Impact of high-intensity ultrasound on physical properties and degree of oxidation of lipase modified menhaden oil with caprylic acid and/or stearic acid

The purpose of this research was to determine the effect of high-intensity ultrasound (HIU) on physical properties, degree of oxidation, and oxidative stability of structured lipids (SLs). Caprylic acid (C) and stearic acid (S) were incorporated into menhaden oil using Lipozyme® 435 lipase to obtain five samples: (1) LC 20 (menhaden oil with 20% of C), (2) LC 30 (menhaden oil with 30% C), (3) LS 20 (menhaden oil with 20% S), (4) LS 30 (menhaden oil with 30% S), and (5) Blend C (menhaden oil with 16.24% C and 13.04% S). Samples were crystallized for 90 min at the following temperatures: (1) LC 20 at 15.5°C, (2) LC 30 at 17.5°C, (3) LS 20 at 24°C, (4) LS 30 at 30°C, and (5) Blend C at 18.0°C, and HIU was applied at the onset of crystallization. Physical properties, degree of oxidation, and oxidative stability were evaluated in sonicated and nonsonicated samples. All SLs had statistically higher G′ after sonication. Sonicated LS 30, LC 30, and Blend C had a higher melting enthalpy than the nonsonicated ones, while enthalpy values in sonicated LS 20 and LC 20 samples were not statistically different than the nonsonicated ones. No significant difference between sonicated and nonsonicated samples was observed in peroxide values (1.2 ± 0.1 meq/kg, p > 0.05) and in the oxidative stability index (6.3 ± 0.2 h, p > 0.05). These results showed that HIU was effective at changing physical properties without affecting the oxidation of the samples.     

Enzymatic modification of trilinolein: Incorporation of n-3 polyunsaturated fatty acids

Two immobilized lipases, IM60 fromMucor miehei and SP435 fromCandida antarctica, were used as biocatalysts for the modification of trilinolein with n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), by using their ethyl esters as acyl donors (EEPA and EDHA, respectively). Transesterification (ester-ester interchange) reactions were carried out in organic solvent. The products were analyzed according to their equivalent carbon number and polarity by reverse-phase high-performance liquid chromatography, and the fatty acid profiles were determined by gas-liquid chromatography. Modified triacylglycerol products contained 1 or 2 molecules of n-3 PUFA. With EEPA as the acyl donor, the total EPA product yields with IM60 and SP435 as biocatalysts were 79.6 and 81.4%, respectively. However, with EDHA as the acyl donor and IM60 and SP435 as biocatalysts, the total DHA product yields were 70.5 and 79.7%, respectively. Effects of reaction parameters, such as type of solvent, enzyme load, time course, and molar ratio of substrates on the n-3 PUFA incorporation, were followed with SP435 as the biocatalyst. High yields were obtained, even in the absence of organic solvent. These lipids do hold promise for specialty nutrition and other therapeutic uses.     

Evaporative light scattering mass detection for high-performance liquid chromatographic analysis of sucrose polyester blends in cooking oils

A high-performance liquid chromatographic method is described to determine the sucrose polyester (SPE) content in seven blends of cooking oils. Four gel-permeation chromatography (GPC) columns were used in series with an evaporative light scattering mass detector to separate the SPE from the acylglycerols in the final chromatogram. The SPE fraction was collected off the GPC column and injected onto a reverse-phase C-18 column for quantitation with sucrose octaacetate as an internal standard and a gradient of nonaqueous solvents as mobile phase. The chromatograms were interference-free, with only two sharp peaks appearing. The standards were linear from 500 to 5000 μg/mL with a correlation coefficient of r=0.999. The mean percent recovery (n=9) and standard deviation were 102±6.7. The detector could detect amounts as low as 5 μg SPE.     

Preparation of trehalose and sorbitol fatty acid polyesters by interesterification

Trehalose octaoleate and sorbitol hexaoleate were prepared by a one-stage solvent-free interesterification of methyl oleate with trehalose octaacetate (TOAC) and sorbitol hexaacetate (SOHA), respectively, in the presence of 1–2.5% sodium metal as catalyst. The greatest yield of trehalose octaoleate, 97.5%, and sorbitol hexaoleate, 96.0%, were obtained at a mole ratio of methyl oleate: SOHA of 6:1 and methyl oleate:TOAC of 8:1 at a synthesis time of 2.5 hr, and temperatures of 105–115°C. The structure of both trehalose octaoleate and sorbitol hexaoleate was confirmed by infrared (IR) and nuclear magnetic resonance (C-13 NMR) spectroscopy. Physical properties such as viscosity, HLB, solubility, color, refractive index, specific gravity and density approximated the physical properties of sucrose polyesters (SPE) and vegetable oils. Trehalose octaoleate and sorbitol hexaoleate, as well as sucrose octaoleate, were not susceptible toin vitro lipolysis, suggesting potential application as low calorie oils. Presented at American Oil Chemists’ Society Annual Meeting in New Orleans, LA, in May, 1987.     

