Effect of cavitation jets on the physicochemical properties and structural characteristics of the okara protein.

The structural andphysicochemical properties of okara protein (OP) subjected to different cavitation jet (CJ) treatment times (0-15 min) were analyzed. In this study, the microstructure and apparent morphology of OP were analyzed by Raman spectrum, fluorescence spectrum, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Physicochemical properties, such as emulsion ability (EA), emulsion stability index (ESI), foaming characteristics (FC), foaming stability (FS), and solubility analysis of the OPs, were characterized. Raman spectrum analysis showed that CJ treatment caused increases in the ordered structure of OPs (α-helix, ß-sheet, and ß-turn), and the disulfide bond g-g-g and g-g-t modes, while it caused a decrease in the t-g-t mode. However, the tertiary structure of OP unfolded and mostly degraded into small subunits because of higher cavitation, shear and temperature effects. AFM observation indicated that CJ resulted in a more uniform distribution of OP. Moreover, changes in the structure of OP significantly affected its functional properties. The results showed that when CJ treatment time was 10 min, the solubility of OP was up to (28.72 ± 1.26)%, the soluble protein content of okara was up to (10.44 ± 0.03) g/100 g, and interface properties were better. In summary, OP has great potential for application in the food area, especially in emulsifying agent and foam system. PRACTICAL APPLICATION: The cavitation jet technology improves the structure and physical and chemical properties of the protein extracted from soybean residue (okara) and provides new ideas for the further development and utilization of soybean residue protein, which may lead to the production of high-value-added functional ingredients from the processing of soybean byproducts.

Soy Protein Nanofiber Scaffolds for Uniform Maturation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.

Effects of native starch and modified starches on the textural, rheological and microstructural characteristics of soybean protein gel.

The effects of native starch (NS), acetylated starch (AS), and acetylated distarch phosphate (ADSP) on the gel properties of soybean protein thermal gel were investigated using texture analysis, low-field nuclear magnetic resonance (LF-NMR) spectroscopy, dynamic rheometry and scanning electron microscopy. The results of the textural profile analysis showed that 10% ADSP increased the hardness and chewiness of the mixed gel, while NS and AS led to decreases in the textural properties. The results of the LF-NMR analysis indicated that the AS improved the water-holding capacity of the mixed gel due to the transformation of weakly bound water to strongly bound water. During heating and cooling, the rheological profiles of the elastic (G') and viscous modulus (G″) of all the samples exhibited a two-stage pattern of decrease and then increase, and the final values of G' and G″ reached maxima when the ADSP content was 10%. The scanning electron microscopy images showed that the ADSP granules dispersed in the gel network. The integrity of the starch granules was crucial for regulating the properties of the soybean protein gel. These results provided information about the further design and preparation of soybean protein foods containing modified starch.

Effects of High-Intensity Ultrasound Pretreatment on Structure, Properties, and Enzymolysis of Soy Protein Isolate.

The objective of this study was to investigate the effects of different high-intensity ultrasonication (HIU) pretreatment on the structure and properties of soybean protein isolate (SPI) as well as enzymatic hydrolysis of SPI by bromelain and antioxidant activity of hydrolysates. The HIU-treated SPI fractions showed a decrease in the proportion of α-helices and ß-turns and an increase in the content of ß-sheets and random coils based on Fourier-transform infrared spectroscopy. Near-infrared spectra and fluorescence spectra analyses provided support for the changes in secondary and tertiary structures of SPI after ultrasound treatment. The particle size of SPI decreased from 217.20 nm to 141.23 nm and the absolute zeta potential increased. Scanning electron microscopy showed that HIU treatment changed apparent morphology. Dynamic and static light scattering of ultrasonicated samples showed that SPI structure had changed from hard-sphere to hollow-sphere or polydisperse and monodisperse gaussian coils. HIU pretreatment significantly increased the hydroxyl-radical scavenging and the degree of hydrolysis of the SPI hydrolysates.

Fabrication, properties and applications of soy-protein-based materials: A review.

