Evolution of Listeria populations in food samples undergoing enrichment culturing

The isolation of Listeria monocytogenes from food is carried out using a double enrichment. It is believed that the double enrichment can allow the overgrowth of Listeria innocua in samples where both species are present. In this study, we have evaluated the impact of overgrowth between Listeria species and strains during each step of the enrichment process. The effect of factors minimizing interactions between strains or phage inhibitory effects has also been estimated. In an artificially contaminated food undergoing enrichment, overgrowth could result from competitive interactions between Listeria spp. resulting from the production of bacteriocins and bacteriophage at high initial contamination levels (>10(4) cfu/g), but not at lower levels (50-100 cfu/g) as generally found in contaminated foods. At high levels of inoculation, the competitive effect could be reduced by solidification of the selective broths, to limit the diffusion of the inhibitors. Overgrowth resulting from differences in growth rate occurred independent of the initial contamination level. However, in naturally contaminated foods undergoing enrichment, there were no absolute correlations between growth rates or inhibitory profiles in terms of strain evolution during enrichment. In fact, Listeria strains which were predominant in the original sample in most cases remained the dominant strains at the end of the enrichment, although the relative proportion of any given strain could change significantly over the enrichment process. Additional factors which have yet to be identified impact on the evolution of Listeria in the two-step enrichment process. Analysis of strain evolution in eight naturally contaminated foods has indicated that the second enrichment step in Fraser broth can be reduced from 48 to 24 h without impacting on the recovery of L. monocytogenes. Our limited survey of naturally contaminated foods also demonstrated that maximum recovery of L. monocytogenes and other Listeria strains was found following 24 h incubation in 1/2 Fraser Broth. This finding suggests that it may be possible to shorten the current two-step isolation method further without reducing method sensitivity.     

Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance

The one-step PCR-mediated technique used for modification of chromosomal loci is a powerful tool for functional analysis in yeast. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe are amenable to this technique. However, the scarce availability of selectable markers for Sz. pombe hampers the easy use of this technique in this species. Here, we describe the construction of new vectors deriving from the pFA6a family, which are suitable for tagging in both yeasts owing to the presence of a nourseothricin-resistance cassette. These plasmids allow various gene manipulations at chromosomal loci, viz. N- and C-terminal tagging with 3HA (haemagglutinin) or 13Myc epitopes, GST (glutathione S-transferase), 4TAP (tandem affinity purification) and several GFP (green fluorescent protein) isoforms. For N-terminal modifications, the use of different promoters allows constitutive (PADH1) or regulatable (PGAL1) promoters for S. cerevisiae and derivatives of Pnmt1 for Sz. pombe expression.     

In vitro comparisons between Carica papaya and pancreatic lipases during test meal lipolysis: Potential use of CPL in enzyme replacement therapy

High levels of lipase activity are known to occur in Carica papaya latex, and this activity is being used in some biotechnological applications. The lipolytic activity of C. papaya lipase (CPL) on dietary triacylglycerols (TAG) has not yet been studied. Hence, the aim of this study was to characterise the specific activity of CPL on dietary TAG present in a crude preparation. Also, we have determined its stability during the lipolysis of a test meal at various pH values mimicking those occurring in the gastro-intestinal tract, with or without bile, and have compared these properties with those of porcine pancreatic extract (PPE) and human pancreatic lipase (HPL). CPL showed maximum stability at pH 6.0, both with and without bile. Some residual activity was still observed at pH 2 (20%), whereas the pancreatic lipases tested were immediately completely inactivated at this pH. In the absence of bile, the highest specific activities were measured at pH 6 in the case of CPL, PPE and HPL. Adding bile slightly decreased the CPL activity in the 4–6 pH range, thus shifting the optimum CPL activity to pH 7, where the presence of bile had no effect. Lipolysis levels decreased with the pH, but CPL was still more active than PPE at pH 5 on a relative basis. These results suggest that CPL might be a promising candidate for use as a therapeutic tool on patients with pancreatic exocrine insufficiency.     

Arachidonic acid autoxidation in an aqueous media effect of α-tocopherol,cysteine and nucleic acids

The autoxidation of arachidonic acid dispersed in aqueous media was evaluated simultaneously with and without different agents, e.g., α-tocopherol at different concentrations, cysteine, DNA and RNA. The autoxidation rate of arachidonic acid was evaluated by quantitative gas liquid chromatography (GLC) determination of the unoxidized acid and by spectrophotometric measurement of conjugated dienes. α-Tocopherol exhibited a prooxidant activity at concentrations of 1.25 × 10⁻⁴ M and 1.25 × 10⁻⁵ M and a weak antioxidant activity at a concentration of 1.25 × 10⁻⁶ M. Cysteine showed antioxidant activity and greatly reduced the prooxidant activity of α-tocopherol. DNA and RNA had no effect in either case. α-Tocopherol oxidation was followed by high pressure liquid chromatography (HPLC). The prooxidant effect was accompanied by a rapid oxidation of α-tocopherol, except in the presence of cysteine, which prevented the oxidation of α-tocopherol.     

