Lipase‐catalyzed production of medium‐chain triacylglycerols from palm kernel oil distillate: Optimization using response surface methodology

Optimization of lipase‐catalyzed esterification for the production of medium‐chain triacylglycerols (MCT) from palm kernel oil distillate and glycerol was carried out in order to determine the factors that have significant effects on the reaction system and MCT yield. Novozyme 435 from Candida antarctica lipase was found to have the highest activity at 52.87 ± 0.03 U/g. This lipase also produced the highest MCT yield, which is 56.67%. The effect of different variables on MCT synthesis was studied with a two‐level five‐factor fractional factorial design. The various variables include (1) reaction temperature, (2) enzyme load, (3) molecular sieves concentration, (4) reaction time and (5) molar substrate ratio. Reaction temperature, reaction time and molar substrate ratio strongly affect MCT synthesis (p <0.05). However, enzyme load and molecular sieve concentration did not have a significant (p >0.05) influence on MCT yield. Therefore, the significant variables such as reaction temperature, reaction time and molar substrate ratio were further optimized through central composite rotatable design (CCRD). Comparisons between predicted and experimental values from the CCRD optimization procedures revealed good correlation, implying that the quadric response model satisfactorily expressed the percentage yield of MCT in the lipase‐catalyzed esterification. The optimum MCT yield is 73.3% by using 2 wt‐% enzyme dosage, a molecular sieves concentration of 1 wt‐%, a reaction temperature of 90 °C, a reaction time of 10 h and a molar substrate ratio of 4 : 1 (medium‐chain fatty acid/glycerol). Experiments to confirm the predicted results using the optimal parameters were conducted and an MCT yield of 70.21 ± 0.18% (n = 3) was obtained.     

Production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor

A laboratory-scale continuous reactor was constructed for production of specific structured triacylglycerols containing essential fatty acids and medium-chain fatty acids (MCFA) in the sn-2 and sn-1,3 positions, respectively. Different parameters in the lipase-catalyzed interesterification were elucidated. The reaction time was the most critical factor. Longer reaction time resulted in higher yield, but was accompanied by increased acyl migration. The concentration of the desired triacylglycerol (TAG) in the interesterification product increased significantly with reaction time, even though there was only a slight increase in the incorporation of MCFA. Increased reactor temperature and content of MCFA in the initial reaction substrate improved the incorporation of MCFA and the yield of the desired TAG in the products. Little increase of acyl migration was observed. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA, or on the resulting composition of TAG products and their free fatty acid content. Therefore, we conclude that the water in the original reaction substrate is sufficient to maintain the enzyme activity in this continuous reactor. Since the substrates were contacted with a large amount of lipase, the reaction time was shorter compared with a batch reactor, resulting in reduced acyl migration. Consequently, the purity of the specific structured TAG produced was improved. Interesterification of various vegetable oils and caprylic acid also demonstrated that the incorporation was affected by the reaction media. Reaction conditions for lipase-catalyzed synthesis of specific structured TAG should be optimized according to the oil in use. Presented in part at Food Science Conference, Copenhagen, Denmark, January 30–31, 1997.     

Production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor

A laboratory-scale continuous reactor was constructed for production of specific structured triacylglycerols containing essential fatty acids and medium-chain fatty acids (MCFA) in the sn-2 and sn-1,3 positions, respectively. Different parameters in the lipase-catalyzed interesterification were elucidated. The reaction time was the most critical factor. Longer reaction time resulted in higher yield, but was accompanied by increased acyl migration. The concentration of the desired triacylglycerol (TAG) in the interesterification product increased significantly with reaction time, even though there was only a slight increase in the incorporation of MCFA. Increased reactor temperature and content of MCFA in the initial reaction substrate improved the incorporation of MCFA and the yield of the desired TAG in the products. Little increase of acyl migration was observed. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA, or on the resulting composition of TAG products and their free fatty acid content. Therefore, we conclude that the water in the original reaction substrate is sufficient to maintain the enzyme activity in this continuous reactor. Since the substrates were contacted with a large amount of lipase, the reaction time was shorter compared with a batch reactor, resulting in reduced acyl migration. Consequently, the purity of the specific structured TAG produced was improved. Interesterification of various vegetable oils and caprylic acid also demonstrated that the incorporation was affected by the reaction media. Reaction conditions for lipase-catalyzed synthesis of specific structured TAG should be optimized according to the oil in use. Presented in part at Food Science Conference, Copenhagen, Denmark, January 30–31, 1997.     

