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Saffron is an ancient spice largely used in traditional medicine. It has been found to be effective in treatment of retinal neurodegenerative diseases like age‐related macular degeneration and Stargardt. In the present manuscript, it is shown that saffron's neuroprotective power is strongly related to the bioactivity of all its chemical components. Nuclear magnetic resonance spectroscopy and "in vitro" experiments confirm the relevance of crocins for saffron efficacy. These results underline the importance of strictly defining the chemical composition of the natural compounds in saffron to optimize their effectiveness in the treatment of diseases. [ABSTRACT FROM AUTHOR]

Lignans are particularly interesting secondary metabolites belonging to the phenyl-propanoid biosynthetic pathway. From the structural point of view, these molecules could belong to the aryltetralin, arylnaphtalene, or dibenzylbutyrolactone molecular skeleton. Lignans are present in different tissues of plants but are mainly accumulated in seeds. Extracts from plant tissues could be characterized by using the NMR-based approach, which provides a profile of aromatic molecules and detailed structural information for their elucidation. In order to improve the production of these secondary metabolites, elicitors could effectively stimulate lignan production. Several plant species are considered in this review with a particular focus on Linum species, well recognized as the main producer of lignans. [ABSTRACT FROM AUTHOR]

In the last years, there was an increasing interest on nuclear magnetic resonance (NMR) spectroscopy, whose applications experienced an exponential growth in several research fields, particularly in food science. NMR was initially developed as the elective technique for structure elucidation of single molecules and nowadays is playing a dominant role in complex mixtures investigations. In the era of the "omics" techniques, NMR was rapidly enrolled as one of the most powerful methods to approach metabolomics studies. Its use in analytical routines, characterized by rapid and reproducible measurements, would provide the identification of a wide range of chemical compounds simultaneously, disclosing sophisticated frauds or addressing the geographical origin, as well as revealing potential markers for other authentication purposes. The great economic value of high‐quality or guaranteed foods demands highly detailed characterization to protect both consumers and producers from frauds. The present scenario suggests metabolomics as the privileged approach of modern analytical studies for the next decades. The large potentiality of high‐resolution NMR techniques is here presented through specific applications and using different approaches focused on the authentication process of some foods, like tomato paste, saffron, honey, roasted coffee, and balsamic and traditional balsamic vinegar of Modena, with a particular focus on geographical origin characterization, ageing determination, and fraud detection. [ABSTRACT FROM AUTHOR]

In previous works on authentic samples of saffron of known history (harvest and processing year, storage conditions, and length of time) some biomarkers were proposed using both FT-IR and NMR metabolomics regarding the shelf life of the product. This work addresses the difficulties to trace back the "age" of commercial saffron samples of unknown history, sets a limit value above which these products can be considered substandard, and offers a useful tool to combat saffron mislabeling and fraud with low-quality saffron material. Investigations of authentic and commercial saffron samples of different origin and harvest year, which had been stored under controlled conditions for different lengths of time, allowed a clear-cut clustering of samples in two groups according to the storage period irrespectively of the provenience. In this respect, the four-year cut off point proposed in our previous work assisted to trace back the "age" of unknown samples and to check for possible mislabeling practices. [ABSTRACT FROM AUTHOR]

Squalene (SQ), a precursor of sterols and terpenoids is a functional lipid of high importance in the food and pharmaceutical sectors. SQ oxidation studies are rather limited compared with those for other olefins. The aim of the present study was to monitor the formation of SQ oxidation products under different conditions (temperature, air supply), to characterise the most abundant of them by spectroscopic techniques and then examine their pro-oxidant activity in a model lipid substrate. Squalane (SQA), the saturated analogue of SQ, was used as a reference compound. FT-MIR analysis indicated the presence of alcohols, epoxides, aldehydes, and ketones. GC-MS was used to characterise SQ primary oxidation and scission products. The presence of epoxides was further confirmed by means of 1H NMR and 13C NMR spectroscopy. It could be argued that SQ stability is due to its stereochemistry and specifically to the presence of methyl groups next to the double bonds. The pro-oxidant activity of SQ oxidation products was evident at 62 and 40°C and suppressed only in the presence of primary antioxidants, not of SQ. The present work adds to the characterisation of SQ oxidised products. To our knowledge their pro-oxidant activity has never been examined before. Practical applications: Characterisation of squalene oxidation products and assessment of their activity as pro-oxidants present both scientific interest regarding the kinetics and product identity as well as a practical impact in case this bioactive lipid is provided for consumption as a functional product. In the past, cholesterol oxidation products and more recently phytosterol ones attracted the interest of researchers, who studied the stability of the respective parent compounds for food safety reasons. Monitoring of the formation of SQ oxidation products under different conditions (temperature, air supply) and chemical characterisation of the most abundant of them by spectroscopic techniques. Examination of their pro-oxidant activity in a model lipid substrate. Squalene may exert a weak antioxidant activity due to competitive oxidation phenomena with the lipid substrate while its oxidation products have a pro-oxidant activity on purified olive oil model substrate that was suppressed only in the presence of primary antioxidants. [ABSTRACT FROM AUTHOR]

Highlights: [•] Preparation and characterization of luminescent branched polyphenylenes. [•] Detailed 1H and 13C NMR characterization and precise signal assignment. [•] Combination of NMR and UV spectroscopies to determine chemical structures. [Copyright &y& Elsevier]

