Despite the scientific evidence supporting their biostimulant activity, the molecular mechanism(s) underlying the activity of protein hydrolysates (PHs) and the specificity among different products are still poorly explored. This work tested five different protein hydrolysates, produced from different plant sources using the same enzymatic approach, for their ability to promote rooting in tomato cuttings following quick dipping. Provided that all the different PHs increased root length (45–93%) and some of them increased root number (37–56%), untargeted metabolomics followed by multivariate statistics and pathway analysis were used to unravel the molecular processes at the basis of the biostimulant activity. Distinct metabolomic signatures could be found in roots following the PHs treatments. In general, PHs shaped the phytohormone profile, modulating the complex interaction between cytokinins and auxins, an interplay playing a pivotal role in root development, and triggered a down accumulation of brassinosteroids. Concerning secondary metabolism, PHs induced the accumulation of aliphatic glucosinolates, alkaloids, and phenylpropanoids, potentially eliciting crop resilience to stress conditions. Here, we confirm that PHs may have a hormone-like activity, and that their application can modulate plant growth, likely interfering with signaling processes. Noteworthy, the heterogenicity of the botanical origin supported the distinctive and peculiar metabolomic responses we observed across the products tested. While supporting their biostimulant activity, these findings suggest that a generalized crop response to PHs cannot be defined and that specific effects are rather to be investigated.

The lentil seed coat is a waste by-product still rich in phenolic compounds, specifically condensed tannins. The effect of different solvents, as well as different processes, namely conventional solid–liquid extraction (CSLE) and ultrasound-assisted extraction (UAE), on the extraction yield of specific phenolic compound classes was studied. Four empirical two-parameter models were examined to select the one that better fit the experimental data obtained under different operating conditions. Additionally, ultra-high-pressure liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-ESI/QTOF-MS) was employed to profile the phenolic compounds obtained under distinct extraction conditions. In the operative conditions adopted here, the bioactive compounds yield achieved using UAE was lower than that obtained with CSLE. The kinetics of polyphenols, flavonoids, and condensed tannins extraction from the lentil seed coat were successfully fitted to the power-law models, yielding mean values of the root mean square < 5.4%, standard error of estimation < 0.53, and coefficient of determination > 0.8. In addition, the UHPLC-ESI/QTOF-MS of the lentil seed coat extracts allowed the putative recognition of nearly 500 compounds, mainly flavonoids and phenolic acids.

Considering that functional components of plant foods are mainly secondary-metabolism products, we investigated the shaping of health-promoting compounds in hydroponically grown butterhead lettuce (Lactuca sativa L. var. capitata) as a function of the strength of the nutrient solution utilized. To this aim, untargeted metabolomics profiling, in vitro antioxidant capacity (total phenolics, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays), and the inhibition of selected enzyme activities were investigated in two butterhead lettuce cultivars with different pigmentation, i.e., green and red Salanova. Full-strength nutrition, together with half- and quarter-strength solutions of macronutrients, was tested. Our results indicate that by reducing the nutrients strength, we could elicit a distinctive shaping of the phenolic profile of lettuce. It is noteworthy that only specific classes of phenolics (namely, lignans and phenolic acids, followed by flavones and anthocyanins) were modulated by the induction of nutritional eustress (fold-change values in the range between −5 and +11). This indicates that specific responses, rather than a generalized induction of phenolic compounds, could be observed. Nonetheless, a genotype-dependent response could be observed, with the red cultivar being much more responsive to nutritional deprivation than the green Salanova lettuce. Indeed, analysis of variance (ANOVA) confirmed a genotype x nutrition interaction in red Salanova (p < 0.001). As a consequence of the changes in phenolic composition, also the antioxidant capacity (p < 0.001) and amylase inhibition (p < 0.001) properties were affected by the growing conditions. However, the effect on cholinesterase and tyrosinase inhibition was poorly affected by the nutritional strength. Provided that yields are not compromised, the application of a controlled nutritional eustress in hydroponically cultivated lettuce may represent a valuable strategy to produce food with tailored functional features in a sustainable manner.

