Monosaccharide Analysis After One-Pot Derivatization Followed by Reverse-Phase Liquid Chromatography Separation and UV/Vis Detection.

Derivatization of monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) introduces two chromophores per sugar molecule. Their separation on a superficially porous C18 reverse-phase column, using common liquid chromatography equipment, results in short analysis times (under 20 min) and high sensitivity (limit of quantitation 1 nmol). This method allows for complex monosaccharide mixtures to be separated and quantified using a reasonably simple and safe derivatization procedure.

Determination of Molecular Dimensions of Carbohydrate Polymers (Polysaccharides) by Size Exclusion Chromatography (SEC).

Calibrated size exclusion chromatography (SEC) is a useful tool for the analysis of molecular dimensions of polysaccharides. The calibration takes place with a set of narrow distributed dextran standards and peak position technique. Adapted columns systems and dissolving processes enable for the adequate separation of carbohydrate polymers. Plant-extracted fructan (a homopolymer with low molar mass and excellent water solubility) and mucilage (differently structured, high molar mass heteropolysaccarides that include existing supramolecular structures, and require a long dissolving time) are presented as examples of the versatility of this technique. Since narrow standards similar to the samples (chemically and structurally) are often unavailable, it must be noted that the obtained molar mass values and distributions by this method are only apparent (relative) values, expressed as dextran equivalents.

Neutral and Pectic Heteropolysaccharides Isolated from Opuntia joconostle Mucilage: Composition, Molecular Dimensions and Prebiotic Potential.

Opuntia joconostle is a semi-wild cactus cultivated for its fruit. However, the cladodes are often discarded, wasting the potentially useful mucilage in them. The mucilage is composed primarily of heteropolysaccharides, characterized by their molar mass distribution, monosaccharide composition, structural features (by vibrational spectroscopy, FT IR, and atomic force microscopy, AFM), and fermentability by known saccharolytic commensal members of the gut microbiota. After fractionation with ion exchange chromatography, four polysaccharides were found: one neutral (composed mainly of galactose, arabinose, and xylose) and three acidic, with a galacturonic acid content from 10 to 35%mol. Their average molar masses ranged from 1.8 × 105 to 2.8 × 105 g·mol-1. Distinct structural features such as galactan, arabinan, xylan, and galacturonan motifs were present in the FT IR spectra. The intra- and intermolecular interactions of the polysaccharides, and their effect on the aggregation behavior, were shown by AFM. The composition and structural features of these polysaccharides were reflected in their prebiotic potential. Lactobacilli and Bifidobacteria were not able to utilize them, whereas members of Bacteroidetes showed utilization capacity. The obtained data suggest a high economic potential for this Opuntia species, with potential uses such as animal feed in arid areas, precise prebiotic, and symbiotic formulations, or as the carbon skeleton source in a green refinery. Our methodology can be used to evaluate the saccharides as the phenotype of interest, helping to guide the breeding strategy.

Prebiotic potential and chemical characterization of the poly and oligosaccharides present in the mucilage of Opuntia ficus-indica and Opuntia joconostle.

The mucilage extracted from the convection-dried cladodes of O. ficus-indica and O. joconostle, two species of economic importance, delivered three fractions after methanol precipitation. Two were composed of high molar mass polysaccharides, and one included water-soluble mono-, di-, and oligosaccharides. The large polysaccharides have a molar mass range of 4.0 × 103 to 8.0 × 105 g·mol-1 and are consistently composed of galactose, arabinose, xylose, and rhamnose; however, the content of galacturonic acid was different between both fractions and species. Their fermentability by selected probiotics was relatively low, 11-27 % compared to glucose, and decreased with increasing levels of galacturonic acid in the molecules. In the third fraction, previously unreported oligosaccharides were found. These include simple- and complex-structured galactooligosaccharides with arabinosyl-, xylosyl- and galacturonosyl acid residues. Their fermentability by prebiotic species can be ascribed more to their structural characteristics and monosaccharide composition than their molecular dimensions.

Self-assembled high molecular weight inulin nanoparticles: Enzymatic synthesis, physicochemical and biological properties.

