Differential scanning calorimetric studies on structured lipids from coconut oil triglycerides containing stearic acid

Triglycerides from coconut oil contain high levels of lauric acid. They were replaced by incremental amounts of stearic acid by interesterification reactions catalyzed by immobilized lipase (IM 60 from Rhizomucor miehei). The reactions were carried out in organic solvents such as hexane. Maximum incorporation of stearic acid was observed by 4 h at 37vv°C or by 2 h at 60vv°C when triglycerides to fatty acid (stearic acid) ratio was maintained at 1v:Ң. The stearic acid level in coconut oil triglycerides was increased from an initial value of 2% to 60% under these conditions. The stearic acid replaced lauric, myristic, and palmitic acids in unmodified triglycerides. A major portion of stearic acid incorporated was found in positions 1 and 3 of triglycerides. Differential scanning calorimetry indicated that stearic acid enrichment increased the solid fat content and also the higher melting polymorphs in modified lipids. The studies also indicated that low melting polymorphic forms of coconut oil triglycerides are converted to higher melting forms by stearic acid enrichment. The modified lipids thus obtained can find use in various food applications.     

Cocoa butter equivalent from enzymatic interesterification of tea seed oil and fatty acid methyl esters

Cocoa butter equivalent (CBE) was prepared by interesterification of tea seed oil, methyl palmitate and methyl stearate with lipase. The lipase was immobilized on macroporous resin selected from eight carriers. The rate of reaction of lipase immobilized on macroporous resin was 6.9 times higher than that of the free enzyme. After repeating application five times, 83.50% activity, of the immobilized lipase, was retained. Factors such as reaction time, temperature, water content, enzyme load and substrate ratio were studied. Three major acyls (palmitoyl, oleoyl and stearoyl) in triacylglycerols of the product were similar to those of cocoa butter. The melting range and dilatation–temperature curves of the prepared CBE were close to that of the cocoa butter.     

Enzyme-catalyzed synthesis of structured lipids via acidolysis of seal (Phoca groenlandica) blubber oil with capric acid

A structured lipid (SL) containing n-3 fatty acids (eicosapentaenoic acid, 20:5n-3; docosahexaenoic acid, 22:6n-3) and capric acid (10:0; a medium chain fatty acid) was prepared using lipase-catalyzed acidolysis of seal blubber oil with capric acid. An immobilized lipase, Lipozyme-IM from Mucor miehei, was used as the biocatalyst. Acidolysis reactions were carried out in hexane and the products were analyzed by gas chromatography. Incorporation of capric acid was affected by mole ratio of substrates, type of organic solvent, reaction temperature, reaction time, water content and the amount of lipase. The optimum reaction mixture and conditions were oil/fatty acid mole ratio of 1:3, hexane, 45 °C, 24 h, 1% (w/w of substrates) water and 10% (w/w of substrates) Lipozyme-IM lipase. Under these conditions, a SL containing 2.3% 20:5n-3, 7.6% 22:6n-3 and 27.1% 10:0 was obtained. Solvents with log P values between 2.5 and 4.5 performed the acidolysis reaction better than those with log P values of less than 2.5. However, in the absence of any organic solvent, Lipozyme-IM afforded a satisfactory incorporation of capric acid into seal blubber oil.     

固定化Enterobacter agglomerans脂肪酶生产菜籽油生物柴油的研究

用硅藻土对实验室筛选得到的成团肠杆菌脂肪酶干燥酶粉进行固定化,固定化酶在有机溶剂体系下催化生产生物柴油。在最佳反应条件,即菜籽油15.47 mL,固定化脂肪酶用量1 000 U,甲醇为酰基受体(7.15 mL,3次等量加入),5 mL正己烷,振荡速度180 r/min,35℃反应48 h时,转化率达91.03%。实验结果表明,油酸含量高有利于生产生物柴油,而芥酸有不利影响。固定化酶稳定性好,重复使用8次,转化率仍大于50%,同时还具有一定的适应性,可催化大豆油和葵花籽油生产生物柴油。研究表明,固定化酶可用于催化生产生物柴油,并有效降低酶催化法的生产成本。     

