Rheological studies on mixtures of agar (Gracilaria changii) and κ-carrageenan

Food Hydrocolloids 20 (2006) 204–217 www.elsevier.com/locate/foodhyd

Rheological studies on mixtures of agar (Gracilaria changii) and k-carrageenan M.H. Norziah*, S.L. Foo, A.Abd. Karim Food Biopolymer Science Group, School of Industrial Technology, Food Technology Department, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia

Abstract The rheological properties of agar and k-carrageenan mixtures (with a total polysaccharide concentration of 1.5% w/w) were investigated using dynamic oscillatory measurements, creep compliance tests and compressive deformation measurements. The effects of different agar/k-carrageenan ratios (100/0, 80/20, 60/40, 40/60 and 20/80) on the formation and properties of the gel mixtures were investigated at different pH values (pH 3.5, 4.5 and 5.5). Temperature dependence of G 0 of mixtures showed one step like change during cooling, however, two-steps like change was observed on heating trace. Significant thermal hysteresis was observed in all mixtures, moreover, the observed hysteresis was influenced by those characteristics of k-carrageenan. The gel point as determined by oscillatory measurements of storage and loss modulus (G 0 and G 00 ) of these mixtures show a monotonic decrease in temperature as the proportion of k-carrageenan increased. Additionally, the gelling temperature of the mixtures resembled that of pure agar system. Incorporation of k-carrageenan caused a large reduction in gel rigidity, which arise from sharp drop in G 0 determined by mechanical spectra. Reduction in gel rigidity has also been demonstrated by a marked increased in instantaneous compliance evaluated by creep compliance tests. Young’s modulus, stress and strain at failure were also monitored. In comparison with agar gel, the mixed gels were much more deformable, with a higher failure strain, but had lower strength indicated by a marked decrease in Young’s modulus and failure stress. On the other hand, a decrease in pH below 4.5 caused a sudden drop in the gelling temperature, G 0 and instantaneous compliance. In general, reduction in pH caused gel to be weaker and more brittle, as shown by lower values for Young’s modulus, failure stress and failure strain. These effects were attributed to an increase in the number of shorter chains, which disfavored the formation of junction zones. The results of this study suggest that the gelation of the mixtures appeared to have occurred through a segregative phase separation where agar-rich phase formed a continuous phase and k-carrageenan richphase formed a discontinuous gelled phase. q 2005 Elsevier Ltd. All rights reserved. Keywords: Rheology; Agar; k-Carrageenan; Dynamic and transient properties

1. Introduction In the realm of food development, combination of more than one type of hydrocolloids is commonly used in food to impart novel organoleptic (mouthfeel) to food product, modify rheological characteristic and satisfy processing requirement in the industry. Agars and carrageenans are important hydrocolloids, which are obtained by extraction of certain species of red seaweed (Rhodophyceae). Agars,

* Corresponding author E-mail address: [email protected] (M.H. Norziah).

0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2005.03.020

the gel-forming polysaccharides extracted from Gelidiaceae and Gracilariaceae species, are linear polymers based on a disaccharide repeat structure of 3-linked b-D-galactopyranosyl and 4-linked 3,6-anhydro-a-L-galactopyranosyl units (Araki, 1966). Gelation of agar occurs only by its agarose content, which is produced exclusively by hydrogen bonds (Glicksman, 1979; Armisen & Galatas, 2000). Agar gel is formed by linked bundles of associated right-handed double helices as a result of a coil-double helix transition (Morris, 1986; Schafer & Steven, 1995). The interaction of the helices among themselves occurs at the junction zones and resulted in a three-dimensional network that was capable of immobilizing water molecules in its interstices (Arnott, Fulner, Scott, Dea, Morehouse, & Rees, 1974). Agar is

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compatible with most of the polysaccharides and proteins in the sense that marked degradation do not occur. Among the mixed systems, interaction between agar and locust bean gum has received the most attention. By inclusion of locust bean gum, gel strength, elasticity and deformability of agar gel was synergistically increased as well as syneresis reduced (Jones, 1972; Selby & Wynne, 1973). When agarose interacts with galactomannan in a co-operative fashion, a distinct gel structure was formed (Dea, Mckinnon, and Rees, 1972; Rees, 1972). Maximal value of elastic modulus was observed for a blending ratio of agarose to galactomannan of 5:1 through viscoelastic study (Tako & Nakamura, 1988). Agar gel has been reinforced in canned food through the addition of 0.1–0.2% alginate (Kojima, Inamasu, & Shiraishi, 1959). The addition of sodium alginate to agar gel may also causes cross-linking between alginate and calcium chloride and diffusing into the gel from outside and tends to decrease the strength of agar gel (Nussinovitch, 1997). At high concentration, agarosegelatin gel tends to interface among one another (Moritaka, Nishinari, Horiuchi, & Watase, 1980). Phase separation of the two polymers occurred and phase inversion took place at specific mixture composition (Clark, Richardson, RossMurphy, & Stubbs, 1983; McEvoy, Ross-Murphy, & Clark, 1985). Carrageenans are anionic polysaccharide extracted from Gigartina, Chondrus, Iridaea and Eucheuma species. They exists in three main forms like k-, i- and l. Among them, kcarrageenan is characterised by its repeating disaccharide units of 3-linked b-D-galactose 4-sulfate and 4-linked 3,6 anhydro-a-D-galactose. k-carrageenan having the ability to form a gel in the presence of specific cations such as potassium. Gelation occurs on cooling below the glasstransition temperature by hydrogen bonding that leads to the development of double helix structures. The mechanism of gelation involves coil-helix transition of k-carrageenan molecules (Morris, Rees, & Robinson, 1980). k-carrageenan in mixtures with l-carrageenan will give elastic cohesive gels like gelatin but less prone to syneresis than the later gel (Igoe, 1982). Locust bean gum interacts synergistically with k-carrageenan and results in increase of gel strength, rigidity, elasticity and cohesiveness at certain ratio (Christensen & Trudsoe, 1980; Arnaud, Chapein, & Lacrox, 1989; Turquois, Rochas, & Taravel, 1992). Incorporation of konjac glucomannan with k-carrageenan also has the ability to form strong elastic gel at least four times higher than the rupture strength of pure k-carrageenan system and reduces syneresis (Baker, 1949; Goycoolea, Richardson, Morris, & Gidley, 1995). The gel is heat stable above boiling point after heating and cooling cycle (Thomas, 1992). Tako and Nakamura (1986) claimed that potassium-k-carrageenan in admixture with locust bean gum forms stronger gel than sodium- and calcium- k-carrageenan. The aim of the present work was to study the influence of k-carrageenan on the rheological behaviour of agar Gracilaria changii as a function of pH, employing small

