Synthesis of alkyl glycoside fatty acid esters in non-aqueous media byCandida sp. lipase

Alkyl glycoside fatty acid esters were successfully synthesized by lipase-catalyzed transesterification of methyl glucoside, methyl glucoside, methyl galactoside and octyl glucoside with methyl oleate. The experiments were carried out in organic media with lipase enzymes fromCandida sp. as biocatalysts. Time course and the effects of temperature, solvent type, substrate concentration, added water and of immobilizedvs. nonimmobilized enzyme were studied. The optimal conditions for the enzymatic synthesis of alkyl glycoside fatty acid esters were: a molar ratio of alkyl glycoside and methyl oleate of 1:4, an immobilized lipase, SP382 fromCandida sp.; benzene/pyridine (2:1, vol/vol) with no added water; temperature, 55°C; reaction time, 48 h; and shaking at 200 rpm. Acceptable levels of oleic acid incorporation (58.6–100 mol%) onto the alkyl glycosides were achieved.     

Enzymatic synthesis of monosaccharide fatty acid esters and their comparison with conventional products

5-O-Acyl-1,2-O-isopropylidene-D-xylofuranose and 6-O-acyl1,2∶3,4-di-O-isopropylidene-D-galactopyranose were enzymatically prepared from the corresponding monosaccharide acetals and commercial (crude) fatty acid mixtures. Subsequent acid-catalyzed hydrolysis of the isopropylidene group(s) gave monosaccharide esters with overall yields of 59–88%, where the monoester content was at least 80% (galactose oleate) and typically 90% for the other preparations. In contrast to sugar fatty acid esters prepared by conventional, high-temperature (trans)esterification, the enzymatically obtained monosaccharide esters contained no appreciable quantities of undersirable side products, and the only contaminants were monosaccharides and fatty acids.     

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.     

Enzymatic production of fatty acid alkyl esters with a lipase preparation from Candida sp. 99‐125

A lipase preparation developed from Candida sp. 99‐125 was used for fatty acid alkyl ester synthesis by both enzymatic esterification of fatty acids, and transesterification of oils and fats. The lipase preparation was chosen based on screening of lipases from commercial sources as well as those produced in the laboratory. The effects of enzyme dosage, solvent types, water absorbent additions, inhibition of short‐chain alcohols, alcohol and acid types, molar ratio of substrates, and reusability of the lipase preparation in esterification were studied. Degree of esterification between oleic acid and methanol under optimal conditions reached 92%. Purity of the methyl ester after washing with water and distillation was 98%. Half‐life of the lipase preparation was calculated to be approximately 340 h. For transesterification of rapeseed oil with the same lipase preparation, the amount of methanol and mode of methanol addition to the reaction were also conducted. Transesterification of the oil with stepwise methanol addition reached 83% after 36 h reaction time.     

Enzymatic synthesis of polyglycerol-fatty acid esters in a solvent-free system

The enzymatic transesterification of fatty acid methyl esters from vegetable oil and polyglycerol has been successfully performed in the presence of Lipozyme without any solvent. The optimal conditions to obtain a mixture with lipophilic tensioactive properties were found to be a molar ratio of fatty acid methyl esters and polyglycerol of 1.33:1 at 60°C. Evaporation of the methanol produced in this reaction increased the yield of the reaction. Moreover, for this reaction, adsorption of the polyol on silica gel prior to the transesterification allowed the completion of the reaction and improved the kinetic properties. Hydrophile-lipophile balance and surface activity of the polyglycerol esters were measured to determine the emulsifying properties of these molecules.     

研磨强化无溶剂法合成蔗糖脂肪酸酯

以蔗糖和脂肪酸甲酯为反应原料,无溶剂条件下采用研磨强化的方法合成了蔗糖脂肪酸酯(即蔗糖酯),考察了研磨时间、乳化剂用量、反应时间、反应压力以及催化剂用量对蔗糖酯收率及脂肪酸甲酯转化率的影响,产品经水洗、醇提精制后,用FTIR和HPLC-ELSD对其进行了表征和分析。结果表明:研磨处理能将反应分散物粒径(D50)降至约5μm,有效地增强了蔗糖与脂肪酸甲酯的无溶剂酯交换反应。得到的最优反应条件为:研磨时间为60min,硬脂酸钾乳化剂质量分数为10%(占体系总质量,下同),K2CO3质量分数为2.0%,反应时间6 h,反应压力0.5 kPa,在该条件下脂肪酸甲酯转化率为91.2%,蔗糖酯收率为61.6%。精制后产品中蔗糖酯质量分数为81.9%,且其游离糖质量分数为0.6%,酸值为3.0mgKOH/g,水分(质量分数)为0.2%和灰分(质量分数)为0.5%,均达到FAO/WHO标准。     

Improved synthesis of sucrose fatty acid monoesters

The base-catalyzed synthesis of four sucrose fatty acid esters (caprylate, laurate, myristate, and palmitate) was performed in dimethylsulfoxide by transesterification of sucrose with the corresponding vinyl esters using disodium hydrogen phosphate as catalyst. In using a molar ratio sucrose/vinyl ester 4∶1 and mild reaction conditions (40°C and atmospheric pressure), yields were higher than 85%. The isolated sucroesters had a higher percentage of monoesters (≥90%) and a lower content of diesters in comparison with commercial derivatives. In all cases, 2-O-acylsucrose was the major product (≥60%) in the monoester fraction.     

