Carotenoids, including beta-carotene, lycopene, and derivatives, such as retinoic acid, have been studied for their significant antiproliferative and differentiating activity on cancer cells in experimental models and in clinics. We are presenting here data on the mechanism of action of a carotenoid-enriched extract obtained from the pumpkin Cucurbita moschata, variety "long of Naples," on two malignant human cell lines, Caco-2 and SAOs, derived from a colon adenocarcinoma and an osteosarcoma, respectively. The carotenoid extract has been obtained from pumpkin pulp and seeds by supercritical CO2 extraction and employed to prepare oil-in-water nanoemulsions. The nanoemulsions, applied at a final carotenoid concentration of 200-400 mu g/ml, were not cytotoxic, but induced a delay in cell growth of about 40% in both SAOs and Caco-2 cell lines. This effect was associated with the activation of a "nonprotective" form of autophagy and, in SAOs cells, to the induction of cell differentiation via a mechanism that involved AMPK activation. Our data suggest the presence of a pool of bioactive compounds in the carotenoid-enriched extract, acting additively, or synergistically, to delay cell growth in cancer cells.

Blanched skins and blanching water, by-products of almond processing, were evaluated as potential ingredients of bakery products. The research included three phases: i) optimization of skin drying; ii) optimization of quali-quantitative determination of phenolic compounds, by comparing three extracting protocols; iii) assessment of the impact of by-products on the rheology of composite doughs with wheat flour. The least time-consuming drying mode (at 60 °C for 30 min) retained better odor notes, higher content of phenolics (814 µg/g d.m. by HPLC, with the most effective extracting method) and greater antioxidant activity than sun-drying. Blanching water showed 917 µg/mL phenolics. Dried almond skins altered alveograph and farinograph indices of dough at doses higher than 30 and 50 g/kg, respectively, whereas blanching water did not cause significant changes. Therefore, almond skins could be used in products tolerating weak gluten network, such as cookies, whereas blanching water could be added to any bakery good.

The aim of this investigation has been the design and validation of an oligonucleotide microarray in order to detect 17 different wine-spoilage microorganisms, i.e. 9 yeasts, 5 lactic bacteria and 3 acetic acid bacteria species. Furthermore, several strains belonging to these species has been found to produce undesirable compounds for wine consumers. Oligonucleotide probes specific for each microorganism were designed to target the intergenic spacer regions (ISR) between18S-5.8S region for yeasts and 16S-ITS1 region for bacteria. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consensus primer. Each oligonucleotide-probes exclusively recognized its target without undesired aspecific cross-hybridizations. Under our experimental condition, the microarray assay analysis was able to detect the amount of DNA equivalent to 24 (Saccharomyces cerevisiae), 160 (Lactobacillus brevis) and 124 (Gluconobacter oxydans) cells, three species chosen as experimental models for the three studied microbial classes. Moreover, a novel procedure that allowed the extraction of genomic DNA from a mixture of eukaryotic and prokaryotic cells from contaminated wine was developed. The obtained results confirm that the microarray assay is able to detect specifically different spoilage microorganisms present in mixture in contaminated wines. For the first time the microarray methodology has been applied for the simultaneous identification of different mixed population of spoilage yeast and bacteria directly isolated from wine, thus indicating the practicability of oligonucleotide microarrays as a contamination control in wine industry.

In questo lavoro è stata condotta la caratterizzazione genetico-molecolare di circa 100 accessioni di olivo campionate in diverse località del Salento. L'obiettivo è stato la caratterizzazione, mediante marcatori molecolari (microsatelliti), di varietà autoctone del Salento a rischio di erosione genetica, e di altre varietà di olivo che, seppure di diversa origine geografica, risultano coltivate nel territorio salentino.

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.

Prunus mahaleb L. is a marginal fruit crop producing cherry-like dark purple drupes with avery bitter taste, rich in polyphenolic compounds.A mahaleb fruit concentrated extract (mfce)has been assayed for its biological activities. In this work, we report results on the in vitroeffects of mfce including: i) anti-proliferative, gap junction intercellular communication (GJIC)modulation and pro-apoptotic properties in a breast cancer cell line (MCF-7), ii) antiinflammatoryproperties in human vascular endothelial cells (HUVEC), iii) anti-mutageniceffect on yeast Saccharomyces cerevisiae strain D7. Mfce exerted a dose-dependent antiproliferativeeffect on MCF-7 cells together with a significant time-dependent increase inGJIC and a pro-apoptotic effect. Furthermore, mfce significantly reduced levels of endothelialinflammatory antigens in a concentration-dependent manner. Finally, mfce protectedS. cerevisiae cells against H2O2 cytotoxicity by direct radical scavenging activity. This studydemonstrated multiple healthful biological effects of mfce, highlighting it as a potentialnutraceutical product.

Thermal properties of eighteen monovarietal extra virgin olive oils from Apulia region in Italy were evaluated by means of a modulated adiabatic scanning calorimeter (MASC) and related to their chemical composition (free acidity, UV absorbance, fatty acid composition, polyphenol and o-diphenol content, oxidation status). MASC was used to study oil sample phase transitions in a temperature scanning mode by using a tailor-made time-temperature protocol. Crystallization kinetics and transition enthalpies were found to be significantly correlated to single free fatty acids (palmitic, oleic and linoleic acids) and to unsaturated/saturated fatty acid ratio. The overall crystallization curves were significantly delayed and occurred over longer time ranges as a function of higher peroxide index and linoleic acid content. Significant correlations were observed between melting profiles and single fatty acids, unsaturated/saturated fatty acid ratio and oleic/linoleic acid ratio. No significant correlations were observed between thermal properties and free acidity, linolenic acid, polyphenol and o-diphenol content.Compared with classical differential scanning calorimetry, MASC was simpler to use. The applied time-temperature protocol allowed to characterize extra virgin olive oils (EVOOs) of different provenience with rapid measurements and friendly outcome data. MASC, with its compact and portable equipment, may promote practical applications of calorimetric test along the olive oil supply chain.

The modern two-phase technology of the Leopard series for olive oil industrial extraction developed by the Pieralisi Group (Pieralisi Maip S.p.A., Jesi, AN, Italy) generates large quantities of a novel by-product (olive paste - OP) constituted of the partially defatted wet drupe pulp without any traces of the kernel. OP is rich in lipophilic and hydrophilic molecules able to exert beneficial effects on human health, including triterpenic acids, phytosterols, tocopherols, carotenoids, polyphenols, minerals and fibers [1]. In the perspective of by-product valorization through a modern biorefinery approach, it is worth noting that dried OP contains more than 10% oil characterized by a well-balanced lipid profile, rich in mono and polyunsaturated fatty acids, and a very good oxidative stability, due to the high concentration of fat-soluble antioxidants. This makes OP oil particularly suitable as functional ingredient for food/feed industry, as well as for the formulation of nutraceutical, cosmeceutical and pharmaceutical products. Supercritical carbon dioxide (SC-CO2) is a green and environmentally friendly technology for the effective extraction of high-value natural molecules. It is gaining a foothold in industrial production of solvent-free vegetable oils and has been also applied to concentrate oil products of lipophilic micronutrients (e.g. vitamin E in soybean and olive oils) [2]. Nevertheless, the industrial application of SC-CO2 technology requires a careful optimization of the operative parameters to make the process efficient from both an economic and a productive point of view. In this work, a response surface methodology approach, based on the Box-Behnken Design, was used to determine the optimal parameters of pressure, temperature and time to simultaneously maximize oil production from the freeze-dried OP of cultivar Leccino and concentrate the most abundant lipophilic bioactives.The experimental data were fitted to a second-order polynomial equation by multiple regression analysis, and examined using appropriate statistical methods. The 3-D response surface plots derived from the mathematical models were applied to determine the optimal extraction parameters, which resulted: temperature 70 °C, pressure 35.5 MPa and time 62 min. Under these conditions, the oil experimental yield was 14.0±0.7%, in close agreement with the predicted value. Compared to the freeze-dried OP, SC-CO2 extracted oil was much concentrated in phytosterols (~13 fold), tocopherols (~6 fold) and squalene (~8 fold); total carotenoid concentration remained almost unchanged, while triterpenic acids, being polar, were substantially reduced (- 79.5%). However, their total content was much higher than in extra virgin olive oil. The biological effects produced by OP oil intake were examined on 3 groups of healthy BALB/c mice (n=5/group) orally administered (for 4 weeks) with 20 or 40 mg/die of OP oil. Untreated animals were used as control. OP oil supplementation had no sig

Plant cell cultures are valuable bioreactors for the production of bioactive compounds. Artemisia annua L. cell suspension cultures established for producing the antimalarial artemisinin (AN) were exploited to investigate the biosynthesis of other healthful phytochemicals. Culture supplementation with the putative elicitors ß-cyclodextrins (ß-CDs) was successful to increase intracellular and extracellular levels of AN. Other bioactive isoprenoid compounds, known for their antioxidant activity, were also investigated in the supplemented A. annua cell cultures by using metabolomic and transcriptomic analyses. In particular, the application of ß-CDs was useful to induce a considerable significant increase of lutein, quinones (Q9 and Q10) intracellular contents compared to untreated cell cultures. Moreover, the release of carotenoids and quinones into the culture medium of ß-CDs-treated cell cultures was significantly enhanced. Real Time PCR analysis revealed an up-regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene, suggesting that the ß-CDs induced increase of isoprenoid compounds could be due to the enhancement of the plastidial isoprenoid biosynthetic route. In addition, protoplasts derived from Artemisia suspension cultures were explored to engineer subcellular compartmentalization using a modified SNARE protein as a molecular tool for biotechnological metabolite accumulation.

Plant cell cultures are valuable tools for the production of bioactive compounds. Recently, many efforts have been made in order to develop and optimize strategies for increasing the yields of the desired plant metabolites by eliciting their biosynthesis or improving the efficiency of product recovery. We established Artemisia annua L. (Asteraceae) cell suspension cultures to investigate the biosynthesis of the antimalarial artemisinin (AN) [1] and other healthful phytochemicals such as carotenoids and quinones. The use of culture supplementation with cyclic oligomers ?-cyclodextrins was successful to significantly increase intracellular and extracellular levels of AN, lutein, Q9 and Q10 [2, 3]. In order to investigate the mechanism underlying these effects, other oligosaccharides having a linear structure were explored as putative elicitors in A. annua cell suspension cultures. For this purpose, oligogalacturonides (OGA) were obtained by hydrolysis of polygalacturonic acid with pectolyase from Aspergillus japonicus. Various OGA fractions were used for culture medium supplementation at different concentrations. The results showed that the 4-5 OGA fraction induced significant increases of AN and the intermediate dihydroartemisinic acid. These results suggest that the ability of ?-CDs to elicit the production of bioactive compounds in A. annua cell cultures could be due to their chemical similarity to pectic oligosaccharides often occurring in plants after fungal infection.

Numerose evidenze scientifiche incoraggiano il consumo di frutta e verdura per prevenire malattie cardiovascolari e tumori. Tuttavia il loro consumo rimane sotto il limite consigliato dall'Organizzazione Mondiale della Sanità. Per questo motivo i consumatori e le aziende alimentari sono alla ricerca di bevande salutistiche "ready-to-drink".I frutti di P. mahaleb, ricchi di composti salutistici (antocianine, flavonoli e cumarine) non sono commestibili a causa del loro sapore amaro, ma l'impiego biotecnologico della fermentazione controllata, utilizzata per migliorare le qualità organolettiche, salutistiche e di serbevolezza di frutta e verdura, potrebbe consentirne l'utilizzo come nuova fonte di probiotici e phytochemicals.

The aim of the present study was to establish a new procedure for the oenological selection of Saccharomyces cerevisiae strains isolated from natural must fermentations of an important Italian grape cultivar, denoted as "Negroamaro". For this purpose, 108 S. cerevisiae strains were selected as they did not produce H2S and then assayed by micro fermentation tests. The adopted procedure made it possible to identify 10 strains that were low producers of acetic acid and hydrogen sulphide and showed that they completed sugar consumption during fermentation. These strains were characterized for their specific oenological and technological properties and, two of them, denoted as 6993 and 6920 strains, revealed themselves to be good candidates as industrial starter cultures. A novel protocol was set up for their biomass production and they were employed for industrial-scale fermentation in two industrial cellars. The two strains successfully dominated the fermentation process and contributed to increasing the wine organoleptic quality. The proposed procedure could be very effective for selecting "company-specific" yeast strains, ideal for the production of typical regional wines. "Winery" starter cultures could be produced on request in a small plant just before or during the vintage season and distributed as a fresh liquid concentrate culture

Betacyanin (betanin), total phenolics, vitamin C and antioxidant capacity (by Trolox-equivalent antioxidant capacity, TEAC and oxygen radical absorbance capacity, ORAC assays) were investigated in two differently coloured cactus pear (Opuntia ficus-indica (L.) Mill.) genotypes, one with purple fruit and the other with orange fruit, from the Salento area, in Apulia (South Italy). In order to quantitate betanin in cactus pear fruit extracts (which is difficult by HPLC because of the presence of two isomers, betanin and isobetanin, and the lack of commercial standard with high purity), betanin was purified from Amaranthus retroflexus inflorescence, characterized by the presence of a single isomer. The purple variety showed very high betanin content, with higher levels of phenolics, vitamin C, and antioxidant capacity (TEAC) than the orange variety. These findings confirm the potential for exploiting the autochthonous biodiversity of cactus pear fruits. In particular, the purple variety could be an interesting source of coloured bioactive compounds which not only have colouring potential, but are also an excellent source of dietary antioxidant components which may have beneficial effects on consumers' health.

Artemisinin is a sesquiterpene antimalarialcompound produced, though at low levels (0.1-1% dryweight), in Artemisia annua in which it accumulates in theglandular trichomes of the plant. Due to its antimalarialproperties and short supply, efforts are being made toimprove our understanding of artemisinin biosynthesis andits production. Native ?-cyclodextrins, as well as thechemically modified heptakis(2,6-di-O-methyl)-?-cyclodextrin(DIMEB) and 2-hydroxypropyl-?-cyclodextrins,were added to the culture medium of A. annua suspensioncultures, and their effects on artemisinin production wereanalysed. The effects of a joint cyclodextrin and methyljasmonate treatment were also investigated. Fifty millimolarDIMEB, as well as a combination of 50 mMDIMEB and 100 ?M methyl jasmonate, was highlyeffective in increasing the artemisinin levels in the culturemedium. The observed artemisinin level (27 ?mol g-1 dryweight) was about 300-fold higher than that observed inuntreated suspensions. The influence of ?-cyclodextrinsand methyl jasmonate on the expression

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.

This work is the first large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy).Yeasts were identified by Internal Transcribed Spacer (ITS) ribotyping and bioinformatic analysis. Thepolymorphism of interdelta elements was used to differentiate Saccharomyces cerevisiae strains. Twentydifferent species belonging to 9 genera were identified. Predominant on the grape surface wereMetschnikowia pulcherrima, Hanseniaspora uvarum and Aureobasidium pullulans, whereas M. pulcherrimaand H. uvarum were dominant in the early fermentation stage. A total of 692 S. cerevisiae isolates wereidentified and a number of S. cerevisiae strains, ranging from 26 to 55, was detected in each of the eightfermentations. The strains were tested for biogenic amines (BAs) production, either in synthetic media orgrape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were able toproduce histamine and cadaverine, during must fermentation. The production of BAs in wine must wasdifferent than that observed in the synthetic medium. This feature indicate the importance of an "ingrape must" assessment of BAs producing yeast. Overall, our results suggest the importance of microbiologicalcontrol during wine-making to reduce the potential health risk for consumer represented bythese spoilage yeasts.

