Selection of egg peptide biomarkers in processed food products by high resolution mass spectrometry.

Food allergy is a growing health problem worldwide; thus, there is an urgent need for robust, specific, and sensitive analytical methods for detecting allergens. Mass spectrometry is an alternative to the existing methods, and it can overcome their limitations. One of the first steps in the development of any analytical method is the identification of the analytes to be further studied. In the case of allergen detection by mass spectrometry, the analytes are peptides. In this study, a strategy was developed for identifying potential peptide biomarkers in processed food products. This strategy was applied to processed egg matrices, and 16 potential peptide biomarkers were identified for the further detection and quantification of egg by means of mass spectrometry. With an empirical approach based on dedicated sample preparation, including tandem Lys-C/trypsin enzymatic digestion and high-resolution mass spectrometry analysis, hundreds of peptides from egg proteins were identified. This list of peptides was further refined with a series of criteria, obtained from empirical evidence, to identify the ideal biomarkers for the development of a quantitative method. These criteria include the resistance to food processing and the specificity of the peptides for eggs but also the effects of amino acid modifications and enzymatic digestion efficiency.

Thermal degradation of cloudy apple juice phenolic constituents.

Although conventional thermal processing is still the most commonly used preservation technique in cloudy apple juice production, detailed knowledge on phenolic compound degradation during thermal treatment is still limited. To evaluate the extent of thermal degradation as a function of time and temperature, apple juice samples were isothermally treated during 7,200s over a temperature range of 80-145 °C. An untargeted metabolomics approach based on liquid chromatography-high resolution mass spectrometry was developed and applied with the aim to find out the most heat labile phenolic constituents in cloudy apple juice. By the use of a high resolution mass spectrometer, the high degree of in-source fragmentation, the quality of deconvolution and the employed custom-made database, it was possible to achieve a high degree of structural elucidation for the thermolabile phenolic constituents. Procyanidin subclass representatives were discovered as the most heat labile phenolic compounds of cloudy apple juice.

Application' and validation of autochthonous lactic acid bacteria starter cultures for controlled leek fermentations and their influence on the antioxidant properties of leek.

Leek (Allium ampeloprasum var. porrum) is one of Belgium's most important outdoor vegetables, mainly cultivated for its white shaft. Fermentation of leek offers opportunities in view of biomass valorization and product diversification. This study deals with the implementation and validation of starter cultures to perform controlled leek fermentations and to ensure a high quality of the end-products. Therefore, a thorough study of the fermentation microbiology and the influence of three starter culture strains (Lactobacillus plantarum IMDO 788, Lactobacillus sakei IMDO 1358, and Leuconostoc mesenteroides IMDO 1347) on the metabolite kinetics of leek fermentation and antioxidant properties of leek was performed. Overall, the application of lactic acid bacteria starter cultures resulted in a fast prevalence of the species involved, coupled to an accelerated acidification. Of the three starter cultures tested, the mixed starter culture of L. plantarum IMDO 788 and L. mesenteroides IMDO 1347 was most promising, as its application resulted in fermented leek of good microbiological quality and in a more extensive carbohydrate consumption, whereby diverse end-metabolites were produced. However, high residual fructose concentrations allowed yeast outgrowth, resulting in increased ethanol and glycerol concentrations, and indicated the lack of a prevailing strictly heterofermentative LAB species. The antioxidant capacity of fermented leek samples, as measured with the oxygen radical absorbance capacity assay, increased when starter cultures were used, whereas with regard to 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity, only leek fermented with L. sakei IMDO 1358 scored higher than spontaneously fermented leek. The total phenolic content was not influenced by the use of starter cultures, while the S-alk(en)yl-L-cysteine sulfoxides content decreased strongly. A preliminary sensory analysis revealed that the spontaneously fermented leek and the one obtained with the mixed starter culture were preferred by consumers, emphasizing again the importance of microbial successions in vegetable fermentations.

An improved mass spectrometric method for identification and quantification of phenolic compounds in apple fruits.

