Cost-effective and controllable synthesis of isomalto/malto-polysaccharides from ß-cyclodextrin by combined action of cyclodextrinase and 4,6-α-glucanotransferase GtfB.

Isomalto/malto-polysaccharides (IMMPs) derived from malto-oligosaccharides such as maltoheptaose (G7) are elongated non-branched gluco-oligosaccharides produced by 4,6-α-glucanotransferase (GtfB). However, G7 is expensive and cumbersome to produce commercially. In this study, a cost-effective enzymatic process for IMMPs synthesis is developed that utilizes the combined action of cyclodextrinase from Palaeococcus pacificus (PpCD) and GtfB-ΔN from Limosilactobacillus reuteri 121 to convert ß-cyclodextrin into IMMPs with a maximum yield (16.19 %, w/w). The purified IMMPs synthesized by simultaneous or sequential treatments, designated as IMMP-Sim and IMMP-Seq, possess relatively high contents of α-(1 â†’ 6) glucosidic linkages. By controlling the release of G7 and smaller malto-oligosaccharides by PpCD, IMMP-Seq was obtained of DP varying from 12.9 to 29.5. Enzymatic fingerprinting revealed different linkage-type distribution of α-(1 â†’ 6) linked segments with α-(1 â†’ 4) segments embedded at the reducing end and middle part. The proportion of α-(1 â†’ 6) segments containing the non-reducing end was 56.76 % for IMMP-Sim but 28.98 % for IMMP-Seq. Addition of G3 or G4 as specific acceptors resulted in IMMPs exhibiting low polydispersity. This procedure can be applied as a novel bioprocess that does not require costy high-purity malto-oligosaccharides and with control of the average DP of IMMPs by adjusting the substrate composition.

Convergent synthesis of oligomannose-type glycans via step-economical construction of branch structures.

Oligomannose-type glycans on glycoproteins are important signaling molecules in the glycoprotein quality control system in the endoplasmic reticulum. Recently, free oligomannose-type glycans generated by the hydrolysis of glycoproteins or dolichol pyrophosphate-linked oligosaccharides were recognized as important signals for immunogenicity. Hence, there is a high demand for pure oligomannose-type glycans for biochemical experiments; however, the chemical synthesis of glycans to achieve high-concentration products is laborious. In this study, we demonstrate a simple and efficient synthetic strategy for oligomannose-type glycans. Sequential regioselective α-mannosylation at the C-3 and C-6 positions of 2,3,4,6-unprotected galactose residues in galactosylchitobiose derivatives was demonstrated. Subsequently, the inversion of the configuration of the two hydroxy groups at the C-2 and C-4 positions of the galactose moiety was successfully carried out. This synthetic route reduces the number of the protection-deprotection reactions and is suitable for constructing different branching patterns of oligomannose-type glycans, such as M9, M5A, and M5B.

Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae ß-galactosidase, followed by purification using silica gel.

The aim of this study was to optimize and compare the production of galactooligosaccharides (GOSs) by free and cotton cloth-immobilized Aspergillus oryzae ß-galactosidase, and perform economical evaluation of production of GOSs (100%) between them. Using the response surface method, the optimal reaction time (3.9 h), initial lactose concentration (57.13%), and enzyme to lactose ratio (44.81 U/g) were obtained for the free enzyme, which provided a GOSs yield of 32.62%. For the immobilized enzyme, the optimal yield of GOSs (32.48%) was obtained under reaction time (3.09 h), initial lactose concentration (52.74%), and temperature (50.0 ℃). And it showed desirable reusability during five successive enzymatic reactions. The recovery rate of GOSs (100%) is 65% using silica gel filtration chromatography. The economical evaluation showed almost no difference in the manufacturing cost for the GOSs (100%) between these two systems, and that the recovery rate had a great impact on the cost.

Development and assessment of GC/MS and HPAEC/PAD methodologies for the quantification of α-galacto-oligosaccharides (GOS) in dry beans (Phaseolus vulgaris).

