Titolo della tesi: High-Resolution NMR-Based Metabolomic Profiling in Plant-Based Foods and Cultural Heritage
Summary Part A
Nuclear Magnetic Resonance spectroscopy is a powerful methodology used to analyse food metabolites. It helps determine the chemical composition, verifying food types and sources and detecting adulteration. Non-target NMR, including metabolic profiling and fingerprinting, enables a comprehensive analysis of specific and non-specific metabolites.
In particular, untargeted NMR-based metabolomics was exploited to acquire the complete metabolic profiles of burdock roots, lemon balm aerial parts, and dandelion roots and aerial parts. This analytical method highlighted the impact of different altitudes and agronomic practices (spontaneous or organic cultivation) on the chemical profile. The analysis of plant samples revealed the presence and quantities of various compounds such as sugars, amino acids, organic acids, polyphenols, and fatty acids. Certain metabolites were found to be specific to particular tissues, for instance, arginine was exclusively found in the roots, while myo-inositol, galactose, glyceryldigalactose, pheophytin, and chlorophyll were identified in the aerial parts. Additionally, specific polyphenols, such as caftaric and chicoric acids, 3,5-di-caffeoylquinic acid, chlorogenic and rosmarinic acids were detected in dandelion, burdock, and lemon Balm, respectively. The quantities of metabolites varied significantly based on the plant's growth conditions, tissue type, and ecotype. Notably, all burdock ecotypes exhibited the highest amino acid content and the lowest organic acid content, while lemon balm displayed the opposite trend. Dandelion parts contained high levels of carbohydrates, except the aerial part, which exhibited the highest content of organic acids. These findings provided valuable insights into the chemistry of medicinal plants, thereby supporting further research in the nutraceutical and phytopharmaceutical fields.
A more in-depth study looked at wild lemon balm, cultivated in Corsica, named Melissa officinalis subsp. altissima (MOA). In this case, in addition to knowledge of the phyto-complex by NMR, studies were conducted on the obtained essential oil of the plant by applying GC-MS. Corsican lemon balm was found to be particularly rich in polyphenols (rosmarinic, chlorogenic, caffeic and caftaric acids), which express its antioxidant activity, and compounds such as Germacren-D, with antibacterial and antifungal properties. Furthermore, the biological activity of plant extracts against neuroinflammation was evaluated. Specifically, hypothalamic Hypoe22 cells were exposed to MOA extracts under basal conditions and were tolerated, demonstrating good biocompatibility and efficacy in protecting cells from H2O2-induced cytotoxicity. The gene expression of inflammatory and neurotrophic mediators such as TNF- α (Tumour necrosis factor), NOS-2 (Nitric oxide synthase-2) and BDNF (Brain-derived neurotrophic factor) in brain samples of C57/BL6 mice (prefrontal cortex and hypothalamus) treated with hydroalcoholic extracts of MOA was then assessed. In addition, the cortical tissues of the mice were exposed to β-amyloid to subsequently assess the expression of IL-6 (Interleukin-6) and ACHE (Acetylcholinesterase) genes during treatment with hydroalcoholic extracts of MOA. All extracts tested were effective in reversing the increased expression of TNF-α and NOS-2 and counteracting the down-regulation of the BDNF gene, suggesting potential protective effects on neuroinflammation. After β-amyloid stimulation, MOA extracts were efficient in inverting the increased expression of IL-6 and ACHE genes, showing potential protective effects on memory impairment and metabolic alterations in Alzheimer's disease.
Metabolomics plays a crucial role in monitoring food safety and quality by providing detailed information on the chemical composition of food products. By analysing metabolite profiles, harmful contaminants can be detected, spoilage markers can be identified, and the authenticity of food can be assessed. This approach allows early identification of potential food safety problems, such as pesticide residues or microbial contamination, thus ensuring consumer protection. Furthermore, metabolomics can help assess the nutritional quality of food, facilitating the development of healthier products. As part of Agritech consortium (National Research Centre for Agricultural Technologies), based on an extensive literature search on traditional and novel plant-based foods, fruit and vegetable matrices were listed and classified. Two products with high safety risks (pesticides, contaminants) were chosen as pilot matrices: tomatoes and apples.
Food packaging plays a critical function in the preservation of fresh food, protecting it from physical, chemical and biological contaminants. Although petroleum-based plastics such as polypropylene are commonly used due to their cost-effectiveness and effectiveness, their non-biodegradable nature poses risks to both humans and the environment. Alternatively, biodegradable materials made from biopolymers such as cellulose, chitosan, starch, polylactic acid (PLA), collagen and casein are being investigated for use in the food industry. These materials must fulfil specific criteria, such as non-toxicity and renewability, to be viable options for food packaging. In this specific case, non-target NMR analysis was used to obtain a complete metabolic profile of samples of Golden Delicious apples supplied by Consorzio Melinda S.c.a. (Cles (TN) - Val di Non - Italy).
