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.     

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.     

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.     

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.     

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.     

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.     

Extraction and Precipitation Chemistry for Mercury Recovery from Aqueous Wastes

Abstract

This paper describes an extraction-precipitation process to recover mercuric chloride from aqueous solutions. The process is a modification of a dicarboxylic acid recovery process that exploits the enhanced solubility of mercuric chloride in organic solvents loaded with water. Mercuric chloride in ethyl acetate has a solubility enhancement of 1.34 moles mercuric chloride per mole of water added to the solvent, consistent with previously reported phase behavior. Solubility enhancement is also reported for formates, in contrast to previously reported results. For example, propyl formate showed 2.91 moles mercuric chloride per mole water enhancement.

The modified process described in this paper relies on (1) favorable liquid-liquid equilibrium (mercuric chloride distribution into ethyl acetate or into propyl formate), (2) favorable liquid-solid equilibrium with enhanced solute solubility on addition of a minor constituent (water), and (3) preferential vaporization of the minor constituent from the solid slurry (relative volatility of water in mercuric chloride loaded ethyl acetate is four). Enhanced solubilities for zinc(II) chloride(3.46) and mercury(II) iodide(1.10) in isopentyl acetate indicate a potential application of the process for the recovery of other metal halides from aqueous wastes.     

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.     

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.     

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.     

Properties of Protein Isolates from Marine Hydrobionts Obtained by Isoelectric Solubilisation/Precipitation: Influence of Temperature and Processing Time

Protein isolates were obtained from marine hydrobionts by the method of isoelectric precipitation with a preliminary stage of protein alkaline solubilisation. Northern blue whiting was chosen as the raw material. Various technological modes of the solubilisation stage were used: the temperature of the reaction mixture was 4 or 20 °C, and the duration was 4 or 16 h. The yield of the product was 44–45% with a high content of the main component (protein) equal to about 95%. It has been shown that a decrease in the temperature and duration of the alkaline solubilisation stage provides the production of protein isolates with good technological properties, a low solubility, high swelling and high emulsifying ability, necessary for its use in the production of functional food products, including therapeutic and prophylactic effects. These technological properties are explained by a change in the composition and structure of the protein, the change being an increase in the content of essential amino acids and the proportion of α-helices in the polypeptide chain. The main patterns obtained will be used to obtain protein isolates from marine molluscs.     

Modeling of Nanofiltration Process for Solvent Recovery from Aqueous Ethanol Solution of Soybean Isoflavones

Abstract

In this paper, the feasibility of recovering the solvent from the aqueous ethanol solution of soybean isoflavones with nanofiltration (NF) was studied. Five commercially available polymeric NF membranes were employed and STARMEM? 122 showed acceptable flux and high retention. The central composite design (CCD) of the response surface methodology (RSM) was applied to model the effects of temperature, pressure, and feed concentration on the permeate flux and the total soybean isoflavone retention. The results indicate that the developed models were in good agreement with the experimental results and they can be used to predict this NF process.     

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.     

An Investigation of pH Mediated Extraction and Precipitation of Phosphorus from Sludge Using Microfiltration: Processing and Costs

Membrane filtration of wastewater sludge is a feasible technology for the recovery of sustainable nutrients in bacteria and particle-free solutions. Diafiltration and acidification strategies were investigated for the recovery of phosphorus (P) from trout farm sludge. Extraction costs were determined using a pilot-scale unit. pH of 2.7 was found to maximize the extraction of P (and metals) and highest concentrations were achieved by acidic leaching of the sludge sediment. Levels of P, Ca, Fe, Mg, and K recovered in soluble fractions were 418 mg/L, 6730 mg/L, 66.8 mg/L, 29 mg/L, and 34.1 mg/L, respectively. Operational costs associated with acidification and power consumption were estimated to be 0.018 kWh/L permeate or 0.037 kWh/g P obtained. The extraction of P by membrane filtration was mainly dependent on pH and cost was considerably lower than that for P-based fertilizers. Owing to the high concentration of Ca and P in the permeate fractions, 99% of the soluble P was precipitated from solution at pH 8. Elemental analysis and FTIR of the precipitate obtained indicated to be carbonated calcium-deficient hydroxyapatite.     

Recovery of Rhamnolipids Produced by Pseudomonas aeruginosa Using Acidic Precipitation,Extraction, and Adsorption on Activated Carbon

Biosurfactants are produced by microorganisms, especially those of the genus Pseudomonas. This study is concerned with the recovery of rhamnolipids produced by Pseudomonas aeruginosa P029-GVIIA, using molasses as substrate. A central compound design 2³ in triplicate at the central point was used to evaluate, the influence of the centrifugation time, the agitation speed, and the pH on the amount of rhamnolipids precipitated by HCl (2 N). A 2⁴ factorial design in triplicate at the central point was used to investigate the influence of the pH (3–10), temperature (30 to 50°C), the concentration of carbon (1–3% w/v), and the agitation speed (100–200 rpm) on the adsorption of rhamnolipids to activated carbon. The tests showed that the adsorption is governed particularly by the pH and the temperature, as well as by the temperature × pH interaction. A pseudo-first order kinetic model successfully fitted the data, showing that the adsorbent had the ability to adsorb approximately 17.16 mg of rhamnolipids/gram of activated carbon.     

Solvent Extraction and Related Studies on Silver Recovery from Aqueous Solutions

Abstract

The main research in the solvent extraction of silver is exhaustively reviewed, together with relevant data on the metal ion complexation properties in one phase. Such features as the transport of competitive cations through liquid membranes and selectivity in silver extraction are also reviewed. The behavior of organic ligands interacting with silver is analyzed on the basis of the atom type or types directly responsible for extraction/complexation processes.     

Nanofiltration Separation of Aqueous Polyethyleneglycol – Salt Mixtures

To investigate nanofiltration (NF) separation for recycling polyethylenglycol (PEG) from an ion partition process using an aqueous two-phase system, fractionation performance of five different NF membranes (NF270, SR3, SR100, SR2, and BW30) with solutions of NaNO₃, KClO₄, and PEG 4000 in water comprising various mixtures were studied. PEG rejections and salt passage were analyzed and explained based on size exclusion as well as electrostatic interactions. The highest permeate flux at high rejection of PEG as well as the lowest salt rejections were obtained with SR2 and NF270 membranes. Similar salt rejections were observed for mixed solute solutions and complex mixtures, all following this trend: SR3 > NF270 > SR2. The PEG rejections were well above 95%. This study also revealed that high salt passage of above 90% could be achieved with the same NF membrane only by unstirred conditions through concentration polarization mechanism; however, at the expense of low flux, especially with high PEG concentrations.     

应用膜技术精制荧光增白剂水溶液的工艺设计

本文对采用卷式纳滤膜作荧光增白剂水溶液的脱盐和浓缩的各种工艺过程进行了预测和比较。将含有1.0mol/L NaCI的0.14mol/L荧光增白剂水溶液浓缩到0.30mol/L再进行连续恒容脱盐,将NaCI浓度降低到0.102mol/L,然后将荧光增白剂水溶液和NaCI的浓度调节到0.25mol/L和0.085mol/L的目标浓度,这种方法所需的膜面积和排放水量都较小。工业运行试验表明该工艺与预测结果吻合良好。