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Biopolymers have gained significant attention due to their environmental advantages, with insects emerging as a promising but underutilized source of chitin and chitosan. In this study, chitosan was extracted from the larval exuviae of Tenebrio molitor through sequential demineralization, deproteinization, and deacetylation steps. For selected analyses, the extracted chitosan was further purified via reprecipitation from an acid solution using a basic precipitant (1 M NaOH). Chitosan was then characterized using chemical and instrumental methods. The results indicated that the chitosan had a medium degree of deacetylation (72.27%) and viscosity-average molecular weight (612 kDa), along with minimal ash (0.33%) and amino acid (0.14%) content, suggesting high product quality. FTIR analysis identified characteristic functional groups present, and SEM analysis highlighted a fibrous and porous microstructure in the purified chitosan. The prepared films exhibited favorable properties, including low thickness (0.0197 mm), high swelling degree (335.07%), moderate water solubility (46.99%), and moisture content of 32.39%, supporting their practical applicability. T. molitor exuviae thus represents a sustainable and environmentally friendly source of high-quality chitosan, with beneficial structural and functional properties, supporting its use in a wide array of value-added applications.

Selenium (Se) biofortification of vegetables can improve dietary Se intake; however, the dose-dependent balance between inorganic Se retention and organic Se assimilation following foliar selenate application remains insufficiently resolved across species. Five leafy vegetable species (garden rocket, wild rocket, dandelion, and two chicory cultivars) were grown under controlled greenhouse conditions and treated twice with foliar sodium selenate at increasing application rates (1 + 1, 2 + 2, 5 + 5, 10 + 0, 10 + 10, and 10 + 50 mg Se L−1) across two experiments. Total Se and Se species were determined by HPLC-UV-HG-AFS following enzymatic extraction and cross-checked on selected extracts by HPLC-ICP-MS. Foliar selenate induced substantial Se accumulation in all species, reaching up to 102 µg g−1 DW in garden rocket. At moderate application rates (notably 2 + 2 and 5 + 5 mg Se L−1), a considerable proportion of extracted Se was converted into organic forms, with selenomethionine (SeMet) accounting for up to ~40% of total extracted Se. In contrast, at the highest application rate (10 + 50 mg Se L−1), inorganic Se(VI) became predominant (often >40%), while SeMet proportion declined sharply to ~2–4%, indicating a saturation of metabolic assimilation capacity under high Se exposure. Leaf biomass was promoted at intermediate treatments (e.g., 5 + 5 and 10 + 0/10 + 10 mg Se L−1), whereas the highest rate reduced growth. Overall, foliar selenate effectively biofortifies chicory, rocket, and dandelion leaves, but excessive application rates shift Se speciation toward inorganic storage and markedly suppress SeMet formation. These findings highlight the importance of dose optimization to maximize nutritional quality while avoiding metabolic overload.

All olive (Olea europaea L.) plant tissues have a high phenolic content. However, the effects of the cultivar and sampling period on the tissue phenolic content remain almost unknown; in addition, the interactions between nutrient uptake and leaf phenol concentrations have not been clarified. This study sampled olive leaves to explore how the cultivar, sampling period, and their interaction affect leaf phenol and nutrient concentrations. Leaves were collected from six cultivars during three seasonal periods: harvest (October; SP1), dormancy (January; SP2), and pruning (March; SP3). Five were Istrian cultivars (‘Bova’, ‘Buža muška’, ‘Buža puntoža’, ‘Istarska bjelica’, ‘Rošinjola’), and one was the Italian cultivar ‘Leccino’. Phenolic profiles in olive leaves were correlated with potassium (K), phosphorus (P), and copper (Cu) concentrations. However, significant correlations between these nutrients and oleuropein, verbascoside, and total phenolic content (TPC) were determined only for ‘Rošinjola’. Oleuropein was the most abundant phenolic compound, while among genotypes, ‘Buža muška’ showed the highest oleuropein levels across all sampling periods, indicating its potential source of oleuropein in olive leaves. Seasonal variations in olive leaf phenolic compounds appear to be strongly influenced by phenological phase, nutrient dynamics, and weather conditions, as confirmed by multivariate analysis across sampling periods and cultivars. The findings emphasise the importance of selecting both an appropriate cultivar and sampling period to maximise the accumulation of olive leaf phenolic compounds. Nevertheless, long-term experimentation on cultivars with a high leaf phenolic potential, like ‘Buža muška’ and ‘Rošinjola’, is necessary in order to develop appropriate farming strategies for maximising phenolic compounds with human or plant health benefits.

