Lipase‐catalysed biosynthesis of hexyl butyrate by direct esterification: optimisation by response surface methodology

The ability of immobilised lipase from Rhizomucor miehei (Lipozyme IM‐77) to catalyse the direct esterification of hexanol and butyric acid was investigated. Response surface methodology (RSM) and a four‐factor/five‐level central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters such as reaction time (2–10 h), temperature (25–65 °C), substrate molar ratio of hexanol to butyric acid (1:1–3:1) and enzyme amount (10–50%; 0.24–1.18 BAUN) on the percentage molar conversion of hexyl butyrate by direct esterification. All the parameters had significant effects on the percentage molar conversion. Based on ridge maximum analysis, the optimal conditions for synthesis were: reaction time 8.0 h, temperature 46.9 °C, substrate molar ratio 1:1.2 and enzyme amount 36.4% (0.87 BAUN). The predicted value was 100% and the actual experimental value 98.2% molar conversion. Copyright © 2003 Society of Chemical Industry     

Optimisation of enzymatic esterification of soybean oil deodoriser distillate

The enzymatic esterification of free fatty acids from soybean oil deodoriser distillate with ethanol, catalysed by immobilised fungal lipase, was studied. The extent of conversion of free fatty acids to ethyl esters was optimised using a response surface methodology obtained through a second‐order factorial experimental design. The variables studied were reaction temperature (30–70 °C), enzyme concentration (7–23%) and ethanol/free fatty acid molar ratio (0.3–3.7:1). The optimal reaction conditions achieved were temperature from 46.4 to 53.6 °C, enzyme concentration from 13.6 to 16.5% and ethanol/free fatty acid molar ratio from 1.7 to 2.3:1, with conversions above 88%. No significant tocopherol losses were observed during the process. In conclusion, enzymatic fatty acid esterification was shown to be a technically viable process. © 2001 Society of Chemical Industry     

Optimal production and structural characterization of erythorbyl laurate obtained through lipase-catalyzed esterification

Erythorbic acid, a stereoisomer of L-ascorbic acid, has been extensively used as an antioxidant but cannot be applied to lipid-based foods due to its poor lipophilicity. For this reason, synthesis of erythorbyl laurate (6-O-lauroyl-erythorbate) was achieved in acetonitrile using an immobilized lipase from Candida antarctica as a biocatalyst to increase its lipophilicity. Response surface methodology was used to optimize the erythorbyl laurate synthesis conditions in terms of enzyme content (1,000–5,000 propyl laurate unit, PLU), molar ratio of lauric acid to erythorbic acid (5–25), and reaction temperature (25–65°C). The central composite experimental results showed the conditions for maximum molar conversion yield were as follows: enzyme content, 2,994 PLU; lauric acid to erythorbic acid molar ratio, 24.23; and reaction temperature, 53.03°C. The maximum molar conversion yield reached 77.81%, which was in agreement with the predicted value (76.92%). The erythorbyl laurate was purified and identified by Fourier transform-infrared spectroscopy (FT-IR). This research could help to develop an economical method of synthesizing erythorbyl laurate for use as a novel foodgrade emulsifier with antioxidative activity.     

Optimization of Sucrose Polyester Synthesis Using Response Surface Methodology

Response Surface Methodology (RSM) was used to evaluate the effects of synthetic variables such as reaction time (6–12 hr), temperature (130–150°C), and substrate molar ratio of fatty acid methyl esters (FAME) of peanut oil to sucrose (10:1 to 14:1) on the % molar conversion to sucrose polyester, utilizing 10g of free sucrose as the reactant. Optimization of the synthetic reaction was performed by canonical analysis to derive the stationary point. Based on contour plots and canonical analysis, optimum conditions were: reaction time 11.5 hr, synthetic temperature 144°C, and substrate molar ratio 11.4:1. Predicted molar conversion was 43.39% (10g sucrose synthesized 29.4g sucrose polyester) at the optimum point. Experimental data indicated up to 48.4% yield based on theoretical % molar conversion.     

