Chemical characterisation of oligosaccharides in commercially pasteurised dromedary camel (Camelus dromedarius) milk

It has been suggested that bactrian camel milk and colostrum may be a good source of biologically significant oligosaccharides but, although the oligosaccharides found in bactrian camel milk and colostrum have been characterised, those in dromedary camel milk have not. In this study, seven oligosaccharides from commercially available pasteurised dromedary camel milk were characterised using ¹H nuclear magnetic resonance spectroscopy. The following oligosaccharides were detected: Gal(β1-3)Gal(β1-4)Glc (3′-galactosyllactose), Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-neohexaose), Neu5Ac(α2-3)Gal(β1-4)Glc (3′-sialyllactose), Neu5Ac(α2-6)Gal(β1-4)Glc (6′-sialyllactose), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl-3′-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyllacto-N-novopentaose a) and Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (monosialyllacto-N-neohexaose).     

Chemical characterization of the oligosaccharides in Bactrian camel (Camelus bactrianus) milk and colostrum

Bactrian camel milk and colostrum are commonly used as foods in Mongolia, whose people believe that these products promote human health. It has been hypothesized that milk oligosaccharides are biologically significant components of human milk, acting as receptor analogs that inhibit the attachment of pathogenic microorganisms to the colonic mucosa, and as prebiotics, which stimulate the growth of bifidobacteria within the infant colon. To evaluate their biological significance, we studied the oligosaccharides present in samples of Bactrian camel milk and colostrum. Using ¹H-nuclear magnetic resonance spectroscopy, we identified and characterized the following oligosaccharides of camel colostrum: Gal(β1–4)[Fuc(α1–3)]Glc (3-fucosyllactose), Gal(β1–3)Gal(β1–4)Glc (3′-galactosyllactose), Gal(β1–6)Gal(β1–4)Glc (6′-galactosyllactose), Neu5Ac(α2–3)Gal(β1–4)Glc (3′-sialyllactose), Neu5Ac(α2–6)Gal(β1–4)Glc (6′-sialyllactose), Neu5Ac(α2–3)Gal(β1–3)Gal(β1–4)Glc (sialyl-3′-galactosyllactose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (sialyllacto-N-tetraose c), Neu5Ac(α2–3)Gal(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (sialyllacto-N-novopentaose a), Gal(β1–3)[Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (sialyllacto-N-novopentaose b); and Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyllacto-N-neohexaose). The oligosaccharides in the mature camel milk were characterized as 3′-galactosyllactose, Gal(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (lacto-N-novopentaose I), and 3′-sialyllactose.     

Concentrations of sialyloligosaccharides in bovine colostrum and milk during the prepartum and early lactation

Sialyloligosaccharides and sialylglycoconjugates in colostrum and milk are regarded to be important biological components with respect to be source of brain gangliosides in infant and to be antiinfectional components for the attack by the pathogenic bacteria and virus. Several acidic oligosaccharides have been characterised in both bovine and human milk or colostrum. The sialyloligosaccharide content of human colostrum and milk has been extensively studied, whereas that of cows milk and colostrum has received less attention. In this study, the concentrations of three sialyloligosaccharides of bovine colostrum and milk were determined at various stages during the prepartum and the first 7 d postpartum. The concentration of 3'SL (Neu5Ac(alpha2-3)Gal(beta1-4)Glc) reached a maximum value of 0.85 mg/ml immediately following parturition while the concentrations of 6'SL (Neu5Ac(alpha2-6)Gal(beta1-4)Glc) and 6'SLN (Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc) of 0.14 and 0.12 mg/ml, respectively, were much lower at this initial stage, although these concentration were maximum immediately following parturition. Bovine colostrum, especially that collected immediately after parturition, may be suitable as a source of 3'SL and other sialyloligosaccharides for use as additives by the food or pharmaceutical industries.     

