Rheological Evaluation of Gelatin-Xanthan Gum System With

Food Hydrocolloids 28 (2012) 141e150

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Food Hydrocolloids

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Rheological evaluation of gelatinexanthan gum system with high levels of co-solutes in the rubber-to-glass transition region

Filiz Altay a, Sundaram Gunasekaran b,* a Istanbul Technical University, Faculty of Chemical and Metallurgical, Department of Food Engineering, Maslak, Istanbul 34469, Turkey b University of WisconsineMadison, Department of Biological Systems Engineering, 460 Henry Mall, Madison, WI 53706, USA article info abstract

Article history: Effects of moisture content, xanthan gum (XG) addition, and glucose syrup (GS):sucrose ratio on the Received 20 October 2011 gelation of gelatin-XG systems with high levels of co-solutes were investigated in the rubbery and the Accepted 8 December 2011 glass transition regions. Frequency sweep tests were performed between 0.1 and 100 rad and the storage (G0) and loss (G00) moduli of the system were measured in the temperature range of 60 to 15 C. The Keywords: onset of glass transition region increased with decreasing moisture content. The timeetemperature Gelatin superposition yielded master curves of G0 and G00 as a function of timescale of measurement. G00 and Xanthan gum 00 G were superimposed with the horizontal shift factor aT, which was temperature dependent according Tg e e WLF equation to the Williams Landel Ferry (WLF) equation. Glass transition temperature (Tg) of the samples were Free volume determined by dynamic mechanical analysis (DMA) from the peak of tan d. Tg decreased with XG addition. The energy of vitriﬁcation of samples with XG increased compared to samples containing only gelatin. Relaxation spectra of the samples were calculated from rheological measurements using the ﬁrst and second approximations. The Rouse theory was more closely followed with the second approximation. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction constructing master curves of mechanical spectra, spanning many decades of frequency. Isothermal data obtained by frequency Dynamic mechanical measurements are widely used to probe sweeps at different temperatures are shifted along the frequency structureeproperty relationships in amorphous synthetic polymers axis and overlaid to obtain a master curve at an arbitrarily chosen during vitrification. The synthetic polymer approach, in which the reference temperature. TTS can express the effects of time and idea of molecular mobility governing the kinetics of phase/state temperature on viscoelastic properties separately by enlarging the transitions and chemical reactions is applied, has been extended to effective time or frequency scale available for experimental food biopolymers (Kasapis, Al-Marhoob, & Deszczynski, Mitchell, & measurements. The superposition of curves from frequency sweeps Abeysekara, 2003; Kasapis, Al-Marhoobi, & Sworn, 2001; Levine & at constant temperature intervals yields the shift factor (aT), which Slade, 1988). This approach has been applied extensively together indicates how much the time scale of measurement shifts with with free volume theory to high-concentration mixtures of sugars temperature (Ferry, 1980). The underlying basis of TTS is the and biopolymers (Deszczynski, Kasapis, MacNaughton, & Mitchell, equivalence between time (or frequency) and temperature as they 2003; Kasapis, Al-Marhoobi, & Giannouli, 1999; Kasapis, Des- affect molecular processes that influence the viscoelastic behavior brieres, Al-Marhoobi, & Rinaudo, 2002; Kasapis et al., 2001; Kasapis of polymeric materials and glass-forming small molecules (Slade & & Sworn, 2000) and it has been reported that small addition of Levine, 1993). The criteria for the applicability of TTS are as follows polysaccharides to sugar-containing systems accelerate their vitri- (Ferry, Fitzgerald, Johnson, & Grandine, 1951): (a) shapes of adja- fication (Kasapis et al., 2001). cent curves should match exactly, (b) the same values of aT must The synthetic polymer approach includes the application of the superpose all the viscoelastic functions, and (c) the temperature principle of timeetemperature superposition (TTS), which is also dependence of aT must have a reasonable form consistent with known as the method of reduced variables. TTS has been used for experience. For the last criterion, Williams, Landel, and Ferry (1955) proposed an empirical relationship known as the Williams-Landel- Ferry (WLF) equation. * Corresponding author. Tel.: þ1 608 262 1019; fax: þ1 608 262 1228. The glass transition is relevant to the behavior of food materials E-mail address: [email protected] (S. Gunasekaran). for several reasons. For both polymers and low molecular weight