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deformation (dynamic and transient) and large deformation (failure) measurements.

2. Materials and methods 2.1. Materials Powdered agar Gracilaria changii was generously supplied by Eugene Chemical Sdn. Bhd (Penang, Malaysia). Commercial-grade k-carrageenan (Colloid 710 H-96) was kindly donated by TIC GUMS INC (Maryland, USA). According to the label, k-carrageenan sample contain 0.23% sodium, 0.17% potassium and 7.46% calcium. Chemicals such as citric acid (C6H8O7$H2O) and sodium citrate (C6H5O7Na3$2H2O) were purchased from Sigma Chemical Company (St Louis, MO, USA). All the hydrocolloids and chemicals were used without further purification in this study. 2.2. Dynamic rheological measurements Mixtures of agar and k-carrageenan at a total polymer concentration of 1.5% wt were prepared by dispersing the powdered hydrocolloids in citrate buffer solutions (pH 3.5, 4.5 and 5.5) at the different agar/k-carrageenan ratios (100/0, 80/20, 60/40, 40/60 and 20/80) under constant stirring at room temperature. Dispersions were then heated to 90 8C and held for 1 min. It was necessary to keep the sample at elevated temperature to obtain a homogeneous solution and prevent gelation before dynamic rheological measurements (temperature and frequency sweeps) and creep compliance tests were conducted. All samples were prepared in duplicates. Rheological measurements using small amplitude oscillatory shear were performed on the agar/k-carrageenan mixtures using controlled stress CarriMed CSL 100 rheometer (TA Instruments, Surrey, England). For all tests, G 0 , G 00 , tan d were computed from raw oscillatory data using TA Instrument Rheology Advantage Data Analysis software (version 3.0.1) (TA Instruments, Surrey, England). All measurements were performed in duplicate with highly reproducible data (relative standard deviation !10%) for each sample. 2.2.1. Temperature sweep Rheological behaviour of the sol-gel transition of the agar/k-carrageenan mixtures was measured by performing temperature sweep. The hot sample solutions were loaded onto the pre-heated peltier plate (90 8C) of the rheometer and allowed to equilibrate for 5 min. The periphery of the samples was coated with light silicon oil and enclosed within a solvent trap equipped with rheometer, in order to minimize water evaporation. Temperature dependence of storage (G 0 ) and loss (G 00 ) moduli as well as loss tangent (tan d) were observed by cooling the systems from 90 8C to 8 8C, and then reheating to 90 8C linearly after 5 min equilibration

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time. The temperature gradient was 2 8C/min both on cooling and heating scan, while the frequency was fixed at 2. 0 Hz. A cone and plate geometry (6 cm diameter, 28 cone angle, and 55 mm gap) was used in the measurements. As biopolymer gels are strain sensitive during gelation, the linear viscoelastic spectra were measured at a constant strain as low as 0.01, which was well within the linear viscoelastic region. Prior to temperature sweep, dynamic torque sweeps were conducted to choose common linear viscoelastic region for all mixed systems. The gel point temperature (Tgel) was determined by extrapolating the rapidly rising G 0 to intercept the temperature axis (Hsieh, Regenstein, & Rao, 1993). The melting temperature (Tmelt) was taken as the point at which phase angle, d, peaks immediately after a sharp increase (Sarabia, Gomez-Guillen, & Montero, 2000). 2.2.2. Frequency sweep The hot solution of agar/k-carrageenan mixtures was loaded onto the plate preheated at 90 8C, which were then rapidly quenched to 8 8C. The effect of oscillatory frequency on the dynamic rheological properties of the final gel network was evaluated at 8 8C after the gels were equilibrated for 5 min. The mechanical spectra were characterized by G 0 , G 00 and tan d as a function of angular frequency in the range of 0.01–10 Hz. Measurements were carried out with a cross-hatched parallel plate (diameterZ 4 cm, gapZ1000 mm) to avoid gel slippage during oscillation. The strain amplitude used were the same as previously described in temperature sweep. 2.3. Creep compliance measurement The examination of viscoelastic properties of the mixtures was followed by shear creep experiments on the fresh sample by using cross-hatched parallel plate (4 cm diameter, gapZ1000 mm). The hot solutions were transferred directly onto the pre-heated peltier plate (90 8C). A solvent trap was used to prevent loss of moisture by evaporation. The temperature was fixed at 8 8C and measurements were performed after a 5 min aging period. A constant shear stress of 300 Pa was applied instantaneously to the sample and the resultant strains were monitored for 5 min. After completion of the run, the imposed stresses were withdrawn and the extents of strain recovery were recorded for 5 min. The creep compliance, J(t) (ratio of strain to stress) as a function of time was computed by TA Instrument Rheology Solution Software (version 1.1.6) (TA Instruments, Surrey, England). The creep parameter, Jo which is the instantaneous compliance was plotted as a function of polysaccharide ratio. 2.4. Compression measurement The mechanical properties of mixed systems were determined by compression tests using TA XT2i Texture

