0 62.1 50.5 50.4 
0.03 56.4 52.6 48.7 63.0 51.1 50.8 
0.04 58.6 53.5 49.6 64.1 51.9 51.4 
0.05 60.5 54.5 50.6 65.2 52.7 52.1 
0.06 62.9 55.5 51.7 66.4 53.8 53.0 
0.08 64.7 56.7 53.0 67.7 55.0 53.9 
0.10 67.4 58.0 54.3 69.0 56.3 55.0 
0.13 69.8 59.4 55.9 70.8 57.8 56.2 
0.16 72.1 61.1 57.5 72.5 59.4 57.7 
0.20 74.9 62.9 59.2 74.5 61.2 59.3 
0.25 77.7 64.6 61.2 76.5 63.1 61.1 
0.32 80.5 66.2 63.3 78.8 64.9 62.8 
0.40 83.4 68.9 65.5 81.2 67.2 64.9 
0.50 86.4 71.1 67.7 83.8 69.5 66.6 
0.63 89.7 73.5 70.1 86.5 71.8 69.0 
0.79 91.7 76.0 72.5 89.3 74.3 71.5 
1.00 96.1 78.7 75.1 92.2 76.9 73.9 
1.26 99.4 81.3 77.7 95.2 79.4 76.4 
1.59 102.7 84.0 80.2 98.2 82.1 78.9 
2.00 105.5 86.8 83.0 101.4 84.8 81.5 
2.51 109.9 89.5 85.7 104.5 87.6 84.1 
3.16 113.5 92.6 88.7 107.9 90.5 86.8 
3.98 117.2 95.6 91.6 111.2 93.5 89.7 
5.01 120.9 98.6 94.5 114.5 96.4 92.5 
6.31 124.7 101.6 97.5 117.9 99.4 95.3 
7.94 128.5 104.8 100.5 121.4 102.4 98.2 
10.00 132.4 108.0 103.6 124.8 105.5 101.2 
12.59 136.3 111.1 106.8 128.4 108.7 104.1 
15.85 140.3 114.4 110.0 132.0 111.8 107.1 
19.95 144.4 117.7 113.2 135.6 115.0 110.2 
25.12 148.5 121.1 116.6 139.3 118.4 113.4 
31.62 152.8 124.5 120.0 143.0 121.6 116.5 
39.81 157.1 127.9 123.5 146.8 125.1 119.8 
50.12 161.5 131.5 127.1 150.6 128.5 123.0 
63.10 166.1 135.1 130.8 154.4 131.9 126.4 
79.43 170.7 138.6 134.5 158.4 135.5 129.7 
100.00 175.3 142.5 138.5 162.4 139.0 133.1 
 
  
 
 100 
 
Table C.8. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 6 over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC at pH = 6 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 67.9 59.8 54.1 63.3 60.6 60.7 
0.03 68.6 60.8 55.0 64.6 61.0 61.1 
0.03 69.4 61.7 56.0 65.9 61.6 61.5 
0.04 70.4 62.7 57.1 67.1 64.4 62.2 
0.05 71.5 63.7 57.9 68.2 65.3 62.9 
0.06 72.9 64.7 58.9 69.5 66.2 63.8 
0.08 74.2 65.8 59.9 70.7 67.3 64.7 
0.10 75.8 66.9 61.1 72.0 68.7 66.0 
0.13 77.6 68.3 62.3 73.6 69.9 67.3 
0.16 79.4 69.8 63.7 75.1 71.5 68.8 
0.20 81.3 71.4 65.3 77.0 73.1 70.5 
0.25 83.0 73.2 66.9 78.8 74.9 72.2 
0.32 85.6 75.0 68.6 80.8 77.0 74.1 
0.40 88.3 77.1 70.5 82.9 79.0 76.0 
0.50 90.9 79.2 72.5 85.2 81.1 78.2 
0.63 93.4 81.4 74.6 87.5 83.5 80.4 
0.79 96.1 83.6 76.7 90.0 85.8 82.7 
1.00 98.8 86.0 79.0 92.5 88.3 85.1 
1.26 101.6 88.4 81.3 95.1 90.8 87.5 
1.59 104.4 90.9 83.6 97.7 93.3 90.0 
2.00 107.3 93.3 86.0 100.3 95.8 92.5 
2.51 110.1 95.9 88.4 103.1 98.5 95.0 
3.16 113.2 98.5 90.9 105.9 101.2 97.6 
3.98 116.2 101.1 93.4 108.7 103.8 100.2 
5.01 119.2 103.8 95.9 111.5 106.5 102.9 
6.31 122.3 106.4 98.5 114.4 109.3 105.7 
7.94 125.6 109.2 101.1 117.3 112.1 108.4 
10.00 128.8 112.0 103.7 120.3 114.9 111.2 
12.59 132.1 114.9 106.4 123.2 117.8 114.0 
15.85 135.5 117.8 109.2 126.3 120.7 117.0 
19.95 139.0 120.8 112.1 129.4 123.8 120.0 
25.12 142.6 123.8 115.0 132.6 126.8 123.1 
31.62 146.3 127.0 118.0 135.9 130.0 126.3 
39.81 150.2 130.2 121.1 139.3 133.2 129.7 
50.12 154.2 133.6 124.4 142.9 136.6 133.2 
63.10 158.3 137.0 127.8 146.6 140.1 136.9 
79.43 162.7 140.7 131.4 150.5 143.8 140.7 
100.00 167.3 144.3 135.3 154.3 147.5 144.7 
 
