A novel process for physically refining rice bran oil through simultaneous degumming and dewaxing

A new process for the physical refining of rice bran oil through combined degumming and dewaxing was developed on a laboratory scale and then demonstrated on a commercial scale. The simultaneous degumming and dewaxing of the crude oil with a solution of water and CaCl₂, followed by crystallization at a low temperature (20°C), facilitated precipitation of the hydratable and nonhydratable phosphatides along with the wax, which enabled its separation and reduction to a greater extent. Bleaching and subsequent winterization (20°C) of this oil further reduced the phosphorus content to less than 5 ppm. Thus, these pretreatment steps enabled the physically refined rice bran oil to meet commercially acceptable levels for color, FFA content, and cloud point values (10–12 Lovibond units in a 1-in, cell, <0.25%, and 4–5°C, respectively) with very low neutral oil loss; this has not been observed hitherto. Rice bran oil is known for its high levels of bioactive phytochemicals, such as oryzanol, tocols, and sterols. The process reported here could retain more than 80% of these micronutrients in the end product. This paper was previously presented at the 95th AOCS Annual Meeting and Expo, Cincinnati, Ohio, May 9–12, 2004     

Enzymatic Degumming of Crude Jatropha Oil: Evaluation of Impact Factors on the Removal of Phospholipids

Jatropha curcas seeds are rich in non‐edible oil, and this plant has received much interest in recent years, especially with respect to biodiesel production. Owing to the high content of phospholipids, crude jatropha oil has to be refined before further use. Conventional refining processes have several environmental and energetic shortcomings. Thus, the search for alternative degumming methods has become relevant. This study compares the enzymatic degumming of screw‐pressed crude jatropha oil with Lecitase Ultra (phospholipase A1) and LysoMax (phospholipase A2). Degumming with phospholipase A2 was less effective that degumming with phospholipase A1. Phospholipase A1 showed the highest reaction rate at 50 °C, 700 rpm stirring, 3 mL of water per 100 g of oil, and with 75 ppm of added phospholipase. To ensure optimum enzyme activity, the pH was adjusted to 5. The phosphorus content was reduced continuously for reaction times up to 3 h. The residual phosphorus content was found to be independent of its initial level. Laboratory experiments showed that enzymatic degumming of jatropha oil with phospholipase A1 at the adapted parameters enables the phosphorus content to be reduced to levels below 4 ppm.     

Degumming rice bran oil using phospholipase‐A1

The efficacy of enzymatic degumming was assessed using the third generation phospholipase‐A₁, Lecitase®‐Ultra (EC 3.1.1.3) from Thermomyces lanuginosa/Fusarium oxysporum with different qualities of crude rice bran oil. The phosphorus content in the oil reduced to ～10 mg/kg from an initial level of 390 mg/kg after 2 h of incubation period at 50°C. However, in the solvent‐phase degumming, there was practically no phospholipid reduction at lower water content (2%) due to the poor contact between the highly nonpolar solvent and the aqueous phase (citric acid, NaOH, and enzyme solutions). Increasing the water content to 20% reduced the phosphorus level in the degummed‐oil to 71 mg/kg but did not match the performance of oil‐phase degumming. The degumming efficiency of Lecitase®‐Ultra was effective in oil‐phase and suitable for practical application. Solvent‐phase enzymatic degumming offers more benefits but needs greater efforts to overcome the challenges.     

Optimization of enzymatic degumming process for rapeseed oil

An enzymatic process optimization and a largescale plant trial for rapeseed oil degumming were carried out by a novel microbial lipase. Response surface methodology was used to obtain the desired data in the process optimization. Enzyme dosage, temperature, and pH were important determining factors affecting oil degumming. The optimal set of variables was an enzyme dosage of 39.6 mg/kg, a temperature of 48.3°C, and a pH of 4.9. The phosphorus content could be reduced to 3.1 mg/kg at the optimal levels of the tested factors. An enzymatic degumming plant trial was performed on a 400 tons/d oil production line. pH was found to play an important role in degumming performance. When the pH was 4.6–5.1, the corresponding phosphorus content of degummed rapeseed oil could be reduced to less than 10 mg/kg, which met the demands of the physical refining process.     