Qualitative and quantitative mass spectrometry comparison of characteristic galactosyl lactose isomers from goat milk at different lactation stages

Galactooligosaccharides are composed mainly of galactosyl lactose, which is important for infant growth and as a functional food additive. Although galactosyl lactose is abundant in goat milk, its complex structure has hindered the separation and analysis of its isomers. In this study, 5 isomers of goat milk galactosyl lactose were separated by HPLC: β6′-galactosyl lactose (β6′-GL), α6′-galactosyl lactose (α6′-GL), β4′-galactosyl lactose (β4′-GL), α3′-galactosyl lactose (α3′-GL), and β3′-galactosyl lactose (β3′-GL). This composition differs from that of commercial galactooligosaccharide products, which comprise mainly β-configuration oligosaccharides. The isomers were then qualitatively and quantitatively compared at different lactation stages using online HPLC-mass spectrometry. Relative quantitative analysis showed that the total content of the 5 galactosyl lactose isomers was highest in transitional goat milk. Specifically, β3′-GL was the main isomer in colostrum and α3′-GL was the main isomer in transitional and mature milk. β6′-Galactosyl lactose and β4′-GL tended to increase and then decrease during lactation. Moreover, α3′-GL content was 2 times higher than in colostrum and 10 times higher in transitional milk than in mature milk; in contrast, for β3′-GL, the values were 5 and 2 times higher, respectively. Absolute quantitative analysis revealed that β3′-GL was the most abundant isomers in colostrum (32.3 mg/L), and α3′-GL was the most abundant in transitional milk (88.1 mg/L) and mature milk (36.3 mg/L). These findings provide an important quantitative basis for understanding the relationship between structure and function of galactosyl lactose in goat milk, as well as its exploitation as a functional food.     

Rapid,quantitative analysis of 3′- and 6′-sialyllactose in milk by flow-injection analysis–mass spectrometry: Screening of milks for naturally elevated sialyllactose concentration

Non-protein-bound oligosaccharides are important bioactive components of cow milk, with potential human-health benefits such as stimulation of the growth of beneficial gut bacteria and defense against pathogens. In bovine milk, the majority of oligosaccharides are sialylated; 3′-sialyllactose (3′-N-acetylneuraminyl-d-lactose; 3′-SL) is the predominant sialylated oligosaccharide, followed by 6′-sialyllactose (6′-N-acetylneuraminyl-d-lactose; 6′-SL). Both 3′-SL and 6′-SL have antimicrobial activity. As bovine milk products such as infant formula can be an important component of the human diet, and the concentrations of 3′-SL and 6′-SL are lower in bovine milk compared with human milk, we aimed to identify cows that naturally produce higher concentrations of sialyllactose in their milk. Milk from such cows could be used to produce foods with an increased sialyllactose content, potentially providing increased health benefits. We speculated that cows overexpressing 3′-SL and 6′-SL would exist at low frequency in the population and, to allow their efficient identification, we developed a novel assay for 3′-SL and 6′-SL utilizing flow-injection analysis-mass spectrometry, which could be used for high-throughput analysis of milk samples. We then determined 3′-SL and 6′-SL concentrations in milk samples from 15,507 cows from Friesian, Jersey, and Friesian-Jersey crossbred animals. We found 329 cows with concentrations of 3′-SL or 6′-SL >2-fold higher than the mean, 26 cows with concentrations of 3′-SL or 6′-SL >3-fold higher than the mean, and 1 cow with concentrations of 3′-SL >4-fold higher than the mean. Although these outliers were observed across the 3 groups of cows, breed had a strong effect on mean 3′-SL and 6′-SL concentrations.     

Identification of Sialylated Oligosaccharides Derived from Ovine and Caprine Caseinomacropeptide by Graphitized Carbon Liquid Chromatography–Electrospray Ionization Ion Trap Tandem Mass Spectrometry

Isolation and characterization of oligosaccharides from caseinomacropeptide (CMP) are important in understanding the biological and functional properties of CMP. However, it is difficult to achieve this goal, due to the high degree of isomerism present in these types of compounds. In this study, the sialylated oligosaccharides derived from ovine and caprine CMP were released as oligosaccharide alditols by reductive β-elimination and subsequently separated and characterized using graphite carbon column liquid chromatography–negative electrospray ionization ion trap tandem mass spectrometry (LC/ESI(?)-MSⁿ). Although, the chromatographic resolution of isomeric oligosaccharides was not achieved perfectly, the characteristic tandem mass spectra of these compounds allowed differentiating and confirming unequivocally the structure of each one of the oligosaccharides. In CMP of both species, four trisaccharides and four tetrasaccharides were identified as O-glycans. Their chemical structures were identified to be Galβ1-3(NeuAcα2-6)GalNAcol, NeuAcα2-3Galβ1-3GalNAcol, Galβ1-3(NeuGcα2-6)GalNAcol, NeuGcα2-3Galβ1-3GalNAcol, NeuAcα2-3Galβ1-3(NeuGcα2-6)GalNAcol, NeuGcα2-3Galβ1-3(NeuAcα2-6)GalNAcol, NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNAcol, and NeuGcα2-3Galβ1-3(NeuGcα2-6)GalNAcol. The LC/MSⁿ methodology using an ion trap-type mass analyzer shown in this study is of general applicability for determination of short O-glycan oligosaccharides.     

