延黄牛脂制备高纯度1,3-甘油二酯工艺

目的:提高延黄牛脂的附加值。方法:采用固定化脂肪酶Lipozyme RM IM为催化剂,牛油脂肪酸乙酯(乙醇解法自制)和甘油为原料制备1,3-甘油二酯,运用氢谱考察脂肪酶添加量、底物摩尔比、反应时间以及反应温度对粗反应混合物中1,3-甘油二酯含量的影响,并阐明醇解酯交换前后牛油与甘油二酯产物的脂肪酸组成变化。结果:1,3-甘油二酯合成最佳条件为脂肪酶Lipozyme RM IM质量分数为1%,底物摩尔比(n_{脂肪酸乙酯}∶n_甘油)2∶1,反应时间6 h,反应温度50℃。此条件下的1,3-甘油二酯生成率为72.5%,甘油酯得率为77%;纯化后纯度提高至90.79%。与原油(延黄牛脂)相比,甘油二酯产物中亚油酸和油酸含量分别升高了13.65%,6.47%,饱和度降低了7.17%。结论:延黄牛脂制备1,3-甘油二酯不仅能改变甘油酯结构,还可以改变牛脂的脂肪酸组成,降低其饱和度。     

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.     

酶法催化制备富含甘油二酯米糠油的研究

以高酸值米糠油为原料,采用无溶剂体系酶法催化制备富含甘油二酯的米糠油.考察了脂肪酶种类及添加量,酯化剂种类,底物质量比,反应时间,反应温度对甘油二酯含量和游离脂肪酸残余量的影响.通过单因素试验和响应面试验,确定酶法催化酯化的最佳工艺条件为:固定化脂肪酶Lipozyme RM IM作为催化剂,油酸甘油一酯作为酯化剂,底物质量比0.25:1,反应温度56℃,反应时间5.75 h,脂肪酶添加量4.77%.在此条件下,产物中甘油二酯含量和游离脂肪酸残余量分别为27.61%和0.25%.     

Enzymatic synthesis of structured lipids using a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were selected to synthesize symmetrical SL since recently NRRL Y-7723 lipase was identified as a novel cold-active lipase. Both lipases showed 1,3-regiospecifity toward the glycerol backbone of borage oil. The effects of enzyme loading and temperature on caprylic acid incorporation into the borage oil were investigated. For Lipozyme RM IM and NRRL Y-7723 lipase, the incorporation of caprylic acid increased as enzyme loading increased up to 4% of total weight of the substrate, but significant increases were not observed when enzyme loading was further increased. The activity of NRRL Y-7723 lipase was higher than that of Lipozyme RM IM in the temperature range between 10 and 20 °C.     

Oxidation Stability of O/W Emulsion Prepared with Linolenic Acid Enriched Diacylglycerol

The sn‐1,3‐regiospecific Rhizomucor miehei lipase (Lipozyme RM IM) was employed to produce structured diacylglycerol (SL‐DAG), which contained 67.3 mol% DAG with 27.2 area% of C18:3. To investigate the oxidative stability of the SL‐DAG in emulsion form, 5% oil‐in‐water (O/W) emulsions were prepared with 200 and 400 ppm sinapic acid. It was shown that the hydroperoxide values of the control (without any antioxidant) was the highest (117.7 meq/L) on day 43 of storage and thereafter the value decreased. However, the emulsions with 200 and 400 ppm sinapic acid resulted in slow oxidation degree until day 64 of storage (30.3 and 7.3 meq/L, respectively). Aldehyde measurements for the 200 ppm sinapic acid emulsion (12.8 mmol/mol) and the 400 ppm sinapic acid emulsion (7.5 mmol/mol) also showed better oxidative stability than that for the 200 ppm catechin emulsion (27.4 mmol/mol) and the control (52.7 mmol/mol). Although the SL‐DAG in the emulsions contains high levels of polyunsaturated fatty acids, the degree of oxidation in the emulsions can be reduced when sinapic acid is used as an antioxidant.     

