Study of the effects of pasteurization and selected microbial starters on functional traits of fermented table olives.

Table olives are one of the most known fruit consumed as fermented food, being a fundamental component of the Mediterranean diet. Their production and consumption continue to increase globally and represent an important economic source for the producing countries. One of the most stimulating challenges for the future is the modernization of olive fermentation process. Besides the demand for more reproducible and safer production methods that could be able to reduce product losses and potential risks, producers and consumers are increasingly attracted by the final product characteristics and properties on human health. In this study, the contribution of microbial starters to table olives was fully described in terms of specific enzymatic and microbiological profiles, nutrient components, fermentation-derived compounds, and content of bioactive compounds. The use of microbial starters from different sources was tested considering their technological features and potential ability to improve the functional traits of fermented black table olives. For each fermentation assay, the effects of controlled temperature (kept at 20 °C constantly) versus not controlled environmental conditions (oscillating between 7 and 17 °C), as well as the consequences of the pasteurization treatment were tested on the final products. Starter-driven fermentation strategies seemed to increase both total phenolic content and total antioxidant activity. Herein, among all the tested microbial starters, we provide data indicating that two bacterial strains (Leuconostoc mesenteroides KT 5-1 and Lactiplantibacillus plantarum BC T3-35), and two yeast strains (Saccharomyces cerevisiae 10A and Debaryomyces hansenii A15-44) were the better ones related to enzyme activities, total phenolic content and antioxidant activity. We also demonstrated that the fermentation of black table olives under not controlled environmental temperature conditions was more promising than the controlled level of 20 °C constantly in terms of technological and functional properties considered in this study. Moreover, we confirmed that the pasteurization process had a role in enhancing the levels of antioxidant compounds.

Highly Efficient Verbascoside Production from Olive (Olea europea L. var. Cellina di Nardò) In Vitro Cell Cultures.

Olive (Olea europea L.) is one of the oldest and most important fruit tree species cultivated in the Mediterranean region. Various plant tissues, drupes, and olive oil contain several phenolics (including verbascoside, although it is present in the plant at a low level) that are well-known for their highly beneficial effects on human health. An in vitro olive cell suspension culture (cultivar Cellina di Nardò, "CdN") was established, characterized for its growth and morphological features. Furthermore, a vital and relatively uniform population of protoplasts was generated from the olive suspension culture to investigate their cellular characteristics during growth. The polyphenolic extract of the in vitro "CdN" olive cells contained almost exclusively verbascoside, as revealed by the UPLC-ESI-MS analysis. The content of verbascoside reached up to 100 mg/g DW, with an average production rate of approximately 50 mg/g DW over one year of culture. This level of production has not been previously reported in a limited number of previous studies. This remarkable production of verbascoside was associated with an exceptionally high antioxidant capacity. The high level of verbascoside production and purity of the extract make this system a promising tool for secondary metabolite production.

Chromogenic Assay Is More Efficient in Identifying α-Amylase Inhibitory Properties of Anthocyanin-Rich Samples When Compared to the 3,5-Dinitrosalicylic Acid (DNS) Assay.

The inhibition of carbohydrate digestion by plant bioactive compounds is a potential dietary strategy to counteract type 2 diabetes. Indeed, inhibition of α-amylase, a key enzyme that carries out the bulk of starch digestion, has been demonstrated for a range of bioactive compounds including anthocyanins; however, sample pigmentation often interferes with measurements, affecting colorimetric assay outcomes. Therefore, the present study compared the performance of a direct chromogenic assay, using 2-chloro-4 nitrophenyl α-D-maltotrioside (CNPG3) as a substrate, with the commonly used 3,5-dinitrosalicylic acid (DNS) assay. The direct chromogenic assay demonstrated a 5-10-fold higher sensitivity to determine α-amylase inhibition in various samples, including acarbose as a reference, pure anthocyanins, and anthocyanin-rich samples. The IC50 values of acarbose presented as 37.6 µg/mL and 3.72 µg/mL for the DNS assay and the direct chromogenic assay, respectively, whereas purified anthocyanins from blackcurrant showed IC50 values of 227.4 µg/mL and 35.0 µg/mL. The direct chromogenic assay is easy to perform, fast, reproducible, and suitable for high-throughput screening of pigmented α-amylase inhibitors.