Origin of problems encountered in rice bran oil processing

Rice bran oil, not being a seed‐derived oil, has a composition qualitatively different from common vegetable oils and the conventional vegetable oil processing technologies are not adaptable without incurring huge losses. The oil's unusual high content of waxes, free fatty acids, unsaponifiable constituents, phospholipids, glycolipids and its dark color, all cause difficulties in the refining process. An attempt was made in this investigation to look into factors that are responsible for such difficulties and to develop suitable methodologies for physical refining of rice bran oil. Special attention was given to dewaxing, degumming and deacidification steps. The high content of glycolipids (∼6%) present in the oil was found to be a central problem and their removal appeared crucial for successful processing of the oil. We have also isolated and identified, for the first time, phosphorus‐containing glycolipids that are unique to this oil. These compounds prevent a successful degumming of the oil and their high surface activity leads to unusually high refining losses during alkali refining. A number of simple processes has been evolved, including 1) a simultaneous dewaxing and degumming process, 2) an unusual enzymatic process to degum the oil, 3) processes for the removal of the glycolipids including the phosphoglycolipids and 4) a process for the isolation of the glycolipids which may have potential applications in the food, cosmetic and pharmaceutical industries. The processing protocol suggested here becomes the first and only one to produce an oil with less than 5 ppm of phosphorus from crude rice bran oil, rendering it thus suitable for physical refining. We believe that the present results are very significant and should contribute to a better utilization of this valuable oil.     

Immobilization of Phospholipase A<Subscript>1</Subscript> and its Application in Soybean Oil Degumming

Phospholipase A₁ (PLA₁), or Lecitase^® Ultra, was immobilized on three different supports, calcium alginate (CA), calcium alginate-chitosan (CAC), and calcium alginate-gelatin (CAG), and crosslinked with glutaraldehyde. The results indicated that PLA₁–CA retained 56.2% of the enzyme’s initial activity, whereas PLA₁–CAC and PLA₁–CAG retained 65.5 and 60.2%, respectively. Compared with free PLA₁, the optimal pH of immobilized PLA₁ shifted to the basic side by 0.5–1.0 pH units and the pH/activity profile range was considerably broadened. Similarly, the temperature-optima of PLA₁–CAC and PLA₁–CAG increased from 50 to 60 °C, and their thermal stability increased with relative activities of more than 90% that covered a wider temperature range spanning 50–65 °C. In a batch oil degumming process, the final residual phosphorus content was reduced to less than 10 mg/kg with free PLA₁, PLA₁–CAC and PLA₁–CA in less than 5, 6 and 8 h respectively while PLA₁–CAG was only able to reduce it to 15 mg/kg in 10 h. When the PLA₁–CAC was applied in a plant degumming trial, the final residual phosphorus content was reduced to 9.7 mg/kg with 99.1% recovery of soybean oil. The recoveries of immobilized PLA₁–CAC and activity of PLA₁ were 80.2 and 78.2% respectively. Therefore, it was concluded that PLA₁–CAC was the best immobilized enzyme complex for the continuous hydrolysis of phospholipids in crude vegetable oils.     

