Optimization of the Aqueous Enzymatic Extraction of Rapeseed Oil and Protein Hydrolysates

An aqueous enzymatic extraction method was developed to obtain free oil and protein hydrolysates from dehulled rapeseeds. The rapeseed slurry was treated by the chosen combination of pectinase, cellulase, and β-glucanase (4:1:1, v/v/v) at concentration of 2.5% (v/w) for 4 h. This was followed by sequential treatments consisting of alkaline extraction and an alkaline protease (Alcalase 2.4L) hydrolysis to both produce a protein hydrolysate product and demulsify the oil. Response surface methodology (RSM) was used to study and optimize the effects of the pH of the alkaline extraction (9.0, 10.0 and 11.0), the concentration of the Alcalase 2.4L (0.5, 1.0 and 1.5%, v/w), and the duration of the hydrolysis (60, 120, and 180 min). Increasing the concentration of Alcalase 2.4L and the duration of the hydrolysis time significantly increased the yields of free oil and protein hydrolysates and the degree of protein hydrolysis (DH), while the alkaline extraction pH had a significant effect only on the yield of the protein hydrolysates. Following an alkaline extraction at pH 10 for 30 min, we defined a practical optimum protocol consisting of a concentration of 1.25–1.5% Alcalase 2.4L and a hydrolysis time between 150 and 180 min. Under these conditions, the yields of free oil and protein hydrolysates were 73–76% and 80–83%, respectively. The hydrolysates consisted of approximately 96% of peptides with a MW less than 1500, of which about 81% had a MW less than 600 Da.     

Extraction and Demulsification of Oil From Wheat Germ,Barley Germ,and Rice Bran Using an Aqueous Enzymatic Method

An aqueous enzymatic method was developed to extract oil from wheat germ. Wheat germ pretreatment, effect of various industrial enzymes, pH, wheat germ to water ratio, reaction time and effect of various methods of demulsification, were investigated. Pretreatment at 180 °C in a conventional oven for 4 min reduced the moisture 12.8–2.2 % and significantly increased the oil yield. Adding a combination of protease (Fermgen) and cellulase (Spezyme CP) resulted in a 72 % yield of emulsified oil from wheat germ (both commercial and laboratory milled wheat germ). Using the same oil extraction conditions optimized for wheat germ, yields of 51 and 39 % emulsified oil were obtained from barley germ (laboratory milled), and rice bran, respectively. Three physical demulsification methods (heating, freeze-thawing, and pH adjustment) and enzymatic methods (Protex 6L, Protex 7L, Alcalase, Fermgen, Lysomax and G-zyme 999) were compared. After demulsification with Protex 6L, free oil yields of 63.8 and 59.5 % were obtained with commercial wheat germ and with laboratory milled wheat germ, respectively. Using the same demulsification conditions optimized for wheat germ, yields of 45.7 % emulsified oil and 35 % free oil were obtained for barley germ and rice bran, respectively.     

水酶法提取大豆油

The procedure of enzymatic aqueous extraction of soybean oil was assessed when two-step controlled enzymatic hydrolysis was applied. With aqueous extraction of soybean oil-containing protein, the highest yield of oil was 96.1% at the optimized conditions studied. Soybean oil-containing protein was hydrolyzed and resulted in releasing part of oil. The separated protein that contained 40% oil was enriched due to its adsorption capacity of released oil, the average oil extraction yeild reached 93.5%. Then the high oil content protein was hydrolyzed again to release oil by enzyme, the oil extraction yeild was 80.4%. As a result, high quality of soybean oil was obtained and the content of total oil vield was 74.4%.     

Efficient and Response Surface Optimized Aqueous Enzymatic Extraction of Camellia oleifera (Tea Seed) Oil Facilitated by Concurrent Calcium Chloride Addition

This paper reports an efficient aqueous enzymatic extraction (AEE) method for Camellia oleifera seed oil with the aid of response surface analysis. A maximum oil recovery of ~93.5% was obtained when a 2‐step AEE process was performed using 0.80% cellulase (v/w) solution at pH 6.0 maintained at 50 °C for 1 h followed by a solution of 0.70% Alcalase® with pH 9.2 at 57 °C for 4.1 h. It was found that the addition of Ca²⁺ during the proteolysis stage improved the free oil yield from ~62.1 to ~86.6%. This was attributed to the removal of tea saponins, cross‐linkage of anionic polysaccharides, and destabilization of cream emulsion by Ca²⁺. This was verified by decreased tea saponin and polysaccharide levels in the cream emulsion and bulk solution as well as lowering of the emulsion fraction. It was determined that addition of CaCl₂ solution in continuous flow to the proteolysate is superior to one‐time or batch addition in inhibiting emulsion formation. The addition of CaCl₂ may provide a means of replacing the more laborious, time‐consuming demulsification process otherwise required.     

