Practical examples of innovative NMR approaches to food analysis

NMR spectroscopy is currently one of the key methods for food characterisation1. Foodstuff is a complex matrix including many different compounds with different chemical structures, concentrations, solubility, properties and nutritional values. Each food type contains primary and secondary metabolites. Primary metabolites, i.e. organic acids, amino acids and sugars, involved in the basic functions of the living cell are ubiquitous although they are present in different species at different concentrations. Secondary metabolites such as phenolic compounds, terpenes and sterols are specific to food type and can be considered markers of the product. Together with primary metabolites, they are important from a nutritional point of view. NMR methodology enables primary and secondary metabolites to be identified and quantified, bringing high-throughput spectroscopic / structural information on a wide range of metabolites simultaneously with high precision.

NMR methodology is especially helpful for molecular identification. The metabolite identification is usually obtained by means of 1D and 2D experiments, addition of standard compounds, literature data and by comparison with a database of standard compounds. Moreover, the identification of metabolites in a mixture is often obtained without separation of individual components. Rather simple, direct and rapid sample preparation procedures without prerequisite derivatisation of components are usually required. 1H NMR is a quantitative technique owing to the proportionality of signal integral to the molar concentration of the corresponding metabolite.

NMR analysis together with chemometrics has allowed some important characteristics of food such as geographical origin, farming, development, genotype-phenotype relation, quality and genuineness to be investigated.

Here, we report some interesting results that we have obtained studying kiwifruits, olive oils, sea bass, truffles, lettuce and tomatoes. For more details, see references.

Kiwifruit

Kiwifruit are an important horticultural crop with a characteristic flavour, fragrance and health properties which are due to the fruit chemical composition. Knowledge of the metabolic profile of kiwifruit is extremely important for fruit commercial value and also for the industries which extract specific compounds from the fruit to obtain specific additives. The metabolic profile of aqueous extracts of kiwifruit (Actinidia deliciosa, Hayward cultivar) as well as the water content of the whole fruit was obtained by NMR methodologies2. The knowledge of the metabolic profiling of kiwifruits at different development stages may have an important role in the determination of the most suitable time of harvesting as well as in the quantitative determination of nutrients at different growth times. The metabolic profile has been monitored from June to December showing ripening associated changes of some com – pounds such as sugars, organic acids and amino acids. The water content, also monitored over the seven month period, was determined directly on the intact fruit, measuring the spinspin relaxation time by a portable unilateral NMR instrument that was fully non-invasive. Clear trends of the relaxation time have been observed during the monitoring period, providing information on the state of kiwifruit growth and ripeness.

Olive oil

Extra virgin olive oil is a ‘natural fruit juice’ with peculiar sensory and nutritional quality. The composition of extra virgin olive oils is the result of complex interactions between olive variety, pedoclimatic conditions, fruit ripening and agronomic factors (Figure 2).

Figure 2: Different factors determining olive oil composition

NMR spectroscopy allowed olive oils to be characterised in terms of geographical origin3-5, genetical origin6,7, authenticity8 and quality9. Using high field proton NMR spectroscopy, it is possible to determine not only the components present in a major amount, i.e. fatty acid chains, but also some minor components such as aldehydes, sterols, terpenes and squalene. Important information on the fatty acid distribution on the glycerol moiety is provided by the 13C NMR spectroscopy10. The NMR protocol affords the selection of some signals belonging to specific compounds present in the 1H NMR spectrum, the measure of the signal intensity and the statistical elaboration of the intensity by means of an appropriate statistical analysis (Figure 3).

Figure 3: NMR resonances useful for olive oil analysis

Sea bass

Sea bass is a very popular ocean-going fish with peculiar sensory and nutritional qualities. Intensive sea bass farming has raised concerns over the quality of cultured fish in comparison with that of wild fish. NMR methodology was used as an analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples (Figure 4). Wild and cultured sea bass have been distinguished by different amounts of fatty acids, cholesterol, phospholipids and some water-soluble metabolites11,12.

