Modeling and Optimization of Lipozyme RM IM-Catalyzed Esterification of Medium- and Long-Chain Triacyglycerols (MLCT) Using Response Surface Methodology

Optimization conditions of Lipozyme RM IM lipase esterification of capric and stearic acids with glycerol for the production of medium- and long-chain triacyglycerols (MLCT) fat suitable for food applications such as margarine and shortening were investigated. Response surface methodology (RSM) was applied to model and optimize the reaction conditions, namely, the reaction time (8–24 h), enzyme load (5–15 wt.%), and fatty acids/glycerol ratio (3:1–4:1) and represented by Ti, En, and Sb, respectively. Best-fitting models were successfully established for both MLCT yield (R ² = 0.9507) and residual FFA (R ² = 0.9315) established by multiple regressions with backward elimination. Optimal reaction conditions were 13.6–14.0 h for reaction time, 7.9–8.0 wt.% for enzyme load, and 3:1 for fatty acids/glycerol molar ratio. Chi-square test showed that there were no significant (P > 0.05) differences between the observed and predicted values of both models. Refined MLCT fat blend had sufficient solid fat at room temperature and made it suitable to use as a hard stock in shortening and margarine production.     

响应面优化脂肪酶催化合成中长碳链甘三酯

采用响应面设计对脂肪酶Novozym 435在无溶剂体系中催化甘油和中长碳链脂肪酸(辛酸、癸酸和油酸混合物)酯化反应合成中长碳链甘三酯进行了研究.研究发现:反应温度、加酶量和反应时间对中长碳链甘三酯得率具有显著性影响(P＜0.05),而底物摩尔比(脂肪酸与甘油摩尔比)对中长碳链甘三酯得率不具有显著性影响.优化得到的最佳条件为:反应温度90℃,加酶量6.5％(以脂肪酸和甘油的总质量计),底物摩尔比3.5∶1,反应时间12.97 h.在此条件下,平均甘三酯得率为78.5％;产品中甘三酯、甘二酯、甘一酯和游离脂肪酸含量分别为85.6％、0.3％、0.1％和14.0％;产品甘三酯中辛酸、癸酸和长碳链脂肪酸含量分别为25.4％、10.7％和63.9％,与目标中长碳链甘三酯产品指标基本一致.     

Deep Frying Performance of Enzymatically Synthesized Palm-Based Medium- and Long-Chain Triacylglycerols (MLCT) Oil Blends

The main aim of this work was to assess the frying strength of the enzymatically synthesized palm-based medium- and long-chain triacylglycerols (MLCT) oil with the aid of different antioxidants under deep-frying conditions. Palm-based MLCT oil in the presence of synthetic or natural antioxidants showed significantly better (P < 0.05) thermal resistance and oxidative strength than refined, bleached, and deodorized (RBD) palm olein throughout the five consecutive days of frying. Rancimat induction period, free fatty acid content, anisidine value, E^\text1% _1\textcm E^{{\text{1\% }}} _{1{\text{cm}}} at 232 and 268 nm, color, percentage of oil uptake, and viscosity measurement can be used as oil quality parameters to indicate the degree of oil deterioration under continuous stressed frying conditions. No significant changes (P > 0.05) in the saturated/unsaturated fatty acids ratio across frying periods indicated good oxidative stability of the palm-based MLCT oil. Due to the polarity of medium- and long-chain triacylglycerols in palm-based MLCT oil, total polar compounds determination may not be a suitable oil quality measures. Sensory evaluation of fried chips showed no significant differences (P > 0.05) between chips fried in RBD palm olein and palm-based MLCT oil over the 3-month storage period.     

Valorization of Olive Pomace Oil with Enzymatic Synthesis of 2‐Monoacylglycerol

2‐Monoacylglycerols (2‐MAG) with a high content of oleic acid at sn‐2 position was synthesized by enzymatic ethanolysis of refined olive pomace oil, which is a byproduct of olive oil processing. Six lipases from different microbial sources were used in the synthesis of 2‐MAG. Immobilized lipase from Candida antarctica gave the highest product yield among the selected lipases. Response surface methodology was applied to optimize reaction conditions; time (4 to 10 h), temperature (45 to 60 °C), enzyme load (10 to 18 wt%), and ethanol:oil molar ratio (30:1 to 60:1). The predicted highest 2‐MAG yield (84.83%) was obtained at 45 °C using 10 (wt%) enzyme load and 50:1 ethanol:oil molar ratio for 5 h reaction time. Experiments to confirm the predicted results at optimum conditions presented a 2‐MAG yield of 82.54%. The purification yield (g 2‐MAG extracted/100 g of total product) was 80.10 and 69.00 for solvent extraction and low‐temperature crystallization, respectively. The purity of the synthesized 2‐MAG was found to be higher than 96%.     

