Effects of polyols on cryostructurization of barley β-glucans

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FOOD

HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 263–277 www.elsevier.com/locate/foodhyd

Effects of polyols on cryostructurization of barley b-glucans A. Lazaridou, H. Vaikousi, C.G. Biliaderis Laboratory of Food Chemistry & Biochemistry, Department of Food Science and Technology, School of Agriculture, Aristotle University, P.O. Box 256, Thessaloniki 541 24, Greece Received 3 October 2006; accepted 18 November 2006

Abstract The effects of polyols (fructose, glucose, sorbitol, sucrose, and xylose) on cryostructurization of three barley mixed-linkage (1-3), (1-4) b-D-glucan isolates (b-glucans) differing in molecular size (apparent molecular weights of 210, 140, and 70  103 Da) were investigated. Polyols were incorporated at concentrations ranging from 5% to 30% (w/w) into aqueous b-glucan solutions (3% w/w) and the dispersions were subjected to 14 or 22 repeated freezing (15 1C for 24 h) and thawing (5 1C for 24 h) cycles. Such a treatment yielded compact, porous cryostructurates that were smoother and less porous with the addition of polyols. Optical microscopy confirmed these observations showing a fibrillar network for the cryostructurates that became less pronounced with increasing polyol concentration. For the high molecular weight (210  103) preparation, with exception of sorbitol, the addition of polyols to b-glucan solutions retarded the cryostructurization, as shown by phenomenological observations after each freezing–thawing cycle, and resulted in weaker and less thermostable cryogels compared to those of control, as determined by small deformation mechanical measurements. Xylose and fructose showed a stronger inhibitory effect on structure formation, compared to sucrose and glucose, whereas sorbitol was a promoter when added up to 20% w/w level. However, the above effects of polyols were largely diminished with decreasing molecular weight of the b-glucan and seemed to be independent of the type of polyol for the 140 and 70  103 b-glucan samples. With increasing sugar concentration there was an increase in the number of cycles, at which cryostructurates were formed, and the resultant gels exhibited lower elastic modulus (G0 ) and melting temperature (Tm), and higher tand values, compared to cryogels without polyol added. On the other hand, with increasing sorbitol level the elasticity of the cryogels decreased, whereas their stability upon heating increased. Moreover, differential scanning calorimetry exhibited a more cooperative gel2sol transition and significantly higher apparent melting enthalpy values (DH), calculated from the endothermic peaks, for b-glucan cryostructurates fortified with polyols compared to control preparations. Apparent yield stress values, as determined by stress sweep and creep tests, showed that addition of polyols resulted in more ‘‘brittle’’ gels, following the order: control4sorbitol4sucrose4glucose4xylose4fructose. On the other hand, large deformation mechanical tests (compression mode) revealed an increase in firmness and strength of b-glucan cryogels with inclusion of polyols in the order: sucrose, fructoseoglucose, xyloseosorbitol. r 2006 Elsevier Ltd. All rights reserved. Keywords: Barley (1-3)(1-4)-b-D-glucan; Polyols; Freeze–thaw; Cryogels; Rheology; Differential scanning calorimetry; Compression test; Microscopy

1. Introduction Mixed-linkage (1-3), (1-4) b-D-glucans (b-glucans) occur in the subaleurone and endosperm cell walls of the cereal grains. These biopolymers are linear homopolysaccharides of D-glucopyranosyl residues (Glcp) arranged as mixed sequences of consecutively (1-4)-linked Glcp units in blocks (i.e., oligomeric cellulose segments) that are Corresponding author. Tel.: +30 2310 991797; fax: +30 2310 471257.

E-mail address: [email protected] (C.G. Biliaderis). 0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.11.012

separated by single (1-3)-linkages. Although most of the cellulose-like segments are trimers and tetramers, longer cellulosic oligosaccharides up to 20 glucosyl units are also present in the polymeric chains. The lichenase digestion of b-glucans from different origins reveals differences in the amounts of 3-O-b-cellobiosyl-D-glucose (DP3) and 3-O-b-cellotriosyl-D-glucose (DP4) in the polymeric chain, while the total amount of cellulosic oligosaccharides with degrees of polymerization DPX5 is rather similar (5–10%) for the cereal b-glucans. The amount of trisaccharide (DP3) for the b-glucans follows the decreasing

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order of wheat (67–72%), barley (52–69%), and oat (53–61%), whereas the relative amount of the tetrasaccharide (DP4) follows the increasing order of wheat (21–24%), barley (25–33%), and oat (34–41%). The differences in triand tetrasaccharide amounts observed among b-glucans from different cereal sources is also reflected in the molar ratio of cellotriose to cellotetraose units (DP3/DP4), following the order of wheat (3.0–4.5), barley (1.8–3.5), rye (1.9–3.0), and oat (1.5–2.3); this ratio is considered as a fingerprint of the structure of cereal b-glucans. Values of molecular weight for cereal b-glucans have been reported in the range of 80–2700  103, 65–3100  103, 209–416  103, and 21–1100  103 for barley, oat, wheat, and rye, respectively (Lazaridou, Biliaderis, & Izydorczyk, 2007). Over the last two decades the acceptance b-glucans as a functional, bioactive ingredient has increased the popularity and human consumption of cereal-based foods. These water-soluble fibers seem to improve blood glucose regulation and reduce serum cholesterol levels in diabetic and hypercholesterolemic subjects, respectively (Wood, 2002). Such beneficial health effects have been attributed to the solubility of b-glucans in water and their capacity to form highly viscous solutions (Kahlon, Chow, Knuckles, & Chiu, 1993; Wood, Braaten, Scott, Riedel, Wolynetz, & Collins, 1994). In 1997, the US Food and Drug Administration (FDA) approved a health claim for the use of oat-based foods for lowering the risk of heart disease and passed a unique ruling that allowed oat bran to be registered as the first cholesterol-reducing food at a dosage of 3 g b-glucan per day with a minimum 0.75 g of b-glucan per serving (Anon, 1997). Recently, a similar health claim for the barley b-glucan has also been approved (Anon, 2005). The potential use of b-glucans as hydrocolloids in the food industry has also been proposed based on their rheological attributes, i.e. their gelling capacity and ability to increase the viscosity of aqueous solutions. The prospective use of cereal b-glucan concentrates or isolates as thickening agents, stabilizers and fat mimetics to modify the texture and appearance in calorie-reduced, low-fat foods, such as gravies, salad dressings, and ice cream formulations has been recently reviewed (Lazaridou et al., 2007). Fresh cereal b-glucan solutions exhibit a typical viscoelastic flow behavior, but under certain conditions can form network structures due to intermolecular interactions (chain aggregation) (Irakli, Biliaderis, Izydorczyk, & Papadoyannis, 2004; Lazaridou & Biliaderis, 2004; Lazaridou, Biliaderis, & Izydorczyk, 2003; Lazaridou, Biliaderis, Micha-Screttas, & Steele, 2004; Vaikousi & Biliaderis, 2005; Vaikousi, Biliaderis, & Izydorczyk, 2004). Cereal b-glucans can thus form hydrogels which can be obtained under isothermal (5–45 1C, 4–12% w/v polymer concentration) conditions (Irakli et al., 2004; Lazaridou et al., 2003, 2004; Vaikousi et al., 2004); the gelling ability of b-glucans cured at temperatures above 0 1C and the properties of the gels were found to depend on molecular size, fine structure (DP3/DP4 ratio) and concentration of the b-glucans, as

