Measurement of total fructan in foods by enzymatic/spectrophotometric method: collaborative study

An AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measuring oligofructans and fructan polysaccharide (inulins) in mixed materials and food products. The sample is extracted with hot water, and an aliquot is treated with a mixture of sucrase (a specific sucrose-degrading enzyme), alpha-amylase, pullulanase, and maltase to hydrolyze sucrose to glucose and fructose, and starch to glucose. These reducing sugars are then reduced to sugar alcohols by treatment with alkaline borohydride solution. The solution is neutralized, and excess borohydride is removed with dilute acetic acid. The fructan is hydrolyzed to fructose and glucose using a mixture of purified exo- and endo-inulinanases (fructanase mixture). The reducing sugars produced (fructose and glucose) are measured with a spectrophotometer after reaction with para-hydroxybenzoic acid hydrazide. The samples analyzed included pure fructan, chocolate, low-fat spread, milk powder, vitamin tablets, onion powder, Jerusalem artichoke flour, wheat stalks, and a sucrose/cellulose control flour. Repeatability relative standard deviations ranged from 2.3 to 7.3%; reproducibility relative standard deviations ranged from 5.0 to 10.8%.     

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 sugars and starches in foods by a modification of the AOAC total dietary fiber method.

A separation scheme for the determination of sugars and starch in processed food was developed. It is based on AOAC Method 985.29 for total dietary fiber with these modifications: carbohydrate starches are separated into soluble and insoluble fractions before they are hydrolyzed; acetonitrile is used instead of ethanol to separate sugars from enzyme-resistant carbohydrates, proteins, and other macromolecules; and a solid-phase extraction filter is included to remove substances that interfere with high-performance liquid chromatography (HPLC). Recovery studies indicate a > 97% sugar recovery. Twenty foods were analyzed. After enzymatic hydrolysis, fructose, glucose, sucrose, maltose, and lactose were extracted and determined by HPLC using a refractive index detector. Starch content was calculated from the increase in the amount of glucose. The results were compared with values listed on the "Nutrition Facts" panel for that food. The analyzed amounts of sugars and starches were 73-96% of declared values.     

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.     

Comparative evaluation of extraction procedures and method validation for determination of carbohydrates in cereals and dairy products by capillary electrophoresis

This work describes the development and application of two optimized electrolytes: 15 mmol/L sorbate, 0.2 mmol/L CTAB, and 35 mmol/L sodium hydroxide for the determination of fructose, glucose, maltose, maltotriose, and sucrose in cereals and 15 mmol/L sorbate, 0.3 mmol/L CTAB, and 55 mmol/L sodium hydroxide for the determination of fructose, glucose, galactose, lactose, and sucrose in dairy products. Both methods were validated with respect to linearity, limits of detection (using both signal-to-noise ratio and the Eurachem approach), recovery tests, and intra- and inter-day precision exhibiting adequate performance. Additionally, statistically similar results were obtained in a comparative study of extraction procedures for carbohydrates using the AOAC protocol, ultrasound extraction, magnetic stirring, and sample dissolution.     

Separation of glucooligosaccharides and polysaccharide hydrolysates by gradient elution hydrophilic interaction chromatography with pulsed amperometric detection

Commercial glucooligosaccharide mixtures (Polycose) and polysaccharide hydrolysates (acid and enzymatic) were fractionated by hydrophilic interaction chromatography and observed by pulsed amperometric detection. Seven peaks were observed when 625 ng of glucose oligomers in Polycose were fractionated. The between-run precision of retention times (n = 10, 100 μg, 15 peaks) ranged from a relative standard deviation (R.S.D.) of 0.09 to 0.40%; between-run precision of peak areas (n = 10) for the same separations had values that ranged from 2.66 to 14.4%. Injection-to-injection time was 48 min. When polysaccharide hydrolysates were fractionated using a gradient program capable of resolving all of the oligosaccharide species, dextran-derived -(1→6)- glucooligosaccharides were retained to a greater degree than amylose-derived -(1→4)-glucooligosaccharides, which were retained to a greater degree than β-(2→1)-fructooligosaccharides derived from inulin. Excluding the peaks that eluted before glucose or fructose, 25 to 35 peaks were observed after fractionation of the hydrolysates. Differences in elution profiles were observed between acid and enzymatic hydrolysis products of the same polysaccharide as well as between hydrolysis products of different polysaccharides. In conjunction with high-performance size-exclusion chromatography, the method demonstrated the effect of preheating starch before hydrolysis with isoamylase.     

Determination of carbohydrates and sweeteners in foods and biologically active additives by high-performance liquid chromatography

A method for the determination of large amounts of carbohydrates (glucose, lactose, maltose, mannose, sucrose, and fructose) and sweeteners (xylitol and sorbitol) by reversed-phase liquid chromatography with refractive index detection without derivatization has been developed. The limits of determination for glucose, fructose, and sucrose in liquid samples were 0.1 g/L, and for xylite, lactose, maltose, mannose, and sorbite, 1 g/L. In solid samples the limits of determination for glucose, fructose, and sucrose were 0.1%, and for xylite, lactose, maltose, mannose, and sorbite, 0.6%. The method is applicable to the analysis of samples of wine, juice, honey, cookies, dairy products, and biologically active additives. The developed method for the determination of carbohydrates and sweeteners in foods and biologically active additives was certified in the Mendeleev Institute for Metrology (St. Petersburg).     

Simultaneous Saccharification of Inulin and Starch Using Commercial Glucoamylase and the Subsequent Bioconversion to High Titer Sorbitol and Gluconic Acid

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.     

Flow-injection determination of sugars with immobilized enzyme reactions and chemiluminescence detection

Glucose is determined by reaction with gluocose oxidase to produce hydrogen peroxide which is quantified via a chemiluminescence reaction with luminol. Sucrose, maltose, lactose and fructose are determined by enzymatic conversion to glucose (using invertase, amyloglucosidase, lactase. and glucose isomerase, respectively) and subsequent determination of the glucose, All enzymes are immobilized on controlled-pore glass and contained in flow-through reactors. For glucose, sucrose, and maltose the linear log-log working range 0.2 μM-1 mM, with a detection limit of 0.1 μM; for lactose and fructose the linear working range is 3 μM-1 mM with a detection limit of 1 μM. Assay time is 2 min.     

红枣中果糖、葡萄糖与蔗糖的高效液相色谱示差检测分析

建立了同时分析鲜红枣中果糖、葡萄糖和蔗糖的高效液相色谱方法.利用鲜红枣样品进行添加回收实验,获得方法对果糖、葡萄糖和蔗糖测定的平均回收率分别为93.5%、91.8%和89.8%,变异系数3.27%、2.34%、5.86%.结果表明:采用高效液相色谱示差分析方法定量检测鲜红枣中果糖、葡萄糖和蔗糖快速、简便.同时得出方法的检出限为:果糖5.73×10-3mg/mL,葡萄糖5.22×10-3mg/mL,蔗糖3.59×10-3mg/mL,测定结果符合食品检测要求.     

Determination of saccharides in fruit juices by capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A fast method for the detection of cheap sweeteners is presented. Detecting the adulteration of foods rich in carbohydrates is complicated by the presence of variety of commercial sweeteners that are designed to match exactly the major carbohydrate profiles of these foods. Electrophoretic and mass spectrometric assays for the determination of fruit juice authenticity were developed. Capillary zone electrophoresis with indirect detection was employed to detect adulteration of juices demonstrated by the ratio of the concentrations of major low molecular mass saccharides (glucose, fructose and sucrose). Traces of oligosaccharides, which are not present in the sugar profiles of citrus fruits but are present in inexpensive sweeteners, were evaluated as the other group of target compounds. The fast determination of oligomeric starch hydrolysates in a complex matrix was tested by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and applied to orange juice. MALDI-TOFMS was shown to be a suitable method for the identification of adulteration of fruit juices by starch hydrolysates. The effects of the presence of salts and low molecular mass saccharides on the detection of oligosaccharides by MALDI-TOFMS were studied. Low molecular mass saccharides and organic acids decrease the detectability of oligosaccharides by MALDI-TOFMS, but the concentration of maltooligosaccharides present in juices sweetened with starch hydrolysates is high enough to be detected with good sensitivity.     

毛细管区带电泳-间接紫外检测法快速测定食品中乳糖、蔗糖、葡萄糖和果糖

建立了毛细管区带电泳-间接紫外检测快速测定食品中乳糖、蔗糖、葡萄糖和果糖的方法。以水或5 mmol/L醋酸为样品提取液,未涂层熔融石英毛细管(30.2 cm(有效长度20 cm)×50 μm)为分离柱,4 mmol/L山梨酸钾+10 mmol/L磷酸钠+30 mmol/L NaOH(pH 12.56)+0.5 mmol/L十六烷基三甲基溴化铵(CTAB)为分离缓冲液,在-8 kV下分离,于254 nm波长下检测,10 min内实现了食品中上述4种糖的同时分离与测定。乳糖、蔗糖、葡萄糖和果糖的检出限(S/N=3)分别为50、75、25和25 mg/L,定量限(S/N=10)分别为150、225、75和75 mg/L,回收率在87.0%~107.0%之间,相对标准偏差在1.2%~4.7%之间。整个实验过程未使用有机溶剂。用该法测定了9种食品样品及1个质控样品,结果表明该法简单、快速、准确,适用于食品中乳糖、蔗糖、葡萄糖和果糖的日常测定。     

Rapid and sensitive determination of carbohydrates in foods using high temperature liquid chromatography with evaporative light scattering detection

In the present work, an evaporative light scattering detector was used as a high-temperature liquid chromatography detector for the determination of carbohydrates. The compounds studied were glucose, fructose, galactose, sucrose, maltose, and lactose. The effect of column temperature on the retention times and detectability of these compounds was investigated. Column heating temperatures ranged from 25 to 175°C. The optimum temperature in terms of peak resolution and detectability with pure water as mobile phase and a liquid flow rate of 1 mL/min was 150°C as it allowed the separation of glucose and the three disaccharides here considered in less than 3 min. These conditions were employed for lactose determination in milk samples. Limits of quantification were between 2 and 4.7 mg/L. On the other hand, a temperature gradient was developed for the simultaneous determination of glucose, fructose, and sucrose in orange juices, due to coelution of monosaccharides at temperatures higher than 70°C, being limits of quantifications between 8.5 and 12 mg/L. The proposed hyphenation was successfully applied to different types of milk and different varieties of oranges and mandarins. Recoveries for spiked samples were close to 100% for all the studied analytes.     

Enzymatic synthesis of prebiotic oligosaccharides

Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrates through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an alpha-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.     

金属卤化物促进糖类催化转化制备5-羟甲基糠醛

研究了碱金属卤化物对AlCl₃催化葡萄糖转化制备5-羟甲基糠醛（HMF）的促进作用. 结果表明,NaF对反应有显著抑制作用,而NaI和NaBr对反应有显著促进作用,而且NaI比NaBr的促进效果更明显. 在N,N-二甲基乙酰胺（DMAC）中,以NaI为添加剂,130 ℃反应15 min,AlCl₃催化葡萄糖转化制备HMF,葡萄糖转化率由71%提高到86%,HMF收率由36%提高到62%. AlCl₃-NaI-DMAC体系也可用于果糖、甘露糖等单糖,蔗糖、麦芽糖、纤维二糖等二糖,以及菊粉等多糖的转化. 以蔗糖为原料,HMF收率可达63%.     

液相色谱法测定蚕卵中的碳水化合物

本文报道了蚕卵样品的前处理方法,它用于提取净化其中的水溶性糖和糖醇;采用Waters μ-Bondapak Carbohydrate柱,麦芽糖为内标,定量分析蚕卵内果糖、葡萄糖、山梨醇、蔗糖和海藻糖的含量。回收率在87%以上。     

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.     

Separation and quantification of beer carbohydrates by high-performance liquid chromatography with evaporative light scattering detection

An HPLC method with an evaporative light scattering detector was optimized and validated for quantification of carbohydrates in beer. The chromatographic separation was achieved using a Spherisorb NH2, 5 microm chromatographic column and gradient elution with acetonitrile/water. The determinations were performed in the linear range of 0.05-5.0 g/L for fructose, 0.05-5.0 g/L for glucose, 0.05-15.0 g/L for maltose, 0.05-10.0 g/L for maltotriose, and 0.05-5.0 g/L for maltotetraose. The detection limits were 0.005 g/L for fructose, 0.008 g/L for glucose, and 0.01 g/L for maltose, maltotriose, and maltotetraose. The reliability of the method in terms of precision and accuracy was evaluated in three beer matrices, low alcohol beer, 6% alcohol beer, and beer made with part of adjuncts (4.5% alcohol). Relative standard deviations (RSDs) ranged between 1.59 and 5.95% (n = 10), and recoveries ranged between 94 and 98.4%.     

A HPLC method for specific determination ofα-amylase and glucoamylase in complex enzymatic preparations

Summary In the hydrolysis of soluble starch by mixtures of α-amylase and glucoamylase, the ratios maltose/glucose and maltoriose/glucose linearly depend, over a wide range, on the relation between both enzymes and are independent on the activity level of the enzymatic preparation. HPLC determination of hydrolysis products (glucose, maltose and maltotriose) of soluble starch by mixtures of these enzymes, after incubation under controlled conditions, is a rapid method for the evaluation of the relative levels of each enzyme in the mixtures. The method, first developed using pure commercial amylases, is applied, with consistent results, to cell free media ofAspergillus niger cultures on a glycogen-rich effluent.     

Kinetics and mechanism of oxidation of some simple reducing sugars by permanganate ion in alkaline medium

The kinetics of oxidation of glucose, galactose, fructose, maltose and sucrose by alkaline permanganate anion has been studied. The reactions studied spectrophotometrically over a wide range of experimental conditions show that the rate of the reactions is enhanced by increase in pH, ionic strength, and temperature as well as the reactant concentrations. The mechanism has been proposed to proceed via the formation of enediol intermediate complexes and the order of reactivities of the sugars is fructose > glucose ≈ galactose > maltose > sucrose. The activation parameters were evaluated and lend further support to the proposed mechanism.