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.     

Reduction of Graininess Formation in Beef Tallow-Based Plastic Fats by Chemical Interesterification of Beef Tallow and Canola Oil

To manufacture beef tallow (BT)-based shortening and margarine with a reduced tendency to developing sandiness, BT/canola oil (CaO) blend (80:20 w/w), selected from the BT and CaO blends mixed in different ratios from 60:40 to 85:15 with 5% increments, was subjected to chemical interesterification (CIE) with sodium methoxide as the catalyst. The interesterified products were compared with the starting mixture in terms of solid fat content (SFC), and contents of high-melting point 1,3-disaturated long-chain fatty acid 2-monounsaturated long-chain fatty acid triacylglycerols (SUS TAGs) including 1,3-distearoyl-2-oleoyl-glycerol (StOSt), 1,3-dipalmitoy-2-oleoyl-glycerol (POP), and 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POSt). Under the selected conditions: 60 °C, 0.6% CH₃ONa, 90 min, the CIE product had a SFC profile that meets suggested bakery fat requirements, besides a content of SUS TAGs which is 22.14% lower than that of the non-interesterified blend. Also the fat produced had stable β′ polymorphs, crystal morphology, crystal sizes (<20 μm), and could resist temperature fluctuations. The CIE product obtained herein has an increased potential for manufacturing bakery shortenings and margarines with reduced graininess formation, increasing the possibilities for the commercial use of BT and CaO.     

Stability of Interesterified Fats

Stability study carried out with interesterified (i) refined cottonseed oil (CSO), blends of (ii) vanaspati and refined groundnut oil (40:60 w/w) (vanaspati-GNO), (iii) refined and bleached sal (Shorea robusta) fat and refined groundnut oil (30:70) (Sal-GNO) and (iv) edible grade sheep tallow and refined groundnut oil (30:70) (ST-GNO) indicated that rearranged CSO and ST-GNO were more and sal-GNO less prone to oxidative rancidity compared to their corresponding starting stocks. Vanaspati-GNO was more or less similar to its starting stock. In other words, the process of interesterification had different effects on different starting stocks vis-à-vis oxidative rancidity. Addition of antioxidant (BHA) improved the stability of interesterified products to a large extent. Development of oxidative rancidity as measured by peroxide value in all the interesterified products during storage was faster than that observed with vanaspati. All the interesterified products were stable towards hydrolytic rancidity during storage. When the linoleic content of a blend was about 25%, the process of interesterification had no or marginal adverse effect on the development of oxidative rancidity. Higher linoleic acid (about 50%) made the stock more unstable as a result of interesterification. Comparatively more instability of interesterified blend containing sheep tallow cannot be explained on the basis of its linoleic acid content and its migration as a result of rearrangement.     

Influence of lipase-catalyzed interesterification on the oxidative stability of melon seed oil triacylglycerols

Melon seeds are rich in oil. However, the stability of melon seed oil (MSO) is low because of its high content of the essential fatty acid, linoleic acid (18:2n-6). MSO was physically blended or enzymatically interesterified with higholeic sunflower oil (HOSO). The fatty acid composition of MSO was remarkably changed after interesterification. Palmitic (16:0), stearic (18:0), and oleic (18:1n-9) acid contents increased at the sn-2 position of triacylglycerols, whereas 18:2n-6 decreased due to interesterification. The oxidative stability of the physical and Pseudomonas sp. (PS30) lipase-interesterified blends was assessed with the Oxidative Stability Instrument, peroxide value, and conjugated diene methods. The stability of MSO increased with increased proportions of HOSO, which was the source of 18:1n-9 in the blends. The ratio of 18:1n-9/18:2n-6 improved from 0.18 in MSO to 1.47 in the enzymatically interesterified blend. Calculated oxidizability and the results of oxidation tests of the blends confirmed the improvement in MSO stability by both physical blending and enzymatic interesterification.     