well as on gel curing temperature. Furthermore, dilute b-glucan solutions (1–4% w/v) subjected to several repeated freeze–thaw cycles were shown to turn into various cryostructurate forms (Lazaridou & Biliaderis, 2004; Vaikousi & Biliaderis 2005). The cryogelation ability, the phenomenological appearance of the cryogels and their properties, as well as the yield of cryostructurates are influenced by the initial solution concentration, the number of freeze–thaw cycles, and the molecular features of the b-glucans. The cryostructurization capacity of b-glucans and the strength of resultant structures increase with decreasing molecular size of the polysaccharide and with increasing initial solution concentration, number of freeze–thaw cycles, and trisaccharide segments (DP3) in the polymeric chains (Lazaridou & Biliaderis, 2004). Further to molecular-structural features of b-glucans and processing factors, formulation could also have an impact on the functional properties of cereal b-glucan gels. Irakli et al. (2004) investigated by large deformation testing the effect on textural properties of barley b-glucan gels (6% w/v) after inclusion of various sugars, fructose, glucose, sucrose, xylose, and ribose at 30% (w/v) concentration and found that gel firmness was affected by the type of the sugar co-solute. The prospective use of cereal b-glucans as hydrocolloids in frozen products is dependent on their cryostructurization ability. Vaikousi and Biliaderis (2005) using rheological measurements revealed that addition of sucrose at 30% (w/w) level to a dilute (4% w/w) barley b-glucan solution, which was subjected to repeated freeze–thaw cycles, impeded the cryogelation process, resulting in weaker cryostructurates. However, the cryogelation potential of cereal b-glucans in aqueous media has not been extensively explored in the presence of polyols that could be used in low-calorie food formulation and/or ‘tooth-friendly’ frozen deserts. In the present study, the effects of polyols, type (fructose, glucose, sorbitol, sucrose, and xylose), and concentration (5–30% w/w) on cryostructurization of barley b-glucans differing in molecular weight were investigated using macroscopic observations and various instrumental techniques, such as microscopy, rheometry, calorimetry, and uniaxial compression testing. 2. Materials and methods 2.1. Materials and molecular characterization of b-glucans Three mixed-linkage (1-3), (1-4) b-D-glucan samples (BGL210, BGL140, and BGL70) differing in molecular size were used in this study. These samples were acidhydrolysates from a b-glucan isolate obtained from whole barley flour. The isolation–purification protocol and the acid hydrolysis procedures followed for the production of these samples is described by Vaikousi et al. (2004). The apparent molecular weights of the b-glucans, estimated with high-performance size exclusion chromatography (HPSEC) combined with a refractive index (RI) detector,

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were found to be 210, 140, and 70  103 Da, for BGL210, BGL140, and BGL70, respectively, whereas the DP3/DP4 molar ratio, determined by lichenase treatment and highperformance anion-exchange chromatography (HPAEC) combined with a pulsed amperometric detector (PAD), was 2.8 for all b-glucan isolates. In addition, the BGL210, BGL140, and BGL70 preparations were characterized for their high b-glucan content (91.5, 91.8, and 90.8% d.b.) and low protein levels (0.8, 0.6, and 1.5% d.b.). Five different polyols, four sugars, fructose (Fru), glucose (Glu) sucrose (Suc), and xylose (Xyl), and a sugar alcohol, sorbitol (Sor), were used in this study as cosolutes. Fructose, glucose, and sucrose were provided from Panreac Quimica SA (Barcelona, Spain), Merck (Darmstadt, Germany), and Riedel-de Haen AG (Seelze, Germany), respectively. Sorbitol and xylose were purchased from Wako Pure Chemical Industries Ltd. (Japan).

2.2. Preparation of cryostructurates Fresh b-glucan solutions (3% w/w) were prepared by stirring of the b-glucan samples in double distilled water at 85 1C until complete solubilization of the material. Following cooling at around 40 1C, polyols were incorporated at different concentrations (5, 10, 15, 20, and 30% w/w) into the b-glucan aqueous dispersions under stirring until total dissolution. The solutions were subsequently cooled at room temperature and loss of water due to its evaporation was determined by weighing the samples and was compensated by addition of the required amount of distilled water. Immediately, 3.5 g of b-glucan–polyols solutions were poured into cylindrical plastic molds with internal diameter of 39 mm. The molds were tightly closed with a lid and rapidly stored in a freezer at 15 1C for 24 h. They were subsequently allowed to thaw in a refrigerator at 5 1C over a period of 24 h. This process of holding periods of samples at 15 1C and at 5 1C for 24 h is defined as one freezing–thawing cycle. Control formulations containing each of the three b-glucan samples (3% w/w) without addition of polyols were also subjected to the same treatment. For the thawed-out samples phenomenological observations were carried out after each freezing–thawing cycle, and instrumental measurements were performed in triplicate after 14 repeated freezing–thawing cycles (14N). The thawed-out samples after 14 cycles of treatment represented themselves as systems with various morphological features, such as liquid-like dispersions, loose or compact cryostructurates, depending on their formulation. The preparations that did not turn in a cryostructurate form up to the 14th freezing–thawing cycle were subjected to additional cycles and the measurements for these formulations were also performed at the 22nd cycle (22N), in which cryostructuration has occurred for all samples. Further to the control formulations of cryostructurates prepared by repeated freezing–thawing cycles, an additional control gel was prepared by curing of a