0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.12.007 142 F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150 glasses, there is a large change in material properties while going 2. Materials and methods through the glass transition. Functional behavior of the material is affected by the dramatic slowing of diffusive processes as a material 2.1. Materials is cooled towards Tg. This slowing will affect stability to crystallization and time-dependent processes associated with crystalliza- Pigskin gelatin (Type B) and laboratory grade sucrose were tion, drying/rehydration and spoilage, when the rate-limiting step purchased from EM Science and Fischer Chemicals, respectively. is the rate of diffusion encountered between substrates or enzyme Glucose syrup, with dextrose equivalent of 43.4 and total solids of and substrate (Noel, Ring, & Whittam, 1993). In addition, it has been 80.5%, was obtained from Cargill, IA, USA (lot number C007138). presumed that at temperatures below Tg amorphous sugars in foods The water content of GS was taken into account in calculating the are stable. Food products are subject to changes in moisture content sample composition. Food-grade XG (lot number 3D0724A) was and temperature during processing and storage, both of which obtained from CP Kelco U.S. Inc., Chicago, IL, USA. decrease stability of amorphous compounds in their rubbery state by increasing temperature difference (T T )(Roos & Karel, 1993). g 2.2. Sample preparation A glass forms when a typical liquid, a state with a disordered molecular structure, is cooled to a temperature generally w100 C Several gelatin-XG systems were prepared. For each, the below its equilibrium crystalline melting temperature (T )or m required amount of gelatin and XG were dissolved separately in freezing point, at a cooling rate sufficiently high to avoid crystalli- deionized water to prepare 10% solution at 75 C and 600 rpm for zation of the liquid. This solidification process, known as vitrifica- 20 min and 4% solution at 60 C and 425 rpm for 2 h, respectively. tion, results in immobilization of the disordered structure of the The required amount of sucrose was mixed with 1/3 part of water in liquid such that the resulting glassy solid is spatially homogeneous, a temperature-controlled kettle. Then GS, gelatin, and XG solution but without any long-range lattice order, and is incapable of were added into the sucrose solution. The mixture was stirred at exhibiting any long-range, cooperative relaxation behavior (e.g., about 90 C for 30e60 min depending on the desired level of total translational mobility) on a practical time scale. The most impor- solids, which was checked by a refractometer (Atago N-3E, Japan). tant distinction between dimensionally extended (a) relaxations, Total solids content of the gels, which were cured overnight in which give rise to the glass transition as translational motions a refrigerator at 0 C, were determined using the AOAC method become restricted at T , and small-scale (b and g) relaxations, for g (AOAC, 1990); the moisture contents were calculated by subtracting which small-scale rotational motions do not become restricted as T total solids content from one hundred. The compositions of all falls below T , is the cooperative nature of a relaxations (Slade & g samples tested are presented in Table 1. Levine, 1993). The gelatin-XG systems were investigated at two moisture Fitting the master curves to WLF equation enables predicting contents (20 and 25%), three gelatin:XG ratios (5:0, 9:1, and 4:1) the T . It has been proposed that the rheological T is a point g g and three GS:sucrose ratios (<1, 1 and >1) at each moisture between the T region and the glassy state. The T can signify the g g content. For each sample, two batches were prepared and tested. transformation from free-volume phenomena of the polymeric backbone in the Tg region to an energetic barrier to motions in the glassy state involving stretching and bending of chemical bonds 2.3. Rheological measurements (Kasapis et al., 2001). Free volume can be defined as holes of the order of molecular (monomeric) dimensions or smaller voids 2.3.1. SAOS associated with packing irregularities. Many properties of liquids, The small amplitude oscillatory shear (SAOS) technique was whether polymeric or not, can be attributed to the presence of used to determine the dependence of viscoelastic behavior on a substantial proportion of free volume. The thermal expansion temperature and time. Freshly prepared samples were loaded onto coefficient of liquids represents the creation of additional free a controlled-stress dynamic rheometer (Bohlin CVOR, Malvern Inc., volume with rising temperature. At high temperatures, where local Southampton, MA) equipped with a 40-mm parallel-plate geom- Brownian motion is rapid in a polymeric fluid or soft solid, lowering etry (1 mm gap). Measurements were performed at a frequency of of temperature is accompanied by collapse in free volume as the 1 rad/s and 1% strain. Samples were loaded at 60 C and cooled molecular adjustments take place freely within a normal experi- down to 15 C at a scan rate of 1 C/min. Mineral oil was used to mental scale. At lower temperatures, the adjustments are slower, cover the exposed edges between the parallel plates to minimize and if crystallization does not occur first, a temperature may be moisture loss. Frequency sweeps tests in the range of 0.1e100 rad/s reached at which the collapse does not occur at all within the were performed interrupting heating runs of 9 Cto24Cat3C experimental time scale. Then the only residual volume contraction intervals. For each batch one measurement was made, and two is of a solid-like character, and whatever free volume is left batches were tested for each sample. presumably remains constant (Ferry, 1980). In synthetic polymer approach, the temperature function 2.3.2. DMA determines how much the frequency scale (i.e., the magnitude of Freshly prepared samples were poured into 17-mm inner the shift factor) changes with temperature. The second character- diameter, 66-mm long aluminum tube molds. The inside surface of istic of the dynamic properties is the time function, which deter- the molds were coated with vegetable oil to prevent the gel from mines how much the storage modulus (G0) and loss modulus (G00) sticking. The ends of the molds were closed with rubber stoppers. are affected by that shift. The estimation of time effect is expressed The tubes were placed vertically in a refrigerator at 0 C for over- by the distribution function of relaxation times, F, which is ob- night. Prior to measurement, the gels were removed from the tained from either G0 or G00 (Kasapis & Sablani, 2000). molds and cut into cylindrical discs. Average aspect ratio (height/ Our objectives were to investigate the gelation kinetics of mixed diameter) of the specimens was 0.42 0.02. system of gelatin and co-solutes glucose syrup (GS) and sucrose in The samples were cooled at a rate of 1 C/min from 0 Cto 0 00 the rubbery and Tg regions as a function of GS:sucrose ratio, 60 C and storage (E ) and loss (E ) moduli were measured using moisture content, and addition of xanthan gum (XG) and to char- a dynamic mechanical analyzer (DMA 7e, PerkineElmer, Chicago, acterize glass (or a) transition as a function of temperature and/or IL) with PyrisÔ software. Experiments were performed in time using the WLF equation and free volume theory. compression mode using a 10-mm diameter parallel plate system F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150 143

Table 1 Composition of each sample.

