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.     

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.     

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 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.     

Flaking as a Pretreatment for Enzyme-Assisted Aqueous Extraction Processing of Soybeans

Soybean moisture content (7.2–12.8%) and conditioning temperature (51–79 °C) during flaking were evaluated to determine their effects on oil and protein extraction and oil distribution among fractions produced in enzyme-assisted aqueous extraction processing (EAEP). Extractions were performed by using two-stage countercurrent EAEP at a 1:6 solids-to-liquid ratio with 0.5% protease (wt/g extruded flakes) at pH 9.0 and 50 °C for 1 h. Oil extraction improved when using soybeans with moisture contents ranging from 8.0 to 12.0% for flaking but was not affected by conditioning temperature. Oil extraction was reduced when moving away from 10% moisture with the lowest values at 7.2 and 12.8% moisture. Free oil extraction increased as soybean moisture content increased from 7.2 to 12.8% although total oil extraction was reduced at 12.8% moisture. Higher (79 °C) and lower conditioning temperatures (51 °C) improved free oil extraction and reduced cream emulsion formation. Skim oil content was not significantly affected by soybean moisture content and the conditioning temperature, although an undesirable high oil content in the skim was observed at 8% moisture and at 55 °C. The cream with a high oil yield was easily demulsified compared with cream containing a low oil yield (95 vs. 76.5% de-emulsification). Due to differences in cream stability, similar oil recoveries (78–80%) were obtained for treatments yielding creams with either low or high oil yields. Mean protein extraction of 95% was achieved for all treatments and was not significantly affected by soybean moisture content at flaking or conditioning temperature.     

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.     

Integration of Aqueous Two‐Phase Extraction into Downstream Processing

Aqueous two‐phase extraction (ATPE) is increasingly considered to be a feasible unit operation, e.g., for the capture of monoclonal antibodies or recombinant proteins. So far, knowledge on the applicability of ATPE in antibody processes has been collected mostly in lab‐scale. In contrast, approaches for the integration of ATPE into a downstream process are investigated. A complete process sequence including extraction, washing, ultrafiltration, and ion‐exchange chromatography is discussed and suggested for antibody purification. Excellent antibody purities can be achieved. Additionally, a model is applied that allows early‐on prediction of a multistage ATPE with high prediction accuracy. Finally, an economic evaluation between ATPE and Protein A chromatography is performed, reaching up to five‐fold cost‐saving factors.     

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.     

Comparison of Lipid Extraction from Microalgae and Soybeans with Aqueous Isopropanol

The extraction efficiency of microalgae lipids with aqueous isopropanol (IPA) was investigated and compared with the extraction of oil from full-fat soy flour. The effects of the type of microalgae (Scenedesmus sp. and Schizochytrium limacinum), cell rupture, and IPA concentration on the yield of oil and non-lipid biomass were determined. The oil yield from intact cells of Scenedesmus was 86–93 % with 70, 88, or 95 % (by wt) IPA. Ultrasonic cell rupture prior to oil extraction decreased the oil yield of Scenedesmus to 74 % when extracting with 70 % IPA. The oil yield from intact cells of S. limacinum was <23 % regardless of the IPA concentration, but ruptured cells gave a 94–96 % oil yield with 88 or 95 % IPA. The different response of the two microalgae to extraction with IPA is possibly caused by differences in the cell wall structure and type and amount of polar lipids. The oil yield from soy flour with 88 and 95 % IPA was 93–95 %, which was significantly greater than yields with 50 and 70 % IPA. Cell rupture had no effect on soy flour extraction. In general, the oil yield from the ruptured cells of both microalgae and soy flour increased with increasing IPA concentration.     

Advances in Aqueous Extraction Processing of Soybeans

Aqueous extraction processing technologies, having advanced in recent years, may be a viable alternative to hexane extraction to separate oil and protein from soybeans. Different extraction strategies incorporating various modes of comminution, extraction buffers, and enzymes allow production of a range of oil and protein products, but also create different processing challenges. Processes capable of achieving high free oil yields often result in a soluble protein fraction difficult to isolate and dilute oil emulsions difficult to break. Other processes can achieve high yields and purities of native soy protein, but with reduced free oil yield or require a high osmotic and ionic strength extraction buffer. This review article discusses these various advanced processes and their relative advantages and disadvantages. In addition, the current understanding of the underlying fundamental concepts of aqueous extraction is discussed in order to help direct future investigations to improve these technologies.     

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.     

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.     