The quantification of α-Galacto-oligosaccharides (GOS) in beans has been increasingly approached through different methodologies. However, reported GOS contents revealed up to 8-times disparity, which cannot be only attributed to the bean cultivar and underlines the need of using validated analytical methodologies. This study aimed to optimize and validate the extraction of the most abundant GOS found in beans, namely raffinose, stachyose and verbascose, and comparatively assess their determination by High-Performance Anion Exchange Chromatography/Pulsed Amperometric Detector (HPAEC/PAD) and Gas Chromatography/Mass Spectrometry (GC/MS). Hot sonication followed by shaking with 70% ethanol resulted in excellent GOS extraction efficiencies (92.54-107.94%). GC/MS determination was more reliable than HPAEC/PAD, with limits of quantification of 4.48-224.31 mg/kg and intra/inter-day repeatabilities <10%. The analysis of six bean varieties proved the feasibility of the GC/MS methodology, displaying total GOS contents from 1453.07 ±â€¯169.31 to 2814.34 ±â€¯95.28 mg/100 g. Stachyose was significantly (p < 0.05) the main GOS in all samples.

Advances in the preparation and assessment of the biological activities of chitosan oligosaccharides with different structural characteristics.

Chitosan oligosaccharides (COSs) are widely used biopolymers that have been studied in relation to a variety of abnormal biological activities in the food and biomedical fields. Since different COS preparation technologies produce COS compounds with different structural characteristics, it has not yet been possible to determine whether one or more chito-oligomers are primarily responsible for the bioactivity of COSs. The inherent biocompatibility, mucosal adhesion and nontoxic nature of COSs are well documented, as is the fact that they are readily absorbed from the intestinal tract, but their structure-activity relationship requires further investigation. This review summarizes the methods used for COS preparation, and the research findings with regard to the antioxidant, anti-inflammatory, anti-obesity, bacteriostatic and antitumour activity of COSs with different structural characteristics. The correlation between the molecular structure and bioactivities of COSs is described, and new insights into their structure-activity relationship are provided.

Maltoheptaoside hydrolysis with chromatographic detection and starch hydrolysis with reducing sugar analysis: Comparison of assays allows assessment of the roles of direct α-amylase inhibition and starch complexation.

The aim was to determine inhibition of human α-amylase activity by (poly)phenols using maltoheptaoside as substrate with direct chromatographic product quantification, compared to hydrolysis of amylose and amylopectin estimated using 3,5-dinitrosalicylic acid. Acarbose exhibited similar IC50 values (50% inhibition) with maltoheptaoside, amylopectin or amylose as substrates (2.37 ± 0.11, 3.71 ± 0.12 and 2.08 ± 0.01 µM respectively). Epigallocatechin gallate, quercetagetin and punicalagin were weaker inhibitors of hydrolysis of maltoheptaoside (<50% inhibition) than amylose (IC50: epigallocatechin gallate = 20.41 ± 0.25 µM, quercetagetin = 30.15 ± 2.05 µM) or amylopectin. Interference using 3,5-dinitrosalicylic acid was in the order punicalagin > epigallocatechin gallate > quercetagetin, with minimal interference using maltoheptaoside as substrate. The main inhibition mechanism of epigallocatechin gallate and punicalagin was through complexation with starch, especially amylose, whereas only quercetagetin additionally binds to the α-amylase active site. Interference is minimised using maltoheptaoside as substrate with product detection by chromatography, potentially allowing assessment of direct enzyme inhibition by almost any compound.

Dietary galactosyl and mannosyl carbohydrates: In-vitro assessment of prebiotic effects.

