Study of the antioxidant fraction in edible olive oils obtained from different technological systems and parameters

Questo lavoro di tesi è stato suddiviso in tre parti. L’argomento principale è stato lo “Studio della componente antiossidante di oli ottenuti da olive mediante l’utilizzo di diversi sistemi e parametri tecnologici”. E’ ben noto come la qualità ossidativa di un olio di oliva dipenda oltre che dalla sua composizione in acidi grassi, dalla presenza di composti caratterizzati da un elevata attività antiossidante, ovvero le sostanze fenoliche. I composti fenolici contribuiscono quindi in maniera preponderante alla shelf life dell’olio extravergine di oliva. Inoltre sono state riscontrate delle forti correlazione tra alcune di queste sostanze e gli attributi sensoriali positivi di amaro e piccante. E’ poi da sottolineare come il potere antiossidante dei composti fenolici degli oli vergini di oliva, sia stato negli ultimi anni oggetto di considerevole interesse, poiché correlato alla protezione da alcune patologie come ad esempio quelle vascolari, degenerative e tumorali. Il contenuto delle sostanze fenoliche negli oli di oliva dipende da diversi fattori: cultivar, metodo di coltivazione, grado di maturazione delle olive e ovviamente dalle operazioni tecnologiche poiché possono variare il quantitativo di questi composti estratto. Alla luce di quanto appena detto abbiamo valutato l’influenza dei fattori agronomici (metodi di agricoltura biologica, integrata e convenzionale) e tecnologici (riduzione della temperatura della materia prima, aggiunta di coadiuvanti in fase di frangitura e di gramolatura, confronto tra tre oli extravergini di oliva ottenuti mediante diversi sistemi tecnologici) sul contenuto in composti fenolici di oli edibili ottenuti da olive (paper 1-3-4). Oltre alle sostanze fenoliche, negli oli di oliva sono presenti altri composti caratterizzati da proprietà chimiche e nutrizionali, tra questi vi sono i fitosteroli, ovvero gli steroli tipici del mondo vegetale, che rappresentano la frazione dell’insaponificabile quantitativamente più importante dopo gli idrocarburi. La composizione quali-quantitativa degli steroli di un olio di oliva è una delle caratteristiche analitiche più importanti nella valutazione della sua genuinità; infatti la frazione sterolica è significativamente diversa in funzione dell’origine botanica e perciò viene utilizzata per distinguere tra di loro gli oli e le loro miscele. Il principale sterolo nell’olio di oliva è il β- sitosterolo, la presenza di questo composto in quantità inferiore al 90% è un indice approssimativo dell’aggiunta di un qualsiasi altro olio. Il β-sitosterolo è una sostanza importante dal punto di vista della salute, poiché si oppone all’assorbimento del colesterolo. Mentre in letteratura si trovano numerosi lavori relativi al potere antiossidante di una serie di composti presenti nell’olio vergine di oliva (i già citati polifenoli, ma anche carotenoidi e tocoferoli) e ricerche che dimostrano invece come altri composti possano promuovere l’ossidazione dei lipidi, per quanto riguarda il potere antiossidante degli steroli e dei 4- metilsteroli, vi sono ancora poche informazioni. Per questo è stata da noi valutata la composizione sterolica in oli extravergini di oliva ottenuti con diverse tecnologie di estrazione e l’influenza di questa sostanza sulla loro stabilità ossidativa (paper 2). E’ stato recentemente riportato in letteratura come lipidi cellulari evidenziati attraverso la spettroscopia di risonanza nucleare magnetica (NMR) rivestano una importanza strategica da un punto di vista funzionale e metabolico. Questi lipidi, da un lato un lato sono stati associati allo sviluppo di cellule neoplastiche maligne e alla morte cellulare, dall’altro sono risultati anche messaggeri di processi benigni quali l’attivazione e la proliferazione di un normale processo di crescita cellulare. Nell’ambito di questa ricerca è nata una collaborazione tra il Dipartimento di Biochimica “G. Moruzzi” ed il Dipartimento di Scienze degli Alimenti dell’Università di Bologna. Infatti, il gruppo di lipochimica del Dipartimento di Scienze degli Alimenti, a cui fa capo il Prof. Giovanni Lercker, da sempre si occupa dello studio delle frazioni lipidiche, mediante le principali tecniche cromatografiche. L’obiettivo di questa collaborazione è stato quello di caratterizzare la componente lipidica totale estratta dai tessuti renali umani sani e neoplastici, mediante l’utilizzo combinato di diverse tecniche analitiche: la risonanza magnetica nucleare (1H e 13C RMN), la cromatografia su strato sottile (TLC), la cromatografia liquida ad alta prestazione (HPLC) e la gas cromatografia (GC) (paper 5-6-7)

