Vol. 86, No. 3, 2009 333

Structural and Physical Changes in Ultrasound-Assisted Frozen Wet Gluten

GuoSheng Song,1,2 Song Qing Hu,1,3 Lin Li,1 Ping Chen,1 and Xing Shen1

ABSTRACT Cereal Chem. 86(3):333–338

Ultrasound-assisted freezing is a novel food-freezing technique which is of benefit to frozen food quality. Studies of the structural and physical changes in the ultrasound-assisted frozen wet gluten system illustrate how the ultrasound irradiation improves the frozen dough quality. The micro-topography of ice crystals in frozen wet gluten was observed indirectly by scanning electron microscopy; the ice crystals were smaller and more uniform in wet gluten frozen using the ultrasound-assisted technique than in wet gluten frozen without ultrasound. Fourier transform infrared spec-troscopy was used to monitor the changes in the secondary structure of a frozen gluten heavy water system during frozen storage. It was deter-mined that there were no noticeable changes in the structure of the wet gluten frozen using the ultrasound-assisted technique. However, the struc-

ture of the wet gluten frozen without ultrasound became unordered. The freezable water content in the frozen wet gluten was determined by dif-ferential scanning calorimetry. Compared with conventional freezing, more water was frozen using ultrasound-assisted freezing. Furthermore, the freezable water content in the wet gluten frozen using the ultrasound-assisted technique did not increase during frozen storage. Ultrasonic cavitation promoted the primary and secondary nucleation of ice in the wet gluten and loosened the weak interactions between the gluten and the water. Consequently, the existence of a microstructure with small and uniform ice crystals was noted, which was determined to be beneficial with regard to reducing the deterioration of the gluten network and, hence, improving the quality of the frozen food.

Frozen storage is an efficient method for preserving food, and

freezing is a popular form of food processing. As an important composition in most food materials, water has three microstates: free water, loosely bound water, and bound water (Higuchi and Iijima 1985). When food materials are frozen and chilled, only the free water and a portion of the loosely bound water form ice, while the bound water and the remaining portion of the loosely bound water do not form ice. As a result, the former is called freezable water and the latter is called unfreezable water. Unfor-tunately, the quality of food materials degrades as a result of the freezing process and frozen storage. Many studies (Lu and Grant 1999; Bhattacharya et al 2003) have shown that ice crystal growth is the main reason for this degradation. As the ice crystals grow and take up a larger volume, the quality of the food suffers due to an increase in the amount of freezable water and water migration (Larsson and Eliasson 1996; Rasanen et al 1997; Esselink et al 2003). To reduce deterioration caused by freezing, many novel freezing methods such as high-pressure-assisted freezing (Ka-lichevsky et al 1995; Otero et al 2000), dehydrofreezing (Spiazzi et al 1998), and ultrasound-assisted freezing (Li and Sun 2002; Zheng and Sun 2006) have been explored.

Power ultrasound, a kind of ultrasound that employs low-frequency, high-intensity waves, has been proven to improve the ice crystallization process. Power ultrasound plays a role in the initiation of crystal nuclei through acoustic cavitation, the forma-tion, growth, and violent collapse of small bubbles or voids in liquids, induced by ultrasonic vibration (Simal et al 1998). Earlier experiments (Inada et al 2001; Zhang et al 2001, 2003) indicate that cavitation and its secondary effects strongly promote phase changes from supercooled water containing air bubbles to ice. In the experiments, power ultrasound not only promoted primary nucleation of ice in a sucrose solution by increasing the tempera-ture at which nucleation takes place but also promoted secondary nucleation by breaking up the preexisting dendritic ice crystals into smaller fragments (Chow et al 2003, 2004, 2005). Indeed, the ultrasound-assisted technique improved the quality of frozen foods (Li and Sun 2002; Zheng and Sun 2006).