Sample number Moisture content (%) Gelatin (%) XGa (%) GSb (%) Sucrose (%) Gelatin:XG ratio GS: sucrose ratio 125 5e 40 30 5:0 1.33:1 2 4.5 0.5 40 30 9:1 1.33:1 3 4 1 40 30 4:1 1.33:1 45e 35 35 5:0 1:1 5 4.5 0.5 35 35 9:1 1:1 6 4 1 35 35 4:1 1:1 75e 30 40 5:0 0.75:1 8 4.5 0.5 30 40 9:1 0.75:1 9 4 1 30 40 4:1 0.75:1 10 20 5 e 45 30 5:0 1.5:1 11 4.5 0.5 45 30 9:1 1.5:1 12 4 1 45 30 4:1 1.5:1 13 5 e 37.5 37.5 5:0 1:1 14 4.5 0.5 37.5 37.5 9:1 1:1 15 4 1 37.5 37.5 4:1 1:1 16 5 e 35 40 5:0 0.88:1 17 4.5 0.5 35 40 9:1 0.88:1 18 4 1 35 40 4:1 0.88:1

a XG: xanthan gum. b GS: glucose syrup. at 1 rad/s; the strains ranged from 0.01% to 5%. The purge gas used decrease in temperature, G00 begins to surpass G0, which is recog- was helium. For each sample, two measurements were performed. nized as the onset of glass transition (Kasapis, Abeysekara, Atkin, Deszczynski, & Mitchell, 2002) and the system begins to be glassy (region III). However, it is hard to clearly distinguish viscous and 2.4. Statistical analysis rubbery regions for samples containing XG (Fig. 2), because G0 and G00, though converge at about 40 C, do not intersect over the entire The means and standard deviations of the replicate measure- temperature range studied. Furthermore G0 > G00 at 60 C, indicating ment data were calculated and factorial ANOVA (analysis of vari- that the gelatin-XG system has elastic character even at that high ance) was used to determine the signiﬁcance of differences among a temperature, contrary to our expectation. the treatment levels at p ¼ 0.05, using commercial statistical soft- Since for gelatin-XG samples T values are not detectable from ware (SAS 9.1, SAS Institute Inc., Cary, NC, USA). m their cooling curves, we attempted to determine Tm by applying the WintereChambon criterion (Winter & Chambon, 1986). However, 3. Results and discussion because G0 and G00 were not congruent at any temperature, the WintereChambon plots (Fig. 3) did not reveal gel point for 4.5% 3.1. Cooling curves of gelatinexanthan systems with co-solutes gelatin þ 0.5% XG þ 40% GS þ 30% sucrose system. It is well known that XG forms only transient weak gels above 2% (w/v) since the The cooling curves of gelatin and gelatin-XG mixture are pre- junction zones are weak (Tombs & Harding, 1998, Chap. 5; 00 sented in Figs. 1 and 2, respectively. For gelatin, as expected G Paradossi, Chiessi, Barbiroli, & Fessas, 2002). Thus, XG concentra- 0 dominates G in the viscous region (labeled as region I). As tions we used are too low to even form weak networks; therefore, 0 00 temperature decreases G begins to take over G at a crossover point samples with XG did not exhibit Tm, except one sample (Table 2, called melting point (Tm), and the system enters the rubbery region sample 14). 0 (region II). Comparing to G in Figs. 1 and 2, network formation However, for the gelatin-XG samples the onset of Tg region was accelerates as gelatin concentration decreases. In a study, it was distinguishable (Fig. 2). The Tm and/or temperature at onset of Tg reported that the onset of network formation as a function of region are listed in Table 2 along with their total solids content. The polymer concentration and it was below 30 C for similar gelatin Tm values for gelatin samples appeared to remain approximately containing systems (Kasapis & Al-Marhoobi, 2005). With further

5 log G'

4 III log G" Pa) 3 II 2

Log (G or G I 1

0 -20 -10 0 10 20 30 40 50 60

Temperature (oC) Fig. 1. Cooling curves of storage (G0) and loss (G00) moduli for 5% gelatin þ 40% glucose syrup þ 30% sucrose showing part of viscous region (I) and rubbery region (II) (scan Fig. 2. Cooling curves of storage (G0) and loss (G00) moduli for 4% gelatin þ 1% xanthan rate: 1 C/min, frequency: 1 rad/s). Tm and the onset of Tg were indicated at the end of gum þ 40% glucose syrupþ30% sucrose showing part of viscous region (I) and rubbery region I and region II, respectively. region (II) (scan rate: 1 C/min, frequency: 1 rad/s). 144 F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150

from 75% to 80%, acts as a stronger antiplasticizing agent for the onset of Tg region. Kasapis et al. (2002) reported a shift in the onset of Tg from 14 Cto38 C for a network of gellan gum (0.5%) þ sucrose (50%) þ GS when GS increased from 30% to 35%. The addition of XG or changing GS:sucrose ratio did not signiﬁ- cantly affect the onset temperature of Tg region of gelatin (Altay, 2006).