Tomato (Solanum lycopersicum L.) is one of the most cultivated vegetable in the worldand it is a large source of bioactive compounds, including lycopene and othercarotenoids, flavanols (quercetin) and flavanones (naringenin). However, theconcentration of flavonoids in tomato is considered sub-optimal, particularly becauseanthocyanins are not generally present. Therefore this species has been the object of anintense metabolic engineering in order to obtain anthocyanin-enriched tomatoes byusing either breeding or transgenic strategies.Some tomato-related wild species, such as S. chilense, S. cheesmaniae, S.lycopersicoides and S. habrochaites, biosynthesize anthocyanins in the sub-epidermaltissue, and some allneles from those genotypes have been introgressed into cultivatedgenetics backgrounds, resultig in a 'purple' skin color. The breeding activity pursued atTuscia University (Viterbo, Italy), aimed at combining different alleles leading toanthocyanin production, have been lead to "Sun BlackTM , a trade mark-protectedtomato line with deep purple pigmentation, due to the biosynthesis of anthocyanins inthe peel. "Sun Black" is therefore a breeding product, not a GMO product. This aspect isconsidered an added-value for the market. In fact "Sun Black" tomato has been availablein July 2015 on the Italian market, thanks to an exclusive agreement between the ownerof the trademark and Unicoop Firenze.We report here the chemical characterization and structure elucidation of the uniqueanthocyanins found in the epiderm of "Sun Black" tomato, as well as other bioactivecompounds (carotenoids, polyphenols, vitamin C) of the whole fruit.The results show the unique features of this tomato genotipe as a 'novel food' withfunctional properties.Keyword: tomato, anthocyanins, functional food

In questo lavoro ci siamo occupati del recupero del germoplasma olivicolo salentino ancora presente in provincia di Lecce, ma a forte rischio di erosione genetica. In particolare, abbiamo studiato il profilo molecolare di varietà antiche, quali: Colozzese, Barone di Monteprofico, Ornella, Usciana, Cornola, Uliva grossa, e di diversi cloni delle cultivar autoctone più diffuse sul territorio, ovvero Cellina di Nardò e Ogliarola di Lecce. Tutte le varietà analizzate in questo studio sono conservate in un campo collezione allestito negli anni '90 in agro di Carpignano salentino.

Higher plants synthesize an amazing diversity of phenolic secondary metabolites.Phenolics are defined secondary metabolites or natural products because, originally, they wereconsidered not essential for plant growth and development. Plant phenolics, like other naturalcompounds, provide the plant with specific adaptations to changing environmental conditions and,therefore, they are essential for plant defense mechanisms. Plant defensive traits are costly for plantsdue to the energy drain from growth toward defensive metabolite production. Being limited withenvironmental resources, plants have to decide how allocate these resources to various competingfunctions. This decision brings about trade-offs, i.e., promoting some functions by neglecting othersas an inverse relationship. Many studies have been carried out in order to link an evaluation of plantperformance (in terms of growth rate) with levels of defense-related metabolites. Available resultssuggest that environmental stresses and stress-induced phenolics could be linked by a transductionpathway that involves: (i) the proline redox cycle; (ii) the stimulated oxidative pentose phosphatepathway; and, in turn, (iii) the reduced growth of plant tissues.

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 olive oil is an unfavorable substrate for microbial survival and growth. Only few microorganisms use olive oil fattyacids as carbon and energy sources, and survive in the presence of olive oil anti-microbial components. In this study,we have evaluated the occurrence of microorganisms in 1-year-stored extra-virgin olive oil samples. We detected thepresence of bacterial and yeast species with a recurrence of the bacterium Stenotrophomonas rhizophila and yeastSporobolomyces roseus. We then assayed the ability of all isolates to grow in a mineral medium supplemented with acommercial extra-virgin olive oil as a sole carbon and energy source, and analyzed the utilization of olive oil fatty acidsduring their growth. We finally focused on two bacterial isolates belonging to the species Pantoea septica. Both theseisolates produce carotenoids, and one of them synthesizes bioemulsifiers enabling the bacteria to better survive/growth in this unfavorable substrate. Analyses point to a mixture of glycolipids with glucose, galactose and xylose ascarbohydrate moieties whereas the lipid domain was constituted by C6-C10 ?-hydroxy carboxylic acids.

The molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood although this information is essential to design rational strategies for industrial strain improvement. In this study, we applied comparative genomics to identify all genetic changes occurring during the development of an erythromycin overproducer obtained using the traditional mutate-and- screen method.Results: Compared with the parental Saccharopolyspora erythraea NRRL 2338, the genome of the overproducing strain presents 117 deletion, 78 insertion and 12 transposition sites, with 71 insertion/deletion sites mapping within coding sequences (CDSs) and generating frame-shift mutations. Single nucleotide variations are present in 144 CDSs. Overall, the genomic variations affect 227 proteins of the overproducing strain and a considerable number of mutations alter genes of key enzymes in the central carbon and nitrogen metabolism and in the biosynthesis of secondary metabolites, resulting in the redirection of common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, affect global expression profile as demonstrated by a comparative analysis of the parental and overproducer expression profiles. Genomic data, finally, suggest that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes. Conclusions: This study helps to clarify the mechanisms underlying antibiotic overproduction providing valuable information about new possible molecular targets for rationale strain improvement.

The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 and the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

Here we describe the encapsulation in ?-cyclodextrins (?-CDs) of wheat bran, pumpkin and tomato oleoresins, extracted by supercritical carbon dioxide, to obtain freeze-dried powders useful as ready-to-mix ingredients for novel functional food formulation. The stability of tocochromanols, carotenoids and fatty acids in the oleoresin/?-CD complexes, compared to the corresponding free oleoresins, was also monitored over time in different combinations of storage conditions. Regardless of light, storage at 25 °C of free oleoresins determined a rapid decrease in carotenoids, tocochromanols and PUFAs. ?-CD encapsulation improved the stability of most bioactive compounds. Storage at 4 °C synergized with encapsulation in preventing degradation of bioactives. Unlike all other antioxidants, lycopene in tomato oleoresin/?-CD complex resulted to be more susceptible to oxidation than in free oleoresin, likely due to its selective sequestration from the interaction with other lipophilic molecules of the oleoresin

This review reports the use of wheat milling by-products for the extraction of high quality oil and vitamin E including our results on the exploitation of durum wheat bran as a valuable source of important healthful compounds. Wheat oil can be used as an ingredient in food, pharmaceutical or cosmetic preparations because it contains important bioactive compounds such as vitamin E, carotenoids and unsaturated fatty acids. Different methods are used for oil recovery from plant materials, such as solvent extraction, mechanical pressing or the ecofriendly supercritical carbon dioxide (SC-CO2) extraction technology. By using SC-CO2, we obtained an oil from durum wheat (Triticum durum Desf.) bran and optimized the extraction conditions to increase oil and vitamin E yields. Wheat bran, which is composed of pericarp, aleurone layer and germ, is discarded during the early stages of durum wheat milling processes to obtain a final product (semolina) that is stable over time. Maximum oil and vitamin E yields were obtained when a durum wheat bran matrix with particle size of *30 mesh and a moisture content of 2.6 % was used. The optimal conditions for oil extraction were: 300-350 bar, 60-70 °C, and 4 l min-1 gaseous CO2 flow rate for 1 h. The chemical composition (vitamin E forms, carotenoids, quinones, lipids and fatty acids) of the SC-CO2 extracted oil was analyzed and compared to that of the oil extracted by Soxhlet using hexane as solvent. The findings here reported highlight the importance of durum wheat bran as a rich source of valuable natural nutrients.

In this work a process for obtaining high vitamin E and carotenoid yields by supercritical carbon dioxide(SC-CO2) extraction from pumpkin (Cucurbita moschata Duch.) is described. The results show that the useof a vacuum oven-dried [residual moisture (8%)] and milled (70 mesh sieve) pumpkin flesh matrixincreased SC-CO2 extraction yields of total vitamin E and carotenoids of 12.0- and 8.5-fold,respectively, with respect to the use of a freeze-dried and milled flesh matrix. The addition of milled(35 mesh) pumpkin seeds as co-matrix (1:1, w/w) allowed a further 1.6-fold increase in carotenoidyield, besides to a valuable enrichment of the extracted oil in vitamin E (274 mg/100 g oil) andpolyunsaturated fatty acids. These findings encourage further studies in order to scale up the processfor possible industrial production of high quality bioactive ingredients from pumpkin useful in functionalfood or cosmeceutical formulation.

Artemisinin is an effective antimalarial compound obtained by thè aerial parts of thè herbalplant Artemisia annua L. Unfortunately, thè production of artemisinin in plants is very low (0.1-1% on a dry weight basis) and its chemical synthesis is very difficult. In recent yearsbiotechnological attempts to improve artemisinin production either in plant or geneticallyengineered microrganisms were made. A. annua celi and tissue cultures were also exploredfor thè production of artemisinin, although thè yieids obtained have been so far quite low. Wehave recently established A. annua celi cultures able to biosynthesize artemisinin and torespond to thè elicitation by methyl jasmonate (MeJA). Interestingly, amounts of artemisininproduced by these cultures were also found in thè spent culture medium. Cyclodextrins (CDs)are known to increase thè water solubility of various compounds, by forming host-guestinclusion complexes. Chemically modified CDs, such as 2,6 dimethyl-p-cyclodextrins (DIMEB)have been successfully used to improve thè production of health-promoting plant metabolitessuch as resveratrol in grape celi cultures. The aim of this work was to evaluate thè ability ofDIMEB to enhance artemisinin production in A. annua suspension celi cultures. HPLCanalysis of artemisinin and Real Time PCR gene expression analysis were carried out inDIMEB treated and untreated cultures. DIMEB induced a 300-fold increase of artemisininlevels in thè spent culture medium after a three-day-treatment. The addition of MeJA induceda further increase of thè artemisinin amounts. The expression levels of artemisininbiosynthetic genes (cytochrome P450 monoxygenase, cytochrome P450 reductase andartemisinic aldehyde A11 reductase) were not altered by thè DIMEB treatment. These resultssuggest that DIMEB improved artemisinin yieids of A. annua celi cultures, probably notaffecting thè biosynthetic pathway but enhancing thè solubility and stability of artemisininmolecules.

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 aim of this study was to investigate the influence of encapsulation on the storage stability of oil extracted by supercritical carbon dioxide from a micronized durum wheat bran fraction. Wheat bran oil was encapsulated in 2% (w/v) sodium alginate beads. Encapsulated and unencapsulated oil samples were stored at 4°C or 25°C, daylight or darkness, over 90 days and, at defined time points, subjected to stability evaluation based on fatty acid hydroperoxide production, and tocopherol (?, ? and ? forms), tocotrienol (?, ? and ? forms) and carotenoid (lutein, zeaxanthin and ?-carotene) degradation. The encapsulation of the oil into alginate beads significantly increased stability, optimally when stored at 4°C, maintaining high levels of isoprenoids and low content of fatty acid hydroperoxides over 30 days of storage.

Table olives fermentation is a process unpredictable and frequently performed using traditional practices often inadequate to obtain products with acceptable quality and safety standards. In the present study, the efficacy of selected yeast strains as starters to drive fermentations of green and black table olives by the Greek method was investigated. Pilot-scale production by spontaneous fermentation as a control, olives started with previously selected Saccharomyces cerevisiae strains; fermentation driven by commercial S. cerevisiae baker's yeast strain were carried out for each one of Manzanilla, Picual, and Kalamàta table olives cultivars.RESULTS:Time of fermentation was significantly shortened to 40 days to complete the transformation process for all the three tested cultivars. Inoculated table olives were enhanced in their organoleptic and nutritional properties in comparison with corresponding samples obtained by spontaneous fermentation. The use of starters was also able to improve safety traits of table olives in terms of biogenic amines reduction as well as of absence of undesired microorganisms at the end of the process.CONCLUSIONS:Autochthonous, but also non-authochthonous yeasts can be used to start and control le olives fermentations and can significantly improve quality and safety aspects of table olives produced by many smallholder farmers. This article is protected by copyright

Table olives fermentation is a process unpredictable and frequently performed using traditional practices often inadequate to obtain products with acceptable quality and safety standards. In the present study, the efficacy of selected yeast strains as starters to drive fermentations of green and black table olives by the Greek method was investigated. Pilot-scale production by spontaneous fermentation as a control, olives started with previously selected Saccharomyces cerevisiae strains; fermentation driven by commercial S. cerevisiae baker"s yeast strain were carried out for each one of Manzanilla, Picual, and Kalamàta table olives cultivars.RESULTS: Time of fermentation was significantly shortened to 40 days to complete the transformation process for all the three tested cultivars. Inoculated table olives were enhanced in their organoleptic and nutritional properties in comparison with corresponding samples obtained by spontaneous fermentation. The use of starters was also able to improve safety traits of table olives in terms of biogenic amines reduction as well as of absence of undesired microorganisms at the end of the process.CONCLUSIONS: Autochthonous, but also non-authochthonous yeasts can be used to start and control le olives fermentations and can significantly improve quality and safety aspects of table olives produced by many smallholder farmers.

Supercritical carbon dioxide (SC-CO2) represents an effective, non-toxic technology for extracting food-grade oleoresins from a range of edible plant materials [1]. The obtained oleoresins are a rich source of bioactive molecules, mainly carotenoids, tocochromanols and polyunsaturated fatty acids (PUFAs). Most bioactives are very reactive to light, oxygen and free radical species, this greatly limits their stability and reduce their shelf-life and health benefits [2,3]. Encapsulation into micro or nano particles is the most promising technique to increase their stability over time. Cyclodextrins (CDs) are biocompatible, non-toxic cyclic oligosaccharides obtained by enzymatic conversion of starch. They can form inclusion complexes with specific guest molecules, contributing to their stabilization against the adverse effects of physicochemical agents. CDs are approved in Europe as soluble dietary fiber and novel food ingredient. In this work is reported the encapsulation in CDs of pumpkin and tomato oleoresins, extracted by SC-CO2. The stability of tocochromanols, carotenoids and fatty acids in the oleoresin/CD complexes, compared to the corresponding free oleoresins, was monitored over time in different storage conditions (25°C, dark; 25°C light; 4°C, dark). Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) analyses, demonstrated that oleoresins/CDs have different physical characteristics from free oleoresins. Regardless of light, storage at 25°C of free oleoresins determined a rapid decrease in carotenoids, tocochromanols and PUFAs. CD encapsulation improved the stability of most bioactive compounds. Upon storage, the retention of tocochromanols, carotenoids and fatty acids was higher in oleoresin/CD complexes stored at 4°C than in the corresponding free oleoresins, indicating that encapsulation and low temperature have a synergistic effect in preventing the degradation against oxidation of bioactives. Unlike all other analyzed antioxidants, lycopene in tomato oleoresin/CD complex resulted to be more susceptible to oxidation than in free oleoresin, likely due to its selective sequestration from the interaction with other lipophilic molecules of the oleoresin. The results suggest that oleoresin/complexes, being generally more stable than free oleoresins, can be used as ready-to-mix high-quality ingredients in the preparation of innovative functional foods simultaneously enriched with carotenoids tocochromanols, PUFAs and fibers.

Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-?-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

Tableoliveshavebeena component of theMediterranean diet for centuries,with the trendfor their consumptioncurrently increasing worldwide. They are rich in bioactive molecules with nutritional, antioxidant, anti-inflammatory orhormone-like properties. In the present study, the concentrations of phenolics, triterpenic acids, carotenoids and vitamins, aswell as fatty acid profiles and antioxidant activity,were analyzed in the edible portion of black table olives (Olea europea L.) fromItalian (Cellina di Nardò and Leccino) and Greek (Kalamàta and Conservolea) cultivars fermented with selected autochthonousstarters and in the corresponding monovarietal olive oils.RESULTS: On a fresh weight basis, Cellina di Nardò and Leccino table olives showed the highest total phenolic content. Nosignificant differences were found with respect to the levels of total triterpenic (maslinic and oleanolic) acids and vitaminE among cultivars. All table olives were characterized by high amounts of oleic, linoleic and palmitic acids. Oils were richerin lipophilic antioxidants (carotenoids and tocochromanols) than table olives, which, instead, showed a higher content ofpolyphenols and triterpenic acids than oils.CONCLUSION: The present study demonstrates that fermented table olives are an excellent natural source of unsaturated fattyacids, as well as being nutritionally important health-promoting bioactive compounds.

Table olives have been a component of the Mediterranean diet for centuries and their consumption is increasing worldwide. They contain several nutritional components such as unsaturated fatty acids, vitamin E, triterpenic acids, sterols, polyphenols and carotenoids [1]. The content of these bioactive molecules depends on the olive variety, the ripening stage of the olive fruit, the cultivation conditions and the processing method [2]. There are numerous commercial preparations of table olives and in a recent work we have developed and validated a new procedure for table olive production based on the use of selected autochthonous microbial starters and a productive process optimized to mimic the microbial evolution observed during spontaneous fermentations. [3]. In this work, we characterized some bioactive compounds and coloring pigments of fermented black table olives, belonging to two Italian (Cellina di Nardò and Leccino) and two Greek (Kalamàta and Conservolea) cultivars. Greek table olives resulted richer in carotenoids, vitamin E and triterpenic acids (maslinic and oleanoic acids) than Italian cv, contents ranged from 2.28 to 6.91 µg/g fw (edible portion), 53.64 to 59.15 µg/g fw and 2.19 to 3.28 mg/g respectively. Polyphenol compounds are known to be responsible for the bitter taste of the olive fruit and most of them are involved in table olives colour changes. In all tested black table olive cultivars, polyphenols content ranged from 0.87 mg/g fw (in Conservolea) to 23.49 mg/g fw (Cellina di Nardò). Moreover, we analysed the content of tyrosol, hydroxytyrosol, oleuropein aglycone, verbascoside, isoverbascoside, rutin, pyrocathecol, luteolin glucoside, quercetin, cyanidine rutinoside and cyniadine glucoside. In particular, Cellina di Nardò is characterized by the highest levels of anthocyanin pigments such as cyanidin 3-O-glucoside (14.77 mg/g fw) and cyanidin-3-O-rutinoside (3.17 mg/g fw) responsible for the characteristic black color. The results indicated that fermented black table olives are an excellent natural source of bioactive compounds and can represent a potential important functional food.

A pressing necessity of the Apulian wine industry is to being able to pilot and to control the wine production to obtain wines with peculiar characteristics and with respect of the typicality guaranteed by the denominations of origin. The employment of selected autochthonous yeast strains would be a potent instrument to improve the organoleptic and sensory characteristics of typical regional wines. In fact, indigenous yeasts are better adapted to a specific must and therefore they are able to exalt the peculiarities of the derived wine. The present work described the genetic diversity of autochthonous Saccharomyces cerevisiae strains derived from natural must fermentations of an important Apulian grape cultivar, denoted as “Primitivo”. The yeast strains showing the best technological and oenological properties were selected and their fermentative performances were assayed by either laboratory tests and industrial scale fermentations. Two autochthonous yeast strains showed to be good candidates as industrial starter cultures, since they dominated the fermentation process and produced wines characterized by peculiar oenological and organoleptic features, that were judged very pleasant by a panel of winemakers.

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.

The recent finding of nutrigenomics and the growing trend towards personalized nutrition encourage research directed towards innovative functional food development [1]. Dry pasta, coming widely and frequently in the Mediterranean diet, is considered an optimal, economic and easy-to-use vehicle for phytochemicals acting as nutrition enhancers or providing specific physiological functions. Pasta, made from refined wheat, lacks many of the nutrients found in whole grains. As such, it has been the object of many supplementation strategies to improve pasta nutritional value and functional attributes.Pumpkin (Cucurbita spp.) fruits and seeds have received considerable attention in recent years because of the nutritional and health benefits of the presence on many lipophylic biologically active compounds, mainly carotenoids, tocochromanols, phytosterols and PUFA [2].Supercritical carbon dioxide (SC-CO2) pumpkin extracted oils contain the broad lipophilic phytocomplex of the starting matrix in a highly bioavailable form [3]. Phytocomplex constituents, alone or synergistically, act as antioxidants, vitamins, anti-inflammatory and anticancer agents, as well as in cell signaling and gene expression regulation, playing a pivotal role in building and maintaining our health [4]. The limited stability of the oils over time and/or during food processing and storage is the main drawback for their usage in the formulation of functional products. Most bioactives are, in fact, very reactive to light, oxygen and free radicals and/or susceptible to thermal degradation. Encapsulation into micro or nano particles is the most promising technique to increase oil stability. The feasibility of chlatrating the oil in ?-Cyclodextrin (?-CD) complexes to improve its chemical stability and prepare powders useful as ready-to-mix ingredients for novel functional food formulations was also demonstrated [5].The aim of this study was to explore the feasibility of producing an innovative supplemented durum wheat pasta enriched in the lipophilic phytocomplex extracted by SC-CO2 from ripe pumpkin peponides. The oil was mixed to the dough as free or as ready-to-mix oil/?-CDs powder. Four types of pasta were prepared: (i) control spaghetti (S-CTRL); (ii) spaghetti supplemented with ?-CDs (S-?-CD); (iii) spaghetti supplemented with pumpkin oil (S-Oil) and (iv) spaghetti supplemented with the pumpkin oil/?-CD powder (S-Oil/?-CD). The biochemical, antioxidant, textural and sensory attributes of pasta types were evaluated and compared. The results showed that S-Oil and S-Oil/?-CD spaghetti were significantly enriched in phytosterols, squalene, carotenoids, tocochromanols and unsaturated fatty acids, providing a substantial proportion of the recommended daily allowance (RDA) for A and E vitamins, as well as increased lipophilic antioxidant capacity. Furthermore, ?-CD pumpkin oil complexation increased the stability of some of the bioactive components. With regard to the sensory evaluation o

Wheat species contain a variety of polyphenols including phenolic acids, flavonoids, proanthocyanidins, condensed tannins, catechins and lignans. Phenolic acids represent the most common phenolic compounds found in whole wheat grains and can be found as soluble free, soluble conjugated bound to low molecular mass components (e.g. saccharides or organic acids); and insoluble bound forms, linked to polymers of the plant cell wall. Despite their high value for human health, only few studies have been carried out on the genetics and genomics of these bioactive compounds in durum wheat. The objectives of the present work were i) the estimation of genetic variability for phenolic acids composition and concentration in a set of 111 tetraploid wheat genotypes (Triticum turgidum L.), comprising durum cultivars, landraces and wild accessions; and ii) the detection of regions attributable to individual phenolic acids and total phenolic acids concentration by the means of a genome wide association study (GWAS). Phenolic acids were extracted from whole-meal flour and identified by DAD-HPLC analysis. A total of six major phenolic acids were quantified across the durum wheat genotypes, namely: ferulic, sinapic, p-coumaric, vanillic, syringic and p-hydroxibenzoic acids. The amount of total bound phenolic acids, ranged from 341 to 1700 ?g g-1 of whole-meal flour, with a mean value of 800 ?g g-1. The soluble free fraction (measured spectrophotometrically after Folin Ciocalteu reaction) ranged from 1280 to 3150 ?g g-1 as ferulic acid equivalents. The analysis of variance (ANOVA) revealed the effects of genotype, year and year x genotype that were significant for all phenolic acids. The ratio of genotypic variance to total variance was moderately high suggesting that phenolic acids concentration can be further improved by breeding approaches in durum wheat.The GWAS was based on the detection of correlations between genotype and phenotype in the durum germplasm collection by using a high-density wheat SNP iSelect array including approximately 90,000 gene associated SNPs. Results showed several significant marker-trait associations (MTA), identifying eight quantitative trait loci (QTL) associated with phenolic acids concentration. The highest number of MTAs was identified on chromosome 7A, where one QTL region was associated with phenolic acids content. The lowest number of MTAs was detected on chromosomes 3A and 5B, where only one MTA identified a single locus. Conservation of synteny between SNPs and the annotated genes and proteins in Brachypodium distachyon, Oryza sativa and Sorghum bicolor allowed the identification of two QTLs coincident with two different candidate genes.

Spontaneous grapes must fermentation, induced by the indigenous micro flora, is believed to be associated with a specific vineyard and to give a distinctive style and quality to that wine. The alcohol-tolerant Saccharomyces cerevisiae strains invariably dominate the latter stage of natural wine fermentation. The S. cerevisiae population and other specific yeasts present in the vineyard niche habitats are considered autochthonous and their involvement in natural fermentation allows the production of wines with particular features in each microclimatic area. The present study was aimed to the individuation of autochthonous yeast strains useful in the improvement of oenological production of Salento, which is a very important wine-producing area of Southern Italy. Grapes were sampled from the most representative areas of Salento region (Ugento e Guagnano for "Negroamaro" and Gioia del Colle for "Primitivo") and separately subjected to natural fermentation in an experimental scale. The identification of micro biota present during the last step of wine fermentation (>1 °Bé) of grapes, was carried out to select autochthonous yeast strains for industrial wine production. Aliquots of must samples of several dilutions were isolated after spreading on YPD agar medium supplemented. As preliminary screening, averages of 1500 colonies for each of the three fermentations were assayed for H2S production on BIGGY agar. The colonies appearing white or light brown (about the 10% of initial 1500 individual clones) were selected and recognized as S. cerevisiae by a microbiological screening based on the implementation of the taxonomical keys for identification of yeasts belonging to Saccharomyces genus. Identification at strain level of S. cerevisiae isolates is a fundamental step to investigate the biodiversity of this yeast and to examine population dynamics during the fermentative process. The criterion for strain differentiation was provided by the amplification of genomic sequence blocks flanked by delta elements of retrotrasposon origin. Interdelta (ID) typing has proved to be a very convenient method that can advantageously replace the other methods for molecular characterization of S. cerevisiae strains. For the first time, a recently developed method has been applied for the ID by the use of fluorescent primers and automatic sequencers. When compared with gel-based analytical methods, the use of capillarity system and automated analysis increase data throughput, scoring and reliability, decreasing the overall experimental error. The results of the genetic characterization of S. cerevisiae strains associated to natural fermentations of must from "Primitivo" and "Negroamaro" grapes and their implications for the selection of autochthonous industrial starters will be discussed.

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.

Phenolic acid intake through the consumption of whole-wheat foods provides important health benefits associated with reduced risks of cardiovascular diseases and colon cancer. The genetic variation for phenolic acids was extensively studied in common wheat, but a comprehensive survey in tetraploid wheat is lacking. In this study we evaluated the genetic variability for individual and total phenolic acids concentration existing in a large collection of tetraploid wheat (Triticum turgidum L.). A two-year evaluation was undertaken on the whole-meal flour of 111 genotypes belonging to seven T. turgidum subspecies including cultivars, landraces and wild accessions. Durum cultivars [T. turgidum subsp. durum (Desf.) MacKey], had the highest average concentration of total phenolic acids (828.7 ug g-1 dm in 2012; 834.5 ug g-1 dm in 2013) with values varying from 550.9 ug g-1 dm to 1,701.2 ug g-1 dm, indicating a variation of >3 fold. The lowest concentration of phenolic acids was found in T. turgidum subsp. dicoccum (Schrank ex Schübler) Thell. Rivet wheat (T. turgidum L. subsp. turgidum) had phenolic acid concentrations similar to those in durum, but less variation was noted among the accessions. On the other hand, the accessions of the four remaining subspecies showed lower phenolic acid concentrations and variation among the accessions as compared to durum. A total of six phenolic acids were identified across the wheat genotypes. The effects of genotype, year and year x genotype were estimated by ANOVA and resulted significant for all phenolic acids. The ratio of genotypic variance to total variance suggested the possibility of improving phenolic acid content in elite wheat germplasm through appropriate breeding programs. Moreover, significant correlations between phenolic acids and other quality characteristics of the grain were detected.

The understanding of the yeast population dynamics during spontaneous alcoholic fermentation allows to preserve the microbial biodiversity, to use as indigenous fermentation starters so and to improve the organoleptic and sensory properties of the produced wines. However, it is similarly important to investigate the safety aspects of microbial biodiversity, in particular, on the undesired production of biogenic amines (BAs), low-molecular-weight organic bases produced in wine by the activity of microbial-specific amino acid decarboxylases. This study is the first large-scale investigation on vineyard-associated yeast strains from Apulia (Southern Italy). Eight natural must fermentations were carried out by sampling grape (Vitis vinifera) in the most significant production areas for Negroamaro and Primitivo cultivars: Torchiarolo, Copertino, Cutrofiano and Melissano for the former, Galatina, Torchiarolo, Manduria and Gioia del Colle areas for the latter. Yeasts isolates were identified by PCR ribotyping and bioinformatic analysis of the rRNA Internal region denoted as Transcribed Spacer (ITS). The Saccharomyces cerevisiae strains were further identified and differentiate as strain level by evaluating the polymorphism of their interdelta elements. The results of the molecular analyses revealed the presence of twenty different species belonging to 9 genera. In particular, Hanseniaspora uvarum, Metschnikowia pulcherrima and Aureobasidium pullulans were the dominant strains on the grape surface, whereas M. pulcherrima and H. uvarum were predominant during the early fermentation stage. We identified 692 S. cerevisiae isolates and a number of different strain in each of the 8 fermentations strains, ranging from 26 to 55, The strains were assayed for BAs production, either in synthetic media or grape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were capable to produce in wine histamine and cadaverine. The production of BAs in the synthetic medium was dissimilar than that detected in wine, thus enhancing significance to assess the yeast BAs production by an "in grape must" assay, in order to reducethe potential health risk for consumer represented by these spoilage yeasts. To the best of our knowledge, this is the primary study regarding the biodiversity and safety aspects of grape-associated yeast strains in this important wine-producing area of Southern Italy.

Phenolic acids are major components of cell walls in wheat and have important implications on human health as antioxidants with anti-tumor activity. Our objectives were to identify phenolic acid genes in wheat by single nucleotide polymorphisms (SNPs) detected within the coding sequences of candidate genes, and to identify chromosomal regions associated with single phenolic acids and total soluble phenolic compounds. A set of candidate genes involved in the biosynthesis of hydroxycinnamic acid derivatives were identified by comparative genomics. SNPs found in the coding sequences of six genes (PAL1, PAL2, C4H, C3H, COMT1 and COMT2) were used to determine their chromosomal location and accurate map position on two reference consensus linkage maps. The genome-wide association study (GWAS), based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs, detected 22 quantitative trait loci (QTL) distributed on almost all durum wheat chromosomes. Two QTL for p-coumaric acid were coincident with the phenylalanine ammonia-lyase (PAL2) and p-coumarate 3-hydroxylase (C3H) genes on chromosome arms 2AL and 1AL, respectively. The availability of candidate gene-based markers can allow elucidating the mechanism of phenolic acids accumulation in wheat kernels and exploiting the genetic variability of phenolic acids content for the nutritional improvementof wheat end-products.

