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.     

Quantity and Quality of Free Oil Recovered from Enzymatically Disrupted Soybean Oleosomes

The operational variables impacting the quantity and quality of free oil recovered from isolated soybean oleosomes by enzymatic extraction were evaluated to optimize this process. Those variables were: protease concentration (0–2.5%), protease hydrolysis time (3 vs. 18 h), and slurry destabilization time (30 min vs. 3 h). Analysis of interactions between these variables and the yield of free oil revealed that the protease concentration was the most significant variable. The quantity of free oil extracted by using 3 h of oleosomes hydrolysis and 30 min of slurry destabilization was not significantly different from that using 18 h of oleosomes hydrolysis and 3 h of slurry destabilization. The optimum conditions, 0.5% Protex 6L, 3 h of hydrolysis, and 30 min of destabilization, resulted in 90% free oil. Oils extracted by the aqueous process had a fatty acid composition similar to conventional hexane-based process with oxidative stability indices ranging from 9 to 12 h. Enzyme assisted aqueous extraction resulted in a high quality oil which has 88% less free fatty acids than hexane-extracted oil. The optimal conditions generated 85.5% soybean storage proteins in skim with peptides smaller than 6.5 kDa and the degree of hydrolysis of 19.5%. The present study demonstrates that oil can be extracted from soybeans efficiently without hexane or other petroleum solvents.     

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.     

The Isolation of Yellow Mustard Oil Using Water and Cyclic Ethers

An aqueous extraction process (AEP) was developed for dehulled yellow mustard flour with the aim of producing yellow mustard oil for industrial applications, as a by-product of food protein production. During AEP, most of the oil extracted was bound in a stable oil-in-water emulsion that must be destabilized to recover free oil. The oil distribution after aqueous extraction and the composition of the emulsion produced were determined. The emulsion was solubilized in organic solvents including tetrahydrofuran (THF) and 1,4-dioxane to fully recover the oil in a single-phase oil–solvent-water miscella. Over 97 and 95% of the oil in the emulsion was successfully recovered using 4:1 THF:oil and 9:1 dioxane:oil weight ratios, respectively. The oil recovery from the emulsion was optimized, based on experimentally prepared ternary phase diagrams of THF/oil/water and dioxane/oil/water. The results suggest that this technically viable approach can successfully recover essentially all of the oil from the emulsion, equivalent to an overall free oil recovery of ~63% from dehulled yellow mustard flour.     

Flaking and extrusion as mechanical treatments for enzyme-assisted aqueous extraction of oil from soybeans

Flaking and extruding dehulled soybeans were evaluated as a means of enhancing oil extraction efficiency during enzyme-assisted aqueous processing of soybeans. Cellulase, protease, and their combination were evaluated for effectiveness in achieving high oil extraction recovery from extruded flakes. Aqueous extraction of extruded full-fat soy flakes gave 68% recovery of the total available oil without using enzymes. A 0.5% wt/wt protease treatment after flaking and extruding dehulled soybeans increased oil extraction recovery to 88% of the total available oil. Flaking and extruding enhanced protease hydrolysis of proteins freeing more oil. Treating extruded flakes with cellulase, however, did not enhance oil extraction either alone or in combination with protease. Discrepancies in oil extraction recoveries were encountered when merely considering crude free fat because some oil became bound to denatured protein during extrusion and/or sample drying. Bound fat was unavailable for determination by using the hexane extraction method, but was accounted for by using the acid hydrolysis method for total oil determination. Oil extraction recovery from extruded soybean flakes was affected by oil determination methods, which was not the case for unextruded full-fat soy flour.     

Enzyme-Assisted Aqueous Extraction of Oil from Isolated Oleosomes of Soybean Flour

Enzyme-assisted aqueous extraction of oil from isolated soybean oleosomes was evaluated as an alternative to the conventional organic solvent extraction. Three different processes: hydrolysis of oleosomes, thermal demulsification of the skim or the slurry, and destabilization of the cream by the churning butter process were examined to enhance the release of free oil from isolated oleosomes. The oil extraction involved incubating the oleosomes with either 0, 2.5 or 5% protease (Protex 6L^®) at 60 °C, pH 9 for 18 h, destabilizing the slurry by three thermal strategies: freeze/thaw, freeze/thaw and heating, and destabilizing the cream by the churning butter process without and with 5% of phospholipase A₂ (Multifect L1 10L^®), at 40 °C, pH 8 for 4 h. The best total free oil yield was 83–88% by hydrolyzing oleosomes with 2.5 or 5% Protex 6L^®, destabilizing the slurries by heating and destabilizing the resulting cream by the churning butter process. The oleosomes treated with 2.5 and 5% proteases generated hydrolyzed soybean storage proteins at 18–20% degree of hydrolysis, with all the storage proteins hydrolyzed to peptides smaller than 6.5 kDa, compared to the oleosomes disrupted without proteases.     