硅胶吸附甘油在无溶剂酶法甘油解合成甘油二酯中的作用研究

以菜籽油、甘油为原料,采用无溶剂体系酶法甘油解生产1,3-甘油二酯(1,3-DAG)。研究结果表明,采用硅胶吸附甘油法可明显减少间歇反应中酶活损失,显著提高酶的生产能力和使用寿命。硅胶/甘油的比例不仅影响降低酶活损失的效果,而且与甘油解反应的初速度、转化率、产物组成密切相关。硅胶/甘油为1∶1(W/W)时,间歇反应酶的生产能力可提高2.3倍,酶的重复使用次数可达20次。     

酶法甘油解制备甘油二酯的研究

以精炼菜籽油为底物,通过酶法甘油解制备甘油二酯,比较了3种常用固定化脂肪酶甘油解制备甘油二酯的能力。结果表明:LipozymeRM IM具有较好的甘油耐受性,采用甘油预吸附的方式进行甘油解反应,可明显减少反应中酶活损失,显著提高酶的重复使用寿命。在菜籽油与甘油摩尔比1∶1,酶添加量为油质量的5%,硅胶与甘油质量比1∶1,反应温度60℃的优化条件下,甘油解反应8 h后,产物中的甘油二酯含量达到57.5%。通过硅胶预吸附甘油可以使LipozymeRM IM酶的多批次操作稳定性得到很大提高,半衰期达到22次,有应用于工业生产的潜力。     

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.     

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.     

无溶剂体系中酶催化合成结构脂质条件初探

以菜籽油和辛酸为原料,在无溶剂体系中用脂肪酶催化酸解合成结构脂质。对6种不同来源的脂肪酶进行筛选,结果表明Lipozyme RMIM催化活性高、Sn-1,3位特异性强。以Lipozyme RMIM为催化用酶,考察了反应时间、反应温度、底物比(菜籽油与辛酸摩尔比)、加酶量、体系水分含量对酸解反应的影响。结果表明,在反应时间15 h,反应温度50℃,底物比1∶4,加酶量10%条件下,辛酸合成率达40%。     

几种醇对Lipozyme RM IM催化卵磷脂乙醇解的影响及其工艺条件优化

研究脂肪酶Lipozyme RM IM催化大豆卵磷脂（phosphatidylcholine,PC）乙醇解反应制备溶血卵磷脂（lysophosphatidylcholine,LPC）过程中其他几种醇对PC乙醇解的影响,并对乙醇解条件进行了优化。发现正丁醇对PC乙醇解无明显影响,叔丁醇对PC乙醇解有一定的抑制作用,1,2-丙二醇和丙三醇呈现较好的促进作用。最后选用丙三醇作为乙醇解的促进剂,通过响应面分析法确定了无溶剂体系中Lipozyme RM IM催化PC醇解的最佳工艺条件为加酶量15%（以PC质量计,m/m）、反应温度28 ℃、加水量8%（水/乙醇溶液,V/V）、丙三醇10% （丙三醇/乙醇溶液,V/V）、底物质量浓度1.49 g/mL（PC/乙醇溶液,m/V）、反应时间11 h,此条件下 LPC转化率高达98.2%。实验证明,丙三醇对无溶剂体系中Lipozyme RM IM催化条件下的PC乙醇解反应有很好的促进作用。     

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%。     

Lipase-catalyzed modification of lard to produce human milk fat substitutes

The objective of the present work was to modify lard into human milk fat substitutes (HMFS) by Lipozyme RM IM-catalyzed acidolysis. Lard and soybean fatty acids were esterified in a solvent-free system. The reaction substrates for HMFS production were specially chosen to mimic human milk fats. Factors such as temperature, time, water content, enzyme load, substrate ratio, and enzyme reusability were investigated. The relationships between initial incorporation rate (Inc/h) and temperature (T, K) were set up, based on the Arrhenius law for both linoleic and for linolenic acids. Scale-up trials were carried out to confirm the feasibility of enzymatic modification for the production of HMFS. The characteristics of the product, produced in the scale-up acidolysis under selected conditions (temperature 61 °C, water content 3.5%, lard:fatty acids 1/2.4 (mol/mol), Lipozyme RM IM load 13.7%, and time 1.0 h), were similar to the fat in Chinese mothers' milk. The results showed that it was possible to produce human milk fat substitutes from lard through enzymatic acidolysis with soybean fatty acids.     