Interesterification and hydrolysis catalyzed by fatty acid‐modified lipases

Native lipases often exhibit poor interesterification activity. We previously developed a fatty acid modification method to improve the activity of lipases. In this study, we applied this fatty acid modification method to several lipases and evaluated their interesterification and hydrolytic activities. The resulting interesterification activity was strongly dependent on the modifying fatty acid used. Of the saturated fatty acids tested, stearic acid modification substantially improved the interesterification activity of three lipases. Hydrolytic activity was affected slightly by the modifying fatty acid used. Substrate specificity of the modified lipase with triglycerides was also investigated and it was found that fatty acid modification changed the substrate specificity of some lipases.     

Production of specific‐structured triacylglycerols by lipase‐catalyzed reactions: a review

Regiospecificity is one of the major advantages of using lipase technology for the modification of oils and fats to produce high‐value added products, such as cocoa butter equivalents, human milk fat substitutes, and other specific‐structured lipids. Due to the high cost of biocatalysts, the mainstream applications of lipases for normal oils and fats are still limited. Therefore, positional specificity of lipases has the priority and will be the target property to be exploited for commercial and industrial developments, because no chemical method has such a specificity and is promising or possible for this task. In this paper, encouraging products resulting from this regiospecificity are reviewed together with the critical evaluation of their reaction schemes, side reactions and by‐products, sources of substrate oils and acyl donors, and production processes.     

Production of structured triacylglycerols in an immobilised lipase packed‐bed reactor: batch mode operation

Structured triacylglycerols with caprylic acid at the sn‐1 and sn‐3 positions of the glycerol backbone and eicosapentaenoic acid (EPA) at the position sn‐2 were synthesised by acidolysis of a commercially available EPA‐rich oil (EPAX4510, Pronova Biocare) and caprylic acid catalysed by the 1,3‐specific immobilised lipase Lipozyme IM. The reaction was carried out in an immobilised lipase packed‐bed reactor by recirculating the reaction mixture through the bed. The exchange equilibrium constants between caprylic acid and the native fatty acids of EPAX4510 were determined. The n‐3 polyunsaturated fatty acids (PUFAs), EPA and docosohexaenoic acid (DHA), were the most easily displaced by the caprylic acid. The exchange equilibrium constants were 3.68 and 3.06 for EPA and DHA, respectively. The influence of the flow rate of the reaction mixture through the packed‐bed and the substrate concentration in the reaction rate were studied. For flow rates between 74 and 196 cm³ h^?1 (bed of 6.6 mm internal diameter and 0.46 porosity) and triacylglycerol concentrations between 0.036 and 0.108 M , the data fitted well to an empirical kinetic model which allowed representative values of the apparent kinetic constant to be obtained. Hence, the average reaction rates and kinetic constants of exchange of caprylic acid and native fatty acids of EPAX4510 could be calculated. In the conditions indicated, the parameter (lipase mass × time/triacylglycerol mass, m_Lt/V[TG]₀) constituted the intensive variable of the process for use in predicting the composition of structured triacylglycerols at different reaction times. At equilibrium, the structured triacylglycerol produced had the following composition: caprylic acid 59.5%, EPA 9.6%, DHA 2.2% and oleic acid 11.8%. Copyright © 2004 Society of Chemical Industry     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

Operational stability of Thermomyces lanuginosa lipase during interesterification of fat in continuous packed‐bed reactors

The operational stability of a commercial immobilized lipase from Thermomyces lanuginosa (“Lipozyme TL IM”) during the interesterification of two fat blends, in solvent‐free media, in a continuous packed‐bed reactor, was investigated. Blend A was a mixture of palm stearin (POS), palm kernel oil (PK) and sunflower oil (55 : 25 : 20, wt‐%) and blend B was formed by POS, PK and a concentrate of triacylglycerols rich in n‐3 polyunsaturated fatty acids (PUFA) (55 : 35 : 10, wt‐%). The bioreactor operated continuously at 70 °C, for 580 h (blend A) and 390 h (blend B), at a residence time of 15 min. Biocatalyst activity was evaluated in terms of the decrease of the solid fat content at 35 °C of the blends, which is a key parameter in margarine manufacture. The inactivation profile of the biocatalyst could be well described by the first‐order deactivation model: Half‐lives of 135 h and 77 h were estimated when fat blends A and B, respectively, were used. Higher levels of PUFA in blend B, which are rather prone to oxidation, may explain the lower lipase stability when this mixture was used. The free fatty acid content of the interesterified blends decreased to about 1% during the first day of operation, remaining constant thereafter.     