Abstract: The aim of this work was the geographical discrimination of honey from three different floral sources on the basis of the saccharide content. In particular, multifloral honey from different countries, “high mountain multifloral” and rhododendron honeys from different regions of the northern part of Italy were investigated by means of 1H NMR spectroscopy and modeled with OPLS-DA for accurate samples classification. NMR data allowed a correct geographical discrimination of honeys highlighting the discriminating saccharides. Our results strongly support the capability of NMR and chemometrics to address the quality requirements, thus suggesting its use in quality assessment of honey in terms of geographical determination. [Copyright &y& Elsevier]

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data. [ABSTRACT FROM AUTHOR]

Abstract: The increasing attention for food quality and safety led to develop several analytical techniques suitable to address these demands. Coffee has been already demonstrated to represent a worldwide appreciated beverage and its high economical value could induce frauds or adulteration practices involving both compositional and geographical aspects. In the last years, metabolic profiling revealed to be suitable to face the quality determination of food matrices and NMR confirmed its potentiality in metabolites characterization. The present study reports the capability of NMR spectroscopy to investigate the metabolite content of roasted Coffea arabica samples from the three main production areas, America, Africa and Asia. OPLS-DA models performed on 1H NMR data led to a clear separation of samples according to their origin: fatty acids, chlorogenic acids and lactate and finally acetate and trigonelline resulted the main compounds characterizing the American, African and Asian samples respectively. The analytical approach here presented confirmed the potentiality of the joined NMR analysis and statistical treatments in quality determination of food matrices. [Copyright &y& Elsevier]

Abstract: The metabolic characterisation of food and beverages nowadays has been largely employed in combination with multivariate data analysis. In this work, the valuable Amarone “Passito” dry red wine produced in Verona area (Italy) was investigated in order to find a possible correlation between metabolic content and vintage/ageing process. A total number of 46 wine samples from 11 different winemakers, three different vintages and ageing process were investigated without sample preparation. 1H NMR spectra were analysed in combination with principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) protocols to investigate vintage and ageing effects on samples differentiation based on the metabolic content. The results indicated amino acids, sugars, and aromatic compounds the most relevant discriminant variables for Amarone wine samples. [Copyright &y& Elsevier]

Abstract: The constitutive expression of the rice Osmyb4 gene in Arabidopsis plants gives rise to enhanced abiotic and biotic stress tolerance, probably by activating several stress-inducible pathways. However, the effect of Osmyb4 on stress tolerance likely depends on the genetic background of the transformed species. In this study, we explored the potential of Osmyb4 to enhance the cold and freezing tolerance of Osteospermum ecklonis, an ornamental and perennial plant native to South Africa, because of an increasing interest in growing this species in Europe where winter temperatures are low. Transgenic O. ecklonis plants were obtained through transformation with the Osmyb4 rice gene under the control of the CaMV35S promoter. We examined the phenotypic adaptation of transgenic plants to cold and freezing stress. We also analysed the ability of wild-type and transgenic Osteospermum to accumulate several solutes, such as proline, amino acids and sugars. Using nuclear magnetic resonance, we outlined the metabolic profile of this species under normal growth conditions and under stress for the first time. Indeed, we found that overexpression of Osmyb4 improved the cold and freezing tolerance and produced changes in metabolite accumulation, especially of sugars and proline. Based on our data, it could be of agronomic and economic interest to use this gene to produce Osteospermum plants capable of growing in open field, even during the winter season in climatic zone Z9. [ABSTRACT FROM AUTHOR]

Abstract: 13C NMR spectroscopy was employed for evaluating glucose and fructose isoforms content in Traditional Balsamic Vinegar of Modena. With the use of reference spectra recorded in water for samples obtained with respect to set rules, a shift determination method for fructose carbon isoforms was introduced to determine the frauds present in unknown Traditional Balsamic Vinegar of Modena samples. No sample preparation yields this approach highly reliable and time saving. Following this approach, for the first time, an objective analytical technique can be used alternatively to the actual procedures for Traditional Balsamic Vinegar certification. [Copyright &y& Elsevier]

Abstract: The Michael addition of ketone-derived enamines, metalated methylene active compounds and N-methyl pyrroles to γ-fluoro-α-nitroalkenes provided in moderate to good isolated yields the corresponding β-fluoroalkyl nitro compounds, which represent new interesting, highly functionalized building blocks in organofluorine chemistry. [Copyright &y& Elsevier]

Ras proteins are small G proteins playing a major role in eukaryotic signal transduction. Guanine nucleotide exchange factors (GEF) stimulate GDP/GTP exchange, resulting in the formation of the active Ras-GTP complex. In mammalian cells, two major Ras-specific GEF exist: Sos-like and Cdc25-1ike. To date, structural data are available only for Cdc25[SUPMm]. We designed and synthesized Cdc25Mm-derived peptides spanning residues corresponding to the hSos1 HI helical hairpin that has been implicated in the GEF catalytic mechanism. NMR experiments on a chemically synthesized Cdc25[SUPMm][SUBl178-1222] peptide proved that helix I readily reaches a conformation very similar to the corresponding helix in hSosl, while residues corresponding to helix H in hSosl show higher conformational flexibility. Molecular dynamics studies with the appropriate solvent model showed that different conformational spaces are available for the peptide. Since helix H is making several contacts with Ras and a Cdc25[SUPMm][SUBl178-1222] peptide is able to bind nucleotidefree Ras in a BIAcore assay, the peptide must be able to obtain the proper Ras-interacting conformation, at least transiently. These results indicate that rational design and improvement of the Ras-interacting peptides should take into account conformational and flexibility features to obtain molecules with the appropriate biochemical properties. [ABSTRACT FROM AUTHOR]

