G ELATION —C RYOGELATION

1.7.2 G ELATION —C RYOGELATION

In addition to solution viscosity enhancement on storage, β-glucans have been shown to gel under certain conditions. 15–21,43,45,46,175,179 Cereal β-glucan hydrogels with different molecular characteristics and properties have been obtained under isothermal (5 to 45˚C, 4 to 12% w/v polymer concentration) conditions 17,18,21,43,45 as well as after repeated freezing and thawing cycles of relatively dilute polysaccharide solutions (1 to 4% w/v) 19,20 ; the later process is called cryogelation and the formed gels are cryogels. The β-glucan hydrogels have been examined by small-strain dynamic rheometry, differential scanning calorimetry (DSC), and large deformation mechanical tests. The gelling ability of β-glucans cured at temperatures above 0˚C and the properties of the gels were found to depend on molecular size, fine structure (DP3/DP4 ratio), and concentration of the β-glucans, as well as on gel-curing temperature. Similarly, the

32 Functional Food Carbohydrates

cryogelation ability, the phenomenological appearance of the cryogels and their properties, and the yield of cryostructurates were influenced by the initial solution

concentration, the number of freeze–thaw cycles, and the molecular features of the β- glucans. The obtained cereal β-glucan gels formed at temperatures above 0˚C, and the cryogels belong to the category of physically cross-linked gels whose three- dimensional structure is stabilized mainly by multiple inter- and intrachain hydrogen bonds in the junction zones of the polymeric network. Gels prepared after freeze–thaw cycling at low initial polysaccharide concentrations, such as those usually found in frozen food formulations, are comparable with the gel networks prepared at room temperature and at much higher β-glucan concentrations. 19

The gelation capacity of aqueous dispersions from cereal β-glucans differing in molecular size and fine structure has been monitored isothermally at different temper-

atures above 0˚C and polymer concentrations; i.e., the time-dependent evolution of G', G'', and tan δ was monitored periodically by applying a frequency of 1 Hz and a strain

level of 0.1%, conditions in the linear viscoelastic region. 17,18,21,43,45 After an induction period, G' and G'' increase with time and the aqueous dispersions begin to adopt gel- like properties (G' > G''). At the end of the gel-curing experiment, the behavior becomes typical of an elastic gel network and the G' attains a pseudo-plateau value, G' max (Figure 1.3). The time where G' crosses G'' is considered the gelation time, and the maximum

″ (Pa), tan

′, G

G 10 ′ (Pa) 100 G

tan δ

20 30 40 50 60 70 80 90 100 Temperature (°C)

Time (h)

FIGURE 1.3 Gelation kinetics for a representative barley β-glucan (apparent molecular weight of the peak fraction calculated as 100 × 10 3 ) preparation at 8% (w/v) (frequency, 1

Hz; strain, 0.1%; 25˚C) and following melting profile (inset) of the formed gel (frequency, 1 Hz; strain, 0.1%; 3˚C/min heating rate). (From Lazaridou, A. et al., Food Hydrocolloids, 18, 837, 2004.)

Cereal β -Glucans: Structures, Physical Properties, and Physiological Functions 33

slope of the G' curve is reported as an index of the gelation rate; the latter, known as “elasticity increment, I E ”, can be calculated as I E = (dlog G'/dt) max . 15 Melting profiles for the β-glucan gels following their formation can be also monitored by dynamic rheometry (Figure 1.3, inset); the melting point (Tm) taken as the temperature where G' becomes equal to G''. All of the above parameters from gelation kinetics of different β-glucan samples are summarized in Table 1.6.

Two different gelation models have been proposed in the literature for mixed- linkage (1 →3),(1→4)-β-glucans. One involves the side-by-side interactions of cel- lulose-like segments of more than three contiguous β-(1→4)-linked glucosyl units, 182 and the other the association of chain segments with consecutive cellotriosyl units

linked by β-(1→3) bonds. 15 Among cereal β-glucans of equivalent molecular weight, the gelation time decreased and the gelation rate increased in the order of oat, barley, and wheat β-glucans, reflecting the order of the molar ratio DP3/DP4 units (Table 1.6). 15,16,18,46 In addition to the fine structure, the molecular size of the polymer seems to have a strong impact on polysaccharide gelation ability. For samples with a similar distribution of cellulose-like fragments, the gelation time decreases and the gelation rate increases with decreasing molecular weight, possibly due to the higher mobility of the shorter chains, which enhances diffusion and lateral interchain associa- tions. 14,15,17,18,21,43,45,47 The gelation rate also increases with increasing concentration and gel-curing temperatures, reaching a maximum at ~25 to 35˚C; at higher tem-

peratures the I E values decrease. 17,18,21,43 Moreover, incorporation of various sugars, at a concentration of 30% (w/v), to the barley β-glucans gels (6% w/v) led to an increase of the gelation time, following the order of control (without sugar) < glucose < fructose < sucrose < xylose < ribose. 45