Interesterification of sesame oil and a fully hydrogenated fat using an immobilized lipase catalyst in both batch and continuous‐flow processes

Lipase‐mediated interesterification of sesame oil and a fully hydrogenated soybean oil was studied at 70 °C in both a batch reactor (BR) and a continuous‐flow packed‐bed reactor (PBR) using four different initial weight ratios of substrates (90 : 10, 80 : 20, 70 : 30 and 60 : 40) with Lipozyme TL IM (Thermomyces lanuginosa) as the biocatalyst. Reaction rates were determined by following the dependence of the profile of the product triacylglycerols (TAG) on the reaction time (BR) or the space time (PBR) via RP‐HPLC‐ELSD. Product TAG identities were confirmed by HPLC‐APCI‐MS. Primary differences between the performances of the two reactors were the maximum level of net hydrolysis (ca. 3 and 10 wt‐% lower acylglycerols at equilibrium for the PBR and BR, respectively), the time or space time required to approach quasi‐equilibrium conditions, and less migration of acyl groups in the PBR trials. For the BR trials, quasi‐equilibrium conditions were approached in 4–6 h, while for the PBR trials short space times (15 min to 2 h) were sufficient to produce effluent compositions similar to equilibrium BR compositions. The predominant TAG families formed by interesterification were LLS, PSO, PSL, SSL, and SSO (L = linoleic; S = stearic; P = palmitic; O = oleic). Oxidative stabilities, melting profiles and solid fat contents were determined for selected reaction products.     

Chemical and enzymatic interesterification of a beef tallow and rapeseed oil equal‐weight blend

A mixture of beef tallow and rapeseed oil (1:1, wt/wt) was interesterified using sodium methoxide or immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (Novozym 435) as catalysts. Chemical interesterifications were carried out at 60 and 90 °C for 0.5 and 1.5 h using 0.4, 0.6 and 1.0 wt‐% CH₃ONa. Enzymatic interesterifications were carried out at 60 °C for 8 h with Lipozyme IM or at 80 °C for 4 h with Novozym 435. The biocatalyst doses were kept constant (8 wt‐%), but the water content was varied from 2 to 10 wt‐%. The starting mixture and the interesterified products were separated by column chromatography into a pure triacylglycerol fraction and a nontriacylglycerol fraction, which contained free fatty acids, mono‐, and diacylglycerols. It was found that the concentration of free fatty acids and partial acylglycerols increased after interesterification. The slip melting points and solid fat contents of the triacylglycerol fractions isolated from interesterified fats were lower compared with the nonesterified blends. The sn‐2 and sn‐1,3 distribution of fatty acids in the TAG fractions before and after interesterification were determined. These distributions were random after chemical interesterification and near random when Novozym 435 was used. When Lipozyme IM was used, the fatty acid composition at the sn‐2 position remained practically unchanged, compared with the starting blend. The interesterified fats and isolated triacylglycerols had reduced oxidative stabilities, as assessed by Rancimat induction times. Addition of 0.02% BHA and BHT to the interesterified fats improved their stabilities.     

Kinetics of lipase‐catalysed interesterification of triolein and caprylic acid to produce structured lipids

The influence of the molar ratio caprylic acid/triolein, enzyme concentration and water content on the kinetics of the interesterification reaction of triolein (TO) and caprylic acid (CA) were studied. The enzyme used was the 1,3‐specific Rhizomucor miehei lipase. Data modelling was based on a simple scheme in which the acid was only incorporated in positions 1 and 3 of the glyceride backbone. In addition, it was assumed that positions 1 and 3 of the triglycerides were equivalent and that the events at position 1 did not depend on the nature of the fatty acid in position 3 and vice versa. Monoglycerides and diglycerides were not detected during the experiments. This was attributed to the low water content of the immobilised enzyme particles. The value of the equilibrium constant, K, for the exchange of caprylic and oleic acids was 2.7, which indicated that the incorporation of caprylic acid into triglycerides was favoured compared with the incorporation of oleic acid. Simple first order kinetics could describe the interesterification reaction. Using this model and the calculated equilibrium constant, the apparent kinetic constants were calculated. The model fitted all the experimental data except for the CA/TO molar ratios larger than 6. Moreover, the interesterification reaction rate had a maximum value at CA/TO molar ratios of 4–6 mol mol^?1. Copyright © 2003 Society of Chemical Industry     