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobactedum Sulfolobus solfataricus. The protein is endowed with DNA binding properties. RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction. as seen by its abnormally high pK[sub a] value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK[sub a] of ∼9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations. [ABSTRACT FROM AUTHOR]

Investigates the thermodynamic stability of Sso7d, a 7-kda DNA-binding protein in Sulfolobus solfataricus. Composition of archaeobacteria and eubacteria; Analysis on the rearrangement of the hydrophobic core; Comparison between the structures of the recombinant wild-type Sso7d and the F31A mutant.

The challenge to manufacture medical devices with specific antibacterial functions, and the growing demand for systems able to limit bacterial resistance growth, necessitates the development of new technologies which can be easily produced at an industrial level. The object of this work was the study and the development of silver, titanium dioxide, and chitosan composites for the realization and/or implementation of biomedical devices. Thermoplastic elastomeric polyurethane was selected and used as matrix for the various antibacterial functions introduced during the processing phase (melt compounding). This strategy was employed to directly incorporate antimicrobial agents into the main constituent material of the devices themselves. With the exception of the composite filled with titanium dioxide, all of the other tested composites were shown to possess satisfactory mechanical properties. The best antibacterial effects were obtained with all the composites against Staphylococcus aureus: viability was efficiently inhibited by the prepared materials in four different bacterial culture concentrations. [ABSTRACT FROM AUTHOR]

In Italy, 42 PDO extra virgin olive oil (EVOOs) are present to date, with the chronologically first one registered in late 1996. PDO EVOOs are broadly distributed along Italy with a higher concentration in the central and southern regions due to the optimal pedoclimatic conditions for olive growth (Olea europaea L.). In this review, NMR (nuclear magnetic resonance) studies focused on Italian PDO EVOOs and the application of NMR spectroscopy in the analysis of other olive‐tree‐derived products are reported. NMR is historically known as the principal analytical technique useful for structural characterization of single molecule in solution. In the past few years, NMR evolved to cover a pivotal role for the analysis of different chemical compounds present in a mixture, without the need of any chemical derivatization of sample. Concerning the 1H NMR spectrum of a typical EVOO sample, the dominant signals due to acylglycerols, together with other minor components like aldehydes, sterols, and terpens, can be characterized within a single experiment in the organic extract of olive oil. The minor component content is particularly interesting because it allows for geographical origin discrimination and cultivar discrimination. Other products derived from the olive tree and investigated by NMR have also been discussed, specifically, table olives, pomace, and olive paste. Practical Applications: The studies here highlight the capability of NMR in discriminating Italian PDO EVOOs from EVOOs of other countries, Italian PDO EVOOs of different restricted area within the same Italian region, and EVOOs obtained from different cultivars. In addition, NMR spectroscopy allows the evaluation of chemical constituents in olive‐tree‐derived products like olive paste, pomace, and wastewater, detecting bioactive compounds. Therefore the NMR approach could be practically applied in the study of EVOO and olive‐tree‐derived products by control agencies, regulatory authorities, for the disclosure of new fraud practices and to protect PDO products. Practical applications could also involve the search of olive‐tree‐based bioactive compounds for their potential use as nutraceuticals. Nuclear magnetic resonance spectroscopy (NMR) is used to discriminate Italian Protected Designation of Origin (PDO) extra‐virgin olive oils (EVOOs) from EVOOs of other countries, Italian PDO EVOOs of different restricted area within the same Italian region, and EVOOs obtained from different cultivars. The chemical constituents of olive‐tree‐derived products are also determined. Results strongly support the use of NMR by control agencies and regulatory authorities. Nuclear magnetic resonance spectroscopy (NMR) is used to discriminate Italian Protected Designation of Origin (PDO) extra‐virgin olive oils (EVOOs) from EVOOs of other countries, Italian PDO EVOOs of different restricted area within the same Italian region, and EVOOs obtained from different cultivars. The chemical constituents of olive‐tree‐derived products are also determined. Results strongly support the use of NMR by control agencies and regulatory authorities. [ABSTRACT FROM AUTHOR]

Abstract Fossa cheese is a typical Italian cheese characterised by ripening in pits dug in tuffaceous rock. In this work, 1H NMR spectroscopy was applied to characterise the lipid fraction and the metabolite profile of Fossa cheese produced with sheep's milk. Samples were collected before and after ripening in different pit environments. The higher levels of free amino acids (FAAs) found in samples suggest that cheeses underwent a strong proteolytic process. Differences in quantity and variety of FAAs were influenced by the two ripening environments. Diacylglycerols and butyric acid, which can be linked to lipolysis, were detected in all the samples. Longer ripened samples had higher contents of rumenic acid (18:2 cis -9, trans -11) and glycerol, and lower contents of lactate and citrate. The presence of tyramine was observed by two-dimensional NMR, while cadaverine was not detected in the samples. [ABSTRACT FROM AUTHOR]