Because gluten is the main protein that maintains the structure of dough, studies on the effect of ultrasound-assisted freezing on the structural and physical properties of the frozen wet gluten would further our understanding of how ultrasound-assisted freez-ing intrinsically improves the quality of frozen food in general. Many processes, such as differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), and cryogenic scanning electron microscopy (cryo-SEM), have been used to monitor the structural and physical properties of frozen dough (Higuchi and Iijima 1985; Pezolet et al 1992; Zounis et al 2002; Bot 2003; Esselink et al 2003; van Velzen et al 2003; Wellner et al 2005; Zhong and Sun 2005). We froze wet gluten both with and without the ultrasound-assisted technique, investigated the de-pendencies of the freezable water content on the frozen storage time using DSC, used FT-IR to monitor the changes in protein secondary structure in the frozen gluten heavy water systems with the frozen storage period, indirectly observed the size and distri-bution of ice crystals at room temperature using SEM (which was more convenient than cryo-SEM), and compared the differences in dependencies between the samples frozen with and without ultrasound irradiation.

MATERIALS AND METHODS

Materials Food-grade gluten was obtained from Runfon Co. Ltd., Guang-

zhou, China. It was made from aged wheat (Jinan 17) and had a moisture content of 10.2%.

Ultrasound-Assisted Freezing Cycle System The schematic of an ultrasound-assisted freezing cycle system,

consisting of an ultrasonic bath (designed by ourselves, and pro-duced by Guangzhou NewPower Ultrasonic Electronic Equip-ment, China), a efficient thermal regulator (Ministat cc1, Peter Huber Company, Germany), and a Digi-Sense’s 12-channel scan-ning thermocouple thermometer (Cole-Parmer Instrument Corpo-ration, Vernon Hills, IL) is shown in Fig. 1. The ultrasonic bath generates ultrasound at a frequency of 25 kHz. The adjustable power is 0–450W. A 50% (v/v) glycol (AR, The Chemical Re-agent Factory, Tianjin, China) water solution was used as the coolant that cycled between the bath and the thermal regulator.

Sample Preparation The wet gluten for DSC and SEM was prepared by mixing as-

received food-grade gluten and distilled water at a weight ratio of 4:6. The wet gluten was mixed for 20 min in a multifunctional

1 Institute of Light Chemistry, South China University of Technology, Guangzhou510641, PR China.

2 Analytical and Testing Center, South China University of Technology, Guangzhou510641, PR China.

3 Corresponding author. Phone and Fax: +86-20-8711-3252. E-mail address: fesqhu@scut.edu.cn

doi:10.1094 / CCHEM-86-3-0333 © 2009 AACC International, Inc.

334 CEREAL CHEMISTRY

dough blender (BL5, Guangzhon Panyu Lifeng Food Machine Shop, China).The wet gluten for FT-IR was prepared by mixing as-received food-grade gluten that was dried in a forced convec-tion oven at 378°K for 24 hr and deuterium oxide (D2O) at a weight ratio of 4:6. The prepared wet gluten was sealed with cling wrap, stored at 277°K for 1 hr, and then divided into portions. Each sample had a diameter of 70 mm and a height of 16 mm. After the division of the gluten, the samples were resealed and again stored at 277°K.

Freezing The sealed samples were immersed into coolant with a tem-

perature of 253°K. This temperature was regulated by the thermal regulator. The velocity of the fluid and the intensity of the ultra-sound varied with different positions in the ultrasound tank. There-fore, care was taken to keep the samples in the same position in each experiment (Li and Sun 2002). The thermometer measured the real-time temperature of the geometric center of each sample (sample temperature) and transmitted this information to the com-puter. In the ultrasound-assisted freezing trials, the samples were irradiated by ultrasound waves with a frequency of 25 kHz and different electrical powers (0, 175, 288, 360, and 440W) (cycle of 30 sec on, 30 sec off). The freezing was stopped when the sample temperature of each sample reached 255°K. The freezing time (that required to decrease the sample temperature from 298 to 255°K) was the shortest at a power of 360W (Song et al 2009). After freezing, the frozen wet gluten was stored at 253 ± 1°K in the refrigerator. The samples frozen with and without ultrasonic irradiation (25 kHz and 360W) were analyzed by DSC, FT-IR, and SEM at room temperature.

