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With metabolomics food analytics goes all the way

In the not-too-distant future, food analysts hope they will be able do more than just detect mycotoxin traces and determine the concentration of individual nutrients such as vitamin C. Their ambition is in fact much broader: “We would like to understand the nutritional and physiological effect of food,” said Prof. Sabine Kulling from the Max Rubner Institute in Karlsruhe. As with other life sciences areas, metabolomics is seen as a key technology for research into issues relating to the quality and safety of food.

Prof. Sabine Kulling, Director of the Department of Safety and Quality of Fruit and Vegetables at the MRI. © MRI

“Nowadays, the determination of food quality and safety is based on far more parameters and compounds than it was in the past,” said Sabine Kulling referring to recent developments in food analysis. Sabine Kulling, director of the Department of Safety and Quality in Fruit and Vegetables, is in charge of establishing metabolomics techniques at the Max Rubner Institute, the German Federal Institute of Nutrition and Food.

The novelty of metabolomics approaches is their ability to give the broadest possible overview of the biochemical composition (metabolites) of complex biological samples. While these approaches can provide information about target and non-target metabolites in one analysis, classical analyses are limited to determining the absolute concentration of target compounds. In contrast, this novel, non-target approach allows the reproducible quantification of all possible metabolites. “One single analysis is used to attempt to characterise a food item as comprehensively as possible, even when the food of interest consists of several hundred compounds,” Kulling explains. Metabolomics has two big advantages: first, it enables the detection of a much larger number of metabolites than other technologies do. Second, it enables unknown metabolites to be identified as important ones and subsequently leads to their unequivocal identification. 

The metabolome of apples differs according to variety, origin, cultivation conditions, maturity, storage conditions and fungal infections. © Claudia Hautumm/pixelio.de

According to Prof. Kulling, metabolomics will be applied to establishing new breeds, the processing of foods and the use of new processing methods. Metabolomics can be used to explore the differences between an existing apple variety and a new variety and to identify the advantages (health-promoting compounds) or disadvantages (contaminants introduced during processing) of the new variety for consumers, to name but one example. Metabolomics also facilitates research into the effect of food in terms of human health; approaches exist that are able to explore what happens with food and its ingredients in the human metabolism and that assess the influence of diet on the human metabolism – issues that have always attracted huge public interest.

Complex analyses bring disciplines closer together

Prof. Kulling has observed a clear trend in the analysis of foodstuffs, namely the attempt to characterise the ingredients and composition of foodstuffs more comprehensively than was previously possible. However, this objective can only be achieved when different disciplines work closely together. The era when a food chemist carried out vitamin C analyses on his or her own is long gone. Metabolome analyses create huge amounts of data, which is why food chemists depend on the cooperation with biostatisticians and bioinformaticians. Plant food analyses depend on the expertise of biologists such as plant physiologists in order to interpret the acquired data.

Nevertheless, the emergence of metabolomics technologies does not make simple and targeted analyses any less important. However, the wealth of information these technologies generate means they have major advantages, especially when it comes to assessing the safety of foodstuffs for human health.

Metabolomics research: NMR and coupled mass spectrometry

Two-dimensional mass spectrum of a tomato. © Kulling/MRI

Metabolomics approaches principally involve the use of two analytical methods: NMR (nuclear magnetic resonance) spectroscopy and mass spectrometry, which is one of the most widely used and powerful methods. Mass spectrometry is often interfaced with chromatographic separation technologies (gas chromatography and high performance liquid chromatography). The combination of chromatographic separation technologies with mass spectrometry allows the more effective separation of food ingredients. 

The Max Rubner Institute is particularly focussed on the development of mass spectrometry-based metabolomics methods. In cooperation with the Karlsruhe Institute of Technology (KIT; Prof. Burkhard Luy), the MRI researchers use NMR-based methods for conducting metabolome analyses. 

Food inspectors are using NMR-based screening methods for determining the authenticity of fruit juices. © Rainer Sturm/Pixelio.de

The CVUA (Investigative Office for Food Control and Animal Health) in Karlsruhe has had access to an NMR spectrometer since 2010. Compared to older devices, this NMR spectrometer can be operated with samples that are relatively simple to prepare; it also has a shorter measurement time and increased sample throughput. Dr. Thomas Kuballa from the CVUA in Karlsruhe calls the NMR spectrometer an analytical allrounder: “The device can be used for a broad range of applications, including the identification of organic and biochemical molecules, the determination of their structure and the quantitative analysis of the components of complex mixtures such as foodstuffs.” Moreover, the device is suitable for testing non-target compounds for their authenticity and origin, making it superior to any other analytical technique (2011 Annual Report, p. 157 f.)

