Arland T. Hotchkiss

Dietary prebiotics: current status and new definition

In November 2008, a group of scientists met at the 6th Meeting of the International Scientific Association of Probiotics and Prebiotics (ISAPP) in London, Ontario, Canada, to discuss the functionality of prebiotics. As a result of this, it was concluded that the prebiotic field is currently dominated by gastrointestinal events. However, in the future, it may be the case that other mixed microbial ecosystems may be modulated by a prebiotic approach, such as the oral cavity, skin and the urogenital tract. Therefore, a decision was taken to build upon the current prebiotic status and define a niche for ‘dietary prebiotics’. This review is co-authored by the working group of ISAPP scientists and sets the background for defining a dietary prebiotic as ‘‘a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health’’.

Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 Å and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, α-D-GalpA-([1→4]-α-D-GalpA)3-(1→4)-D-GalpA, is composed of five galacturonopyranose units (D-GalpA) linked by α-(1→4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein–oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC–galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the β elimination cleavage of polygalacturonic acid.

In Vitro Determination of Prebiotic Properties of Oligosaccharides Derived from an Orange Juice Manufacturing By-Product Stream

ABSTRACT Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host.

Global Structure of Microwave-Assisted Flash-Extracted Sugar Beet Pectin

The global structure of microwave-assisted flash-extracted pectins isolated from fresh sugar beet pulp has been studied. The objective was to minimize the disassembly and possibly the degradation of pectin molecules during extraction. These pectins have been characterized by high-performance size exclusion chromatography with light scattering, viscometric detection, and atomic force microscopy (AFM). Analysis of molecular parameters was performed on 15 and 8 microm size column packings. Samples analyzed with 15 microm packing gave weight-average molar masses that ranged from 532,000 to 1.2 million Da, radii of gyration from about 35 to 51 nm, polydispersities from 1.78 to 2.58, intrinsic viscosities from about 3.00 to 4.30 dL/g, and recoveries from 8.40 to 14.81% of dry weight. Chromatography revealed that a bimodal distribution of high molar mass spherical particles and lower molar mass coils was obtained. AFM images of pectin corroborated this conclusion and further revealed that these strands and spherical particles were integrated into networks. It is demonstrated that microwave-assisted extraction of sugar beet pulp under moderate pressure and at relatively low temperature could extract under acid conditions high molar mass, moderate-viscosity pectin in minutes rather than hours as required by conventional heating.

Identification of extensin protein associated with sugar beet pectin.

Several studies have suggested that the emulsification properties associated with pectin obtained from sugar beet (Beta vulgaris) are due to the presence of a protein-pectin complex. Nevertheless, the identity of the protein has remained elusive. Pectin, extracted from sugar beet pulp by microwave-assisted extraction, and a commercial sample were both subjected to protease digestion with trypsin. The resulting peptides were separated from the pectin solution by ultrafiltration using a 3 kDa molecular weight cutoff (MWCO) membrane and analyzed using matrix-assisted laser desorption ionization with tandem time-of-flight mass spectrometry. The partial sequences derived from the mass spectrometry analyses of the resulting tryptic peptides are found to be highly consistent with extensin protein matched from the B. vulgaris Genetic Index database and also correspond to previously reported extensin peptides found in sugar beet cell suspension cultures. Further attempts were made to disassociate the protein from pectin using 1 M NaCl and a 100 kDa MWCO membrane; however, no peptides were observed following trypsin digestion of the permeate solution. This evidence suggests the existence of a complex between the pectin and extensin that is not due to ionic interactions. Trypsin digestion of commercial sugar beet pectin also produced the peptide profile observed with the microwave-assisted extracted pectin sample. Atomic force microscopy established that the number of rod-like elements decreased following protease treatment compared to the untreated sample.

Progressive dissociation of pectin.

The structural organization of alkaline soluble peach pectin was investigated over size ranges extending from micrometers to tenths of nanometers. Analysis was by electron microscopy and high-performance anion-exchange chromatography (HPAEC). Superimposed and individual circular microgels in the micrometer size range were isolated from mesocarp tissue of cell walls and visualized by rotary shadowing. Dilute NaCl and 50% aqueous glycerol disaggregated these microgels into rods, segmented rods, and kinked rods, which collectively comprised the internal gel network of the microgels. Image analysis of the shadowed specimens before and after disaggregation followed by curve fitting of the smoothed distributions revealed a multimodal distribution of lengths. HPAEC revealed that the multimodal aggregates were stable for the most part to further dissociation by increasing ionic strength.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro.

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment was determined in the human HT29 cell line by viable count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coil and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected, but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 microg ml(-1), respectively.

Activation of Human T-Helper/Inducer Cell, T-Cytotoxic Cell, B-Cell, and Natural Killer (NK)-Cells and induction of Natural Killer Cell Activity against K562 Chronic Myeloid Leukemia Cells with Modified Citrus Pectin

BackgroundModified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets like T, B and NK-cells.MethodsMCP treated human blood samples were incubated with specific antibody combinations and analyzed in a flow cytometer using a 3-color protocol. To test functionality of the activated NK-cells, isolated normal lymphocytes were treated with increasing concentrations of MCP. Log-phase PKH26-labeled K562 leukemic cells were added to the lymphocytes and incubated for 4 h. The mixture was stained with FITC-labeled active form of caspase 3 antibody and analyzed by a 2-color flow cytometry protocol. The percentage of K562 cells positive for PKH26 and FITC were calculated as the dead cells induced by NK-cells. Monosaccharide analysis of the MCP was performed by high-performance anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD).ResultsMCP activated T-cytotoxic cells and B-cell in a dose-dependent manner, and induced significant dose-dependent activation of NK-cells. MCP-activated NK-cells demonstrated functionality in inducing cancer cell death. MCP consisted of oligogalacturonic acids with some containing 4,5-unsaturated non-reducing ends.ConclusionsMCP has immunostimulatory properties in human blood samples, including the activation of functional NK cells against K562 leukemic cells in culture. Unsaturated oligogalacturonic acids appear to be the immunostimulatory carbohydrates in MCP.

Isolation of oligogalacturonic acids up to DP 20 by preparative high-performance anion-exchange chromatography and pulsed amperometric detection

Milligram quantities of oligogalacturonic acids up to a degree of polymerization (DP) of 20 were purified by high-performance anion-exchange chromatography utilizing a preparative-scale (21-mm i.d.) CarboPac PA1 column and a nonlinear potassium acetate (pH 7.5) gradient. Detection was accomplished by pulsed amperometry without post-column addition of hydroxide. Pulsed amperometry at near-neutral pH is an excellent detection method for preparative separations of carbohydrates because it avoids base-catalyzed degradation reactions that can occur at high pH. This method was simpler, faster, had higher sample loading capacity and allowed for the isolation of higher DP oligogalacturonic acids than other methods reported previously. With this improved method, multi-milligram quantities of valuable oligogalacturonic acids (up to DP 20) can be readily isolated.

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two‐layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non‐cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid‐modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4‐linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi‐purified by DEAE Sephacel (Cl−) anion‐exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4‐xylosyl backbone and 2,4‐xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3‐linked galactan.