La presentazione avrà come temi principali: i) alimenti funzionali ossia alimenti capaci di influire positivamente sullo stato di salute umana; ii) ortaggi biofortificati con sali minerali utili alla salute umana; iii) nutraceutici di natura fenolica, cioè composti bioattivi naturalmente presenti negli alimenti. In particolare saranno illustrate le attività ed i risultati di alcuni studi scientifici, condotti presso ISPA-CNR, su alimenti presenti nella Dieta Mediterranea come olive da tavola, carciofo, mirtillo, melograno. Saranno presentati nuovi alimenti vegetali biofortificati mediante l'utilizzo di tecniche agronomiche innovative (idroponica) e nuovi alimenti funzionalizzati mediante l'aggiunta della componente bioattiva (polifenoli) in matrici cerealicole come pasta e pane. Infine, saranno descritti dei modelli di digestione gastrointestinale in vitro accoppiati a sistemi cellulari, che permettono di valutare la stabilità chimica dei composti bioattivi, la loro bioaccessibilità (frazione di composti bioattivi rilasciata dall'alimento dopo il processo di digestione), biodisponibilità (frazione di composti bioattivi disponibile nel circolo ematico e potenzialmente attiva sui tessuti target) e quindi il loro potenziale effetto benefico sulla salute umana.

Whole wheat foods are significant source of compounds exhibiting health-promoting properties. One of the most abundant class of phytochemicals in the wheat grain is represented by phenolic acids that are typically localized in the bran and germ portions. The objective of this study was to estimate the extent of variation for phenolic acids in durum wheat (T. turgidum L. ssp. durum). In addition, this study aimed at evaluating the anti-inflammatory activity of phenolic acids contained in whole-meal flour extracts. Phenolic acids were recovered from the whole meal flours of 65 durum cultivars and subsequently identified and quantified by HPLC-DAD analysis. Then, the anti-inflammatory activity of phenolic acids extracts was evaluated on LPS-stimulated HT-29 human colon cells by measuring the levels of interleukin 8 (IL-8) and transforming growth factor ?1 (TGF- ?1). A large variation for the content of phenolic acids was observed among genotypes and, on average, it accounted for 830 ?g/g dry weight. Whole meal flour extract significantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 µg/mL of phenolic acids. Conversely, the secretion of the anti-inflammatory mediator TGF-?1 was not modified by addition of phenolic acids to HT-29 cells. Results showed that durum cultivars have different contents of phenolic acids, suggesting that a number of elite varieties could be used for breeding purposes. Moreover, results provide further insight into the health-related benefits of durum wheat foods as depending on the anti-inflammatory activity of phenolic acids.

Artichoke (Cynara cardunculus (L.) subsp. scolymus Hayek) is an important component of theMediterranean diet, and a good source of health-promoting compounds, including polyphenols.The main phenolic compounds of artichoke are 5-O-caffeoylquinic acid (chlorogenicacid), and 3,5-O-, 1,5-O-dicaffeoylquinic acids and its beneficial effects are linked totheir antioxidant activity that can be affected by their stability under gastro-intestinal conditions,during digestion. This study aimed to assess the antioxidant activity induced byartichoke head extract, and its main individual polyphenols, on a stimulated HT-29 cell line,using a DCFH-DA probe. In addition, influences of in vitro digestion on artichoke head andits individual polyphenols towards antioxidant activity,were tested. Artichoke extract showedhigher antioxidant activity than the individual polyphenols, even after in vitro gastrointestinaldigestion. In addition, in vitro digestion did not modify the antioxidant activity ofartichoke polyphenols, except for 1,5-O-dicaffeoylquinic acid,which proved to be the least active.

A very large amount of phenol-polluted waters is formed from the productionof olive oil (olive mill waste water, OMWW) and their disposal represents a seriousproblem. The aim of this study was to investigate the use of peroxidase (POD) fromartichoke (Cynara cardunculus L. subsp. scolymus (L.) Hayek) leaves to removephenols present in OMWW. The POD catalyzes the polymerization andprecipitation of aqueous phenols. This application could be useful, because in theMediterranean region a large amount of OMWW as well as a lot of artichoke scrapsresulting from artichoke processing industry, are produced. So, it is possible toconsider this biomass (leaves and external bud bracts) as a useful and cheap sourceof peroxidase. The enzyme extraction was performed by homogenization andseparation of pigments using an aqueous two phase polymer system (polyethyleneglycol/(NH4)2SO4), that allowed the accumulation of POD in the salt phase. Theoptimal reaction conditions were evaluated. The phenolic concentration was assayedby Folin-Ciocalteu method. Preliminary results showed that the optimal conditionsfor enzyme activity were in a broad range of both pH (from 4 to 7) and temperature(from 5 to 50°C). In optimal enzymatic conditions, a reduction of more than 60% ofinitial phenolic concentration was observed.

Artichoke is a characteristic crop of the Mediterranean area, recognized for its nutritional value and therapeutic properties due to the presence of bioactive components such as polyphenols, inulin, vitamins and minerals. Artichoke is mainly consumed after home and/or industrial processing, and the undersized heads, not suitable for the market, can be used for the recovery of bioactive compounds, such as polyphenols, for cosmetic applications. In this paper, the potential skin anti-age effect of a polyphenolic artichoke extract on endothelial cells was investigated. The methodology used was addressed to evaluate the antioxidant and anti-inflammatory activities and the improvement of gene expression of some youth markers. The results showed that the artichoke extract was constituted by 87% of chlorogenic, 3,5-O-dicaffeoylquinic, and 1,5-O-dicaffeoylquinic acids. The extract induced important molecular markers responsible for the microcirculation and vasodilatation of endothelial cells, acted as a potential anti-inflammatory agent, protected the lymphatic vessels from oxidative damage by ROS formation, and enhanced the cellular cohesion by reinforcing the tight junction complex. In addition, the artichoke extract, through the modulation of molecular pathways, improved the expression of genes involved in anti-ageing mechanisms. Finally, clinical testing on human subjects highlighted the enhancement by 19.74% of roughness and 11.45% of elasticity from using an artichoke extract cosmetic formulation compared to placebo cream

