赤灵芝中三萜类活性成分的提取筛选和质谱分析

研究赤灵芝总三萜最佳提取工艺条件,评价三萜类化合物对脂肪氧化酶的抑制效果。采取单因素实验和响应面法分析提取时间、提取次数、液料比和乙醇浓度对赤灵芝三萜得率的影响,筛选最佳工艺参数;利用高效液相色谱-电喷雾质谱联用技术（HPLC-ESI-MS）对赤灵芝中三萜类提取物化学成分进行分析鉴定,以脂肪氧化酶为生物靶分子,采用超滤质谱技术筛选酶抑制剂,探究三萜类化合物对脂肪氧化酶的抑制效果。结果显示：最佳工艺条件为料液比1:30、乙醇浓度75%、提取次数3次和提取时间1.5 h;在这些条件下,总三萜得率为1.04%;从赤灵芝中筛选出5种脂肪氧化酶抑制剂,5种化合物对1.0 U/mL脂肪氧化酶活性有较强抑制作用,抑制效果的强弱顺序为灵芝酸B（52.64%）>灵芝酸C2（40.21%）>灵芝酸D2（31.25%）>灵芝酸F（12.69%）>灵芝酸A（10.82%）。实验结果表明,响应面法筛选赤灵芝三萜的提取工艺参数准确可靠,可用以赤灵芝三萜的生产制备;赤灵芝中三萜类活性成分可抑制脂肪氧化酶,表明有缓解炎症的功效,研究结果可为赤灵芝中抗炎成分的进一步研究提供一定理论基础。     

蛋白质氧化对白斑狗鱼肌肉蛋白理化性质变化的影响

以白斑狗鱼为研究对象,提取肌浆蛋白和肌原纤维蛋白,通过三种氧化系统(Fe-H_2O_2-Asc氧化系统、亚油酸脂肪氧化系统和氧化酶氧化系统),对蛋白质进行不同程度的体外模拟氧化,发现两种蛋白质对Fe-H_2O_2-Asc氧化系统敏感。在Fe-H_2O_2-Asc氧化系统中,羰基含量和表面疏水性随着H_2O_2浓度和氧化时间的增加而增加;且氧化时间越长,表面疏水性增加越明显;总巯基含量和游离氨含量随着H_2_O2浓度和氧化时间的增加而下降,且游离氨基在氧化5h时变化更显著。由此可见,氧化可以改变鱼肉蛋白的理化性质,可能会进一步导致其结构和功能性质的改变。     

大豆发芽过程中酶的含量变化及营养变化研究

探讨大豆发芽过程中脂肪氧化酶、内源蛋白酶和游离氨基酸及部分营养物质的变化情况。采用不同发芽时间的发芽大豆进行脂肪氧化酶和内源蛋白酶含量的检测和分析。结果表明:沈农8号大豆中,脂肪氧化同功酶L-1的含量最高,在大豆发芽过程中,脂肪氧化酶的活性逐渐降低;内源蛋白酶和游离氨基酸的含量变化基本一致;脂肪含量降低;维生素C含量增高。      

大豆的脂肪氧化酶与正己醛含量的关系

<正> 大豆的腥味与多价不饱和脂肪酸的氧化有关,在氧化过程中脂肪氧化酶起着催化作用。本文以正己醛作为代表探讨了脂肪氧化酶与致腥物质含量的关系。发现大豆在生长和贮存期间就有正己醛的生成,其中以脂肪氧化酶引起的酶促氧化为主。干破碎豆粒成粉,不管酶活     

高压促进猪肉肌内脂肪氧化过程中脂肪氧化酶的作用

为研究高压促进猪肉肌内脂肪氧化过程中脂肪氧化酶(Lipoxygenase,LOX)的作用,以去除LOX的猪背最长肌为原料,在其中加入事先从猪肉中提取的或外源LOX(大豆脂肪氧化酶),经600MPa、50℃或350MPa、20℃处理并经6d冷藏后,测定各样品高压处理和冷藏后LOX活性和TBARS值。结果表明:LOX对高压下肌内脂肪氧化的启动有重要作用,但其不会影响样品冷藏后的最终氧化状态(TBARS值);在高压处理后的冷藏中,LOX的作用不明显,主要以脂肪自动氧化为主,即使加入5倍浓度的外源LOX,也只引起样品最终TBARS值的少量增加。因此,高压促进猪肉肌内脂肪氧化中主要以自动氧化为主,而LOX的作用很小。      

