Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21

Culture conditions of Schizochytrium limacinum SR21 for the purpose of microbial docosahexaenoic acid (DHA) production were investigated. The strain SR21 showed a wide tolerance to salinity; that is, the optimum salinity was between 50% and 200% that of sea water. Monosaccharides (glucose and fructose) and glycerol supported good cell growth and DHA yield. Di- and polysaccharides, oleic acid, and linseed oil gave low DHA yields. A high content of DHA (more than 30% of total fatty acids) was obtained from culture on glucose, fructose, and glycerol, and also the strain had simple polyunsaturated fatty acid profiles. The major polyunsaturated fatty acids other than DHA were n-6 docosapentaenoic acid only, and the contents of icosapentaenoic acid and arachidonic acid were less than 1%. Using corn steep liquor as a nitrogen source, a high total fatty acid content was obtained. The total fatty acid content in the dry cell weight increased as the concentration of the nitrogen source decreased, reached more than 50%. An increase in carbon source concentration led to a high DHA yield. A maximum DHA yield of more than 4 g/l was obtained in both glucose and glycerol media at 9% and 12% respectively. S. limacinum SR21 was thought to be a promising resource for microbial DHA production yielding a good level of productivity as well as a simple polyunsaturated fatty acid profile. Received: 26 June 1997 / Received revision: 29 August 1997  / Accepted: 19 September 1997     

Production of structured triacylglycerols (STAG) rich in docosahexaenoic acid (DHA) in position 2 by acidolysis of tuna oil catalyzed by lipases

This work deals with the production of structured triacylglycerols (STAG) with caprylic acid (CA) located in positions 1 and 3 of the molecule of glycerol and docosahexaenoic acid (DHA) in position 2, by acidolysis of tuna oil and CA, catalyzed by several lipases. To this end several lipases and immobilization supports were tested with the aim of avoiding the acyl-migration observed in previous works. The determination of the best catalyst (i.e. the lipase and the immobilization support as a whole) was carried out by experiments of acidolysis of cod liver oil and CA in a bath reactor. The best results were obtained with the lipases from Rhizopus oryzae (Lipase D) and Rhizopus delemar (Lipase Rd), immobilized on Accurel MP1000 (a microporous polypropylene) with a lipase/support ratio 1:1.5 (w/w). The activity of these immobilized lipases was stable for a minimum of 5 days in the operational conditions (up to 40 °C).Lipase Rd was selected for the next step in which it was immobilized on Acurrel MP1000 to obtain STAG enriched in DHA by acidolysis of tuna oil (20% DHA) with CA. The experiments were carried out by recirculating the reaction mixture through an immobilized lipase packed bed reactor at different substrate/hexane ratios, as well as in absence of solvent. In the latter case, STAG with 51% CA and 13% DHA were obtained at 73 h. This result indicates that with this catalyst an acceptable reaction rate was attained in absence of solvent. A structural analysis by the pancreatic lipase method carried out to STAG with 45% CA and 16% DHA indicated that 91% of the CA incorporated is located in positions 1 and 3, and that 51% of the DHA is located in position 2 (MLM structure). This position is also rich in palmitic, eicosapentaenoic and oleic acids.After the acidolysis reaction a mixture of STAG and free fatty acids was obtained. The recovery of STAG from this reaction mixture is difficult because of the high content of free fatty acids. A separation method based on the neutralization of the free fatty acids with a KOH hydroalcoholic solution has been developed. By this procedure pure (100%) STAG were obtained with a recovery yield of 80%.     

Production of triacylglycerols rich in palmitic acid at position 2 as intermediates for the synthesis of human milk fat substitutes by enzymatic acidolysis

