Importance of enzyme purity and activity in the measurement of total dietary fiber and dietary fiber components

A study was made of the effect of the activity and purity of enzymes in the assay of total dietary fiber (AOAC Method 985.29) and specific dietary fiber components: resistant starch, fructan, and beta-glucan. In the measurement of total dietary fiber content of resistant starch samples, the concentration of alpha-amylase is critical; however, variations in the level of amyloglucosidase have little effect. Contamination of amyloglucosidase preparations with cellulase can result in significant underestimation of dietary fiber values for samples containing beta-glucan. Pure beta-glucan and cellulase purified from Aspergillus niger amyloglucosidase preparations were used to determine acceptable critical levels of contamination. Sucrose, which interferes with the measurement of inulin and fructooligosaccharides in plant materials and food products, must be removed by hydrolysis of the sucrose to glucose and fructose with a specific enzyme (sucrase) followed by borohydride reduction of the free sugars. Unlike invertase, sucrase has no action on low degree of polymerization (DP) fructooligosaccharides, such as kestose or kestotetraose. Fructan is hydrolyzed to fructose and glucose by the combined action of highly purified exo- and endo-inulinases, and these sugars are measured by the p-hydroxybenzoic acid hydrazide reducing sugar method. Specific measurement of beta-glucan in cereal flour and food extracts requires the use of highly purified endo-1,3:1,4 beta-glucanase and A. niger beta-glucosidase. Beta-glucosidase from almonds does not completely hydrolyze mixed linkage beta-glucooligosaccharides from barley or oat beta-glucan. Contamination of these enzymes with starch, maltosaccharide, or sucrose-hydrolyzing enzymes results in production of free glucose from a source other than beta-glucan, and thus an overestimation of beta-glucan content. The glucose oxidase and peroxidase used in the glucose determination reagent must be essentially devoid of catalase and alpha- and beta-glucosidase.     

Measurement of sugar content by multidimensional analysis and mid-infrared spectroscopy

The advent of more and more powerful micro-computers has allowed the introduction of multidimensional analysis in research laboratories. Complex mathematical treatments are now possible within a few seconds. Prediction equations that linked sucrose, fructose, glucose, total sugars and reducing sugars concentrations to the spectral data, were established by regression on the principal components. Very high correlation coefficient values between the first ten axes and the chemical values were obtained. The bias and standard deviation (S.D.) values obtained between reference and predicted values were good. From such aqueous biological samples containing a ternary mixture of sucrose, fructose and glucose it was possible to (i) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars, total sugars)-using spectral pattern; and (ii) to specifically measure their concentrations with good accuracy.     

Measurement of alpha-amylase activity in white wheat flour,milled malt,and microbial enzyme preparations,using the Ceralpha assay: collaborative study

This study was conducted to evaluate the method performance of a rapid procedure for the measurement of alpha-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing alpha-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable alpha-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, alpha-glucosidase, and beta-amylase. When the substrate is cleaved by endo-acting alpha-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of alpha-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 micromol p-nitrophenyl (in the presence of excess quantities of alpha-glucosidase) in 1 min at 40 degrees C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal alpha-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.     

Analysis of mono- and disaccharides in milk-based formulae by high-performance liquid chromatography with refractive index detection

A simple and reproducible method for the qualitative and quantitative analysis of free mono- and disaccharides (fructose, glucose, galactose, sucrose, lactulose and lactose) in milk-based formulae by high-performance liquid chromatography (HPLC) with refractive index (RI) detection was developed and validated. The method showed good linearity with determination coefficients exceeding 0.99. The limits of detection (DL) in these sugars were 0.17, 0.13, 0.06, 0.16, 0.05 and 0.25 mg/ml, respectively; and the limits of quantification (QL), 0.27, 0.24, 0.20, 0.26, 0.22 and 0.38 mg/ml. The relative standard deviations (R.S.D.s) for repeatability in fructose, sucrose, lactulose and lactose were 0.78, 0.99, 2.91 and 0.46 and the R.S.D.s for reproducibility were 4.8, 6.15, 7.04 and 2.49, respectively. Recoveries in all sugars were between 93 and 113%.     

Enzymeless determination of total sugar by luminol–tetrachloroaurate chemiluminescence on chip to analyze food samples

Chemiluminescence (CL) emission from luminol–tetrachloroaurate ([AuCl₄]^?) system studied in presence of monosaccharide sugars such as glucose and fructose was investigated on a microfluidic chip fabricated by the soft lithography technique. CL emission from the luminol–[AuCl₄]^? system at 430?nm was intensified remarkably by the catalytic activity of glucose and fructose at room temperature. Under optimized conditions, the CL emission intensity of the system was found to be linearly related to the concentration of the sugars. Based on this observation, nonenzymatic determination of total sugar (glucose, fructose, or hydrolyzable sucrose) was performed in a rapid and sensitive analytical method. The results revealed that the linearity ranged from 9 to 1,750?μM for glucose and 80 to 1,750?μM for fructose, with a limit of detection of 0.65 and 0.69?μM, respectively. The relative standard deviations determined at 250?μM based on six repetitive injections were 1.13 and 1.15?% for glucose and fructose, respectively. The developed method was successfully applied for determination of the total sugar concentration in food and beverages.

