Selective recovery of anthocyanins and hydroxycinnamates from a byproduct of citrus processing

The adsorption-desorption performances of commercial resins (two polystyrene-divinylbenzene copolymers and one methacrylic) were tested in column studies for the selective recovery of anthocyanins and hydroxycinnamates from pigmented pulp wash (PW), a byproduct of blood orange juice processing. Methanol, ethanol, and their mixtures with different percentages of water as eluents were tested in order to investigate the selective desorption of these natural antioxidants with the goal of minimizing the presence of other compounds, mostly flavanones and limonoids, in the concentrated eluates. The results indicated that polystyrene-divinylbenzene resins were able to adsorb a major amount of anthocyanins and hydroxycinnamates and to desorb them in more-concentrated fractions. No selectivity was observed using pure methanol and ethanol which resulted in concentrates with no further separation of components from the starting raw PW. The ethanol/water concentrates showed higher yields than the fractions attained using methanol/water; however, a lower selectivity toward anthocyanin pigments was observed. The best performing resin, EXA-118, together with the mixture methanol/water 50:50 (v/v) as best eluent, appears to be the most suitable system to obtain highly concentrated extracts. Thus, it was chosen to perform a larger experiment, to analyze the selectivity of the removal upon an increase in elution volume.     

Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics

Culex quinquefasciatus (the southern house mosquito) is an important mosquito vector of viruses such as West Nile virus and St. Louis encephalitis virus, as well as of nematodes that cause lymphatic filariasis. C. quinquefasciatus is one species within the Culex pipiens species complex and can be found throughout tropical and temperate climates of the world. The ability of C. quinquefasciatus to take blood meals from birds, livestock, and humans contributes to its ability to vector pathogens between species. Here, we describe the genomic sequence of C. quinquefasciatus: Its repertoire of 18,883 protein-coding genes is 22% larger than that of Aedes aegypti and 52% larger than that of Anopheles gambiae with multiple gene-family expansions, including olfactory and gustatory receptors, salivary gland genes, and genes associated with xenobiotic detoxification.     

Effect of lime-induced leaf chlorosis on ochratoxin A, trans-resveratrol, and epsilon-viniferin production in grapevine (Vitis vinifera L.) berries infected by Aspergillus carbonarius

Berries of Vitis vinifera L. cv. Merlot, grown on a neutral or calcareous soil, were infected, at phenological phases of veraison and ripening, by a conidial suspension of Aspergillus carbonarius to control ochratoxin A production and trans-resveratrol- and epsilon-viniferin-induced synthesis as affected by the soil lime content. Chlorosis occurrence was evaluated by a visual rating scale at veraison, and the leaves from vines growing on the calcareous soil showed the typical yellowing, whereas those grown on the neutral soil were dark green. Berry mineral element yield was recorded at veraison and ripening. Infection symptoms on berries were more severe at ripening in bunches collected from vines grown in calcareous soil. Ochratoxin A concentration increased at phenological phase of veraison in berries harvested from vines cultivated in calcareous soil. A. carbonarius enhanced trans-resveratrol and epsilon-viniferin production in infected berries more than in the control samples. Moreover, at veraison their concentration in the berries collected from vines grown in calcareous soil was greater than that recorded from berries collected from vines grown in the neutral soil. The lowest symptom severity was observed on berries containing the highest copper concentration.     

Effect of ochratoxin A-producing Aspergilli on stilbenic phytoalexin synthesis in grapes

Berries of Vitis vinifera L. cv. Barbera were infected, at veraison and during ripening, by a conidial suspension of A. japonicus, A. ochraceus, A. fumigatus and two isolates of A. carbonarius to control ochratoxin A production and stilbene induced synthesis. The experimental design provided also for intact and punctured berries and incubation temperature of 25 degrees C and 30 degrees C. All the tested fungi, except A. fumigatus, significantly increased trans-resveratrol synthesis over the control, while trans-piceid was not affected; only A. ochraceus significantly elicited the berries to synthesize piceatannol. The two isolates of A. carbonarius produced higher amounts of ochratoxin A than did the other fungi. A positive correlation between ochratoxin A and trans-resveratrol synthesis occurred. trans-Resveratrol and piceatannol showed fungicidal activity against A. carbonarius, being able to completely inhibit fungal growth at a concentration of 300 microg/g and 20 microg/g, respectively.     

Recovery of anthocyanins from pulp wash of pigmented oranges by concentration on resins

A pulp wash (PW) coming from a plant for citrus processing of pigmented oranges was utilized as a starting material to recover anthocyanins, using the procedure of concentration on resin. Six commercial food-grade resins were tested to find the more suitable ones for adsorbing anthocyanins, and 96% ethanol was used as eluent for desorbing them. An automated experimental apparatus was developed to perform the adsorption-desorption procedure on column. The kinetics results in a batch system and experiments on column showed that the more efficient resins are those made of styrene-divinylbenzene having a pore radius ranging from 70 to 150 A and a surface area from 600 to 800 m(2)/g, namely, Sepabeads SP 70 and Relite EXA 90. The richest fractions collected from these resins contained about 95% of the anthocyanins in a volume of about 2% of the loaded PW. The HPLC profile of the desorbed anthocyanins is the same as that in PW. These fractions contain other phenol compounds, such as hesperidin and derivatives of hydroxycinnamic acids, in remarkable amounts. Ethanol can be easily removed from the solution and recycled, thus affording a much more concentrated product which can find application as a food colorant or antioxidant ingredient for a nutritional integrator.     