不同氧化酶体系对阿拉伯木聚糖溶液流变学性质影响的研究

本文采用小振幅振荡流变学测量法研究了三种不同氧化酶-过氧化物酶（POX）、葡萄糖氧化酶（GOX）、脂肪氧合酶（LOX）对小麦面粉中水可提取阿拉伯木聚糖（WEAX）流变学性质的影响.结果显示：1）在POX/H2O2反应体系中,一定酶用量范围内（2.4～4.8U/g）,增加POX的用量可提高WEAX发生氧化胶凝的速率及增强凝胶的弹性,进一步增加酶用量则仅可缩短凝胶形成时间;2）在GOX/葡萄糖（Glc）/POX反应体系中,GOX用量对WEAX溶液流变性质的影响与POX的相似,但与POX相比,由GOX催化的氧化胶凝反应有一迟滞期;3）在LOX/亚油酸（LA）反应体系中,LOX对于WEAX的氧化胶凝反应有一最适用量,酶用量过低或过高均不利于凝胶的形成,且LOX的作用要弱于前两种氧化酶.     

通电加热条件对豆浆中脂肪氧化酶活性的影响

采用通电加热和传统(油浴)加热两种方式处理豆浆样品,研究通电加热过程中电源电压、电源频率和保温时间对豆浆中脂肪氧化酶活性的影响。结果表明:采用的电源电压越高,通电加热处理后的豆浆中脂肪氧化酶活性越高;较高的电源频率对脂肪氧化酶的钝化具有显著的促进作用,但是当电源频率超过500 Hz后,电源频率的提高对脂肪氧化酶的活性影响较小;延长保温时间会促进脂肪氧化酶的钝化,通电加热过程中电场的存在会促进脂肪氧化酶的钝化。各因素对脂肪氧化酶活性的作用规律,可以对通电加热设备的设计与生产产生积极的指导作用。     

玉米脂肪氧化酶同工酶缺失对种子发芽率的影响

用不同存贮时间及老化处理的玉米种子,采用脂肪氧化酶偶联氧化β-胡萝卜素产生显色反应,研究了脂肪氧化酶同工酶不同位点缺失对玉米种发芽率影响及其缺失体在玉米种子中的丰富度.结果表明:同缺lox-1、 lox-2能提高玉米种子发芽率,延缓玉米种子衰老;玉米种子资源中具有较丰富的脂肪氧化酶缺失体资源,其中,糯质型、硬粒型、北方的玉米种子中同缺lox-1、 lox-2的种质比例较高.     

脂肪氧化酶对啤酒抗氧化性能的影响及其特性研究

分析了目前国内广泛使用的麦芽中的脂肪氧化酶的活性,并研究了脂肪氧化酶对啤酒抗氧化性能的影响。同时对麦芽中的脂肪氧化酶的基本酶学性质进行了调查,跟踪了工业化生产规模的制麦和糖化过程中的脂肪氧化酶的活性。结果表明麦芽含有脂肪氧化酶,在通常的糖化工艺条件下,仍有催化活性,导致麦汁和啤酒TBA增加。麦芽脂肪氧化酶的最适pH为6.5,在20～40℃之间表现出较高的活性,温度高于50℃时,热稳定性迅速下降。大麦经发芽之后脂肪氧化酶大幅度增加,峰值为原大麦的5倍左右,但是经过焙焦之后,酶活又大幅度下降。在糖化过程中,当醪液的温度低于60℃时,醪液中能检测出脂肪氧化酶的活性,是脂肪氧化酶催化氧化脂肪酸的关键阶段。      

钙果种仁油的提取及成分分析

用不同的提取溶剂对钙果种仁中的脂肪进行提取,石油醚的提取效果最好;研究了提取温度、提取时间和料液比3个因素对仁油提取的影响,通过正交试验确定了最佳工艺参数:提取温度60℃,提取时间6h,料液比1:10。在此工艺下进行提取,提油率为97.62%。用气相色谱法测定其脂肪酸组成,共检测出5种主要脂肪酸,分别为棕榈酸、棕榈烯酸、硬脂酸、油酸和亚油酸,其中以油酸(68.53%)亚油酸(27.43%)为主。     

Carotene cooxidation activity of tomato lipoxygenase-2

The tomato isoenzyme lipoxygenase-2 presented a carotene cooxidation activity; it showed more activity with linoleic acid as substrate than with linolenic acid. The lipoxygenase was inhibited by reagents of the disulphide groups (dithiothreitol and 2-mercaptoethanol) and —SH groups ((4-hydroxymercuri)benzoic acid and iodoacetamide) and some antioxidants.     