SEM The frozen samples were prepared using a freeze dryer (Alpha

1-4, Christ Co., Germany) and the unfrozen wet gluten was dried at 298°K, with a relative humidity (RH) of 40%. All the samples were then ground to particles. The particles with a diameter of 165–198 µm from the 100–80 meshes were photographed using SEM. These studies were conducted using a scanning electron mi-croscope (1530 VP, Leo Co., Germany). Each sample was placed on the sample holder with the help of double-sided scotch tape and sputter-coated with gold (5 min) using a sputter coater (1D-3, RMC-Eiko Corp., Japan) before observation.

FT-IR FT-IR experiments were recorded on a spectrometer equipped

with a single-reflection diamond-attenuated total reflection acces-sory (Vector 33, Bruker Company, Germany). Opus 4.2 software was used for the spectral processing. The use of D2O was an im-portant prerequisite because H2O was characterized by strong

overlapping absorption, with the carbonyl stretching band of glu-ten protein at ≈1,640 cm–1. It should be noted that this experiment was based on the assumption that the conformational behavior of gluten proteins in D2O was more or less similar to that in H2O, because both solvents possess corresponding liquid properties, volume properties, densities, and dielectric constants at room temperature (van Velzen et al 2003; Wellner et al 2005). The rela-tive contents of α-helix, intramolecular β-sheet, intermolecular β-sheet, and β-turns were calculated following previously described methods by examining the FT-IR spectra and band fitting (Pezolet et al 1992; van Velzen et al 2003; Wellner et al 2005; Mejia et al 2007).

Measuring Water Content The total water contents in each of the samples were deter-

mined by the dry weight loss method. The samples were dried to a constant weight in a forced convection oven at 378°K. Total water content was expressed as the percentage of loss on drying compared with the total weight of the sample before drying. The freezable water in the sample was determined by DSC (Netzch Co., Germany). The instrument was calibrated with indium and zinc standards before sample measurements and all measurements were performed under an atmosphere of nitrogen. All samples were first quenched to 223°K and held for 3 min. Then, the sam-ples were scanned at 5°K/min to 463°K. The melting peak of ice in the sample was analyzed with a software program produced by the manufacturer. The point at which the ice began to melt was set as the temperature where the curves began to deviate from the baseline to the endothermic side. The melting enthalpy was inte-grated from the beginning of ice melting to 283°K. From the de-termined melting enthalpy and total water in the sample, the freezable water content could be calculated by the following for-mula (Baier-Schenk et al 2005)

FW = ΔHmelting/(ΔHice × TW) (1)

where FW is the freezable water content in 100 g of total water; ΔHmelting is the melting enthalpy of ice in the sample determined by DSC in J/g; ΔHice is the fusion enthalpy of ice, equal to 335 J/g; and TW is the total water content in the sample determined by the dry weight loss method in 100 g of sample. All measure-ments were repeated three times. The reported values are the av-erage of the three measurements.

RESULTS AND DISCUSSION

Microstructure of Ice Crystals As shown in the SEM photograph (×500) in Fig. 2, the interior

of wet gluten that was not processed by freezing is very compact and there are no obvious pores. As the wet gluten samples were frozen with and without ultrasound irradiation before freeze-drying, many obvious pores are visible in the SEM photograph at the same magnification (Figs. 3 and 4). During the freezing proc-ess, the freezable water in the wet gluten formed ice crystal nuclei that eventually grew to occupy a larger volume. After freeze-drying, the pores were left as the ice crystals in the frozen wet gluten sublimed. The size and distribution of the ice crystals formed in the frozen wet dough could be analyzed by SEM pho-tography of the freeze-dried samples (Figs. 3 and 4).