Analyzer (Stable Micro Systems Ltd., Surrey, England) interfaced with microcomputer and equipped with 5 kg loading cell and 75 mm compression platen. For compression measurements, the solution were prepared in the same way as describe above. The hot solutions were filled into 35 ml Teflon syringes (inner diameter, 2.30 cm; length, 10.30 cm) whose inner surface was pre-coated with parafin grease to prevent sticking. The syringes employed in these experiments have been modified by removing the bottom parts and plugged using rubber stoppers. Finally, the syringes were covered with parafilm to avoid dehydration. The samples were kept at 10 8C for 24 h to allow curing before use. The gels were then removed from the syringes and cut into cylinders of 2.0 cm length. The cylindrical gels were placed between flat surface fitted to the instrument and compressed constantly at crosshead speed of 0.5 mm/s with 70% deformation until failure. The mechanical properties of the gels were evaluated at room temperature by measuring the initial linear slope of the force-deformation curve of the gels giving Young’s modulus (E) which is an indicator of gel firmness as described by Mao, Tang, and Swanson (2000). Failure strain (3f) as an indicator of brittleness of gels and failure stress (sf) an an indicator of gel strength were also determined. The reported mean value for each type of gel was determined with two individually prepared gels as the replicated experimental units and 5 sub-samples tested for each gel. 2.5. Statistical anlaysis Where necessary, the test data were statistically analyzed by Two-way analysis of Variance (ANOVA) using SPSS Version 10.0.1 For Windows (SPSS Inc., Chicago, Illinois). Duncan test was also carried out to perform comparison of means at the 5% significance level.

3. Results and discussion 3.1. Sol-gel transition The viscoelastic studies of agar/k-carrageenan mixtures were made using a constant polymer concentration of 1.5% wt, at pH values of 3.5, 4.5 and 5.5. Fig. 1 shows the temperature dependence of the dynamic moduli (G 0 and G 00 ) during cooling from 90 to 8 8C of different ratios of agar/kcarrageenan mixtures and of pure agar system at pH 3.5. Similar traces were also obtained for mixtures at pH 4.5 and 5.5. The viscoelastic measurements on 1.5% wt agar/kcarrageenan mixtures at all pH showed a maximum in G 0 and G 00 as temperature was reduced from 90 to 8 8C with G 0 being larger than G 00 . The mixtures and pure agar system showed similar G 0 trend upon cooling down to 8 8C, although the temperature at which G 0 increased steeply shifted to lower temperatures, depending on the

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Fig. 1. Temperature dependence of (a) storage (G 0 ) and (b) loss (G 00 ) moduli of agar/k-carrageenan mixtures (1.5%wt total polymer concentration): 100/0 (6), 80/20 (B), 60/40 (7), 40/60 (,) and 20/80 (&) at pH 3.5 during cooling trace. G 0 in open symbols and G 00 in filled symbols.

k-carrageenan content in the mixtures. This may be related to the fact that agar and k-carrageenan undergo similar gelation mechanism. It is widely accepted that the gelation of agarose and k-carrageenan involves a coil-helix transition of the galactan molecules (Viebke & Piculell, 1996). The formation of helices and subsequent association of the helices are essential features in the gelation schemes of agar and k-carrageenan (Stephen, 1995). On heating, two distinct thermal transitions were obtained in all mixtures at pH 3.5 but not in the pure agar system (Fig. 2) and similar traces were also obtained at the other pHs studied. There was a significant change in

the melting behaviour of the mixtures as the proportion of kcarrageenan varied. During heating process, both moduli initially decreased sharply at the lower temperature range and subsequently decreased gradually with increasing temperature. Similar tendency was also observed in kcarrageenan/locust bean gum mixtures (Lundin & Hermansson, 1997) and k-carrageenan/konjac glucomannan (Kohyama, Iida, & Nishinari, 1993). This would indicate that two levels of structure were exhibited in the gel networks and this suggested existence of segregative phase separation. Fig. 2 shows the first melting step occurred in the temperature range between 15 and 38 8C which is close to

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Fig. 2. Temperature dependence of (a) storage (G 0 ) and (b) loss (G 00 ) moduli of agar/k-carrageenan mixtures (1.5% wt total polymer concentration): 100/0 (6), 80/20 (B), 60/40 (7), 40/60 (,) and 20/80 (&) at pH 3.5 during heating trace. G 0 in open symbols and G 00 in filled symbols.