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b-glucan (3% w/w) dispersion isothermally at 5 1C in order to compare the two systems. 2.3. Optical microscopy Microphotographs of cryostructurates were taken using an Olympus BX61 microscope equipped with dry lenses and a microscope digital camera Olympus DP70 (Japan), using the Olympus micro DP70 software. The cryostructurates obtained after 14 freezing–thawing cycles were placed under the lenses in the form of a thin layer without any other preparation. 2.4. Rheology The rheological properties of barley b-glucan cryostructurates were studied by a rotational Physica MCR 300 rheometer (Physica Messtechnic GmbH, Stuttgart, Germany) using a parallel plate geometry (25 mm diameter and 1 mm gap); the temperature was regulated by a Paar Physica circulating bath and a controlled peltier system (TEZ 150 P/MCR) with an accuracy of 70.1 1C. The data of the rheological measurements were analyzed with a supporting the rheometer software US200 V2.21. The thawed-out material was placed onto the plate fixture of the rheometer, and small deformation oscillatory measurements of G0 (storage modulus), G00 (loss modulus), and tand (G00 /G0 ) were performed with a strain of 0.1% and a range of frequencies (0.1–10 Hz) at 5 1C. Additional rheological measurements were also performed for the cryostructurates: (a) melting profiles (heating rate at 3 1C/min) under dynamic conditions at a strain level of 0.1% and a frequency of 1 Hz; (b) stress sweep tests in a range of 0.08–1000 Pa at a frequency of 1 Hz and 5 1C; and (c) creep tests at 5 1C by applying a constant stress for 60 s on the sample and allowing strain recovery in 180 s after removal of load. The latter creep-recovery cycle was repeated several times with successively increasing level of constant applied stress at each cycle. During all rheological measurements, the periphery of the samples was coated with a thin layer of paraffin oil in order to prevent evaporation. 2.5. Calorimetry Differential scanning calorimetry (DSC) measurements were carried out with a PL DSC—Gold calorimeter (Polymer Labs. Ltd, Epsom, UK). Cryostructurates obtained after 14 freezing–thawing cycles were first lyophilized. The freeze-dried samples were then pulverized under liquid nitrogen. Dry powders containing 5.5 mg b-glucan were subsequently placed in the DSC crucibles (Mettler, ME-29990, SS) and hydrated at 25 1C with water up to a final solids concentration of 20% (w/w). The crucibles were hermetically sealed and allowed to equilibrate with the added water for 72 h (at 25 1C) before the

ARTICLE IN PRESS DSC measurements (thermal scans at a heating rate of 5 1C/min). 2.6. Compression tests For large deformation mechanical tests (compression mode), the cryostructurates obtained after 14 freezing–thawing cycles were examined. Cryogels were prepared from 9 g of b-glucan–polyols solutions poured into cylindrical plastic molds of internal diameter 30 mm and subjected to the same freezing–thawing protocol described above. Compression tests were performed with a TA-XT2i instrument (Stable Micro systems, Godalming, Surrey, UK) at 25 1C, using a 75 mm diameter plunger. Cryogels were equilibrated at 25 1C and compressed up to a deformation level of 50% at a crosshead speed of 0.3 mm/s. The initial force–displacement curves were converted into stress (s)—‘Hencky’ strain (eH) curves and used to calculate the compression modulus (E) from the initial slope of the curves and true stress (sTR), at 40% deformation. These compression parameters were determined according to Lazaridou et al. (2003). 3. Results and discussion 3.1. Macroscopic and microscopic characteristics of cryostructurates Dilute b-glucan solutions (1–4% w/v) when they are subjected to several repeated freeze–thaw cycles have shown to turn into cryostructurates (Lazaridou & Biliaderis, 2004; Vaikousi & Biliaderis, 2005) having various morphological features, including cryoprecipitates (small particles), and loose or compact gels. The phenomenological appearance of the gelled materials obtained after this process is influenced by the initial solution concentration, the number of freeze–thaw cycles, as well as by the molecular features of the b-glucan isolates, such as the molecular size and the molar ratio of cellotriose to cellotetraose segments in the polysaccharide chains (Lazaridou & Biliaderis, 2004). In the present study, various polyols were incorporated into aqueous solutions of bglucans with different molecular weight, and the blends were subjected to repeated freezing–thawing cycles. Phenomenological observations on the thawed-out samples after each number of freezing–thawing cycle were carried out and the number of cycles that required for formation of a cryostructurate form was determined. The effects of polyol type and concentration on cryostructurate formation of b-glucans are depicted in Fig. 1. For the b-glucan sample with the high molecular size (BGL210), with exception of sorbitol, the addition of polyols to b-glucan solutions significantly retarded the formation of cryostructurates (Fig. 1). Generally, xylose and fructose showed a stronger inhibitory effect on structure formation, compared to sucrose and glucose, whereas sorbitol did not substantially alter the cryogelation

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Fig. 1. Number of repeated freeze–thaw cycles required for formation of cryostructurates in 3% (w/w) aqueous dispersions of b-glucans: (a) BGL210 isolate with different polyols at various concentrations and (b) BGL210, BGL140, and BGL70 samples with different polyols at 15% (w/ w) concentration.

ability of the high molecular b-glucan. For instance, the 3% (w/w) dispersion of BGL210 transformed into a cryostructurate after five freezing–thawing cycles, whereas addition of polyols at 15% (w/w) increased this number to 11 when sucrose and glucose were incorporated and to 15 and 16 when fructose and xylose were added, respectively. Moreover, with increasing sugar concentration there was an increase in the number of cycles required for cryostructurization (Fig. 1a). On the other hand, sorbitol did not alter or slightly affected the cryogelation ability of b-glucans. Incorporation of sorbitol at low concentration (5% w/w) into the BGL210 b-glucan solution slightly promoted the cryogelation behavior; cryostructurates were formed after four freezing–thawing cycles. Upon further

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addition of sorbitol to 10–20% (w/w) there was a small increase in the number of cycles (5) required for cryostructurate formation; the gelation ability in this concentration range seemed to be similar to that of the respective control preparation. However, when sorbitol was added to the b-glucan solution at high concentrations (30% w/w) the cryogelation ability was slightly inhibited; the transformation of aqueous dispersions to a cryostructurate form occurred after six freezing–thawing cycles. Similar to the present findings, Vaikousi and Biliaderis (2005) showed that addition of sucrose at 30% level to a dilute (4% w/w) barley b-glucan dispersion impeded the cryogelation process as indicated by the increasing number of required freezing–thawing cycles for cryostructurate formation, compared to that for the control preparations. Moreover, Irakli et al. (2004) reported that incorporation of various sugars (glucose, fructose, sucrose, xylose, and ribose) at 30% (w/w) concentration into a barley b-glucan dispersion (6% w/w) cured at room temperature increased the gelation time of the polysaccharide as shown by small oscillatory measurements carried out isothermally. Further to the type and concentration of polyols, the impact of polyols on cryostructurization of b-glucans was also strongly dependent on the molecular size of the polysaccharide (Fig. 1b). The inhibitory effect of sugars on cryogelation capacity of b-glucans considerably diminished when incorporated in the low molecular size preparations, BGL140 and BGL70. When polyols were added into the formulations with the low molecular weight b-glucans, the number of freezing–thawing cycles at which cryostructurization occurred was just slightly larger compared to the respective control preparations. Moreover, the small inhibitory effect of polyols on cryogelation of low molecular size b-glucans seemed to be independent of the type of polyol. The macroscopic images in Fig. 2 show that dilute dispersions (3% w/w) of b-glucans without (Fig. 2a and b) or with polyols (Fig. 2c and d) submitted to repeated freezing–thawing cycles give compact, self-supporting, translucent gels. A fundamental characteristic feature of cryogels is their heterogeneous and highly porous structure arising from the formation of ice crystals during freezing. The ice crystals induce pore formation and, after thawing, macropores of various size and ‘architecture’ remain in the cryogel body (Lozinsky & Plieva, 1998); through repeating freeze–thaw cycling, additional pores are formed and interconnected, leading to an increase of pore sizes and finally result in a three-dimensional (3D) fibrillar network (Willcox et al., 1999). Moreover, differences in morphology of the cryostructurates exist, depending on the b-glucan molecular weight and cryogel formulation (Fig. 2); samples with low molecular size had a fibrillar and porous macrostructure (Fig. 2a) that became less intense with increasing molecular weight of the b-glucan (Fig. 2b) and even less pronounced with incorporation of the polyols (Fig. 2c and d). Overall, cryostructurates with added sugars were smoother and less porous compared to those of