3.2. Temperature dependence of dynamic properties

Mechanical spectra of samples containing 5% gelatin þ 40% GS þ 30% sucrose and 4% gelatin þ 1% XG þ 40% GS þ 30% sucrose are presented in Figs. 4 and 5, respectively. Both moduli increase with frequency, but the changes in G00 with log frequency are more linear than those of G0, especially at lower temperatures. The samples can be considered elastic because G0 > G00 at all temperatures. To apply TTS, a reference temperature To was arbitrarily chosen (Table 3). The mechanical spectra at temperatures other than To were shifted to the left and right of To along the log frequency axis. This allows relating the viscoelasticity at any stage of the thermal run to that at To as long as the frequency of the former is multiplied by a shift factor, aT. As noted earlier, exact matching of the shape of adjacent curves is a prerequisite for TTS. Figs. 6 and 7, show that e þ Fig. 3. The Winter Chambon plots of sample containing 4.5% gelatin 0.5% xanthan conditions for TTS are met by G0, G00 and tan d (¼G00/G0) within our gum þ 40% glucose syrup þ 30% sucrose (a) at 24 and 30 C, and (b) at 40, 50 and 60 C. experimental temperature range. In Figs. 6 and 7, the left side of the point at which G0 > G00 (the the same at given moisture content; however, they were higher for plateau zone, where log(ua ) < 1) is where the gel-like character is samples containing w25% moisture than w20% moisture content. T intensified with decreasing frequency hence it shows an increasing The pyrolidine-rich regions of gelatin chains act as nucleation separation between the moduli. Kasapis et al. (2001) reported sites for the formation of potential junction zones, which are similar pattern for 1% high-acyl gellan þ 70% GS mixture, which stabilized by interchain hydrogen bonds. Breakage of these was explained as due to the minimum contribution of configura- hydrogen bonds is temperature sensitive and responsible for gel tional rearrangements between junction zones (short) and beyond melting (Ledward, 2000). The increases in T of samples without m junction zones (long) of the high-acyl gellan network to the XG, with an increase in water content, can be explained by more relaxation process. This minimum contribution of configurational interchain hydrogen bonds within gelatin molecules in the abun- rearrangements between junction zones may have been the reason dance of water. Gelatin gels having more stabilized junction points for the separation of G0 and G00 in the plateau zone for the gelatin- by interchain hydrogen bonds probably melt at higher tempera- containing sample. When XG was added to the mixture (Fig. 7), this tures. Statistically, the most important factor affecting the onset of Tg region is moisture content (F < 0.0001) (Altay, 2006). 5.2 -12°C The onset temperature of Tg region changed from below zero to a above zero values when the moisture content was lowered from -9°C -6°C w w 25 to 20% (Table 2). Clearly, higher amount of co-solute, i.e., 4.7 -3°C 0°C

Table 2 , Pa) 4.2 3°C 6°C Total solids content (TSC), melting temperature (Tm) and onset temperature of Tg a 9°C region (Tg-onset) of the samples.

Log (G 3.7 12°C b Sample number TSC (%) Tm ( C) Tg-onset ( C) 15°C 1 74.68 0.77 35.45 0.95 12.57 1.06 18°C 3.2 21°C 2 74.03 0.27 e 7.35 2.65 -1 0 1 2 24°C 3 74.93 0.58 e 8.45 3.05 Log (frequency, rad/s) 4 75.41 1.37 34.65 0.85 7.50 3.50 e 5 75.67 1.40 11.56 1.76 5.5 -12°C e b 6 75.71 0.92 1.75 2.75 -9°C 7 75.93 0.70 34.50 0.50 12.50 0.50 -6°C

e ) 4.5 8 76.29 0.19 8.25 0.95 -3°C e 9 75.30 0.54 4.75 2.25 0°C , Pa 10 81.47 0.12 30.00 0.70 16.25 1.25 3.5 3°C 11 79.16 1.08 e 16.80 3.80 6°C 12 80.03 0.03 e 6.20 2.50 9°C 13 82.18 0.68 30.15 0.35 14.85 1.15 Log (G 2.5 12°C 14 81.14 0.12 38.50 1.00 9.60 2.30 15°C e 15 80.90 0.01 0.95 2.95 1.5 18°C 16 81.54 0.10 32.15 0.15 6.05 0.65 -1 0 1 2 21°C 17 81.82 0.44 e 10.15 1.45 24°C Log (frequency, rad/s) 18 81.1 1.01 e 8.40 0.30

a values reported are mean standard deviation. Fig. 4. Frequency sweeps of sample containing 5% gelatin þ 40% glucose syrup þ 30% b See Table 1 for compositions of different samples. sucrose at different temperatures: (a) storage modulus (G0) and (b) loss modulus (G00). F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150 145

o C ðT ToÞ log a ¼ 1 (1) 10 T o þ C2 T To o o Where, C1 and C2 are WLF constants. Fitting the shift factors of the rubbery/Tg region to the WLF/free-volume framework was done by plotting 1/log aT against 1/(T To) and obtaining the two parame- o o ﬁ ters C1 and C2 from the slope and intercept of the linear t, respectively (Figs. 8 and 9). In terms of the free volume theory, the o o parameters C1 and C2 correspond to B/2.303fo and fo/af, respectively. Where fo is the fractional free volume and af is the coefﬁcient of thermal expansion. For simplicity, value of B was taken as unity (Williams et al., 1955). The form of Eq. (1) is independent of the choice of To. It is useful o o 1 1 to double check the values of C1 and C2 by re-computing C1 andC2 at a second reference temperature, T1 according to the following relationship (Ferry, 1980):