This paper studies the synthesis of triacylglycerols (TAGs) rich in palmitic acid (PA) at position 2, from palm oil stearin (POS), a vegetable oil highly rich in this acid (60%). The developed process consists of two steps: (1) obtaining PA enriched free fatty acids (FFAs), and (2) enrichment of POS in PA by acidolysis of this oil with PA enriched FFAs, catalyzed by lipase Novozym 435. In step (1) two PA enriched FFA mixtures were obtained: one by saponification of POS, and a PA concentrate (75.1% PA) obtained by crystallization at low temperature in solvents. The latter was obtained carrying out two crystallizations in acetone at −24 and −20 °C, from which PA was recovered in the solid phases with a total yield of 84%. These PA enriched FFA mixtures were used in step (2) of acidolysis of POS, along with commercial PA (98% PA). In this acidolysis step four factors were studied: temperature, hexane/reaction mixture ratio, FFA/POS molar ratio and the intensity of treatment (IOT = lipase amount × reaction time/POS amount). The best results (TAGs with 79% PA and 75% PA at position 2) were obtained with commercial PA, at 37 °C, 10 mL hexane/g reaction mixture, a FFA/POS molar ratio 3:1 (1:1, w/w) and an IOT = 9.6 g lipase × h/g POS (for example 48 h, 10 g lipase and 50 g POS). PA enriched TAGs were purified neutralizing the FFAs by KOH hydroethanolic solutions and extracting the TAGs with hexane. In this way 99% pure acylglycerols (TAG + DAG) were obtained; the recovery yield of this purification step was 95%. The experiments carried out with POS demonstrated that it is possible to use only this oil (60% PA, 23% PA at position 2) as a source of PA to obtain a TAG with 70.7% PA and 70.5% PA at position 2. This process consists of four steps: (1) saponification of POS, (2) crystallization of FFAs to obtain PA enriched FFAs (75.1% PA), (3) acidolysis of POS with these FFAs, catalyzed with Novozym 435, to produce PA enriched TAGs at position 2 (70.5% PA) and (4) purification of TAGs to obtain approximately 95% purity and yield. These PA enriched TAGs could be used to obtain structured TAGs rich in PA at position 2 and in oleic acid at positions 1 and 3 (OPO), which is the principal TAG of human milk fat.     

酶法合成2-氧-α-D-葡萄糖基-L-抗坏血酸(AA-2G)条件的研究

采用单因素优化法对环糊精葡萄糖苷转移酶(CGTase)合成糖基抗坏血酸(AA-2G)条件进行优化,AA-2G的产量为2.76 g/L,比未优化前0.46g/L提高了500%。再采用响应面法对AA-2G合成条件进行优化。由Plackett-Burman法筛选出三个主要因素为:pH、V_C和麦芽糊精浓度;由最陡爬坡实验得出最佳响应面区域;最后由Box-Behnken实验,得到最优条件为:pH 5.51,V_C36.16g/L,麦芽糊精28.54 g/L,转化时间24 h,温度37℃。在此条件下,AA-2G的理论产量为3.15 g/L,通过验证实验,得出AA-2G的产量为3.13 g/L,与预测的理论值接近,比单因素优化的结果(2.76g/L)提高了14%。     

Optimization of ultrasound-accelerated synthesis of enzymatic octyl hydroxyphenylpropionate by response surface methodology

The ultrasound-accelerated enzymatic synthesis of octyl hydroxyphenylpropionate (OHPP) from p-hydroxyphenylpropionic acid (HPPA) and octanol was investigated in this study. A commercially available immobilized lipase from Candida antarctica, Novozym 435, was used as the biocatalyst. A three-level-three-factor Box-Behnken design experiment and response surface methodology were used to evaluate the effects of temperature, reaction time, and enzyme activity on percent yield of OHPP. The results indicated that temperature and enzyme activity significantly affected percent yield, whereas reaction time did not. A model for the synthesis of OHPP was established. Based on a ridge max analysis, the optimum conditions for OHPP synthesis were predicted to use a reaction temperature of 58.8°C, a reaction time of 14.6 h, and an enzyme activity of 410.5 PLU with a yield of 98.5%. A reaction was performed under these optimal conditions, and a yield of 97.5% ± 0.1% was obtained.     

响应面法优化酶法转化合成维生素C糖苷（AA-2G）条件的研究

采用Design—Expert软件的Central Composite Design（CCD）响应面设计对环糊精葡萄糖苷转移酶转化合成糖基抗坏血酸（AA-2G）的五个主要因素（转化时间、转化温度、pH、Vc浓度、β-环糊精浓度）进行了研究。采用降维分析方法对pH与转化时间、转化温度、Vc浓度、β-环糊精浓度以及反应温度与反应时间的交互作用对酶法转化合成AA-2G的影响进行了分析。建立了影响因素与响应值之间的回归方程,根据回归方程优化得到最佳转化条件为：转化时间25h,温度36．5℃,pH5．4,Vc72dL,β-环糊精55g／L。在此条件下,AA-2G的理论产量为10．06g／L,在验证实验中AA-2G的产量为9．76g／L,与预测的理论产量接近,比优化前提高了33％。     

Synthesis of structured lipid with balanced omega-3: Omega-6 ratio by lipase-catalyzed acidolysis reaction: Optimization of reaction using response surface methodology