Effect of Oral and Parenteral N Nutrition vs N-Free Nutrition on the Endogenous Amino Acid Flow at the Ileum of the Pig*

Two methods were tested for suppressing the depressive effect of N-free diets on the digestive secretions in pigs: the blood perfusion of amino acids (AA) or the peptide alimentation method. In the latter, enzymically hydrolysed casein (EHC), composed of oligopeptides and free AA, was used as the source of nitrogen. The unabsorbed dietary N molecules were discarded from the ileal digesta by ultrafiltration or gel filtration, assuming that the endogenous fraction did not contain significant amounts of small molecules. The AA supply by blood perfusion had no effect on the ileal endogenous AA losses (8·0 g AA kg⁻¹ DM intake) in growing pigs (±50 kg), compared with the N-free diet alone (8·3 g), whereas the EHC supplementation significantly increased them (18·0 g). The increase was due to both endogenous and dietary N. The presence of unabsorbed dietary AA in the ileal digesta was confirmed by the AA profile of the soluble molecules with a very low molecular mass (<3 kDa), which was close to that of EHC. Both ultrafiltration (cut-offs of 3 or 10 kDa) and gel filtration methods, utilised to discard the remaining dietary molecules, also eliminated a significant proportion of endogenous AA.     

Individual cell lag time distributions of Cronobacter (Enterobacter sakazakii) and impact of pooling samples on its detection in powdered infant formula

Cells of six strains of Cronobacter were subjected to dry stress and stored for 2.5 months at ambient temperature. The individual cell lag time distributions of recovered cells were characterized at 25 °C and 37 °C in non-selective broth. The individual cell lag times were deduced from the times taken by cultures from individual cells to reach an optical density threshold. In parallel, growth curves for each strain at high contamination levels were determined in the same growth conditions. In general, the extreme value type II distribution with a shape parameter fixed to 5 (EVIIb) was the most effective at describing the 12 observed distributions of individual cell lag times. Recently, a model for characterizing individual cell lag time distribution from population growth parameters was developed for other food-borne pathogenic bacteria such as Listeria monocytogenes. We confirmed this model’s applicability to Cronobacter by comparing the mean and the standard deviation of individual cell lag times to populational lag times observed with high initial concentration experiments. We also validated the model in realistic conditions by studying growth in powdered infant formula decimally diluted in Buffered Peptone Water, which represents the first enrichment step of the standard detection method for Cronobacter. Individual lag times and the pooling of samples significantly affect detection performances.     

On-road emissions of light-duty vehicles in europe

For obtaining type approval in the European Union, light-duty vehicles have to comply with emission limits during standardized laboratory emissions testing. Although emission limits have become more stringent in past decades, light-duty vehicles remain an important source of nitrogen oxides and carbon monoxide emissions in Europe. Furthermore, persisting air quality problems in many urban areas suggest that laboratory emissions testing may not accurately capture the on-road emissions of light-duty vehicles. To address this issue, we conduct the first comprehensive on-road emissions test of light-duty vehicles with state-of-the-art Portable Emission Measurement Systems. We find that nitrogen oxides emissions of gasoline vehicles as well as carbon monoxide and total hydrocarbon emissions of both diesel and gasoline vehicles generally remain below the respective emission limits. By contrast, nitrogen oxides emissions of diesel vehicles (0.93 ± 0.39 grams per kilometer [g/km]), including modern Euro 5 diesel vehicles (0.62 ± 0.19 g/km), exceed emission limits by 320 ± 90%. On-road carbon dioxide emissions surpass laboratory emission levels by 21 ± 9%, suggesting that the current laboratory emissions testing fails to accurately capture the on-road emissions of light-duty vehicles. Our findings provide the empirical foundation for the European Commission to establish a complementary emissions test procedure for light-duty vehicles. This procedure could be implemented together with more stringent Euro 6 emission limits in 2014. The envisaged measures should improve urban air quality and provide incentive for innovation in the automotive industry.     