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Fig. 2. Macroscopic images of b-glucan (3% w/w) cryostructurates obtained after 14 repeated freeze–thaw cycles of samples: (a) BGL70control, (b) BGL140-control, (c) BGL140 with sucrose (15% w/w), and (d) BGL210 with fructose (5% w/w).

polyol-free b-glucan formulations. These observations are in agreement with our previous macroscopic observations on morphology of cereal b-glucan dispersions (3% w/w) submitted to successive freezing–thawing cycles (Lazaridou & Biliaderis, 2004); samples with low molecular size gave fibrilar structures, whereas their higher molecular size counterparts gave gelatinous cryoprecipitates. Moreover, in aggreement with these findings, Ahmad and Williams (1999), studying the effects of various sugars (ribose, fructose, glucose, maltose, and sucrose) on the physicochemical properties of sago starch gels subjected to freeze–thaw cycling treatments, showed that the gels became spongy after one or two freeze–thaw cycles in the absence of polyols, while with added sugars the gels were smooth even after a few freezing–thawing cycles. The microstructure of a b-glucan gel formed by isothermal curing at 5 1C for 60 days (Fig. 3a) appeared homogeneous with some nodes, similar in microstructural features with some cereal b-glucan gels aged also at 5 1C and reported by Tosh, Brummer, Wood, Wang, and Weisz (2004), using phase contrast microscopy. On the other hand, the microstructure of b-glucan gels obtained after 14 freeze–thaw cycles (Fig. 3b–f) showed the presence of a fibrillar network, which is consistent with the macroscopic observations on the cryostructurates. With decreasing molecular size of the b-glucans the fibrillar structures became more intense (Fig. 3b–d), whereas addition of polyols eliminated these features (Fig. 3e and f). Microscopic images (Fig. 4) showed that with increasing polyol concentration the fibrillar network of cryostructurates became less apparent and at high concentrations even disappear giving a smoother homogenous microstructure with nodes (Fig. 4b), comparable to that of b-glucan gels cured at temperatures above zero. In a previous study (Normand, Aymard, Lootens, Amici, & Plucknett, 2003),

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Fig. 4. Optical microscopy images of b-glucan (3% w/w) cryostructurates obtained after 14 repeated freeze–thaw cycles of BGL210 sample with glucose at different concentrations (w/w): 5% (a), 10% (b), 15% (c), and 20% (d).

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transmission electron microscopy (TEM) unveiled similar changes in the microstructure of 2% (w/w) agarose gels with increasing sucrose concentration from 20% to 60% (w/w). In a pure agarose gel, the aggregated chains formed fibers leading to an inhomogeneous microstructure with polymer-poor and polymer-rich regions, whereas when sucrose was added the microheterogeneity diminished progressively and a finer with smaller pore size gel network eventually appeared.

G′, G′′ (Pa)

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Fig. 5. Mechanical spectra (0.1% strain, 5 1C) of 3% (w/w) b-glucan (BGL210-control) cryostructurate obtained after 14 repeated freeze–thaw cycles and gels obtained after isothermal curing at 5 1C for specified time periods.

The cereal b-glucan gels obtained after isothermal curing at temperatures above zero or after the freeze–thaw cycling both belong to the category of physically cross-linked gels, whose 3D structure is stabilized mainly by multiple interand intrachain hydrogen bonds in the junction zones of the polymeric network (Lazaridou & Biliaderis, 2004; Lazaridou et al., 2003, 2004). Fig. 5 shows the mechanical spectrum of a 3% (w/w) b-glucan dispersion that was submitted to the cryogenic treatment of 14 freeze–thaw

cycles, in comparison with corresponding mechanical spectra for b-glucan dispersions at the same concentration, but aged at 5 1C for specified time periods. The b-glucan preparation subjected to the temperature cycling process gave a mechanical spectrum of a strong gel network after 14 freeze–thaw cycles; G0 is independent of frequency and much greater than G00 , G0 410G00 . In contrast, the b-glucan dispersions at equivalent concentration cured at above zero

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cycling. Similarly, it has been shown that locust bean gum solutions cured at room temperature on a timescale of several months converted into gels (Richardson, Clark, Russell, Aymard, & Norton, 1999), whereas the same solutions could gel after just one freeze–thaw cycle (Lozinsky, Damshkaln, Brown, & Norton, 2000; Tanaka, Hatakeyama, & Hatakeyama, 1998). Moreover, the formation of strong gels from xanthan solutions has been reported via a freeze–thaw process, and this does not occur in unfrozen solutions of the same polymer concentration, even over a much longer timescale (Giannouli & Morris, 2003). The reinforcement of b-glucan network structures induced by cryostructurization is usually attributed to the cryoconcentration of the polysaccharide in the unfrozen bulk phase. This phenomenon results in the promotion of associative interactions among the polysaccharide chains. The formation and properties of cereal b-glucan gels

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temperatures for 28 days, i.e. the analogous time period which lasts the freeze–thaw process of 14 cycles, displayed the typical viscoelastic behavior of concentrated polymer solutions. Thus, the response was liquid-like at lower frequencies, where G00 was larger than G0 and both moduli increased with increasing frequency, whilst the behavior approached that of solid-like materials at higher frequencies, i.e. the G0 was greater than G00 . The rheological behavior of this preparation became gel-like only after 44 days of curing at 5 1C as shown in Fig. 5; the G0 was greater than G00 , and both moduli were almost independent of frequency, but their difference was small, much less than one logarithmic cycle. Even after curing of the b-glucan preparations at infinite time, which is a fairly long time (135 days) enough for G0 to reach a ‘‘pseudoplateau’’ value, the gel strength of the sample cured at 5 1C was also lower than that of preparations subjected to the freeze–thaw

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Fig. 6. Effects of polyols (15% w/w) on mechanical spectra (0.1% strain, 5 1C) of 3% (w/w) b-glucan (BGL210) dispersions submitted to 14 repeated freeze–thaw cycles.

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prepared after repeated freeze–thaw cycles at relatively low (3% w/v) initial polysaccharide concentrations (Lazaridou & Biliaderis, 2004) were comparable with gel networks prepared at room temperature and at much higher (10% w/ v) b-glucan concentration (Lazaridou et al., 2004); in fact, the apparent melting enthalpy (DH) values were similar for both type of gels.

The effects of polyols on the cryogelation ability of b-glucan were also investigated by dynamic rheological measurements. Confirming the macroscopic observations (Fig. 1), oscillatory tests showed that the cryogelation ability, strength and elasticity of the gels obtained from the high molecular size b-glucans (BGL210) followed the order: water, sorbitol4sucrose, glucose4fructose, xylose.