Lipase‐catalyzed production of biodiesel from rice bran oil

Biodiesel has attracted considerable attention as an alternative fuel during the past decades. The main hurdle to the commercialization of biodiesel is the cost of the raw material. Use of an inexpensive raw material such as rice bran oil is an attractive option to lower the cost of biodiesel. Two commercially available immobilized lipases, Novozym 435 and IM 60, were employed as catalyst for the reaction of rice bran oil and methanol. Novozym 435 was found to be more effective in catalyzing the methanolysis of rice bran oil. Methanolysis of refined rice bran oil and fatty acids (derived from rice bran oil) catalyzed by Novozym 435 (5% based on oil weight) can reach a conversion of over 98% in 6 h and 1 h, respectively. Methanolysis of rice bran oil with a free fatty acid content higher than 18% resulted in lower conversions (<68%). A two‐step lipase‐catalyzed methanolysis of rice bran oil was developed for the efficient conversion of both free fatty acid and acylglycerides into fatty acid methyl ester. More than 98% conversion can be obtained in 4–6 h depending on the relative proportion of free fatty acid and acylglycerides in the rice bran oil. Inactivation of lipase by phospholipids and other minor components was observed during the methanolysis of crude rice bran oil. Simultaneous dewaxing/degumming proved to be efficient in removing phospholipids and other minor components that inhibit lipase activity from crude rice bran oil. Copyright © 2005 Society of Chemical Industry     

Enzymatic Synthesis of Biodiesel from Transesterification Reactions of Vegetable Oils and Short Chain Alcohols

Biodiesel synthesis by alcoholysis of three vegetable oils (soybean, sunflower and rice bran) catalyzed by three commercial lipases (Novozym 435, Lipozyme TL-IM and Lipozyme RM-IM), and the optimization of the enzymes stability over repeated batches is described. The effects of the molar ratio of alcohol to oil and the reaction temperature with methanol, ethanol, propanol and butanol were also studied. All three enzymes displayed similar reaction kinetics with all three oils and no significant differences were observed. However, each lipase displayed the highest alcoholysis activity with a different alcohol. Novozym 435 presented higher activity in methanolysis, at a 5:1 methanol:oil molar ratio; Lipozyme TL-IM presented higher activity in ethanolysis, at a 7:1 ethanol:oil molar ratio; and Lipozyme RM-IM presented higher activity in butanolysis, at a 9:1 butanol:oil molar ratio. The optimal temperature was in the range of 30–35 °C for all lipases. The assessment of enzyme stability over repeated batches was carried out by washing the immobilized enzymes with different solvents (n-hexane, water, ethanol, or propanol) after each batch. When washing with n-hexane, approximately 90% of the enzyme activity remained after seven synthesis cycles.     

Effect of method of stabilization on aqueous extraction of rice bran oil

Rice bran oil is widely used in pharmaceutical, food and chemical industries due to its unique properties and high medicinal value. In this study aqueous extraction of rice bran oil from rice bran available in Sri Lanka, was studied. Key factors controlling the extraction and optimal operating conditions were identified. Several methods of bran stabilization were tested and the results were analyzed. The yield and quality of aqueous extracted oil was compared with hexane extracted oil.Aqueous extraction experiments were conducted in laboratory scale mixer–settler unit. Steaming, hot air drying, chemical stabilization and refrigeration better controls the lipase activity compared to solar drying. Steaming is the most effective stabilization technique. The extraction capacity was highest at solution pH range 10–12. Higher oil yield was observed at higher operating temperatures (60–80 °C). Kinetic studies revealed that extraction was fast with 95% or more of the extraction occurring within first 10–15 min of contact time. Parboiling of paddy increases the oil yield. Highest oil yield of 161 and 131 mg/g were observed for aqueous extraction of parboiled bran and raw rice bran respectively. The aqueous extracted oil was low in free fatty acid content and color compared to hexane extracted rice bran oil and other commonly used oils. Major lipid species in rice bran oil were oleic, linoleic and palmitic.     

