Enzymatic synthesis of structured lipids: Transesterification of triolein and caprylic acid ethyl ester

Structured lipids were successfully synthesized by lipase-catalyzed transesterification (ester interchange) of caprylic acid ethyl ester and triolein. The transesterification reaction was carried out in organic solvent as reaction media. Eight commercially-available lipases (10% w/w substrates) were screened for their ability to synthesize structured lipid by incubating with 100 mg triolein and 78.0 mg caprylic acid ethyl ester in 3 mL hexane at 45°C for 24 h. The products were analyzed by reverse-phase high-performance liquid chromatography with evaporative light-scattering detector. Immobilized lipase IM60 fromRhizomucor miehei converted most triolein into structured lipids (41.7% dicapryloolein, 46.0% monocapryloolein, and 12.3% unreacted triolein). However, lipase SP435 fromCandida antarctica had a higher activity at higher temperature. The reaction catalyzed by lipase SP435 yielded 62.0% dicapryloolein, 33.5% monocapryloolein, and 4.5% unreacted triolein at 55°C. Time course, incubation media, added water, and substrate concentration were also investigated in this study. The results suggest that lipase-catalyzed transesterification of long-chain triglycerides and medium-chain fatty acid ethyl ester is feasible to synthesize structured lipids.     

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     

Enzymatic modification of canola/palm oil mixtures: Effects on the fluidity of the mixture

The bioreactor system to interesterify edible oils and fats at an ultra-micro aqueous phase of 100 ppm and less was investigated. The adsorption of lecithin, together with lipase onto a carrier, was effective for conducting the interesterifying reaction efficiently for oils and fats in micro aqueous phase. To improve the handling properties of palm oil at rather low temperature, palm oil was blended with canola or soybean oil, and then these blended oils were modified by enzymatic selective interesterification in a solvent-free, ultra-micro aqueous bioreactor system with an immobilized lipase that had 1,3-positional specificity. The effects of enzymatic interesterification were confirmed by triglyceride determination, by solid fat content profiles and by cloud point profiles, which were also compared to products of chemical interesterification. The improvement in the fluidity of blended oils with canola oil by the enzymatic reaction was bigger than with soybean oil, and chemical interesterification had no effects on the fluidity of blended oils.     

Production of structured lipids by lipase-catalyzed interesterification in a flat membrane reactor

The application of membrane technology to the enzymatic production of specific structured lipids has been investigated in this work. Membrane screening was carried out in a membrane diffusion cell. Twenty-six flat membranes of different materials were tested using rapeseed oil and capric acid. The suitable membranes were selected in terms of higher fatty acid and lower rapeseed oil permeation rates. The stability of membranes and the effect of fatly acid chain length on effluent fluxes were also investigated. Reaction experiments were carried out in a membrane reactor between medium-chain triacylglycerols and n−3 polyunsaturated fatty acids (PUFA) from fish oil. Lipozyme IM was used as the biocatalyst. The incorporation of PUFA into medium-chain triacylglycerols was increased by about 15% in a PUFA 90-h reaction by simultaneous separation of the released medium-chain fatty acids, compared to no separation under the same reaction conditions. It has thus clearly been demonstrated that membrane-assisted separation improved the incorporation of acyl donors into oils beyond the reaction equilibrium defined by the original substrate concentration.     

Extraction of aminoglycoside antibiotics with reverse micelles

The reverse micelle system of sodium di-2-ethylhexyl phosphate was used to extract aminoglycoside antibiotics, neomycin and gentamicin. The aminoglycosides can be efficiently extracted into a reverse micelle solution, and the antibiotics extracted into the micelle phase can readily be recovered back to a divalent cation aqueous solution, such as Ca²⁺. The transfer efficiency, %E, is heavily dependent on pH and salt concentration in the aqueous feed solution. %E decreases drastically with pH in the pH range 8·5–11, and declines with increasing (NH₄)₂SO₄ concentration. A simple transfer mechanism was proposed which suggests that the antibiotic molecules were extracted into the inner water cores of reverse micelles through attractive electrostatic interaction during forward transfer. In backward transfer, the antibiotics loaded in the micelle phase are released back to an aqueous phase through breaking up of the reverse micelles by using divalent cation solutions. The model is supported by the results of dynamic light scattering and infra-red spectroscopy study.     

