人乳脂替代品1,3-二油酸-2-棕榈酸三甘酯的研究进展

1,3-二油酸-2-棕榈酸三甘酯是一种重要的人乳脂替代品,其脂肪酸组成及脂肪酸在甘油三酯中的位置分布均与天然母乳脂非常相似,将其添加到婴幼儿奶粉中,可确保为婴儿提供接近母乳的甘油三酯营养供应。因此,采用绿色酶催化技术开发1,3-二油酸-2-棕榈酸三甘酯有着非常广阔的前景。概述了1,3-二油酸-2-棕榈酸三甘酯酶催化合成技术的研究进展,对合成工艺中的反应底物、酶、反应介质、反应类型以及催化反应中存在的问题进行了探讨。     

液相色谱-串联质谱法定量分析莱茵衣藻1,3-二油酸-2-棕榈酸甘油三酯

研究建立了基于超高效液相色谱串联三重四级杆质谱技术的1,3-二油酸-2-棕榈酸甘油三酯(1,3-Dioleoyl-2-palmitoylglycerol, OPO)分析方法。在正离子模式下, 以[876>577]为定量离子对, 通过多反应监测模式(Multiple Reaction Monitoring, MRM)扫描, 采用内标法对OPO进行定量分析。对缺氮胁迫条件下一株野生型莱茵衣藻(Chlamydomonas reinhardtii)cc-5325及其半乳糖基水解酶基因敲降突变体M08的OPO进行了定量分析, 研究结果发现莱茵衣藻中OPO在缺氮胁迫期间显著积累, 且不同遗传背景的莱茵衣藻OPO积累量不同。在缺氮第1至第3天, M08的OPO含量相较于cc-5325分别提高了3.70、3.04和2.74倍, M08的OPO产量相较于cc-5325分别提高了1.13、1.53和1.33倍, 说明通过遗传改造莱茵衣藻的某些脂质代谢途径相关基因可以增加其OPO含量, 因此莱茵衣藻具有改成为商业化油脂新食品原料的潜在应用价值。研究建立的功能脂质OPO的定量分析方法对进一步研究不同遗传改造的莱茵衣藻中OPO结构脂的检测提供了技术支持和分析基础。     

生物法合成维生素C棕榈酸酯

研究了不同的脂肪酶在有机溶剂体系中催化合成L-维生素C棕榈酸酯的反应。针对维生素C在有机溶剂中溶解度较低这一问题,对催化合成维生素C棕榈酸酯反应的脂肪酶和反应介质进行比较,同时对影响合成维生素C棕榈酸酯反应的因素（温度、底物浓度、底物摩尔比、反应时间和酶量等）进行探讨,优化了反应条件：在10mL的丙酮中,1.094g棕榈酸与0.107g维生素C在酶量为20％（W/W, 固定化酶/维生素C）的固定化脂肪酶催化下,初始含0.4nm分子筛20%,温度为60℃,转速为200r/min,反应48h转化率可以达到80%,产物维生素C棕榈酸酯的浓度可达20g/L。     

微藻中棕榈油酸的研究进展

棕榈油酸是一种ω-7单不饱和脂肪酸,它具有重要的营养和医药保健价值,同时也是制备生物柴油和1-辛烯的优质原料。棕榈油酸普遍存在油料作物中,但是含量较低,而含量较高的野生植物又存在资源少问题,因此利用富含棕榈油酸且适合大规模培养的微藻是解决棕榈油酸来源的一条新型途径。本文重点介绍了微藻中棕榈油酸来源的优势、棕榈油酸的生理功能、微藻中棕榈油酸的合成途径及分离纯化方法,并且展望了棕榈油酸进一步的研究方向。     

利用微藻生产单不饱和脂肪酸——棕榈油酸

正棕榈油酸(C16:1Δ9)具有重要的营养和医药价值,进行相应产品的开发已是势在必行。目前棕榈油酸主要来源于生物合成。大田栽培的普通油料作物中棕榈油酸含量很低,而棕榈油酸含量高的野生植物存在资源少的问题,因此利用棕榈油酸含量高且能够规模培养的微藻来进行棕榈油酸的生产已成为生物技术和可再生资源研究的一个热点领域。文章介绍了棕榈油酸的原料来源,并结合笔者实验室对黄丝藻棕榈油酸的研究,综述了微藻棕榈油酸的优势及其合成途径,最后初步展望了微藻棕榈油酸研究的发展方向。     