Efficient and Response Surface Optimized Aqueous Enzymatic Extraction of Camellia oleifera (Tea Seed) Oil Facilitated by Concurrent Calcium Chloride Addition

This paper reports an efficient aqueous enzymatic extraction (AEE) method for Camellia oleifera seed oil with the aid of response surface analysis. A maximum oil recovery of ~93.5% was obtained when a 2‐step AEE process was performed using 0.80% cellulase (v/w) solution at pH 6.0 maintained at 50 °C for 1 h followed by a solution of 0.70% Alcalase® with pH 9.2 at 57 °C for 4.1 h. It was found that the addition of Ca²⁺ during the proteolysis stage improved the free oil yield from ~62.1 to ~86.6%. This was attributed to the removal of tea saponins, cross‐linkage of anionic polysaccharides, and destabilization of cream emulsion by Ca²⁺. This was verified by decreased tea saponin and polysaccharide levels in the cream emulsion and bulk solution as well as lowering of the emulsion fraction. It was determined that addition of CaCl₂ solution in continuous flow to the proteolysate is superior to one‐time or batch addition in inhibiting emulsion formation. The addition of CaCl₂ may provide a means of replacing the more laborious, time‐consuming demulsification process otherwise required.     

Influence of Coagulant Salt Addition on the Treatment of Oil‐in‐Water Emulsions by Centrifugation,Ultrafiltration, and Vacuum Evaporation

Abstract

Droplet size is a key factor in the treatment of oil‐in‐water (O/W) emulsions, because of its influence on emulsion properties. The addition of a coagulant salt generally causes emulsion destabilization, increasing the droplet size, and enhancing coalescence between oil droplets, which helps its further treatment. The influence of CaCl₂ addition on droplet size distribution of a commercial O/W emulsion used in machining processes was studied in order to facilitate oil removal and to improve its further treatment by centrifugation, ultrafiltration (UF) and vacuum evaporation. The critical coagulation concentration (CCC) was observed at a CaCl₂ concentration of 0.05 M. The quality of the final aqueous effluent, expressed as its chemical oxygen demand (COD) value, was compared for all treatments. The highest COD values were obtained for centrifugation, while the COD of the UF permeate was approximately constant for all UF trials. The best effluent quality was obtained by vacuum evaporation. A combination of these techniques should be appropriate for most industrial treatments of O/W emulsions, depending on the subsequent use of the resulting aqueous effluent.     

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.     

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.     

Influence of the vegetable oil refining process on free and esterified sterols

The influence of the refining process on the distribution of free and esterified phytosterols in corn, palm, and soybean oil was studied. Water degumming did not affect the phytosterol content or its composition. A slight increase in the content of free sterols was observed during acid degumming and bleaching due to acid-catalyzed hydrolysis of steryl esters. A significant reduction in the content of total sterols during neutralization was observed, which was attributed to a reduction in the free sterol fraction. Free sterols probably form micelles with soaps and are transferred into the soapstock. The steryl ester content remained constant during all neutralization experiments, indicating that hydrolysis of steryl esters did not take place during neutralization. During deodorization, free sterols are distilled from the oil, resulting in a gradual reduction in the total sterol content as a function of the deodorization temperature (220–260°C). A considerable increase in the steryl ester fraction was found during physical refining, probably owing to a heat-promoted esterification reaction between free sterols and FA.     

Pilot Plant Recovery of Soybean Oleosome Fractions by an Enzyme-Assisted Aqueous Process

An aqueous enzymatic procedure for oleosome fractionation from 25 g of soy flour was developed in our laboratory. This fractionation procedure was evaluated with 75 kg using pilot plant equipment to evaluate the effect of the scale-up on the recovery, proximate composition, soybean storage protein profiles, and subcellular microstructure of oleosome fractions. The process included enzymatic hydrolysis, grinding, and centrifugation, respectively. Pilot-scale grinding and centrifugation of the slurry were accomplished with a Stephan^® Microcut mill grinder and a three phase decanter. A blender and swinging bucket rotor were used for the laboratory-scale fractionation. The oleosome fractions recovered in the pilot plant were similar in oil and protein content to those obtained in the laboratory. The pilot-scale process resulted in a significantly higher oil yield of 93.40% as total oleosomes compared to that of 76.83% achieved in the laboratory. Urea–SDS gel electrophoresis of proteins extracted from the oleosomes and supernatant from the pilot-scale fractionation had similar profiles to those obtained in the laboratory. Electron microscopy verified that the structure of isolated oleosomes was virtually identical with that of in situ oleosomes. This work confirms that large-scale fractionation of oleosomes from full fat soybean flour can be accomplished.     

