Enzymatic preparation of L‐α‐glycerylphosphorylcholine in an aqueous medium

L ‐α‐Glycerylphosphorylcholine (L ‐α‐GPC) was successfully prepared from phosphatidylcholine (PC) of food‐grade soy lecithin powder using a novel enzymatic reaction in an aqueous medium. 94.5% yield of L ‐α‐GPC was obtained under the optimal conditions of 55°C, 6.67 mg/mL substrate, 2 mM CaCl₂, and 33.4 U/mL phospholipase A₁ (Lecitase Ultra). L ‐α‐GPC at 98% purity, 73.4% (wt%) recovery, and specific rotation ( ) of ?2.5° was achieved by silica gel column chromatography. Owing to its excellent catalytic efficiency, low cost, and ready availability, phospholipase A₁ (Lecitase Ultra) provides a very satisfactory option for converting PC to L ‐α‐GPC. Practical applications: L ‐α‐Glycerylphosphorylcholine (L ‐α‐GPC) has been studied recently for its potential use as a supplement that may support neurological functions, but it is only found in trace amounts in nature. The present results indicate that Lecitase Ultra can be used for producing L ‐α‐GPC from aqueous PC and suggest encouraging prospects for practical or industrial applications utilizing its notable catalytic performance, economy, and convenience.     

Evaluation of the Oxidative Stability of Diacylglycerol-Enriched Soybean Oil and Palm Olein Under Rancimat-Accelerated Oxidation Conditions

The oxidative stability of diacylglycerol (DAG)-enriched soybean oil and palm olein produced by partial hydrolysis using phospholipase A1 (Lecitase Ultra) and molecular distillation was investigated at 110 °C by the Rancimat method with and without addition of synthetic antioxidants. Compared with triacylglycerol oils, the DAG-enriched oils displayed lower oxidative stability due to a higher content of unsaturated fatty acids and a lower level of tocopherols. With the addition (50–200 mg/kg) of tert-butylhydroquinone (TBHQ) or ascorbyl palmitate (AP), the oxidative stability indicated by induction period (IP) of these DAG-enriched oils under the Rancimat conditions was improved. The IP of the diacylglycerol-enriched soybean oil increased from 4.21 ± 0.09 to 12.64 ± 0.42 h when 200 mg/kg of TBHQ was added, whereas the IP of the diacylglycerol-enriched palm olein increased from 5.35 ± 0.21 to 16.24 ± 0.55 h when the same level of AP was added. Addition of TBHQ, alone and in combination with AP resulted in a significant (p ≤ 0.05) increase in oxidative stability of diacylglycerol-enriched soybean oil. AP had a positive synergistic effect when used with TBHQ.     

Fast synthesis of 1,3‐DAG by Lecitase® Ultra‐catalyzed esterification in solvent‐free system

Lecitase® Ultra, a phospholipase, was explored as an effective biocatalyst for direct esterification of glycerol with oleic acid to produce 1,3‐DAG. Experiments were carried out in batch mode, and optimal reaction conditions were evaluated. In comparison with several organic solvent mediums, the solvent‐free system was found to be more beneficial for this esterification reaction, which was further studied to investigate the reaction conditions including oleic acid/glycerol mole ratio, temperature, initial water content, enzyme load, and operating time. The results showed that Lecitase® Ultra catalyzed a fast synthesis of 1,3‐DAG by direct esterification in a solvent‐free medium. Under the optimal reaction conditions, a short reaction time 1.5 h was found to achieve the fatty acid esterification efficiency of 80.3 ± 1.2% and 1,3‐DAG content of 54.8 ± 1.6 wt% (lipid layer of reaction mixture mass). The reusability of Lecitase® Ultra was evaluated via recycling the excess glycerol layer in the reaction system. DAG in the upper lipid layer of reaction mixture was purified by molecular distillation and the 1,3‐DAG‐enriched oil with a purity of about 75 wt% was obtained. Practical applications: The new Lecitase® Ultra catalyzed process for production of 1,3‐DAG from glycerol and oleic acid described in this study provides several advantages over conventional methods including short reaction time, the absence of a solvents and a high product yield.     

