Two-Stage Countercurrent Enzyme-Assisted Aqueous Extraction Processing of Oil and Protein from Soybeans

Enzyme-assisted aqueous extraction processing (EAEP) is an increasingly viable alternative to hexane extraction of soybean oil. Although considered an environmentally friendly technology where edible oil and protein can be simultaneously recovered, this process employs much water and produces a significant amount of protein-rich aqueous effluent (skim). In standard EAEP, highest oil, protein and solids yields are achieved with a single extraction stage using 1:10 solids-to-liquid ratio (extruded flakes/water), 0.5% protease (wt/g extruded flakes), pH 9.0, and 50 °C for 1 h. To reduce the amount of water used, two-stage countercurrent EAEP was evaluated for extracting oil, protein and solids from soybeans using a solids-to-liquid ratio of 1:5–1:6 (extruded flakes/water). Two-stage countercurrent EAEP achieved higher oil, protein and solids extraction yields than using standard EAEP with only one-half the usual amount of water. Oil, protein and solids yields up to 98 and 96%, 92 and 87%, and 80 and 77% were obtained when using two-stage countercurrent EAEP (1:5–1:6) and standard single-stage EAEP (1:10), respectively. Recycling the second skim obtained in two-stage countercurrent EAEP enabled reuse of the enzyme, with or without inactivation, in the first extraction stage producing protein with different degrees of hydrolysis and the same extraction efficiency. Slightly higher oil, protein and solids extraction yields were obtained using unheated skim compared to heated skim. These advances make the two-stage countercurrent EAEP attractive as the front-end of a soybean biorefinery.     

Scale-up of Enzyme-Assisted Aqueous Extraction Processing of Soybeans

The effects of scaling-up enzyme-assisted aqueous extraction process (EAEP) using 2 kg of flaked and extruded soybeans as well as the effects of different extrusion and extraction conditions were evaluated. Standard single-stage EAEP at 1:10 solids-to-liquid ratio (SLR) was used to evaluate the effects of different extruder screw speeds and whether or not collets were extruded directly into water. Increasing extruder screw speed from 40 to 90 rpm improved oil extraction yield from 85 to 95%. Oil, protein, and solids extraction yields of 97, 86, and 78% were obtained when extruding directly into water and 95, 84, and 77% when not extruding into water. When not extruding into water, standard single-stage EAEP (1:10 SLR) yielded 95, 84, and 77% of total oil, protein, and solids extraction, respectively, and two-stage countercurrent EAEP (1:6 SLR) yielded 99, 94, and 83% total oil, protein, and solids extraction, respectively. These yields were similar to those previously obtained in the laboratory (0.08 kg soybeans), but higher oil contents were observed in the skim fractions produced at pilot-plant scale for both processes. Modifying processing parameters improved the oil distribution among the fractions, increasing oil yield in the cream fraction (from 76 to 86%) and reducing oil yield in the skim fraction (from 23 to 12%). Steady-state oil extraction was achieved after two 2-stage extractions. Two-stage countercurrent EAEP is particularly attractive due to reduced water usage compared to conventional single-stage extraction.     

Functional Properties of Protein Isolates Produced by Aqueous Extraction of De-hulled Yellow Mustard

Two types of protein isolates were prepared from de‐hulled yellow mustard flour by aqueous extraction, membrane processing and isoelectric precipitation. The precipitated and soluble protein isolates had 96.0 and 83.5% protein content on a moisture and oil free basis, respectively. Their functional properties were evaluated and compared with commercial soybean and other Brassica protein isolates. The soluble protein isolate exhibited high values for all properties. The precipitated protein isolate showed excellent oil absorption and emulsifying properties but poor solubility, water absorption and foaming properties due to its high lipid content (~25%). Storage temperature had limited effect on lipid oxidation, and hence the stability of the precipitated protein isolate at 25–45 °C. Flavor of wieners and bologna prepared with 2% of this isolate as binder was comparable to those prepared with soy protein isolate.     