Inulin has interesting physicochemical and functional properties, and therefore a wide range of applications in the food and medical industries. It has gained great traction due to its ability to form nanoparticles and its possible application as nanovehicle for drug delivery. In this work, we demonstrated that the enzymatically-synthesized high molecular weight (HMW) inulin forms stable spherical nanoparticles with an average diameter of 112 ± 5 nm. The self-assemblage of HMW inulin nanoparticles is carried out during enzymatic synthesis of the polymer, and become detectable after a certain critical aggregation concentration (CAC) is reached. Both, the CAC and nanoparticle size are influenced by the reaction temperature. These nanoparticles are not toxic for peripheral blood mononuclear cells, at concentrations below 200 µg/mL; no significant prebiotic potential was detected in cultures of 13 probiotic strains. This work contributes to a better understanding of the formation of HMW inulin nanoparticles and their biological properties.

Fermentation of non-digestible raffinose family oligosaccharides and galactomannans by probiotics.

Due to their prebiotic potential indigestible oligosaccharides became a major focus of research interest. In this study the growth of selected probiotic strains including lactobacilli, bifidobacteria, Lactococcus lactis, Streptococcus salivarius ssp. thermophilus, Pediococcus ssp. and Enterococcus faecium with the, raffinose family oligosaccharides (RFOs) raffinose, stachyose and verbascose and galactomannan from guar bean Cyamopsis tetragonoloba (total guar carbohydrates, oligosaccharides (dp 2-4) and polysaccharides (dp > 5), obtained by size exclusion chromatography) were tested by means of turbidity measurements. RFOs were used by 75% of all strains, with some delay for the trisaccharide raffinose and the tetrasaccharide stachyose and a limited fermentation of the pentasaccharide verbascose. L. reuteri, P. pentosaceus and B. lactis HNO19™ were able to ferment not only raffinose and stachyose but also verbascose. Guar oligosaccharides were fermented by 15 out of 20 strains; P. acidilactici, L. acidophilus, L. rhamnosus GG and B. animalis ssp. lactis BB12 metabolized them comparably well as glucose or galactose. Isolated guar polysaccharides were not fermented whereas total guar carbohydrates were fermented by 7 strains, apparently caused by the oligosaccharide content. The findings of this study may be important for functional food products especially for indigestible oligosaccharides which may cause adverse effects in the gut when not cleaved.

Molecular dimensions and structural features of neutral polysaccharides from the seed mucilage of Hyptis suaveolens L.

The seed mucilage of Hyptis suaveolens L. includes acid - and neutral heteropolysaccharides in a ratio of about 1:1. The anionic charged fraction responsible for swelling and viscous behaviour possesses an average molar mass of Mw=350kg/mol, Mn=255kg/mol. The neutral polysaccharide fraction shows an average molar mass of Mw=47kg/mol and Mn=28kg/mol and is composed of d-Galp-, d-Glcp- and d-Manp residues in a molar ratio of about 3:2:1. The structural features present galactoglucan (30%) and galactoglucomannan (70%) with a high level of terminal ß-linked d-Galp residues (18%). Structural details of galactoglucomannan are derived by combined enzymatic and chemical methods as well as NMR spectroscopy. Sequences of octa/nonasaccharide ß-d-Glcp-(1→4)[ß-d-Galp-(1→2)-α-d-Galp-(1→6)]-ß-d-Manp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)[ß-d-Galp-(1→2)-α-d-Galp-(1→6)]-ß-d-Manp and lower mass tetrasaccharide repeating units ß-d-Glcp-(1→4)[ß-d-Galp-(1→2)-α-d-Galp-(1→6)]-ß-d-Manp were found. The level of the prebiotic activity is related to the availability of ß-linked d-Galp residues in the side chains of the molecules.

Prebiotic potential of neutral oligo- and polysaccharides from seed mucilage of Hyptis suaveolens.

Prebiotics are selectively fermented by the gastrointestinal microflora, resulting in benefits to human health. The seed mucilage of Hyptis suaveolens contains neutral and acidic polysaccharides in a ratio of 1:1. The neutral polysaccharides consist of galactose, glucose and mannose whereas the acidic polysaccharides contain fucose, xylose and 4-O-methylglucuronic acid -residues. The growth of probiotics in the presence of total, acidic or neutral polysaccharides and oligosaccharides was tested using turbidity measurements. The majority (11 out of 14) of the tested probiotic strains significantly grew in the neutral fraction. Growth occurred with some time delay, but may be longer lasting than with other lower molecular prebiotics. The extent of growth increased with neutral polysaccharides from H. suaveolens corresponding to the externally available galactose units (20%). In conclusion, neutral poly- and oligosaccharides from H. suaveolens have a prebiotic potential characterized by a delayed but long lasting effect.

Characterization of water and alkali extractable arabinoxylan from wheat and rye under standardized conditions.