Short‐path distillation of palm olein and characterization of products

Short‐path distillation (SPD) has been a technique used to purify products containing monoacylglycerols (MAG), diacylglycerols (DAG), etc. Palm oil and its fractions contain high contents of DAG, typically 5–8%, some of which have significant effects on the crystallization behavior of the fats. A possible way of reducing the DAG to lower levels using SPD is evaluated. Distillation of refined, bleached and deodorized palm olein was performed at different temperatures (220–250 °C) and flow rates (500 and 1000 g/h). Feed oil, residue oil and distillates were characterized in terms of composition and melting and cooling behavior. The DAG content of the feed oil was 6.5%. At high evaporating temperatures, the free fatty acid (FFA) concentration in the residue oil and the distillate oil decreased for the same flow rate. Increasing the feed flow rate while maintaining constant temperature led to a greater FFA concentration in both streams. The DAG content in the distillate increased at higher temperature, reaching 68% at 250 °C, while the residue oil achieved a level of 2.8% at lower flow feeding rates. Melting and cooling behavior were influenced by the composition of DAG and triacylglycerols. Thus, the distillate oils had higher melting profiles in contrast to the feed oil and the residue oil, which had similar profiles despite the removal of higher‐melting components.     

Physical properties of palm‐based diacylglycerol and palm‐based oils in the preparation of shelf‐stable margarine

Ternary mixtures containing palm olein (POL), palm kernel oil (PKO) and palm oil‐based diacylglycerol (PO‐DAG) were designed using mixture design. The corresponding physical properties such as solid fat content (SFC) as well as deviation from SFC (ΔSFC) using nuclear magnetic resonance (NMR) and melting and crystallization properties using differential scanning calorimetry (DSC) were studied. Ternary phase behaviour was analysed using isosolid diagrams. The most intensive eutectic interaction among the three binary blends studied was observed along the binary line of PKO/PO‐DAG followed by POL/PKO and POL/PO‐DAG. The higher ΔSFC did not always lead to the more intensive eutectic behaviour among the blends. Addition of pure POL, 33.33 and 66.66% POL, and no POL to 50/50 mixture of PKO/PO‐DAG decreased heat of crystallization (ΔHc) as well as crystallization onset (T_O). However, as the same amounts of PO‐DAG and PKO were added to the 50/50 mixtures of POL/PKO and POL/PO‐DAG, respectively, blend containing the equi‐mixture of POL, PKO and PO‐DAG (33.33/33.33/33.33) was found to have the lowest ΔHc. This was also reflected in the corresponding eutectic effect observed at 20–25 and 5–10°C, respectively. Palm‐based DAG‐enriched shelf‐stable margarine consisting of POL/PKO/PO‐DAG (42.5/42.5/15 w/w) was optimally formulated through analysis of multiple isosolid diagrams and was found to have quite similar SFC profile with commercial shelf‐stable margarine. Practical applications: In this study, valuable information about complicated interactions among the palm oil‐based diacylglycerol (PO‐DAG) and palm‐based oils with different FA chain length was obtained in the ternary system. These informative data may be useful in future exploitation of solid fat‐based DAG in blend with natural fats for various DAG‐enriched plastic fat products. Furthermore, Design Expert software was found to be a valuable tool to optimize the new fat blend formulation using the minimum number of blend preparation. By using this tool, assessment of complicated behaviour among the blend components through construction of the corresponding phase diagrams which are critical for optimization purposes as well as fat product development, would also be possible.     

Preparation of Diacylglycerol-Enriched Oil from Free Fatty Acids Using Lecitase Ultra-Catalyzed Esterification

Production of diacylglycerol-enriched oil by esterification of free fatty acids (FFA) with glycerol (GLY) using phospholipase A1 (Lecitase Ultra) was investigated in this work. The variables including reaction time (2–10 h), water content (2–14 wt%, FFA and GLY mass), enzyme load (10–120 U/g, FFA and GLY mass), reaction temperature (30–70 °C) and mole ratio of GLY to FFA (0.5–2.5) were studied. The optimum conditions obtained were as follows: reaction temperature 40 °C, water content 8 wt%, reaction time 6 h, molar ratio of GLY to FFA 2.0, and an enzyme load of 80 U/g. Under these conditions, the esterification efficiency (EE) of free fatty acids was 74.8%. The compositions of the FFA and acylglycerols of the upper oil layer (crude diacylglycerol) of the reaction mixture were determined using a high temperature gas chromatograph (GC). The crude diacylglycerol from the selected conditions was molecularly distilled at 170 °C evaporator temperatures to produce a diacylglycerol-enrich oil (DEO) with a purity of 83.1% and a yield of 42.7%.     