The environmental crisis caused by the use of petroleum-based nondegradable polymers and the impending petroleum finite resources have directly threatened human being's sustainable development. Therefore, ecofriendly polymers based on natural renewable resources are attracting more and more attention. As the byproducts of soy oil industries, soy protein, is regarded as a viable alternative for petroleum-based polymeric products. In order to improve the physical properties, especially the mechanical properties and water resistance that limit their extensive applications, different modifications were adopted. Among these efforts, incorporating nanoparticles and blending with other polymers are proved to be effective ways. The properties of the resulting materials are highly dependent on the processing methods, nature of the components, dispersion status and the compatibility. This review intends to provide a clear overview on preparation, properties, and applications of soy-protein-based materials. These biodegradable materials will find more and more potential applications in biodegradable foams, edible films, packaging materials, biomedical materials, etc.

High-Pressure Homogenization Pretreatment before Enzymolysis of Soy Protein Isolate: the Effect of Pressure Level on Aggregation and Structural Conformations of the Protein.

The high-pressure homogenization (HPH) treatment of soybean protein isolate (SPI) before enzymatic hydrolysis using bromelain was investigated. Homogenization pressure and cycle effects were evaluated on the enzymatic degree of hydrolysis and the antioxidant activity of the hydrolysates generated. The antioxidant activity of SPI hydrolysates was analyzed by 1,1-dipheny-2-picrylhydrazyl (DPPH). The sizes and structures of the SPI-soluble aggregate after HPH treatment were analyzed using dynamic and static laser light scattering. The changes in the secondary structure, as measured by Fourier transform infrared spectroscopy (FTIR) and the macromorphology of SPI, were measured by scanning electron microscope (SEM). These results suggested that the HPH treatment (66.65%) could increase the antioxidant activities of the SPI hydrolysates compared with the control (54.18%). SPI hydrolysates treated at 20 MPa for four cycles obtained higher DPPH radical-scavenging activity than other samples. The control was predicted to be a hard sphere, and SPI treatment at 10 MPa was speculated to be Gaussian coil, polydisperse, and then the high-pressure treated SPI became a hollow sphere. Changes in the secondary structures showed protein aggregate formation and rearrangements. The image of SPI varied from a globular to a clump structure, as observed by the SEM. In conclusion, combining HPH treatment and enzymolysis could be an effective way to improve the antioxidant activity of the SPI.

On characterization of anisotropic plant protein structures.

In this paper, a set of complementary techniques was used to characterize surface and bulk structures of an anisotropic Soy Protein Isolate (SPI)-vital wheat gluten blend after it was subjected to heat and simple shear flow in a Couette Cell. The structured biopolymer blend can form a basis for a meat replacer. Light microscopy and scanning electron microscopy provided a detailed view of structure formation over the visible surfaces of the SPI-gluten blend. Protein orientation in the direction of the flow was evident and fibrous formation appeared to exist on the macro- and micro-scale. Furthermore, according to texture analysis, the structured biopolymer obtained from the Couette Cell after processing at 95 °C and 30 RPM for 15 min has high tensile stress and strain anisotropy indices (∼2 and ∼1.8, respectively), comparable to those of raw meat (beef). The novel element in this work is the use of the neutron refraction method, utilizing spin-echo small angle neutron scattering (SESANS), to provide a look inside the anisotropic biopolymer blend complementing the characterization provided by the standard techniques above. With SESANS, it is possible to quantify the number of fibre layers and the orientation distribution of fibres. For a specimen thickness of 5 mm, the obtained number of fibre layers was 36 ± 4 and the standard deviation of the orientation distribution was 0.66 ± 0.04 radians. The calculated thickness of one layer of fibres was 138 µm, in line with SEM inspection.

Bacterial filtration efficiency of green soy protein based nanofiber air filter.

