Potentially Health-Promoting Spaghetti-Type Pastas Based on Doubly Modified Corn Starch: Starch Oxidation via Wet Chemistry Followed by Organocatalytic Butyrylation Using Reactive Extrusion.

Extruded spaghetti-type pasta systems were obtained separately either from native or oxidized starch prepared via wet chemistry with the aim of evaluating the effect of oxidation modification of starch. In addition to this, the butyrylation reaction (butyrate (Bu) esterification-short-chain fatty acid) using native or oxidized starch was analyzed under reactive extrusion (REx) conditions with and without the addition of a green food-grade organocatalyst (l(+)-tartaric acid) with the purpose of developing potentially health-promoting spaghetti-type pasta systems in terms of increasing its resistant starch (RS) values. These would be due to obtaining organocatalytic butyrylated starch or not, or the manufacture of a doubly modified starch (oxidized-butyrylated-starch oxidation followed by organocatalytic butyrylation) or not. To this end, six pasta systems were developed and characterized by solid-state 13C cross-polarization magic angle spinning nuclear magnetic resonance (CP MAS NMR) spectroscopy, degree of substitution (DS), attenuated total reflectance Fourier transform infrared (ATR/FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), pancreatic digestion, free Bu content analysis and in vitro starch digestibility. The results obtained here suggest that starch oxidation hydrolytically degrades starch chains, making them more susceptible to enzymatic degradation by α-amylase. However, the oxidized starch-based pasta systems, once esterified by Bu mainly on the amylose molecules (doubly modified pasta systems) increased their RS values, and this was more pronounced with the addition of the organocatalyst (maximum RS value = ~8%). Interestingly, despite the checked chemical changes that took place on the molecular structure of starch upon butyrylation or oxidation reactions in corn starch-based spaghetti-type pasta systems, and their incidence on starch digestibility, the orthorhombic crystalline structure (A-type starch) of starch remained unchanged.

Bifidobacterial ß-Galactosidase-Mediated Production of Galacto-Oligosaccharides: Structural and Preliminary Functional Assessments.

In the current study the ability of four previously characterized bifidobacterial ß-galactosidases (designated here as BgaA, BgaC, BgaD, and BgaE) to produce galacto-oligosaccharides (GOS) was optimized. Of these enzymes, BgaA and BgaE were found to be promising candidates for GOS production (and the corresponding GOS mixtures were called GOS-A and GOS-E, respectively) with a GOS concentration of 19.0 and 40.3% (of the initial lactose), respectively. GOS-A and GOS-E were partially purified and structurally characterized. NMR analysis revealed that the predominant (non-lactose) disaccharide was allo-lactose in both purified GOS preparations. The predominant trisaccharide in GOS-A and GOS-E was shown to be 3'-galactosyllactose, with lower levels of 6'-galactosyllactose and 4'-galactosyllactose. These three oligosaccharides have also been reported to occur in human milk. Purified GOS-A and GOS-E were shown to be able to support bifidobacterial growth similar to a commercially available GOS. In addition, GOS-E and the commercially available GOS were shown to be capable of reducing Escherichia coli adhesion to a C2BBe1 cell line. Both in vitro bifidogenic activity and reduced E. coli adhesion support the prebiotic potential of GOS-E and GOS-A.

Enzymatic Synthesis and Structural Characterization of Novel Trehalose-Based Oligosaccharides.

Trehalose, α-d-glucopyranosyl-(1↔1)-α-d-glucopyranoside, is a disaccharide with multiple effects on the human body. Synthesis of new trehalose derivatives was investigated through transgalactosylation reactions using ß-galactosidase from four different species. ß-galactosidases from Bacillus circulans (B. circulans) and Aspergillus oryzae (A. oryzae) were observed to be the best biocatalysts, using lactose as the donor and trehalose as the acceptor. Galactosyl derivatives of trehalose were characterized using nuclear magnetic resonance spectroscopy. Trisaccharides were the most abundant oligosaccharides obtained followed by the tetrasaccharide fraction (19.5% vs 8.2% carbohydrates). Interestingly, the pentasaccharide [ß-Galp-(1→4)]3-trehalose was characterized for the first time. Greater oligosaccharide production was observed using ß-galactosidase from B. circulans than that obtained from A. oryzae, where the main structures were based on galactose monomers linked by ß-(1→6) and ß-(1→4) bonds with trehalose in the ending. These results indicate the feasibility of commercially available ß-galactosidases for the synthesis of trehalose-derived oligosaccharides, which might have functional properties, excluding the adverse effects of the single trehalose.

Biosynthesis of Nondigestible Galactose-Containing Hetero-oligosaccharides by Lactobacillus plantarum WCFS1 MelA α-Galactosidase.