C1C1 o ¼ 1 2 C1 (2) 1 þ C2 To T1

o ¼ 1 þ C2 C2 To T1 (3)

Ferry (1980) also stated that To can be replaced by Tg in Eqs. (1)e(3), o o g g in which case aT, C1 and C2 are referred to as aTg, C1 andC2, respectively. A somewhat more objective procedure is based on the obser- Fig. 5. Frequency sweeps of sample containing 4% gelatin þ 1% xanthan gumþ40% vation from Eq. (3) that glucose syrup þ 30% sucrose at different temperatures: (a) storage modulus (G0) and (b) loss modulus (G00). o ¼ 1h To C2 T1 C2 TN (4)

Where, TN is a fixed temperature at which, regardless of the arbi- separation was smaller, probably due to increasing contribution of trariness of To, log aT becomes infinite per WLF equation. The XG molecules. In Figs. 6 and 7, the right side of the point at which temperature TN is called the Vogel temperature after Vogel, who in 00 > 0 > G G (when log(uaT) 1) is the Tg region which is partially seen. 1921 used a similar characteristic temperature in an empirical Here, the viscoelastic behavior is dominated by the configurational equation for temperature dependence of viscosity. As a rule of rearrangements of the protein and polysaccharide backbones that thumb, TN is usually about 50 C below Tg (Ferry, 1980). are shorter than the distance between the junction zones. The The temperature dependency of viscosity is determined by an temperature/frequency dependence of relaxation processes asso- energy barrier for hole formation, which must be related to the ciated with the chain backbone motions is considered the primary average free volume present. The apparent activation energy for mechanism and it is denoted by a. The characteristic feature of a- viscoelastic relaxation times, DHa is (Ferry, 1980): transition is dissipation of energy seen in the values of tan d exceeding one (partly seen in Figs. 6 and 7)(Kasapis et al., 2001). À Á dðlnaT Þ 2 The upper range of the reduced frequency in Figs. 6 and 7 should DH ¼ R ¼ 2:303RCoCoT2 Co þ T T (5) a dð1=TÞ 1 2 2 o cover the other transitions such as b, g, etc. (if they exist) and eventually the glassy state, which is beyond the temperature range Where, R is the gas constant. DHa is also called as the energy of of this study. Secondary transitions have not been observed in the vitrification (Ev)(Kasapis, Al-Marhoobi, & Mitchell, 2003). The mechanical properties of linear biopolymers (Kasapis et al., 2001). dramatic increase in DHa with decreasing temperature, especially The success of TTS of moduli suggests that no morphological near Tg, can be explained by the drastic decrease in relative free changes have occurred within the material during cooling, and the volume. We defined the activation energies calculated from Eq. (5) moduli displayed similar temperature dependence (Ferry, 1980). as the activation energy for the temperatures at the onset of Tg and This behavior is similar to that of an amorphous polymeric material the energy of vitrification for temperatures at Tg. rather than that of a conventional network (Papageorgiou, Kasapis, The onset temperature of the Tg region (Tg-onset), arbitrarily o o & Richardson, 1994). We used TTS at temperatures below Tm, chosen reference temperature (To), WLF constants C1 and C2 1 1 supposedly after morphological changes occurred around Tm; calculated at To and C1 and C2 , calculated at Tg-onset, activation therefore, TTS is successfully applied to our systems. The phase energies for viscoelastic relaxation times at Tg-onset (Ea), free volume behavior of agarose/getain mixtures in the presence of glucose at Tg-onset (free volume calculated at Tg-onset)(fg-onset), and thermal syrup as co-solute was also modeled successfully using TTS expansion coefficient (thermal expansion coefficient calculated at (Sharma, George, Button, May, & Kasapis, 2011). Tg-onset)(af)atTg-onset are summarized in Table 3 for all samples. o o Although universal values for C1 and C2 are 17.44 and 51.6 K, o ¼ o ¼ respectively C1 8.86 and C2 101.6 K are somewhat better 3.3. Modeling the mechanism of a-transition approximations (Williams et al., 1955). Yıldız and Kokini (2001) stated that WLF constants appear to be material-specific and can The method of reduced variables includes fitting of the function be affected by moisture content therefore should be determined aT(T) to certain analytical expressions. The WLF equation is used to experimentally for food polymers. However, in Table 3 we see that 1 1 w fit empirically derived shift factors in the a-transition: C1 and C2 values for samples containing only gelatin at 20% 146 F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150