High bacterial filtration efficiency (BFE) filters, based on nanofibers derived from blends of grain proteins and poly-ethylene-oxide (PEO), were produced by an electrospinning process. Specifically, polymer blends consisting of purified soy flour/PEO with a ratio of 7/3 were spun into nanofibers and characterized. A new laboratory based experimental apparatus for testing BFE was designed and used to test BFE of bacterial aerosols consisting of Escherichia coli (E. coli). Performances of soy protein based nanofiber filters with nanofiber mass varying from 1 to 5 g/m2 as well as a nanofiber filters prepared from pure PEO were compared. The results showed that BFE values for filters containing 5 g/m2 protein based nanofibers and PEO nanofiber filter were 100 and 81.5%, respectively. The results also indicated that the BFE increased as the protein content in the nanofiber filter increased. These novel protein based nanofiber filters have demonstrated a clear potential for effective removal and retention of E. coli bacteria during air-filtration. These filters can be effectively deployed in environments such as hospitals and senior residential areas to reduce bacterial infections.

The impact of anthocyanin-rich red raspberry extract (ARRE) on the properties of edible soy protein isolate (SPI) films.

UNLABELLED: To modify the properties of edible soy protein isolate (SPI) films, 0.5% anthocyanin-rich red raspberry (Rubus strigosus) extract (ARRE) (0.5 g raspberry powder in 95% ethyl alcohol/water/85% lactic acid [80:19:1. v/v/v]) was incorporated into film-forming solutions. ARRE resulted in an SPI film having significantly enhanced tensile strength (P < 0.05) and % elongation at break (P < 0.05), as well as increased water swelling ratio (P < 0.05) and in vitro pepsin digestibility (P < 0.05). The resultant films also showed significantly decreased water solubility and water vapor permeability (P < 0.05). In addition, ARRE increased darkness, redness, and yellowness film appearance as evidenced by a lower L* (P < 0.05), greater positive a* (P < 0.05), and a higher b* (P < 0.05) than the control film. Scanning electron microscopy images revealed that extract-added films had denser and more compact cross-section microstructure. Fourier transform infrared spectra illustrated that ARRE-created hydrogen bonding involved conformational changes of soy protein without destroying its backbone structure. SDS-PAGE electrophoretograms revealed that the extract induced intermolecular interaction of the soy protein monomers. Natural plant extracts would be a promising ingredient to make SPI films with different physicochemical properties and applications. PRACTICAL APPLICATION: This study characterizes the potential physicochemical changes of SPI film with incorporated raspberry extract. Upon the above modification, the resultant film was found to enhance the applications of pure SPI film in food packaging. For example, SPI-ARRE film could prolong the usage life of SPI film due to increased strength, or could be useful as a desiccant (drying agent) such as a water-absorbing sheet for preserving dried foods due to its increased hydrophilic surface and water-swelling ratio. SPI-ARRE film could also be alternately used as a food wrap with unique color.

Biodegradable soy protein isolate-based materials: a review.

Recently, there is an increasing interest of using bio-based polymers instead of conventional petroleum-based polymers to fabricate biodegradable materials. Soy protein isolate (SPI), a protein with reproducible resource, good biocompatibility, biodegradability, and processability, has a significant potential in the food industry, agriculture, bioscience, and biotechnology. Up to now, several technologies have been applied to prepare SPI-based materials with equivalent or superior physical and mechanical properties compared with petroleum-based materials. The aim of this review is focused on discussion of the advantages and limitations of native SPI as well as the bulk and surface modification strategies for SPI. Moreover, some applications of SPI-based materials, especially for food preservation and packaging technology, were discussed.

Physicochemical properties of soy protein: effects of subunit composition.