In this study, we investigated the influence of pedological parameters and variation of altitude on the mineral nutrients, phenolic compounds, and antioxidant activities of olive leaves. Samples of the Chetoui cultivar were collected from eight geographical locations with different altitudes. Levels of phenolic compounds varied according to the altitude. Classification of the locations revealed that altitude 1 (>500 m) was characterized by high levels of secoiridoids and simple phenols, while altitude 2 (500–300 m) and altitude 3 (<300 m) were higher in flavonoids. Levels of Mn, Ca and B in the leaves and level of Zn in the soil were significantly correlated with the abundance of oleuropein and luteolin-7-O glucoside, the most important phenols in Chetoui olive leaves. The results suggest that, in addition to pedological criteria, environmental conditions also influence the formation of phenolic compounds.

The foliar application of various biostimulants, such as protein hydrolysates (PHs), has been associated with improved nutrient uptake efficiency and stress tolerance in perennial crops, like olive (Olea europaea L.). In this study, PHs obtained by enzymatic hydrolysis by Alcalase Pure (referred to as treatment H1), Alcalase Pure and Flavourzyme (referred to as treatment H2), or Alcalase Pure and Protana™ Prime (referred to as treatment H3) with proteins from pumpkin seed cake were tested for their potential beneficial growth, performance, and nutrition effects in one-year-old olive seedlings grown under controlled conditions. Amino acid and element compositions were evaluated in the PHs, which were used for foliar application six times at eight-day intervals. Control (C) plants were treated the same way, but without PHs. Shoot and root growth, leaf reflectance indices, and the composition of micro and macronutrients in different organs and leaf tissues were determined. Plants in the H2 treatment grew significantly better than C plants. They had the highest Photochemical Reflectance Index and a Chlorophyll-Normalized Difference Vegetation Index similar to that of C plants, indicating an optimal growth/photosynthesis balance. A decrease in the concentration of several mineral elements in the lower epidermis in H2- and H3-treated plants compared to C and H1-treated plants was accompanied by their increase in the spongy mesophyll, indicating their redistribution to support increased metabolism, resulting in increased shoot growth in these two treatments. Arguably, these observed effects could be attributed to the amino acid profile of the H2 mixture, which had the highest concentration of L-proline, L-arginine, and L-lysine among the three PH mixtures, and a higher L-asparagine concentration than the H1 mixture. Overall, the results highlight the applicative potential of tailored PH formulations for the optimization of growth, mineral element composition, and physiological performance in olive cultivation.

Olive (Olea europaea L.) cultivars often exhibit genotype-specific responses to micronutrient management. In this study, we investigated the metabolic leaf fingerprinting of three cultivars ‘Rošinjola’, ‘Leccino’, and ‘Istarska bjelica’ at two sampling periods (SP-I = 64 days after treatment (DAT) and SP-II = 118 DAT), following boron foliar fertilisation (+B = 41.62 mM B; −B = 0 mM B) applied 50 days after anthesis. To our knowledge, this is the first study to provide such a detailed evaluation of boron-induced shifts in phenolic metabolism in olive leaves. At harvest (SP-II), all three cultivars showed higher concentrations of total identified phenolic compounds in +B plants compared with the −B controls. Notably, the concentration of verbascoside at harvest was higher in +B plants of ‘Istarska bjelica’ and ‘Leccino’, but not in ‘Rošinjola’. Oleuropein content increased in +B plants at harvest to a level higher than 4870 mg/100 g DW, irrespective of cultivar. Conversely, apigenin-7-glucoside declined from SP-I to SP-II in ‘Leccino’ regardless of treatment, whereas in ‘Istarska bjelica’, this decrease occurred only in control plants, with boron preventing the seasonal decline. These findings confirm the prolonged effect of boron foliar fertilisation on phenolic metabolism in olive leaves and highlight cultivar-specific differences in metabolic responses. Further research is needed to clarify how these metabolic shifts relate to primary plant metabolism and how they influence olive oil quality traits among cultivars grown under Croatian conditions.

Silicon is one of the most abundant elements in the Earth’s crust and, although it is not an essential element for plant growth, it is considered beneficial as it can help in defense against biotic and abiotic stresses. Research on the effects of foliar sprays containing silicon has been well documented for different plants. However, in olive plants there is considerably less research focused on silicon soluble foliar formulations, which differs from the application of inert materials, like kaolin. The purpose of this study was to evaluate the effects of three increasing levels of foliar-applied soluble silicon on the characteristics of olive fruit and olive oil parameters. Fruit morphology, contents of multiple polyphenols, fatty acid profiles and mineral content in olive oil, olive oil quality parameters, fruit yield, and olive oil production parameters were assessed. Results show significant effects of silicon treatments on palmitic, palmitoleic, and linoleic acids and on the content of mono- and polyunsaturated fatty acids. Polyphenols were generally more influenced by the cultivar rather than by silicon. Apigenin, phenolic acids, and flavonoids were impacted by silicon treatments. Fruit morphology was affected by silicon treatment and, generally, parameters (fruit and seed length, width, and mass) increased at higher silicon application. This was concomitantly seen in both fruit and extra virgin olive oil (EVOO) yield increase, but only for the Leccino cultivar, suggesting specific cultivar x treatment interplay for those traits. Increases in mineral contents in olive oil due to Si treatments were not found, except for silicon, where Si2 and Si3 treatments yielded higher silicon content in olive oil compared to control. Results of this study indicate that Silicon Foliar Application could be used to enhance olive yield and only slightly modifies the fatty acid composition of olive oil and the content of some of the phenols. Although silicon application did affect several parameters, the effect of cultivar was more pronounced in affecting most of the parameters. Further research could be driven towards the effect of foliar silicon on these parameters under environmental stress factors.

