Lipase‐catalysed production and chemical composition of diacylglycerols from soybean oil deodoriser distillate

Diacylglycerols (DAG) were enzymatically produced by lipase‐catalysed esterification of glycerol with fatty acids from soybean oil deodoriser distillate (SODD). Effects of reaction parameters such as reaction time, temperature, enzyme type, enzyme load, substrate molar ratio and water content, as well as the effect of molecular sieves as water adsorbent were studied. Lipozyme RM IM was determined to be the most effective among the lipases screened. The following conditions yielded 69.9% DAG (all percentages are wt/wt): 4 h reaction time, 65 °C reaction temperature, 10% Lipozyme RM IM, 2.5:1 fatty acid to glycerol molar ratio, and 30% molecular sieves. DAG synthesis of 11.9% was still observed at 10% water content. After purification, the product oil contained 86.3% DAG. This oil consisted predominantly of 1,3‐diolein (19.1%), 1‐oleoyl‐3‐linoleoyl‐glycerol (18.2%) and 1‐oleoyl‐2‐linoleoyl‐glycerol (16.6%). The fatty acid profile of the oil was similar to that of refined, bleached and deodorised (RBD) soybean oil. The % ratio of 1,3‐ to 1,2‐positional isomers of DAG was at 56:44.     

高纯度EPA/DHA甘油三酯的酶法合成

利用脂肪酶分别催化游离型EPA和DHA与甘油发生酯化反应生成甘油三酯,考察了合成的影响因素. 结果表明,正己烷6 mL,甘油/EPA(DHA)摩尔比为1:3(以0.4 mmol甘油为基准),Novozym 435添加量为100 mg,反应温度40℃,振荡频率150 r/min,反应24 h后添加1 g分子筛,反应时间48 h,EPA甘油三酯与DHA甘油三酯的得率分别可达88.64%和88.07%,EPA与DHA酯化度分别可达95.0%和94.5%. 分析结果表明,所得产物为EPA甘油三酯和DHA甘油三酯.     

Enzymatic esterification of θ-3 fatty acid concentrates from seal blubber oil with glycerol

Enzymatic synthesis of acyglycerols directly from glycerol and an θ-3 fatty acid concentrate, prepared from seal blubber oil, in organic solvents was studied. Seven lipases were used as biocatalysts for esterification, Lipase LP-401-AS from Chromobacterium viscosum showed the highest activity for esterification. Effects of reaction parameters, namely, temperature, time course, type of solvent, water content, amount of glycerol, enzyme load and solvent volume, were followed with lipase IP-401-AS as the biocatalyst of choice. Optimal reaction conditions were established, and the maximal degree of acylglycerol synthesis reached was 94.3%. The concentrations of monoacylglycerols, diacylglycerols, and triacylglycerols were 13.8, 43.1, and 37.4%, respectively. Therefore, acylglycerols containing predominantly θ-3 fatty acid concentrates may be easily synthesized directly via their reaction with glycerol.     

Concentration and purification of stearidonic,eicosapentaenoic, and docosahexaenoic acids from cod liver oil and the marine microalgaIsochrysis galbana

n-3 Polyunsaturated fatty acids (n-3 PUFA) from the marine microalgaIsochrysis galbana were concentrated and purified by a two-step process—formation of urea inclusion compounds followed by preparative high-performance liquid chromatography. These methods had been developed previously with fatty acids from cod liver oil. By the urea inclusion compounds method, a mixture that contained 94% (w/w) stearidonic (SA), eicosapentaenoic (EPA), plus docosahexaenoic (DHA) acids (4:1 urea/fatty acid ratio and 4°C crystallization final temperature) was obtained from cod liver oil fatty acids. Further purification of SA, EPA, and DHA was achieved with reverse-phase C₁₈ columns. These isolations were scaled up to a semi-preparative column. A PUFA concentrate was isolated fromI. galbana with methanol/water (80:20, w/w) or ethanol/water (70:30, w/w). With methanol/water, a 96% EPA fraction with 100% yield was obtained, as well as a 94% pure DHA fraction with a 94% yield. With ethanol/water as the mobile phase, EPA and DHA fractions obtained were 92% pure with yields of 84 and 88%, respectively.     