Influence of lipase-catalyzed interesterification on the oxidative stability of melon seed oil triacylglycerols

Melon seeds are rich in oil. However, the stability of melon seed oil (MSO) is low because of its high content of the essential fatty acid, linoleic acid (18:2n-6). MSO was physically blended or enzymatically interesterified with higholeic sunflower oil (HOSO). The fatty acid composition of MSO was remarkably changed after interesterification. Palmitic (16:0), stearic (18:0), and oleic (18:1n-9) acid contents increased at the sn-2 position of triacylglycerols, whereas 18:2n-6 decreased due to interesterification. The oxidative stability of the physical and Pseudomonas sp. (PS30) lipase-interesterified blends was assessed with the Oxidative Stability Instrument, peroxide value, and conjugated diene methods. The stability of MSO increased with increased proportions of HOSO, which was the source of 18:1n-9 in the blends. The ratio of 18:1n-9/18:2n-6 improved from 0.18 in MSO to 1.47 in the enzymatically interesterified blend. Calculated oxidizability and the results of oxidation tests of the blends confirmed the improvement in MSO stability by both physical blending and enzymatic interesterification.     

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.     

Valorization of Beef Tallow by Lipase-Catalyzed Interesterification with High Oleic Sunflower Oil

Although beef tallow (BT) has been considered a hard low-trans fat convenient to be used in several bakery applications, it has some undesirable characteristics like fatty acid composition, crystallization behavior, graininess formation and poor plastic range. This work studied the modification of BT by blending at different percentages with high oleic sunflower oil (HOSFO) followed by the enzyme-catalyzed interesterification of the blends. The reduction in the solid fat content achieved by the simple blending was enhanced by the interesterification process, as a result of the increase in the concentration of the diunsaturated monosaturated type triacylglycerols. Interesterification strongly impacted too on the crystallization behavior of the blends, since products showed more homogeneous and regular crystals than the starting mixture. Results show that lipase catalyzed interesterification of BT with HOSFO offers a useful tool for the design of fats with adjustable physicochemical properties, improved with respect to that of the starting fats.     

Alteration of steryl ester content and positional distribution of fatty acids in triacylglycerols by chemical and enzymatic interesterification of plant oils

Steryl ester content of refined and interesterified corn, soybean, and rapeseed oils has been measured via clean-up on a short silica gel column, followed by high performance liquid chromatography with evaporative light-scattering mass detector. Chemical interesterification, catalyzed by sodium methoxide, led to random positional distribution of fatty acids in triacylglycerols and some increase in the steryl ester content of all three oils. Enzymatic interesterification, catalyzed by the immobilized lipase from Rhizomucor miehei (Lipozyme), resulted in a distinct reduction in steryl ester content, but essentially no alteration in positional distribution of fatty acids in triacylglycerols occurred. Formation of steryl esters during chemical and enzymatic interesterification was also examined by radioactive tracer technique with [4-¹⁴C]β-sitosterol added as marker to refined rapeseed oil and measurement of the radioactive steryl esters formed. Chemical interesterification of rapeseed oil resulted in moderate formation (10% of total radioactivity) of radioactive β-sitosteryl esters. Enzymatic interesterification of the oil, catalyzed by Lipozyme, led to little formation of radioactive β-sitosteryl esters, whereas with the lipase from Candida cylindracea high proportions (>90% of total radioactivity) of ¹⁴C-labeled β-sitosteryl esters were formed. Part of doctoral thesis of Roseli Ap. Ferrari to be submitted to Faculdade de Engenharia de Alimentos, Universidade de Campinas, Campinas, Brazil.     

Pseudomonas fluorescens Lipase-catalysed Interesterification of Butter Fat

Butter fat was subjected to interesterification reactions catalysed by Pseudomonas fluorescens lipase in media of variable water content. The reaction products were analysed by gas chromatography on an immobilised phenylmethylsilicone capillary column. Triacylglycerol compositions were determined by normalisation, and free fatty acid and mono-and diacylglycerol compositions and contents by internal standardisation. In general, the triacylglycerol compositions of interesterification products were similar to each other and distinctly different from those of untreated butter fat. The compositions of triacylglycerols of the reaction products were similar to the composition calculated according to random distribution, except for the higher proportions of saturated triacylglycerols with 36 and 42–50 acyl carbons and monoene triacylglycerols with 38 acyl carbons in the reaction products. Enzymatic deacylation of reaction products showed the fatty acyl groups to be nearly randomly distributed among the three positions of glycerol. The contents of the hydrolysis products (MAGs, DAGs and FFAs) depended on the water content of the reaction medium.     