Production of olive oil enriched with medium chain fatty acids catalysed by commercial immobilised lipases

Structured triacylglycerols, containing medium chain fatty acids, were produced by acidolysis of virgin olive oil with caprylic or capric acid, at a molar ratio of olive oil:fatty acid of 1:2, at 45 °C for 24 h, in solvent-free media or in n-hexane, catalysed by Thermomyces lanuginosa (Lipozyme TL IM), Rhizomucor miehei (Lipozyme RM IM) and Candida antarctica (Novozym 435) immobilised lipases. Incorporations were always greater for capric than for caprylic acid. For both acids, higher incorporations were always attained in solvent-free media: the highest caprylic acid incorporations were obtained with Novozym 435 (25.5 mol%) and Lipozyme RM IM (25.7 mol%), while similar capric acid incorporations were obtained with all biocatalysts (27.1–30.4 mol%).     

Studies of reaction variables for lipase-catalyzed production of alpha-linolenic acid enriched structured lipid and oxidative stability with antioxidants

Alpha-linolenic acid (ALA) enriched structured lipid (SL) was produced by lipase-catalyzed interesterification from perilla oil (PO) and corn oil (CO). The effects of different reaction conditions (substrate molar ratio [PO/CO 1:1 to 1:3], reaction time [0 to 24 h], and reaction temperature [55 to 65 °C]) were studied. Lipozyme RM IM from Rhizomucor miehei was used as biocatalyst. We obtained 32.39% of ALA in SL obtained under the optimized conditions (molar ratio-1:1 [PO:CO], temperature-60 °C, reaction time-15 h). In SL, the major triacylglycerol (TAG) species (linolenoyl-linolenoyl-linolenoyl glycerol [LnLnLn], linolenoyl-linolenoyl-linoleoyl glycerol [LnLnL]) mainly from PO and linoleoyl-linoleoyl-oleoyl glycerol (LLO), linoleoyl-oleoyl-oleoyl glycerol (LOO), palmitoyl-linoleoyl-oleoyl glycerol (PLO) from CO decreased while linolenoyl-linolenoyl-oleoyl glycerol (LnLnO) (18.41%), trilinolein (LLL) (9.06%), LLO (16.66%), palmitoyl-linoleoyl-linoleoyl glycerol (PLL) (9.69%) were increased compared to that of physical blend. Total tocopherol content (28.01 mg/100 g), saponification value (SV) (192.2), and iodine value (IV) (161.9) were obtained. Furthermore, oxidative stability of the SL was also investigated by addition of 3 different antioxidants (each 200 ppm of rosemary extract [SL-ROS], BHT [SL-BHT], catechin [SL-CAT]) was added into SL and stored in 60 °C oven for 30 d. 2-Thiobabituric acid-reactive substances (TBARS) value was 0.16 mg/kg in SL-CAT and 0.18 mg/kg in SL-ROS as compared with 0.22 mg/kg in control (SL) after oxidation. The lowest peroxide value (POV, 200.9 meq/kg) and longest induction time (29.88 h) was also observed in SL-CAT.     

脂肪酶催化鱼油醇解富集EPA和DHA的研究

研究了固定化脂肪酶Lipozyme RM IM催化鱼油部分醇解反应,探讨了油醇比、酶加量等因素对反应的影响,在优化条件下,可以使鱼油中EPA、DHA的含量由26.1%升至43.O%,得率大于75%,对鱼油醇解产物进行了分子蒸馏提纯,得到了富含EPA、DHA的甘油酯型鱼油产品.     

