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.     

Protein Recovery in Aqueous Extraction Processing of Soybeans Using Isoelectric Precipitation and Nanofiltration

Isoelectric precipitation and whey nanofiltration were evaluated in recovering protein from skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes. Countercurrent two-stage EAEP was performed at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h to extract oil and protein from soybeans. Two protein recovery strategies were applied to skim fractions produced by different extraction treatments: Treatment 1 using 0.5% protease (wt/g extruded flakes) in both extraction stages; Treatment 2 using 0.5% protease only in the 2nd extraction stage; and Treatment 3 using no enzyme in either extraction stage. Protein recovery by using isoelectric precipitation was inversely related to the extent of hydrolysis with recoveries of 27, 61, and 87% of skim proteins from Treatments 1, 2, and 3, respectively. Overall protein recoveries of 26, 54, and 57% of the original protein in the extruded full-fat flakes were achieved when combining extraction treatments and isoelectric precipitation. Nanofiltering isoelectric wheys (500-Da membrane) achieved protein retentate yields of 96.3, 94.5, and 91.8% (1.9–2.8 concentration factor) with permeate fluxes up to 1.35 kg/h m². About 97, 98, and 99% of skim protein were recovered by isoelectric precipitation and whey nanofiltration for Treatments 1, 2, and 3, respectively. Overall protein recoveries of 93, 87, and 65% of the protein in the extruded flakes were achieved for Treatments 1, 2, and 3, respectively. Although high protein retentions were achieved, very low permeate fluxes were observed for whey nanofiltration.     

Effect of moisture and temperature on the phosphorus content of crude soybean oil extracted from fine flour

Analysis of total oil content in soybeans is usually done by extracting flours, whereas commercial extraction for recovery of oil is done by extracting flakes. It has recently become apparent that phosphorus content of crude soybean oil extracted from flours can vary depending on extraction temperature and flour moisture. In this study, flour moistures below 6% yielded crude oil with low phosphorus (15 ppm), but phosphorus in the oil increased rapidly to 260 ppm at 9% moisture. When temperature of the extraction was increased from 25 to 60°C, the phosphorus in extracted oil also increased for moisture contents of 6.6% and 8.3%, but not for moisture contents of 5% and 3%. In addition to the effects of extraction temperature, it was found that preheating whole soybeans at various temperatures affected phosphorus in oil from extracted flour. Preheating at 130°C caused high phosphorus content regardless of how dry the flour was, whereas preheating at 100°C or below caused phosphorus content that increased with increased moisture. The response of phosphorus content in crude oil to temperature and moisture may be useful in improving the quality of commercially extracted soy oil.     

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.     

Properties of Soy Protein Produced by Countercurrent,Two-Stage,Enzyme-Assisted Aqueous Extraction

Enzyme-assisted aqueous extraction processing (EAEP) is an environmentally friendly technology where oil and protein can be simultaneously extracted from soybeans by using water and protease. Countercurrent, two-stage, EAEP was performed at a 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h to extract oil and protein from soybeans. The skim fractions were produced by three methods: (1) by treating with 0.5 % protease (wt/g extruded flakes) in both extraction stages; (2) by treating with 0.5 % protease in the 2nd extraction stage only; and (3) by using the same two-stage extraction procedure without enzymes in either extraction stages. Countercurrent, two-stage, protein extraction of air-desolventized, hexane-defatted, soybean flakes was used as a control. Solubility profiles of the skim proteins were the typical U-shaped curves with the lowest solubility at the isoelectric point of soy protein (pH 4.5). The use of the enzyme slightly improved solubility of the recovered protein with hydrolyzed proteins having higher solubilities at acid pH. Emulsification and foaming properties were generally reduced by the use of enzyme during EAEP extractions. The skims produced with protease-extracted (hydrolyzed) proteins gave gels with lower hardness than did unhydrolyzed proteins when heated at 80 °C. The essential amino acid compositions and protein digestibilities were not adversely affected by either extrusion or extraction treatments.     