the onset of melting for pure Na-k-carrageenan (1 wt%) at about 20 8C (Lundin & Hermansson, 1997) while the other transition occurred in the range of 72–84 8C, coincident with the melting point of agar i.e around 85 8C (Armisen, 1993). Thereby, during the heating process, it was considered the first decrease in G 0 observed may be caused by the melting of weaker junction zones of non-aggregated k-carrageenan helices while the second step decrease in G 0 may be induced by the melting of stronger junction zones formed by the aggregated agar helices. This observation was characteristically similar to those of gellan gel (Manning, 1992). This

phenomenon can also be explained by Zipper model approach (Nishinari, Koide, Williams, & Philips, 1990). In terms of a Zipper model approach, the appearance of two steps melting in the mixtures suggests that the zippers with different bonding energies or different rotational freedoms may be formed in the presence of k-carrageenan. The lower melting process is attributed to the melting of zippers with lower bonding energies or with higher rotational freedoms while the higher melting process corresponds to the melting of zippers with higher bonding energies or lower rotational freedoms. This again shows that in the mixtures each

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hydrocolloid formed its own junction zones and no specific associative interaction between chains was occurring prior to gel formation. Strong thermal hysteresis between cooling and heating traces was observed in all mixtures, but the extent of thermal hysteresis for the mixtures was clearly reduced with increasing proportion of k-carrageenan (data is not shown). Similar thermal hysteresis was also reported for k-carrageenan/galactomannan mixed systems (Fernandes, Goncalves, & Doublier, 1992) and k-carrageenan/konjac glucomannan systems (Kohyama et al., 1993). Thermal hysteresis is an expected consequence of aggregation (Morris & Norton, 1983). Hence, the origin of these effects may be ascribed to the absence of the helix aggregation of kcarrageenan. According to Viebke, Borgstrom, and Picullel (1995), when a dispersion of k-carrageenan is cooled, the random coil forms of k-carrageenan molecules transformed into double helical conformers. It is widely accepted that the helices associate into rigid rods in the presence of specific gel promoting salts such as KCl (Viebke, Piculell, and Nilsson, 1994). Such interhelical association is usually evident from coil to helix and from helix to coil (Nilsson &Piculell, 1991; Piculell, 1995; Ikeda & Kumagai, 1998). Thermorheological measurements on these mixtures allowed us to correlate to a certain extent the temperature dependence of their structural development and to suggest possible molecular mechanisms. Fig. 3 shows the variation of the gelation temperature, Tgel of the mixtures as a function of polysaccharide ratio at 46.0

pH 3.5

44.0

pH 4.5 pH 5.5

Gelation temperature, Tgel(˚C)

42.0

40.0

38.0

36.0

34.0

32.0

30.0 100/0

80/20

60/40

40/60

20/80

Ratio of agar to κ-carrageenan Fig. 3. Gelation temperature, Tgel of agar/k-carrageenan mixtures (1.5% wt total polymer concentration) as a function of polysaccharide ratio at pH 3.5 (B); pH 4.5 (,); and pH 5.5 (6), (vertical bar represents plus one standard deviation from the mean).

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various pHs studied. As illustrated in Fig. 3, Tgel decreased linearly as the proportion of k-carrageenan increased, and at each combination of the mixtures, Tgel decreased with decreasing pH in general. It is generally accepted that increase in Tgel results from the existence of associative phase separated network or interpenetrating network in a single non-separated phase and on the basis of this consideration it is possible to affirm that such phenomenon is not occurring in the mixtures. Therefore, it is suggested that k-carrageenan does not interact associatively with agar to promote the formation of an ordered structure and that phase arrangement of the two components in the system is a bicontinous arrangement. A possible explanation is the thermodynamic incompatibility and excluded effects, in the absence of favourable intermolecular chains between agar and k-carrageenan, which lead to a decrease in the hydrodynamic volume of each component in the mixtures. Thus segregative phase separation was exhibited in the mixed systems. Tgel of the mixtures (between 38.4 and 43.8 8C) obtained was close to that of simple agar gel (w40 8C) even though the proportion of agar was as low as 20%. The gelation of 1.0% wt of pure Na-k-carrageenan occurred at 19 8C (Lundin & Hermansson, 1997). Therefore, it is likely that the initial three-dimensional network of the mixtures was formed by agar that constituted as the continuous phase, with k-carrageenan forming a discontinuous gelled phase upon cooling. The agar-rich phase forms the continuous phase even at low agar concentrations probably because it attracts more solvent and gels at higher temperature. According to Loren and Hermansson (2000), biopolymer that gels first will mainly determine the topology of the gel structure and consequently govern its gelling properties. Papageorgiou, Kasapis, and Richardson (1994), working with gellan/gelatin mixed gel, reported that the temperature course of gel formation supported the concept of a continuous gellan phase with a discontinous gelatin filler, even at a polymer concentration of 0.075% gellan and 5% gelatin. The mechanism of the gelation of the mixtures was influenced by pH in that Tgel decreased progressively with decreasing pH (Fig. 3). The observed effects could be explained through an understanding of reduction in molecular weight of agar and k-carrageenan induced by acid hydrolysis during thermal processing. Agar and k-carrageenan are relatively less stable to hydrolytic cleavage; and undergo depolymerization which result in lower molecular weight (BeMiller & Whistler, 1996). Carrageenan in the acid form cleaves the molecule at the 3,6-anhydrogalactose linkage and looses gel strength below pH value of about 4.3 (Imeson, 2000) under extensive heating. As a consequence, shorter chains (i.e. lower molecular weight chains) would disfavour the formation of junction zone and the number of hydrogen bonds formed within a junction zones would be comparatively lower (Yoshimura & Nishinari, 1999). This effect tends to delay the development of gel networks through interhelical association.