偏甘油酯脂肪酶Lipase G50催化酯化法制备甘油二酯

利用偏甘油酯脂肪酶Lipase G50催化甘油和脂肪酸酯化反应合成甘油二酯.探讨了酶加量、底物摩尔比、反应温度及加水量对酯化反应的影响.结果表明最佳反应条件为:脂肪酶Lipase G50加量为350 U/g,甘油和脂肪酸的摩尔比5∶1,加水量为底物总质量的5％,反应温度30℃,反应时间24h.在最佳反应条件下脂肪酸的酯化率为75.02％,甘油二酯的含量达到44.74％,产物中没有甘油三酯生成.     

Optimisation of Novozym‐435‐catalysed esterification of fatty acid mixture for the preparation of medium‐ and long‐chain triglycerides (MLCT) in solvent‐free medium

Novozym‐435‐catalysed esterification of caprylic acid, capric acid and oleic acid with glycerol for the synthesis of medium‐ and long‐chain triglycerides (MLCT) in vacuum and solvent‐free system was investigated in this study. Response surface methodology with a three‐level, four‐factorial design was applied to optimise the enzymatic esterification for the synthesis of MLCT. The optimum conditions were as follows: reaction temperature 90 °C, 4.80 wt% enzyme load (relative to the weight of total substrates) and substrate molar ratio (fatty acids/glycerol) of 3:1 and 12.37 h. Under above‐mentioned conditions, Triglycerides (TG) yield, MLCT and the residual free fatty acids (FFA) content in the product were 93.54%, 72.19% and 4.21%, respectively. The content of caprylic acid, capric acid and long‐chain fatty acids of TG was 24%, 10% and 66%, respectively. Novozym 435 in the study showed no selectivity for the different fatty acids and also could be used 14 times without obvious loss of enzyme activity.     

非水介质中脂肪酶催化合成正戊酸异戊酯的研究

德氏根霉菌(Rhizopusdelemar) 经固态发酵生产脂肪酶,以此酶为催化剂,在非水介质中合成了正戊酸异戊酯。研究了反应温度、溶剂、底物浓度、底物摩尔比、吸水剂用量等因素对酯化反应的影响,确定了正戊酸异戊酯的最佳合成条件为:反应温度为5 0℃,异辛烷为反应介质,底物浓度为0 15mol/L ,酸醇摩尔比为1∶1 4。在反应体系中需加入0 2 5 g/mL的5 分子筛,以吸收酯化反应生成的水。在优化的条件下,反应6h后,酯合成转化率达98%。     

Monoacylglycerol Production from Butteroil by Glycerolysis with a Gel-Entrapped Microbial Lipase in Microaqueous Media

Reaction parameters were optimized to support polyethyleneglycol-based and polypropyleneglycol-based gel-entrapped lipase-mediated glycerolysis of butteroil to yield monoacylglycerol. Single liquid-phase reactive mixtures contained 25% (w/w) butteroil dissolved in t-butanol, to which glycerol and water was added. Optimum reaction conditions were 35–40 °C, and 5–10 mg enzyme reagent and 0.10g glycerol/mL substrate solution. Optimum acyl group:glycerol molar ratio was about 0.64. The optimum water content was dependent on glycerol content and was about 6% (w/w) of the glycerol at 0.10g glycerol/mL substrate solution. The gel-entrapped lipases had half-lives of about 35–46 cycles of 72 hr each.     

无溶剂直接酯化法合成亚油酸甾烷醇酯的研究

为了得到甾烷醇与亚油酸在无溶剂条件下,直接酯化法合成亚油酸甾烷醇酯的最佳工艺条件,以酯化率和体系氧化程度为考察指标,单因素考察筛选利于合成的底物酸醇摩尔比、催化剂添加量、反应温度和反应时间,并采用L9(34)正交实验进一步优化得出最佳合成工艺:亚油酸与甾烷醇的摩尔比3∶1,催化剂用量3%,反应时间5h,反应温度150℃,在此条件下,亚油酸甾烷醇酯的酯化率达到81.23%。采用薄层色谱,高效液相色谱和近红外光谱对亚油酸甾烷醇酯进行了鉴定,并研究了亚油酸甾烷醇酯的热力学特性。     

