Relation between the structure of matrices and their mechanical relaxation mechanisms during the glass transition of biomaterials: A review

Food Hydrocolloids 26 (2012) 464e472

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Relation between the structure of matrices and their mechanical relaxation mechanisms during the glass transition of biomaterials: A review Stefan Kasapis* School of Applied Sciences, RMIT University, City Campus, Melbourne, Vic 3001, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 May 2010 Accepted 30 September 2010

It has been demonstrated that industrial polysaccharides (agarose, deacylated gellan and k-carrageenan) form networks of reduced enthalpic content in the presence of high levels of non-crystallizing co-solute (e.g., glucose syrup) that exhibit timeetemperature dependent behaviour of a typical rubberlike polymer. In contrast, amylose holds its structural characteristics unaltered and does not reach a state of molecular mixing with glucose syrup, with morphological features being those of a micro phase-separated mixture. Variation in phase morphology and density of intermolecular associations leads to entropic or enthalpic viscoelasticity in systems, and it was utilised to define distinct classes of food related biomaterials exhibiting an extensive glass transition region or absence of vitrification phenomena. The approach was extended to encompass the experimental parameters of a porous matrix and the application of hydrostatic pressure. In the former, work discusses discrepancies in the Tg e porosity relationship attributable to the different extent to which the two techniques of calorimetry and mechanical spectroscopy respond to degrees of molecular mobility. In the latter, it was shown that the timeetemperatureepressure equivalence of synthetic amorphous polymers is not operational in the glass-like behaviour of high sugar systems in the presence of gelatin or gelling polysaccharides. The existing body of evidence allowed quantitative treatment of results based on the asymmetric distribution theory of molecular relaxation time that identifies the chemical fingerprint of the local motions operating at the vicinity of Tg. Furthermore, the diffusional mobility of a bioactive compound within a glassy matrix could be followed in relation to temperature induced changes in free volume using the timeetemperature superposition principle. Ó 2010 Published by Elsevier Ltd.

Keywords: Mechanical Tg Thermomechanical complexity Hydrostatic pressure Porosity Caffeine diffusion

1. The passage from elastic to entropic networks with increasing levels of co-solute in biopolymer matrices Over the past few years a considerable amount of work has been done in this research group on the measurement of dynamic mechanical and stress relaxation properties of biopolymers in mixture with increasing levels of co-solute. The main object of this work has been to establish correlations between these properties and the three-dimensional structures of the macromolecules. A stage has now been reached at which it appears possible to formulate several working protocols capable of relating structural features and the chemical nature of the polymeric repeat unit to changes of magnitude in observations obtained at the glass transition region. Furthermore, “complications” that may arise by adding to the effect of temperature and molecular weight of

* Tel.: þ61 3 9925 5244; fax: þ61 3 9925 5241. E-mail address: [email protected]. 0268-005X/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.foodhyd.2010.09.019

polymers that of hydrostatic pressure and porous matrices can be reasonably explored (Blaszczak, Valverde, & Fornal, 2005). In model systems of biopolymer/co-solute with relevance to confectionery products, formulations include polysaccharide concentrations below 2.0% in the presence of high levels of sugar (up to 90.0%). Fig. 1 reproduces normalized data of the storage modulus recorded for various polysaccharide-sugar systems at ambient temperature. It constitutes an attempt to summarize the variation in mechanical properties, as found in the literature, in a single succinct graph presentation. Addition of sucrose, fructose, glucose or glucose syrup up to 40% (0.5e2 mol L
1) creates stronger and more thermally stable structures for the industrially important polysaccharides of k-carrageenan, agarose and deacylated gellan gum (Watase, Nishinari, Williams, & Phillips, 1990), and this trend of network reinforcement is also seen in the amylose matrix. The nature of modulus development is entirely different at intermediate levels of co-solute broadly defined between 40.0% and 65.0% solids. There is an abrupt drop in the values of storage modulus in mixtures of sugar with the industrial polysaccharides

S. Kasapis / Food Hydrocolloids 26 (2012) 464e472

Log (Normalised shear modulus)

5 4 3 2

amylose + sugar 1 0

sugar

-1 -2

coil-to-helix polysaccharide + sugar

-3 0

10

20

30

40

50

60

70

80

90

100

Sugar Concentration (%) Fig. 1. Variation of normalized storage modulus on shear (frequency of 100 rad s
1) as a function of sugar concentration for amylose/sugar, coil-to-helix polysaccharides (agarose/sugar, k-carrageenan/sugar, deacylated gellan/sugar), and single sugar preparations.

