Enzyme-assisted Aqueous Extraction Processing Research Articles

The Integral and Extrinsic Bioactive Proteins in the Aqueous Extracted Soybean Oil Bodies

Soybean oil bodies (OBs), naturally pre-emulsified soybean oil, have been examined by many researchers owing to their great potential utilizations in food, cosmetics, pharmaceutical, and other applications requiring stable oil-in-water emulsions. This study was the first time to confirm that lectin, Gly m Bd 28K (Bd 28K, one soybean allergenic protein), Kunitz trypsin inhibitor (KTI), and Bowman-Birk inhibitor (BBI) were not contained in the extracted soybean OBs even by neutral pH aqueous extraction. It was clarified that the well-known Gly m Bd 30K (Bd 30K), another soybean allergenic protein, was strongly bound to soybean OBs through a disulfide bond with 24 kDa oleosin. One steroleosin isoform (41 kDa) and two caleosin isoforms (27 kDa, 29 kDa), the integral bioactive proteins, were confirmed for the first time in soybean OBs, and a considerable amount of calcium, necessary for the biological activities of caleosin, was strongly bound to OBs. Unexpectedly, it was found that 24 kDa and 18 kDa oleosins could be hydrolyzed by an unknown soybean endoprotease in the extracted soybean OBs, which might give some hints for improving the enzyme-assisted aqueous extraction processing of soybean free oil.

Oxidative stability of soybean oil in oleosomes as affected by pH and iron

The oxidative stability of oil in soybean oleosomes, isolated using the Enzyme-Assisted Aqueous Extraction Process (EAEP), was evaluated. The effects of ferric chloride, at two concentration levels (100 and 500μM), on lipid oxidation, was examined under pH 2 and 7. The peroxide value (PV) and thiobarbituric acid-reactive substance (TBARS) value of oil, in oleosome suspensions stored at 60°C, were measured over a 12day period. The presence of ferric chloride significantly (P<0.05) affected the oxidative stability of oil in the isolated oleosome, as measured by the PV and TBARS. Greater lipid oxidation occurred under an acidic pH. In the pH 7 samples, the positively charged transition metals were strongly attracted to the negatively charged droplets. However, the low ζ-potential and the high creaming rate at this pH, may have limited the oxidation. Freezing, freeze–drying or heating of oleosomes have an insignificant impact on the oxidative stability of oil in isolated soybean oleosomes. Manufacturers should be cautious when adding oleosomes as ingredients in food systems containing transition metal ions.

Characteristics of Oil and Skim in Enzyme‐Assisted Aqueous Extraction of Soybeans

AbstractThe 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 &lt;20 kDa MW molecules. About 71 % of the skim peptides were &lt;20 kDa MW, with 49 % being &lt;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.

Enzyme-Assisted Aqueous Extraction of Lipid from Microalgae

An improved lipid extraction process has been established for microalgal using enzyme-assisted aqueous extraction processing (EAEP), which mainly involved in sonication and enzyme treatment. As compared to cellulase, neutral protease and alkaline protease, significantly higher lipid recovery was achieved by snailase and trypsin. The highest lipid recovery of 49.82% was obtained by a combined sonication-enzyme treatment at pH 4. The enhancement mechanism of the EAEP was analyzed in terms of the particle size of cream and zeta potential. In addition, microalgal lipid recovery was also affected by lipid class composition and the type of algae. The present study demonstrates a promising alternative to conventional lipid extraction of microalgae and the quantitative information on EAEP of oleaginous alga can provide valuable data for process design at pilot and industrial scale.

Combined effect of ohmic heating and enzyme assisted aqueous extraction process on soy oil recovery.

This research describes a new technological process for soybean oil extraction. The process deals with the combined effect of ohmic heating and enzyme assisted aqueous oil extraction process (EAEP) on enhancement of oil recovery from soybean seed. The experimental process consisted of following basic steps, namely, dehulling, wet grinding, enzymatic treatment, ohmic heating, aqueous extraction and centrifugation. The effect of ohmic heating parameters namely electric field strength (EFS), end point temperature (EPT) and holding time (HT) on aqueous oil extraction process were investigated. Three levels of electric field strength (i.e. OH600V, OH750V and OH900V), 3 levels of end point temperature (i.e. 70, 80 and 90°C) and 3 levels of holding time (i.e. 0, 5 and 10min.) were taken as independent variables using full factorial design. Percentage oil recovery from soybean by EAEP alone and EAEP coupled with ohmic heating were 53.12% and 56.86% to 73% respectively. The maximum oil recovery (73%) was obtained when the sample was heated and maintained at 90°C using electric field strength of OH600V for a holding time of 10min. The free fatty acid (FFA) of the extracted oil (i.e. in range of 0.97 to 1.29%) was within the acceptable limit of 3% (oleic acid) and 0.5-3% prescribed respectively by PFA and BIS.

