H HRMAS-NMR metabolomic to assess quality and traceability of mozzarella cheese from Campania buffalo milk

The production of Mozzarella di Bufala Campana (MBC) is relevant for the agro-food economy of the Campania Region of Italy and the mark of Protected Designation of Origin (PDO) has been assigned to MBC in relation to its geographical origin. Advanced analytical methods must be then employed to assess authenticity, traceability, and quality of MBC. ¹H HRMAS-NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Resonance) spectroscopy was applied here to directly identify specific metabolites in MBC intact samples without time-consuming sample pre-treatments. Overcrowded conventional ¹H HRMAS-NMR spectra were selectively simplified with two NMR pulse sequences: eCPMG and eDiff, by modulating spin-spin relaxation times and diffusion of MBC molecular components, respectively. Signal elaboration of edited spectra was combined with multivariate analyses to enable significant metabolic differentiation between MBC samples from two different production sites in Campania. Principal Components Analysis (PCA) for eCPMG spectra explained 97.54% of total variance between the two MBC groups for four metabolites (β-galactose, β-lactose, acetic acid, and glycerol). Less efficient was groups distinction by PCA for eDiff spectra, although differences in polyunsaturated acids, such as linoleic and linolenic acids, were highlighted. Similarly, Discriminant Analysis (DA) provided MBC group classification with 100% success in validation tests for eCPMG spectra, while DA prediction ability was reduced to 94.12% for eDiff spectra. Hierarchical Cluster Analysis (HCA) gave a totally correct classification between the two MBC groups only for eCPMG spectra. eCPMG spectra were also used to identify metabolites during MBC aging. As compared to fresh samples, 2 days old MBC samples showed increasing signals for isobutylic alcohol, lactic acid, and acetic acid. This work shows that ¹H HRMAS-NMR spectroscopy can rapidly characterise the metabolic profile of intact MBC samples and statistically distinguish the geographical origin of buffalo milk mozzarella and its freshness.     

Solid‐state NMR characterization of cyclomaltoheptaose (β‐cyclodextrin) polymers using high‐resolution magic angle spinning with gradients

Solid‐state nuclear magnetic resonance (NMR) techniques were used to characterize cyclomaltoheptaose (β‐cyclodextrin, β‐CD) polymers. These insoluble materials have been investigated by cross‐polarization magic angle spinning with dipolar decoupling (CP/MAS), magic angle spinning without dipolar decoupling (MAS), and high‐resolution magic angle spinning with gradients (HRMAS). These NMR spectra allow the assignment of the principal ¹H and ¹³C signals. The presence of two distinct components (cross‐linked β‐CD and polymerized epichlorohydrin) in the materials was clearly demonstrated. These polymers were used as sorbents and the resulting NMR spectra are presented and discussed. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1288–1295, 2000     

Study of the suitability of HRMAS NMR for metabolic profiling of tomatoes: Application to tissue differentiation and fruit ripening

The feasibility of HRMAS NMR as an efficient technique for metabolic studies of tomato fruit and tomato tissues is described. A good-taste tomato variety from Almería (Spain) (commercial name Zayno) was chosen for this study. Fruits of three different ripening stages (green, turning and red) were selected, based on their external appearance. NMR data of whole fruits were obtained through ¹H HRMAS NMR spectra of tomato purée. Flesh, peel and seeds from mature red fruits were separately analysed. ¹H HRMAS NMR spectra showed resolutions similar to that of solution ¹H NMR with the advantages of minimal sample manipulation and possibility of analysing polar and non-polar compounds simultaneously. Seeds could be clearly distinguished by visual comparison of the spectra. Tryacylglycerols, in which linoleic (47%) and oleic (28%) acyl chains preponderate, were the major components of seeds. By contrast, signals of fructose, glucose, citric acid, and the amino acids GABA, glutamine and glutamate dominated the spectra of flesh, peel and tomato purée. Principal components analysis, performed on the covariance matrix, revealed a clear separation between peel and flesh. Apart form the presence of the cutin biopolymer, peel showed a specific accumulation of sugar and sugar moieties related to pectin degradation, as well as glycosylated metabolites. Chemometric analysis also allowed the metabolic trajectory from green to red mature stages to be visualised. However, sample heterogeneity in the analysis of the whole fruit proved to be an important issue. The source of such heterogeneity was assigned to the presence of tissues of different hardness in the small amount of sample used. The presence of two clusters of samples at the turning stage indicated that the levels of certain metabolites could diverge significantly in fruits with almost identical appearance.     

High resolution magic angle spinning NMR spectroscopy for metabolic assessment of cancer presence and Gleason score in human prostate needle biopsies

Objectives  Histopathology of prostate needle biopsies (PNBs) is an important part in the diagnosis, prognosis and treatment evaluation of prostate cancer. The determination of metabolite levels in the same biopsies may have additional clinical value. Here, we demonstrate the use of non-destructive high resolution magic angle spinning (HRMAS) proton NMR Spectroscopy for the assessment of metabolic profiles of prostate tissue in PNBs as commonly obtained in standard clinical practice. Materials and methods  PNBs that were taken routinely from 48 patients suspected of having prostate cancer were subjected to HRMAS proton NMR spectroscopy. Subsequent histopathology of the same biopsies classified the tissue either as cancer (n = 10) or benign (n = 30). Results  Some practical aspects of this assessment were evaluated, such as typical spectral contamination caused by the PNB procedure. Significant metabolic differences were found between malignant and benign tissue using a small set of ratio’s involving signals of choline compounds, citrate and lactate. Moreover, significant correlations were observed between choline, total choline, and citrate over creatine signal ratios and the Gleason scores of tumor in PNBs and of tumor in the whole prostate. Conclusion  This preliminary study indicates that HRMAS NMR of routinely obtained PNBs can provide detailed metabolic information of intact prostate tissue with clinical relevance. Albert Verhofstad died before publication of this article.     

HRMAS-nuclear magnetic resonance spectroscopy characterization of tomato “flavor varieties” from Almería (Spain)

¹H HRMAS NMR spectroscopy has been applied to investigate differences at a metabolic level between “flavor varieties” of high quality tomatoes marketed as Protected Geographical Indication from Almería (Spain). The varieties studied include the non-hybrid Raf tomato, one of the most tasteful commercial tomatoes that is traditionally grown in this Spanish region, together with other two hybrid varieties of commercial names Rambo and Zayno. Standard quality parameters were poorly reliable for establishing specific correlations with a given variety. In contrast, ¹H HRMAS NMR spectroscopy, in combination with principal component analysis (PCA) and assigned signal analysis (ASA) provided a clear differentiation between varieties as a function of the ripening process and revealed the existence of variety-dependent relationships between external appearance and metabolic content. ??-Aminobutyric acid (GABA) is envisioned as a good marker for monitoring the ripening process. The content in other taste-relevant metabolites (fructose and organic acids) proved to be variety-dependent. The results show a preferential accumulation of citric acid and fructose in Rambo and Raf varieties, respectively. Raf fruits are characterized by high fructose to glucose and fructose to citric acid ratios. The content of malic acid during ripening also remains high for Raf tomato. These combined features are responsible of the unique characteristic and exceptional taste of this traditional line of tomato.