Phenolic acids are major components of cell walls in wheat and have important implicationson human health as antioxidants with anti-tumor activity. Understanding the genetic basis ofphenolic acids content and composition of wheat grain, and identifying associated markers providethe basis for improving wheat quality by molecular breeding. The objective of this study was: a) toidentify phenolic acids genes in wheat by exploiting genomic resources and SNPs detected withinthe coding sequences of candidate genes; and b) to identify chromosomal regions associated tosingle phenolic acids and total soluble phenolic compounds by genome-wide association study(GWAS) and candidate gene approaches using a tetraploid wheat collection coupled with the 90KiSelect SNP genotyping array. A set of candidate genes involved in the biosynthesis ofhydroxycinnamic acid derivatives (p-coumaric acid, ferulic acid and sinapic acid) and in the totalsoluble phenolics have been identified in wheat by comparative genomics. Single nucleotidepolymorphisms (SNPs) found in the coding sequences of six candidate genes (PAL1, PAL2, C4H,C3H, COMT1 and COMT2) allowed to determine their chromosomal location and accurate mapposition on two reference consensus linkage maps. The genome-wide association study based ongenotyping a tetraploid wheat collection with 81,587 gene-associated SNPs revealed severalsignificant marker-trait associations, identifying 22 quantitative trait loci (QTLs) associated withindividual phenolic acids and total soluble phenol compounds distributed on almost all durumwheat chromosomes. A QTL for p-coumaric acid was found to be coincident with the candidategene phenylalanine ammonia-lyase (PAL2) on chromosome arm 2AL. The availability of perfect,candidate gene-based markers can allow elucidating the mechanism of phenol acids accumulation inthe wheat kernels and exploiting the genetic variability of phenol acids content for the nutritionalimprovement of wheat end-products.

Phenolic acids are major components of plant cell walls in wheat and have important implications on human health as antioxidants with anti-tumor activity. They occur in wheat grains as: i) soluble free phenolic acids; ii) soluble conjugated phenolics bound to low molecular mass components such as saccharides or organic acids; iii) insoluble bound forms of phenolics, linked to polymers of the plant cell wall (Li et al., 2008). Despite their high value for human health, few studies have been carried out on the genetics of phenolic acids in durum wheat. The genetic variability of phenolic acids composition and concentration was investigated, over two years, in a set of 111 tetraploid wheat genotypes, belonging to seven Triticum turgidum L. subspecies, including cultivars, landraces and wild accessions. Regions attributable to individual phenolic acids and total phenolic acids concentration were identified through a genome wide association study (GWAS). A total of six phenolic acids were identified by DAD-HPLC analysis across the 111 wheat genotypes, namely: ferulic, sinapic, p-coumaric, vanillic, syringic and p-hydroxibenzoic acids. The amount of total bound phenolic acids, ranged from 341 to 1700 ?g g-1 of whole-meal flour, with a mean value of 800 ?g g-1 (Laddomada et al, 2016). The soluble free fraction (measured spectrophotometrically after Folin Ciocalteu reaction) ranged from 1280 to 3150 ?g g-1 as ferulic acid equivalents. The effects of genotype, year and year x genotype were estimated by ANOVA and resulted significant for all phenolic acids. The ratio of genotypic variance to total variance was moderately high suggesting that a breeding approach could be considered to increase phenolic acids concentration in durum wheat.The GWAS revealed a total of 29 significant marker-trait associations (MTA), identifying eight quantitative trait loci (QTL) associated with phenolic acids content. The highest number of MTAs was identified on chromosome 7A, where one QTL region was associated with phenolic acids content, while the lowest number of MTAs was detected on chromosomes 3A and 5B, where only one MTA identified a single locus. Conservation of synteny between SNP marker sequences and the annotated genes and proteins in Brachypodium distachyon, Oryza sativa and Sorghum bicolor allowed the identification of two QTLs coincident with two different candidate genes.

Lycopene is a carotenoid pigment found in a wide variety of vegetables and fruits. More than six hundred carotenoids have been identified in nature, but only twenty are present in human blood and tissues, including lycopene (the most abundant in plasma), ?-carotene, lutein and zeaxanthin [1]. Several exogenous pigmented antioxidants act on cancer progression and have immune-modulating effects by free radical scavenging activity and/or still unknown mechanisms. However, the controversy recently arisen for some of them requires a careful investigation in relation to their source and synergistic effects [2]. The purpose of this work was: i) to optimize the extraction of lycopene from ripe fruits of tomato, watermelon and GAC by supercritical CO2 in order to obtain natural, solvent-free lycopene containing oleoresins suitable as ingredients for the formulation of innovative functional foods and/or nutraceuticals; ii) to characterize each oleoresin with respect to carotenoids, tocochromanols, lipids, lipophilic and hydrophilic antioxidant activities, micro and macro elements; iii) the preparation of inclusion complexes (ICs) of the oleoresins in food grade ?-cyclodextrins (?-CDs) as hydrophilic nanocarriers [3]; iv) to assess, in-vitro, the effects of administration of each oleoresin/?-CD IC at different concentrations (0.5 to 10 µM) on cell apoptosis and on Interleukin-8 (IL-8) release by A549 human lung adenocarcinoma cells, for 24, 48 and 72 hours.The results showed that all oleoresins represent a healthy and high added-value source of lycopene characterized by: i) the absence of toxic impurities such as solvents, pesticides and heavy metals; ii) a high lycopene bioavailability, increased by the presence of co-extracted natural lipophilic components; iii) a high lipophilic antioxidant activity, improved by synergy with other biomolecules. When administered at a concentration of 10 µM for 72 hours, the three lycopene extracts showed conflicting behaviors: watermelon oleoresin increased cell apoptosis and decreased the release of IL-8, a pro-inflammatory cytokine involved in cancer progression; GAC and tomato oleoresins, instead, decreased cell apoptosis and increased IL-8 release. This discrepancy is likely related to a different lycopene/vitamin E ratio, with watermelon oleoresin showing the highest value among the extracts. Shorter incubation time and lower lycopene concentrations did not affect cell viability and IL-8 release. Further studies are required to assess the possible use of the lycopene containing oleoresin/?-CD ICs in health promoting functional foods and nutraceuticals.

This study, for the first time describes the volatiles profile of sparkling wine produced by the traditional method, using a base wine obtained from the "Maresco" cultivar . Maresco cultivar, is one of the minor autochthonous vines in Apulia region, with the largest cultivation area located in Valle d'Itria (Central Apulia, Southern Italy). The grapes are characterized by a good balance of acidity and structure intense, a floral aroma and great a potential for the development of aromatic sparkling wine. Volatile fraction was investigated using an optimized HS-SPME method combined with the gas chromatography/mass spectrometry (GC-MS). The procedure was optimized for the following parameters: i) SPME fiber selection, ii) sample amount; iii) NaCl addition, iv) pre-incubation and extraction time, v) incubation and extraction temperature. Two-factors three-level designs were used in the optimization of pre-incubation/extraction time and temperature. As resulting by the optimization analyses, the following conditions were selected for volatiles extraction required the use of DVB/CAR/PDMS 50/30 ?m as fiber, 5 ml of wine, 2 gr of NaCl, stirring, incubation at 40°C for 30 min and extraction at 40°C for 10 min. Twenty-two compounds were identified, including alcohols, esters, volatiles acids and terpens. The results obtained by the GC-MS analyses for the characterization of volatile compounds in Maresco sparkling wine will be discussed.

Senescence is an integrated response of plants to various internal (developmental) and external (environmental) signals. It is a highly regulated process leading eventually to the death of cells, single organs such as leaves, or even whole plants. In cereals, which are monocarpic plants, senescence represents the final stage of development. In order to study senescence in durum wheat (Triticum turgidum subsp. durum), a cDNA-AFLP analysis was performed. The transcription profiles of plants at different developmental stages (flowering and senescent) were compared. About 2000 cDNA fragments, ranging in size from 160 to 1900 bp, were reproducibly detected. This allowed the identification of 57 differentially expressed cDNAs corresponding to genes belonging to different functional categories related to cellular metabolism, transcription, maintenance of DNA structure, transport and signal transduction. This paper reports the identification of novel durum wheat candidate genes involved in the senescence process, and provides new information about the senescence programme of this important crop species.

Spontaneous grapes must fermentations are promoted by the indigenous yeasts, that are able to confer a distinctive style and quality to the produced wine. The spontaneous fermentations of grape must are at first dominated by non-Saccharomyces yeasts and, in a final stage the alcoholic fermentation process is completed by dominant S. cerevisiae strains (Bauer and Pretorius, 2000).The autochthonous yeast strains are associated to a specific vineyard niche habitat and their role in natural fermentation allows the production of wines with particular features in each microclimatic area (Pérez-Coello et al., 1999). However, in order to avoid the unpredictability of must spontaneous fermentation, the winemakers employ commercial dry active yeast culture for wine industrial productions.Increasing interest in the application of locally selected yeasts for fermentation management has been reported (Tristezza et al., 2012). The employment of autochthonous strains of S. cerevisiae as starters seems to be preferable since they are adapted to all the constraints related to a specific wine-production area (Lopes et al., 2007) and are thus capable to dominate more efficiently the indigenous microflora during the fermentation process. Moreover, autochthonous yeast strains can assure the preservation and/or the enhancement of the typical oenological and sensory features which could be considered representative of an oenological region (Rodríguez et al., 2010). In the present study, we developed and applied a strategy to select S. cerevisiae strains from a larger number of yeast isolates. This was achieved adopting a number of key parameters indicative of the strains technological and enological properties. S. cerevisiae population has been isolated from natural fermentations of grape musts, which derive from grapes sampled from the six most representative Negroamaro and Primitivo producing-areas in Apulia (Southern Italy). The yeast populations were identified by molecular assays (AFLP and sequencing) and some selected representative strains were subjected to physiological, oenological and technological characterization.At the end of the selection procedure, that lasted three years, three indigenous S. cerevisiae strains (one for Primitivo and two for Negroamaro), characterized by interesting technological and oenological properties, were selected. The three selected strains were evaluated by both laboratory tests and semi-industrial scale fermentations to confirm their ability to act as autochthonous fermentation starters. An optimized procedure was worked out for the production of starter biomasses, in order to test them in Negroamaro and Primitivo wine production, at an industrial scale, in six different wineries. The employment of autochthonous starter cultures for the industrial production of typical wines in Apulia will be discussed.

L'articolo illustra gli obiettivi ed i risultati più salienti relativi al PSR Regione Puglia progetto SaVeGraINPuglia.

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.

Oenococcus oeni is the most important LAB species involved in MLF. Highgenotypic heterogeneity of O. oeni strains and a further divergence within thebacterial strain population has been demonstrated. Genotypic diversityseems to be correlated with different metabolic characteristics of bacteria andthis may affect the organoleptic properties of obtained wines. The aim ofthis work was to detect several genes involved in important metabolic pathways(i.e. citrate, sulphur and arginine metabolisms) in 10 indigenous O. oeni strainsselected from Negroamaro wine, a red table wine (Apulia, Italy).This work revealed several new genetic markers that made possible to use a PCRdetection approach to investigate strain heterogeneity for a large range ofgenes encoding enzymes of oenological relevance.

In conventional winemaking, grape must fermentations are carried out by a succession of different yeast species. The Saccharomyces cerevisiae completes the process, whereas its initial stage is dominated by non-Saccharomyces strains, whose by-products contribute to the composition of the wine bouquet. In this study, we evaluated the performance of two selected strains of Hanseniaspora uvarum and S. cerevisiae as multistarters for inoculation of Negroamaro must vinification.

Saccharomyces cerevisiae is the yeast species predominating the alcoholic fermentation of grape must. The aim of this research was to evaluate the impact of indigenous S.cerevisiae strains biodiversity on the aroma of wines from Negroamaro grapes. Grapes collected in two different Negroamaro producing micro districts in Salento (Southern Italy), were subjected to natural fermentation and two indigenous S.cerevisiae populations were isolated. Fifteen strains for each of the two populations were selected and tested by micro fermentation assay in order to evaluate their specific contribute to the volatiles composition and sensory impact of the produced wines. The aromatic profile of wines obtained by each selected strain was characterized by different contents of acetates, ethyl esters of fatty acids, higher alcohols, thus showing to be related to the strains geographical origin. The sensorial analysis of wines produced by the six best performing strains confirmed that they are good candidates as industrial starter cultures, This study indicates that the use of a "microarea-specific" starter culture is a powerful tool to enhance the peculiarity of wines deriving from specific areas. © 2014 Elsevier Ltd.

The polyphenol oxidase (PPO) enzyme, which can catalyze the oxidation of phenolics to quinones, has been reported to be involved in undesirable browning in many plant foods. This phenomenon is particularly severe in artichoke headswounded during the manufacturing process. A full-length cDNA encoding for a putative polyphenol oxidase (designated as CsPPO) along with a 1432 bp sequence upstream of the starting ATG codon was characterized for the first time from [Cynara cardunculus var. scolymus (L.) Fiori]. The 1764 bp CsPPO sequence encodes a putative protein of 587 amino acids with a calculated molecular mass of 65,327 Da and an isoelectric point of 5.50. Analysis of the promoter region revealed the presence of cis-acting elements, some of which are putatively involved in the response to light and wounds. Expression analysis of the gene in wounded capitula indicated that CsPPO was significantly induced after 48 h, even though the browning process had started earlier. This suggests that the early browning event observed in artichoke heads was not directly related to de novo mRNA synthesis. Finally, we provide the complete gene sequence encoding for polyphenol oxidase and the upstream regulative region in artichoke

Table olives are one of the most important fermented food in the Mediterranean countries. Apart from lactic acid bacteria and yeasts that mainly conduct the olive fermentation, molds can develop on the brine surface, and can have either deleterious or useful effects on this process. From the food safety point of view, occurring molds could also produce mycotoxins, so, it is important to monitor and control them. In this respect, identification of molds associated to two Italian and two Greek fermented black table olives cultivars, was carried out. Sixty strains were isolated and molecularly identified as Penicillium crustosum (21), P roqueforti (29), P paneum (1), P expansum (6), P. polonicum (2), P commune (1). A group of 20 selected isolates was subjected to technological (beta-glucosidase, cellulolytic, ligninolytic, pectolytic, and xylanolytic activities; proteolytic enzymes) and safety (biogenic amines and secondary metabolites, including mycotoxins) characterization. Combining both technological (presence of desired and absence of undesired enzymatic activities) and safety aspects (no or low production of biogenic amines and regulated mycotoxins), it was possible to select six strains with biotechnological interest. These are putative candidates for future studies as autochthonous co-starters with yeasts and lactic acid bacteria for black table olive production.

Artemisia annua L., is a herbal plant traditionally used for the treatment of fever. The glandular trichomes of this plant accumulate, although at low levels, artemisinin which is highly effective against malaria. Due to the great importance of this compound many efforts have been made to improve the knowledge on artemisinin production both in plants and in cell cultures. In this study A. annua suspension cultures were established in order to investigate the effects of methyl jasmonate (MeJA) and miconazole treatments on artemisinin biosynthesis. Twenty-two mM MeJA induced a three-fold increase of artemisinin production soon after 30 min. Two hundred mM miconazole induced a 2.5 fold increase of artemisinin production after 24 h treatment but had severe effects on cell viability. The influence of these treatments on the expression of biosynthetic genes was also investigated. MeJA induced an up-regulation of CYP71AV1 while miconazole induced an up-regulation of CPR and DBR2.