Table 3 o o 1 1 The onset temperature of the Tg region (Tg-onset), reference temperature (To), C1, C2, C1 and C2 , activation energy (Ea), fg-onset and af at Tg-onset for all samples. o o 1 1 1 4 Sample Tg-onset ( C) Tg-onset (K) To (Tref) (K) C1 C2 (K) C1 C2 (K) Ea (kJ mol ) fg-onset af at Tg-onset 10 a 1 number at Tg-onset (K ) T1 ¼ Tg-onset (K) 1 13 260 273 4.93 63.56 6.20 50.56 159 0.070 13.85 2 7 266 285 5.83 76.14 7.77 57.14 184 0.056 9.79 3 8 265 273 10.59 125.81 11.31 117.81 129 0.038 3.26 4 8 265 273 5.18 67.13 5.88 59.13 134 0.074 12.49 5 12 261 273 7.34 88.53 8.49 76.53 145 0.051 6.68 6 2 271 273 9.75 115.21 9.92 113.21 123 0.044 3.87 7 13 260 273 4.77 63.87 5.99 50.87 152 0.073 14.26 8 8 265 273 7.16 82.41 7.93 74.41 143 0.055 7.36 9 5 268 273 10.53 97.43 11.10 92.43 165 0.039 4.23 10 16 289 285 10.78 115.54 10.42 119.54 139 0.042 3.49 11 17 290 282 7.87 72.85 7.09 80.85 141 0.061 7.58 12 6 279 273 9.02 89.93 8.45 95.93 131 0.052 5.35 13 15 288 282 10.04 119.06 9.56 125.06 121 0.045 3.63 14 10 283 285 7.01 88.58 7.18 86.58 127 0.061 6.99 15 1 274 273 9.36 105.72 9.27 106.72 125 0.047 4.39 16 6 279 273 11.09 112.59 10.53 118.59 132 0.041 3.48 17 10 283 282 6.35 72.47 6.27 73.47 131 0.070 9.43 18 8 281 273 10.80 107.63 10.05 115.63 131 0.043 3.74

a See Table 1 for compositions of different samples.

moisture content are somewhat closer to 8.86 and 101.6 K than to 14 and 17) compared to those of samples containing only gelatin the universal values. (samples 10, 13 and 16), respectively. At this reduced moisture Activation energies at the onset of Tg region at w20% moisture content, XG molecules may have some positive afﬁnity to gelatin content were smaller than at w25% moisture content (Table 3). It is molecules, leading the total volume occupied by the polymers to probably because network has less free volume for molecular decrease whereas fg to increase. Further addition of XG resulted in motions at lower moisture content than at higher moisture content. 15%, 23% and 39% decrease in free volume for samples 12, 15 and 18 Reduced molecular motions at lower moisture content may lead to compared to samples 11, 14 and 17, respectively, but they are still smaller activation energies. higher than the fg of samples containing only gelatin. At 0.5% XG As expected, free volume of samples containing only gelatin level, fg of the system may increase to a certain point and further (samples 1, 4, and 7 compared to samples 10, 13, and 16) increased addition of XG may occupy more space in this expanded volume of with moisture content at the onset of Tg region (fg-onset), probably the system, therefore decrease in fg. af of samples followed the addition of water gives mobility to the system. Free volume of same pattern as that of fg for both moisture contents (Table 3). At samples at w25% moisture content decreased with the addition of 25% moisture content, af of samples without XG are higher than for XG. This decrease in fg was 46%, 41% and 47% for samples containing samples containing XG, implying that samples without XG collapse 1% XG (samples 3, 6 and 9) compared to those of samples con- at a faster rate (Nickerson, Paulson, & Speers, 2004). At 20% mois- taining only gelatin (samples 1, 4 and 7), respectively. It is probably ture content, samples with 1% XG are less hydrated, ordered and because XG is a very large molecule and therefore increasing levels stable, resulting in larger fg and af (Nickerson et al., 2004). XG lowers fg of the system. However, at w20% moisture content, fg The Tg of the samples were beyond the temperature range of increased with the addition of 0.5% XG. The increase in free volume rheometer measurements. Therefore, Tg was measured using DMA was 45%, 36% and 71% for samples containing 0.5% XG (samples 11, and was deﬁned as the maximum peak of tan d (Table 4). In Figs. 10

6 3 6 3

log G' log G' log G" 5 log G" tan delta 5 tan delta 2 2 4 4 Tan δ or G Pa) ,

3 Tan δ 3 1 1 Log (G or G Pa) , Log (G 2 2

1 0 1 0 -4 -3 -2 -1 0 1 2 3 4 -4 -3 -2 -1 0 1 2 3 4

Log ωaT Log ωaT

Fig. 6. Master curve of the sample containing 5% gelatin þ 40% glucose syrup þ 30% Fig. 7. Master curve of the sample containing 4% gelatin þ 1% xanthan gum þ 40% sucrose. Reference temperature: 0 C. glucose syrup þ 30% sucrose. Reference temperature: 0 C. F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150 147