固定化脂肪酶合成维生素A棕榈酸酯

研究了有机溶剂中脂肪酶催化维生素A棕榈酸酯的合成工艺。采用维生素A醋酸酯和棕榈酸乙酯作为反应底物, 对催化合成维生素A棕榈酸酯反应介质进行了比较, 同时对影响合成维生素A棕榈酸酯反应的因素(温度、初始水含量、底物摩尔比、反应时间和酶量等)进行了探讨, 优化了反应条件: 在10 mL的石油醚中, 体系初始含水量0.2%(体积比V/V), 0.100 g 维生素A醋酸酯和0.433 g 棕榈酸乙酯在酶量为1.1 g的固定化酶催化下, 在30°C、190 r/min下反应12 h, 转化率可以达到83%, 固定化酶可连续使用5次以上。     

固定化脂肪酶合成维生素A棕榈酸酯

研究了有机溶剂中脂肪酶催化维生素A棕榈酸酯的合成工艺。采用维生素A醋酸酯和棕榈酸乙酯作为反应底物, 对催化合成维生素A棕榈酸酯反应介质进行了比较, 同时对影响合成维生素A棕榈酸酯反应的因素(温度、初始水含量、底物摩尔比、反应时间和酶量等)进行了探讨, 优化了反应条件: 在10 mL的石油醚中, 体系初始含水量0.2%(体积比V/V), 0.100 g 维生素A醋酸酯和0.433 g 棕榈酸乙酯在酶量为1.1 g的固定化酶催化下, 在30°C、190 r/min下反应12 h, 转化率可以达到83%, 固定化酶可连续使用5次以上。     

神经网络优化非水相脂肪酶催化合成抗坏血酸油酸酯的条件研究

抗坏血酸油酸酯具有强抗氧化作用．为了获得脂肪酶催化合成抗坏血酸油酸酯的最适条件,主要研究了反应温度、脂肪酶量、油酸量对抗坏血酸油酸酯合成效果的影响．采用中心组合设计和动量梯度下降神经网络对反应条件网络进行训练仿真,并利用训练好的网络对催化酯化工艺条件进行预测．研究结果表明：经过训练的网络可以很好的模拟反应条件,得到了脂肪酶催化反应的最佳工艺参数．当抗坏血酸0．8g时,反应温度56℃,油酸量0．95g,固定化脂肪酶量0．74g,添加分子筛条件下,抗坏血酸油酸酯的转化率为46.5％．该方法为抗坏血酸酯化催化效果的预测提供了一条可行的途径．     

麦红吸浆虫中脂肪酸的气相色谱分析

建立了一种测定麦红吸浆虫中脂肪酸的微量气相色谱分析方法,该方法具有称样量小,操作简单,灵敏度高,重现性好等特点,称样量仅为3－5mg,各脂肪酸RSD均小于5．7％,用湿法研磨麦红吸浆虫以破碎细胞和提取脂肪,水溶浓缩提取液后进行甲酯化反应,上清液用于气相色谱分析,检出十种脂肪酸,主要脂肪酸有豆蔻酸,棕榈酸,棕榈油酸,硬脂酸,油酸,亚油酸,不同产地的红吸浆虫样品中C16：0,C17：1,C18：0,C18：2和不饱和脂肪酸含量存在明显差异。     

