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sucrose gels
Food Hydrocolloids 16 (2002) 89±94
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The effect of centrifugation on agar/sucrose gels q Jin-E. Shin, Paul Cornillon*, Linda Salim Purdue University, Whistler Center for Carbohydrate Research, Food Science Department, 1160 Food Science Building, West Lafayette, IN 47907-1160, USA Received 1 May 2000; revised 22 January 2001; accepted 6 April 2001
Abstract The viscoelascity and water mobility of 3% agar gels containing 5, 10 and 20% sucrose, before and after centrifugation, were studied by using dynamic mechanical spectroscopy and nuclear magnetic resonance relaxometry. Centrifugation was found to induce damage in the gel by phase separation of water. However, results suggest that water can recombine after centrifugation with closely packed macromolecules to provide a similar molecular structure to that of the gel before centrifugation. Sucrose helped reduce exudation of water at low centrifugation speeds through hydrogen bonding. The effects of centrifugation at high speed on water mobility were compensated by increasing sucrose concentration. Centrifugation created water compartments in the gel with distinct motion properties. On the contrary, sucrose concentration did not affect the hydraulic radius of these compartments. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Agar/sucrose gels; Water mobility; NMR; Centrifugation
1. Introduction The hydrocolloid agar is mainly composed of agarobiose (3-b-d-galactopyranosyl-(1- . 4)-3,6-anhydro-a-l-galactose, Matsuhashi, 1990). The principal sources of commercial agar include the gelidiales and gracilariales that are aquatic plants found primarily in Asia (Matsuhashi, 1990; Stanley, 1990). Due to differences in species, habitat, harvesting season, extraction and processing conditions during extraction, various 3,6-anhydro-galactose (3,6-AG) contents and molecular sizes are found for agar extracts, which accordingly possess different gel characteristics (Matsuhashi, 1990). Based on the theory of rubber elasticity, the rheological properties of a thermoreversible gel were correlated with the molecular characteristics of junction zones formed by ordered helices (Oakenfull, 1984; Oakenfull & Scott, 1984, 1988). For example, the storage modulus (G 0 ) of a gel is proportional to the average number of junction zones within the gel network. Clark (1990) showed that the dependence of the modulus on polysaccharide concentration increased with the average number of double helices participating in a junction zone. Recently, changes in dynamic storage and loss moduli (G 0 and G 00 ) accompanying the development of a gel structure have been examined to q Journal paper no. 16270 of the Purdue University Agricultural Experiment Station. * Corresponding author. Tel.: 11-765-494-1749; fax: 11-765-494-7953. E-mail address: [email protected] (P. Cornillon).
clarify the sol±gel transition mechanism of the gel (Clark, 1990; Da Silva & Rao, 1995). Nuclear magnetic resonance (NMR) relaxometry is a powerful technique to study gel structure and analyze the mechanisms associated with gel formation. Indeed, gels contain a large amount of trapped water and the mobility of these water molecules are governed by a proton relaxation behavior. A gel is composed of multiple networks formed through various interactions between polysaccharide chains, in which water is entrapped. The extent of network formation is most probably responsible not only for the mechanical properties of the gel but also for the behavior (in terms of mobility) of the incorporated water. Investigations of gels formed by the interaction of biological macromolecules using pulsed NMR spectroscopy have been carried out by determining either the longitudinal (T1) or the transverse (T2) relaxation times, both indicators of the mobility of water within a gel (Cooke & Kuntz, 1974; Fung, 1977). When water is tightly associated with a substrate, it is highly immobilized and shows reduced T2, whereas bulk water is very mobile with a relatively long T2. Thus, useful information on the strength or degree of water interaction with macromolecules can be obtained. In NMR studies on food systems, Leung, Steinberg, Nelson, and Wei (1976) found that T1 and T2 were characteristics of the water binding properties of the food materials. In this work, it also is reported that water in hydrated corn starch samples could be separated into two species with different T2 values, corresponding to a mobile and a so-called `bound' water
0268-005X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0268-005 X(01)00 058-3
90
J.-E. Shin et al. / Food Hydrocolloids 16 (2002) 89±94
Table 1 Water loss of agar±sucrose gels. Note: (1) Average of six replicates; (2) Average of two replicates; (w.b.) wet basis Centrifugation
Gel
Water loss (% w.b.)
