Synthesis of triacylglycerol containing conjugated linoleic acid by esterification using two blended lipases

We have developed an efficient esterification for the synthesis of triacylglycerol (TAG) containing conjugated linoleic acids (CLA) using a blend of two powdered lipases. Two pairs of blended lipases promoted the esterification. Rhizomucor miehei lipase, plus Alcaligenes sp. lipase and Penicillium cammembertii MAG and DAG lipase plus Alcaligenes sp. lipase were used. At the optmal ratio of two lipases, the content of TAG containing CLA (TAG-CLA) in all glycerols reached 82–83% after 47 h using 1 wt% of lipases. With R. miehei lipase plus Alcaligenes sp. lipase, the reaction time to obtain ca. 60% of TAG-CLA was one-third of that needed with R. miehei lipase alone. The optimal ratio of two lipases differed between these two pairs. The optimal ratio was 70–80 wt% of R. miehei lipase in the last stage of the reaction, whereas it was over a wide range of 10–90 wt% for P. camembertii lipase. In the blend of R. miehei lipase plus Alcaligenes sp. lipase, activity remained very high after 10 cycles of esterification (every 47 h) and could be used in the industrial production of TAG-CLA.     

Conjugated linoleic acid production from linoleic acid by lactic acid bacteria

After screening 14 genera of lactic acid bacteria, Lactobacillus plantarum AKU 1009a was selected as a potential strain for CLA production from linoleic acid. Washed cells of L. plantarum with high levels of CLA production were obtained by cultivation in a nutrient medium with 0.06% (wt/vol) linoleic acid (cis-9,cis-12-octadecadienoic acid). Under the optimal reaction conditions with the free form of linoleic acid as the substrate, washed cells of L. plantarum produced 40 mg CLA/mL reaction mixture (33% molar yield) from 12% (wt/vol) linoleic acid in 108 h. The resulting CLA was a mixture of two CLA isomers, cis-9,trans-11 (or trans-9,cis-11)-octadecadienoic acid (CLA1, 38% of total CLA) and trans-9,trans-11-octadecadienoic acid (CLA2, 62% of total CLA), and accounted for 50% of the total FA obtained. A higher yield (80% molar yield to linoleic acid) was attained with 2.6% (wt/vol) linoleic acid as the substrate in 96 h, resulting in CLA production of 20 mg/mL reaction mixture [consisting of CLA1 (2%) and CLA2 (98%)] and accounting for 80% of total FA obtained. Most of the CLA produced was associated with the cells (ca. 380 mg CLA/g dry cells), mainly as FFA.     

Effect of gamma irradiation on allelopathic potential ofSorghum bicolor against weeds and nitrification

The effect of low doses of gamma irradiation on the allelopathic potential ofSorghum bicolor against weeds and nitrification was investigated. The results revealed that all test doses (500, 1000 and 1500 rad) significantly increased the allelopathic activity of root exudates, aqueous extracts, and decaying residues aganst seed germination and seedling growth ofAmaranthus retroflexus. The results also indicated that all test doses stimulated the allelopathic potential of aqueous extracts and decaying residues against nitrification activity. The possible application of this approach in biological control is briefly discussed.     

Monooxygenase system of Bacillus megaterium ALA2: Studies on linoleic acid epoxidation products

Bacillus megaterium ALA2 produces many oxygenated FA from linoleic acid: 12,13-dihydroxy-9(Z)-octadecenoic acid; 12,13,17-trihydroxy-9(Z)-octadecenoic acid; 12,13,16-trihydroxy-9(Z)-octadecenoic acid; 12-hydroxy-13,16-epoxy-9 (Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9 (Z)-octadecenoic acid. Recently, we studied the monooxygenase system of B. megaterium ALA2 by comparing its palmitic acid oxidation products with those of the well-studied catalytically self-sufficient P450 monooxygenase of B. megaterium ATCC 14581 (NRRL B-3712) and of B. subtilis strain 168 (NRRI B-4219). We found that their oxidation products are identical, indicating that their monooxygenase systems (hydroxylation) are similar. Now, we report that strain ALA2 epoxidizes linoleic acid to 12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12 (Z)-octadecenoic acid, the initial products in the linoleic acid oxidation. The epoxidation enzyme did not oxidize specific double bond of the linoleic acid. The epoxidation activity of strain ALA2 was compared with the above-mentioned two Bacillus strains. These two Bacillus strain also produced 12,13-expoxy-9 (Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid, indicating that their epoxidation enzyme systems might be similar. The ratios of epoxy FA production by these three strains (A1 A2, NRRI B-3712, and NRRI B-4219) were, respectively, 5.56∶0.66∶0.18 for 12,13-epoxy-9(Z)-octadecenoic acid and 2.43∶0.41∶0.57 for 9,10-epoxy-12(Z)-octadecenoic acid per 50 mL medium per 48 h.     

