With new genetically modified crops being constantly developed, many new targets need to be analysed for regulatory compliance. Classical PCR-based approaches currently used are however reaching their limits in terms of target throughput and new approaches are needed to adapt to the evolving situation. The Ion AmpliSeqTM HD technology was here adapted for the simultaneous detection of 108 GMO targets by Next Generation Sequencing (NGS) on the Ion GeneStudio S5 platform, using PCR primers mostly retrieved from the official European GMO methods. Tested on a wide array of reference materials, the method delivered specific detection of the expected GMO targets and reached a sensitivity down to 0.01% (w/w) as required by GMO analytical guidelines. Numerous proficiency test samples were successfully analysed which confirmed the method performance and its applicability on real test samples. With its straightforward library preparation and ready-to-use bioinformatics analysis, the developed method is well tailored for routine GMO testing laboratories.

Herbs and spices are food categories known to be at high risk of adulteration. Presence of undeclared foreign plant species has often been reported in oregano and may have a direct impact on its organoleptic quality and potentially the safety of this aromatic herb. A droplet digital PCR approach was developed to assess the purity of oregano by quantifying the DNA copies of oregano versus the total plant DNA copies. Nuclear single-copy genes were selected by targeting the terpene synthase 5 gene from oregano and the plant phosphoenolpyruvate carboxylase 2 gene. The reactions were specific to the Origanum genus and plant materials respectively, whereas trueness and precision data confirmed the reliability of the method to quantify oregano. The applicability of the method was further verified on proficiency test samples before being applied on commercial oregano samples.

To enhance health benefits, a probiotic can be co-administered with a metabolizable prebiotic forming a synergistic synbiotic. We assessed the synergies resulting from combining Bifidobacterium longum subsp. infantis LMG 11588 and an age-adapted blend of six human milk oligosaccharides (HMOs) in ex vivo colonic incubation bioreactors seeded with fecal background microbiota from infant and toddler donors. When HMOs were combined with B. infantis LMG 11588, they were rapidly and completely consumed. This resulted in increased short chain fatty acid (SCFA) production compared to the summed SCFA production from individual ingredients (synergy). Remarkably, HMOs were partially consumed for specific infant donors in the absence of B. infantis LMG 11588, yet all donors showed increased SCFA production upon B. infantis LMG 11588 supplementation. We found specific bacterial taxa associated with the differential response pattern to HMOs. Our study shows the importance of carefully selecting pre- and probiotic into a synergistic synbiotic that could benefit infants.

Herbs and spices are known to be prone to food fraud and accurate analytical tools are needed to detect adulterants. Amongst the potential adulteration, dilution with bulking agents has regularly been reported, especially with inorganic materials such as talc or brick powder. Energy Dispersive X-Ray Fluorescence (ED-XRF) spectrometry is a well-established non-destructive analytical technique for qualitative and quantitative elemental analysis of a wide variety of samples. ED-XRF was here evaluated for the detection of inorganic adulterants in turmeric, paprika and oregano, which were selected as representative for the herbs & spices food category. Magnesium, silicon, and calcium were identified as elements to detect talc, soapstone, brick/clay powder, and chalk inorganic adulterants. ED-XRF successfully detected adulterated samples when spiked down to 5% (w/w) in the selected herbs and spices. With its ease-of-use and speed, ED-XRF is well adapted for the monitoring of inorganic adulteration of herbs and spices along the supply chain.

Contamination of vegan products and vegan shrimps with real shrimps and crustaceans must be avoided to comply with vegan claims, even more for preventing strong allergenic reactions. Therefore, the detection of crustaceans must be reliable and sensitive enough for authenticity, traceability, and food safety purposes. A new real-time PCR assay was developed targeting the mitochondrial 16S rRNA gene of crustaceans and was optimised to avoid critical mismatches with primers and probe. By testing several crustacean species and common food ingredients, the method was demonstrated to be specific to crustaceans only. To comply with the limit of non-vegan contamination established at 0.1% (w/w) by the European Vegetarian Union, dedicated cut-off CT values were determined on vegan raw materials and on vegan shrimps spiked with crustacean materials. The method reached a sensitivity ≤ 0.0005% (w/w), which was further confirmed on reference materials containing a similar amount of crustacean.