Conjugated linoleic acid (CLA) production and lipase‐catalyzed interesterification of purified CLA with canola oil

In this study, two important isomers of CLA, i.e. c9,t11 and t10,c12, were produced up to ca. 73% of total fatty acids, employing alkali isomerization of safflower oil, followed by purification with only one‐step urea crystallization to 85.6%, while the recovery of the purification process was 35%. Interesterification (acidolysis) of purified CLA with canola oil was then conducted by Thermomyces lanuginosus lipase. The CLA content incorporated into the triacylglycerols (TG) was 26.6 mol‐% after 48 h of reaction time. Physical and chemical properties of the TG were then changed according to the degree of substitution of oleic acid in canola oil with CLA.     

Enzymatic interesterification of structured lipids containing conjugated linoleic acid with palm stearin for possible margarine production

Structured lipids containing conjugated linoleic acid as a functional ingredient were blended with palm stearin in the ratios of 30 : 70, 40 : 60, 50 : 50, 60 : 40 and 70 : 30 (wt/wt). The blends were subjected to enzymatic interesterification by Candida antarctica lipase. After interesterification of the blends, changes in the physical properties of the products, including lower melting points and solid fat contents along with different melting behaviors, were evidenced. Analysis of triacylglycerols (TAG) of the interesterified blends showed a decrease in the concentration of high‐melting TAG. X‐ray diffraction analysis revealed, that all the reacted blends were predominantly in the β' crystal form. The mixture could be used for the formulation of margarines or other, similar products.     

Enzymatic alcoholysis of triolein to produce wax ester

An alcoholysis reaction between triolein and oleyl alcohol catalyzed by Lipozyme and Novozyme was carried out to produce oleyl oleate, a wax ester. The effects of various reaction parameters such as time, reaction temperature, amount of enzyme, molar ratio of substrates (oleyl alcohol/triolein), various organic solvents used and the initial water activity, a_w of the reaction system were studied. The best conditions tested to produce wax ester were respectively, incubation time, 5 h; temperature, 50 °C for Lipozyme and 60 °C for Novozyme; weight of enzyme, 0.30 g and molar ratio of oleyl alcohol to triolein, 6:1. The use of organic solvents greatly influenced the activity of lipase. Generally, the activity of lipase was high in nonpolar solvents with log P values greater than 2.50. Heptane and hexane were the best solvents tested. The enzymatic synthesis of oleyl oleate was best carried out at a_w 0.32. Analysis of the yield of the products of the reaction at optimized reaction condition using Lipozyme showed that 75.66% oleyl oleate was produced. © 2001 Society of Chemical Industry     

The evolution of enzymatic interesterification in the oils and fats industry

Immobilised lipases have now become accepted as a mainstream technology for fat modification. This paper presents the development of this technology in particular for the production of trans‐free fats. One of the factors influencing this development has been concern for health, which has made trans fats a major issue for food manufacturers and consumers. Enzymatic interesterification is a relatively new method for producing trans‐free alternatives for fats used in conventional margarines and shortenings. The development of this technology is examined from the perspective of an eventual industrial application rather than operation only at the laboratory scale. This paper also covers the practical means of operating immobilised enzyme columns and gives examples of how formulations can be adapted to match existing specifications. There are environmental benefits when choosing enzyme technology in comparison with chemical‐based routes. Life cycle assessments have been used to quantify the differences in environmental impact of this new technology. The final process is both capable of providing fats with the correct melting properties but without trans fats and of reducing the environmental impact of fat processing. Finally, the future developments that are anticipated in the applications of this technology are considered.     

Enzymatic esterification of DL‐menthol with propionic acid by lipase from Candida cylindracea

This work deals with the resolution of DL ‐menthol with propionic acid by Candida cylindracea lipase (Ccl) in organic solvent reaction systems and a reverse micelles system of sodium 1,4‐bis (2‐ethylhexyl) sulfosuccinate (AOT). The activity and stability as well as enantioselectivity of the lipase in two systems were studied. The results indicate that the lipase showed higher stability in reverse micelles than in organic solvent, which proved that the reverse micelles system has potential application for maintaining the activity of the enzyme for a long time. This is because lipase molecules can be entrapped in water‐containing micro‐drops of reverse micelles, avoiding direct‐contract with unfavorable organic medium. The enantioselectivity (E > 30, ee_p = 92.5) in the two systems is relatively high, although the conversion is moderate. The influence of the characteristic parameters of the two systems, such as pH, temperature, w₀ (molar ratio of water to AOT in reverse micelles systems) and water content (organic solvent) on the conversion of DL ‐menthol was also investigated. Copyright © 2005 Society of Chemical Industry     

