植物组织中低聚糖乙酰化及毛细管气相色谱分析

建立一种用乙酰化衍生处理低聚糖并用毛细管气相色谱-FID进行分析的方法。以1-甲基咪唑为催化剂并以乙酸酐为乙酰化试剂,同时对植物样品中蔗糖、棉子糖和水苏糖等低聚糖乙酰化产物进行毛细管气相色谱分离和FID检测。确定了低聚糖乙酰化衍生物的毛细管气相色谱分析条件,并对低聚糖乙酰化反应条件及色谱分离条件进行了优化。结果表明,在80–1000ng·μL–1范围内线性关系良好,蔗糖、棉子糖和水苏糖的相关系数（R）分别为0.9952、0.9957和0.9877,并且精准度与回收率均较高。使用该方法对低聚糖进行乙酰化反应重现性好、所需样品材料及试剂量少且污染毒害小,能够得到理想的分离、检测和定量分析效果,适用于少量植物组织中低聚糖的定量分析。该方法在食品、医药检测和基础科学研究领域均具有广泛的适用性及参考价值。     

植物组织中糖与糖醇乙酰化及毛细管气相色谱分析

介绍了一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中糖与糖醇进行乙酰化衍生化后的气相色谱分离和质谱鉴定的分析方法。并对糖与糖醇乙酰化影响较大的反应温度、反应时间、反应物组成和反应物浓度等条件进行了比较研究,确定了糖与糖醇乙酰化各步反应的最佳反应温度和反应时间,分析了各组分间相互作用及其用量对衍生化效率的影响。对多种糖与糖醇乙酰化产物进行毛细管气相色谱分离、FID检测及GC-MS结构鉴定。研究证明, 在合适条件下应用此方法对糖与糖醇进行乙酰化,反应完全,产物单一,能得到理想的分离、检测和定量分析效果。适用于微量植物组织中多种单糖、双糖及其糖醇的定量分析。     

植物组织中糖与糖醇乙酰化及毛细管气相色谱分析

介绍了一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中糖与糖醇进行乙酰化衍生化后的气相色谱分离和质谱鉴定的分析方法.并对糖与糖醇乙酰化影响较大的反应温度、反应时间、反应物组成和反应物浓度等条件进行了比较研究,确定了糖与糖醇乙酰化各步反应的最佳反应温度和反应时间,分析了各组分间相互作用及其用量对衍生化效率的影响.对多种糖与糖醇乙酰化产物进行毛细管气相色谱分离、FID检测及GC-MS结构鉴定.研究证明,在合适条件下应用此方法对糖与糖醇进行乙酰化,反应完全,产物单一,能得到理想的分离、检测和定量分析效果.适用于微量植物组织中多种单糖、双糖及其糖醇的定量分析.     

分析植物组织中海藻糖的气质联用及毛细管气相色谱法

介绍一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中海藻糖等糖类物质进行乙酰化衍生化后的气相色谱分离、质谱鉴定的分析方法.以核糖醇为内标,通过校准曲线对植物组织中的海藻糖进行定量分析.此法测定海藻糖的最低量可达8.17×10-11g,适于植物样品中微量海藻糖的分析测定.     

大豆低聚糖对肠道双歧杆菌和肠杆菌的促生长作用

目的:观察大豆低聚糖和所含糖对肠道菌群中双歧杆菌、肠杆菌体外促生长作用。方法:按1％大豆低聚糖、0．36％水苏糖、0．52％蔗糖、0．11％棉子糖含量配制培养液,分别接种各试验菌株,在0、12、24小时测定各培养液吸光度A值(分光光度计．550nm)和pH值。结果:经24小时培养,加大豆低聚糖的双歧杆菌标准株和临床分离株培养液A值分别为>1．5和1．307,大于肠杆菌标准株和临床分离株的1．1和1．173,而其pH值小于肠杆菌;加水苏糖的双歧杆菌培养液A值为1．47,大于蔗糖的1．4和棉子糖的0．53。结论:大豆低聚糖促双歧杆菌生长作用大于促肠杆菌生长,水苏糖是促双歧杆菌增殖生长的主要成分。     