Standard deviation
5 min @ 5000 rpm 5 min @ 5000 rpm 5 min @ 5000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm 5 min @ 5000 rpm 5 min @ 5000 rpm 5 min @ 5000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm
1% agar 3% agar 5% agar 1% agar 3% agar 5% agar 3% agar±5% sucrose 3% agar±10% sucrose 3% agar±20% sucrose 3% agar±5% sucrose 3% agar±10% sucrose 3% agar±20% sucrose
1.8 (1) 0 (1) 0 (1) 21.3 (1) 7.6 (1) 0.1 (1) 0 0 0 2.23 (2) 2.7 (2) 2.9 (2)
1.8 0.0 0.0 6.0 5.7 0.1 0 0 0 0.04 0.4 0.6
fraction. It is now accepted that such `bound' water does not really exist but refers to water with restricted motion capability due to the presence of nearby macromolecules. Similarly, Capelin and Blanshard (1977) reported a pulsed NMR study to characterize the interaction behavior of water with gluten and starch in bread. The main objective of the present work was to characterize the changes of water mobility and viscoelastic properties induced by centrifugation on agar/sucrose gels by using NMR and dynamic mechanical spectroscopy measurements, respectively. Variables taken into account were the centrifugation speed and time, and the sucrose concentration. 2. Materials and methods 2.1. Preparation of agar gels Agar was purchased from Sigma Chemical Company (St Louis, MO) and sucrose from Aldrich (Milwaukee, WI). No further puri®cation was done on these chemicals. The concentration of agar was 1, 3 and 5% (w/w). Sucrose was added into the 3% agar gel at levels of 5, 10 and 20% (wet basis) to determine its effects on gel properties. To prepare the gels, water was heated to 908C in a beaker on a hot plate. Then, agar and sucrose were poured gradually into the beaker and heated to 958C on a hot plate for 15 min. Then the solution was cooled down to 608C and the amount of water loss (determined by weight) due to evaporation was added back to the solution. Twenty grams of the solution were poured in a centrifuge tube (27 mm diameter) and then cooled to room temperature to allow the gel to form. A maximum of 12 h (one night) was waited for the centrifugation tests to occur depending on the availability of the equipment. Samples after centrifugation were stored in a refrigerator at 48C before further rheological and NMR analyses. Measurements were made on samples within 3 days. 2.2. Centrifugation Twenty grams of sample were placed in a 50 ml centri-
fugation tube (27 mm diameter). The centrifugation conditions were either 5000 rpm (i.e. 3020 g) for 5 min or 15,000 rpm (i.e. 27,200 g) for 30 min. After centrifugation at room temperature, the exudate was weighed (Table 1). Measurements were made in duplicate. Each remainder of the gel (i.e. a total of two for each gel) after exudate removal was used for rheological and NMR experiments. The remainder of the gels could be identi®ed as damaged since the removal of water created cracks and lack of structure to the gel. For rheological and NMR analyses, care was taken to transfer this damaged section of the gel to either the rheometer or NMR tube without breaking or disturbing the damaged structure. The damaged gels were not remelted prior to measurement but rather used `as is' to fully evaluate the level of damage created by water removal from centrifugation. 2.3. Rheological measurements A controlled-stress ViscoTech rheometer (ReoLogica Instruments, Lund, Sweden) was used to measure the rheological properties of agar/sucrose gels. Operations including temperature control and data handling were conducted using a PC-based software provided by the manufacturer. Agar/ sucrose gels were placed between two parallel circular plates (5 mm spacing thickness) and trimmed to have the same diameter as the upper plate (20 mm). All measurements were performed at constant temperature (268C) under linear behavior and with a normal force of 0.1 N for the 3 and 5% agar gels and 0.05 N for the 1% agar gels. The storage modulus (G 0 ) was monitored as a function of time and frequency. Frequency sweep was carried out between 0.01 and 10 Hz. This property was measured during 25 min for each sample. 2.4. NMR experiments NMR relaxation properties were measured using a Maran Ultra NMR benchtop spectrometer (Resonance Instruments Ltd., Witney, UK) running at 23 MHz with constant temperature set to 358C. The gels were placed in 2.5 cm
J.-E. Shin et al. / Food Hydrocolloids 16 (2002) 89±94
91
Fig. 1. Effect of centrifugation on the variation of G 0 vs frequency for the 3% agar, 5% sucrose gel. A control sample before centrifugation; V sample after centrifugation at 5000 rpm for 5 min; O sample after centrifugation at 15,000 rpm for 30 min.
diameter NMR tubes. Spin±spin and spin±lattice relaxation curves were measured for each sample. The relaxation delay was 15 s, and the number of scans was 16 so that a good signal to noise ratio could be obtained (usually greater than 10). For spin±spin relaxation curves, the standard CarrPurcell-Meiboom-Gill pulse sequence was used with a number of echoes collected of 1024. The pulse spacing time was ®xed to 500 ms. For spin±lattice relaxation times measurement, the inversion recovery pulse sequence was used with time intervals between the 180 and 908 pulses varying between 1 ms and 15 s. The relaxation curves were analyzed with either a multi-exponential curve ®t or a distribution of relaxation times using software provided by the manufacturer. The multi-exponential curve ®t program was based on non-linear least square procedure to ®t experimental data to a mathematical model. The distribution of relaxation times software was based on a similar approach to that described elsewhere (Provencher, 1982). When performing both analyses, the results of the curve ®t were similar to that of the continuous analysis of relaxation behavior. Hence, we will only be reporting values obtained by
curve ®tting. All measurements were made in duplicate and the maximum standard deviation for T2 was 6.2 ms (i.e. about 14.2%) and for T1, 95 ms (i.e. 5.6%). 3. Results and discussion 3.1. Rheological properties Figs. 1±3 present the variations of the average storage modulus (G 0 ) as a function of frequency. As can be seen from the error bars (which represent a 95% con®dence interval), the variability in the measurements has been a major problem especially for the samples that were subjected to centrifugation. Indeed, centrifugation resulted in highly heterogeneous samples (i.e. in appearance) that lost a rather large amount of water in some cases (Table 1). These differences in texture made the rheological measurement fairly unpredictable. However, by analyzing the average modulus, it appears that centrifugation at 5000 rpm for 5 min caused the gels (for all sucrose
Fig. 2. Effect of centrifugation on the variation of G 0 vs frequency for the 3% agar, 10% sucrose gel. A control sample before centrifugation; V sample after centrifugation at 5000 rpm for 5 min; O sample after centrifugation at 15,000 rpm for 30 min.
