Nils Wax

Abstract

DNA-basierte Authentifizierung von pflanzlichen und tierischen Lebensmitteln anhand extrachromosomaler Sequenzunterschiede/ DNA-based authentication of plant and animal foods based on extrachromosomal sequence differences

Betreuer: Prof. Dr. Markus Fischer (UHH, Hamburg School of Food Science)

Food fraud occurs when food is intentionally counterfeited and placed on the market for economic or financial gain. Although food laws provide a target state through regulations and guidelines, suitable verification procedures are needed to guarantee protection against fraudulent practices. Molecular biological methods are suitable for highly processed foodstuff due to the chemical stability of the analyte (DNA). To detect species- or variety-specific differences in the DNA sequence, the genomes of foodstuffs usually need be partially or, in some cases, fully sequenced. Sequence comparisons can then be used to identify the relevant species or varieties. In routine analysis, it is then sufficient to detect these differences using suitable amplification methods.

The above-described approach can be used to address a wide variety of issues related to the biological identity of food ingredients. The dissertation is therefore divided into two sections: “Differentiation of plant foods” (detection of bitter almonds and differentiation of cocoa varieties) and “Differentiation of animal foods” (identification offish species). In detail, the initial objective was to develop a technique for identifying bitter almonds in almond products. Using next-generation sequencing technology, the plastid genomes of six sweet almond varieties and six bitter almonds of different origin were sequenced. The raw data (reads) were assembled using an already known sweet almond plastid reference genome (NC_034696.1). Identified sequence differences were validated in the next step using Sanger sequencing in additional almond samples. Based on the validated sequence differences, specific molecular biological downstream methods were then developed to detect these differences in almonds and marzipan. Double mismatch allele-specific qPCR (DMAS-qPCR) was used to detect the two identified polymorphic loci (rpoB, rps4). Using DMAS-qPCR, it was possible to make a statement about the allele distribution of rpoB and rps4 in a larger sample set and estimate the bitter almond content in processed products such as marzipan. The results showed that sweet and bitter almonds share the same haplotype, but additional sequence variations were present in most bitter almond populations. The rps4 sequence variant of bitter almond could be detected in marzipan with (debittered) bitter almonds if the samples contained at least 8 % bitter almonds. Analyzing the genetic profile thus made it possible to detect bitter almonds in marzipan. Secondly, to implement a laboratory-independent test system for the rapid on-site analysis of genetic profiles, the suitability of in-vitro CRISPR-Cas12a diagnostic was evaluated. By using two test systems developed for the exemplary matrix cocoa, admixtures of 5% of the cocoa variety CCN-51 (P < 0.01) could be reliably detected in short time. In the second section, simplified DNA extraction and detection methods were developed for animal foodstuffs (fish products: Plaice (Pleuronectes platessa and sole (Solea solea)). As part of this work, species-specific isothermal amplification using loop-mediated isothermal amplification (LAMP) on DNA extracted from P. platessa and S. solea and simplified detection using a lateral flow assay (LFA) and a portable fluorescence analyzer was achieved.