Enzyme-Catalyzed Production and Chemical Composition of Diacylglycerols from Corn Oil Deodorizer Distillate

Diacylglycerols (DAG) were enzymatically synthesized by lipase-catalyzed esterification of glycerol with fatty acids from corn oil deodorizer distillate (CrODD). Effects of reaction parameters such as reaction time, temperature, enzyme type, enzyme load, substrate mole ratio, and water content, as well as the effect of molecular sieves as a water adsorbent were investigated. Rhizomucor miehei lipase (Lipozyme RM IM) was found to be most effective among the lipases screened. The following conditions yielded 70.0% (w/w) DAG: 5 h reaction time, 65°C reaction temperature, 10% (w/w) Lipozyme RM IM, 2.5:1 fatty acid to glycerol molar ratio, and 30% (w/w) molecular sieves. DAG synthesis of 12.4% (w/w) was still observed at 10% (w/w) water content. 84.2% (w/w) of DAG was obtained after purification. The DAG oil comprised predominantly of 1-oleoyl-3-linoleoyl-glycerol (28.5%), 1,3-diolein (22.7%), 1-oleoyl-2-linoleoyl-glycerol (17.9%), and 1,2-diolein (10.9%). Fatty acid profile was similar to that of refined, bleached and deodorised (RBD) corn oil. The ratio of 1,3- to 1,2-positional isomers of DAG was at 1.82:1.     

Optimization of lipase-catalyzed enantioselective esterification of (±)-menthol in ionic liquid

Response surface methodology was successfully applied to optimize lipase-catalyzed enantioselective esterification of (±)-menthol. The effects of various reaction conditions, including reaction time, temperature, enzyme loading, substrate molar ratio and water activity, were investigated. A Central Composite Rotatable Design was employed to search for the optimal conversion of (±)-menthol and enantiomeric excess. A quadratic polynomial regression model was used to analyze the experimental data at a 95% confidence level (p < 0.05). The analysis confirmed that reaction temperature, enzyme loading and reaction time were the significant factors affecting the conversion of (±)-menthol. Moreover, reaction temperature, enzyme loading, substrate molar ratio and reaction time were found to affect the enantiomeric excess significantly. The coefficient of determination of these two models was found to be 0.980 and 0.967, respectively. Two sets of optimum reaction conditions were established and the verified experimental trials were performed for validating the optimum points. Under the optimum conditions, the conversion of (±)-menthol and the enantiomeric ratio exceeded 53% and 40%, respectively.     

酶法全酯化合成富含EPA/DHA甘油酯工艺研究

以富含EPA/DHA的脂肪酸为底物,采用两步酶法合成富含EPA和DHA的甘油酯。首先,以T1脂肪酶为催化剂催化富含EPA/DHA的脂肪酸和甘油反应;在最优条件为:反应温度40℃,水分添加量为底物混合物的3%、甘油与脂肪酸摩尔比3∶1和酶添加量50 U/g底物混合物时,富含EPA/DHA的脂肪酸的转化率达到62%以上,此时产物中甘油三酯、甘油二酯、甘油单酯的质量分数分别为10.52%、38.15%、25.64%。将游离酶催化酯化反应产物中的油相回收,利用自制的固定化CALB(LipozymeCALB L固定于环氧树脂ECR8285上)为催化剂,在真空条件下继续催化未反应的脂肪酸与偏甘油酯(甘油单酯和甘油二酯)继续酯化反应12 h,此时产物中甘油三酯、甘油二酯和甘油单酯的质量分数分别达到38.34%、51.02%、10.63%,没有检测到脂肪酸的存在。     

Simultaneous saccharification and fermentation of furfural residues by mixed cultures of lactic acid bacteria and yeast to produce lactic acid and ethanol