Lunasin and Bowman-Birk Protease Inhibitor Concentrations of Protein Extracts from Enzyme-Assisted Aqueous Extraction of Soybeans

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.

The Research on Freeze-Thaw De-Emulsification Technology in Enzyme-Assisted Aqueous Extraction Processing

Experiments were carried out to analysis optimization of freeze-thaw regression model with Response Surface Analysis Method. The optimal response result is: freeze temperature: -18°C, freeze time: 12.5h, thaw temperature: 57°C, released oil yield: 93.16%, total free oil yield rate: 89.28%. The mechanism of demulsification was studied using Micro-imaging, SEM cooling stage, DSC, FTIR spectrometer. DSSP was changed and part of hydrogen bond was broken and α－helix structure conversed to random coil during the freeze process were found the effective freeze-thaw method to destabilize the emulsion.

Protein Recovery in Aqueous Extraction Processing of Soybeans Using Isoelectric Precipitation and Nanofiltration

AbstractIsoelectric 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.

Efficiency of pretreatments for optimal enzymatic saccharification of soybean fiber

The effectiveness of several pretreatments [high-power ultrasound, sulfuric acid (H2SO4), sodium hydroxide (NaOH), and ammonium hydroxide (NH3OH)] to enhance glucose production from insoluble fractions recovered from enzyme-assisted aqueous extraction processing of extruded full-fat soybean flakes (FFSF) was investigated. Sonication of the insoluble fraction at 144μmpp (peak-to-peak) for 30 and 60s did not improve the saccharification yield. The solid fractions recovered after pretreatment with H2SO4 [1% (w/w), 90°C, 1.5h], NaOH [15% (w/w), 65°C, 17h], and NH3OH [15% (w/w), 65°C, 17h] showed significant lignin degradation, i.e., 81.9%, 71.2%, and 75.4%, respectively, when compared to the control (7.4%). NH3OH pretreatment resulted in the highest saccharification yield (63%) after 48h of enzymatic saccharification. A treatment combining the extraction and saccharification steps and applied directly to the extruded FFSF, where oil extraction yield and saccharification yield reached 98% and 43%, respectively, was identified.

Integrated Countercurrent Two‐Stage Extraction and Cream Demulsification in Enzyme‐Assisted Aqueous Extraction of Soybeans

AbstractCountercurrent 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.

Microbial Fermentation Simultaneous Extraction Processing of Oil and Protein from Soybean

Although enzyme-assisted aqueous extraction processing (EAEP) is an environmentally friendly alternative technology to hexane extraction of soybean oil，it’s application is limited by enzyme price. This processing employs microbial fermentation to extract oil and protein from a variety of pretreatment full fat soy flours, and optimizes the fermentation conditions using the Response Surface Methodology (RSM). The results showed that extrusion combination with wet- thermal treatment were beneficial to improving oil and protein extraction, and total oil extraction yield achieve the highest value 95.1% at 7.4% extruded full fat soy flour, 5.4% inoculation volume, the initial pH8.4. At this condition, protein extraction yield reached to 86.1%. These advances make the microbial fermentation extraction processing attractive channel as the front-end of a soybean biorefinery.

Effect of Extruding Parameters on Soybean Oil Yield of Enzyme-Assisted Aqueous Extraction Processing

Experiments were carried out to investigate effects of extruding parameters on soybean oil yield extracted with enzyme-assisted aqueous extraction. It was found that extrusion and expansion parameters affected significantly on total soy oil yield. Optimal extrusion and expansion parameters for highest total soy oil yield were obtained using response surface methodology. Effect of extrusion and expansion pretreatment on soy cells was studied using light microscope and transmissive microscope. State of free oil and protein during hydrolysis was observed using light microscope. Ultrasonic assistant ethanol de-emulsification was found the effective method to destabilize the emulsion.