The increased consumption of whole wheat grains and whole wheat products has been associated with reduced risk of developing chronic diseases, such as cardiovascular disease, type 2 diabetes and colon cancer. These beneficial effects have been ascribed to the presence in whole wheat kernels of bioactive compounds which may vary for total content and composition among different wheat species and wheat varieties. In this work we present the profile of hydrophilic and lipophilic bioactive compounds of whole wheat semolina from five durum wheat elite cultivars. Whole semolina samples were analyzed to evaluate the total content and composition of phenolic acids (hydrophilic extract) and the total content and composition of carotenoids, tocopherols and tocotrienols (lipophilic extract). The total phenolic acid content was variable among the cultivars and ranged from 488 ?g/g to 1490 ?g/g whole flour. Among the detected compounds, ferulic acid was the most abundant, followed by sinapic acid and p-coumaric acid. Total carotenoid content varied from 2.64 µg/g whole flour and 4.75 µg/g whole flour and were mostly represented by lutein and zeaxanthin, while ?- e ?-carotene were present in trace amounts. Three different homologues of tocotrienols were detected (?, ? and ?), varying in a range between 18.3 and 28.6 µg/g whole flour, while tocopherols were detected in trace amounts. Duilio and Svevo cultivars, exhibited the highest content of hydrophilic and lipophilic bioactive compounds, respectively, and were selected to test the anti-inflammatory activity of extracts on human intestinal HT-29 cells. Preliminary experiments were carried out in order to assess the highest not cytotoxic concentration of lipophilic and hydrophilic extracts by using MTT test. Both extracts will be used to assess the anti-inflammatory activity on HT-29 cells stimulated by LPS mitogen, by quantification of IL-8. This research shows that whole wheat semolina flours of these five cultivars varied significantly in their contents of bioactive compounds and differences in their anti-inflammatory potential might suggest the possibility that durum wheat varieties could be selected based on potential health benefits.

Selection of artichoke cultivars with specific physical, physiological and biochemical characteristics is required by processors and retailers in order to commercialize artichokes as fresh or fresh-cut products. In this work six artichoke cultivars were evaluated for their cold storage suitability byfollowing the variations in their main physical (browning index), physiological (respiration rate) and biochemical (antioxidant activity, total phenol content, polyphenol oxidase and peroxidase activity) parameters at 1 °C for 12 days. In addition, linear regressions among antioxidant activity, total phenols and enzymatic activities were assessed with the aim of selecting the most suitable cultivar for storage and processing. The low values for the physical, physiological and biochemical parameters measured in Romolo led us to indicate this cultivar as the best for storage. On the other hand, it may be preferable to designate cultivars, with higher values for the same parameters for fresh consumption, partly due to their high antioxidants content.

Introduction. A healthy and balanced diet prevents the occurrence of many age-related diseases. On the basis of this claim, new types of food, defined novel foods, such as potato selenium, carrot, onion, tomato iodine, have been developed.Objectives. Considering the biological activities induced by silicon (antioxidant activity, increased mineralization of bone, development of connective tissue), the aim of this study was to obtain different silicon biofortificated vegetables (Tatsoi, Mizuna, Portulaca, Bietola and Cicoria) used as mix for the IV gamma product (ready to use), in order to have an useful food for post-menopausal women. In addition, the assessment of bioaccessibility of crops, by in vitro gastro-digestion process, and antioxidant activity on human intestinal cell line, were carried out.Methodology. For the biofortified plants production, a soilless system able to monitoring the effective absorption of the silicon was used. The plants were digested using the protocol described by Ferruzzi et al., (2001) in order assess the bioaccessibility. The antioxidant activity of digesta samples was evaluated on Caco-2 cell line using DCFH-DA probe.Results and conclusions. No modifications on the production and/or quality of the plant were found. A species-related accumulation of silicon was recorded with an mean value of 130 mg/kg fresh weight respect to average value blank of 22 mg/Kg. In addition, the silicon was bioaccessible in a range from 36 to 75% in relation to vegetable species studied. The biofortification significantly (p<0.001) improved the antioxidant activity induced by crops.

Olive oil production generates large amounts of recalcitrant compounds, the olive oil mill wastewater (OMWW), which represent one of the most contaminating effluents among those produced by the agrofood industries. Nowadays, this view has changed to one that recognizes the waste as a lowcost starting material rich in bioactive compounds, particularly biophenols, that can be extracted and applied as natural antioxidants for the food and pharmaceutical industries. The data reported in this paper indicate that the OMWW extracts, besides low molecular weight antioxidant phenolics such as tyrosol and hydroxytyrosol, also contain phenolics with a molecular weight in the range of 600-5000 Da, which exhibit efficient scavenging activities against hydroxyl and peroxyl radicals. This group of phenolics includes, besides verbascoside, isoverbascoside, and an oxidized form of verbascoside, a number of higher molecular weight phenolics arising from oxidative polymerization of hydroxytyrosol and caffeic acid. Overall, these higher molecular weight phenolics prove to be, in some in vitro tests, more efficient scavengers of hydrophilic hydroxyl radicals than hydroxytyrosol, which could be used for industrial applications as natural nontoxic antioxidants.

In this study, the naturally debittered table olives cv Bella di Cerignola were studied in order to (i) characterizetheir phenolic composition; (ii) evaluate the polyphenols bioaccessibility; (iii) assess their absorption and transport, acrossCaco2/TC7. LC-MS/MS analysis has confirmed the presence of hydroxytyrosol acetate, caffeoyl-6?-secologanoside, andcomselogoside. In vitro bioaccessibility ranged from 7% of luteolin to 100% of tyrosol, highlighting the flavonoids sensitivity tothe digestive conditions. The Caco2/TC7 polyphenols accumulation was rapid (60 min) with an efficiency of 0.89%; the overallbioavailability was 1.86% (120 min), with hydroxytyrosol and tyrosol the highest bioavailables, followed by verbascoside andluteolin. In the cells and basolateral side, caffeic and coumaric acids metabolites, probably derived from esterase activities, weredetected. In conclusion, the naturally debittered table olives cv Bella di Cerignola can be considered as a source of bioaccessible,absorbable, and bioavailable polyphenols that, for their potential health promoting effect, permit inclusion of table olives as afunctional food suitable for a balanced diet.

The main objective of the present study was the characterization of phenolic compounds from "Bella di Cerignola" table olive cultivar, one of the most important variety in Apulia (Southern Italy), that received the "Protected Denomination of Origin" (DOP) in 2000 by EU. In addition, the assessment of bioaccessibility of these polyphenols was investigated by using in vitro gastro- intestinal digestion model1. Methodologies: After extraction, the phenolic concentration was determined by HPLC according to Lattanzio 1982(2). Moreover, the olives were subjected to successive gastric and pancreatic digestion, following the method of Versantvoort et al. (2005)1 .The polyphenols were identified by HPLC-DAD and LC-HRMS analysis in order to assess their bioaccessibility. Results and conclusion: The main polyphenols identified were: hydroxytyrosol, tyrosol, caffeic acid, verbascoside, isoverbascoside, caffeoyl-6-secologanoside, comselogoside, luteolin. Interesting is the presence of comselogoside and caffeoyl-6-secologanoside, already identified in OMWW3 and olive fruits4 . After in vitro digestion process, all total phenols identified were bioaccessible, apart luteolin that was absent in the digestive fraction. In particular, the bio-accessibilities were: 84.2% for hydroxytyrosol, 100% for tyrosol and caffeic acid, 47 % for verbascoside, 75% for isoverbascoside, 77% for caffeoyl-6-secologanoside, 81% for comselogoside. Regarding tyrosol, caffeic acid and isoverbascoside, their amount, in the chyme fraction, was higher than no digested olives, probably for hydrolysis and isomerizations phenomena occurred in the gastro-intestinal conditions. In conclusion, the data obtained provide preliminary insight on the potential for bioavailability of the polyphenols present in a complex matrix such as "Bella di Cerignola" cultivar.