Enzyme-Assisted Aqueous Extraction of Oil and Protein from Soybeans and Cream De-emulsification

The effects of two commercial endoproteases (Protex 6L and Protex 7L, Genencor Division of Danisco, Rochester, NY, USA) on the oil and protein extraction yields from extruded soybean flakes during enzyme-assisted aqueous extraction processing (EAEP) were evaluated. Oil and protein were distributed in three fractions generated by the EAEP: cream + free oil, skim and insolubles. Protex 6L was more effective for extracting free oil, protein and total solids than Protex 7L. Oil and protein extraction yields of 96 and 85%, respectively, were obtained using 0.5% Protex 6L. Enzymatic and pH treatments were evaluated to de-emulsify the oil-rich cream. Cream de-emulsification generated three fractions: free oil, an intermediate residual cream layer and an oil-lean second skim. Total cream de-emulsification was obtained when using 2.5% Protex 6L and pH 4.5. The extrusion treatment was particularly important for reducing trypsin inhibitor activity (TIA) in the protein-rich skim fraction. TIA reductions of 69 and 45% were obtained for EAEP skim (the predominant protein fraction) from extruded flakes and ground flakes, respectively. Protex 6L gave higher degrees of protein hydrolysis (most of the polypeptides being between 1,000 and 10,000 Da) than Protex 7L. Raffinose was not detected in the skim, while stachyose was eliminated by α-galactosidase treatment.     

Characteristics of Oil and Skim in Enzyme-Assisted Aqueous Extraction of Soybeans

The economic viability of enzyme-assisted aqueous extraction processing (EAEP) of soybeans depends on properties and potential applications of all fractions (skim and insolubles as well as oil). EAEP oil contained lower free fatty acid, phosphorus, and tocopherol contents, similar unsaponifiable matter levels, and higher degrees of oxidation (peroxide and p-anisidine values) than hexane-extracted oil. The phospholipid profile of EAEP fractions was mainly composed of phosphatidic acid, followed by phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Most of phospholipids were present in the skim, except for phosphatidic acid, which was the major phospholipid in the cream fraction. Skim and cream contained 55 and 3 % of the soluble carbohydrates in the original extruded flakes, respectively. Soluble carbohydrates of the skim were mainly composed of stachyose (5.8 ± 0.8 mg/mL) and sucrose (9.9 ± 0.8 mg/mL), which were hydrolyzed into glucose, galactose, and fructose after addition of α-galactosidase. Skim and cream peptides contained <20 kDa MW molecules. About 71 % of the skim peptides were <20 kDa MW, with 49 % being <1.35 kDa MW, 22 % being 17–1.35 kDa MW, and 29 % being 44–670 kDa MW. Skim protein and carbohydrate contents make this fraction suitable for replacing water in ethanol fermentations, thereby improving the fermentation rate/production and the nutritional quality of distiller’s dried grains with solubles.     

Separating Oil from Aqueous Extraction Fractions of Soybean

Previous research has shown that enzyme-assisted aqueous extraction processing (EAEP) extracts 88–90% of the total soybean oil from extruded full-fat soy flakes into the aqueous media, which is distributed as cream (oil-in-water emulsion), skim, and free oil. In the present work, a simple separatory funnel procedure was effective in separating aqueous skim, cream and free oil fractions allowing mass balances and extraction and recovery efficiencies to be determined. The procedure was used to separate and compare liquid fractions extracted from full-fat soy flour and extruded full-fat soy flakes. EAEP extracted more oil from the extruded full-fat soy flakes, and yielded more free oil from the resulting cream compared to unextruded full-fat soy flour. Dry matter partitioning between fractions was similar for the two procedures. Mean oil droplet sizes in the cream and skim fractions were larger for EAEP of extruded flakes compared to non-enzymatic AEP of unextruded flour (45 vs. 20 μm for cream; 13 vs. 5 μm for skim) making the emulsions from EAEP of extruded flakes less stable. All major soy protein subunits were present in the cream fractions, as well as other fractions, from both processes. The cream could be broken using phospholipase treatments and 70–80% of total oil in the extruded full-fat flakes was recovered using EAEP and a phospholipase de-emulsification procedure.     

Integrated Countercurrent Two-Stage Extraction and Cream Demulsification in Enzyme-Assisted Aqueous Extraction of Soybeans

Countercurrent two-stage extraction and cream demulsification were fully integrated and demonstrated on laboratory scale (2 kg soybeans) wherein the enzyme used for demulsifying the cream was used in the extraction steps of enzyme-assisted aqueous extraction processing (EAEP). Protease enzyme (Protex 6L) entered the integrated EAEP process in the demulsification step and the skim, which contained the enzyme, resulting from breaking the cream emulsion was recycled upstream into the second extraction stage and then to the first extraction stage. Oil, protein and solids extraction yields of 96.1 ± 1.4%, 89.3 ± 1.0%, and 81.2 ± 2.0%, respectively, were achieved with steady-state operation of integrated EAEP. Higher degrees of protein hydrolysis (DH) were obtained when using the integrated process compared with the process when not recycling the enzyme. Higher extents of hydrolysis probably increased emulsion formation thereby affecting lipid distribution among the fractions. Overall free oil recovery was reduced due to more oil shifting to the skim fraction.     

