高效阴离子交换色谱-脉冲安培法测定W135群和Y群脑膜炎球菌多糖中半乳糖和葡萄糖含量

目的采用高效阴离子交换色谱-脉冲安培法(High-performance anion-exchange chromatography with pulsedamperometric detection,HPAEC-PAD)测定W135群和Y群脑膜炎球菌荚膜多糖中半乳糖和葡萄糖的含量,并对该方法进行验证及初步应用。方法多糖蛋白结合物原液的混合液经HCl水解处理后,进样CarboPac PA10柱,以氢氧化钠-醋酸钠梯度流洗,脉冲积分安培检测器检测。筛选样品的水解条件;对HPAEC-PAD法进行专属性、精密性及准确性验证,并进行初步应用。结果确定的样品水解条件为以2 mol/L HCl于85℃处理1 h。载体蛋白破伤风类毒素对检测结果无干扰;重复性、日内精密性和不同操作者之间精密性试验结果的RSD均小于5%;准确性试验中葡萄糖和半乳糖的回收率在90%～110%之间。HPAEC-PAD法测定多糖蛋白结合物原液的多糖含量与传统间苯二酚比色法测定结果相当;成品疫苗中的W135群和Y群多糖含量测定结果符合《A、C、W135、Y群脑膜炎球菌结合疫苗制造及检定规程草案》的规定(均≥4μg/剂)。结论 HPAEC-PAD法专属性、精密性和准确性良好,可用于A、C、W135、Y群脑膜炎球菌结合疫苗中W135群和Y群多糖含量的测定。     

Effects of Solvent, Temperature, Time, Solvent-to-Sample Ratio, Sample Size, and Defatting on the Extraction of Soluble Sugars in Soybean

ABSTRACT Extraction solvent, temperature, time, and solvent‐to‐sample ratio were studied to identify the optimal conditions for extracting sugars from nondefatted soybean. Water and 10%, 50%, and 80% (v/v) ethanol were used as the solvents. Extraction temperatures of 25 °C, 50 °C, and 80 °C, for 15, 30, and 60 min and solvent‐to‐sample ratios of 5:1, 10:1, and 15:1 (v/w) were combined with different solvents. The sugar composition in the soybean extracts was analyzed by high‐performance anion‐exchange chromatography with pulsed amperometric detection. The sugar appropriate to be used as the standard in the phenol‐sulfuric acid method in sugar quantification, and effects of sample size and defatting on sugar extraction were also evaluated. The optimum extraction protocol concluded from this study includes the use of water as the solvent at a solvent‐to‐sample ratio of 5:1 at 25 °C or 50 °C for 15 min. Sucrose was shown to be the best sugar as the standard to quantify the total soluble sugars in soybean. Extractions from sample size of 1 g and 0.1 g yielded similar soluble sugars. A greater amount of sugars was extracted with the defatted soybean meal than with the nondefatted sample.     

高效阴离子色谱法测定当归果胶多糖中的糖醛酸含量

建立高效阴离子色谱-脉冲安培检测(HPAEC-PAD)当归果胶多糖中糖醛酸的定性、定量方法,并对测试样品的预处理方法进行研究。分别对多糖样品进行不同预处理,采用外标法测定糖醛酸含量。结果表明,多糖样品需经脱脂处理并结合弱酸水解和酶解才能获得准确、真实的结果;建立了半乳糖醛酸(GalA)和葡萄糖醛酸(GlcA)的线性回归方程,相关系数均为0.9990。经计算,HPAEC-PAD法测定当归多糖的GalA含量为53.62%,与比色法(58.27%)的相对误差为8.67%,并具有良好的重现性。采用HPAEC-PAD法对糖醛酸定性、定量,无需衍生化,且灵敏度高、重现性好,是快速测定糖醛酸含量的有效方法。     

b型流感嗜血杆菌结合疫苗游离多糖含量检测高效阴离子交换色谱-脉冲安培法的建立及验证

目的 建立b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)结合疫苗游离多糖含量检测的超速离心联合高效阴离子交换色谱-脉冲安培(high performance anion exchange chromatography with pulsed amperometric detect...     