Calcium is an essential nutrient for human health, because it is a structural component and takes part in a variety of biological processes. The aim of this study was to increase Ca content of baby leaf vegetables (BLV: basil, mizuna, tatsoi and endive), as fresh-cut products. For the production of biofortified BLV, a floating system with two level of Ca (100 and 200 mg L-1) in the nutrient solution was used. In addition, the assessment of bioaccessibility of Ca, by in vitro digestion process, was performed. In all vegetables, the Ca biofortification (200 mg L-1) caused a significant Ca enrichment (9.5% on average) without affecting vegetables growth, oxalate contents and marketable quality. Calcium bioaccessibility ranged from 25% (basil) to 40% (endive) but the biofortified vegetables showed more bioaccessible Ca. These results underline the possibility to obtain Ca biofortified BLV by using agronomic approaches.

Pasta represents one of the most well-known products all over the world. It is a traditional food of Mediterrean diet and it is generally manufactured with durum wheat, that confers the excellent rheologicalproperties of dough, the superior color, the appearance and the cooking quality compared to other flours. The World Health Organization (WHO) and Food and Drug Administration (FDA) consider spaghetti pastaa good vehicle for the addition of nutrients and consumers are increasingly interested in foods containing health promoting ingredients. In this contest, the objective of the present study was to characterize thebioactive components, such as carotenoids, triterpenic acids and polyphenols, present in durum wheat semolina spaghetti enriched with an olive oil patè coming from olive oil processing chain, and dried inorder to obtain a flour (OPF) suitable for the pasta process. In particular, OPF was added to the pasta dough at 10% and 15% (w/w). Finally, spaghetti with 10% OPF, considered acceptable to the sensory panel test, were characterized for their bioactive components. Further, their bioaccessibility, after simulated gastro-intestinal digestion, was also assessed. Results showed that 10% OPF addition enriched the spaghetti in ?-tocopherol, ?- and ?-carotene, maslinic and eonolic acids. The ratio of polyunsaturated (PUFA) to saturated fatty acids (SFA) resulted higher (1.16) than spaghetti used as control (0.69). The total polyphenols content (free, conjugated and bound) in dry spaghetti samples increased of about 3 times respect to the control. In particular, the amount of total free phenolics in the enriched sample, was almost 50 times higher respect to the control spaghetti, with tyrosol and oleuropein as the most abundant. In addition, it is interesting to underline that the enriched spaghetti showed an high amount of flavonoids, such as apigenin, luteolin and quercetin already present in OPF. The phenolic acids were mainly present in the conjugated and bound fractions with the highest amount in the spaghetti with 10% OPF. Finally, the bioactive components showed a good level of biaccessibility, although during the cooking process, the 50% was naturally lost.

The prickly pear cactus, Opuntia ficus-indica (OFI) is a tropical or subtropical plant originally grown in South America; its cladodes represent a by-product that generally is used for animal feed. Moreover, they are rich in pectin, mucilage and minerals, vitamins and antioxidants and the use as food medicine and cosmetic ingredients, could be proposed. In the present study, the characterization of bioactive compounds (polyphenols, sugars and minerals) in dehydrated OFI cladodes were performed. Further, antioxidant activity and polyphenols/minerals stability after simulated gastro-intestinal digestion, were also evaluated.

Previously we presented the purification, biochemical characterization, and cloning of a cationic peroxidase isoenzyme (CysPrx) from artichoke (Cynara cardunculus subsp scolymus (L.) Hegi) leaves. The protein was shown to have some interesting properties, suggesting that CysPrx could be a considered as a potential candidate for industrial application. In addition, from the CysPrx sequence, two full-lengh cDNAs: CysPrx1 and CysPrx2, differing for three amino acids, were isolated. A three-dimensional model was predicted from CysPrx1 by homology modeling, using two different computational tools. Herein we discuss the roles of particular amino acid residues and structural motifs or regions of both deduced sequences with the aim to find new understandings between the new plant peroxidase isoenzymes and their physiological substrates. Additionally, the obtained information may lead to new methods for improving the stability of the enzyme in several processes of biotechnological interest for peroxidase applications.

This investigation shows the influence of the fermentation process started with the probiotic Lactobacillusparacasei LMG P-22043 (PF) on the fate of health promoting polyphenols in artichoke in comparison with notstarted fermented artichokes (NSF). The fermentation process did not influence significantly the polyphenolcompositions. However, low variations mainly related to the isomerization process of dicaffeoylquinic acids,probably due to the different pH values of the two samples, were observed. Further, the total polyphenolbioaccessibility of PF artichoke was lower (34%) than of NSF artichoke (64%) in small intestine. Moreover, bothpolyphenol isomerization and bioaccessibility influenced the antioxidant activity that was significantly lower forPF artichoke than for NSF ones. These effects were probably related to the bioprotective process exerted by theprobiotic strain on the food matrix, with the consequent limited release of polyphenols in the digestion liquids,making them available for further metabolism and absorption in the colon.

Table olives are a typical food of the Mediterranean diet and an important source of phenolic compoundswith high biological potential for human health. Their concentrations (?1-2% of FW) confer to table olivesantioxidant, anti-inflammatory, and antitumoral properties. The polyphenols content and composition in table olives can be affected by several factors, such as cultivars, climate, fruits ripeness, and, mainly, theprocessing methods. Among the de-bittering processes, the Greek method represents a spontaneous fermentation procedure that is driven by a mixed population of microorganisms, mainly consisting of yeastsand lactic acid bacteria (LAB). In this work, the effects of fermentation by autochthonous yeast and LAB starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto(TDB) and Cellina di Nardò (CEL) were studied in comparison with the commercial products. The samples were characterized by HPLC-DAD for their polyphenols composition; 18 compounds were identified andthe cultivar related effect was highlighted. The main identified phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucosideand quercetin in CEL. Further, the fermentation using selected LAB and yeasts influenced differently the composition and amount of polyphenols of the three cultivars, in particular the BDC amount was doubledcompared with the commercial sample. Instead, for TDB and CEL, the treatment did not influence the polyphenols composition. It is noteworthy that the use autochthonous microbial starter to drive table olivesfermentation process allows to maintain stable or increases polyphenols concentration in fermented table olives, significantly reducing the time necessary for de-bittering and improving organoleptic and sensorycharacteristics of the final product. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW).Moreover, the polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted.Finally, fermented table olives are excellent source of health promoting compounds. Indeed, hydroxytyrosol and tyrosol are almost 8 times more than in olive oil for which a nutritional EU claim (No433/2012 of 23 May 2012) that attributes the protective effect from oxidative stress by polyphenols on blood lipids, has been established.

