Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have ...been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group. DNA-based approaches (using either nuclear or mitochondrial DNA) are most commonly used in this case, due to their high specificity and to the high resilience of the target molecules to food processing techniques. In this article, we identified, using an automated screening approach, novel barcode regions and their associated primers in the nuclear genome, to be used for the efficient identification of Gadoids. The barcode regions were tested on official and commercial samples, raw or mildly treated products, like frozen, or salted, as well as pre-cooked complex mixtures and processed samples, using next-generation sequencing (NGS) technique. The method proposed could complement existing fish identification strategies in establishing an efficient framework to detect and prevent frauds along the food chain.
Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the ...present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion. The results of this study indicate that for each crop at least one taxon-specific assay can be identified as having a single DNA target. A short list of taxon-specific reference assays is proposed as best candidates for the relative quantification of GM events for soybean, maize, cotton and oilseed rape. The investigated assays could be in most cases transferred to ddPCR without further optimisation. The use of DNA digestion did not improve ddPCR characteristics such as rain and resolution at the conditions tested.
•DdPCR was used to test number of targets of validated reference assays for 4 GM crops.•At least 1 taxon-specific assay for a single DNA target was identified per crop.•Most qPCR based assays tested can be transferred to ddPCR without optimisation.•DNA digestion does not improve ddPCR characteristics at the conditions tested.
Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or ...shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the "top-five" serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform.
Discarded leaves of red chicory (Radicchio “Rosso di Chioggia” IGP) were fermented with one
Saccharomyces
yeast and four lactic acid bacteria chosen on the basis of their ability to grow on plant ...material without any need of supplements. Antiradical and antimicrobial activities of the resulting products were assessed. Among the strains tested,
Lactobacillus plantarum
and
L. hilgardii
gave the best performances and also provided fermented substrates with antiradical and antimicrobial activities. In particular the latter compounds were found only in fermented samples, confirming that the choice of appropriate microorganisms for fermentation could be useful when the aim is to target specific functional foods starting from by-products or waste material.
Katiki Domokou is a traditional Greek cheese, which has received the Protected Designation of Origin recognition since 1994. Its microfloras have not been studied although its structure and ...composition may enable (or even favor) the survival and growth of several pathogens, including Listeria monocytogenes. The persistence of L. monocytogenes during storage at different temperatures has been the subject of many studies since temperature abuse of food products is often encountered. In the present study, five strains of L. monocytogenes were aseptically inoculated individually and as a cocktail in Katiki Domokou cheese, which was then stored at 5, 10, 15, and 20°C. Pulsed-field gel electrophoresis was used to monitor strain evolution or persistence during storage at different temperatures in the case of the cocktail inoculum. The results suggested that strain survival of L. monocytogenes was temperature dependent since different strains predominated at different temperatures. Such information is of great importance in risk assessment studies, which typically consider only the presence or absence of the pathogen.
The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially ...outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi‐urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real‐time PCR analysis. Our results suggest that this ‘GM pollen monitoring by bioaerosol sampling and PCR screening’ approach might represent an useful aid in the surveillance of GM‐free areas, centres of origin and natural reserves.
As part of the risk assessment (RA) requirements for genetically modified (GM) plants, according to Regulation (EU) No 503/2013 and the EFSA guidance on the RA of food and feed from GM plants (EFSA ...GMO Panel 2011), applicants need to perform a molecular characterisation of the DNA sequences inserted in the GM plant genome. This Technical Note to the applicants puts together requirements and recommendations for the quality assessment of the methodology, analysis and reporting when DNA sequencing is used for the molecular characterisation of GM plants. In particular, it applies to the use of Sanger sequencing and next‐generation sequencing for the characterisation of the inserted genetic material and its flanking regions at each insertion site, the determination of the copy number of all detectable inserts and the analysis of the genetic stability of the inserts. This updated document replaces the EFSA 2018 Technical Note and reflects the current knowledge in scientific‐technical methods for generating and verifying, in a standardised manner, DNA sequencing data in the context of RA of GM plants. It does not take into consideration the verification and validation of the detection method which remains under the remit of the Joint Research Centre (JRC).
EFSA was requested by the European Commission (in accordance with Article 29 of Regulation (EC) No 178/2002) to provide a scientific opinion on the application of new developments in biotechnology ...(new genomic techniques, NGTs) to viable microorganisms and products of category 4 to be released into the environment or placed on the market as or in food and feed, and to non‐viable products of category 3 to be placed on the market as or in food and feed. A horizon scanning exercise identified a variety of products containing microorganisms obtained with NGTs (NGT‐Ms), falling within the remit of EFSA, that are expected to be placed on the (EU) market in the next 10 years. No novel potential hazards/risks from NGT‐Ms were identified as compared to those obtained by established genomic techniques (EGTs), or by conventional mutagenesis. Due to the higher efficiency, specificity and predictability of NGTs, the hazards related to the changes in the genome are likely to be less frequent in NGT‐Ms than those modified by EGTs and conventional mutagenesis. It is concluded that EFSA guidances are ‘partially applicable’, therefore on a case‐by‐case basis for specific NGT‐Ms, fewer requirements may be needed. Some of the EFSA guidances are ‘not sufficient’ and updates are recommended. Because possible hazards relate to genotypic and phenotypic changes introduced and not to the method used for the modification, it is recommended that any new guidance should take a consistent risk assessment approach for strains/products derived from or produced with microorganisms obtained with conventional mutagenesis, EGTs or NGTs.
This study explored the microbiota of Formaella, Kopanisti, Feta and Mana cheeses. A total of 133 wild lactic acid bacteria were isolated and classified phenotypically. Mesophilic lactobacilli were ...the most abundant group. Thermophilic lactobacilli and thermophilic cocci were the best milk acidifiers, whereas thermophilic lactobacilli were the most proteolytic isolates. Higher peptidolytic and esterolytic activities were obtained with thermophilic cocci. Only five isolates were lipolytic, whereas none was able to catabolize citrate. Fast gas chromatography−mass spectrometry analysis of the metabolites produced and subsequent principal components analysis revealed segregated groups of isolates in accordance with the phenotypic ones. Electronic nose analysis revealed similar results.
Lactobacillus rennini and
Lactobacillus acidipiscis were found to be the sole microbial species in Kopanisti cheese and Mana. These isolates produced alcohols and aldehydes as major volatile compounds, as a result of secondary amino acid catabolism.
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