Assessment of safety of enterococci isolated throughout traditional Terrincho cheesemaking: virulence factors and antibiotic susceptibility

Enterococci account for an important fraction of the adventitious microflora of traditional cheeses manufactured in Mediterranean countries from small ruminants' raw milk and play an important role in the development of suitable organoleptic characteristics of the final product. It has been suggested that animals used for food or animals that supply edible products are a reservoir of antibiotic-resistant enterococci. The main purpose of this research effort was thus to identify, to the species level, a total of 73 enterococci with high tolerance to acidic pH and bile salts (as prevailing environmental conditions in the first portion of the gastrointestinal tract), which were previously isolated from the milk feedstock to the final product of Terrincho cheesemaking, and to determine their profiles of antibiotic susceptibility, coupled with the occurrence of specific virulence factors (especially in those that might eventually be claimed to exhibit suitable probiotic and technological performances). Isolates, identified by both API 20 STREP and PCR methods, were found to belong to the following Enterococcus species: E. casseliflavus, E. durans, E. faecalis, E. faecium, and E. gallinarum. Susceptibility of those isolates was observed to most antibiotics tested, whereas none harbored aminoglycoside resistance genes. PCR screenings for cytolysin genes (cylL(L), cylL(s), cylM, cylB, and cylA), surface adhesin genes (efaA(fs), efaA(fm), and esp), the aggregation protein gene (agg), and the extracellular metalloendopeptidase gene (gelE) were performed. All isolates proved negative for cylL(L), cylM, cylB, and agg genes. Both E. faecalis strains were positive for the cell wall-associated protein Esp and the cell wall adhesin efaA(fs), whereas the cell wall adhesin efaA(fm) was detected in 11 of the 12 E. faecium strains. Only one strain possessed the cylL(s) determinant, and another possessed the cylA gene. Incidence of virulence determinants was thus very low; hence, the enterococcal adventitious microflora tested is essentially safe.     

The methionine precursor dl-2-hydroxy-(4-methylthio)butanoic acid protects intestinal epithelial barrier function

dl-2-hydroxy-(4-methylthio)butanoic acid (HMTBA) is a source of dietary methionine (Met) that is widely used in poultry nutrition. We have previously shown that HMTBA is preferentially diverted to the transsulfuration pathway, which gives antioxidant metabolites such as taurine and glutathione. Therefore, here we hypothesize that this Met source can protect epithelial barrier function in an in vitro model of intestinal inflammation of Caco-2 cells. The results show that HMTBA prevents the increase in paracellular permeability induced by H₂O₂ or tumour necrosis factor-α. This effect can be attributed to the increased production of taurine and reduced glutathione. Similar results were obtained for dl-Met, although the protective role of the amino acid was less pronounced than that of the hydroxy analogue. In conclusion, the diversion to the transsulfuration pathway means that this Met precursor is of greater value than previously thought, due to its capacity to improve intestinal homeostasis and the quality of poultry products destined for human consumption.     

Genetic diversity of Oenoccoccus oeni isolated from wines treated with phenolic extracts as antimicrobial agents

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds.     

Effect of heating temperature and sodium chloride concentration on ultrastructure and texture of gels made from giant squid (Dosidicus gigas) with addition of starch,l-carrageenan and egg white

This paper seeks to compare the ultrastructure of gels made from frozen muscle of giant squid (Dosidicus gigas) at various temperatures with a number of different rheological parameters, with reference to a variety of added ingredients (non-muscle proteins and hydrocolloids) and to NaCl concentration. Interesting data on gel rheological properties were found where formulae containedl-carrageenan, starch and egg white, with a low salt concentration (1.5%). This seems to be because carrageenan forms an independent network which supports the principal structure formed by the fish protein; starch is incorporated into the network and retains water; and egg white forms a supplementary network which helps to improve rheological properties.     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Stereospecificity of premature human infant lingual lipase

The lingual lipase in gastric aspirates from premature infants was found to be partially stereospecific forsn-3 esters of synthetic enantiometric triacylglycerols containing 18∶1 and 16∶0. Thesn-3 ester was hydrolyzed about 4 times faster than the acid at thesn-1 position with no difference in rates between 18∶1 and 16∶0. Thesn-2 was also hydrolyzed to some extent. Scientific contribution no. 949, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, Ct 06268.     

Active nano-CuPt3 electrocatalyst supported on graphene for enhancing reactions at the cathode in all-vanadium redox flow batteries

Graphene-supported monometallic (Pt) and bimetallic (CuPt₃) cubic nanocatalysts have been investigated as new positive electrode materials for improving the VO₂⁺/VO²⁺ redox process occurring in the vanadium redox flow batteries (VRB). High-resolution transmission electron microscopy (HRTEM) and scanning electron microscopy (SEM) have been employed to characterize the electrodes. The presence of the CuPt₃ nanocubes on graphene conferred higher electrocatalytic activity due to the much higher electroactive area compared to that obtained with the Pt nanoparticles. The electrochemical surface area of the nano-(CuPt₃)-decorated graphene electrode was 105% higher compared to non-decorated graphene, being then a promising alternative for improving the VRB.     

The effect of pH on the competition between polyphosphate-accumulating organisms and glycogen-accumulating organisms

In enhanced biological phosphorus removal (EBPR) processes, glycogen-accumulating organisms (GAOs) may compete with polyphosphate-accumulating organisms (PAOs) for the often-limited carbon substrates, potentially resulting in disturbances to phosphorus removal. A detailed investigation of the effect of pH on the competition between PAOs and GAOs is reported in this study. The results show that a high external pH ( approximately 8) provided PAOs with an advantage over GAOs in EBPR systems. The phosphorus removal performance improved due to a population shift favouring PAOs over GAOs, which was shown through both chemical and microbiological methods. Two lab-scale reactors fed with propionate as the carbon source were subjected to an increase in pH from 7 to 8. The phosphorus removal and PAO population (as measured by quantitative fluorescence in situ hybridisation analysis of "Candidatus Accumulibacter phosphatis") increased in each system, where the PAOs appeared to out-compete a group of Alphaproteobacteria GAOs. A considerable improvement in the P removal was also observed in an acetate fed reactor, where the GAO population (primarily "Candidatus Competibacter phosphatis") decreased substantially after a similar increase in the pH. The results from this study suggest that pH could be used as a control parameter to reduce the undesirable proliferation of GAOs and improve phosphorus removal in EBPR systems.