Biological effects of bioactive components and extracts derived from edible plants commonly used in human nutrition

The main aim of this PhD research project was the evaluation of the biological effects of bioactive compounds derived from edible plants, with particular attention on their possibility to counteract oxidative damage and inflammation. After a preliminary study of in vitro antioxidant activity, regarding the modification eventually occurring after home freezing and cooking of edible vegetables, cultured mammalian cells were used as experimental model systems. Soluble extract and essential oils derived from different cultivars of Brassicaceae and Lamiaceae were tested as possible tools for the counteraction of the oxidative damage due to reactive oxygen species (ROS), underlining differences related to cultivar and agronomic techniques. Since accumulating evidence indicates that phytochemicals exhibit several additional properties in complex biological systems, a nutrigenomic approach was used to further explain the biological activity of a green tea extract, and to evidence the anti-inflammatory role of bioactive compounds derived from different foods. Overall, results obtained could contribute to a better understanding of the potential health benefit of plant foods.

Multifactorial analysis and food safety issues of edible marine gastropods intended for human consumption

The present thesis aims to evaluate a method to assess the viability; estimate the bacterial and viral (Hepatitis A and Norovirus) contamination; describe how some parameters change during a week in refrigerated condition and after 24 hours of immersion; estimate indole-producing bacteria and biogenic amines; evaluate the presence of saxitoxin and tetrodotoxin. The method to assess the viability using sea salt is easy to apply. Marine gastropods did not accumulate fecal contaminants, but vibrios due to their feeding. The Vibrio spp. load was even higher than the one registered on Ruditapes philippinarum belonging to the same area For what to concern the evaluation during a week in refrigerated condition and after 24 hours of immersion, non-re-immersed gastropods exceeded the acceptable mortality (10%) after three days in refrigerated conditions, but the Vibrio spp. load did not show a significant increase within three days. The TVC was already high from the beginning and its major part consisted of SSOs, which could be explained by gastropods’ feed, such as the Pseudomonas spp. load and the abundance of IPB. The BAs amount was also correlated with viability and had a statistically significant difference within a week on refrigerated conditions, principally because putrescine, tyramine, spermidine, and cadaverine rise in non-re-immersed samples. It also should be noted that the BAs amount was higher on average than the recommendation of literature. Moreover, re-immersed batches showed acceptable viability even after 3 days, and the Vibrio spp. load, TVC, SSOs, and biogenic amines remained almost constant within a week contrary to non-re-immersed samples. Finally, T. mutabilis and B. brandaris did not accumulate NoVs and TTX. We obtained only one positivity of the HAV sample and traces of STX (not at levels toxic to humans). Our results contribute to identifying food-borne hazards for T. mutabilis and B. brandaris.