Enzymatic synthesis of geranyl acetate inn-hexane withCandida antarctica lipases

Geranyl acetate is an important flavor and fragrance compound. Two immobilizedCandida antarctica lipases, SP382 and SP435, were investigated for their use in the synthesis of geranyl acetate by direct esterification. Yields between 95 and 99% molar conversion were obtained with 2 and 15% (w/w reactants) of SP435 and SP382 lipases, respectively. Optimum yields were obtained at 0.1M acetic acid and 0.12M geraniol after 16-h incubation. No inhibitory effect was observed at increasing concentrations of geraniol. Addition of 60% (w/w reactants) water led to 50 and 60% reduction in the esterification activity of SP382 and SP435 lipases, respectively. The best yields were obtained at added water contents between 0–5% (w/w reactants). Solvents with a logP value of 0.85 or more gave reaction yields of more than 80% molar conversion. Higher logP values did not necessarily lead to higher conversion yields. The immobilized lipase SP382 was still active after reusing ten times in the direct esterification reaction.     

Enzymatic interesterification of triolein and tristearin: Chemical structure and differential scanning calorimetric analysis of the products

The structural composition and thermal properties of the products of enzymatic interesterification of triolein and tristearin were investigated. The biocatalyst for the reaction was an immobilized Candida antarctica lipase, SP435. Enzyme load of 10% (w/w reactants) produced 72% of desired total products. Oleoyl-distearoyl triglycerides (SSO, OSS) had higher melting points than dioleoyl-stearoyl triglycerides (OOS, SOO) because the sample contained larger amounts of stearic acid than oleic acid residues. SOS and OSO were hardly produced (0.2 to 1.2%), which indicates that SP435 acted as a nonspecific lipase when catalyzing the interesterification of triolein and tristearin. The maximal yield of OSS and SSO (46.9%) was achieved with a 1.2 mole ratio of triolein to tristearin. As the proportion of tristearin was increased, the production of SOO and OOS decreased, the melting profile of the interesterified triglycerides shifted toward higher melting forms, and the solid fat content increased, indicating formation of hard fats.     

A comprehensive study of extraction of hyperoside from Hypericum perforatum L. using CTAB reverse micelles

BACKGROUND: Hyperoside is a valuable natural pharmaceutical that has many biomedical functions. By the traditional method of alcohol extraction, only hyperoside solution of very low concentration can be obtained, so a new method is urgently needed to produce pure hyperoside more effectively. Reverse micellar extraction has been widely used in the purification of biological macromolecules. Theoretically, this method could also be used to purify materials with small molecules. Therefore it would seem appropriate to consider the extraction of hyperoside, a material with small molecules, using reverse micelles. In this study the factors affecting hyperoside extraction using cetyl trimethylammonium bromide (CTAB) reverse micelles were comprehensively investigated. RESULTS: The results showed that the partition coefficient increases with an increase in aqueous phase pH or CTAB concentration before reaching a saturated state. The existence of anions in the system can affect the extraction in several ways. For example, SO and PO ions can form hydrogen bonds with hyperoside and thereby improve the extraction significantly. CONCLUSION: This study proved that extraction using CTAB reverse micelles is a very effective method to separate hyperoside from Hypericum perforatum L. Under optimal conditions the extraction efficiency can reach 68.3% and the single‐strip‐extraction efficiency can reach 85.9%. The proportion of hyperoside among the flavonoids left in the strip‐extract can reach 93.3 wt%. This finding is very significant, because it demonstrates that reverse micellar extraction is a promising way to separate and purify materials with small molecules. Copyright © 2008 Society of Chemical Industry     

Enzymatic hydrolysis of canola oil with hydrodynamic cavitation

A hydrodynamic cavitation system based on a venturi was used to test the effectiveness of cavitation for enhancing the enzymatic hydrolysis of canola oil using lipase from Candida rugosa. Cavitation led to the production of fine oil-in-water and water-in-oil emulsions with the enzyme in the water phase. Using venturi inlet pressures of up to 8 bar, the yield of fatty acids was only about 60% of the maximum possible. In contrast, a simple stirred batch reactor produced over 90% of the maximum possible yield with reaction rates equal to, or better than, those obtained in a cavitating system. It was concluded that cavitation inhibited the reaction in some way and is not effective for intensification of hydrolysis.     