Arabinoxylans (AXs) are an important component of wheat and rye dough. They bind water, contribute to the formation of viscous dough and improve the quality of bread. For the application of AX fractions in bread making process, it is useful to record a quality profile of wheat fractions compared to the quality profile of rye fractions under standardized conditions. In this work water and alkali extractable AX containing fractions, from wheat- and rye wholemeal, were extracted under standardized conditions and characterized. For analysis of composition, structural features, and molecular dimension a combination of chemical, physicochemical, enzymatic and chromatographic techniques was applied. The molar mass distributions obtained by means of an innovative colorimetric pentose detection in the eluted SEC fractions were comparable for all under standardized conditions extracted AXs. The determined molar masses of AXs extracted both from wheat- and from rye grain were close to 2.0 × 10(5) g/mol for water extractable AXs and 3.0 × 10(5) g/mol for alkali extractable AXs. Different susceptibility to endoxylanase treatment, having been observed as differences in the SEC profiles, may be evidence of structural differences between AXs depending on their origin. The viscosities of AX solutions were strongly influenced by their molar mass and structure; samples being less susceptible to endoxylanase provided solutions of higher viscosity.

Bioconversion of piceid to resveratrol by selected probiotic cell extracts.

Resveratrol exerts several pharmacological activities, including anti-cancer, anti-inflammatory, cardioprotective, or antioxidant effects. However, due to its occurrence in plants more in glycosidic form as piceid, the bioavailability and bioactivity are limited. The enzymatic potential of probiotics for the transformation of piceid to resveratrol was elucidated. Cell extract from Bifidobacteria (B.) infantis, B. bifidum, Lactobacillus (L.) casei, L. plantarum, and L. acidophilus was evaluated for their effect in this bioconversion using high-performance liquid chromatography (HPLC) as analytical tool. Cell extract of B. infantis showed the highest effect on the deglycosylation of piceid to resveratrol, already after 30 min. Cell extracts of all other tested strains showed a significant biotransformation with no further metabolization of resveratrol. The conversion of piceid to resveratrol is of importance to increase bioavailability and bioactivity as shown for anti-inflammation in this study. Cell extracts from probiotics, especially from B. infantis, may be added to piceid containing products, for achieving higher biological effects caused by the bioactivity of resveratrol or by health promoting of the probiotics. These findings open a new perspective of novel combination of cell extracts from probiotics and piceid, in health-promoting pharmaceutical and food products.

Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var. azul.

Fructo-oligosaccharides (FOSs) of a six year old agave plant variety, Agave tequilana, were isolated and fractionated by 2D preparative chromatography (SEC and rpHPLC). Structural analyses of different FOS-fractions were performed by reductive methylation analysis connected to GC/FID identification and NMR-analysis. FOSs from leaves (d.p. 3-8) contain single α-d-Glcp residues as well in terminal as internal position, however (2→1)-linked ß-d-Fruf residues only. FOSs from stem, however, contain as well (2→1)- and (2→6)-linked ß-d-Fruf residues with branched oligomeric repeating units. These characteristics indicate an enzymatically catalyzed metabolic regulation for the biosynthesis and transformation of fructans in A. tequilana which strongly depends on location and transport activities.

Characterization and in vitro immunomodulatory screening of fructo-oligosaccharides of Asparagus racemosus Willd.

Asparagus racemosus Linn. (Fam. Liliaceae) is an ethno-pharmacologically acclaimed Ayurvedic medicinal plant. In the present study, aqueous extract of A. racemosus (ARC) was fractionated and screened for the polysaccharide fraction (ARP). The characterization was done by enzymatic, Size Exclusion, gas chromatography with flame ionization detector (GC-FID), high pressure anion exchange chromatography (HPAEC) and thin layer chromatographic analyses. Phyto-chemical evaluation confirmed the presence of 26.7% of 2→1 linked fructo-oligosaccharides (FOS). They have a degree of polymerization (DP) of nearly 7-8. Cytotoxicity evaluation on P388 cell lines was consistent with low cytotoxicity of the extracts. In vitro Natural Killer (NK) cell activity was evaluated using human peripheral blood mononuclear cells (PBMC) isolated from whole blood on a ficoll-hypaque density gradient. K562 a myeloid leukemia cell line, were used as target cells. ARC, tested over the range 0.2-50 µg/ml, showed a dose-related stimulation of NK cell activity with a peak increase of 16.9±4.4% at 5.6 µg/ml. However, ARP demonstrated a higher stimulatory activity of 51.8±1.2% at 25 µg/ml. The results indicate that the FOS from A. racemosus potentiates the NK cell activity and this could be an important mechanism underpinning the 'Rasayana' properties of this plant.