Conjugated linoleic acid concentration in processed cheese

The conjugated linoleic acid (CLA) concentration of a variety of processed cheese products ranged between 3.2 to 8.9 mg/g fat. Processing cheddar cheese at temperatures of 80°C and 90°C under atmospheric conditions increased (p < 0.05) CLA content, while processing under nitrogen (70°C, 85°C) had no effect. Increasing concentrations of whey protein concentrate (WPC) and its low molecular weight (LMW) fraction from 0 to 6% increased CLA formation. Six percent WPC and LMW fraction produced a 35% and 19% increase in CLA concentration, respectively, compared to processed cheese. The high molecular weight fraction of WPC did not increase CLA concentration. These results suggest that processing conditions and whey components play a role in CLA formation in processed cheese.     

Enzymatic enrichment of conjugated linoleic acid isomers and incorporation into triglycerides

A method was developed for the enrichment of either the cis9,trans11 or the trans10,cis12 isomer of conjugated linoleic acid (CLA) from a synthetic CLA mixture consisting predominantly of these isomers in equal amounts. Lipases were screened for their ability to selectively esterify one isomer at a significantly greater rate than the other isomer. An immobilized lipase from Rhizomucor miehei was nonselective, but a lipase from Geotrichum candidum esterified the cis9,trans11 isomer more rapidly than the trans10,cis12 isomer. This selectivity was exploited at the kilogram scale to prepare an ester fraction with a content of 91% cis9,trans11 CLA and an unreacted free fatty acid fraction consisting of 82% trans10,cis12 CLA, based on total CLA content. The components of the reaction mixture were separated by molecular distillation. Each enriched fraction was then incorporated into palm oil triglycerides by interesterification with the non-selective lipase from R. miehei. Two triglyceride fats resulted, which were enriched in either cis9,trans11 CLA (26.5% cis9,trans11 and 1.7% trans10,cis12) or trans10,cis12 CLA (3.5% cis9,trans11 and 22.9% trans10,cis12).     

Positional and geometrical isomers of linoleic acid in partially hydrogenated oils

The geometrical and positional isomers of linoleic acid of a partially hydrogenated canola oil-based spread were isolated and identified. Through partial hydrazine reduction and mass spectral studies,cis-9,trans-13 octadecadienoic acid was identified as the major isomer. Other quantitatively important isomers characterized werecis-9,trans-12;trans-9,cis-12 andcis-9,cis-15. These four were also the major isomers in margarine based on common vegetable oils. A number of minor isomers were detected and some structures identified weretrans-9,trans-12;trans-8,cis-12;trans-8,cis-13;cis-8,cis-13;trans-9,cis-15;trans-10,cis-15 andcis-9,cis-13. The proportions of the various isomers are given for some margarines in the Canadian retail market. The amounts oftrans-9,trans-12 isomer in Canadian margarines were generally below 0.5% of the total fatty acids.     