Cider is a slightly alcoholic beverage and is an important and promising segment of the fruit industry. In this study, a still cider produced by fermentation of apples juice was investigated. A simultaneous characterization of bioactive compounds in apple juice and the intermediate products of the cidermaking process was performed for the first time using Nuclear Magnetic Resonance spectroscopy. Alongside sugar consumption, a progressive increase in trigonelline, chalcone, fumarate, caffeic acid, uracil, tyrosol and xanthine content was detected during the alcoholic fermentation process. Of note was the relatively high tyrosol content which imparts health benefits in the cider. The concomitant changes in phenol, amino acid and organic acid (malic, lactic, quinic, pyruvic, citric and succinic) content, was important when monitoring the fermentation process and assessing quality control of the final product. [ABSTRACT FROM AUTHOR]

Crocus sativus L. is largely cultivated because it is the source of saffron, a well-appreciated and valued spice, not only for its culinary use but also because of its significant biological activities. Stigmas are the main product obtained from flowers, but in addition, tepals, largely considered a waste product, represent a big source of flavonoids and anthocyanins. This study aimed to delve into the phytochemical composition of saffron tepals and investigate whether the composition was influenced by the extraction technique while investigating the main analytical techniques most suitable for the characterization of tepal extracts. The research focuses on flavonoids, a class of secondary metabolites, and their health benefits, including antioxidant, anti-inflammatory, and anticancer properties. Flavonoids occur as aglycones and glycosides and are classified into various classes, such as flavones, flavonols, and flavanones. The most abundant flavonoids in tepals are kaempferol glycosides, followed by quercetin and isorhamnetin glycosides. Overall, this review provides valuable insights into the potential uses of tepals as a source of bioactive compounds and their applications in various fields, promoting a circular and sustainable economy in saffron cultivation and processing. [ABSTRACT FROM AUTHOR]

In recent years, fermented foods have attracted increasing attention due to their important role in the human diet, since they supply beneficial health effects, providing important sources of nutrients. In this respect, a comprehensive characterization of the metabolite content in fermented foods is required to achieve a complete vision of physiological, microbiological, and functional traits. In the present preliminary study, the NMR-based metabolomic approach combined with chemometrics has been applied, for the first time, to investigate the metabolite content of Phaseolus vulgaris flour fermented by different lactic acid bacteria (LAB) and yeasts. A differentiation of microorganisms (LAB and yeasts), LAB metabolism (homo- and heterofermentative hexose fermentation), LAB genus (Lactobacillus, Leuconostoc, and Pediococcus), and novel genera (Lacticaseibacillus, Lactiplantibacillus, and Lentilactobacillus) was achieved. Moreover, our findings showed an increase of free amino acids and bioactive molecules, such as GABA, and a degradation of anti-nutritional compounds, such as raffinose and stachyose, confirming the beneficial effects of fermentation processes and the potential use of fermented flours in the production of healthy baking foods. Finally, among all microorganisms considered, the Lactiplantibacillus plantarum species was found to be the most effective in fermenting bean flour, as a larger amount of free amino acids were assessed in their analysis, denoting more intensive proteolytic activity. [ABSTRACT FROM AUTHOR]

In this paper, we report the metabolic and molecular changes in response to cold and drought induced in Osmyb4 transgenic Arabidopsis thaliana compared with the wildtype (WT). The rice Osmyb4 gene codes for a transcription factor (Myb4) induced by cold treatment and, in Arabidopsis transgenic plants, improves cold and freezing tolerance [Vannini C, Locatelli F, Bracale M, Magnani E, Marsoni M, Osnato M, Mattana M, Baldoni E, Coraggio I (2004) Plant J 37: 115–127]. Here, we report the ability of Myb4 to induce also drought tolerance in Arabidopsis transgenic plants. By the use of nuclear magnetic resonance (NMR) and enzymatic assays, we showed that several compatible solutes (glucose, fructose, sucrose, proline, glycine betaine and sinapoyl malate) accumulate in higher amount in Osmyb4-overexpressing plants with respect to the WT, both under normal and stress conditions. Considering proline, we also found that in transgenic plants the levels of the mRNAs coding for Δ1-pyrroline-5-carboxylate synthase (EC not assigned) and for Δ1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) were higher and lower, respectively. The constitutive activation of several stress-inducible pathways and different kinetics in the accumulation of several metabolites, in Myb4 transgenic plants, may represent an advantage to prepare plants to face the stress condition. Moreover, these results taken together suggest that Myb4 integrates the activation of multiple components of stress response. [ABSTRACT FROM AUTHOR]