BACKGROUND MicroRNAs (miRNAs) are small noncoding RNAs involved in posttranscriptional regulation. miRNAs can be secreted and found in many body fluids, and although they are particularly abundant in breastmilk, their functions remain elusive. Human milk (HM) miRNAs start to raise considerable interest, but a comprehensive understanding of the repertoire and expression profiles along lactation has not been well characterized. OBJECTIVES This study aimed to characterize the longitudinal profile of HM miRNA between the second week and third month postpartum. METHODS We used a new sensitive technology to measure HM miRNAs in a cohort of 44 French mothers [mean ± SD age: 31 ± 3.5; BMI (in kg/m2) 21.8 ± 2.3] who delivered at term and provided HM samples at 3 time points (17 ± 3 d, 60 ± 3 d, and 90 ± 3 d) during follow-up visits. RESULTS We detected 685 miRNAs, of which 35 showed a high and stable expression along the lactation period analyzed. We also described for the first time a set of 11 miRNAs with a dynamic expression profile. To gain insight into the potential functional relevance of this set of miRNAs, we selected miR-3126 and miR-3184 to treat undifferentiated Caco-2 human intestinal cells and then assessed differentially expressed genes and modulation of related biological pathways. CONCLUSIONS Overall, our study provides new insights into HM miRNA composition and, to our knowledge, the first description of its longitudinal dynamics in mothers who delivered at term. Our in vitro results obtained in undifferentiated Caco-2 human intestinal cells transfected with HM miRNAs also provide further support to the hypothesized mother-to-neonate signaling role of HM miRNAs. This trial was registered at clinicaltrials.gov as NCT01894893.

Background Spices and herbs are food categories regularly cited as highly susceptible to be adulterated. To detect potential adulteration with undeclared species, DNA-based methods are considered the most suitable tools. Objective In this study, the performance of the ready-to-use Thermo Scientific (TM) NGS Food Authenticity Workflow (Thermo Fisher Scientific)-a commercial DNA metabarcoding approach-is described. The tool was further applied to analyze 272 commercial samples of spices and herbs. Method Pure samples of spices and herbs were analyzed with the Thermo Scientific NGS Food Authenticity Workflow to assess its specificity, and spikings down to 1% (w/w) allowed evaluation of its sensitivity. Commercial samples, 62 and 210, were collected in Asian and European markets, respectively. Results All tested species were correctly identified often down to the species level, while spikings at 1% (w/w) confirmed a limit of detection at this level, including in complex mixtures composed of five different spices and/or herbs. The analysis of 272 commercial samples showed that 78% were compliant with the declared content, whereas the rest were shown to contain undeclared species that were in a few cases allergenic or potentially toxic. Conclusions The Thermo Scientific NGS Food Authenticity Workflow was found to be suitable to identify food plant species in herbs and spices, not only when tested on pure samples, but also in mixtures down to 1% (w/w). The overall workflow is user-friendly and straightforward, which makes it simple to use and facilitates data interpretation.

Consumption of plant-based products and meat alternatives has been rising for a few years, with vegetarian and vegan diets gaining more and more popularity. To comply with legislation and maintain consumers? trust, food manufacturers of vegan products must ensure the absence of animal residues in these products. A real-time PCR method targeting the 16S mitochondrial rRNA gene from vertebrates has been developed in order to detect the presence of vertebrate material in plant-based ingredients and finished products. The reaction successfully amplified on mammals, birds, reptiles and fish samples only. The analysis of plant-based samples spiked at 0.1% (w/w) with meat or fish materials confirmed the sensitivity of the method at this level, considered as the limit for unintended presence of animal residues by the European Vegetarian Union. The method was applied to commercialized vegan finished products from European markets and confirmed the absence of animal material in all the tested samples.

Food fraud and species substitution are topical issues that must be addressed for quality and safety purposes and to maintain consumers' trust. DNA-based methods are considered as the reference approach for species identification, but they are usually limited to a few species only. In this study, the performance of a commercial NGS solution has been evaluated as an untargeted tool to identify meat species. Tested on pure meat samples, all species were correctly identified including several exotic species, and close-related species were correctly differentiated. Species were successfully detected and identified in mixtures down to 1% (w/w). The reliability of the tool was further confirmed on several proficiency test samples, and promising quantification data were obtained. Finally, 45 ground meat samples sourced from local European and Asian markets were analyzed and 18% of them showed cases of adulteration with undeclared meat species.

The growing population worldwide is associated with an increasing demand for food and a corresponding growth of the agri-food sector along with an elevated risk of economically motivated food adulteration. Recent food adulteration crises had consequences worldwide due to the globalization of the raw material supply. Crises highlighted the fact that food adulteration did not receive sufficient attention for many years. Identification of poor supply chain transparency and raw material traceability as major vulnerabilities triggered a rapid progress in new software tools enabling the mapping of food supply chains. In contrast to safety evaluations performed on newly identified food contaminants or "new ingredients" for novel food products, no assessment of the potential vulnerabilities of food raw materials have been properly performed in the past. However, food companies started recently to conduct systematic analysis and mapping of raw materials vulnerabilities in order to identify and prioritize major risks and develop food fraud mitigation plans. The type of analytical approaches applied to detect nutrients or food contaminants are not suitable to detect the infinite possibilities associated with the criminal intentional act of food adulteration. Therefore, food adulteration requires further development of untargeted methodologies, in addition to targeted ones, for rapid screening of abnormalities and subsequent identification of adulterants.

