Aqueous extracts of Agave sisalana boles have prebiotic potential

Abstract

This work aimed at evaluating the prebiotic potential of the aqueous extract and crude polysaccharides from Agave sisalana boles by an in vitro screening. Crude polysaccharides were obtained from the aqueous bole extract by precipitation with acetone and resuspension in water. The liquid extract and the polysaccharide solution were then spray dried and submitted to thermal analysis and quantification of metabolites. Prebiotic activity was checked on probiotic strains belonging to the Lactobacillus genus using inulin, fructo-oligosaccharides, fructose and glucose as positive controls. The powder of A. sisalana bole extract, which has recently been identified as a rich source of inulin, exhibited higher potential of fermentation compared with crude polysaccharides.     

Germplasm evaluation to obtain inulin with high degree of polymerization in Mediterranean environment

Abstract

Long-chain length (DP) inulin-type fructans show pronounced beneficial effects to colon microbiota. In the past, cardoon roots for inulin production to fortify spaghetti gave very good results, but their harvest requires high costs. The aim of the work was to evaluate different types of inulin, extracted from cardoon roots and buds, topinambur tubers and chicory roots local populations in order to identify crops more suitable for high DP inulin production in Mediterranean environment. The inulin amount resulted in 115 (chicory), 390 (cardoon) and 550?g kg^?1 of d.m. (topinambur). Both in cardoon roots and in topinambur tubers the DP was very high. With the aim to produce a functional food, substituting inulin in staple food tubers of topinambur could be a good alternative to cardoon roots, due to the biological cycle of the plant (annual vs poliennal), the lowest costs of harvest and the good amounts of high DP inulin.     

Synthetic Studies on Selectin Ligands/Inhibitors: One-Pot Synthesis of the Mono- and Oligo-Sulfated 2-(Tetradecyl)Hexadecyl β-D-Galacto- and Lactopyranosides as the Sulfatide Mimetics 1

Abstract

The ¹³C NMR spectra of inulin oligomers in D₂O with degree of polymerization (DP) of 3 through 9, along with two other inulin oligomer mixtures of average DP = 17 and DP = 31 were recorded. Significant variations in the chemical shift of some fructofuranose carbon signals indicates that unlike glucans, simple helical structures are not the predominant conformation for inulin oligomers—at least up to DP = 9. Models of the DP = 5 oligomer show that it should prefer a single helical conformation which however, would not be accessible to longer DP oligomers due to severe steric interactions.     

MD Calculations on Nystose Combined with NMR Spectroscopy on Inulin Related Oligosaccharides

Abstract

Molecular Dynamics (MD) calculations have been performed on nystose in water. According to these calculations the glycosidic linkages of the molecule are flexible. Structures obtained with MD calculations are compared with NMR data of several inulin related oligosaccharides and inulin, resulting in a model for the conformation of their fructofuranosyl residues. To extend the set of available NMR data of inulin related oligosaccharides, the complete assignment of the ¹H and ¹³C NMR signals of β-D-fructofuranosyl-(2->l)-ß-D-fructofuranosyl-(2->l)-ß-D-fructofuranosyl-(2->l)-ß-D-fructofuranosyl-(2->l)-α-D-glucopyranoside has been given here, using several 2D homo- and heteronuclear NMR experiments. Accurate coupling constants have been obtained by simulation of the 600 MHz 1D NMR spectra.     

Characterisation of different inulin samples by DSC: Influence of polymerisation degree on melting temperature

Melting behaviour of powder inulin has been studied by differential scanning calorimetry. DSC curves show two endothermic peaks, relative to water elimination and to inulin melting, respectively. The second peak is dependent on inulin type and a shift to a higher temperature is observed with increasing average polymerisation degree (DP) of the sample. For similar crystallinity index, linear relations have been underlined and so predicting inulin mean DP can be done by DSC analysis. The study shows that a relatively high heating rate (25°C min^–1) can be used and brings a supplementary interest by an important reduction of analysis time.This revised version was published online in November 2005 with corrections to the Cover Date.     