Pretreatment of corn oil for physical refining

Crude corn oil that contained 380 ppm of phosphorus and 5% of free fatty acids was degummed, bleached, and winterized for physical refining. The pretreatment and the steam-refining conditions were studied in pilot plant scale (2 kg/batch). The efficiency of wet degumming and of the total degumming processes, at different temperatures, was evaluated. TriSyl silica was tested as an auxiliary agent in the reduction of the phosphorus content before bleaching. The experimental conditions of the physical refining were: temperature at 240 or 250°C; 8 to 18 mbar vacuum, and distillation time varying from 1 to 3 h. Degumming at 10 or 30°C resulted in the removal of more phosphorus than at 70°C. Water degumming was more efficient than the processes of total degumming or acid degumming. Corn oil, degummed at 10 or 30°C, after bleaching passed the cold test, irrespective of the degumming agent used. Degumming and winterization took place simultaneously at these temperatures. The pretreatment was able to reduce the phosphorus content to less than 5 ppm. The amount of bleaching earth was reduced by carrying out dry degumming or by using silica before bleaching. Corn oil acidity, after physical refining, varied from 0.49 to 1.87%, depending on the residence time. Contrary to alkali refining, physical refining did not promote color removal due to the fixation of pigments present in the crude corn oil.     

Effects of Degumming and Bleaching on 3-MCPD Esters Formation During Physical Refining

Crude palm oil (CPO) was physically refined in a 200-kg batch pilot refining plant. A study of the possible role of degumming and bleaching steps in the refining process for a possible critical role in the formation of 3-chloropropane-1,2-diol (3-MCPD) esters was evaluated. For the degumming step, different percentages of phosphoric acid (0.02–0.1%) as well as water degumming (2.0%) were carried out. Six different types of bleaching clays, mainly natural and acid activated clays were used for bleaching process at a fixed dose of 1.0%. Deodorization of the bleached oils was performed at 260 °C for 90 min. Analyses showed that 3-MCPD esters were not detected in the CPO. Phosphoric acid degumming (0.1%) in combination with acid activated clays produced the highest levels (3.89 ppm) of 3-MCPD esters in the refined (RBD) oil. The esters were at the lowest levels (0.25 ppm) when the oil was water degummed and bleached with natural bleaching clays. However, the refined oil qualities were slightly compromised. Good correlation of 0.9759 and 0.9351 was obtained when concentration of the esters was plotted against acidity of the bleaching earths for the respective acid and water degumming processes. The findings revealed the contribution of acidic conditions on the higher formation of 3-MCPD esters. In order to lower the esters formation, it is important to reduce acid dosage based on the crude oil qualities or to find alternatives to acid degumming process. Neutralization of the acidity prior to deodorization was effective in reducing the formation of 3-MCPD esters.     

Ambient-temperature extraction of rice bran oil with hexane and isopropanol

Hexane and isopropanol were compared as solvents for use in ambient-temperature equilibrium extraction of rice bran oil (RBO). Isopropanol was as effective as hexane in extracting RBO when 20 mL of solvent was used to extract 2 g of bran. Free fatty acid levels were 2–3% in both solvents and similar to that previously reported for hexane extraction of RBO hexane extraction by this method. Larger-scale extractions with 30 g of bran and 150 mL of solvent produced oil with a similar free fatty acid content and a phosphorus level of approximately 500 ppm. The oil extracted with isopropanol was significantly more stable to heat-induced oxidation than hexane-extracted oil. Antioxidants that are more easily extracted by isopropanol than hexane may be responsible for the increased stability.     

Immobilization of β-glucosidase and its aroma-increasing effect on tea beverage

β-Glucosidase was effectively immobilized on alginate by the method of crosslinking–entrapment–crosslinking. After optimization of the immobilized conditions, the activity recovery of immobilized β-glucosidase achieved to 46.0%. The properties of immobilized β-glucosidase were investigated. Its optimum temperature was determined to be 45 °C, decreasing 10 °C compared with that of free enzyme, whereas the optimum pH did not change. The thermal and pH stabilities of immobilized β-glucosidase increased to some degree. The K_m value for immobilized β-glucosidase was estimated to be 1.97 × 10^?3 mol/L. The immobilized β-glucosidase was also applied to treat the tea beverage to investigate its aroma-increasing effect. The results showed that after treated with immobilized β-glucosidase, the total amount of essential oil in green tea, oolong tea and black tea increased by 20.69%, 10.30% and 6.79%, respectively. The storage stability and reusability of the immobilized β-glucosidase were improved significantly, with 73.3% activity retention after stored for 42 days and 93.6% residual activity after repeatedly used for 50 times.     