A decreasing molecular weight and an increasing DP3/DP4 ratio in the β-glucan chains yielded increased G' max and decreased tan δ values of the gels (Table 1.6) 18,46 ; i.e., with increased amounts of the cellotriosyl units there are longer and more junction zones giving denser cross-linked networks. The dependence of storage modulus on β-glucan concentration (C) followed power law relationships; G' varied as C 7.2–7.5 . When the experimental data of G' max vs. concentration for two representative oat

β-glucan samples with apparent molecular weights of 35 and 110 × 10 3 were fitted into the exponential equation, estimates of 3.5 and 4.4% (w/w), respectively, for the critical gelling concentration, C o , were obtained by extrapolation of this function; C o is defined as the concentration below which no macroscopic gel is formed. 17

The Tm values of gels seem to increase with increasing molecular size and amount of DP3 units in the polysaccharide; for cereal β-glucan gels cured at room temperature, the Tm varies within the narrow range of 65 to 72˚C (Table 1.6). Although a slower gelation process is noted for the high molecular size β-glucans, the gel network structure consists of structural elements (microaggregates) with better organization, or it involves interchain associations over longer chain seg- ments. 17,18,21 Curing of the gels at higher temperatures (45˚C) than room temperature results in increased values for the gelation time and Tm and a decrease in gelation rate and G' max . 17

All β-glucan isolates from cereals were able to form cryogels at such low initial polysaccharide concentrations (even at 1% w/v), at which these polysaccharides could exist in frozen products. The mechanical spectrum of the solution before

34 Functional Food Carbohydrates

freezing was typical of a liquid-like system, but after repeated cryogenic cycles, a gradual transition occurred to the behavior of a weak gel and finally to that of a

strong elastic gel (Figure 1.4). These changes were promoted by high initial solution concentration and amount of DP3 units in the polymeric chain, and by low β-glucan molecular size; i.e., it happened at a lower number of cycles with increasing con- centration and DP3 segments and decreasing molecular size. Moreover, the G' values for cereal β-glucan cryogels, obtained from their mechanical spectra at 5˚C, increase with increasing number of freeze–thaw cycles and the trisaccharide units (DP3), and

with decreasing molecular size of the polysaccharide. 19 The concentration at which cryogelation took place was lower than c** of the examined β-glucan isolates 19 and lower than the critical gelling concentrations estimated for oat β-glucan preparations cured at room temperatures. 17 The major reason for the apparent shifting of critical gelling concentration toward lower values, compared to the same process at gel- curing temperatures above zero, is cryoconcentration. This phenomenon happens during freezing of the polymer solutions, which causes an increase of the β-glucan concentration in the still unfrozen regions of the system’s bulk phase and results in

the promotion of associative interactions among polysaccharide chains. 19 Cryogela- tion ability is also influenced by food formulation. The presence of sucrose (30%

w/v) in a β-glucan dispersion, submitted to repeated cycles of freezing–thawing, induced a significant delay in the transition of β-glucan solutions to a gel state, and

the resultant cryogels were weaker. 20 The DSC thermal scans of cereal β-glucan hydrogels formed after storage at temperature above 0˚C and, by cryogelation, exhibited rather broad endothermic gel

″ (Pa) 100 ′, G

G 10

1 G′ fresh solution

G″ fresh solution

G′ 3rd cycle

G″ 3rd cycle

G′ 9th cycle

G″ 9th cycle 0.1 0.1 1 10

Frequency (Hz)

FIGURE 1.4 Mechanical spectra (0.1% strain, 5 o C) of unfrozen fresh solution, and cryogels obtained after specified number of freeze–thaw cycles for wheat β-glucan (apparent molecular

weight calculated as 200 × 10 3 ) at 3% w/v initial concentration. (From Lazaridou, A. and Biliaderis, C.G., Food Hydrocolloids, 18, 933, 2004. With permission.)