In this work, different commercial extra-virgin olive oils (EVOO) were subjected to in vitro gastrointestinal digestion and the changes in bioactive compounds were evaluated by ultra-high-pressure liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry, using untargeted metabolomics. As expected, raw EVOO samples were abundant in total sterols (on average: 3007.4 mg equivalents/kg) and tyrosol equivalents (on average: 334.1 mg equivalents/kg). However, the UHPLC-QTOF screening allowed us to annotate 309 compounds, with a large abundance of sterols (219 compounds), followed by polyphenols (67 compounds) and terpenoids. The in vitro gastrointestinal digestion was found to affect the phytochemical composition of the different EVOO samples. In particular, both unsupervised and supervised statistics depicted the modifications of the bioactive profile following gastric and pancreatic phases. Overall, the compounds which resulted as the most affected by the in vitro digestion were flavonoids (cyanidin and luteolin equivalents), whilst relatively high % bioaccessibility values were recorded for tyrosol equivalents during the pancreatic phase (on average, 66%). In this regard, oleuropein-aglycone (i.e., the major phenolic compound in EVOO) was converted to hydroxytyrosol, moving from an average value of 1.3 (prior to the in vitro digestion) up to 9.7 mg equivalents/kg during the pancreatic step. As proposed in the literature, the increase in hydroxytyrosol might be the result of the combined effect of lipase(s) activity and acidic conditions. Taken together, the present findings corroborate the suitability of untargeted metabolomics coupled to in vitro digestion methods to investigate the bioaccessibility of phenolic compounds. In this regard, a significant impact of in vitro gastrointestinal digestion on polyphenolic profiles has been detected, thus suggesting the need to account for actual bioaccessibility values rather than just considering the amounts in the raw commodity.

Biostimulants are gaining increasing interest because of their ability to provide a green and effective strategy towards sustainable crop production. Nonetheless, their mode of action remains often unknown. The object of this work was to unravel the mechanisms through which 4-Vita, a biostimulant plant extract, can mitigate drought stress in tomato. To this aim, tomato plants were treated with two foliar applications of 4-Vita and drought stress imposed to both treated and control plants. Phenomics investigations were coupled to mass spectrometric untargeted metabolomics, and raw data were elaborated by multivariate statistics and pathway analysis. The biostimulant elicited a broad reprogramming of the tomato’s secondary metabolism, including its phytohormones profile, corroborating an improved ability to cope with drought stress. A series of mechanisms could be identified in response to the biostimulant treatment under drought, pointing to the preservation of photosynthetic machinery functionality. The modulation of thylakoid membrane lipids, the increase in xanthins involved in ROS detoxification, and the modulation of chlorophylls synthesis could also be observed. Overall, a series of coordinated biochemical mechanisms were elicited by the biostimulant treatment, supporting the increased resilience to drought stress in tomato.

Biostimulants are becoming increasingly popular in agriculture for their ability to induce beneficial effects in crops, paving the way towards the identification of new materials with biostimulant potential. This study evaluated the potential of different concentrations of an aqueous extract (0.25%, 0.50%, and 1.00%, dry weight/water volume, respectively) obtained from duckweed (Lemna minor L.) to stimulate olive plants. Leaf net photosynthesis (Pn), leaf transpiration rate (E), stomatal conductance (gs), sub-stomatal CO₂ concentration (Ci), chlorophyll content and other plant growth parameters were investigated. As a result, the extract improved Pn, gs, Ci, chlorophyll content and plant biomass production (leaf fresh and dry weight). Furthermore, the duckweed extract generally increased the uptake of nitrogen (N), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe) and zinc (Zn), while it did not influence the content of sodium (Na), manganese (Mn) and copper (Cu). The untargeted metabolomic profiling of the extract revealed the presence of signalling compounds (including phytohormones), phenolics and glutathione. Such broad diversity of bioactives may support the stimulatory potential observed in olive. In summary, this study revealed for the first time that duckweed could be seen as a promising species to obtain extracts with biostimulant properties in olive trees.