Thirty-nine phenolic compounds were analysed using ultra high performance liquid chromatography (UHPLC) coupled with diode array and accurate mass spectrometry detection using electrospray ionisation (DAD/ESI-am-MS). Instrumental parameters such as scan speed, resolution, and mass accuracy were optimised to establish accurate mass measurements. The method was fully validated in terms of model deviation (r(2)>0.9990), range (typically 10-3500 ngg(-1)), intra/inter-day precision (<6% and <8%, respectively) and accuracy (typically 100 ± 10%). The mass accuracy of each selected phenolic compound was below 1.5 ppm. The results confirmed that the UHPLC-DAD/ESI-am-MS method developed here was convenient and reliable for the determination of phenolic compounds in apple extracts.

Species diversity, community dynamics, and metabolite kinetics of spontaneous leek fermentations.

Leek (Allium ampeloprasum var. porrum) is one of Belgium's most important vegetables. All or part of the green leek parts are often left on the fields because of their limited cooking applications compared to the white leek parts. Therefore, the possibility to perform leek fermentations in view of product valorization and diversification was investigated. This study deals with the community dynamics, species diversity, and metabolite kinetics of spontaneous leek fermentations, thereby studying the influence of added NaCl concentration, harvesting season, and duration of the fermentation. The combination of a culture-dependent and culture-independent approach revealed the prevalence of lactic acid bacteria (LAB) from the third day of fermentation onwards, which was not influenced by the fermentation conditions applied. Enterobacteriaceae, Pseudomonadaceae, and yeasts disappeared after one week of fermentation. Leuconostoc mesenteroides, Lactobacillus sakei, and Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus parabrevis were the most frequently isolated LAB species. Both added NaCl concentrations were suitable to perform successful fermentations within three weeks. By that time, glucose and fructose, the main leek carbohydrates, were metabolized into mainly lactic acid, acetic acid, ethanol, and mannitol. A sensory analysis revealed that the fermented white leek parts were generally more appreciated than the fermented green leek parts.

Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA.

Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used.

ELISA detection of hazelnut proteins: effect of protein glycation in the presence or absence of wheat proteins.

Hazelnuts are widely used in the food industry, especially confectionary foods. Nevertheless, these nuts contain several allergenic proteins that may be unexpectedly present as contaminants in various foods and may pose a serious threat to allergic consumers. The enzyme-linked immunosorbent assay (ELISA) is the preferred method to assess the level of hazelnut protein contamination. It is commonly used by both the food industry and enforcement agencies. Several ELISA kits are commercially available. However, protein detectability by ELISA may be affected by severe changes that proteins undergo during processing. The aim of this study is therefore to investigate the impact of processing on the ability to detect hazelnut protein by four commercial ELISA kits. Hazelnut proteins in the presence or absence of soluble wheat proteins were modified with glucose via the Maillard reaction. Changes in hazelnut proteins, such as the formation of protein-bound carbonyls, losses of reactive lysine residues and free amino groups, and severe aggregation dramatically affected the hazelnut protein detection by the commercial kits. The observed impact was highly dependent on the type of ELISA kit used.

Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.).

To unravel the relationship between the European wild apple, Malus sylvestris (L.) Mill., and its domesticated relative M. domestica Borkh., we studied chloroplast DNA variation in 634 wild and 422 domesticated accessions originating from different regions. Hybridization between M. sylvestris and M. domestica was checked using 10 nuclear microsatellites and a Bayesian assignment approach. This allowed us to identify hybrids and feral plants escaped from cultivation. Sixty-eight genotypes belonging to 12 other wild Malus species, including 20 M. sieversii (Ledeb.) Roem. accessions were also included in the analysis of chloroplast diversity. Marker techniques were developed to type a formerly described duplication and a newly detected transversion in the matK gene. Chloroplast DNA variation was further investigated using PCR-RFLP (Polymerase Chain Reaction-Random Fragment Length Polymorphism), and haplotypes were constructed based on all mutational combinations. A closer relationship than presently accepted between M. sylvestris and M. domestica was established at the cytoplasmic level, with the detection of eight chloroplast haplotypes shared by both species. Hybridization between M. sylvestris and M. domestica was also apparent at the local level with sharing of rare haplotypes among local cultivars and sympatric wild trees. Indications of the use of wild Malus genotypes in the (local) cultivation process of M. domestica and cytoplasmic introgression of chloroplast haplotypes into M. sylvestris from the domesticated apple were found. Only one of the M. sieversii trees studied displayed one of the three main chloroplast haplotypes shared by M. sylvestris and M. domestica. This is surprising as M. sieversii has formerly been described as the main maternal progenitor of the domesticated apple. This study hereby reopens the exciting discussion on the origin of M. domestica.

Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable beta-glucuronidase accumulation levels.

This study aimed at determining whether transgene expression variability is observed in single-copy T-DNA plants and whether it can be correlated with the T-DNA integration position. Among a population of 135 Arabidopsis thaliana transformants, selected on the basis of antibiotic resistance marker expression, 21 single-copy T-DNA transformants were identified and characterized. In 19 of these 21 lines, 35S-beta-glucuronidase transgene expression, measured in two subsequent generations, was similar. This observation means that the intra-transformant variability was as high as the inter-transformant variability. Integration into an intergenic or genic region, into an exon or intron, in sense or antisense orientation, did not result in differential transgene expression. Remarkably, single-copy transformants were not always the highest expressers, implying that low transgene expression is not always induced by multicopy transformants. In only 2 of the 21 single-copy plants was the transgene expression more than 20-fold lower. However, characteristics of the insertion position in one of these lines did not differ significantly when compared to high-expressing lines. In the remaining line, methylation of the transgene was clearly demonstrated. In conclusion, screening for single-copy T-DNA transformants greatly enriches for stable and high transgene expression, because the integration position is not a major determinant of transgene expression variability in Arabidopsis.

Cross-pollination between genetically modified and conventional oilseed rape fields.

Since the introduction of genetically modified crops in Europe, gene flow from those crops to conventional crops and wild relatives has been a key element in the safety assessment. In this experiment cross-pollination frequencies from a genetically modified herbicide tolerant oilseed rape crop to a conventional one were measured at six defined distances from the GM crop by taking seed samples in the conventional crop and analysing the progeny for presence of the herbicide tolerance gene. Levels of cross-pollination tend to decrease with increasing distance from the pollen source. Transgenic volunteers emerging in the subsequent crop can however be readily controlled with normal agricultural practices.

Fingerprinting and quantification of GMOs in the agro-food sector.

Most strategies for analyzing GMOs in plants and derived food and feed products, are based on the polymerase chain reaction (PCR) technique. In conventional PCR methods, a 'known' sequence between two specific primers is amplified. To the contrary, with the 'anchor PCR' technique, unknown sequences adjacent to a known sequence, can be amplified. Because T-DNA/plant border sequences are being amplified, anchor PCR is the perfect tool for unique identification of transgenes, including non-authorized GMOs. In this work, anchor PCR was applied to characterize the 'transgene locus' and to clarify the complete molecular structure of at least six different commercial transgenic plants. Based on sequences of T-DNA/plant border junctions, obtained by anchor PCR, event specific primers were developed. The junction fragments, together with endogeneous reference gene targets, were cloned in plasmids. The latter were then used as event specific calibrators in real-time PCR, a new technique for the accurate relative quantification of GMOs. We demonstrate here the importance of anchor PCR for identification and the usefulness of plasmid DNA calibrators in quantification strategies for GMOs, throughout the agro-food sector.

AFLP markers demonstrate local genetic differentiation between two indigenous oak species [ Quercus robur L. and Quercus petraea (Matt.) Liebl.] in Flemish populations.

The nuclear genetic variation within and between four sessile ( Q. petraea) and six pedunculate ( Q. robur) autochthonous Flemish oak populations was investigated with AFLP markers. One sessile and one pedunculate oak population were additionally screened for detailed leaf characteristics using an image analysis system. Principal coordinate analysis on the AFLP data classified the oaks in two main groups, according to their taxonomic status. No species-specific AFLP markers were found using four primer combinations, but marker frequency differences up to 71% were recorded between both species. Analysis of the genetic structure showed that the divergence between species, as observed by ordination, was significant. Both species revealed similar diversity levels. A smaller though significant differentiation was also revealed for both species among populations within species. Molecular and morphology based approaches showed a high degree of consistency. Screening of 60 AFLP primer combinations using a bulking strategy did not allow identifying species-specific markers, which supports the conclusions reached in previous studies. The distribution of genetic variability at the species and at the population level is discussed.

