Enzyme-assisted synthesis of structured lipids containing long-chain omega-3 and omega-6 polyunsaturated fatty acids

Senanayake, S. P. J. Namal (2000) Enzyme-assisted synthesis of structured lipids containing long-chain omega-3 and omega-6 polyunsaturated fatty acids. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Lipase-catalyzed acidolysis of borage (Borago officinalis L.) and evening primrose (Oenothera biennis L.) oils with long-chain ω3-polyunsaturated fatty acids (LCω3-PUFA), namely docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA), in organic solvents was studied. Six microbial lipases from Candida antarctica, Mucor miehei, Pseudomonas sp., Aspergillus niger, Candida rugosa and Thermomyces lanuginosus were initially used as biocatalysts for the acidolysis reaction. Among the enzymes tested, an immobilized lipase, Novozym-435 from Candida antarctica showed the highest degree of DHA incorporation (25.8-28.7%, after 24 h) in borage oil (BO) and evening primrose oil (EPO). However, the maximum incorporation of EPA (28.7-30.7%, after 24 h) in both oils was achieved with lipase PS-30 from Pseudomonas sp. In another study, incorporation of EPA+DHA into BO and EPO was carried out by first screening of lipases listed above; lipase PS-30 from Pseudomonas sp. was the most efficient enzyme examined (31.7-32.7% EPA+DHA incorporation, after 24 h). -- Effects of variation of reaction parameters, namely enzyme load, temperature, time course, mole ratio of substrates and type of organic solvents were monitored for the most effective enzymes, those from Candida antarctica and Pseudomonas sp., as the biocatalysts of choice. Incorporation of DHA and/or EPA increased significantly (p ≤ 0.05) with increasing the amount of enzyme. As the incubation time progressed, incorporation of these fatty acids was also increased, similar to that observed when the temperature and mole ratio of substrates increased. The highest DHA (37.4-39.7%) or EPA (37.4-39.9%) incorporation occurred at a mole ratio of 1:3 (oil/ DHA or EPA). However, maximum incorporation of EPA+DHA (54-57.5%) occurred at a mole ratio of 1:2:2 (oil/EPA/DHA) and then remained constant between mole ratios of 1:2:2 and 1:3:3. Among solvents examined, n-hexane served best in giving rise to 25.5-27.4% DHA, 25.2-26.8% EPA and 27.8-33.3% EPA+DHA incorporation in the oils. However, solvent-free reactions also gave satisfactory incorporation of 18.1-20.5% DHA, 18.6-20.4% EPA and 23.4-28.8% EPA+DHA in the oils tested. -- Response surface methodology (RSM) was used to obtain a maximum yield of DHA, EPA and EPA+DHA incorporation while using the minimum amount of enzyme possible. Process parameters studied were the amount of enzyme (100-350 units), reaction temperature (20-60°C) and reaction time (6-30 h). All experiments were carried out according to a face-centred cube design. Under optimum conditions (162-165 units of Candida antarctica enzyme: 43-50°C; 25-27 h), incorporation of DHA was 35.6% in BO and 33.5% in EPO. Optimization of acidolysis of oils with EPA, gave rise to a maximum of 35.4 and 33.9% EPA incorporation in BO and EPO, respectively, at 299-309 units of Pseudomonas sp. enzyme, reaction temperature of 40-44°C and reaction time of 25-27 h. Similarly, maximum incorporation of EPA+DHA in BO (35.5%) and EPO (33.6%) was attained at 278-299 units of Pseudomonas sp enzyme, at 42-43°C after 24-26 h. -- In another study, enzymatically modified oils, produced under optimum reaction conditions, were classified using thin-layer chromatography-flame ionization detection (TLC-FID). The results showed that the content of TAG (85.1-95.1%) was much higher than that of the DAG (2.4-11.2%) and MAG (0.3-9.7%) in the structured lipids so produced. Because free fatty acids were removed by NaOH after the acidolysis reaction, they were not detected by TLC-FID. The products were also separated by TLC and the fatty acid compositions of their corresponding isolated bands analyzed by gas chromatography. Results showed that