月桂酸单甘酯的催化合成

文章以磷钨酸为催化剂,4分子筛为脱水剂,在无溶剂条件下月桂酸和甘油为原料合成月桂酸单甘酯,考察了反应温度、摩尔比（月桂酸/甘油）、反应时间、催化剂用量、分子筛用量对反应产率的影响,并确定了反应最佳条件是：反应温度200℃,摩尔比（月桂酸/甘油）1∶2.5,反应时间为2 h,磷钨酸用量为3%,分子筛用量为5%（月桂酸与...     

Lipase-Catalysed Enrichment of γ-Linolenic Acid from Evening Primrose Oil in a Solvent-Free System

The enrichment of γ-linolenic acid (GLA) was carried out in a solvent-free system by lipase-catalysed esterification of free fatty acids from evening primrose oil (EPO-FA) and 1-butanol (BtOH). The lipase employed to conduct this study was a free preparation of Candida rugosa. Variables evaluated were: substrate molar ratio (1:4, 1:6, 1:8, 1:10 and 1:12, EPO-FA:BtOH), temperature (10, 20, 30, 40, 50 and 60 °C), and enzyme loading (5, 10, 15 and 20 %, based on the total weight of substrates). GLA was highly enriched in the non-esterified fatty acid fraction since C. rugosa showed very low selectivity for this fatty acid. We were able to increase the content of GLA to ca. 70 wt.% under the following optimal conditions: 30 °C, 10 % enzyme loading and a 1:10 molar ratio (EPO-FA:BtOH), after 24 h. An additional set of experiments was conducted whereby the amount of water was controlled by addition of molecular sieves to the reaction mixture. The latter experiments produced a higher GLA concentrate (83.74 wt.%), under the optimal conditions described above and by adding 10 % molecular sieves (based on the total weight of substrates) after 36 h.     

Production of n-3 polyunsaturated fatty acid-enriched fish oil by lipase-catalyzed acidolysis without solvent

Fish oil rich in n-3 polyunsaturated fatty acid (n-3 PUFA) was prepared by nonsolvent enzymic acidolysis. n-3 PUFA-enriched fish oil contained 25% eicosapentaenoic acid (EPA) and 40% docosahexaenoic acid (DHA). In acidolysis of cod liver oil, EPA content of the original fish oil was reduced at 5 h, but DHA content of the fish oil increased. It was assumed that EPA in the fish oil was replaced by DHA to reach a new chemical equilibrium. Two-stage acidolysis, which was carried out under CO₂ replacement early (about 3 h) and also in vacuum at 5–24 h, was effective for reduction in the content of diacylglycerol, which was formed by reverse reaction, hydrolysis. This method has industrial significance because PUFA-enriched triacylglycerol is easily separated from the reaction mixture by molecular distillation. Bioreactors for fats and their derivatives, Part XIV.     

Solvent-free enzymatic glycerolysis of marine oils

Marine triglyceride oils (cod liver oil and oils from blubber of harp seal and minke whale) were reacted with glycerol using lipase as a catalyst at low temperature. A solvent-free batch system with magnetic stirring was used. Solidification of the reaction mixture occurred, and a mixture of mono-, di-, and triglycerides was obtained in all cases. The recovered glyceride mixtures were solid at room temperature. The yield of monoglyceride (MG) and the fatty acid profile of the MG fractions were dependent on oil and the type of lipase used as a catalyst. Of the commercially-available lipases investigated, lipase AK fromPseudomonas sp. synthesized the highest yield of MG (42–53%) at 5°C. These MG fractions were low in saturated fatty acids (4–11%) and high in long-chain monounsaturated fatty acids (52–69%). The concentration of n-3 polyunsaturated fatty acids was 12–20%.     

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.     