Effect of Chlorophytum Borivilianum Santapau and Fernandes on sexual dysfunction in hyperglycemic male rats.

OBJECTIVE: To investigate the effects of Chlorophytum borivilianum (CB) on sexual dysfunction, loss of body weight, and lack of libido in hyperglycemic rats induced with streptozotocin or alloxan. METHODS: Wistar strain male albino rats were divided into five groups of six animals each: the control group (2% polyvinylpyrollidone solution), the streptozotocin control group (50 mg/kg), the alloxan control group (100 mg/kg), the streptozotocin + CB treated group (200 mg/kg), and the alloxan + CB treated group (200 mg/kg). Only after confirming the induction of diabetes, the animals of test groups were treated with CB. The sexual behavior of male rats of in presence of female rat in a special cage was recorded. The effects of induced diabetes in control groups and on simultaneous extract treatment in CB treated groups were tested for sexual parameters. The parameters evaluated included mount, ejaculation, and intromission latencies/frequencies, hesitation time, and penile erection index. Parallel to this, using a separate set of similarly treated animals, the influence of diabetes and CB treatment on anabolism and weight of secondary sexual organs were determined on day 0 and day 28 of the treatment. RESULTS: CB extract treatment ameliorated the diabetes-induced dysfunction at 200 mg/kg dose. There was very low weight loss (P<0.05) in CB-treated animals as compared to the diabetic control. There was a very high latency time (P<0.05) in the diabetic animals, whereas the latency time was very low in CB-treated animals. Mount, intromission, and ejaculation frequencies were very high (P<0.01) in CB-treated animals, while streptozotocin and alloxan groups animals had a very significantly lower sexual behavior (P<0.05) compared to the normo-glycemic control group animals. CONCLUSION: CB can significantly ameliorate diabetes-induced sexual dysfunction. Polysaccharide and saponin-rich aqueous extract appears to have the most suitable effects on diabetes and its associated effects on sexual functionality.

Effect of seasonal changes on content and profile of soluble carbohydrates in tubers of different varieties of Jerusalem artichoke (Helianthus tuberosus L.).

A high content (60-65% of dry mass DM) of water soluble carbohydrates was found in early harvested varieties (Bella and Bianka) and middle early varieties (Topstar and Gigant) harvested 22-25 weeks after plantation. In late varieties (Waldspindel, Violet de Rennes, Rote Zonenkugel) a similar amount was obtained (55-60% of DM) when harvested 29-33 weeks after planting. There was a distinctive impact on maturing process as well as frost period alterations which resulted in conversion of high polymer inulin to low polymer inulin as well as to sucrose. In early/middle early varieties a correlation between sucrose and inulin level (r = - 0.952**) with a linear regression of y = - 1.35x + 62.32 was observed, whereas the dpn of inulin decreased from 12-14 to 6-8. In late cultivars this correlation was not as exact (r = - 0.502**); dpn of inulin decreased from 12-16 to 7-10. This knowledge about carbohydrate profiles for different varieties of Jerusalem artichoke offers the possibility of selecting suitable cultivars and deciding the appropriate harvest time for an optimum processing of tubers for their application as prebiotic and novel food component.

De facto molecular weight distributions of glucans by size-exclusion chromatography combined with mass/molar-detection of fluorescence labeled terminal hemiacetals.

A major capability of polysaccharides in aqueous media is their tendency for aggregation and dynamic formation of supermolecular structures. Even extended dissolution processes will not eliminate these structures which dominate many analytical approaches, in particular absolute molecular weight determinations referring to light scattering data. An alternative approach for determination of de facto molecular weight for glucans with free terminal hemiacetal functionality (reducing end group) has been adjusted from carbohydrates for midrange and high-dp glucans: quantitative and stabilized labeling as aminopyridyl-derivatives (AP-glucans) and subsequent analysis of SEC-separated elution profiles based on simultaneously monitored mass and molar fractions by refractive index and fluorescence detection. SEC-DRI/FL of AP-glucans proved as an appropriate approach for determination of de facto molecular weight of constituting glucan molecules even in the presence of supermolecular structures for non-branched (pullulan), branched (dextran), narrow distributed and broad distributed and for mixes of compact and loose packed polymer coils (starch glucan hydrolizate).