Applications of NMR Spectroscopy

Food is a complex system formed by several chemical compounds and physical structures at different organization levels. For food analysis and characterization, it is not only important the study of the chemical composition, which will define the nutrient content, but also the physical distribution of the different compartments and structures that will define the physical properties of food products. Physical properties of food will define the palatability and texture of the food product and thus, the acceptance by the consumers. When talking about Nuclear Magnetic Resonance (NMR) spectroscopy we refer to several techniques that study the interaction of electromagnetic radiation with matter. Nuclear magnetic spectroscopy is the use of the NMR phenomenon to study physical, chemical and biological properties of matter, from the microscopic to the macroscopic. NMR spectroscopy is a very successful and multipurpose technique which is very suitable combined with chemometrics, for the analysis of food products [1]. In this chapter, we will review several NMR techniques that are related to both chemical and physical characterization. Such techniques are 1H High-Resolution Magic Angle Spin (1H HR-MAS), which provides a high resolution chemical spectrum without component extraction [2], relaxometry, which gives information about the water compartmentation, structure and integrity [3], magnetic resonance imaging (MRI) and chemical shift imaging (CSI), which is an efficient tool for the physiological analysis of fruit and vegetables [4]. The following chapter will address, first of all, what needs to be measured on food, as well as several NMR techniques that have been used for the analysis of food products. These techniques are 1H High Resolution Magic Angle Spin (1H HR-MAS), MRI, 1D and 2D relaxometry, relaxometry mapping and chemical shift imaging. We further focus on the explanation of multicomponent analysis and finally offer some remarks about prospects in the field.

During storage and use of edible oils and other lipid-containing foods, reactions between lipids and oxygen occur, resulting in lipid oxidation and the subsequent development of off-flavors and odors. Accurate and timely assessment of lipid oxidation is critical for effective quality control of food products. NMR spectroscopy techniques including 1H, 13C, and 31P NMR have been used to determine the oxidation stage and quality of lipids, to elucidate chemical structures of oxidation products, and to verify oxidation mechanisms. NMR spectroscopy methods have been successfully employed to identify oxidation products, including primary oxidation products such as hydroperoxides and conjugated dienes and secondary products such as aldehydes, alcohols, epoxides and their derivatives. 1H NMR can also be used to determine the extent of lipid oxidation during frying and storage by monitoring the decrease in peak area of protons located in reactive sites of oil molecules including olefinic, bisallylic and allylic protons. 13C NMR has been used to identify oxidation products along with 1H NMR, gas chromatography-mass spectroscopy (GC-MS) and other methods. 31P NMR also has been utilized to assess the oxidation of edible oils along with 1H and 13C NMR spectroscopies. These methods correlate well with traditional methods and offer highly reliable, non-destructive, fast analysis of lipid oxidation. These analytical methods will be summarized in this chapter.

Anthocyanins are an important class of natural pigments widely distributed throughout the Plant Kingdom and foodstuffs. They are rather reactive compounds that are involved in several equilibria in aqueous solution and give rise to new classes of anthocyanin-derived pigments. This reactivity creates changes in the exhibited color that is especially interesting with regards to foods and beverages. Among the anthocyanin-derived pigments, pyranoanthocyanins have gained attention in the last two decades because of their higher stability, mainly the resistance to discoloration by changing pH or after the addition of bisulfite, in comparison to their precursor anthocyanins. One of the first challenges regarding pyranoanthocyanins was their structural elucidation, leading to an increasing difficulty due to the finding of several new subclasses of chemical structures, beginning with one the first discovered, relatively simple, vitisin-type pyrano-anthocyanins and continuing with other more complex structures like those of flavanyl-vinyl-pyranoanthocyanins or pyranoanthocyanin dimers. Multidimensional Mass Spectrometry (MSn) and Nuclear Magnetic Resonance (NMR) spectroscopy have been crucial tools for the structure elucidation of pyranoanthocyanins. In addition, NMR spectroscopy has contributed to the gaining of knowledge about the aqueous solution equilibria in which pyranoanthocyanins are involved, thus helping the interpretation of the molecular basis behind the higher stability of such anthocyanin-derived pigments. This review introduces the pyranoanthocyanins by means of a description of the anthocyanin reactivity and the formation of anthocyanin-derived pigments in foods and beverages. The review continues with an overview of the current knowledge about different structures of pyranoanthocyanins, their main properties related to exhibited color, especially their behavior against changing pH and bisulfite bleaching, their reactivity, and the occurrence of pyranoanthocyanins in foods and beverages. Finally, this review deals with the application of NMR spectroscopy to several interesting issues related to pyranoanthocyanin chemistry, namely the structure elucidation of the different classes of pyrano-anthocyanins, the behavior of these compounds in aqueous equilibria of hydration and proton transfer, and the formation of new pyranoanthocyanin-related compounds.

