Enzyme-assisted Aqueous Extraction Research Articles

Economic Feasibility of Soybean Oil Production by Enzyme-Assisted Aqueous Extraction Processing

Aqueous oil extraction is an approach that could replace organic solvent extraction with water. Compared to typical solvent extraction and mechanical pressing processes, aqueous extraction has higher oil recovery (over 80%) than the mechanical pressing process, and resolve issues resulted from chemical loading and remaining in the hexane extraction. Proteases are used to assist free oil release from oil bodies by hydrolyzing cotyledon cell walls in aqueous extraction process. The resulting enzyme-assisted aqueous extraction process (EAEP) includes dehulling, flaking, extruding, enzymatic extraction, and enzymatic demulsification processes. SuperPro Designer was used to conduct a techno-economic analysis (TEA) of the extraction process. The total capital investment, operation cost, and profits were evaluated. During EAEP, insolubility of water and oil allows the simultaneous extraction of protein and oil. This decreases operation costs, especially the oil purification process, and therefore increases profits made from the main product (soybean oil). This simultaneous extraction also increases the profit towards the coproduct, i.e., protein in skim. Additionally, the absence of chemical and enzyme recycling contribute to the better economic value of EAEP. Despite the increase in facility costs due to extraction and demulsification units, the value-added coproduct extraction and high free oil yield contribute to the economic feasibility of EAEP in industrial- and commercial-scale productions when skim and insoluble fiber are used as water and carbohydrate supplies for integrated soy/corn ethanol biorefinery processing.

Effect of Enzymatic Aqueous Extraction on Quality Characteristics of Maize Germ Oil During Storage

Enzyme-assisted aqueous extraction (EAAE) method is among the emerging technologies and eco-friendly process for oil extraction. The aim of this study was to evaluate the effect of oil extraction methods and packaging material on shelf life of maize germ oil. Maize germ oil extraction was done by EAAE and solvent extraction method. HDPE and glass bottles were used as packaging materials. Quality characteristics evaluated were acid number, pH value, total phenolics content, colour change, unsaponifiable matter, saponification value, peroxide value and density at 15-day intervals for 90 days of storage period. The quality characteristics of oil changed with the passage of storage periods, and maximum change upto 190 % increase in peroxide value occurred when oil was solvent extracted and stored in HDPE bottle. The quality parameters of EAAE extracted oil were superior to solvent extracted oil. During storage acid number, pH, total phenolics, unsaponifiable matter and peroxide value of oil were significantly affected (P< 0.05) by packaging material, storage period and method of oil extraction.

Simultaneous extraction and enrichment of polyphenol and lutein from marigold (Tagetes erecta L.) flower by an enzyme-assisted ethanol/ammonium sulfate system.

Enzyme-assisted aqueous two-phase extraction (EA-ATPE) using ethanol/ammonium sulfate system was investigated for total polyphenol (TP) and lutein from marigold flowers. The important factors were investigated by single factor experiment and response surface methodology combined with Box-Behnken design to optimize the operating parameters of EA-ATPE. The maximum yields of TP and lutein were 83.56 ± 0.69 mg g-1 and 5.59 ± 0.13 mg g-1, respectively. Compared with aqueous two-phase extraction and Soxhlet extraction (SE), the extraction yield of TP by EA-ATPE is 64.91% higher and the extract of EA-ATPE has better antioxidant activity. The pretreatment effect was also researched by scanning electron microscopy. Thus, EA-ATPE is an efficient method for extracting bioactive components from plants.

Enzyme-assisted Aqueous Extraction of Oil from Rice Germ and its Physicochemical Properties and Antioxidant Activity

Enzyme-assisted aqueous extraction of rice germ oil (RGO) was performed in this study. The physicochemical properties, fatty acid composition, bioactive substances and antioxidant activity of RGO were analyzed. An enzyme composed of alcalase and cellulase (1:1, w/w) was found to be the most effective in the extraction yield of oil. The optimal oil yield of 22.27% was achieved under the conditions of an enzyme concentration of 2% (w/w), incubation time of 5 h, incubation temperature of 50°C, water to seed ratio of 5:1, and pH 6.0. The predominant fatty acids of RGO were oleic acid (39.60%), linoleic acid (34.20%) and palmitic acid (20.10%). The total saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) composition of RGO were 22.50%, 39.60% and 36.00%, respectively. RGO yielded a high content of γ-oryzanol (530 mg/100 g oil), tocotrienol (62.96 mg/100 g oil), tocopherol (23.24 mg/100 g oil) and a significant amount of phytosterol (372.14 mg/100 g oil). It exhibited notable antioxidant activities with IC50 values of 32.37 and 41.13 mg/mL, according to the DPPH radical scavenging assay and β-carotene/linoleic acid bleaching test, respectively.