Synthesis of palm oil‐based polyester polyol for polyurethane adhesive production

Palm oil‐based polyester polyol is synthesized by ring opening reaction on epoxidized palm olein by phthalic acid. The reaction is carried out in a solvent free and noncatalyzed condition with the optimal reaction condition at 175°C for 5 h reaction time. The physical state of the product is a clear bright yellowish liquid with low viscosity value of 5700–6700 cP at 25°C and pour point of 15°C. The chemical structure and molecular weight of the polyester polyol were characterized by FTIR, ¹H‐NMR, ¹³C‐NMR, and GPC. The optimal polyol with molecular weight of 36,308 dalton and hydroxyl value of 78.17 mg KOH/g sample was reacted with polymeric 4,4′‐methylene diphenyl diisocyanate (pMDI) at isocyanate index of 1.3 to produce polyurethane adhesive. The lap shear strength of the polyurethane adhesive showed two times higher than the commercial wood adhesives. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2014 , 131, 39967.     

Application of palm olein in the production of zero‐trans Iranian vanaspati through enzymatic interesterification

The utilization of palm olein in the production of zero‐trans Iranian vanaspati through enzymatic interesterification was studied. Vanaspati fat was made from ternary blends of palm olein (POL), low‐erucic acid rapeseed oil (RSO) and sunflower oil (SFO) through direct interesterification of the blends or by blending interesterified POL with RSO and SFO. The slip melting point (SMP), the solid fat content (SFC) at 10–40 °C, the carbon number (CN) triacylglycerol (TAG) composition, the induction period (IP) of oxidation at 120 °C (IP₁₂₀) and the IP of crystallization at 20 °C of the final products and non‐interesterified blends were evaluated. Results indicated that all the final products had higher SMP, SFC, IP of crystallization and CN 48 TAG (trisaturated TAG), and lower IP₁₂₀, than their non‐interesterified blends. However, SMP, SFC, IP₁₂₀, IP of crystallization and CN 48 TAG were higher for fats prepared by blending interesterified POL with RSO and SFO. A comparison between the SFC at 20–30 °C of the final products and those of a commercial low‐trans Iranian vanaspati showed that the least saturated fatty acid content necessary to achieve a zero‐trans fat suitable for use as Iranian vanaspati was 37.2% for directly interesterified blends and 28.8% for fats prepared by blending interesterified POL with liquid oils.     

High‐pressure enzymatic hydrolysis of oil

The hydrolysis of sunflower and soybean oil, catalyzed by two enzymes, non‐immobilized Candida rugosa and immobilized Candida antarctica lipase, was performed at atmospheric and high‐pressure. The results showed that at atmospheric pressure between 40 °C and 60 °C initial reaction rates were influenced by the temperature variation, as expected. Due to favorable physico‐chemical properties of dense gases as reaction media, hydrolysis of soybean oil was performed in non‐conventional solvents: in supercritical (SC) CO₂ and near‐critical propane. In SC CO₂ the activity of non‐immobilized Candida rugosa lipase decreased while the reaction rates of hydrolysis catalyzed by immobilized Candida antarctica lipase were 1.5‐fold higher than at atmospheric pressure. However, the reaction rates for the hydrolyses catalyzed by both lipases, were much higher in propane than at atmospheric pressure.     

Enzymatic Glycerolysis of Palm and Palm Kernel Oils

Glycerolysis of palm and palm kernel oils were carried out using commercial lipases from Candida antarctica (Novozym 435) and Mucor miehei (Novozym 388) as catalyst (500 units lipase/g oil) at 40°C and with an oil:glycerol molar ratio of 1:2 in a solvent-free system. Novozym 435 catalyzed the glycerolysis of palm and palm kernel oils giving reaction products in similar compositions. Partial acylglycerols contents of the glycerolysis products obtained from palm and palm kernel oils were 64% (wt) and 66% (wt), respectively. However, partial acylglycerols contents of the glycerolysis products obtained from palm and palm kernel oils conducted with Novozym 388 as catalyst at the same conditions were 44% (wt) and 56% (wt), respectively. On the other hand, free fatty acid contents of the glycerolysis products of palm and palm kernel oils obtained using Novozym 388 were higher, 25–30% (wt), than those obtained by Novozym 435, 4–5% (wt). The monoacylglycerols fraction with the highest content of oleic acid, 62.7% (wt), was obtained from the palm kernel oil glycerolysis reaction catalyzed by Novozym 435.     

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.     