The development of fast, reliable and culture-independent molecular tools to detect bacteria producingbiogenic amines deserves the attention of research and ultimately of the food industry in order to protectconsumers' health. Here we present the application of a simple, low-cost, fast and sensitive method toperform microdroplet-based multiplex PCR, directly on a food matrix, for the simultaneous detection ofbacterial genes involved in biogenic amine biosynthesis. After inoculating wine with Lactobacillus brevisIOEB 9809, cell lysis and DNA amplification are performed in one single step, without preliminary nucleicacid extraction or purification treatments. The assay is performed in about 30 min, requiring 150 nL ofstarting sample and it enables the detection of down to 15 bacterial cells. With respect to traditionalculture techniques, the speed, the simplicity and the cheapness of this procedure allow an effectivemonitoring of microbial cells during food-making and processing.

Laccases are biotechnologically interesting enzymes belonging to the polyphenol oxidases family. They are widely distributed throughout the phylogenetic scale from bacteria to mammals. In fungi the analysis of three-dimensional crystal structures of laccases indicate that ascomycete laccases are processed at their C-termini, at a conserved cleavage site, resulting in the proteolytic removal of C-terminal residues. We have isolated and cloned in expression vectors the cDNAs encoding two laccase isoforms (Ery3 and Ery4) from the basidiomycete Pleurotus eryngii. The Ery3 enzyme expressed in Saccharomyces cerevisiae, is functional, whereas the recombinant Ery4 protein does not show enzymaticactivity. In order to explain this evidence, we investigated the relationship between the structure of the C-terminal extension and laccase enzymatic activity. The tasks of the present study were to determine the biological role of laccase C-terminal, and to validate a "molecular engineering" approach for the production of recombinant laccases with novel biochemical properties. Genetically engineered mutant genes were produced from Ery4 by: i) progressive 3'-terminal deletions, ii) point mutations, iii) Ery3/Ery4 chimeras. The mutant genes were expressed in S. cerevisiae and active recombinant laccase isoforms were produced, exhibiting each a different biological behaviour. The correlations between the structural information deriving from both biochemical and bioinformatic analyses shed light on the role of Cterminal region in determining laccase functions. The obtained data also indicated that our approach could represent an efficient method for laccase genetic engineering. To our knowledge, this study has produced the first evidences obtained by biotechnological approach of the involvement of the C-terminal tail in the inactivation/activation process of a basidiomycete laccase.

The characterization of autochthonous Saccharomyces cerevisiae strains is an important step towards the conservation and employment of microbial biodiversity.The utilization of selected autochthonous yeast strains would be a powerful tool to enhance the organoleptic and sensory properties of typical regional wines. In fact, indigenous yeasts are better tailored to a particular must and because of this they are able to praise the peculiarities of the derived wine. The present study described the biodiversity of indigenous S. cerevisiae strains isolated from natural must fermentations of an ancient and recently rediscovered Apulian grape cultivar, denoted as "Susumaniello." The yeast strains denoted by the best oenological and technological features were identified and their fermentative performances were tested by either laboratory assay. Five yeast strains showed that they could be excellent candidates for the production of industrial starter cultures, since they dominated the fermentation process and produced wines characterized by peculiar oenological and organoleptic features.

DNA-based approaches were used to characterize a strain (Salento-1) of Xylella fastidiosa obtained from an olive plant suffering from the syndrome of quick decline in Apulia (South Italy). Salento-1 was indistinguishable from strain CoDiRO previously isolated from olive in Apulia and assigned to X. fastidiosa subsp. pauca. Based on our results and comparative analysis with reported data, the subspecies pauca, multiplex, and fastidiosa may invade olive throughout the world (California, Italy, Argentina and Brazil). The strain Salento-1 has been deposited in the National Collection of Plant Pathogenic Bacteria (NCPPB), England, and in the Belgian Coordinated Collections of Microorganisms (BCCM), Belgium.

Wheat is a staple cereal and an important source of minerals in human diets. Mineral content in the wheat grain has a genetic basis, but the trait is also influenced by environmental factors [1]. Many efforts have been devoted to increase the content of some elements such as Fe, Zn, Mn and Cu, which have a physiological function in the organism. In particular, mapping populations and genetic linkage maps have been suggested as a useful tool in breeding programs [2]. The present work was undertaken to evaluate by ICPOES the variation in macronutrients (Ca, Mg, K), micronutrients (Zn, Fe, Cu, Mn) and toxic trace elements (Cd, Pb) in the whole grains of 29 wheat accessions and cultivars, representative of old and modern wheat germplasm. The wheat genotypes were grown in the experimental field of the University of Bari at Valenzano (Bari, Italy) in 2013/14 (site A) and at Policoro (Matera) in 2014/15 (site B), in a randomized complete block design with three replications and plots [3]. Multivariate Analysis (MVA) of data applied on the whole data (Figure1) showed interesting differences in the mineral content for the considered sites and harvesting seasons. Lower content of Mn and Cd and higher content of Zn, K and Cu were found in sites A and B, respectively.

Oenococcus oeni is the main lactic acid bacterium involved in Malolactic Fermentation since its high adaptation capacity in wine (1). Extensive studies, carried out over the years, furnished a considerable amount of information on the genetic, physiology and metabolism of this bacterium (2-4). The increasing acquisition of data about the secondary metabolic activities exhibited by O. oeni, which impact strongly to sensory properties of wine, stimulated the investigations on variability within indigenous populations isolated from various winemaking environments.Indeed, strains can modified differently the flavour, quality and safety of wine according to their to metabolic diversity and, due to the economic importance of wine, great interest has been addressed to the study of intraspecific heterogeneity of O. oeni (5, 6).In this work, the detection of several genes involved on important metabolic pathways (i.e. citrate, sulphur and arginine metabolism) was performed on 10 indigenous O. oeni strains of Negroamaro wine, a table red wine (Apulia, Italy). These strains were selected from 95 isolates, collected during a spontaneous malolactic fermentation, according to the results of Amplified Fragment Length Polymorphism (AFLP) analysis. It was screened a total of 16 genes, most of them (11) never assayed before on O. oeni. All strains possessed 10 genes encoding enzymes such as malolactic enzyme (mleA), esterase (estA), citrate lyase ?citD, citE and citF), citrate transporter (maeP), ?-acetolactate decarboxylase (alsD), ?-?acetolactatesynthase (alsS), S-adenosylmethionine synthase (metK) and cystathionine ?-lyase (metC) and resulted negative in the detection of genes encoding cystathionine ?-lyase (metB), ornithine transcarbamylase (arcB) and carbamate kinase (arcC) (table 1.). The sequence of PCR fragments of 11 genes of a representative strain (ITEM 15929) were compared to those of three reference O. oeni strain. The indigenous strain phylogenetically resulted more similar to PSU-1 and ATCC BAA1163 than AWRI B429. The present study provides information on population structure of the species and describes new genetic markers useful for detecting the genetic potential of O. oeni strains to contribute to aroma production and to improve the quality of wine.Table 1. Results of PCR detection of different enzyme-encoding genes in a population of 10 O.oeni strains.Target geneAccession number protein activities

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is increasing. In the Greek system, table olives are produced by natural fermentation process, that is not predictable and strongly influenced by the physical-chemical conditions and by the presence of microorganisms contaminating the olives, In this study , we have developed and validated a novel procedure for table olive production based on the use of a mixed yeast/bacteria starter.

Table olives represent one important fermented product in Europe and, in the world, their demand is constantly increasing. At the present time, no systems are available to control black table olives spontaneous fermentation by the Greek method. During this study, a new protocol for the production of black table olives belonging to two Italian (Cellina di Nardò and Leccino) and two Greek (Kalamàta and Conservolea) cultivars has been developed: for each table olive cultivar, starter-driven fermentations were performed inoculating, firstly, one selected autochthonous yeast starter and, subsequently, one selected autochthonous LAB starter. All starters formulation were able to dominate fermentation process. The olive fermentation was monitored using specific chemical descriptors able to identify a first stage (30 days) mainly characterized by aldehydes; a second period (60 days) mainly characterized by higher alcohols, styrene and terpenes; a third fermentation stage represented by acetate esters, esters and acids. A significant decrease of fermentation time (from 8 to 12 months to a maximum of 3 months) and an significant improvement in organoleptic characteristics of the final product were obtained. This study, for the first time, describes the employment of selected autochthonous microbial resources optimized to mimic the microbial evolution already recorded during spontaneous fermentations.

Two approaches were developed in order to select microorganisms suitable to be used in olive millwastewaters bioremediation. By the first approach, three hundred yeasts were isolated from fiveindustrial mills and identified by molecular analysis. The different strains were selected accordingto their capacity to grow in OMW (olive mill wastewaters) as the sole carbon source and to reducephenolics, chemical oxygen demand (COD) and antimicrobial compounds. One Geotrichum candidumisolate was used to set up a whole-cell immobilization system in calcium alginate gel andthe COD and phenolic reduction obtained using the immobilized cells showed respectively a 2.2-and 2-fold increase compared to the removal obtained using free cells. By the second approach, anew protocol was developed to isolate and select aerobic microorganisms from different industrialsamples and environmental niches (soils, OMWs) and able to detoxify olive mill wastewaters.

Egypt is the most important producer and consumer Country of table olives in the world. Within MARSADEV Project, a survey on table olives commercial products collected in the Northwestern region of Matrouh Governorate in Egypt was performed. Low safety conditions were observed in these products. Some of them are affected by the occurrence of high counts of Pseudomonas, Staphylococci and Enterobacteriaceae and some of them by the presence of biogenic amines. All tested samples resulted very poor in compounds related to microbial activities, such as organic acids and olives-derived phenols. Also, from the organoleptic point of view, odour profiles of tested olives are very modest. Scarce hygienic conditions were registered within domestic or small industrial plants along the whole production chain: from the harvest, to the processing and to the table olives packaging and storage. Several operations, such as the use of microbial starters, the monitoring of the fermentation process, together with some operational precautions, i.e. the heat treatment of water before olive soaking and the use of gloves during all operations, have been proposed to improve the process safety. The use of selected yeasts as starter for table olives production and the control of physical-chemical parameters during fermentation allowed to control the process, to sensitively improve organoleptic and safety traits of table olives, to reduce time required to end the transformation process from 8 to 2 months.Guidelines suggesting good practices and describing the most important operational steps to be followed for table olives processing have been produced for Bedouin rural communities living in this area, in particular for women within the concerned communities.

We report the effect of heat, drought and combined stress on the expression of a group of genes that are up-regulated under these conditions in durum wheat (Triticum turgidum subsp. durum) plants. Modulation of gene expression was studied by cDNA-AFLP performed on RNAs extracted from flag leaves. By this approach, we identified several novel durum wheat genes whose expression is modulated under different stress conditions. We focused on a group of hitherto undescribed up-regulated genes in durum wheat, among these, 7 are up-regulated by heat, 8 by drought stress, 15 by combined heat and drought stress, 4 are up-regulated by both heat and combined stress, and 3 by both drought and combined stress. The functional characterization of these genes will provide new data that could help the developing of strategies aimed at improving durum wheat tolerance to field stress.

Two approaches were developed in order to select microorganisms suitable to be used in olive mill wastewaters bioremediation. By the first approach, three hundred yeasts were isolated from five industrial mills and identified by molecular analysis. The different strains were selected according to their capacity to grow in OMW (olive mill wastewaters) as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One Geotrichum candidum isolate was used to set up a whole-cell immobilization system in calcium alginate gel and the COD and phenolic reduction obtained using the immobilized cells showed respectively a 2.2- and 2-fold increase compared to the removal obtained using free cells. By the second approach, a new protocol was developed to isolate and select aerobic microorganisms from different industrial samples and environmental niches (soils, OMWs) and able to detoxify olive mill wastewaters.

The high content of bioactive molecules in bran and germ portions of the kernel makes whole wheat grain a functional food to prevent several degenerative and chronic diseases. In particular, whole wheat caryopsides contain high amounts of phenolic acids having favourable effects on human health due to their antioxidant and anti-inflammatory activity. Genetic variation for phenolic acid content and composition has been documented among different cereal grains with particular regard to soft, medium and hard wheat (Triticum aestivum L.). Less attention has been devoted to tetraploid wheats (Triticum turgidum L.). The objective of this study was to evaluate the variability for phenolic acid content and composition in a core collection of 112 genotypes of tetraploid wheat, including cultivars, landraces and wild accessions. In particular, the core collection comprised 65 durum cultivars (T. turgidum L. ssp. durum), 12 accessions of ssp. turgidum, 8 of ssp. turanicum, 8 of ssp. polonicum, 3 of ssp. carthlicum, 9 of ssp. dicoccum and 7 of ssp. dicoccoides.Phenolic acids extracted from the whole semolina flour were identified and quantified by HPLC-DAD analysis. The results showed a significant genotype influence on the content of phenolic acids. The ssp. durum cultivars showed a higher content of phenolic acids compared to landraces and wild accessions. On average, total phenolic acid content ranged from about 830 ?g/g dry weight (ssp. durum) to 570 ?g/g dry weight (ssp. dicoccum and ssp. turgidum). Large and significant variation for total phenolic acid content was observed among the durum wheat cultivars, with cv. Ambral, Pedroso, Primadur, Mida and Tito largely exceeding 1000 ?g/g dry weight of total phenolic acids. Ferulic acid was the most abundant phenolic acid in all genotypes, particularly in the ssp. durum (on average 654 ?g/g dry weight), whereas the ssp. dicoccum, turgidum and diccoides showed the least content (less than 450 ?g/g dry weight). Sinapic acid was the second phenolic acid for abundance and it varied from ~54 ?g/g dry weight (ssp. carthlicum) to ~140 ?g/g dry weight (ssp. durum). Other phenolic acids identified in this work were p-coumaric acid (19 ÷ 26 ?g/g dry weight), vanillic acid (5 ÷ 9 ?g/g dry weight), syringic acid (2 ÷ 7 ?g/g dry weight) and 2,4- dihydroxybenzoic acid (1 ÷ 3 ?g/g dry weight). In conclusion, the results showed that the content of phenolic acids in the studied tetraploid wheat collection varied significantly, suggesting that a number of durum wheat elite varieties could be used for breeding purposes. Also, the use of cultivars with high phenolic acid content could allow the production of whole durum wheat food products with enhanced health-related benefits.

Phenolic acids are a specific class of phenolic compounds present in all cereals. The antioxidant properties of phenolic acids have been demonstrated both in vivo and in vitro, suggesting that these components may play an important role in preventing human diseases related to aging. The genetic variation for phenolic acid concentration was extensively studied in bread and durum wheat collections in Europe and in some areas of the Mediterranean Region, but a comprehensive survey in Mexican wheat materials is lacking. The aim of the present research has been to evaluate the variability present for individual and total phenolic acids present in the wholemeal flour of a large collection of Mexican durum wheat landraces and varieties developed by CIMMYT. Environmental effects, including full and reduced irrigation, mild and severe heat stress were considered to estimate potential association between phenolic acid content and different level of water-deficit and heat stress conditions. Seven phenolic acids were identified in all tested samples, of which ferulic acid resulted the most abundant, followed by sinapic, p-coumaric, vanillic, syringic, p-hydroxybenzoic and caffeic acids. Considering the whole wheat collection, the mean value for total phenolic acid content was about 600 ?g/g d.m., and a large variation was observed among the genotypes. In particular, the landraces showed a threefold range of variation, from a minimum of 300 ?g/g d.m. to a maximum of 840 ?g/g d.m., which was larger as compared to that of varieties. About the effect of the environment, PCA analysis of the data showed that drought and heat stress significantly increased phenolic acid concentration. These results could be useful for breeders to develop varieties with relevant levels of phenolic acids, which in turn could show high antioxidant activity and enhance the health promoting value of durum wheat based products.