Large agarose-lipase beads for the hydrolysis of triglycerides

Candida cylindracea lipase could be immobilized by binding to large agarose beads or by entrapment within polyacrylamide polymer. Large agarose beads, prepared from native agarose, were preferred for their higher binding capacity and easy intact separation for reuse. The agarose-lipase complex is stable for 6 months at 4°C, maintaining 80% of its initial activity despite eight-fold re-use. Consequently, the lipase complex is recommended for semi-continuous hydrolysis of olive oil and milk fat triglycerides. The extent of total hydrolysis was 30·8 ± 1·25% for olive oil as against 23·1 ± 0·95% for milk fat.     

零反式脂肪酸涂抹脂基料油酶法酯交换制备研究

利用固定化脂肪酶催化棕榈油硬脂和葵花籽油进行酯交换反应,制备零反式脂肪酸涂抹脂基料油。考察了不同搅拌速度、反应温度、酶加量和反应时间对酯交换反应的影响,并对反应前后油脂的熔点、晶型和结晶速率等结晶行为进行了分析和比较。结果表明:搅拌速度200 r/min,酶加量6%,在70℃下反应3 h为最优的酯交换条件。酯交换产物的熔点大幅下降,晶型主要以β’晶型为主,结晶速率变慢。     

A comparison between olive oil and extra-virgin olive oil used as covering liquids in canned dried tomatoes: hydrolytic and oxidative degradation during storage

The hydrolytic and oxidative degradation of olive oil and extra‐virgin olive oil, used as covering liquids in canned dried tomatoes, was studied during storage by means of conventional (acidity, peroxide value, p‐anisidine value) and non conventional (polar compounds) analyses. The effects of the addition of spices were also considered. The hydrolysis and oxidation of olive oil increased faster and was higher than that of extra‐virgin olive oil in terms of absolute values but some other indices, such as percentage of oligopolymers and percentage of oxidized triglycerides, increased faster in extra‐virgin olive oil than in olive oil. The antioxidant effect given by a higher concentration of polyphenols in the extra‐virgin olive oil was shown by a reduced amount of secondary oxidation. However, olive oil and extra‐virgin olive oils showed similar behaviour in terms of peroxide formation.     

Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase

Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40 degrees C in a mixture of tuna oil and 1 3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2 3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40 degrees C for 12 h in a mixture of tuna oil and 1 3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2 3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1 3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively.     

单辛酸甘油酯的酶法合成

以Novo435固定化脂肪酶为催化剂,在无溶剂条件下催化甘油和辛酸合成单辛酸甘油酯。通过单因素试验确定加酶量、反应温度、反应时间三个因素的取值范围,并用响应面实验设计和分析方法对合成反应条件进行了优化。结果表明,在甘油辛酸摩尔比1:1、反应温度66.8℃、脂肪酶与反应底物质量比0.88%、无水的条件下反应11.5h,辛酸转化率达93.53%,单辛酸甘油酯含量为47.69%。     

Enzymatic synthesis of cocoa butter analog through interesterification of lard and tristearin in supercritical carbon dioxide by lipase

Substrate oil composition, reaction time, acyl donor, temperature, and pressure affected the triacylglycerol (TG) content of cocoa butter analog during the interesterification reaction catalyzed by lipase in a supercritical carbon dioxide (SC-CO₂) system. Among oil sources used to interact with tristearin, the content of 1(3)-palmitoyl-3(1)-stearoyl-2-monoolein (POS) (P, palmitate; O, oleate; S, stearate) and 1-palmitoyl-2, 3-dioleoylglycerol (POO) in analog was most similar to the corresponding TG content of cocoa butter when analog was prepared with lard. The optimized interesterification reaction using lard and tristearin (at a mole ratio of 1.4) as substrates to produce cocoa butter analog in a SC-CO₂ system was at 17 MPa, 50 °C, pH 9, for 3 h with an immobilized lipase, Lipozyme IM-20, from Mucor miehei. The lyophilized enzyme facilitated the production of cocoa butter analog in anhydrous substrates (a_w 0.33). The yield and melting point of the purified cocoa butter analog by a silica column was 63% and 34.5 °C, respectively, when the analog was produced under optimal conditions.     