Pilot-Plant Proof-of-Concept for Integrated, Countercurrent, Two-Stage, Enzyme-Assisted Aqueous Extraction of Soybeans

Proof-of-concept for integrated, countercurrent, two-stage, enzyme-assisted aqueous extraction processing of soybeans was demonstrated on a pilot-plant scale (75 kg extruded flaked soybeans) where the protease used to demulsify the cream was recycled into upstream extraction stages. Oil, protein, and solids extraction yields of 98.0 ± 0.5%, 96.5 ± 0.4%, and 86.8 ± 0.5% were achieved by using the integrated countercurrent process. A three-phase horizontal decanter centrifuge efficiently separated the solids from the two liquid fractions (skim and cream). Fine separation between the two liquid fractions was important to reducing the volume of skim contaminating the cream fraction, thereby reducing the amount of enzyme used for cream demulsification and subsequent extraction. We were able to reduce enzyme use when moving from the laboratory to the pilot-plant scale, which reduced the degree of protein hydrolysis and improved cream demulsification. Enzyme-catalyzed cream demulsification was 91.6% efficient and 93.0% free oil recovery from cream was achieved by using the integrated approach.     

Characterization of Fractionated Soy Proteins Produced by a New Simplified Procedure

It was possible to fractionate soy protein into two soy protein isolate fractions (>90% protein) enriched in either glycinin or β-conglycinin by using a new simplified procedure (referred to as the Deak procedure) employing CaCl₂ and NaHSO₃. The Deak procedure produced fractions with higher yields of solids, protein, and isoflavones, and similar protein purities as well as improved functional properties compared to fractions recovered by established, more complex soy protein fractionation procedures. The Deak glycinin-rich fraction comprised 15.5% of the solids, 24.4% of the protein, and 20.5% of the isoflavones in the starting soy flour, whereas the glycinin-rich fraction of the established procedure (Wu procedure) comprised only 11.6% of the solids, 22.3% of the protein, and 9.6% of the isoflavones. The Deak β-conglycinin-rich fraction comprised 23.1% of the solids, 37.1% of the protein, and 37.5% of the isoflavones in the starting soy flour, whereas the Wu β-conglycinin-rich fraction comprised only 11.5% of the solids, 18.5% of the protein, and 3.3% of the isoflavones. Protein purities were >80% for both fractions when using both procedures. The Wu procedure produced protein fractions with slightly higher solubilities and similar surface hydrophobicities; whereas, the fractions produced by the Deak procedure had superior emulsification and foaming properties and similar dynamic viscosity behaviors.     

Nutrient Enhancement of Corn Distillers Dried Grains by Addition of Coproducts of the Enzyme-Assisted Aqueous Extraction Process of Soybeans in Corn Fermentation

Corn distillers dried grains with solubles (DDGS) are not nutritionally complete as a nonruminant ingredient owing to poor essential amino acid profile, and high fat and fiber contents. Coproducts of soybean enzyme-assisted aqueous extraction process, skim (wastewater) and insoluble fiber (IF; solid residue), and/or enzymes (pectinase, cellulase, and acid protease; referred to as PCF) were evaluated as distillers dried grains (DDG) nutritional quality enhancers in corn fermentation. Corn-soy DDG had ~10% higher protein, ~3% lower fat, and ~2% lower fiber contents compared to corn DDG; fiber content was further reduced with PCF treatment (~4% total decrease). Concentrations of all essential amino acids in corn-soy DDG showed at least a threefold increase, except for allo-isoleucine and tryptophan, compared to corn DDG. Corn-soy DDG had ~25% decrease in total fatty acid (TFA) and ~6% decrease in free fatty acid (FFA) contents compared to corn DDG; TFA and FFA contents further decreased with PCF treatment. Corn-soy DDG had ~15%, 3%, and 1.7% lower hemicellulose, cellulose, and lignin contents, respectively, compared to corn DDG; hemicellulose content further decreased with PCF treatment. Mineral composition of corn-soy DDG was in the recommended range, except Na and S were out of range by 0.79% and 0.74%, respectively. All results, except for Na and S, suggest strong potential of using skim and IF as DDG nutritional quality enhancers.     