Effects of allelopathic compounds produced by native and invasive marine macroalgae on the associated communities

In this study, the production of bioactive secondary metabolites called "allelochemicals" by algae has been investigated, specifically focusing on polyunsaturated aldehydes (PUAs). PUAs are known to have adverse effects on planktonic grazers and on phytoplankton; however, their effect on benthic communities has been poorly studied. Macroalgae are ecosystem engineers that play an important role in the structure of the habitat and associated communities, presenting a great variability in their morphology and structural complexity, which is a primary factor in the structuring of associated communities. In recent decades, it has been seen how the introduction of invasive species can modify the benthic habitat structure, causing cascading effects on the trophic chain. The thesis includes several field and laboratory studies. Field studies examined aldehyde production by native and invasive macroalgal species (Sargassum muticum, in the Adriatic Sea, and Rugulopterix okamurae in the Strait of Gibraltar), their structural complexity, together with their associated phyto and meiobenthos. Two laboratory studies were conducted. The first one, based on microcosms experiments, evaluated the effect of PUA (produced by the diatom Skeletonema marinoi, or as decadienal analytical standard) on meiofauna. The second one evaluated the inhibitory effect of dilkamural, an allelopathic compound isolated from R. okamurae, on unicellular phototrophs. Our results showed that PUAs produced by macroalgae were species-specific and had a significant impact on the benthic community. The morphology of macroalgae was an important factor in shaping associated communities, particularly for microphytobenthos. Invasive species, such as S. muticum and R. okamurae, could reduce the biodiversity of native benthic communities and simplify the habitat. Dilkamural was hypothesized to be an allelochemical defense, and laboratory toxicity tests confirmed this hypothesis. Overall, this thesis sheds light on the importance of allelochemicals and macroalgal structural complexity in the benthic environment and highlights the potential impact of invasive species.

Effects of habitat characteristics on cryptic fish assemblages

Habitat structure is known to influence the abundance of fishes on temperate reefs. Biotic interactions play a major role in determining the distribution and abundance of species. The significance of these forces in affecting the abundance of fishes may hinge on the presence of organisms that either create or alter habitat. On temperate reefs, for example, macroalgae are considered autogenic ecosystem engineers because they control resource availability to other species through their physical structure and provide much of the structure used by fish. On both coral and temperate reefs, small cryptic reef fishes may comprise up to half of the fish numbers and constitute a diverse community containing many specialized species. Small cryptic fishes (<100 mm total length) may be responsible for the passage of 57% of the energy flow and constitute ca. 35% of the overall reef fish biomass on coral reefs. These benthic fish exploit restricted habitats where food and shelter are obtained in, or in relation to, conditions of substrate complexity and/or restricted living space. A range of mechanisms has been proposed to account for the diversity and the abundance of small fishes: (1) lifehistory strategies that promote short generation times, (2) habitat associations and behaviour that reduce predation and (3) resource partitioning that allows small species to coexist with larger competitors. Despite their abundance and potential importance within reef systems, little is known of the community ecology of cryptic fishes. Specifically on habitat associations many theories suggested a not clear direction on this subject. My research contributes to the development of marine fish ecology by addressing the effects of habitat characteristics upon distribution of cryptobenthic fish assemblages. My focus was on the important shallow, coastal ecosystems that often serve as nursery habitat for many fish and where different type of habitat is likely to both play important roles in organism distribution and survival. My research included three related studies: (1) identification of structuring forces on cryptic fish assemblages, such as physical and biological forcing; (2) macroalgae as potential tools for cryptic fish and identification of different habitat feature that could explain cryptic fish assemblages distribution; (3) canopy formers loss: consequences on cryptic fish and relationship with benthos modifications. I found that: (1) cryptic fish assemblages differ between landward and seaward sides of coastal breakwaters in Adriatic Sea. These differences are explained by 50% of the habitat characteristics on two sides, mainly due to presence of the Codium fragile, sand and oyster assemblages. Microhabitat structure influence cryptic fish assemblages. (2) Different habitat support different cryptic fish assemblages. High heterogeneity on benthic assemblages reflect different fish assemblages. Biogenic components that explain different and diverse cryptic fish assemblages are: anemonia bed, mussel bed, macroalgal stands and Cystoseira barbata, as canopy formers. (3) Canopy forming loss is not relevant in structuring directly cryptic fish assemblages. A removal of canopy forming algae did not affect the structure of cryptic fish assemblages. Canopy formers algae on Conero cliff, does not seem to act as structuring force, probably due to its regressive status. In conclusion, cryptic fish have been shown to have species-specific associations with habitat features relating to the biological and non biological components afforded by fish. Canopy formers algae do not explain cryptic fish assemblages distribution and the results of this study and information from the literature (both from the Mediterranean Sea and elsewhere) show that there are no univocal responses of fish assemblages. Further exanimations on an non regressive status of Cystoseira canopy habitat are needed to define and evaluate the relationship between canopy formers and fish on Mediterranean sea.