Table olives are one of the most important traditional fermented vegetables in Europe and their world consumption is constantly increasing. In the Greek style, table olives are obtained by spontaneous fermentations, without any chemical debittering treatment. Evolution of sugars, organic acids, alcohols, mono and polyphenol compounds and volatile compounds associated with the fermentative metabolism of yeasts and bacteria throughout the natural fermentation process of the two Italian olive cultivars Cellina di Nardò and Leccino were determined. A new protocol was developed and applied aimed at the technological characterization of LAB and yeast strains as possible candidate autochthonous starters for table olive fermentation from Cellina di Nardò and Leccino cultivars. The study of the main physical, chemical and aromatic parameters during fermentation helped to determine chemical descriptors that may be suitable for monitoring olive fermentation. In both the analyzed table olive cultivars, aldehydes proved to be closely related to the first stage of fermentation (30 days), while higher alcohols (2-methyl-1-propanol; 3-methyl-1-butanol), styrene, and o-cymene were associated with the middle stage of fermentation (90 days) and acetate esters and acetic acid with the final step of olive fermentation (180 days).

Table olives are one of the most important traditional fermented vegetables in Europe and their worldconsumption is constantly increasing. Conservolea and Kalam
ata are the most important table olivesGreek varieties. In the Greek system, the final product is obtained by spontaneous fermentations,without any chemical debittering treatment. This natural fermentation process is not predictable andstrongly influenced by the physical-chemical conditions and by the presence of microorganismscontaminating the olives. Natural fermentations of Conservolea and Kalam
ata cultivars black olives were studied in order to determine microbiological, biochemical and chemical evolution during the process. Following the process conditions generally used by producers, in both cultivars, yeasts were detected throughout the fermentation, whereas lactic acid bacteria (LAB) appeared in the last staged of the process. A new optimized specific protocol was developed to select autochthonous yeast and LAB isolates that can be good candidates as starters. These microorganisms were pre-selected for their ability to adapt to model brines, to have beta-glucosidase activity, not to produce biogenic amines. Chemical compounds deriving by microbiological activities and associated to the three different phases (30, 90 and 180 days) of the fermentation process were identified and were proposed as chemical descriptors to follow the fermentation progress.

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 identification of novel plant-based functional foods or nutraceutical ingredients that possess bioactive properties with antioxidant function has recently become important to the food, nutraceutical and cosmetic industries. This study evaluates the polyphenolic composition, identifies bioactive compounds and assays the total antioxidant capacity of Prunus mahaleb L. fruits collected from different populations and sampling years in the countryside around Bari (Apulia Region, Italy).RESULTS: We identified nine polyphenolic compounds including major anthocyanins, coumaric acid derivatives and flavonols from P. mahaleb fruits. The anthocyanin content (in some populations > 500 mg/100 g fresh weight; FW) in the fruit was comparable to that reported for so-called superfruits such as bilberries, chokeberries and blackcurrants. Coumaric acid derivatives comprised a large portion of the total polyphenolic content in the P. mahaleb fruits. Antioxidant activities, assessed using oxygen radical absorbance capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) assays, measured up to 15 and 4.5 mmol/100 g FW, respectively. CONCLUSION: Our findings suggest that mahaleb fruit (currently not consumed fresh or used in other ways) could serve as a source of bioactive compounds and therefore find interest from the functional food and nutraceutical industries, as a natural food colorant and antioxidant ingredient in the formulation of functional foods.

Almonds (Prunus dulcis (Mill.) D.A. Webb or Amygdalus communis L.) are used in the preparation of bakery and confectionery products including almond cookies, marzipan and almond milk. The first productive step consists of blanching and peeling the almonds, so as to remove the skin. Skins account for 6-8% of the seed and are mainly destined to cattle feeding. Blanching water represents a waste, involving a disposal expense for the producers.Therefore, in this work we evaluated the potential of by-products of almond processing, namely blanched skins and blanching water, as ingredients of functional bakery products, due to the presence of phenolic compounds and, in the skin, also fiber. We started by optimizing skin drying; then we quali-quantitative determined the phenolic compounds; finally we assessed the impact of by-products on the rheology of composite dough with wheat flour.Oven-drying at 60 °C for 30 min was less-time consuming than other time-temperature combinations tested and retained good odor notes. Moreover, this drying condition allowed to maintain higher content of phenolics (814 µg/g d.m. by HPLC) and higher antioxidant activity than sun-drying. Blanching water, on the other hand, had a content of phenolic compounds accounting for 917 µg/mL.The alveograph and farinograph indices of composite dough was altered by adding dried almond skins at doses higher than 30 and 50 g/kg, respectively. Blanching water did not changed significantly these indices.In conclusion, almond skins could be used at percentages lower than 30% or in bakery products tolerating a weak gluten network, such as cookies. Blanching water, instead, could be added to any kind of bakery good with no rheological negative effect.

There is an increasing consumer and retailer demand for new natural colorants. Anthocyanins are a class of molecules already used as water-soluble natural color- ant and for their health effects. Prunus mahaleb L. is a tree native to the Mediterranean area, producing highly pig- mented small-stone fruits currently not used for fresh con- sumption due to their astringent and sour taste. In the pre- sent study, we set up a protocol to obtain a "mahaleb fruit concentrated extract" (mfce) and characterized it chemi- cally by high-performance liquid chromatography/diode array detection. Anthocyanins, flavonols, flavanols, cou- marin, sugars, ascorbic acid and organic acids were identi- fied and quantified. After characterization, the antioxidant capacity of the mfce was assayed in vitro by three different methods: Trolox equivalent antioxidant capacity, Oxygen Radical Absorbance Capacity and Folin-Ciocalteu assays. The concentrated extract produced and characterized in this study showed a high content of anthocyanins, flavonols and coumarin and a strong antioxidant capacity. Therefore, mfce may be of interest as a natural food colorant and as an ingredient in the formulation of functional foods.

Regina tomato, a locally cultivated Italian landrace, is listed as an item in the 'List ofTraditional Agri-Food Products' of the Italian Department for Agriculture and itemised as 'SlowFood presidium' by the Slow Food Foundation. It is classified as a long-storage tomato since it can bepreserved for several months after harvest thanks to its thick and coriaceous skin. Three ecotypeswere investigated for main physical and chemical traits both at harvest and after three months ofstorage. Experimental results indicate that this tomato landrace has a qualitative profile characterizedby high concentrations of tocopherols, lycopene and ascorbic acid (maximum 28.6 and 53.7 mg/kgfresh weight, FW, and 0.28 mg/g FW, respectively) even after a long storage time, together withlower average Total Soluble Solids. The initial and post-storage contents of the bioactive compoundschanged at a different rate in each ecotype (i.e., in Monopoli Regina tomato the highest content of-Tocopherol, thereafter reduced to the same level of the other two ecotypes). These results indicateunique and unmistakable features of this long-storage tomato, closely linked to the geographicorigin area that include both natural (available technical inputs) and human (specific culturalpractices) factors.

Ochratoxin A (OTA) undergoes to enzymatic biodegradation by proteolytic enzymes able to hydrolyze its amide bond with consequent formation of ochratoxin ? (OT?) and L-?-phenylalanine. This mechanism can be regarded as a detoxification method since OT? and L-?-phenylalanine are considered as less and non-toxic, respectively. Different microorganisms belonging to bacterial, yeast and fungal species have been reported to degrade OTA. Several enzymes may be involved in microbiological degradation of OTA, such as carboxypeptidase A, lipase, and acid proteases. Also Aspergillus carbonarius, one of the most important fungal producer of OTA and the major responsible of OTA contamination of grapes, wine and by-products, turned out to be able to degrade OTA. In the attempt to identify the enzyme able to degrade OTA in this microorganism, a protease encoding gene, located in the genomic region recognized as OTA cluster, has driven our attention. In particular, this gene, namely Acap1 of A. carbonarius strain ITEM 5010, encodes for an aspartic protease and is located downstream of the core genes involved in OTA biosynthesis. Acap1gene was isolated and cloned for its characterization. The gene is 1367 bp long and the in silico analyses of the deduced protein sequence of 421 aa revealed that the AcAP1 protein shows the functional typical structure of aspartic protease enzymes. Aspartyl proteases are a highly specific family of proteases that tend to cleave dipeptide bond and they are optimally active at acidic pH. Heterologous recombinant production of the AcAP1 protein has been carried out in order to verify the involvement of AcAP1 in the ability of A. carbonarius in OTA degradation and to analyze its structural and functional properties for a potential biotechnological use of the enzyme. Acap1 gene was cloned in two expression vectors (p426 and pYES), carrying a constitutive and an inducible promoter, respectively, in fusion with a sequence encoding for a His-tag at the 3'-terminus. Three different strains of Saccharomyces cerevisiae, carrying diverse genotypes, have been transformed. Data concerning the protein expression by yeast, evaluation of the protease activity, and purification of the recombinant protein will be produced.

The ERY4 laccase gene of Pleurotus eryngii is not biologically active when expressed in yeast. To explain this finding, we analysed the role of the C-terminus of Ery4 protein by producing a number of its different mutant variants. Two different categories of ERY4 mutant genes were produced and expressed in yeast: (i) mutants carrying C-terminal deletions and (ii) mutants carrying different site-specific mutations at their C-terminus. Investigation of the catalytic properties of the recombinant enzymes indicated that each novel variant acquired different affinities and catalytic activity for various substrates. Our results highlight that C-terminal processing is fundamental for Ery4 laccase enzymatic activities allowing substrate accessibility to the enzyme catalytic core. Apparently, the last 18 amino acids in the C-terminal end of the Ery4 laccase play a critical role in enzyme activity, stability and kinetic and, in particular biochemical and structural data indicate that the K532 residue is fundamental for enzyme activation. These studies shed light on the structure/function relationships of fungal laccases and will enhance the development of biotechnological strategies for the industrial exploitation of these enzymes.

Sea fennel (Crithmum maritimum L.) is aperennial halophyte species typical of coastal ecosystems,used fresh in traditional cuisine and folkmedicine due to its sensory properties and a goodcontent of healthy compounds. Although consideredas a promising biosaline crop, this halophyte isunderutilized for commercial cultivation possiblydue to a shortage of its consumer demand. Forpromoting a full exploitation of this species, a newfood product was obtained by drying sea fennel usingdifferent treatments (air-drying, microwave-drying,microwave-assisted air-drying and freeze-drying).Water activity, essential oil content, chlorophylls,surface colour, colouring power and sensory evaluationwere analyzed. All drying treatments allow toobtain a good water activity but significantly reducedthe content of essential oils and chlorophylls. Freezedryingand microwaving preserved the surface colourparameters more than other drying treatments, whilefreeze-drying gave the product the best colouringpower. Based on sensory analysis, microwave-drying,microwave-assisted air-drying and freeze-dryingshowed the highest scores among the drying methods.Taken together the results indicate that microwavingand freeze-drying are optimal for preserving qualitativetraits, including organoleptic properties, in driedsea fennel for food use. Furthermore, dried sea fennelcan be usefully exploited in human food not only forits aromatic traits but also for its food colouring powerlike other plant derived natural colorants. It could beconcluded that this underutilized crop could play abetter role for making up a sustainable food productionsystem.

Pomegranate, tomato and grape seeds are quantitatively relevant agri-food by-products rich in molecules beneficialto human health. To valorize this resource, the composition and antioxidant activity of seeds and derivingsupercritical CO2 (SC-CO2) extracted oleoresins were evaluated. Grape seeds showed the highest content of totalphenolic compounds (33.9 mg GAE/g), flavonoids (15.6 mg CE/g) and condensed tannins (14.0 mg CE/g), whiletomato seeds presented the highest content of tocochromanols (159.6 ?g/g). Grape seeds showed the highesttotal antioxidant activity (178.2 ?mol TE/g), as evaluated by TEAC assay, followed by pomegranate (19.8 ?molTE/g) and tomato (9.8 ?mol TE/g). Oleoresin yields obtained by SC-CO2 extraction from the seeds ranged between3.1 (pomegranate) to 7.8 (tomato) g oleoresin/100 g. Total tocochromanols were abundant in pomegranate(2008 ?g/g) and tomato (1769 ?g/g) oleoresins; a relatively low amount was instead detected in theoleoresin extracted from grape seeds (636 ?g/g). Carotenoids were not detected in all oleoresins. Pomegranateoleoresin had a higher antioxidant activity than the others. Mono- and polyunsaturated fatty acids were moreabundant than saturated in all oleoresins, with the highest percentage of unsaturated fatty acids detected inpomegranate seed oleoresin (~90%), mainly due to punicic acid (~70%).

In recent years, the interest towards functional properties of foods has increased progressively and a relevant role has been played by antioxidant compounds, such as anthocyanins, carotenoids and phenolic acids, able to scavenge free radicals. Purple wheat contains higher levels of anthocyanins than conventional wheat cultivars. Durum wheat is also characterized by relevant levels of carotenoids, compared to soft wheat. The aim of this work has been to breed durum wheat lines characterized by high anthocyanin and high carotenoid levels. This strategy enhances the antioxidant activity of the derived whole meal and processed products (Pasqualone et al., 2015). Purple wheat line CItr 14629 (Triticum turgidum ssp. durum (Desf.) Husnot), derived from an Ethiopic landrace kindly provided by the United States Department of Agriculture (USDA), and the Italian durum wheat cv. Grecale, characterized by high carotenoid content, have been crossed and the segregant generations grown and evaluated according to the pedigree method. Twenty-five F6 lines has been selected on the basis of pericarp color, plant height, 1000-seed weight, protein content, and yellow index of whole meal flour (known to be related to carotenoid pigments). The total anthocyanins content of these 25 lines, all characterized by maximum score for visual inspection of pericarp color (score = 5, range 1-5 from yellow to dark purple), ranged from 0.94 to 43.06 mg/kg cyanidin-3-glucoside (Cy-3-Glu) on dry matter, whereas the purple parent showed a mean value of 37.13 mg/kg Cy-3-Glu (d.m.). The variability of this range evidenced that the visual evaluation of pericarp color, usually considered for selecting purple wheats (Knievel et al., 2009), is not sufficiently precise compared to spectrophotometric measure of total anthocyanins in the extracts obtained from whole meal. The lines showing the highest anthocyanin content will be characterized for the single anthocyanins and for the major phenolic acids by means of HPLC analysis. No correlation was found between total anthocyanin content and the a* colorimetric index of whole meal. Protein content of the selected lines was comprised between 13.3% and 17.9% d.m., whereas 1000-seed weight ranged from 33.3 g to 56.3 g. Yellow index was in the range 12.9-14.6. Plant height of the selected lines was lowered to 80-90 cm in comparison to 130 cm of the Ethiopian parental line. The selected lines are currently under agronomic evaluation in different field trials and different nutritional traits will be particularly considered to assess the grain and flour quality.

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, Chemical Oxygen Demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.