The Preparation and the Assignment of the 1H and 13C NMR Spectra of Methylated Derivatives of Inulin Oligosaccharides

Abstract

The complete assignment of the ¹H and ¹³C NMR spectra for permethylated sucrose, 1-kestose and nystose are given. Methylation of these inulin oligosaccharides did not change the conformation of the ring structures. A partially methylated derivative of nystose has been prepared using water as the solvent. The difference in reactivity of the various hydroxyl groups of nystose appeared to be very small.     

Statistical Spectral Analysis of liquid X-ray Diffraction Data

Abstract

The experimental procedure of determining the structure of a liquid by diffraction techniques is reformulated herein as a stochastic experiment subject to the data analysis formalism of statistical spectral analysis. Observed in such an experiment are averaged local microscopic fluctuations from the bulk density. The intensity function then represents a stochastic spectrum and it becomes necessary to statistically estimate a minimum bias, minimum variance covariance function which is the net radial distribution function. A low-pass tapered data window produces such an optimum estimate.     

Determination of Chlorpyrifos and its Metabolite 3,5,6-Trichloro-2-Pyridinol in Tap Water and Bananas by Quantitative TLC on Preadsorbent Silica Gel

Abstract

Chlorpyrifos insecticide and its metabolite TCP were determined in tap water and banana samples by TLC of extracts on preadsorbent silica gel layers, detection with silver nitrate reagent, and densitometric scanning. Cleanup steps were required for the fruit sample extracts. Recovery of chlorpyrifos from tap water at 5 ppb averaged 87.5% and from banana at 0.05 ppm was 84.6%. Recovery of TCP from water at 5 ppb averaged 84.0% and from banana at 0.05 ppm was 86.8%. The sensitivity and precision of the method were shown to be adequate for routine residue analysis.     

Water state diagram and thermal properties of fructans powders

The thermal analyses and the water state diagrams of agavin and inulin were investigated. The thermogravimetric analysis and differential scanning calorimetry (DSC) were used to know the difference between fructans. Linear inulin (220.0 °C) showed a higher thermal stability than the branched agavins (206.7 °C). The samples displayed different physical states and lightness depending on the water activity of storage (a _w). The agavin showed different physical states: powder (a _w < 0.33), soft lump (a _w ≈ 0.43), sticky (0.55 < a _w < 0.77), and liquid (a _w > 0.85), while inulin showed two physical states: an amorphous powder at a _w < 0.55 and a semicrystalline solid at a _w > 0.69. These physical states decreased the lightness of fructans. The GAB equation and the Gordon–Taylor equation showed a good fit to the experimental data (R ² > 0.95). The K constant of GAB equation predicted a lower water affinity of inulin powder, while the agavin powder presented a higher water affinity. The DSC showed a glass transition temperature of 118.1 and 135.3 °C for agavin and inulin anhydrous powders, respectively. An increase in water content in agavin showed a higher depression in the glass transition temperature than in inulin. Also, the inulin exhibited a melting point while agavin not. The water state diagrams showed the critical water activity at which the agavin (0.50) and inulin (0.59) powders were stable to physical changes. This knowledge is used to understand the physical and structural behaviors of agavin and inulin at the different water activities.     

NMR Study of the Inclusion Complexes of Carboxy-Phenoxathiin Derivatives with β-Cyclodextrin

The inclusion complexes of the carboxylate forms of 3-carboxy-(I) and 2-carboxy-phenoxathiin (II) with -cyclodextrin were studied by bothone- and two-dimensional NMR spectroscopy. The analysis of the induced chemical shifts of theguests in the presence of different amounts of the host indicates the formation of complexes with 1:1stoichiometry and association averaged pK values of 3.75 (I) and 4.4 (II). Thequalitative analysis of cross peaks in the ROESY spectra support the inclusion of the guests in the cavitywith the substituted phenyl ring, the COO^- group being in the proximity of the primary rim.     