Vegetables biofortified with iodine: chemical, agronomic and qualitative aspects of raw and fried potatoes

Iodine is an essential microelement for human health because it is a constituent of the thyroid hormones that regulate growth and development of the organism. Iodine Deficiency Disorders (IDDs) are believed to be one of the commonest preventable human health problems in the world today, according to the World Health Organization: that diseases include endemic goiter, cretinism and fetal abnormalities, among others, and they are caused by lack of iodine in the diet, that is the main source of iodine. Since iodine intake from food is not enough respect to human needs, this can be remedied through dietary diversification, mineral supplementation, food fortification, or increasing the concentration and/or bioavailability of mineral elements in the edible portions of crops through agricultural intervention or genetic selection (biofortification). The introduction of iodized salt is a strategy widely used and accepted to eradicate iodine deficiency, because it is an inexpensive source of stable iodine. Since the intake of salt, though iodized, must still be limited according to the risk of cardiovascular disease, so the increase of iodine content in plants for the production of functional foods is representing a field of study of particular interest and a potential market. In Italy potatoes enriched with iodine are produced by a patented procedure of agronomic biofortification for the fresh market since several years, furthermore they are recently accepted and recommended by Italian Thyroid Association, as an alternative source of iodine. Researches performed during the PhD course intended to characterize this innovative vegetables products, focusing the attention on different aspects, such as chemistry, agriculture, and quality of fresh and fried potatoes. For this purpose, lipid fraction of raw material was firstly investigated, in order to assess whether the presence of iodine in plant metabolism can affect fatty acid or sterol biosynthesis, according to the hypothesis that iodine can be bounded to polyunsaturated fatty acids of cell membranes, protecting them from peroxydation; phytosterols of plant sterol are also studied because their importance in reducing serum cholesterol, especially in potato plant sterols are also involved in synthesis of glycoalkaloid, a family of steroidal toxic secondary metabolites present in plants of the Solanaceae family. To achieve this goal chromatographic analytical techniques were employed to identify and quantify fatty acids and sterols profile of common and iodine enriched row potatoes. Another aim of the project was to evaluate the effects of frying on the quality of iodine-enriched and common potatoes. Since iodine-enriched potatoes are nowadays produced only for the fresh market, preliminary trials of cultivation under controlled environment were carried out to verify if potato varieties suitable for processing were able to absorb and accumulate iodine in the tuber. In a successive phase, these varieties were grown in the field, to evaluate their potential productivity and quality at harvest and after storage. The best potato variety to be destined for processing purposes, was finally subjected to repeated frying cycles; the effects of lipid oxidation on the composition and quality of both potatoes and frying oil bath were evaluated by chromatographic and spectrophotometric analytical techniques. Special attention were paid on volatile compounds of fried potatoes.

