Physicochemical and Functional Properties of Soy Protein Substrates Modified by Low Levels of Protease Hydrolysis

ABSTRACT: Endo-protease treatments achieving low degrees of hydrolysis (DH 2% and 4%) were used to improve functional properties of hexane-extracted soy flour (HESF), extruded-expelled partially defatted soy flour (EESF), ethanol-washed soy protein concentrate (SPC), and soy protein isolate (SPI). These substrates had protein dispersibility indices ranging from 11% to 89%. Functional properties, including solubility profile (pH 3 to 7), emul-sification capacity and stability, foaming capacity and stability, and apparent viscosity were determined and related to surface hydrophobicity and peptide profiles of the hydrolysates. Protein solubilities of all substrates increased as DH increased. Emulsification capacity and hydrophobicity values of the enzyme-modified HESF and EESF decreased after hydrolysis, whereas these values increased for SPC and SPI. Emulsion stability was improved for all 4% DH hydrolysates. Hydrolyzed SPC had lower foaming capacity and stability. For substrates other than SPC, foaming properties were different depending on DH. Hydrolysis significantly decreased the apparent viscosities regardless of substrate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated differences in the molecular weight profiles of the hydrolysates. HESF and EESF, which had high proportions of native-state proteins, showed minor changes in the peptide profile due to hydrolysis compared with SPC and SPI.     

Preparation and Functional Properties of Enzymatically Deamidated Soy Proteins

Heat-denatured soy protein was hydrolyzed by Alcalase to 2.0% or 4.0% degree of hydrolysis (DH), heated again at 100°C and deamidated with B. circulans peptidoglutaminase. The extent of deamidation was 6.0% and 8.2% for 2.0 DH hydrolysates and 12.8% and 16.0% for 4.0 DH hydrolysates heated for 15 and 30 min, respectively. Deamidation increased protein solubility and substantially enhanced emulsifying activity under mildly acidic (pH 4–6) as well as alkaline conditions. Deamidation improved emulsion stability and foaming power of heat-denatured hydrolysed soy proteins. Enzymatically deamidated soy protein hydrolysates had improved functional properties compared to nondeamidated hydrolysates and the native soy protein.     

Effect of combined treatment of hydrolysis and polymerization with transglutaminase on β-lactoglobulin antigenicity

The effect of combined treatments of hydrolysis with different proteases, and subsequent polymerization with transglutaminase on the antigenic activity of β-Lg was studied. For the hydrolysis of β-Lg using Alcalase, Neutrase or bromelain, the reaction conditions were 3?% β-Lg and enzyme:substrate 25?U?g^?1 of protein, as was defined using factorial study. Under these conditions, the degree of hydrolysis (DH) of the hydrolysates was 12.6?% when obtained with Alcalase and approximately 4?% with Neutrase or bromelain. Post-hydrolysis polymerization did not result in an increase in molecular mass of the protein, but these samples presented a lower DH, determined by trinitrobenzenosulfonic acid (TNBS) method, suggesting that polymerization had occurred. Hydrolysis with the three enzymes reduced the β-Lg antigenicity, as evaluated by ELISA and immunoblotting analyses. The IgE-binding responses were practically null (<9?μg?mL^?1), 22.82 and 55.73?μg?mL^?1 towards the hydrolysates obtained with Alcalase, bromelain, and Neutrase, respectively. The post-hydrolysis polymerization increased or had no significant effect (P?≥?0.05) on the antigenic response of the hydrolysates.     

Effect of lipoxygenase activity in defatted soybean flour on the gelling properties of soybean protein isolate

