Pharmacological screening and biotecnological production of alkaloids from tissues and cells cultured by plants of the Amaryllidaceae family.

In this work particular attention was given to the study of secondary metabolites produced by some plants belonging to the Amaryllidaceae family, in the specific case isoquinoline alkaloids. At the first instance were characterized both qualitatively and quantitatively three different plants belonging to Amaryllidaceae family, such as: Crinum angustum Steud., Pancratium illyricum L., and Leucojum nicaeense Ard. The alkaloids extracts obtained were separately tested against enzymes involved in specific diseases or liable in multifactorial pathologies, like: MMPs, AChE,and PPO. From leaves extract of P.illyricum was isolated a new compound, 11α-hydroxy-O-methylleucotamine, with important role in AChE inbition. Considering the protection role against external bodies carried out by these metabolites in plant, extracts were also assayed against ATCC microorganisms and clinical isolates. Plants with promising pharmacological activities have been the basis for development of in vitro plant models.

Epigenetics of nutrition in aging

There is a need to identify factors that are able to influence health in old age and to develop interventions that could slow down the process of aging and its associated pathologies. Lifestyle modifications, and especially nutrition, appear to be promising strategies to promote healthy aging. Their impact on aging biomarkers has been poorly investigated. In the first part of this work, we evaluated the impact of a one-year Mediterranean-like diet, delivered within the framework of the NU-AGE project in 120 elderly subjects, on epigenetic age acceleration measures assessed with Horvath’s clock. We observed a rejuvenation of participants after nutritional intervention. The effect was more marked in the group of Polish females and in subjects who were epigenetically older at baseline. In the second part of this work, we developed a new model of epigenetic biomarker, based on a gene-targeted approach with the EpiTYPER® system. We selected six regions of interest (associated with ELOVL2, NHLRC1, SIRT7/MAFG, AIM2, EDARADD and TFAP2E genes) and constructed our model through a ridge regression analysis. In controls, estimation of chronological age was accurate, with a correlation coefficient between predicted and chronological age of 0.92 and a mean absolute deviation of 4.70 years. Our model was able to capture phenomena of accelerated or decelerated aging, in Down syndrome subjects and centenarians and offspring respectively. Applying our model to samples of the NU-AGE project, we observed similar results to the ones obtained with the canonical epigenetic clock, with a rejuvenation of the individuals after one-year of nutritional intervention. Together, our findings indicate that nutrition can promote epigenetic rejuvenation and that epigenetic age acceleration measures could be suitable biomarkers to evaluate their impact. We demonstrated that the effect of the dietary intervention is country-, sex- and individual-specific, thus suggesting the need for a personalized approach to nutritional interventions.

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.

Soil application of Meliaceae derivatives: effect on carbon and nitrogen dynamics in the soil-plant system

The effect of soil incorporation of 7 Meliaceae derivatives (6 commercial neem cakes and leaves of Melia azedarach L.) on C and N dynamics and on nutrient availability to micropropagated GF677 rootstock was investigated. In a first laboratory incubation experiment the derivatives showed different N mineralization dynamics, generally well predicted by their C:N ratio and only partly by their initial N concentration. All derivatives increased microbial biomass C, thus representing a source of C for the soil microbial population. Soil addition of all neem cakes (8 g kg-1) and melia leaves (16 g kg-1) had a positive effect on plant growth and increased root N uptake and leaf green colour of micropropagated plants of GF677. In addition, the neem cakes characterized by higher nutrient concentration increased P and K concentration in shoot and leaves 68 days after the amendment. In another experiment, soil incorporation of 15N labeled melia leaves (16 g kg-1) had no effect on the total amount of plant N, however the percentage of melia derived-N of treated plants ranged between 0.8% and 34% during the experiment. At the end of the growing season, about 7% of N added as melia leaves was recovered in plant, while 70% of it was still present in soil. Real C mineralization and the priming effect induced by the addition of the derivatives were quantified by a natural 13C abundance method. The real C mineralization of the derivatives ranged between 22% and 40% of added-C. All the derivatives studied induced a positive priming effect and, 144 days after the amendment, the amount of C primed corresponded to 26% of added-C, for all the derivatives. Despite this substantial priming effect, the C balance of the soil, 144 days after the amendment, always resulted positive.

Bio-preservation and bio-active compounds as sustainable strategies to improve quality and safety of fresh and fermented products

The development of new “green” and sustainable approaches to reduce food wastes, guaranteeing food quality, microbiological safety and the environmental sustainability, is of great interest for food industry. This PhD thesis, as part of the European project BioProMedFood (PRIMA–Section2 Programme), was focused on two strategies: the use of natural antimicrobials and the application of microbial strains isolated from spontaneously fermented products. The first part concerned the valorisation of microbial biodiversity of 15 Mediterranean spontaneously fermented sausages, through the isolation of autochthonous lactic acid bacteria (LAB) strains, mainly Latilactobacillus sakei, that were characterised regarding their safety and technological aspects. The most promising strains were tested as bio-protective cultures in fresh sausages, showing promising anti-listerial activity, or as starter cultures in fermented sausages. The second part of the research was focused on the use of natural compounds (phenolic extracts and essential oils from Juniperus oxycedrus needles and Rubus fruticosus leaves) with antimicrobial potential. They were tested in vitro against List. monocytogenes and Enterococcus faecium, showing differences in relation to species and type of extracts, but they hint at important possibilities for applications in specific foods. Concluding, this PhD thesis highlighted the great potential of traditional meat products as an isolation source of new strains with industrial importance. Moreover, the antimicrobial potential of compounds obtained from plant matrices opened promising perspectives to exploit them as “green” strategies to increase fresh food safety. The last topic of research, carry out in collaboration with Department of Nutrition and Food Sciences (University of Granada), investigated the effect of LAB fermentation on avocado leaves by-products, focusing on the bio-availability of phenolic compounds in the plant extracts, caused by microbial metabolism.