Comparison of Fig. 3A with Fig. 3B indicates that a greater number of smaller and more uniform ice crystals formed during ultrasound-assisted freezing than during the conventional freezing process without ultrasonic irradiation; the sizes of most crystals were <5 µm in ultrasound-assisted freezing, whereas the sizes were >6 µm (some as large as >15 µm) in conventional freezing. After the frozen wet gluten was freeze-stored for 75 days, the difference in the microtopography of the ice crystals became even more apparent (Fig. 4). In the wet gluten frozen with and without ultrasound assistance, most ice crystals grew up to <20 and >30

Fig. 1. Schematic of ultrasound-assisted freezing cycle system. 1, 6, 7, 9: coolant; 2: sample column; 3: thermometer probe; 4: scanning thermo-couple thermometer; 5: ultrasonic tank; 8: connect to ultrasonic generator;10: efficient thermal regulator; 11: ultrasonic transducer; 12: computer.

Vol. 86, No. 3, 2009 335

µm, respectively. Ultrasonic cavitation and its secondary effects induced intragluten primary ice nucleation by increasing the tem-perature at which the nucleation took place. Regular intragluten primary nucleation was inhibited during the conventional freezing process and the primary nucleation rate in the extragluten region

simultaneously increased (Arthey 1993; Zheng and Sun 2006). Furthermore, in ultrasound-assisted freezing, ultrasonic cavitation broke up the preexisting dendrite ice crystals to promote secon-dary nucleation. Consequently, the interior and exterior water in the gluten network structure was induced to form a greater num-ber of smaller and more uniform ice crystals. The formed ice crystals continued to grow due to the migration of moisture dur-ing frozen storage. As expected, after frozen storage, the ice crys-tals that grew in the wet gluten frozen with ultrasound were smaller than those in conventional freezing because the original size was smaller and also because there was no evident excess water available to freeze during the frozen storage of these sam-ples.

Changes in Secondary Structure A series of gluten samples in heavy water (D2O) were measured

on a 55° ZnSe ATR plate to investigate structural changes. FT-IR spectra were not useful for absolute amount determination of the secondary structures. Instead, relative intensities at 1,668 (β-turns), 1,650 (α-helix), 1,631 (intramolecular β-sheet), and 1,612 cm–1 (intermolecular β-sheet) were used to estimate the secondary structures (Pezolet et al 1992; van Velzen et al 2003). The gluten heavy water systems were frozen with and without ultrasound irradiation and then stored for 0, 3, 30, 56, and 75 days. Reason-able differences in the secondary structures between the samples processed with and without ultrasound were observed. Compared with the sample frozen without the assistance of ultrasound, there

Fig. 2. Scanning electron microscopy (SEM) photograph of dried wetgluten without freeze-processing. At ×500 magnification, bar correspondsto 20 µm.

Fig. 3. Scanning electron microscopy (SEM) photographs of freeze-driedwet gluten frozen A, with and B, without ultrasonic irradiation. At ×500 magnification, bar corresponds to 10 µm.

Fig. 4. Scanning electron microscopy (SEM) photographs of freeze-driedwet gluten freeze-stored for 75 days after freezing A, with and B, without ultrasonic irradiation. At ×400 magnification, bar corresponds to 20 and 10 µm in A and B, respectively.

336 CEREAL CHEMISTRY

were no evident changes in the content of any protein secondary structures in the gluten heavy water sample that was frozen with ultrasound (Figs. 5–8). When the gluten heavy water sample fro-zen without ultrasound was stored at 253 ± 1°K, the contents of intramolecular β-sheet and β-turn increased gradually, whereas the content of α-helix evidently decreased with prolonged frozen storage time (Figs. 6–8). In protein secondary structures, α-helix is more ordered than β-sheet and β-turn; therefore, the experimen-tal results indicated that the contents of secondary structures in the samples frozen without ultrasound became unordered during frozen storage, whereas there was no evident change over storage time in the contents of secondary structures in the samples frozen with the assistance of ultrasound (Figs. 5–8).

