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The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates enormous quantities of waste and by-products, which can be exploited as new raw materials to obtain innovative ingredients and therefore make the olive production more sustainable. In a previous study, we decided to foster olive seeds by generating two protein hydrolysates using food-grade enzymes, alcalase (AH) and papain (PH). These hydrolysates have shown, both in vitro and at the cellular level, antioxidant and antidiabetic activities, being able to inhibit the activity of the DPP-IV enzyme and modulate the secretion of GLP-1. Given the multifunctional behavior of peptides, both hydrolysates displayed dual hypocholesterolemic activity, inhibiting the activity of HMGCoAR and impairing the PPI of PCSK9/LDLR, with an IC 50 equal to 0.61 mg/mL and 0.31 mg/mL for AH and PH, respectively. Furthermore, both samples restored LDLR protein levels on the membrane of human hepatic HepG2 cells, increasing the uptake of LDL from the extracellular environment. Since intestinal bioavailability is a key component of bioactive peptides, the second objective of this work is to evaluate the capacity of AH and PH peptides to be transported by differentiated human intestinal Caco-2 cells. The peptides transported by intestinal cells have been analyzed using mass spectrometry analysis, identifying a mixture of stable peptides that may represent new ingredients with multifunctional qualities for the development of nutraceuticals and functional foods to delay the onset of metabolic syndrome, promoting the principles of environmental sustainability.

Extra virgin olive oil (EVOO) is a valuable product and is highly appreciated by consumers for its great nutritional value. However, to date, there has been a lack of uniform systems capable of ranking the nutritional value of EVOO based on its chemical composition in terms of macro- and micronutrients (including phenolic compounds and tocopherols). The aim of this study was to propose a scoring algorithm to rank the nutritional value of EVOO samples, considering their chemical composition in macro- and micronutrients and their sensitivity to oxidation phenomena. Data from more than 1000 EVOO samples were used to assess the variability of the data, considering the selected negative parameters (free acidity, peroxide value, spectrophotometric indices) and positive components (composition in tocopherols via HPLC-DAD, phenolic compounds via HPLC-DAD, and fatty acids via GC-MS) so as to ensure the universal validity of the scoring algorithm. The dataset included samples from the main producing countries worldwide, in addition to Australia, across several production years; data were selected to represent different production realities. A mathematical model was set up for each chemical component, resulting in six variable values. By combining these values with a dimensionless constant value, the algorithm for computing the nutritional value score (NVS) was defined. It allows the nutritional value of an oil to be ranked on a scale of 0 to 100 based on its chemical composition. The algorithm was then successfully tested using chemical data from about 300 EVOO samples obtained from laboratories from different Italian regions. The proposed NVS is a simple and objective tool for scoring the nutritional value of an EVOO, easy to understand for both producers and consumers.

Fruits and vegetables processing produces significant amounts of by-products rich in valuable bioactive compounds such as polyphenols and dietary fiber. Food by-product re-use promotes the eco-sustainability of several crops. This study aimed to apply green extractions of bioactive compounds from by-products of basil, tomato, and red bell pepper production. Tests were performed by applying extraction procedures both at laboratory scale and using the Timatic ® extractor. Water and ethanol 10% and 20% were used for extraction of red bell pepper and tomato, testing different temperatures (30, 50, and 90 °C; water at 90 °C and ethanol 20% were applied for basil. The obtained phenolic extracts were analyzed by HPLC-DAD-MS. Polysaccharides of tomato and red bell pepper were extracted at laboratory scale and chemically characterized using 1 H-NMR to define the methylation and acylation degree, and DLS to estimate the hydrodynamic volume. Laboratory extraction tests allowed efficient scaling-up of the process on the Timatic ® extractor. Phenolic content in the dried extracts (DE) ranged 8.0-11.2 mg/g for tomato and red bell pepper and reached 240 mg/g for basil extracts. Polysaccharide yields ( w / w on DM) reached 6.0 and 10.4% for dried tomato and red bell pepper, respectively. Dry extracts obtained using the Timatic ® extractor and water can be useful sources of bioactive phenols. The study provided new data on tomato and red bell pepper polysaccharides that may be useful for future applications.