Enzymatic Production of ABA-Type Structured Lipids Containing Omega-3 and Medium-Chain Fatty Acids: Effects of Different Acyl Donors on the Acyl Migration Rate

Enzymatic production of ABA-type structured lipids (SLs) containing marine-derived long-chain polyunsaturated fatty acid (n-3 PUFA) and medium-chain fatty acids was investigated. Response surface methodology was applied to optimize the reaction system and evaluate the effects of reaction factors and different acyl donors namely n-3 PUFA and n-3 PUFA ethyl esters (n-3 PUFA-EE). Well-fitting models were obtained by multiple regressions with backward elimination. For both n-3 PUFA and n-3 PUFA-EE systems, both reaction time and enzyme load had significant (P < 0.05) positive effects on incorporation and acyl migration rate. Water content were found to have significant (P < 0.05) negative effects on incorporation but positive effects on acyl migration in the n-3 PUFA-EE system. Although there were no significant difference in terms of incorporation rate, n-3 PUFA were found to produce five times higher acyl migration rate than n-3 PUFA-EE. The optimal reaction conditions to produce high yield of ABA-type SLs of 49.6% with low acyl migration rate of 2.6% were as follows: n-3 PUFA-EE as acyl donors; enzyme load, 6 wt.%; reaction time, 6 h; and water content, 3 wt.%. Thus, n-3 PUFA-EE was found to be a suitable acyl donor to produce high yield of ABA-type SLs with low acyl migration rate.     

酶催化亚麻籽油甘油解制备富含α-亚麻酸的甘油二酯的研究

在无溶剂体系中,以亚麻籽油和甘油为反应底物,Lipozyme435为催化剂,制备富含α-亚麻酸的甘油二酯,采用单因素实验与响应面分析法考察了制备过程中底物摩尔比、反应时间、加酶量和反应温度对甘油二酯得率的影响。结果表明,反应的最佳条件为底物摩尔比(亚麻籽油∶甘油)=5∶3,加酶量8.8wt%,反应温度为58.3℃,反应时间为9.1h。在此反应条件下反应所得产物中甘油二酯含量约达50.21%,纯化后的甘油二酯的理论纯度可达60.12%,α-亚麻酸的含量达46.41%。     

Optimization of omega-3 enriched-diacylglycerol production by enzymatic esterification using a response surface methodology

Incorporation of an eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) moiety into diacylglycerol (DAG) oil using lipase-catalyzed esterification was optimized using an ethyl ester form of EPA/DHA. A response surface methodology (RSM) was used to optimize reaction parameters (time, temperature, and substrate mole ratio) for incorporation of DHA and EPA into DAG oil. Predictive models for DHA+EPA contents of DAG and the amount of DAG produced after esterification were adequate and reproducible. DHA+EPA contents of DAG significantly increased with reaction time and substrate mole ratio (p<0.05). In contrast, the reaction temperature negatively affected the amount of DAG after esterification. Synthesis of DHA+EPA-enriched DAG was optimized for a maximum DAG content with the highest DHA+EPA content, in which 630.0 mg of DAG containing 34.8% DHA and EPA was predicted using the RSM model. The optimal reaction conditions were predicted at 20.6 h, 57.9 and a DHA/EPAenriched ethyl ester: DAG oil ratio of 2.5:1.     

Production of lipase‐catalyzed solid fat from mustard oil and palm stearin with linoleic acid by response surface methodology