The prebiotic activities of hydrolyzed guar gum (GMOS, <1 kDa; GMPS, 1-10 kDa), manno-oligosaccharide (MOS, <1 kDa), and galacto-oligosaccharide (GOS, <1 kDa) were evaluated by in vitro fermentation. The tested carbohydrates showed selective prebiotic effects on bacterial growth, short-chain fatty acid (SCFA)-production, and substrate consumption. GOS and GMOS markedly promoted the growth of bifidobacteria and Clostridium butyricum, respectively, whereas MOS showed the strongest butyrogenic effect. Moreover, SCFA production in the hydrolyzed guar gum groups was closely related to the varied molecular weight (Mw) of the hydrolysate. During in vitro fermentation with human fecal inocula, GMOS gave the highest yields of lactate, propionate, and butyrate after 48 h fermentation. Combined application of MOS and C. butyricum increased the abundance of Clostridiaceae_1. Overall, our results indicate that galactosyl and mannosyl carbohydrates have individualized prebiotic effects which are associated with their chemical structures including their glycoside composition and Mw.

Fast and Low-Cost Purification Strategy for Oligosaccharide Synthesis Based on a Hop-On/Off Carrier.

A feasible and convenient strategy for oligosaccharide synthesis, which realizes reaction in solution while product purification occurs only by solid-liquid filtration, has been developed. By using a hop-on/off carrier (polytetrafluoroethylene particle), rapid synthesis of tumor-associated antigen Globo-H hexasaccharide has been successfully achieved within 5 steps in 48% overall yield without any intermediate purification by column chromatography. Also, global deprotection, including the cleavage of the tag, proceeded simultaneously only by one-step hydrogenolysis.

Production, Structural Characterization, and In Vitro Assessment of the Prebiotic Potential of Butyl-Fructooligosaccharides.

Short-chain fatty acids (SCFAs), especially butyrate, produced in mammalian intestinal tracts via fermentation of dietary fiber, are known biofunctional compounds in humans. However, the variability of fermentable fiber consumed on a daily basis and the diversity of gut microbiota within individuals often limits the production of short-chain fatty acids in the human gut. In this study, we attempted to enhance the butyrate levels in human fecal samples by utilizing butyl-fructooligosaccharides (B-FOS) as a novel prebiotic substance. Two major types of B-FOS (GF3-1B and GF3-2B), composed of short-chain fructooligosaccharides (FOS) bound to one or two butyric groups by ester bonds, were synthesized. Qualitative analysis of these B-FOS using Fourier transform infrared (FT-IR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), nuclear magnetic resonance (NMR) and low-resolution fast-atom bombardment mass spectra (LR-FAB-MS), showed that the chemical structure of GF3-1B and GF3-2B were [O-(1-buty-ß-D-fru-(2→1)-O-ß-D-fru-(2→1)-O-ß-D-fru-O-α-D-glu] and [O-(1-buty)-ß-D-fru-(2→1)-O-ß-D-fru-(2→1)-O-(4-buty)-ß-D-fru-O-α-D-glu], respectively. The ratio of these two compounds was approximately 5:3. To verify their biofunctionality as prebiotic oligosaccharides, proliferation and survival patterns of human fecal microbiota were examined in vitro via 16S rRNA metagenomics analysis compared to a positive FOS control and a negative control without a carbon source. B-FOS treatment showed different enrichment patterns on the fecal microbiota community during fermentation, and especially stimulated the growth of major butyrate producing bacterial consortia and modulated specific butyrate producing pathways with significantly enhanced butyrate levels. Furthermore, the relative abundance of Fusobacterium and ammonia production with related metabolic genes were greatly reduced with B-FOS and FOS treatment compared to the control group. These findings indicate that B-FOS differentially promotes butyrate production through the enhancement of butyrate-producing bacteria and their metabolic genes, and can be applied as a novel prebiotic compound in vivo.

Docking proteins and peptides under evolutionary constraints in Critical Assessment of PRediction of Interactions rounds 38 to 45.