(Kasapis, Al-Marhoobi, Deszczynski, Mitchell, & Abeysekera, 2003). Amylose, on the other hand, keeps or accelerates the strengthening of the network, an outcome which yields a wide loop between the structural profiles of the two types of systems. In the former, these observations were rationalized by considering that stabilization of stress-supporting polymerepolymer interactions require hydrogen bonding from the surrounding (aqueous) environment (Chandrasekaran & Radha, 1995). The increasing shortage of water molecules and their relatively stable hydrogen bonding with sugar deprives potential polymeric associations with a stabilizing hydration layer thus increasing the disordered component of the polysaccharide network. In contrast, partial polymer disaggregation is not evident in the amylose sequences that maintain cohesion in the glucose-syrup environment. The structural disparity between the two types of networks was further probed using differential scanning calorimetry at a low scan rate (1  C/min). Cooling runs of aqueous solutions of the industrial polysaccharides produce well-defined and sharp exothermic events that vary in size, shape and temperature range (Nitta et al., 2003). The area underneath these peaks can be used to obtain values of change in enthalpy (DH) of the coil-to-helix transition induced by cooling. Addition of co-solute at intermediate levels of solids (and beyond) results in broad exotherms of reduced DH values arguing for a gradual ordering process and a reduction in the cooperativity of helix formation (Al-Marhoobi & Kasapis, 2005). None of the above is observed in the amylose thermograms where the temperature variation of heat flow for aqueous and intermediate-solid amylose preparations exhibits a monotonic baseline drift down to 0  C (Shrinivas, & Kasapis, in press). It was concluded that the absence of a coil-to-helix transition for amylose argues against the necessity of hydrogen bonding with the aqueous environment as an essential part of the process of structure development and stabilization in this system. The contrast between the phase behavior of amylose/co-solute mixture and industrial polysaccharides can serve as a guide for similar work in other highsolid preparations of contemporary interest, and to facilitate a direct link with mechanistic models that follow glass transition phenomena (Li et al., 2003; Ngai, Magill, & Plazek, 2000).

Referring back to Fig. 1, it is clear that the drop in the mechanical strength of industrial polysaccharides is followed by an equally spectacular increase in the values of shear storage modulus at the upper range of co-solute (>65.0% solids). This is associated with an increasing vitrification of the high-solids system where macromolecules form structures of reduced enthalpy of cross-linking (Kasapis, 2006). The increased flexibility of chain segments between intermolecular associations renders important the entropic contribution to network’s elasticity thus shaping up a rubber-to-glass transition (Ferry, 1991). Indeed, only at very high levels of solids (90%) the kinetics of sugar vitrification match those of the polysaccharideesugar mixtures and this has been attributed to extreme removal of water molecules from the polysaccharide domains so that formation of a three-dimensional structure is abandoned. This pictorial manifestation of vitrification in the examples of Fig. 1 is not exclusive but has been reproduced for a plethora of high sugar/biopolymer mixtures thus begging the question of the universality of viscoelasticity in these systems (Kasapis, 2001). Such uniformity in viscoelastic patterns should allow pinpointing the glass transition temperature (Tg), which is one of the main indicators utilised to control organoleptic quality and consistency in processed food products (Slade & Franks, 2002, p. x). In doing so, the theory of free volume has been widely employed in a “quantitative fashion” to interpret glassy phenomena in terms of molecular processes (Dlubek et al., 2003). The term “quantitative fashion” refers to the WLF equation and the timeetemperature superposition principle that are utilised to pinpoint Tg. Conventionally, this should be located at the end of the glass transition region where the free volume declines to insignificant levels, i.e. about 3% of the total volume of the material (Rieger, 2001). Fig. 2 summarizes the values of Tg obtained mechanically using the above mentioned protocol and calorimetrically for biopolymer/ sugar mixtures ranging in level of solids from 73 to 93% (Kasapis, 2008). There is a hundred-and-twenty degree increase in the DSC

60

DSC sugar

50

agarose/sugar

κ-carrag/sugar

40 30

Rheology sugar 4MW gelatin/sugar

HM pectin/sugar

deac gellan/sugar 2DE pectin/sugar

deac gellan/sugar

LBG-guar/sugar

κ-carrag/sugar agarose/sugar chitosan/sugar

20 10

Tg (°C)

6

465

0 -10 -20 -30 -40 -50 -60 70

75

80

85

90

95

100

Solids content (%) Fig. 2. Variation of the calorimetric glass transition temperature with solids content of sugar (B), 0.7% agarose þ sugar (,), 0.7% k-carrageenan þ sugar (6), 1% pectin (DE of 92) þ sugar (>), and 1% deacylated gellan þ sugar (); and of the rheological glass transition temperature for single sugar samples (C) and in mixture with 25% gelatin (four Mn of 68, 55.8, 39.8 and 29.2 kDa; -), 0.5% deacylated gellan (þ), 1e1.3% pectin (two DE of 92 and 22; d), 0.5e1% LBG or guar gum (:), 0.5e1% k-carrageenan (A), 0.7% agarose ( ), and 2% chitosan (
), as indicated in the inset.