Enzymatic synthesis of wax esters from rapeseed fatty acid methyl esters and a fatty alcohol

Wax esters were transesterified from fatty acid methyl esters of rapeseed and a fatty alcohol (1-hexadecanol, 16:0). The amounts of both the substrates were fixed to 0.1 mmol and an immobilized enzyme, Lipozyme, was used as catalyst. The experiment was performed following a statistic central composite design with five variables. The enzyme/lipid ratio was varied between 0.3–0.9 of the substrate weight and the enzyme was equilibrated to different water activities varying from 0.11 to 0.44. A temperature range of 50–80°C was investigated and the reaction time lasted up to 40 min. A solvent, isooctane, constituted 0–30% of the substrate weight. The first experimental series was performed in small closed test tubes. In the second series the caps of the test tubes were off to evaporate the methanol produced during the reaction. The highest initial reaction rate was 9.6 g_{wax esters}/g_enzyme · h. It appeared when: the enzyme/lipid ratio was low, 0.3, the temperature was high, 80°C; no isooctane was present; and the water activity was below 0.11. The initial reaction rate was independent of the caps on the test tubes. With the large amount of enzyme the yield of wax esters was above 70% after 10 min in both experimental series. In the reaction with caps, the reaction reached equilibrium at 83% after 20 min at 80°C. However, without caps the continuous evaporation of methanol increased the equilibrium constantly, and after 40 min at 80°C a yield of 90% was reached.     

Chemo-enzymatic synthesis of disaccharide fatty acid esters

A novel enzymatic method for the synthesis of disaccharide fatty acid esters was developed with immobilizedMucor miehei lipase (Lipozyme IM-60; Novo Nordisk, Bagsvaerd, Denmark) as a catalyst. A range of lactose and maltose monoesters was prepared in overall yields of 48–77% from the corresponding sugar acetals and fatty acids.     

长链脂肪酸淀粉酯合成及应用研究进展

长链脂肪酸淀粉酯是一类具有特殊的热塑性、疏水性、乳化性和可生物降解性的变性淀粉,在食品、纺织、化妆品、医药等行业有着广泛的应用。本文系统地介绍了长链脂肪酸淀粉酯的性质、合成方法和应用领域;分析了各种制备方法的特点,并指出了今后研究方向和发展前景。     

Enzymatic direct synthesis of acrylic acid esters of mono‐ and disaccharides

BACKGROUND: There is an increased need to replace materials derived from fossil sources by renewables. Sugar‐cane derived carbohydrates are very abundant in Brazil and are the cheapest sugars available in the market, with more than 400 million tons of sugarcane processed in the year 2007. The objective of this work was to study the preparation of sugar acrylates from free sugars and free acrylic acid, thus avoiding the previous preparation of protected sugar derivatives, such as glycosides, or activated acrylates, such as vinyl acrylate. RESULTS: Lipase catalyzed esterification of three mono‐ and two disaccharides with acrylic acid, in the presence or absence of molecular sieves was investigated. The reactions were monitored by high‐performance liquid chromatography (HPLC) and the products were analyzed by matrix‐assisted laser desorption ionization–time of flight (MALDI‐TOF) mass spectrometry. The main products are mono‐ and diacrylates, while higher esters are formed as minor products. The highest conversion to sugar acrylates was observed for the D ‐glucose and D ‐fructose, followed by D ‐xylose and D ‐maltose. Molecular sieves had no pronounced effect on the conversion CONCLUSIONS: A feasible method is described to produce and to characterize sugar acrylates, including those containing more than two acrylate groups. The process for production of these higher esters could potentially be optimized further to produce molecules for cross‐linking in acrylate polymerization and other applications. The direct enzymatic esterification of free carbohydrates with acrylic acid is unprecedented. Copyright © 2008 Society of Chemical Industry     

Enzymatic transesterification of sunflower oil in an aqueous-oil biphasic system

A biphasic oil-aqueous system for FAME production by enzymatic catalysis was studied. The transesterification of sunflower oil with methanol was catalyzed by free or immobilized lipases from Rhizomucor miehei (Palatase 20 000 L) and Humicola insolens (Lipozyme TL 100 L). The effects of protein amount, temperature, pH, and molar ratio of methanol to sunflower oil on the enzymatic reaction using free lipase were evaluated; the best results were obtained with H. insolens, at pH 5, 40°C, and 36.8 mg of protein. By using this lipase immobilized in Hypol^® 2002 (64. 8 mg of protein) at a 6∶1 methanol/oil molar ratio and a 2∶1 volumetric oil/water phase ratio, an ester content of 96.1% and a conversion of 91.2% were achieved. The immobilized lipase could be reused, although a 30% reduction in conversion efficiency was observed after four uses.     