Computational structural prediction of macromolecular interactions is a fundamental tool toward the global understanding of cellular processes. The Critical Assessment of PRediction of Interactions (CAPRI) community-wide experiment provides excellent opportunities for blind testing computational docking methods and includes original targets, thus widening the range of docking applications. Our participation in CAPRI rounds 38 to 45 enabled us to expand the way we include evolutionary information in structural predictions beyond our standard free docking InterEvDock pipeline. InterEvDock integrates a coarse-grained potential that accounts for interface coevolution based on joint multiple sequence alignments of two protein partners (co-alignments). However, even though such co-alignments could be built for none of the CAPRI targets in rounds 38 to 45, including host-pathogen and protein-oligosaccharide complexes and a redesigned interface, we identified multiple strategies that can be used to incorporate evolutionary constraints, which helped us to identify the most likely macromolecular binding modes. These strategies include template-based modeling where only local adjustments should be applied when query-template sequence identity is above 30% and larger perturbations are needed below this threshold; covariation-based structure prediction for individual protein partners; and the identification of evolutionarily conserved and structurally recurrent anchoring interface motifs. Overall, we submitted correct predictions among the top 5 models for 12 out of 19 interface challenges, including four High- and five Medium-quality predictions. Our top 20 models included correct predictions for three out of the five targets we missed in the top 5, including two targets for which misleading biological data led us to downgrade correct free docking models.

Provitamin D3 modulation through prebiotics supplementation: simulation based assessment.

Vitamin D is important in multiple health conditions. Vitamin D deficiency is prevalent globally even with exposure to adequate sunlight. Reduction in provitamin D3 (7-dehydrocholesterol, 7-DHC) is an important cause of vitamin D3 deficiency. Vitamin supplementation, food fortification, and use of probiotics are some approaches to reduce vitamin D3 deficiency. This study investigates plausibility of 7-DHC biosynthesis through dietary prebiotics supplementation. Furthermore, it reports mechanistic details and constraints for the biosynthesis using flux balance analysis (FBA) simulations. The FBA simulations using co-metabolism models comprising human host and a resident bacterium (Faecalibacterium prausnitzii or Bacteroides thetaiotamicron) indicated increased flux of 7-DHC with short-chain fructooligosaccharide (scFOS) or inulin supplementation. We observed around 2-fold increase in flux compared to the baseline. Biosynthesis of 7-DHC was primarily modulated through acetate, pyruvate and lactate secreted by the bacterium. We observed diverse mechanisms and dose dependent responses. We extended this assessment to 119 resident bacteria and investigated the metabolites profiles with prebiotics supplementation. In summary, the current study suggests the potential use of applying prebiotics in enhancing 7-DHC biosynthesis. Furthermore, performance of the different gut bacteria with prebiotic supplementation for secreted metabolites profile is reported. These results may be useful to design future clinical studies.

Quantitative Assessment of the Conformational Heterogeneity in Amylose across Force Fields.

The inherent flexibility and conformational heterogeneity of a carbohydrate pose a challenge for its modeling and sampling by the existing classical force field. This work quantitatively assesses the quality of four popular carbohydrate force fields (CHARMM36, GLYCAM06, OPLS-AA, GROMOS53A6CARBO_R) against their ability to accurately model the conformational landscape of a dodecamer of single-stranded amylose, the key constituent of starch. While past NMR and X-ray studies have hinted at evidence of a helical structure of amylose and its spontaneous helix-coil transition, it remains to be seen how existing force fields fare against modeling its structural transition. Toward this end, we perform a multimicrosecond long extensive molecular dynamics simulation of dodecamer of a single-stranded amylose chain in explicit water in each of the four force fields and assess these force fields' ability to model relative structural transitions via analyzing the radius of gyration, glycosidic linkage orientation, and pyranose ring puckering of the amylose. In particular, the simulations show that while GLYCAM06 and CHARMM36 force fields predict a significant helix-coil transition in the amylose, GROMOS53A6CARBO_R and OPLS-AA majorly favor extended conformation. The Markov State Model (MSM), built using the simulation trajectories, for each force field, provides a comparative quantification of the population of key macrostates of amylose and elucidates an underlying network of pathways of their mutual interconversion. The macrostates obtained from MSM revealed that metastable helixlike and semicoil intermediate conformations are more probable for CHARMM36, whereas elongated or helixlike conformations are more probable in OPLS-AA and GROMOS53A6CARBO_R. GLYCAM06 showed significant probability for both helix and coil conformations along with intermediate conformations. We find that the differences in the conformations across force fields are governed by differences in the kinetics of glycosidic linkages and pyranose ring pucker conformers. All four force fields share one common point that the majority of α(1 → 4) glycosidic linkages preferred syn conformation, which is found to be energetically more favorable than anti. However, except for GROMOS53A6CARBO_R, all other force fields predicted non-negligible minor anti conformation. The multimicrosecond long simulations on the single-chain amylose, in combination with MSM, described here, suggest that sampling of α(1 → 4) linked oligosacharides on microsecond time scales enable quantitative predictions of helix-coil, glycosidic linkage, and pyranose ring exchange kinetics. These exchange kinetics have otherwise remained inaccessible to quantification by experiments or nanosecond time scale simulations which might have hindered the comparison of the possibility of helix-coil exchange across different force fields on equal footing.