Production of margarine fats by enzymatic interesterification with silica-granulated Thermomyces lanuginosa lipase in a large-scale study

Interesterification of a blend of palm stearin and coconut oil (75∶25, w/w), catalyzed by an immobilized Thermomyces lanuginosa lipase by silica granulation, Lipozyme TL IM, was studied for production of margarine fats in a 1- or 300-kg pilot-scale batch-stirred tank reactor. Parameters and reusability were investigated. The comparison was carried out between enzymatic and chemical interesterified products. Experimentally, Lipozyme TL IM had similar activity to Lipozyme IM for the interesterification of the blend. Within the range of 55–80°C, temperature had little influence on the degree of interesterification for 6-h reaction, but it had slight impact on the content of free fatty acids (FFA). Drying of Lipozyme TL IM from water content 6 to 3% did not affect its activity, whereas it greatly reduced FFA and diacylglycerol contents in the products. Lipozyme TL IM was stable in the 1-kg scale reactor at least for 11 batches and the 300-kg pilot-scale reactor at least for nine batches. Due to regiospecificity of the lipase (sn-1,3 specific), enzymatically interesterified products had different fatty acid distribution at sn-2 position from the chemically randomized products, implying the potential nutritional benefits of the new technology. Presented at the 91st American Oil Chemists' Society Annual Meeting in San Diego, April 28, 2000.     

Enzymatic interesterification of olive oil with fully hydrogenated palm oil: Characterization of fats

Seven different reaction products were prepared via enzymatic interesterification of extra‐virgin olive oil (EVOO) and fully hydrogenated palm oil (FHPO), by varying the initial weight ratio of EVOO to FHPO from 80 : 20 to 20 : 80. The chemical, physical and functional properties of both the semi‐solid reaction products and the corresponding physical blends of the precursor starting materials were characterized. Fats prepared using large proportions of FHPO contained high levels of TAG species containing only saturated fatty acid residues. By contrast, high levels of TAG species containing both saturated and unsaturated fatty acid residues were found in fat products obtained with the lowest proportions of FHPO. Independently of the initial weight ratio of EVOO to FHPO, the interesterified products were characterized by a higher molar ratio of unsaturated to saturated fatty acid residues at the sn‐2 position, were softer over a wide temperature range, exhibited lower oxidative stabilities and were completely melted at lower temperatures than the corresponding physical blends. Potential applications of the reaction products range from margarines (highest weight ratios of EVOO to FHPO) to frying fats (lowest weight ratios of EVOO to FHPO).     

Oxidative stability during storage of structured lipids produced from fish oil and caprylic acid

Structured lipids produced by enzymatic or chemical methods for different applications have been receiving considerable attention. The oxidative stability of a randomized structured lipid (RFO), produced by chemical interesterification from fish oil (FO) and tricaprylin, and a specific structured lipid (SFO), produced by enzymatic interesterification from the same oil and caprylic acid, was compared with the stability of FO. Oils were stored at 2°C for 11 wk followed by storage at 20°C for 6 wk. In addition, the antioxidative effect of adding the metal chelators EDTA or citric acid to SFO was investigated. FO contained the largest amount of PUFA and RFO the lowest. However, SFO had a higher PV initially and during storage at 2°C, whereas the PV of FO was highest during storage at 20°C. The level of volatile oxidation products was highest in SFO during the entire storage period, and off-flavors were more pronounced in SFO. The lower oxidative stability of SFO was probably related to the initially lower quality (regarding oxidation products), which is apparently a result of the long production procedure required. Addition of metal chelators did not reduce the oxidation of the SFO.     

