Cristiano Bello

Identification and Quantification of Phenolic Compounds in Grapes by HPLC-PDA-ESI-MS on a Semimicro Separation Scale

Reversed phase high performance liquid chromatography (RP-HPLC) on a semimicro separation scale was employed to develop a straightforward method for the simultaneous separation, identification, and quantification of phenolic compounds occurring in whole berries of Vitis vinifera, which comprise phenolic acids, flavonols, catechins, stilbenes, and anthocyanins. A C-18 narrow bore column of 150 x 2.0 mm I.D. and a semimicro photodiode array detector (PDA) cell of 2.5 microL, in conjunction with a mass spectrometry detector equipped with an electrospray ionization source (ESI-MS) to confirm peak identification, were employed. The C-18 narrow bore column was eluted by a multisegment gradient of increasing concentration of acetonitrile in water-formic acid solution that was optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their retention times and both UV-visible and mass spectra, acquired by a mass spectrometer (MS) equipped with an electrospray ionization (ESI) source, employed in conjunction with the PDA detector. Libraries comprising retention times, UV-visible, and mass spectra for major phenolic compounds expected in grape berries were made by subjecting solutions of each phenolic standard to the optimized RP-HPLC method. Quantification of individual compounds was performed by the external standard method using a six point regression graph of the UV-visible absorption data collected at the wavelength of maximum absorbance of each analyte determined by the PDA spectra. The RP-HPLC method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. The developed method was successfully applied to identify the phenolic compounds occurring in the whole berries of nine red and one white grape of different varieties of Vitis vinifera, comprising some autochthonous varieties of south Italy such as Aglianico, Malvasia Nera, Uva di Troia, Negroamaro, Primitivo, and Susumaniello. Large differences in the content of phenolic compounds was found in the investigated grape varieties. As expected, only glycosilated flavonols were quantified, and the total amount of these compounds was higher in the whole berries of red grapes than in the white Moscato, where the most abundant phenolic compound was quercetin 3-O-glucoside. In almost all samples, the most and least abundant anthocyanins were malvidin 3-O-glucoside and cyanidin 3-O-glucoside, respectively, with the exception of Uva di Troia where the least abundant anthocyanin was delphinidin 3-O-glucoside.

Fusarium verticillioides and maize interaction in vitro: relationship between oxylipin cross-talk and fumonisin synthesis

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize. Even if frequently asymptomatic, it can produce a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins are factors able to modulate the secondary metabolism in fungi. To uncover the existence of the relationship between oxylipin production and fumonisin synthesis in F. verticillioides, we analysed some molecular and physiological parameters, such as the expression of genes whose products are related to oxylipin synthesis (i.e. lipoxygenase, diol synthases and fatty acid oxidase), the oxylipin profile of both cracked maize and the pathogen by using a lipidomic approach (i.e. combining LC-TOF and LC-MS/MS approaches with a robust statistical analysis) and the synthesis of fumonisin B1. The results suggested a close relationship between the modification of the path...

Aflatoxin Control in Maize by Trametes versicolor

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1.

Grape variety related trans-resveratrol induction affects Aspergillus carbonarius growth and ochratoxin A biosynthesis.

The paper reports the results of a study performed to investigate the influence of the grape variety on the growth of Aspergillus carbonarius on grape berries and the correlation between the amount of ochratoxin A (OTA) and the content of trans-resveratrol produced after fungal contamination. Variations in the amount of OTA produced by the fungus are observed depending on both grape variety and on the induction of trans-resveratrol determined during the infection. The obtained data suggest that if an increase in trans-resveratrol production in grape berries occurs early after the fungal infection, the berry exploits this compound to control OTA synthesis. If the increase in trans-resveratrol concentration is delayed after fungal infection (40 h), a control of OTA accumulation can not be achieved. The possibility of exerting significant control of OTA biosynthesis by this phytoalexin seems to rely in the promptness of its production, as occurs also in other fungus plant interactions and, in turn, seems to be dependent also on grape cultivar. In this fungus-plant system, trans-resveratrol appears to represent a defence-related compound toward A. carbonarius and OTA contamination.

Functional roles of the fatty acid desaturases encoded by KlOLE1, FAD2 and FAD3 in the yeast Kluyveromyces lactis

Functional properties of cell membranes depend on their composition, particularly on the relative amount of saturated, unsaturated and polyunsaturated fatty acids present in the phospholipids. The aim of this study was to investigate the effect of cell membrane composition on cell fitness, adaptation and stress response in Kluyveromyces lactis. To this purpose, we have deleted the genes FAD2 and FAD3 encoding Δ12 and ω3 desaturases in Kluyveromyces lactis, thus generating mutant strains with altered fatty acid composition of membranes. These strains were viable and able to grow in stressing conditions like hypoxia and low temperature. Deletion of the Δ9 desaturase-encoding gene KlOLE1 resulted in lethality, suggesting that this enzyme has an essential role in this yeast. Transcription of the desaturase genes KlOLE1, FAD2 and FAD3 and cellular localization of the corresponding enzymes, have been studied under hypoxia and cold stress. Our findings indicate that expression of these desaturase genes and membrane composition were modulated by hypoxia and temperature stress, although the changes induced by these and other assayed conditions did not dramatically affect the general cellular fitness.

Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.

Trametes versicolor bioactive compounds stimulate Aspergillus flavus antioxidant system and inhibit aflatoxin synthesis

Aflatoxins are bioactive compounds, which are toxic and carcinogenic for humans and animals, and are produced mainly by Aspergillus flavus and A. parasiticus. We studied the effect of some bioactive compounds produced into the culture filtrates of the basidiomycete Trametes versicolor on A. flavus growth and aflatoxin synthesis. Some aspects of the mechanism of action of different filtrate fractions were investigated.The results showed that these compounds stimulate the antioxidant system of the Aspergillus mycelia by the induction of a transient and modulated oxidative burst (peroxides and superoxide anions formation) perceived by Afyap1 transcription factor. T. versicolor culture filtrate fractions, significantly inhibited aflatoxin biosynthesis (A: 75% and B: 55%) apparently by stimulating endogenous antioxidant system. In conclusion, bioactive compounds present in the T. versicolor filtrates probably act by modulating the fungal antioxidant system, eventually leading to aflatoxin inhibition. T. versicolor could represent a novel promising tool to control aflatoxins in foods and feeds.