Toxicological safety assessment of the human-identical milk oligosaccharide 3'-sialyllactose sodium salt.

Human breastmilk is a mixture of nutrients, hormones and bioactive molecules that are vital for infant growth and development. Infant formula (IF) lacks many of these compounds, most notably human milk oligosaccharides (HMOs), which are abundant in breastmilk but scarce in IF. Sialyllactoses, such as 3'-sialyllactose, constitute a large portion of the HMO fraction. To produce IF that matches breastmilk more closely, biosynthesized human-identical milk oligosaccharides (structurally identical to HMOs) such as 3'-sialyllactose sodium salt (3'-SL) are proposed for use in IF and foods for the general population. The safety assessment of 3'-SL comprised in vitro genotoxicity tests and a 90-day oral (gavage) toxicity study. This is the first 90-day study conducted with 3'-SL using neonatal rats (7 days old at the start of dosing-equivalent age to newborn human infants in terms of central nervous system and reproductive development), demonstrating the safety of 3'-SL for consumption by infants, the most sensitive age group. The neonatal rats received 3'-SL at doses up to 5,000 mg/kg body weight (BW)/day and reference controls received 5,000 mg/kg BW/day of fructooligosaccharide (an ingredient approved for use in IF) for comparison with the high-dose 3'-SL group, followed by a 4-week recovery period. There was no evidence of genotoxicity in vitro. In the absence of any test item-related adverse effects in the 90-day study, the high dose (5,000 mg/kg BW/day) was established as the no-observed-adverse-effect level. This confirms the safety of 3'-SL for use in IF for infants, as well as in functional foods for the general population.

Futile Encounter Engineering of the DSR-M Dextransucrase Modifies the Resulting Polymer Length.

The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains. Nuclear magnetic resonance analysis shows that this mutation influences both the dynamics of the active site and the water accessibility. Monte Carlo simulation of the elongation process allows interpretation of these results in terms of enhanced futile encounters, whereby the less effective binding increases the pool of effective seeds for the dextran chains and thereby directly determines the length distribution of the final polymers.

Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose⁻Oligosaccharide Synthesis.

Isomaltose-oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0-10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl ß-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.

De novo structural determination of mannose oligosaccharides by using a logically derived sequence for tandem mass spectrometry.

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.

Anticoagulant mechanism, pharmacological activity, and assessment of preclinical safety of a novel fibrin(ogen)olytic serine protease from leaves of Leucas indica.