Development and Scale-up of Aqueous Surfactant-Assisted Extraction of Canola Oil for Use as Biodiesel Feedstock

Aqueous surfactant-assisted extraction (ASE) has been proposed as an alternative to n-hexane for extraction of vegetable oil; however, the use of inexpensive surfactants such as sodium dodecyl sulfate (SDS) and the effect of ASE on the quality of biodiesel from the oil are not well understood. Therefore, the effects on total oil extraction efficiency of surfactant concentration, extraction time, oilseed to liquid ratio and other parameters were evaluated using ASE with ground canola and SDS in aqueous solution. The highest total oil extraction efficiency was 80 %, and was achieved using 0.02 M SDS at 20 °C, solid–liquid ratio 1:10 (g:mL), 1,000 rpm stirring speed and 45 min contact time. Applying triple extraction with three stages reduced the amount of SDS solution needed by 50 %. The ASE method was scaled up to extract 300 g of ground canola using the best combination of extraction conditions as described above. The extracted oil from the scale-up of the ASE method passed the recommendation for biodiesel feedstock quality with respect to water content, acid value and phosphorous content. Water content, kinematic viscosity, acid value and oxidative stability index of ASE biodiesel were within the ASTM D6751 biodiesel standards.     

Simplex-Centroid Mixture Design Applied to the Aqueous Enzymatic Extraction of Fatty Acid-Balanced Oil from Mixed Seeds

A one-step method was developed to extract oil from a mixture of soybeans, peanuts, linseeds, and tea seeds using an aqueous enzymatic method. The proportion of the four seeds was targeted in accordance with a fatty acid ratio of 0.27 (SFA, saturated fatty acid(s)): 1 (MUFA, monounsaturated fatty acid(s)): 1 (PUFA, polyunsaturated fatty acid(s)), and the oil extraction yield was maximized by applying the simplex-centroid mixture design method. Three models were developed for describing the relationship between the proportion of the individual seeds in the mixture, the fatty acid ratio in the extracted oil, and the oil extraction yield, respectively. The developed models were then analyzed using an ANOVA and were found to fit the data quite well, with R ² values of 0.98, 0.93, and 0.93, respectively. The three models were validated experimentally. The results indicated that the ratio of fatty acids in the oil ranged between 0.98 and 1.12 (MUFA:PUFA) and between 0.26 and 0.28 (SFA:MUFA), which were quite close to the target values of 1 and 0.27, respectively. The oil extraction yield of 62.13 % was slightly higher than the predicted value (60.32 %).     

Optimization of the Aqueous Enzymatic Extraction of Oil from Iranian Wild Almond

The seeds of wild almond, Amygdalus scoparia, contain a relatively high quantity of oil. In the current study, aqueous enzymatic extraction of the oil from Iranian wild almond was investigated using a protease and a cellulase to assist the extraction process. The effects of temperature, incubation time and pH on the oil recovery were evaluated using Box?Behnken design from response surface methodology (RSM). A 77.3 % recovery was predicted for oil using aqueous enzymatic extraction procedure at the optimized conditions of RSM (pH 5.76; 50 °C/5 h) when both enzymes were used at 1.0 % level (v/w). In practice, when both enzymes were used, a maximum of 77.8 % oil recovery was achieved at pH 5; 50 °C/4 h. Fatty acid profile, refractive index and saponification value of the aqueous enzymatic extracted oil in the current study were similar to those of the oil extracted with hexane. However, acid value, unsaponifiable matter and p‐anisidine value were higher when compared to those with hexane extracted oil. Peroxide value of the aqueous enzymatic oil was lower than that of oil extracted by hexane. Aqueous enzymatic extraction can be suggested as an environmentally‐friendly method to obtain oil from wild almond.     

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.     

Destabilization of the Emulsion Formed during Aqueous Extraction of Soybean Oil

Characterization and destabilization of the emulsion formed during aqueous extraction of oil from soybean flour were investigated. This emulsion was collected as a cream layer and was subjected to various single and combined treatments, including thermal treatments and enzymatic treatments, aimed at recovery of free oil. The soybean oil emulsion formed during the aqueous extraction processing of full fat flour contains high molecular weight glycinin and β-conglycinin proteins and smaller oleosin proteins, which form a multilayer interface. Heat treatment alone did not modify the free oil recovery but freeze–thaw treatment increased the oil yield from 3 to 22%. After enzymatic treatment of the emulsion, its mean droplet size changed from 5 to 14 μm and the oil recovery increased to 23%. This increase could be attributed to the removal (due to enzymatic hydrolysis) of large molecular weight polypeptides from the emulsion interface, resulting in partial emulsion destabilization. When enzymatic treatment was followed by a freeze–thaw step, the oil recovery increased to 46%. This result can be attributed to the thinner interfacial membrane after enzymatic hydrolysis, partial coalescence during freeze–thaw, and coalescence during centrifugation. Despite the reduction in emulsion stability achieved, additional demulsification approaches need to be pursued to obtain an acceptably high conversion to free oil.