Figure 4: Principal component analysis of wild and cultured sea bass

Truffle

Due to their typical sensorial characteristics, truffles, the ‘black diamonds’, have a relevant gastronomic interest due to their being widely used in European and Chinese cooking. Their NMR analysis has shown the presence of water-soluble metabolites belonging to different classes such as sugars, polyols, amino acids and organic acids, as well as cell membrane components such as sterols, lipids and fatty acids13. Moreover, NMR analysis has been applied to study the effect of small doses (1–2.5 kGy) of γ-rays used to prolong the shelf life of truffles14.

Tomato

Tomatoes are an essential crop in terms of nutritional quality and economic relevance. Intensive conventional agriculture of tomatoes based on nonrenewable resources has to become more sustainable in order to avoid the depletion of natural resources and the loss of fertile agricultural lands. Cultural practices that utilise organic cultivation can subscribe to sustainable agriculture by limiting chemical usage and reduced tillage. In the case of tomatoes, a reduced tillage, cover crop based sustainable cultivation system was proposed. NMR spectroscopy has contributed to monitoring the metabolic changes of tomato fruits associated with different cultivation practices15,16. According to NMR results, the cultivation of tomatoes using Leguminous hairy vetch (Vicia villosa Roth) mulch significantly stimulated the accumulation of asparagine, glutamate, glutamine, choline, and citrate concomitant with a decrease in glucose in tomato fruits during ripening as compared to conventional black polyethylene mulch.

Lettuce

Leaf vegetables constitute an important part of a healthy diet and it is of the utmost interest to determine their composition as completely as possible. A comprehensive NMR-based approach to the study of lettuce leaves has been undertaken in order to identify their metabolic composition and the influence of different factors, such as developmental stage, and genotype on metabolic profile. NMR analysis has allowed the content of water-soluble and lipo-soluble metabolites to be determined17. The maturation process has been found to be the major cause of metabolic changes in lettuce leaves18.

References

1. L. Mannina, A.P. Sobolev, S. Viel, “Liquid state 1H high field NMR in food” Progress in Nuclear Magnetic Resonance Spectroscopy, in press doi:10.1016/j.pnmrs.2012.02.001

2. D. Capitani, L. Mannina, N. Proietti , A.P. Sobolev , A. Tomassini, A. Miccheli, M.E. Di Cocco, G. Capuani, R. De Salvador, M. Delfini, “Monitoring of metabolic profiling and water status of Hayward kiwifruits by Nuclear Magnetic Resonance” Talanta, Vol. 82, pp. 1826-1838, 2010

3. L. Mannina F. Marini, M. Gobbino, A.P. Sobolev, D. Capitani, “NMR and chemometrics in tracing European olive oils: the case study of Ligurian samples” Talanta, Vol. 80, pp. 2141-2148, 2010

4. M. D’Imperio, L. Mannina, D. Capitani, O. Bidet, E. Rossi, F.M. Bucarelli, G.B. Quaglia, A.L. Segre, “NMR and statistical study of olive oils from Lazio: a geographical, ecological and agronomic characterization” Food Chemistry, Vol. 105, No. 3, pp. 1256-1267, 2007

5. L. Mannina, M. Patumi, N. Proietti, D. Bassi, A.L. Segre, “Geographical characterization of Italian extra virgin olive oils using high-field 1H NMR spectroscopy” Journal of Agricultural and Food Chemistry, Vol. 49, No. 6, pp. 2687-2696, 2001

6. L. Mannina, G. Dugo, F. Salvo, L. Cicero, G. Ansanelli, C. Calcagni, A. L. Segre, “Study of the cultivar-composition relationship in Sicilian olive oils by GC, NMR, and statistical methods” Journal of Agricultural and Food Chemistry, Vol. 51, No. 1, pp. 120-127, 2003

7. L. Mannina, G. Fontanazza, M. Patumi, G. Ansanelli, A. L. Segre, “Italian and Argentine olive oils: a NMR and gas chromatographic study” Grasas y Aceites, Vol. 52, No. 6, pp. 380-388, 2001.

