基于部分酸水解-亲水作用色谱-质谱的黄芪多糖结构表征

来自中药的水溶性多糖具有广谱治疗和低毒性特点,是天然药物及保健品研发中的重要组成部分。针对中药多糖结构复杂、难以表征的问题,本文以中药黄芪中的多糖为研究对象,采用"自下而上"法完成对黄芪多糖的表征。首先使用部分酸水解方法水解黄芪多糖,分别考察了水解时间、酸浓度和温度的影响。在适宜条件(4 h、1.5 mol/L三氟乙酸、80 ℃)下,黄芪多糖被水解为特征性的寡糖片段。接下来,采用亲水作用色谱与质谱联用对黄芪多糖部分酸水解产物进行分离和结构表征。结果表明,提取得到的黄芪多糖主要为1→4连接线性葡聚糖,水解得到聚合度4~11的葡寡糖。本研究对其他中药多糖的表征具有一定的示范作用。     

基于部分酸水解-亲水作用色谱的黄芪多糖指纹图谱分析及结合反相指纹图谱全面质量评价方法的建立（英文）

多糖是黄芪的重要成分,但多糖相对分子质量大、极性强,难以用色谱方法直接分析,导致目前缺乏能够反映黄芪多糖组成差异的质量评价方法。首先通过部分酸水解方法,将多糖水解成可供分析的寡糖,建立基于部分酸水解-亲水作用色谱的黄芪多糖指纹图谱。通过正交实验选取最佳水解条件:温度80℃、酸浓度1.5 mol/L,水解时间4 h。该方法重复性好,对20批黄芪药材的多糖指纹图谱分析显示相似度为0.258~0.949,反映出黄芪多糖组成的明显差异。同时建立了黄芪的反相液相色谱指纹图谱,用于控制除多糖以外的其他成分,对同样的20批黄芪药材进行分析,实现对黄芪全面的质量评价。实验表明,基于部分酸水解-亲水作用色谱的黄芪多糖指纹图谱可对黄芪多糖的质量进行有效评价,是对黄芪质量评价方法的重要补充。     

人参多糖部分酸水解物的HPLC-ESI-QTOF-MS分析

本文采用酸水解方式,获得人参多糖的部分水解产物,通过高效液相色谱-电喷雾电离高分辨飞行时间质谱(HPLC-ESI-QTOF-MS)对人参多糖部分酸水解产物进行分析,建立人参多糖糖谱研究的方法。同时结合几种标准二糖的MS和MS/MS分析,建立依靠质谱分析确定多糖糖苷键类型的方法。研究结果确定了5种糖苷键链接类型在MS/MS中的断裂规律,并发现人参多糖的糖苷键链接类型为1,3糖苷键和1,4糖苷键。本文研究方法,具有简单快速、稳定性好、准确度高等优点,为中草药的指纹性糖谱研究提供了新的借鉴方法。     

基于高效阴离子色谱-脉冲安培法优化阿拉伯木聚精单精组成分析

以大粒车前子来源阿拉伯木聚糖为研究对象,采用完全酸水解结合高效阴离子色谱-脉冲安培法测定单糖组成,系统优化酸的种类和浓度、水解温度和时间、水解后样品放置时间等水解条件.结果表明,将车前子多糖置于2 mol/L H2 SO4、120℃常压油浴条件下水解2 h时,效果较好,但水解后稀释液放置时间不宜超过6 h.大粒车前子多糖主要由阿拉伯糖(8.89%)和木糖(41.52%)组成,同时检测出半乳糖醛酸(0.73%)、葡萄糖醛酸(3.44%)和微量的半乳糖、葡萄糖,结果重现性良好.利用上述最优条件水解黍子壳、燕麦麸皮和青稞来源阿拉伯木聚糖并分析单糖组成,均获得较好结果.本研究为分析各种来源阿拉伯木聚糖的单糖组成提供了参考.     