NMR based metabolic profiling was used to investigate metabolic changes in ciders fermented by yeast species as valuable tool for the identification of metabolites responsible for yeast activities. 1H NMR spectra of apple ciders produced by Saccharomyces cerevisiae EC1118, Torulaspora delbrueckii TD291, Starmerella bacillaris YR21, Hanseniaspora osmophila HO16, Hanseniaspora uvarum Y4M, and Saccharomyces uvarum SU3, revealed to be dominated by glycerol and organic acids signals. The aromatic region showed the presence of low intense signals referred to epicatechin, catechin, chlorogenate, xanthine, uracil, tyrosol, fumarate, histamine, and histidine. The anomeric region showed signals related to xylose, arabinose, glucose, fructose and sucrose. Finally, in the aliphatic region, signals due to amino acids like alanine, aspartate, asparagine, isoleucine and threonine, organic acids like acetate, lactate, malate, pyruvate, quinate, succinate, and alcohols like 2,3 butanediol, ethanol, and glycerol and sterol were detected. Principal component analysis performed considering NMR data from either all spectral regions and only aromatic region revealed the potentiality to discriminate the yeast action. Signals due to glucose, fructose, glycerol, malate, fumarate, tyrosol, histidine, and histamine resulted discriminant for sample differentiation. This study, the first one by NMR, provides preliminary insights on the metabolic differences among yeast species involved in cider production. • Apple juice fermented by 6 yeast species. • NMR characterization of metabolite profiles of fermented apple juice. • Specie-specific biomarkers related to apple juice fermentation. [ABSTRACT FROM AUTHOR]

Abstract Nuclear magnetic resonance (NMR) spectroscopy, which is one of the most powerful "omics" analytical platforms, has been broadly adopted recently in foodomics. 1H NMR has been applied to the study of the metabolite profile of dairy products throughout the supply chain, in relation to different aspects such as animal health, milk quality, geographical origin and cheese ripening process. This review reports and discusses the literature on the topic, also collecting the identified metabolites in a descriptive table and depicting them in a Venn diagram for both milk and cheese; moreover, experimental details of the reviewed papers have been reported. The present review provides an exhaustive state-of-the-art in the field of dairy products, addressing both NMR experts and non-experts to the still unexplored potential applications of NMR in dairy characterisation, and in general in foodomics. [ABSTRACT FROM AUTHOR]

Saffron, the dried red stigmas of Crocus sativus L., is considered as one of the most expensive spices worldwide, and as such, it is prone to adulteration. This study introduces an NMR-based approach to identify and determine the adulteration of saffron with Sudan I–IV dyes. A complete 1 H and 13 C resonance assignment for Sudan I–IV, achieved by two-dimensional homonuclear and heteronuclear NMR experiments, is reported for the first time. Specific different proton signals for the identification of each Sudan dye in adulterated saffron can be utilised for quantitative 1 H NMR (qHNMR), a well-established method for quantitative analysis. The quantification of Sudan III, as a paradigm, was performed in varying levels (0.14–7.1 g/kg) by considering the NMR signal occurring at 8.064 ppm. The high linearity, accuracy and rapidity of investigation enable high resolution 1 H NMR spectroscopy to be used for evaluation of saffron adulteration with Sudan dyes. [ABSTRACT FROM AUTHOR]

In this paper, we report on the postpolymerization modification (PPM) of a polymer to introduce new functionalities that enable click chemistry reactions for microarray applications. The parent polymer, named copoly(DMA-NAS-MAPS), is composed of N,N-dimethylacrylamide (DMA), a monomer that self-adsorbs onto different materials through weak interactions such as hydrogen bonding or van der Waals forces, 3-(trimethoxysilyl)propyl methacrylate (MAPS) that strengthens the stability of the coating through the formation of covalent bonds with siloxane groups on the surface to be coated, and N-acryloyloxysuccinimide (NAS), an active ester group, highly reactive toward nucleophiles, which enables bioprobe immobilization. This copolymer has been widely exploited to coat surfaces for microarray applications but exhibits some limitations because of the potential hydrolysis of the active ester (NHS ester). The degradation of the NHS ester hampers the use of this coating in some situations, for example, when probe immobilization cannot be accomplished through a microspotting situation, but in large volumes, for example, in microchannel derivatization or micro-/nanoparticle functionalization. To overcome the limitations of NHS esters, we have developed a family of polymers that originate from the common copolymer precursor, by reacting the active ester contained in the polymer chain with a bifunctional amine. In particular, the functional groups introduced in the polymer using PPM enable click chemistry reactions such as azide/alkyne or thiol/maleimide "click" reactions, with suitably modified biomolecules. The advantages of such reactions are quantitative yields, orthogonality of functional groups, and insensitivity of the reaction to pH. The new click functionalities, inserted with quantitative yields, improve the stability of the coating, enabling the attachment of biomolecules directly from a solution and avoiding the spotting of reduced volumes (pL) of probes. Finally, we have demonstrated the applicability of the click surfaces in a highly effective solid-phase PCR for the genotyping of the G12D KRAS mutation. [ABSTRACT FROM AUTHOR]

Breast milk (BM) feeding is the gold standard in neonate nutrition. When BM is not available it can be substituted or integrated with commercial formula milk (FM) usually sold under different brands and formulations. In this work, the low-molecular-weight hydrophilic compounds in milk were studied by gas chromatography electronic impact mass spectrometry (GC–MS), comparing eight different FM brands with BM samples. With the aid of multivariate statistical data analysis, a marked variability among FM brands, especially driven by the presence of prebiotics in their formulation, was highlighted. Quali-quantitative differences were found between FM and BM. Orotic acid and isomaltulose were found exclusively in FM, while phenylalanine and tyrosine levels were high in two FM brands. Moreover, higher levels of malic acid, sugars (glucose, fructose and galactose), and mannitol were detected in FM. On the other hand, BM showed a higher amino acid content. In conclusion, GC–MS proved to be a very sensitive analytical technique for the study of FM, highlighting metabolite differences among FM brands, and between FM and BM, that may have a possible strong impact on neonatal nutrition. [ABSTRACT FROM AUTHOR]

