Application of nuclear magnetic resonance spectroscopy and vibrational spectroscopy for the characterisation of fruit products and beverages

O trabalho apresentado nesta tese pretendeu desenvolver e aplicar métodos espectroscópicos, em especial espectroscopia de ressonância magnética nuclear e técnicas acopladas, e também espectroscopia de infravermelho, para estudar a composição e a bioquímica de frutos e bebidas. No Capítulo I, apresenta-se o estado do conhecimento sobre a composição e bioquímica dos alimentos em análise neste trabalho, evidenciando-se os aspectos que justificam o seu estudo mais aprofundado e completo, para o qual a presente tese pretende contribuir. O Capítulo II descreve, de forma resumida, os fundamentos das técnicas analíticas utilizadas, focando nomeadamente a espectroscopia de RMN de estado líquido e de estado sólido, o acoplamento da RMN com cromatografia líquida e espectrometria de massa (HPLCRMN/ MS) e a espectroscopia de infravermelho. Os métodos de análise multivariada usados encontram-se também sumariamente descritos neste Capítulo. No Capítulo III, descreve-se a aplicação de técnicas de RMN de alta resolução para caracterizar a composição de sumo e polpa de manga. O espectro de RMN-1H do sumo (600 MHz) apresenta elevada complexidade e contém informação sobre uma vasta gama de compostos. Recorrendo a espectros bidimensionais, conseguem identificar-se cerca de 50 compostos, incluindo vários açúcares, ácidos orgânicos e aminoácidos. O fruto intacto (polpa) é estudado directamente e de forma não-invasiva por RMN de estado sólido (400 e 500 MHz), utilizando a técnica de rotação segundo o ângulo mágico (MAS) e uma sonda de alta resolução (HR-MAS). A técnica de HRMAS permite obter espectros com uma resolução seis vezes maior do que a obtida com MAS, sendo possível registar espectros 2D e caracterizar detalhadamente a composição da polpa. Comparando os espectros da polpa e do sumo respectivo, encontram-se algumas diferenças na composição em lípidos e pectinas, que se encontram em maior abundância no fruto intacto, demonstrando que a usual extrapolação da composição do sumo como composição global do fruto pode não ser totalmente aplicável. No Capítulo IV apresenta-se um estudo sistemático do processo de amadurecimento da manga (cv. Tommy Atkins), levado a cabo através de medições físico-químicas e de espectroscopia de RMN-1H de alta resolução aplicada a sumos e polpas. A quantificação de alguns componentes do sumo é efectuada por integração dos sinais de RMN, usando uma referência interna (TSP). As principais alterações de composição com o amadurecimento consistem no aumento dos teores de sacarose, alguns aminoácidos (ex. valina, alanina, ácido ?-aminobutírico), constituintes pécticos e polifenóis e na diminuição dos teores de glucose, ácido cítrico, ácido xiquímico, arginina, tirosina e fenilalanina. Uma segunda variedade estudada (cv. Haden) apresenta diferenças no processo de amadurecimento sobretudo ao nível da variação de alguns aminoácidos e compostos aromáticos. Ainda neste capítulo, um estudo preliminar sobre o efeito da refrigeração mostra que a maior parte das alterações indicadas acima são retardadas ou não ocorrem a baixa temperatura, daí resultando frutos com baixa qualidade organoléptica. O Capítulo V descreve a quantificação dos três principais açúcares do sumo de manga em função do amadurecimento, usando regressão parcial dos mínimos quadrados (PLS) aplicada a espectros de infravermelho (FTIR-ATR) e de RMN-1H. A calibração é efectuada com base em soluções padrão de glucose, frutose e sacarose, seguindo um desenho experimental triangular, com seis níveis de concentração para cada açúcar. O método de PLS-FTIR permite prever a concentração dos três açúcares com elevada exactidão, excepto no caso da glucose quando, nos últimos estágios de amadurecimento, esta atinge teores abaixo da gama de concentrações usada na calibração. Por outro lado, a aplicação de PLS aos espectros de RMN das soluções padrão e dos sumos de manga apresenta erros elevados, cuja magnitude parece depender do alinhamento dos espectros, necessitando este método de optimização. No Capítulo VI, a espectroscopia de RMN-1H de alta resolução é usada para monitorizar e identificar alterações de composição em sumos de manga sujeitos a degradação natural, aquecimento e contaminação microbiológica. Permitindo identificar muitos dos compostos que se alteram em função destes efeitos, de forma rápida e simultânea, e seguir a sua variação ao longo do tempo, a técnica de RMN revela-se promissora para a detecção de indicadores de degradação/contaminação