Chemoenzymatic synthesis of structured triacylglycerols with conjugated linoleic acids (CLA) in central position

A chemoenzymatic process for the production of structured triacylglycerols (TAG) containing CLA at sn2 position and lauric acid at external ones is proposed. First, castor bean oil was chemically dehydrated and isomerised to obtain a new modified oil with very high proportion of CLA (>95%). Then, this new oil was used for enzymatic transesterification allowing the grafting of lauric acid at external positions of the TAG backbone by using 1,3 regioselective enzymes. Among these, Aspergillus niger lipase was not satisfactory giving very low lauroyl incorporation (<5%) On the contrary, lipases from Thermomyces lanuginosa (Lipozyme TL IM) and from Carica papaya latex allowed good reaction yields. The effect of the type of acyl donor was studied. With alkyl esters T. lanuginosa lipase provided a final incorporation of 58.9% after 72 h corresponding to 88.4% transesterification yield. Concerning C. papaya lipase, incorporation of lauroyl residues was lower than Lipozyme TL IM. This lipase exhibited higher performance with lauric acid accounting for 44.7% lauroyl incorporation at the end of reaction for a 67.1% transesterification yield. The effect of the substrates mole ratio was also evaluated. It was observed that a 1:3 TAG/acyl donor mole ratio was the most efficient for both lipases. Finally, fatty acids regiodistribution of the newly formed structured TAG was determined. With Lipozyme TL IM, the proportion of lauric acid incorporated at the sn2 position did not exceed 5.4% after 72 h while with C. papaya lipase a more pronounced incorporation of lauroyl residues at the central position (8.8%) was observed.     

Lipase-catalyzed acidolysis of sunflower oil: Kinetic behavior

In the present study, sunflower oil was modified with a palmitic–stearic acids blend by means of an immobilized sn-1,3 specific lipase (Lipozyme RM IM) to produce structured lipids. Products were analyzed to determine fatty acid incorporation (F_M) into triacylglycerol structure and to quantify by-products as monoglycerides (MG) and diglycerides (DG). The effects of the reaction conditions (temperature, time, incorporated water) on enzymatic acidolysis were studied. Nonlinear regression methods were employed to fit experimental data with kinetic models proposed in the literature. The disappearance of reactant fatty acids (RF) over time was successfully modeled by a Ping-Pong Bi-Bi mechanism. F_M was also represented with a lumped parameter model of the enzymatic mechanism. Maximum RF disappearance and F_M onto sunflower oil glycerides increased with increasing reaction temperature. MG and DG concentrations in water-free systems were stabilized in low levels, while the incorporation of water to the reaction mixture produced a considerable increase in DG formation principally. Kinetic and equilibrium parameters showed temperature dependencies.     

Thermal and crystal characteristics of enzymatically interesterified fats of fatty acid-balanced oil and fully hydrogenated soybean oil in supercritical CO2 system

Blends of fatty acid-balanced oil that was prepared by the aqueous enzymatic extraction, and with fully hydrogenated soybean oil in different weight ratios from 30:70 to 80:20 (wt%) were interesterified using Lipozyme RM IM in a supercritical CO₂ system. The optimal immobilized enzyme dosage, pressure, substrate ratio, temperature, and time were 6% (w/w) of initial substrates, 8 MPa, blend ratio with 60:40 (wt%) of fatty acid-balanced oil and fully hydrogenated soybean oil, a temperature of 70°C, and reaction time of 2 h, respectively. It was observed that at the optimal conditions, under supercritical CO₂ conditions, the reaction time of the interesterification was shorter than that of conventional enzymatic interesterification. The slip melting point, solid fat content, fatty acid composition, differential scanning calorimetry, polymorphic form and crystal morphology of the enzymatically interesterified fats were evaluated. The results indicated that the interesterified fats showed desirable physical properties with lower slip melting point and solid fat content, suitable crystal form (β′ polymorph), and without trans-fatty acid for possible use as a shortening and margarine stock.     

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。