Enzymatic Modification of Anhydrous Milkfat with n-3 and n-6 Fatty Acids for Potential Use in Infant Formula: Comparison of Methods

A structured lipid (SL) with a high amount of sn‐2 palmitic acid was synthesized from anhydrous milkfat and was then enriched with docosahexaenoic (DHA) and arachidonic (ARA) acids using an immobilized lipase. Three different methods were compared including physical blending, enzymatic interesterification, and enzymatic acidolysis. Products were compared with respect to differences in fatty acid profiles, reaction times, antioxidant contents, oxidative stability, melting and crystallization profiles, and reaction yields. The acidolysis method was the least suitable for the synthesis of desired product because of a low reaction yield, low incorporation of DHA, low oxidative stability, and the extra processing steps required. The physical blending and interesterification methods were suitable, but the interesterification product (IE‐SL) had higher amounts of ARA at the sn‐2 position. The IE‐SL contained total ARA and DHA of 0.63 and 0.50 mol%, and 0.55 and 0.46 mol% at the sn‐2 position, respectively. The IE‐SL also contained 44.97 mol% sn‐2 palmitic acid. The reaction yield for the IE‐SL was 91.84 %, and its melting completion and crystallization onset temperatures were 43.1 and 27.1 °C, respectively. This SL might be totally or partially used in commercial fat blends for infant formula.     

Production of Structured Triacylglycerol via Enzymatic Interesterification of Medium-Chain Triacylglycerol and Soybean Oil Using a Pilot-Scale Solvent-Free Packed Bed Reactor

Oils rich in medium- and long-chain triacylglycerols (MLCT) serve as functional oils to help reduce body fat accumulation and weight gain. However, most of the MLCT-rich products on the market are physical blends of medium- and long-chain triacylglycerols (MCT and LCT, respectively) that are not structured triacylglycerols (TAG). In this study, an efficient pilot-scale packed bed reactor (PBR) of immobilized lipase from Thermomyces lanuginosus (Lipozyme® TL IM, Novozymes, Bagsvaerd, Denmark) was employed for producing structured MLCT via 1,3-specific interesterification of TAG enriched in caprylic and capric acyl groups and soybean oil (SBO). The PBR was operated under continuous recirculation mode in the absence of solvent. Optimal reaction conditions were determined to be: caprylic/capric TAG: SBO ratio (45:55 w/w), reaction temperature (75 °C) and residence time (16.0 min) on MLCT production were studied. When employing a pilot-scale PBR (100 kg day⁻¹) under optimal conditions, a product containing 76.61 wt% MLCT was produced. Lipozyme TL IM was reused for 25 successive batch reactions (125 kg substrates) with no significant reduction in catalytic efficiency. The light yellow MLCT-enriched product had a high level of saturated fatty acids (SFA, 82.74 wt%) in its sn-2 position as a result of the enzyme's 1,3-positional specificity. One-stage molecular distillation and methanol extraction were used to remove the free fatty acids, mono-, and diacylglycerols generated from hydrolysis. With distillation temperature of 150 °C and oil-to-methanol ratio of 1:3 v/v, MLCT content was further increased to 80.07 wt%. The enzymatic PBR was therefore effective in producing structured MLCT at a pilot-scale under solvent-free conditions.     

Human milk fat substitutes: Advances and constraints of enzyme‐catalyzed production

The production of triacylglycerols (TAGs) with similar structures to the human milk fat TAGs is only possible using sn‐1,3 regioselective lipases as biocatalysts. The advances on the processes currently performed for the production of human milk fat substitutes (HMFS), namely the biocatalysts used, the best operation conditions, reactor types and operation modes, as well as the major drawbacks of these enzymatic systems, are addressed in this article.     

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.     

Lipozyme IM‐catalyzed interesterification for the production of margarine fats in a 1 kg scale stirred tank reactor