Glyoxalase I Assay as a Possible Tool for Evaluation of Biological Activity of Antioxidant-Rich Plant Extracts.

The health-promoting properties of natural plant bioactive compounds are mainly attributable to their ability to counteract oxidative stress. This is considered a major causative factor in aging and aging-related human diseases, in which a causal role is also ascribed to dicarbonyl stress. This is due to accumulation of methylglyoxal (MG) and other reactive dicarbonyl species, leading to macromolecule glycation and cell/tissue dysfunction. The glyoxalase (GLYI) enzyme, catalyzing the rate-limiting step of the GSH-dependent MG detoxification pathway, plays a key role in cell defense against dicarbonyl stress. Therefore, the study of GLYI regulation is of relevant interest. In particular, GLYI inducers are important for pharmacological interventions to sustain healthy aging and to improve dicarbonyl-related diseases; GLYI inhibitors, allowing increased MG levels to act as proapoptotic agents in tumor cells, are of special interest in cancer treatment. In this study, we performed a new in vitro exploration of biological activity of plant bioactive compounds by associating the measurement of their antioxidant capacity (AC) with the evaluation of their potential impact on dicarbonyl stress measured as capability to modulate GLYI activity. AC was evaluated using TEAC, ORAC, and LOX-FL methods. The GLYI assay was performed using a human recombinant isoform, in comparison with the recently characterized GLYI activity of durum wheat mitochondria. Different plant extracts were tested, obtained from plant sources with very high phytochemical content ('Sun Black' and wildtype tomatoes, black and 'Polignano' carrots, and durum wheat grain). Results showed high antioxidant properties of the tested extracts, associated with different modes (no effect, activation, and inhibition) and effectiveness in modulating both GLYI activity sources. Overall, results indicate the GLYI assay as an advisable and promising tool for researching plant foods as a source of natural antioxidant compounds acting as GLYI enzymatic regulators to be used for dietary management associated the treatment of oxidative/dicarbonyl-promoted diseases.

The Chelating Ability of Plant Polyphenols Can Affect Iron Homeostasis and Gut Microbiota.

In the past decades, many studies have widely examined the effects of dietary polyphenols on human health. Polyphenols are well known for their antioxidant properties and for their chelating abilities, by which they can be potentially employed in cases of pathological conditions, such as iron overload. In this review, we have highlighted the chelating abilities of polyphenols, which are due to their structural specific sites, and the differences for each class of polyphenols. We have also explored how the dietary polyphenols and their iron-binding abilities can be important in inflammatory/immunomodulatory responses, with a special focus on the involvement of macrophages and dendritic cells, and how they might contribute to reshape the gut microbiota into a healthy profile. This review also provides evidence that the axes "polyphenol-iron metabolism-inflammatory responses" and "polyphenol-iron availability-gut microbiota" have not been very well explored so far, and the need for further investigation to exploit such a potential to prevent or counteract pathological conditions.

The Green Leaf Volatile (Z)-3-Hexenyl Acetate Is Differently Emitted by Two Varieties of Tulbaghia violacea Plants Routinely and after Wounding.

While studying aromas produced by the edible flowers of Tulbaghia violacea, we noticed a different production of (Z)-3-Hexenyl acetate (a green-leaf volatile, GLV) by purple (var. 'Violacea') and white (var. 'Alba') flowers. The white Tulbaghia flowers constantly emits (Z)-3-Hexenyl acetate, which is instead produced in a lower amount by the purple-flowered variety. Thus, we moved to analyze the production of (Z)-3-Hexenyl acetate by whole plants of the two varieties by keeping them confined under a glass bell for 5 h together with a SPME (Solid Phase Micro Extraction) fiber. Results show that six main volatile compounds are emitted by T. violacea plants: (Z)-3-Hexenyl acetate, benzyl alcohol, nonanal, decanal, (Z)-3-Hexenyl-α-methylbutyrate, and one unknown compound. By cutting at half-height of the leaves, the (Z)-3-Hexenyl acetate is emitted in high quantities from both varieties, while the production of (Z)-3-Hexenyl-α-methylbutyrate increases. (Z)-3-Hexenyl acetate is a GLV capable of stimulating plant defenses, attracting herbivores and their natural enemies, and it is also involved in plant-to-plant communication and defense priming. Thus, T. violacea could represent a useful model for the study of GLVs production and a 'signal' plant capable of stimulating natural defenses in the neighboring plants.