酿酒酵母脂酰-△9脱氢酶亚细胞定位表达及其对烟草脂肪酸合成的影响

以烟草Nicotiana tabacum L.为宿主植物,分别在细胞质内质网和质体内定位表达酿酒酵母Saccharom,ees cerevisiae脂酰-CoA-△9脱氢酶(Sc△9D),以期提高植物组织中棕榈油酸(16∶1△9)的积累量和分析该酶不同亚细胞定位表达对油脂代谢的影响.与野生型和空载体(对照)植物相比,转基因烟草植株叶片中单不饱和的棕榈油酸及顺式十八碳烯酸(18∶1△11)含量明显提高,而饱和的棕榈酸(16∶0)含量相应减少,多不饱和的亚油酸(18∶2△9,12)和亚麻酸(18∶3△9,12,15)含量亦降低.Sc△9D质体定位表达烟叶中棕榈油酸及顺式十八碳烯酸含量分别是Sc△9D细胞质内质网定位表达烟叶的2.7和1.9倍.这表明酵母脂酰-△9脱氢酶能在高等植物细胞中正确催化棕榈酸(16∶0)转化为棕榈油酸(16∶1△9),而且在质体内表达的效应显著高于在细胞质内质网上的效应.新建立了一种应用脂酰-CoA-△9脱氢酶代谢工程培育植物组织高水平合成积累棕榈油酸等ω-7脂肪酸的策略,有助于在生物量大的烟叶等营养器官中组装ω-7脂肪酸合成途径以生产优质生物燃油.     

野生艾草黄酮的含量及抗氧化性研究

目的：测定艾草中总黄酮含量,并对其抗氧化性、羟自由基（-OH）的清除能力和过氧化值（POV）进行了研究。方法：有机溶剂提取法从野生艾草中提取黄酮,以芦丁为对照品测定艾草中总黄酮含量。采用邻苯三酚自氧化测定抗氧化性;用新鲜猪油测定过氧化值（POV）。结果：艾草中总黄酮含量高达5．5％,对O2^-抑制率为36．8％,黄酮浓度超过2mg／mL时对羟自由基（-OH）清除能力逐渐增强,0．50％黄酮的POV较强,仅次于0．05％的维生素C,说明艾草黄酮有较好的开发价值。     

脂肪酶二步法催化鱼油下脚料富集DHA

采用Pseudomonas sp．脂肪酶水解,Geotrichum sp．脂肪酶选择性酯化的两步法显著提高游离脂肪酸（FFA）中二十二碳六烯酸（DHA）的含量。通过筛选合适的底物醇,优化水解和选择性酯化反应条件达到富集DHA的目的。结果表明月桂醇为选择性酯化的最适底物醇。确定的最佳水解条件：4g反应底物,m（水）／m（粗鱼油）=1,1000 U Pseudomonas sp．脂肪酶,40℃,搅拌速度200r／min,反应24h;最佳酯化条件：3g反应底物,n（FFA）／n（醇）=1／2,1000 U固定化Geotrichum sp．脂肪酶,1g正己烷,30℃,200r／min搅拌,反应20h。经过水解和一次选择性酯化反应后,DHA含量从原料粗鱼油中的18．9％提纯到72．8％;经二次选择性酯化后,DHA含量上升到92％。脂肪酶水解-选择性酯化的两步法是富集DHA的有效方法。     

Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats

Cell growth, lipid accumulation and cellular lipid composition of Yarrowia lipolytica growing on mixtures of industrial fats containing stearic, oleic, linoleic and palmitic acid have been studied. During growth, the strain incorporated oleic and linoleic acids more rapidly than the saturated fatty acids. Relatively high lipid accumulation (up to 0.44 g of lipids per g of dry matter) was observed when stearic acid was included in the culture medium. In contrast, substrates rich in oleic acid did not favor cellular lipid accumulation. The accumulated lipids, mainly composed of triacylglycerols (45-55% w/w), demonstrated a different total fatty acid composition compared with that of the substrate; in all cases, the microorganism showed the unusual capacity to increase its cellular stearic acid level, even if this fatty acid was not found in high concentrations in the substrate. This permitted the synthesis of interesting lipid profiles with high percentages of stearic acid and non-negligible percentages of palmitic and oleic acid, with a composition resembling that of cocoa-butter.     