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In all cases, however, well-defined Tmelt could not be located. Substantial variations in the onset of Tmelt were observed in the heating trace. The uncertainty in determining the precise moment at which dissociation of the crosslinked junction occurs while melting is likely due to the changes in the magnitude of the applied strain during measurement and variation in gel morphology especially when the sample became very thin at a temperature range of 15–50 8C. Therefore, it can be speculated that a very large deformation might have occurred and hindered the helix to coil transition on heating to some extent. Similar phenomenon was also reported in the rheological studies of aqueous dispersions of non-aggregated k-carrageenan helices (Ikeda & Nishinari, 2001). Variations in Tmelt may also probably be due to segregative phase separation in the systems leading to a gel structure with a very wide range of length scale (Loren & Hermansson, 2000). It was found that the temperature band of melting becomes broader which may attribute to a less co-operative process of coil to helix formation during the gelation. Therefore, to elucidate the Tmelt fully, thermal analysis such as differential scanning calorimetry could be performed. 3.2. Mechanical spectra The mechanical spectra shows the dominant character (viscous or elastic) as a function of frequency. Fig. 4 depicts an overlay of the mechanical spectra of agar/k-carrageenan mixtures at different ratios at pH 3.5. The mechanical spectra of the mixtures at pH 4.5 and 5.5 were very similar to their corresponding ones at pH 3.5. The corresponding spectra of the mixtures also possessed characteristic similar in behaviour to those of pure agar system. All mixtures showed highly elastic behaviour with G 0 significantly larger than G 00 , with both moduli being independent over the

Fig. 4. Mechanical spectra of storage (G 0 ) and loss (G 00 ) moduli of agar/k carrageenan mixtures (1.5%wt total polymer concentration): 100/0 (6), 80/20 (B), 60/40 (7), 40/60 (,) and 20/80 (&) at pH 3.5; G 0 in open symbols and G 00 in filled symbols.

frequency studied (0.01–10 Hz), irrespective of the incorporation of k-carrageenan and pH. Furthermore, G 0 of the mixtures was at least two orders of magnitude higher than G 00 confirming true gel characters were perceived in all mixtures. These mechanical spectra also provide the experimental evidence that the overall chain mobility of molecules within the gel network is very low. However, the mechanical spectra of 20/80 mixtures at pH range studied showed a slight concavity in G 00 . This has been associated with existence of a relaxation of a mechanism, which is normally present at lower angular frequencies. The existence of concavity is characteristic of k-carrageenan component since it is observed in pure k-carrageenan but not in agar. Physical gels (i.e. non-covalent gels) like PVC, gelatin and amylose-k-carrageenan mixtures have been reported to exhibit similar behaviour (te Nijenhuis, 1997; Tecante & Doublier, 2002). In addition, it is also noted that inconsistent results i.e. a wide range of variation in G 0 and G 00 were obtained for 20/80 mixtures at pH 5.5. As proposed by Richardson and Goycoolea (1994), inconsistent result could be attributed to the syneresis of k-carrageenan, from which the released water creates slip when conducting dynamic measurements. Slippage may result in erroneous measurements and syneresed water could have caused a sharp drop in G 0 (Richardson & Goycoolea, 1994). However, in the present study, existence of G 0 peak and water losses were not observed and a perfect sinusoidal torque was obtained during measurements. Furthermore Arnaud et al. (1989) reported that a perfect sinusoidal torque is an indication that no sliding effect could have occurred. Therefore, irreproducible results were not likely caused by practical problem. On the basis of evidence from mechanical spectra, however, it seems reasonable to conclude the underlying structure of the mixtures under the influence of pH appears somewhat more complex. The G 0 values obtained for agar/k-carrageenan mixed systems at a frequency of 1 Hz are plotted against the agar/k-carrageenan ratio at the 1.5% total polymer concentration in Fig. 5. G 0 of the mixtures progressively decreased as the proportion of k-carrageenan increased to 40%, but subsequently increased again when the proportion of k-carrageenan was further raised. Nevertheless, the presence of k-carrageenan in different proportions, resulted in a lower rigidity indicated by G 0 as compared to pure agar gel of equal concentration; therefore, it is apparent that enhancement of the mechanical properties did not occur. This would suggest that in agar/k-carrageenan systems no interaction between chains occurred (mutual incompatibility) and each polysaccharide formed its own junction zones. It is generally accepted that the enhancement of G 0 results from the existence of interpenetrating network (Piculell, Nilsson, & MuhrDeck, 1992; Nishinari, Watase, Rinaudo & Milas, 1996) in a single non separated phase or associative phase separated network; and on the basis of this consideration, it is possible to affirm that such phenomenon

M.H. Norziah et al. / Food Hydrocolloids 20 (2006) 204–217 20.0 pH 3.5

18.0

pH 4.5 pH 5.5

Storage modulus, G’ (kPa)