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Glu

Suc

Xyl

10000 14N

22N

14N

22N

14N

22N

14N

22N

G′ (Pa)

1000

100

10

a 10000

1

G′ (Pa)

1000

100

10

1 Control

Fru polyols (% w/w)

Glu 5

Sor 10

15

Suc 20

Xyl

30

b 100000

G′ (Pa)

10000

1000

100

10

1 BGL210 Control

BGL140 Fru

Glu

BGL70 Sor

Suc

Xyl

Fig. 7. Storage modulus (G0 ) values recorded at 5 1C, 1.06 Hz, and 0.1% strain of dispersions submitted to 14 freeze–thaw cycles of: (a) BGL210 with different polyols at various concentrations (inset shows the G0 values of the formed cryostructurates obtained after 14 and 22 cycles) and (b) BGL210, BGL140, and BGL70 with different polyols at 15% (w/w) concentration.

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Thus, mechanical spectra of aqueous dispersions of the high molecular size b-glucans with polyols added at 15% (w/w) level and subjected to 14 freeze–thaw cycles (Fig. 6) showed strong elastic gels similar to those of control samples when sorbitol was added, weak gels with glucose and sucrose, and liquid-like behavior in the case of fructose and xylose inclusion. The values of elastic modulus, G0 (Fig. 7) and the corresponding tand values (Fig. 8) of the high molecular weight b-glucan cryogels obtained from their mechanical spectra indicated that the addition of

tan δ

l Xy

r

c Su

So

lu G

C

on

Fr

tro

l

0.8

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 u

1

tan δ

a

0.6

0.4

0.2

0 Control

Fru polyols (% w/w)

Glu 5

Sor 10

15

Suc 20

Xyl

30

b 0.8 0.7

tan δ

0.6 0.5 0.4 0.3 0.2 0.1 0 BGL210 Control

BGL140 Fru

Glu

BGL70 Sor

Suc

Xyl

Fig. 8. Values of tand recorded at 5 1C, 1.06 Hz, and 0.1% strain of cryostructurates obtained after 14 freeze–thaw cycles of: (a) BGL210 with different polyols at various concentrations (inset shows the tand values of all preparations, gelled and liquid-like dispersions, submitted to 14 freeze–thaw cycles) and (b) BGL210, BGL140, and BGL70 samples with different polyols at 15% (w/w) concentration.

271

xylose and fructose resulted in formation of weaker (lower G0 ) and less elastic (higher tand) gel structures, compared to those formed when sucrose and glucose were added at equivalent concentration levels. Addition of sorbitol to the high molecular size b-glucan formulations up to 20% (w/w) resulted in slightly stronger cryostructurates, compared to the corresponding control cryogel, whereas a further increase of polyol level (30% w/w) gave a weaker network structure (Fig. 7). Moreover, with increasing concentration of polyols in the high molecular size b-glucan preparation there was a decrease in G0 (Fig. 7a) and an increase in the tand values (Fig. 8a) of the resultant cryostructurates obtained after 14 freeze–thaw cycles. This trend of decreasing gel strength with polyol concentration was also observed for those preparations that were additionally examined after 22 cycles (22N) (inset of Fig. 7). These were the samples which did not give a cryostructurate after 14 cycles (14N) of treatment, as indicated by macroscopic observations (Fig. 1), and their tand values (41) are shown in the inset of Fig. 8. However, if one compares the G0 values of cryostructurates obtained after 14N to those formed after 22N for each type of sugar, a tendency of increase in G0 values with concentration is evident. This could be explained by a strengthening of the cereal b-glucan cryogels with increasing number of cycles which can even reach a plateau value after several cycles of cryogenic treatment (Lazaridou & Biliaderis, 2004). The increase of cryogel strength with increasing number of cycles is attributed to the increasing cross-link density (intermolecular hydrogen bonds of homologous chain sequences) at each repeated cycle and has been also reported for cryostructurates of PVA, locust bean gum, xanthan, and ice cream model systems containing galactomannans (Giannouli & Morris, 2003; Nagura, Hamano, & Ishikawa, 1989; Patmore, Goff, & Fernandes, 2003; Stauffer & Peppas, 1992; Tanaka et al., 1998; Watase & Nishinari, 1988). This view is consistent with the findings of the BGL210 formulation with 20% (w/ w) sucrose, for which the gel strength was measured at both numbers of cycles (inset of Fig. 7). Therefore, the observed trend of gel weakening with increasing polyol concentration could be ascribed to the inhibitory action of co-solutes on b-glucan cryostructurization that probably leads to a postponed increase of G0 . Possibly, a plateau G0 value is attained upon successive subzero temperature cycling which seems to increase with increasing polyol concentration. Thus, the strength of the b-glucan cryostructurates depends on polyol concentration as well as on the intensity of applied cryogenic treatment. Moreover, consistent with the phenomenological observations of Fig. 1, the strength (Fig. 7b) and elasticity (Fig. 8b) of the cryostructurates from the low molecular size b-glucans (BGL140 and BGL70) were not largely affected by the inclusion of polyols into formulation. However, there was a small decrease in cryostructurate strength with addition of sugars, regardless of the type of sugar used for the BGL140 formulations; this was

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eliminated with further decrease in b-glucan molecular size (BGL70) (Fig. 7b). All preparations of BGL140 and BGL70 samples with added polyols gave strong elastic gels, having tand values around 0.1, slightly higher than those of the respective control formulations (Fig. 8b). Similarly, in previous studies rheological measurements revealed weakening effects of sugars on polysaccharide gels obtained after cryotropic treatment (Giannouli & Morris, 2003; Vaikousi & Biliaderis, 2005). Vaikousi and Biliaderis (2005) found that addition of sucrose at 30% level into a dilute (4% w/w) barley b-glucan dispersion subjected to several repeated freeze–thaw cycles resulted in cryostructurates with lower values of elastic modulus (G0 ) and of apparent viscosity (Z), compared to those of polyol-free preparations. Giannouli and Morris (2003) reported that incorporation of sugars (sucrose, glucose, fructose, and maltose) into xanthan (0.5 wt%) dispersions submitted to one freeze–thaw cycle had no effect on the cryogel strength up to 10 wt% sugar concentration; however, higher levels of sugars caused a progressive reduction of both dynamic moduli (G0 and G00 ) of xanthan cryogels, with no cryogelation being observed at sugar concentrations above 20 wt%. In both studies this behavior was attributed to the possible depression of the freezing point of the system by addition of sugars that can lead to a decrease in the extent of ice crystallization and hence in the extent of alignment and association of the polysaccharide chains. The resultant increase in the volume of the unfrozen liquid microphase regions of the system’s bulk phase, due to the presence of sugars, causes a decrease in the concentration of the macromolecules in the unfrozen microphases, thus weakening the effective polymer–polymer interactions. On the other hand, Giannouli and Morris (2003) reported that cryogels of galactomannans (locust bean gum) showed increasing strength when sucrose or other sugars were added up to a concentration of 50 wt%, whereas a reduction in moduli occured only at higher sugar concentrations. In a recent study, Doyle, Giannouli, Martin, Brooks, and Morris (2006) investigated the effect of various polyols (sucrose, glucose, fructose, and sorbitol) at quite high concentrations (40–60 wt%) on galactomannan cryogelation using large deformation compression testing. The gel strength showed an initial increase and subsequent a decrease with increasing concentration of sugar; maximum strength was attained at 45 wt% fructose, 50 wt% sucrose or sorbitol, and 55 wt% glucose, and increased in the same order (i.e., fructoseosucroseEsorbitologlucose). Similarly, it has been shown that incorporation of sucrose at high concentrations promotes gelation phenomena of locust bean gum, without the need to go through the freeze–thaw process (Richardson & Norton, 1998). Overall, in a number of different studies (Ahmad & Williams, 1999; Biliaderis & Prokopowich, 1994; Nishinari & Watase, 1992; Nishinari, Watase, Miyoshi, Takaya, & Oakenfull, 1995; Nishinari, Watase, Williams, & Phillips, 1990, 1991; Prokopowich & Biliaderis, 1994; Watase, Nishinari, Williams, & Phillips, 1990), various views have