不同来源肝细胞在体外降脂药物评价中药效反应的对比

目的:通过对比不同来源的人肝癌细胞系HepG2和原代大鼠肝细胞在体外降脂药物评价中药效反应,指导两种肝细胞在体外降脂药物评价中的实际应用。方法:用游离脂肪酸(油酸/棕榈酸,2:1)诱导HepG2细胞、原代大鼠肝细胞脂肪变性,并用100μmol·L-1苯扎贝特干预,检测细胞内甘油三酯(TG)、总胆固醇(TC)、活性氧(ROS)含量,细胞内脂滴数目、并检测细胞上清液中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果:FFA刺激使HepG2细胞和原代大鼠肝细胞脂质沉积(TG、脂滴)和氧化应激(ROS、MDA、SOD)水平上升。苯扎贝特对HepG2细胞1 mmol·L-1FFA造模组和原代大鼠肝细胞0.5 mmol·L-1FFA造模组脂质沉积和氧化应激水平改善显著;而HepG2细胞0.5 mmol·L-1FFA造模组和原代大鼠肝细胞1 mmol·L-1FFA造模组脂质沉积和氧化应激水平在苯扎贝特干预后变化不明显。结论:在相同FFA造模浓度,原代大鼠肝细胞病理特征变化更为明显;苯扎贝特对两种肝细胞在脂质沉积和氧化应激水平的作用也不完全相同。因而HepG2细胞和原代大鼠肝细胞在体外降脂药物评价中药效反应是不完全相同的。     

Production of the infant formula ingredient 1,3-olein-2-palmitin in Arabidopsis thaliana seeds

In human milk fat, palmitic acid (16:0) is esterified to the middle (sn-2 or β) position on the glycerol backbone and oleic acid (18:1) predominantly to the outer positions, giving the triacylglycerol (TG) a distinctive stereoisomeric structure that is believed to assist nutrient absorption in the infant gut. However, the fat used in most infant formulas is derived from plants, which preferentially esterify 16:0 to the outer positions. We have previously showed that the metabolism of the model oilseed Arabidopsis thaliana can be engineered to incorporate 16:0 into the middle position of TG. However, the fatty acyl composition of Arabidopsis seed TG does not mimic human milk, which is rich in both 16:0 and 18:1 and is defined by the high abundance of the TG molecular species 1,3-olein-2-palmitin (OPO). Here we have constructed an Arabidopsis fatty acid biosynthesis 1-1 fatty acid desaturase 2 fatty acid elongase 1 mutant with around 20% 16:0 and 70% 18:1 in its seeds and we have engineered it to esterify more than 80% of the 16:0 to the middle position of TG, using heterologous expression of the human lysophosphatidic acid acyltransferase isoform AGPAT1, combined with suppression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE. Our data show that oilseeds can be engineered to produce TG that is rich in OPO, which is a structured fat ingredient used in infant formulas.     

The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities.

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.     

Esterification of fatty acids using Candida antarctica lipase A in water-abundant systems

The feasibility of using native lipase A from Candida antarctica (CAL-A) to esterify fatty acids with water-insoluble alcohols in the presence of excess water was investigated in stirred-tank reactors. For high reaction rates, a ratio of water:substrates of 0.6-1.4:1 (v/v) was required. CAL-A showed higher substrate selectivity for the esterification of saturated palmitic acid with branched-chain 2-ethyl-1-hexanol than for unsaturated oleic acid with linear alcohol (1-decanol). After 18 h at 70 °C in a 1.5 l bulk stirred-tank reactor, an 2-ethyl-1-hexyl palmitic acid ester was obtained near 100 % yield [molar ratio palmitic acid:2-ethyl-1-hexanol ~1:1.25, with 1.11 % (w/w) Novocor ADL (based on palmitic acid weight)].     

Long-term effects of cis and trans monounsaturated (18:1) and saturated (16:0) fatty acids on the synthesis and secretion of apolipoprotein A-I- and apolipoprotein B-containing lipoproteins in HepG2 cells