Rapid degradation of new disinfection by-products in drinking water by UV irradiation: N-Nitrosopyrrolidine and N-nitrosopiperidine

Disinfection by-products (DBPs) have caused a great threat to drinking water security. Nitrosoamines that are potent carcinogenic, mutagenic, and teratogenic have been discovered in disinfection process as a new kind of DBPs. Accordingly, nitrosoamines elimination from water should be of particular concern. In this paper, UV irradiation was used to degrade the model DBPs: N-nitrosopyrrolidine (NPyr) and N-nitrosopiperidine (NPip). The results showed that UV irradiation was an effective method for the removal of both NPyr and NPip from water. NPip was easier to be degraded compared with NPyr. Water pH had little effect on NPyr photodegradation, but had great influence on NPip photodegradation. The characteristic of the natural water influenced the removal efficiency of NPyr and NPip slightly, suggesting that UV irradiation was effectual to eliminate NPyr and NPip in practice. Due to N–N bond fission, pyrrolidine and piperidine were generated as the primary products of NPyr and NPip photodegradation, respectively. Then aliphatic amines with low molecular weight were formed as further degradation products of NPyr, including methylamine, dimethylamine, ethylamine, diethylamine, n-propylamine and n-methylethylamine. Methylamine, dimethylamine, n-propylamine, n-methylethylamine, diethylamine, n-butylamine and n-amylamine were formed as the further degradation products of NPip.     

A novel compound, 12,13,17-trihydroxy-9(Z)-Octadecenoic acid,from linoleic acid by a new microbial isolateClavibacter sp. ALA2

A novel compound, 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA), was produced from linoleic acid by microbial transformation at 25% yield. The newly isolated microbial strain that catalyzed this transformation was identified asClavibacter sp. ALA2. The product was purified by high-pressure liquid chromatography, and its structure was determined by¹H and¹³C nuclear magnetic resonance, Fourier transform infrared, and mass spectroscopy. Maximum production of THOA was reached after 85 h of reaction. THOA was not further metabolized by strain ALA2. This is the first report on 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation.     

亚油酸乳状液体系氧化模型与求解

研究了恒表面氧压静置式无限氧补偿条件下乳状液中亚油酸的氧化,通过综合考虑气液边界传质阻力、水相扩散、油水边界乳化剂膜边界层阻力、多不饱和脂肪酸(PUFA)自催化氧化反应动力学,建立了扩散-氧化数学模型。采用非对称正交配置法处理特殊边界,求解偏微分方程组,计算了氧化过程中乳状液中氧和亚油酸在垂直于液膜方向的浓度分布,计算方法快捷、有效;结合数学模拟试验和实验值,确定了气液界面氧的液膜传质系数和水相中氧的扩散系数。实验验证该模型能较好地拟合静置式无限氧补偿条件下乳状液中氧的扩散和亚油酸的氧化过程。     

Oxidation of linoleic acid in emulsions: Effect of substrate, emulsifier, and sugar concentration

Linoleic acid oxidation in oil-in-water emulsions stabilized by a nonionic surfactant (Tween-20) was studied. The emulsion composition was varied at a constant oil droplet size. Lipid oxidation was measured as a function of time in the presence of a catalyst (FeSO₄ corbic acid) by two methods: gas chromatographic determination of residual substrate and ultraviolet-visible spectrophotometric determination of conjugated dienes. Rate of oxidation was influenced by the emulsion composition (relative concentrations of substrate and emulsifier) and especially by the partition of the emulsifier between the interface and water phase. Concentrations of emulsifier exceeding the critical micelle concentration protected the fatty acid against oxidation. Excess surfactant formed micelles and mixed micelles with linoleic acid, which retarded oxidation by diluting the substrate or perhaps by replacing linoleic acid at the interface, making it less accessible to radical attack. The addition of sucrose also had a protective effect, but only up to a certain concentration, indicating the effect may involve factors other than viscosity.     

A rapid synthesis of poly (p-dioxanone) by ring-opening polymerization under microwave irradiation

Summary A rapid synthesis of poly(p-dioxanone) (PPDO) was carried out smoothly and effectively from the monomer p-dioxanone (PDO) with constant microwave powers of 90, 180, 270, and 360 W, respectively, in a microwave oven at a frequency of 2.45 GHz. The temperature of the polymerization ranged from 158 to 198 °C. PPDO with a viscosity-average molecular weight (Mv) of 156,000g/mol and yield of 63% was obtained at 270W for 25 min using 1/1000 (mol/mol) Sn(Oct)₂ as a catalyst, while it took more than 14h to obtain PPDO with high molecular weight and monomer conversion by conventional heating method when Sn(Oct)₂ used as a catalyst. Therefore, it is obvious that the polymerization rate is faster than that of the conventional polymerization method when microwave irradiation is used in polymerization process.     