Aqueous enzymatic sesame oil and protein extraction

In the present work we evaluated five enzyme-mixtures (Protex 7L, Alcalase 2.4L, Viscozyme L, Natuzyme, and Kemzyme) for their effectiveness in extracting the oil and protein recovery from sesame seeds during an enzyme-assisted aqueous extraction (EAAE) process. Alcalase 2.4L was found to be the best for attaining a high oil yield (57.4% of the total oil content in the seed), whereas, the maximum amount of protein (87.1% of the total seed protein), was recovered in the aqueous phase with Protex 7L. The quality attributes such as fatty acids profile, density, refractive index, free fatty acid contents, iodine value, colour, saponification number and unsaponifiable matter of the sesame oil, extracted by aqueous enzymatic process, were comparable with that of the control (oil extracted without enzyme treatment) and hexane-extracted oil (HEO), revealing no significant ( p > 0.05) variations among oils, produced by either of the methods. The oxidative stability state of the enzyme-extracted oil (EEO) was noted to be considerably improved relative to the control and HEO. The amount of tocopherols for the oils, produced by the enzyme–adjuvant was found to be higher than the control and HEO. An appreciable increase in the antioxidant activity as assessed by determinations of total phenolic contents, DPPH radical scavenging capacity, and inhibition of linoleic acid oxidation of EEO was also established. Overall, the present results revealed improvement in the quality of the EEO while a major portion of the food grade protein was also extracted in the aqueous phase.

Comparison and optimization of enzymatic saccharification of soybean fibers recovered from aqueous extractions

Soybean insoluble fractions recovered from aqueous extraction processing (AEP) and enzyme-assisted AEP (EAEP) of full-fat soybean flakes (FFSF) and extruded FFSF were evaluated as a feedstock for the production of fermentable sugars using enzymes. Among the four insoluble fractions (AEP FFSF, EAEP FFSF, AEP extruded FFSF and EAEP extruded FFSF), the composition analysis revealed that the one recovered from EAEP of extruded FFSF had the highest glucan content, 16% [dry basis (db)], as compared to about 10% (db) for the other fractions. Thirty-three percent of the initial glucan of the insoluble recovered from AEP and EAEP of FFSF were converted into glucose using 33 FPU of Accellerase 1000/g-glucan. This saccharification yield was increased to 44% with extruded fibers. The higher saccharification yield of 49% was obtained at 45 °C, 1% glucan loading, and 101 FPU/g-glucan enzymes loading after 27 h of hydrolysis.

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

AbstractSoybean 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.

AQUEOUS EXTRACTION OF OIL AND PROTEIN FROM SOYBEAN AND LUPIN: A COMPARATIVE STUDY

ABSTRACT This study compares the effects of extrusion pretreatment and protease addition during aqueous extraction processing (AEP) of soybean and lupin flakes. AEP of flakes resulted in the lowest yields of oil (56%), protein (71%) and [cream + free oil] (8%) for soybean, while for lupin, yields were 48, 69 and 2%, respectively. AEP protein extraction yields were decreased by extrusion pretreatment, but this pretreatment improved enzymatic action, increasing protein extractability from soybean and lupin by 47 and 26%, respectively. For both protein crops, enzyme-assisted AEP (EAEP) of extruded flakes yielded the highest oil, protein and [cream + free oil] yields, which were 96, 85 21%, respectively, for soybean. Yields for lupin were 81, 77 and 10%, respectively. Extrusion followed by enzyme addition positively impacted demulsification yield, the creams from EAEP of soybean and lupin extruded flakes being the less stable toward enzymatic demulsification. PRACTICAL APPLICATIONS The vegetable oil industry is looking for alternatives to the traditional solvent extraction of oil-bearing seeds, and there is a need to increase the inefficient conventional aqueous extraction of protein from residual defatted meal, a by-product of the oil extraction process. The concept of enzyme-assisted aqueous extraction processing (EAEP) has been successfully developed for extruded soybean material, but its efficiency on other oil-bearing seeds still needs to be demonstrated. By determining the oil and protein extraction yields recovered during EAEP of extruded lupin flakes, the feasibility of transferring this process from soybean to other oilseeds will be established.