the addition of 1% XG at w20% moisture content. Taking the onset of Tg values (Table 3) into account, the differences between the onset of Tg and Tg values (Table 4) can be evaluated. The differences between the onset of Tg obtained from rheological measurements and Tg obtained from DMA measurements increased with XG addition at w25% moisture content, indicating an extended introduction to glass transition region. These differences decreased with the addition of 1% XG at w20% moisture content when glucose syrup:sucrose ratios were 1.5:1 and 1:1. This may indicate that the addition of 1% XG started the glass transition earlier compared to other samples at w20% moisture content. In addition, the differences between the onset of Tg and Tg are higher at w20% moisture Fig. 8. The WLF plot of sample containing 5% gelatin þ 40% glucose syrup þ 30% content than the values at w25% moisture content. Presumably, sucrose. lack of moisture plays an important role as a plasticizing agent during glass transition. In Table 4, Tm/Tg ratios are also listed to help determine the and 11, cooling curves of the sample containing 5% gelatinþ40% nature of the mixtures. According to Slade and Levine (1993),ifTm/ GSþ30% sucrose and the sample containing 4% gelatinþ1% XGþ40% Tg < 1.5 and/or (Tm Tg) < 100 C, this type of biopolymers can be GSþ30% sucrose obtained from DMA are presented, respectively. classified as third class of polymers characterized by highly Within the experimental temperature range, the samples generally unsymmetrical structures. The samples containing only gelatin, showed two distinctive regions, the Tg region and the glassy state except for one sample (4.5% gelatin þ 0.5% XG þ 37.5% GS þ 37.5% 7 7.5 with modulus values of 10 e10 Pa at 50 C. Typically values for sucrose), which exhibited Tm values had Tm/Tg < 1.5 and (Tm- high-sugar biopolymer mixtures in the glassy state are between Tg) w 60 C(Table 4). Therefore, these samples may belong to 107.5 and 109.5 Pa, so there appears to be a displacement of about a third class of polymers, with highly unsymmetrical structures. two orders of magnitude, which might be due to instrument Their Tg was near Tm. Furthermore, TN was not 50 C below Tg for o z z compliance (Kasapis, 2008). In the Tg region the viscous component these polymers because C2 50 is true when Tm/Tg 1.5 (Slade & is dominant (when tan d starts to peak), which is related to the Levine, 1993). Therefore, Eq. (4) cannot be applied to our samples. 0 immobilization of the polymeric backbone; in the glassy state E Instead, experimental Tg values were applied as the reference g g becomes dominant (Deszczynski et al., 2003). From the cooling temperature in Eqs. (2) and (3), and the values of C1 and C2 were curves, it can be seen that samples without XG exhibit broader tan d calculated for each samples, and then fg, af at Tg and Ev were than samples containing XG. According to Kasapis et al. (1999), calculated (Table 4). Sharma et al. (2011) were used the WLF a broader transition zone is evident for the heterogeneity in the equation for predicting mechanical Tg for agarose/gelatin mixture protein-sugar mixture. It is interesting to note that samples with in the presence of glucose syrup as co-solute using the theory of XG had narrower transition zones, even though the system had four free volume. components. This may be attributed to the presence of XG, which The Ev of gelatin samples at 20% moisture level was 260, 218 and accelerated the vitrification process leading to narrowing of the 214 kJ/mol at their Tg values 243, 241 and 241 K, respectively. transition zone. Kasapis and Al-Marhoobi (2005) observed similar Kasapis et al. (2003) calculated Ev of 266 and 311 kJ/mol for gelatin/ results for k-carrageenan containing system comparing to gelatin co-solute mixtures at 80% total solids with different molecular containing system. weight at Tg ¼ 234 and Tg ¼ 258.5 K, respectively. For amorphous According to statistical analysis, the most important factor synthetic polymers it is of the order of 260 kJ/mol if Tg ¼ 200 K and affecting Tg is gelatin:XG ratio (F < 0.0001) (Altay, 2006), and Tg 1047 kJ/mol if Tg ¼ 400 Kasapis et al. (2003). The Ev of samples decreased with XG addition. Possibly because addition of XG increased when the addition of XG at 0.5% (Table 4), perhaps due to renders chain segments more flexible, even though free volume of increasing cooperative motions with the addition of XG (Roudaut, system is decreased (Table 4). XG is known for forming weak gels Simatos, Champion, Contreras-Lopez, & Le Meste, 2004). If activa- with weak junction zones (Tombs & Harding, 1998, Chap. 5; tion energies calculated at Tg-onset in Table 3 and Ev calculated at Tg Paradossi et al., 2002). The second important factor affecting Tg was in Table 4 were compared, it could be seen that there was a rise in dependent on both gelatin:XG ratio and moisture content terms of required energy for arrangements of molecules when (F < 0.0001) (Altay, 2006). The Tg decreased with the addition of temperature decreased from the onset of Tg to Tg, meaning that the 0.5% XG at w25% moisture content. The Tg values decreased with difficulty increased for transverse vibrations flexing over several atoms to occur (Kasapis & Sworn, 2000). At w25% moisture content, free volume of gelatin samples was higher than 0.025 reported for polymeric materials at their Tg 6 (Williams et al., 1955); at w20% moisture content, the free volume was lower and was closer to 0.025. At w25% moisture level, the 4 y = -11.876x - 0.0944 2 addition of XG decreased free volume of the system. This is also true R = 0.9991 T 2 for af of samples. Larger af values of gelatin samples at w25% moisture content indicate that these samples collapse at a faster 0 rate. In addition the system is less hydrated, ordered, and stable, 1 / Log a -2 which results in larger fg and af values (Nickerson et al., 2004).