Production of structured triglycerides rich in n-3 polyunsaturated fatty acids by the acidolysis of cod liver oil and caprylic acid in a packed-bed reactor: equilibrium and kinetics

Structured triglycerides (ST) enriched in n-3 polyunsaturated fatty acids (PUFAs) (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA) in position 2 of the triglyceride backbone were synthesised by acidolysis of cod liver oil (CLO) and caprylic acid (CA) catalysed by the 1,3-specific immobilised lipase Lipozyme IM. The reaction was carried out in three ways: (1) in a batch reactor (where the influence of temperature on the incorporation of CA into the CLO triglyceride was studied); (2) in an immobilised lipase packed-bed reactor (PBR) by recirculating the reaction mixture from the exit of the bed to the substrate reservoir (product recirculation) to determine the equilibrium composition; and (3) in a PBR without recirculation. A “lag” period of duration inversely proportional to the initial water amount of the lipase, was observed when new lipase was used. Apparently, during this “lag” period the hydro-enzymatic layer that surrounds the lipase surface reaches its water equilibrium content. A reaction scheme, where only the fatty acid in the positions 1 and 3 of the glycerol backbone were exchanged by CA, was proposed. The exchange equilibrium constants between CA and the native fatty acids of CLO were determined. The n-3 PUFAs (EPA and DHA) were the most resistant native fatty acids to exchange with exchange equilibrium constants of 1.32 and 0.28, respectively. Also, average reaction rates and kinetic constants of exchange of CA and native fatty acid of CLO were calculated. Low kinetic constants were observed for EPA, DHA and palmitic acid. For acidolysis reaction in the continuous mode PBR, the lipase amount/(flow rate × substrate concentration) ratio (m_L/q[TG]₀) could be considered as the intensive variable of the process for use in scale up of the PBR. A simple equation was proposed for the prediction of the fatty acid composition of the ST at the exit of the PBR as a function of the intensive variable m_L/q[TG]₀. At equilibrium, the ST produced had the following composition: CA 57%, EPA 5.1%, DHA 10.0% and palmitic acid 6.3% (only considering the major fatty acids). In addition, the proportion of EPA and DHA that esterified the position 2 of the ST was 13.5%, which represented 44% of the total fatty acids in the position 2 of the resultant ST.     

Enzymatic modification of evening primrose oil: Incorporation of n−3 polyunsaturated fatty acids

Immobilized lipase SP435 fromCandida antaractica was used as a biocatalyst for the modification of the fatty acid composition of evening primrose oil by incorporating n−3 polyunsaturated fatty acid (PUFA) and eicosapentaenoic acid (EPA). Transesterification (ester-ester interchange) was conducted in organic solvent or without solvent, with EPA ethyl ester (EEPA) as the acyl donor. Products were analyzed by gas-liquid chromatography (GLC). After 24-h incubation in hexane, the fatty acid composition of evening primrose oil was markedly changed to contain up to 43% EPA. The amount of 18:2n−6 PUFA was reduced by 32%, and the saturated fatty acid content was also reduced. The effects of incubation time, molar ratio, enzyme load, and reaction medium on mol% EPA incorporation were also studied. Generally, as the incubation time (up to 24 h), molar ratio, and enzyme load increased, EPA incorporation also increased. Evening primrose oil, containing EPA and γ-linolenic acid (18:3n−6) in the same glycerol backbone, was successfully produced and may be more beneficial for certain applications than unmodified oil.     

Concentrating PUFA from mackerel processing waste

Mackerel processing waste comprising skins, viscera, and muscle tissue was evaluated for concentrating PUFA by urea complexation. Fish oil was extracted using either chloroform/methanol (2∶1, vol/vol) or hexane/isopropanol (3∶2, vol/vol). The yield of oil, as well as iodine, peroxide, and acid values, was determined for fresh fish oil extracts, and oil samples were storel at −70°C in the presence of 100 ppm α-tocopherol. PUFA concentrates were prepared from saponified fish oil. The mean oil yields were 9.18±2.3, 9.2%±2.4, and 38.1±3% for viscera, muscle, and skin, respectively. The mean baseline iodine value was 134±5, which increased to 296±7 after urea complexation. It was possible to concentrate PUFA from mackerel processing waste. The type of tissue used did not affect the amount of PUFA concentrated. Mackerel skin was most desirable because of its high oil content.     