The effects of fermentation by autochthonous microbial starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto (TDB) and Cellina di Nardò (CEL) were studied, highlighting also the cultivars influence. In BDC with starter, polyphenols amount doubled compared with commercial sample, while in TDB and CEL, phenolics remain almost unchanged. The main phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucoside and quercetin in CEL. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW). The polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted. Finally, fermented table olives are excellent source of health promoting compounds, since hydroxytyrosol and tyrosol are almost 8 times more than in olive oil.

Oil industry produces large volume of wastes, both solid and liquid, which represent a disposal and potentially environmental pollution problem. Nevertheless, they are also promising sources of compounds that can be recovered and used as valuable substances. The aim of this work is to exploit solid olive by-products, in particular dry olive paste flour (DOPF) coming from Coratina cultivar, to enrich fish burger and enhance its quality characteristics. The addition of olive by-products leads to an increase of the phenolic content and antioxidant activity of the enriched food product; however, this also involves a deterioration of its sensory quality. To balance the quality and sensory characteristics of the investigated food, the study has been organized in three subsequent phase: first, the quality value of DOPF in terms of phenolic compounds content and antioxidant activity, has been assessed; afterward, DOPF has been added to fish burger to enhance its quality characteristics and, finally, the formulation of DOPF loaded fish burger has been optimize in order to improve its sensory quality. Results recorded in this study suggested that the enriched burger with 10% of DOPF showed a considerably increase in aliquot of polyphenols and antioxidant activity, but it negatively influenced its sensory quality. Pre-treating DOPF by hydration/extraction with milk, significantly improved the burger sensory quality by reducing the concentration of bitter components such as polyphenols.

Whole wheat foods are significant source of compounds exhibiting health-promoting properties. One of the most abundant class of phytochemicals in the wheat grain is represented by phenolic acids that are typically localized in the bran and germ portions. The objective of this study was to estimate the extent of genetic variation for phenolic acids in durum wheat (T. turgidum L. ssp. durum). In addition, this study aimed at evaluating the anti-inflammatory activity of phenolic acids contained in whole-meal flour extracts. Phenolic acids were recovered from the whole meal flours of 65 durum cultivars and subsequently identified and quantified by HPLC-DAD analysis. Then, the anti-inflammatory activity of phenolic acids extracts was evaluated on LPS-stimulated HT-29 human colon cells by measuring the levels of interleukin 8 (IL-8) and transforming growth factor ?1 (TGF- ?1). A large variation for the content of phenolic acids was observed among genotypes and, on average, it accounted for 830 ?g/g dry weight. Whole meal flour extract significantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 µg/mL of phenolic acids. Conversely, the secretion of the anti-inflammatory mediator TGF-?1 was not modified by addition of phenolic acids to HT-29 cells. Results showed that durum cultivars have different contents of phenolic acids, suggesting that a number of elite varieties could be used for breeding purposes. Moreover, results provide further insight into the health-related benefits of durum wheat foods as depending on the anti-inflammatory activity of phenolic acids.

Food plants biofortification for micronutrients is a tool for the nutritional value improvement of food. Soilless cultivation systems, with the optimal control of plant nutrition, represent a potential effective technique to increase the beneficial element content in plant tissues. Silicon (Si), which proper intake is recently recommended for its beneficial effects on bone health, presents good absorption in intestinal tract from green bean, a high-value vegetable crop. In this study we aimed to obtain Si biofortified green bean pods by using a Si-enriched nutrient solution in soilless system conditions, and to assess the influence of boiling and steaming cooking methods on Si content, color parameters and Si bioaccessibility (by using an in vitro digestion process) of pods. The Si concentration of pods was almost tripled as a result of the biofortification process, while the overall crop performance was not negatively influenced. The Si content of biofortified pods was higher than unbiofortified also after cooking, despite the cooking method used. Silicon bioaccessibility in cooked pods was more than tripled as a result of biofortification, while the process did not affect the visual quality of the product. Our results demonstrated that soilless cultivation can be successfully used for green bean Si biofortification.

Olive oil mill waste waters (OMWWs), a polluting byproduct of the olive oil production process, contain large amount of organics and inorganics compounds (polyphenols, salts, etc) that, if inappropriately discharged, can cause environmental pollution. Polyphenols have several biological activity, but at high concentration they are toxic and their removal from OMWW is an important matter. In this work the detoxification of OMWW, performed by membrane filtration systems, was investigated with the dual aims, of reducing the organic load of OMWW andrecovering polyphenols. The OMWWs were processed by ceramic and polymeric membranes with different porosity, to obtain three fractions: microfiltrate (MF, 100,000 Da), ultrafiltrate (UF, 5000 Da) and nanofiltrate (NF, 200 Da). The fractions were characterized by HPLC analysis, to quantify the main polyphenols present: Hydroxytyrosol, Tyrosol, Caffeic and Coumaric Acids, Verbascoside, Isoverbascoside, Secologanoside, and Comselogoside. MF and UF fractions exhibited antimicrobial activity against Escherichia coli, as well as against Pseudomonas aeruguinosa and Staphylococcus aureus pathogen strains. No activity was given by NF fraction. Thus, MF and UF might be considered as natural antibiotics, taking into account mainly their effect in inhibiting the growth of E. coli and S. aureus. Phytotoxic and genotoxic effects were analysed in Vicia faba L. plants germinated in MF, UF and NF fractions. The major phytotoxic effects were shown inseeds germinated in MF fraction. Genotoxicity analysis relied on mitotic activity (MI), aberration index (AI) and micronucleus frequency (MCN): MI was generally reduced, cytogenetic aberrations were found in all the treatments and MCN were mostly present in MF fraction. Results generally indicated the efficiency in polyphenols recovery and natural antibiotic activity of MF and UF fractions. Genotoxicity and phytotoxicity effects of filtrated OMWWs suggested the need of further treatments/ dilutions before their discharge in the environment.

Olive quick decline syndrome (OQDS) caused by X. fastidiosa is currently causing severe damages to the production and reducing the life span of the plants in the Salento peninsula of Apulia (Italy). No effective means of control of X. fastidiosa is currently available. The objective of this study was to evaluate in vitro antimicrobial activities against X. fastidiosa (strain Salento-1) of different classes of compounds having diverse origins, i.e. traditional antibiotics, plant-derived natural products, and microbial metabolites. A preliminary bioassay, performed by the agar disc diffusion method, revealed that 17 of the 31 antibiotics tested did not affect bacterial growth at a dose of 5 ?g. Olive mill wastewaters (OMWs), which are known to possess a broad range of antimicrobial activity, are able to inhibit X. fastidiosa in vitro. Most interestingly when we analysed different OMWs derived micro, ultra and nano-filtered fractions as well as some of the single phenolic compounds that they contain, we found that the OMWs micro-filtered fraction is the most effective against the bacterium but only few phenolics are active in their pure form. Also some fungal extracts and bacteria toxins showed noteworthy inhibitory effect to strain Salento-1 growth. The possible use of some of these products for curative/preventive treating OQDS-affected or at-risk olive plants will be discussed.