发酵法精制大豆低聚糖的研究

研究了三种面包酵母对大豆低聚糖碳源利用的选择性。结果表明 :面包酵母C可选择性地利用蔗糖 ,而水苏糖和棉子糖的保留率大于 96 %;通过添加酵母膏 ,经过 3 6h培养 ,面包酵母C可全部利用大豆低聚糖中的蔗糖。进一步研究表明 ,以大豆乳清废糖浆为原料直接发酵再经下游处理 ,可得到蔗糖含量低于 1 3 %的精制大豆低聚糖干粉。     

植物中棉子糖系列寡糖代谢及其调控关键酶研究进展

棉子糖系列寡糖代谢与植物生长发育、逆境胁迫、种子耐贮性及脱水耐性等关系密切.棉子糖系列寡糖的合成从棉子糖的合成开始,由半乳糖苷肌醇上的半乳糖基的转移依次生成棉子糖、水苏糖、毛蕊花糖等.寡糖代谢是一个复杂的调控体系,其中肌醇-1-磷酸合成酶、肌醇半乳糖苷合成酶、蔗糖合成酶、棉子糖合成酶、水苏糖合成酶和毛蕊花糖合成酶等参与了棉子糖系列寡糖的生物合成过程.本文对植物中棉子糖系列寡糖的代谢及其重要调控酶的特性、功能及分子生物学研究进展进行综述.     

不同品种的地黄低聚糖含量的比较研究

采用HPLc-ELSD法,对6个不同品种(包括野生和栽培)地黄的低聚糖进行含量测定.测定结果表明:不同的地黄品种低聚糖含量差异比较显著;栽培品种‘85 -5’、‘9302’和‘北京1号’低聚糖含量比较接近;野生品种‘BA3’的水苏糖含量普遍较低,而蔗糖含量比较高;‘北京1号’水苏糖含量较高,总低聚糖含量比较均衡,占干重...     

应用毛细管气相色谱法检测棉铃虫细胞色素P450 O-脱甲基活性

依据毛细管气相色谱法灵敏度高 ,样品用量少 ,分辨率高的特点 ,建立了一个用于检测棉铃虫Helicoverpaarmigera幼虫中肠和脂肪体细胞色素P45 0O -脱甲基活性产物的小型反应体系 ( 875 μL) ,其中含 0 1mol L ,pH 7 8Tris HCl缓冲液 ,0 5 μmol,NADPH ,7 5 μmolMgCl2 ,0 0 85 %BSA ,0 1 5 7mmol对硝基苯甲醚及酶液。利用毛细管气相色谱直接检测该反应体系中棉铃虫细胞色素P45 0的O -脱甲基产物对硝基苯酚。     

气相色谱-质谱联用技术及其在代谢组学中的应用

代谢组学是以高通量、高灵敏度、高分辨率的现代仪器分析方法为手段,对细胞、体液、组织中所有代谢物进行无偏向的定性与定量分析的一门学科。气相色谱-质谱联用技术具有较高的检测灵敏度和鉴定准确度,通过标准谱图库的比对可对代谢物进行快速的鉴定,因此被广泛应用于生物样品的代谢产物的检测中。文中对近年来气相色谱-质谱联用技术的发展以及在代谢组学研究中取得的成果进行了综述。首先介绍了气相色谱-质谱联用技术的分类和常用的样品衍生化方法;继而从样品预处理、定性与定量分析、数据分析三方面介绍了气相色谱-质谱联用技术分析代谢物的方法,并系统地对该技术在微生物、植物、疾病诊断领域的应用实例进行了评述;最后提出了当前气相色谱-质谱联用技术在代谢组学研究中存在的问题并对后续的研究进行了展望。     

Chain Elongation of raffinose in pea seeds. Isolation, characterization, and molecular cloning of mutifunctional enzyme catalyzing the synthesis of stachyose and verbascose.

Raffinose oligosaccharides are major soluble carbohydrates in seeds and other tissues of plants. Their biosynthesis proceeds by stepwise addition of galactose units to sucrose, which are provided by the unusual donor galactinol (O-alpha-d-galactopyranosyl-(1-->1)-l-myo-inositol). Chain elongation may also proceed by transfer of galactose units between raffinose oligosaccharides. We here report on the purification, characterization, and heterologous expression of a multifunctional stachyose synthase (EC ) from developing pea (Pisum sativum L.) seeds. The protein, a member of family 36 of glycoside hydrolases, catalyzes the synthesis of stachyose, the tetrasaccharide of the raffinose series, by galactosyl transfer from galactinol to raffinose. It also mediates the synthesis of the pentasaccharide verbascose by galactosyl transfer from galactinol to stachyose as well as by self-transfer of the terminal galactose residue from one stachyose molecule to another. These activities show optima at pH 7.0. The enzyme also catalyzes hydrolysis of the terminal galactose residue of its substrates, but is unable to initiate the synthesis of raffinose oligosaccharides by galactosyl transfer from galactinol to sucrose. A minimum reaction mechanism which accounts for the broad substrate specificity and the steady-state kinetic properties of the protein is presented.     

Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport

Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds.     

Enzymatic control of the accumulation of verbascose in pea seeds

Verbascose, the pentasaccharide of the raffinose family of oligosaccharides, consists of galactose units joined to sucrose. In pea (Pisum sativum) seeds, the content of verbascose is highly variable. In a previous study on a high‐verbascose pea cultivar, the present authors have demonstrated that verbascose is synthesized by a multifunctional stachyose synthase (EC 2.4.1.67), which utilizes raffinose as well as stachyose as a galactosyl acceptor. Herein the results of a study of the cloning and functional expression of stachyose synthase from the low‐verbascose genotype SD1 are reported and it is demonstrated that this line contains a protein with a reduced ability to synthesize verbascose. Analysis of seeds from seven pea lines revealed a positive correlation between verbascose synthase activity and verbascose content. Among these genotypes, only the SD1 line showed low verbascose synthase activity when the data were normalized to stachyose synthase activity. These results suggest that differences in the level of verbascose synthase activity could be caused by mutations in the stachyose synthase gene as well as by variation in the amount of the protein. The lines were also analysed for activity of α‐galactosidase, a catabolic enzyme that could limit the extent of verbascose accumulation. No relationship was found between α‐galactosidase activity and the amount of raffinose family oligosaccharides.     

Preparation of partially benzylated mono-, di-, and trisaccharides by selective cleavage of the beta-fructofuranosidic linkage in fully benzylated sucrose and sucrose-related oligosaccharides under acidic conditions

Several partially benzylated mono-, di-, and trisaccharides having an anomeric hydroxyl group were successfully prepared by selective cleavage of the β-fructofuranosidic linkage in fully benzylated sucrose and sucrose-related oligosaccharides derived from lactosucrose, raffinose, melezitose, stachyose, and nystose under acidic conditions using 1:10 75% aqueous sulfuric acid–dioxane at room temperature for 1 h.     

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides

Derivatisation/FABMS strategies applicable to the structure analysis of low microgramme quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitropus acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.     

Cold-induced accumulation of raffinose family oligosaccharides in somatic embryos of Norway spruce (<Emphasis Type="Italic">Picea abies</Emphasis>)

Summary Exposure of mature cotyledonary somatic embryos of Picea abies to low temperature (4°C) resulted in the accumulation of raffinose family oligosaccharides (RFOs)—raffinose and stachyose. The RFO content represented approximately 20% of the total soluble saccharides with the RFO: sucrose ratio being almost 1∶3 (molar basis) after 3 wk of cold exposure. This treatment, like desiccation, brings the endogenous saccharide spectrum nearer to that of mature zygotic embryos of the same species (zygotic embryos, RFO: sucrose ratio 1∶1.5 on a molar basis). Based on indications that RFOs are at least partly responsible for the positive effects of desiccation, we propose cold treatment as an alternative to slow desiccation for conifer somatic embryogenesis protocols.     

The metabolism of galactose and the raffinose oligosaccharides by germinating bambarra groundnut seeds

Stachyose is present in the highest amount in the soluble sugar fraction of dry bambarra groundnut cotyledons, followed in descending order by raffinose, sucrose and verbascose. During germination in the dark, the stachyose and raffinose content decrease rapidly, but there is little change in the relatively small amount of verbascose present. The sucrose content increases rapidly during the first two weeks and decreases thereafter. Free glucose and fructose were present in the cotyledons after the 7th day and gradually increased in amount with time of germination. Free galactose and other galactose-containing oligosaccharides were not detected in either the dry or germinated bambarra seeds. During germination, galactose was the only identifiable sugar, aside from traces of sucrose, glucose and fructose, in the extracted soluble sugar fraction in the embryonic axes of all ages when the tissue was incubated with D-[1¹⁴C] galactose. With the cotyledons, however, most of the radioactivity was in glucose and fructose during the early period of germination and in sucrose later. A small fraction of the radioactivity was lost as CO₂.