液相色谱法分析植物多糖中单糖组成及含量

植物多糖中单糖组成检测中存在加标回收率低、数据平行性差等问题。本文旨在探讨液相色谱分析多糖中单糖组成与含量方法的关键点。通过分析多糖水解效率及水解损失率,确定最佳水解时间,对比酸碱中和法和真空干燥法的损失率,确定水解后最佳除酸方式,通过分析单糖1-苯基-3-甲基-5-吡唑啉酮衍生物的稳定性,确定最佳分析时间。以海带为样品,选择三氟乙酸进行水解,最佳水解时间为2 h,最佳除酸方式为碱中和法,最佳分析时间为衍生后12 h内。7种单糖连续进样6针的相对标准偏差(RSD)均小于2%,标准曲线相关系数r²均大于0.999。优化条件下利用1-苯基-3-甲基-5-吡唑啉酮衍生-液相色谱检测单糖组成及含量,方法的回收率较高,数据平行性好。     

当归多糖的水解特征及其水解产物分析

对当归多糖ASP3的水解特征及其水解产物的组成和红外光谱特征进行了研究。分别采用0.05、0.2和0.5mol/L三氟乙酸(trifluoroacetic acid,TFA)和内切-α-(1→4)-聚半乳糖醛酸酶对ASP3进行部分酸水解和酶水解,并研究其水解特征;结合GC、FT-IR等方法对其水解产物的组成和红外光谱特征进行分析。实验结果表明,ASP3是一种果胶多糖,主要由光滑区(半乳糖醛酸聚糖)和毛发区(富含中性糖侧链的鼠李半乳糖醛酸聚糖)两部分组成:GalA和Rha位于多糖分子的主链,由于Rha含量较低,大部分GalA相连形成半乳糖醛酸聚糖光滑区;Gal、Ara、Man及Glc位于多糖分子的支链,其中Gal以较高的聚合度(半乳聚糖)与主链相连,Ara以还原性末端或低聚寡糖的形式与主链相连或与半乳聚糖末端相连形成阿拉伯半乳聚糖。     

气相色谱-质谱联用同时分析新型细菌多糖中的单糖和糖醛酸

对纯化的新型细菌多糖进行酸水解,用乙硫醇-三氟乙酸和醋酐-吡啶体系先后对酸水解物进行衍生,与之前报道不同的是糖醛酸得到有效衍生化。以木糖为内标,采用气相色谱-质谱联用(GC-MS)定量分析该多糖酸水解物中单糖和糖醛酸衍生物发现,该多糖的糖链由岩藻糖、葡萄糖、葡萄糖醛酸和半乳糖组成,其相对物质的量比为1.50:1.0:0.79:2.06;中性糖比例与糖醇乙酸酯化分析岩藻糖、葡萄糖和半乳糖的相对物质的量比(1.76:1.0:1.98)接近;糖醛酸咔唑法与该方法分析葡萄糖醛酸的含量分别为16.19%和14.85%。以上结果表明所建立的衍生化方法及GC-MS同时定量分析多糖酸水解物中单糖和糖醛酸的方法可行。此外还对葡萄糖醛酸的质谱裂解机理进行了阐述。     

斯氏花群海葵多糖Ⅰ和Ⅱ的组成研究

从生长于我国南海西沙群岛的斯氏花群海葵中分离得到两种具有心血管活性的物质—多糖(Ⅰ)和多糖(Ⅱ),运用化学分析,光谱分析及酸水解—双向纸层析等对它们的组成进了研究。结果表明多糖(Ⅱ)由核糖、半乳糖和乙酰氨基半乳糖以分子比约为3.2:2.0:1.0组成,多糖(Ⅰ)则由核糖和半乳糖以分子比约为2.2:1.0组成。     

HPLC-ESI-MS联用法测定灵芝胶囊多糖的单糖组成

建立了测定灵芝胶囊中多糖含量与单糖组分的高效液相色谱-电子喷雾离子源-质谱联用(HPLC-ESI-MS)检测方法,研究了灵芝多糖酸水解为单糖及1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生化的条件,三氟乙酸(TFA)的浓度在0.5 mol/L、水解温度70℃、水解时间30 min时能将灵芝多糖完全水解为单糖,在70℃PMP衍生化反应30 min,4℃冷却,得到双分子PMP单糖衍生物。5种水解后的单糖线性范围为0.1~50 mg/L,检出限为25μg/L(S/N≥3),在3个加标水平(0.1,10,50 mg/g)下,5种单糖平均回收率为95%~105%,精密度(RSD)为0.5%~3.5%。方法能满足对灵芝胶囊质量控制的检测要求。     