The interaction of carbohydrates with a variety of biological targets, including antibodies, proteins, viruses and cells are of utmost importance in many aspects of biology. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins. In this study, a novel slide is reported for the fabrication of glycan arrays that combines the higher sensitivity of a layered Si-SiO 2 with a novel approach to form a polymeric coating easily modifiable by subsequent click reaction. The alkyne-containing copolymer, adsorbed from an aqueous solution, produces a coating by a single step procedure and serves as a soft, tridimensional support for the oriented immobilization of carbohydrates via azide/alkyne Cu(I) catalyzed “click” reaction. The equilibrium and kinetics parameters of the interaction of Concanavalin A with eight synthetic glycans were determined using fluorescence microarray and Reflective Phantom Interface (RPI), a recently proposed optical label-free detection approach. The enhancement of fluorescence provided by the Si-SiO 2 slides enabled to extend the limit of detection at lower surface densities of lectins, in turn enabling the study of the interaction for a wide range of glycans surface density. Equilibrium dissociation constants of a few nM were extracted for multivalent glycan-lectin binding, mimicking the conditions of biological membranes, whereas hundreds of nM were observed at the lower glycan surface densities. [ABSTRACT FROM AUTHOR]

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters. [ABSTRACT FROM AUTHOR]

In the present work, a preliminary study for the detection of adulterated saffron and the identification of the adulterant used by means of 1 H NMR and chemometrics is reported. Authentic Greek saffron and four typical plant-derived materials utilised as bulking agents in saffron, i.e., Crocus sativus stamens, safflower, turmeric, and gardenia were investigated. A two-step approach, relied on the application of both OPLS-DA and O2PLS-DA models to the 1 H NMR data, was adopted to perform authentication and prediction of authentic and adulterated saffron. Taking into account the deficiency of established methodologies to detect saffron adulteration with plant adulterants, the method developed resulted reliable in assessing the type of adulteration and could be viable for dealing with extensive saffron frauds at a minimum level of 20% (w/w). [ABSTRACT FROM AUTHOR]

1 H NMR-based metabolomics is proposed here for the quality control of saffron upon storage using the ability to detect a wide range of chemical compounds with minimal sample preparation. Saffron quality deterioration is the result of enzymatic, oxidative and hydrolytic reactions that change the content and structure of the glycosides of crocetin and picrocrocin, which are the metabolites responsible for the major sensory properties of this most expensive spice. Several authentic saffron samples (n = 98) of known storage history after processing were examined. The Principal Component Analysis shows a clear-cut separation of samples into two groups based on the storage period regardless of the sample origin. The S-plot derived from the Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA) model shows the markers for quality deterioration, i.e., sugars bound to crocetin, glucose in picrocrocin, free sugars and fatty acids. These new markers become critical for the samples that were stored for more than 4 years. The OPLS-DA model was validated with a test set and proved to be properly designed to predict the length of storage after harvest. The answer to the question set in the market of “when” and “why” a saffron sample can no longer be considered fresh is supported by our findings. [ABSTRACT FROM AUTHOR]

High-resolution NMR spectroscopy was employed to analyze Italian Protected Designation of Origin (PDO) saffron from L'Aquila, S. Gimignano and Sardinia and commercial saffron samples available on the Italian market. An extensive resonance assignment of DMSO saffron extract was reported, including glucose and gentiobiose in bound and unbound form for the first time. A multivariate statistical analysis of NMR data led to a clustering of samples by performing unsupervised PCA. OPLS-DA model was successively performed to highlight the markers responsible for this discrimination. An analysis of the corresponding S-plot indicated that picrocrocin and crocins were the most relevant compounds for characterizing Italian PDO saffron. By contrast, commercial saffron barely contained these characteristic compounds, and they were primarily enriched in fatty acids. [ABSTRACT FROM AUTHOR]

Abstract: One of the major objectives of rice (Oryza sativa L.) breeding programs is the development of new varieties with higher tolerance/resistance to both abiotic and biotic stresses. In this study, Italian rice cultivars were subjected to osmotic stress or benzothiadiazole (BTH) treatments. An analysis of the expression of selected genes known to be involved in the stress response and 1H nuclear magnetic resonance (1H NMR) metabolic profiling were combined with multivariate statistical analyses to elucidate potential correlations between gene expression or metabolite content and observed tolerant/resistant phenotypes. We observed that the expression of three chosen genes (two WRKY genes and one peroxidase encoding gene) differed between susceptible and resistant cultivars in response to BTH treatments. Moreover, the analysis of metabolite content, in particular in the osmotic stress experiment, enabled discrimination between selected cultivars based on differences in the accumulation of some primary metabolites, primarily sugars. This research highlights the potential usefulness of this approach to characterise rice varieties based on transcriptional or metabolic changes due to adverse environmental conditions. [Copyright &y& Elsevier]

