Finding Answers with Foodomics

LCGC recently spoke to Miguel Herrero of the Institute of Food Science Research (CIAL-CSIC) at the Spanish National Research Council, in Madrid, Spain, about his research in foodomics-based approaches, the evolution of food analysis, and the benefits of two-dimensional (2D) liquid chromatography (LC) in this application.

What areas of food analysis is your group focusing on?

Herrero: We work mainly in the application of foodomics-based approaches to study the bioactivity of dietary compounds. Our research group defined the term foodomics for the first time in 2009 as "a new discipline that studies the food and nutrition domains through the application and integration of advanced –'omics' technologies to improve consumers' well-being, health, and confidence" (1). Basically, we believe that foodomics can help to provide new answers to some of the important challenges (such as food safety and quality, traceability, and new foods for health improvement and disease prevention) that society is facing at present. In this regard, we maintain two parallel and interconnected research lines. One is focused on the development of new environmentally green processes to obtain bioactive compounds from natural sources, and the other is directed to the development and application of new advanced analytical methods to assess the bioactivity of those compounds.

Why is food analysis important and what are you currently working on?

Herrero: There is no doubt that food analysis is crucial nowadays because it is essential to maintain and control food safety, traceability, and food legislation compliance as well as food quality of marketed products. More recently, food analysis has become important for the study of the beneficial health effects that some foods may have. The increase in the application of "omics" approaches in food science and technology is further proof of its importance.

Currently, we are involved in two research projects. The first of these is at a national level and we are looking for sound scientific evidence using a foodomics approach on the bioactivity of food polyphenols (from rosemary, olive leaves, and algae) against colon cancer using in vitro and in vivo models. To do that we are using a wide array of novel approaches such as the use of new integrated extraction processes based on pressurized fluids to obtain polyphenols and selected fractions from the natural samples, as well as a combination of advanced analytical methods (mainly metabolomics and transcriptomics but also proteomics) with in vitro and in vivo assays.

The second one is a European project based on the development of biorefinery processes of microalgae. We are collaborating with 25 other research groups working on very different fields of application. Our main input is to develop green strategies to extract specialties from the studied microalgae and the different fractions generated during their biorefinery processing. The final goal of the project is to provide a proof-of-concept about the possibility to develop integrated, multiple-product biorefinery processes (involving cultivation, harvesting, extraction, processing, and application) for valuable specialties from algae for application in food, aquafeeds, and nonfood products.

How has food analysis evolved and what are the most recent trends?

Herrero: Traditional food analysis has been progressively displaced by instrumental analysis and the use of hyphenated techniques, which are routinely applied in research laboratories to a certain extent. However, the great advances produced as a result of the "postgenomic era" have produced huge developments in analytical instruments, which are today capable of resolving problems unattainable not so long ago. Thanks to the use of those technologies, food science is working closely with other disciplines such as pharmacology, medicine, and biotechnology as the boundaries between them increasingly blend. A good example of this new reality is the great number of ongoing research projects dealing with the relationship between food and health.

This is where foodomics stands and why modern food analysis is so different to classical food analysis. In my opinion, this is the most important trend in food analysis and we will surely see an increase in the type of research that will take advantage of these technologies to reach more global conclusions. Besides, the greater knowledge that may be gained through the application of transcriptomics, proteomics, and metabolomics will be essential to fulfill the strict requirements from many regulation agencies (such as the European Food Safety Authority [EFSA]) to demonstrate and understand the beneficial action of a food or food component in the body.

How would you like the field of food analysis to evolve in the future? In your opinion, what is important for the future of food analysis?

Herrero: I would like to see a stronger integration of the huge amount of data that is now generated using the different omics approaches to help obtain stronger and more robust evidence of the potential health benefits that certain foods and food ingredients may provide. I think this is the way that food analysis will evolve as society's (consumers, the media, and industry) interest in healthy foods increases.

The most important thing as a researcher will probably be to fully exploit all the capabilities that technology offers. In this regard, I think that the merging of different fields and the already mentioned diffusion of boundaries is very positive. Based on the interdisciplinary approach that most ambitious research projects have nowadays, the interaction between researchers with different backgrounds (analytical chemistry, microbiology, biochemistry, bioinformatics, and so on) will give a definitive boost to food-related research.

What are the benefits of using 2D liquid chromatography (LC) in food analysis, particularly in complex food samples? How easy is it to apply in this area?

