Protection of probiotic bacteria in a synbiotic matrix following aerobic storage at 4 °C.

The survival of single strains of Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, and Lactobacillus reuteri was investigated in synbiotics that included 10 mg/ml of fructo-oligosaccharides, inulin and pectic-oligosaccharides in an alginate matrix under refrigerated (4 °C) aerobic storage conditions. When the matrices were cross-linked with calcium (45 mM), 102-103 cfu/ml of L. acidophilus and L. reuteri, and 0-103 cfu/ml of B. breve and B. longum survived refrigerated aerobic storage for 28 days. Following refrigerated storage, acetic (3-9 mM), butyric (0-2 mM), propionic (5-16 mM) and lactic acids (1-48 mM) were produced during the growth of probiotics in BHI broth at 37 °C, suggesting their metabolic activity after storage was stressed. When calcium cross-linking was not used in synbiotics, the matrix remained more gel-like after inoculation when compared to the calcium cross-linked matrix. At least 107 cfu/ml of probiotic bacteria survived after 21 days of storage within these gel-like alginate matrices. Significantly higher levels of B. breve, L. acidophilus and L. reuteri were obtained from the synbiotic matrices supplemented with fructo-oligosaccharides, inulin and pectic-oligosaccharides compared to alginate alone. B. longum survival was the same (~7 logs) in all gel-like synbiotic matrices. These results show that synbiotics protected probiotic bacteria and extended their shelf-life under refrigerated aerobic conditions. Synbiotics represent a viable delivery vehicle for health-promoting bacteria.

Inhibition by pectic oligosaccharides of the invasion of undifferentiated and differentiated Caco-2 cells by Campylobacter jejuni.

The ability of pectic oligosaccharides (POS) to inhibit adherence to and invasion of undifferentiated (UC) and differentiated (DC) Caco-2 cells by Campylobacter jejuni (C. jejuni) was investigated. It was observed that both adherence and invasion were significantly higher in UC than in DC. POS (2.5mg/ml) had no significant effect on the number of bacteria which can adhere to cells, but they significantly inhibited cell invasion. The extent of the anti-invasive effect of POS was dependent on the concentration, although the entire range tested (from 2.5mg/ml to 0.05 mg/ml) was capable of inhibiting the invasion of Caco-2 cells by Campylobacter to some degree. The pre-incubation or not of C jejuni with POS did not influence the behaviour observed. The results obtained in this work suggest that POS could be potentially useful as alternatives to antibiotics in the control of C. jejuni.

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.

In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream.

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.

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.

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 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(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 beta elimination cleavage of polygalacturonic acid.

Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum.

DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum. Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA. Mature PehA accumulated primarily in the periplasm of A. vitis and pehA+ Escherichia coli cells during exponential growth. A. vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E. carotovora and R. solanacearum polygalacturonases.

Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection.

Underivatized oligogalacturonic acids with a degree of polymerization (DP) ranging from 2 to 50 have been separated for the first time on a high-performance CarboPac PA1 pellicular anion-exchange stationary phase column. Baseline separation of these pectic fragments was accomplished using a nonlinear gradient of pH 6 potassium oxalate buffer as the mobile phase. Acetate buffer linear gradients were also useful as mobile phases, but only for separations of oligogalacturonic acids that were soluble in this solvent (DP less than 20). Additionally, oligogalacturonic acid separations were accomplished on a lower capacity AS4A stationary phase column. Triple pulse amperometric detection was selective, sensitive, and reproducible, nevertheless, oligogalacturonic acid response factors were affected by DP and compositional changes in the mobile phase.

CONSPECIFICITY OF SOME CHAROPHYTES(1).

Sweets Lake, British Columbia, is the type locality for plants originally described as Chara buckellii. Plants from this locality liave been crossed successfully to a second clone from Laguna La Drava, Argentina, the type locality for Nitellopsis bulbillifern. Plants from both clones were also crossed with others from Texas, New Mexico, and Wyoming that agree in most respects with. Robinson's original description of C. longifolia. On the basis of such crosses we suggest that all 3 taxa are biologically conspecific. There is no evidence, either from the field or experimental crosses, of gene flow between C. longifolia and C. hornemannii; the two should be considered separate species.