HPLC Determination of Carbendazim in Formulations

Abstract

A rapid high performance liquid chromatographic method has been developed to determine Carbendazim (methyl 2-benzimidazole carbamate) in formulations using reverse phase μ Bondapak C₁₈ column and ultraviolet detection. The sample is extracted from formulations with methanol, carbaryl is used as an internal standard. Absorbance is measured at 254 nm and the compound is quantitated by peak height ratios. The method is simple and recoveries averaged between 91–93%.     

Determination of Cyclohexylamine in Water by Solid Phase Extraction and Quantitative High Performance Thin Layer Chromatography

Abstract

Cyclohexylamine (CHA) was isolated from water by solid phase extraction on a C₁₈ microcolumn. The CHA in the column eluate was chromatographed on a high performance preadsorbent silica gel layer, detected with ninhydrin reagent and exposure to UV light, and quantified by reflectance scanning. Recovery from fortified distilled water samples at 10 ppm averaged 95.0% and at 1 ppm averaged 94.9%. Quantitative recoveries from tap water at 10 ppm and from lake water at 1 ppm were also demonstrated. Other boiler water additives permitted by FDA do not interfere.     

Rapid simultaneous quantification of fructooligosaccharides in Morinda officianalis by ultra‐high performance liquid chromatography

Many Chinese herbal medicines with tonifying effects contain high levels of inulin fructooligosaccharides. These herbal medicines have high development and utilization value because of their effects against dementia, depression, and oxidative stress; on improving learning and memory ability; and on enhancing immunity. In this study, a method was developed for the separation and simultaneous quantitation of fructose, glucose, sucrose, and ten inulin fructooligosaccharides by ultra‐high‐performance liquid chromatography with evaporative light scattering detection within 10 min. Separation was performed on an Amide column with gradient elution. The calibration curves for the 13 constituents showed good linearity (R² > 0.9991). The limits of detection and quantification were 10.78–33.44 and 35.94–124.81 μg/mL, respectively, and the recoveries ranged from 98.90 to 103.67%. This method was successfully used to quantify the 13 constituents in the Chinese herbal medicine Morinda officinalis. The contents of the ten inulin fructooligosaccharides ranged from 56.28 to 60.71%. This method is accurate, rapid and simple and can be used for quantitative analysis in the quality control of herbal medicines and functional foods.     

Controlled Production of Fructose by an Exoinulinase from <Emphasis Type="Italic">Aspergillus Ficuum</Emphasis>

An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg⁻¹(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography. The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4 and 50 °C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation for 12 h and 72 h respectively. The respective K _m and V _max values were 4.75 mM and 833.3 μmol min⁻¹ ml⁻¹, respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 °C, 150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 °C, 150% inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose from inulin hydrolysis is biotechnologically feasible.     

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (<Emphasis Type="Italic">Arctium lappa</Emphasis>L.)

Background  

We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.     

Determination of Cefpodoxime Levels and Cefpodoxime Stability In Human Urine By Direct Injection HPLC with Column-Switching

Abstract

A semi-automated method providing on-line sample extraction and quantitative analysis for cefpodoxime in human urine, injected directly into the HPLC, is reported.

Samples were filtered by the analyst, injected into the HPLC system with an autosampler and loaded onto a 3 cm RP-18 precolumn with a mobile phase consisting of 10% methanol in 0.2% phosphoric acid and then automatically eluted onto a RP-18 analytical column using a mobile phase containing 7% acetonitrile in pH 5.2 sodium acetate buffer. the mean between-day precision of the standards was ± 4.29%. Spiked urine control recovery averaged 96 ± 6% for controls ranging from 1.0 to 20.0 μg/mL. the limit of quantitation for the method was 0.11 μg/mL.     