do sumo, sendo potencialmente útil para fins de controlo de qualidade. No Capítulo VII, alguns componentes minoritários de sumo de manga, sumo de uva e vinho são investigados através do acoplamento das técnicas de RMN, cromatografia líquida e espectrometria de massa (HPLC-RMN/MS). Em particular, o sumo de manga é analisado com vista ao estudo dos hidratos de carbono menos abundantes, tendo-se alcançado uma melhor caracterização da fracção péctica. A análise de sumo de uva e vinho (extracto fenólico) destina-se a caracterizar os constituintes aromáticos destas amostras, tendose conseguido identificar vários compostos fenólicos de difícil atribuição só com base na análise directa por RMN. Os compostos identificados na uva incluem o ácido gálico e vários ácidos cinâmicos (ex. p-cumárico, transcutárico e trans-caftárico), enquanto que no vinho se detectam catequina e epicatequina, trans-resveratrol, tirosol e ácido cafeico. O Capítulo VIII centra-se na caracterização da composição química da cerveja através das técnicas de RMN e de HPLC-RMN/MS. A análise directa por RMN uni- e bidemsional (500 MHz) permite a identificação de cerca de 30 compostos, entre os quais vários ácidos orgânicos, aminoácidos e álcoois. Os oligossacarídeos de maltose (dextrinas) dão origem aos sinais mais largos e intensos do espectro, mas o elevado grau de sobreposição impede a sua caracterização estrutural. A identificação de dextrinas de diferentes tamanhos é conseguida através da análise da cerveja por HPLC-RMN/MS, que requer apenas a desgaseificação da amostra. A análise por HPLC-RMN/MS permite também confirmar a presença de vários compostos aromáticos e revelar outros não identificados só com base na análise por RMN, tais como os álcoois aromáticos 2-feniletanol, tirosol e triptofol. Finalmente, no Capítulo IX, descreve-se a análise de componentes principais (PCA) dos espectros de RMN-1H e de FTIR-ATR de um conjunto de 50 cervejas de diferentes marcas, tipos e países de origem. Por PCA dos espectros de RM N as cervejas são separadas de acordo com a sua composição em hidratos de carbono, embora alguns componentes minoritários (em particular compostos aromáticos) também contribuam para a distinção das amostras, nomeadamente em termos dos dois tipos principais ‘ale’ e ‘lager’. A aplicação de PCA aos espectros de FTIR origina a distinção das cervejas com base no seu teor alcoólico e na sua composição em hidratos de carbono. A análise canónica de correlação (CCA) entre os espectros de RMN e de FTIR mostra uma correlação elevada entre os dois domínios, permitindo identificar os sinais que variam no mesmo sentido.The work reported in this thesis aimed at developing and applying spectroscopic methods, principally nuclear magnetic resonance (NMR) spectroscopy and hyphenated techniques (HPLC-NMR and HPLC-NMR/MS), along with infrared spectroscopy, for studying the complex composition and biochemistry of different fruit products and beverages. Chapter I summarises some of the present knowledge about the food systems studied, giving particular emphasis to the aspects that frame the problems investigated in this thesis. The fundamentals of the analytical methods employed are shortly presented in Chapter II, focusing on the principles underlying the experiments that have been carried out in this work, namely solution and solid state high resolution 1H NMR spectroscopy, HPLC-NMR/(MS) and Fourier-transform infrared spectroscopy. The multivariate analysis methods that have been applied to the spectroscopic data are also briefly presented. In Chapter III, the application of high resolution NMR to characterise the composition of mango juice and intact pulp is described. The high field (600 MHz) 1H spectra of juices show a high degree of complexity, revealing the potential of the technique for the simultaneous detection of a wide range of components. Spectral assignment is carried out with basis on 1D and 2D spectra, resulting in the identification of about 50 components, including several sugars, organic acids and amino acids. Mango pulps are studied directly and non-invasively by techniques derived from solid state NMR, namely 1H HR-MAS NMR (400 and 500 MHz), which results in a six-fold resolution improvement compared to standard 1H MAS. HR-MAS enables 2D experiments to be carried out, thus allowing for the pulp composition to be thoroughly characterised. The pulp is found to have a richer composition in lipids and pectins than the corresponding juice, showing that the composition of the liquid phase is not fully representative of the composition of the whole fruit as