Destabilization of the Emulsion Formed during Aqueous Extraction of Soybean Oil

Characterization and destabilization of the emulsion formed during aqueous extraction of oil from soybean flour were investigated. This emulsion was collected as a cream layer and was subjected to various single and combined treatments, including thermal treatments and enzymatic treatments, aimed at recovery of free oil. The soybean oil emulsion formed during the aqueous extraction processing of full fat flour contains high molecular weight glycinin and β-conglycinin proteins and smaller oleosin proteins, which form a multilayer interface. Heat treatment alone did not modify the free oil recovery but freeze–thaw treatment increased the oil yield from 3 to 22%. After enzymatic treatment of the emulsion, its mean droplet size changed from 5 to 14 μm and the oil recovery increased to 23%. This increase could be attributed to the removal (due to enzymatic hydrolysis) of large molecular weight polypeptides from the emulsion interface, resulting in partial emulsion destabilization. When enzymatic treatment was followed by a freeze–thaw step, the oil recovery increased to 46%. This result can be attributed to the thinner interfacial membrane after enzymatic hydrolysis, partial coalescence during freeze–thaw, and coalescence during centrifugation. Despite the reduction in emulsion stability achieved, additional demulsification approaches need to be pursued to obtain an acceptably high conversion to free oil.     

Ethanol-Assisted Aqueous Enzymatic Extraction of Peony Seed Oil

An ethanol-assisted aqueous enzymatic extraction was performed for peony seed oil (content of 30%). This method included cooking pretreatment, pectinase hydrolysis, and aqueous ethanol extraction, and the corresponding variables in each step were investigated. The changes in viscosity and dextrose equivalent values of the reaction medium as a function of changing enzymatic hydrolysis time were compared to the oil yield. The microstructures of peony seeds were analyzed using confocal laser scanning microscopy to understand the process of oil release as a result of cooking and grinding. The highest oil yield of 92.06% was obtained when peony seeds were cooked in deionized water with a solid–liquid ratio of 1:5 (w/v) at 110°C for 1 hour, ground to 31.29 μm particle size, treated with 0.15% (w/w) pectinase (temperature 50°C, pH 4.5, time 1 hour), and then extracted with 30% (v/v) aqueous ethanol (temperature 60°C, pH 9.0, time 1 hour). After processing with pectinase followed by ethanol extraction, the residual oil content in water and sediment phase decreased to 5% and 3%, respectively. The quality of the oil obtained by ethanol-assisted aqueous enzymatic extraction was good, complying with the Chinese standard.     

Aqueous Enzymatic Extraction of Oil and Protein Hydrolysates from Roasted Peanut Seeds

To evaluate the effects of the roasting process on the extraction yield and oil quality, peanut seeds were roasted at different temperatures (130–220 °C) for 20 min prior to the aqueous extraction of both oil and protein hydrolysates with Alcalase 2.4 L. Roasting temperatures did not significantly affect the yields of free oil, whereas the temperature of 220 °C led to a reduced recovery of protein hydrolysates. The color and acid values of peanut oils did not change significantly with roasting temperatures. The enzyme-extracted oil with roasting at 190 °C had a relatively low peroxide value, a strong oxidative stability, and the best flavor score. Using the same seed-roasting temperature (190 °C), quality attributes such as color, acid and peroxide values, phosphorus content and oxidative stability of the enzyme-extracted oil were better than those of the oil obtained by an expeller. After the peanut seeds were roasted at 190 °C for 20 min, with a seeds-to-water ratio of 1:5, an enzyme concentration of 2%, and an incubation time of 3 h, the yields of free oil and protein hydrolysates were 78.6 and 80.1%, respectively. After demulsification of the residual emulsion by a freezing and thawing method, the total free oil yield increased to 86–90%.     