In a cellulosic ethanol production system, yeasts cannot be reused, and it is difficult to avoid the formation of lactic acid. A novel mixed fermentation system based on water-rinsed furfural residue was designed to produce lactic acid and ethanol simultaneously by yeast and lactic acid bacteria. The fermentation broth can be used for the production of lactic acid, ethanol, or ethyl lactate, which is a very suitable alternative green solvent. Simultaneous saccharification and fermentation by mixed cultures of lactic acid bacteria and yeast (MSSF) in different conditions were carried out. Acid/alcohol molar ratios (molar ratio of lactic acid to ethanol) were investigated to determine the effects of temperature, substrate concentration, and mass rate of yeast to lactic acid bacterial cells on MSSF. Cellulose conversion rate was also calculated to evaluate the effectiveness of MSSF. The cellulose conversion rate of MSSF was higher than those of ethanol simultaneous saccharification and fermentation and lactic acid simultaneous saccharification and fermentation. The cellulose conversion rate increased with increasing substrate concentration, while the acid/alcohol molar ratio decreased with increasing substrate concentration. These results indicate that yeast cells could provide nutrients for lactic acid bacteria. MSSF at 38 °C is also apt to obtain a low acid/alcohol molar ratio. A 1:1 lactic acid/alcohol molar ratio will be obtained at a fluctuating temperature (38 °C between 0 and 54 h, 42 °C after 54 h) and a substrate concentration of 9%, while keeping a high cellulose conversion rate and final lactic acid concentration. A probable bottleneck for MSSF and its potential solution are also proposed in this paper.     

酶法合成共轭亚油酸单甘酯的研究

采用脂肪酶G50(来源于Penicillium camembertii)作为生物催化剂,催化共轭亚油酸(CLA)和甘油酯化生成共轭亚油酸单甘酯(MAG)。研究了在无溶剂体系中,底物摩尔比、酶加量、体系含水量、反应温度和反应时间对产物中MAG含量和CLA酯化率的影响。结果表明,最佳反应条件为:底物摩尔比n(甘油)∶n(CLA)=4∶1,加酶量300U/g(基于反应底物总质量),体系含水量1%,反应温度15℃,反应时间24h。在最佳反应条件下,CLA的酯化率达到84.98%,MAG的含量为68.40%,共轭亚油酸双甘酯(DAG)含量为16.58%。通过分析产物的脂肪酸组成,发现Penicillium脂肪酶G50对CLA异构体没有拆分效果。     

根霉脂肪酶有机相合成己酸乙酯条件的研究

用根霉ZM-10脂肪酶为催化剂,在有机相中催化合成己酸乙酯。文中研究了温度、底物浓度、酸醇比、溶剂、吸水方法等对己酸乙酯合成转化率的影响。结果表明,以环己烷为溶剂,以摩尔比为1∶1.3的己酸和乙醇为底物,己酸浓度为0.2 mol/L,在40℃条件下振荡反应14h,合成己酸乙酯的的转化率可达到91.5%。     

Study of lipase‐catalysed synthesis of ascorbyl benzoate in cyclohexanone using response surface methodology

BACKGROUND: Compared with ascorbic acid, ascorbyl esters exhibit many advantages. Their biosynthesis in organic media has been widely studied owing to its many advantages over chemosynthesis. However, since the natural substrates of lipase are fatty acid triacylglycerols, few data are available on the lipase‐catalysed esterification of ascorbic acid with aromatic acids. Furthermore, although many reports can be found on the biosynthesis of ascorbic acid with fatty acids, few deal with the interactions between important reaction factors. The aim of the present study was to optimise the biosynthesis process of ascorbic acid with an aromatic acid, benzoic acid, in cyclohexanone using a statistical approach and to investigate the interactions among variables. RESULTS: Among the five factors studied, only substrate concentration, water activity and temperature had a significant effect on the reaction. Enzyme concentration affected the process slightly, while the effect of reaction time became minor after equilibrium. For the first time, using a partial derivative method, it was quantitatively demonstrated how the optimum of the variable studied depended on the constant values of fixed variables used in a single‐factor experiment. Statistical analysis predicted a maximal conversion rate (47.66%) at enzyme concentration 10 g L^?1, substrate concentration 0.1031 mol L^?1, water activity 0.4896, reaction time 48 h and temperature 66.63 °C. CONCLUSION: This study illustrated possible factor interactions during the lipase‐catalysed synthesis of ascorbyl benzoate in cyclohexanone, and a maximal conversion rate was achieved under optimal reaction conditions. Copyright © 2007 Society of Chemical Industry     