Characterization of Thermo-stable Endoinulinase from a New Strain <Emphasis Type="Italic">Bacillus Smithii</Emphasis> T7

A new thermophilic inulinase-producing strain, which grows optimally at 60 °C, was isolated from soil samples with medium containing inulin as a sole carbon source. It was identified as a Bacillus smithii by analysis of 16s rDNA. Maximum inulinase yield of 135.2 IU/ml was achieved with medium pH7.0, containing inulin 2.0%, (NH₄)H₂PO₄ 0.5%, yeast extract 0.5%, at 50 °C 200 rpm shaker for 72-h incubation. The purified inulinase from the extracellular extract of B. smithii T7 shows endoinulinolytic activity. The optimum pH for this endoinulinase is 4.5 and stable at pH range of 4.0–8.0. The optimum temperature for enzyme activity was 70 °C, the half life of the endoinulinase is 9 h and 2.5 h at 70 °C and 80 °C respectively. Comparatively lower Michaelis–Menten constant (4.17 mM) and higher maximum reaction velocity (833 IU/mg protein) demonstrate the endoinulinase’s greater affinity for inulin substrate. These findings are significant for its potential industrial application.     

Efficient utilization of inulin and glycerol as fermentation substrates in erythritol and citric acid production using <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> expressing inulinase

Inulin and glycerol were used as substrates for efficient erythritol and citric acid production by newly engineered Yarrowia lipolytica strains. Hydrolysis of inulin by the Y. lipolytica Wratislavia K1 strain was established by expressing the Kluyveromyces marxianus INU1 gene. Erythritol was produced in two stages: inulin was used for biomass formation, followed by erythritol biosynthesis initiated by glycerol addition. The highest titer of erythritol obtained, 120.9 g L^?1 with the yield of 0.6 g g^?1, was produced by the K1 INU 6 strain. Moreover, the K1 INU 6 strain in fed-batch culture produced a high amount of citric acid: 105.2 g L^?1 after 235 h from 200 g L^?1 of inulin. Maximum activity of inulinase during this culture was 14000 U g^?1 of cell dry mass. The presented study proves the potential of new Y. lipolytica transformants for efficient erythritol and citric acid production from inexpensive raw materials such as inulin and glycerol.     

Polysaccharides as Effective Sorbents for Lead and Cadmium

Sorption of lead and cadmium cations from solutions of their salts on amorphous cellulose, potato starch, apple pectin, and chicory root inulin was studied. Sample preparation was performed in conditions (pH and stirring time and intensity) similar to those in the stomach and intestines of a typical person. The sorption properties of the studied polysaccharides with respect to both the individual heavy metals and their mixtures is variable. The sorption properties series are cellulose ≈ pectin < starch< inulin for Pb²⁺ and cellulose < pectin < starch ≈ inulin for Cd²⁺.     

Isolation and identification of inulooligosaccharides resulting from inulin hydrolysis

In this study, inulooligosaccharides (F_n-type inulin) resulting from the endo-inulinase hydrolysis of globe artichoke inulin were purified and characterized. The aim was to produce F_n oligomer standards with the intention of identifying them in the complex inulin chromatogram. Inulin was extracted from globe artichoke and presented a high average degree of polymerization (DP) of about 80 as determined by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). This inulin was hydrolyzed by a commercial endo-inulinase yielding a product with a very high F_n/GF_n molecule ratio, thus limiting the interference of GF_n during the purification process. High-performance size exclusion chromatography was used to individually isolate and collect each retention peak corresponding to a specific oligomer. The purity of these fractions was checked by HPAEC-PAD and showed that relatively pure molecules were produced. Electrospray ionization mass spectrometry allowed the molecular weight determination of these purified oligomers and ascertained their DP as F₂, F₃ and F₄. These F_2-4 standards were used with glucose, fructose, sucrose and GF_2-4 (commercially available) to spike commercial oligofructose products in order to determine the elution profile in the HPAEC-PAD chromatogram.