Enzyme-Assisted Aqueous Oil Extraction from High Oleic Sunflower Seeds in a Scalable Prototype Reactor

Enzyme assisted aqueous extraction processing (EAEP) facilitates the production of high quality oils without the need for chemical refining. However, up to date, oil recovery remains remarkably below that found in mechanical and solvent extraction. In this study, response surface methodology (RSM) was applied, to optimize oil extraction from high oleic sunflower seeds via EAEP in a prototype automatic continuous stirred-tank extractor. Temperature, enzyme mass, water mass and proton concentration were varied for the exploration of optimum process conditions. The fit of a full second-order model was significant (p = 0.003), while the lack of fit was non-significant (p = 0.111). A local maximum was found at an extraction efficiency of 82.18%. A subsequent mass balance succeeded in reproducing the results from the RSM optimization. 93.03 ± 2.80% of the initial total mass was recovered, of which 90.90 ± 4.3% could be related to the components of the fractions.

Physical-Chemical Properties of Edible Film Made from Soybean Residue and Citric Acid

The effect of citric acid on the properties of soybean enzyme-assisted aqueous extraction processing (EAEP) residue edible film was studied. The washed soybean EAEP residue was produced by the process of EAEP. It was determined that the washed soybean EAEP residue is rich in fibers (76.10 ± 1.03%) and has lower oil and protein contents (7.74 ± 0.11% and 3.50 ± 0.20%, resp.). Edible films intended for food packaging have been produced from the washed EAEP residue combined with glycerol, different concentrations of citric acid (0%, 10%, 15%, 20%, 25%, and 30%), and sodium hypophosphite. The spectra have evidenced that the cross-linking reaction of citric acid and fibers has taken place in the residue. 30-CA films showed the highest tensile strength (17.52 MPa) and the lowest water vapor permeability (7.21 g·cm−1·s−1·Pa−1). Also, it indicated that citric acid can cross-link with the hydroxyls of polysaccharide and improve the compatibilization between the polymeric molecules to improve the intermolecular interaction between polysaccharide molecules, so that the water uptake is reduced. The smooth surface and better translucency of the films suggest that the EAEP residue films treated with citric acid are suitable for application in food packaging.

Effect of co-products of enzyme-assisted aqueous extraction of soybeans, enzymes, and surfactant on oil recovery from integrated corn-soy fermentation

Integrated corn-soy fermentation, utilizing co-products of soybean enzyme-assisted aqueous extraction process (EAEP) of soybeans in corn fermentation, has shown potential for enhancing bioethanol production compared to corn only fermentation. To maximize economic returns, oil may be recovered. In the present study, the effect of skim and insoluble fiber, and oil extraction aids on ethanol yield, oil partition, and oil recovery, and quality of Distillers Dried Grains (DDG) was investigated. Two fiber hydrolyzing enzymes (pectinase and cellulase), an acid protease (Fermgen®), and a surfactant (Tween 80) were evaluated. Addition of skim, mixture of skim and insoluble fiber, or Fermgen® to corn fermentation resulted in a ∼32 h decrease in fermentation time. Addition of soy co-products also resulted in ∼10–28% increase in oil partition in thin stillage with no additional enzyme or surfactant treatment. Addition of insoluble fiber alone resulted in ∼19% decrease in solids partition in thin stillage. Maximum free oil recovery, 22.5 ± 4.5%, was achieved from corn-insoluble fiber thin stillage with a combined treatment of enzymes (pectinase, cellulase, and Fermgen®) and surfactant (Tween 80). Maximum extractable oil recovery, 67 ± 3.2%, was achieved with the enzyme treatment alone. Corn-soy DDG has ∼11% higher protein, ∼2% lower fiber, and ∼2% lower fat contents compared to corn DDG. The fiber content was further reduced to ∼2% after enzyme treatment. This study demonstrates an efficient use of soy EAEP co-products and enzymes to maximize oil partition in thin stillage, and produce a high quality corn-soy DDG.