Ochratoxin a in Grapes and Wine

The mycotoxin ochratoxin A is a potent nephrotoxin and a possible human carcinogen. It occurs in a variety of plant products, including wine, grape juice and dried vine fruits. Several surveys have shown that the range of ochratoxin A contents detected in wine produced in Europe varied between 0.01 and 3.4gl^–1. Both incidence and concentration of the toxin were higher in wines from southern regions and increased in the order white < rosè < red. In Italy, field trials were conducted in 1999 and 2000 to study fungi associated with grapes and their ability to produce ochratoxin. Aspergillus and/or Penicillium strains were present on grapes, starting from setting in a few vineyards. The highest level of grape colonisation was found at early veraison in 1999 and at ripening in 2000. In both years, 95% of strains belonged to the genus Aspergillus. Aspergillus niger aggregate was dominant, with about 50% of the ochratoxin-positive strains identified as A carbonarius. Other authors have confirmed the relevance of these fungi and underlined the contribution of A. carbonarius to the ochratoxin contamination of wine. This species is very invasive and colonises and penetrates berries, even without skin damage. It emerges that temperature, rain and relative humidity are the main factors that influence ochratoxin production in grapes.     

The precision and robustness of published protocols for disc diffusion assays of antimicrobial agent susceptibility: an inter-laboratory study

The precision of the disc diffusion protocols previously published by Alderman and Smith (Aquaculture 196 (2002) 211) was analysed in a seven-laboratory trial using Escherichia coli ATCC 25922 as the test strain. Discs containing seven antimicrobial agents were employed and 2899 zone size measurements were generated. The total data generated in the trial was used to quantify the intra- and inter-laboratory precisions. The study design also facilitated the investigation of the influence of the source of media and the source of discs on zone sizes. A smaller two-laboratory trial was employed to investigate the influence of incubation time of zone size.

The intra-laboratory precision was relatively high with the mean of the coefficients of variation calculated for each laboratory and each agent being 4.7%. In contrast, the inter-laboratory precision was very much lower with the mean of the values for each agent being 11.1%. Significant influences on zone size were detected for all three parameters of the protocol. Media source effects were particularly notable with respect to oxytetracycline and oxolinic acid discs, disc source effects with respect to ampicillin and sulphamethoxazole/trimethoprim discs and incubation times with the ampicillin and amoxycillin discs. ANOVA analysis of the total data set confirmed that inter-laboratory variation was the major factor influencing the low precision of the protocol.

The overall precision of the protocols used here was found to be significantly lower than that implied by the control limits associated with the same bacterium in other validated disc diffusion protocols. The implications of these results, for the further development of the protocols under investigation, are discussed.     

Rapid detection of kernel rots and mycotoxins in maize by near-infrared reflectance spectroscopy

Near-infrared (NIR) spectroscopy is a practical spectroscopic procedure for the detection of organic compounds in matter. It is particularly useful because of its nondestructiveness, accuracy, rapid response, and easy operation. This work assesses the applicability of NIR for the rapid identification of micotoxigenic fungi and their toxic metabolites produced in naturally and artificially contaminated products. Two hundred and eighty maize samples were collected both from naturally contaminated maize crops grown in 16 areas in north-central Italy and from ears artificially inoculated with Fusarium verticillioides. All samples were analyzed for fungi infection, ergosterol, and fumonisin B1 content. The results obtained indicated that NIR could accurately predict the incidence of kernels infected by fungi, and by F. verticillioides in particular, as well as the quantity of ergosterol and fumonisin B1 in the meal. The statistics of the calibration and of the cross-validation for mold infection and for ergosterol and fumonisin B1 contents were significant. The best predictive ability for the percentage of global fungal infection and F. verticillioides was obtained using a calibration model utilizing maize kernels (r2 = 0.75 and SECV = 7.43) and maize meals (r2 = 0.79 and SECV = 10.95), respectively. This predictive performance was confirmed by the scatter plot of measured F. verticillioides infection versus NIR-predicted values in maize kernel samples (r2 = 0.80). The NIR methodology can be applied for monitoring mold contamination in postharvest maize, in particular F. verticilliodes and fumonisin presence, to distinguish contaminated lots from clean ones, and to avoid cross-contamination with other material during storage and may become a powerful tool for monitoring the safety of the food supply.     

Adsorption of flavonoids on resins: cyanidin 3-glucoside

Adsorption of cyanidin-3-glucoside in aqueous batch solutions was studied at pH 3.5 and 20 degrees C using 13 commercial resins with different hydrophobicity, surface areas (SA, 330-1200 m2/g), and pore radii (PR, 20-260 A). The solute affinity toward different resins was described in terms of Langmuir and Freundlich parameters; experimental data were well-fitted to the two isotherms, which were both utilized to compare resins adsorption capacity. The styrene-divinylbenzene EXA-118 resin (SA, 1200 m2/g; PR, 90 A) showed the maximum effectiveness among the tested resins; a good efficacy for removing cyanidin 3-glucoside was shown also by EXA-90 (SA, 630 m2/g; PR, 105 A). Some experiments on a styrene-divinylbenzene resin and an acrylic one demonstrated that adsorption was not influenced by pH variations of the solution within the range of 1.0-4.5. For the 10 resins having PR < or = 105 A, correlation analysis evidenced the linear increasing dependence of Freundlich constant KF on physical characteristics of surface area and pore radius. The adsorption of cyanidin 3-glucoside at 20 degrees C was compared with that of hesperidin performed in batch runs on the same resins. Moreover, the best resin (EXA-118) was tested with a sample of pigmented orange juice to assess its performance in terms of selectivity, adsorption, and desorption capabilities on a real matrix.