Lipase PS-catalyzed transesterification of citronellyl butyrate and geranyl caproate: Effect of reaction parameters

Pseudomonas sp. lipase PS was immobilized by adsorption and tested for its ability to catalyze the synthesis of citronellyl butyrate and geranyl caproate by transesterification in n-hexane. The reaction parameters investigated were: enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse. Yields as high as 96 and 99% were obtained for citronellyl butyrate and geranyl caproate, respectively, with 300 units (approx. 15% w/w of reactants) of lipase PS. Increasing amounts of terpene alcohol inhibited lipase activity, while excess acyl donor (triacylglycerol) concentration enhanced ester production. Optimal yields were obtained at temperatures from 30–50°C after 24-h incubation time. Yields of 90 and 99% were obtained for citronellyl and geranyl esters, respectively, with 2% added water. Solvents with log P values ≥ 2.5 showed the highest conversion yields. pH 7 and 6–8 seemed to be ideal for citronellyl butyrate and geraniol caproate, respectively. The lipase remained active after reusing 12 times.     

Emulsification properties of polyesters and sucrose ester blends II: Alkyl glycoside polyesters

Surface active properties such as surface and interfacial tension reductions and stability of oil-in-water (o/w) and water-in-oil (w/o) emulsions by alkyl glycoside fatty acid polyesters, a potential fat substitute, and emulsifier blends of alkyl glycoside polyesters and Ryoto sugar esters were investigated. Our results indicate that blending of lipophilic and hydrophilic emulsifying agents produces a synergistic effect on reduction of surface and interfacial tensions and may, in some cases, result in more stable emulsions. Alkyl glycoside polyesters may be suitable for w/o emulsions, such as margarine and butter, and their blends with hydrophilic emulsifiers will produce reduced calorie emulsifiers suitable for use in o/w emulsions, such as salad dressing. There appears to be great potential for using such emulsifier blends in food, cosmetics and pharmaceutical applications.     

Enzymatic Interesterification of High Oleic Sunflower Oil and Tripalmitin or Tristearin

The objective of this study was to produce low saturated, zero‐trans, interesterified fats with 20 or 30 % saturated fatty acids (SFA) such as C16:0 or C18:0. Tripalmitin (TP) or tristearin (TS) was blended with high oleic sunflower oil (HOSO) at different ratios (0.1:1, 0.3:1, and 0.5:1 [w/w]). Total C16:0 and C18:0 compositions of the resulting TP/HOSO and TS/HOSO blends, respectively, were plotted against blending ratios. Linear interpolation was used to estimate blending ratios that would yield physical blends (PB) with 20 or 30 % SFA. Interesterified blends (IB) were then synthesized from the customized PB using Lipozyme TL IM as the biocatalyst. Total and sn‐2 fatty acid compositions, triacylglycerol (TAG) molecular species, thermal behavior, and oxidative stability of PB and IB were compared. The total fatty acid compositions of PB and IB were similar but fatty acid positional distributions and TAG molecular species composition differed. IB contained 5–10 % more SFA at the sn‐2 position than corresponding PB. Furthermore, interesterification generated mono‐ and disaturated TAG species which resulted in broader melting profiles for IB. However, IB had lower oxidative stability than PB. The reformulation of food products with zero‐trans interesterified fats may be advantageous to the reduction of cardiovascular disease burden in the population.     

Protein engineering and applications of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase isoforms

Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.     

Enzymatic interesterification of lard and high-oleic sunflower oil with Candida antarctica lipase to produce plastic fats

Lard and high-oleic sunflower oil (Trisun® Extra) were interesterified at 55°C for 24 h with SP435 lipase from Candida antarctica to produce plastic fats. As the amount of trisun increased, percentage free fatty acid, unsaturated fatty acid/saturated fatty acid value, oxidizability, and the amount of 18:1 found at the sn-2 position of triglyceride products increased. Differential scanning calorimetry showed that the low-melting components in the product contained more 18:1 than the high-melting components. A 60:40 (w/w) ratio of lard to trisun had the widest plastic range (3–26°C). The scaled-up reaction to produce this blend resulted in a product that had 60.1% 18:1 at the sn-2 position compared to 44.9% for the physical blend. The solid fat content of the 60:40 interesterified mixture resembled soft-type margarine oil.