A Separation‐Integrated Cascade Reaction to Overcome Thermodynamic Limitations in Rare‐Sugar Synthesis

Enzyme cascades combining epimerization and isomerization steps offer an attractive route for the generic production of rare sugars starting from accessible bulk sugars but suffer from the unfavorable position of the thermodynamic equilibrium, thus reducing the yield and requiring complex work‐up procedures to separate pure product from the reaction mixture. Presented herein is the integration of a multienzyme cascade reaction with continuous chromatography, realized as simulated moving bed chromatography, to overcome the intrinsic yield limitation. Efficient production of D ‐psicose from sucrose in a three‐step cascade reaction using invertase, D ‐xylose isomerase, and D ‐tagatose epimerase, via the intermediates D ‐glucose and D ‐fructose, is described. This set‐up allowed the production of pure psicose (99.9 %) with very high yields (89 %) and high enzyme efficiency (300 g of D ‐psicose per g of enzyme).     

Flow injection spectrophotometric determination of reducing sugars using a focalized coiled reactor in a domestic microwave oven

A flow injection (FI) spectrophotometric procedure for determining reducing sugars content in sugar cane juices using a focalized PTFE coiled reactor positioned at the output antenna of a domestic microwave oven at 700 W is proposed. In this system, sample solution converge to 1.0 mol l(-1) NaOH and 5.2 mmol l(-1) K(3)Fe(CN)(6) solutions previously mixed and the decrease of hexacyanoferrate(III) concentration was monitored at 420 nm. Under best analytical conditions, there was a direct relationship between absorbance decrease and reducing sugar content (fructose plus glucose concentrations) in the concentration range from 50 to 1200 micromol l(-1) with a detection limit of 15 micromol l(-1). The relative standard deviations (rds) were less than 1.4% for ten injection of 400 and 800 micromol l(-1) fructose solution and the analytical frequency was 70 h(-1). A paired t-test showed that all results obtained for sugar cane juices using this FI procedure and the Somogyi-Nelson batch procedure agree at the 95% confidence level.     

Inulin and oligofructose are part of the dietary fiber complex

Dietary fiber has been defined as the remnants of plant cells resistant to hydrolysis by human alimentary enzymes. Its main chemical constituents are hemicelluloses, celluloses, lignin, pectins, gums, and waxes. The U.S. Food and Drug Administration and the U.S. Department of Agriculture determine compliance with nutritional labeling regulations for dietary fiber by use of the existing AOAC INTERNATIONAL methods for total dietary fiber. The above compounds are readily detected by these methods. However, some oligo- and polysaccharides are resistant to human alimentary enzymes and do not precipitate in 78% ethanol, the usual reagent for precipitating dietary fiber in analytical procedures. Some of these saccharides, termed fructans, are inulin and oligofructose. They possess many physiological attributes normally associated with dietary fiber. Inulin is a mixture of oligo- and polysaccharides composed of fructose moieties joined by beta(2-->1) linkages in linear chains. Almost each chain ends with a glucose moiety. Oligofructose is a synonym for fructo-oligosaccharides, with fructose moieties joined by beta(2-->1) linkages, as in inulin. Not all molecules have a glucose unit, and the chain length is less than 10 units. A method for inulin and oligofructose was developed and approved official first action by AOAC INTERNATIONAL in early 1997. It involves extraction of sample and treatment of the extract with amyloglucosidase followed by fructozyme (Fructozyme Enzyme Process Division, Novo Nordisk, Novo Industry, Copenhagen, Denmark). The sugars released in each of the 3 steps are measured by anion-exchange chromatography. The concentration of fructans is calculated as the difference of sugars, glucose and fructose, after the enzymatic treatments and the initial sample. The repeatability standard deviations for inulin and oligofructose ranged from 2.9 to 5.8% and the reproducibility standard deviations ranged from 4.7 to 11.1%. The method was accepted by AOAC INTERNATIONAL.     

高效毛细管电泳－间接电导法快速分离混合糖

测定;间接电导检测法;高效毛细管电泳－间接电导法快速分离混合糖     

Determination of some carbohydrates with N-bromophthalimide and N-bromosaccharin

Two better titrimetric methods have been developed for determination of carbohydrates such as glucose, fructose, lactose and sucrose, and sugars present in honey and milk. They involve reduction of Cu(II) to Cu(I) by the carbohydrate concerned, and oxidative titration of the Cu(I) with a standard solution of N-bromophthalimide or N-bromosaccharin.     