The present study deals with the production of structured lipid containing omega-3 and omega-6 fatty acids in the ratio of 1:1 by incorporating omega-3 fatty acids (α-linolenic acid) from linseed oil into groundnut oil using lipase (Lipozyme IM from Rhizomucor miehei) catalyzed acidolysis reaction in hexane. The reaction conditions were optimized by response surface methodology with a four-variable five-level central composite rotatable experimental design. The influence of four independent parameters, namely ratio of fatty acid concentrate from linseed to groundnut oil (0.66–1.98, w/w), reaction temperature (30–60 °C), enzyme concentration (1–5%) and reaction time (2–54 h) on omega-3 fatty acids incorporation into groundnut oil were optimized. Optimal conditions for the structured lipid containing omega-3 to omega-6 fatty acids in the ratio of 1:1 were determined to be; enzyme concentration 3.75% (w/w), temperature 37.5 °C, incubation time 30.81 h and ratio of free fatty acid concentrate from linseed oil to groundnut oil 1.16 (w/w).     

Optimization of the Emerson-Trinder enzymatic reaction by response surface methodology

The Emerson-Trinder reaction has been optimized in this work using an initial rate spectrophotometric method and response surface methodology (RSM). In this investigation, the variation range of critical variables along with the fixed parameters were selected based on a preliminary 'one at a time' (OVAT) procedure for the subsequent RSM chemometric analysis as follows: pH (6-10), buffer concentration (50-250 mM), 4-aminoantipyrine (4-AAP) concentration (1-5 mM), temperature (25-45°C). The optimum values of fixed parameters were: 4-fluorophenol (4-FP, 30 mM), horseradish peroxidase (HRP) enzyme activity (0.12 U mL^-1), and the fixed concentration of the H₂O₂ in the chemometric experiments was 11.4 µM. The non-linear nature of the experimental response of the reaction system was explained by a second-order polynomial equation, which revealed the impact of the experimental factors, their interactions and also their optimum values. The results of the reported RSM analysis proved to be quite appropriate for the design and optimization of this reaction, as illustrated by the relatively high value of the determination coefficient (R²=96.7%) for the fitting of quadratic model, along with the satisfactory results generated by the analysis of variance (ANOVA). All the evaluated analytical characteristics of this method: typical reaction progress curves, resulting linear calibration curve, within-day precisions at low and at high levels, and the upper and lower detection limits were, also, reported. In addition, to check the quality of the optimization and validity of the model, the assay of H₂O₂, in pooled serum matrix and in cosmetic samples, was performed.     

酶法制备1-磷酸葡萄糖合成条件的响应曲面法优化

以葡萄糖为原料,三聚磷酸钠为磷酰化试剂,马铃薯磷酸化酶为催化剂,制备1-磷酸葡萄糖.利用Box-Behnken实验设计原理,采用三因素三水平的响应面分析法,以产物含量为响应值考察温度、物料比(葡萄糖/三聚磷酸钠)、时间3个因素影响.采用氢核磁共振波谱仪对产品进行了分析.结果表明,马铃薯磷酸化酶制备葡萄糖磷酸酯的最佳工艺条件为:温度35℃,葡萄糖与三聚磷酸钠物料比为1.35∶1(mol/mol),时间19h.     

Optimization of enzymatic hydrolysis of mango kernel starch by response surface methodology

Amyloglucosidase (EC 3.2.1.3) from Aspergillus niger was employed for the saccharification of mango (Mangifera indica Linn) kernel starch. Response surface methodology based on a three-level three-factor Box-Behnken design was employed to optimize the important process variables such as substrate concentration (137.5-412.5 mg), enzyme concentration (4-12 mg) and temperature (35-55 °C). The sugar yield increased with both enzyme concentration and temperature, and decreased with substrate concentration. The response surface model indicated optimum conditions (substrate, 137.5 mg; enzyme, 12 mg; temperature, 55 °C) for obtaining 0.4851 mg sugar/mg substrate, which was also verified experimentally.     

Optimization of fermentation parameters for the biomass and DHA production of Schizochytrium limacinum OUC88 using response surface methodology

Fermentation parameters for biomass and DHA production of Schizochytrium limacinum OUC88 in a fermenter (working volume 7 L) were optimized using Plackett–Burman and central composite rotatable design. Out of 10 factors studied by Plackett–Burman design, 4 influenced the biomass production significantly. Central composite rotatable design was used to optimize the significant factors and response surface plots were generated. Using these response surface plots and point prediction, optimized values of the factors were determined as follows temperature (°C) 23 °C, aeration rate 1.48 L min⁻¹ L⁻¹, agitation 250 rpm and inoculum cells in mid-exponential phase, the maximum yield of DCW and DHA were 24.1 and 4.7 g L⁻¹, respectively. These predicted values were also verified by validation experiments.     