Olive quick decline syndrome (OQDS) is causing severe damages to the olive trees in Salento (Apulia, Italy) and poses a severe threat for the agriculture of Mediterranean countries. DNA-Based Typing Methods have pointed out that OQDS is caused by a single outbreak strain of Xylella fastidiosa subsp. pauca referred to as CoDiRO or ST53. Since no effective control measures are currently available, the objective of this study was to evaluate in vitro antimicrobial activities of different classes of compounds against Salento-1 isolated by a OQDS affected plant and classified as ST53. A bioassay based on agar disc diffusion method, revealed that 17 out of the 32 tested antibiotics did not affect bacterial growth at a dose of 5 g disk-1. When we assayed micro-, ultra- and nano-filtered fractions of olive mill wastewaters (OMWs) we found that the micro-filtered fraction resulted the most effective against the bacterium. Moreover, some phenolics (4-methylcathecol, cathecol, veratric acid, caffeic acid, oleuropein) were active in their pure form. Noteworthy, also some fungal extracts and fungal toxins showed inhibitory effects on bacterial growth. Some of these compounds can be further explored as potential candidate in future applications for curative/preventive treating OQDS-affected or at-risk olive plants.

White table grape cv. Italia is a typical component of the Mediterranean diet and a source of phenolic compounds, particularly abundant in the skin portion. The aim of this study was to characterize the phenolic profile of the table grape skin and to assess its stability after the in vitro digestion process. The main phenolic compounds identified by the HPLC-DAD analysis were: procyanidin B1, caftaric acid, catechin, coutaric acid, quercetin 3-glucuronide and quercetin 3-glucoside. All compounds showed a good stability after in vitro digestion (from 43 to 80%). Moreover, the influence of grape skin polyphenols on the modulation of ROS and GSH levels was evaluated in basal and in stressed conditions on human intestinal cells (HT-29). In basal conditions, a higher polyphenol concentrations exerted pro-oxidant effect corresponding to high ROS level and low GSH content. This effect was probably due to the polyphenolic oxidation in cell culture condition with consequent production of hydrogen peroxide. Otherwise, in stressed conditions, grape skin polyphenols exerted antioxidant effects up to 1.3×10-6 ?g/g and restored the stress-related GSH reduction. The in vitro digestion process attenuated the biological effect of grape skin polyphenols on intestinal cell line (HT-29). In conclusion, grape skin polyphenols showed different behavior in relation to their concentrations and to the intracellular ROS levels.

In this study, the effects of pure olive phenolic compounds and olive mill wastewater (OMWW) (after membrane filtration treatments) on Aspergillus flavus growth and aflatoxin B1 (AFB1) production, were investigated. Five OMWWs coming from Greek (Lianolia, Koroneiki and Asprolia) and Italian (Cellina di Nardò and Coratina) olive oil cultivars, opportunely filtered using a membrane system, were added at two concentrations (5 and 15%) to growth medium, in order to evaluate their effect on A. flavus growth and AFB1 production. The OMWW fractions treatment, after 6 days of incubation, did not inhibit the fungal growth rate, but at 15% concentration significantly reduced the AFB1 production (ranging from 88 to 100%). A similar approach was used for caffeic acid, hydroxytyrosol, tyrosol and verbascoside, the major pure phenolic compounds identified in OMWW fractions. They were evaluated at increasing doses (10, 50 and 100 ?g/ml) following both AFB1 production and fungal growth. At the highest concentration (100 ?g/ml) all pure compounds showed a reduction of about 99% of AFB1 production without any influence on fungal growth. This is the first time in which OMWWs and their main phenolics were used in the treatments against AFB1 production. The results obtained could provide possible new strategies for preventing AFB1 food contamination using olive polyphenols and OMWW fractions with anti-aflatoxigenic effect, and permitting to harness in a sustainable way an olive oil by-product.

Food industries are increasingly oriented towardnew foods to improve nutritional status and/or to combat nutritionaldeficiency diseases. In this context, siliconbiofortification could be an innovative tool for obtainingnew foods with possible positive effects on bone mineralization.In this paper, an alternative and quick in vitro approachwas applied in order to evaluate the potential healthpromotingeffects of five silicon-biofortified leafy vegetables(tatsoi, mizuna, purslane, Swiss chard and chicory) on bonemineralization compared with a commercial silicon supplement.The silicon bioaccessibility and bioavailability of thefive leafy vegetables (biofortified or not) and of the supplementwere assessed by applying a protocol consisting ofin vitro gastrointestinal digestion coupled with a Caco-2 cellmodel. Silicon bioaccessibility ranged from 0.89 to 8.18 mg/Land bioavailability ranged from 111 to 206 ?g/L of Si for bothvegetables and supplement. Furthermore, the bioavailablefractions were tested on a human osteoblast cell model following the expression of type 1 collagen and alkaline phosphatase.The results obtained highlighted that the bioavailablefraction of biofortified purslane and Swiss chard improved theexpression of both osteoblast markers compared with the supplementand other vegetables. These results underline the potentiallybeneficial effect of biofortified leafy vegetables andalso indicate the usefulness of in vitro approaches for selectingthe best vegetable with positive bone effects for further in vivoresearch.

Artichoke is a characteristic crop of the Mediterranean area recognized for its nutritional value and therapeutic properties due to the presence of bioactive components such as polyphenols, inulin, vitamins and minerals. The objective of this paper is related to the evaluation of the potential role of an artichoke ethanolic extract (AEE), recovered from undersized heads, in promoting the endothelial cell functions and stimulating the gene expression of some youth associated markers, with the main goal of characterizing it as cosmetic anti-age ingredient.

In this study, the quali-quantitative composition of hydrophilic (phenolic acids)and lipophilic (isoprenoids) extracts from whole-meal flour of five elite Italian durum wheatcultivars was determined. Significant differences in the content of bioactive compoundswere observed among the wheat extracts, in particular concerning the content of boundphenolic acids, lutein and ?-tocotrienols. The cultivars Duilio and Svevo showed the highestamount of phenolic acids and isoprenoids, respectively. Extracts were evaluated for theiranti-inflammatory activity on HT-29 human colon cells by measuring the levels ofinterleukin 8 (IL-8) and transforming growth factor ?1 (TGF-?1). Durum wheat extractssignificantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 ?g/mLof phenolic acids and at 0.2 ?g/mL of isoprenoids. Conversely, the secretion of theanti-inflammatory mediator TGF-?1 was not modified by neither hydrophilic nor lipophilicextracts. These results provide further insight into the potential of durum wheat on humanhealth suggesting the significance of varieties with elevated contents of bioactive components.