Oregano: chemical analysis and evaluation of its antimalarial, antioxidant, and cytotoxic activities

Abstract: GC‐FID and GC‐MS analysis of essential oil from oregano leaves (Origanum compactum) resulted in the identification of 46 compounds, representing more than 98% of the total composition. Carvacrol was the predominant compound (36.46%), followed by thymol (29.74%) and p‐cymene (24.31%). Serial extractions with petroleum ether, ethyl acetate, ethanol, and water were performed on aerials parts of Origanum compactum. In these extracts, different chemical families were characterized: polyphenols (gallic acid equivalent 21.2 to 858.3 g/kg), tannins (catechin equivalent 12.4 to 510.3 g/kg), anthocyanins (cyanidin equivalent 0.38 to 5.63 mg/kg), and flavonoids (quercetin equivalent 14.5 to 54.7 g/kg). The samples (essential oil and extracts) were subjected to a screening for antioxidant (DPPH and ABTS assays) and antimalarial activities and against human breast cancer cells. The essential oil showed a higher antioxidant activity with an IC₅₀= 2 ± 0.1 mg/L. Among the extracts, the aqueous extract had the highest antioxidant activity with an IC₅₀= 4.8 ± 0.2 mg/L (DPPH assay). Concerning antimalarial activity, Origanum compactum essential oil and ethyl acetate extract showed the best results with an IC₅₀ of 34 and 33 mg/mL, respectively. In addition, ethyl acetate extract (30 mg/L) and ethanol extract (56 mg/L) showed activity against human breast cancer cells (MCF7). The oregano essential oil was considered to be nontoxic.     

Tailor made OSB for special application

The purpose of this study was to develop speciality oriented strand board (OSB) with high stiffness for use in products such as engineered wood flooring (EWF). Three-layer oriented strand boards were manufactured from two feedstocks of strands: a mixture of 90% aspen (Populus tremuloides) and 10% of paper birch (Betula papyrifera), and 100% of small diameter ponderosa pine logs (Pinus ponderosa). The OSB panels were manufactured under a factorial design of three resin contents, two density profiles, and three weight ratios for the face and core layers. Tests to determine density, bending modulus of elasticity (MOE), internal bond (IB) and thickness swelling (TS) were performed according to ASTM standard D 1037-06a. The results showed that the higher values of bending MOE for panels made from aspen/birch mixture and ponderosa pine, 8190 and 9050?MPa, respectively, were obtained for the same combination of factors. Such high bending MOE values are very close to Baltic birch (Betula pendula) plywood, a product known for its high stiffness. The effect of resin content on IB is more pronounced for panels made from ponderosa pine than panels made from the aspen/birch mixture. Thickness swelling of panels made from ponderosa pine strands is higher than thickness swelling of panels made from a mixture of aspen and birch strands. The results indicate the potential to tailor an OSB for a specific application such as EWF.     

Isolation of Alicyclobacillus and the influence of different growth parameters

Alicyclobacillus species are thermo-acidophilic, endospore-forming bacteria that are able to survive pasteurisation and have been implicated in a number of spoilage incidents involving acidic foods and beverages. The aim of this study was to compare three isolation methods used for the detection of Alicyclobacillus acidoterrestris and to investigate the influence of incubation temperature on the growth of A. acidoterrestris and A. acidocaldarius. Peach juice samples inoculated with A. acidoterrestris K47 were analysed using either the International Federation of Fruit Juice Producers (IFU) Method No. 12 (Method A), which involved spread plating onto Bacillus acidoterrestris (BAT) agar at pH 4.0; Method B, which involved pour plating using potato dextrose agar (PDA) at pH 3.7; or Method C, which made use of membrane filtration followed by incubation on K agar at pH 3.7. The performance of the three methods differed significantly, with the IFU Method No. 12 recovering the highest percentage of cells at 75.97%, followed by Method B at 66.79% and Method C at 3.43%. These findings strengthen the proposal of the IFU for the use of the IFU Method No. 12 as a standard international method for the detection of Alicyclobacillus. To investigate the effect on growth of different incubation temperatures A. acidoterrestris (three strains) and A. acidocaldarius (two strains) were incubated at either 45 °C or 25 °C. Growth at 25 °C was slower and maximum cell concentrations were lower (1 × 10⁵-10⁶ cfu/mL compared to 1 × 10⁷-10⁸ cfu/mL) than at 45 °C for A. acidoterrestris. A. acidocaldarius was unable to grow at 25 °C and cell concentrations decreased by 1-2 logs. Since a growth temperature of 25 °C could not inhibit growth of A. acidoterrestris, cooling to room temperature (20°-25 °C) is not an effective control measure for A. acidoterrestris inhibition.