猪肉12-脂肪氧合酶催化结构域的表达、纯化及其酶学性质分析

研究脂肪氧合酶（lipoxygenase,LOX）在猪肉贮藏、加工过程中对脂肪氧化及风味形成的作用机制。通过序列分析和聚合酶链式反应扩增获得了猪肉12-脂肪氧合酶催化结构域（12-lipoxygenase catalytic domain,12-LOXcd）的编码基因,采用大肠杆菌表达系统,经镍柱亲和层析和Superdex G200凝胶过滤层析纯化得到12-LOXcd蛋白,并研究其酶学性质。结果表明,构建的原核表达载体pMBP-12-LOXcd在大肠杆菌中成功可溶性表达了猪肉12-LOXcd,该重组蛋白经纯化可达电泳纯。12-LOXcd以亚油酸为底物的比活力为2 826.7 U/mg,最适pH值为6.0,最适作用温度为30 ℃。亚油酸Km为0.40 mmol/L,亚麻酸Km为0.55 mmol/L,花生四烯酸Km为4.15 mmol/L,表明最适底物为亚油酸。与大豆LOX相比,该酶在较高NaCl质量分数（9%）时仍保持活性稳定;对热较不稳定,在60 ℃条件下失活,但优于大豆LOX的热稳定性;此外,12-LOXcd的pH值稳定性也优于大豆LOX,在碱性条件下仍能保留部分活力。     

Characterization of factors that transform linoleic acid into di- and trihydroxyoctadecenoic acids in mash

The qualities of beer are deteriorated by the presence of either di- or trihydroxyoctadecenoic acids, which reduce the beer 'head' and produce an astringent flavor. In this study we found that native extracts of malt mash transformed linoleic acid into di- and trihydroxyoctadecenoic acids, but this transforming activity and lipoxygenase activity were inactivated by heating the mash at 70 degrees C for 30 min. Recombinant barley lipoxygenase 1 was not able to transform linoleic acid into di- and trihydroxyoctadecenoic acids. The transforming activity of mash extract heated at 70 degrees C for 30 min could be restored by the addition of recombinant barley lipoxygenase 1; in contrast, the activity of boiled mash extract was not substantially restored by the recombinant enzyme. These results indicate that di- and trihydroxyoctadecenoic acids are generated from linoleic acid by both lipoxygenase and a heat-stable enzymatic factor present in the mash.     

Influence of lipid content and lipoxygenase on flavor volatiles in the tomato peel and flesh

Ten different varieties of tomatoes were separated into peel and flesh and each portion was measured separately. Headspace volatiles were measured in real time using selected ion flow tube mass spectrometry. Lipoxygenase activity was measured using the adsorption of conjugated dienes formed by lipoxygenase. Lipid was extracted and fatty acids were quantified using a gas chromatograph. Volatiles were significantly greater in the peel than flesh when there was a significant difference. The lipoxygenase activity of flesh and peel correlated with the volatiles produced by the lipoxygenase pathway. There was no correlation with other volatiles, which are not dependent on lipid oxidation by lipoxygenase. The lipoxygenase activity, total fatty acid content, and linolenic acid of the peel were greater than the flesh, which is directly related to an increase in fresh, green volatiles. Addition of exogenous lipoxygenase had no effect on lipoxygenase-derived volatiles formed. The addition of linoleic acid caused an increase in hexanal, 1-hexanol, and (E)-2-heptenal in the flesh and (E)-2-heptenal in the peel. Stored unrefrigerated peel had higher volatile concentrations, whereas refrigerated peel had significantly lower concentration than day 0. Storage decreased lipoxygenase activity in the unrefrigerated and refrigerated peel, but had no effect on the fatty acid content. Overall, linolenic acid was the most important to the formation of headspace volatiles, but lipoxygenase activity and unknown factors are also important. PRACTICAL APPLICATION: The peel of a tomato is most beneficial to the production of volatiles associated with the fresh aroma of tomatoes; therefore, it should be used in the processing of tomato products to produce a fresh, green aroma rather than being removed. Knowledge of the effects of lipoxygenase activity, total fatty acid content, and fatty acid profile on flavor volatiles will allow for better selection of a variety for raw consumption.     

Partial characterization of a lipoxygenase from fusarium proliferatum

Summary

Biomass of the fungus Fusarium proliferatiim was produced and used to obtain a crude extract (FI) of lipoxygenase. The enzyme was further purified by ammonium sulfate precipitation at 40% of saturation (FII). The enzymatic extract (FII) showed its optimum activity at pH 6.0. The apparent K _m and V _max values for the lipoxygenase (FII) were calculated to be 5.15 × 10^?5 M and 1.61 μmol hydroperoxide/mg protein/min, respectively. Enzyme activity remained relatively stable at potassium cyanide concentrations as high as 60 mM. The presence of 5 mM ethylenediaminetetraacetate activated the enzyme by 50%, whereas the use of 1.2 mM hydroquinone resulted in a 2‐fold increase in lipoxygenase activity. The partially purified enzyme (FII) showed a three‐fold enhancement of activity towards linoleic acid compared to linolenic acid as well as mono‐, di‐ and trilinolein.     