Solubilization of methanol and ethanol in palm oil stabilized by medium‐ and long‐chain alkanols

Solubilization of methanol and ethanol in crude palm oil, refined, bleached and deodorized palm oil (RBD PO) and RBD palm olein (PO_L) was studied using medium‐ and long‐chain alkanols (C₄–C₁₂). Ternary phase diagrams were constructed to determine the solubilization (isotropic) region. The results showed that methanol and ethanol are solubilized to a greater extent in an unsaturated palm olein than the saturated CPO and RBD PO in the presence of long‐chain alkanols. The minima of the solubilization curves for dodecanol, decanol and octanol were 27%, 30% and 33% of alkanol respectively in the methanol system, whereas in the ethanol system, the minima for the same alkanols were found at 22%, 24% and 27%. The longer chain‐length alkanol (dodecanol) requires a lesser amount (21% and 32%) to achieve miscibility compared with 53% and 57% for butanol in mixtures containing 70:30 and 50:50 wt ratios respectively. The kinematic viscosity of the isotropic solutions increases with the chain‐length and percentage of alkanols. Solubilization using a PO_L/methanol/butanol system significantly reduced the kinematic viscosity of PO_L from 72.7 mm² s^?1 to the value allowable for No 2 diesel fuel (1.9–4.1 mm² s^?1) or about a 96% reduction from the initial kinematic viscosity of PO_L. © 2002 Society of Chemical Industry     

Physico‐chemical properties of palm olein fractions as a function of diglyceride content in the starting material

Crude olein preparations with different amounts of diacylglycerols (DAG) were refined, bleached and deodorized (RBD) prior to the dry fractionation process. The RBD olein samples with different amounts of DAG were then individually fractionated into low‐melting (super olein) and high‐melting fractions (soft stearin). Physical and chemical characteristics, i.e. iodine value, cloud point, slip melting point, triacylglycerol (TAG) and DAG profile, fatty acid composition, thermal profile and solid fat content, of the super olein and soft stearin fractions were analyzed. The TAG profile obtained from the RBD olein having a low DAG content (0.89%) showed a higher amount of the diunsaturated TAG, i.e. dioleyl pamitoyl glycerol, in the olein fraction (57.3%). This, consequently, led to super olein fractions with a better iodine value (IV 65) and the cloud point at 1.3 °C, compared to non‐treated super olein (DAG 5%) with an IV of 60.5 and the cloud point at 4.1 °C.     

Lipase/acyltransferase‐catalysed interesterification of fat blends containing n‐3 polyunsaturated fatty acids

The lipase/acyltransferase from Candida parapsilosis is an original biocatalyst that preferentially catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. In this study, the performance of the immobilised biocatalyst in the interesterification in solvent‐free media of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA) was investigated. The interesterification activity of this biocatalyst at a water activity (a_w) of 0.97 was similar to that of commercial immobilised lipases at a_w values lower than 0.5. Thus, the biocatalyst was further used at an a_w of 0.97. Response surface modelling of interesterification was carried out as a function of medium formulation, reaction temperature (55–75 °C) and time (30–120 min). Reaction media were blends of palm stearin (PS), palm kernel oil and triacylglycerols (TAG) rich in n‐3 PUFA (“EPAX 4510TG”; EPAX AS, Norway). The best results in terms of decrease in solid fat content were observed for longer reaction time (>80 min), lower temperature (55–65 °C), higher “EPAX 4510TG” content and lower PS concentration. Reactions at higher temperature led to final interesterified fat blends with lower free fatty acid contents. TAG with high equivalent carbon number (ECN) were consumed while acylglycerols of lower ECN were produced.     

Noncatalytic Hydrolysis of Iminodiacetonitrile in Near‐Critical Water – A Green Process for the Manufacture of Iminodiacetic Acid

The hydrolysis of iminodiacetonitrile (IDAN) in near‐critical water, without added catalysts, has been successfully conducted with temperature and residence time ranges of 200–260 °C and 10–60 min, respectively. The effects of temperature, pressure, and initial reactant/water ratio on the reaction rate and yield have been investigated. The final reaction products primarily included iminodiacetic acid (IDA) and ammonia associated with other by‐products; gas formation was negligible. The maximum yield of IDA was 92.3 mol.‐% at 210 °C and 10 MPa, with a conversion of almost 100 %.The apparent activation energy and ln A of IDAN hydrolysis were evaluated as 45.77 ± 5.26 kJ/mol and 8.6 ± 0.1 min^–1, respectively, based on the assumption of first‐order reaction. The reaction mechanism and scheme were similar to those of base‐catalyzed reactions of nitriles examined in less severe conditions.     