usually assumed. Chapter IV presents a systematic study of the ripening process of mango (cv. Tommy Atkins), monitored by physicochemical parameters and by high resolution 1H NMR of both juices and pulps. Some juice components are quantified by integration of NMR signals, using an internal reference (TSP). Mango ripening is found to be characterised by increases in the contents of sucrose, some amino acids (e.g. valine, alanine, GABA), pectic constituents and polyphenols, and by decreases in the contents of glucose, citric acid, shikimic acid, arginine, tyrosine and phenylalanine, reflecting the complex biochemistry of ripening. The other cultivar studied (cv. Haden) is found to differ mainly in the variation patterns of some amino acids and aromatic compounds. Furthermore, a preliminary study of the effect of refrigeration on ripening shows that most changes indicated above do not occur under low temperature, resulting in fruits with poor edible quality. In Chapter V, partial least squares (PLS1) regression is applied to FTIRATR and to 1H NMR data in order to quantify the three main sugars in mango juices as a function of ripening. Calibration is based on standard solutions of glucose, fructose and sucrose, following a triangular experimental design with six concentration levels for each sugar. The PLS -FTIR method shows good predictive ability, although the accuracy of glucose determination decreases at later ripening stages, when concentrations fall below the lower limit of the concentration range used for calibration. The PLS -NMR method shows much higher prediction errors, which seem to depend on spectral alignment, and needs further optimisation. In Chapter VI, the application of high resolution NMR to monitor the complex compositional changes of mango juices subject to natural degradation, heat treatment and microbial contamination is described. Enabling the identification of many compounds undergoing changes upon these effects, in a rapid and simultaneous manner, and their variations to be followed throughout time, the NMR technique may be regarded as a sensitive and powerful tool for detecting indicators of spoilage/contamination, being potentially useful for quality control purposes. Chapter VII describes the application of hyphenated NMR methods (HPLC-NMR and HPLC-NMR/MS) to investigate minor components in mango juice, grape juice and wine. In particular, the carbohydrate composition of mango juice is investigated and further advances in the characterisation of the pectic fraction achieved. The analysis of grape juice and wine (phenolic extract) aims at characterising their complex aromatic composition and results in the identification of several phenolic compounds, overcoming the difficulties found when using standard NMR. Gallic acid and several cinnamic acids (e.g. pcoumaric, trans-coutaric and trans-caftaric) are identified in grape juice, whereas catechin, epicatechin, trans -resveratrol, tyrosol and caffeic acid are found in the wine phenolic extract. Chapter VIII is focused on the characterisation of beer chemical composition by high resolution NMR and HPLC-NMR/MS. Direct analysis of beer by 1D and 2D NMR (500 MHz) is shown to enable the identification of about 30 components, such as several organic acids, amino acids and alcohols. Malto-oligosaccharides (dextrins) give rise to the broadest and most intense signals in the spectrum, but the strong spectral overlap hinders their structural characterisation. The identification of dextrins of different sizes is achieved by HPLC-NMR/MS of beer, preceded only by degassing of the sample. In addition, HPLC-NMR/MS is shown to be useful for confirming the identity of some aromatic compounds previously assigned by NMR alone, and for revealing new ones, such as the aromatic alcohols 2-phenylethanol, tyrosol and tryptophol. Finally, in Chapter IX, principal component analysis (PCA) is applied to the 1H NMR spectra and to the FTIR-ATR spectra of a set of 50 beers differing in label, type and country of origin. PCA of NMR data results in the separation of beers mainly according to their carbohydrate composition, although minor components (aromatic compounds in particular) are also found to contribute for the distinction of beers, namely in terms of the two main types ale and lager. PCA of FTIR data separates beers with basis on differences in their alcoholic content and carbohydrate composition. Canonical correlation analysis of NMR and FTIR spectra shows a high correlation between the two domains, enabling the identification of the spectral features varying in the same direction

PEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles

Silk fibroin nanoparticles are emerging as promising nanomedicines, but their full therapeutic potential is yet to be realized. These nanoparticles can be readily PEGylated to improve colloidal stability and to tune degradation and drug release profiles; however, the relationship between silk fibroin nanoparticle PEGylation and macrophage activation still requires elucidation. Here, we used in vitro assays and nuclear magnetic resonance based metabolomics to examine the inflammatory phenotype and metabolic profiles of macrophages following their exposure to unmodified or PEGylated silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles, but they showed different phenotypic and metabolic responses to each nanoparticle type. Unmodified silk fibroin nanoparticles induced the upregulation of several processes, including production of proinflammatory mediators (e.g., cytokines), release of nitric oxide, and promotion of antioxidant activity. These responses were accompanied by changes in the macrophage metabolomic profiles that were consistent with a proinflammatory state and that indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. By contrast, PEGylated silk fibroin nanoparticles induced milder changes to both inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is PEGylation-dependent. Overall, PEGylation of silk fibroin nanoparticles reduced the inflammatory and metabolic responses initiated by macrophages, and this observation could be used to guide the therapeutic applications of these nanoparticles. © 2019 American Chemical Society

Metabolic reprogramming of macrophages exposed to silk, poly(lactic-co-glycolic acid) and silica nanoparticles

Monitoring macrophage metabolism in response to nanoparticle exposure provides new insights into biological outcomes, such as inflammation or toxicity, and supports the design of tailored nanomedicines. We describe the metabolic signature of macrophages exposed to nanoparticles ranging in diameter from 100 to 125 nm and made from silk, poly(lactic-co-glycolic acid) or silica. Nanoparticles of this size and type are currently at various stages of pre-clinical and clinical development for drug delivery applications. We used 1H NMR analysis of cell extracts and culture media to quantify the changes in the intracellular and extracellular metabolomes of macrophages in response to nanoparticle exposure. Increased glycolytic activity, an altered tricarboxylic acid cycle and reduced ATP generation were consistent with a pro-inflammatory phenotype. Furthermore, amino acids possibly arising from autophagy, the creatine kinase/phosphocreatine system and a few osmolytes and antioxidants emerged as important players in the metabolic reprogramming of macrophages exposed to nanoparticles. This metabolic signature was a common response to all nanoparticles tested; however, the direction and magnitude of some variations were clearly nanoparticle specific, indicating material-induced biological specificity. Overall, metabolic reprogramming of macrophages can be achieved with nanoparticle treatments, modulated through the choice of the material, and monitored using 1H NMR metabolomics

Comparative Metabolomics Study of the Impact of Articaine and Lidocaine on the Metabolism of SH-SY5Y Neuronal Cells

Articaine (ATC) and lidocaine (LDC) are the local anesthetics (LAs) currently most employed in dentistry. Cases of paresthesia, reported more frequently for ATC, have raised concerns about their potential neurotoxicity, calling for further investigation of their biological effects in neuronal cells. In this work, the impact of ATC and LDC on the metabolism of SH-SY5Y cells was investigated through 1H NMR metabolomics. For each LA, in vitro cultured cells were exposed to concentrations causing 10 and 50% reductions in cell viability, and their metabolic intracellular and extracellular profiles were characterized. Most effects were common to ATC and LDC, although with varying magnitudes. The metabolic variations elicited by the two LAs suggested (i) downregulation of glycolysis and of glucose-dependent pathways (e.g., one-carbon metabolism and hexosamine biosynthetic pathway), (ii) disturbance of branched chain amino acids (BCAA) catabolism, (iii) downregulation of TCA cycle anaplerotic fueling and activation of alternative energy producing pathways, (iv) interference with choline metabolism and (v) lipid droplet build-up. Interestingly, LDC had a greater impact on membrane phospholipid turnover, as suggested by higher phosphatidylcholine to phosphocholine conversion. Moreover, LDC elicited an increase in triglycerides, whereas cholesteryl esters accumulated in ATC-exposed cells, suggesting a different composition and handling of lipid droplets.publishe

Silk Hydrogel Substrate Stress Relaxation Primes Mesenchymal Stem Cell Behavior in 2D

Tissue-mimetic silk hydrogels are being explored for diverse healthcare applications, including stem cell delivery. However, the impact of stress relaxation of silk hydrogels on human mesenchymal stem cell (MSC) biology is poorly defined. The aim of this study was to fabricate silk hydrogels with tuned mechanical properties that allowed the regulation of MSC biology in two dimensions. The silk content and stiffness of both elastic and viscoelastic silk hydrogels were kept constant to permit direct comparisons. Gene expression of IL-1β, IL-6, LIF, BMP-6, BMP-7, and protein tyrosine phosphatase receptor type C were substantially higher in MSCs cultured on elastic hydrogels than those on viscoelastic hydrogels, whereas this pattern was reversed for insulin, HNF-1A, and SOX-2. Protein expression was also mechanosensitive and the elastic cultures showed strong activation of IL-1β signaling in response to hydrogel mechanics. An elastic substrate also induced higher consumption of glucose and aspartate, coupled with a higher secretion of lactate, than was observed in MSCs grown on viscoelastic substrate. However, both silk hydrogels changed the magnitude of consumption of glucose, pyruvate, glutamine, and aspartate, and also metabolite secretion, resulting in an overall lower metabolic activity than that found in control cells. Together, these findings describe how stress relaxation impacts the overall biology of MSCs cultured on silk hydrogels.