In the meantime, the first commercial NMR-based screening method (Bruker BioSpin GmbH) has become available for classification analyses. This method enables official food control authorities to screen fruit juices and determine the origin of the fruit as well as its variety, and differentiate between juice made from concentrate and juice not from concentrate. The method can also be used for the screening of wine and oils. “I am sure that, once it is validated, this method will become part of official inspections,” Sabine Kulling says. She believes that the mass spectrometric screening of juices and other samples is more difficult and more time-consuming than the NMR-based method. Moreover, mass spectrometry-based metabolomics methods have not yet been standardised. “I am sure this will happen within the next few years,” says Sabine Kulling who bases her belief on the observation that big companies are increasingly turning to mass spectrometry-based screening methods. 

DNA-based analytics methods

Like in many other life sciences areas, molecular biology has also become an integral part of food analyses. Real-time PCR (polymerase chain reaction) has been used for this purpose for quite some time. In the meantime, other DNA-based analysis methods are moving into the focus of food analysts, notably methods that are able to identify small sequence differences, something PCR is not able to do.

The detection of DNA is the method of choice for screening foodstuffs for the presence of genetically modified organisms. DNA-based analyses are also used to test the authenticity of animal and plant products and for determining allergens. In addition, such analyses are used for screening foodstuffs for the presence of microbial contaminants and for testing the quality of microorganisms (production strains) used for food production.

The use of aptamers, i.e. oligonucleic acid or peptide molecules that bind to a specific target molecule, are also gaining in importance in the analysis of foods. Intramolecular interactions between DNA and RNA sequences lead to the formation of three-dimensional structures. In the same way as antibodies, these structures can bind to other molecules, including proteins, cells and low-molecular substances. Analytical methods make use of this phenomenon in order to use artificial aptamers for capturing, enriching and identifying target molecules of interest. In the field of food analyses, aptamers are used for screening food pathogens and mycotoxins (Haase et al. p. 350).

New molecular detection systems

The application of targeted molecular biology methods only works when the target DNA sequence of the organism of interest is known. Huge progress has been made with the development of next-generation sequencing technologies. The genomes of numerous microorganisms and of plants and animals used for the production of human food have been deciphered. Collections of reference sequences of a plethora of living species (e.g. the collection maintained by the International Consortium for the Barcode of Life, see link in the top right-hand corner) represent an excellent basis for food analyses. Research has shown that no more than one or two biological barcodes are needed for the unequivocal identification of a species. Barcodes therefore efficiently expand the existing range of molecular detection systems.

Originally used in medical diagnostics, high-resolution melting analyses (HRMA) are now also used for assessing the authenticity of basmati rice or sweet cherries and for screening GMO (Haase et al., p. 348).

Loop-mediated isothermal amplification (LAMP) is a relatively new method and experts believe that its importance in the analysis of food will grow considerably. LAMP is an enrichment method that has the potential to be used as screening assay in the field or at the point of clinical care. In contrast to PCR it only uses a single temperature incubation, which obviates the need for thermal cyclers. LAMP is currently used for the detection of pathogenic microorganisms and genetically modified organisms.

Need for methods that allow the identification of the effectiveness of functional components

The Dechema (Society for Chemical Engineering and Biotechnology) published its position on the issue of food and nutrition research in 2012. Although touching on the subject of metabolomics, the word metabolomics itself was not used: Food ingredients that promote human health, foodstuffs and diets for maintaining physical health and cognitive abilities in old age – the future trends of the nutritional and life sciences – also pose a new challenge for the field of food analysis. The Dechema experts are of the opinion that new methods that enable the identification of the effectiveness of functional components need to be developed alongside methods and procedures that enable the identification of the effect of complex foodstuffs on the human metabolism and on the physiological processes after food consumption. And last but not least, experts are calling for the development of biomarkers that are suitable for food analyses. What they effectively want is new screening/high-throughput systems (bioassays, in vitro analysis) that facilitate the discovery and development of new substances with detectable mechanisms of action.

References:

Annual Report 2011 (in German) - Überwachung Lebensmittel · Bedarfsgegenstände · Kosmetika · Trinkwasser · Futtermittel, Baden-Württemberg Ministry of Rural Areas and Consumer Protection.

Haase, Ilka/Vaagt, Franziska/Fischer, Markus (in German): Trendbericht Lebensmittelchemie 2011, in: Nachrichten aus der Chemie, 60/March 2012, p. 346-351.

Schubert, Rainer/Wimmer, Barbara, Pferd im Rind: Was steckt drin? - moderne DNA-Analytik zur Speziesbestimmung, in: labor & more, 2.13, 2013, p. 43f.

Dechema Biotechnology: Lebensmittel- und Ernährungsforschung - Aktuelle Handlungsfelder und Forschungsbedarf. Position paper of the Food Biotechnology section, Frankfurt am Main, 2012.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/with-metabolomics-food-analytics-goes-all-the-way