Effect of processing on sphingolipid content in soybean products

Soybeans are believed to be a rich source of sphingolipids, a class of polar lipids that has received attention for their possible cancer-inhibiting activities. The effect of processing on the sphingolipid content of various soybean products has not been determined. Glucosylceramide (GlcCer), the major sphingolipid type in soybeans, was measured in several processed soybean products to illustrate which product(s) GlcCer is partitioned into during processing and where it is lost. Whole soybeans were processed into full-fat flakes, from which crude oil was extracted. Crude oil was refined by conventional methods, and defatted soy flakes were further processed into alcohol-washed and acid-washed soy protein concentrates (SPC) and soy protein isolates (SPI) by laboratory-scale methods that simulated industrial practices. GlcCer was isolated from the samples by solvent extraction, solvent partition, and TLC and was quantified by HPLC. GlcCer remained mostly within the defatted soy flakes (91%) rather than in the oil (9%) after oil extraction. Only 52, 42, and 26% of GlcCer from defatted soy flakes was recovered in the acid-washed SPC, alcohol-washed SPC, and SPI products, respectively. All protein products had a similar GlcCer concentration of about 281 nmol/g (dry wt basis). The minor quantity of GlcCer in the crude oil was almost completely removed by water degumming.     

Mechanical oil expression from extruded soybean samples

Soybean is generally recognized as a source of edible and industrial oil, and the deoiled meal is seen as a source of protein in animal feed. In recent years, however, more interest has been directed toward using soy meal as a protein source for human consumption. Extrusion-expelling of soybean provides an opportunity in this direction. The main focus of this study was to maximize the oil recovery from extruded soybean processed using three different kinds of extruders and processing conditions. These extruded samples were later pressed uniaxially in a specifically designed test-cell and the oil recovery was recorded over time. The effects of process variables, including applied pressure, pressing temperature and sample height, were investigated. Results indicated that over 90% of the available oil could be recovered from pressing of extruded soy samples. The information generated is likely to be useful in interpreting the effect of process variables and extruding equipment for pretreatment of soybean for subsequent mechanical oil expression.     

The comparative value of heated ground unextracted soybeans and heated dehulled soybean flakes as a source of soybean oil and energy for the chick

Experiments were conducted to evaluate heated unextracted soybean fractions as sources of soybean oil and protein for the growing chick. Heated dehulled unextracted soybean flakes produced growth rate and feed efficiency equal to that obtained with the combination of soybean oil meal and degummed soybean oil while heated ground unextracted soybeans were less satisfactory in this respect. The poorer results obtained with ground unextracted soybeans were shown to be related to a poorer absorbability of the oil in them. Flaking the soybeans markedly improved the absorbability of the oil by the chick, probably by causing a greater disruption of cellular structure than was obtained by the grinding of the soybeans. The metabolizable energy of ground unextracted soybeans was substantially less than that of unextracted soybean flakes. Most of the differences in metabolizable energy were accounted for by differences in absorbability of the oil. Soybean hulls at a level equivalent to that contained in soybeans were found to have no effect on growth rate and only a slight effect on feed efficiency. Autoclaving soybean oil did not lower its value for the chick. The relationship between the poorer growth obtained with ground unextracted soybeans and the low absorbability of the oil in them was discussed. To obtain maximum efficiency in the use of unextracted soybean products in chick rations, some such means as flaking must first be employed to increase the availability of the oil.     

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.     

Protein Extraction and Membrane Recovery in Enzyme-Assisted Aqueous Extraction Processing of Soybeans

Enzyme-assisted aqueous extraction processing (EAEP) is an environmentally friendly process in which oil and protein can be simultaneously recovered from soybeans by using water and enzymes. The significant amount of protein-rich effluent (skim) constitutes a challenge to protein recovery. Countercurrent two-stage EAEP at a 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h was used to extract oil and protein from dehulled, flaked and extruded soybeans. Different enzyme use strategies were used to produce different skim fractions: 0.5% protease (wt/wt extruded flakes) in both extraction stages; 0.5% protease only in the 2nd extraction stage; and no enzyme in either stage. Dead-end, stirred-cell membrane filtration was evaluated with each skim. About 96, 89, and 66% of the protein were extracted with the three enzyme treatments, respectively. Protein retentate yields of 91, 96, and 99% were obtained for the three enzyme treatments, respectively, by using double membrane filtration (30 kDa/500 Da) of the skims, achieving permeate fluxes up to 1.24 kg/m² h at 3.9–4.8 concentration factors (CF) and 0.56 kg/m² h at 1.9–2.9 CF for 30 kDa ultrafiltration and 500 Da nanofiltration, respectively. For cross-flow ultrafiltration with the 3-kDa membrane, pH and presence of insoluble protein aggregates significantly affected permeate flux. Maximum permeate flux occurred at high pH and in the presence of protein aggregates, achieving a mean value of 4.1 kg/m² h at 1.7 bar transmembrane pressure.     