When the wet glutens were freshly frozen, both with and with-out ultrasound irradiation, the size of most ice crystals formed in the network structure of the glutens was <20 µm and did not dam-age the gluten. The evident differences of the secondary structure contents in the wet gluten frozen with and without ultrasound were not detected by FT-IR. As the frozen wet gluten samples were freeze-stored, the ice crystals grew (Fig. 4). In the samples of wet gluten frozen without ultrasound, most ice crystals grew to >30 µm after the wet gluten was freeze-stored for 75 days (Fig.

4B). The gluten could be damaged, to some extent, due to the larger ice crystals expanding to take up a larger volume. The sec-ondary structures in the gluten became unordered. However, in wet gluten frozen with ultrasound, the ice crystals grew to only <20 µm after the wet gluten was freeze-stored for 75 days (Fig. 4a) and deterioration of the network structure in the frozen wet gluten was not evident.

Changes in Freezable Water Content Approximately 70% of the water in the wet gluten samples

froze during the freezing process (Fig. 9). The freezable water content was higher in the samples frozen with ultrasound than in the samples frozen without ultrasound. Freezable water contains free water and some loosely bound water. The loosely bound wa-ter interacted with the hydrophilic amino residues in the gluten by weak interactions such as hydrogen bonding and Van Der Waals interaction. Because the ultrasonic effects loosened the weak in-teractions between the molecules (Feng and Li 1992), more loosely bound water was able to ice up when the wet gluten was frozen with ultrasound irradiation.

When the wet gluten sample frozen without ultrasound was stored at 253 ± 1°K, the freezable water increased gradually with

Fig. 5. Relative contents of intermolecular β-sheet in frozen wet gluten after frozen storage for 0, 3, 30, 56, and 75 days. Mean values ± standard error of three replicates.

Fig. 6. Relative contents of intramolecular β-sheet content in frozen wet gluten after frozen storage for 0, 3, 30, 56, and 75 days. Mean values ±standard error of three replicates.

Fig. 7. Relative contents of α-helix content in frozen wet gluten afterfrozen storage for 0, 3, 30, 56, and 75 days. Mean values ± standard error of three replicates.

Fig. 8. Relative contents of β-turns content in frozen wet gluten afterfrozen storage for 0, 3, 30, 56, and 75 days. Mean values ± standard error of three replicates.

Vol. 86, No. 3, 2009 337

prolonged frozen storage time. The fastest increase in the freez-able water content occurred during the first three days (Fig. 9); this result agreed with the study of Bot (2003). The increase in the amount of freezable water might be explained by loosely bound water that did not freeze in the processing without ultrasound and continued to ice up during the first period of frozen storage; or by unordered secondary structures of the wet gluten frozen without ultrasound being damaged due to the growth of ice crystals during frozen storage; or the unordered structure change resulted in the continuous liberation of water molecules that were originally bound to the protein residue and these molecules gathered to form ice nuclei or to cause the preexisting ice crystals to grow larger as the period of frozen storage continued (Fig. 4B). The growing ice crystals destroyed the gluten network and irreversibly depressed the qualities of the gluten water system.

When the wet gluten was frozen with ultrasound, many small and uniform crystals formed (Fig. 3A); deterioration of the sec-ondary structures was not evident during frozen storage, little bound water was liberated, and, consequently, there was no evi-dence that the freezable water content increased during the frozen storage period (Fig. 9). However, the crystals also grew when the wet gluten frozen with ultrasound was freeze-stored (Fig. 4A), which should be recrystallized due to temperature fluctuation during storage.