BACKGROUND: Solid fat was produced from mustard oil and palm stearin through lipase‐catalyzed reaction, in which linoleic acid was intentionally incorporated. For optimizing the reaction condition of melting point and ω6/ω3 fatty acids, response surface methodology (RSM) was employed with three reaction variables such as substrate mole ratio of mustard oil (MO) to palm stearin (PS) (X₁), reaction temperature (X₂) and reaction time (X₃). RESULTS: The predictive model for melting point of solid fat was adequate and reproducible due to no significant lack of fit (P = 0.0764), P‐value (0.0037) of the model, and satisfactory level of coefficient of determination (R² = 0.92). For the ω6/ω3 ratio model, R² and P‐value were 0.89 and 0.0132, respectively, but lack of fit was significant (P = 0.0389). The melting point of the produced solid fat was affected by substrate mole ratio, whereas reaction temperature and time had no significant effect. The ω6/ω3 ratio of solid fat was influenced by substrate mole ratio and reaction temperature but not by reaction time. Based on ridge analysis, lower ω6/ω3 ratio was predicted by decreasing substrate mole ratio and reaction time, and by increasing reaction temperature. CONCLUSIONS: For producing solid fat with a specific melting point of 34.57 °C, a combination of 1:2 (X₁), 65.17 °C (X₂) and 21.46 h (X₃) was optimized, and the optimization was confirmed under the same reaction conditions. The solid fat contained palmitic (37.8%), linoleic (24.8%), oleic (21.3%), and erucic acid (9.7%), and its solid fat content was 30.3% and 10.3% at 20 and 30 °C, respectively. Copyright © 2009 Society of Chemical Industry     

Enrichment of Rice Bran Oil with ��-Linolenic Acid by Enzymatic Acidolysis: Optimization of Parameters by Response Surface Methodology

Lipase-catalyzed enrichment of rice bran oil with n-3 fatty acid in order to obtain a structured lipid containing essential fatty acids has been optimized by response surface methodology. In this process, α-linolenic acid was used as an acyl donor using lipase-catalyzed acidolysis in hexane in presence of immobilized lipase from Rhizomucor miehei. The effect of incubation time and temperature, enzyme concentration and substrates mole ratio and their complex interaction on percentage incorporation of n-3 fatty acid, ratios of saturated fatty acid to polyunsaturated fatty acids, monounsaturated fatty acids to polyunsaturated fatty acids and n-6 to n-3 (18:2 to 18:3) fatty acids have been studied using a central composite rotatable design of experiments. The results showed that at the optimum conditions such as reaction time 4.5 h and reaction temperature 37. 5°C, substrate ratio ranging from 1.0 to 1.9, enzyme concentration varying from 1.0% to 2.0% are needed to fulfill the conditions such as percentage incorporation of n-3 fatty acid ≤18%, ratio of saturated fatty acid to poly unsaturated fatty acid ≥0.42, ratio of mono unsaturated fatty acid to poly unsaturated fatty acid ≥0.8, and ratio of n-6 to n-3 ≥1.30.     

LIPASE-CATALYZED PRODUCTION OF STRUCTURED LIPIDS VIA ACIDOLYSIS OF FISH OIL WITH CAPRYLIC ACID

Structured lipids containing eicosapentaenoic and docosahexaenoic acids were manufactured in a batch reactor by lipase-catalyzed acidolysis of fish oil with caprylic acid. The following free lipases (Lipase AP, Aspergillus niger ; Lipase P, Pseudomonus sp. ; Lipase AY, Candida rugosa ; Lipase AK, Pseudomonas fluoresescens ; Lipase F, Rhizopus oryzae ; Lipase D, Rhizopus delemar ) were screened under selected reaction conditions. The conditions were enzyme load 5%, substrate mole ratio 1:6 (fish oil: caprylic acid), and reaction temperature of 50C. Lipase AK had the highest activity and was suitable for production of structured lipids from fish oil. The optimal mole substrate ratio of fish oil to caprylic acid for Lipase AK was 1:6 to 1:8. The time course of the reaction at different enzyme loads demonstrated that 40% incorporation of caprylic acid could be obtained for Lipase AK in 5 h with 10% enzyme load. Addition of water had little effect on the activity of the lipase. Lipase AK and Lipozyme IM were further compared under the same conditions, in which Lipase AK had a slightly higher incorporation of caprylic acid, similar acyl migration of caprylic acid from sn-1,3 positions to the sn-2 position, and a slightly lower selectivity towards docosahexaenoic acid.     