16.0 14.0 12.0 10.0 8.0 6.0 4.0

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The values of G 0 dramatically decreased with decreasing pH (Fig. 5), with the exception of 80/20 mixtures, presumably due to the degradation of both polysaccharides by acid hydrolysis. As previously described in Sub-Section 3.1, molecular weight of k-carrageenan decreased as a consequence of acid hydrolysis. For some polymer gel, the rigidity was found to decrease with decreasing molecular weight (Mitchell, 1980). On reduction in molecular weight of components, the probability that junction zones formed decreased, and the flexible molecular chains connecting junction zone will become shorter. Thereby, the number of elastically active chains decreases and hence the rigidity of gels decreases with decreasing molecular weight. However, at particular ratio 80/20, the rigidity of the gel network (G 0 ) are less pH-dependent. 3.3. Transient properties

2.0 0.0 100/0

80/20

60/40

40/60

20/80

Ratio of agar to κ -carrageenan Fig. 5. Effect of agar/k-carrageenan ratio on the storage modulus (G 0 ) of mixed gels at 1.5% total polymer concentration at pH 3.5 (B); pH 4.5 (,) and pH 5.5 (6). Frequency at 1 Hz, (vertical bar represents plus one standard deviation from the mean).

did not occur in these mixtures. In other words, the results suggested the existence of segregative phase-separated network in the systems. It has been known that only small differences in the chemical structures of the polysaccharides can lead to incompatibility. This mutual incompatibility generally leads to phase separation (Garnier, Schorsch, & Doublier, 1995). Furthermore, the rigidity (G 0 ) of the mixtures progressively decreased as k-carrageenan gradually increased in proportion revealing what can be referred to as a dilution effect of the k-carrageenan. It is likely that in the mixtures, when the proportion of k-carrageenan increased, the number of junction zones substantially decreased resulting in a reduction in the number of elastically active chains by a competitive effect for solvent between agar and k-carrageenan. Similar effects were also reported in gellan/k-carrageenan and gellan/i-carrageenan (Nishinari, Watase, Rinaudo, & Milas., 1996; Rodriguez-Hernandez, & Tecante, 1999). G 0 increased substantially at ratio of O40% for k-carrageenan suggests that k-carrageenan being in the greater proportion began to contribute to the rheological behaviour as it gradually approached its gel forming concentration. Gel forming concentration of kcarrageenan in KCl- free medium is nearly 1.0 wt% at 25 8C (Rochas & Rinaudo, 1980). This is confirmed by the fact that at 80% of k-carrageenan, the mechanical spectra (Fig. 4) are no longer similar in shape to the spectra of simple agar and mixtures with low concentration of kcarrageenan.

In addition to the above oscillatory measurements, viscoelastic behaviour of agar/k-carrageenan mixtures was also studied by performing creep-compliance tests. Creep experiments for physical gels are complicated by a number of experimental factors and thus there are surprisingly few data in the literature, most of which were on single component biopolymer gels such as cold-set gelatin (Higgs & Ross-Murphy, 1990; Normand & Ravey, 1997). For mixed biopolymer gels creep measurements are still sparse. The creep-compliance tests for the agar/k-carrageenan mixtures were carried out by using a constant and low amplitude stress (300 Pa). Fig. 6 shows the typical creep and recovery curves for the mixtures reflecting transient properties. Both the creep and recovery phases of the mixtures were qualitatively similar to those of simple agar.

Fig. 6. Typical creep/recovery response of agar/k-carrageenan mixtures (1. 5% wt total polymer concentration).

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The creep phase is relatively flat and close to horizontal and there is an instantaneous elastic response followed by a constant compliance during recovery period (almost fully recoverable deformation in the recovery region). As shown in Fig. 6, all mixtures exhibited a pattern typical of viscoelastic solids. In all cases the gels were almost recovered to their original states, and permanent deformation was small after removal of stress, indicating an elastic gel structure. The higher the recovery phase, the higher is the viscoelastic solid character of the sample. In addition, it is obvious that the systems very quickly approached the equilibrium compliance conditions, whereby its magnitude reflects the number and density of permanent cross-links, confirming that all mixtures have apparently more elastic and flexible structures. An initial damped shear strain oscillations induced by instantaneous stress was observed in all mixtures during the beginning of the creep and recovery phase in w1.0 s (Fig. 7). The damping effect revealed that the viscoelastic

response can be acquired in short time scale wless than 1 s of data. This damping effect is induced by the sudden increase in stress, coupled with the instrument and sample inertia. This is consistent with that reported earlier by Normand and Ravey (1997); Lefebvre, Renard, and Sanchez-Gimeno (1998); Gilsenan and Ross-Murphy (2001). According to Zolzer and Eike (1993), damped shear strain oscillations is likely due to the mechanical coupling between viscoelasticity of the sample and the inertia of the rotating part of the rheometer.The damped strain oscillations showing ringing behaviour is also known as creep ringing. As illustrated in Fig. 7, the efficiency of the damped shear strain oscillations decreased as the proportion of k-carrageenan increased (i.e. increases in J(t)). Conversely, the increase in the proportion of k-carrageenan in the mixtures gave rise to higher amplitude of the damped shear strain oscillations. In other words, concomitant with the increase in amplitude of creep ringing was a decline in the efficiency of the damping as the proportion of k-carrageenan in the mixtures was increased. Thereby, decrease in rigidity (1/J(t)) of the mixtures may reflect in a decrease in the efficiency of the creep ringing, accompanied by an increase in the amplitude of the creep ringing. Fig. 8 shows the creep parameter as a function of polysaccharide ratio across the pH range studied. An increase in the Jo parameter of the mixtures was observed with an increase in k-carrageenan proportion up to a critical point before decreasing, i.e. a peak was observed (Fig. 8). The analysis of Jo data suggested that the presence of 1.80 pH 3.5

Instantaneous compliance, Jo x 10-4 (Pa-1)

1.60

pH 4.5 pH 5.5

1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 100/0

80/20

60/40

40/60

20/80

Ratio of agar to κ -carrageenan

Fig. 7. Creep compliance curves of agar/k-carrageenan mixtures (1.5% wt total polymer concentration) on an expanded scale.