466

S. Kasapis / Food Hydrocolloids 26 (2012) 464e472

measured Tg of sugar and sugar/biopolymer samples. This is in direct contrast to the increase in the values of the mechanical Tg due to the addition of biopolymers to sugar samples. Thus, addition of gelatin fractions of increased molecular weight (Mn), pectin of increased degree of esterification (DE), deacylated gellan with added sodium ions and k-carrageenan with potassium counterions, i.e. characteristics that enhance gelation, accelerate the mechanical manifestation of vitrification in these systems. It was concluded that the apparent acceleration of vitrification is related to the ability of the biopolymer to form a network (Tolstoguzov, 2003), thus making the mechanical Tg synonymous to a network Tg. Network formation is a macromolecular process which rheology is well qualified to follow (Windhab, 1996/7). In contrast, calorimetry provides information primarily on the mobility of the sugar molecules (Aubuchon, Thomas, Theuerl, & Renner, 1998), and the small addition of biopolymer in this case is a mere cross-contamination. 2. Molecular mobility of polymeric associations in relation to the manifestation of glass transition phenomena The glass transition temperatures discussed thus far were recorded for biopolymer/sugar mixtures that exhibit a strong temperature dependence of shear storage modulus. An example of this spectacular development of viscoelastic functions is illustrated in Fig. 3 for the cooling profile of 0.5%-carrageenan in the presence of 85% co-solute (Evageliou, Kasapis, & Hember, 1998). This is a typical case of an extensive master curve of viscoelasticity spanning the entirety of the rubber-to-glass transition (Zhao, Morgan, & Harris, 2005), and we felt that it merits detailed discussion. The onset of network formation of k-carrageenan occurs at temperatures higher than 80  C, as judged by the frequency-dependent index of G’ overtaking G", during cooling. The second thermal transition (G" becomes greater than G’), which demarcates the onset of the glass transition region, occurs at about 60  C. Between the temperature range of 60 and e10  C in the k-carrageenan/

Fig. 3. Cooling run of G0 for 0.5% k-carrageenan, and cooling/heating profiles of G0 and G00 for 85% glucose syrup, and 0.5% k-carrageenan with 85% glucose syrup (scan rate 1  C/min; frequency of 1.6 Hz; 0.1% strain; 0.01 M added KCl).

glucose-syrup sample, both shear moduli rise rapidly and the small-deformation response remains predominantly liquid-like. At the very end of the cooling run, however, G’ manages to overtake G" once more with its values approaching 108.5 Pa. The full picture of the glass transition extends to over four orders of magnitude, with the final crossing of traces signifying the mechanical glass transition temperature (Tg ¼
10  C) and the onset of the glassy state (G’ > G"). As expected for the rubberto-glass transition, changes are fully reversible on heating creating overlapping spectra in Fig. 3 (Hrma, 2008). In the absence of cosolute, k-carrageenan systems undergo a sharp solegel transition at low temperatures (z28  C), with the solid component of the network levelling off towards the end of the cooling run (Fig. 3). As in Funami et al. (2007), values of G" remain low to monitor accurately (tan d < 0.01). At subzero temperatures, ice formation causes catastrophic slippage of the sample on the plate thus preventing further experimentation. This, of course, is very different from the behaviour of the mixture of k-carrageenan with 85% glucose syrup whose structure follows a gradual pattern of network reinforcement during cooling from high temperatures and eventually converts into a glass at subzero temperatures. For the purpose of comparison, Fig. 3 also reproduces the viscoelastic spectrum of a single glucose-syrup preparation (85% solids). The rubbery region (G’ > G") is absent in glucose syrup, which exhibits a predominant viscous response throughout the accessible temperature range. Thus the sample is transformed from a pourable solution at high temperature to a liquid of extremely high viscosity at
20  C. It was suggested that the polydispersity of glucose syrup inhibits crystallisation and encourages vitrification of the system (Kasapis, 2002). On the other hand, the presence of k-carrageenan results in a rubbery structure which undergoes rapid vitrification with the glass transition temperature being captured at the onset of the glassy state at approximately
10  C. Implementation of the combined WLF/free-volume analysis predicted a value of the mechanical Tg for the 85% glucose syrup at much lower temperatures (
27  C in Tsoga, Kasapis, & Richardson, 1999). This is a similar result to what has been described in Fig. 2 and argues that small additions of polysaccharide can accelerate dramatically the mechanical manifestation of vitrification properties in sugar samples. The high sugar/polysaccharide and gelatin mixtures that find application in confectionery formulations (e.g., wine gums and gummy bears) exhibit classic viscoelastic behaviour (Veiga-Santos, Oliveira, Cereda, & Scamparini, 2007), which facilitates application of the aforementioned basic approach for pinpointing the glass transition temperature. In terms of food processing and storage, however, interesting examples related to vitrification phenomena include the dehydration/preservation of fruits, vegetables and meat or fish in relation to the product quality. Food dehydration increases dramatically the concentration of solids in the remaining water thus bringing the system close to its rubber-to-glass transition (Bai, Rahman, Perera, Smith, & Melton, 2001; Matveev, Grinberg, & Tolstoguzov, 2000). This, of course, imparts heavily to the textural properties of dried foodstuffs and the accurate identification of Tg should offer insights into the quality control and the ultimate acceptability of these products. Fig. 4 reproduces mechanical profiles of this second category of materials that exhibit partial vitrification and, as such, the glass transition temperature is increasingly difficult to define and locate. This is illustrated in Fig. 4a for the thermal profile of abalone meat dried to 70% solids (Sablani, Kasapis, Rahman, Al-Jabri, & Al-Habsi, 2004). To start with, the magnitude of the plateau at temperatures above 0  C is unusually high reaching values close to 108 Pa for the storage modulus, as compared, for example, with the corresponding values of the rubbery gel in Fig. 3 for the k-carrageenan/