N-丙酰-唾液酸衍生物的化学酶法合成及在测定禽蛋中唾液酸含量的应用

为实现禽类蛋黄和蛋清中N-乙酰神经氨酸(N-acetylneuraminic acid,Neu5Ac)的准确定量,消除禽蛋样品中分析物以外的物质产生的基质效应对分析结果造成的影响,本研究以甘露糖胺为底物采用化学酶法合成了非天然唾液酸衍生物5-溴-4-氯-3-吲哚-β-D-半乳糖苷-N-丙酰-唾液酸(5-Bromo-4-...     

牛免疫球蛋白G的体外人源唾液酸化及Fc片段的制备

建立将牛免疫球蛋白G (bovine immunoglobulin G,bIgG)糖链末端N-羟乙酰神经氨酸酶切并连接人源N-乙酰神经氨酸(N-acetylneuraminic acid,Neu5Ac)的方法,在实现bIgG转化为人源IgG (human IgG,hIgG)的基础上,研究hIgG可结晶(Fc)片段的制备...     

鹿蹄橐吾多糖LW21的分离纯化及其结构分析

目的:确定鹿蹄橐吾多糖LW21的结构,为探讨鹿蹄橐吾多糖的药理活性,合理利用鹿蹄橐吾这种植物资源提供依据。方法:水提取醇沉获得鹿蹄橐吾水溶性粗多糖LW,经酸性乙醇分级和DEAE-SephdexA-25纯化得多糖LW21。纸层析、醋酸纤维薄膜电泳和Sepharose CL-4B柱层析进行纯度鉴定,LW21结构分析采用高碘酸氧化、Smith降解、甲基化分析及IR、NMR、GC和GC-MS等方法。结果表明:LW21为均一多糖,相对分子质量约为1.1×106。其单糖组成为鼠李糖(Rha)、阿拉伯糖(Ara)、甘露糖(Man)、葡萄糖(Glc)、半乳糖(Gal),物质的量比为7.4:11.9:25.7:40.0:14.9。多糖LW21为有分枝结构,主链由Glc和Man构成,其中其中Man主要以β(1→2)和β(1→6)糖苷键连接,β(1→2)糖苷键在3-O处和6-O处有分枝,β(1→6)糖苷键在2-O和4-O处有分枝,Glc也主要以β(1→2)及β(1→6)糖苷键连接,β(1→2)糖苷键在6-O处有分枝,β(1→6)糖苷键连接,在2-O处有分枝。分子支链由Ara、Rha、Gal构成。末端残基为Gal、Ara、Rha、Glc。结论:鹿蹄橐吾多糖LW2 1是一新结构多糖,为首次从鹿蹄橐吾中分离得到。     

Dietary intervention with sialylated lactulose affects the immunomodulatory activities of mice

Four sialylated lactuloses [N-acetylneuraminic acid-α2,3-lactulose (Neu5Acα2,3lactulose), N-acetylneuraminic acid-α2,6-lactulose (Neu5Acα2,6lactulose), deaminoneuraminc acid-α2,3-lactulose (Kdnα2,3lactulose), and deaminoneuraminc acid-α-2,6-lactulose (Kdnα2,6lactulose)] were reported to modulate the immunity of mice. The influences of cytokine expression, cell immunity, humoral immunity, and nonspecific immunity were investigated in our study using several techniques. Analysis via ELISA showed that cytokine expression was induced by sialylated lactulose treatment consistently in the serum and spleen. Among the 4 tested sialylated lactuloses, Neu5Acα2,6lactulose performed the best, simultaneously and appropriately promoting the expression of proinflammatory and anti-inflammatory factors in the serum and spleen. Kdnα2,3lactulose showed the best antioxidant activity according to detection of the activity of superoxide dismutase, myeloperoxidase, peroxidase, and alkaline phosphatase. Flow cytometry revealed that only Kdnα2,3lactulose significantly boosted the CD3⁺ T lymphocyte ratio similarly to that of lactulose. Analysis of the hemolysin content to characterize humoral immunity revealed that Kdnα2,3lactulose notably increased hemolysin content compared with that in the control group. To evaluate the nonspecific immune effects of the 4 sialylated lactuloses, a fluorescence microsphere phagocytosis assay was used to analyze the phagocytosis of macrophages. Kdnα2,3lactulose still performed the best in enhancing the phagocytosis of macrophages, showing markedly increased phagocytic percentage and phagocytic index values compared with those in the control and lactulose groups. Comparing the differences of these 4 sialylated lactuloses in affecting immunity in mice revealed that Kdnα2,3lactulose had the best overall performance in influencing cytokine expression, cell immunity, humoral immunity, and nonspecific immunity. This study provides critical support for use of sialylated lactuloses as potential immunomodulators in foods.     