The objective of this study was to compare the long-term effects of oleic (cis 18:1), elaidic (trans 18:1), and palmitic (16:0) acids on hepatic lipoprotein production, using HepG2 cells as an experimental model. The net accumulation in the medium of apolipoprotein A-I (apoA-I) was not significantly altered by fatty acids, whereas that of apoB was increased with oleic and elaidic acids. Oleic acid, and to a lesser extent elaidic and palmitic acids, increased the mass of triglycerides in the medium and the incorporation of [(3)H]glycerol into secreted triglycerides. The incorporation of [(14)C]acetate into cellular and secreted total cholesterol was stimulated by 96% and 83%, respectively, with elaidic acid but was not significantly modified by oleic or palmitic acid. Relative to oleic acid, the secretion of (14)C-labeled phospholipids and triglycerides was decreased 28% to 31% with elaidic and palmitic acids whereas that of free cholesterol and cholesteryl esters was enhanced 93% and 73%, respectively, with elaidic acid but remained unchanged with palmitic acid. Compared with oleic acid, elaidic acid stimulated the secretion of very low density lipoprotein cholesterol (VLDL-Chol), low density lipoprotein cholesterol (LDL-Chol), and high density lipoprotein cholesterol (HDL-Chol) by 43%, 70%, and 34%, respectively, whereas palmitic acid decreased VLDL-Chol but had no significant effect on LDL-Chol and HDL-Chol. The ratios of total cholesterol to HDL-Chol were 3.17, 3.60, and 3.25 with oleic, elaidic, and palmitic acids, respectively; the corresponding ratios of LDL-Chol to HDL-Chol were 0.87, 1.10, and 0.93, respectively. Compared with oleic and palmitic acids, the LDL and HDL particles secreted in the presence of elaidic acid contained higher levels of free cholesterol and cholesteryl esters and a lower content of phospholipids. The phospholipid-to-total cholesterol ratios of HDL were 1.05, 0.40, and 0.76 with oleic, elaidic, and palmitic acids, respectively.Our results indicate that in comparison with cis monounsaturated and saturated fatty acids, trans fatty acids have more adverse effects on the concentration and composition of lipoproteins secreted by HepG2 cells.     

Highly selective synthesis of 1,3-oleoyl-2-palmitoylglycerol by lipase catalysis.

1,3-Oleoyl-2-palmitoylglycerol (OPO), an important structured triglyceride in infant nutrition, was synthesized by a two-step process in high yields and purity using sn1,3-regiospecific lipases. In the first step, tripalmitin (TP) was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn1,3-regiospecific lipases yielding the corresponding 2-monopalmitin (2-MP). The 2-MP was isolated in up to 85% yield and >95% purity by crystallization and esterified in the second step with oleic acid using the same lipases to form the structured triglyceride OPO in up to 78% yield containing 96% palmitic acid in the sn2-position. Water activity, solvent, as well as carrier for lipase immobilization strongly influenced the yield and purity of products in both steps. The best results were achieved with lipases from Rhizomucor miehei and Rhizopus delemar immobilized on EP 100 and equilibrated to a water activity of 0.43. Special emphasis was given to develop this process in solvents that are allowed to be used in foodstuffs and to perform the second step in a solvent-free system.     

Replacement of the aliphatic chains of Clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition.

The membrane lipid aliphatic chains of Clostridium acetobutylicum ATCC 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids. Growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains. Growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and C19 chains containing cyclopropane rings. When cells were grown with mixtures of palmitic and oleic acids, the ether-linked chains of the plasmalogens were greater than or equal to 64% 18:1 plus C19 chains containing cyclopropane rings at all ratios of oleic to palmitic acid in the medium. The acyl chains reflected the palmitic acid content of the medium more closely. Marked changes were observed in both phospholipid and glycosyldiglyceride compositions as the lipid acyl and ether-linked chains became more enriched with unsaturated and cyclopropane chains. The ratio of the glycerol acetal of plasmenylethanolamine to phosphatidylethanolamine increased, the ratio of cardiolipin to phosphatidylglycerol decreased, and the ratio of diglycosyldiglyceride to monoglycosyldiglyceride increased. However, the monoglycosyldiglyceride/diglycosyldiglyceride ratio was lower for cells grown on 100% oleic acid than for cells grown on 60 or 80% oleic acid. In the membranes of cells grown on 100% oleic acid, the ratio of glycolipids to phospholipids was lower than that found in cells grown on 60% oleic acid. These results indicate that C. acetobutylicum regulates its polar lipid composition in a complex manner involving phospholipids and glycosyldiglycerides. These changes can affect the equilibria between those lipids that form bilayers and those lipids that tend to form nonlamellar phases when enriched with unsaturated aliphatic chains. Phosphoglycolipids of unknown structure were also observed in cells grown either with biotin or with fatty acids. The content of the most abundant phosphoglycolipid also varied with the degree of unsaturation of the cellular lipids.