been presented about the effect of individual sugars on gelation of polysaccharides, which may play either an inhibitory or a promoting role on the polysaccharide chain aggregation events; these effects seem to depend on several factors, such as hydration characteristics and structure of the co-solutes (compatibility with the water structure), water content of the system, polyol concentration, and storage conditions. In the present study, carried out under a subzero temperature cycling process, the tested sugars, xylose, fructose, sucrose, and glucose, hindered the b-glucan network formation and this might reflect compatibility of these solutes with the hydrated polymer, probably greater for xylose and fructose than for glucose and sucrose. This may decrease the extent of phase separation between the polymer chains and sugars and thereby reduce the effective concentration of the b-glucan in its microdomains. On the other hand, sorbitol, acting as a promoter of b-glucan cryostructurization, might reflect greater incompatibility of the polysaccharide–sorbitol mixtures which leads to enhancement of polymer–polymer interactions and chain aggregation events. 3.3. Thermal properties of cryostructurates Fig. 9 shows typical melting profiles of b-glucan (BGL210) cryostructurates without or with polyols obtained after 14 freeze–thaw cycles, which are similar with analogous melting profiles obtained from cereal b-glucan gels formed by isothermal curing at temperatures above zero (Irakli et al., 2004; Lazaridou et al., 2003, 2004; Vaikousi et al., 2004). For all cryostructurates there was a sharp sigmoidal one-step transition of the G0 as a function of temperature, implying melting of the network structure. The temperature at which G0 ¼ G00 is defined as the melting temperature, Tm; the melting temperatures of the cryostructurates are summarized in Fig. 10. The Tm values as 10000 control Fru Glu Sor Suc Xyl

1000

G′ (Pa)

272

100

10

1

0.1 0

20

40

60

80

100

Temperature (°C)

Fig. 9. Effects of polyols (10% w/w) on melting profiles of 3% (w/w) bglucan (BGL210) cryostructurates obtained after 14 freeze–thaw cycles; heating rate 3 1C/min, frequency 1 Hz, and strain 0.1%.

ARTICLE IN PRESS A. Lazaridou et al. / Food Hydrocolloids 22 (2008) 263–277 Control 65

14 N

Fru 14 N

Glu

Suc

Xyl

22 N 14 N 22 N 14 N 22 N 14 N 22 N

a 75

Tm (°C)

60 55 50

70

45

Tm (°C)

65

polyols (% w/w) 5

60

10 15 55

20 30

50

45 Control

b

Fru

Glu

Sor

Suc

Xyl

75 70

Tm (°C)

Control 65

Fru

60

Glu Sor

55

Suc 50

Xyl

45 BGL210

BGL140

BGL70

Fig. 10. Melting temperature (Tm) of cryostructurates obtained after 14 freeze–thaw cycles of: (a) BGL210 with different polyols at various concentrations (inset shows the Tm values of the formed cryostructurates obtained after 14 and 22 cycles) and (b) BGL210, BGL140, and BGL70 with different polyols at 15% (w/w) concentration; heating rate 3 1C/min, frequency 1 Hz, and strain 0.1%.

well as the melting profiles of the cryogels showed that the gel-sol transition depends on the type and concentration of polyols, and on b-glucan molecular size as well. A more thermally stable gel points to a larger extension of the junction zones and/or a better organization of the domains of ordered chain segments in the network structure. With the exception of sorbitol, inclusion of all sugars into the two higher molecular weight b-glucan preparations, BGL210 and BGL140, resulted in less thermostable gel structures (Figs. 9 and 10). Among the different cryogels tested, the least thermostable were those containing xylose and fructose, followed by the glucose and sucrose formulations. For the BGL210 preparations, the Tm of cryogels followed the same order as for the cryogelation ability, strength, and elasticity; i.e. water, sorbitol4sucrose, glucose4fructose, xylose (Figs. 9 and 10). These trends were less pronounced for the cryostructurates obtained from the BGL140 preparation (Fig. 10b).

273

On the other hand, the effects of polyols on the thermostability of b-glucan cryostructurates obtained from the lowest molecular weight sample (BGL70) differed from those of the higher molecular size preparations. With the exception of xylose that also significantly reduced the Tm of the BGL70 gels, fructose and glucose slightly increased the Tm, while addition of sucrose and sorbitol considerably increased the thermostability of these cryostructurates. With the exception of sorbitol, incorporation of all sugars into the BGL210 formulations and with increase of their concentration resulted in less thermostable gel structures (Fig. 10a). Among the various cryogel formulations, the least thermostable were those containing xylose and fructose, followed by glucose and sucrose. In contrast, when sorbitol was added, up to a 15% level, there was no impact on Tm of the respective gels; however, with further increase of sorbitol concentration a significant increase in the thermostability was noted. Comparing the Tm values of some of the cryostructurates obtained after 14N to those formed after 22N for each type of sugar at the same polyol level, a tendency for increase in melting temperature with sugar concentration was seen (e.g., sucrose at 20%, inset of Fig. 10). This is consistent with the observations on increasing G0 values with increased concentration of sugars in the cryostructurates when the measurements were carried out after several freezing–thawing cycles, enough for the development of a strong gel network. With increasing number of cycles, the cross-links may increase both in number and in length resulting in a network with higher density of the ordered junctions zones and consequently in more thermostable structures; e.g. the Tm for the sucrose cryostructurates largely increased from 50 to 60 1C with increasing number of freeze–thaw cycles from 14 to 22 (inset of Fig. 10a). Overall, the trend of decreasing thermostabily with increasing sugar concentration that was initially observed could be ascribed to the inhibitory action of the co-solutes on b-glucan cryostructurization. However, in the case that the cryogenic treatment is enough for the development of an extensive 3D network with high density of physical cross-links, possibly the dependence of cryostructurate thermostability on polyol concentration would show the reverse trend. Fig. 11 shows the effects of polyols at 5% (w/w) concentration on DSC thermal scans of b-glucan cryostructurates obtained by repeated freeze–thaw cycles. All scans showed rather broad endothermic gel-sol transitions at around 60–75 1C, which implies melting of the network structures. Similar endothermic events on DSC heating profiles have been also found for several cereal b-glucan cryogels submitted to similar cryogenic treatments (Lazaridou & Biliaderis, 2004) as well as for cereal b-glucan gels formed at gel curing temperatures above zero (Lazaridou et al., 2003, 2004; Tosh et al., 2004; Vaikousi et al., 2004). Incorporation of polyols into b-glucan cryostructurates, even at low concentration (5% w/w), considerably modified the DSC profiles. Thus, with addition of polyols the