  
 
 101 
 
Table C.9. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 7 over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC at pH = 7 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 73.6 70.9 74.7 64.1 67.5 66.1 
0.03 75.1 72.4 75.7 65.1 68.4 66.6 
0.03 76.6 74.0 76.6 66.5 69.5 67.3 
0.04 77.7 75.3 78.2 67.5 70.4 68.1 
0.05 79.5 76.6 79.6 68.7 71.4 70.4 
0.06 80.9 77.9 81.5 70.0 72.9 71.4 
0.08 82.8 79.4 83.1 71.7 74.3 72.6 
0.10 84.9 81.0 85.0 73.5 75.5 73.8 
0.13 87.3 82.4 86.9 75.3 77.3 75.2 
0.16 89.7 84.1 89.1 77.3 79.0 76.8 
0.20 92.1 86.1 91.3 79.3 80.9 78.7 
0.25 94.7 88.2 93.6 81.4 83.0 80.7 
0.32 97.4 90.4 96.1 83.7 85.3 82.8 
0.40 100.2 92.8 98.8 86.1 87.7 85.1 
0.50 102.9 95.3 101.5 88.5 90.2 87.4 
0.63 106.2 97.8 104.4 91.1 92.7 89.9 
0.79 109.1 100.5 107.3 93.8 95.4 92.4 
1.00 112.5 103.7 110.3 96.5 98.2 95.1 
1.26 115.6 106.4 113.4 99.4 101.0 97.8 
1.59 118.9 109.3 116.4 102.2 103.8 100.5 
2.00 122.2 112.3 119.6 105.1 106.7 103.2 
2.51 125.6 115.3 122.7 108.0 109.6 106.1 
3.16 129.0 118.3 126.0 111.0 112.5 109.0 
3.98 132.4 121.4 129.0 114.0 115.4 111.8 
5.01 135.8 124.5 132.7 117.0 118.4 114.7 
6.31 139.2 127.5 136.1 120.1 121.4 117.7 
7.94 142.8 130.7 139.5 123.2 124.5 120.6 
10.00 146.4 133.8 142.9 126.4 127.6 123.7 
12.59 149.9 136.9 146.4 129.6 130.8 126.7 
15.85 153.6 140.2 149.9 132.9 134.1 129.9 
19.95 157.4 143.5 153.5 136.2 137.4 133.0 
25.12 161.2 146.8 157.2 139.6 140.7 136.3 
31.62 165.1 150.2 161.0 143.1 144.2 139.6 
39.81 169.1 153.6 164.7 146.6 147.8 143.1 
50.12 173.2 157.1 168.6 150.3 151.4 146.6 
63.10 177.5 160.7 172.7 154.2 155.2 150.4 
79.43 182.0 164.4 176.7 158.2 159.1 154.3 
100.00 186.8 168.2 181.1 162.6 163.3 158.5 
 
  
 