Soybean lipoxygenase was inactivated to different degrees by dry heating of defatted soybean flour for 0, 5, 10, 15, 20 and 25 min and soy protein isolates were prepared thereof by isoelectric precipitation of the water extract of the defatted soybean flour. The fluorescence emission intensity at 420 nm of the chloroform–methanol extract of soy protein isolates, which was indicator of the existence of peroxidized lipid, varied in parallel with the lipoxygenase residual activity in defatted soybean flours. The dispersion of soy protein isolate showed an increasing turbidity with the increase of lipoxygenase residual activity in the starting defatted soybean flour, suggesting an elevated tendency to form insoluble aggregates during the preparation of soy protein isolate. Small deformation rheological test revealed that the gelling times were shorter for those soy protein isolates derived from low lipoxygenase activity defatted soybean flours than that of high lipoxygenase activity. Frequency sweep showed that G′ of soy protein isolate derived from low lipoxygenase defatted soybean flour was independent of oscillation frequency in contrast to that of derived from non dry-heated defatted soybean flour, the latter showed a marked frequency dependence. Large deformation test revealed that the gel hardness increased about 10 times after dry heating of defatted soybean flour for 20 min. As the increase of the lipoxygenase residual activity, the gel permeability increased markedly, suggesting that soy protein isolate from high lipoxygenase defatted soybean flour produced coarser textured gel, which corresponded well with the results of scanning electron microscopy.     

Effect of acid hydrolysis on rheological and thermal characteristics of lentil starch slurry

A comparative rheological and thermal study was carried out between acid hydrolyzed and unhydrolyzed (control) lentil starch dispersions (25-33.3 g starch per 100 g water) as function of temperature. After acid hydrolysis, the peak gelatinization temperature (T_p) shifted to higher temperature than the corresponding starch without hydrolysis whereas the gelatinization enthalpy remained unaffected by hydrolysis. The starch gelatinization kinetics was evaluated by a non-isothermal technique as function of elastic modulus (G′) and G′ vs. time (t) data up to the gelatinization peak value was considered for rate estimation. A 2nd-order reaction kinetics described well the starch gelatinization process and the process activation energy was ranged between 241 and 434 kJ/mol. Acid hydrolysis strongly affected the rheological properties by lowering gel strength compared to unhydrolyzed starch. The creep analysis further revealed that starch gel was significantly affected by hydrolysis and exhibited less resistant to the stress. A 4-parameters Burgers model well-described creep curves and supported oscillatory rheological data.     

Modifications of soy protein isolates using combined extrusion pre-treatment and controlled enzymatic hydrolysis for improved emulsifying properties

Effects of combined extrusion pre-treatment and controlled enzymatic hydrolysis on the physico-chemical properties and emulsifying properties of soy protein isolates (SPI) have been investigated. Results showed that extrusion pre-treatment caused a marked improvement in the accessibility of SPI to enzymatic hydrolysis, resulting in changes in degree of hydrolysis (DH), protein solubility (PS), surface hydrophobicity (H₀) and molecular weight distributions (MWD) for ESPIH (extrusion pre-treated SPI hydrolysates). It was observed that emulsion systems formed by control SPI or SPIH (SPI hydrolysates) (20% v/v oil, 1.6% w/v emulsifier, and pH 7.0) were unstable over a quiescent storage period of 21 days, due to bridging flocculation and creaming. However, ESPIH (9.1% DH) was capable of producing a very fine emulsion (d₃₂ = 0.42 μm, d₄₃ = 2.01 μm) which remained stable over a long term quiescent storage. Various surface properties of ESPIH products have also been studied in relation to DH and emulsifying functionalities. It was suggested that significantly increased protein solubility and decreased molecular weight could be the main reasons for the greatly improved emulsifying capability of ESPIH. This study demonstrated that modified soy protein could be an excellent emulsifying agent for food and other applications. It also demonstrated that combined extrusion pre-treatment and enzymatic hydrolysis could be a highly effective method for functionality modification of globular proteins.     

Gelation behaviour and rheological properties of acid-induced soy protein-stabilized emulsion gels

The protein concentration dependence on the rheological properties of acid-induced gels formed with unheated and heated soy protein-stabilized emulsions (UHSPE and HSPE) was investigated at different acidification temperatures. Pre-heat treatment on soy protein solutions resulted in a higher storage modulus (G′) and a shorter gelation time (t_gel) of acid-induced emulsion gels. A maximum in tan δ was observed in the UHSPE gels but no maximum was detected in the HSPE gels. Increasing the acidification temperature decreased the G′ and t_gel. The dependence of the G′ on the protein concentration (c) can be scaled with a power law: G′ ∼ c^A. The exponent (A) increased with pre-heat treatment and acidification temperature. The experimental data.fitted the fractal scaling model (G′ ∼ φ^A) and the simple time- scaling model above very well for the acid-induced soy protein-stabilized HSPE gels with varying oil volume fraction. The large deformation and fracture properties were significantly affected by soy protein concentration, pre-heat treatment, acidification temperature and volume fraction of oil droplets (p < 0.05).     