Soy protein elastomer (SPE) exhibits elastic, extensible, and sticky properties in its native state and displays great potential as an alternative to wheat gluten. The objective of this study was to better understand the roles of soy protein subunits (polypeptides) contributing to the functional properties of SPE. Six soy protein samples with different subunit compositions were prepared by extracting the proteins at various pH values on the basis of the different solubilities of conglycinin (7S) and glycinin (11S) globulins. Soy protein containing a large amount of high molecular weight aggregates formed from α' and α subunits exhibited stronger viscoelastic solid behavior than other soy protein samples in terms of dynamic elastic and viscous modules. Electrophoresis results revealed that these aggregates are mainly stabilized through disulfide bonds, which also contributed to higher denaturation enthalpy as characterized by DSC and larger size protein aggregates observed by TEM. Besides, the most viscoelastic soy protein sample exhibited flat and smooth surfaces of the protein particles as observed by SEM, whereas other samples had rough and porous particle surfaces. It was proposed that the ability of α' and α to form aggregates and the resultant proper protein-protein interaction in soy proteins are the critical contributions to the continuous network of SPE.

Development of a probiotic delivery system from agrowastes, soy protein isolate, and microbial transglutaminase.

UNLABELLED: Probiotic delivery system was developed via the use of microbial transglutaminase (MTG) cross-linked soy protein isolate (SPI) incorporated with agrowastes such as banana peel (BE), banana pulp (BU), and pomelo rind (PR). Inoculums of Lactobacillus bulgaricus FTDC 1511 were added to the cross-linked protein matrix. The incorporation of agrowastes had significantly (P<0.05) reduced the strength, pH value, and the lightness of the SPI gel carriers, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the occurring cross-links within the SPI gel carriers were attributed to the addition of MTG. Scanning electron microscope micrographs illustrated that SPI carriers containing agrowastes have exhibited a less-dense protein matrix. All the SPI carriers possessed maximum swelling ratio at 4 to 4.5 within 15 min in simulated gastric fluid (SGF), whereas the maximum swelling ratios of SPI/BE, SPI/BU, and SPI/PR were higher compared to that of control in simulated intestinal fluid (SIF). Additionally, SPI carriers in SGF medium did not show degradation of structure, whereas a major collapse of network was observed in SIF medium, indicating controlled-release in the intestines. The addition of agrowastes into SPI carriers led to a significantly (P<0.0001) lower release of L. bulgaricus FTDC 1511 in SGF medium and a higher release in SIF medium, compared to that of the control. SPI carriers containing agrowastes may be useful transports for living probiotic cells through the stomach prior to delivery in the lower intestines. PRACTICAL APPLICATION: Agrowastes could be utilized as a new probiotic carrier for enhanced gastrointestinal transit and during storage. This also reduces the amount of agrowastes accumulated.

Physicochemical properties of ß and α'α subunits isolated from soybean ß-conglycinin.

Soy protein has shown great potential for use in biobased adhesives. ß-Conglycinin is a major component of soy protein; it accounts for 30% of the total storage protein in soybean seeds. ß-Conglycinin was isolated and purified, and its subunits' (ß, α'α) physicochemical and adhesive properties were characterized. Crude ß-conglycinin was isolated from soy flour and then purified by the ammonium sulfate precipitation method. The α'α and ß subunits were isolated from the purified ß-conglycinin by anion exchange chromatography. Yields of α'α subunits and ß subunits from 140 g of soy flour were 1.86 g (1.3%) and 0.95 g (0.67%), respectively. The minimum solubility for α'α subunits, ß subunits, and ß-conglycinin occurred in pH ranges of 4.1-5.4, 3.5-7.0, and 4.8-5.3, respectively. Transmission electron microscopy showed that the ß subunits existed as spherical hydrophobic clusters, whereas α'α subunits existed as uniformly discrete particles at pH 5.0. Differential scanning calorimetry showed that ß subunits had higher thermal stability than α'α subunits. The pH had a lesser effect on adhesion strength of the ß subunits than on that of the α'α subunits. The adhesives made from ß subunits also showed greater water resistance than those from α'α subunits and ß-conglycinin. Soy protein rich in ß subunits is likely a good candidate for developing water-resistant adhesives.

Characterization and evaluation of the Ag+-loaded soy protein isolate-based bactericidal film-forming dispersion and films.