Accurate quantitative methods are needed to determine the amount of transgenic material in ingredients and comply with labelling GMO thresholds. Quantitative real-time PCR methods are usually applied for GMO quantification, but since a few years, digital PCR (dPCR) has been described as a potential alternative by quantifying DNA molecules directly without any standard curves. In this study, the performance of dPCR to quantify 9 GM-soya events and 15 GM-maize events was assessed. Following GMO validation guidelines, the trueness and precision were determined on high, medium and low levels of transgenic content. Results showed biases below ± 25% and satisfactory precision data. Limits of quantification were determined for each GM-event and were between 12 and 31 target copies. The reliability of GMO quantification by dPCR was further confirmed by analysing several proficiency test samples. Overall, dPCR showed accurate and precise GMO quantification on all the tested GM-events, from high to low transgenic amount. With its ease-of-use, dPCR was found to be an appealing alternative technology for routine GMO testing laboratories.

Meat has been identified as one of the food categories at most risk of food fraud. Meat species substitution has been in the spotlight with the European horse meat scandal in 2013. Analysis of cases reported on the web shows that incidents of meat substitution are still recurring worldwide. Altogether these cases highlight significant weaknesses in the supply chain transparency and traceability of raw meat materials. This has triggered recent progress from the food industry to apply new software tools enabling the mapping of meat supply chains. Nevertheless, a meat vulnerability assessment showed that meat and derivatives are highly susceptible to many fraudulent malpractices. Therefore, more effective measures are needed to manage the risk and new analytical solutions are required to increase the deterrence of meat adulteration and rapid detection of fraud. DNA-based methods have evolved rapidly as shown with the application of the new LCD array and Next Generation Sequencing (NGS) in order to detect broad meat species adulteration. Moreover, new technologies such as NGS together with the Rapid Evaporative Ionization Mass Spectrometry (REIMS) are emerging as a really promising association of analytical approaches for rapid detection of several malpractices.

Methods to detect the presence of genetically modified organisms (GMOs) are evolving constantly to comply with legislation and include new transgenic traits developed by biotechnology companies. Since traditional screening methods target only a limited number of markers, not all GM-events can be detected easily. To cover a broader range, a new GMO screening method was developed, based on two real-time PCR reactions, targeting i) six major GM-markers and ii) six GM-events that do not contain these markers. This method used the FAST PCR technology and allowed a further reduction in time-to-result. Using a wide array of reference materials and proficiency test samples, the method showed a broad screening capacity and the absolute limit of detection was consistently below 20 copies, thereby demonstrating the method is fit-for-purpose.

Considering the recent cases of meat adulteration and fraud, efficient and accurate analytical methods are needed for the specific identification of meat species as a key importance to maintain consumers' trust and to comply with various local labelling legislations. As a reference approach, the application of PCR to meat speciation is always limited to few species, whereas there is a strong need to broaden the number of identified meat species. PCR was here associated with a commercial DNA macro-array spotted with meat-specific capture probes, allowing the simultaneous identification of 32 meat species on 8 samples per chip support. Tested on pure meat samples, spiked samples, proficiency test samples, and processed samples, the method showed high specificity on the targeted species and allowed a sensitivity down to 1% (w/w). Easy-to-use, fast and cost-efficient, this multi-screening approach is fit-for-purpose as a tool to detect meat substitution or adulteration for meat testing laboratories.

Crises related to the presence of melamine in milk or horse meat in beef have been a wake-up call to the whole food industry showing that adulteration of food raw materials is a complex issue. By analysing the situation, it became clear that the risk-based approach applied to ensure the safety related to chemical contaminants in food is not adequate for food fraud. Therefore, a specific approach has been developed to evaluate adulteration vulnerabilities within the food chain. Vulnerabilities will require the development of new analytical solutions. Fingerprinting methodologies can be very powerful in determining the status of a raw material without knowing the identity of each constituent. Milk adulterated by addition of adulterants with very different chemical properties could be detected rapidly by Fourier-transformed mid-infrared spectroscopy (FT-mid-IR) fingerprinting technology. In parallel, a fast and simple multi-analytes liquid-chromatography tandem mass-spectrometry (LC/MS-MS) method has been developed to detect either high levels of nitrogen-rich compounds resulting from adulteration or low levels due to accidental contamination either in milk or in other sensitive food matrices. To verify meat species authenticity, DNA-based methods are preferred for both raw ingredients and processed food. DNA macro-array, and more specifically the Meat LCD Array have showed efficient and reliable meat identification, allowing the simultaneous detection of 32 meat species. While the Meat LCD Array is still a targeted approach, DNA sequencing is a significant step towards an untargeted one.

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers’ perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.