Effect of degree of hydrolysis on the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates

Loach (Misgurnus anguillicaudatus) proteins were hydrolysed by papain and Protamex, the antioxidant activity of loach protein hydrolysates (LPH) was investigated. The results demonstrated that extensive hydrolysis by papain and Protamex led to the browning of the hydrolysates. When the degree of hydrolysis (DH) was 23%, hydrolysates prepared by papain (HA) exhibited the strongest antioxidant activity. The maximum values of the hydroxyl, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging activities and the reducing power were 56.1%, 95.5%, 2.80 mM and 1.46, respectively. The hydrolysates prepared by Protamex (HB) showed the strongest hydroxyl radical scavenging activity (55.0%) at DH 28%, DPPH radical scavenging activity (92.2%) and ABTS radical scavenging activity (2.81 mM) at DH 23%, and the reducing power (1.17) at DH 33%. At the same DH value, there were significant (p < 0.05) differences between HA and HB. Several antioxidant amino acid residues, especially Trp and His, contributed significantly to the antioxidant activity of the hydrolysates. An increase of peptides with molecular weight below 500 Da was observed as the DH increased for all LPH. The above results indicated that DH and protease greatly influenced the molecular weight and amino acid residue composition of LPH, and further influenced the antioxidant activity.Industrial relevanceLoach has long been employed as a traditional Chinese medicine for the treatment of many kinds of diseases. From our previous work, loach was determined to be a good source of protein (accounts for approximately 17% (w/w) of the body weight). In this work, loach proteins were hydrolyzed by papain and Protamex to specific extent. The effect of DH on the antioxidant activities of hydrolysates was investigated. The results indicated that loach protein hydrolysates were potent antioxidants which were significantly affected by DH. This research is helpful for extensive development of loach product.     

Effect of limited hydrolysis of soy protein on the interactions with polysaccharides at the air–water interface

The objective of the work was to study the effect of limited hydrolysis of soy protein on the interactions with polysaccharides with and without surface activity at the air–water interface at neutral pH where a limited incompatibility between macromolecules can occur. The surface pressure and phase angle as a function of time were evaluated with a drop tensiometer at 20 °C, pH 7 and ionic strength 0.05 M.Hydrolysates of 2% (H1) and 5.4% (H2) degree of hydrolysis (DH) with neutral protease from Aspergillus oryzae were obtained from a commercial soy protein isolate. The polysaccharides used were: hydroxypropylmethylcellulose (HPMC) as surface active polysaccharide; lambda carrageenan (λC) and locust bean gum (LB) as non-surface active polysaccharides.It was found that increasing DH decreased the surface pressure and increased film viscoelasticity (determined as the phase angle, θ) of soy protein hydrolysates and the nature of protein–polysaccharide interactions was strongly affected by DH. The presence of polysaccharides led to an increase of surface pressure of H1 but when added to H2, HPMC and λC decreased the surface pressure.The less hydrolyzed protein H1 gave rise to a higher surface pressure and film viscoelasticity in combination with the polysaccharides. This result points out that a limited protein hydrolysis was sufficient to improve the surface properties of soy proteins if used in combination with polysaccharides.Polysaccharides used in admixture with hydrolyzed soy proteins could control and improve the stability of foams and emulsions not only by increasing bulk viscosity but also by improving film viscoelasticity.     

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe

Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe, hydrolysed by Alcalase 2.4 L (RPH) with different degrees of hydrolysis (DH) at various concentrations were examined. As DH increased, the reduction of DPPH, ABTS radicals scavenging activities and reducing power were noticeable (p < 0.05). The increases in metal chelating activity and superoxide scavenging activity were attained with increasing DH (p < 0.05). However, chelating activity gradually decreased at DH above 30%. All activities except superoxide anion radical scavenging activity increased as the concentration of hydrolysate increased (p < 0.05). Hydrolysis using Alcalase could increase protein solubility to above 80% over a wide pH range (2–10). The highest emulsion ability index (EAI) and foam stability (FS) of hydrolysates were observed at low DH (5%) (p < 0.05). Concentrations of hydrolysates determined interfacial properties differently, depending on DH. The molecular weight distribution of RPH with 5%DH (RPH5) was determined using Sephadex G-75 column. Two major peaks with the molecular weight of 57.8 and 5.5 kDa were obtained. Fraction with MW of 5.5 had the strongest metal chelating activity and ABTS radical scavenging activity. The results reveal that protein hydrolysates from defatted skipjack roe could be used as food additives possessing both antioxidant activity and functional properties.     