-4 3.4. Relaxation distribution functions of the gelatin-XG systems -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 1 / (T-T ) o In Fig. 12a and b, the dependence of mechanical behavior on 0 Fig. 9. The WLF plot of the sample containing 4% gelatinþ1% xanthan gumþ40% frequency (or time) at constant temperature are represented by G 00 glucose syrup þ 30% sucrose. and G in terms of the ﬁrst and second approximations, 148 F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150

Table 4 Experimental Tg values ( C and K) of all samples obtained from DMA, the Tm/Tg ratios, Tm Tg differences, the WLF coefﬁcients, energy of vitriﬁcation (Ev), fg and ag at Tg. a g g 1 4 1 Sample number Tg ( C) Tg (K) Tm/Tg Tm Tg C1 C2 (K) Ev (kJ mol )atTg fg af at Tg 10 (K ) 1 23.91 0.33 249 1.24 59 7.92 39.56 238 0.055 13.86 2 35.23 0.23 238 ee 15.23 29.14 567 0.029 9.78 3 37.40 1.50 236 ee 15.00 88.81 180 0.029 3.26 4 18.14 1.94 255 1.21 53 7.08 49.13 179 0.061 12.49 5 35.51 1.16 237 ee 12.37 52.53 253 0.035 6.68 6 38.74 1.46 234 ee 14.74 76.21 203 0.029 3.87 7 22.32 0.62 251 1.23 57 7.28 41.87 210 0.060 14.25 8 46.04 2.66 227 ee 16.21 36.41 439 0.027 7.36 9 41.43 0.81 232 ee 18.18 56.43 332 0.024 4.23 10 29.86 2.09 243 1.25 60 16.94 73.54 260 0.026 3.49 11 27.91 0.04 245 ee 15.99 35.85 513 0.027 7.57 12 37.57 0.86 235 ee 15.62 51.93 318 0.028 5.35 13 31.71 2.66 241 1.26 62 15.31 78.06 218 0.028 3.63 14 38.29 2.13 235 1.33 77 16.10 38.58 441 0.027 6.99 15 34.86 2.24 238 ee 13.99 70.72 215 0.031 4.39 16 32.21 1.92 241 1.27 64 15.49 80.59 214 0.028 3.48 17 20.15 0.31 253 ee 10.59 43.47 298 0.041 9.44 18 41.71 0.51 231 ee 17.71 65.63 276 0.025 3.74

a See Table 1 for compositions of different samples.

respectively. For comparing the effect of time on gelatin-XG In the first approximation, the values of F, from the G0 and G00 systems, the distribution function of relaxation times (F), derived data, converge at a line with theoretical slope of 0.5, where, from SAOS data, was used. Although at any frequency G0 and G00 are according to Rouse (1953) and Bueche (1954), the relaxation independent, they are connected through the F transform and spectrum exhibits fairly long-range cooperative motions of the good quality mechanical measurements should return the same chain backbone and these cooperative motions are responsible for value of the distribution function. Details on the derivation of F free-volume phenomena (Kasapis & Al-Marhoobi, 2002). However, have been given previously (Ferry, 1980; Kasapis & Sablani, 2000). it appears that this approach failed to follow the entire derived Master curves of mechanical spectra of shear moduli during curves of F. Therefore, the calculation of the second approximation cooling were obtained spanning several decades of relaxation time. (Kasapis et al., 2003) was used. In doing so, the slope, m (dlog F/ In Fig. 12a, the first-approximation calculations (Ferry, Fitzgerald, dlog s), was obtained at various points of the log F versus log s Johnson, & Grandine, 1951) produced relaxation spectra for curve (Fig. 12a). Matlab (version 7.0.4, MathWorks Inc., Natick, MA, gelatin-XG system. With decreasing time, the values of shear USA) was used to calculate the gamma function for each value of m moduli increased almost four orders of magnitude, and the using related equations (Kasapis et al., 2003), which led to the network exhibited considerable viscous behavior e both are char- estimation of the numerical factors A and B. These were employed acteristics of vitrification. Fig. 12a displays three distinct regions: to shift the F curves to the second approximation (Fig. 12b). It a relatively steep portion at short timescales, which correspond to appears that in the experimental part of the Tg region, values of F 0 0 00 the beginning of Tg region; a flatter portion at longer times where G from G and G are closer to each other than the values of F from the and G00 converge; and the last portion exhibiting complete relaxa- first-approximation calculations and the theoretical slope of 0.5 tion of the system in the flow region where G0 and G00 deviate. according to the Rouse theory was more closely observed.

Fig. 10. Cooling curve of storage (E0), loss (E00) and tan d modulus for the sample Fig. 11. Cooling curve of storage (E0), loss (E00) and tan d modulus for the sample containing 5% gelatin þ 40% glucose syrup þ 30% sucrose. containing 4% gelatin þ 1% xanthan gum þ 40% glucose syrup þ 30% sucrose. F. Altay, S. Gunasekaran / Food Hydrocolloids 28 (2012) 141e150 149

Acknowledgements

We are grateful to Prof. Stefan Kasapis of RMIT University, Australia for his guidance during the initial stages of the study and to Prof. Richard Hartel of University of WisconsineMadison for allowing access to DMA in his laboratory. We also acknowledge Prof. John Lucey of University of WisconsineMadison for his comments during manuscript preparation.

References

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