Lycopene is a red carotenoid thought effective in preventingsome types of cancers and modulating immune and inflammatory responses.Though some fruits share the common feature of synthesizing and storinglycopene as main carotenoid within chromoplasts, they significantlydiffer in the lipophilic phytocomplex. The purpose of this work was: i)to extract solvent-free lycopene-rich oleoresins from gac, tomato andwatermelon ripe-fruits by supercritical CO2 suitable for functional foodand nutraceutical formulations; ii) characterize the oleoresins withrespect to carotenoids, tocochromanols, lipids, metals and antioxidantactivity; iii) prepare stable aqueous suspensions through oleoresinclathration into ?-cyclodextrins (?-CDs); iv) investigate the effect ofeach lycopene-containing suspension on viability of human lungadenocarcinoma cells.The results showed that all oleoresins represent a safe added-valuesource of lycopene characterized by high lipophilic antioxidant activity,improved by the synergy with other biomolecules. Interestingly, whenadministered at identical concentrations (0.5-10 ?M) of lycopene, theextracts showed conflicting behaviors: gac and tomato suspensionssignificantly increased cell viability, whereas watermelon oleoresin/?-CDsuspension significantly decreased cell viability. Although furtherstudies are required, this research suggests a fundamental role ofphytocomplex in affecting the responses of cancer cell survival to the invitro administration of lycopene.

Stimulated production of secondary phenolic metabolites and proline was studied by using cell cultures of artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] submitted to nutritional stress. Artichoke cell cultures accumulated phenolic secondary metabolites in a pattern similar to that seen in artichoke leaves and heads (capitula). This paper shows that both callus and cell suspension cultures under nutritional stress accumulated phenolic compounds and proline, at the same time their biomass production was negatively affected by nutrient deficiency. The results obtained strongly suggest that plant tissues respond to nutrient deprivation by a defensive costly mechanism, which determines the establishment of a mechanism of trade-off between growth and adaptive response. Furthermore, the results of this research suggest that perception of abiotic stress and increased phenolic metabolites are linked by a sequence of biochemical processes that also involves the intracellular free proline and the oxidative pentose phosphate pathway. The main conclusion of this paper is that, once calli and cell suspension cultures respond to nutrient deficiency, in acclimated cells the establishment of a negative correlation between primary metabolism (growth) and secondary metabolism (defence compounds) is observed.

Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). 27However, the inoculation of lactic acid bacteria together with yeast starter cultures is a promising system to enhance the quality and safety of wine. In recent years, the use of immobilized cell systems has been investigated, with interesting results, for the production of different fermented foods and beverages. In this study we have carried out the simultaneous immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used them in microvinifications tests to produce Negroamaro wine. The process was monitored by chemical and sensorial analyses and dominance of starters and cell leaking from beads were also checked. Co-immobilization of S. cerevisiae and O. oeni allowed to perform an efficient fermentation process, producing low volatile acidity levels and ethanol and glycerol concentrations comparable with those obtained by cell sequential inoculum and co-inoculum of yeast and bacteria cells in free form. More importantly, co-immobilization strategy produced a significant decrease of the time requested to complete AF and MLF. The immobilized cells could be efficiently reused for the wine fermentation at least three times without any apparent loss of cell metabolic activities. This integrated biocatalytic system is able to perform simultaneously AF and MLF, producing wines improved in organoleptic traits in comparison with wines fermented following traditional sequential AF and MLF with free cell starters. The immobilized-cell system, that we here describe for the first time in our knowledge, offers many advantages over conventional free cell fermentations, including: (i) elimination of non-productive cell growth phases; (ii) feasibility of continuous processing; (iii) regeneration and re-use of the biocatalyst.

Traditional vinification process is undertaken with the inoculation of the lactic acid bacteria (LAB) at the end of alcoholic fermentation (AF) to induce malolactic fermentation (MLF). MLF is an important phase during winemaking and the LAB co-inoculation with yeast starter represents a promising approach to enhance the quality and safety of wine. In this investigation we have studied: i) the effect of timing of LAB inoculation on the vinification dynamics and chemical features of Negroamaro wines; ii) the interactions between two commercial yeast and two commercial Oenococcus oeni strains. The fermentations dynamics were monitored by microbial counts, quantifying L-malic acid concentration and analyzing the volatile compounds contents in the obtained wines. Our results indicate that simultaneous yeasts/bacteria inoculation at the beginning of vinification reduces the processes duration and simultaneously lowers of volatile acidity. Wine obtained after co-inoculum showed a profile dominated by red and ripe fruits notes associated to esters and to buttery and creamy notes linked to diethyl succinate and ethyl lactate. Furthermore, compatibility specification between commercial yeasts and LAB strains were observed, suggesting the importance of the assessment of microbial-compatibility before their utilization in large-scale vinification.

Strain SPC-1T was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide and formed slimy, viscous, orange-pigmented colonies and grew optimally at around pH 6.0-6.5 and 26-30°C in the presence of 0-0.5% NaCl. Phylogenetic analysis based on comparisons of 16S rRNA gene sequences demonstrated that SPC-1T clustered together with species of the genus Sphingomonas sensu strictu. The G+C content of the DNA (66.1 mol%), the presence of Q-10 as the predominant ubiquinone, sym-homospermidine as the predominant polyamine, and 2-hydroxymiristic acid (14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomical position. 16S rRNA gene sequence analysis showed that SPC-1 was most closely related to Sphingomonas hankookensis ODN7T, Sphingomonas insulae DS-28T and Sphingomonas panni C52T (98.19%, 97.91% and 97.11% similarity, respectively). However, DNA-DNA hybridization analysis did not reveal any relatedness at the species level. Further differences were apparent in biochemical traits, and fatty acid, quinone and polyamine profiles leading us to conclude that strain SPC-1T (JCM 17498; ITEM 13494) represents a new species of Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed. A component analysis of the exopolysaccharide (named SPC-1T EPS) suggested that it represents a novel type of sphingan containing glucose, rhamnose, mannose and galactose, while glucuronic acid, which is commonly found in sphingans, was not detected.

Celiac disease is an immune-mediated enteropathy precipitated by the ingestion of gluten-containing foodsin genetically predisposed children and adults. After a positive diagnosis for celiac disease, the only availabletreatment is to adopt a gluten-free diet, and evaluation of the absence of gluten in foods is crucial for the health ofceliac patients. In the present study, a recombinant glutamine-binding protein (GlnBP) from Escherichia coli showedits ability to recognize peptides deriving from digested wheat flour. GlnBP and the commercially available 4F3monoclonal antibody, raised against a region of the ?-gliadin peptide 33-mer from wheat, demonstrated the abilityto detect gliadin extracted from wheat flour. Recombinant GlnBP and 4F3 monoclonal antibody were used as newcapture agents for the development of a protein chip able to detect gluten in foods. The protein microarray systemhas proven to detect the presence of gliadin in a range of concentrations between 500 and 5 ppm.

Table olives are one of the most important traditional fermented vegetables in Southern European countries and their consumption is constantly increasing throughout the world. Today, the industrial production of black table olives is carried out by spontaneous fermentation processes which are not predictable and are strongly influenced by the autochthonous microflora, the physical-chemical conditions, the availability of fermentable substrates and salt content. Evolution of sugars, organic acids, alcohols, mono and polyphenol compounds and volatile compounds associated with the fermentative metabolism of yeasts and bacteria throughout the natural fermentation process of the two Italian olive cultivars Cellina di Nardò and Leccino were determined. A new protocol was developed and applied aimed at the selection of LAB and yeast strains as candidate autochthonous starters for table olive fermentation from Cellina di Nardò and Leccino cultivars.

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging.Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from Endoplasmic Reticulum to Vacuole trafficking.The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

Carotenoids are well known for their nutritional properties and health promoting effects representing attractive ingredients to develop innovative functional foods, nutraceutical and pharmaceutical preparations. Pumpkin (Cucurbita spp.) flesh has an intense yellow/orange color owing to the high level of carotenoids, mainly ?-carotene, ?-carotene, ?-cryptoxanthin, lutein and zeaxanthin. There is considerable interest in extracting carotenoids and other bioactives from pumpkin flesh. Extraction procedures able to preserve nutritional and pharmacological properties of carotenoids are essential. Conventional extraction methods, such as organic solvent extraction (CSE), have been used to extract carotenoids from plant material for a long time. In recent years, supercritical carbon dioxide (SC-CO2) extraction has received a great deal of attention because it is a green technology suitable for the extraction of lipophylic molecules and is able to give extracts of high quality and totally free from potentially toxic chemical solvents. Here, we review the results obtained so far on SC-CO2 extraction efficiency and quali-quantitative composition of carotenoids from pumpkin flesh. In particular, we consider the effects of (1) dehydration pre-treatments; (2) extraction parameters (temperature and pressure); the use of water, ethanol and olive oil singularly or in combination as entrainers or pumpkin seeds as co-matrix.

This study investigates the effects of tomato puree fortification (ftp) with several anthocyanin-rich food colorants on bioactive compound content (phenolics, isoprenoids), antioxidant capacity, in vitro biological activities and consumer acceptance. Tomato puree (tp) was added with different anthocyanin extracts from Daucus carota L. var. atrorubens (Anthocarrot), Vitis vinifera L. fruit skins (Enocolor), Sambucus nigra L. (Elderberry) or Prunus mahaleb L. (Mahaleb) fruits. The consumer acceptance (colour, flavor, taste, visual appearance) was at high level, except for Mahaleb-added extracts. Compared to the control (tp), the addition of colouring extracts increased significantly the total phenolic content, before pasteurization, in addition to the expected anthocyanin content. However, after pasteurization, only Anthocarrot-ftp preserved increased phenolic (+53%) content, as well as higher antioxidant capacity (50%). Consistently, against tp, Anthocarrot-ftp exhibited an increased anti-inflammatory capacity as showed by the reduced expression of vascular cell adhesion molecule (VCAM)-1 in human cultured endothelial cells, under inflammatory conditions.

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is a well-known refreshing and thirst-quenching typical summer fruit. It has attracted considerable interest for the presence of lycopene and citrulline, biomolecules with undisputed health-promoting effects. Numerous epidemiological studies have revealed the involvement of lycopene in prevention of many degenerative diseases, including arteriosclerosis, diabetes and cancer, likely acting as antioxidant and/or through still poorly understood different molecular mechanisms [1]. Likewise, citrulline, a water-soluble, non-essential amino acid, was recently claimed as having great potential in prevention and treatment of Alzheimer's disease, dementia, erectile dysfunction, high blood pressure, diabetes and, as food supplement, in improving athletic performance [2]. Given the nutritional importance of these bioactive molecules, the preparation of natural concentrated extracts, useful as high-value ingredients for the production of nutraceuticals and/or pharmaceuticals, could represent a viable alternative to the usual production and distribution chain of watermelon and a sustainable process for non-marketable fruits and processing byproducts valorization, opening new opportunities for agri-food industry development.In the last decades the interest towards green, environmentally friendly and safe extraction technologies has strongly increased. Supercritical CO2 extraction has been successfully used to obtain lycopene containing oleoresins from freeze-dried tomato matrices [3,4]. The homogenized flesh of red watermelon (cv. Dumara) peponides was centrifuged to prepare a lycopene rich freeze-dried matrix suitable for supercritical CO2 extraction from the pellet and a citrulline containing powder from the supernatant. The optimal physico-chemical parameters to maximize lycopene extraction yields from the watermelon matrix have been identified. The presence of lipids from watermelon seeds in the matrix has a positive effect on oleoresin extraction. The addition of an oleaginous co-matrix consisting of roughly grinded hazelnut seeds did not further increase the extraction yield. The use of ethanol as co-solvent resulted in a significant increase in lycopene extraction but not of citrulline. The biochemical analyses showed that the extract is a healthy and high added-value source of lycopene and other valuable lipophilic antioxidants useful as bioactive color additive in functional food, nutraceutical, cosmeceutical and pharmaceutical formulations.

Lycopene is a carotenoid pigment found in a wide variety of vegetables and fruits. More than six hundred carotenoids have been identified in nature, but only twenty are present in human blood and tissues, including lycopene (the most abundant in plasma), ?-carotene, lutein and zeaxanthin [1]. Several exogenous pigmented antioxidants act on cancer progression and have immune-modulating effects by free radical scavenging activity and/or still unknown mechanisms. However, the controversy recently arisen for some of them requires a careful investigation in relation to their source and synergistic effects [2]. The purpose of this work was: i) to optimize the extraction of lycopene from ripe fruits of tomato, watermelon and GAC by supercritical CO2 in order to obtain natural, solvent-free lycopene containing oleoresins suitable as ingredients for the formulation of innovative functional foods and/or nutraceuticals; ii) to characterize each oleoresin with respect to carotenoids, tocochromanols, lipids, lipophilic and hydrophilic antioxidant activities, micro and macro elements; iii) the preparation of inclusion complexes (ICs) of the oleoresins in food grade ?-cyclodextrins (?-CDs) as hydrophilic nanocarriers [3]; iv) to assess, in-vitro, the effects of administration of each oleoresin/?-CD IC at different concentrations (0.5 to 10 µM) on cell apoptosis and on Interleukin-8 (IL-8) release by A549 human lung adenocarcinoma cells, for 24, 48 and 72 hours.The results showed that all oleoresins represent a healthy and high added-value source of lycopene characterized by: i) the absence of toxic impurities such as solvents, pesticides and heavy metals; ii) a high lycopene bioavailability, increased by the presence of co-extracted natural lipophilic components; iii) a high lipophilic antioxidant activity, improved by synergy with other biomolecules. When administered at a concentration of 10 µM for 72 hours, the three lycopene extracts showed conflicting behaviors: watermelon oleoresin increased cell apoptosis and decreased the release of IL-8, a pro-inflammatory cytokine involved in cancer progression; GAC and tomato oleoresins, instead, decreased cell apoptosis and increased IL-8 release. This discrepancy is likely related to a different lycopene/vitamin E ratio, with watermelon oleoresin showing the highest value among the extracts. Shorter incubation time and lower lycopene concentrations did not affect cell viability and IL-8 release. Further studies are required to assess the possible use of the lycopene containing oleoresin/?-CD ICs in health promoting functional foods and nutraceuticals.

In oenology, the utilization of mixed starter cultures composed by Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers in order to enhance sensory quality and complexity of the final product without compromising the general quality and safety of the oenological products. In fact, several non-Saccharomyces yeasts are already commercialized as oenological starter cultures to be used in combination with Saccharomyces cerevisiae, while several others are the subject of various studies to evaluate their application. Our aim, in this study was to assess, for the first time, the oenological potential of H. uvarum in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for industrial wine production. Three previously characterized H. uvarum strains were separately used as multi-starter together with an autochthonous S. cerevisiae starter culture in lab-scale micro-vinification trials. On the basis of microbial development, fermentation kinetics and secondary compounds formation, the strain H. uvarum ITEM8795 was further selected and it was co- and sequentially inoculated, jointly with the S. cerevisiae starter, in a pilot scale wine production. The fermentation course and the quality of final product indicated that the co-inoculation was the better performing modality of inoculum. The above results were finally validated by performing an industrial scale vinification The mixed starter was able to successfully dominate the different stages of the fermentation process and the H. uvarum strain ITEM8795 contributed to increasing the wine organoleptic quality and to simultaneously reduce the volatile acidity. At the best of our knowledge, the present report is the first study regarding the utilization of a selected H. uvarum strain in multi-starter inoculation with S. cerevisiae for the industrial production of a wine. In addition, we demonstrated, at an industrial scale, the importance of non-Saccharomyces in the design of tailored starter cultures for typical wines.