Secondary metabolites in olive (Olea europaea L.) leaves constitute a complex framework wherein phenylpropanoids, terpenoids, and secoiridoids in particular, serve as major contributors to olive plant resilience. Silicon (Si) stands as a mediator of defense mechanisms in plants, enhancing their protective responses and adaptability. A field trial on one-year-old plantlets of two metabolically distinct olive genotypes was conducted to investigate the effects of foliar-applied Si on the phytochemical profiles of locally treated leaves. Silicon’s systemic effects in juvenile leaves were also appraised. We accounted for intervarietal differences in nutrient uptake and conducted in situ measurements of physiological indices. The peak of the summer season and the onset of autumn were chosen as the two sampling time points. Intense summer conditions prompted metabolic adjustments that resulted in phytochemical profiles unique to each cultivar. These profiles were further significantly altered by Si while remaining genotype-specific, with substantial increases in prominent compounds like oleuropein (105% and 252%) and verbascoside (62% and 126%), depending on the genotype. As the pressure from environmental factors eased, the differences in Si-mediated phytochemical responses emerged. Silicon had a limited effect on the phytochemical profile of the resilient cultivar which acquired a metabolic steady-state, while it significantly altered the profile of its metabolically more versatile counterpart, resulting with a progressive increase in its oleuropein (37%) and verbascoside (26%) levels. These effects extended to untreated, juvenile leaves as well. While effective in altering and improving the phytochemical composition of olive leaves, Si acted in a manner that adhered to each genotype’s metabolic foundation. The intensity of environmental constraints, along with each cultivar’s inherent sensitivity to them, seems to be tied to silicon’s capacity to mediate significant phytochemical alterations. The extent of silicon’s prophylactic function may therefore be dependent on a genotype’s metabolic foundation and overall sensitivity, and as such it seems inseparable from stress and its intensity.

It is not yet clear how adding silicon foliar fertilisation affects olive leaf (OL) phenolics and their potential to impact different cancer cells. Thus, we conducted a field trial to study the effect of foliar Si biostimulant fertilisation on the OL phenolic content of the ‘Leccino’ (LE) and ‘Istarska Bjelica’ (IB) cultivars. The experiment compared untreated Control (C) and three distinct levels of silicon (Si1, Si2, Si3) with Si concentrations of 0.55 g/L, 1.1 g/L, and 2.2 g/L, respectively. Si3 application resulted in the highest levels of oleuropein, apigenin-7-O-glucoside, luteolin-4-O-glucoside, rutin, and tyrosol compared to the C treatment. The polyphenols showed high cytotoxic activity in three cancer cell lines tested: cervical adenocarcinoma (HeLa), colon cancer (HCT116), and osteosarcoma (U2OS). The strongest inhibition of cell growth was observed in the HCT116 cell line. All cancer cells tested were more sensitive to treatment with polyphenols isolated from plants with added Si than those without added Si. The cytotoxic activity of the extracts on the healthy cell line RPE1 was similar to that on the cancer cell line HCT116 and U2OS.

For the first time the effects of different sampling periods and their interaction with five major autochthonous Croatian Istrian olive cultivars and the Italian cultivar ‘Leccino’ on the quantity and composition of olive leaf phenolic compounds and mineral nutrients were investigated. For that purpose, olive leaves were sampled in two collecting periods, in October and March, coinciding with the harvesting and pruning periods, respectively. All selected cultivars had a higher oleuropein leaf content in the pruning collecting period, with the highest levels noted for the ‘Leccino’ and ‘Buža’ cultivars. Cultivar significantly affected almost all the investigated phenols, with higher concentrations of these valuable compounds in the pruning than in the harvesting period. Differences observed in leaf mineral composition were closely related to the differences in phenolic profiles and were significantly affected by genotype. Some of the studied mineral nutrients, such as P, Cu and B, were found to be significantly correlated with the most abundant olive leaf phenolic compounds, oleuropein and verbascoside.