Factors affecting emulsion stability and quality of oil recovered from enzyme-assisted aqueous extraction of soybeans

The objectives of the present study were to assess how the stability of the emulsion recovered from aqueous extraction processing of soybeans was affected by characteristics of the starting material and extraction and demulsification conditions. Adding endopeptidase Protex 6L during enzyme-assisted aqueous extraction processing (EAEP) of extruded soybean flakes was vital to obtaining emulsions that were easily demulsified with enzymes. Adding salt (up to 1.5 mM NaCl or MgCl 2) during extraction and storing extruded flakes before extraction at 4 and 30 °C for up to 3 months did not affect the stabilities of emulsions recovered from EAEP of soy flour, flakes and extruded flakes. After demulsification, highest free oil yield was obtained with EAEP of extruded flakes, followed by flour and then flakes. The same protease used for the extraction step was used to demulsify the EAEP cream emulsion from extruded full-fat soy flakes at concentrations ranging from 0.03% to 2.50% w/w, incubation times ranging from 2 to 90 min, and temperatures of 25, 50 or 65 °C. Highest free oil recoveries were achieved at high enzyme concentrations, mild temperatures, and short incubation times. Both the nature of enzyme (i.e., protease and phospholipase), added alone or as a cocktail, concentration of enzymes (0.5% vs. 2.5%) and incubation time (1 vs. 3 h), use during the extraction step, and nature of enzyme added for demulsifying affected free oil yield. The free oil recovered from EAEP of extruded flakes contained less phosphorus compared with conventional hexane-extracted oil. The present study identified conditions rendering the emulsion less stable, which is critical to increasing free oil yield recovered during EAEP of soybeans, an environmentally friendly alternative processing method to hexane extraction.

Scale‐up of Enzyme‐Assisted Aqueous Extraction Processing of Soybeans

AbstractThe 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.

Destabilization of the emulsion formed during the enzyme-assisted aqueous extraction of oil from soybean flour

Soybean oil extraction from soybean flour by a neutral metallo-endopeptidase enzyme-assisted aqueous extraction process, optimized for effectiveness in reducing oil content of the solid residue, yields a small fraction of the oil as free oil whereas most is emulsified in a cream layer. Analysis of the cream showed the presence of peptides/proteins and phospholipids, either of which could serve as emulsifiers. Several demulsification treatments targeting these components – protease addition, phospholipase addition, and acidification (pH 4.5) – were evaluated for effectiveness against the targeted emulsifiers and in conversion of the cream to free oil. Acidification increased the oil yield from 2% to 83%. After a two-stage enzymatic demulsification process with an alkaline endopeptidase, the oil recovery increased to 95%. A lysophospholipase A 1 treatment at pH 4.5 provided complete conversion of emulsified to free oil. The phospholipids (PL) present in the original and post-treatment cream were quantified using phosphorus-31 nuclear magnetic resonance spectroscopy. PL contained phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and their lyso-derivatives. Protein size distribution, PL proportions and fatty acid composition changed after the demulsification treatments. Recycle of the endopeptidase is feasible as more than 90% of its activity was retained.

Low temperature dry extrusion and high-pressure processing prior to enzyme-assisted aqueous extraction of full fat soybean flakes

Oil, protein and solid extraction yields obtained during aqueous extraction processing (AEP) of full fat soybean flakes (FFSF), FFSF extruded at a die temperature of 100 °C and FFSF pressurised at 200 and 500 MPa for 15 min at 25 °C, were compared to those obtained during enzyme-assisted aqueous extraction processing (EAEP) using 0.5% of protease Protex 7L. Without enzyme addition, pretreatment of the FFSF with extrusion and 500 MPa increased and decreased, respectively, the oil extraction yield while protein extraction yield was significantly decreased after both treatments. The best treatment in terms of oil and protein recovery was EAEP of extruded flakes with 90% and 82% of oil and protein extraction yield, respectively, and 17% of free oil. Addition of protease during extraction significantly decreased the yield of isolated soy protein (ISP) due to an increased solubility of the proteins at pH 4.5. ISP from extruded EAEP had higher solubility at pH 7.0 and better functionality. The DSC results, combined with the protein extraction yields, showed that a proportion of the proteins became insoluble after extrusion and 500 MPa treatment, while only those extracted from 500 MPa FFSF had a reduced native state.