The aim of this work was to recover and identify the phenolic compounds from olive mill waste water (OMWW) samples belonging to two Italian (Cellina and Coratina) and three Greek (Asprolia, Lianolia and Koroneiki) olive cultivars. The OMWWs were processed using membrane technologiesto obtain three fractions: microfiltrate (MF), ultrafiltrate (UF) and nanofiltrate (NF). These steps allowed to purify the OMWWs in order to achieve fractions with different profile and concentrations of polyphenols. In particular, the amount of polyphenols ranged from 2456 ?g/mL to 5284 ?g/mL in MF; from 1404 ?g/mL to 3065 ?g/mL in UF and from 373 ?g/mL to 1583 ?g/mL in NF. Among the cultivars analyzed Coratina followed by Lianolia showed the highest amount of verbascoside (VB) (308?g /mL in Coratina versus 145?g/mL in Lianolia, respectively) in UF fractions. Furthermore, UF fractions that showed adequate purification degree and polyphenol enrichments, were used for the identification of the phenolic compounds by liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MSn) analysis. Twenty three compounds, belonging to the following classes of constituents: secoiridoids and their derivatives, phenyl alcohols phenolic acid and derivatives, and flavonoids, were identified in almost all the UF fractions of the different cultivars. Finally, differences were observed among the cultivars regarding the presence of elenolic acid derivatives, hydroxytyrosol glucoside, and ?-hydroxyverbascoside diasteroisomers. The results obtained showed that OMWW can be considered as raw material for the isolation of valuable bioactive compounds able to be used in food, cosmetic and pharmaceutical industry.

Artichoke is a rich source of health promoting compounds such as polyphenols, important for their pharmaceutical and nutritional properties. In this study, the potential for bioavailability of the artichoke polyphenols was estimated by using both in vitro digestion and Caco-2 human intestinal cell models. In vitro digestive recoveries (bio-accessibility) were found to be 55.8% for total artichoke phenolics and in particular, 70.0% for chlorogenic acid, 41.3% for 3,5-O-dicaffeoylquinic acid, and 50.3% for 1,5-O-dicaffeoylquinic acid, highlighting potential sensitivity of these compounds to gastric and small intestinal digestive conditions. Uptake of artichoke polyphenols was rapid with peak accumulation occurring after 30 min with an efficiency of 0.16%, according to the poor uptake of dietary polyphenols. Some compounds, such as coumaric acid, caffeic acid and caffeic acid derivatives, were also detected in the basolateral side assuming an extra and intracellular esterases activities on chlorogenic acid. Only apigenin-7-O-glucoside was absorbed and transported through the Caco-2 monolayer demonstrating its bioavailability in the extent of 1.15% at 60 min. In addition, permeability coefficient (Papp=2.29 x 10-5 cm/sec), involving apical to basolateral transport of apigenin 7-O-glucoside, was calculated to facilitate estimation of absorption and transport through Caco-2 monolayer. Finally, the mono and dicaffeoylquinic acids present in artichoke heads, exert an antioxidant activity on human low density lipoprotein system correlated to their chemical structure. In conclusion, the utilized in vitro models, although not fully responding to the morphological and physiological features of human in vivo conditions, could be a useful tool for investigating mechanistic effects of polyphenols released from food matrix.

Artichoke (Cynara cardunculus L. subsp. scolymus Hayek) is an important component of the Mediterranean diet and a good source of health-promoting compounds well-known antioxidant and anti-inflammatory molecules, such as polyphenols(1). Recent evidences indicated that probiotic strains play an important role in modulating activities in the gut-associated lymphoid tissue. In this study the beneficial effects of artichoke was modulated by adding probiotic cells during in vitro digestion process. The artichoke intestinal digesta (containing a range from 0.033 to 67.13 ppm of total polyphenols), in presence or absence of the probiotic Lactobacillus paracasei strain LMG P-22043, was evaluated for antioxidant activity on human intestinal cell line (HT-29) using DCF-DA probe. Although the recovered bioaccessibility of total polyphenols did not change in the presence of L. paracasei (71± 17.6 % with respect to 66 ± 14.4%), the antioxidant effect was significantly (p<0.05) improved by the presence of probiotic cells (IC50 value= 1.0 ± 0.7 ppm of total polyphenols) respect to the digested artichoke (IC50= 2.7 ± 0.7ppm). These preliminary results indicate a possible interactive action between the strain and the active components of artichoke toward antioxidant activity in intestinal cells and suggest to further investigate similar interaction toward inflammatory mediators, considering the anti-inflammatory properties evidenced for other strains belonging to L. pacarasei (2).

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

A cationic soluble peroxidase isoenzyme (CysPrx) has been purified and characterized from artichoke (Cynara cardunculus subsp. scolymus (L.) Hegi) leaves by combination of aqueous two phase extraction, ion exchange chromatography, and gel filtration. The purification fold was 149 and the activity recovery 5.5%. CysPrx was stable from 5 to 45 °C with a pH optimum around 5.5; the pI was 8.3 and the MW of 37.7 ± 1.5 kDa. MALDI-TOF MS analysis provided partial peptide sequences and resolved CysPrx isoenzyme into two putative isoforms. The presence of these isoforms was confirmed by the isolation of full-length cDNA encoding CysPrx that generate two slightly different sequences coding for two putative CysPrx: CysPrx1 and CysPrx2. The obtained MS peptides showed a 35% coverage with 100% identity with the two CysPrx deduced protein sequences. A molecular modeling analysis was carried out to predict in silico the protein structure and compare it with other plant Prx structures. Considering that CysPrx is quite stable, the study carried out in this paper will offer new insights for the production of the recombinant protein for utilization of CysPrx as an alternative Prx for food technology, biomedical analysis and bioremediation.

An integrated device for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is described in this study. The SB is composed of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor, all located in the basolateral compartment (BC) of a cell culture plate. Caco-2TC7 cells, differentiated on culture inserts, separated the apical compartment that simulates the intestinal lumen, from the BC which represented the bloodstream. The system recorded currents relative to glucose (1mM) absorption, obtaining bioavailability values (5.1%) comparable to HPLC analysis (4.8%). Phloridzin and phloretin, specific phenolic inhibitors of SGLT1 and GLUT2 glucose transporters, reduced the glucose transport of almost 10 times. They were minimally absorbed in the BC with a bioavailability of 0.13% and 0.49% respectively. The hypoglycemic potential of blueberry and pomegranate juices was also studied. In particular, the amount of glucose absorbed through the Caco-2TC7 monolayer was 8? for pomegranate and 1.7? for blueberry, demonstrating the potential hypoglycemic effect of the juices. Polyphenols absorption was also monitored by the SB and an increase was recorded during the first 50min in presence of both blueberry and pomegranate juices, then a constant decrease occurred. The proposed device has been developed as innovative tool for the dynamic monitoring of natural compounds effects on glucose absorption, in order to manage postprandial hyperglycemia.