S. Kasapis / Food Hydrocolloids 26 (2012) 464e472

a

10.0

Log (Modulus/Pa)

9.5

9.0

G'

8.5

8.0 G" 7.5

7.0 -50

-40

-30

-20

-10

0

10

20

30

40

Temperature (°C)

b

9.0

5

2

dlogG"/dT -1

dlogG'/dT

8.0

-4

G'

(dlogModulus / dT) x 100

Log (Modulus / Pa)

8.5

7.5 -7

Mechanical Tg

G"

7.0

-10

-80 -75 -70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10

Temperature (°C) Fig. 4. (a) Heating profile of storage (G0 ) and loss (G00 ) modulus for dried abalone at 70% solids (scan rate: 1  C/min, frequency: 0.1 rad/s, strain: 0.002%), and (b) Heating profile of storage (G0 ) and loss (G00 ) modulus (left y axis), and the corresponding firstderivative plot of G0 and G00 as a function of sample temperature versus the sample temperature (right y axis) for dried shark at 62% solids (scan rate: 1  C/min, frequency: 0.1 rad/s, strain: 0.00072%).

glucose-syrup formulation (<104 Pa). Second, the change in moduli at subzero temperatures is about an order of magnitude from one step to another, as compared to the four orders of magnitude for the glass transition in Fig. 3. Third, the liquid-like response, which is the primary indication of glassy relaxation processes (Farhat, Mousia, & Mitchell, 2003), is substantially diminished with the values of tand throughout the experimental range remaining well below one. In contrast, the rubber-to-glass transition in Fig. 3 develops from about 104 to 108 Pa and, within this range, the values of tan d are much higher than one. Fig. 4b reproduces another example within the second category of partially vitrified materials where the effect of temperature on linear viscoelasticity is even less pronounced. Shark muscle was dehydrated to 62% solids, which required work to be performed at subzero temperatures (Sablani & Kasapis, 2006). The elastic component of the network (G’) is about an order of magnitude higher than the viscous element (G"), with both moduli changing moderately with temperature. At the lowest range of temperatures (<
63.5  C), the trace of storage modulus approaches an upper bound of rigidity characteristic of the glassy state and that of loss

467

modulus declines rapidly. Frequency sweeps between 0.1 and 100 rad/s at the glassy state verified the predominance of the solidlike element of the network, with modulus traces remaining relatively constant with changing frequency (data not shown). It was suggested that dehydration of abalone and shark muscle produces tightly packed aggregates of reduced molecular mobility, an outcome which diminishes the opportunity for a full manifestation of the glass transition in these systems. In support of this, it was difficult to obtain discernable glass transition spectra using the standard technique of modulated differential scanning calorimetry (MDSC) at various scan rates and annealing temperatures or times for dried foodstuffs (Kalichevsky, Blanshard, & Marsh, 1993). On the other hand, one is able to obtain and contrast Tg values from MDSC with those of the mechanical Tg for mixtures of high sugar in the presence of low to medium levels of gelatin or polysaccharide, which are materials of the first category of fully vitrified systems (Deszczynski, Kasapis, MacNaughton, & Mitchell, 2002). To circumvent the problem of a diminished glass transition in dehydrated fish (and fruits where the same behavior was observed) and to identify an objective way to assess the temperature dependence of molecular processes in these systems, a start was made by stating that the thermal effect on the structure of dried materials is less pronounced thus resulting in partial vitrification in terms of temperature band and viscoelastic functions (Rahman, 1999). It was then argued that the fundamentals of a fully developed glass transition in amorphous polymers originate from the same dynamics of molecular chains found in systems of curtailed vitrification, which coexists with partially ordered or crystalline arrangements (Maltini & Anese, 1995). Based on this postulate, a potentially useful device was conceived for the estimation of the extent of vitrification in partially glassy (composite) materials. This entailed plotting of the first derivative of shear storage modulus as a function of sample temperature, versus the sample temperature during vitrification (Kasapis, 2004). In doing so, the shear storage modulus becomes the appropriate parameter for consideration, since its trace is reduced to a minimum at the conjunction of the WLF/free volume fit that models the glass transition region and the Andrade equation that models the glassy state (Peleg, 1992; Pomeranz, 1991), i.e. the precise point where the fundamental Tg was defined for the fully vitrified systems. Based on this principle, similar treatments were pursued for the viscoelastic behaviour of partially amorphous tuna, apple, apricot, pear, plum and dates. These produced smooth first-derivative curves, with the relaxations spectrum of the viscous component of the network (G") unraveling rapidly, as compared to the solid element (G’) of the dehydrated foodstuffs. The minimum of the storage modulus trace clearly demarcated the mechanical glass transition temperature. The approach should be extended to other partially amorphous materials in order to document the universality of the function of storage modulus on shear as the relevant indicator of molecular mobility for this type of systems. Finally, the literature offers a single example as part of a class of materials that are devoid all together of glass transition phenomena. This is the date-pit powder that has been roasted and presently contains 10% water on a wet basis (Hamada, Hashim, & Sharif, 2002; Sawaya, Khalil, & Safi, 1984). It is not feasible to detect changes in the nature of a second order transition either by oscillatory rheometry or differential calorimetry (DSC). In order to increase the sensitivity of the thermal analysis, modulated differential scanning calorimetry (MDSC) was subsequently employed. The technique is capable of resolving the total heat flow into a reversing and a non-reversing (kinetic) component. Phenomena such as glass transition and melting are reversing or heat capacity events. Non-reversing signals contain kinetic events such as