The Controlled Semi-Solid Fermentation of Seaweeds as a Strategy for Their Stabilization and New Food Applications.

For centuries, macroalgae, or seaweeds, have been a significant part of East Asian diets. In Europe, seaweeds are not considered traditional foods, even though they are increasingly popular in Western diets in human food applications. In this study, a biological processing method based on semi-solid fermentation was optimized for the treatment of the seaweed Gracilaria gracilis. For the first time, selected lactic acid bacteria and non-conventional coagulase-negative staphylococci were used as starter preparations for driving a bio-processing and bio-stabilization of raw macroalga material to obtain new seaweed-based food prototypes for human consumption. Definite food safety and process hygiene criteria were identified and successfully applied. The obtained fermented products did not show any presence of pathogenic or spoilage microorganisms, thereby indicating safety and good shelf life. Lactobacillus acidophilus-treated seaweeds revealed higher α-amylase, protease, lipase, endo-cellulase, and endo-xylanase activity than in the untreated sample. This fermented sample showed a balanced n-6/n-3 fatty acid ratio. SBM-11 (Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus) and PROMIX 1 (Staphylococcus xylosus) treated samples showed fatty acid compositions that were considered of good nutritional quality and contained relevant amounts of isoprenoids (vitamin E and A). All the starters improved the nutritional value of the seaweeds by significantly reducing the insoluble indigestible fractions. Preliminary data were obtained on the cytocompatibility of G. gracilis fermented products by in vitro tests. This approach served as a valid strategy for the easy bio-stabilization of this valuable but perishable food resource and could boost its employment for newly designed seaweed-based food products.

Ensiling Grape Pomace With and Without Addition of a Lactiplantibacillus plantarum Strain: Effect on Polyphenols and Microbiological Characteristics, in vitro Nutrient Apparent Digestibility, and Gas Emission.

The present study investigated the effects of different grape pomace storage techniques on the effectiveness as feed on in vitro ruminant digestion efficiency. Grape pomace from an autochthonous red grape variety (cv Nero di Troia) was used as fresh (GP) or ensiled, both without additives (SIL) and with the addition of a bacterial strain, Lactiplantibacillus plantarum 5BG (SIL+). All the different storage treatments were subject to chemical and microbiological evaluation, as well as in vitro digestibility, and gas production. Microbiological data revealed the good quality of grape pomace and silages due to the lactic acid bacteria populations and low presence, or absence, of undesirable microorganisms. The addition of L. plantarum 5BG influenced the chemical characteristics of the silage (SIL+). Ensiling technique deeply changed the polyphenolic composition, reducing anthocyanins, flavonols, and flavanols (condensed tannins precursors), particularly when L. plantarum 5BG was added. Antioxidant capacity was reduced by ensiling, in correlation with the polyphenolic content decrease. The oxygen radical absorbance capacity (ORAC) value of SIL+ was the lowest (P < 0.01) and its total phenol content was lower than SIL (P < 0.01). No statistical differences were observed between GP, SIL, and SIL+ on the antioxidant capacity by TEAC assay (P > 0.05). Ensiling did not affect the grape pomace nutrient profile, except for the reduction in NFC content. Apparent in vitro digestibility showed how ensiling increased dry matter (DM), organic matter (OM), neutral detergent fiber (NDF), crude protein (CP), ether extract (EE), and non-fiber carbohydrates (NFC) disappearance (P < 0.01), particularly with the L. plantarum 5BG inoculation. Moreover, SIL+ showed the lowest propionic acid (P < 0.05) and the highest methane (P < 0.01), butyric acid (P < 0.01), and nitrogen (P < 0.05) in vitro production. Ensiling GP resulted in a better in vitro digestibility, particularly if L. plantarum 5BG strain is added, probably due to the reduction of flavanols and their lower microbial activity inhibition.