ARTICLE IN PRESS A. Lazaridou et al. / Food Hydrocolloids 22 (2008) 263–277

274

Endothermic Heat Flow

Control ΔH: 0.71 mJ/mg

Fru

0.2 mWatts

Glu

ΔH: 4.21 mJ/mg

Sor

ΔH: 4.11 mJ/mg

Suc

Xyl

ΔH: 4.75 mJ/mg ΔH: 4.48 mJ/mg ΔH: 4.55 mJ/mg

40

45

50

55

60 65 70 75 Temperature (°C)

80

85

90

Fig. 11. Effects of polyols (5% w/w) on DSC thermal curves of 3% (w/w) b-glucan (BGL210 sample) cryostructurates obtained after 14 freeze–thaw cycles; heating rate 5 1C/min.

broadness of the endothermic peaks decreased, reflecting an increase of the cooperativity of the gel2sol transition which was stronger in the case of sorbitol (Fig. 11). Moreover, inclusion of polyols into the b-glucan cryostructurates increased significantly the apparent melting enthalpy values (DH), determined from the DSC curves, that indicate the required energy for disruption of H-bonding within the junction zones; the DH values increased almost independently of the type of polyol from 0.7 mJ/mg b-glucan for formulations with pure polysaccharide to the range of 4–4.5 mJ/mg b-glucan in the cryostructurates involving polyols. Similarly, other researchers (Nishinari et al., 1990, 1995; Watase et al., 1990) have found an increase of the DH values for gel-sol processes when sugars and other polyols, up to a certain concentration, were incorporated into agarose or k-carrageenan gels. The increase of melting enthalpy with inclusion of polyols in b-glucan cryostructurates, at a first glance, seems to be opposite to the general inhibitory action of sugars on cryostructurization observed by macroscopic observations and dynamic rheometry. However, it is well known that DSC and dynamic rheological testing examine different properties of a gel network; the latter shows the density of junctions zones, while the former reflects upon their nature and ‘quality’ of organization. Thus, inclusion of polyols might promote intermolecular interactions and chain associations of localized nature, as indicated by the increase of DH values, whereas extensive cross-linking that could lead to network formation is impeded. Similar observations have been reported in previous studies on gelation of cereal b-glucans (Lazaridou & Biliaderis, 2004; Lazaridou et al., 2003, 2004) as well as of locust bean galactomannans (Richardson et al., 1999), in which for some preparations a significant level of short-range interchain associations develops (DSC endotherm), but

the formation of an extensive cross-linked network (as probed by small deformation testing) is retarded. The DSC endothermic peak reflecting the gel-sol transition of b-glucan cryostructurates shifted to lower temperatures with addition of polyols (Fig. 11). Thus, the peak Tm, (i.e., the melting point of cryostructurates) decreased from approximately 74 1C to a slightly lower range of 67–71 1C by adding polyols. These results are in disagreement with previous studies on the effect of sugars and other polyols on agarose or k-carrageenan gels (Nishinari & Watase, 1992; Nishinari et al., 1990, 1991, 1995; Watase et al., 1990) that showed an increase in Tm with polyol addition. In the case of b-glucan cryostructurates the increase of DH and the decrease of Tm with inclusion of polyols in the cryogel formulations suggest that these co-solutes might promote the lateral chain aggregation locally, but this is not enough for formation of a well-organized thermostable gel network. The thermostability of b-glucan cryostructurates containing polyols as shown by DSC measurements followed the order sorbitol4sucrose4glucose4fructose4xylose, which seems to be consistent with the results for the Tm values derived from the small oscillatory mechanical measurements. 3.4. Apparent yield stress of cryostructurates The yield stress of b-glucan cryostructurates can be referred to the applied shear stress, at which the material flow starts. In this study, this critical stress was estimated by two different rheological tests, stress sweep and creep tests. Because the obtained yield stress value is dependent on the applied technique, it is preferable to use the term apparent yield stress. By gradually increasing the stress amplitude applied to the cryostructurates a linear viscoelastic region at low stress values is observed (Fig. 12). Deviation from linearity occurs when the gelled material starts to deform, implying a destruction of the weak physical network structure. The stress amplitude at which G0 begins to decrease by 5% from its maximum values was taken as a measure of the apparent yield stress of the cryostructurate (inset of Fig. 12); these values are summarized in Table 1. The addition of polyols in b-glucan cryogels resulted in decrease of the range of linearity, and thus of the apparent yield stress (Fig. 12 and Table 1). Similarly, Dickinson and Matia Merino (2002) found that incorporation of sugars into acid-induced caseinate gels promoted a strain-weakening behavior, thus showing a shortening of the linear viscoelastic regions as determined with strain sweep tests. Repeated creep-recovery tests were also conducted on b-glucan cryostructurates with the constant applied stress successively increasing after each creep-recovery cycle (Fig. 13). For low levels of applied stress, the resistance of gelled material to deformation was high, as shown by the low values of maximum creep % strain (strain at the end of creep phase), and when the load was removed the sample recovered completely; this behavior is indicative of

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275

10000

10 9

1000

7

100

G′/G′o

10

1

1 0.8 0.6

Control Fru 0.95

Suc

Strain (%)

G′ (Pa)

8

0.4 Critical stress 0.2 0 0.1 10 100 1000 1

5 4

2 1 0

Fig. 12. Stress sweep curves recorded at 5 1C and 1 Hz frequency of 3% (w/w) b-glucan (BGL210 sample) cryostructurates without or with polyols (10% w/w) obtained after 14 freeze–thaw cycles; the inset shows how the apparent yield stress was calculated. G0 o is the G0 at the linear region.