468

S. Kasapis / Food Hydrocolloids 26 (2012) 464e472

crystallisation, crystal perfection and reorganization, cure, and decomposition (Schawe, 1996). Fig. 5 illustrates the heat flow of MDSC and its two components for the roasted date-pit sample at 10% moisture content (Rahman, Kasapis, Al-Kharusi, Al-Marhubi, & Khan, 2007). The expanded scale of the non-reversing response exhibits an endothermic event which reflects the profile of total heat flow of lipid melting on the right y axis. Significantly, the heat capacity trace shows no signs of a stepwise decrease upon heating the material. This outcome is rather unexpected and contravenes previous thermal observations on the structural properties of biopolymer/sugar based materials, as mentioned earlier. In the latter, “classic” sigmoidal spectra follow endothermic changes in heat capacity upon heating hence demarcating the temperature range of glass transitions (Kasapis & Al-Marhoobi, 2005). It appears, therefore, that the literature has documented a third type of structural response in biomaterials, although such an assertion would require identifying and extending research to several examples within this category. The response should be the outcome of a high ratio of low-mobility tightly packed regions to amorphous domains that exhibit thermal motions at the macromolecular level. The former regions would enhance strong interactions and a percolation effect (clustering) between adjacent sequences thus rendering free-volume effects insignificant within the accessible temperature range of polymeric chemical stability. Clearly, there is a divide between the high conformational freedom of polymeric networks in the presence of co-solute, the dehydrated matrices of fish and fruit, and the last example of roasted date-pits whose restricted mobility stabilises conformation and prevents vitrification. 3. Further considerations in the investigation of the thermomechanical Tg: the porous matrix and the application of hydrostatic pressure i) The Effect of Porosity on Glass Related Structural Changes

-25

Glass Transition Temperature (°C)

The discussion thus far explored the application of the network Tg in model confections, and dried fish muscle and fruit leathers, which relate to the more established concepts of state diagram and adsorption isotherms in this field (Roos, 1995; Sablani, Kasapis, & Rahman, 2007). These advances allowed consideration of additional aspects of glassy behavior including the effect of a porous

matrix. In this particular case, the hypothesis examined was that pore formation, which affects irreversibly cellular structure during dehydration (Bengtsson, Rahman, Stanley, & Perera, 2003; Mayor & Sereno, 2004), should be reflected in mechanical measurements. Consequently, changes in structural properties due to porosity should relate to the mechanical glass transition temperature thus affording comparisons once more with the DSC Tg. Fig. 6 reproduces the variation in the values of the mechanical and thermal glass transition temperature with increasing apparent porosity for a well documented example in the literature, which in this case, is the dehydrated apple tissue (Kasapis, Sablani, Rahman, Al-Marhoobi, & Al-Amri, 2007). Care was taken during that investigation to keep the moisture content of the matrix constant (z81%) while the volume fraction of total pores ranged from 0.38 to 0.79. Reproducible mechanical profiles identified the first derivative of shear storage modulus as a function of temperature to be the appropriate indicator of the mechanical Tg at the conjunction of the WilliameLandeleFerry/free-volume theory and the modified Arrhenius equation. That was in accordance with the working protocol described in the preceding section for the structural properties of dehydrated fish muscle and fruit leathers at the vicinity of the glass transition temperature. Information on the microstructural characteristics and morphology of porous apple preparations was also made available via modulated differential scanning calorimetry and scanning electron microscopy (SEM). Variation in mechanical glass transition temperature is highlighted in Fig. 6 and values for all systems exhibited a constant and negative gradient with increasing apparent porosity. In contrast to mechanical spectroscopy, the values of DSC Tg were independent of the level of porosity and remained fixed well below the rheological counterparts. Results also suggested that under conditions of extremely high pore content both DSC and rheology should yield comparable estimates of the glass transition temperature. Rationalisation of the above findings was assisted using SEM and it was evidenced from the micrographs that prolonged processing and the creation of high levels of intercellular spaces (i.e. higher values of apparent porosity) led to the disintegration of the apple matrix and the destruction of its continuity. The magnitude of the structural weakening is probed in the mechanical profile as a reduction in the values of the