双有机溶剂中黑曲霉脂肪酶催化阿魏酸的酯化反应

在双有机溶剂体系中,用黑曲霉脂肪酶催化合成阿魏酸油醇酯,考察酶浓度、温度和底物摩尔比等因素对酯化反应的影响。结果表明:在异辛烷/丁酮体系中,当反应温度为60℃,阿魏酸和油醇的摩尔比为1∶8,即阿魏酸浓度为0.39 mg/mL和油醇浓度为4.3 mg/mL,脂肪酶浓度为0.2 g/mL时,转化率为97.6%;而在环己烷/丁酮体系中,当反应温度为60℃,阿魏酸和油醇的摩尔比为1∶8,即阿魏酸浓度为0.49 mg/mL和油醇浓度为5.37 mg/mL,脂肪酶浓度为0.25 g/mL时,转化率为91.0%。     

Effects of kojic acid on changes in the microstructure and myofibrillar protein of duck meat during superchilled storage

This study investigated the effect of 0.8% (m/v) kojic acid treatment on changes in the microstructure and myofibrillar protein of duck meat covered with oxygen-permeable polyvinylchloride (PVC) film (9 ± 0.5 µM) during superchilled storage (−1.65 ± 0.5°C). The superchilled samples exhibited wider gaps between muscle fibers at 5 weeks storage compared with kojic acid–treated groups. Based on the variation of water status, the water-holding capacity decreased significantly (p < 0.05), and bound water and immobilized water were gradually converted into free water during superchilled storage. For kojic acid–treated samples, however, no major changes were observed with respect to muscle structure, water status, and protein degradation at 6 weeks. The 0.8% (m/v) kojic acid treatment increased the water-holding capacity, reduced carbonyl content and protein degradation, and decreased the α-helix contents loss of myofibrillar proteins. Kojic acid treatment effectively protected myofibrillar protein structure from being destroyed during superchilled storage, suggesting that this method was a good way to reduce protein oxidation and prolonged its shelf life.     

有机介质中酶催化合成己酸乙酯的研究

本实验研究了有机介质中CRL 脂肪酶催化己酸与无水乙醇合成己酸乙酯的酯化反应条件,并利用红外光谱、GC-MS、气相色谱等现代方法对合成的产物进行了表征。结果显示,合成的产物为己酸乙酯,其纯度为96.91%,分子量144。酯化反应在以正庚烷为反应介质,反应温度为311K,己酸与无水乙醇的摩尔比为1:1.3,己酸浓度为0.2mol/L 的条件下,反应24h 后,转化率达到94.6%。     

Production of structured lipids containing conjugated linolenic acid: optimisation by response surface methodology

Structured lipids containing conjugated linolenic acid (CLNA) were produced separately by enzymatic acidolysis reaction of corn and canola oils (CAO) with bitter gourd (Momordica charantia L) seed oil fatty acids [bitter gourd seed oil fatty acids (BGFA)]. Reactions were conducted using a commercial immobilised sn‐1,3‐specific lipase from Thermomyces lanuginosa (Lipozyme TL IM) in hexane. The effects of reaction time, substrate molar ratio, temperature and enzyme amount on incorporation yield of CLNA were investigated and optimised by response surface methodology with three‐level, two‐factor face‐centred cube design. When reactions were conducted using 10% enzyme for 3 h, the optimum reaction conditions were found for corn oil (CO) as 53.5 °C and 5.9:1 BGFA/CO molar ratio. At these conditions, the incorporation of CLNA into CO was determined as 41.4%. However, CLNA incorporation into CAO was resulted as 37% at optimum conditions which were 54.2 °C and 6.8:1 BGFA/CAO molar ratio.