Small Molecules as Modulators of Different Targets Involved in Tumor Progression

Tumor is a lesion that may be formed by an abnormal growth of neoplastic cells. Many factors increase the risk of cancer and different targets are involved in tumor progression. Within this thesis, we have addressed two different biological targets, independently connected with tumor formation, e.g. Hsp90 and androgen receptor. The ATP-dependent chaperone Hsp90 is responsible for the conformational maturation and the renaturation of proteins. “Client” proteins are associated with the cancer hallmarks, as cell proliferation and tumor progression. Consequently, Hsp90 has evolved into promising anticancer target. Over the past decade, radicicol has been identified as potential anticancer agent targeting Hsp90, but it is not active in vivo. With that aim of obtaining radicicol-related derivatives, we developed the design and synthesis of new chalcones analogs. Chalcones, which are abundant in edible plants, own a diverse array of pharmacological activities and are considered a versatile scaffold for drug design. Antiproliferative assays and western blot analysis on the new compounds showed that some of those display an interesting cytotoxic effect and the ability to modulate Hsp90 client proteins expression. Androgen Receptor (AR) hypersensitivity plays crucial role in prostate cancer, which progression is stimulated by androgens. The therapy consists in a combination of surgical or chemical castration, along with antiandrogens treatment. Casodex® (bicalutamide), is the most widespread antiandrogen used in clinic. However, hormonal therapy is time-limited since many patients develop resistance. Commercially available antiandrogens show a common scaffold, e.g. two substituted aromatic rings linked by a linear or a cyclic spacer. With the aim of obtaining novel pure AR antagonists, we developed a new synthetic methodology, which allowed us to introduce, as linker between two suitably chosen aromatic rings, a triazole moiety. Preliminary data suggest that the herein reported new molecules generally decrease PSA expression, thus confirming their potential AR antagonistic activity.

Veterinary drugs in drinking water used for pharmaceutical treatments in breeding farms

Water is susceptible to be used for numerous purposes, including edible, both for humans and animals. In the food animal production, drinking water is frequently used as a way to carry out the most common pharmacological treatments. In these cases, there are many variables which could degrade drugs dissolved in this mean, even when properly arranged pharmaceutical formulations are used. In fact, although a product obtains a Marketing Authorization through appropriate laboratory studies both drug stability and solubility, on the other hand the solubility of the same drug in natural water used as a drinking water is not documented. In the present study has been evaluated the dissolution kinetics (at 0 hours and 24 hours) of products, having oxytetracycline and tylosin as active ingredient, used in drinking water samples in order to see how the different physical and chemical factors that characterize the drinking water may affect therapeutic efficacy. In fact, multiple factors, also of little relevance if individually considered, are able to adversely affect the pharmacological treatment carried out in drinking water.

Effects of temperature and acidity on Mediterranean benthic calcifiers, in the face of globale climate change

The Mediterranean Sea is expected to react faster to global change compared to the ocean and is already showing more pronounced warming and acidification rates. A study performed along the Italian western coast showed that porosity of the skeleton increases with temperature in the zooxanthellate (i.e. symbiotic with unicellular algae named zooxanthellae) solitary scleractinian Balanophyllia europaea while it does not vary with temperature in the solitary non-zooxanthellate Leptopsammia pruvoti. These results were confirmed by another study that indicated that the increase in porosity was accompanied by an increase of the fraction of the largest pores in the pore-space, perhaps due to an inhibition of the photosynthetic process at elevated temperatures, causing an attenuation of calcification. B. europaea, L. pruvoti and the colonial non-zooxanthellate Astroides calycularis, transplanted along a natural pH gradient, showed that high temperature exacerbated the negative effect of lowered pH on their mortality rates. The growth of the zooxanthellate species did not react to reduced pH, while the growth of the two non-zooxanthellate species was negatively affected. Reduced abundance of naturally occurring B. europaea, a mollusk, a calcifying and a non-calcifying macroalgae were observed along the gradient while no variation was seen in the abundance of a calcifying green alga. With decreasing pH, the mineralogy of the coral and mollusk did not change, while the two calcifying algae decreased the content of aragonite in favor of the less soluble calcium sulphates and whewellite (calcium oxalate), possibly as a mechanism of phenotypic plasticity. Increased values of porosity and macroporosity with CO2 were observed in B. europaea specimens, indicating reduces the resistance of its skeletons to mechanical stresses with increasing acidity. These findings, added to the negative effect of temperature on various biological parameters, generate concern on the sensitivity of this zooxanthellate species to the envisaged global climate change scenarios.