DHA and/or EPA were mainly located in TAG fractions of enzymatically modified oils (33.2-35.4% DHA; 32.5-33.2% EPA and 33.6-35.5% EPA+DHA in DHA, EPA and EPA+DHA-enriched oils, respectively). The TAG fractions also contained appreciable proportions of γ-linolenic acid (GLA) (11.0-17.1 and 7.5-7.6% in DHA, EPA and EPA+DHA-enriched BO and EPO, respectively). -- Stereospecific analysis was carried out to establish positional distribution of fatty acids in the TAG of DHA, EPA and EPA+DHA-enriched oils. In DHA-enriched BO, DHA was randomly distributed over the three positions of TAG while GLA was mainly esterified at the sn-2 and sn-3 positions. In DHA-enriched EPO, however, DHA and GLA were concentrated in the sn-2 position. In EPA-enriched BO, EPA was randomly distributed over the three positions of TAG, similar to that observed for DHA. In EPA-enriched EPO, however, this fatty acid was mainly located at the primary positions (sn-1 and sn-3) of TAG. In both oils, GLA was preferentially esterified at the sn-2 position. In EPA+DHA-enriched BO, EPA and DHA were mainly esterified at the sn-1 and sn-3 positions of TAG while GLA was mainly located at the sn-2 position. In EPA+DHA-enriched EPO, GLA was mainly located at the sn-2 and sn-3 positions; EPA was preferentially esterified at the sn-1 and sn-3 positions, while DHA was found mainly at the sn-3 position. -- The oxidative stability of enzymatically modified oils as well as their unmodified counterparts was evaluated under Schaal oven conditions at 60°C over a 96 h storage period. Conjugated dienes (CD), 2-thiobarbituric acid reactive substances (TBARS) and headspace volatiles were determined. In addition, proton nuclear magnetic resonance (¹H NMR) spectroscopy was used to monitor relative changes in the proton absorption pattern of the fatty acids of oils during storage. Among the oils examined, enzymatically modified products gave rise to higher CD and TBARS as compared to those of their unmodified counterparts. The main volatile compounds identified in enzymatically modified oils were acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal, octanal and nonanal. However, the main volatile compound found in unmodified oils was hexanal. The contents of propanal and hexanal produced by enzymatically modified oils were significantly higher (p ≤ 0.05) than those produced by their unmodified counterparts. These results suggested that the modified oils were more prone to oxidation than their unmodified counterparts. -- The double bond index (DBI) and methylene bridge index (MBI), represent the number of double bonds and bis allylic methylene bridge positions in PUFA, respectively, were calculated. DBI and MBI of enzymatically modified oils were significantly (p ≤ 0.05) higher than those of their unmodified counterparts. During oxidation of oils, DBI and MBI were decreased. Regression analysis was carried out to correlate various parameters of oxidation (CD, TBARS, hexanal and propanal contents) with DBI and MBI of oils; a negative correlation (r = 0.574-0.975; p ≤ 0.1-0.05) existed between these variables. -- Relative changes of aliphatic to olefinic (Rao) and aliphatic to diallylmethylene (Rad) protons ratios, during oil oxidation, were determined by ¹H NMR spectroscopy. An increase in Rao and Rad values was obtained over the entire storage period. A highly significant correlation (r = 0.930-0.992; p ≤ 0.005) existed between the CD values and changes in Rao and Rad during oxidation of all oils. The correlation coefficient between TBARS and changes in Rao and Rad values was in the range of 0.779 -0.983 (p ≤ 0.05). A high correlation (r = 0.948-0.996; p ≤ 0.005) was found between hexanal content and Rao and Rad of oils. Propanal content was also highly correlated (r = 0.950-0.990; p ≤ 0.005) with Rao and Rad. This suggests that ¹H NMR could be used to simultaneously estimate both primary and secondary oxidation changes in native and enzymatically modified oils.

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