Olive oil processing industries generate substantial quantities of phenolic-rich by-products, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterisation of antioxidant compounds from olive mill wastewater (OMWW), a polluting by-product of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to a low-pressure gel filtration chromatography on a Sephadex LH-20. The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, ?-hydroxyverbascoside, ?-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 human colon carcinoma cell lines, an established model system for studying drug transport properties, was also assayed.

Fresh-cut and whole iceberg lettuce were stored in refrigerated condition and the main sensory (overall appearance: OA, browning index: BI), physical (colour variation: ?E*), chemical (respiration activity: RA, ammonia content: A) and biochemical (total chlorophyll: TC, antioxidant activity: AA, total phenols: TP, o-quinones: o-q, polyphenol oxidase: PPO, phenylalanine ammonia-lyase: PAL and peroxidase: POD) traits were followed during the trial. Significant relationships among these parameters were assessed by principal component analysis (PCA) in order to find the main traits related to OA, in fresh-cut lettuce. Results from PCA showed that OA was inversely correlated with BI (r=-1.00), DE* (r= -0.98), A (r= -0.94), AA (r= -0.86), TP (r= -0.82), o-q (r= -0.89), and PAL (r= -0.80). Moreover, OA was positively related with RA (r= 1.00) and PPO activity (r= 0.96). Ammonia resulted significantly correlated with the main sensory, physical, chemical and biochemical parameters. Thus, this parameter, with DE* could be used as indicator of the product quality and acceptability in control quality procedures. In conclusion, rating scale used to estimate OA, resulted a valid method to evaluate the iceberg lettuce quality, since it showed significant relationships with the main analytical parameters.

The mineral silicon (Si) is an essential element for humans and a general component of the diet found mainly in plant-based foods. The aim of this study was to obtain Si biofortificated leafy vegetables (tatsoi, mizuna, purslane, basil, Swiss chard, and chicory) to use for the fresh-cut products (ready to use). For the production of biofortified plants, a floating system with 0, 50 and 100 mg L-1 of Si in nutrient solution, was used. In addition, the assessment of bioaccessibility of biofortified plants, by in vitro gastro-digestion process, was performed.RESULTSThe added silicon in nutrient solution did not influence yield and colour of vegetables but a species-related accumulation of Si (expressed as SiO2) was found: from 18 to 69 mg kg-1 fresh weight (FW) in tatsoi, from 19 to 106 mg kg-1 FW in mizuna, from 15 to 93 mg kg-1 FW in purslane, from 41 to 294 mg kg-1 FW in basil, from 17 to 76 mg kg-1 FW in Swiss chard, and from 23 to 76 mg kg-1 FW in chicory. The Si became bioaccessible in all species considered in a range from 23% (basil) to 64% (chicory).CONCLUSIONThe application of Si to the nutrient solution in the range of 50-100 mg L-1 allows biofortification of leafy vegetables. In addition, the biofortified vegetables showed, on average, more bioaccessible Si, with respect to unbiofortified vegetables. © 2015 Society of Chemical Industry

Fruits and vegetables contain a number of components (polyphenols, carotenoids, vitamins, folate, minerals such as magnesium, calcium, silicon and other compounds) that could affect the bone health. Considering the biological activities induced by silicon (antioxidant activity, increased mineralization of bone, development of connective tissue), this study aims to compare the biological activity induced by the silicon biofortificated and unbiofortificated vegetables (tatsoi, mizuna, purslane, Swiss chard and chicory) respect to the commercial silicon supplement. In addition, the assessment of silicon bioaccessibility, bioavailability and biological activity in human intestinal cell line (Caco-2) and human osteoblasts (hOBs) were performed. After in vitro gastrointestinal digestion of plant edible leaves and the silicon supplementation, the silicon bioaccessibility ranged from 7.01 to 13.22 mg/L. On Caco-2 cell line, the antioxidant activity of the bioaccessible fraction was assessed as reduction of induced ROS levels and ranged from 25 to 44% respect to stimulated control. The silicon biofortification significantly (p<0.001) improved the antioxidant activity induced by the vegetables. The bioavailability of digested samples, evaluated on Caco-2 differentiated cell line, ranged from 237 to 440 µg/L with percentages ranged from 27% to 61% in relation to the considered vegetable species. The expression of collagen type 1 and alkaline phosphatase induced by the bioavailable fraction after 12 and 36 h of treatment was evaluated in hOBs by real-time PCR. The bioavailable fraction of tatsoi, purslane and Swiss chard (biofortificated leafy vegetables), improved (after 36 hours of incubation) the expression of collagen type 1 and alkaline phosphatase with respect to the silicon supplement and unbiofortifated vegetables in hOBs.

Verbascoside (VB) is a phenolic antioxidant present in different plants, but, as reported for other polyphenols, could be unstable at different pH conditions. The aim of this work was to assess how chemical modification of VB, occurring during incubation at pH values similar to gastric (pH 3) and intestinal (pH 7) conditions, may impact antioxidant activity in two human intestinal cells lines. Quali-quantitative approaches were performed in order to evaluate the specific interactions between VB and its derivatives. HPLC analysis was performed to assess possible VB transformation into derivative products. The antioxidant activity of a mixture of VB, Isoverbascoside (IsoVB) and oxidized products (obtained after incubation at pH 7), or purified VB and IsoVB, was assessed in two human intestinal cell lines (HT-29 and Caco-2) using a DCFH-DA probe. VB was stable at pH 3 with a recovery of ~100% after 24 hrs. In contrast, VB was unstable at pH 7, with a loss of 62.4%, transforming into IsoVB and other oxidative products. Cellular antioxidant assays found that the mixture of oxidized VB products resulted less active (EC50 ranging from 2.7 to 3.4 ?M) than VB (EC50 ranging from 0.24 to 0.29 ?M), IsoVB (EC50 ranging from 0.85 to 1.4 ?M), and VB+IsoVB (EC50 ranging from 0.12 to 0.21 ?M). Cellular uptake of IsoVB was found to differ between cell lines, with higher uptake by Caco-2 compared to HT-29. Both graphical and mathematical methods identified different interactions between VB and IsoVB on the two cell lines. In conclusion, theantioxidant activity of VB can be modified by the conditions present in intestinal environment.

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress.

The use of peroxidase (POD) from artichoke (Cynara cardunculus L. subsp. scolymus (L.) Hayek) leaves to remove phenols present in olive mill waste water (OMWW) was studied. The enzyme extraction was performed using an aqueous two phase polymer system. The phenol concentration was assayed using the Folin-Ciocalteu method. The optimal reaction conditions were evaluated. The highest phenol removal was obtained using 4 mM H(2)O(2) and an incubation of 24 hours. The enzyme showed a good activity in a broad range of pH (from 4 to 7) and temperature (from 5 to 50 degrees C). In optimal enzymatic conditions, a reduction of more than 60% of initial phenol concentration was observed. High pressure liquid chromatography (HPLC) analysis of OMWW untreated and treated with POD extracts revealed that enzyme treatment brought about changes in the original OMWW polyphenolic profile. In particular the removal of verbascoside, hydroxytyrosol, and two caffeic acid derivatives was noticed. The results showed that the soluble POD from artichoke leaves could have a promising perspective in OMWW phenol removal, due to its broad pH and temperature working range and its cheap source.