CONCLUSIONS

As the frozen wet gluten was freeze-dried, pores were created due to the sublimation of ice crystals. These pores were used to analyze the microtopography of ice crystals by SEM at room temperature. Ultrasonic cavitation and its secondary effects pro-moted the primary and secondary nucleation of ice in the wet gluten; numerous small and uniform crystals formed in the wet gluten when it was frozen with suitable ultrasound irradiation. The changes in relative content of the secondary structures in the frozen wet gluten were monitored by FT-IR. The results indicated that the application of power ultrasound was beneficial in main-taining the secondary structures during frozen storage because the ice crystals could not grow enough to damage the gluten network. The freezable water content in the frozen wet gluten was deter-mined using the melting enthalpy from DSC thermograms. The ultrasonic effects loosened the weak interactions between the water molecules and the gluten amino residences, and more water was able to transform to the ice phase when the wet gluten was frozen with ultrasound assistance.

Furthermore, when the wet gluten was frozen with ultrasound, few water molecules bound with the gluten were liberated and there was very little gluten structure damage by the ice crystals. As a result, there was no evidence that the freezable water content changed during frozen storage. The differences of structural and physical changes in the wet gluten frozen with and without ultra-sound, monitored by SEM, FT-IR, and DSC, suggested that ultra-sound-assisted freezing could improve the structure and the quality of frozen food materials consisting of gluten (or even those of protein).

ACKNOWLEDGMENTS

We are grateful for financial support from the National Natural Science Fund of China (No. 20436020, 20506006).

LITERATURE CITED

Arthey, D. 1993. Freezing of vegetables and fruits. Pages 237-269 in: Frozen Food Technology. C.-P. Mallett, ed. Blackie Academic and Pro-fessional: Glasgow, UK.

Baier-Schenk, A., Handschin, S., and Conde-Petit, B. 2005. Ice in pre-fermented frozen bread dough—An investigation based on calorimetry and microscopy. Cereal Chem. 82:251-255.

Bhattacharya, M., Langstaff, T. M., and Berzonsky, W. A. 2003. Effect of frozen storage and freeze-thaw cycles on the rheological and baking properties of frozen doughs. Food Res. Int. 36:365-372.

Bot, A. 2003. Differential scanning calorimetric study on the effects of frozen storage on gluten and dough. Cereal Chem. 80:366-370.

Chow, R., Blindt, R., Chivers, R., and Povey, M. 2003. The sonocrystalli-sation of ice in sucrose solutions: Primary and secondary nucleation. Ultrasonics 41:595-404.

Chow, R., Blindt, R., Kamp, A., Crocutt, P., and Chivers, R. 2004. The microscopic visualization of the sonocrystallisation of ice using a novel ultrasonic cold stage. Ultrasonics Sonochem. 11:245-250.

Chow, R., Blindt, R., Chivers, R., and Povey, M. 2005. A study on the primary and secondary nucleation of ice by power ultrasound. Ultra-sonics 43:227-230.

Esselink, E. F. J., van Aalst, H., Maliepaard, M., and van Duynhoven, J. P. M. 2003. Long-time storage effect in frozen dough by spectroscopy and microscopy. Cereal Chem. 80:396-403.

Feng, R., and Li, H. M. 1992. Sonochemistry and Its Application. Science & Technology Publishing: China.

Higuchi, A., and Iijima, T. 1985. DSC investigation of the states of water in poly(vinyl-alcohol) membranes. Polymer 26:1207-1211.

Inada, A., Zhang, X., Yabe, A., and Kozawa, Y. 2001. Active control of phase change from supercooled water to ice by ultrasonic vibration. 1. Control of freezing temperature. Int. J. Heat Mass Transfer 44:4523-4531.

Kalichevsky, M. T., Knorr, D., and Lillford, P. J. 1995. Potential food applications of high-pressure effects on ice-water transitions. Trends Food Sci. Technol. 6:253-258.

Larsson, H., and Eliasson, A. C. 1996. Phase separation of wheat flour dough studied by ultracentrifugation and stress relaxation. I. Influence of water content. Cereal Chem. 73:18-24.