-30

Mechanical T g

-35

-40

-45 Thermal T g -50 0.3

0.4

0.5

0.6

0.7

0.8

0.9

Porosity Fig. 5. MDSC thermogram of roasted date-pit powder at 10% (wet basis) water content.

Fig. 6. Variation of the mechanical and thermal glass transition temperature with increasing apparent porosity of the dehydrated apple tissue.

S. Kasapis / Food Hydrocolloids 26 (2012) 464e472

macromolecular glass transition temperature. Thus the lower the volume fraction of total pores the more intact the cell walls and the greater the extent to which the mechanical Tg differs from the measurements of calorimetry. The latter appears to be insensitive to the macromolecular (network) morphology of the dried apple tissue. This work supports further an underlying process that becomes increasingly apparent in the literature. It relates to the observation that besides a thorough description of the material in terms of its composition or preparation history, the glass transition temperature depends on the analytical method and protocol employed (Lewicki & Porzecka-Pawlak, 2005; Schmidt, 1999; 2004). Thus the discrepancies observed in the values of mechanical and DSC Tg in Fig. 6 as a function of apparent porosity in apple tissue should not be considered to be an experimental artifact but, rather, a reflection of the distinct property and distance scale being probed by the two techniques. ii) High Hydrostatic Pressure In contrast to data on temperature effects dealt extensively in here, there appear to be very few phenomenological observations on the vitrification properties of high sugar/biopolymers under pressure and no data at all in relation to treatment with various theoretical approaches. It was felt, therefore, that this technology of the future would be of some interest to the reader as discussed briefly below. The examples obtained from the literature are marine and bacterial polysaccharides that have come to prominence as gelatin replacers, with agarose, carrageenans and deacylated gellan gum being the most notable ingredients in high sugar formulations (Fonkwe, Narsimhan, & Cha, 2003). This section will address the dependence of relaxation processes, as manifest in changes of the glass transition temperature, for a range of hydrostatic pressures (0.1e700 MPa). Table 1 reproduces the values of Tg for polysaccharide/co-solute preparations (78% total level of solids) determined using MDSC. In general, there is a progressive decrease in recorded data from about
55 to
62  C with increasing the experimental hydrostatic pressure. It appears that the treatment of gelling polysaccharide in the presence of sugar with high pressure resulted in the disruption of the three-dimensional structure formed in these systems. At extreme levels of applied pressure (700 MPa), vitrification points approach those for the single sugar systems. For example, values between
62 and
65  C have been reported in the glass transition curve of the state diagram of glucose preparations at 78% solids (Roos, 1995a).

Table 1 Glass transition temperatures (Tg) of polysaccharide/co-solute preparations (78% total level of solids) determined using modulated differential scanning calorimetry. 0.1 MPa 3% agarose Tg replications
56.3
56.8 ( C)
55.0

150 MPa

300 MPa

þ 75% glucose syrup
57.0
60.4
56.4
58.1
59.4
60.5

2% gellan þ 76% glucose
56.4 Tg replications
56.5
57.0
55.5 ( C)
56.2
53.5

500 MPa

700 MPa


59.4
63.5
61.2


61.3
63.9
59.9

syrup (6.7 mM CaCl2)
57.3
57.4
56.6
59.2
56.6
60.6


62.2
62.3
61.5

1.5% k-carrageenan þ 76.5% glucose syrup (30 mM KCl) Tg replications
52.9
54.3
53.5
59.5
60.3 
53.2
55.6
60.6
62.9
63.6 ( C)
52.6
57.4
57.0
59.0
60.2 Average Tg