The harnessing of medicinal plants containing a plethora of bioactive molecules may lead to the discovery of novel, potent and safe therapeutic agents to treat thrombosis-associated cardiovascular diseases. A 35 kDa (m/z 34747.5230) serine protease (lunathrombase) showing fibrin(ogen)olytic activity and devoid of N- and O- linked oligosaccharides was purified from an extract of aqueous leaves from L. indica. The LC-MS/MS analysis, de novo sequencing, secondary structure, and amino acid composition determination suggested the enzyme's novel characteristic. Lunathrombase is an αß-fibrinogenase, demonstrating anticoagulant activity with its dual inhibition of thrombin and FXa by a non-enzymatic mechanism. Spectrofluorometric and isothermal calorimetric analyses revealed the binding of lunathrombase to fibrinogen, thrombin, and/or FXa with the generation of endothermic heat. It inhibited collagen/ADP/arachidonic acid-induced mammalian platelet aggregation, and demonstrated antiplatelet activity via COX-1 inhibition and the upregulation of the cAMP level. Lunathrombase showed in vitro thrombolytic activity and was not inhibited by endogenous protease inhibitors α2 macroglobulin and antiplasmin. Lunathrombase was non-cytotoxic to mammalian cells, non-hemolytic, and demonstrated dose-dependent (0.125-0.5 mg/kg) in vivo anticoagulant and plasma defibrinogenation activities in a rodent model. Lunathrombase (10 mg/kg) did not show toxicity or adverse pharmacological effects in treated animals.

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate-based cholera toxin inhibitors.

Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B-subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1-oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene-dextran sulfate-polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (Kd ) determination. The Kd values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC50 values of an ELISA-type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen-induced secretory diarrhea.

Analytical characterization of human milk oligosaccharides - potential applications in pharmaceutical analysis.

Human breast milk is the gold standard for infant feeding and the best possible nourishment a new-born could have. Breastfeeding is the natural way to provide optimal nutritional, immunological and emotional nurturing for the healthy growth and development of infants. Human milk is a complex and dynamic biofluid comprised of many hundreds to thousands of distinct bioactive structures, among which one of the most abundant substances are the non-conjugated complex carbohydrates referred to as human milk oligosaccharides (HMOs). Due to their structural diversity and abundance, HMOs possess many beneficial biological functions. In order to understand human milk composition and HMO functions, state-of-the-art glycomic methods are inevitable. The industrial, large scale chemoenzymatic production of the most abundant HMOs became a reality in the last years and it evokes the need for straightforward and genuine analytical procedures to monitor the synthetic process and the quality of the products. It is obvious, that HMOs represent the next breakthrough in infant nutrition, as the addition of HMOs (such as 2'-fucosyllactose or lacto-N-neotetraose) to infant- and follow-on formulas, processed cereal-based food and baby foods for infants and young children etc. will revolutionize this field. This review highlights the potential applications of HMOs in the (bio)pharmaceutical industry, also summarizes the analytical methods available for the characterization of HMOs. An overview of the structure and function of HMOs along with their determination methods in complex matrices are provided. Various separation methods including liquid- and gas chromatography and capillary electrophoresis for the characterization and novel approaches for the quantitation of HMOs are discussed.

Oral delivery of ursolic acid-loaded nanostructured lipid carrier coated with chitosan oligosaccharides: Development, characterization, in vitro and in vivo assessment for the therapy of leishmaniasis.

Visceral leishmaniasis (VL) is a life-threatening disease caused by Leishmania donovani due to uncontrolled parasitisation of liver, spleen, and bone marrow. Ursolic acid (UA), a promising anti-inflammatory, anti-bacterial and anti-diabetic drug used successfully for treatment of ailments. Development of new delivery system is extremely urgent for UA with better efficacy and fewer side effects. The aim of present research work was to formulate and evaluate the potential anti-leishmanial activity of UA loaded N-octyl-chitosan surface decorated nanostructured lipid carrier system (UA-NLC) for delivery to the macrophages for VL. UA-NLC were prepared and characterized for shape, size, fourier transforms scanning electron microscopy (FESEM), transmittance electron microscopy (TEM), entrapment efficiency and in vitro drug release. The results indicate that the formulated UA-NLC had nano size range (103.7±2.8nm to 143.0±3.8nm) with high drug loading capacity (12.05±0.54%) and entrapment efficiency (88.63±2.7%). Ex vivo drug uptake by macrophage was also evaluated. The UA-NLC was more effective against AG83 wild type (12 fold), SSG-R (4 fold), PMM-R (4 fold) and GE1 field isolated (3 fold) cellular amastigotes than its free form. In vivo study showed orally effective UA-NLC could suppress the parasite burden to 98.75%.