Aqueous Surfactant Two‐Phase Systems for the Continuous Countercurrent Cloud Point Extraction

Aqueous nonionic surfactant solutions split into two phases if the temperature is increased beyond a certain temperature, the so‐called cloud point temperature. Presently many different types of nonionic surfactants are produced commercially, out of these numerous have been considered as potential solvent for the cloud point extraction. In this work the crucial thermophysical properties of nonionic surfactants are investigated to determine the potential of surfactant systems for extraction processes. Phase equilibria of the binary system Triton X‐114/water and the ternary system Triton X‐114/water/phenol were measured. Based on these data the cloud point extraction was implemented in a continuous stirred extraction column. It was found, that increasing temperature within the column reduces the loss of surfactant and leads to an increasing enrichment factor. This work demonstrates that surfactant/water systems represent a suitable alternative to conventional solvents and can effectively be processed in continuous extraction columns.     

Downstream Processes for Aqueous Enzymatic Extraction of Rapeseed Oil and Protein Hydrolysates

Downstream processes following aqueous enzymatic extraction (AEE) of rapeseed oil and protein hydrolysates were developed to enhance the oil and protein yields as well as to purify the protein hydrolysates. The wet precipitate (meal residue) from the AEE was washed with twofold water at 60 °C, pH 11 for 1 h. Emulsions from the AEE and the washing step were pooled and submitted to a stepwise demulsification procedure consisting of storage-centrifugation and freezing–thawing followed by centrifugation. Aqueous phases were pooled and adsorbed onto macroporous adsorption resins (MAR) to remove salts and sugars. Following extensive rinsing with deionized water (pH 4), desorption was achieved by washing with 85% ethanol (v/v) to obtain crude rapeseed peptides (CRPs). In a separate experiment, stepwise desorption was carried out with 25, 55, and 85% ethanol to separate the bitter peptides from the other peptides. Using a combination of the AEE process, washing and demulsification steps, the yields of the total free oil and protein hydrolysates were 88–90% and 94–97%, respectively. The protein recovery was 66.7% and the protein content was enriched from 47.04 to 73.51% in the CRPs. No glucosinolates and phytic acid were detected in the CRPs. From the stepwise desorption, a non-bitter fraction RP25 (containing 64–66% of total desorbed protein) had a bland color and significantly higher protein content (81.04%) and hence was the more desirable product.     

Effects of Extraction Temperature and Preservation Method on Functionality of Soy Protein

The effects of extraction temperature and preservation method on the functional properties of soy protein isolate (SPI) were determined. Four extraction temperatures (25, 40, 60, and 80 °C) were used to produce SPI and yields of solids and protein contents were determined. Three preservation methods were also tested (spray-drying, freeze-drying, and freezing–thawing) and compared to fresh (undried) samples for each extraction temperature. No differences in yields of solids and protein were observed among SPIs extracted at 25, 40, and 60 °C; however, SPI extracted at 80 °C yielded significantly less solids and protein. Extraction temperature significantly affected SPI functionality. As extraction temperature increased, solubility and emulsification capacity decreased; surface hydrophobicities, emulsification activities and stabilities, and dynamic viscosities increased; and foaming properties improved. Preservation method also significantly affected SPI functionality. Drying method did not affect the denaturation enthalpies of SPIs, but spray-dried SPIs had higher solubilities, surface hydrophobicities, and emulsification stabilities, and lower viscosities, emulsification activities and rates of foaming than freeze-dried SPI exhibited. Emulsification and foaming capacities and foaming stabilities were similar for both methods of drying. There was significant interaction between extraction temperature and preservation method for all functional properties except emulsification capacity.     

Functional Properties of Soy Protein Isolates Prepared from Gas-Supported Screw-Pressed Soybean Meal