Chlorogenic acids, the esters of caffeic and quinic acids, are the main phenolic acids detected in Acmella oleracea extracts and have gained increasing interest in recent years due to their important biological activities. Given their structural similarity and instability, the correct analysis and identification of these compounds in plants is challenging. This study aimed to propose a simple and rapid determination of the A. oleracea caffeoylquinic isomers, applying an HPLC-MS/MS method supported by a mathematical algorithm (Linear Equation of Deconvolution Analysis (LEDA)). The three mono- and the three di-caffeoylquinic acids in roots of Acmella plants were studied by an ion trap MS analyzer. A separation by a conventional chromatographic method was firstly performed and an MS/MS characterization by energetic dimension of collision-induced dissociation mechanism was carried out. The analyses were then replicated using a short HPLC column and a fast elution gradient (ten minutes). Each acquired MS/MS data were processed by LEDA algorithm which allowed to assign a relative abundance in the reference ion signal to each isomer present. Quantitative results showed no significant differences between the two chromatographic systems proposed, proving that the use of LEDA algorithm allowed the distinction of the six isomers in a quarter of the time.

Proso millet has been proposed as an effective anti-diabetic food thanks to the combined action of polyphenols and starch. This study aimed to characterize the chemical composition of twenty-five accessions, in order to enhance this cereal as an alternative to available starch for food applications or to propose new food ingredients with health benefits. Proso millet contained a high percentage of starch, reaching values of 58.51%. The amylose content showed high variability, with values ranging from 1.36 to 42.70%, and significantly higher values were recorded for the white accessions than for the colored ones. High-resistant starch content (13.41-26.07%) was also found. The HPLC-MS analysis showed the same phenolic pattern in all the samples. Cinnamic acids are the most abundant compounds and significant differences in their total content were found (0.69 to 1.35 mg/g DW), while flavonoids were only detected in trace amounts. Statistical results showed significantly higher antiradical activity in the colored accessions than in the white ones.

Twenty oenotannins from different botanical origins were studied in model wine solution (1 g/L, 12% ethanol, pH 3.5). An original device was created for measuring Oxidation-Reduction potential ( ORp ) of the solutions at 20 °C in strict anoxic condition by the electrochemical method of the platinum electrode zero-current potential. Reactivity against proteins and antioxidant properties were related to the chemical structure and, consequently, to the botanical origin of the oenotannins. The highest turbidity after BSA addition (ΔNTU > 1000) values were measured for the gallic hydrolysable tannins. The ORp versus standard hydrogen electrode ranged from 420 to 260 mV. The ellagitannins had the highest antioxidant power ( AP %), followed by condensed tannins and gallotannins, highlighting a correlation with the phenolic profile. Based on these findings, two formulations were prepared as a blend of some of the tested oenotannins, with the ability to increase (MIX1) and decrease (MIX2) the ORp of the model wine.

The effects of the phenolic compounds of extra virgin olive oil (EVOO) on AGS cells have never been studied so far, which is the aim of this study. The profiles of the main phenolic components in EVOOs, mainly secoiridoid compounds derived from the transformation of oleuropein during the olive milling process, were evaluated and compared. Oils of different origins were evaluated aiming at verifying whether chemical differences in the phenolic composition of the dry extracts played a role in the metabolism and in maintaining the cellular redox state of AGS cells. The following key enzymes of some metabolic pathways were studied: lactate dehydrogenase, enolase, pyruvate kinase, glucose 6-phosphate dehydrogenase, citrate synthase, 3-Hydroxyacyl-CoA dehydrogenase and hexokinase. As confirmed through PCA analysis, pretreatments with the dry extracts of EVOOs at different concentrations appeared to be able to counteract the enzymatic activity alterations due to oxidative stress induced by H 2 O 2 1 mM and 2 mM. The studied phytocomplexes showed the ability to protect AGS cells from oxidative damage and the secoiridoid derivatives from both oleuropein and ligstroside contributed to the observed effects. The results suggested that EVOOs with medium to high concentrations of phenols can exert this protection.