Influence of soybean protein isolates-phosphatidycholine interaction on the stability on oil-in-water emulsions

Soybean protein isolates and phospholipids present specific surface properties with synergistic or antagonistic effects on emulsion stability. Oil-in-water emulsions (25∶75 w/w) were prepared using native and denatured soybean isolates (NSI and DSI, respectively) with the addition of phosphatidylcholine (PC) (protein/PC ratio 100∶1 to 10∶1). The effect of ionic strength was also studied by adding sodium chloride (0–100 mM) to the aqueous phase. Analysis of NSI/PC and DSI/PC emulsions showed that the creaming rate diminished upon addition of PC, with the creamed phase showing more stability than those of the control systems. In DSI/PC systems, the coalescence process was partially controlled, as evidenced by a decrease in the size of oil droplets. Both systems were altered by the presence of sodium chloride, with an increase in the creaming rate attributable to flocculation and the coalescence of droplets. Under these conditions, DSI/PC emulsions exhibited a stronger protein-phospholipid interaction than those of NSI/PC.     

Physicochemical Characteristics and Composition of Indian Soybean Oil Deodorizer Distillate and the Recovery of Phytosterols

The deodoriser distillate (DOD) of Indian soybean oil obtained from two industries processing soybean oil was investigated for its physicochemical characteristics, its composition of tocopherols, phytosterols, fatty acids and recovery of phytosterols for use in nutraceutical products. It was found that the two DOD samples studied were dark in color and had higher amounts of free fatty acids (22.7 and 49.9%), unsaponifiable matter (11.8 and 21.9%) (5–10 times found in soybean oil), total tocopherols (1957–2256 mg/100 g) (20 times the amount in soybean oil), and 6–10% of phytosterols (12–20 times the soybean oil). The fatty acids found were palmitic (23.2–25.5%), stearic (1.4–2.4%), oleic (23.8–26.1%), linoleic (40.4–41.1%) and linolenic (2.7–3.2%) acids. The unsaponifiable matter (21.9%) and phytosterols (8.7%) content of DOD-2 were higher than in DOD-1 and hence was more suited for isolation of phytosterols. Using hexane and water for crystallisation, the DOD-2 yielded a phytosterol fraction with lower recovery of 13.2–17.8% while treatment with alkali to remove FFA and the glycerides followed by organic solvent extraction yielded unsaponifiable matter containing phytosterols with a recovery of 74.6%. Later the unsaponifiable matter was purified by double crystallisation into a mixture of phytosterols of 87% purity containing β-sitosterol (34.3%), stigmasterol (3.1%) and campesterol (50.1%). The product may find use in foods, pharmaceuticals, cosmetics and allied industries probably as a nutraceutical.     

Destabilization of Emulsion Formed During Aqueous Extraction of Peanut Oil: Synergistic Effect of Tween 20 and pH

To destabilize the emulsion formed during aqueous extraction processing (AEP) of peanuts, Tween and Span series surfactants (Tween 20, Tween 80, Span 20, and Span 80) were used alone or in combination to break the emulsion. Results indicate that only Tween surfactants had a pronounced demulsifying effect that was dependent on Tween concentration and system pH. When 1.2 wt% Tween 20 aqueous solution was used for oil extraction at pH 10.0, the highest free oil yield was achieved at 76.1 %, which was similar to the oil recovery of using proteases as a destabilization agent. The results obtained using a model emulsion system containing peanut oil and Tween 20/peanut protein isolates (PPI) showed that when Tween 20 and PPI coexisted in extraction medium at pH 10.0, the dynamic interfacial tension and droplet size distribution curves were very similar to those when Tween 20 was used alone, suggesting that Tween 20 dominated at the interface, instead of PPI. Destabilization of the model emulsions relied on three important factors: inclusion of Tween 20 at the initial mixing stage, high pH, and a gentle mixing speed. A synergistic destabilization mechanism of using Tween 20 at high pH during AEP was proposed. The discovery of Tween 20 as an effective demulsifier significantly contributes to the development of AEP of oilseeds.     