White flakes (WFs) are obtained from dehulled flaked soybeans by extracting oil with hexane and flash- or downdraft-desolventizing the (defatted) flakes, and WFs are the normal feedstock used to produce soy protein ingredients. Gas-supported screw pressing (GSSP) is a new oilseed crushing technology in which traditional screw pressing is combined with injecting high-pressure CO₂, thereby producing hexane-free, low-fat, high-PDI soybean meal. The objectives of the present study were to evaluate yields, compositions, and functional properties of soy protein isolates (SPIs) produced from GSSP soybean meal and to compare these properties to those of SPIs produced from WFs. GSSP meals produced SPIs in significantly higher yields (59.7–63.1% vs. 51.6–61.1%), with greater free (0.05–0.40%) and bound fat (3.70–4.92%) contents than did WFs. There were no significant differences in protein contents of the SPI; all exceeded 90% protein content (db). SPIs prepared from GSSP meals had similar or slightly lower water-solubilities compared to SPIs prepared from WFs. SPIs prepared from GSSP meals had higher water-holding capacities and viscosities, and significantly better emulsifying and fat-binding properties compared to SPIs prepared from WFs. SPIs prepared from WFs had significantly better foaming properties compared to SPIs prepared from GSSP meals, which were attributed to the lower fat contents of SPIs prepared from WFs.     

Optimization of the Aqueous Enzymatic Extraction of Rapeseed Oil and Protein Hydrolysates

An aqueous enzymatic extraction method was developed to obtain free oil and protein hydrolysates from dehulled rapeseeds. The rapeseed slurry was treated by the chosen combination of pectinase, cellulase, and β-glucanase (4:1:1, v/v/v) at concentration of 2.5% (v/w) for 4 h. This was followed by sequential treatments consisting of alkaline extraction and an alkaline protease (Alcalase 2.4L) hydrolysis to both produce a protein hydrolysate product and demulsify the oil. Response surface methodology (RSM) was used to study and optimize the effects of the pH of the alkaline extraction (9.0, 10.0 and 11.0), the concentration of the Alcalase 2.4L (0.5, 1.0 and 1.5%, v/w), and the duration of the hydrolysis (60, 120, and 180 min). Increasing the concentration of Alcalase 2.4L and the duration of the hydrolysis time significantly increased the yields of free oil and protein hydrolysates and the degree of protein hydrolysis (DH), while the alkaline extraction pH had a significant effect only on the yield of the protein hydrolysates. Following an alkaline extraction at pH 10 for 30 min, we defined a practical optimum protocol consisting of a concentration of 1.25–1.5% Alcalase 2.4L and a hydrolysis time between 150 and 180 min. Under these conditions, the yields of free oil and protein hydrolysates were 73–76% and 80–83%, respectively. The hydrolysates consisted of approximately 96% of peptides with a MW less than 1500, of which about 81% had a MW less than 600 Da.     

Extraction,Composition and Functional Properties of Pennycress (Thlaspi arvense L.) Press Cake Protein

This study compared two methods for extracting the protein in pennycress (Thlaspi arvense L.) press cake and determined the composition and functional properties of the protein products. Proteins in pennycress press cake were extracted by using the conventional alkali‐solubilization–acid‐precipitation (AP) method or saline‐based (SE) procedure (0.1 M NaCl at 50 °C). The extraction method has a major influence on the purity and functional properties of press cake protein products. AP had a lower protein yield (23 %) but much higher purity (90 % crude protein) compared with SE (45 % yield, 67 % crude protein). AP protein isolate had high foam capacity (120 ml), high foam stability (96 % foam volume retention) and high emulsion stability (24–35 min), and it was resistant to heat denaturation (3 % loss of solubility at pH 2 and pH 10). On the other hand, SE protein concentrate showed remarkably high solubility (>76 %) between pH 2 and 10 and exceptional emulsifying activity (226–412 m²/g protein), but was more susceptible to heat denaturation at pH 7 and pH 10 (65–78 % loss of solubility). These results strongly demonstrate that higher purity pennycress press cake protein can be produced by either saline extraction or acid precipitation and have functional properties that are desirable for non‐food uses.     

Effects of Tween 20 and Transglutaminase Modifications on the Functional Properties of Peanut Proteins

The effects of Tween 20 on the emulsification and gelation properties of peanut protein isolates (PPI) were investigated. The functional properties of different peanut protein products, including PPI, Tween 20-assisted aqueous extraction peanut proteins (TPP), and their transglutaminase-modified products (TG-TPP), were then compared. The results indicate that the addition of Tween 20 to the PPI resulted in higher emulsifying activity index (EAI) and emulsion stability index (ESI) values than PPI without Tween 20; however, the emulsions produced by the PPI–Tween 20 mixtures were easier to destabilize during storage. As the amount of Tween 20 was increased, both the surface hydrophobicity and gel strength of the PPI–Tween mixtures significantly decreased. TPP (containing approximately 11% Tween 20) exhibited significantly different functional properties from PPI. Compared with PPI, TPP had higher EAI and ESI values but a weaker heat-induced gelation ability. The protein solubility of TPP was markedly higher than that of PPI. Modification of TPP with transglutaminase (TGase) significantly enhanced their gelation and oil-binding properties but reduced the protein solubility and ESI value. The remarkable improvement in the gelation ability of TG-TPP was mainly attributed to the formation of high-molecular-weight protein aggregates and their conformational changes.     