The gastrointestinal tract is the main target ofexposure to mycotoxin fumonisin B1 (FB1), common naturalcontaminant in food. Previous studies reported thatproliferating cells are more sensitive than confluent cells tothe toxic effect of FB1. This study aims to investigate, bydose- and time-dependent experiments on human colonproliferating intestinal cell line (HT-29), the modificationsinduced by FB1 at concentrations ranging from 0.25 to69 lM. The choice of highest FB1 concentration consideredthe low toxicity previously reported on intestinal celllines, whereas the lowest one corresponded to the lowerFBs levels permitted by European Commission Regulation.Different functional parameters were tested such as cellproliferation, oxidative status, immunomodulatory effectand changes in membrane microviscosity. In addition FB1-FITC localization in this cell line was assessed by usingconfocal laser scanning microscopy. Lipid peroxidationinduction was the main and early (12 h) effect induced byFB1 at concentrations ranging from 0.5 to 69 lM, followedby inhibition of cell proliferation (up to 8.6 lM), theimmunomodulatory effect (up to 17.2 lM), by assessingIL-8 secretion, and increase in membrane microviscosity(up to 34.5 lM). The toxic effects observed in differentfunctional parameters were not dose-dependent and couldbe the consequence of the FB1 intracytoplasmatic localizationas confirmed by confocal microscopy results. Thedifferent timescales and concentrations active of differentfunctional parameters could suggest different cellular targetsof FB1.

The objective of the present study was to use an in vitro digestion/Caco-2 human intestinal cell model to assess the bioaccessibility and intestinal absorption/metabolism of verbascosides from a partially purified OMWW extract. In vitro digestive recoveries (bioaccessibility) of verbascoside and isoverbascoside following simulated gastro/intestinal digestion, were found to be 35.5 ± 0.55% and 9.2 ± 0.94% respectively. Accumulation of verbascoside and isoverbascoside from OMWW extract by Caco-2 monolayers was found to be linear between 10 and 100?M and rapid with peak accumulation occurring by 30min. Verbascoside was found to be metabolized by Caco-2 primarily by O-methylation generating two distinct metabolite forms with m/z of 634.5 and 353. Combined, these data support the notion that verbascosides from OMWW extract are bioaccessible and should be considered biologically relevant phenolic forms from OMWW.

During recent years food industries generally produce a large volume of wastes both solid and liquid, representing a disposal and potential environmental pollution problem.Objective: The goal of the study was to optimize, from both sensory and nutritional points of view, the formulation of durum wheat spaghetti enriched with an olive oil industrial by-product,indicated as olive paste. Methods: Three consecutive steps were carried out. In the first one, the olive paste was air-dried at low temperature, milled to record olive paste flour and properly analyzedfor its biochemical composition. In the second step, the olive paste flour was added to the pasta dough at 10% and 15% (w/w). In the last step, different concentrations of transglutaminase wereadded to enriched pasta (10% olive paste) to further improve the quality. Sensory properties and nutritional content of enriched and control pasta were properly measured. Results: Spaghetti with10% olive paste flour and 0.6% transglutaminase were considered acceptable to the sensory panel test. Nutritional analyses showed that addition of 10% olive paste flour to pasta considerably increased content of flavonoids and total polyphenols. Conclusions: The proper addition of olive paste flour and transglutaminase for pasta enrichment could represent a starting point to valorize olive oil industrial by-products and produce new healthy food products.

During the last years the interest on the recovery, recycling and upgrading of residues from plant food processing, has increased drastically. Generally, food industries produce large volume of by products, both solid and liquid, which represent a problem for disposal and for environmental pollution. Nevertheless, they are also promising sources of bioactive compounds that can be recovered and used as valuable substances by developing new enriched food, such pasta and bread. Because bread and pasta are foods consumed every day, they can be considered as a carrier of functional substances that have a beneficial role on consumer health. Therefore, the aim of this study was to study the impact of agronomic by-product incorporation, such as olive cake flours, on the sensorial qualities of cereal-based food as pasta and bread. Commercial durum wheat semolina for pasta and commercial soft wheat flour for bread, were mixed with 10% w/w of two different olive cake flours. The bioactive compounds present in olive cake, pasta and bread were identified and characterized, and their stability, after the processing, was evaluated. Pasta and bread based on durum wheat semolina and wheat flour (100%), respectively were produced without olive cake flours and used as controls. Dry spaghetti and bread samples were submitted to a panel of 10 trained tasters in order to evaluate the sensorial attributes, since the incorporation of the olive cake flours, could determined a decline of sensory quality. Besides, the effect of olive cake flours particle size on the quality of food, was also studied, thus demonstrating that samples enriched with fine particle size showed the greatest overall quality score.

Olive oil processing industries generate substantial quantities of phenolic-rich byproducts, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterization of antioxidant compounds from olive mill wastewater (OMWW), a polluting byproduct of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to low-pressure gel filtration chromatography on a Sephadex LH-20.The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, ?-hydroxyverbascoside, ?-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 cells, an established model system for studying drug transport properties, was also assayed. Finally, the antioxidant activities of the LH-20 fraction and verbascoside were characterized by two different techniques. Individual verbascoside was more active as a scavenger of reactive oxygen species and as a chemopreventive agent protecting low-density lipoproteins from oxidative damage than the LH-20 fraction.

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1IUmL(-1) of FSH/LH, and 100M cysteamine, in 5% CO2 in air at 38.5°C for 24h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03nM, and 2 incubation times (24 and 2h) were tested. In the 2-h exposure group, after 2h of exposure to VB, oocytes were washed and cultured up to 24h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P<0.05. Compared to controls, VB treatment at the concentration of 1.03nM and 24h of exposure had no effect on MII rates (196/268, 73% v. 226/323, 70% MII/cultured oocytes; P>0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P<0.05), blastocyst yield/cleaved embryos (59/156, 38% v. 45/165, 27%, respectively; P<0.05), and total blastocyst cell numbers (108.62±19.87 v. 89.61±26.32, respectively; P<0.05) compared to control oocytes. The VB treatment at the same concentration but for 2h induced only significantly higher cleavage rate (196/210, 93% v. 165/226, 73%; P<0.05). In conclusion, our results showed that VB treatment at 1.03nM during 24h of IVM exerted a positive effect on in vitro embryo development of prepubertal ovine oocytes by increasing the blastocyst yield and their quality. The hypothesis that VB at nanomolar concentrations may improve cumulus-oocyte energy/redox status is under investigation.