Herrero: One of the techniques that we use in the lab is comprehensive two-dimensional LC (LC × LC). This technique is able to provide increased resolving and identification power, which is really interesting for food analysis. Food samples are usually complex mixtures of different components of a diverse nature. Even when a food sample is rich in a particular class of those components, its composition is often very heterogeneous: Here is where LC × LC gives its best. There are natural mixtures that cannot be separated using monodimensional approaches; they are simply too complex - for example, polyphenols, carotenoids, or lipids. In those cases, a careful selection of each of the two dimensions involved in LC × LC separations may provide orthogonal and complementary separation mechanisms that could allow the separation of closely related but different components that cannot be separated in one dimension. Furthermore, this technique allows sample treatments that sometimes conceal the real native composition in food to be avoided. For instance, using this approach it has been possible to describe the native carotenoid composition (free and esterified carotenoids) of oranges, avoiding the need of saponification (2).

Obviously, as in life, nothing is perfect. The setup of these methods can be very complicated because there are some technical and physical issues that should be overcome, including solvent incompatibilities between dimensions, the need for fast second dimension separations, and appropriate on-line transfers from first to second dimension, among others. On-line coupling to mass spectrometry (MS) is also another tricky point. Therefore, some requirements have to be met to make these separations worthy, but once one has everything in place, the obtained results are really beautiful.

You recently studied the phenolic compound pattern of apples using 2D LC. Could you talk a little about this research?

Herrero: In this work we applied an LC × LC–mass spectrometry (MS) method to separate and identify apple procyanidins and other polyphenols simultaneously, obtaining a complete polyphenolic profile typical from different apple varieties (3). The most relevant part of this work was the development of a two-dimensional method coupling a hydrophilic-interaction chromatography (HILIC) separation in the D1 to a reversed-phase separation in the D2 that was capable of providing a complete polyphenolic profile that included flavan-3-ols and oligomeric procyanidins (with a degree of polymerization up to 8 units), several dihydrochalcones, flavonols, and phenolic acids in a single run. By combining the information coming from the diode-array and MS detectors we could tentatively identify some 65 polyphenols in those samples, which I think is a nice result.

Regarding the apple varieties studied, we could confirm that the polyphenolic profile was significantly different both qualitatively and quantitatively speaking. Just out of curiosity for those particularly worried about antioxidant intake, granny smith apples were those richer in procyanidins closely followed by reinette apples.

You are the only national research group working with 2D LC in Spain at the moment - why do you think that is?

Herrero: Right now, it is true that there is no other Spanish research group using LC × LC, although it is also true that there are not a huge amount of groups worldwide when compared to other analytical approaches. The main reason for this is undoubtedly the relative complexity of setting up a reliable LC × LC instrument. Commercial 2D LC instruments have recently entered the market, having overcome some of the limitations of building a system from the beginning. Previously, extensive expert experience was needed to build a robust instrument using different pumps, valves, connections, software integration, and data elaboration, which created a stumbling block to the take-up of the technique. In general, although one may suppose that the situation is similar, for LC × LC's sister technique 2D gas chromatgoraphy (GC × GC), but it is rather different. GC × GC is strongly established and is more user-friendly. Anyway, I am sure that in the future more laboratories will discover the potential of this technique for their applications as the arrival of commercial 2D systems makes this approach easier.

Where will your research with 2D LC take you in the future?

Herrero: We are working right now on a variety of new separations of complex food-related samples. I am eager to continue with this line of research in my lab because there are quite a lot of underexplored analytes for which separation could be greatly improved using LC × LC. I would also like to have the chance to use this technique within metabolomics approaches, as the potential there is massive. However, in that latter group of applications, sensitivity must be high. For this reason I would also like to explore some instrumental alternatives to make the LC × LC coupling less prone towards sensitivity problems.

Of course, the development of these new ideas is inevitably related to getting more support from our government for science in general and young researchers in particular. Right now for young researchers like myself, it is quite difficult to keep doing what we are educated and prepared for because there is not a clear tenure track to allow the effective incorporation of researchers in the Spanish system. In any case, I will keep going on and hope for the best.

References

(1) A. Cifuentes, J. Chromatogr. A 1216, 7109 (2009).

(2) P. Dugo, M. Herrero, D. Giuffrida, T. Kumm, G. Dugo, and L. Mondello, J. Agric. Food Chem. 56, 3478–3485 (2008).

(3) L. Montero, M. Herrero, E. Ibáñez, and A. Cifuentes, J. Chromatogr. A 1313, 275–283 (2013).