Effects of green tea extract and α-tocopherol on the lipid oxidation rate of omega-3 oils, incorporated into table spreads, prepared using multiple emulsion technology.

This study examined the effectiveness of fat and water soluble antioxidants on the oxidative stability of omega (ω)-3 rich table spreads, produced using novel multiple emulsion technology. Table spreads were produced by dispersing an oil-in-water (O/W) emulsion (500 g/kg 85 camelina/15 fish oil blend) in a hardstock/rapeseed oil blend, using sodium caseinate and polyglycerol polyricinoleate as emulsifiers. The O/W and oil-in-water-in-oil (O/W/O) emulsions contained either a water soluble antioxidant (green tea extract [GTE]), an oil soluble antioxidant (α-Tocopherol), or both. Spreads containing α-Tocopherol had the highest lipid hydroperoxide values, whereas spreads containing GTE had the lowest (P < 0.05), during storage at 5°C, while p-Anisidine values did not differ significantly. Particle size was generally unaffected by antioxidant type (P < 0.05). Double emulsion (O/W/O) structures were clearly seen in confocal images of the spreads. By the end of storage, none of the spreads had significantly different G' values. Firmness (Newtons) of all spreads generally increased during storage (P < 0.05).

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

Effects of argon enriched low-oxygen atmospheres and of high-oxygen atmospheres on the kinetics of polyphenoloxidase (PPO).

The reported benefits of enrichment of air atmospheres with argon or oxygen for control of enzymatic browning were investigated by determining the effects of these atmospheres on PPO kinetics. Kinetics of purified apple PPO and a commercially available mushroom PPO were studied in an in vitro model system. Enrichment with argon produced greater inhibitory effects than the current industry practice of enrichment with nitrogen. Km(app) values (mM) for apple PPO in 3%O(2)/97%Ar, 3%O(2)/97%N(2), and air, were 133, 87, and 48, respectively. The data indicate that inhibition by both gases is competitive, and also support the hypothesis that the greater inhibitory effect of argon was proportional to the size of the Van der Waals radius of argon against nitrogen (1.91 Å against 1.54 Å). Much smaller inhibitory effects were observed in the presence of 80% O(2) (Km(app) 57 mM), and the nature of this inhibition was less clear. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh-cut products, and may allow less anoxic atmospheres to be used. Practical Application: Control of enzymatic browning without sulfites continues to be a challenge in some fresh-cut products. While sporadic benefits of these atmospheres in control of enzymatic browning have been reported, results have been inconsistent in commercial practice. The results suggest that the benefits of argon enrichment may be relatively small, and may require critical enzyme, substrate, and gas levels to be successful. However, these benefits may be exploitable commercially in some fresh-cut products, and allow less anoxic atmospheres to be used.

Active immunization of hamsters against Clostridium difficile infection using surface-layer protein.

Clostridium difficile is the leading cause of infectious antibiotic-associated diarrhoea, particularly among the elderly. Its surface-layer protein (SLP) was tested as a vaccine component in a series of immunization and challenge experiments with Golden Syrian hamsters, combined with different systemic and mucosal adjuvants. Some regimens were also tested in a nonchallenge BALB/c mouse model, enabling closer monitoring of the immune response. None of the regimens conferred complete protection in the hamster model, and antibody stimulation was variable within regimens, and generally modest or poor. Mice displayed stronger antibody responses to SLP compared with hamsters. Two hamsters of five given SLP with Ribi (monophosphoryl lipid A and synthetic trehalose dicorynomycolate) survived the challenge, as did two of three given SLP with Ribi and cholera toxin. This modest trend to protection is interpreted with caution, because the survivors had low anti-SLP serum antibody titres. The hamsters were an outbred line, and subject to more genetic variability than inbred animals; however, BALB/c mice also showed strongly variable antibody responses. There is a clear need for better adjuvants for single-component vaccines, particularly for mucosal delivery. The hamster challenge model may need to be modified to be useful in active immunization experiments with SLP.

Sequence and phylogenetic analysis of the gene for surface layer protein, slpA, from 14 PCR ribotypes of Clostridium difficile.

Clostridium difficile is the commonest cause of antibiotic-associated diarrhoea, with the hospitalized elderly being at particular risk. The organism makes a crystalline surface protein layer (S-layer), encoded by the slpA gene, the product of which is cleaved to give two mature peptides which associate to form the layer. The larger peptide (high molecular weight; HMW), derived from the C-terminal portion of the precursor, is relatively conserved, whereas the smaller peptide (low molecular weight; LMW), derived from the N-terminal portion of the precursor, is a dominant antigen which substantially forms the basis for serotyping of isolates. PCR ribotyping is a more discriminatory typing method, based on the intergenic rRNA. We obtained the sequence for slpA and some flanking DNA from a collection of C. difficile strains of 14 ribotypes isolated from elderly patients. Sequences from different ribotypes were compared with one another and with published sequences. Sequences from C. difficile ribotypes 046 and 092 were identical. Sequences from ribotype pairs 005 and 054, 012 and 046/092, 014 and 066 and 031 and 094 differed by 1-3 nt in the slpA gene. There were ultimately nine ribotypes or groups of ribotypes with very different slpA sequences, particularly in the region encoding the LMW peptide. The sequence from ribotype 002 was very different from previously published sequences. The DNA segment sequenced included the 5' 315 bp of a secA homologue, encoding a putative transport protein required for peptide secretion across the plasma membrane. The amino acid sequences of the predicted HMW peptides were aligned and a neighbour-joining tree was produced using 10,000 bootstrap replicates. The predicted SecA N-terminal region was similarly analysed. For both SlpA and SecA, a strong association was found between ribotypes 012, 046/092, 017, 031 and 094. Ribotypes 001 and 078 formed part of this clade for SlpA but not SecA, indicating independent evolution for slpA and secA, presumably because they come under different selection pressures.

Passive immunisation of hamsters against Clostridium difficile infection using antibodies to surface layer proteins.

Clostridium difficile is a major cause of antibiotic-associated diarrhoea and the primary cause of pseudomembraneous colitis in hospitalised patients. We assessed the protective effect of anti-surface layer protein (SLP) antibodies on C. difficile infection in a lethal hamster challenge model. Post-challenge survival was significantly prolonged in the anti-SLP treated group compared with control groups (P=0.0281 and P=0.0283). The potential mechanism of action of the antiserum was shown to be through enhancement of C. difficile phagocytosis. This report indicates that anti-SLP antibodies can modulate the course of C. difficile infection and may therefore merit closer investigation for use as constituents of multi-component vaccines against C. difficile associated diarrhoea.