Tannins in the skin and seeds of grapes used to make red wine are responsible for the two dominant sensory perceptions of astringency and bitterness. Astringency is a tactile sensation causing a dry, rough and puckering mouth-feel, while bitterness triggers an unpalatable harsh taste. Although these flavors are both associated with tannins, their mechanisms of action differ greatly. Astringency results from an interaction between the tannins and the saliva proteins, whereas bitterness is the result of an interaction between the tannins and the taste receptors located on the tongue. In the last decade, various studies using NMR spectroscopy have revealed new clues to the understanding of astringency perception at the molecular level. We now know the three-dimensional structure and the colloidal state of tannins are key factors in the mechanism of tannin-saliva protein interactions. Although the latter are undeniably related to astringency, it is only very recently we have learned that the lipids of oral cavity membranes and a fortiori provided by fat foods could also play a role in this complex sensory phenomenon. Indeed, strong interactions between tannins and membrane lipids have been highlighted in recent research supported by a fluidizing effect on membranes depending on the tannin structure. These findings show lipids interfere with tannin-saliva protein and tannin-taste receptor interactions involved in astringency and bitterness respectively. In addition to their role in taste, tannins as antioxidant molecules and in a larger extent polyphenolic compounds provided by foods are strongly suspected to have a positive role in many pathologies. Whereas their antioxidant properties have been widely demonstrated, their protective effect on membrane against lipid oxidation has been shown for the first time by NMR investigations. New insights into the location of tannins within the membrane have been proposed to explain their inhibitory effects on free radicals. Moreover, a synergistic effect has been evidenced proving the beneficial effect of food polyphenols as shown by epidemiological studies.

Quantitative NMR (qNMR) is a powerful tool to quantify an analyte without the need for an identical standard, which is considered to be a primary ratio method. In particular, the use of 1H NMR has been widely applied in the quantification of medicines, beverage components, and natural products in medicinal plant extracts because of its high sensitivity and the widespread presence of 1H nuclei in organic molecules. Our group has previously reported a novel 1H qNMR technique that is able to determine quantitative values of the analyte with metrological traceability to the International System of Units with a certified reference material as the internal standard (IS); this is called AQARI (accurate quantitative NMR with internal reference substance).

There are other advantages to qNMR using 1H such as simple sample preparation, low sample consumption, and rapid, non-destructive measurement. Furthermore, it is widely recognized as a reliable technique due to the development of high-field magnets, and improvements in probe technology and gradient shimming techniques contributed to the enhancement of sensitivity, resolution, and precision. In Japan, qNMR using 1H NMR has been set as an official method for assessing the purities of reference substances for pharmaceuticals and food additives.

In this review, we describe our recent studies regarding the applicability of qNMR using 1H NMR for the purity assessment of organic compounds and the analysis of complex mixtures. The first half of the review introduces qNMR using 1H NMR with IS, while the second half describes our recent purity assessments of commercial reagents for food analysis, and the quantification of several preservatives in processed foods by qNMR.

Cell-free protein expression systems offer many advantages over cell-based approaches for the expression and isotope labeling of proteins for NMR analysis. Cellfree systems allow for the rapid single day production of target proteins at milligram production levels with inexpensive isotope enrichment. The open nature of these systems also allows the addition of molecules that can aid the folding and stabilization of target proteins. In this chapter we briefly discuss the available cell-free expression systems and whether they can be used for the isotope-labeling of a target protein, and how new PCR-directed cell-free expression approaches can aid the rapid identification of expression constructs with enhanced yield and solubility. We then focus on recent advances in the cell–free production of proteins for NMR structural analysis of large proteins and macromolecular complexes (including membrane proteins). The NMR spectra of such molecules are often problematic to assign because of their large number of cross peaks and line broadening resulting in loss in signal resolution and intensity. A range of selective, combinatorial and segmental isotope labeling strategies based on cell-free protein synthesis are now available to enable residue-specific and sequencespecific assignment of NMR spectra. Cell-free deuteration of target proteins can reduce line broadening issues, and stereo-array isotope labeled (SAIL) amino acids can be incorporated to provide NOE constraints for structure determination. Cell-free protein synthesis also allows the incorporation of unnatural amino acids which can act as NMR probes to provide long distance information.