Biotechnological production and application of fructooligosaccharides

Currently, prebiotics are all carbohydrates of relatively short chain length. An important group is the fructooligosaccharides, which are a special kind of prebiotics associated to their selective stimulation of the activity of certain groups of colonic bacteria that have a positive and beneficial effect on intestinal microbiota, reducing incidence of gastrointestinal infections, respiratory and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of fructooligosaccharides, such as solid-state fermentation utilizing various agroindustrial by-products. By optimizing the culture parameters, fructooligosaccharides yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of fructooligosaccharides. This paper is an overview on the results of recent studies on fructooligosacharides biosynthesis, physicochemical properties, sources, biotechnological production and applications.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

Consumer Focused Review of the Pork Supply Chain

Pig meat production was valued at €290 (£198) million at farm gate in Republic of Ireland (ROI) in 2007. In Northern Ireland (NI) in 2006, pig meat was estimated to account for almost seven percent of gross turnover in the food and drinks processing sector at £190 (€280) million. Whilst researching for this report it emerged that comparable figures for the value of the pig meat industry on ROI and NI are not available. This report showed that pig production on the IOI has changed from a small-scale enterprise carried out by a large number of mixed farmers to a modern industry comprised of a small number of specialist producers operating large-scale units. Most products for retailers are prepared and packed in specialised cutting and processing units which may or may not be integrated in the slaughter plant. For some pork products, various additives such as salt, herbs and flavour enhancers are added. Pork products are then stored and transported, frozen or chilled to wholesale, retail and catering facilities for ultimate sale to consumers.

Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins.

The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death. Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications. A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands. Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification. In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization. Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively. The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system. Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.

The Peroxisomal Enzyme L-PBE Is Required to Prevent the Dietary Toxicity of Medium-Chain Fatty Acids.

Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10 and Cyp4a14 to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and β-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe(-/-) mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber but does not improve protection of the plant against Pythium root rot

The biocontrol strain CHA0 of Pseudomonas fluorescens produces small amounts of indole-3-acetic acid via the tryptophan side chain oxidase and the tryptophan transaminase pathways. A recombinant plasmid (pME3468) expressing the tryptophan monooxygenase pathway was introduced into strain CHA0; this resulted in elevated synthesis of indole-3-acetic acid in vitro, especially after addition of -tryptophan. In natural soil, strain CHA0/pME3468 increased fresh root weight of cucumber by 17-36%, compared to the effect of strain CHA0; root colonization was about 106 cells per g of root. However, both strains gave similar protection of cucumber against Pythium ultimum. In autoclaved soil, at 6×107 cells per g of root, strain CHA0 stimulated growth of roots and shoots, whereas strain CHA0/pME3468 caused root stunting and strong reduction of plant weight. These results are in agreement with the known effects of exogenous indole-3-acetic acid on plant roots and suggest that in the system examined, indole-3-acetic acid does not contribute to the biocontrol properties of strain CHA0.

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Primary dengue virus infections induce differential cytokine production in Mexican patients

Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF) and dengue haemorrhagic fever (DHF) from dengue virus (DENV) 1 (n = 116) or 2 (n = 55) were compared. DF and DHF were defined according to the patient’s clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcription-polymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL)-12p70, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

Induction of tolerogenic vs immunogenic dendritic cells (DCs) in the presence of GM-CSF is regulated by the strength of signaling from monophosphoryl lipid A (MPLA) in association with glutathione and fetal hemoglobin gamma-chain.

Previous studies showed a fetal sheep liver extract (FSLE), in association with monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS), could interact to induce the development of dendritic cells (DCs) which regulated production of Foxp3+ Treg. This interaction was associated with an altered gene expression both of distinct subsets of TLRs and of CD200Rs. Prior studies had suggested that major interacting components within FSLE were gamma-chain of fetal hemoglobin (Hgbgamma) and glutathione (GSH). We investigated whether differentiation/maturation of DCs in vitro in the presence of either GM-CSF or Flt3L to produce preferentially either immunogenic or tolerogenic DCs was itself controlled by an interaction between MPLA, GSH and Hgbgamma. At low (approximately 10 microg/ml) Hgbgamma concentrations, DCs developing in culture with GSH and MPLA produced optimal stimulation of allogeneic CTL cell responses in vitro (and enhanced skin graft rejection in vivo). At higher concentrations (>40 microg/ml Hgbgamma) and equivalent concentrations of MPLA and GSH, the DCs induce populations of Treg which can suppress the induction of allogeneic CTL and graft rejection in vivo. These different populations of DCs express different patterns of mRNAs for the CD200R family. Addition of anti-TLR or anti-MD-1 mAbs to DCs developing in this mixture (Hgbgamma+GSH+MPLA), suggests that one effect of (GSH+Hgbgamma) on MPLA stimulation may involve altered signaling through TLR4.

Characterization of an interaction between fetal hemoglobin and lipid A of LPS resulting in augmented induction of cytokine production in vivo and in vitro.

A previously described extract of sheep fetal liver was reported to reverse many of the cytokine changes associated with aging in mice, including an augmented spleen cell ConA-stimulated production of IL-4 and decreased production of IL-2. Similar effects were not seen with adult liver preparations. These changes were observed in various strains of mice, including BALB/c, DBA/2 and C57BL/6, using mice with ages ranging from 8 to 110 weeks. Preliminary characterization of this crude extract showed evidence for the presence of Hb gamma chain, as well as of lipid A of LPS. We show below that purified preparations of sheep fetal Hb, but not adult Hb, in concert with suboptimally stimulating doses of LPS (lipid A), cooperate in the regulation of production of a number of cytokines, including TNFalpha and IL-6, in vitro. Furthermore, isolated fresh spleen or peritoneal cells from animals treated in vivo with the same combination of Hb and LPS, showed an augmented capacity to produce these cytokines on further culture in vitro. Evidence was also obtained for a further interaction between CLP, LPS and fetal Hb itself in this augmented cytokine production. These data suggest that some of the functional activities in the fetal liver extract reported earlier can be explained in terms of a novel immunomodulatory role of a mixture of LPS (lipid A) and fetal Hb.

A Case Study of China’s Commercial Pork Value Chain, August 2005

In China, with the cost of improved technology rising, surplus labor shrinking, and demand for food quality and safety increasing, it will be just a matter of time before the country’s hog production sector will be commercialized like that of developed countries. However, even if China’s cost of production converges to international levels, as shown in this case study, China may continue to retain some competitive advantage because of the labor-intensive nature of the marketing services involved in hog processing and meat distribution. The supply of variety meats offers the most promising market opportunity for foreign suppliers in China. The market may open further if the tariff rate for variety meats is reduced from 20% and harmonized with the pork muscle meat rate of 12%, and if the value-added tax of 13% is applied equally to both imported and domestic products. The fast-growing Western-style family restaurant and higher-end dining sector is another market opportunity for high-quality imported pork.

Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Logistics-production, logistics-marketing and external integration: Their impact on performance

Highly competitive environments are leading companies to implement SupplyChain Management (SCM) to improve performance and gain a competitiveadvantage. SCM involves integration, co-ordination and collaborationacross organisations and throughout the supply chain. It means that SCMrequires internal (intraorganisational) and external (interorganisational)integration. This paper examines the Logistics-Production and Logistics-Marketing interfaces and their relation with the external integrationprocess. The study also investigates the causal impact of these internaland external relationships on the company s logistical service performance.To analyse this, an empirical study was conducted in the Spanish Fast MovingConsumer Goods (FMCG) sector.