 102 
 
Table C.10. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions at pH = 8 over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC at pH = 8 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 80.1 64.8 57.7 44.1 63.4 59.4 
0.03 80.5 66.4 58.0 45.6 65.4 60.3 
0.03 81.2 67.6 58.7 47.0 66.6 61.2 
0.04 82.0 68.9 59.2 48.4 67.6 61.7 
0.05 84.2 70.1 60.2 49.7 69.0 62.6 
0.06 86.1 71.3 60.9 51.1 70.3 63.6 
0.08 88.4 72.7 62.3 52.7 71.9 64.5 
0.10 90.4 74.3 63.6 54.1 73.5 65.7 
0.13 92.4 75.9 65.2 55.8 75.1 67.0 
0.16 94.5 77.8 66.6 57.5 76.9 68.6 
0.20 96.7 79.8 68.5 59.2 78.8 70.1 
0.25 99.2 81.9 70.3 61.0 80.8 71.8 
0.32 101.8 84.2 72.3 62.9 83.1 73.6 
0.40 104.5 86.5 74.2 64.8 85.5 75.5 
0.50 107.3 88.9 76.6 66.8 87.8 77.6 
0.63 110.1 91.4 78.7 68.9 90.3 79.7 
0.79 113.0 94.0 80.9 71.1 92.8 81.9 
1.00 116.0 96.7 83.4 73.2 95.5 84.3 
1.26 119.2 99.4 85.7 75.5 98.1 86.6 
1.59 122.2 102.0 88.2 77.6 100.7 89.0 
2.00 125.4 104.9 90.7 80.0 103.5 91.4 
2.51 128.6 107.7 93.1 82.2 106.3 93.9 
3.16 131.9 110.6 95.8 84.7 109.1 96.4 
3.98 135.2 113.6 98.3 87.1 111.9 98.9 
5.01 138.6 116.6 100.9 89.5 114.8 101.5 
6.31 142.0 119.6 103.5 91.9 117.6 104.0 
7.94 145.5 122.6 106.1 94.4 120.6 106.6 
10.00 149.1 125.7 108.8 96.9 123.5 109.2 
12.59 152.7 128.9 111.6 99.5 126.4 111.9 
15.85 156.4 132.2 114.4 102.1 129.4 114.6 
19.95 160.1 135.5 117.2 104.8 132.5 117.4 
25.12 164.2 138.9 120.1 107.5 135.6 120.3 
31.62 168.1 142.5 123.1 110.3 138.9 123.2 
39.81 172.4 146.1 126.2 113.3 142.2 126.2 
50.12 176.7 150.0 129.3 116.3 145.6 129.4 
63.10 181.2 153.9 132.7 119.4 149.2 132.6 
79.43 185.9 158.1 136.1 122.8 152.7 136.1 
100.00 190.6 162.4 139.8 126.3 156.3 139.8 
 
  
 