Bioactive Compounds and Antioxidant Capacity in Anthocyanin-Rich Carrots: A Comparison between the Black Carrot and the Apulian Landrace "Polignano" Carrot.

The carrot is one of the most cultivated vegetables in the world. Black or purple carrots contain acylated anthocyanins which are of special interest to the food industry for their stability and nutraceutical characteristics. Anthocyanin-rich fruits and vegetables have gained popularity in the last ten years, due to the health benefits they provide. In this paper, the characterizations of the bioactive compounds and antioxidant capacities of different anthocyanin-containing carrots (a black carrot-BC, and a local purple carrot, the "Polignano" carrot-PC), compared to the commercial orange carrot (OC) (lacking of anthocyanins), are reported. The anthocyanin profiles of the polyphenolic extracts of BC and PC were similar, but differences were observed at quantitative levels. The total anthocyanin content in BC was more than twice that in PC (13.84 ± 0.61 vs. 5.64 ± 0.48 mg K Eq. g-1 DW). Phenolic acids (mostly chlorogenic acid) were also present at high level in anthocyanin-rich carrots compared to OC. High polyphenol content accounted also for a high reducing capacity (evaluated by Folin-Ciocalteu reagent, FCR), and antioxidant capacity (evaluated by TEAC and ORAC assays) which were the highest for BC (FCR value: 16.6 ± 1.1 mg GAE. g-1 DW; TEAC: 76.6 ± 10.6 µmol TE. g-1 DW; ORAC: 159.9 ± 3.3 µmol TE. g-1 DW). All carrot genotypes (mostly OC) were rich in carotenoids (BC 0.14 ± 0.024; PC 0.33 ± 0.038; OC 1.29 ± 0.09 mg. g-1 DW), with predominance of α and ß-carotene, in OC, and lutein in BC. PC showed the highest malic acid and sugar (glucose plus fructose) content. In conclusion, while BC is remarkable for nutraceutical features, the local genotype ("Polignano" carrot) is worth considering in genetic biodiversity conservation programme.

Nutraceutical Characterization of Anthocyanin-Rich Fruits Produced by "Sun Black" Tomato Line.

Tomato (Solanum lycopersicum L.) is one of the most cultivated vegetable in the world and it represents a large source of bioactive compounds, including carotenoids and polyphenols (phenolic acids and flavonoids). However, the concentration of flavonoids in tomato is considered sub-optimal, particularly because anthocyanins are not generally present. Therefore, this crop has been the object of an intense metabolic engineering in order to obtain anthocyanin-enriched tomatoes by using either breeding or transgenic strategies. Some wild tomato species, such as S. chilense and S. cheesmaniae, biosynthesize anthocyanins in the fruit sub-epidermal tissue, and some alleles from those genotypes have been introgressed into a new developed purple tomato line, called "Sun Black" (SB). It is a tomato line with a purple skin color, both in green and in red fruit stages, due to the biosynthesis of anthocyanins in the peel, and a normal red color pulp, with a taste just like a traditional tomato. SB is the result of a breeding programme and it is not a genetically modified (GM) product. We report the chemical characterization and structure elucidation of the attractive anthocyanins found in the peel of SB tomato, as well as other bioactive compounds (carotenoids, polyphenols, vitamin C) of the whole fruit. Using one- and two-dimensional NMR experiments, the two main anthocyanins were identified to be petunidin 3-O-[6″-O-(4‴-O-E-p-coumaroyl-α-rhamnopyranosyl) -ß-glucopyranoside]-5-O-ß-glucopyranoside (petanin) and malvidin 3-O-[6″-O-(4‴-O-E-p-coumaroyl-α-rhamnopyranosyl)-ß-glucopyranoside]-5-O-ß-glucopyranoside (negretein). The total anthocyanins in the whole ripe fruit was 1.2 mg/g dry weight (DW); 7.1 mg/100 g fresh weight (FW). Chlorogenic acid (the most abundant phenolic acid) was 0.6 mg/g DW; 3.7 mg/100 g FW. The main flavonol, rutin was 0.8 mg/g DW; 5 mg/100 g FW. The total carotenoid content was 211.3 µg/g DW; 1,268 µg/100 g FW. The total phenolic content was 8.6 mg/g DW; 52.2 mg/100 g FW. The vitamin C content was 37.3 mg/100 g FW. The antioxidant activities as measured by the TEAC and ORAC assays were 31.6 and 140.3 µmol TE/g DW, respectively (193 and 855.8 µmol TE/100 g FW, respectively). The results show the unique features of this new tomato genotype with nutraceutical properties.