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J.-E. Shin et al. / Food Hydrocolloids 16 (2002) 89±94
Fig. 3. Effect of centrifugation on the variation of G 0 vs frequency for the 3% agar, 20% sucrose gel. A control sample before centrifugation; V sample after centrifugation at 5000 rpm for 5 min; O sample after centrifugation at 15,000 rpm for 30 min.
concentrations) to become less elastic as compared to the control, since its G 0 value was smaller. The same results were obtained with the gels centrifuged at 15,000 rpm and 30 min only for sucrose concentrations higher than 10%. After removal of the samples from the centrifuge, a phase separation in the gels was observed that consisted of a weaker gel (less elastic) and a non-continuous water phase. On the other hand, the 3% agar±5% sucrose gel centrifuged at 15,000 rpm for 30 min tended to have a similar G 0 value as for control and a higher average G 0 value than when centrifuged at 5000 rpm. Thus, high centrifugation speeds may not change dramatically the texture of a gel possibly due to phase separation of water from the gel followed by recombination of some water in the modi®ed gel network (see discussion thereafter). Centrifugation would create `small' pools of water inside the network without affecting the mechanical properties too much. Water loss (as high as 21% as indicated in Table 1) was observed at high centrifugation speed indicating a loss of binding capability from the polymer network. From Table 1, water loss also was related to the initial concentration of agar and slightly related to sucrose
concentration, especially when centrifuged at 15,000 rpm. The centrifugation conditions had a more important effect on water loss. The effect of centrifugation could be quanti®ed by analyzing the relative storage modulus (RG 0 ), de®ned as being the ratio of the storage modulus of a centrifuged agar-sucrose gel over the storage modulus of the same agar±sucrose gel prior to centrifugation. Fig. 4 indicates the variation of RG 0 as a function of sucrose concentration in the gel. At high centrifugation speeds and low sucrose content, RG 0 is rather constant indicating that it does not have an effect on the rheological behavior of the gel. However, when sucrose concentration increases up to 20%, G 0 of the centrifuged gel becomes much larger indicating a stiffer gel (most likely due to water loss and increase in polymer packing in the gel). At lower speeds, the decrease of RG 0 was more dramatic indicating that more structural and textural changes occurred to these gels as opposed to high speeds. However, sucrose content did not seem to in¯uence much the texture of the gel during centrifugation at low speed (most likely due to minimal or no water loss). Having sucrose in the polymer network could
Fig. 4. In¯uence of sucrose concentration on the relative G 0 (RG 0 ) of a centrifuged 3% agar gel. Frequency 1 Hz. The control sample is the corresponding gel prior to centrifugation. RG 0 was calculated using the average values of G 0 for each sample.
J.-E. Shin et al. / Food Hydrocolloids 16 (2002) 89±94
93
Fig. 5. Effects of centrifugation and sucrose content on T1 values of 3% agar gels.
have prevented exudation of water from the network as compared to pure agar gels because of the binding properties of sucrose through hydrogen bonding (Table 1). 3.2. NMR properties 3.2.1. Spin±lattice relaxation Fig. 5 presents the variation of the spin±lattice relaxation time (T1) of agar±sucrose gels before and after centrifugation. For the control gel (no centrifugation) and the gel centrifuged at low speed, T1 decreased as sucrose concentration increased due to a decrease in water mobility from more interactions with sugars and macromolecules, and a competitive effect on water binding between sucrose and agar. The decrease in T1 is fairly linear with sucrose concentration. However, centrifugation at high speed produced a gel that had a much lower T1 value when no sucrose was in the gel. When sucrose was added to agar in the gel, the relaxation times were consistent with the results of the other gels i.e. T1 decreased as sucrose concentration increased. Hence, the effect of centrifugation at high speed on water mobility can be compensated by the addition of sucrose to retain interactions with water molecules. 3.2.2. Spin±spin relaxation Table 2 presents the values of spin±spin relaxation times (T2) as a function of sucrose concentration for all 3% agar gels. Curve ®tting and continuous analysis indicated that the relaxation behavior was governed by two different mobility states. Depending on the nature and treatment of the gel, the ®rst mobility state had a T2a between 27 and 41 ms (representing between 69 and 97% of the total signal) and the second mobility state had a T2b value between 80 and 850 ms (representing the rest of the signal). Before centrifugation, sucrose helped decrease T2a but increased its overall importance in the signal. This result is consistent with the increase in water interaction in the gel through hydrogen bonding. After centrifugation, both relaxation times did not seem to be affected. However, the shorter component
(T2a) had a slightly smaller importance in the overall signal indicating that centrifugation freed up some water molecules from their interaction with sucrose. This also indicates an averaging effect of interactions through hydrogen bonds between water, agar, and sucrose. The presence of two relaxation times in the relaxation behavior of these gels indicates that the macromolecules in the gel had a certain degree of packing, or even crosslinking, creating two water compartments with restricted motion. Also, T2a value for centrifuged samples was similar to that of not-centrifuged samples, which could explain why the storage modulus G 0 of that particular gel was relatively close to that of the control agar gel. 3.2.3. Spin±lattice relaxation (T1) distribution Chui, Phillips, and McCarthy (1995) analyzed the microstructure of gels of agar at various concentrations from 1 to 5.72% (w/w) using a ®ber model associated with the Brownstein and Tarr theory applied to spin±lattice relaxation phenomena (Brownstein & Tarr, 1979). They found that an increase in the concentration of agar decreased the