 103 
 
Table C.11. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 5 mM NaCl over 5 FTCs (n=3). 
Storage Modules (Pa) Mean for XCHC with 5 mM NaCl 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 79.0 66.5 87.6 87.3 78.9 94.1 
0.03 77.3 64.4 83.9 86.6 77.4 90.8 
0.03 76.9 63.4 82.0 87.4 76.0 88.4 
0.04 76.4 63.2 82.5 88.4 75.6 87.3 
0.05 77.1 63.7 83.5 89.8 76.3 86.5 
0.06 78.1 64.8 84.9 90.9 77.1 86.4 
0.08 78.9 66.1 86.8 93.7 78.6 86.9 
0.10 81.0 67.6 88.9 96.9 80.4 87.5 
0.13 81.5 69.3 91.1 99.5 82.0 88.5 
0.16 85.2 71.3 93.8 102.4 84.1 89.7 
0.20 87.6 73.2 96.3 105.3 86.2 91.2 
0.25 90.0 75.5 99.2 108.3 88.5 92.8 
0.32 92.7 77.7 102.0 111.4 90.9 94.8 
0.40 95.4 80.2 105.1 114.6 93.3 96.8 
0.50 98.2 82.5 108.2 117.8 95.9 99.1 
0.63 101.0 85.0 111.3 121.0 98.7 101.7 
0.79 104.0 87.5 114.6 124.4 101.5 104.1 
1.00 106.9 90.1 117.9 127.9 104.4 106.8 
1.26 110.0 92.8 121.2 131.3 107.4 109.5 
1.59 112.9 95.4 124.5 134.8 110.3 112.3 
2.00 116.0 98.2 127.9 138.4 113.3 115.0 
2.51 119.1 101.0 131.3 141.9 116.4 117.8 
3.16 122.3 103.8 134.8 145.6 119.5 120.7 
3.98 125.4 106.6 138.2 149.2 122.6 123.5 
5.01 128.6 109.4 141.7 152.9 125.8 126.4 
6.31 131.8 112.3 145.2 156.6 129.0 129.3 
7.94 135.2 115.2 148.7 160.4 132.3 132.2 
10.00 138.5 118.1 152.3 164.2 135.6 135.2 
12.59 141.9 121.1 155.8 167.9 138.9 138.1 
15.85 145.2 124.2 159.4 172.2 142.3 141.1 
19.95 148.7 127.2 163.0 176.4 145.7 144.1 
25.12 152.1 130.3 166.7 180.5 149.1 147.0 
31.62 155.6 133.4 170.3 184.6 152.6 150.1 
39.81 159.2 136.6 174.0 188.8 156.2 153.2 
50.12 162.8 139.9 177.6 193.0 159.8 156.3 
63.10 166.6 143.2 181.3 197.4 163.4 159.4 
79.43 170.3 146.5 185.0 201.7 166.9 162.5 
100.00 174.0 149.8 188.6 206.4 170.2 165.6 
 
 
 
 
 
 104 
 
Table C.12. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 15 mM NaCl over 5 FTCs 
(n=3). 
Storage Modules (Pa) Mean for XCHC with 15 mM NaCl 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 117.9 99.5 124.1 109.3 91.8 90.5 
0.03 116.0 97.9 121.3 106.7 89.9 97.5 
0.03 116.4 97.5 120.3 106.4 90.1 97.6 
0.04 117.4 98.2 119.9 106.5 90.6 98.1 
0.05 119.7 99.3 120.9 107.2 92.3 100.1 
0.06 122.2 100.9 122.6 108.8 94.2 102.4 
0.08 124.8 102.8 124.9 110.4 95.8 103.6 
0.10 128.0 105.1 127.1 113.3 98.1 106.8 
0.13 131.5 107.6 130.3 116.1 100.5 110.1 
0.16 135.1 110.1 133.7 119.5 103.7 113.3 
0.20 138.8 113.1 137.2 123.1 106.8 116.6 
0.25 142.9 116.5 141.0 126.8 110.5 119.8 
0.32 147.0 119.9 145.2 130.6 113.8 123.4 
0.40 151.2 123.4 149.3 134.5 117.3 127.4 
0.50 155.4 127.0 153.7 138.4 119.5 131.6 
0.63 160.0 130.6 158.3 142.5 124.6 135.6 
0.79 164.5 134.6 162.8 146.8 128.9 140.0 
1.00 169.0 138.5 167.4 151.0 132.8 143.4 
1.26 173.7 142.6 172.2 155.1 136.8 148.8 
1.59 178.3 146.5 176.9 159.6 140.7 153.1 
2.00 182.9 150.7 181.7 164.1 144.8 157.6 
2.51 187.7 154.8 186.5 168.5 148.8 162.0 
3.16 192.5 159.0 191.5 172.9 153.1 166.4 
3.98 197.3 163.1 196.3 177.4 157.2 170.8 
5.01 202.1 167.4 201.2 181.9 161.4 175.2 
6.31 207.0 171.6 206.0 186.4 165.6 179.8 
7.94 211.8 175.8 210.9 190.9 169.8 184.4 
10.00 216.8 180.0 215.9 195.5 174.0 189.0 
12.59 221.8 184.3 220.9 200.0 178.4 193.5 
15.85 226.8 188.7 225.9 204.6 182.7 198.1 
19.95 231.8 193.0 230.8 209.2 187.2 202.7 
25.12 237.0 197.4 236.0 213.9 191.6 207.1 
31.62 242.1 201.8 241.1 218.5 196.0 211.9 
39.81 247.3 206.2 246.3 223.3 200.5 216.8 
50.12 252.7 210.6 251.5 228.1 205.0 221.1 
63.10 258.0 215.1 256.7 232.9 209.6 226.4 
79.43 263.5 219.6 262.1 237.8 214.5 231.0 
100.00 269.3 223.7 267.7 242.8 219.2 235.6 
 