Study on antibacterial activity of silver nanoparticles synthesized by gamma irradiation method using different stabilizers

Colloidal solutions of silver nanoparticles (AgNPs) were synthesized by gamma Co-60 irradiation using different stabilizers, namely polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), alginate, and sericin. The particle size measured from TEM images was 4.3, 6.1, 7.6, and 10.2 nm for AgNPs/PVP, AgNPs/PVA, AgNPs/alginate, and AgNPs/sericin, respectively. The influence of different stabilizers on the antibacterial activity of AgNPs was investigated. Results showed that AgNPs/alginate exhibited the highest antibacterial activity against Escherichia coli (E. coli) among the as-synthesized AgNPs. Handwash solution has been prepared using Na lauryl sulfate as surfactant, hydroxyethyl cellulose as binder, and 15 mg/L of AgNPs/alginate as antimicrobial agent. The obtained results on the antibacterial test of handwash for the dilution to 3 mg AgNPs/L showed that the antibacterial efficiency against E. coli was of 74.6%, 89.8%, and 99.0% for the contacted time of 1, 3, and 5 min, respectively. Thus, due to the biocompatibility of alginate extracted from seaweed and highly antimicrobial activity of AgNPs synthesized by gamma Co-60 irradiation, AgNPs/alginate is promising to use as an antimicrobial agent in biomedicine, cosmetic, and in other fields.     

Assessment of the Photosynthetic Apparatus Functions by Chlorophyll Fluorescence and P700 Absorbance in C3 and C4 Plants under Physiological Conditions and under Salt Stress

Functions of the photosynthetic apparatus of C3 (Pisum sativum L.) and C4 (Zea mays L.) plants under physiological conditions and after treatment with different NaCl concentrations (0–200 mM) were investigated using chlorophyll a fluorescence (pulse-amplitude-modulated (PAM) and JIP test) and P₇₀₀ photooxidation measurement. Data revealed lower density of the photosynthetic structures (RC/CSo), larger relative size of the plastoquinone (PQ) pool (N) and higher electron transport capacity and photosynthetic rate (parameter R_Fd) in C4 than in C3 plants. Furthermore, the differences were observed between the two studied species in the parameters characterizing the possibility of reduction in the photosystem (PSI) end acceptors (REo/RC, REo/CSo and δRo). Data revealed that NaCl treatment caused a decrease in the density of the photosynthetic structures and relative size of the PQ pool as well as decrease in the electron transport to the PSI end electron acceptors and the probability of their reduction as well as an increase in the thermal dissipation. The effects were stronger in pea than in maize. The enhanced energy losses after high salt treatment in maize were mainly from the increase in the regulated energy losses (Φ_NPQ), while in pea from the increase in non-regulated energy losses (Φ_NO). The reduction in the electron transport from Q_A to the PSI end electron acceptors influenced PSI activity. Analysis of the P₇₀₀ photooxidation and its decay kinetics revealed an influence of two PSI populations in pea after treatment with 150 mM and 200 mM NaCl, while in maize the negligible changes were registered only at 200 mM NaCl. The experimental results clearly show less salt tolerance of pea than maize.     

Specific determination of malonaldehyde by N-methyl-2-phenylindole or thiobarbituric acid

Reactivity of a commercially available test kit (LPO-586), based on N-methyl-2-phenylindole, toward aldehydes was characterized and compared with that of thiobarbituric acid (TBA). In hydrochloric acid, LPO-586 produced a violet pigment with malonaldehyde (MA) but not with other tested aldehydes. In methane sulfonic acid, LPO-586 produced the violet pigment with MA and 4-hydroxynonenal (HNE), but not with other tested aldehydes. Pigment formation with MA was not inhibited by other aldehydes, but that with HNE was inhibited by alka-2,4-dienals. TBA produced a red pigment with MA but not with other tested aldehydes in hydrochloric acid or in acetate with ethylenediaminetetraacetic acid (EDTA). Both the LPO-586 test in hydrochloric acid and the TBA test in hydrochloric acid or in acetate with EDTA can be used for specific measurement of MA in oxidized lipid samples.     