Synthesis of mesoporous silica templated by Pluronic F68 and its application in the immobilization of lipase

Mesoporous silica templated by Pluronic F68 was synthesized and characterized by TEM, N₂ adsorption–desorption isotherms and FT–IR spectra. The sample had a high specific surface area (761 m² g⁻¹) and the mean pore diameter was 4.7 nm, indicating that it can be used as porcine pancreatic lipase (PPL) support. The physical adsorption of PPL on this mesoporous material in phosphate buffer solution with different pH values has been studied. The maximum adsorbed amount was observed at pH 7.0 and amounted to 826 mg g⁻¹ and the maximum activity value of immobilized PPL was 227 μmol g⁻¹ min⁻¹. The optimal pH and temperature of the hydrolysis of triacetin for the immobilized PPL were at 8.0 and 45 °C, while they were at pH 7.0 and 35 °C for free PPL. The immobilized PPL showed excellent adaptability in higher pH and excellent heat resistance compared to free PPL. The retained activity of immobilized PPL was found to be ca. 50% of its original activity after the 5th reuse.     

Fatty Acid Composition,Oxidative Stability,and Radical Scavenging Activity of Vegetable Oil Blends with Coconut Oil

Coconut (Cocos nucifera) contains 55–65% oil, having C12:0 as the major fatty acid. Coconut oil has >90% saturates and is deficient in monounsaturates (6%), polyunsaturates (1%), and total tocopherols (29 mg/kg). However, coconut oil contains medium chain fatty acids (58%), which are easily absorbed into the body. Therefore, blends of coconut oil (20–80% incorporation of coconut oil) with other vegetable oils (i.e. palm, rice bran, sesame, mustard, sunflower, groundnut, safflower, and soybean) were prepared. Consequently, seven blends prepared for coconut oil consumers contained improved amounts of monounsaturates (8–36%, p < 0.03), polyunsaturates (4–35%, p < 0.03), total tocopherols (111–582 mg/kg, p < 0.02), and 5–33% (p < 0.02) of DPPH (2,2-diphenyl-1-picrylhydrazyl free radicals) scavenging activity. In addition, seven blends prepared for non-coconut oil consumers contained 11–13% of medium chain fatty acids. Coconut oil + sunflower oil and coconut oil + rice bran oil blends also exhibited 36.7–89.7% (p < 0.0005) and 66.4–80.5% (p < 0.0313) reductions in peroxide formation in comparison to the individual sunflower oil and rice bran oil, respectively. It was concluded that blending coconut oil with other vegetable oils provides medium chain fatty acids and oxidative stability to the blends, while coconut oil will be enriched with polyunsaturates, monounsaturates, natural antioxidants, and a greater radical scavenging activity.     

Membrane degumming of crude rice bran oil: Pilot plant study

The procedure for the classical chemical refining of vegetable oils consists of degumming, alkali neutralization, bleaching, and deodorization. Conventional refining of rice bran oil using alkali gives oil of acceptable quality, but the refining losses are very high. A critical work has been carried out to study the application of membrane technology in the pretreatment of crude rice bran oil. Oils intended for physical refining should have a low phosphorus content, and this is not readily achievable by the conventional acid/water degumming process. The application of membrane technology for the pretreatment of rice bran oil has been investigated. The process has already been successfully applied to other vegetable oils. Ceramic membranes, which are important from the commercial point of view, were examined for this purpose. The results showed that the membrane‐filtered oils met the requirements of physical refining, with a substantial reduction in color. It was observed that most of the waxy material was also rejected. Experiments were carried out to establish the relationship between permeate flux and rejection with membrane pore size, trans‐membrane pressure and micellar solute concentration.     