Biodiesel Production from Mustard Emulsion by a Combined Destabilization/Adsorption Process

Tetrahydrofuran, added to the oil‐in‐water emulsions formed by the aqueous processing of yellow mustard flour, produced oil/water/THF miscellas containing 1–2 % water. The high water content prevented the direct conversion of the system to fatty acid methyl esters (FAME) through a single‐phase base‐catalyzed transmethylation process. Dehydration of these miscellas by adsorption on 4A molecular sieves at room temperature using either batch or continuous fixed‐bed systems successfully reduced the water content to the quality standards needed for biodiesel feedstock (0.3 %). Equilibrium adsorption studies for the uptake of water from oil/THF/water miscella phases at room temperature allowed quantitative comparison of the water adsorption capacity based on the oil and THF concentrations of the miscellas. Batch contact was used to investigate the dominant parameters affecting the uptake of water including miscella composition, adsorbent dose and contact time. The adsorption of the water was strongly dependent on adsorbent dose and miscella oil concentrations. The regeneration of molecular sieves by heating under nitrogen at reduced pressure for 6 h at 275 °C resulted in incomplete desorption of miscella components. The adsorption breakthrough curves in terms of flow rates, initial water and oil miscella concentrations were determined. The dehydrated miscella phases were reacted with methanol in a single‐phase base‐catalyzed transmethylation process with high yields (99.3 wt%) to FAME. The resulting FAME met the ASTM international standard in terms of total glycerol content and acid number.     

分子蒸馏富集海狗油中多不饱和脂肪酸

海狗油中富含多不饱和脂肪酸,如二十碳五烯酸(EPA)、二十二碳五烯酸(DPA)、二十二碳六烯酸(DHA)等。用分子蒸馏法对海狗油中多不饱和脂肪酸进行富集,通过控制适宜的温度和压力等条件,得到较为满意的分离效果。当进料速率为80 mL/h,预热温度为80℃,刮膜器转速为250r/min,蒸馏温度为120℃,压力为15 Pa时,经过一级分子蒸馏,得到EPA、DPA和DHA的总含量为54.86%(质量分数)的海狗油产品,收率为92.7%,并用气相色谱法测定了产品的脂肪酸组成。     

固定化Candida sp.99-125脂肪酶催化大豆油合成脂肪酸乙酯

探讨了酶法合成脂肪酸乙酯作为生物柴油的可行性. 以大豆油和乙醇为原料,利用本实验室自制的固定化Candida sp. 99-125脂肪酶催化反应,深入研究水含量、溶剂量、脂肪酶量及反应温度等因素对酶法合成脂肪酸乙酯的影响. 结果表明,以大豆油质量为基准,在水含量为 12.5%(w)、溶剂正己烷为3 mL/g、脂肪酶量为20%(w)、温度40℃的优化反应条件下,3次流加乙醇,170 r/min摇瓶反应,12 h后可以达到96.8%的最高酯得率. 进一步研究表明,在此优化反应条件下,连续使用14批脂肪酶酯得率可保持70%以上.     

Enzymatic synthesis of steryl esters of polyunsaturated fatty acids

Steryl esters of long-chain fatty acids have water-holding properties, and polyunsaturated fatty acids (PUFA) have various physiological functions. Because steryl ester of PUFA can be expected to have both features, we attempted to synthesize steryl esters of PUFA by enzymatic methods. Among lipases used, Pseudomonas lipase was the most effective for the synthesis of cholesteryl docosahexaenoate. When a mixture of cholesterol/docosahexaenoic acid (3:1, mol/mol), 30% water, and 3000 units/g of lipase was stirred at 40°C for 24 h, the esterification extent attained 89.5%. Under the same reaction conditions, cholesterol, cholestanol, and sitosterol were also esterified efficiently with docosahexaenoic, eicosapentaenoic, arachidonic, and γ-linolenic acids.     