Pectolytic enzyme reduces the concentration of colloidal particles in wine due to changes in polysaccharide structure and aggregation properties.

To explore the effect of pectolytic enzyme application on polysaccharide size and colloidal interactions, rosé wines were prepared from pressed Shiraz grape juice, and red wines were made from the same juice by the addition of skins and seeds from fresh pressings. A pectolytic enzyme preparation containing primarily polygalacturonase and side-activities of arabinase and pectin lyase was used. Enzyme treatment enhanced the contribution of high molecular weight (≈ 200 kDa) polysaccharides rich in mannose and glucose (mannoproteins) and removed arabinose-rich polysaccharides of intermediate (≈ 40 kDa) size. Enzyme application reduced the molecular weight average of a class of smaller polysaccharides of approximately 6 kDa (rhamnogalacturonan II). All wines had similar particle sizes, but enzyme treatment markedly reduced particle concentration in wines, particularly in rosé wines. Wine polysaccharides were purified and when reconstituted in model wine, showed reduced particle concentration in response to enzyme treatment. Aggregation of polysaccharide in the presence of seed tannin was also markedly reduced by enzyme treatment. The results indicated that changes in structure introduced by the enzyme affected wine colloidal properties, potentially increasing polysaccharide solubility.

Phenolic Profile and Antioxidant Activity of Italian Monovarietal Extra Virgin Olive Oils.

In the last years, the interest in Italian monovarietal oils has increased due to their specific organoleptic qualities. Extra virgin olive oils (EVOOs) are rich in phenolic compounds, secondary metabolites well known and studied for their nutraceutical properties. However, among EVOOs, there is great variability in phenolic composition due to the origin, the production technique, and mainly, the genotype. The aim of this work was to evaluate the different phenolic profiles and the antioxidant activities of monovarietal oils. The results confirm this variability. In fact, the overall content of oleuropein varies up to four times between the different genotypes (from 33.80 to 152.32 mg/kg oil), while the oleocanthal content is significant only in two oils. The antioxidant activity, determined with 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays, is correlated with the content of total phenolic substances, with half maximal inhibitory concentration (IC50) values for the DPPH test ranging from 160 to 91 mg of oil, while the ORAC test shows values between 5.45 and 8.03 µmol Trolox equivalent (TE)/g oil.

Antimicrobial and Antibiofilm Activity against Staphylococcus aureus of Opuntia ficus-indica (L.) Mill. Cladode Polyphenolic Extracts.

Plant extracts are a rich source of natural compounds with antimicrobial properties, which are able to prevent, at some extent, the growth of foodborne pathogens. The aim of this study was to investigate the potential of polyphenolic extracts from cladodes of Opuntia ficus-indica (L.) Mill. to inhibit the growth of some enterobacteria and the biofilm formation by Staphylococcus aureus. Opuntia ficus-indica cladodes at two stages of development were analysed for total phenolic content and antioxidant activity by Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) (in vitro assays) and by cellular antioxidant activity in red blood cells (CAA-RBC) (ex vivo assay). The Liquid Chromatography Time-of-Flight Mass Spectrometry (LC/MS-TOF) analysis of the polyphenolic extracts revealed high levels of piscidic acid, eucomic acid, isorhamnetin derivatives and rutin, particularly in the immature cladode extracts. Opuntia cladodes extracts showed a remarkable antioxidant activity (in vitro and ex vivo), a selective inhibition of the growth of Gram-positive bacteria, and an inhibition of Staphylococcus aureus biofilm formation. Our results suggest and confirm that Opuntia ficus-indica cladode extracts could be employed as functional food, due to the high polyphenolic content and antioxidant capacity, and used as natural additive for food process control and food safety.

Antioxidant Activity and Anthocyanin Contents in Olives (cv Cellina di Nardò) during Ripening and after Fermentation.