pore size of the ®ber due to a closer packing of macromolecules. At 1%, the hydraulic radius of the ®ber was nearly 200 nm whereas at 5.72% it was around 40 nm. This approach was used in this study to determine the in¯uence of sucrose concentration and centrifugation conditions on the hydraulic radius of the ®bers. When water molecules diffuse in pores, they can bounce on the walls of the pore and the energy exchange involved in this molecule/wall interaction is characterized by a surface `sink' parameter (Chui et al., 1995). In this analysis, the sink effect from sugars was not taken into account since they do not represent any `sink' for water to relax on, but rather are solvated in water. Thus, sucrose would be dissolved in the interspace between macromolecules. After inversion of the data as indicated by Chui et al. (1995), the distribution of the hydraulic radius could be obtained. For all agar±sucrose gels, the radius was between 10 and 25 nm, indicating that the concentration
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J.-E. Shin et al. / Food Hydrocolloids 16 (2002) 89±94
Table 2 Short and long components of T2 relaxation with their respective populations for all 3% agar gels studied with and without sucrose 3% agar gel
T2a (ms)
% T2a
T2b (ms)
% T2b
No centrifugation, no sucrose No centrifugation, 5% sucrose No centrifugation, 5% sucrose Average Std dev No centrifugation, 10% sucrose No centrifugation, 10% sucrose Average Std dev No centrifugation, 20% sucrose No centrifugation, 20% sucrose Average Std dev 5000 rpm for 5 min, 5% sucrose 5000 rpm for 5 min, 5% sucrose Average Std dev 5000 rpm for 5 min, 10% sucrose 5000 rpm for 5 min, 10% sucrose Average Std dev 5000 rpm for 5 min, 20% sucrose 5000 rpm for 5 min, 20% sucrose Average Std dev 15,000 rpm for 30 min, 5% sucrose 15,000 rpm for 30 min, 5% sucrose Average Std dev 15,000 rpm for 30 min, 10% sucrose 15,000 rpm for 30 min, 10% sucrose Average Std dev 15,000 rpm for 30 min, 20% sucrose 15,000 rpm for 30 min, 20% sucrose Average Std dev
40.9 32.5 29.4 31.0 2.2 30.6 31.1 30.9 0.4 38.5 29.4 34.0 6.4 36.3 30.9 33.6 3.8 30.1 33.1 31.6 2.1 41.0 38.1 39.6 2.1 32.3
68.8 97.4 97.1 97.2 0.2 94.7 94.6 94.6 0.0 89.8 88.4 89.1 1.0 91.0 79.5 85.2 8.2 78.2 96.6 87.4 13.1 86.2 78.1 82.2 5.7 78.8
131 422.9 395.4 409.2 19.4 350.1 341.7 345.9 5.9 257.3 221.2 239.3 25.5 90.8 72.8 81.8 12.7 97.1 841.7 469.4 526.5 208 132.1 170.1 53.7 82.1
31.2 2.6 2.9 2.8 0.2 5.3 5.4 5.4 0.0 10.2 11.6 10.9 1.0 9.0 20.5 14.8 8.2 21.8 3.4 12.6 13.1 13.8 21.9 17.8 5.7 21.2
27.4
85.7
76.1
14.3
29.9 3.5 37.8
82.3 4.9 85.9
79.1 4.2 117.7
17.7 4.9 14.1
30.8
78.7
103.2
21.3
34.3 4.9 31.1
82.3 5.1 81.2
110.5 10.3 131.5
17.7 5.1 18.8
32.4
77.6
124.9
22.4
31.8 0.9
79.4 2.5
128.2 4.7
20.6 2.5
of sucrose did not in¯uence the pore dimension. This dimension would then be more dependent on the macromolecular structure of agar. The range of radius found in this research differs somewhat from Chui et al. (1995). One reason for this difference might be due to the value of the spin±lattice relaxation time of bulk water that was selected in the calculation between the present study and Chui et al. (1995).
4. Conclusion The water loss for the 3% agar gels (with or without sucrose) after being centrifuged at 5000 rpm for 5 min or 15,000 rpm for 30 min is related to: (1) the rheological properties and (2) the water proton longitudinal (T1) and transverse (T2) relaxation times. There was no water loss for the gels centrifuged at 5000 rpm for 5 min. As a result, their G 0 value was lower, so the T1 and T2 values were greater as compared to the gel centrifuged at 15,000 rpm for 30 min. Therefore, the agar gel centrifuged at 5000 rpm for 5 min had higher water mobility. The agar gel centrifuged at 15,000 rpm for 30 min presented a reorganization of the polymer network probably through closer packing of polymer chains. They also had a greater water loss. In the presence of sucrose, less packing could be observed due to solvation of sucrose in water. References Brownstein, K. R., & Tarr, C. E. (1979). Importance of classical diffusion in NMR studies of water in biological cells. Physical Review A, 19 (6), 2446±2453. Capelin, S. C., Blanshard, J. M. V., & Pulsed, N. M. R. (1977). studies of the interaction of water with gluten and starch in bread. Cereal Foods World, 22, 471±485. Chui, M. C., Phillips, R. J., & McCarthy, M. J. (1995). Measurement of the porous microstructure of gels by nuclear magnetic resonance. Journal of Colloid and Interface Science, 174, 336±344. Clark, A. H. (1990). Gels and gelling. In H. G. Schwartzberg & R. W. Hartel, Physical chemistry of foods (pp. 263±305). New York: Marcel Dekker. Cooke, R., & Kuntz, I. D. (1974). The properties of water in biological systems. Annual Reviews of Biophysics and Bioengineering, 3, 95±126. Da Silva, J. A. L., & Rao, M. A. (1995). Rheology of structure development in high methoxyl pectin/sugar system. Food Technology, 49 (10), 70±73. Fung, B. M. (1977). Proton and deuteron relaxation of muscle water over wide ranges of resonance. Biophysics Journal, 18 (2), 235±239. Leung, H. K., Steinberg, M. P., Nelson, A. I., & Wei, L. W. (1976). Water binding of macromolecules determined by pulsed NMR. Journal of Food Science, 41, 297±300. Matsuhashi, T. (1990). Agar. In P. Harris, Food gels (pp. 1±51). London: Elsevier Science. Oakenfull, D. (1984). A method for using measurements of shear modulus to estimate the size and thermodynamic stability of junction zones in noncovalently cross-linked gels. Journal of Food Science, 49 (4), 1103±1104. Oakenfull, D., & Scott, A. (1984). Hydrophobic interaction in the gelation of high methoxyl pectins. Journal of Food Science, 49 (4), 1093±1098. Oakenfull, D. G., & Scott, A. (1988). Size and stability of the junction zones in gels of iota and kappa carrageenan. In G. O. Phillips, P. A. Williams & D. J. Wedlock, Gums and stabilizers for the food industry (pp. 127±134). Vol. 4. Oxford: IRL Press. Provencher, S. W. (1982). A constrained regularization method for inverting data represented by linear algebraic or integral equations. Computational and Physical Communications, 27, 213±227. Stanley, N. F. (1990). Carrageenan. In P. Harris, Food gels (pp. 79±119). London: Elsevier Science.
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The effect of centrifugation on agar/sucrose gels q Jin-E. Shin, Paul Cornillon*, Linda Salim Purdue University, Whistler Center for Carbohydrate Research, Food Science Department, 1160 Food Science Building, West Lafayette, IN 47907-1160, USA Received 1 May 2000; revised 22 January 2001; accepted 6 April 2001
Abstract The viscoelascity and water mobility of 3% agar gels containing 5, 10 and 20% sucrose, before and after centrifugation, were studied by using dynamic mechanical spectroscopy and nuclear magnetic resonance relaxometry. Centrifugation was found to induce damage in the gel by phase separation of water. However, results suggest that water can recombine after centrifugation with closely packed macromolecules to provide a similar molecular structure to that of the gel before centrifugation. Sucrose helped reduce exudation of water at low centrifugation speeds through hydrogen bonding. The effects of centrifugation at high speed on water mobility were compensated by increasing sucrose concentration. Centrifugation created water compartments in the gel with distinct motion properties. On the contrary, sucrose concentration did not affect the hydraulic radius of these compartments. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Agar/sucrose gels; Water mobility; NMR; Centrifugation
1. Introduction The hydrocolloid agar is mainly composed of agarobiose (3-b-d-galactopyranosyl-(1- . 4)-3,6-anhydro-a-l-galactose, Matsuhashi, 1990). The principal sources of commercial agar include the gelidiales and gracilariales that are aquatic plants found primarily in Asia (Matsuhashi, 1990; Stanley, 1990). Due to differences in species, habitat, harvesting season, extraction and processing conditions during extraction, various 3,6-anhydro-galactose (3,6-AG) contents and molecular sizes are found for agar extracts, which accordingly possess different gel characteristics (Matsuhashi, 1990). Based on the theory of rubber elasticity, the rheological properties of a thermoreversible gel were correlated with the molecular characteristics of junction zones formed by ordered helices (Oakenfull, 1984; Oakenfull & Scott, 1984, 1988). For example, the storage modulus (G 0 ) of a gel is proportional to the average number of junction zones within the gel network. Clark (1990) showed that the dependence of the modulus on polysaccharide concentration increased with the average number of double helices participating in a junction zone. Recently, changes in dynamic storage and loss moduli (G 0 and G 00 ) accompanying the development of a gel structure have been examined to q Journal paper no. 16270 of the Purdue University Agricultural Experiment Station. * Corresponding author. Tel.: 11-765-494-1749; fax: 11-765-494-7953. E-mail address: [email protected] (P. Cornillon).
clarify the sol±gel transition mechanism of the gel (Clark, 1990; Da Silva & Rao, 1995). Nuclear magnetic resonance (NMR) relaxometry is a powerful technique to study gel structure and analyze the mechanisms associated with gel formation. Indeed, gels contain a large amount of trapped water and the mobility of these water molecules are governed by a proton relaxation behavior. A gel is composed of multiple networks formed through various interactions between polysaccharide chains, in which water is entrapped. The extent of network formation is most probably responsible not only for the mechanical properties of the gel but also for the behavior (in terms of mobility) of the incorporated water. Investigations of gels formed by the interaction of biological macromolecules using pulsed NMR spectroscopy have been carried out by determining either the longitudinal (T1) or the transverse (T2) relaxation times, both indicators of the mobility of water within a gel (Cooke & Kuntz, 1974; Fung, 1977). When water is tightly associated with a substrate, it is highly immobilized and shows reduced T2, whereas bulk water is very mobile with a relatively long T2. Thus, useful information on the strength or degree of water interaction with macromolecules can be obtained. In NMR studies on food systems, Leung, Steinberg, Nelson, and Wei (1976) found that T1 and T2 were characteristics of the water binding properties of the food materials. In this work, it also is reported that water in hydrated corn starch samples could be separated into two species with different T2 values, corresponding to a mobile and a so-called `bound' water
0268-005X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0268-005 X(01)00 058-3
90
J.-E. Shin et al. / Food Hydrocolloids 16 (2002) 89±94
Table 1 Water loss of agar±sucrose gels. Note: (1) Average of six replicates; (2) Average of two replicates; (w.b.) wet basis Centrifugation
Gel
Water loss (% w.b.)
Standard deviation
5 min @ 5000 rpm 5 min @ 5000 rpm 5 min @ 5000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm 5 min @ 5000 rpm 5 min @ 5000 rpm 5 min @ 5000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm 30 min @ 15,000 rpm
1% agar 3% agar 5% agar 1% agar 3% agar 5% agar 3% agar±5% sucrose 3% agar±10% sucrose 3% agar±20% sucrose 3% agar±5% sucrose 3% agar±10% sucrose 3% agar±20% sucrose
1.8 (1) 0 (1) 0 (1) 21.3 (1) 7.6 (1) 0.1 (1) 0 0 0 2.23 (2) 2.7 (2) 2.9 (2)
1.8 0.0 0.0 6.0 5.7 0.1 0 0 0 0.04 0.4 0.6
fraction. It is now accepted that such `bound' water does not really exist but refers to water with restricted motion capability due to the presence of nearby macromolecules. Similarly, Capelin and Blanshard (1977) reported a pulsed NMR study to characterize the interaction behavior of water with gluten and starch in bread. The main objective of the present work was to characterize the changes of water mobility and viscoelastic properties induced by centrifugation on agar/sucrose gels by using NMR and dynamic mechanical spectroscopy measurements, respectively. Variables taken into account were the centrifugation speed and time, and the sucrose concentration. 2. Materials and methods 2.1. Preparation of agar gels Agar was purchased from Sigma Chemical Company (St Louis, MO) and sucrose from Aldrich (Milwaukee, WI). No further puri®cation was done on these chemicals. The concentration of agar was 1, 3 and 5% (w/w). Sucrose was added into the 3% agar gel at levels of 5, 10 and 20% (wet basis) to determine its effects on gel properties. To prepare the gels, water was heated to 908C in a beaker on a hot plate. Then, agar and sucrose were poured gradually into the beaker and heated to 958C on a hot plate for 15 min. Then the solution was cooled down to 608C and the amount of water loss (determined by weight) due to evaporation was added back to the solution. Twenty grams of the solution were poured in a centrifuge tube (27 mm diameter) and then cooled to room temperature to allow the gel to form. A maximum of 12 h (one night) was waited for the centrifugation tests to occur depending on the availability of the equipment. Samples after centrifugation were stored in a refrigerator at 48C before further rheological and NMR analyses. Measurements were made on samples within 3 days. 2.2. Centrifugation Twenty grams of sample were placed in a 50 ml centri-
fugation tube (27 mm diameter). The centrifugation conditions were either 5000 rpm (i.e. 3020 g) for 5 min or 15,000 rpm (i.e. 27,200 g) for 30 min. After centrifugation at room temperature, the exudate was weighed (Table 1). Measurements were made in duplicate. Each remainder of the gel (i.e. a total of two for each gel) after exudate removal was used for rheological and NMR experiments. The remainder of the gels could be identi®ed as damaged since the removal of water created cracks and lack of structure to the gel. For rheological and NMR analyses, care was taken to transfer this damaged section of the gel to either the rheometer or NMR tube without breaking or disturbing the damaged structure. The damaged gels were not remelted prior to measurement but rather used `as is' to fully evaluate the level of damage created by water removal from centrifugation. 2.3. Rheological measurements A controlled-stress ViscoTech rheometer (ReoLogica Instruments, Lund, Sweden) was used to measure the rheological properties of agar/sucrose gels. Operations including temperature control and data handling were conducted using a PC-based software provided by the manufacturer. Agar/ sucrose gels were placed between two parallel circular plates (5 mm spacing thickness) and trimmed to have the same diameter as the upper plate (20 mm). All measurements were performed at constant temperature (268C) under linear behavior and with a normal force of 0.1 N for the 3 and 5% agar gels and 0.05 N for the 1% agar gels. The storage modulus (G 0 ) was monitored as a function of time and frequency. Frequency sweep was carried out between 0.01 and 10 Hz. This property was measured during 25 min for each sample. 2.4. NMR experiments NMR relaxation properties were measured using a Maran Ultra NMR benchtop spectrometer (Resonance Instruments Ltd., Witney, UK) running at 23 MHz with constant temperature set to 358C. The gels were placed in 2.5 cm
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Fig. 1. Effect of centrifugation on the variation of G 0 vs frequency for the 3% agar, 5% sucrose gel. A control sample before centrifugation; V sample after centrifugation at 5000 rpm for 5 min; O sample after centrifugation at 15,000 rpm for 30 min.
diameter NMR tubes. Spin±spin and spin±lattice relaxation curves were measured for each sample. The relaxation delay was 15 s, and the number of scans was 16 so that a good signal to noise ratio could be obtained (usually greater than 10). For spin±spin relaxation curves, the standard CarrPurcell-Meiboom-Gill pulse sequence was used with a number of echoes collected of 1024. The pulse spacing time was ®xed to 500 ms. For spin±lattice relaxation times measurement, the inversion recovery pulse sequence was used with time intervals between the 180 and 908 pulses varying between 1 ms and 15 s. The relaxation curves were analyzed with either a multi-exponential curve ®t or a distribution of relaxation times using software provided by the manufacturer. The multi-exponential curve ®t program was based on non-linear least square procedure to ®t experimental data to a mathematical model. The distribution of relaxation times software was based on a similar approach to that described elsewhere (Provencher, 1982). When performing both analyses, the results of the curve ®t were similar to that of the continuous analysis of relaxation behavior. Hence, we will only be reporting values obtained by
curve ®tting. All measurements were made in duplicate and the maximum standard deviation for T2 was 6.2 ms (i.e. about 14.2%) and for T1, 95 ms (i.e. 5.6%). 3. Results and discussion 3.1. Rheological properties Figs. 1±3 present the variations of the average storage modulus (G 0 ) as a function of frequency. As can be seen from the error bars (which represent a 95% con®dence interval), the variability in the measurements has been a major problem especially for the samples that were subjected to centrifugation. Indeed, centrifugation resulted in highly heterogeneous samples (i.e. in appearance) that lost a rather large amount of water in some cases (Table 1). These differences in texture made the rheological measurement fairly unpredictable. However, by analyzing the average modulus, it appears that centrifugation at 5000 rpm for 5 min caused the gels (for all sucrose
Fig. 2. Effect of centrifugation on the variation of G 0 vs frequency for the 3% agar, 10% sucrose gel. A control sample before centrifugation; V sample after centrifugation at 5000 rpm for 5 min; O sample after centrifugation at 15,000 rpm for 30 min.
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Fig. 3. Effect of centrifugation on the variation of G 0 vs frequency for the 3% agar, 20% sucrose gel. A control sample before centrifugation; V sample after centrifugation at 5000 rpm for 5 min; O sample after centrifugation at 15,000 rpm for 30 min.
concentrations) to become less elastic as compared to the control, since its G 0 value was smaller. The same results were obtained with the gels centrifuged at 15,000 rpm and 30 min only for sucrose concentrations higher than 10%. After removal of the samples from the centrifuge, a phase separation in the gels was observed that consisted of a weaker gel (less elastic) and a non-continuous water phase. On the other hand, the 3% agar±5% sucrose gel centrifuged at 15,000 rpm for 30 min tended to have a similar G 0 value as for control and a higher average G 0 value than when centrifuged at 5000 rpm. Thus, high centrifugation speeds may not change dramatically the texture of a gel possibly due to phase separation of water from the gel followed by recombination of some water in the modi®ed gel network (see discussion thereafter). Centrifugation would create `small' pools of water inside the network without affecting the mechanical properties too much. Water loss (as high as 21% as indicated in Table 1) was observed at high centrifugation speed indicating a loss of binding capability from the polymer network. From Table 1, water loss also was related to the initial concentration of agar and slightly related to sucrose
concentration, especially when centrifuged at 15,000 rpm. The centrifugation conditions had a more important effect on water loss. The effect of centrifugation could be quanti®ed by analyzing the relative storage modulus (RG 0 ), de®ned as being the ratio of the storage modulus of a centrifuged agar-sucrose gel over the storage modulus of the same agar±sucrose gel prior to centrifugation. Fig. 4 indicates the variation of RG 0 as a function of sucrose concentration in the gel. At high centrifugation speeds and low sucrose content, RG 0 is rather constant indicating that it does not have an effect on the rheological behavior of the gel. However, when sucrose concentration increases up to 20%, G 0 of the centrifuged gel becomes much larger indicating a stiffer gel (most likely due to water loss and increase in polymer packing in the gel). At lower speeds, the decrease of RG 0 was more dramatic indicating that more structural and textural changes occurred to these gels as opposed to high speeds. However, sucrose content did not seem to in¯uence much the texture of the gel during centrifugation at low speed (most likely due to minimal or no water loss). Having sucrose in the polymer network could
Fig. 4. In¯uence of sucrose concentration on the relative G 0 (RG 0 ) of a centrifuged 3% agar gel. Frequency 1 Hz. The control sample is the corresponding gel prior to centrifugation. RG 0 was calculated using the average values of G 0 for each sample.
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Fig. 5. Effects of centrifugation and sucrose content on T1 values of 3% agar gels.
have prevented exudation of water from the network as compared to pure agar gels because of the binding properties of sucrose through hydrogen bonding (Table 1). 3.2. NMR properties 3.2.1. Spin±lattice relaxation Fig. 5 presents the variation of the spin±lattice relaxation time (T1) of agar±sucrose gels before and after centrifugation. For the control gel (no centrifugation) and the gel centrifuged at low speed, T1 decreased as sucrose concentration increased due to a decrease in water mobility from more interactions with sugars and macromolecules, and a competitive effect on water binding between sucrose and agar. The decrease in T1 is fairly linear with sucrose concentration. However, centrifugation at high speed produced a gel that had a much lower T1 value when no sucrose was in the gel. When sucrose was added to agar in the gel, the relaxation times were consistent with the results of the other gels i.e. T1 decreased as sucrose concentration increased. Hence, the effect of centrifugation at high speed on water mobility can be compensated by the addition of sucrose to retain interactions with water molecules. 3.2.2. Spin±spin relaxation Table 2 presents the values of spin±spin relaxation times (T2) as a function of sucrose concentration for all 3% agar gels. Curve ®tting and continuous analysis indicated that the relaxation behavior was governed by two different mobility states. Depending on the nature and treatment of the gel, the ®rst mobility state had a T2a between 27 and 41 ms (representing between 69 and 97% of the total signal) and the second mobility state had a T2b value between 80 and 850 ms (representing the rest of the signal). Before centrifugation, sucrose helped decrease T2a but increased its overall importance in the signal. This result is consistent with the increase in water interaction in the gel through hydrogen bonding. After centrifugation, both relaxation times did not seem to be affected. However, the shorter component
(T2a) had a slightly smaller importance in the overall signal indicating that centrifugation freed up some water molecules from their interaction with sucrose. This also indicates an averaging effect of interactions through hydrogen bonds between water, agar, and sucrose. The presence of two relaxation times in the relaxation behavior of these gels indicates that the macromolecules in the gel had a certain degree of packing, or even crosslinking, creating two water compartments with restricted motion. Also, T2a value for centrifuged samples was similar to that of not-centrifuged samples, which could explain why the storage modulus G 0 of that particular gel was relatively close to that of the control agar gel. 3.2.3. Spin±lattice relaxation (T1) distribution Chui, Phillips, and McCarthy (1995) analyzed the microstructure of gels of agar at various concentrations from 1 to 5.72% (w/w) using a ®ber model associated with the Brownstein and Tarr theory applied to spin±lattice relaxation phenomena (Brownstein & Tarr, 1979). They found that an increase in the concentration of agar decreased the pore size of the ®ber due to a closer packing of macromolecules. At 1%, the hydraulic radius of the ®ber was nearly 200 nm whereas at 5.72% it was around 40 nm. This approach was used in this study to determine the in¯uence of sucrose concentration and centrifugation conditions on the hydraulic radius of the ®bers. When water molecules diffuse in pores, they can bounce on the walls of the pore and the energy exchange involved in this molecule/wall interaction is characterized by a surface `sink' parameter (Chui et al., 1995). In this analysis, the sink effect from sugars was not taken into account since they do not represent any `sink' for water to relax on, but rather are solvated in water. Thus, sucrose would be dissolved in the interspace between macromolecules. After inversion of the data as indicated by Chui et al. (1995), the distribution of the hydraulic radius could be obtained. For all agar±sucrose gels, the radius was between 10 and 25 nm, indicating that the concentration
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Table 2 Short and long components of T2 relaxation with their respective populations for all 3% agar gels studied with and without sucrose 3% agar gel
T2a (ms)
% T2a
T2b (ms)
% T2b
No centrifugation, no sucrose No centrifugation, 5% sucrose No centrifugation, 5% sucrose Average Std dev No centrifugation, 10% sucrose No centrifugation, 10% sucrose Average Std dev No centrifugation, 20% sucrose No centrifugation, 20% sucrose Average Std dev 5000 rpm for 5 min, 5% sucrose 5000 rpm for 5 min, 5% sucrose Average Std dev 5000 rpm for 5 min, 10% sucrose 5000 rpm for 5 min, 10% sucrose Average Std dev 5000 rpm for 5 min, 20% sucrose 5000 rpm for 5 min, 20% sucrose Average Std dev 15,000 rpm for 30 min, 5% sucrose 15,000 rpm for 30 min, 5% sucrose Average Std dev 15,000 rpm for 30 min, 10% sucrose 15,000 rpm for 30 min, 10% sucrose Average Std dev 15,000 rpm for 30 min, 20% sucrose 15,000 rpm for 30 min, 20% sucrose Average Std dev
40.9 32.5 29.4 31.0 2.2 30.6 31.1 30.9 0.4 38.5 29.4 34.0 6.4 36.3 30.9 33.6 3.8 30.1 33.1 31.6 2.1 41.0 38.1 39.6 2.1 32.3
68.8 97.4 97.1 97.2 0.2 94.7 94.6 94.6 0.0 89.8 88.4 89.1 1.0 91.0 79.5 85.2 8.2 78.2 96.6 87.4 13.1 86.2 78.1 82.2 5.7 78.8
131 422.9 395.4 409.2 19.4 350.1 341.7 345.9 5.9 257.3 221.2 239.3 25.5 90.8 72.8 81.8 12.7 97.1 841.7 469.4 526.5 208 132.1 170.1 53.7 82.1
31.2 2.6 2.9 2.8 0.2 5.3 5.4 5.4 0.0 10.2 11.6 10.9 1.0 9.0 20.5 14.8 8.2 21.8 3.4 12.6 13.1 13.8 21.9 17.8 5.7 21.2
27.4
85.7
76.1
14.3
29.9 3.5 37.8
82.3 4.9 85.9
79.1 4.2 117.7
17.7 4.9 14.1
30.8
78.7
103.2
21.3
34.3 4.9 31.1
82.3 5.1 81.2
110.5 10.3 131.5
17.7 5.1 18.8
32.4
77.6
124.9
22.4
31.8 0.9
79.4 2.5
128.2 4.7
20.6 2.5
of sucrose did not in¯uence the pore dimension. This dimension would then be more dependent on the macromolecular structure of agar. The range of radius found in this research differs somewhat from Chui et al. (1995). One reason for this difference might be due to the value of the spin±lattice relaxation time of bulk water that was selected in the calculation between the present study and Chui et al. (1995).
4. Conclusion The water loss for the 3% agar gels (with or without sucrose) after being centrifuged at 5000 rpm for 5 min or 15,000 rpm for 30 min is related to: (1) the rheological properties and (2) the water proton longitudinal (T1) and transverse (T2) relaxation times. There was no water loss for the gels centrifuged at 5000 rpm for 5 min. As a result, their G 0 value was lower, so the T1 and T2 values were greater as compared to the gel centrifuged at 15,000 rpm for 30 min. Therefore, the agar gel centrifuged at 5000 rpm for 5 min had higher water mobility. The agar gel centrifuged at 15,000 rpm for 30 min presented a reorganization of the polymer network probably through closer packing of polymer chains. They also had a greater water loss. In the presence of sucrose, less packing could be observed due to solvation of sucrose in water. References Brownstein, K. R., & Tarr, C. E. (1979). Importance of classical diffusion in NMR studies of water in biological cells. Physical Review A, 19 (6), 2446±2453. Capelin, S. C., Blanshard, J. M. V., & Pulsed, N. M. R. (1977). studies of the interaction of water with gluten and starch in bread. Cereal Foods World, 22, 471±485. Chui, M. C., Phillips, R. J., & McCarthy, M. J. (1995). Measurement of the porous microstructure of gels by nuclear magnetic resonance. Journal of Colloid and Interface Science, 174, 336±344. Clark, A. H. (1990). Gels and gelling. In H. G. Schwartzberg & R. W. Hartel, Physical chemistry of foods (pp. 263±305). New York: Marcel Dekker. Cooke, R., & Kuntz, I. D. (1974). The properties of water in biological systems. Annual Reviews of Biophysics and Bioengineering, 3, 95±126. Da Silva, J. A. L., & Rao, M. A. (1995). Rheology of structure development in high methoxyl pectin/sugar system. Food Technology, 49 (10), 70±73. Fung, B. M. (1977). Proton and deuteron relaxation of muscle water over wide ranges of resonance. Biophysics Journal, 18 (2), 235±239. Leung, H. K., Steinberg, M. P., Nelson, A. I., & Wei, L. W. (1976). Water binding of macromolecules determined by pulsed NMR. Journal of Food Science, 41, 297±300. Matsuhashi, T. (1990). Agar. In P. Harris, Food gels (pp. 1±51). London: Elsevier Science. Oakenfull, D. (1984). A method for using measurements of shear modulus to estimate the size and thermodynamic stability of junction zones in noncovalently cross-linked gels. Journal of Food Science, 49 (4), 1103±1104. Oakenfull, D., & Scott, A. (1984). Hydrophobic interaction in the gelation of high methoxyl pectins. Journal of Food Science, 49 (4), 1093±1098. Oakenfull, D. G., & Scott, A. (1988). Size and stability of the junction zones in gels of iota and kappa carrageenan. In G. O. Phillips, P. A. Williams & D. J. Wedlock, Gums and stabilizers for the food industry (pp. 127±134). Vol. 4. Oxford: IRL Press. Provencher, S. W. (1982). A constrained regularization method for inverting data represented by linear algebraic or integral equations. Computational and Physical Communications, 27, 213±227. Stanley, N. F. (1990). Carrageenan. In P. Harris, Food gels (pp. 79±119). London: Elsevier Science.