Lipase‐catalysed production and chemical composition of diacylglycerols from soybean oil deodoriser distillate

Diacylglycerols (DAG) were enzymatically produced by lipase‐catalysed esterification of glycerol with fatty acids from soybean oil deodoriser distillate (SODD). Effects of reaction parameters such as reaction time, temperature, enzyme type, enzyme load, substrate molar ratio and water content, as well as the effect of molecular sieves as water adsorbent were studied. Lipozyme RM IM was determined to be the most effective among the lipases screened. The following conditions yielded 69.9% DAG (all percentages are wt/wt): 4 h reaction time, 65 °C reaction temperature, 10% Lipozyme RM IM, 2.5:1 fatty acid to glycerol molar ratio, and 30% molecular sieves. DAG synthesis of 11.9% was still observed at 10% water content. After purification, the product oil contained 86.3% DAG. This oil consisted predominantly of 1,3‐diolein (19.1%), 1‐oleoyl‐3‐linoleoyl‐glycerol (18.2%) and 1‐oleoyl‐2‐linoleoyl‐glycerol (16.6%). The fatty acid profile of the oil was similar to that of refined, bleached and deodorised (RBD) soybean oil. The % ratio of 1,3‐ to 1,2‐positional isomers of DAG was at 56:44.     

Fast Production of Diacylglycerol in a Solvent Free System via Lipase Catalyzed Esterification Using a Bubble Column Reactor

In this study, diacylglycerols (DAG) were synthesized rapidly (~30 min) in a solvent‐free system via esterification of glycerol with fatty acids (FA, the mixture of 60 wt% palm oil deodorizer distillate and 40 wt% oleic acid) catalyzed by Lipozyme 435 (Novozymes A/S, Copenhagen, Denmark) using a bubble column reactor. The content of DAG, monoacylglycerols (MAG), triacylglycerols (TAG) and free fatty acids (FFA) in the crude product were 57.94 ± 1.60 wt%, 24.68 ± 2.08 wt%, 2.67 ± 1.72 wt% and 14.69 ± 1.22 wt%, respectively under the selected conditions, which were enzyme load of 5.0 wt%, glycerol/FA mole ratio of 7.5, initial water content of 2.5 wt%, reaction temperature of 60 °C, reaction time of 30 min and N₂ gas flow of 10.6 cm min^?1. The final product containing 91.30 ± 1.10 wt% of DAG was obtained by one‐step molecular distillation at 200 °C. The reusability of Lipozyme 435 was investigated by evaluating the esterification degree (ED) and the DAG content in the crude products in 30 successive runs. The enzyme retained 95.10 % of its original activity during 30 successive runs according to comparison of the ED. The new process showed a very high efficiency in production of DAG with a high purity. The ratio of positional isomers 1,3‐DAG to 1,2 ‐DAG was 2:1 in the final product. The certain plasticity (melting point of 44 °C) and content of unsaturated fatty acids made the product a valuable food ingredient.     

Synthesis of water‐reducible acrylic–alkyd resins based on modified palm oil

Water‐reducible acrylic–alkyd resins were synthesized from the reaction between monoglycerides prepared from modified palm oil and carboxy‐functional acrylic copolymer followed by neutralization of carboxyl groups with diethanolamine. Modified palm oil was produced by interesterification of palm oil with tung oil at a weight ratio of 1 : 1, using sodium hydroxide as a catalyst, whereas carboxy‐functional acrylic copolymer was prepared by radical copolymerization of n‐butyl methacrylate and maleic anhydride. The amount of acrylic copolymer used was from 15 to 40% by weight, and it was found that homogeneous resins was obtained when the copolymer content was 20–35 wt %. All of the prepared water‐reducible acrylic–alkyd resins were yellowish viscous liquids. Their films were dried by baking at 190°C and their properties were determined. These films showed excellent water and acid resistance and good alkali resistance. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1170–1175, 2005     

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.     

Improvement of mono and diacylglycerol production via enzymatic glycerolysis in tert‐butanol system

In this work we report experimental data regarding the glycerolysis of olive oil using Novozym 435 in tert‐butanol organic system aiming at the production of monoacylglycerols (MAG) and diacylglycerols (DAG). Experiments were performed in batch mode, recording the reaction kinetics and evaluating the effects of temperature, enzyme concentration, tert‐butanol:oil/glycerol volume ratio and using solvent to substrates ratio of 1:1 and 5:1 v/v. Experimental results showed that lipase‐catalyzed glycerolysis in tert‐butanol might be a potential route for the production of high contents of MAG and DAG. The results also showed that it is possible to maximize the production of MAG and/or DAG, depending on the glycerol to oil molar ratio employed in the reactional system. Higher contents of MAG (53 wt%) and DAG (50 wt%) were achieved using glycerol to oil molar ratio of 3:1/6:1 and 0.5:1.5, respectively, both in 8 h of reaction at 70°C, 600 rpm and enzyme concentration of 10 wt%.