Lawrence A. Johnson

Lunasin has demonstrated antioxidative, anti-inflammatory, and chemopreventive properties. The objectives were to evaluate the concentration of lunasin in different lunasin-based commercial dietary supplements, to produce a lunasin-enriched soy extract (LESE) using a two-step pilot-plant-based ultrafiltration process, and to evaluate their biological potential in vitro. LESE was produced using 30 and 1 kDa membranes in a custom-made ultrafiltration skid. Lunasin was quantified in eight products and LESE. Lunasin concentrations of the lunasin-based products ranged from 9.2 ± 0.6 to 25.7 ± 1.1 mg lunasin/g protein. The LESE extract contained 58.2 mg lunasin/g protein, up to 6.3-fold higher lunasin enrichment than lunasin-based dietary supplements. Antioxidant capacity ranged from 121.5 mmol Trolox equivalents (TE)/g in Now^® Kids to 354.4 mmol TE/g in LESE. Histone acetyltransferase (HAT) inhibition ranged from 5.3% on Soy Sentials^® to 38.3% on synthetic lunasin. ORAC and lunasin concentrations were positively correlated, and HAT and lunasin concentrations were negatively correlated (p < 0.05). Melanoma B16-F10 and A375 cells treated with lunasin showed dose-dependent inhibitory potential (IC₅₀ equivalent to 330 and 370 μM lunasin, respectively). Lunasin showed protein kinase B expression (57 ± 14%) compared to the control (100%) in B16-F10. Lunasin concentration found in commercial products and lunasin-enriched soy extract could exert benefits to consumers.

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.

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.

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.

Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89–96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.

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.

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.

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.

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.

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127−139 and 138−155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.