Fig. 8. Instantaneous compliance of agar/k-carrageenan mixtures (1.5% wt total polymer concentration) as a function of polysaccharide ratio at pH 3.5 (B); pH 4.5 (,) and pH 5.5 (6), (vertical bar represents plus one standard deviation from the mean).

M.H. Norziah et al. / Food Hydrocolloids 20 (2006) 204–217 50.0 pH 3.5 pH 4.5 45.0

35.0

30.0

25.0

20.0

15.0 100/0

80/20

60/40

40/60

20/80

Ratio of agar to κ-carrageenan Fig. 9. Young’s modulus, E of agar/k-carrageenan mixtures (1.5%wt total polymer concentration) as a function of polysaccharide ratio at pH 3.5 (B); pH 4.5 (,) and pH 5.5 (6), (vertical bar represents plus one standard deviation from the mean).

3.4. Large deformation mechanical properties

45.0 pH 3.5 40.0

pH 4.5 pH 5.5

35.0

Failure stress,σ f (kPa)

Fig. 9 shows Young’s modulus (E) of the mixtures as a function of polysaccharide ratio across pH range studied. Generally, E was drastically reduced when the proportion of k-carrageenan increased from 20–60%, but subsequently increased again as the proportion of k-carrageenan was further raised to 80%. This tendency was consistent with the results obtained from small deformation measurements. This is probably true because, in theory, E characterizes the linear elastic behaviour at small deformation. On the other hand, effect of the presence of k-carrageenan on the gel strength (indexed by sf) appeared to be pH dependent (Fig. 10). For instance, at pH 3.5, the sf initially decreased with increasing k-carrageenan proportion to a minimum value and then increased slightly with further increase in kcarrageenan proportion. While at pH 4.5 and 5.5, the sf decreased markedly as the k-carrageenan proportion is raised and then gradually leveled off when the kcarrageenan proportion within the region 40–60%, but subsequently increased again on further addition of kcarrageenan. Increase in gel strength as proportion of kcarrageenan is raised to 80% is likely attributed to the large amount of k-carrageenan that began to contribute to the gel properties. At all k-carrageenan proportion in the mixtures studied, however, the gel strength of mixed gels were far below the gel strength of pure agar gel. The decrease in

pH 5.5

40.0

Young's Modulus, E (kPa)

k-carrageenan significantly affect the viscoelastic properties of the mixtures. Since Jo is the reciprocal of a viscoelastic constant (G 0 ), lower Jo means higher G 0 and higher rigidity of the gel. As the proportion of k-carrageenan increased, decreases in gel rigidity (i.e., increases in Jo) was observed. It is suggested that the addition of k-carrageenan seems to lower the rigidity of the mixtures. The pattern of the structure rigidity (G 0 ) observed from small amplitude oscillatory testing is in agreement with the monitored Jo. An exception was observed in 20/80 mixture at pH 5.5. As mentioned before, inconsistent G 0 were also observed in the frequency sweep. However, this phenomenon was not observed in creep measurements. It is likely that the gels were not in true equilibrium and the cross-links underwent a slow change with time during oscillatory measurements. Long time creep experiments might be able to eliminate the contribution of topological entanglements to the measured rigidity of a networks, but they can leave intact the remaining physical interaction (permanent junction zones) to support the stress (Chronakis, 1996). The creep behaviour of the mixtures was also studied as a function of pH. The reduction in pH lowered the rigidity (1/Jo) of the mixtures (Fig. 8). As noted before, there is some evidence in the data reported previously, that reduction in pH gave rise to the depolymerization of the polysaccharide molecules and thus led to a decrease in the number of elastically active chains connecting the junction zones.

213

30.0

25.0

20.0

15.0

10.0

5.0

0.0 100/0

80/20

60/40

40/60

20/80

Ratio of agar to κ -carrageenan Fig. 10. Failure stress, 7f of agar/k-carrageenan mixtures with 1.5% wt total polymer concentration as a function of polysaccharide ratio at pH 3.5 (B); pH 4.5 (,) and pH 5.5 (6), (vertical bar represents plus one standard deviation from the mean).

M.H. Norziah et al. / Food Hydrocolloids 20 (2006) 204–217 14.0

Moduli (kPa)

12.0 10.0 8.0 6.0 4.0 2.0 0.0 100/0 80/20 60/40 40/60 20/80 Ratio agarto κ-carrageenan 20.0