Abstract: This study reports direct quantification of arabica in roasted and ground coffee blends of Coffea arabica and Coffea canephora var. robusta. 1H–NMR analysis of water extracts of coffee blends were combined with multivariate statistical analysis to obtain an OPLS model with high predictive capability. This approach allowed to evaluate the composition of coffee blends of unknown arabica and robusta content, on the basis of multiple chemical components. Differences in geographical origin of the analyzed samples did not affected the compositional determination of coffee blends. This approach represents a valid tool in authentication procedures of arabica and robusta blends of roasted and ground coffee. [Copyright &y& Elsevier]

Abstract: Understanding how proteins are approached by surrounding molecules is fundamental to increase our knowledge of life at atomic resolution. Here, the surface accessibility of a multifunctional small protein, the archaeal protein Sso7d from Sulfolobus solfataricus, has been investigated by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes. The DNA binding domain of Sso7d appears very accessible both to TEMPOL and Gd(III)(DTPA-BMA). Differences in paramagnetic attenuation profiles of 1H–15N HSQC protein backbone amide correlations, observed in the presence of the latter paramagnetic probes, are consistent with the hydrogen bond acceptor capability of the N-oxyl moiety of TEMPOL to surface exposed Sso7d amide groups. By using the gadolinium complex as a paramagnetic probe a better agreement between Sso7d structural features and attenuation profile is achieved. It is interesting to note that the protein P-loop region, in spite of the high surface exposure predicted by the available protein structures, is not approached by TEMPOL and only partially by Gd(III)(DTPA-BMA). [Copyright &y& Elsevier]

The analytical performance of the microarray technique in screening the affinity and reactivity of molecules towards a specific target, is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule, thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here we introduce a new N, N -dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N -acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bi-functional copolymer has been obtained by partial post polymerization modification of the functional groups of a common precursor. A NMR characterization of the copolymer was conducted to quantify the percentage of NAS that has been transformed into azido groups. The polymer was used to coat surfaces onto which both native antibodies and alkyne modified peptides were immobilized, to perform the phenotype characterization of extracellular vesicles (EVs). This strategy represents a convenient method to reduce the number of analysis, thus possible systematic or random errors, besides offering a drastic shortage in time, reagents and costs. [Display omitted] • A bi-functional copolymer for microarray applications is introduced. • The bi-functional copolymers bear active esters and azido groups simultaneously. • Native antibodies and oriented peptides are immobilized on the same microarray slide. • The copolymer is obtained by partial post polymerization modification of NAS groups. • Co-immobilization of probes allows the phenotype characterization of EVs. [ABSTRACT FROM AUTHOR]

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-β-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-β-glucoside. [ABSTRACT FROM AUTHOR]

We have characterized the high-temperature molten globule state of bovine α-lactalbumin by a combined use of photochemically induced dynamic nuclear polarization and nitroxide surface perturbation. Both techniques are extremely well suited to follow the progressive increase of exposed surfaces in the native state and the appearance of partially or completely unfolded species. Our results suggest that the molten globule state obtained at high temperature and pH 7. and the state obtained at pH 2 are not only thermodynamically but also structurally very similar. [ABSTRACT FROM AUTHOR]

We have suggested elsewhere the use of surface mapping by spin label probes (Esposito et at., 1992). According to this approach, soluble nitroxides are added to a protein solution. Resonances of protons that are accessible to the nitroxide are broadened and bleached out of the spectrum, while resonances in the protein interior remain unaffected. This approach is, in principle, complementary to another technique, photochemically induced dynamic nuclear polarization, which maps the position of aromatic protons on the protein surface. A detailed comparison between the two techniques is necessary for a confident use of the more. recent suggested nitroxide perturbation approach. In the present study, we show that the results obtained by the two techniques for the native state of bovine α-lactalbumin are fully consistent and may therefore be combined for the study of protein surfaces. [ABSTRACT FROM AUTHOR]

Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes. [ABSTRACT FROM AUTHOR]

Conventional proton nuclear magnetic resonance (1H-NMR) has been widely used for identification and quantification of small molecular components in food. However, identification of major soluble macromolecular components from conventional 1H-NMR spectra is difficult. This is because the baseline appearance is masked by the dense and high-intensity signals from small molecular components present in the sample mixtures. In this study, we introduced an integrated analytical strategy based on the combination of additional measurement using a diffusion filter, covariation peak separation, and matrix decomposition in a small-scale training dataset. This strategy is aimed to extract signal profiles of soluble macromolecular components from conventional 1H-NMR spectral data in a large-scale dataset without the requirement of re-measurement. We applied this method to the conventional 1H-NMR spectra of water-soluble fish muscle extracts and investigated the distribution characteristics of fish diversity and muscle soluble macromolecular components, such as lipids and collagens. We identified a cluster of fish species with low content of lipids and high content of collagens in muscle, which showed great potential for the development of functional foods. Because this mechanical data processing method requires additional measurement of only a small-scale training dataset without special sample pretreatment, it should be immediately applicable to extract macromolecular signals from accumulated conventional 1H-NMR databases of other complex gelatinous mixtures in foods. [ABSTRACT FROM AUTHOR]