Cereal β -Glucans: Structures, Physical Properties, and Physiological Functions 35

Temperature (°C)

H (mJ/mg)

Oat 65

Endothermic heat flow ←

0.2 mWatts Oat 65 0.5 Oat 100

Time (h) Temperature (°C)

FIGURE 1.5 DSC thermal curves of oat β-glucan gels obtained from solutions of 10% w/v concentration cured at 25˚C for specified time periods (left, top) and of oat β- glucan cryogels obtained from solutions of 3% w/v concentration submitted to nine freeze–thaw cycles (left, bottom), and time dependence of apparent melting enthalpy of the β-glucan gels cured at 25˚C (concentration, 10% w/v) (right); heating rate = 5˚C/min. Samples named as oat 65 and

oat 100 had apparent molecular weights of 65 and 100 × 10 3 , respectively. (From Lazaridou A. et al., Food Hydrocolloids, 17, 693, 2003; Lazaridou, A. and Biliaderis, C.G., Food Hydrocolloids , 18, 933, 2004.)

sol transitions at 55 to 80˚C (Figure 1.5, left). 17–19,21,43 Similar to the rheological responses, DSC kinetic data showed that the rate of endotherm development during

gel curing at room temperature (Figure 1.5, right), 17,18 as well as the apparent melting enthalpy values (plateau ΔH) of the gels, increases with decreasing molecular size 17,21 and with increasing the DP3/DP4 ratio (Table 1.6). 17,18 The ΔH values of the cryogels also increase with increasing the DP3/DP4 ratio and with decreasing molecular size

of the cereal β-glucan. 20 Moreover, the melting temperature of all gel networks studied, as obtained by DSC, seem to increase with the molecular size and the amount of cellotriosyl units of the β-glucans. 17–19,21

Variations in mechanical properties of cereal β-glucan gels have also been revealed by large deformation compression tests. For samples with similar molecular size, an increase in the DP3/DP4 ratio resulted in higher values of firmness and gel strength and a decrease in brittleness; compression modulus (E), true stress ( σ TR ), and strain ( ε TR ) obtained from the stress–Hencky strain curves (Figure 1.6) define the firmness, strength, and brittleness of the gel, respectively. An increase in strength and a decrease in brittleness of the β-glucan gels cured at room temperature were found with increasing concentration, molecular size, and DP3/DP4 ratio of the polysaccharide (Table 1.6). 17,18,21 Similar to the gels cured at room temperature,

36 Functional Food Carbohydrates

FIGURE 1.6 Compression stress ( σ)–Hencky strain (ε H ) curves for gels of cereal β-glucans (concentration, 8% w/v; gel-curing temperature, 25˚C). The samples are named oat (oat

β-glucan), bar (barley β-glucan), and whe (wheat β-glucan), and their calculated apparent molecular weights were 100 and 200 × 10 3 . (From Lazaridou, A. et al., Food Hydrocolloids, 18, 837, 2004. With permission.)

cryogels from high molecular size β-glucans seem to exhibit better organized and stronger structures than their low molecular size counterparts, implying that networks obtained from high molecular size β-glucans involve interchain associations over longer chain segments. Therefore, high molecular size β-glucans can form better organized and stronger gels than low molecular size samples, from the point of view of macrostructure. In contrast with the results obtained for gels formed at 25˚C, the strength of cryogels submitted to large deformations was the highest for oat, lowest for wheat, and intermediate for barley β-glucans — a fact that was attributed to

differences in the nature of the network microstructure among samples. 20 Micro- scopic images of cereal β-glucan gels aged 7 days at 5˚C revealed that the micro- structure was not homogenous and there was a coarsening of gel structure as the β-(1→3)-linked cellotriosyl unit content increased. 47

Other factors having an impact on textural properties of cereal β-glucan gels are polysaccharide concentration, gel-curing temperature, and formulation. Generally, gel strength and firmness increase and brittleness decreases with concentra- tion. 15,17,21,173 Moreover, gel structure formation at a higher temperature (45˚C) than

room temperature results in less firm, weaker, and less brittle gels. 17 Compression tests of mixed gels from barley β-glucan (6% w/v) and low molecular weight sugars

(30% w/v) revealed that the mechanical properties of gels are affected by the type of added sugar. The strength of the gels, as expressed by the true stress, increased with the addition of glucose, fructose, and sucrose, but decreased with ribose. Moreover, the Young’s modulus of most mixed gels increased with the addition of

Cereal β -Glucans: Structures, Physical Properties, and Physiological Functions 37

hexose sugars, such as glucose, fructose, and sucrose, but declined significantly with the pentose sugars, such as xylose and ribose. 45