Pâté is a by-product of olive oil production which represents an abundant source of phenolic compounds and can be used for food formulation, reducing its environmental impact and promoting a circular economy. In this context, the effects of a hydroalcoholic extract of pâté were evaluated for the first time in an AGS human cell line commonly used as model of gastric mucosa. Pâté was obtained from Tuscan olives; the total phenolic content was 16.6 mg/g dried extract, with verbascoside and secoiridoid derivatives as the most abundant phenols. The phenolic pâté extract did not alter viability, distribution of cell cycle phases or proliferation and migration of AGS cells at the tested concentrations. Seven enzymes were chosen to investigate the metabolic effect of the pâté extract in the context of oxidative stress. Pâté produced a statistically significant increase in the activity of key enzymes of some metabolic pathways: Lactate dehydrogenase, Enolase, Pyruvate kinase, Glucose 6-phosphate dehydrogenase, Citrate synthase, 3-Hydroxyacyl-CoA dehydrogenase and Hexokinase. Pre-treatments with the extract of pâté at 100 µg/mL or 200 µg/mL, as observed through PCA analysis, appeared able to counteract the enzymatic activity alterations due to oxidative stress induced by H 2 O 2 1 mM and 2 mM. The results indicate that dried pâté, due to its phenolic components, can be proposed as a new functional food ingredient., Competing Interests: The authors declare no conflict of interest.

Olives are very rich in phenolic compounds with important health-promoting properties. The profile and content of phenols in olive pulp and virgin olive oil are strongly influenced by the fruit ripening degree, but little is known concerning the evolution of phenolic compounds in the seed. In this work, the phenolic composition of seed from Tuscan cultivars (Frantoio, Moraiolo, Leccino) was studied over maturation. Starting from each seed sample, a phenolic extract was prepared and analyzed by HPLC-DAD-MS. Nüzhenide and nüzhenide 11-methyl oleoside were by far the most abundant phenolic compounds; their content reached up to 46 g/kg in dry seeds, although this diminished in the final stage of fruit maturation. At the same time, the phenolic composition of the pulp was also characterized over the course of maturation, showing that oleuropein was by far the most abundant compound, with concentrations comparable to those of nüzhenide and nüzhenide 11-methyl oleoside in the seeds. Overall, the total amount of phenols in seed dry extracts was significant, reaching approx. 100 g/kg. The chemically characterized dry phenolic extracts from seeds could be used for future biological assays aimed at evaluating the potential bioactivities of these phytocomplexes.

Millet is the sixth-highest yielding grain in the world and a staple crop for millions of people. Fermentation was applied in this study to improve the nutritional properties of pearl millet. Three microorganism combinations were tested: Saccharomyces boulardii (FPM1), Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius (FPM2) and Hanseniaspora uvarum plus Fructilactobacillus sanfranciscensis (FPM3). All the fermentation processes led to an increase in minerals. An increase was observed for calcium: 254 ppm in FPM1, 282 ppm in FPM2 and 156 ppm in the unfermented sample. Iron increased in FPM2 and FPM3 (approx. 100 ppm) with respect the unfermented sample (71 ppm). FPM2 and FPM3 resulted in richer total phenols (up to 2.74 mg/g) compared to the unfermented sample (2.24 mg/g). Depending on the microorganisms, it was possible to obtain different oligopeptides with a mass cut off ≤10 kDalton that was not detected in the unfermented sample. FPM2 showed the highest resistant starch content (9.83 g/100 g) and a prebiotic activity on Bifidobacterium breve B632, showing a significant growth at 48 h and 72 h compared to glucose ( p < 0.05). Millet fermented with Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius can be proposed as a new food with improved nutritional properties to increase the quality of the diet of people who already use millet as a staple food.

For improving the management of the production chain of PGI Mantua pears (which comprises many varieties, including Abate Fetel), applying the cardinal principles of circular economy and sustainability, the fruits with diseases or defects were recovered for producing dried rounds of pears from the Abate Fetel cultivar, a new product with high nutritional value that extends the remaining life. This process led to the production of secondary and residual by-products, which are mainly composed of the highest and lowest part of the fruits, comprising seeds, pulps, peels and petioles. Hence, this study was focused on the valorization of these secondary by-products of Abate Fetel pears through the production of pear extracts using traditional and "green" extraction methods that involve the use of supercritical CO 2 fluid extraction. The produced extracts, together with a reference solvent-derived extract, were analyzed by HPLC-ESI-MS, and in parallel, their direct and cellular antioxidant activity were assessed. Evidence has indicated that all the tested extracts reduced the H 2 O 2 -induced reactive oxygen species (ROS), lipid peroxidation and nitric oxide (NO) levels, respectively, in human intestinal Caco-2 cells. Hence, this study clearly suggests that extracts obtained from Mantuan PGI pear by-products may be used as valuable sources of bioactive upcycled phytocomplex for the development of dietary supplements and/or functional foods.