Determining Neu5Ac in infant formula with ultra‐performance liquid chromatography–tandem mass spectrometry

The aim of this work was to measure N‐acetylneuraminic acid (Neu5Ac) in milk‐based infant formulas. The analysis was performed by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS). The total Neu5Ac were released using trichloroacetic acid and hydrochloric acid and purified using a HLB column. The linearity from 0.05 to 5.0 μg/mg Neu5Ac was adequate. Sialic acid recoveries ranged from 91.8% to 112.4%. The detection and quantification limits (limit of detection, 0.01 μg Neu5Ac/mg; limit of quantitation, 1.08 μg Neu5Ac/mg) were low enough to determine the sialic acid in infant formulas. The validated method is highly reproducible and sensitive, and it is easy to perform.     

气相色谱法同时测定多糖中的中性糖、糖醛酸、氨基糖和唾液酸

采用甲醇解和硅烷衍生化,用气相色谱法同时测定样品中的中性糖、糖醛酸、N-乙酰氨基糖和唾液酸,取得了很好的效果。3种标准单糖混合物(包含阿拉伯糖、鼠李糖、岩藻糖、木糖、甘露糖、半乳糖、葡萄糖、葡萄糖醛酸、半乳糖醛酸、N-乙酰半乳糖、N-乙酰葡萄糖、5-乙酰唾液酸)和2种多糖样品(豆腐渣多糖DFP、鸡腿菇多糖F32)用1 mol/L盐酸甲醇在85℃反应18～24 h,碳酸银中和,加入乙酸酐室温暗处反应24 h,使氨基糖重新引入N-乙酰基,除银盐,干燥,加入硅烷化试剂[V(吡啶)∶V(六甲基二硅氨烷)∶V(三甲基氯硅烷)=5∶1∶1),室温放置30 min,最后用气相色谱进行分析。     

Cholesterol Oxides in Swedish Foods and Food Ingredients: Milk Powder Products

Cream-, whole milk-and skim milk powders had 1.6, 2.0, and 18.9 mg cholesterol/g fat, respectively. About 90% of the cholesterol was unesterified. Analyses of fresh spray-dried whole-and skim milk powders from “low and medium heat” conditions and fresh roller-dried whole milk powder from “low heat” conditions contained less than 0.1 ppm cholesterol oxides in lipids. However, spray-dried whole-and skim milk powders from “high heat” conditions had substantial levels of some important cholesterol oxides. Skim-milk powders, stored in big sacks for 11–37 months and small consumer packages for 2–23 months, contained variable amounts of 5, 6α-epoxy-5β-cholestan-3β-ol (0.3–4.0 ppm), 5,6β-epoxy-5α-cholestan-3β-ol(0.7–10.40ppm), cholest-5-ene-3β,7α-diol(0.5–16.2 ppm), cholest-5-ene-3β,7β-diol(1.3–20.8 ppm), cholest-5-ene-3β,20α-diol(0.6–2.7 ppm), cholest-5-ene-3,25-diol(0.3–0.8 ppm), 5α-cholestane-3β,5,6β-triol(1.3–2.5 ppm) and 3-hydroxy-cholest-5-en-7one(1.8-24.9 ppm).     