55.2  1.8
56.2  1.2
57.8  2.0
60.3  1.5
61.7  1.9

469

It was put forward that quantification of the effect of pressure on the structural properties of individual polysaccharide/co-solute samples documents a break in the timeetemperatureepressure superposition, which is operational in the vitrification of amorphous synthetic materials. Thus destabilization of thermal and viscoelastic relaxation processes is monitored under pressure in Table 1, as opposed to the positive temperature or pressure effect on the corresponding behaviour of elastomers with hydrophobic interactions (e.g., styrene or butadiene rubbers in Mpoukouvalas, Floudas, Zhang, & Runt, 2005). Recent work demonstrated that rubbery polysaccharide gels maintain a degree of intermolecular aggregation in a high co-solute environment (e.g., glucose syrup, sucrose, etc.), albeit substantially diminished in comparison with the extensive volume of enthalpic associations of the aqueous counterparts (Kasapis et al., 2003). Considering this, it seems that pressure-induced vitrification patterns appear to be largely irreversible due to the destabilization of the remaining aggregated assemblies of the high co-solute polysaccharide networks. Thus disruption and the slow kinetics of recovery of the brittle polysaccharide agglomerates lead to lower values in the micro and macro-examination of the glass transition temperature. 4. First application of the coupling model to the structural relaxation of high sugar/biopolymer mixtures Experimental observations and their treatment with the combined framework of WLF/free-volume theory offer basic insights into the molecular dynamics of vitrified biopolymer/sugar systems (Nickerson & Paulson, 2005). Nevertheless, the inherent complexity of these multi-component systems has resulted in a certain opposition in the use of free volume, since, in physics, intermolecular interactions are more fundamental and the ultimate determining factor of molecular dynamics in densely packed polymers (Alves, Mano, Gomez Ribelles, & Gomez Tejedor, 2004). Furthermore, the WLF approach, which is also known as thermorheological simplicity (TS), implies that all relaxation processes have the same temperature dependence, i.e. a change in temperature shifts the time or frequency scale of all of them by the same amount (Plazek, Chay, Ngai, & Roland, 1995). However, this is not a universal observation and, instead, thermorheological complexity (TC) has been reported on the superposition of viscoelastic functions in a number of amorphous synthetic polymers and epoxy resins (Plazek & Ngai, 1991). For a densely packed system, thermorheological complexity in the softening transition is the result of a variety of motions ranging from the Rouse modes of Guassian submolecules, to sub-Rouse, and local segmental motions (Robertson & Palade, 2006). The viscoelastic behaviour at the extreme short-time side of the softening transition that accounts for the glass transition temperature is mainly contributed by local segmental motions. The newly introduced “coupling model” attempts to follow progression in viscoelasticity at the vicinity of the glass transition temperature. It stipulates that the local segmental motions are heavily affected by the interactions between polymeric segments and neighbouring environment, with the distribution of relaxation times resembling a stretched exponential function, or the function of Kohlrausch, Williams and Watts (KWW):

i h ØðtÞ ¼ exp
ðt=sÞ1
n

(1)

where, s is the coupled relaxation time, and n is the coupling constant taking values between 0 and 1.0 (Ngai et al., 2000). Equation (1) finds physical significance through its close relationship with the predictions of the coupling theory. The coupling

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S. Kasapis / Food Hydrocolloids 26 (2012) 464e472

constant, n, reflects the intensity of interactions between the underlying relaxation and the physicochemical environment of the surrounding materials. A second relationship exists between the coupled relaxation time s and the primitive relaxation time s0, which is also governed by the interactions between neighbouring segments. For measurements of stress relaxation modulus within the normal time constraints of a rheological experiment, the above mathematical expression recasts to the following form (Ngai, 2000; Ngai, Capaccioli, & Shinyashiki, 2008):

i h   GðtÞ ¼ Gg
Ge exp
ðt=sÞ1
n þ Ge

(2)

where, Gg and Ge are the glassy and equilibrium moduli of the local segmental motions, and t is the time after application of a fixed strain. Fig. 7 illustrates the first application of this school of thought to the vitrification properties of sugar preparations in the presence of agarose, deacylated gellan and k-carrageenan (Jang et al., unpublished results). Master curves of stress relaxation with time were produced using the corresponding glass transition temperatures as the reference temperature. The extreme short time of the glass transition region in the master curves, which is dominated by the local segmental motions, was fitted using the KWW function in the form of Equation (2). By way of example, the glassy (Gg) and equilibrium (Ge) moduli of the local segmental motions for the high sugar/agarose system were taken to be 1.61  109 and 1  108 Pa, respectively.

As shown in Fig. 7, the asymmetric KWW function described well the progression of stress relaxation modulus at the short-time region of glass transition phenomena (non-linear analysis was carried out with the regression coefficients of all the fits being above 0.99). These fits were used to obtain values of the coupling constant, which ranged from 0.60 to 0.69 for the three polysaccharide systems. Physical significance to those values should be assigned in relation to previously reported data for amorphous synthetic polymers and high-sugar gelatin gels (Kasapis, 2006a; Ngai & Rendell, 1993), in order to assess the extent of sterically interfering or electrostatically interacting polysaccharide segments in this particular solvent environment.

5. Kinetics of bioactive compound release in a high-solid carbohydrate matrix The final section of this treatise considers that the increasing overlap between contemporary food and nutritional sciences will stimulate research of complex materials (biopolymer plus small organic compounds) with a nutraceutical function. Understanding the molecular dynamics of high-solid carbohydrates and proteins at the vicinity of Tg leads to the desire to improve the stability and quality of processed foods at which chemical reaction pathways and enzymatic processes are critical considerations. For example, these are formulations where biopolymeric matrices act as inactive

Fig. 7. Short-time region of the stress relaxation master curve for 2% agarose þ 78% glucose syrup (a), 1% deacylated gellan þ 79% glucose syrup (0.0075 M CaCl2) (b), and 0.5% kcarrageenan þ 79.5% glucose syrup (0.01 M KCl) (c) using the corresponding Tg values as the reference temperature, with empty circles indicating experimental data and solid lines following the predictions of the KWW function.