Enzymatic hydrolysis pretreatment for mechanical expelling of soybeans

Mechanical expelling of soybeans with enzymatic hydrolysis as pretreatment was investigated, and the process parameters were optimized by means of response surface methodology. Enzyme pretreatment enhanced both the amount of extractable oil in soybeans and oil extractability. A second-order response surface model was developed to predict the expelled oil as a function of the six process parameters investigated. The optimum was found at: Moisture content during hydrolysis, 23.00% wet basis (w.b.); enzyme concentration, 11.84% vol/wt; incubation period, 13.24 h; moisture content during pressing, 9.36% w.b.; pressing pressure, 75 MPa; and pressing time, 5.36 min. The parameters had no interactive effects on expelled oil. Pressing pressures above 75 MPa caused extrusion. Under the optimal conditions, oil expelled from dehulled cracked soybeans by static pressing at room temperature (18°C) was 63.5% of the total extractable oil. Much higher oil recovery would be expected in actual screw expellers due to dynamic pressing and higher operating temperature. Oil recovery could be further increased by adding one or more conventional pretreatments to the enzymatic hydrolysis pretreatment investigated in this study.     

Structure,Protein Interactions and In Vitro Protease Accessibility of Extruded and Pressurized Full-Fat Soybean Flakes

The objectives of the present study were to determine how extrusion (barrel temperature of 100 °C) and high-pressure processing (HPP, 200 and 500 MPa, 15 min, 25 °C) of full-fat soybean flakes (FFSF) modified the structure of soybean cotyledon cells, the protein interactions and the in vitro protease accessibility. Cellular disruption of the cotyledon cells was only observed for extruded FFSF. Extrusion and HPP at 500 MPa favored formation of insoluble protein aggregates, in which oil was entrapped. High pressure size exclusion chromatography (HPSEC) and extraction methods using buffers containing SDS and 2-mercaptoethanol suggested that noncovalent interactions were the main forces in protein aggregate formation during HPP 500 MPa and extrusion. Intermolecular cross-linking by disulfide bonding was also involved in insoluble aggregates, but at a lesser extent than noncovalent interactions. Extrusion and HPP 500-MPa treatment enhanced the proteolytic attack, while treatment at 200 MPa had no impact. Drastic changes in the peptide profile of the extracted proteins were, however, only observed for the enzyme-treated 500-MPa FFSF. Optimal oil and protein extraction yields required cellular disruption of cotyledon cells and hydrolysis of protein aggregates, which were obtained with enzyme-assisted aqueous extraction of extruded FFSF.     

Effect of equilibrium oil extraction on the chemical composition and sensory quality of soy flour and concentrates

Previous studies have shown that ambient-temperature equilibrium, hexane extraction of soy flour yielded the same amount of oil as was extracted from soy flakes by conventional high-temperature processing. The oil obtained at ambient temperatures contained less phospholipid than commercial crude oils obtained by traditional processing. In this study, chemical composition, flavor and odor of soy flour obtained after oil extraction by the equilibrium procedure were evaluated before and after toasting. Results were compared with those obtained for commercial untoasted food-grade soy flakes. Chemical and sensory analyses were performed on soy protein concentrates (SPC) prepared from defatted flour, defatted toasted flour and commercial defatted white food-grade flakes. SPC were made by acid and ethanol-extraction methods. Ethanol extraction of soy flour produced SPC with similar protein, lipid and sensory qualities to those obtained from commercial flakes. Acid extraction produced SPC with more lipid than was obtained by ethanol extraction. Toasted soy flour and flakes had similar sensory properties, as did the SPC prepared from them.     

Solvent extraction of oil from soybean flour I— extraction rate,a countercurrent extraction system,and oil quality

Measurements of rates of oil extraction from either fine flour or soybean flakes in a column showed that oil extraction from flour was dependent on the volume of solvent, but oil extraction from flakes depended on time of contact rather than volume of solvent. We interpreted the data to mean that oil was being washed out of the fine flour with little diffusion involved, whereas in flakes, the limit on rate was diffusion of the solvent into and out of the tissue. Fine full fat flour worked well in a batchwise countercurrent extraction system with mixing and centrifugal separation. Because the oil dissolved immediately and reached equilibrium rapidly, the actual material balance was close to calculated values. However, due to the large hold-up volume, the separation of miscella from the meal required several mixing and separation stages. The oil resulting from this countercurrent extraction system had a superior quality with 37 ppm phosphorus, 0.08% free fatty acids, and a light color.     