Antioxidant activity of protein hydrolysates derived from threadfin bream surimi byproducts

Antioxidant activities of protein hydrolysates from threadfin bream surimi wastes, including frame, bone and skin (FBS) and refiner discharge (RD), were investigated. FBS and RD were rich in Lys, Glu, Gly, Pro, Asp, Leu, His, Tyr and Phe. FBS was hydrolysed to a greater extent than RD regardless of proteinases tested (Virgibacillus sp. SK33 proteinase, Alcalase, pepsin and trypsin). Pepsin-hydrolysed FBS, at a 5% degree of hydrolysis (DH), showed the highest antioxidant activity based on 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) radical (0.455 ± 0.054 mg Trolox equivalents/mg leucine equivalents), ferric reducing antioxidant power (FRAP) (0.221 ± 0.005 mM Trolox equivalents) and inhibition of β-carotene bleaching assays. FBS hydrolysates showed higher antioxidant activity based on chemical assays than their RD counterparts. However, FBS and RD hydrolysates protected HepG2 cells against tert-butyl hydroperoxide-induced oxidative damage to a similar extent. Therefore, FBS and RD hydrolysates have a potential as antioxidative neutraceutical ingredients.     

Influence of the degree of hydrolysis on the bioactive properties of whey protein hydrolysates using Alcalase®

Whey protein concentrate was enzymatically hydrolysed at several time courses using the commercial preparation Alcalase^® 2.4 L, different hydrolysates were achieved, and the effect of degree of hydrolysis (DH) on both technological and biological properties was studied. Results have shown that solubility, antioxidant and ACE inhibition activities were increased as DH was also augmented from about 8 to 17%. RP‐HPLC studies also revealed a decrease in hydrophobicity when samples were hydrolysed in comparison with controls. When the enzyme hydrolytic action was augmented, it stimulated both the bioactivity of whey protein and relevant technological properties, allowing these hydrolysates to be employed as additives in the development of food formulations.     

Hydrolysates of native and modified soy protein isolates: structural characteristics,solubility and foaming properties

The effect of enzymatic hydrolysis on structure characteristics (surface aromatic hydrophobicity and molecular size) of both native and modified soy protein isolates was studied. Effects on thermal behavior and functional properties (solubility and foam formation and stabilization at pH 4.5) were also analysed. Hydrolysates were obtained by bromelain digestion at pH 8 of native (N) and of thermally treated isolates at pH 7 (T7) and at pH 1.6 (T1.6). The differential effect of bromelain on the three isolates produced partially hydrolyzed structures, which exhibited an enhancement of their protein solubility (S_TCA and S_{pH 4.5}). Bromelain digestion was more effective on isolate T7 resulting hydrolysates with improved capacity to form foams at pH 4.5. The different functional behavior at pH 4.5 of hydrolysates was explained through the changes in thermal behavior, surface aromatic hydrophobicity and molecular mass distribution.     

Pilot-scale production of hydrolysates with altered bio-functionalities based on thermally-denatured whey protein isolate

Whey protein isolate (WPI) solutions (100 g L⁻¹ protein) were subjected to a heat-treatment of 80 °C for 10 min. Unheated and heat-treated WPI solutions were hydrolysed with Corolase^® PP at pilot-scale to either 5 or 10% degree of hydrolysis (DH). Hydrolysates were subsequently processed via cascade membrane fractionation using 0.14 μm, and 30, 10, 5 and 1 kDa cut-off membranes. The compositional and molecular mass distribution profiles of the substrate hydrolysates and membrane processed fractions were determined. Whole and fractionated hydrolysates were assayed for both angiotensin-I-converting enzyme (ACE) inhibitory activity and ferrous chelating capabilities. A strong positive correlation (P < 0.01) was established between the average molecular mass of the test samples and the concentration needed to chelate 50% of the iron (CC₅₀) in solution. The lowest ACE inhibition concentration (IC₅₀ = 0.23 g L⁻¹ protein) was determined for the 1 kDa permeate of the heat-treated 10% DH hydrolysate.     