Comparison of Antioxidant Activities of Isoflavones from Kudzu Root (Pueraria lobata Ohwi)

ABSTRACT: Isoflavones and their glycosides were isolated from kudzu root, and formononetin-7- O -glucoside (ononin) was identified from the plant for the first time. Among isolated compounds, the antioxidant potency of 5 major compounds was determined using (1) 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging assay, (2) thiocyanate assay in the linoleic acid model system, and (3) lipoxygenase inhibition assay. The study indicates that 5 compounds act as free-radical scavengers and inhibit both linoleic acid peroxidation and lipoxygenase activity. The 3 different assays for determining antioxidant activity revealed that activity in one test did not necessarily correlate with activity in another test. Arachidonic acid release in the intact HL-29 cancer cell, biochanin A, showed strong inhibition of about 70%, at a concentration of 50 μ.M.     

Characterization of Seed Lipoxygenase of Phaseolus vulgaris cv, Haricot

Phaseolus vulgaris cv, haricot seed lipoxygenase was extracted and partially purified. The precipitation of an active lipoxygenase fraction with solid ammonium sulfate (at 20–50% of saturation) increased its activity by a factor of 3. The pH for optimum activity was 7.3. The addition of 40 mM potassium cyanide resulted in a doubling of the enzyme activity. Enzyme activity was completely lost on storage at 4°C for 48 hr. The activity of haricot lipoxygenase extract was considerably greater on linoleic acid than on its esters, and decreased in the order: mono- > di > trilinolein. The haricot lipoxygenase shares many of the characteristics of the corresponding enzyme found in several seed sources.     

Characterisation of lipoxygenase from pea seeds (Pisum sativum var. Telephone L.)

Lipoxygenase (LOX) from pea seeds (Pisum sativum var. Telephone L.) was extracted and studied of biochemical properties. The molecular mass of purified lipoxygenase was 93 kDa. The effects of substrate specificity, pH, and sensibility to various inhibitors: caffeic acid, ferulic acid, benzoic acid, catechin, quercetin and kaempferol of LOX were investigated. Lipoxygenase showed the highest activity toward linoleic acid and the lowest toward oleic acid as substrates. Kinetic studies indicated that V_max of the LOX activity was 151.5 U/min and corresponding K_m value of 0.44 × 10⁻³ M. Optimum pH of lipoxygenase was reported at 5.5. Caffeic acid was the most effective inhibitor and kaempferol was the least effective.     

脂质氧化诱导的大豆蛋白质聚集机理的研究

利用脂肪氧合酶、亚油酸和大豆分离蛋白构建的模拟体系，研究了脂质氧化诱导的大豆蛋白质聚集的机理。结果表明，在制备大豆分离蛋白的模拟体系中同时添加亚油酸和脂肪氧合酶会使样品的蛋白质氧化值与表面疏水性增加，巯基与二硫键的含量下降，说明脂肪氧合酶催化的脂质氧化可使大豆蛋白结构发生明显的改变。SDS-PAGE电泳表明大豆蛋白7S和11S的各个亚基均参与了反应，尤其是7S部分反应更为明显。HPLC凝胶色谱和激光光散射分析进一步证实反应后的大豆蛋白（RSPI4和5）形成了相对高分子质量和颗粒粒径较大的聚集体。现有的实验证据表明，高度聚集蛋白质的形成可能是通过包括二硫键在内的共价变联，以及诸如氢键、盐桥，尤其是疏水相互作用等的非共价相互作用，形成较大的聚集体。由脂肪氧合酶诱导的蛋白质一脂质相互作用模拟实验的结果说明。脂质氧化对大豆蛋白具有明显的影响；在大豆分离蛋白加工的过程中，控制脂肪氧合酶的活力或脂质的含量是非常重要的。     

Volatiles derived from lipoxygenase-catalysed reactions in winged beans (Psophocarpus tetragonolobus)

Volatiles formed by the decomposition of hydroperoxides generated by lipoxygenase-catalysed oxidation of linoleic acid contribute a beany flavour to legumes. Eleven volatiles have been identified after treatment of linoleic acid by lipoxygenase extracted from winged bean seeds. Five of these volatiles; namely, hexanal, 2-heptanone, 2-pentyl furan, undecane and tridecane, have been detected in the headspace of a sample of winged bean flour after storage. Moisture levels below 10% appear to inhibit lipoxygenase activity, since headspace samples from raw and heat treated samples had very similar composition, and the peroxide value remained low after 10 months' storage at 37°C. Incubation of a lipoxygenase extract with linoleic acid gave rise to a larger concentration of volatiles at pH 9 than at pH 6·5 due to the increased lipoxygenase activity at the higher pH.