Proposed kinetic mechanism of biodiesel production through lipase catalysed interesterification with a methyl acetate acyl acceptor and ionic liquid [BMIM][PF6] co‐solvent

The production of biodiesel was investigated using a lipase‐catalysed (Novozym 435) reaction involving methyl acetate and ionic liquid [BMIM][PF₆] as a co‐solvent to produce an environmentally friendly, “green” process. Experiments were conducted at various amounts of ionic liquid. The reaction mechanism was examined through use of kinetic modelling and the effect of ionic liquid was studied for the first time. Studies indicated that the reaction followed a Ping–Pong Bi–Bi mechanism, and that the ionic liquid present in the system led to reduced initial reaction rates due to mass transfer limitations.     

Formation of short‐chain glycerol‐bound oxidation products and oxidised monomeric triacylglycerols during deep‐frying and occurrence in used frying fats

Heating fats and oils at high temperature in the presence of air, a common procedure in culinary practices such as frying, results in a complex mixture of oxidation products. These compounds may impair the nutritional value of the food. Among them, there is a growing interest in the group of oxidised triacylglycerol monomers because of their high absorbability. The main structures in this group include triacylglycerols (TG) containing short‐chain acyl groups formed by homolytic β‐scission of the alkoxy radicals coming from allylic hydroperoxides. In addition there are TG containing oxidised fatty acyl groups of molecular weight similar to that of their parent TG, i.e., epoxy, keto and hydroxy fatty acyl groups. In this review, the main routes of formation of oxidised TG monomers are detailed. Also, the most relevant advances in the analysis of intact TG molecules by high‐performance liquid chromatography coupled with mass spectrometry are discussed. Special attention is paid to the present analytical possibilities for accurate quantification of the most important oxidised compounds formed at high temperature. Both the need to convert fats and oils into simpler derivatives, thus concentrating the compounds bearing the oxidised structure, and the methylation procedure selected to avoid artefact formation are justified. Typical concentrations of short‐chain fatty acids, short‐chain aldehydic acids, short‐chain diacids, and monoepoxy fatty acids, ketoacids and hydroxyacids in frying oils from restaurants and fried‐food outlets, with polar lipids levels at the limit of rejection for human consumption, are given.     

Alcoholysis of waste fats with 2‐ethyl‐1‐hexanol using Candida antarctica lipase A in large‐scale tests

Commercial native lipase A from Candida antarctica was used to produce alkyl esters through the alcoholysis of (waste) fats with 2‐ethyl‐1‐hexanol. The process was carried out in batch stirred tank reactors (from 100 mL up to 3000 L). The content of alkyl esters in reaction mixtures was determined by gradient HPLC using an evaporative light scattering detector and the reaction progress was controlled by determining the ratio of the palmitic acid ester peak area to the oleic acid ester peak area in HPLC chromatograms. The results show that alcoholysis is the favoured reaction in presence of excess water and water‐insoluble alcohols in comparison with hydrolysis (fatty acid content <5%). The optimum amount of water for the alcoholysis was found to be 80–100% of the amount of fat. In the presence of low quantities of water both alcoholysis and hydrolysis are slow. Conversion rate increases with increasing temperature to 65–70 °C. Based on these results a large‐scale test to produce 3000 L of alkyl ester (to be used as lubricant coolant) was carried out. The experiments have proved that alcoholysis is completed after about 7–10 h depending on temperature.     

