Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology

We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.     

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of ochratoxin A

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the determination of ochratoxin A (OTA) was developed. Fluorescein‐labelled OTA derivative (tracer) was synthesized and purified by thin‐layer chromatography. The optimized OTA FPIA had a dynamic range from 5 to 200 ng mL^?1 with IC₅₀ value of 30 ng mL^?1 and a detection limit of 3 ng mL^?1. The method developed was characterized by high specificity and reproducibility. Cross‐reactivity with other mycotoxins (zearalenone, aflatoxins, patulin and T‐2 toxin) was negligible (<0.1%). Methanol extracts of barley samples were used for the analysis. The results of OTA determination in barley were compared with those determined by indirect competitive enzyme‐linked immunosorbent assay (ELISA). Recoveries for the samples spiked at 50, 100 and 500 ng g^?1 levels were 91, 90 and 97%, respectively, for FPIA, and 98, 98 and 102%, for ELISA. Naturally contaminated barley samples were analysed by these methods but some disagreement was observed between the results. The FPIA method can be applied for screening of food samples for OTA residues without a complicated clean‐up.     

A novel and enhanced approach for the assessment of the total carotenoid content of foods based on multipoint spectroscopic measurements

We have devised a more sensible approach to estimate the carotenoid content of orange juices, which can be regarded as a model system of food with intricate carotenoid pattern. For this purpose spectroscopic information at several wavelengths and spectra of the juices and not from their carotenoid extracts were considered, such that more accurate and rapid quantitative assessments can be achieved. The wavelengths proposed on the basis of the characteristic vector method were 420, 455, 515, 545 and 610 nm or 420, 445, 510, 545 and 605 nm, depending on the measurement conditions. The correlations between the carotenoid content and the reflectances at these wavelengths were very good (R = 0.94 and 0.90, respectively). Additionally, it was demonstrated that the colour of the juices could be assessed with very good accuracy considering them. Due to its simplicity and rapidity, this method is intended to facilitate the quality control of the carotenoid content of foodstuffs in the industry and/or in the field.     

Set-up of a multivariate approach based on serum biomarkers as an alternative strategy for the screening evaluation of the potential abuse of growth promoters in veal calves

A chemometric class modelling strategy (unequal dispersed classes – UNEQ) was applied for the first time as a possible screening method to monitor the abuse of growth promoters in veal calves. Five serum biomarkers, known to reflect the exposure to classes of compounds illegally used as growth promoters, were determined from 50 untreated animals in order to design a model of controls, representing veal calves reared under good, safe and highly standardised breeding conditions. The class modelling was applied to 421 commercially bred veal calves to separate them into ‘compliant’ and ‘non-compliant’ with respect to the modelled controls. Part of the non-compliant animals underwent further histological and chemical examinations to confirm the presence of either alterations in target tissues or traces of illegal substances commonly administered for growth-promoting purposes. Overall, the congruence between the histological or chemical methods and the UNEQ non-compliant outcomes was approximately 58%, likely underestimated due to the blindness nature of this examination. Further research is needed to confirm the validity of the UNEQ model in terms of sensitivity in recognising untreated animals as compliant to the controls, and specificity in revealing deviations from ideal breeding conditions, for example due to the abuse of growth promoters.     

Effect of the region of origin on the perceived quality of olive oil: An experimental approach using a control group

This study attempts to measure the overall effect of the image of an olive oil producing region and the effect of the components of this image on consumers’ perceived quality of an olive oil. Two successive experiments were performed in two different cultural contexts (France and Tunisia). To test the overall effect of the image, a quasi-experimental, pre-test/post-test type research design was developed, using a control group while the discrete choice method was used to study the effect of the components. The results confirm that the image of the product’s region of origin influences perceived overall quality regardless of the respondents’ nationality. The results partially confirm that each dimension of the image of the region of origin has a specific influence on consumers’ selection behaviors. The authors discuss the limitations of the results and the measurements commonly used to assess perceived quality.     

Expanding the diversity of oenococcal bacteriophages: Insights into a novel group based on the integrase sequence

Temperate bacteriophages are a contributor of the genetic diversity in the lactic acid bacterium Oenococcus oeni. We used a classification scheme for oenococcal prophages based on integrase gene polymorphism, to analyze a collection of Oenococcus strains mostly isolated in the area of Bordeaux, which represented the major lineages identified through MLST schemes in the species. Genome sequences of oenococcal prophages were clustered into four integrase groups (A to D) which were related to the chromosomal integration site. The prevalence of each group was determined and we could show that members of the int_B- and int_C-prophage groups were rare in our panel of strains. Our study focused on the so far uncharacterized members of the int_D-group. Various int_D viruses could be easily isolated from wine samples, while int_D lysogens could be induced to produce phages active against two permissive O. oeni isolates. These data support the role of this prophage group in the biology of O. oeni. Global alignment of three relevant int_D-prophages revealed significant conservation and highlighted a number of unique ORFs that may contribute to phage and lysogen fitness.     

Beyond water activity: recent advances based on an alternative approach to the assessment of food quality and safety.

Water, the most abundant constituent of natural foods, is a ubiquitous plasticizer of most natural and fabricated food ingredients and products. Many of the new concepts and developments in modern food science and technology revolve around the role of water, and its manipulation, in food manufacturing, processing, and preservation. This article reviews the effects of water, as a near-universal solvent and plasticizer, on the behavior of polymeric (as well as oligomeric and monomeric) food materials and systems, with emphasis on the impact of water content (in terms of increasing system mobility and eventual water "availability") on food quality, safety, stability, and technological performance. This review describes a new perspective on moisture management, an old and established discipline now evolving to a theoretical basis of fundamental structure-property principles from the field of synthetic polymer science, including the innovative concepts of "water dynamics" and "glass dynamics". These integrated concepts focus on the non-equilibrium nature of all "real world" food products and processes, and stress the importance to successful moisture management of the maintenance of food systems in kinetically metastable, dynamically constrained glassy states rather than equilibrium thermodynamic phases. The understanding derived from this "food polymer science" approach to water relationships in foods has led to new insights and advances beyond the limited applicability of traditional concepts involving water activity. This article is neither a conventional nor comprehensive review of water activity, but rather a critical overview that presents and discusses current, usable information on moisture management theory, research, and practice applicable to food systems covering the broadest ranges of moisture content and processing/storage temperature conditions.     

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.