 
 
 
 
 105 
 
Table C.13. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 20 mM NaCl over 5 FTCs 
(n=3). 
Storage Modules (Pa) Mean for XCHC with 20 mM NaCl 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 89.0 122.4 102.9 83.4 95.7 98.7 
0.03 99.4 120.9 101.4 82.3 94.5 94.0 
0.03 100.9 121.3 101.0 84.6 94.5 96.3 
0.04 102.1 122.1 101.5 87.0 96.5 96.2 
0.05 103.6 123.4 102.9 88.9 99.3 97.2 
0.06 105.7 125.1 104.7 91.1 102.1 98.7 
0.08 108.4 127.0 106.8 93.3 105.5 100.7 
0.10 111.4 129.1 109.1 95.6 108.4 103.0 
0.13 114.3 131.7 111.6 98.1 111.5 105.6 
0.16 117.5 134.7 114.3 100.6 114.6 108.3 
0.20 120.7 137.9 117.2 103.4 117.8 111.3 
0.25 124.3 141.4 120.1 106.2 121.0 114.4 
0.32 128.0 144.9 123.3 109.2 124.4 117.6 
0.40 131.7 148.6 126.5 112.0 127.8 120.9 
0.50 135.4 152.4 129.7 115.1 131.2 124.3 
0.63 139.3 156.2 133.2 118.3 134.7 127.7 
0.79 143.1 160.2 136.7 121.6 138.3 131.2 
1.00 147.1 164.2 140.1 124.9 142.0 134.9 
1.26 151.0 168.2 143.8 128.1 145.6 138.5 
1.59 155.1 172.3 147.4 131.4 149.3 141.7 
2.00 159.2 176.3 151.0 134.8 153.1 145.9 
2.51 163.3 180.5 154.6 138.2 156.8 149.6 
3.16 167.4 184.6 158.3 141.6 160.6 153.3 
3.98 171.5 188.8 161.9 145.0 164.3 157.0 
5.01 175.7 192.9 165.6 148.5 168.1 161.0 
6.31 179.9 197.1 169.3 151.9 171.9 164.8 
7.94 184.0 201.4 173.0 155.4 175.7 168.6 
10.00 188.2 205.6 176.7 158.9 179.6 172.4 
12.59 192.5 209.9 180.3 162.4 183.4 176.2 
15.85 196.7 214.2 184.1 165.9 187.3 180.0 
19.95 201.1 218.5 187.8 169.4 191.3 183.9 
25.12 205.4 222.8 191.5 173.0 195.3 187.8 
31.62 209.9 227.2 195.3 176.6 199.3 191.6 
39.81 214.2 231.6 199.1 180.3 203.3 195.6 
50.12 218.7 236.1 202.9 184.1 207.4 199.5 
63.10 223.3 240.6 206.8 188.0 211.6 203.5 
79.43 227.8 245.2 210.7 237.8 215.9 207.6 
100.00 232.6 250.1 215.0 242.8 220.6 211.7 
 
 
 
 
 