S. Kasapis / Food Hydrocolloids 26 (2012) 464e472

drug additives (excipients) and enzymatic activity relates to the diffusion-controlled substrate/enzyme interaction (Burin, Jouppila, Roos, Kansikas, & Buera, 2004). Chemical considerations in these systems are mainly interested in the prevention of flavor/color degradation, diffusional mobility of bioactive compounds and oxidative reactions such as in fat rancidity (Burin, Jouppila, Roos, Kansikas, & del Pilar Buera, 2000). Given the above, this write-up attempts to provide brief insights into the wealth of information to be gleaned in nutraceutical systems from a combination of mechanical and spectroscopic measurements using as an example the psychoactive compound of caffeine. Many food materials like tea, coffee, sodas and chocolate contain this mild stimulant in amounts that vary widely from 100 to over 1000 mg/ml (Belay, Ture, Redi, & Asfaw, 2008). Monitoring the diffusional mobility of caffeine in glassy carbohydrate matrices requires first to obtain readings of its UVevis absorbance over a certain period of time. The experimental temperature range should also be chosen to coincide with the glass transition region of the sample matrix as identified by DSC and theoretical modeling of mechanical data. Control of the 80% glucose-syrup glassy matrix over the diffusional mobility of caffeine is assessed in Fig. 8 via the shift factor, aT, for the timeetemperature superposition of mechanical spectra of the matrix and UVevis absorbance spectra for caffeine (Shrinivas et al., unpublished results). Thus the illustration summarizes the molecular dynamics of sample matrix and bioactive compound, with data being normalized at an arbitrary taken reference temperature within the glass transition region (To ¼
30  C). Relaxation data of glucose syrup within the glass transition region are described by the WLF/free-volume theory, which produces a Tg value of
43  C for 80% solids. In contrast, the rates of molecular diffusion of caffeine were plotted against the predictions of the modified Arrhenius equation, which yields a good quality fit and a constant value for the energy of activation (Ea ¼ 0.47 kJ/mol). This is a relatively low estimate and reflects the freedom of mobility of the compound within the carbohydrate matrix. In contrast, the activation energy of the latter ranges from 98 to 140 kJ/mol, thus indicating a strong temperature

6.0

4.5

Arrhenius fit matrix

3.0

Log a T

WLF fit matrix 1.5 Arrhenius fit caffeine 0.0

-1.5 Tg -3.0 -80

-60

-40

-20

0

20

40

Temperature (°C) Fig. 8. Temperature variation of the shift factor aT within the glass transition region (,) and the glassy state (B) for 80.0% glucose syrup, and for UVevis kinetic data of 0.4% caffeine (:) diffused from the carbohydrate matrix to dichloromethane (reference temperature for both systems is
30  C).

471

effect on the rubber-to-glass transition of polysaccharides or the melt-to-glass transformation of glucose syrup (Kasapis, 2001). These results have a generic impact and can be utilised to evaluate the statement found in the literature that an operational Arrhenius function is a sign of a comparatively high level of free volume. However, it has been proved difficult to quantify this both for the matrix and the dispersed bioactive compound. The glucose syrup/caffeine work allows estimation of the fractional free volume for the 80.0% glucose-syrup sample that varies from 0.031 at To to 0.024 at Tg. In addition, a WLF fit on the kinetic data of caffeine yielded a fractional free volume of 0.209 at the reference temperature of
30  C. Conditions of molecular relaxation and diffusional mobility correspond to a free volume towards 20 and 30 percent of the total volume of the molecule (Cangialosi, Schut, van Veen, & Picken, 2003), and the value of 20.9% indicates that this is indeed the case for caffeine within the glassy carbohydrate matrix. The latter, on the other hand, is characterized by a collapsing free volume (e.g., 3.1% at To) that makes volume related or WLF considerations the overriding molecular mechanism of vitrification. In conclusion, we have dealt with the main objective of this treatise, which has been to raise awareness that phenomenological and theoretical aspects of polymeric structure and functionality can be correlated and extended to biopolymer/co-solute systems in a high-solid environment (>70% solids in the formulation). It is further anticipated that research will increasingly incorporate bioactive compounds in the polymeric matrix in order to achieve fundamental understanding of chemical, biological and physical processes occurring in real foodstuffs, nutraceuticals and biomedical applications (e.g., topical drug delivery systems). The work on glucose syrup/caffeine mixtures outlines a tool of attack to achieve such understanding in complex molecular arrangements with potential for industrial exploitation.

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