可德兰寡糖的制备及其组分分析

目的：可德兰多糖是目前发现的唯一不含分支的线性β-1,3- 葡聚糖,由于难溶于水而限制其生物活性的研究,为此对可德兰寡糖的制备及其组分进行研究。方法：采用化学法酸解可德兰多糖。结果：通过红外光谱与基质辅助激光解析/ 电离飞行时间质谱(MALDI-TOF MS)的检测,结果表明：可德兰多糖降解后结构未发生变 化,降解产物的聚合度为2～19,其中聚合度为5 的成分含量最高;高效阴离子交换色谱脉冲安培检测法分析结果表明：反应温度105℃、反应时间150～180min 是降解可德兰寡糖的合理反应条件,降解效果最佳。结论：本研究建立的方法可简单、高效的降解可德兰多糖,制备其寡糖。     

Structural Characterization of Oat Bran (1→3), (1→4)-β-D-Glucans by Lichenase Hydrolysis Through High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection

Cereal mixed linkages (1 → 3) (1 → 4)-β-D-glucan is a linear polysaccharide composed of glucose units. Oat β-glucan is a natural polymer. The main products of β-glucanase are oligosaccharides with DP3 and DP4, i.e., 3-Ob-cellobiosyl-D-glucose and 3-Ob-cellotriosyl-D-glucose, which represent over 90% of the molecule. Keeping in mind all the benefits of oat bran, the present study was planned to investigate the structural properties of oat bran, high-performance anion exchange chromatography with pulsed amperometric detection was used to examine these oligosaccharides. The structural analysis of oat bran of two oat varieties revealed that the ratio of soluble and insoluble triose to tetraose in β-glucan fraction was 1.44 and 1.78, respectively, for Avon variety; while the ratio of soluble and insoluble triose to tetraose in β-glucan fraction for Sargodha-81 was 1.49 and 1.77. The major units determined were cellotriose and cellotetraose. Other units cellopentaose and hexaoses were also existed but in minor fractions. Lichenase hydrolysis high-performance anion exchange chromatography with pulsed amperometric detection appeared to be the best choice for structural analysis of purified samples of mixed-linkage β-glucan.     

Mapping of Saccharomyces cerevisiae metabolites in fermenting wheat straight-dough reveals succinic acid as pH-determining factor

Fermenting yeast does not merely cause dough leavening, but also contributes to the bread aroma and might alter dough rheology. Here, the yeast carbon metabolism was mapped during bread straight-dough fermentation. The concentration of most metabolites changed quasi linearly as a function of fermentation time. Ethanol and carbon dioxide concentrations reached up to 60 mmol/100 g flour. Interestingly, high levels of glycerol (up to 10 mmol/100 g flour) and succinic acid (up to 1.6 mmol/100 g flour) were produced during dough fermentation. Further tests showed that, contrary to current belief, the pH decrease in fermenting dough is primarily caused by the production of succinic acid by the yeast instead of carbon dioxide dissolution or bacterial organic acids. Together, our results provide a comprehensive overview of metabolite production during dough fermentation and yield insight into the importance of some of these metabolites for dough properties.     

HPLC sugar profiles of Algerian honeys

Sugar profiles of fifty honey samples from different regions of Algeria are analysed by HPLC with pulsed amperometric detection. These samples consisted of 25 multifloral and 25 unifloral honeys. Eleven sugars (two monosaccharides, nine oligosaccharides) are quantified. The mean values of fructose and glucose are in the range 35.99–42.57% and 24.63–35.06%, respectively. These monosaccharides are the main sugars of all honey samples. The sucrose, maltose, isomaltose, turanose and erlose are present nearly in all the samples, while raffinose and melezitose are detected in few samples. Furthermore, trehalose is present only in two samples and none of the samples contain melibiose. Low amounts of melezitose, raffinose and erlose are present in the range of 0.03–2.14%, 0.03–0.35% and 0.01–2.35%, respectively. PCA (Principal Component Analysis) showed that the cumulative variance was approximately 40% and Apiaceae honeys are correctly classified using FDA (Factorial Discriminant Analysis).     

Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose

Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids.     

Hydrolysis of β-glucan

The acid and enzymatic hydrolyses of oat β-glucan were compared for commercial oat β-glucan and β-glucan isolated from oat grain and oat bran. The resulting mono- and oligosaccharides were analysed by high-performance anion-exchange chromatography, combined with pulsed amperometric detection. The acid hydrolysis was studied with HCl, TFA and H₂SO₄ at two concentrations, with three durations of hydrolysis and at three temperatures.