The supplementation of goat diets with natural products to obtain milk with nutraceutical components is a common practice. In these last years, the influence of supplementation of specifically designed diets has been studied with different analytical tools in order to explore possible beneficial effects in human consumption of animal milk and milk-derived products. In this study, the lipid fraction of milk from Alpine goats undergoing different dietary regimens was studied by 1H-NMR spectroscopy. Alpine goats were fed with linseed or hempseed supplements, and after 14 weeks of treatment, milk was collected and analyzed. Results showed that feeding diets supplemented with seeds positively affected the fatty acid composition with a pronounced increase in unsaturated fatty acids for both diets compared to a control diet. Specifically, linolenic acid content was more than doubled for linseed diet compared with the hempseed and control groups, while linoleic acid greatly increased only upon hempseed supplementation. However, a number of conjugated linoleic acid (CLA) isomers and higher levels of fatty acids with trans configuration were found in supplemented diets, particularly in the linseed diet. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the impact of the addition of two lactic acid bacteria (LAB) strains, namely Lactobacillus casei -VC199 and Enterococcus lactis -BT161, on the composition of goat cheese at 30 and 60 d of ripening. In the metabolite and lipid profiles, as determined by 1H NMR spectroscopy, we found the primary products of lipolysis: free fatty acids (FFA) and diacylglycerols; of glycolysis: organic acids; and of proteolysis: free amino acids (FAA). The cheese volatilome, monitored over the ripening period by SPME-CG-MS, indicates the mechanism by which these primary metabolic products are converted to flavor compounds. Principal Component Analysis (PCA) suggests that the two LAB strains differently affect the metabolite and volatile compound content of the cheeses, the difference being the greatest at 60 d of cheese ripening. Cheese produced with Lb. casei had greater quantities of FAA, FFA, and organic acids than E. lactis cheese. The volatilome of samples inoculated with E. lactis was characterized by higher ketone content, while Lb. casei produced more acids and esters. The results of this work highlight the potential and complementarity of NMR and SPME-GC-MS analytical techniques to investigate the impact of LAB on cheese compositional characteristics. • Lactobacillus casei and Enterococcus lactis differently influenced cheese ripening. • Lactobacillus casei induced stronger catabolism than Enterococcus lactis. • Enterococcus lactis produced more volatile ketones than Lactobacillus casei. [ABSTRACT FROM AUTHOR]

Cannabis sativa L. is one of the most-studied species for its phytochemistry due to the abundance of secondary metabolites, including cannabinoids, terpenes and phenolic compounds. In the last decade, fiber-type hemp varieties have received interest for the production of many specialized secondary metabolites derived from the phenylpropanoid pathway. The interest in these molecules is due to their antioxidant activity. Since secondary metabolite synthesis occurs at a very low level in plants, the aim of this study was to develop a strategy to increase the production of such compounds and to elucidate the biochemical pathways involved. Therefore, cell suspensions of industrial hemp (C. sativa L. var. Futura) were produced, and an advantageous elicitation strategy (methyl jasmonate, MeJA) in combination with precursor feeding (tyrosine, Tyr) was developed. The activity and expression of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) increased upon treatment. Through 1H-NMR analyses, some aromatic compounds were identified, including, for the first time, 4-hydroxyphenylpyruvate (4-HPP) in addition to tyrosol. The 4-day MeJA+Tyr elicited samples showed a 51% increase in the in vitro assay (2,2-diphenyl-1-picrylhydrazyl, DPPH) radical scavenging activity relative to the control and a 80% increase in the cellular antioxidant activity estimated on an ex vivo model of human erythrocytes. Our results outline the active metabolic pathways and the antioxidant properties of hemp cell extracts under the effect of specific elicitors. [ABSTRACT FROM AUTHOR]

Antibodies to the first loop (ECL1) of CCR5 have been identified in human immunodeficiency virus (HIV)-exposed uninfected individuals (ESN) and in HIV-positive nonprogressing subjects. Thus, these antibodies may confer resistance against HIV infection. To define which amino acids are involved in antibody binding to CCR5, we performed a peptide-scanning assay and studied the immunogenicity of peptides in animal models. A panel of synthetic peptides spanning the CCR5-ECL1 region and displaying glycine or alanine substitutions was assayed for antibody binding with a pool of natural anti-CCR5 antibodies. We used mice and chickens to study the immunogenicity of mutagenized peptide. Structural characterization by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations were performed to better understand the structural and conformational features of the mutagenized peptide. Amino acid substitutions in positions Ala95 and Ala96 (A95-A96) increased antibody-peptide binding compared to that of the wild-type peptide (Asp95-Phe96). The Ala95-96 peptide was shown to induce, in mice and chickens, antibodies displaying biological activity at very low concentrations. Strikingly, chicken antibodies to the Ala95-96 peptide specifically recognize human CCR5 molecules, downregulate receptors from lymphocytes, inhibit CCR5-dependent chemotaxis, and prevent infection by several R5 viruses, displaying 50% inhibitory concentrations of less than 3 ng/ml. NMR spectroscopy and molecular dynamics simulations proved the high flexibility of isolated epitopes and suggested that A95-A96 substitutions determine a slightly higher tendency to generate helical conformations combined with a lower steric hindrance of the side chains in the peptides. These findings may be relevant to the induction of strong and efficient HIV-blocking antibodies. [ABSTRACT FROM AUTHOR]