Insight into the Enzymatic Degumming Process of Soybean Oil

An enzymatic degumming trial of soybean oil was carried out at a capacity of 400 tons/day by applying microbial phospholipase A₁ from Thermomyces lanuginosus/Fusarium oxysporum. When the pH was kept in the range of 4.8–5.1, less than 10 mg/kg of phosphorous content of The oil was obtained. The gum and oil were easily separated after centrifugation and the oil loss was minimal under the process conditions. Through analysis of phospholipids compounds in the gum by Electrospray Ionization-Mass Spectrometer and phosphorous content, it could be seen that both glycerophospholipids and lysophospholipids existed with contents of 45.7 and 54.3%, respectively. The performance of enzymatic degumming was found to be related to the production of glycerophospholipids.     

Isolation and quantitative determination of sterol oxides in plant-based foods: Soybean oil and wheat flour

Soybean oil and wheat flour were analyzed for the content of sitosterol oxides. The method involved chromatography on a Lipidex-5000 column and enrichment on a disposable NH₂-column, yielding a sterol fraction and a sterol oxide fraction. Each fraction was separated as trimethylsilyl-ethers on a methyl silicone capillary column. Analysis of crude and freshly refined soybean oil showed no detectable levels of the isomeric 5,6-epoxysitosterols, the epimeric 7-hydroxysitosterols and 5,6-dihydroxysitosterol at the detection limit of 0.2 ppm. Storage of a refined soybean oil for one year at 4°C caused no significant increase in the level of free sitosterol oxides when compared to the freshly refined soybean oil. Analysis of three wheat flours (at 2, 8 and 36 months) revealed that the samples contained variable levels of 5α,6α-epoxysitosterol (5.4–55 ppm in the lipids), 5β,6β-epoxysitosterol (0.2–29 ppm), 7α-hydroxysitosterol (9.3–118 ppm) and 7β-hydroxysitosterol (9.7–126 ppm).     

Biodiesel Feedstock from Emulsions Produced by Aqueous Processing of Yellow Mustard

The multi-stage treatment of stable oil-in-water emulsions produced during non-enzymatic aqueous processing of dehulled yellow mustard flour with cyclic ethers [tetrahydrofuran (THF) and 1,4-dioxane] was investigated to produce a single-phase oil-solvent-water miscella suitable for biodiesel production. While the single-stage treatment of yellow mustard emulsion recovered 97 % and 95 % of the oil by using 4:1 THF:oil and 9:1 dioxane:oil weight ratios, respectively, miscella phases containing more than 7 % water formed, which made them unsuitable as biodiesel feedstock. Multi-stage treatments of the emulsion using lower THF:oil and dioxane:oil weight ratios were further developed to produce oil-solvent-water miscella phases with low water content. While three-stage extraction of emulsions using 0.5:1, 1:1, 1.5:1, and 2:1 dioxane:oil weight ratios did not destabilize the emulsion, three-stage extraction using 0.5:1 and 0.75:1 THF:oil weight ratios effectively recovered over 97 % of the oil, resulting in the production of oil-rich miscella phases containing only 1 % and 1.5 % water, respectively. These miscella phases were analyzed for free fatty acid and phosphorus contents and proved to be excellent feedstocks for the preparation of high-purity methyl esters through single-phase base-catalyzed transmethylation.     

Effects of soybean pretreatments on crude oil quality

The effects of soybean pretreatments, including infrared (IR) radiation, oven toasting, microwave heating and live steam treatment on crude oil quality were investigated. Free fatty acid, oxidation value, carbonyl value and tocopherol content were used to monitor crude soybean oil quality. All soybean pretreatments were effective in improving the quality of oils from 15 and 18% moisture beans. Based on the analyses, recommended treatments are 3–4 min for IR at 220V–250W; 1 min for microwave heating at 650 W–2450 mHz; 1–1.5 min for steam heating; and 100–120°C, 30 min for oven toasting. Heat treatment of high-moisture soybeans before extraction yielded crude oil with a lower content of phosphatidic acid as compared to that of the untreated beans.     

水酶法提取大豆油

The procedure of enzymatic aqueous extraction of soybean oil was assessed when two-step controlled enzymatic hydrolysis was applied. With aqueous extraction of soybean oil-containing protein, the highest yield of oil was 96.1% at the optimized conditions studied. Soybean oil-containing protein was hydrolyzed and resulted in releasing part of oil. The separated protein that contained 40% oil was enriched due to its adsorption capacity of released oil, the average oil extraction yeild reached 93.5%. Then the high oil content protein was hydrolyzed again to release oil by enzyme, the oil extraction yeild was 80.4%. As a result, high quality of soybean oil was obtained and the content of total oil vield was 74.4%.