UNLABELLED: This study aims to prepare bactericidal films developed from soy protein isolate (SPI) based film-forming dispersions (FFDs) for use in the food and medical fields. The FFD and films were prepared after the incorporation of different concentrations of AgNO3 as a bactericidal agent. The transparency, tensile strength, and antimicrobial features were evaluated. Structural characterizations were also performed by Fourier transform infrared spectroscopy, scanning electron microscope, and atomic force microscopy analysis. Results showed that the opacity of these FFD was greatly decreased after the incorporation of AgNO3. The SPI-5 film has the largest tensile strength (P < 0.05) compared with that of the other ones. Micro structural imaging analysis showed an increase in the surface irregularities with the addition of AgNO3. The minimum inhibitory concentration of AgNO3 was 336 µg/mL FFD for both Escherichia coli ATCC 25923 and Staphylococcus aureus ATCC 25922. The SPI-AgNO3 films developed from the FFD with the minimal AgNO3 concentration at 336 µg/mL FFD also showed bactericidal effects for both the strains. These results may prove promising for the use of SPI-AgNO3 films in the food or medical industries. PRACTICAL APPLICATION: The films prepared in this study are biodegradable and will be used in medical and food fields.

Structural and thermal investigations of biomimetically grown casein-soy hybrid protein fibers.

A hybrid protein fiber from different protein sources such as casein and soybean using wet-spinning technique was prepared. The casein/soybean hybrid fibers were synthesized at different weight ratios such as 100/0 (casein), 75/25, 50/50, 25/75, and 0/100 (soy) and characterized. Electron microscopic analysis confirmed the growth of pure and hybrid fibers and shows an increased surface roughness as the soy concentration increases in the hybrid fibers. Infrared spectra did not exhibit any significant changes in the functional groups between pure and hybrid fibers. X-ray diffraction pattern indicates slight increase in the diffraction peak values of hybrid fibers compared with the neat fibers. Thermal analyses show a moderate increase in the thermal stability of hybrid fibers when compared with the pure fibers. These results implicitly indicate that the casein and soy proteins are homogeneous in the hybrid fiber form. It has been demonstrated that the hybrid fiber with ≥50 wt.% casein content exhibits better morphology and increased thermal stability, which has scope for application in technical and medical industries.

Characterization of amino cross-linked soy protein hydrogels.

This study focused on amino cross-linking as means of forming soy protein hydrogels with modifiable properties. The efficiency of glyceraldehyde, a potential food-grade cross-linking agent, was compared to glutaraldehyde, a well-known dialdehyde. The influence of the concentration of these agents on the degree of cross-linking as well as macroscopic and supramolecular properties was studied. The effect of increasing the cross-linker concentration was mainly an increase in degree of cross-linking and gel storage modulus (G') and a decrease in gel swelling ratio and increase in gel deswelling ratio. However, the cross-linking influence was less pronounced in the case of glyceraldehyde. Glutaraldehyde displayed greater ability to form hydrogels with modifiable properties. Finally, electron micrographs suggested that cross-linking agent type had no impact on gel microstructure.

Effect of continuous flow high-pressure throttling on rheological and ultrastructural properties of soymilk.

Soymilk processing uses a filtration or centrifugation step to remove coarse solids in the comminuted soy. The objective was to utilize the whole beans and determine the effect of continuous flow high pressure throttling (CFHPT) process in reducing particle size and narrowing down the particle size distribution. The rheological and ultrastructural properties of such soymilk were also determined. Whole dehulled soybeans and deionized water were ground in a food processor before comminution in a Megatron (process M) or a Fitzmill (process F) or a Stonemill (process S). The comminuted slurry was homogenized at pressures of 69, 103, 138, 207, and 276 MPa using a CFHPT system, heated to 80 degrees C in a tubular heat exchanger prior to depressurization, and held at elevated temperatures of 97, 106, 114, 131, and 148 degrees C, respectively, for each applied pressure after throttling. To avoid flashing, back pressure was applied after the holding tube and soymilk was cooled immediately. Process M produced soymilk with smallest particle size and the highest apparent viscosity. All soymilk samples showed non-Newtonian pseudoplastic flow behavior. Ultrastructural images showed a clear protein network with very small fat globules entrapped in the protein matrix. Particles were uniformly distributed when the highest pressure treatment was applied for process M, which was considered as the best process.

Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein isolate.

Long, fibrillar semiflexible aggregates were formed from soy glycinin and soy protein isolate (SPI) when heated at 85 degrees C and pH 2. Transmission electron microscopy analysis showed that the contour length of the fibrils was approximately 1 microm, the persistence length 2.3 microm, and the thickness a few nanometers. Fibrils formed from SPI were more branched than the fibrils of soy glycinin. Binding of the fluorescent dye Thioflavin T to the fibrils showed that beta-sheets were present in the fibrils. The presence of the fibrils resulted in an increase in viscosity and shear thinning behavior. Flow-induced birefringence measurements showed that the behavior of the fibrils under flow can be described by scaling relations derived for rodlike macromolecules. The fibril formation could be influenced by the protein concentration and heating time. Most properties of soy glycinin fibrils are comparable to beta-lactoglobulin fibrils.

Morphology and properties of soy protein and polylactide blends.

Blends of soy protein (SP) and a semicrystalline polylactide (PLA) were prepared using a twin-screw extruder. The melt rheology, phase morphology, mechanical properties, water resistance, and thermal and dynamic mechanical properties were investigated on specimens prepared by injection molding of these blends. The melt flowability of soy-based plastics was improved through blending with PLA. Scanning electron microscopy revealed that a co-continuous phase structure existed in the blends with soy protein concentrate (SPC) to PLA ratios ranging from 30:70 to 70:30. SPC/PLA blends showed fine co-continuous phase structures, while soy protein isolate (SPI)/PLA blends presented severe phase coarsening. At the same SP to PLA ratios, SPC/PLA blends demonstrated a higher tensile strength than SPI/PLA blends. The water absorption of soy plastics was greatly reduced by blending with PLA. The compatibility was improved by adding 1-5 phr poly(2-ethyl-2-oxazoline) (PEOX) in the blends, and the resulting blends showed an obvious increase in tensile strength and a reduction in water absorption for SPI/PLA blends. The compatibility between SP and PLA was evaluated by mechanical testing, dynamic mechanical analysis (DMA), water absorption, and scanning electron microscopy (SEM) experiments. Differential scanning calorimetry (DSC) revealed that PLA in the blends was mostly amorphous in the injection molded articles, and SP accelerated the cold crystallization and could increase the final crystallinity of PLA in the blends.

New evidences of glass transitions and microstructures of soy protein plasticized with glycerol.

Soy protein isolate (SPI) and glycerol were mixed under mild (L series) and severe (H series) mixing conditions, respectively, and then were compression-molded at 140 degrees C and 20 MPa to prepare the sheets (SL and SH series). The glass transition behaviors and microstructures of the soy protein plasticized with glycerol were investigated carefully by using differential scanning calorimetry and small-angle X-ray scattering. The results revealed that there were two glass transitions in the SPI/glycerol systems. When the glycerol contents ranged from 25 to 40 wt.-%, all of the SL- and SH-series sheets showed two glass transition temperatures (T(g1) and T(g2)) corresponding to glycerol-rich and protein-rich domains, respectively. The T(g1) values of the sheets decreased from -28.5 to -65.2 degrees C with an increase of glycerol content from 25 to 50 wt.-%, whereas the T(g2) values were almost invariable at about 44 degrees C. The results from wide-angle X-ray diffraction and small-angle X-ray scattering indicated that both protein-rich and glycerol-rich domains existed as amorphous morphologies, and the radii of gyration (R(g)) of the protein-rich domains were around 60 nm, a result suggesting the existence of stable protein domains. The results above suggest that protein-rich domains were composed of the compact chains of protein with relatively low compatibility to glycerol and glycerol-rich domains consisted of relative loose chains that possessed good compatibility with glycerol. The significant microphase separation occurred in the SPI sheets containing more than 25 wt.-% glycerol, with a rapid decrease of the tensile strength and Young's modulus. [illustration in text].