Solvent-free enzymatic synthesis of 1,3-diconjugated linoleoyl glycerol optimized by response surface methodology

An operation mode with N(2) bubbling under vacuum was employed for the solvent-free synthesis of 1,3-diconjugated linoleoyl glycerol (1,3-dCLG) from conjugated linoleic acid (CLA) catalyzed by Novozym 435. The response surface methodology (RSM) was adopted for the optimization of the reaction conditions with five major factors (incubation time, temperature, enzyme load, substrate mole ratio, and system vacuum) and three responses (CLA conversion, 1,3-dCLG yield, and acyl migration). Two sets of optimal conditions were recommended. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of 1,3-dCLG yield. Under the optimal conditions, the yield of 1,3-dCLG up to 93% was obtained. The reaction was scaled up to a production level of 100 g of 1,3-dCLG at a yield of 90.7%, indicating a promising feature of the technology in industrial applications.     

Optimization of lipase-catalyzed synthesis of acetylated EGCG by response surface methodology

(−)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives, which can be widely used as a natural antioxidant in both lipid containing food and cosmetic applications, were prepared by lipase catalyzed acylation of EGCG with vinyl acetate. Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (6–10 h), temperature (30–50 °C), enzyme amount (1.5–2.5% (w/w) of substrate), and substrate molar ratio of EGCG to vinyl acetate (0.5–1.5) on conversion of EGCG. By using multiple regression analysis, the experimental data were fitted to a second order polynomial model. The most suitable combination of variables was 40 °C, 2.12%, 10 h and 1.13 for the reaction temperature, the enzyme amount, the reaction time, and EGCG/vinyl acetate mole ratio, respectively. At these optimal conditions, the conversion yield reached 87.37%. The presence of mono-, di- and tri-acetylated derivatives in acetylated EGCG was confirmed by LC–MS-MS and identified as 5″-O-acetyl-EGCG, 3″, 5″-2-O-acetyl-EGCG and 5′, 3″, 5″-3-O-acetyl-EGCG by NMR.     

Production of structured lipids by acidolysis of an EPA-enriched fish oil and caprylic acid in a packed bed reactor: analysis of three different operation modes

Structured triacylglycerols (ST) enriched in eicosapentaenoic acid (EPA) in position 2 of the triacylglycerol (TAG) backbone were synthesized by acidolysis of a commercially available EPA-rich oil (EPAX4510, 40% EPA) and caprylic acid (CA), catalyzed by the 1,3-specific immobilized lipase Lipozyme IM. The reaction was carried out in a packed bed reactor (PBR) operating in two ways: (1) by recirculating the reaction mixture from the exit of the bed to the substrate reservoir (discontinuous mode) and (2) in continuous mode, directing the product mixture leaving the PBR to a product reservoir. By operating in these two ways and using a simple kinetic model, representative values for the apparent kinetic constants (kX) for each fatty acid (native, Li or odd, M) were obtained. The kinetic model assumes that the rate of incorporation of a fatty acid into TAG per amount of enzyme, rX (mole/(h g lipase)) is proportional to the extent of the deviation from the equilibrium for each fatty acid (i.e., the difference of concentration between the fatty acid in the triacylglycerol and the concentration of the same fatty acid in the triacylglycerol once the equilibrium of the acidolysis reaction is reached). The model allows comparing the two operating modes through the processing intensity, defined as mLt/(V[TG]0) and mL/(q[TG]0), for the discontinuous and continuous operation modes, respectively. In discontinuous mode, ST with 59.5% CA and 9.6% EPA were obtained. In contrast, a ST with 51% CA and 19.6% EPA were obtained when using the continuous operation mode. To enhance the CA incorporation when operating in continuous mode, a two-step acidolysis reaction was performed (third operation mode). This continuous two-step process yields a ST with a 64% CA and a 15% EPA. Finally, after purifying the above ST in a preparative silica gel column, impregnated with boric acid, a ST with 66.9% CA and 19.6% EPA was obtained. The analysis by reverse phase and Ag+ liquid chromatography of the EPA-enriched ST demonstrated that the CA was placed in positions 1 and 3 and the EPA was occupying position 2 of the final ST.     