Inoculum strategies for Penicillium simplicissimum lipase production by solid‐state fermentation using a residue from the babassu oil industry

Two alternative inoculation strategies for lipase production by the fungus Penicillium simplicissimum were tested in solid‐state fermentation using a residue from the babassu oil industry (babassu cake). Conventional spore inoculation was compared with fungal pellets grown in liquid medium and with inocula consisting of fermented cake. Fungal pellets delayed lipase production whereas fermented cake accelerated enzyme synthesis, yielding a productivity of 0.45 U g^?1 h^?1, which is equivalent to the highest values obtained with conventional inocula. Therefore, a 2² factorial design was used to determine the best conditions for lipase production with fermented cake as inoculum strategy, varying the inoculum propagation time and inoculum concentration. Lipase activity and productivity reached 30 U g^?1 and 0.63 U g^?1 h^?1, respectively, with 10% inoculum and 36 h. Thus, fermented cake inocula increased production 1.5‐fold with 10 times fewer spores than in conventional inoculation, indicating that fermented solids are an interesting alternative for inoculum development in solid‐state fermentation, mainly for large‐scale processes. Copyright © 2007 Society of Chemical Industry     

Novel lipase isolated from a Pseudomonas strain and its application in the synthesis of S(+)‐2‐O‐benzylglycerol‐1‐acetate

Isolation of a novel microbial lipase (EC 3.1.1.3) having specific catalytic activity for the synthesis of optically pure 2‐O‐benzylglycerol‐1‐acetate, the building block for the preparation of many β‐blockers, phospholipase A2 inhibitors and other biologically active compounds was the aim of this investigation. A Pseudomonas (strain G6), recently isolated from soil, produced an extracellular lipase. SDS–PAGE analysis showed that the lipase protein was a hexamer. The molecular weight of the sub‐units of the lipase protein were 10, 19, 29, 30, 47 and 53. The catalytic activity of the lipase was exploited for the synthesis of 2‐O‐benzylglycerol‐1‐acetate from 2‐O‐benzylglycerol through transesterification using vinyl acetate as acylating agent. High selectivity of the lipase towards the monoacetate product was demonstrated. A 97% enantiomeric excess (ee) of S(+)‐2‐O‐benzylglycerol‐1‐acetate was obtained when the reaction was carried out at room temperature with shaking. The lipase was highly active in anhydrous organic microenvironments and in non‐polar organic solvents with log P values above 2.5. © 2002 Society of Chemical Industry     

Architecture and performance of mesoporous silica‐lipase hybrids via non‐covalent interfacial adsorption

To investigate the effects of surface property of mesoporous supports on the lipase immobilization and the performance of immobilized lipase, the mesoporous molecular sieve SBA‐15 is functionalized with three organic moieties, dimethyl (DM), diisopropyl (DIP), and diisobutyl (DIB), respectively, by post‐synthesis grafting and one‐pot synthesis methods. Porcine pancreas lipase (PPL) is immobilized on SBA‐15 supports through hydrogen bonding and hydrophobic interaction. The hydrophobic adsorption involves no active sites of PPL, and neither hyper‐activation nor total inactivation occurs. The study on the intrinsic stability of PPL, including thermal stability, pH stability, and storage stability, indicates that the entrapment in mesoporous supports, and especially in organic‐functionalized supports, makes PPL more resistant to temperature increment but more sensitive to pH change. The reusability investigation shows that the organic modification of mesoporous surface inhibits the enzyme leaching to some extent, resulting in a better operational stability. © 2009 American Institute of Chemical Engineers AIChE J, 2010     

Evaluation of a novel Bacillus strain from a north‐western Spain hot spring as a source of extracellular thermostable lipase

BACKGROUND: Thermophilic microorganisms are receiving significant attention as a source of useful thermostable enzymes. However, the number of known strains is still limited, and very often their most interesting biocatalysts are intracellular or membrane‐bound and produced at low levels. Thus, the isolation and study of novel extracellular enzyme‐producing thermophilic microorganisms is very interesting. Moreover, the assessment of bioreactor performance is crucial, given the scarce information on the large‐scale culture of these strains. RESULTS: The production of a thermostable extracellular lipase in submerged cultures of a thermophilic microorganism, recently isolated in north‐west Spain, was investigated. The strain was identified by 16S rDNA sequencing as belonging to genus Bacillus. The influence of operating variables (i.e. pH, temperature, aeration) on lipase biosynthesis was analysed. Enzyme production at bioreactor scale was investigated, special attention being paid to the effect of aeration and agitation rates. CONCLUSION: The best conditions for the studied process were determined in shake flasks as pH 7.0, 55 °C and high aeration levels. Also, the non‐association between lipase production and cell growth was ascertained. The culture of this novel strain was successfully carried out in laboratory‐scale bioreactors, thus proving its potential for further applications. Copyright © 2009 Society of Chemical Industry