Rapid and simple method for the determination in sialic acid of yak milk fat globule membrane

A rapid and simple method for the determination of sialic acid (Neu5Ac) of yak milk fat globule membrane (MFGM) by HPLC with a diode array detector was developed and validated. Samples were cleaned up just by hydrolysis and derivatization before HPLC analysis. Separation was achieved with an Agilent TC-C18 column. The method showed a good linearity (r=0.997), the sensitivity results showed that the limits of detection and limits of quantification for sialic acid were 10.0 and 21.0 μg/mL, respectively. The recovery of Neu5Ac was 95–97%. The method proved very simple and rapid for Neu5Ac analysis since separation was completely achieved at 12 min.     

The Behaviour and Identification of a Range of Starch and Starch-Related Oligosaccharides Series on Gel Permeation Chromatography as a Function of Their Molecular Shapes

The elution behaviour on gel permeation chromatography of a wide range of oligosaccharides which differ in glycosidic linkage has been investigated using a high resolution column of Bio-Gel P2 (-400 mesh). The elution behaviour could be explained on size exclusion mechanisms resulting from the secondary and tertiary structures of oligosaccharides brought about by the nature of the glycosidic linkages concerned. The various series of oligosaccharides followed an order in which starch-derived materials were more included in the gel than oligosaccharides derived from other polysaccharides. The overall order was cyclomalto- < malto-< (2 → 6) -β-D-fructo- < isomalto < (2 → 1)-β-D-fructo- < gentio- < cello-oligosaccharides more specifically cyclo- (1 → 4)-α-D-gluco- < linear (1 → 4)-α-D-gluco- < (2 → 6)-β-D-fructo- < (1 → 6)-α-D-gluco- < (2 → 1)-β-D-fructo- <( 1 → 6)-β-D-gluco- < (1 → 4)-β-D-gluco-oligosaccharides. The particular behaviour of linear malto-oligosaccharides and the different behaviour of isomaltose makes the method still of great use for the assessment of starch-derived materials, including branch point residues. Specific size exclusion mechanisms were found not to operate for methyl ether or glycoside derivatives of monosaccharides although Bio-Gel P2 could be of limited use in methylation analysis and methyl glycoside separations.     

Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria

The bovine Muc1 protein is synthesized by mammary epithelial cells and shed into milk as an integral component of the milk fat globule membrane; however, the structure and functions of this mucin, particularly in relation to lactation, are poorly defined. The objectives of this investigation were to investigate the Muc1 gene and protein structures in the context of lactation and to test the hypothesis that Muc1 has a role in innate immune defense. Polymerase chain reaction analysis of genomic DNA from 630 cattle revealed extensive polymorphism in the variable number of tandem repeats (VNTR) in the bovine Muc1 gene. Nine allelic variants spanning 7 to 23 VNTR units, each encoding 20 AA, were identified. Three alleles, containing 11, 14, and 16 VNTR units, respectively, were predominant. In addition, a polymorphism in one of the VNTR units has the potential to introduce a unique site for N-linked glycosylation. Statistical analysis indicated weak associations between the VNTR alleles and milk protein and fat percentages in a progeny-tested population of Holstein-Friesian dairy cattle. No association with somatic cell count could be demonstrated. Bovine Muc1 was purified from milk fat globule membranes and characterized. The protein was highly glycosylated, primarily with O-linked sialylated T-antigen [Neu5Ac(α2-3)-Gal(β1-3)-GalNAcα1] and, to a lesser extent, with N-linked oligosaccharides, which together accounted for approximately 60% of the apparent mass of Muc1. Purified bovine Muc1 directly bound fluorescently labeled Escherichia coli BioParticles (Invitrogen, Mount Waverley, Australia) and inhibited their binding to bovine mammary epithelial cells grown in vitro. It was also demonstrated that the expression of Muc1 mRNA in bovine mammary epithelial cells was markedly upregulated by lipopolysaccharide. Muc1 may be a pattern recognition protein that has the capacity to sequester bacteria and prevent their attachment to epithelial surfaces by immobilizing and subsequently shedding Muc1-bound bacteria from the cell surface. It was concluded that bovine Muc1 is probably an inducible innate immune effector and an important component of the first line of defense against bacterial invasion of epithelial surfaces, particularly mammary epithelial surfaces and the neonatal gut.     