Allozyme segregation and inter-cytotype reproductive barriers in the polyploid complex Centaurea jacea.

In eastern Belgium, diploid and tetraploid knapweeds (Centaurea jacea L. s. l.) show a parapatric distribution with a contact zone. To compare the success of inter- and intra-cytotype crosses, and to investigate the pattern of allozyme segregation, controlled crosses involving plants from this contact zone were performed. Polysomic inheritance at two loci was observed in tetraploids, suggesting an autopolyploid origin. Two crosses allowed the detection of double reduction events in tetraploids at one locus, but no such event was detected among the 217 progenies. Null alleles were detected in tetraploids at two loci. Both cytotypes were highly self-incompatible. Inter-cytotype crosses were much less successful than intra-cytotype crosses in terms of seed set (five-fold reduction) and germination rates (three-fold reduction), suggesting a 'triploid block' effect. Progenies from inter-cytotype crosses most often had the maternal cytotype and resulted from selfing or pollen contamination. However, six triploids were detected. The significance of these results for the understanding of the geographical distribution of the cytotypes and inter-cytotype gene flow is discussed.

Linkage map construction in the outcrossing species Lolium perenne.

Genetic maps have proven useful tools in several fields of application. First of all, they allow to get insight into the genome organization of a species, and to compare the genome structures of different species. Genetic maps are also useful for the identification of genomic regions involved in physiological processes, and are valuable tools for the positional cloning of genes. In the framework of a project with the aim of identifying genomic regions involved in disease resistance in Lolium spp., we constructed genetic maps using different marker systems. In the ryegrass species L. perenne and L. multiflorum self-pollination is prevented by a very efficient self incompatibility system. This has clear implications in mapping studies: inbred lines and double haploids are difficult to produce, and if produced low seed yields are obtained. For this reason, we created several mapping populations by crossing two highly heterozygous unrelated plants. This implies that at any given locus, up to four different alleles might be segregating in the offspring. Different marker systems were used for the construction of the genetic maps. In first instance, AFLP was used as a high throughput marker system. This allowed to generate a high number of DNA-markers useful for map construction in a quick way. The drawback of the AFLP technique is twofold: AFLP markers are 'anonymous', and are dominant (not all alleles at a locus can be detected). Co-dominant marker systems, which allow to detect all alleles present at any locus, are much more informative and are required for the construction of detailed linkage maps in outcrossers. In this study, we used three co-dominant marker systems: SSR, STS and RFLP. The RFLP markers were generated using heterologous probes derived from other monocots, what should allow to analyze the syntenic relationships between ryegrass and other monocots. Using these markers generated with different techniques, a genetic map of ryegrass has been constructed suitable for QTL analysis and comparative genetics.

Gene flow from a large scale release of genetically modified herbicide tolerant and hybrid winter oilseed rape (Brassica napus L.).

One of the major concerns regarding the cultivation of transgenic crops is the uncontrolled spread of transgenes among fields or to related wild species. To address this concern a long-term study has been set up to assess the amount of gene flow that is occurring from a large scale field trial of genetically modified herbicide tolerant and hybrid (GMHTH) oilseed rape. During the first year of the project, outcrossing of the herbicide tolerance gene from a large scale release of GMHTH winter oilseed rape to a non-transgenic oilseed rape crop in the vicinity and to wild relatives in the environment has been monitored. After harvesting seeds on oilseed rape plants or wild relatives, the seeds are sown in the greenhouse. Seedlings are sprayed with the herbicide on two different occasions. In this way escapes of the first treatment that are not transgenic will be eliminated. To confirm the transgene state of the surviving plants, leaf material is collected from resistant plants. DNA is extracted and the material is examined for the presence of the herbicide tolerance gene by means of a PCR assay. Results on the levels of cross pollination will be discussed.