蒲黄多糖的研究

蒲黄经热水提取,脱除蛋白质、乙醇沉淀,Sephadex G50柱层析分离,DEAE-Sephadex A25和DEAE-Cellulose离子交换柱层析分离得3种白色粉末状多糖TAA、TAB、TAC。经凝胶柱层析、醋酸纤维素薄膜电泳证明,三者为均一体。3种多糖经酸全水解、纸层析和气相色谱分析,确定了糖基的组成及摩尔组成比。用核磁共振、甲基化分析、NaIO_4氧化等方法对多糖结构进行了初步探讨。     

毛细管型离子色谱-脉冲安培法检测枸杞多糖的单糖组成

采用ICS-5000毛细管离子色谱仪对枸杞多糖中的10种单糖进行了分离测定.优化了前处理过程中影响多糖水解的酸种类、酸浓度、水解温度和时间等参数,优化条件为使用2 mol/L三氟乙酸溶液在100℃下水解120min,在该条件下,果糖回收率约为50％,其余单糖回收率在84％～104％之间.采用新型淋洗液自动发生装置电解产生淋洗液,Capillary CarborPac PA20色谱柱分离,毛细管安培池检测,10种单糖成分标准曲线线性关系良好,相关系数均大于99.9％;检出限在2.5～75 μg/L之间,为枸杞多糖中单糖组分测定提供了新的可行方法.     

Preparation of cellulose nano-crystals through a sequential process of cellulase pretreatment and acid hydrolysis

In this paper, a cellulase pretreatment was studied prior to the acid hydrolysis to decrease the total acid usage during the cellulose nano-crystals (CNC) preparation from a bleached softwood kraft pulp. Cellulase pretreatment facilitates the subsequent acid hydrolysis to produce CNC with similar quality to that of the control, but at a lower sulfuric acid concentration. The underline mechanism is that cellulase pretreatment led to the formation of more carbonyl groups which can be oxidized into carboxyl groups in the subsequent acid hydrolysis, furthermore, more hydroxyl groups are exposed, thus esterification into sulfonic groups can be enhanced. The results showed that with a cellulase dosage of 4.8 u/g (based on dry pulp) in the pretreatment stage, the sulfuric acid concentration can be decreased from 64 to 40 wt% without compromising the quality of resulting CNC particles. Other results from charge properties, Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM) analyses also supported the conclusions.     

Summary of findings from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI): corn stover pretreatment

The Biomass Refining Consortium for Applied Fundamentals and Innovation, with members from Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, has developed comparative data on the conversion of corn stover to sugars by several leading pretreatment technologies. These technologies include ammonia fiber expansion pretreatment, ammonia recycle percolation pretreatment, dilute sulfuric acid pretreatment, flowthrough pretreatment (hot water or dilute acid), lime pretreatment, controlled pH hot water pretreatment, and sulfur dioxide steam explosion pretreatment. Over the course of two separate USDA- and DOE-funded projects, these pretreatment technologies were applied to two different corn stover batches, followed by enzymatic hydrolysis of the remaining solids from each pretreatment technology using identical enzyme preparations, enzyme loadings, and enzymatic hydrolysis assays. Identical analytical methods and a consistent material balance methodology were employed to develop comparative sugar yield data for each pretreatment and subsequent enzymatic hydrolysis. Although there were differences in the profiles of sugar release, with the more acidic pretreatments releasing more xylose directly in the pretreatment step than the alkaline pretreatments, the overall glucose and xylose yields (monomers + oligomers) from combined pretreatment and enzymatic hydrolysis process steps were very similar for all of these leading pretreatment technologies. Some of the water-only and alkaline pretreatment technologies resulted in significant amounts of residual xylose oligomers still remaining after enzymatic hydrolysis that may require specialized enzyme preparations to fully convert xylose oligomers to monomers.     