Ethanol Production by Soy Fiber Treatment and Simultaneous Saccharification and Co-Fermentation in an Integrated Corn-Soy Biorefinery

Insoluble fiber (IF) recovered from the enzyme-assisted aqueous extraction process (EAEP) of soybeans is a fraction rich in carbohydrates and proteins. It can be used to enhance ethanol production in an integrated corn-soy biorefinery, which combines EAEP with traditional corn-based ethanol processing. The present study evaluated IF as a substrate for ethanol production. The effects of treatment of IF (soaking in aqueous ammonia (SAA), liquid hot water (LHW), and enzymatic hydrolysis), primarily simultaneous saccharification and co-fermentation (SSCF), as well as scaling up (250 mL to 60 L) on ethanol production from IF alone or a corn and IF slurry were investigated. Enzymatic hydrolysis (pectinase, cellulase, and xylanase, each added at 5% soy solids during simultaneous saccharification and fermentation/SSCF) was the best treatment to maximize ethanol production from IF. Ethanol yield almost doubled when SSCF of IF was performed with Saccharomyces cerevisiae and Escherichia coli KO11. Addition of IF in dry-grind corn fermentation increased the ethanol production rate (~31%), but low ethanol tolerance of E. coli KO11 was a limiting factor for employing SSCF in combination corn and IF fermentation. Nonlinear Monod modeling accurately predicted the effect of ethanol concentration on E. coli KO11 growth kinetics by Hanes-Woolf linearization. Collectively, the results from this study suggest a potential of IF as a substrate, alone or in dry-grind corn fermentation, where it enhances the ethanol production rate. IF can be incorporated in the current bioethanol industry with no added capital investment, except enzymes.

3D confocal Raman imaging of oil-rich emulsion from enzyme-assisted aqueous extraction of extruded soybean powder

The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing (EAEP) was a critical step to break the oil-rich emulsion structure in order to recover oil. Albeit EAEP method has been applied as an alternative way to conventional solvent extraction method, the structure morphology of oil-rich emulsion was still unclear. The current study aimed to investigate the structure morphology of oil-rich emulsion from EAEP using 3D confocal Raman imaging technique. With increasing the enzymatic hydrolysis duration from 1 to 3 h, the stability of oil-rich emulsion was decreased as visualized in the 3D confocal Raman images that the protein and oil were mixed together. The subsequent Raman spectrum analysis further revealed that the decreased stability of oil-rich emulsion was due to the protein aggregations via SS bonds or protein-lipid interactions. The conformational transfer in protein indicated the formation of a compact structure.

Response surface methodology to optimize enzyme-assisted aqueous extraction of maize germ oil.

The present study was aimed at optimizing the enzyme assisted aqueous extraction (EAAE) process for extraction of maize germ oil. The different EAAE process parameter viz., pH of the slurry, seed to water ratio, the temperature of incubation and time of hydrolysis were optimized for improving oil recovery and oil quality. The combined effect of independent variables on recovery of oil, time taken for oil extraction and various quality parameters were studied using response surface methodology. The designed experimental runs were conducted to obtain the optimal conditions as 5.85pH of slurry, 1:6.92 seed to water ratio, 45.12°C temperature of incubation and 1h time of hydrolysis. Oil extracted under these conditions was light yellowish in color and had a pleasant nutty taste, with maximum oil recovery of 70%. The extraction variables viz., pH of slurry, seed to water ratio, the temperature of incubation had a significant effect on recovery of oil and various quality characteristics, however the time of hydrolysis had a non significant effect.

Enzymatic Extraction of Oil fromBalanites Aegyptiaca(Desert Date) Kernel and Comparison with Solvent Extracted Oil

Aqueous enzymatic extraction of oil from Balanites aegyptiaca (desert date) kernel was optimized using a mixture of four enzymes (alcalase, cellulase, viscozyme L and protease). The physicochemical properties and fatty acid composition of the oil obtained by enzyme assisted aqueous extraction and byorganic solvent extraction were compared. Using 2% of each enzyme and 1:10 of water-to-kernel flour ratio, the yield of oil extraction was 38%, estimated in comparison with extraction by Soxhlet method. Linoleic acid was the most prominent fatty acid, followed by oleic acid, and palmitic acid. No significant differences (P > 0.05) were observed in the fatty acid composition of the oil extracted using enzymes mixtures and organic solvents. However, the oil obtained by aqueous enzymatic extraction presented a significantly lower (P < 0.05) acid value, peroxide value, color intensity and phospholipids content than the oil obtained by solvent extraction. Practical Application The enzymatic process described for oil extraction from desert date kernel is environment-friendly as it does not require organic solvents and does not produce harmful waste, which is desirable not only for the safety of employees, but also for environmental protection. Oil with superior characteristics than those obtained by organic solvent extraction is produced, due to its lower FFAs and peroxide values and lower color intensity. Additionally, the overall costs of the refining process are reduced because the phospholipids are retained in the residual solid phase and there is no need of degumming.