The olive tree "Cellina di Nardò" (CdN) is one of the most widespread cultivars in Southern Italy, mainly grown in the Provinces of Lecce, Taranto, and Brindisi over a total of about 60,000 hectares. Although this cultivar is mainly used for oil production, the drupes are also suitable and potentially marketable as table olives. When used for this purpose, olives are harvested after complete maturation, which gives to them a naturally black color due to anthocyanin accumulation. This survey reports for the first time on the total phenolic content (TPC), anthocyanin characterization, and antioxidant activity of CdN olive fruits during ripening and after fermentation. The antioxidant activity (AA) was determined using three different methods. Data showed that TPC increased during maturation, reaching values two times higher in completely ripened olives. Anthocyanins were found only in mature olives and the concentrations reached up to 5.3 g/kg dry weight. AA was determined for the four ripening stages, and was particularly high in the totally black olive fruit, in accordance with TPC and anthocyanin amounts. Moreover, the CdN olives showed a higher TPC and a greater AA compared to other black table olives produced by cultivars commonly grown for this purpose. These data demonstrate the great potential of black table CdN olives, a product that combines exceptional organoleptic properties with a remarkable antioxidant capacity.

Radical Scavenging and Anti-Inflammatory Activities of Representative Anthocyanin Groupings from Pigment-Rich Fruits and Vegetables.

Anthocyanins, the naturally occurring pigments responsible for most red to blue colours of flowers, fruits and vegetables, have also attracted interest because of their potential health effects. With the aim of contributing to major insights into their structure-activity relationship (SAR), we have evaluated the radical scavenging and biological activities of selected purified anthocyanin samples (PASs) from various anthocyanin-rich plant materials: two fruits (mahaleb cherry and blackcurrant) and two vegetables (black carrot and "Sun Black" tomato), differing in anthocyanin content (ranging from 4.9 to 38.5 mg/g DW) and molecular structure of the predominant anthocyanins. PASs from the abovementioned plant materials have been evaluated for their antioxidant capacity using Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. In human endothelial cells, we analysed the anti-inflammatory activity of different PASs by measuring their effects on the expression of endothelial adhesion molecules VCAM-1 and ICAM-1. We demonstrated that all the different PASs showed biological activity. They exhibited antioxidant capacity of different magnitude, higher for samples containing non-acylated anthocyanins (typical for fruits) compared to samples containing more complex anthocyanins acylated with cinnamic acid derivatives (typical for vegetables), even though this order was slightly reversed when ORAC assay values were expressed on a molar basis. Concordantly, PASs containing non-acylated anthocyanins reduced the expression of endothelial inflammatory antigens more than samples with aromatic acylated anthocyanins, suggesting the potential beneficial effect of structurally diverse anthocyanins in cardiovascular protection.

Polyphenolic composition and antioxidant activity of the under-utilised Prunus mahaleb L. fruit.

BACKGROUND: 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 > 5 g kg(-1) 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 ORAC and TEAC assays, measured up to 150 and 45 mmol Trolox equivalents kg(-1) FW, respectively. Therefore antioxidant capacity of P. mahaleb fruits is relatively high and comparable to that of superfruit varieties that are often used in commercial nutraceutical products. 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. © 2015 Society of Chemical Industry.

Betalains, Phenols and Antioxidant Capacity in Cactus Pear [Opuntia ficus-indica (L.) Mill.] Fruits from Apulia (South Italy) Genotypes.

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 colored 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 cactus pear 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 colored bioactive compounds which not only have coloring potential, but are also an excellent source of dietary antioxidant components which may have beneficial effects on consumers' health.

Enhanced production of bioactive isoprenoid compounds from cell suspension cultures of Artemisia annua L. using ß-cyclodextrins.

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-ß-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.

Purification and chemical characterisation of a cell wall-associated ß-galactosidase from mature sweet cherry (Prunus avium L.) fruit.

Using four different chromatographic steps, ß-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium ß-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and ß-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing ß-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry ß-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple ß-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry ß-galactosidase exhibited a strict specificity towards p-nitrophenyl ß-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening.

Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development.

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8-10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits.