Influence of reaction medium composition on enzymatic synthesis of galactooligosaccharides and lactulose from lactose concentrates prepared from whey permeate

The paper presents the results of investigations into the technological possibilities of controlling the transgalactosylation process of lactose in permeate after whey ultrafiltration in order to improve the efficiency of galactooligosaccharides or lactulose synthesis. The synthesis efficiency was influenced by the selection of a β-galactosidase preparation, substrate concentration and, in the synthesis of lactulose, also by the ratio of lactose and fructose added to the reaction mixture. The obtained synthesis efficiency of GOS and, most of all, of lactulose (65 g L⁻¹), may be found satisfactory. The study also resulted in a proposed GOS or lactulose concentrates (concentrated or dried) production technology using permeate after ultrafiltration of milk or whey as lactose sources. Presented at the 35th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 26–30 May 2008.     

A spectrophotometric method for quantitative determination of lactulose in pharmaceutical preparations

A simple spectrophotometric assay for the quantification of lactulose in pharmaceutical preparations was developed. The method is based on hydrolysis of lactulose under acidic conditions. The hydrolyzed product reacts with resorcinol, giving absorption peaks at 398 and 480 nm. Both absorption wavelengths can be used for the determination of lactulose. The limit of detection of lactulose at 398 nm and 480 nm was 0.075 μg mL⁻¹ and 0.65 μg mL⁻¹, respectively. The calibration was linear in the range of 5–25 μg mL⁻¹. Analytical conditions were optimized, and the method was validated for analysis of pharmaceutical preparations. The determined amount of lactulose was found to be in good agreement with labeled claims in commercial products. The proposed method is economical, convenient, and suitable for the quantification of lactulose in pharmaceutical preparations. The text was submitted by the authors in English.     

Analysis of free carbohydrates in milk using micropacked columns

Summary A gas chromatographic method using a micropacked column is described for the analysis of lactose, galactose, lactulose and epilactose in processed milks. The method is evaluated for precision and accuracy using phenyl-β-glucoside as an internal standard. Recoveries near 100% were found for lactulose concentrations higher than 0.1 mgml⁻¹, showing coefficients of variation from 5.9 to 9.4%.     

Rapid determination of lactulose in milk by microdialysis and biosensors

A simple and rapid flow system for the determination of lactulose in milk samples was developed. It is based on the hydrolysis of lactulose to galactose and fructose by the enzyme beta-galactosidase immobilised in a reactor. The amount of fructose produced was measured with an electrochemical biosensor based on the fructose dehydrogenase enzyme, K3[Fe(CN)6] as mediator and a platinum based electrochemical transducer. Parameters such as the enzyme immobilisation in the reactor and under the electrode surface, the lifetime of the beta-galactosidase reactor and of the dehydrogenase biosensor and the flow parameters were studied and optimised. Fructose was determined in the range 1 x 10(-6)-5 x 10(-3) mol l-1 with an RSD of about 2% and a detection limit of 5 x 10(-7) mol l-1. The use of a microdialysis probe as the sampling system permitted the direct measurement of lactulose in milk samples without pre-treatment in the range 1 x 10(-5)-5 x 10(-3) mol l-1. The sensitivity of the procedure allowed pasteurised, UHT and in-container sterilised milk to be distinguished.     

Ready detection of small amounts of lactulose in dairy products by thin-layer chromatography

Summary Ready detection of lactulose in milk and in mixtures with other sugars is achieved by diluting the sample ten times with acetone and successive thin-layer chromatographic separation on silica gel-boric acid, developed with acetonitrile: water 5020. The method allows detection of up to 0.02% lactulose on total carbohydrates and permits a semi-quantitative estimation of lactulose in milk.     

Methyl methacrylate-co-itaconic acid (MMA-co-IA) hydrogels for controlled drug delivery

In the present work methyl methacrylate-co-itaconic acid (MMA-co-IA) hydrogels were synthesized by free radical copolymerization of methyl methacrylate (MMA) and itaconic acid (IA) using ethylene glycol dimethacrylate (EGDMA) and methylene bisacrylamide (MBAAm) as crosslinkers and benzoyl peroxide as initiator. Selected samples were loaded with model drug lactulose. For the lactulose release, the effect of pH, monomeric compositions, degree of crosslinking were investigated. The release of lactulose was studied for 8 h period in USP phosphate buffer solutions of varying pH 1.2, 5.5, 6.5 and 7.0. The drug release data were fitted into various kinetics models like the zero order, first order, Higuchi and Peppas. The release kinetics of lactulose from MMA/IA hydrogels was found to be best described by the Peppas model. Results showed that drug release increased by increasing IA content in the hydrogels but the effect of changing of crosslinking ratio on drug release was not significant. The surface morphology of MMA/IA drug loaded hydrogel was studied by SEM which revealed uniform distribution of the drug in the hydrogels. In conclusion, it can be said that lactulose can be successfully incorporated into crosslinked MMA/IA hydrogels and its release can be modulated by changing the mole fraction of the acid component in the gels.     

Determination of lactulose and mannitol in urine by HRGC

Alteration of intestinal permeability towards compounds like sugars is an indication of abnormalities observed in some diseases. Measurement of the dual sugar absorption of lactulose and mannitol is a non-invasive test of intestinal permeability. The use of this test is however limited by difficulties in the analysis of sugars in urine at low concentrations. In this study we have developed an analytical procedure for quantitative assay of the lactulose–mannitol pair in urine by high resolution gas chromatography. The minimum detectable amounts are 7.5 pg for lactulose and 4.4 pg for mannitol. The calibration curves are linear in the range of 100–6400 mg L^?1. Recoveries are within the range 86–102 % for mannitol and 84–105 % for lactulose.     

Simple and sensitive multi-sugar-probe gut permeability test by high-performance liquid chromatography with fluorescence labelling

Enteral intake of a mixture of inert, non-metabolic monosaccharide and disaccharide probes, followed by measurement of their urinary probe ratio, is a well known method to investigate gut permeability. However, most applications lack sensitivity, thus a large amount of especially the disaccharide lactulose has to be ingested. This may cause diarrhoea, which influences the outcome of the test.

Recently, a new fluorescent label 9-fluorenylmethyl chloroformate hydrazine (FMOC-hydrazine) was introduced, which reacts with reducing sugars to form stable and highly fluorescent single peak derivatives in organic medium. We applied this reagent to develop a sensitive measurement of reducing sugar probes in aqueous samples (e.g. urine).

The presented method has a linear response for each sugar derivative between 1 and 1250 pmol with an R² ranging from 0.9997 for lactulose to 0.9999 for rhamnose. The limit of detection, calculated as a signal-to-noise ratio of three, was 0.05 pmol for lactulose and 0.01 pmol for rhamnose, xylose and 3-O-methyl-D-glucose, corresponding to urine concentrations of 0.11 μmol/1 for lactulose and 0.02 μmo1/l for rhamnose, xylose and 3-O-methyl-D-glucose. Compared to other tests, the limit of detection is very low. This enabled a reduction in the enteral intake of the disaccharide lactulose from 6–10 g to 1.5 g, thereby minimizing the chance of introducing diarrhoea. The coefficient of variation was below 3% both in standards and urine samples. After spiking the urine with the saccharides a recovery of 102% for lactulose, 101% for rhamnose, xylose and 3-O-methyl-D-glucose was found.

In order to evaluate the presented method we compared the lactulose rhamnose ratio measured in urine of healthy human volunteers and kept the ingested dose in agreement with literature values. Furthermore, the ratio was measured after 3, 6 and 9 h to establish the minimal response time required to measure correct ratios. We found that even after 3 h the ratio was stable at a value of 0.0133 which is comparable to literature values (0.008-0.052).     

离子色谱法测定大肠癌患者尿液中的甘露醇和乳果糖

建立了大肠癌患者尿液中甘露醇和乳果糖的高效阴离子交换分离-脉冲积分安培(HPAEC-PAD)检测方法.样品经稀释、净化、过滤后进行色谱分析,采用Carbopac PA1(250 mm×4mm)阴离子交换柱分离,以NaOH-乙酸钠梯度淋洗液为流动相,流速1.0 mL/min,脉冲积分安培检测器(PAD)检测,外标法定量.结果表明,甘露醇和乳果糖的的线性范围为0.2～20 mg/L,相关系数分别为0.9995和0.9998,回收率为91.7％～102.6％,相对标准偏差均小于2.1％,检出限为0.05和0.06 mg/L(S/N=3).本方法前处理简便、灵敏度高、重现性好,可用于大肠癌患者尿液中的甘露醇和乳果糖的测定.     

A Simple,Robust, and Convenient HPLC Assay for Urinary Lactulose and Mannitol in the Dual Sugar Absorption Test

Background: Heterogeneous laborious analytical methodologies for the determination of urinary lactulose and mannitol limit their utility in intestinal permeability testing. Methods: We developed an assay using a Shimadzu HPLC system, an Aminex HPX87C column, and refractive index detection. The test was calibrated using a series of dilutions from standard stock solutions of lactulose and mannitol ‘spiked’ into urine samples. The utility to quantify urinary excretion during the dual sugar absorption test over 6 h was also determined. Results: Lactulose and mannitol were eluted isocratically at 5.7 and 10.1 min, respectively, with water as a mobile phase at a flow rate of 0.3 mL min⁻¹, 858 psi, 60 °C. The calibration curves for both sugars were linear up to 500 µg mL⁻¹ with a limit of detection in standard solutions at 4 µg mL⁻¹ and in ‘spiked’ urine samples at 15 µg mL⁻¹. The intra-assay and inter-assay CVs were between 2.0–5.1% and 2.0–5.1% for lactulose and 2.5–4.4% and 2.8–3.9% for mannitol. The urinary profiles of the 6 h absorption of lactulose and mannitol showed similar peak-retention times to standard solutions and were well-resolved at 5.9 and 10.4 min, respectively. Conclusions: The assay was easy to automate, using commonly available equipment and convenient requiring no prior laborious sample derivatization. The simplicity, reproducibility, and robustness of this assay facilitates its use in routine clinical settings for the quantification of intestinal permeability.     

Effect of synthesis parameters on polymethacrylic acid xerogel structures and equilibrium swelling

Hydrogels based on crosslinked polymethacrylic acid were synthesized via free-radical polymerization in aqueous solution, using N,N′-methylene bisacrylamide as a crosslinking agent and 2,2′-azobis-[2-(2-imidazolin-2-yl)propane] dihydrochloride as an initiator. The influence of the reaction parameters (the neutralization degree of methacrylic acid and the initial monomer concentration) on the equilibrium swelling degree, the swelling kinetic parameters and the basic structural properties of xerogels was investigated. The change of synthesis parameters leads to the change of the basic structural parameters of xerogel, as well as the equilibrium swelling degree and the initial swelling rate of the hydrogels. It is found that there are power form relationships between the equilibrium swelling degree, the initial swelling rate and the structural xerogel’s properties and the change of the neutralization degree of monomer, i.e. the monomer concentration. The examined correlations proved that the crosslinking density is the crucial parameter which determines all the other investigated structural and swelling parameters. The article is published in the original.     

高效阴离子交换色谱-脉冲安培法检测小鼠尿液中的甘露醇、单糖和乳果糖

采用高效阴离子交换色谱-脉冲安培检测法(HPAEC-PAD)建立了小鼠尿液中甘露醇、单糖(包括半乳糖、葡萄糖、甘露糖和果糖)和乳果糖的分析方法。样品经离心沉淀除去蛋白并过分子膜,以CarboPacTM PA1阴离子交换柱为分离柱,采用NaOH梯度淋洗,脉冲安培四电位检测。结果表明,甘露醇、半乳糖、葡萄糖、甘露糖、果糖和乳果糖在0.1～5.0 mg/L内线性良好,线性相关系数r2为0.988～0.999,样品加标回收率为95.5%～104.2%,检出限为0.0013～0.0048 mg/L。此法准确、快速、简便,能同时对6种糖类化合物进行分析,可以跟踪检测整个糖类代谢过程中甘露醇、单糖和乳果糖之间的代谢关系。     

A Lactulose Bienzyme Biosensor Based on Self‐Assembled Monolayer Modified Electrodes

A bienzyme biosensor in which the enzymes β‐galactosidase (β‐Gal), fructose dehydrogenase (FDH), and the mediator tetrathiafulvalene (TTF) were coimmobilized by cross‐linking with glutaraldehyde atop a 3‐mercaptopropionic acid (MPA) self‐assembled monolayer on a gold disk electrode, is reported. The working conditions selected were E_app=+0.10 V and (25±1) °C. The useful lifetime of one single TTF‐β‐Gal‐FDH‐MPA‐AuE was surprisingly long, 81 days. A linear calibration plot was obtained for lactulose over the 3.0×10^?5–1.0×10^?3 mol L^?1 concentration range, with a limit of detection of 9.6×10^?6 mol L^?1. The effect of potential interferents (lactose, glucose, galactose, sucrose, and ascorbic acid) on the biosensor response was evaluated. The behavior of the SAM‐based biosensor in flow‐injection systems in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining lactulose in a pharmaceutical preparation containing a high lactulose concentration, and in different types of milk. Finally, the analytical characteristics of the TTF‐β‐Gal‐FDH‐MPA‐AuE are critically compared with those reported for other recent enzymatic determinations of lactulose.     

Description of the adsorption equilibrium of organic substances absorbed from a flow of moist air by a fixed bed of active carbon moistened to equilibrium

Isotherms of the adsorption of benzene vapor from moist air on active carbon (AC) moistened to equilibrium are obtained from dynamic experiments. The experimental data on the adsorption equilibrium of some organic substances from a flow of moist air by a fixed bed of AC moistened to equilibrium are obtained. The data on the equilibrium adsorption of benzene vapor is analyzed using the Dubinin-Radushkevich equation (the theory of volume filling of micropores). It is revealed that the characteristic adsorption energy of benzene vapor decreases as the filling of the microporous volume with water molecules increases. The characteristic adsorption energy depends on the following factors: polarizability of a substance in the adsorption field created by micropores, the number of carbon atoms in the adsorbate molecules, and parameters of the porous structure. The equation for the calculation of the parameters of equilibrium adsorption of organic substances from moist air on AC moistened to equilibrium are obtained.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 434–438, March, 1995.     

The Influence of Preparation Methods on the Swelling and Network Properties of Acrylamide Hydrogels with Crosslinkers

Abstract

Using different types and concentrations of crosslinkers, acrylamide (AAm) hydrogels have been prepared with chemical initiation and gamma irradiation techniques. The effects of the preparation method, crosslinkers type and concentration on swelling behavior of AAm hydrogels were investigated. Swelling was performed in distilled water and followed gravimetrically. Swelling parameters such as equilibrium swelling degree, equilibrium water content (EWC), maximum swelling, initial swelling rate, diffusion exponent and coefficient, and network parameters such as molecular mass between crosslinks, crosslink density, mesh size, and porosity were calculated and evaluated. The range of equilibrium swelling degree of AAm hydrogels was varied from 255% to 1450% depending upon the preparation method, crosslinker type, and crosslinker concentration. The diffusion of water into AAm hydrogels was found to be nonFickian.     

Extracting parameters for base-pair level models of DNA from molecular dynamics simulations

 A method is described to extract a complete set of sequence-dependent energy parameters for a rigid base-pair model of DNA from molecular dynamics (MD) simulations. The method is properly consistent with equilibrium statistical mechanics and leads to effective inertia parameters for the base-pair units as well as stacking and stiffness parameters for the base-pair junctions. We give explicit formulas that yield a complete set of base-pair model parameters in terms of equilibrium averages that can be estimated from a time series generated in an MD simulation. The expressions to be averaged depend strongly both on the choice of coordinates used to describe rigid-body orientations and on the choice of strain measures at each junction. Received: 12 July 2000 / Accepted: 5 January 2001 / Published online: 3 May 2001     

Non-equilibrium capillary electrophoresis of equilibrium mixtures--appreciation of kinetics in capillary electrophoresis

We describe a new electrophoretic method (patent pending), Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM), and demonstrate its application to the study of protein-DNA interactions. A single NECEEM experiment allows for the determination of equilibrium and kinetic parameters of protein-DNA complex formation. The equilibrium mixture is prepared by mixing protein and DNA; it contains three components: free protein, free DNA, and the protein-DNA complex. A small plug of such a mixture is injected onto a capillary and the three components are separated under non-equilibrium conditions using a run buffer that does not contain the components of the equilibrium mixture. The protein-DNA complex decays during the NECEEM separation; the resulting electropherograms contain characteristic peaks and exponential curves. A simple analysis of a single electropherogram reveals two parameters: the equilibrium dissociation constant of the protein-DNA complex and the monomolecular rate constant of complex decay. The bimolecular rate constant of complex formation can then be calculated as the ratio of the two experimentally-determined constants. NECEEM was applied to find the equilibrium and kinetic parameters of interaction between an E. coli single-stranded DNA binding protein and a fluorescently-labeled oligonucleotide. The constants determined by NECEEM are in good agreement with those obtained by other methods. The new method is simple, fast, and accurate. It can be equally applied to other non-covalent molecular complexes.     

Ion-exchange chromatography of lactose-lactulose isomerization mixtures using a boric acid-borate eluent

Mixtures obtained by the isomerization of lactose on anion exchangers have been analysed by ion-exchange chromatography. With a 57-mm column of Aminex A-25 and 0.400 M H₃BO₃-0.005 M Na₂B₄O₇ as eluent, a complete separation of actose, epilactose (4-O-β-d-galactopyranosyl-d-mannose), lactulose, galactose and tagatose is obtained in about 50 min. The influence of various factors on the qualitative and quantitative results is given. The ratio of Na₂B₄O₇ to H₃BO₃ in the eluent has an important influence on the order of elution of ketoses and aldoses, especially at low ratios. A special device is described for removing excess of lactose during the analysis. The method describe is also applicable to the analysis of lactulose syrups.