Polyphenol oxidase in reverse micelles of AOT/cyclohexane: A thermostability study

Polyphenol oxidase catalysing the oxidation of 4‐methylcatechol in reverse micelles of AOT/cyclohexane had an optimum temperature 15 °C higher than in aqueous medium. However, the enzyme lost stability when it was preincubated in reverse micelles in the absence of substrate regardless of the temperature although the effect was more pronounced at higher temperatures. The thermostability of polyphenol oxidase is higher when it is injected in reverse micelles containing buffer than when injected in initially empty micelles. Moreover, the thermostability of polyphenol oxidase in reverse micelles is strongly dependent on the size of the micelles, the bigger the micelle the greater the stability. The thermoinactivation of the enzyme follows a monomolecular process characteristic of a conformational change so the protein is protected by ligands towards inactivation. p‐Nitrophenol as competitive inhibitor and acetyl tyrosine ethyl ester as alternate substrate increase the half‐life of the polyphenol oxidase by about 2.5 and 4 times respectively. This finding may allow the use of the enzyme at higher temperatures with a gain in its stability. © 2001 Society of Chemical Industry     

Reactivity of medium-chain substrates in the interesterification of tripalmitin catalyzed by papaya lipase

Reactivity of different medium-chain substrates, i.e., n-octanol, caprylic acid, and its alkyl (methyl, ethyl, n-propyl, and n-butyl) esters, was assessed in the interesterification of tripalmitin catalyzed by papaya (Carica papaya) lipase. Alcoholysis with n-octanol was the fastest reaction leading to the highest conversion of tripalmitin to n-octyl palmitate and concomitant formation of di- as well as monopalmitoylglycerols. This was followed by transesterification of tripalmitin with n-butyl and n-propyl caprylates, which in turn were faster than transesterification with ethyl and methyl caprylates, yielding in each case the corresponding alkyl palmitates and triacylglycerols containing palmitoyl and capryloyl moieties as the major reaction products. Acidolysis of tripalmitin with caprylic acid yielded palmitic acid and triacylglycerols containing palmitoyl and capryloyl moieties as the major reaction products, however, with the lowest conversion among the three interesterification reactions studied. In each case, interesterification was accompanied by some hydrolysis of tripalmitin.     

Enzymatic kinetics for lipase‐catalyzed hydrolysis of water‐insoluble substrate in AOT reverse micelles,involving native and activated lipases

The addition of short chain polyethylene glycols (PEGs) activated Chromobacterium viscosum lipase in AOT reverse micelles. In this study, it was assumed that when AOT reverse micelles contained PEG molecules, native and activated lipases contributed to the reaction according to their weight fraction in the system. The fractions of individual lipases and their specific activities were estimated by using kinetics parameters determined by previous kinetic models. The maximum specific activity of PEG‐activated lipase appeared to be 2.7 times higher than that of native lipase. The predicted weighted‐average activity agreed well with experimental data. The results showed that the previous kinetic models could be used to estimate the fraction of lipase activated by PEG molecules and their specific activity. Copyright © 2004 Society of Chemical Industry     

Enzymatic modification of triolein: Incorporation of caproic and butyric acids to produce reduced-calorie structured lipids

Lipase-catalyzed acidolysis of triolein with caproic and butyric acids was performed to produce reduced-calorie structured lipids (SL). The SL were obtained by incubating a 1:4:4 mole ratio of triolein, caproic acid, and butyric acid, respectively, with 10% of lipase (w/w of total substrates) in 1.5 mL hexane at 55°C for 24 h. Of nine commercially avaialble lipases screened, IM60, which contains the lipase from Rhizomucor miehei, was the most effective and produced 13 mol% unreacted triolein, 49% disubstituted, and 38% monosubstituted triacylglycerols that contained short-chain fatty acids. The products were analyzed by reverse-phase high performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters studied included time course, temperature, enzyme load, and substrate mole ratio. The yields obtained demonstrate that a structured lipid with long-chain and short-chain fatty acids can be synthesized by using IM60 lipase in organic medium.     

Liquid–liquid extraction of a recombinant protein with reverse micelles

Recombinant cytochrome b₅ was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase. The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein. This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis. The back-extraction of cytochrome b₅ to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1–2 mol dm^?3 NaCl) and low temperatures (4°C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.     

Synthesis of macroporous poly(styrene-divinyl benzene) microspheres by surfactant reverse micelles swelling method

Macroporous poly(styrene-divinyl benzene) microspheres with pore size of about 500 nm were prepared by a new method, surfactant reverse micelles swelling method. The macroporous microspheres were prepared by convenient suspension polymerization. The difference from conventional suspension polymerization was that a higher concentration of surfactant was added in the oil phase. The effects of the amount and type of surfactants on the morphology of microspheres were investigated, and the formation mechanism was also discussed. Macropores were formed when the concentration of surfactant was much higher than critical micelle concentration (cmc). It was proposed that a large amount of reverse micelles formed by adding a large amount of surfactant in the oil droplet phase, and the reverse micelles could absorb water from the external aqueous phase. The water in the oil phase formed macropores after polymerization. The method developed in this study was convenient to prepare microspheres with larger pore size than the conventional method such as agglomeration method of nanoparticles.