Characterization and demusification of cream emulsion from aqueous extraction of peanut

In this study, the effect of aqueous extraction processing (AEP) and enzyme-assisted aqueous extraction processing (EAEP) on peanut oil yield was investigated. Only 34.53 ± 4.07% of free oil was recovered when using AEP without demulsification. In the case of EAEP, 89.31 ± 0.90% of free oil was obtained (1.5% w/w Alcalase 2.4L) without demulsification. Meanwhile, the protein yields of AEP and EAEP were similar, 84.10 ± 0.22% and 80.62 ± 0.92%, respectively. The efficiencies of enzymes (Protex 6L, Protex 7L, Protex 50FP, Alcalase 2.4L, Papain and Lecitase® Ultra) and pH of the released oil from peanut pastes were evaluated. Papain and Protex 50FP were effective in destabilizing the cream emulsion (93.10 ± 0.79% and 94.89 ± 0.19%, respectively). Adjusting the pH of the cream emulsion to the isoelectric point of peanut proteins also destabilized the cream with 85.71% of free oil recovery. Furthermore, the changes in the microstructure of cream emulsion during enzymatic demusification were observed by confocal laser scan microscope (CLSM). The interface tension decreased with the increase in enzymatic treatment time. For surface hydrophobicity (H0), cream emulsions treated by Protex 50FP and Papain had a remarkable decrease. Papain treated cream emulsion had a smaller H0 than that of Protex 50FP due to the protein aggregations formed during adjusting pH value to 4.5. This might have hindered the efficiency of protein hydrolysis and left more basic arachin (MW 18–24 kDa) which have more hydrophobic groups.

Parameters affecting enzyme-assisted aqueous extraction of extruded sunflower meal

Microscopic observation of sunflower meal before and after extraction indicated that extensive cellular disruption was achieved by extrusion, but that unextracted oil remained sequestered as coalesced oil within the void spaces of disrupted cotyledon cells. A full factorial design experiment was defined to develop aqueous extraction processing (AEP) with and without enzymes to improve vegetable oil extraction yields of extruded sunflower meal. This experimental design studied the influence of four parameters, agitation, liquid/solid (L/S) ratio, and cellulase and protease addition, on extraction yield of lipid and protein. Agitation and addition of cellulases increased oil extraction yield, indicating that emulsification of oil and alteration of the geometry of the confining cellular matrix were important mechanisms for improving yields. Protease and liquid-solid ratio of the extraction mixture did not have significant effects, indicating key differences with previously established soy oil extraction mechanisms. Maximum yields attained for oil and protein extraction were 39% and 90%, respectively, with the aid of a surfactant.

Counter-current carbon dioxide extraction of fat from soy skim

This research aims to investigate the use of counter-current carbon dioxide extraction method as a means to reduce residual fat in soy skim after the enzyme-assisted aqueous extraction of soybeans. Extractions with liquid CO2 at 25°C and 10.34MPa and supercritical CO2 at 50°C and 25.16MPa are compared. The effects of solvent-to-feed ratio (2.5–10), addition of ethanol as a modifier (5% w/w) and introductions of packing in the column are also analyzed. Results show that the highest reduction of fat content is obtained without modifier and with packing in the column at 50°C and 25.16MPa. At these conditions, the total fat content present in soy skim was reduced from 4.4 to 0.7%, with the protein content practically unaffected. ANOVA was applied to determine effects on fat and protein in soy skim, being the addition of packing in the column the most significant one. The fatty acid profile was also analyzed, with C18:2 being the predominant fatty acid present in all the soy skim samples. Gel electrophoresis indicated that supercritical CO2 settings did not affect the polypeptide band patterns; however, higher pressure, temperature, and CO2 flow significantly increased protein solubility in aqueous media (pH 2–10) compared with un-treated samples.