Lipase production by yeasts from extra virgin olive oil

Newly produced olive oil has an opalescent appearance due to the presence of solid particles and micro-drops of vegetation water from the fruits. Some of our recent microbiological research has shown that a rich micro-flora is present in the suspended fraction of the freshly produced olive oil capable of improving the quality of the oil through the hydrolysis of the oleuropein. Present research however has, for the first time, demonstrated the presence of lipase-positive yeasts in some samples of extra virgin olive oil which can lower the quality of the oil through the hydrolysis of the triglycerides. The tests performed with yeasts of our collection, previously isolated from olive oil, demonstrated that two lipase-producing yeast strains named Saccharomyces cerevisiae 1525 and Williopsis californica 1639 were able to hydrolyse different specific synthetic substrates represented by p-nitrophenyl stearate, 4-nitrophenyl palmitate, tripalmitin and triolein as well as olive oil triglycerides. The lipase activity in S. cerevisiae 1525 was confined to the whole cells, whereas in W. californica 1639 it was also detected in the extracellular fraction. The enzyme activity in both yeasts was influenced by the ratio of the aqueous to the organic phase reaching its maximum value in S. cerevisiae 1525 when the water added to the olive oil was present in a ratio of 0.25% (v/v), whereas in W. californica 1639 the optimal ratio was 1% (v/v). Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil, produced in four different geographic areas, demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected, according to the source of oil samples. The discovery of lipase-positive yeasts in some extra virgin olive oils leads us to believe that yeasts are able to contribute in a positive or negative way towards the organological quality of the olive oil.     

辛癸酸甘油酯和橄榄油的极性调控研究

本文以添加了不同浓度表面活性剂司盘80(Sp80)的辛癸酸甘油酯(Octanoic acid glyceride,ODO)和橄榄油作为模型油脂,测定在不同剪切速率下ODO和橄榄油的表观黏度,分析不同司盘80添加量对ODO和橄榄油界面张力的影响,并对其抗聚并稳定性进行了评价,从而探讨不同浓度表面活性剂下油脂极性的变化规律。结果表明,ODO和橄榄油体系的表观黏度随司盘80添加量的增加而增大;当司盘80浓度小于5%时,ODO-水界面张力降低,ODO极性增大;而在橄榄油体系中,橄榄油-水的界面张力达到平衡所需司盘80用量较ODO少,当司盘80浓度大于1%时,橄榄油极性与界面张力无明显变化,且司盘80可增强ODO和橄榄油油滴的抗聚并稳定性;当司盘80浓度大于5%时,由于油相极性较大,油水间作用力增强且过量胶束生成,反而促进了橄榄油油滴的聚并。     

茶油酶促改性一步反应影响因素研究

中国南方几省茶油有一定产量，而国外茶油产量极小，基本上没有市场。利用茶油的特殊结构进行酶促改性制类可可脂很少见有报道。选择１，３位定向脂肪酶，在茶油、硬脂酸、软脂酸混合非水溶剂（己烷）体系中进行一步酯交换反应，最终制出了类似可可脂结构和组成的代用品。本研究主要对茶油酶促改性的主要影响因素进行了探讨。     

Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase

Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40°C in a mixture of tuna oil and 1/3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40°C for 12 h in a mixture of tuna oil and 1/3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2/3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1/3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively.     

