Devin Maurer

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.

The effectiveness of soaking in aqueous ammonia (SAA) as a pretreatment method for the conversion of soybean fiber to ethanol via simultaneous saccharification and fermentation (SSF) was investigated. Insoluble fiber is a co‐product from oil and protein extraction using two‐stage, countercurrent, enzyme‐assisted, aqueous extraction processing of full‐fat soybean flakes (FFSF) and extruded FFSF. The fiber fractions were soaked in 15 wt% aqueous ammonia at 1:10 solid‐to‐liquid ratio. The effects of operating variables, including treatment times (6, 12, and 24 h), treatment temperatures (60 and 80 °C), and cellulase loadings (15 and 60 FPU/g‐glucan) on the degree of enzymatic hydrolysis were determined. The best SAA conditions were 80 °C for 12 h followed by an enzyme loading of 15 FPU/g‐glucan, which produced a 152‐mg/g glucose yield after 48 h of hydrolysis. This was 8.7 times the amount produced from the same fiber not pretreated with SAA. The glucose yield increased to 381 mg/g when fiber obtained from extruded FFSF was submitted to SAA. SAA (80 °C, 12 h) on extruded fiber subjected to SSF increased ethanol yield from 0.06 g of ethanol/g [40% of theoretical yield] (for non SAA pretreated fiber) to 0.25 g of ethanol/g [92% of theoretical yield]. The combination of extrusion and SAA was an efficient means for converting the fiber‐rich soybean fraction into ethanol.

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.

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.

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.