Physical Properties of Rice Bran Wax in Bulk and Organogels

Differential scanning calorimetry (DSC), optical microscopy, and X-ray diffraction (XRD) were used to examine the thermal behavior, crystal structure, and crystal morphology of rice bran wax (RBX) in bulk and oil–wax mixtures, and to compare them with those of carnauba wax (CRX) and candellila wax (CLX). The RBX employed in the present study was separated from rice bran oil by winterization, filtration, refinement, bleaching, and deodorization. The RBX crystals melted in the bulk state at 77–79 °C with ΔH _melting = 190.5 J/g, which is quite large compared with CLX (129 J/g) and CRX (137.6 J/g). XRD data of the RBX crystals revealed O_⊥ subcell packing and a long spacing value of 6.9 nm. Thin long needle-shaped crystals were observed in the mixtures of RBX and liquid oils [olive oil and salad oil (canola:soy bean oil = 50:50)]; therefore, the dispersion of RBX crystals in these liquid oils was much finer than that of CRX and CLX crystals. Organogels formed when the mixture of every plant wax and liquid oil was melted at elevated temperature and cooled to ambient temperature. However, the mixture of RBX and olive oil at a concentration ratio of 1:99 wt.% formed an organogel at 20 °C, whereas the lowest concentration necessary for CRX to form an organogel in olive oil was 4 wt.% and that for CLX was 2 wt.%. Observation of the rate of gel formation using DSC and viscosity measurements indicated that the gel structure formed soon after RBX crystallized, whereas a time delay was observed between the organogel formation and wax crystallization of CRX and CLX. These results demonstrate RBX’s good organogel-forming properties, mostly because of its fine dispersion of long needle like crystals in liquid oil phases.     

Extraction of rice bran oil using aqueous media

Aqueous extraction of oil from rice bran was studied on a laboratory scale and the resulting product was examined. The following process parameters influencing oil extraction were individually investigated: pH of aqueous media, extraction temperature, extraction time, agitation speed and rice bran‐to‐water ratio. Extraction temperature and pH were found to be the main factors influencing oil extraction. The highest oil yield was obtained at pH 12.0, extraction temperature 50 °C, extraction time 30 min, agitation speed 1000 rpm, and rice bran‐to‐water ratio 1.5‐to‐10. The quality of aqueous‐extracted oil in terms of free fatty acid, iodine value and saponification value was similar to a commercial sample of rice bran oil and hexane‐extracted oil, but the peroxide value was higher. Furthermore, the colour of aqueous‐extracted oil was paler than solvent‐extracted oil. © 2000 Society of Chemical Industry     

Immobilization of proteolytic enzyme on highly porous activated carbon derived from rice bran

Highly porous activated carbon (HPAC) was used as carrier matrix for immobilization of acid protease (AP). Immobilization of acid protease on mesoporous activated carbon (AP-HPAC) performs as best enzyme carrier. At pH 6.0, 250 mg acid protease g⁻¹ HPAC was immobilized. The optimum temperature for both free and immobilized AP activities were 50 °C. After incubation at 50 °C, the immobilized AP maintained about 50% of its initial activity, while the free enzyme was completely inactivated. When testing the reusability of AP-HPAC combination immobilized system, a significant catalytic efficiency was maintained along more than five consecutive reaction cycles. The highly porous nature of the carbon permits significant higher loadings of enzyme, which results in a higher enzyme-support strength and increased stability. The changes in the AP, HPAC and AP-HPAC were confirmed by Fourier Transform Infrared spectroscopy (FT-IR). Furthermore, scanning electron microscopy (SEM) allowed us to observe that the morphology of the surface of HPAC and the AP-HPAC.     

Immobilization and stabilization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K _M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V _max of immobilized enzyme (142.8 μmol min⁻¹ mg⁻¹), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.