Optimization of lipase-catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids by response surface methodology

Lipase-catalyzed caffeic acid phenethyl ester (CAPE) synthesis in ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf₂N]), was investigated in this study. The effects of several reaction conditions, including reaction time, reaction temperature, substrate molar ratio of phenethyl alcohol to caffeic acid (CA), and weight ratio of enzyme to CA, on CAPE yield were examined. In a single parameter study, the highest CAPE yield in [Emim][Tf₂N] was obtained at 70 °C with a substrate molar ratio of 30:1 and weight ratio of enzyme to CA of 15:1. Based on these results, response surface methodology (RSM) with a 3-level-4-factor central composite rotatable design (CCRD) was adopted to evaluate enzymatic synthesis of CAPE in [Emim][Tf₂N]. The four major factors were reaction time (36–60 h), reaction temperature (65–75 °C), substrate molar ratio of phenethyl alcohol to CA (20:1–40:1), and weight ratio of enzyme to CA (10:1–20:1). A quadratic equation model was used to analyze the experimental data at a 95 % confidence level (p < 0.05). A maximum conversion yield of 99.8 % was obtained under the optimized reaction conditions [60 h, 73.7 °C, substrate molar ratio of phenethyl alcohol to CA (27.1:1), and weight ratio of enzyme to CA (17.8:1)] established by our statistical method, whereas the experimental conversion yield was 96.6 ± 2 %.     

Optimization of mycophenolic acid production in solid state fermentation using response surface methodology

Mycophenolic acid (MPA) can be produced in solid state fermentation. An isolate of Penicillium brevi-compactum ATCC 16024 grown on moist wheat bran produced a titre of 425 mg per kg of wheat bran. Central composite rotatable design and response surface methodology were employed to derive a statistical model for media optimization towards production of mycophenolic acid. Five levels with a five factorial design were adopted. The correlation coefficient was 0.82, ensuring a satisfactory adjustment of the model to the experimental values. This statistical design was very effective in improving the titre of mycophenolic acid up to 3286 mg per kg of wheat bran. Received 24 July 1998/ Accepted in revised form 4 December 1998     

响应面法对红法夫酵母合成虾青素主要影响因素的优化

在单因素试验确定了红法夫酵母生物合成虾青素培养基组份的基础上,用响应面法对其浓度进行优化。首先用分式析因设计评价了培养基的各组份对虾青素产量的影响,并找出主要影响因子为蔗糖和酵母粉,二者分别达到了极显著和显著水平。用最陡爬坡路径逼近最大响应区域后,运用旋转中心复合设计及响应面分析,确定了主要影响因子的最佳浓度。其中,蔗糖的最佳浓度为49.8g/L,酵母粉的浓度为9.6g/L。菌株在优化培养基中的虾青素产量为9861μg/L,比优化前增加了近1倍。     

响应面法优化加压提取石榴籽油工艺

以乙醇为溶剂,采用加压溶剂法提取石榴籽油,对其主要影响参数(提取温度、压力、液固比和提取时间)进行优化研究。通过响应面法得到的最优提取工艺条件为液固比33∶1(m L/g),提取温度98℃,在压力0.25 MPa下提取30 min。与加热回流法相比,加压溶剂提取法提取时间缩短5倍,提取率提高了2倍。     

Inhibition of lecithin:cholesterol acyltransferase activity by synthetic phosphatidylcholine species containing eicosapentaenoic acid or docosahexaenoic acid in the sn-2 position.

Phospholipids isolated from the plasma of monkeys fed a diet enriched in fish oil were poor substrates for cholesteryl ester (CE) synthesis by the lecithin:cholesterol acyltransferase (LCAT) reaction relative to those from animals fed a lard containing diet when the phospholipids were used for the preparation of recombinant particles by cholate dialysis (Parks, J. S., B. C. Bullock, and L. L. Rudel. 1989. J. Biol. Chem. 264: 2545-2551). The purpose of the present study was to directly test the influence of eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) in the sn-2 position of phosphatidylcholine (PC) on the activity of LCAT. PC species containing 1-palmitoyl-2-oleoyl PC (POPC), 1-palmitoyl-2-linoleoyl PC (PLPC), 1-palmitoyl-2-arachidonoyl PC (PAPC), 1-palmitoyl-2-eicosapentaenoyl PC (PEPC), or 1-palmitoyl-2-docosahexaenoyl PC (PDPC) were purchased or synthesized and made into recombinant particles of uniform size and composition with [14C]cholesterol and apoA-I using the cholate dialysis procedure. The recombinant particles (PC:cholesterol:apoA-I molar ratio = 42:1.9:1) exhibited the following order of reactivity towards purified human LCAT in vitro: POPC greater than PLPC greater than PEPC = PAPC greater than PDPC. The apparent Vmax/Km for recombinant particles containing PEPC and PDPC was 17% and 7% that of particles containing POPC, respectively. There was a linear decrease in CE formation when the percentage of PEPC or PDPC was increased from 0 to 100% relative to POPC in recombinant particles with a constant PC:cholesterol:apoA-I molar ratio, suggesting that the PEPC and PDPC were competitive inhibitors of the LCAT reaction.(ABSTRACT TRUNCATED AT 250 WORDS)