Studying the effects of whole-wheat flour on the rheological properties and the quality attributes of whole-wheat saltine cracker using SRC,alveograph, rheometer,and NMR technique

Quality attributes of soft wheat products are affected by physicochemical characteristics and rheological properties of wheat flour. Whole-wheat flour has a significant impact on baking qualities (stack height, stack weight, specific volume, and breaking strength) of whole-wheat saltine crackers due to its high water absorption capacity. SRC profiles, alveograph and rheometer parameters were determined to observe the effect of whole-wheat flour on whole-wheat cracker flour blends. NMR technique was utilized to demonstrate the water migration and competition in whole-wheat dough components. Results of SRC testing revealed that the water absorption of whole-wheat flour blends increased with the addition level of whole-wheat flour. The rheological properties (G′, G″, P, L, W values) were influenced significantly by the presence of whole-wheat flour. Results of NMR indicated that water migrated from gluten network into arabinoxylans matrix in whole-wheat dough system, resulting in inferior saltine cracker-baking qualities of whole-wheat flour, i.e., small breaking strength, stack height and specific volume. The stack height, specific volume, and breaking strength of end products showed significant correlations with the arabinoxylans, dough extensibility, and gluten index of whole-wheat flour.     

Modification of the nitrogen solubility properties of soy protein isolate following proteolysis and transglutaminase cross-linking

The effect of (a) limited hydrolysis [0.5–2.0% degree of hydrolysis (DH)] with Alcalase™, (b) cross-linking with transglutaminase (TGase) and (c) combinations of these modifications on the nitrogen solubility (pH 3–8) of soy protein isolate (SPI) was investigated. Between pH 3.0 and 5.0, SPI hydrolysates, hydrolysates of cross-linked SPI and the cross-linked products of SPI hydrolysates displayed significant (P<0.05) increases in solubility compared to unmodified SPI. Cross-linking pre- or post hydrolysis did not alter the overall trend of increased (P<0.05) solubility relative to the unmodified control at low pH. At 2% DH, cross-linking pre- or post-hydrolysis resulted in greater solubility (P<0.05) than that observed in hydrolysates per se at low pH. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the 22 kDa 11S basic polypeptide was relatively resistant to Alcalase hydrolysis and that the 18 and 22 kDa 11S basic polypeptides were not susceptible to TGase cross-linking. The results demonstrate that a combination of enzymatic treatments and the order in which they are applied may have potential for creating novel food ingredients with improved functional properties, especially those properties that are dependant on high solubility at low pH.     

Influence of the extent of enzymatic hydrolysis on the functional properties of protein hydrolysate from grass carp (Ctenopharyngodon idella) skin

Protein hydrolysates from grass carp skin were obtained by enzymatic hydrolysis using Alcalase^®. Hydrolysis was performed using the pH-stat method. The hydrolysis reaction was terminated by heating the mixture to 95 °C for 15 min. At 5.02%, 10.4%, and 14.9% degree of hydrolysis (DH), the hydrolysates were analyzed for functional properties. The protein hydrolysates had desirable essential amino acid profiles. Results demonstrated that the hydrolysates had better oil holding and emulsifying capacity at low DH. The water holding capacity increased with increased levels of hydrolysis. Enzymatic modification was responsible for the changes in protein functionality. These results suggest that grass carp fish skin hydrolysates could find potential use as functional food ingredients as emulsifiers and binder agents.     