10

10

100

75

1000

10

0

Shear stress (Pa)

50

40

30

10000

1

Applied Stress (Pa)

3

Shear stress (Pa)

0.1 0.1

6

20 0.3%

200

400

600

800 1000 1200 1400 1600 Time (s)

9 8 7 Strain (%)

Table 1 Effects of polyols (10% w/w) on apparent yield stress calculated by stress sweep and creep tests of 3% (w/w) b-glucan (BGL210 sample) cryostructurates obtained after 14 freeze–thaw cycles; measurement temperature 5 1C

6

Creep test (Pa)

3

Control Fru Glu Sor Suc Xyl

149.0 3.2 25.0 124.0 45.0 10.7

75.0 1.0 10.0 50.0 20.0 4.0

2

a fully elastic behavior. However, when the stress exceeded the region of linearity, and applied for 60 s, sufficient for the sample to reach steady-state flow, the preparations exhibited a typical viscoelastic response combining both viscous fluid and elastic components. As a result, with increasing stress above a certain value the resistance of cryostructurates to deformation decreased significantly and after removal of the stress the sample did not recover fully, indicating disruption of the structure network and flow. The amplitude of applied stress that resulted in greater than 0.3% recovery strain (strain at the end of recovery phase) was considered as the apparent yield stress of the cryogel (Fig. 13). The values of apparent yield stress for b-glucan cryostucturates estimated from the creep tests are also summarized in Table 1. Consistently with the findings from the stress sweep tests, the addition of polyols to bglucan cryostructurates decreased the apparent yield stress of the respective networks. Moreover, comparing the maximum creep strain values among the different preparations at equivalent stresses, but lower than the apparent yield stresses, it was shown that resistance of cryostructu-

20

4

Stress sweep test (Pa)

(710.0) (70.5) (75.0) (725.0) (710.0) (71.0)

30

5

Samples

(70.21) (70.29) (70.28) (710.22) (70.43) (71.58)

Applied Stress (Pa)

10 5

1

0.3%

0 0

200

400 600 Time (s)

800

1000

Fig. 13. Creep-recovery curves at 5 1C of cryostructurates obtained after 14 freeze–thaw cycles of 3% (w/w) b-glucan (BGL210) without (a) and with sucrose (10% w/w) (b); the constant applied stress was successively increased after each creep-recovery cycle.

rates to deformation also decreased with inclusion of polyols indicating their weakening effect on gel network structure (Fig. 12). Overall, the apparent yield stress values determined by stress sweep tests were higher than those obtained from the creep measurements (Table 1). Nevertheless, both tests showed that addition of polyols resulted in more ‘‘brittle’’ gels and unravel the same order of apparent yield stress values of the b-glucan cryostructurates: control4sorbitol4sucrose4glucose4xylose4fructose. 3.5. Uniaxial compression properties of cryostructurates Beyond the shear deformation experiments, uniaxial compression up to 50% deformation was employed to evaluate the effects of polyols on the mechanical properties of barley b-glucan cryogels. Fig. 14 illustrates the

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276

4

Table 2 Effects of polyols (15% w/w) on compression modulus (E), and true stress (sTR) at 40% deformation of 3% (w/w) b-glucan (BGL140 sample) cryostructurates obtained after 14 freeze–thaw cycles; measurement temperature 25 1C

Sor

3.5 Xyl

3

σ (kPa)

2.5

Glu

2

Fru

1.5

Samples

E (kPa)

sTR (kPa)

Control Fru Glu Sor Suc Xyl

2.47 4.53 7.36 7.48 4.89 5.48

0.75 1.49 1.96 2.52 1.12 2.03

(70.53) (70.26) (70.05) (71.35) (70.27) (70.95)

(70.07) (70.15) (70.06) (70.46) (70.18) (70.29)

Suc

1

Control

0.5 0 0

0.2

0.4 εH

0.6

0.8

Fig. 14. Effects of polyols (15% w/w) on compression stress (s)–‘Hencky’ strain (eH) curves (at 25 1C) of 3% (w/w) b-glucan (BGL140) cryostructurates obtained after 14 freeze–thaw cycles subjected to deformation up to 50%; curves are means of three measurements for each gel type.

compression curves, by plotting stress as a function of ‘Hencky’ strain, of the cryostructurates that were similar with typical compression–strain curves obtained in a previous study (Lazaridou & Biliaderis, 2004) for cereal b-glucan cryogels formed after repeated freeze–thaw cycles. The differences among the formulations are reflected on the calculated from these curves parameters of compression modulus (E) and true stress (sTR) at 40% deformation, which are summarized in Table 2. Incorporation of polyols into b-glucan cryostructurates resulted in more firm and strong gel structures, as shown by the higher E and sTR values of preparations containing polyols, compared to those of control sample. The increased firmness with addition of polyols could be attributed to the higher level of solids included in preparations with polyols than those of the control formulations. Moreover, as already has been discussed in Section 3.1, the macro- and microstructures of the control (polyol-free) cryostructurates are more porous compared to networks containing polyols, which might explain their lower firmness and strength under large deformation mechanical testing. Normand et al. (2003) using large deformation compression tests reported an increase of Young’s modulus and true stress with increasing sucrose concentration from 20% to 70% w/w in agarose gels. Recently, Doyle et al. (2006), studying by the same technique the effects of polyols on cryogelation of galactomannans, found an initial increase and a following decrease for these parameters with increasing of polyol levels in the range of 40–60% w/w. Among the different polyols, incorporation of sorbitol into b-glucan cryostructurates gave the highest firmness

and strength to the gel network structures (Table 2), while the addition of fructose and sucrose resulted in weaker and less firm cryogels. According to the results of Doyle et al. (2006), the order by which various polyols affect the mechanical strength and firmness of galactomannan cryogels may also vary depending on polyol concentration. 4. Conclusions Different type (fructose, glucose, sorbitol, sucrose, and xylose) and concentration (5–30% w/w) of polyols were incorporated into dispersions of barley b-glucans differing in their molecular weight (210, 140, and 70  103 Da) and submitted to repeated freeze–thaw cycles in an effort to explore the effects of formulation on b-glucan cryostructurization. Inclusion of polyols affected the cryogelation ability, macro and microstructures, as well as the mechanical and thermal properties of the b-glucan cryostructurates. Addition of polyols resulted in smoother, less porous macrostructure, which was further reflected to the less fibrillar microstructure observed with increasing polyol concentration. With exception of sorbitol, the addition and increasing concentration of polyols to b-glucan solutions retarded the cryostructurization of the polysaccharide and resulted in weaker, more brittle, and less thermostable cryogels, compared to those of control formulations, as determined by rheological measurements. Xylose and fructose showed a stronger inhibitory effect on structure formation, compared to sucrose and glucose, whereas sorbitol was a promoter when added up to 20% w/w level. Even though incorporation of polyols into b-glucan aqueous dispersions affected considerably the cryostructurization of b-glucans of high molecular size (210  103), for the low molecular weight preparations (140 and 70  103), at which cryogelation is accelerated, the addition of polyols had little inhibitory impact on the cryogelation behavior of the polysaccharide, and additionally was rather independent of the type of polyol used. Moreover, as shown by calorimetric measurements, the addition of polyols increased the cooperativity of gel2sol transition and the melting enthalpy required for disruption of H-bonding in the junction zones, implying that polyols

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promote intermolecular interactions and chain associations of localized nature. On the other hand, with inclusion of polyols, an increase in firmness and strength of the b-glucan cryogels was observed in the order of sucrose, fructoseoglucose, xyloseosorbitol, under large deformation mechanical testing.

Acknowledgments This work has been carried out with the financial support from the Greek Ministry of Education (PYTHAGORAS program, 97436 8, 2005–2007).

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