The aim of the study was to evaluate the effect of two biodegradable packaging materials (ECO- PLA) on the quality and safety of fresh apples during cold storage (21 days, 5 °C) compared to packaging in conventional polymer bags (REF).
Metabolic profiling of the 1H NMR spectra of the hydroalcoholic and organic extracts identified 23 compounds in the apple samples at 14- and 21-days cold storage in the three packages. The metabolic components identified included primary and secondary sugars, metabolites of amino acids and organic acids, fatty acids, sterols and other compounds. In principle, cold storage delayed ripening and senescence processes, in particular the reduction of catalytic activities, including enzymes involved in the respiration pathways, which influence primary and secondary metabolism.
This included the “Pomodoro Riccio” tomato, an old cultivar, which is particularly suited to growing on clay soils, with a low water demand. As water is a primary source of heavy metals and pesticide residues, this cultivar had the potential to improve tomato quality, which emerges as a priority issue for consumer safety. Samples of “Pomodoro Riccio” (whole tomato, tomato paste and tomato waste), collected in 2022 and 2023 years, were provided by the company ‘La Sbecciatrice’ (Caserta, Italy). Considering all the characteristics of the cultivar of interest, the aim of the study was to delineate the metabolic profile and possible identification of new and (re)emerging hazards of a particular tomato ecotype. Specifically, NMR analysis was applied to delineate the metabolic profiles of the same cultivar grown in two different years. Indeed, in addition to ripeness, which may be related to the time of harvest, factors influencing the nutritional composition of tomatoes may be variety, climate, location and agricultural practices. Higher concentrations of amino acids were found in tomatoes grown in 2022. In contrast, tomatoes from the second year of harvest had the highest sugar levels, the most abundant metabolite class measured in all samples. The organic acids total content was more or less similar between the samples according to the years. Considering that the content of sugars and organic acids are the main factors in the taste of tomato fruits, and that catabolites of organic acids are involved in the biosynthesis of amino acids, the “Pomodoro Riccio” harvested in the second year were sweeter than those grown in the first year. Furthermore, although in lower concentrations, the tomato waste had significant amounts of bioactive compounds, which could be used to improve sustainability, e.g. in fermentation processes.
In a different study, NMR metabolomic was applied for the analysis of protein hydrolysates of cauliflower and artichoke waste, which were exploited for the formulation of biostimulants. Specifically, hydrolysates (HY) were characterised chemically and biologically, identifying and quantifying amino acids, organic acids, amines, polyols, mineral elements, phenols, tannins, flavonoids and sulphur compounds by NMR, inductively coupled plasma Mass Spectrometry and UV-visible analysis. The HY of cauliflower leaves and flowers showed the highest concentrations of free amino acids, while the stems were rich in calcium. In the case of artichoke, asparagine, glutamine and aspartic acid were found exclusively in the stems, while the leaves showed high levels of magnesium and manganese, as well as significant antioxidant activity. Diluted HYs were tested as biostimulants at five concentrations to evaluate their effects on seed germination and early growth of clover, alfalfa, durum wheat and maize. It was observed that the artichoke biostimulant positively influenced the germination speed of alfalfa, clover and durum wheat, whereas the cauliflower biostimulant significantly increased the germination speed of maize.
Summary Part B
Cultural heritage (CH) monuments in outdoor environments are highly susceptible to degradation by microorganisms that can cause aesthetic and physicochemical damage to stone surfaces. Lichens, among the most common biodeterioration agents, thrive on a wide range of substrates in different climates. They consist of a microbial community composed mainly of a mycobiont (a fungus) and a photobiont (green algae or cyanobacteria). This study focuses on the Italian archaeological site of Motya Island (Sicily, Italy). This island is vulnerable to climate change, with projections indicating significant sea level rise by 2100, which could lead to flooding of key archaeological areas. The study aimed to simulate the potential impact of future sea level rise on Motya's archaeological ruins. Preliminary metabolomic analyses using nuclear magnetic resonance (NMR) provide insights into the role of lichens in biodeterioration and highlight broader environmental threats to the preservation of archaeological remains. Indeed, it was possible to determine the quantification of metabolites present on lichen rocks sampled directly from Motya Island.
These were experimentally immersed in sterile water (L1), artificial salt water(L2), and Stagnone water (L3) taken directly near the island and monitored at seven (t1), thirty (t2), and sixty days (t3). The results showed a significant reduction in amino acids, sugars, organic acids, and other metabolites from time t0 to t3.