Improvement of emulsifying properties of Maillard reaction products from β-conglycinin and dextran using controlled enzymatic hydrolysis

A novel emulsifier was prepared by conjugating soy β-conglycinin and dextran (MW 67 kDa) under dry-heated Maillard reaction followed by trypsin hydrolysis with the degree of hydrolysis (DH) at 2.2% and 6.5%. The emulsifying properties of β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates (DH 2.2% and DH 6.5%) were investigated using zeta-potential, droplet size and creaming index of the emulsions. The results showed that hydrolysates of β-conglycinin–dextran conjugates (DH 2.2%) were capable of forming a fine emulsion (d₄₃ = 0.62 ± 0.04 μm, pH 7.0) which remained stable during 4 weeks of storage. A variety of physicochemical and interfacial properties of β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates were investigated. Hydrolysates of β-conglycinin–dextran conjugates (DH 2.2%) had a much higher fraction of protein adsorption (F_ads) and a significantly lower saturation surface load (Γ_sat) compared with β-conglycinin, β-conglycinin–dextran conjugates and hydrolysates of β-conglycinin–dextran conjugates (DH 6.5%). This might be due to its higher molecular flexibility, which benefited the adsorption and unfolding of peptide molecules at the droplet interface. These might explain its markedly improved emulsifying capability. The conjugation of β-conglycinin and dextran effectively enhanced the hydrophilicity of the oil droplets surfaces and improved the steric repulsion between the oil droplets. Therefore the emulsions were still stable after 4 weeks of storage against pH, ionic strength and thermal treatment. This study demonstrated that controlled enzymatic hydrolysis of protein–polysaccharide conjugates could be an effective method for preparing favourable emulsifiers.     

Screening of whey protein isolate hydrolysates for their dual functionality: Influence of heat pre-treatment and enzyme specificity

Heat pre-treated and non heat pre-treated whey protein isolate (WPI) were hydrolysed using α-chymotrypsin (chymotrypsin), pepsin and trypsin. The in vitro antioxidant activity, ACE-inhibition activity and surface hydrophobicities of the hydrolysates were measured in order to determine if peptides with dual functionalities were present. Dual functional peptides have both biological (e.g. antioxidant, ACE-inhibition, opioid activities) and technological (e.g. nanoemulsification abilities) functions in food systems. Heat pre-treatment marginally enhanced the hydrolysis of WPI by pepsin and trypsin but had no effect on WPI hydrolysis with chymotrypsin. With the exception of the hydrolysis by trypsin, heat pre-treatment did not affect the peptide profile of the hydrolysates as analysed using size exclusion chromatography, or the antioxidant activity (P > 0.05). Heat pre-treatment significantly affected the ACE-inhibition activities and the surface hydrophobicities of the hydrolysates (P < 0.05), which was a function of the specificity of the hydrolysing enzyme. Extended hydrolysis (up to 24 h) had no significant effect on the DH and the molecular weight profiles (P > 0.05) but in some instances caused a reduction in the antioxidant activity of WPI hydrolysates. The chymotrypsin hydrolysate showed a broad MW size range, and was followed by pepsin and then trypsin. The bioactivities of the hydrolysates generally decreased in the order; chymotrypsin > trypsin > pepsin. This study showed that by manipulating protein conformation with pre-hydrolysis heat treatment, combined with careful enzyme selection, peptides with dual functionalities can be produced from WPI for use as functional ingredients in the manufacture of functional foods.     

THERMAL BEHAVIOR, SOLUBILITY AND STRUCTURAL PROPERTIES OF SOY CONCENTRATE HYDROLYZED BY NEW PLANT PROTEASES

A soy concentrate prepared by alcoholic extraction of defatted soy flour was hydrolyzed with three plant proteases: hieronymin and macrodontin, cysteine proteases, and pomiferin, a serine protease. A commercial microbial protease (alcalase) was included for comparative purposes. Working at optimal conditions for each enzyme, 5–15% degree of hydrolysis (DH) values were obtained. Hydrolysates exhibited a characteristic SDS-PAGE pattern: the plant proteases attacked the polypeptides of 7S and 11S proteins with different intensity and selectivity, especially the A and B polypeptides of the 11S protein. Intermediate molecular weight peptides (24 and 60 kDa) were produced as the hydrolysis progressed. Differential Scanning Calorimetry (DSC) thermograms of flour. concentrate and hydrolysates were analyzed to evaluate the thermal stability and denaturation enthalpies of the major proteins. An increase in the degree of protein denaturation resulting from enzymatic action and a lower thermal stability at low pH were detected. The surface hydrophobicity of all hydrolysates, unlike expected, did not increase. Solubility at pH 7.0 is closely related to the DH, independent of the protease used. Solubility at pH 4.5 appeared to be related to the extent of hydrolysis of polypeptide A by each protease.