Odd–even effect in the formation and extraction performance of Ionic-Liquid-Based aqueous biphasic systems

Ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been extensively investigated in the separation of high-value biomolecules. However, the understanding of the molecular-level mechanisms ruling phase separation and extraction performance of these systems is crucial to successfully design effective separation processes. In this work, IL-based ABS composed of K2HPO4 and cholinium carboxylate ILs ([Ch][CnCO2] with n = 1 to 7, comprising anions with odd and even alkyl chain length) were investigated. The respective ternary phase diagrams, including binodal curves, tie-lengths, tie-line lengths and critical points, as well as the Setschenow salting-out coefficients (ks) that is a quantitative measure of the two-phase formation ability, were determined at 298 K. The extraction performance of these systems was then evaluated for four amino acids (L-tryptophan, L-phenylalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine/L-dopa). It was found that ILs composed of anions with even alkyl chains display slightly higher ks values, meaning that these ILs are more easily salted-out or more easily phase separate to form ABS. On the other hand, ABS formed by ILs with anions comprising odd alkyl chains lead to slightly higher partition coefficients of amino acids. Beyond the neat ILs odd-even effect resulting from their nanostructuration, being this a well-known phenomenon occurring in a series of their thermophysical properties, it is here shown the existence of an odd-even effect displayed by the IL anion aliphatic moiety in aqueous solution, visible both in the two-phase formation ability and extraction performance of ABS. These findings contribute to elucidate the molecular-level mechanisms governing ABS formation and partitioning of biomolecules, ultimately allowing the design of effective separation platforms.publishe

On the Relevance of Soft Tissue Sarcomas Metabolic Landscape Mapping

Soft tissue sarcomas (STS) prognosis is disappointing, with current treatment strategies being based on a "fit for all" principle and not taking distinct sarcoma subtypes specificities and genetic/metabolic differences into consideration. The paucity of precision therapies in STS reflects the shortage of studies that seek to decipher the sarcomagenesis mechanisms. There is an urge to improve STS diagnosis precision, refine STS classification criteria, and increase the capability of identifying STS prognostic biomarkers. Single-omics and multi-omics studies may play a key role on decodifying sarcomagenesis. Metabolomics provides a singular insight, either as a single-omics approach or as part of a multi-omics strategy, into the metabolic adaptations that support sarcomagenesis. Although STS metabolome is scarcely characterized, untargeted and targeted metabolomics approaches employing different data acquisition methods such as mass spectrometry (MS), MS imaging, and nuclear magnetic resonance (NMR) spectroscopy provided important information, warranting further studies. New chromatographic, MS, NMR-based, and flow cytometry-based methods will offer opportunities to therapeutically target metabolic pathways and to monitorize the response to such metabolic targeting therapies. Here we provide a comprehensive review of STS omics applications, comprising a detailed analysis of studies focused on the metabolic landscape of these tumors.publishe

PM2.5 chemical composition and health risks by inhalation near a chemical complex

Particulate matter (PM2.5) samples were collected in the vicinity of an industrial chemical pole and analysed for organic and elemental carbon (OC and EC), 47 trace elements and around 150 organic constituents. On average, OC and EC accounted for 25.2% and 11.4% of the PM2.5 mass, respectively. Organic compounds comprised polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, anhydrosugars, phenolics, aromatic ketones, glycerol derivatives, aliphatic alcohols, sterols, and carboxyl groups, including aromatic, carboxylic and dicarboxylic acids. Enrichment factors > 100 were obtained for Pb, Cd, Zn, Cu, Sn, B, Se, Bi, Sb and Mo, showing the contribution of industrial emissions and nearby major roads. Principal component analysis revealed that vehicle, industrial and biomass burning emissions accounted for 66%, 11% and 9%, respectively, of the total PM2.5-bound PAHs. Some of the detected organic constituents are likely associated with plasticiser ingredients and thermal stabilisers used in the manufacture of PVC and other plastics in the industrial complex. Photooxidation products of both anthropogenic (e.g., toluene) and biogenic (e.g., isoprene and pinenes) precursors were also observed. It was estimated that biomass burning accounted for 13.8% of the PM2.5 concentrations and that secondary OC represented 37.6% of the total OC. The lifetime cancer risk from inhalation exposure to PM2.5-bound PAHs was found to be negligible, but it exceeded the threshold of 10?6 for metal(loi)s, mainly due to Cr and As.3518-5DB7-75B0 | M?rio Tom?N/