Li, B., and Sun, D. W. 2002. Effect of power ultrasound on freezing rate during immersion freezing of potatoes. J. Food Eng. 55:277-282.

Lu, W., and Grant, L. A. 1999. Effects of prolonged storage at freezing temperature on starch and baking quality of frozen doughs. Cereal Chem. 76:656-662.

Mejia, C. D. Mauer, L. J., and Hamaker, B. R. 2007. Similarities and differences in secondary structure of viscoelastic polymers of maize α-zein and wheat gluten proteins. J. Cereal Sci. 45:353-359.

Otero, L., Martino, M., Zaritzky, N., Solas, M., and Sanz, P. D. 2000. Preservation of microstructure in peach and mango during high-pressure-shift freezing. J. Food Sci. 65:466-470.

Pezolet, M., Bonenfant, S., Dousseau, F., and Popineau, Y. 1992. Con-formation of wheat gluten proteins—Comparison between functional and solution states as determined by infrared spectroscopy. FEBS Lett. 3:247-250.

Rasanen, J., Blanshard, J. M. V., Siitari-kauppi, M., and Autio, K. 1997. Water distribution in frozen lean wheat flour dough. Cereal Chem. 74:806-813.

Fig. 9. Freezable water content in frozen wet gluten after frozen storagefor 0, 3, 30, 56, and 75 days. Mean values ± standard error of three repli-cates.

338 CEREAL CHEMISTRY

Simal, S., Benedito, J., Sanchez, E. S., and Rossello, C. 1998. Use of ultrasound to increase mass transport rates during osmotic dehydration. J. Food Eng. 36:323-336.

Song, G. S., Hu, S. Q., Li, L., and Guo, S. Y. 2009. Effects of freezing conditions on freezable water in wet gluten. J. South China Univ. Technol. 37(4):120-124.

Spiazzi, E. A., Raggio, Z. I., Bignone, K. A., and Mascheroni, R. H. 1998. Experiments on dehydrofreezing of fruits and vegetables: mass transfer and quality factors. Advances in the Refrigeration Systems, Food Technologies and Cold Chain, IIF/IIR. 6:401-408.

van Velzen, E. J. J., van Duynhoven, J. P. M., Pudney, P., Weegels, P. L., and van der Maas, J. H. 2003. Factors associated with dough stickiness as sensed by attenuated total reflectance infrared spectroscopy. Cereal Chem. 80:378-382.

Wellner, N., Mills, C. E. N., Brownsey, G., Wilson, R. H., Brown, N., Freeman, J., Halford, N. G., Shewry, P. R., and Belton, P. S. 2005. Changes in protein secondary structure during gluten deformation

studied by dynamic Fourier transform infrared spectroscopy. Biomac-romolecules 6:255-261.

Zhang, X., Inada, A., Yabe, A., Lu, S., and Kozawa, Y. 2001. Active con-trol of phase change from supercooled water to ice by ultrasonic vibra-tion. 2. Generation of ice slurries and effect of bubble nuclei. Int. J. Heat Mass Trans. 44:4533-4539.

Zhang, X., Inada, T., and Tezuka, A. 2003. Ultrasonic-induced nucleation of ice in water containing air bubbles. Ultrasonics Sonochem. 10:71-76.

Zheng, L. Y., and Sun, D. W. 2006. Innovative applications of power ultrasound during food freezing process—A review. Trends Food Sci. Technol. 17:16-23.

Zhong, Z., and Sun, X. S. 2005. Thermal characterization and phase be-havior of cornstarch studied by differential scanning calorimetry. J. Food Eng. 69:453-459.

Zounis, S., Quail, K. J., Wootton, M., and Dickson, M. R. 2002. Studying frozen dough structure using low-temperature scanning electron mi-croscopy. J. Cereal Sci. 35:135-147.

[Received December 23, 2008. Accepted March 18, 2009.]