Table olives are one of the most important traditional fermented vegetables in Southern European (Italy, Greece and Spain) countries. In the Greek-style production system, the fruits are placed directly into the brine, thus allowing the natural fermentation to take place. The spontaneous fermentations, that can last 8-12 months, are driven by mixed populations of microorganisms, mainly the epiphytic microbial population of yeasts and lactic acid bacteria (LAB) (Romero et al., 2004). At present, the industrial table olive process is not predictable and depends on the empirical experience of the producers. In order to avoid the unpredictability of the olive spontaneous fermentation, to improve the productive process and to constantly produce high-quality final products, the use of strains of LAB as starter cultures for olive production has been proposed (Sabatini and Marsilioet al., 2008; Panagou et al., 2008; Blana et al., 2014). However, in the last years, the importance and the potential applications of yeasts as starters for table olive processing has been recognized (Arroyo-López et al., 2008, 2012; Bevilacqua et al., 2012). Objectives: In the present work, we have studied the main physical, chemical and aromatic parameters of natural fermentations of Cellina di Nardò, Leccino, Kalamata and Conservolea table olives in order to determine chemical descriptors correlated to microbiological activities and the dynamics of microorganisms in order to select LAB and yeast strains as candidate autochthonous starter cultures. Conclusions: The identified chemical descriptors can be suitable to follow the trend and to control the outcome of the fermentation and a new protocol aimed to the selection of LAB and yeast strains as candidates autochthonous starters has been developed and applied (Bleve et al. 2014 a, b). Selected microbial starters have been successfully used to ferment olives in pilot and industrial-scale and a new method for table olive production has been set up (Bleve et al. 2103). The use of selected autochthonous starter cultures produced fermented table olives with improved organoleptic, sensorial and nutritional characteristics.

Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF), but winemakers has shown great interest about co-inoculation of yeast and malolactic bacteria at the beginning of AF. In this study we have produced a mixed starter bu co-immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used it in microvinifuications tests

Plants respond to environmental stimuli, such as heat shock, by re-programming cellular activity through differential gene expression, mainly controlled at the transcription level. The current study refers to two sunflower small heat shock protein (sHSP) genes arranged in tandem in head-to-head orientation and linked by a 3809 bp region. These genes exhibit only slight structural differences in the coding portion. They code for cytosolic class I sHSPs and are named HaHSP17.6a and HaHSP17.6b according to the molecular weight of the putative proteins. The genomic organization of these genes is consistent with the idea that many HSP genes originate from duplication events; in this case, probably an inversion and duplication occurred. The HaHSP17.6a and HaHSP17.6b genes are characterized by different expression levels under various heat stress conditions; moreover, their expression is differently induced by various elicitors. The differential regulation observed for HaHSP17.6a and HaHSP17.6b genes differs from previous observations on duplicated sHSP genes in plants.

IntroduzionePrunus mahaleb L. è un arbusto deciduo che cresce in boschi radi anche su terreni marginali e in climi aridi. L'albero è nativo dei paesi del mediterraneo e dell'Asia centrale e produce dei piccoli frutti simili a ciliegie di un colore quasi nero, non commestibili a causa del loro sapore amaro. Studi precedenti hanno dimostrato un elevato contenuto in antocianine in questi frutti, che suggerisce il loro utilizzo come fonte di coloranti alimentari dotati di proprietà salutistiche.ObiettiviCaratterizzazione chimica di un estratto concentrato di frutti di P. mahaleb e studio in vitro dei suoi effetti anticancerogeni, antiinfiammatori e antimutagenici.MetodologiaI frutti di mahaleb sono stati estratti con solventi consentiti per uso alimentare (etanolo + 1% acido citrico 1M) e l'estratto, concentrato sino a 60 °Brix, analizzato utilizzando metodiche HPLC. Sono stati effettuati saggi in vitro per studiare la capacità dell'estratto di inibire la proliferazione di linee cellulari tumorali umane (MCF-7), le sue proprietà antinfiammatorie su cellule endoteliali vascolari umane e infine i suoi effetti antimutagenici su linee di lievito Saccharomyces cerevisiaeD7.Risultati e DiscussioneL'estratto concentrato ha mostrato un elevato contenuto in antocianine, flavonoli e cumarina ed una elevata attività antiossidante simile o superiore a quella di altri noti estratti concentrati di frutti rossi. Lo studio delle attività biologiche dell'estratto ha mostrato: i) un significativo effetto antiproliferativo e capacità di modulare le GJIC in cellule MCF-7; ii) una inibizione dell'espressione di markers infiammatori tipici dell'attivazione endoteliale (ICAM-1, VCAM-1, E-selectine) in cellule endoteliali ottenute da vena ombelicale umana in coltura; iii) una riduzione di circa tre volte dell'effetto mutageno di H2O2 su cellule di S. cerevisiae. Questi risultati suggeriscono l'utilizzo dei frutti di P. mahaleb processati, come fonte di coloranti alimentari naturali e di composti bioattivi con proprietà salutistiche.

Agri-food by-products produced during handling and processing of fruits and vegetables, represent a major waste disposal problem for industry, but they are also promising sources of bioactive compounds that can be recovered and used as valuable substances to develop new enriched food, such as pasta and bakery products. Tarallini are a typical Italian bakery products, that has currently become very popular worldwide as a savory snack or bread substitute. These products can be considered an optimal, cheap and easy-to-use carrier of phytochemicals.Olive fruit is an excellent natural source of unsaturated fatty acids, as well as other nutritionally important health- promoting bioactive compounds [1]. The modern two-phase technology of the Leopard series for olive oil industrial extraction developed by the Pieralisi Group (Pieralisi Maip S.p.A., Jesi, AN, Italy) generates large quantities of a novel by-product (olive paste - OP) made up of the partially defatted wet drupe pulp without any traces of the kernel. OP can be incorporated into wheat based products [2].In this work the biotechnological aptitude of selected microorganisms, yeast and Lactic Acid Bacteria from different sources, were tested to transform OP in a new fermented product. Furthermore, some bioactive compounds, such as polyphenols, triterpenic acids, phytosterols, squalene, tocopherols and carotenoids, of fermented OP obtained from black olives belonging to Leccino and Cellina di Nardò cultivars, were characterized. Finally, new types of tarallini were produced by adding 10% of fermented and non-fermented OP to dough. The levels of bioactive compounds were compared with conventional control tarallini produced with the same semolina but without supplementations.The polyphenols content of olive paste, found in the olive paste Leccino (OPL) was 7.2 mg/g fresh (FW) while for olive paste Cellina di Nardò (OPC) the concentration was 18.1 mg/g FW. The fermentation of OP produces a decrease of phenolic concentration 20% for OPL and 16% for OPC. Among the oleuropein derivatives, 3,4- DHPEA-EDA, showed the higher reductions corresponding to the increase in their hydrolysis products (hydroxytyrosol), due to the enzymatic activity of the strains used for the fermentation of the OP [3].Total triterpenic acid content was about 1.5 and 1.7 mg/g fresh weight in OPL and OPC respectively. These values did not differ significantly compared with the respective fermented OP. The total phytosterols and squalene content ranged between 1.7 mg/g FW (OPC) and 1.9 mg/g FW (OPL) and between 29.10 ?g/g FW (OPL) and 38.95 ?g/g FW (OPC), respectively. After fermentation, total phytosterols content was reduced from 53% to 49% whereas squalene level was reduced from 47% to 23% compared with not fermented OP. The content of tocochromanol and carotenoid did not differ significantly after fermentation process, their content ranged from 22.64 ?g/g FW (fermented OPL) to 24.94 ?g/g FW (not fermented OPL) for tocochrom

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.

Valorization of the neglected Prunus mahaleb L., fruits as a source of functional moleculesGerardi C.1, Frassinetti S.3, Leone A.1, Calabriso N.2, Carluccio M.A.2, F. Blando1 and Mita G. 11 Institute of Sciences of Food Production, CNR, Lecce, Italy2 Institute of Clinical Physiology, CNR, Lecce, Italy3 Institute of Biology and Agricultural Biotechnology, CNR, Pisa, ItalyAbstract: Prunus mahaleb L. is a tree producing dark-red small stone fruits, not used for fresh consumption due to their astringent and sour taste. In this communication will be described a "mahaleb fruit concentrated extract" (mfce) and its chemical characterization. The extract showed high anthocyanin, flavonol and coumarin content. Moreover, the results of the in vitro assays for mfce biological activities will be reported. Mfce showed strong antioxidant capacity and anti-proliferative, pro-apoptotic, anti-inflammatory and anti-mutagenic effects. This study demonstrated several healthful effects of mfce that may be of interest as natural food colorant and as source of molecules for the formulation of functional foods.

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.

Wheat is a staple cereal and an important source of minerals in human diets. Mineral content in the wheat grain has a genetic basis, but the trait is also influenced by environmental factors [1]. Many efforts have been devoted to increase the content of some elements such as Fe, Zn, Mn and Cu, which have a physiological function in the organism. In particular, mapping populations and genetic linkage maps have been suggested as a useful tool in breeding programs [2]. The present work was undertaken to evaluate by ICPOES the variation in macronutrients (Ca, Mg, K), micronutrients (Zn, Fe, Cu, Mn) and toxic trace elements (Cd, Pb) in the whole grains of 29 wheat accessions and cultivars, representative of old and modern wheat germplasm. All 29 lines were the parentals of existing mapping populations [3] and were grown in two different sites in Italy, in 2013/14 and in 2014/15 seasons. Multivariate Analysis (MVA) of data showed interesting differences in the mineral content for the considered sites, harvesting seasons and grain cultivars.[1] Gomez-Becerra HF, Yazici A, Ozturk L, Budak H, Peleg Z, Morgounov A, Fahima T, Saranga Y, Cakmak I. Genetic variation and environmental stability of grain mineral nutrient concentrations in Triticum dicoccoides under five environments. Euphytica 2010, 171:39-52. [2] Srinivasa J, Arun B, Mishra VK, Singh GP, Velu G, Babu R, Vasistha NK, Joshi AK. Zinc and iron concentration QTL mapped in a Triticum spelta × T. aestivum cross. Theor Appl Genet 2014, 127:1643-1651 [3] Pasqualone A, Piarulli L, Mangini G, Gadaleta A, Blanco A, Simeone R. Quality characteristics of parental lines of wheat mapping populations. Agric Food Sci 2015, 24: 118-127.

Phenolic compounds are a widespread group of substances in the plant kingdom, but their dietary intake greatly varies depending on the type and quantity of vegetable foods consumed. Interest in plant polyphenols has focused on their favourable effects on human health, arising from their antioxidant activity and capacity to protect critical macromolecules (such as chromosomal DNA, structural proteins and enzymes, low-density lipoproteins, and membrane lipids) from damages induced by active species of oxygen. Phenolic substances are present also in wheat, with ferulic acid present in the highest amount. Various studies analysed the polyphenol content of soft, medium and hard wheat (Triticum aestivum L.). Marginal attention has been devoted to durum wheat (Triticum turgidum L. spp. turgidum var. durum), the essential raw material to produce high quality pasta, one of the basic foods in the Italian diet. The aim of this work has been to evaluate the variability of total phenolic content (TPC) in Italian durum wheat, in view of selecting the best materials to produce phenolic extracts for functional pasta enrichment. A set of 20 cultivars was considered, chosen among the most cultivated ones. The determination of TPC of the free phenolic fraction, spectrophotometrically assessed after Folin-Ciocalteau reaction, showed levels ranging from 1.28 to 1.94 mg ferulic acid equivalent (FAE)/g wholemeal. The TPC was significantly correlated to the polyphenol oxidase activity of the examined cultivars.A subset of 5 cultivars was subjected to detailed survey of phenolic acids in whole flour. Three separate extractions were carried out to determine the composition and amount of soluble free, soluble conjugated, and bound phenolic acids. Two different mesh size (60 mesh or 0.251 mm; and 10 mesh or 2 mm) were used for milling whole grain samples to evaluate the extraction efficiency of phenolic acids depending on the particle size of whole flour samples. The evaluation of free, conjugated and bound extracts by the means of HPLC-DAD analysis lead to the characterisation of each fraction in terms of composition and amount of major phenolic acids. Sinapic acid resulted the most abundant compound present in the bound fraction, ranging from 335 and 438 ug/g dm, while o-coumaric acid resulted the major component of the conjugated fraction, ranging from 72 and 80 ug/g dm. Other phenolic acids were detected both in the bound and conjugated fractions, like ferulic, 2,4-dihydroxybenzoic, syringic and vanillic acids. The free fraction showed the smallest contribution to the total phenolic acid content (<0.5%), sinapic acid being the most abundant, followed by syring, ferulic and o-coumaric acid respectively.

Xylella fastidiosa is a gram-negative, xylem-limited, bacterium which is responsible, in Italy, for the Olive Quick Decline Syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In the present study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analyzing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars "Ogliarola salentina" and "Cellina di Nardò", and the tolerant cultivar "Leccino". Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.

Volatile metabolites from mold contamination have been proposed for the early identification of toxigenic fungi to prevent toxicological risks, but there are no such data available for Fusarium poae. F. poae is one of the species complexes involved in Fusarium head blight, a cereal disease that results in significant yield losses and quality reductions. The identification of volatile organic compounds associated with F. poae metabolism could provide good markers to indicate early fungal contamination. To this aim, we evaluated the volatile profile of healthy and F. poae-infected durum wheat kernels by SPME-GC/MS analysis. The production of volatile metabolites was monitored for seven days, and the time course analysis of key volatiles was determined. A total of 29 volatile markers were selected among the detected compounds, and multivariate analysis was applied to establish the relationship between potential volatile markers and fungal contamination. A range of volatile compounds, including alcohols, ketones, esters, furans and aromatics, were identified, both in contaminated and in healthy kernels. However, the overall volatile profile of infected samples and controls differed, indicating that the whole volatile profile, rather than individual volatile compounds, could be used to identify F. poae contamination of durum wheat grains.

Wheat bran is generally considered a byproduct of the flour milling industry, but it is a great source of fibers, minerals, and antioxidants that are important for human health. Phenolic acids are a specific class of wheat bran components that may act as antioxidants to prevent heart disease and to lower the incidence of colon cancer. Moreover, phenolic acids have anti-inflammatory properties that are potentially significant for the promotion of gastrointestinal health. Evidence on the beneficial effects of phenolic acids as well as of other wheat bran components is encouraging the use of wheat bran as an ingredient of functional foods. After an overview of the chemistry, function, and bioavailability of wheat phenolic acids, the discussion will focus on how technologies can allow the formulation of new, functional whole wheat products with enhanced health-promoting value and safety without renouncing the good-tasting standards that are required by consumers. Finally, this review summarizes the latest studies about the stability of phenolic acids in wheat foods fortified by the addition of wheat bran, pearled fractions, or wheat bran extracts.

The invention relates to a method for table olive fermentation comprising the steps of: a. soaking the olives in brine;b. inoculating the product obtained in step a. with a yeast culture;c. incubating the product obtained in step b. in order to perform the alcoholic fermentation; d. inoculating the fermented product obtained in step c. using a Lactic Acid Bacterium (LAB) culture;e. incubating the product obtained in step d. to in order to perform the lactic fermentation.Procedures for the selection of starter cultures and their use in fermentation of two cultivars of table olives are described. Some claims are directed to specific starter cultures and the uses thereof for preparing fermented table olives.