Effects of SPORL and Dilute Acid Pretreatment on Substrate Morphology, Cell Physical and Chemical Wall Structures, and Subsequent Enzymatic Hydrolysis of Lodgepole Pine

The effects of pretreatment by dilute acid and sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on substrate morphology, cell wall physical and chemical structures, along with the subsequent enzymatic hydrolysis of lodgepole pine substrate were investigated. FE-SEM and TEM images of substrate structural morphological changes showed that SPORL pretreatment resulted in fiber separation, where SPORL high pH (4.2) pretreatment exhibited better fiber separation than SPORL low pH (1.9) pretreatment. Dilute acid pretreatment produced very poor fiber separation, consisting mostly of fiber bundles. The removal of almost all hemicelluloses in the dilute acid pretreated substrate did not overcome recalcitrance to achieve a high cellulose conversion when lignin removal was limited. SPORL high pH pretreatment removed more lignin but less hemicellulose, while SPORL low pH pretreatment removed about the same amount of lignin and hemicelluloses in lodgepole pine substrates when compared with dilute acid pretreatment. Substrates pretreated with either SPORL process had a much higher cellulose conversion than those produced with dilute acid pretreatment. Lignin removal in addition to removal of hemicellulose in SPORL pretreatment plays an important role in improving the cellulose hydrolysis of the substrate.     

Predicted effects of mineral neutralization and bisulfate formation on hydrogen ion concentration for dilute sulfuric acid pretreatment

Dilute acid and water only hemicellulose hydrolysis are being examined as part of a multiin stitutional cooperative effort to evaluate the performance of leading cellulosic biomass pretreatment technologies on a common basis. Cellulosic biomass, such as agricultural residues and forest wastes, canhave a significant mineral content. It has been shown that these minerals neutralize some of the acid during dilute acid pretreatment, reducing its effectiveness, and the higher solids loadings desired to minimize costs will require increased acid use to compensate. However, for sulfuric acid in particular, an equilibrium shift to formation of bisulfate during neutralization can further reduce hydrogen ion concentrations and compound the effect of neutralization. Because the equilibrium shift has a more pronounced effect at lower acid concentrations, additional acid is needed to compensate. Coupled with the effect of temperature on acid dissociation, these effects increase acid requirements to achieve a particular reaction rate unless minerals are removed prior to hydrolysis.     

Optimizing Dilute-Acid Pretreatment of Rapeseed Straw for Extraction of Hemicellulose

Biological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars prior to fermentation. Hydrolysis can be performed enzymatically or with mineral acids. In this study, dilute sulfuric acid was used as a catalyst for the pretreatment of rapeseed straw. The purpose of this study is to optimize the pretreatment process in a 15-mL bomb tube reactor and investigate the effects of the acid concentration, temperature, and reaction time. These parameters influence hemicellulose removal and production of sugars (xylose, glucose, and arabinose) in the hydrolyzate as well as the formation of by-products (furfural, 5-hydroxymethylfurfural, and acetic acid). Statistical analysis was based on a model composition corresponding to a 3³ orthogonal factorial design and employed the response surface methodology to optimize the pretreatment conditions, aiming to attain maximum xylan, mannan, and galactan (XMG) extraction from hemicellulose of rapeseed straw. The obtained optimum conditions were: H₂SO₄ concentration of 1.76% and temperature of 152.6 °C with a reaction time of 21 min. Under these optimal conditions, 85.5% of the total sugar was recovered after acid hydrolysis (78.9% XMG and 6.6% glucan). The hydrolyzate contained 1.60 g/L glucose, 0.61 g/L arabinose, 10.49 g/L xylose, mannose, and galactose, 0.39 g/L cellobiose, 0.94 g/L fructose, 0.02 g/L 1,6-anhydro-glucose, 1.17 g/L formic acid, 2.94 g/L acetic acid, 0.04 g/L levulinic acid, 0.04 g/L 5-hydroxymethylfurfural, and 0.98 g/L furfural.     