Lipase catalyzed modification of fish oil to incorporate capric acid

Immobilized lipase, IM60, from Rhizomucor miehei, was used as a biocatalyst for the incorporation of capric acid (C10:0) into menhaden fish oil concentrate containing 34.7 mol% eicosapentaenoic acid (20:5n-3) and 34.4 mol% docosahexaenoic acid (22:6n-3). Transesterification (acidolysis) was performed in hexane and solvent-free media. Tocopherol content was analyzed before and after enzymatic modification. Products were analyzed by gas liquid chromatography. After 24 h incubation in hexane, there was an average of 31.1±4.6 mol% incorporation of C10:0 into fish oil, while 20:5 and 22:6 were reduced to 12.6±3.1 and 13.7±4.4, respectively. The solvent-free reaction produced an average of 28.8±4.7 mol% capric acid incorporation; 20:5 and 22:6 decreased to 16.1±5.7 and 13.5±3.0 mol%. The effect of incubation time, substrate mole ratio, enzyme load, and added water were also studied. Generally, as enzyme load, mole ratio, and incubation time increased, mol% capric acid incorporation also increased. Time course of reaction indicated that the highest C10:0 incorporation occurred at 72 h, for both the reaction in hexane (33.5 mol%) and the solvent-free reaction (36.0 mol%). The highest C10:0 incorporation for the substrate mole ratio reaction occurred at a mole ratio of 1:8 in hexane (50.7 mol%) and the solvent-free reaction (36.7 mol%). Although the highest C10:0 incorporation (31.8 and 48.6 mol%) occurred at an enzyme load of 15% in hexane and 20% for the solvent-free reaction respectively, the values were not significantly different (P<0.05) after 5% enzyme load. Mol% incorporation of C10:0 declined with increasing amounts of water. At 1% added water, high C10:0 incorporation was achieved for the reaction in hexane (39.3 mol%) and the solvent-free reaction (26.0 mol%). Pancreatic lipase catalyzed sn-2 positional analysis was performed on the fish oil before and after enzymatic modification. Fish oil containing capric acid was successfully produced and may be beneficial in certain food and nutritional applications.     

PREPARATION OF STRUCTURED LIPIDS WITH SPECIAL FUNCTIONAL PROPERTIES

Enzymatic acidolysis of rapeseed oil with capric acid was carried out to obtain structured lipids. The reaction was catalyzed by Lipozyme IM lipase from Rhizomucor miehei. The enzyme preparations contained 2.8 and 10% water. The reaction conditions were enzyme load of 8% (w/w total substrates), substrate mole ratio of 1:6 (rapeseed oil:capric acid), and reaction temperature of 65C. The results showed that triacylglycerols (TAG) after transesterification contained mainly oleic, linoleic and linolenic acids (about 90%) in the internal sn-2 position, whereas capric acid was mostly in the external sn-1,3 positions (approximately 40%). The quantity of water in the reaction medium had a significant influence on the yield and quality of the TAG fraction.     

Optimization of Edible Oil Extraction from Ofada Rice Bran Using Response Surface Methodology

Effects of roasting temperature and duration on yield and quality (free fatty acid, peroxide value, color) of oil extracted from ofada rice bran was studied using response surface methodology. Roasting temperature and duration were 160, 170, 180, 190, and 200 °C and 5, 10, 15, 25, and 35 min respectively. Data were analyzed by ANOVA and regression analysis. The oil yield ranged between 11.31% and 14.4%, free fatty acid (7.10–12.75%), peroxide values (8.25–13.25 mEq/kg) and color (1.51–1.58 abs). The treatments have significant effects on oil yield, free fatty acid, peroxide values, and color at p < 0.05. Coefficient of determination R ² of oil yield, free fatty acid, color, and peroxide value models were 0.79, 0.91, 0.99, and 0.99 respectively. Optimum temperature and duration of roasting were 190 °C and 10.75 min, respectively. This combination gave 14.45% oil yield, 5.80% free fatty acid, 8.25 mEq/kg peroxide values and 1.51 abs oil color. Desirability of optimization was 0.99.     

Enzymatic synthesis of medium‐ and long‐chain triacylglycerols–enriched structured lipid from Cinnamomum camphora seed oil and camellia oil by Lipozyme RM IM

Medium‐ and long‐chain triacylglycerols (MLCTs)–enriched structured lipid (SL) was synthesised through enzymatic interesterification from Cinnamomum camphora seed oil (CCSO) and camellia oil (CO) using Lipozyme RM IM from Rhizomucor miehei as a biocatalyst. Effects of different reaction conditions including substrate molar ratio, reaction time and reaction temperature were investigated. Results showed that 55.81% of total MLCT species (CCO/LaCL, LaCO/LCL, COO/OCO and LaOO/OLaO) was obtained in the interesterified product under the optimal conditions of substrate molar ratio of 1:1.5 (CCSO/CO) at 60 °C for 3 h. Thereafter, fatty acid profiles, tocopherol contents and physiochemical characteristics of the interesterified product and physical blend were comparatively investigated. The fatty acid composition of the interesterified product consisted of capric acid (26.33%), lauric acid (21.29%) and oleic acid (42.33%). It should be mentioned that the interesterified product contained predominantly oleic acid (88.69%) at Sn‐2 position, while MCFAs (68.05%) at Sn‐1,3 positions. Compared with physical blend, the reduction in tocopherol contents and changes of physiochemical characteristics occurred in SL. The smoke point of the interesterified product was much higher than that of the physical blend, which meant that such MLCTs‐enriched SL could be better for cooking purpose.     