Amperometric Determination of Sugars at Activated Barrel Plating Nickel Electrodes

We report amperometric determination of sugars by using a disposable barrel plating nickel electrode (Ni‐BPE) in this study. The activated Ni‐BPE possesses good reproducibility in flow injection analysis of sugars with a relative standard deviation of 3.16% for 10 consecutive injections of glucose. The electrocatalytic mechanism for the detection of sugars as well as the use as a detector in high‐performance liquid chromatography (HPLC) is investigated. We achieve a good separation of four sugars (glucose, fructose, sucrose, and maltose) in HPLC with favorable sensitivity at a detection potential of +0.55 V vs. Ag/AgCl. The results of wide linear calibration ranges and detection limits in the μM range meet the need of real sample analysis. This detection method is successfully used for quantitative determination of sugars in honey to identify its authentication.     

Simultaneous determination of glucose and sucrose by a glucose-sensing enzyme electrode combined with an invertase-attached cell

Glucose and sucrose are simultaneously determined by using a glucose-sensing enzyme electrode combined with a cell that contains immobilized invertase. The electrode current changes linearly with time for several minutes from ca. 1 min after the addition of a glucose-sucrose mixture. The concentration of sucrose (60 μM-6 mM) is determined from the rate of current change in the linear region, and that of glucose (5 μM-1 mM) is determined by extrapolating the straight current-time line to t=0.45 min and by measuring the intercept on the vertical (current) axis at t=0.45 min. The relative standard deviations are 1.8% for glucose and 3.7% for sucrose (n=10). More than 20 food samples can be analysed in 1 h.     

Focused-microwave-assisted reaction in flow injection spectrophotometry: a new liquid-vapor separation chamber for determination of reducing sugars in wine

The usefulness of an unsegmented liquid/vapor phases flow injection system for determining reducing sugars in wines using a focalized poly(tetrafluoroethylene) (PTFE) coiled reactor positioned at the output antenna of a domestic microwave oven at 700 W is demonstrated. In this system, sample or reference solution is mixed to 2.5 mol l(-1) sodium hydroxide and 6.8 mmol l(-1) potassium hexacyanoferrate(III) solutions previously merged in a confluence point and the reducing sugars (glucose plus fructose) were oxidized by potassium hexacyanoferrate(III) solution into the PTFE coiled reactor positioned in the microwave oven cavity. After phases separation at a proposed separation chamber (SC), the decrease of K(3)Fe(CN)(6) concentration in the liquid phase was measured at 420 nm. Using this SC, a favorable dispersion profile and a decrease of hydrodynamic pressure were observed. The flow procedure presented similar sensitivities to glucose and fructose in the experimental conditions adopted, allowing the determination of the reducing sugars content (fructose plus glucose concentrations) in the concentration range from 40 to 400 mumol l(-1) with a detection limit of 9 mumol l(-1). The RSD was 1.9% for a 120 mumol l(-1) fructose solution (n=12) and the analytical frequency was 54 h(-1). The results obtained for reducing sugars in several wines using the proposed flow system and those obtained using the Somogyi-Nelson batch procedure were in agreement at the 95% confidence level.     

高效阴离子交换色谱-积分脉冲安培检测法同时测定黄酒中的单糖和低聚糖及其指纹图谱的构建

建立了黄酒中葡萄糖、果糖、异麦芽糖、异麦芽三糖、麦芽糖、潘糖、麦芽三糖共7种糖类的高效阴离子交换色谱分离-积分脉冲安培检测的方法。以氢氧化钠和醋酸钠为淋洗液进行梯度洗脱,CarboPac^TM10阴离子交换柱进行分离。该方法可在26 min内实现黄酒中7种糖类的定量分析,在0.5~50 mg/L浓度范围内线性关系良好,样品定量限为0.1 g/L,加标回收率为76.5%~108.4%,精密度(RSD)为3.02%~8.23%。将该法用于不同厂家不同批次的绍兴加饭酒中各种糖类含量的检测,基于所得的检测结果,利用中位数法构建了标准指纹图谱,并采用夹角余弦法评价了各个样品与标准指纹图谱之间的相似度。结果初步显示,不同厂家加饭酒之间的相似度差别较大,该法可作为区别不同厂家酒的依据之一。     

Determination of inulin in foods

A method was developed for determining fructan inulin in various foods (yogurts, honey cakes, chocolates). Warm water was applied for extraction of samples, and mono- and dissacharides were determined by a thin-layer chromatographic densitometric method. A portion of the test solution was hydrolyzed 30 min with 1% oxalic acid in a boiling water bath. Fructose was determined in the hydrolysate. The amount of inulin in a sample was calculated as the difference between the amount of fructose in the sample before and after hydrolysis. The fructose from sucrose formed during the hydrolysis was also considered. The mean recovery from yogurt fortified with 4% inulin was 95.5 +/- 4.5% (mean +/- standard deviation); from honey cakes extract fortified with 10% inulin, 97.3 +/- 5.5%; and from chocolate extract fortified with 30% inulin, 98.6 +/- 6.6% (6 replicates in all cases). Determination of glucose is not necessary for analyzing fructans with the composition expressed shortened to GFn-1 (G, glucose; F, fructosyl) with the average degree of polymerization 8 < or = n < or = 15.     