Synthesis of Fatty Acid Ethyl Ester from Acid Oil in a Continuous Reactor via an Enzymatic Transesterification

Synthesis of a fatty acid ethyl ester via the lipase‐catalyzed transesterification of acid oil and ethanol was investigated in a continuous reactor. Lipozyme TL IM was employed as the immobilized lipase. This immobilized lipase derived from Thermomyces lanuginosus was purchased from Novozymes (Seoul, Korea). The acid oil was prepared by the acidification of soapstock formed as a by‐product during the refining of rice bran oil. The parameters investigated were water content, temperature, and molar ratio of substrates. The relative activity of Lipozyme TL IM was assessed during the repeated use of the immobilized lipase. The water content of the substrate had a considerable effect on the yield and the optimum water content was 4 %. The optimum temperature and molar ratio of acid oil to ethanol were 20 °C and 1:4, respectively. The maximum yield of approximately 92 % was achieved under the optimum conditions. The corresponding compositions were 92 % fatty acid ethyl esters, 3 % fatty acids, and 5 % acylglycerols. When glycerol formed during the reaction was removed by intermittent washing with ethanol, the relative activity of lipase was maintained over 82 % for a total usage of 27 cycles. For a mean residence time of 4 h, the half‐life times of Lipozyme TL IM on the control (unwashed) and treatment (washed) were 39 and 45 cycles, respectively.     

Continuous synthesis of glycerides by lipase in a microporous membrane bioreactor

A membrane bioreactor was developed for continuous synthesis of glycerides by lipase to overcome the drawbacks associated with the usual operation in an emulsion system. One unit (total area: 726 cm²) of flat, plate-type dialyzer was used as the membrane bioreactor at 40 C. The glycerol solution, containing bacterial lipase and water, was supplied continuously to 1 side of a sheet of microporous polypropylene membrane (strongly hydrophobic) and the effluent was recycled, while undiluted liquid fatty acid (oleic or linoleic) was fed continuously to the opposite side of the membrane and came in contact with a glycerol-water-lipase solution to cause the reaction. The product, glycerides, was obtained at the outlet, in a pure state, with no other phase. Highest conversion (ca. 90%) was obtained when the water content of the glycerol solution was 3–4%. As the accumulation of water produced by the reaction lowered the conversion, molecular sieves in a column that the glycerol solution passed through were used for optimal water content. The reaction could be continued at least for 1 month, yielding a conversion above 70% when 1% CaCl₂ was added in the glycerol solution. The main component of glycerides formed was almost equimolar amounts of mono-and diglycerides.     

Specificity of a lipase fromGeotrichum candidum forcis-octadecenoic acid

A lipase fromGeotrichum candidum released mostly oleic acid from glyceryl 1-elaidate-2,3-dioleate and very littletrans fatty acid from margarine. When cod liver, Macadamia nut, peanut and safflower oils were substrates, the oleic acid content of the free acids was always in excess of the amount of the acid in the intact triglycerides. Congo palm oil was digested by bothG. candidum and pancreatic lipases and the fatty acid compositions of the products of hydrolysis compared. The results obtained with the aid ofG. candidum lipase tend to substantiate existence of some of the triglyceride isomers predicted from pancreatic lipase data. Scientific contribution No. 134, Agricultural Experiment Station, University of Connecticut, Storrs.     

脂肪酶催化合成D-异抗坏血酸月桂酸酯

用固定化脂肪酶Lipozyme 435作催化剂,由D-异抗坏血酸和月桂酸合成D-异抗坏血酸月桂酸酯。考察了反应温度、反应溶剂、反应物配比、酶用量、反应时间、月桂酸浓度、分子筛用量、摇床转速对产物收率的影响。结果表明,以20mL叔戊醇为反应溶剂、0.10g脂肪酶为催化剂、2.0g 4A分子筛为脱水剂,在55℃、170r/min转速的恒温摇床中,0.88g D-异抗坏血酸和2.50g月桂酸反应36h,产物收率68%。产物结构经IR、1H NMR、13C NMR和MS表征。