Enzyme-Assisted Aqueous Extraction Of Cashew Nut (Anacardium occidentale L.) Oil

Enzyme-assisted aqueous extraction method was applied to extract oil from cashew nut ( Anacardium occidentale L.). The commercial enzyme (Viscozyme cassava C) was tested for effectiveness in releasing oil during the aqueous extraction. The effect of several parameters such as material/water ratio, enzyme concentration and duration for enzyme incubation on the oil yield was investigated. The conditions for maximum oil release were found with the material/water ratio of 1:9, enzyme concentration of 1% (v/w E/S), and in 3 h of enzyme incubation at 50 o C with constant shaking. The maximum oil yield obtained at those conditions (38.88 % raw material) was significantly (p <0.05) higher than that of the control (without enzyme) (35.92 %), and it represented 86.28 % recovery of the total oil in seed. No hexane and other organic solvents were needed for this process. The cashew nut oil by enzyme-assisted aqueous extraction was relatively stable. Both peroxide value and free fatty acid value were lower than those in the oil obtained by Soxhlet method. Total un-saturated fatty acid in the cashew nut oil was about 84.43 %, in which the most abundant was oleic acid (65.0 %), followed by linoleic acid (18.53%). Cashew nut oil is a good dietary source of un-saturated fatty acids.

Enzyme-Assisted Extraction of Oil from Wet Microalgae Scenedesmus sp. G4

The enzyme-assisted aqueous extraction of oil from wet microalgae was employed to avoid the energy consumption of a dewatering process. In this paper, oil-rich microalgae Scenedesmus sp. G4 was hydrolyzed by enzyme mixtures for oil extraction. The results showed that the algae concentration had the greatest influence on yield of extracted oil, and the temperature and the ratio of enzyme mixtures affected the results as follows: the maximum yield of oil extracted from Scenedesmus sp. G4 reached 86.1% under optimal conditions with an algae concentration at 2.5 g/L, temperature 30/50 °C, pH 3.5/4.5 and a cellulase:pectinase:hemicellulase ratio of 1:1:1 or 1:2:1 (w/w/w). The results also indicated that the enzyme mixtures had a significant impact on the integrity of microalgae cells and the crystallization index increased from 30.7% to around 36.0% after enzymatic hydrolysis treatment. The cell wall of Scenedesmus sp. G4 has a high content of cellulose Iα and low crystallization, which is beneficial to the oil extraction by the enzyme-assisted hydrolysis method.

Effect of co-products of enzyme-assisted aqueous extraction of soybeans on ethanol production in dry-grind corn fermentation

Enzyme-assisted aqueous extraction processing (EAEP) is an environmentally-friendly alternative to solvent and mechanical oil extraction methods, and can achieve ∼97% oil recovery from soybeans. The present study utilized soy skim (protein rich) and insoluble fiber (IF; carbohydrate rich), both co-products of EAEP, in dry-grind corn fermentation. The effects of adding soy skim and untreated IF (UIF), either separately or together, and adding pretreated IF (TIF), on ethanol production were investigated. Maximum ethanol production was achieved when UIF and skim were slurried together (corn-to-UIF ratio 1:0.16; skim-to-UIF ratio 6.5:1) and when fiber-hydrolyzing enzymes were added to corn fermentation. This modification to corn fermentation increased ethanol yield by 20%, ethanol production rate by 3%, and decreased fermentation time by 38h compared to corn-only fermentation. An attempt was also made to utilize pentoses (from soy skim and IF) in integrated corn-soy fermentation slurry by an additional Escherichia coli KO11 fermentation step.

Enzyme-assisted aqueous extraction of oil from Forsythia suspense seed and its physicochemical property and antioxidant activity

In order to exploit the byproduct of Forsythia suspense, its seed oil was first studied for the extraction, physicochemical property and antioxidant activity. The green and effective enzyme-assisted aqueous extraction processing under microwave irradiation was applied to obtain the oil. The optimal oil yield of 21.62±0.51% (dry weight of F. suspense seed basis) was achieved by the treatment of 1.5% (w/w) enzyme cocktail (cellulase/pectinase/proteinase=1/1/1, w/w/w) under 90min. The fatty acid composition of FSSO exhibited the predominance of linoleic acid (72.96%) along with oleic acid (18.66%) and palmitic acid (5.59%), which was quite similar to that of sunflower oil. Moreover, FSSO showed the notable concentration-dependent antioxidant activity with IC50 values of 22.35 and 29.51mg/mL, according to the DPPH radical scavenging assay and β-carotene/linoleic acid bleaching test, respectively. Thus, FSSO may serve as a potential source of valuable product for the food, pharmaceutical, or cosmetics industries.

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.