Moduli(kPa)

the sf and E with the incorporation of k-carrageenan indicates that the gel becomes easier to fracture. The results suggest that the depression in gel properties was likely due to the mutual incompatibility between agar and kcarrageenan that gave rise to segregative phase separation. Hence, the formation of the gel network did not involve strands of both types of polymer. In other words, agar and kcarrageenan may form the polymer network separately. This was confirmed by dynamic oscillation measurements and creep measurements. In contrast to the shape of curves of sf and E with varying polysaccharide ratio, the 3f of the mixtures monotonically increased with increasing k-carrageenan proportion (Figs. 11 and 12). This indicates that the mixed gel also become less brittle and permit substantial elongation of the gel strips without breakage in comparison with agar gels only as the kcarrageenan gradually increased in proportion. Similar tendency was also observed in k-carrageenan/konjac glucomannan mixtures (Kohyama et al., 1993) and kcarrageenan/locust bean gum mixtures (Dunstan, Chen, Liao, Salvatore, Boger, & Prica, 2001). The above can be rationalized by considering a transformation of the aqueous polysaccharide network to a structure of reduced crosslinking where substantial parts of the chains are flexible entities capable of extensive stretching before relaxing as the k-carrageenan gradually increased in proportion. As a conclusion, addition of k-carrrageenan caused mixed gels to be less brittle but easily fracture. According to Hamann and

15.0 10.0 5.0 0.0 100/0 80/20 60/40 40/60 20/80 Ratio agar to κ-carrageenan

Moduli (kPa)

214

18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 0.0 100/0 80/20 60/40 40/60 20/80 Ratio agarto κ-carrageenan

Fig. 12. G 0 and one-third of Young’s modulus (1/3E) for agar/kcarrageenan mixtures (with 1.5% wt total polymer concentration at (a) pH 3.5, (b) pH 4.5 and (c) pH 5.5 G 0 in filled sysmbols and 1/3 E in open symbols.

0.55

0.50

Failure strain, εf

0.45

0.40

0.35

0.30

0.25 100/0

80/20

60/40

40/60

20/80

Ratio of agar to κ-carrageenan Fig. 11. Failure strain, 3f of agar/k-carrageenan mixtures with 1.5% wt total polymer concentration as a function of polysaccharide ratio at pH 3.5 (B); pH 4.5 (,) and pH 5.5 (6), (vertical bar represents plus one standard deviation from the mean).

MacDonald (1992), gel strength and brittleness can vary independently. The effect of pH on the sf, 3f and E of the mixtures was also studied in present work. The trend that sf, 3f and E decreased with decreasing pH was similar with that observed with pure agar. Across the pH range studied, pH 3.5 gave a more pronounced effect on the sf, 3f and E. The mixed gels became more brittle at acidic pH. The gel strength and firmness of the mixed gels was also considerably lower at lower pH. One reason for the difference could be the differing contour length of flexible chains, which connect junction zone (Normand, Lootens, Eleonora, Kevin, & Aymard, 2000). The mixed gels formed at higher pH had longer and more flexible chains than the gels at acidic pH. A long, flexible chain can be extended further before it breaks, thus explaining the higher strain at fracture at high pH. The short chains formed at acidic pH appeared stiff, which may be correlated to the fragile behaviour of these gels. This could be attributed to the fact that under acidic conditions, both polymer suffer acid hydrolysis, reducing its molecular weight and consequently

M.H. Norziah et al. / Food Hydrocolloids 20 (2006) 204–217

decreasing the elasticity of the gel network and also the connectivity of the gel network. G 0 values at 1 Hz obtained from mechanical spectra were also compared with Young’s modulus (E) data as shown in Table 1. It was found that G 0 values reported are consistent with one-third of E. This result satisfied the Poisson relationship between G 0 and E. Theoretically, for a perfectly incompressive gels, E and G 0 of the gel should satisfy the relationship of Ez3 G 0 when the Poisson’s ratio is found to be near 0.5 (Chen, Liao, Boger, & Dunstan, 2001).

215

cross-linking chains, which enhanced chains alignment but disfavoured the formation of junction zones, as a result of acid hydrolysis.

Acknowledgements The financial support by the Ministry of Science, Technology and Innovation, Malaysia under the IRPA grant is gratefully acknowledged.

4. Conclusion The results of the present study show that the addition of k-carrageenan appeared to affect the rheological properties of agar only. The effect was not of a synergistic type. In the presence of k-carrageenan, the gelation characteristics of agar gel only are still clearly evident in the mixtures, but the gelation points are displaced to lower temperatures. The melting of these mixtures proceeded in two discrete steps, corresponding to the transitions of the individual components. Mechanical spectra obtained also showed that the presence of k-carrageenan resulted in a substantial loss in gel rigidity. Solid elastic behaviour was observed in these mixtures. Reduction in rigidity was also evident by transient properties determined over long relaxation times. Furthermore, addition of k-carrageenan led to a marked decrease in the firmness, gel strength and the brittleness of the resulting gels. These effects revealed that the mixtures undergo phase separation, with agar-rich phase forming a continuous phase and k-carrageenan rich-phase forming a discontinuous gelled phase. The rheological properties of the mixtures in both small deformation and large deformation rheology, significantly decreased with decreasing pH. This, in principle, could be due to the reduction in the length of Table 1 The values of G 0 (at 1 Hz) and one-third of E for agar/k-carrageenan mixtures (1.5 wt% total polymer concentration) across pH range studied Sample PH

Ratio

3.5

100/0 80/20 60/40 40/60 20/80 100/0 80/20 60/40 40/60 20/80 100/0 80/20 60/40 40/60 20/80

4.5

5.5

G 0 (kPa)

1/3E (kPa)

13.03 11.50 4.46 5.05 6.27 17.19 12.33 7.09 6.66 9.75 17.03 12.15 7.91 9.71 –

13.31 9.34 5.92 5.34 7.12 15.14 10.32 7.87 7.87 11.46 15.20 11.05 7.75 8.65 12.75

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