This work describes the high capacity of MelA α-galactosidase from Lactobacillus plantarum WCFS1 to transfer galactosyl residues from melibiose to the C6-hydroxyl group of disaccharide-acceptors with ß-linkages (lactulose, lactose, and cellobiose) or α-linkages (isomaltulose and isomaltose) to produce novel galactose-containing hetero-oligosaccharides (HOS). A comprehensive nuclear magnetic resonance characterization of the transfer products derived from melibiose:lactulose reaction mixtures revealed the biosynthesis of α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose as the main component as well as the presence of α-d-galactopyranosyl-(1 → 3)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose and α-d-galactopyranosyl-(1 → 6)-α-d-galactopyranosyl-(1 → 6)-ß-d-galactopyranosyl-(1 → 4)-ß-d-fructose. Melibiose-derived α-galactooligosaccharides (α-GOS), manninotriose and verbascotetraose, were also simultaneously synthesized. An in vitro assessment of the intestinal digestibility of the novel biosynthesized HOS revealed a high resistance of α-galactosides derived from lactulose, lactose, cellobiose, and isomaltulose. According to the evidence gathered for conventional α-GOS and certain disaccharides used as acceptors in this work, these novel nondigestible α-galactosides could be potential candidates to selectively modulate the gut microbiota composition, among other applications, such as low-calorie food ingredients.

Hydrolysis and transglycosylation activities of glycosidases from small intestine brush-border membrane vesicles.

In order to know the catalytic activities of the disaccharidases expressed in the mammalian small intestinal brush-border membrane vesicles (BBMV) high concentrated solutions of sucrose, maltose, isomaltulose, trehalose and the mixture sucrose:lactose were incubated with pig small intestine disaccharidases. The hydrolysis and transglycosylation reactions generated new di- and trisaccharides, characterized and quantified by GC-MS and NMR, except for trehalose where only hydrolysis was detected. In general, α-glucosyl-glucoses and α-glucosyl-fructoses were the most abundant structures, whereas no fructosyl-fructoses or fructosyl-glucoses were found. The in-depth structural characterization of the obtained carbohydrates represents a new alternative to understand the potential catalytic activities of pig small intestinal disaccharidases. The hypothesis that the oligosaccharides synthesized by glycoside hydrolases could be also hydrolysed by the same enzymes was confirmed. This information could be extremely useful in the design of new non-digestible or partially digestible oligosaccharides with potential prebiotic properties.

Optimization of a Class of Tryptophan Dendrimers That Inhibit HIV Replication Leads to a Selective, Specific, and Low-Nanomolar Inhibitor of Clinical Isolates of Enterovirus A71.

Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range.

Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers.

The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1.

Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41.

Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity.

Linear and branched alkyl-esters and amides of gallic acid and other (mono-, di- and tri-) hydroxy benzoyl derivatives as promising anti-HCV inhibitors.

Linear and branched compounds that contain two, three or five units of galloyl (3,4,5-trihydroxybenzoyl) or its isomer 2,3,4-trihydroxybenzoyl, as well as other mono- or dihydroxybenzoyl moieties have been synthesized. These molecules have been evaluated for their in vitro inhibitory effects against a wide panel of viruses showing preferential activity against HIV and HCV. Our structure-activity relationship studies demonstrated that the 2,3,4-trihydroxybenzoyl moiety provides better antiviral activities than the galloyl (3,4,5-trihydroxybenzoyl) moiety that is present in natural green tea catechins. This observation can be of interest for the further rational exploration of compounds with anti-HCV/HIV properties. The most notable finding with respect to HIV is that the tripodal compounds 43 and 45, with three 2,3,4-trihydroxybenzoyl moieties, showed higher activities than linear compounds with only one or two. With respect to HCV, the linear compounds, 52 and 41, containing a 12 polymethylene chain and two 2,3 di- or 2,3,4 tri-hydroxybenzoyl groups respectively at the ends of the molecule showed good antiviral efficiency. Furthermore, the anti-HCV activity of both compounds was observed at concentrations well below the cytotoxicity threshold. A representative member of these compounds, 41, showed that the anti-HCV activity was largely independent of the genetic make-up of the HCV subgenomic replicon and cell lines used.

Substrate channelling in an engineered bifunctional aldolase/kinase enzyme confers catalytic advantage for C-C bond formation.

A new bifunctional enzyme that displays both aldolase and kinase activities has been designed and successfully used in the synthesis of aldol adducts, employing DHA as initial donor, with an increase in the reaction rate of 20-fold over the parent enzymes, which can be interpreted in terms of substrate channelling.

Origins of the double asymmetric induction on proline-catalyzed aldol reactions.

Computational studies to elucidate the origin of the double asymmetric induction on proline-catalyzed aldol reaction have been performed using HF/6-31G(d) calculations. The computed transition structures explain the experimental data obtained.

Asymmetric aldol reaction catalyzed by a heterogenized proline on a mesoporous support. The role of the nature of solvents.

A heterogenized (S)-proline on mesoporous support MCM-41 catalyzes the asymmetric aldol reaction in a wide range of solvents. The progress of the reaction is dependent on the nature of the solvent. Reactions proceed more efficiently in hydrophilic polar solvents; however, the addition of a small amount of water has a positive effect on the rate and the stereoselectivity of the reaction performed in hydrophobic toluene. The reaction under heterogeneous conditions has also been performed on chiral aldehydes, furnishing useful intermediates for the synthesis of azasugars.

Synthesis of azasugars through a proline-catalyzed reaction.

We report an efficient route to obtain azasugars from the enantiomerically pure L- and D-diethyltartrate. The key step is a proline-catalyzed aldol condensation, in which both enantiomers of proline have been used as catalyst, affording complementary anti-aldol products.