DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

To date, studies of the toxicity of engineered nanoparticles (NPs) in fish have not fully considered effects on olfactory-mediated behaviours, despite their ecological importance. In this study the effects of copper NPs (Cu NPs) on the anti-predator behavioural responses of juvenile rainbow trout (Oncorhynchus mykiss) to trout alarm substance was investigated. Individual fish were exposed for 12h to a control (no added Cu), 50μgl(-1) of Cu as Cu NPs, or 50μgl(-1) Cu as CuSO4, after which fish behaviours were analyzed in 10min periods before and after the addition of the alarm substance stimulus. The response of control fish to deionised water (negative control, no alarm substance stimulus) was also analyzed. The alarm substance elicited a behavioural response in the control fish characterized by an immediate freeze response and the slower resumption of swimming activity compared to negative controls exposed to the sham deionised water stimuli. In fish exposed to Cu NPs, the behavioural response to alarm substance was eliminated, with no significant difference in behaviours compared to negative controls. In comparison, exposure to 50μgl(-1) Cu as CuSO4 decreased, but did not eliminate the response of fish to alarm substance, which indicated a significantly greater effect of Cu NPs on olfactory mediated behaviours than of the equivalent concentration of Cu as CuSO4. Measurement of total Cu concentrations in the tissues of fish demonstrated no significant accumulation of Cu from any treatment in gill, liver or brain, confirming the effects of Cu NPs, and to a lesser extent CuSO4, on behavioural responses were mostly associated with the interaction of the materials with the external surfaces of the fish. Scanning electron microscopy revealed that Cu as CuSO4 caused a pronounced depletion of ciliated sensory and non-sensory cells in the olfactory rosette surrounding the midline raphe, whereas Cu NPs had no impact on the structure of the rosette. However, exposure to Cu NPs caused a significant increase in the ratio of oxidized to reduced glutathione in brains of fish, indicating some systemic oxidative stress that was not observed in either controls or fish exposed to CuSO4. Overall, the study showed that the olfactory mediated behaviours of fish were potentially more sensitive to Cu NPs than CuSO4 and NPs elicited effects via a mechanism that is distinct from that of the metal salt.

He Y.-Q., Ma Ch.-Y., Pan Y., Yin L.-J., Zhou J., Duan Y., Zhang H., Ma H. (2018): Bioavailability of corn gluten meal hydrolysates and their effects on the immune system. Czech J. Food Sci., 36: 1-7.The bioavailability of food is central to human nutrition, health and wellbeing. Here, we tested the bioavailability of hydrolysed corn gluten meal using a protein efficiency ratio method, and then analysed differences in bodyweight, weight of organs, routine blood tests and histological sections. The results indicate that the average protein intake of the hydrolysed corn gluten meal (HCGM) group was higher than that of the crude corn gluten meal (CCGM) group, and was associated with an increase in average bodyweight. The corrected protein efficiency ratios (PERs) of the HCGM and CCGM groups were 0.374 and 0.217, respectively; the corrected PER of the HCGM group was 1.72 times higher than that of the CCGM group. These results show that hydrolysis increased the bioavailability of corn gluten meal. Furthermore, there was a significant difference in organ weights (salivary gland P < 0.01; thymus gland P < 0.05; spleen P < 0.01) between the HCGM and CCGM groups. Finally, no inflammatory cell infiltrates nor cell necrosis could be found in any of the histological sections. We speculate that hydrolysed protein preparations can improve immunity.

Oat (Avena sativa L.) is considered to be a healthy food. In contrast to other grain crops, oat is high in protein, lipids, dietary fiber, antioxidants, and uniquely in avenanthramides. The question of whether it can also be consumed by people suffering from celiac disease is still unresolved. The main aim of this study was to extract and sequence genes for potentially harmful avenins, globulins, and α-amylase/trypsin inhibitors in six oat varieties and to establish their variability using PacBio sequencing technology of enriched libraries. The results were compared with sequences of the genes already present in databases. In total, 21 avenin, 75 globulin, and 25 α-amylase/trypsin inhibitor genes were identified and mapped in the hexaploid oat chromosomes. In all of the three gene families, only marginal sequence differences were found between the oat varieties within the individual genes. Avenin epitopes were found in all four types of avenin genes occurring in all oat varieties tested within this study. However, the number of avenin genes was nearly four times lower than of globulin genes and, on the protein level, formed only 10% of storage proteins. Therefore, the question of whether oat is safe to celiac disease people is a question of boundary values.

The presented plasmid DNA is a suitable and inexpensive possibility for evaluating PCR inhibition.

We have developed and validated post-PCR (polymerase chain reaction) high resolution melting (HRM) based assays that allow identifying the presence of two major allergenic foods – soybean and bovine milk – in food products. A new set of primers for PCR was developed for detection of the gene encoding the soybean protein lectin. The assay was validated using reference samples and used for the analysis of artificially prepared mixed samples of dairy and soy products of different matrices, as well as of real products available in the market (spray creams). The limits of detection (LODs) of the soybean (8 copies) and bovine milk (4 copies) assays were lower compared to LODs of other previously published PCR-based assays. The analysis of several commercial spray creams revealed an undeclared presence of soybean in one of the samples. The newly developed HRM assays are precise and robust alternatives for the control of food composition and falsification by competent authorities.

Sovová T., Křížová B., Drábková L., Ovesná J. (2017): Detection of PCR inhibition in food and feed with a synthetic plasmid. Czech J. Food Sci., 35: 160-164.We present a successful use of the plasmid inhibition detection and DNA isolation protocol optimisation for four food/feed samples in qPCR analysis of the sequence coding for chloroplast tRNA-Leu: two meat meal samples and two samples made of cranberries (jam and dried fruit). The quantitative real-time polymerase chain reaction (qPCR) can be inhibited by various substances and the DNA content in the sample can be underestimated. It is necessary to identify the PCR inhibition and choose an optimal DNA isolation protocol to correctly evaluate the sample. In a previous study, we have developed an assay using plasmid DNA carrying a non-homologous random sequence identifying possible inhibitors in qPCR in food/feed samples. The plasmid assay allowed to effectively reveal the PCR inhibition in all of the different sample matrices and to choose an optimal DNA isolation protocol.