8. L. Mannina, M. D’Imperio, D. Capitani, S. Rezzi, C. Guillou, T. Mavromoustakos, M.D. Molero Vilchez, A.H. Fernández, F. Thomas, R. Aparicio, “1H NMR-based protocol for the detection of adulterations of refined olive oils with refined hazelnut oil” Journal of Agriculture and Food Chemistry, Vol. 57, pp. 11550 -11556, 2009

9. L. Mannina, A.P. Sobolev, “High resolution NMR characterization of olive oils in terms of quality, authenticity and geographical origin” Magnetic Resonance in Chemistry, Vol 49, pp. S3-S11, 2011

10. L. Mannina, C. Luchinat, M. Patumi, M.C. Emanuele, E. Rossi. A.L. Segre, “Concentration dependence of 13C NMR spectra of triglycerides: implications for the NMR analysis of olive oils” Magnetic Resonance in Chemistry, Vol. 38, No. 10, pp. 886-890, 2000

11. L. Mannina, A.P. Sobolev, D. Capitani, N. Iaffaldano, M.P Rosato, P. Ragni, A. Reale, E. Sorrentino, I. D’Amico, R. Coppola, “NMR metabolic profiling of organic and aqueous sea bass extracts: implications in the discrimination of wild and cultured sea bass” Talanta, Vol. 77, No. 2, pp. 433-444, 2008

12. A. Reale, E. Sorrentino, N. Iaffaldano. M.P. Rosato, P. Ragni, R. Coppola, D. Capitani, A. P. Sobolev, P. Tremonte, M. Succi, L. Mannina, “Effects of ionizing radiation and modified atmosphere packaging on the shelf life of aqua-cultured sea bass (Dicentrarchus labrax)” World Journal of Microbiology and Biotechnology, Vol. 24, No. 12, pp. 2757-2765, 2008

13. L. Mannina, M. Cristinzio, A.P. Sobolev, P. Ragni, A.L. Segre, “High-field NMR study of truffles (Tuber aestivum vittadini)” Journal of Agricultural and Food Chemistry, Vol. 52, pp. 7988-7996, 2004

14. M. Adamo, D. Capitani, L. Mannina, M. Cristinzio, P. Ragni, A. Tata, R. Coppola, “Truffles decontamination treatment by ionizing radiation” Radiation Physics and Chemistry, Vol. 71, No. 1-2, pp. 167-170, 2004

15. A. P. Sobolev, A. L. Segre, R. Lamanna, “Proton high-field NMR study of tomato juice” Magnetic Resonance in Chemistry, Vol. 41, pp. 237-245, 2003

16. A. Neelam, T. Cassol, R. A. Mehta, A. A. Abdul-Baki, A. P. Sobolev, R. K. Goyal, J. Abbott, A. L. Segre, A. K. Handa, A. K. Mattoo, “A field-grown transgenic tomato line expressing higher levels of polyamines reveals legume cover crop mulch specific perturbations in fruit phenotype at the levels of metabolite profiles, gene expression, and agronomic characteristics” Journal of Experimental Botany, Vol. 59, pp. 2337-2346, 2008

17. A. P. Sobolev, E. Brosio, R. Gianferri, A. L. Segre, “Metabolic profile of lettuce leaves by highfield NMR spectra” Magnetic Resonance in Chemistry, Vol. 43, pp. 625-638, 2005

18. A. P. Sobolev, G.Testone, F. Santoro, C. Nicolodi, M. Iannelli, M.E. Amato, A.Iannello, E. Brosio, D. Giannino, L. Mannina, “Quality traits of conventional and transgenic lettuce (Lactuca sativa L.) at harvesting by NMR metabolic profiling” Journal of Agriculture and Food Chemistry, Vol.58, pp. 6928-6936, 2010

About the authors

Luisa Mannina is an Associated Professor of Food Chemistry at the Sapienza University of Rome. She is co-author of more than 130 papers published on international and national journals. She has been invited for plenary lectures and academic lectures regarding the application of NMR in food chemistry.

Anatoly Petrovich Sobolev is a chemist with a PhD obtained at the Novosibirsk State University, Russia in 1999. He joined the Institute of Chemical Methodologies of CNR, Rome as a researcher in 2007. He is an expert in the application of NMR spectroscopy in food chemistry and metabolomic studies.