Aqueous Extraction of Oil and Protein from Soybeans with Subcritical Water

Aqueous extraction using subcritical water is an environmentally friendly alternative to extracting oil and protein from oilseeds with flammable organic solvents. The effects of solids-to-liquid ratio (1:3.3–1:11.7), temperature (66–234 °C), and extraction time (13–47 min) were evaluated on the extraction of oil and protein from soybean flakes and from extruded soybeans flakes with subcritical water. A central composite design (2³) with three center points and six axial points was used. Subcritical water extractions were carried out in a 1-L high-pressure batch reactor with constant stirring (300 rpm) at 0.03–3.86 MPa. In general, oil extraction was greater for extruded soybean flakes than with soybean flakes. More complete oil extraction for extruded soybean flakes was achieved at around 150 °C and extraction was not affected by solids-to-liquid ratios over the range tested, while oil extraction from soybean flakes was more complete at 66 °C and low solids-to-liquid ratio (1:11.7). Protein extraction yields from flakes were generally greater than from extruded flakes. Protein extraction yields from extruded flakes increased as temperature increased and solids-to-liquid ratio decreased, while greater protein extraction yields from soybean flakes were achieved when using low temperatures and low solids-to-liquid ratio.     

Characterization of extruded-expelled soybean flours

In recent years there has been widespread growth in extruding-expelling (E-E) facilities for small-scale processing of soybeans. To compete in a highly competitive market, these E-E operations are looking for ways to optimize production of their oil and meal products for values to their customers. The objective of this study was to determine the ranges of residual oil contents and protein dispersibility indices (PDI) possible with E-E processing of soybeans. We also characterized the partially defatted meal for other factors important in food and feed applications. Residual oil and PDI values ranged from 4.7 to 12.7% and 12.5 to 69.1%, respectively. E-E conditions significantly influenced residual lipase, lipoxygenase (L1–L3), and trypsin inhibitor activities. Chemical compositions were different for whole, dehulled, and reduced-moisture soybeans, with dehulled soybeans tending to produce meals having higher residual oil contents at higher PDI values. It was possible to process soybeans with different characteristics (e.g., moisture content, whole, dehulled) to produce meals and flours with wide ranges of properties, providing E-E operators with opportunities to market value-added products.     

Functionality of soy protein produced by enzyme-assisted extraction

This study investigated the potential of enzymes to increase soy protein extractability without causing protein degradation. The aqueous extraction of protein was performed from defatted soy flakes on a laboratory-and pilot-plant scale. Yields of protein and reducing sugars were determined in the alkali extracts obtained with cellulases and pectinase, added alone or as cocktails. Using 5% (wt/g of protein) Multifect pectinase resulted in the best improvement of protein yields, which were 50 and 17% greater than the controls in laboratory- and pilot-plant-scale trials, respectively. This enhanced protein extraction was accompanied by an increased reducing sugar content in the aqueous extract compared with the control. Under the conditions tested, no enzyme cocktail markedly increased the protein yield compared with the use of single enzymes. The solubility curve for Multifect pectinase-treated soy protein isolate (SPI) was typical of SPI at pH 2–10. Its foam stability significantly improved, but the emulsification properties declined. Multifect pectinase markedly reduced the viscosity of SPI. SDS-PAGE showed that the α’ and α subunits of β-conglycinin were modified, and glycoprotein staining showed that these modifications were probably due to a protease secondary activity in the pectinase preparation. One cellulase and one pectinase were identified as effective in modifying the protein and reducing sugar extractablity from the defatted soy flakes.     

Oilseed pretreatment in connection with physical refining

Tests have shown that the nonhydratable phosphatides (NHP) arising by the action of phospholipases are not present in significant quantities in commercial soybeans, but that they are formed predominantly only during extraction. By a moisture-heat treatment of the soy flakes prior to the extraction, this enzyme activity can be almost completely eliminated so that, during the subsequent extraction, an enzymatic change of the oil no longer occurs. In comparison with the extraction of untreated soy flakes, the yield of soy lecithin is doubled; the lecithin has a higher content of phosphatidylchol ine; the crude, degummed soy oil has extraordinarily low NHP contents; and the soy meal tastes less bitter.     

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.     

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.