Ethyl esterification of docosahexaenoic acid in an organic solvent-free system with immobilized Candida antarctica lipase

Ethyl docosahexaenoate (EtDHA) is regarded as a potentially useful pharmaceutical substance on account of its beneficial physiological activities. We attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organic solvent-free system using Candida antarctica lipase, which acts strongly on DHA and ethanol. Esterification of 88% was attained by shaking a mixture of DHA/ethanol (1:1, mol/mol) and 2 wt% immobilized C. antarctica lipase at 30 degrees C for 24 h. However, even in the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (1:1, mol/mol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30 degrees C for 24 h with 5 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield.     

Optimization of the enzyme-catalyzed synthesis of amino acid-based surfactants from palm oil fractions

The feasibility of using palm oil fractions as cheap and abundant sources of raw material for the synthesis of amino acid surfactants was investigated. Of a number of enzymes screened, the best results were obtained with the immobilized enzyme, Lipozyme. The effects of temperature, solvent, incubation period, fatty substrate/amino acid molar ratio, enzyme amount, and water removal on the reactions were analyzed and compared to those on reactions with free fatty acids and pure triglycerides as fatty substrates. All reactions were most efficient when carried out at high temperatures (70-80 degrees C) in hexane as a solvent. However, while reactions with free fatty acids proceeded better when a slight excess of the free fatty acids over the amino acids was used, reactions with triglycerides and palm oil fractions were best performed at equimolar ratios. Also, the addition of molecular sieves slightly enhanced reactions with free fatty acids but adversely affected reactions with triglycerides and palm oil fractions. Although reactions with palm oil fractions took longer (6 d) to reach equilibrium compared to reactions with free fatty acids (4 d) and pure triglycerides (4 d), better yields were obtained. Such lipase-catalyzed transacylation of palm oil fractions with amino acids is potentially useful in the production of mixed medium- to long-chain surfactants for specific applications.     

无溶剂系统脂肪酶促POMF酯交换生产CBE的动力学研究

研究了无溶剂系统脂肪酶促棕榈油中间熔点物酯交换生产类可可脂的动力学，在优化的反应条件下，建立了初始底物浓度对反应初速度的影响和三种主要甘三酯浓度随时间变化的两个动力学模型。实验结果表明，计算值与实验值较吻合。     

Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil

The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25-0.4 h intervals.     

有机溶剂体系固定化脂肪酶催化合成生物柴油

探讨了有机溶剂体系固定化Candida antarctica脂肪酶催化大豆色拉油合成生物柴油的过程。将固定化Candida antarctica脂肪酶置于有机溶剂体系中催化合成生物柴油的效果较好。研究发现,在40℃下反应10 h,固定化Candida antarctica脂肪酶以石油醚作为有机溶剂转化率最高,当总醇油物质的量比为3∶1,固定化酶占5%(相对于油质量),加入5%质量分数的水时固定化酶反应活性最高,酯化率可以达到88.4%。固定化酶重复使用10次仍具有较高活性。     

Compositional and textural properties of milkfat–soybean oil blends following enzymatic interesterification

Milkfat–soybean oil blends were enzymatically interesterified (EIE) by Aspergillus niger lipase immobilized on SiO₂–PVA hybrid composite in a solvent free system. An experimental mixture design was used to study the effects of binary blends of milkfat–soybean oil (MF:SBO) at different proportions (0:100; 25:75; 33:67; 50:50; 67:33; 75:25; 100:0) on the compositional and textural properties of the EIE products, considering, as response variables, the interesterification yield (IY), consistency and hardness. Lipase-catalysed interesterification reactions increased the relative proportion of TAGs’ C₄₆–C₅₂ and decreased the TAGs’ C₄₀–C₄₂ and C₅₄ concentrations. The highest IY was attained (10.8%) for EIE blend of MF:SBO 67:33 resulting in a more spreadable material at refrigerator temperature in comparison with butter, milkfat or non-interesterified (NIE) blend. In this case, consistency and hardness values were at least 32% lower than values measured for butter. Thus, using A. niger lipase immobilized on SiO₂–PVA improves the textural properties of milkfat and has potential for development of a product incorporating unsaturated and essential fatty acids from soybean oil.