Lipozyme IM‐catalyzed interesterification of the oil blend between palm stearin and coconut oil (75/25 w/w) was studied for the production of margarine fats in a 1 kg scale batch stirred tank reactor. Parameters such as lipase load, water content, temperature, and reaction time were investigated. The reusability of Lipozyme IM was also studied under optimized conditions. The interesterification products were monitored by analysis of triacylglycerol profiles, the contents of diacylglycerols, free fatty acids (FFA), and solid fat contents. The contents of some triacylglycerol species, which were categorized by equivalent carbon number (ECN), namely ECN34, 36, 48, and 50, decreased by 6.0, 5.9, 5.8, and 13.7%, respectively, after enzymatic interesterification, similar to the reduction of those species after chemical interesterification, 6.6, 6.0, 7.1, and 12.9%, respectively. On the other hand, those of ECN38, 40, 42, 44, and 46 increased by 1.1, 1.6, 6.8, 16.7, and 6.5%, respectively, in comparison with the increase of those species after chemical interesterification, 0.2, 1.5, 6.5, 17.0, and 9.2%, respectively. Lipase load and reaction time had great influence on the degree of interesterification. A Lipozyme IM load of 6% was required for a reaction of 6 h and at 60 °C, to reach a stable degree of interesterification. Temperature variation in the range of 50—75 °C did not affect the reaction degree as well as the contents of diacylglycerols, but the content of FFA slightly increased with higher temperature. Addition of water to the enzyme increased the contents of diacylglycerols and FFA in the products linearly. However, it had no effect on the degree of interesterification for the first batch when the enzyme was reused. Lipozyme IM was stable in the 10‐batch test after adjusting the water content in the system. The relationship between the content of water in the system and that of FFAs in the products was evaluated and discussed.     

Stability of Emulsions Containing Interesterified Fats Based on Mutton Tallow and Walnut Oil

In this work, modified fats were produced by enzymatic interesterification of mutton tallow with walnut oil. As a result of forcing the fat hydrolysis process by addition of water to the enzymatic preparation (11.5, 13.0, 14.5, 16.0 wt %), additional levels of polar fractions (MAGs, DAGs, and FFAs) were observed. The aim of this work was to evaluate the stability of emulsions of modified fats containing natural emulsifiers resulting from enzymatic interesterification of mutton tallow with walnut oil. The physical‐chemical parameters of obtained fats were determined in this study. Using several methods, the stability of the formed emulsions was also evaluated. The results showed that the fats resulting from interesterification in the presence of Lipozyme RM IM (immobilized lipase from Rhizomucor miehei, Novozymes Bagsvaerd, Denmark) with 13.0, 14.5, and 16.0 wt % of water in the enzymatic preparation could form stable emulsion systems. On the other hand, the emulsion of the interesterification system where the amount of water in the enzymatic preparation was 11.5 % showed very low stability. The number of natural emulsifiers (MAGs and DAGs) that arose after interesterification was insufficient to stabilize the emulsion system. The work has shown the possibility of using interesterified fats as the fat phase. Emulsions formed on the basis of interesterified fats without any additional emulsifiers such as sunflower lecithin had properties comparable to emulsions containing mixed non‐interesterified fat containing additional emulsifier. The natural emulsifiers formed as part of enzymatic interesterification allow formation of stable emulsion systems.     

Oxidative stability of soybean oil products obtained by regioselective chemical interesterification

Oxidative stability of products produced as potential margarine basestock from soybean oil and methyl stearate by a novel chemical regioselective interesterification was evaluated. The oxidative stability of the products was evaluated by peroxide formation and volatile analysis during storage in the dark with oxygen at 60°C for 72 h. The product obtained by regioselective interesterification resulted in the lowest peroxide formation and volatile concentration sample in comparison with soybean oil and the randomized product of the regioselective interesterified product. Regioselective interesterification of soybean oil with methyl stearate produced a product with increased oxidative stability. Presented at 84th American Oil Chemists’ Society Annual Meeting, April 26–30, 1993, Anaheim, California.     

Solubility of simple and mixed triacylglycerols in supercritical CO2

A simple mixture of triacylglycerols (TAGs) was synthesized enzymatically from palmitic acid and oleic acid. The mixture contained tripalmitoylglycerol (PPP), trioleoylglycerol (OOO), and various isomers of palmitoyl-dioleoylglycerol (POO) and oleoyl-dipalmitoylglycerol (PPO). The behavior of this TAG mixture in equilibrium with supercritical carbon dioxide (SF-CO₂) was studied at 40 and 60°C between 172 and 310 bar. Generally, the partition coefficients varied inversely with species molecular weight. The solubilities of PPP and OOO in SF-CO₂ were determined at the same conditions. PPP was markedly more soluble in SF-CO₂ when present in the TAG mixture than as a pure compound.