Impact of metal and metal oxide engineered nanoparticles in soil and plant systems

Nanotechnology promises huge benefits for society and capital invested in this new technology is steadily increasing, therefore there is a growing number of nanotechnology products on the market and inevitably engineered nanomaterials will be released in the atmosphere with potential risks to humans and environment. This study set out to extend the comprehension of the impact of metal (Ag, Co, Ni) and metal oxide (CeO2, Fe3O4, SnO2, TiO2) nanoparticles (NPs) on one of the most important environmental compartments potentially contaminated by NPs, the soil system, through the use of chemical and biological tools. For this purpose experiments were carried out to simulate realistic environmental conditions of wet and dry deposition of NPs, considering ecologically relevant endpoints. In detail, this thesis involved the study of three model systems and the evaluation of related issues: (i) NPs and bare soil, to assess the influence of NPs on the functions of soil microbial communities; (ii) NPs and plants, to evaluate the chronic toxicity and accumulation of NPs in edible tissues; (iii) NPs and invertebrates, to verify the effects of NPs on earthworms and the damaging of their functionality. The study highlighted that NP toxicity is generally influenced by NP core elements and the impact of NPs on organisms is specie-specific; moreover experiments conducted in media closer to real conditions showed a decrease in toxicity with respect to in vitro test or hydroponic tests. However, only a multidisciplinary approach, involving physical, chemical and biological skills, together with the use of advanced techniques, such as X-ray absorption fine structure spectroscopy, could pave the way to draw the right conclusions and accomplish a deeper comprehension of the effects of NPs on soil and soil inhabitants.

Valorisation of organic waste: new developments from proton nuclear magnetic resonance characterization

The last half-century has seen a continuing population and consumption growth, increasing the competition for land, water and energy. The solution can be found in the new sustainability theories, such as the industrial symbiosis and the zero waste objective. Reducing, reusing and recycling are challenges that the whole world have to consider. This is especially important for organic waste, whose reusing gives interesting results in terms of energy release. Before reusing, organic waste needs a deeper characterization. The non-destructive and non-invasive features of both Nuclear Magnetic Resonance (NMR) relaxometry and imaging (MRI) make them optimal candidates to reach such characterization. In this research, NMR techniques demonstrated to be innovative technologies, but an important work on the hardware and software of the NMR LAGIRN laboratory was initially done, creating new experimental procedures to analyse organic waste samples. The first results came from soil-organic matter interactions. Remediated soils properties were described in function of the organic carbon content, proving the importance of limiting the addition of further organic matter to not inhibit soil processes as nutrients transport. Moreover NMR relaxation times and the signal amplitude of a compost sample, over time, showed that the organic matter degradation of compost is a complex process that involves a number of degradation kinetics, as a function of the mix of waste. Local degradation processes were studied with enhanced quantitative relaxation technique that combines NMR and MRI. The development of this research has finally led to the study of waste before it becomes waste. Since a lot of food is lost when it is still edible, new NMR experiments studied the efficiency of conservation and valorisation processes: apple dehydration, meat preservation and bio-oils production. All these results proved the readiness of NMR for quality controls on a huge kind of organic residues and waste.

Assessing Household Food Waste in Italy: A Methodology for Detecting Drivers and Quantities

It has been estimated that one third of edible food destined for human consumption is lost or wasted along the food supply chain globally. Much of the waste comes from Global North, where consumers are considered as the bigger contributors. Different studies tried to analyze and estimate the Household Food Waste (HFW), especially in UK and Northern Europe. The result is that accurate studies at national level exist only in UK, Finland and Norway while no such studies are available in Italy, except for survey- based researches. Though, there is a widespread awareness that such methods might be not able to estimate Food Waste. Results emerging from literature clearly suggest that survey estimate inferior amounts of Food Waste as a result, if compared to waste sorting and weighting analysis or to diary studies. The hypothesis that household food waste is under-estimated when gathered through questionnaires has been enquired into. First, a literature review of behavioral economics and heuristics has been proposed; then, a literature review of the sector listing the existing methodologies to gather national data on Household Food Waste has been illustrated. Finally, a pilot experiment to test a mixed methodology is proposed. While literature suggests that four specific cognitive biases might be able to affect the reliability of answers in questionnaires, results of the present experiment clearly indicate that there is a relevant difference between how much the individual thinks to waste and he/she actually does. The result is a mixed methodology based on questionnaire, diary and waste sorting, able to overcome the cons of each single method.