高效液相色谱法测定母乳中唾液酸含量

建立荧光高效液相色谱(fluorescence detector-high performance liquid chromatography,HPLC-FLD)测定母乳中唾液酸N-乙酰神经氨酸(N-acetylneuraminic acid,Neu5Ac)和N-羟乙酰神经氨酸(N-glycolyl neuraminic acid,Neu5Gc)含量的分析方法。利用酸水解法释放出母乳中的唾液酸,以4,5-亚甲二氧基-1,2-邻苯二胺盐(4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride,DMB)为衍生化试剂,50℃避光衍生150min,采用荧光高效液相色谱仪检测。色谱条件：LiChrosorb RP-18柱(250mm×4mm,5μm),流动相为甲醇-乙腈-超纯水(7:8:85),流速0.9mL/min,进样体积10μL,柱温30℃,荧光检测器激发波长373nm,发射波长448nm。结果表明：唾液酸在50～400μmol/L范围内与唾液酸峰面积的线性关系良好,平均回收率为94.0%,精密度的相对标准偏差(relative standard deviation,RSD)为0.4%,稳定性RSD为1.0%,重复性RSD为0.8%,Neu5Ac的最低检出限为0.02μmol/L,Neu5Gc的最低检出限位0.03μmol/L。该方法简单、重复性好、灵敏度高,可广泛用于奶粉、牛奶及母乳中唾液酸含量测定。     

莱菔子化学成分的分离与鉴定

目的：研究莱菔子化学成分,探索生品莱菔子的物质基础。方法：采用大孔吸附树脂HP-20、聚苯乙烯型大孔吸附树脂（MCI gel）、ODS、硅胶及LiChroprep RP-18等色谱技术进行分离纯化,运用光谱学方法对得到的化合物进行结构鉴定。结果：从莱菔子甲醇提取物中分离鉴定了16?个化合物：顺-13-二十二碳烯酸（1）、豆甾-4-烯-3-酮（2）、（22E,24R）-麦角甾-5,22-二烯-3β-醇（3）、豆甾-5-烯-3β-醇（4）、反-阿魏酸甲酯（5）、反-芥子酸甲酯（6）、β-豆甾醇-3-O-β-D-葡萄糖苷（7）、α-D-吡喃半乳糖基-（1-6）-α-D-吡喃葡萄糖基-（1-2）-β-D-呋喃果糖苷（8）、β-D-呋喃果糖基-α-D-（6-芥子酰基）葡萄糖苷（9）、β-D-（3-芥子酰基）呋喃果糖基-α-D-葡萄糖苷（10）、β-D-（3-芥子酰基）呋喃果糖基-α-D-（6-芥子酰基）葡萄糖苷（11）、β-D-（3,4-二芥子酰基）呋喃果糖基-α-D-（6-芥子酰基）葡萄糖苷（12）、异鼠李素-3-O-β-D-葡萄糖苷（13）、异鼠李素-3,4’-O-β-D-二葡萄糖苷（14）、异鼠李素-3-O-β-D-葡萄糖-7-O-α-L-鼠李糖苷（15）、3’-O-甲基-（－）-表儿茶素-7-O-β-D-葡萄糖苷（16）。结论：化合物2、3、7～10、13～16为首次在该植物中得到。     

Comparison of spectrophotometric and HPLC methods for determining sialic acid in infant formulas

Two methods for determining sialic acid in infant formulas – spectrophotometry and HPLC with fluorescence detection – have been optimised and validated, the first one allows to determine total sialic acid while the second allows to differentiate the two main forms of sialic acid (N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc)). A common sample preparation procedure (hydrolysis and purification) for both methods has been proposed. The linearity (from 6 to 150 μg of total sialic acid in the assay for spectrophotometry, and from 12.5 to 250 ng and 1 to 5 ng of Neu5Ac and Neu5Gc, respectively, for HPLC) is adequate. The detection and quantification limits (0.29 and 0.97 mg of total sialic acid/L of reconstituted sample, respectively, for spectrophotometry, and 0.03 and 0.08 mg Neu5Ac/L; 0.003 and 0.009 mg Neu5Gc/L of reconstituted sample, respectively, for HPLC) are low enough for the determination of sialic acid in infant formulas. The precision of both methods, expressed as relative standard deviation, is less than 6%, and the accuracy evaluated by recovery assays show 104% recovery for spectrophotometry; 95% for Neu5Ac and 109% for Neu5Gc for HPLC. Samples analysed show no significant differences (α < 0.05) attributable to the method used; consequently, both of them could be applied after common sample preparation, the choice of technique depending on the facilities available in the laboratory.     

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.