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.     

Production of palm oil-based diacylglycerol using Lecitase Ultra-catalyzed glycerolysis and molecular distillation

Diacylglycerol (DAG) was prepared via glycerolysis of palm oil catalyzed by Lecitase Ultra (LU), a novel phospholipase from the fusion of lipase genes from Thermomyces lanuginose and phospholipase genes from Fusarium oxysporum. Glycerolysis was performed in a solvent-free system. The optimized reaction conditions were: a glycerol/palm oil mole ratio of 7.5:1, initial substrate water content of 5%, substrate enzyme load of 2%, reaction temperature of 40°C, and reaction time of 8 h. In a scale-up reaction, a DAG content of 59.5% in the lipid layer was achieved. Through a two-step molecular distillation, the composition of the target product was 88.1% DAG, 2.8% TAG, 9.0% MAG, and 0.1% FFA. The fatty acid composition of the DAG oil, determined using GC-MS, was enriched compared with the original palm oil.     

Lipase-catalyzed acidolysis of lard for the production of human milk fat substitute

Human milk fat substitute (HMFS) was prepared by Lipozyme RM IM-catalyzed acidolysis of lard and fatty acids obtained from palm kernel oil, tea seed oil and soybean oil, in a solvent-free system. The effects of substrate mass ratio, Lipozyme RM IM load and reaction time on the total fatty acids composition and sn-2 fatty acids composition were investigated. To optimize the reaction conditions, an orthogonal design was selected with three levels and three factors. Substrate mass ratio, Lipozyme RM IM load and reaction time were the factors employed. Under the given reaction temperature of 60 °C, the optimal reaction conditions were, 1/2 mass ratio of lard/fatty acids blend, 7% Lipozyme RM IM load and 1-h reaction time. By the “deducting score” principle, the produced HMFS in comparison with the reported HMFS possessed highest degree of similarity with local HMF from Guangzhou mothers. The results showed that it was possible to produce HMFS through the scale-up enzymatic acidolysis.     

Optimization of Conditions for Enzymatic Production of ACE Inhibitory Peptides from Collagen

Enzymatic condition for producing angiotensin I-converting enzyme (ACE) inhibitory peptides from collagen was optimized with the aid of response surface methodology, which also derived a statistical model for experimental validation. The results showed that the optimal condition for the hydrolysis by pepsin was at pH 2, temperature 37 °C, and in enzyme to substrate ratio (E/S) of 2 when 8.23% collagen (w/v) and 3.82 h of hydrolysis time were applied. Through the single-enzyme hydrolysis, the ACE inhibitory activity could reach an average of 78.06%. In contrast, when a combination of pepsin and trypsin was used for a multiple-proteases hydrolysis, the ACE inhibitory activity could be significantly improved to an average of 88.25%. Furthermore, the IC₅₀ (μg/mL) value of the enzyme combination by pepsin and trypsin (141.64 ± 22.11) was significantly lower than that of the combinations of pepsin and papain (438.59 ± 84.37) or pepsin and protease M (336.76 ± 87.88; p<0.05). Our results have shown that collagen can be used for enzyme-mediated production of ACE inhibitory peptides.     

均匀设计在酯化樟树籽油制备单甘酯中的应用

以樟树籽油为原料、对甲基苯磺酸为催化剂,通过酯化将樟树籽油中的游离脂肪酸转化为单甘酯。采用均匀设计和偏最小二乘回归法,以酯化率(Y1)和单甘酯的含量(Y2)的最大值为优化目标,考察反应温度、催化剂的用量、醇油物质的量配比、反应时间对樟树籽油酯化反应的影响。对两个试验指标进行回归建模,模型拟合的决定系数分别为0.9801、0.9739。最优目标函数值Y1、Y2 分别为98.2%、12.2%,实测值分别为95.6%、14.4%,最佳酯化反应条件为反应温度132℃、催化剂的用量3.5%(油重)、醇油物质的量配比1.8:1、反应时间2h。     

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K _m 5.6 mg/mL, and V _max 925 μmol mg⁻¹ min⁻¹ with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.