Lipase-catalyzed modification of phospholipids: Incorporation of n-3 fatty acids into biosurfactants

Phospholipids were successfully modified by lipase-catalyzed transesterification to incorporate n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6). The phospholipid modification was carried out in organic media with lipase fromMucor miehei (lipozyme) as biocatalyst. The parameters studied were the effect of different solvents, enzymes, acyl donor type, phospholipid class, water, enzyme and substrate concentrations. Hydrolysis of phosphatidylcholine to yield lysophosphatidylcholine and the synthesis of phosphatidylcholine from lysophosphatidylcholine was also carried out. The optimal conditions for the modification of phospholipids by transesterification were obtained with phosphatidylcholine and free eicosapentaenoic acid EPA 45 as acyl donor in the presence of 15% w/w nonimmobilizedMucor miehei lipase (lipozyme) in hexane with no added water. The maximum incorporation of EPA 45 was 17.7 mol%. Hydrolysis was easily achieved with phospholipase A₂ in benzene and Tris-HCl buffer. The synthesis of phosphatidylcholine was difficult, and when it was achieved, not enough phosphatidylcholine was obtained for quantitation.     

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.     

脂肪酸酯的技术与市场发展动向

论述了脂肪酸一元醇酯,多元醇酯的特性、合成及用途,并介绍了由脂肪酸酯开发“绿色”表面活性剂的最新技术动向。     

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.     

Lipase-catalyzed esterification of glycerol and n-3 polyunsaturated fatty acid concentrate in organic solvent

Enzymatic synthesis of glycerides from glycerol and n-3 polyunsaturated fatty acid in organic solvent was studied. Optimal conditions for glyceride synthesis by lipases were established. Of the commercially available lipases that were investigated, lipase PS-30 fromPseudomonas sp. and lipase IM-60 fromMucor miehei resulted in the highest extent of esterification. Isooctane and hexane were particularly useful organic solvents in glyceride synthesis. The water content in the reaction mixture was of primary importance. For lipase PS-30 and lipase IM-60, optimal water contents were 5 and 1%, respectively. Lipases PS-30 and IM-60 manifested contrasting positional specificities in glyceride synthesis. Glycerides containing predominantly eicosapentaenoic acid and docosahexaenoic acid can be easily synthesized.     

Enzymatic synthesis of fatty alcohol esters by alcoholysis

Lipase-catalyzed conversions of some minor oils and fats like mowrah (Madhuca latifolia), mango (Mangifera indica) kernel, and sal (Shorea robusta) fats into low, medium, and high molecular weight alcohol esters have been investigated. In solvent-free medium, alcoholysis of the above-mentioned fats with 10% (w/w) Mucor miehei lipase produced alcohol esters in good yield. The percentage molar conversions of C₄, C₈, C₁₀, C₁₂, C₁₄, C₁₆, C₁₈, and C_18:1 alcohols into corresponding alcohol esters ranged from 86.8 to 99.2, while the percentage molar conversions on the basis of oil were in the range of 108.0 to 123.5.     

Convenient preparation of picolinyl derivatives from fatty acid esters

It is essential to have simple rapid methods for the determination of fatty acid structures. Traditionally, fatty acids are analysed by gas chromatography using their methyl ester derivatives (FAME). However, their corresponding mass spectra exhibit molecular ions but are usually devoid of ions indicative of structural features and, notably, the position of double bounds on the aliphatic chains [1]. In the most useful approach to structure determination, the carboxyl group is derivatised with a reagent containing a nitrogen atom. Recently, a convenient method for preparing picolinyl esters from intact lipids has been published [2]. However, some problems occurred in our laboratory when this method was used, leading to some modifications and optimisation. Thus, hexane and water have been added while sodium bicarbonate has been removed in order to lower contamination. Temperature and length of the reaction have then been optimised in order to get 100% derivatisation for different kinds of lipids (45 °C and 45 min for FAME). Finally, a comparison of the response factors has confirmed the better sensitivity of the picolinyl derivative against FAME (five times more).     

Emulsification properties of polyesters and sucrose ester blends I: Carbohydrate fatty acid polyesters

A number of carbohydrate fatty acid polyesters, potential fat substitutes, were screened for their ability to reduce surface and interfacial tensions alone or as blends with commercial emulsifiers. Commercial sucrose ester emulsifiers (Ryoto, Mitsubishi-Kasei Food Corporation, Tokyo, Japan) were evaluated alone or blended with other sucrose esters or with other carbohydrate fatty acid polyesters, and their surfactant properties were examined in terms of their ability to reduce surface and interfacial tensions at different concentrations and to stabilize oil-in-water (o/w) and water-in-oil (w/o) food emulsions at room and refrigeration temperatures, respectively. In general, when used alone the carbohydrate polyesters were excellent stabilizers of w/o emulsions and poor stabilizers of o/w emulsions. Blending lipophilic carbohydrate polyesters with hydrophilic commercial emulsifiers, such as S-1670, produced stable o/w emulsions and unstable w/o emulsions. Our results suggest that emulsifier blends of potential fat substitutes with FDA-approved commercial sugar ester emulsifiers can be prepared for possible use in low-calorie foods, cosmetics and pharmaceuticals as o/w and w/o emulsifiers.