Simplex-Centroid Mixture Design Applied to the Aqueous Enzymatic Extraction of Fatty Acid-Balanced Oil from Mixed Seeds

A one-step method was developed to extract oil from a mixture of soybeans, peanuts, linseeds, and tea seeds using an aqueous enzymatic method. The proportion of the four seeds was targeted in accordance with a fatty acid ratio of 0.27 (SFA, saturated fatty acid(s)): 1 (MUFA, monounsaturated fatty acid(s)): 1 (PUFA, polyunsaturated fatty acid(s)), and the oil extraction yield was maximized by applying the simplex-centroid mixture design method. Three models were developed for describing the relationship between the proportion of the individual seeds in the mixture, the fatty acid ratio in the extracted oil, and the oil extraction yield, respectively. The developed models were then analyzed using an ANOVA and were found to fit the data quite well, with R ² values of 0.98, 0.93, and 0.93, respectively. The three models were validated experimentally. The results indicated that the ratio of fatty acids in the oil ranged between 0.98 and 1.12 (MUFA:PUFA) and between 0.26 and 0.28 (SFA:MUFA), which were quite close to the target values of 1 and 0.27, respectively. The oil extraction yield of 62.13 % was slightly higher than the predicted value (60.32 %).     

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.     

Optimization of an Aqueous Extraction Process for Pomegranate Seed Oil

Response surface methodology employing a five-level, four-variable central composite rotatable design was applied to study the effects of extraction time, extraction temperature, pH and water/solid ratio on the extraction yield of pomegranate seed oil using an aqueous extraction approach. In addition, quality indices, fatty acid composition and antioxidant activity of the obtained oil were studied and compared with those of typical hexane-, cold press- and hot press-extracted oil. Aqueous extraction resulted in the maximum oil recovery of 19.3% (w/w), obtained under the following critical values: water/solid ratio (2.2:1.0, mL/g), pH 5.0, extraction temperature = 63 °C and extraction time = 375 min. This yield is lower than that obtained via hexane extraction (26.8%, w/w) and higher than the yields from cold press (7.0%, w/w) and hot press (8.6%, w/w) extraction. A comparison of the characteristics of the oils based on extraction method revealed that the unsaturated fatty acid content was highest for the oil obtained by aqueous extraction. In addition, higher levels of iodine and peroxide and lower levels of acid, p-anisidine and unsaponifiable matter were observed. The oil obtained with aqueous extraction also exhibited higher antioxidant activity than oils obtained by hexane or hot press extraction.     

Destabilization of Emulsion Formed During Aqueous Extraction of Peanut Oil: Synergistic Effect of Tween 20 and pH

To destabilize the emulsion formed during aqueous extraction processing (AEP) of peanuts, Tween and Span series surfactants (Tween 20, Tween 80, Span 20, and Span 80) were used alone or in combination to break the emulsion. Results indicate that only Tween surfactants had a pronounced demulsifying effect that was dependent on Tween concentration and system pH. When 1.2 wt% Tween 20 aqueous solution was used for oil extraction at pH 10.0, the highest free oil yield was achieved at 76.1 %, which was similar to the oil recovery of using proteases as a destabilization agent. The results obtained using a model emulsion system containing peanut oil and Tween 20/peanut protein isolates (PPI) showed that when Tween 20 and PPI coexisted in extraction medium at pH 10.0, the dynamic interfacial tension and droplet size distribution curves were very similar to those when Tween 20 was used alone, suggesting that Tween 20 dominated at the interface, instead of PPI. Destabilization of the model emulsions relied on three important factors: inclusion of Tween 20 at the initial mixing stage, high pH, and a gentle mixing speed. A synergistic destabilization mechanism of using Tween 20 at high pH during AEP was proposed. The discovery of Tween 20 as an effective demulsifier significantly contributes to the development of AEP of oilseeds.     

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.     