Physicochemical Properties and ACE-I Inhibitory Activity of Protein Hydrolysates from a Non-Genetically Modified Soy Cultivar

Arkansas‐grown non‐genetically modified soybean cultivar, R08‐4004, was selected to prepare a protein isolate, which was treated with Alcalase for limited enzymatic hydrolysis. The objective was to optimize the Alcalase hydrolysis condition to produce soy protein hydrolysate (SPH) with high protein yield, low bitterness, and clarity for beverage applications. The degree of hydrolysis ranged between 14 and 52 % during the study at varying incubation times using two different concentrations of Alcalase enzyme. Recovery of soluble protein, between 21 and 53 %, was achieved with a decrease in turbidity. There was an increase in surface hydrophobicity (S₀) which is correlated with bitterness of SPH treated with 1.0 AU (3.2 µL/g) of Alcalase 2.4 L. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis showed a distinct hydrolysis pattern in which 7S globulin and the two acidic sub‐units of 11S globulin were hydrolyzed extensively in comparison to the two basic sub‐units of 11S globulin. Limited enzymatic hydrolysis produced low molecular weight peptides <17 kDa. Among these SPHs, the one derived after 120 min incubation had the highest soluble protein yield (43 %), low S₀ value (35.4), low turbidity (0.88), and highest angiotensin‐I converting enzyme (ACE‐I) inhibition activity (66.6 %). This hydrolysate has potential use as protein rich nutraceutical for developing many non‐genetically modified food product applications.     

Enzymatic Demulsification of the Oil-Rich Emulsion Obtained by Aqueous Extraction of Peanut Seeds

This study details the enzymatic destabilization of the emulsion formed during aqueous extraction of peanut seeds and the quality of the resulting oil. The emulsion was exposed to enzymatic treatment and pH adjustment. The experimental results suggest that the alkaline endopeptidase Mifong^®2709 was the most effective demulsifier, while Phospholipase A2 and pH adjustment had little effect on emulsion stability. The demulsifying conditions of Mifong^®2709 were optimized by response surface methodology (RSM). The optimal conditions which produced a free oil yield of ~94 % were: 1:1 water-to-emulsion ratio, enzyme concentration of 1,600 IU/g of emulsion and 70 min hydrolysis time at 50 °C. We found that these conditions resulted in a positive relationship (R ² = 0.9671) between free oil yield and the degree of protein hydrolysis. Increased protease treatment produced a smaller number of oil droplets, but the size of these droplets increased significantly. When compared to demulsified oil products obtained by using thermal treatment, the oil obtained by Mifong^®2709 exhibited lower acid and peroxide values, contained more tocopherols and had a longer induction time as determined in the Rancimat test. The high yield and quality of peanut oil obtained by enzymatic treatment makes enzyme demulsification a promising approach to recovering free oil in aqueous extractions of peanuts.     

Enzyme Processing of Soy Flour with Minimized Protein Loss

The enzymatic treatment of defatted soy flour (SF) to reduce indigestible carbohydrates can result in undesirable protein loss. Here protein loss was minimized with quantitation of the effects of ionic strength (IS), protease activity, and SF toasting. At the enzyme processing condition (25% w/v SF, 50 °C, pH 4.8, 48 hours), protein loss increased linearly with the IS in enzyme broths (EB); e.g., contacting untoasted SF with water or heat-deactivated EB showed protein loss of 28% in water but up to 40% when IS was increased in the range of 0.04–0.19 M. Protein loss also increased with protease in EB (nondeactivated): after adjusted for IS-related loss, approximately 10% and 25% additional protein loss occurred in EB of 73 and 490–557 U/(g SF) protease, respectively. SDS-PAGE results showed that proteolysis was not extensive, mainly on β-conglycinin α'/α and glycinin acidic 37-kDa subunits; and most of the proteolytic products could be recovered by heat-induced precipitation. SF toasting effects were studied, particularly at 2-hours 160°C, with material balances, protein distributions, and monosaccharide yields in hydrolysates. Overall, protein loss was minimized to 5.2% and the conversion of carbohydrate to monomeric sugars increased to 89.2%.