 106 
 
Table C.14. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 75 mM NaCl over 5 FTCs 
(n=3). 
Storage Modules (Pa) Mean for XCHC with 75 mM NaCl 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 111.0 110.7 100.7 117.4 137.1 131.8 
0.03 109.8 110.0 100.5 114.7 133.9 131.0 
0.03 109.0 110.4 101.2 112.3 131.7 131.2 
0.04 109.4 111.1 102.3 112.0 130.6 132.3 
0.05 111.1 112.1 103.9 112.4 130.1 134.0 
0.06 113.6 113.3 105.8 113.6 130.2 136.1 
0.08 116.5 115.4 108.0 115.6 131.0 138.8 
0.10 119.3 117.7 110.5 117.9 132.4 141.9 
0.13 122.5 120.1 113.1 120.7 134.4 145.1 
0.16 125.9 122.5 115.9 123.9 136.6 148.5 
0.20 129.3 125.1 118.7 127.0 139.4 152.2 
0.25 132.8 128.0 121.8 130.6 142.5 155.9 
0.32 136.5 130.9 124.9 134.2 146.0 159.9 
0.40 139.9 134.0 128.1 137.8 149.5 163.9 
0.50 143.6 137.5 131.5 141.3 153.1 168.0 
0.63 147.3 141.1 134.8 144.9 157.0 172.2 
0.79 151.0 144.6 138.3 148.6 160.9 176.4 
1.00 154.8 148.2 141.9 152.3 164.7 180.8 
1.26 158.7 152.0 145.4 156.3 168.8 185.0 
1.59 162.5 155.7 148.9 160.1 172.8 189.4 
2.00 166.2 159.4 152.5 163.9 176.7 193.8 
2.51 170.1 163.1 156.0 167.8 180.8 198.2 
3.16 173.9 166.9 159.6 171.6 184.9 202.5 
3.98 177.7 170.7 163.3 175.4 188.9 207.0 
5.01 181.6 174.6 166.9 179.4 193.0 211.3 
6.31 185.5 178.3 170.5 183.3 197.0 215.7 
7.94 189.4 182.1 174.1 187.2 201.0 220.2 
10.00 193.3 186.0 177.8 191.0 205.0 224.6 
12.59 197.2 189.8 181.4 194.9 209.1 229.1 
15.85 201.1 193.6 185.0 198.9 213.2 233.6 
19.95 205.2 197.4 188.7 202.9 217.3 238.1 
25.12 209.1 201.3 192.4 206.9 221.5 242.6 
31.62 213.2 205.3 196.1 211.0 225.5 247.2 
39.81 217.2 209.2 200.0 215.1 229.7 251.7 
50.12 221.4 213.1 203.7 219.4 233.8 256.4 
63.10 225.7 217.3 207.6 223.8 238.0 261.2 
79.43 229.9 221.4 211.6 228.3 242.2 265.9 
100.00 234.6 225.9 215.9 233.0 246.8 270.8 
 
 
 
 
 
 107 
 
Table C.15. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 100 mM NaCl over 5 FTCs 
(n=3). 
Storage Modules (Pa) Mean for XCHC with 100 mM NaCl 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 117.7 120.5 111.7 137.8 143.3 137.4 
0.03 116.5 121.1 113.1 134.7 140.9 135.5 
0.03 116.4 122.3 114.8 132.4 139.8 135.3 
0.04 117.0 124.0 116.9 132.9 139.9 135.5 
0.05 118.2 125.7 119.6 134.4 140.7 136.0 
0.06 119.9 127.8 122.4 137.1 141.9 136.8 
0.08 121.9 130.3 125.4 140.0 143.6 138.2 
0.10 124.5 133.1 128.7 143.2 145.8 139.6 
0.13 127.2 136.2 131.9 146.7 148.4 141.3 
0.16 130.1 139.8 135.5 150.5 151.2 143.1 
0.20 133.4 143.6 138.9 154.6 154.5 144.9 
0.25 136.8 147.5 142.6 158.7 158.2 148.5 
0.32 140.3 151.6 146.3 162.9 162.4 152.3 
0.40 144.0 155.7 150.1 167.1 166.5 156.2 
0.50 147.7 159.9 154.2 171.3 170.5 160.2 
0.63 151.5 164.3 158.4 175.7 174.9 164.5 
0.79 155.4 168.7 162.5 180.1 179.2 168.6 
1.00 159.4 173.1 166.7 184.3 183.6 173.0 
1.26 163.5 177.6 171.1 174.3 188.0 177.3 
1.59 167.5 182.2 175.4 178.5 192.4 181.7 
2.00 171.6 186.7 179.6 182.8 196.8 186.1 
2.51 175.7 191.3 184.0 187.1 201.3 190.5 
3.16 179.8 195.9 188.3 191.5 205.7 194.9 
3.98 183.9 200.4 192.6 195.8 210.1 199.3 
5.01 188.0 205.1 197.0 200.2 214.7 203.7 
6.31 192.2 209.7 201.4 204.6 219.1 208.1 
7.94 196.3 214.3 205.8 208.9 223.6 212.6 
10.00 200.5 218.9 210.2 213.4 228.0 217.0 
12.59 204.8 223.6 214.6 217.8 232.6 221.4 
15.85 209.0 228.3 219.1 222.3 237.1 225.7 
19.95 213.4 233.1 223.5 226.7 241.7 230.3 
25.12 217.8 237.9 228.0 231.2 246.3 235.6 
31.62 222.3 242.7 232.6 235.7 251.0 240.2 
39.81 226.7 247.7 237.1 240.3 255.7 244.8 
50.12 231.2 252.8 241.7 244.9 260.4 249.5 
63.10 236.0 257.9 246.4 249.6 265.3 254.3 
79.43 240.8 263.2 251.2 254.2 270.3 259.1 
100.00 246.0 268.9 215.9 258.9 275.8 264.2 
 