Strategies for the Removal of Polysaccharides from Biorefinery Lignins: Process Optimization and Techno Economic Evaluation

The utilization of biorefinery lignins as a renewable resource for the production of bio-based chemicals and materials remain a challenge because of the high polysaccharide content of this variety of lignins. This study provides two simple methods; (i) the alkaline hydrolysis-acid precipitation method and (ii) the acid hydrolysis method for the removal of polysaccharides from polymeric biorefinery lignin samples. Both purification strategies are optimized for two different hardwood hydrolysis lignins, HL1 and HL2, containing 15.1% and 10.1% of polysaccharides, respectively. The treated lignins are characterized by polysaccharide content, molecular weight, hydroxyl content, and Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR). Preliminary techno-economic calculations are also carried out for both purification processes to assess the economic potential of these technologies. The results indicate that both protocols could be used for the purification of HL1 and HL2 hydrolysis lignins because of the minimal polysaccharide content obtained in the treated lignins. Nevertheless, from an industrial and economic perspective the acid hydrolysis technology using low acid concentrations and high temperatures is favored over the alkaline hydrolysis-acid precipitation strategy.     

Gas Chromatographic Determination of Poly(3‐hydroxybutyrate) with Alkaline Hydrolysis and Acid Esterification

Abstract

A new pretreatment method for the gas chromatographic determination of poly(3‐hydroxybutyrate) (PHB) was developed based on a combination of alkaline hydrolysis and acid esterification. The determination principle is as follows: PHB is hydrolyzed to its monomer 3‐hydroxybutyrate by alkaline solution, followed by the esterification with methanol to generate the methyl ester of 3‐hydroxybutyrate catalyzed by acid, which is detected by a gas chromatography. From the comparison of effects of alkali and acid on PHB hydrolysis and 3‐hydroxybutyrate esterification, alkali resulted in a better performance for the hydrolysis, while acid was better for the esterification. The pretreatment conditions for PHB were optimized and the determination performance was characterized.     

Optimized hydrolysis and analysis of Radix Asparagi polysaccharide monosaccharide composition by capillary zone electrophoresis

Using orthogonal design, optimized conditions for the hydrolysis of the polysaccharide from Radix Asparagi were determined, as well as its monosaccharide composition. Optimized hydrolysis conditions were a temperature of 100°C in 1.5 M sulfuric acid solution for 5 h. The resulting monosaccharides were derivatized with 1‐phenyl‐3‐methyl‐5‐pyrazolone, then separated by capillary zone electrophoresis in 40 mM sodium tetraborate buffer (pH 10.1), and detected by ultraviolet absorption at 245 nm. Results indicate that the polysaccharide from Radix Asparagi is composed of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid, and galacturonic acid, which differs from published findings. Moreover, xylose, glucuronic acid, and galacturonic acid have not been previously reported in Radix Asparagi polysaccharide. This method is simple, fast, and yields a highly efficient separation. As well, these findings can be applied to quality control of Radix Asparagi and for in‐depth study of the biological activity of Radix Asparagi polysaccharide.     

A technical and economic analysis of acid-catalyzed steam explosion and dilute sulfuric acid pretreatments using wheat straw or aspen wood chips

Lignocellulosic biomass is one of the most plentiful and potentially cheapest feedstocks for ethanol production. The cellulose component can be broken down into glucose by enzymes and then converted to ethanol by yeast. However, hydrolysis of cellulose to glucose is difficult, and some form of pretreatment is necessary to increase the susceptibility of cellulose to enzymatic attack. An analysis has been completed of two pretreatment options, dilute sulfuric acid hydrolysis and sulfur dioxide impregnated steam explosion, for two feedstocks, wheat straw and aspen wood chips. Detailed process flow sheets and material and energy balances were used to generate equipment cost information. A technical and economic analysis compared the two feedstocks for each of the two pretreatments. For the same pretreatment, sugars produced from aspen wood hydrolysis were cheaper because of the higher carbohydrate content of aspen, whereas dilute acid pretreatment is favored over acid-catalyzed steam explosion.