高效阴离子交换色谱-脉冲安培检测法测定烤烟中的水溶性葡萄糖、果糖和蔗糖

用高效阴离子交换色谱-脉冲安培检测法（HPAEC-PAD）测定了烤烟中的水溶性葡萄糖、果糖和蔗糖。采用水浸取及膜过滤法处理烤烟样品,以Dionex CarboPac PA-1阴离子交换柱为色谱柱,0.2 mol/L NaOH水溶液为淋洗液进行分离测定。葡萄糖、果糖和蔗糖的含量与其峰面积的线性关系良好,回收率均在97%以上。方法简便易行,灵敏度高,重现性良好,可以实现对烟草中单糖的快速分离和测定。     

Partial Molar Volumes and Refractions of Aqueous Solutions of Fructose,Glucose, Mannose,and Sucrose at 15.00, 20.00, and 25.00 °C

The partial molar volumes and refractions of aqueous solutions of fructose, glucose, mannose and sucrose were determined at 15.00, 20.00 and 25.00 °C over a wide concentration range. A model is proposed to describe the deviations of the partial molar volumes at higher concentrations from those at infinite dilution. In addition, the partial molar volumes of the sugars at infinite dilution were fit to quadratic relations in temperature.     

高效液相色谱法测定果中的山梨醇和糖类

 用高效液相色谱外标法测定了木　奈果中的可溶性糖和山梨醇,采用μ-Spherogel carbohydrate柱和示差折光检测器,以水为流动相,柱温85 ℃。相关系数在0.970 9以上,加标回收率为75.00%～88.37％。测定结果表明,可溶性糖在果肉中的累积依果肉不同的生长发育阶段有特征性的变化。     

Hydrolytic Methods for the Quantification of Fructose Equivalents in Herbaceous Biomass

A low, but significant, fraction of the carbohydrate portion of herbaceous biomass may be composed of fructose/fructosyl-containing components (“fructose equivalents”); such carbohydrates include sucrose, fructooligosaccharides, and fructans. Standard methods used for the quantification of structural-carbohydrate-derived neutral monosaccharide equivalents in biomass are not particularly well suited for the quantification of fructose equivalents due to the inherent instability of fructose in conditions commonly used for hemicellulose/cellulose hydrolysis (>80% degradation of fructose standards treated at 4% sulfuric acid, 121°C, 1 h). Alternative time, temperature, and acid concentration combinations for fructan hydrolysis were considered using model fructans (inulin, β-2,1, and levan, β-2,6) and a grass seed straw (tall fescue, Festuca arundinacea) as representative feedstocks. The instability of fructose, relative to glucose and xylose, at higher acid/temperature combinations is demonstrated, all rates of fructose degradation being acid and temperature dependent. Fructans are shown to be completely hydrolyzed at acid concentrations well below that used for the structural carbohydrates, as low as 0.2%, at 121°C for 1 h. Lower temperatures are also shown to be effective, with corresponding adjustments in acid concentration and time. Thus, fructans can be effectively hydrolyzed under conditions where fructose degradation is maintained below 10%. Hydrolysis of the β-2,1 fructans at temperatures ≥50°C, at all conditions consistent with complete hydrolysis, appears to generate difructose dianhydrides. These same compounds were not detected upon hydrolysis of levan, sucrose, or straw components. It is suggested that fructan hydrolysis conditions be chosen such that hydrolysis goes to completion; fructose degradation is minimized, and difructose dianhydride production is accounted for.     

Trace analysis of sugars by HPLC and post-column derivatization

Summary A HPLC system with post-column derivatization for the quantitative analysis of sugars in complex matrices is described. As reagent a 0.2% solution of thymol in concentrated sulfuric acid has been used. The reaction is sensitive with reducing as well as non reducing sugars whereas sugar alcohols are discriminated. With this reagent and separation of sugars at high pH values with an anion exchange column it is possible to detect sugars in the ng range. Hence, it is possible to characterize wines not only by their fructose and glucose content but also by differences in the distribution of the other not fermentable sugars like trehalose, arabinose, galacturonic and glucuronic acid.