 
 
 
 
 
 108 
 
Table C.16. Average storage modulus Gd6bG729G522Gd25 Gd6f for 2.0% aqueous xanthan curdlan 
hydrogel complex (XCHC) gum solutions with 200 mM NaCl over 5 FTCs 
(n=3). 
Storage Modules (Pa) Mean for XCHC with 200 mM NaCl 
Freeze Thaw Cycles 
? 
(rad/s) 
0 1 2 3 4 5 
0.02 97.7 93.8 97.4 104.1 101.9 121.9 
0.03 97.2 92.8 96.1 102.6 101.5 122.5 
0.03 96.5 93.3 95.5 103.3 102.4 124.0 
0.04 95.7 94.1 95.3 105.1 103.8 125.1 
0.05 97.4 95.0 95.9 107.4 106.0 127.1 
0.06 100.6 96.3 96.7 110.1 108.9 129.4 
0.08 104.5 98.6 98.1 112.7 111.6 132.6 
0.10 108.1 101.7 100.1 115.6 115.1 135.9 
0.13 111.9 105.0 102.3 118.5 117.1 139.6 
0.16 115.8 108.4 104.8 121.8 121.6 143.6 
0.20 119.7 111.8 107.5 125.2 124.9 147.8 
0.25 123.7 115.4 110.3 128.8 128.2 152.3 
0.32 127.4 118.9 113.3 132.6 131.9 156.7 
0.40 132.0 122.6 116.6 136.4 135.6 161.3 
0.50 136.1 126.3 119.9 140.4 139.5 166.0 
0.63 140.4 130.1 123.3 144.5 143.4 170.7 
0.79 144.5 133.9 126.8 148.6 147.3 175.6 
1.00 148.9 137.8 130.4 152.7 151.2 180.5 
1.26 153.2 141.6 134.0 157.0 155.7 185.4 
1.59 157.4 145.6 137.7 161.4 159.8 190.2 
2.00 161.8 149.5 141.4 165.3 163.9 195.1 
2.51 166.2 153.5 145.0 169.9 168.0 199.9 
3.16 170.5 157.4 148.7 174.1 172.1 204.7 
3.98 174.8 161.4 152.4 178.2 176.2 209.5 
5.01 179.2 165.4 156.1 182.4 180.4 214.3 
6.31 183.5 169.3 159.8 186.9 184.5 219.7 
7.94 187.9 173.4 163.6 191.2 188.5 224.7 
10.00 192.3 177.3 167.4 195.4 192.4 229.5 
12.59 196.8 181.4 171.1 199.7 196.8 234.5 
15.85 201.2 185.5 174.9 204.0 201.2 239.4 
19.95 205.7 189.6 178.9 208.4 205.4 244.4 
25.12 210.2 193.8 182.8 212.7 209.8 249.5 
31.62 214.8 198.2 186.7 217.1 214.1 254.6 
39.81 219.5 202.5 191.0 221.5 218.5 259.8 
50.12 224.2 206.9 195.2 226.0 222.9 265.0 
63.10 229.1 211.6 199.4 230.6 227.5 270.5 
79.43 234.1 216.4 203.9 235.5 232.2 275.9 
100.00 239.8 221.8 208.8 240.8 237.3 282.0 
 
 109 
 
 
  
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