Behavior of phenolic antioxidants in a partitioned medium: Focus on linoleic acid peroxidation induced by iron/ascorbic acid system

The peroxidation of linoleic acid was induced by the Fe²⁺/ascorbic acid system in a micellar medium, and the kinetics of the produced conjugated dienes to a weak level (0.7 μM) in order to initiate only the sequential oxidative reactions without superimposed antioxidant activity. Other conditions were established to obtain a simple kinetic profile. With a linoleic acid/Fe²⁺ ratio of 10 and an Fe²/ascorbic acid ratio of 23 in the medium emulsified by Tween and saturated in oxygen, no limiting effect of substrates was observed for several hours. Fe²⁺ disappeared in less than 5 min, while a linear propagation rate was reached after 1 h for up to 15 h. Two parameters were chosen to quantify the level of the oxidation: the slope of the linear phase, and the extrapolated absorbance of this step at zero time. These two parameters were used in order to compare the antioxidant power of different phenolic compounds in this partitioned medium.     

Wet oxidation of wastewater containing hydrocarbons by novel supported Pd catalysts

Pd catalysts supported on TiO₂, ZrO₂, ZSM-5, MCM-41 and activated carbon were used in catalytic wet oxidation of hydrocarbons such as phenol, m-cresol and m-xylene. It was found that the Pd/TiO₂ catalyst was highly effective in the wet oxidation of hydrocarbon. The activities of catalysts with various hydrocarbon species, catalyst support, oxidation state of catalyst performed in a 3-phase slurry reactor show that reaction on Pd surface is more favorable than that in aqueous phase and that the active site is oxidized Pd in catalytic wet air oxidation of hydrocarbons. Based on the experimental results, a plausible reaction mechanism of wet oxidation of hydrocarbons catalyzed over Pd/TiO₂ catalyst was proposed. This catalyst is superior to other oxide catalysts because it suppressed the formation of hardly-degradable organic intermediates and polymer.     

Lipid and fatty acid biosynthesis by Rhodotorula minuta

Demand for fatty acids is increasing at an annual rate of 17%, due to their increased use in the oleochemical and transport industries. Presently, vegetable oils are the major source of fatty acids, whereas lipids with fatty acids similar to those of some vegetable oils have been reported to be synthesized by oleaginous microorganisms. In the present study, the culturing conditions for the oleaginous yeast Rhodotorula minuta IIP-33 have been optimized. In contrast to the lipid accumulation characteristics of most oleaginous yeasts, a carbon-to-nitrogen ratio of 30 was favorable for maximal accumulation of lipids (48%) with 22.5% conversion of glucose as carbon substrate. The lipids contained fatty acids in the C₇–C₁₈ range, the relative composition of which varied with culture temperature.     

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.     

Preparation of poly(acrylic acid)–chitosan hydrogels by gamma irradiation and in vitro drug release

Biocompatible and biodegradable pH‐responsive hydrogels based on poly(acrylic acid) (AAc) and chitosan were prepared for controlled drug delivery. These interpolymeric hydrogels were synthesized by a gamma irradiation polymerization technique. The degree of gelation was over 96% and increased as the chitosan or acrylic acid content increased. The equilibrium swelling studies of hydrogels prepared in various conditions were carried out in an aqueous solution, and the pH sensitivity in the range of pH 1–12 was investigated. The AAc/chitosan hydrogels showed the highest water content when the 30 vol % AAc and 0.1 wt % chitosan were irradiated with a 30‐kGy radiation dose. Also, an increase of swelling degree with an increase in the pH was noticed and showed the highest value at pH 12. The drug, 5‐fluorouracil, was loaded into these hydrogels and the release studies were carried out in simulated gastric and intestinal fluids. The in vitro release profiles of the drugs showed that more than 90% of the loaded drugs were released in the first 1 h at the intestinal pH and the rest of the drug had been released slowly. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3660–3667, 2003