Separating Oil from Aqueous Extraction Fractions of Soybean

Previous research has shown that enzyme-assisted aqueous extraction processing (EAEP) extracts 88–90% of the total soybean oil from extruded full-fat soy flakes into the aqueous media, which is distributed as cream (oil-in-water emulsion), skim, and free oil. In the present work, a simple separatory funnel procedure was effective in separating aqueous skim, cream and free oil fractions allowing mass balances and extraction and recovery efficiencies to be determined. The procedure was used to separate and compare liquid fractions extracted from full-fat soy flour and extruded full-fat soy flakes. EAEP extracted more oil from the extruded full-fat soy flakes, and yielded more free oil from the resulting cream compared to unextruded full-fat soy flour. Dry matter partitioning between fractions was similar for the two procedures. Mean oil droplet sizes in the cream and skim fractions were larger for EAEP of extruded flakes compared to non-enzymatic AEP of unextruded flour (45 vs. 20 μm for cream; 13 vs. 5 μm for skim) making the emulsions from EAEP of extruded flakes less stable. All major soy protein subunits were present in the cream fractions, as well as other fractions, from both processes. The cream could be broken using phospholipase treatments and 70–80% of total oil in the extruded full-fat flakes was recovered using EAEP and a phospholipase de-emulsification procedure.     

水酶法提取肉桂油的工艺研究

探索了肉桂油的水酶法提油工艺.研究了不同酶系以及酶解条件对肉桂油得率的影响.结果表明,果胶酶的作用效果好;酶解条件中,加酶量、酶解时间都对肉桂油得率有显著的影响,通过单因素分析及正交试验的结果,得出在固液比1∶6、酶解pH=3.5、酶解温度40℃、加酶量1.5％、酶解时间2h条件下,肉桂油得率能达到1.72％.经检测得到处理得到的肉桂油中桂皮醛含量达到83.6％.     

Properties of Soy Protein Produced by Countercurrent,Two-Stage,Enzyme-Assisted Aqueous Extraction

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.     

Effect of Aqueous Enzymatic Processes on Sunflower Oil Quality

The use of enzymes in aqueous vegetable oilseed extraction for simultaneous recovery of high quality oil and protein is gaining recognition. In the present work, five enzyme preparations [Protex 7L by Genencor (Rochester, NY USA), Alcalase 2.4L, and Viscozyme L by Novozymes (Bagsvaerd, Denmark), Natuzyme by Bioproton Pty Ltd (Australia) and Kemzyme by Kemin Europa N·V. (Belgium)] were studied to evaluate their effects on the extraction of oil and protein from sunflower seeds. Preliminary experiments were conducted for the selection of enzymes, optimum enzyme concentration, incubation time and pH. Maximum oil yield (87.25% of the total oil in the seed) was obtained with Viscozyme L, whereas, Protex 7L offered the highest level of protein in the aqueous phase. The comparison of the quality attributes of enzyme-assisted aqueous extracted (EAAE) oil with those of solvent-extracted and control (oils extracted without enzyme treatment) oils revealed no significant (P > 0.05) variations for iodine value, density, refractive index, unsaponifiable matter, and fatty acid composition among the extraction methods. The control and EAAE oils also exhibited a better oxidation state. The tocopherol concentration for the oils, produced with the enzymes, was noted to be quite improved relative to the control and solvent-extracted oils. A higher antioxidant activity in terms of total phenolic contents, 2,2′-diphenyl-1-picrylhydrazyl scavenging capacity and inhibition of linoleic acid peroxidation was also observed for the EAAE oils as against control and the solvent-extracted oils.     

Enzymatic Demulsification of the Oil-Rich Emulsion Obtained by Aqueous Extraction of Peanut Seeds

This study details the enzymatic destabilization of the emulsion formed during aqueous extraction of peanut seeds and the quality of the resulting oil. The emulsion was exposed to enzymatic treatment and pH adjustment. The experimental results suggest that the alkaline endopeptidase Mifong^®2709 was the most effective demulsifier, while Phospholipase A2 and pH adjustment had little effect on emulsion stability. The demulsifying conditions of Mifong^®2709 were optimized by response surface methodology (RSM). The optimal conditions which produced a free oil yield of ~94 % were: 1:1 water-to-emulsion ratio, enzyme concentration of 1,600 IU/g of emulsion and 70 min hydrolysis time at 50 °C. We found that these conditions resulted in a positive relationship (R ² = 0.9671) between free oil yield and the degree of protein hydrolysis. Increased protease treatment produced a smaller number of oil droplets, but the size of these droplets increased significantly. When compared to demulsified oil products obtained by using thermal treatment, the oil obtained by Mifong^®2709 exhibited lower acid and peroxide values, contained more tocopherols and had a longer induction time as determined in the Rancimat test. The high yield and quality of peanut oil obtained by enzymatic treatment makes enzyme demulsification a promising approach to recovering free oil in aqueous extractions of peanuts.